Foodborne Disease Handbook Second Edition, Revised and Expanded Volume 4: Seafood and Environmental Toxins
edited by
Y. H. Hui Science Technology System West Sacramento, California
David Kitts University of British Columbia Vancouver, British Columbia, Canada
Peggy S. Stanfield Dietetics Resources Twin Falls, Idaho
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D E K K E R
MARCEL DEKKER, INC.
NEWYORK BASEL
ISBN: 0-8247-0344-8
This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York. NY 10016 tell 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekkcr AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzcrland tel: 41-61-261-8482: fax: 41-61-261-8896 World Wide Web http:llwww.dekker.com
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Introduction to the Handbook
The Foodborne Disease Handbook, Second Edition, Revised and Expanded, could not be appearing at a more auspicious time. Never before has the campaign for food safety been pursued so intensely on so many fronts in virtually every country around the world. This new edition reflects at least one of the many aspects of that intense and multifaceted campaign: namely, that research on food safety has been very productive in the years since the first edition appeared. The Handbook is now presented in four volumes instead of the three of the 1994 edition. The four volumes are composed of 86 chapters, a 22% increase over the 67 chapters of thefirstedition. Much of the information in thefirst edition has been carried forward to this new edition because that information is still as reliable and pertinent as it was in 1994. This integration of the older data with the latest research findings gives the readera secure scientific foundation on which to base important decisions affecting the public’s health. We are not so naive as to think that only scientific facts influence decisions affecting food safety. Political and economic factors and compelling national interests may carry greater weight in the minds of decision-makers than the scientific findings offered in this new edition. However,if persons in the higher levelsof national governments and international agencies, such as the Codex Alimentarius Commission, the World Trade Organization, the World Health Organization, and the Food and Agriculture Organization, who must bear the burden of decision-making need and are willingto entertain scientific findings, then the information in these four volumes will serve them well indeed. During the last decade of the previous century, we witnessed an unprecedentedly intense and varied program of research on food safety, as we have already noted. There are compelling forces driving these research efforts. The traditional food-associated pathogens, parasites, and toxins of forty years ago still continue to cause problems today, but newer or less well-known species and strains present extraordinary challenges to human health. These newer threats may be serious even for the immunocompetent, but for the immunocompromised they can be devastating. The relative numbers of the immunocompromised in the world population are increasing daily. We include here not just those also the elderly; thevery young; affected by the human inmunodeficiency virus (HIV), but the recipients of radiation treatments, chemotherapy, and immunosuppressive drugs; paiii
iV
Introduction to the Handbook
tients undergoing major invasive diagnostic or surgical procedures; and sufferers of debilitating diseases such as diabetes.To this daunting list of challenges must be added numerous instances of microbial resistance to antibiotics. Moreover, it is not yet clear how the great HACCP experiment will play out on the worldwide stage of food safety. Altruism and profit motivation have always made strange bedfellows in the food industry. It remains to be seen whether HACCP will succeed in wedding these two disparate motives into a unifying force for the benefitofall concerned-producers, manufacturers, retailers, and consumers. That HACCP shows great promise is thoroughly discussed in Volume 2, with an emphasis on sanitation in a public eating place. All the foregoing factors lend a sense of urgency to the task of rapidly identifying toxins, species, and strainsof pathogens and parasites as etiologic agents, and of determining their roles in the epidemiology and epizootiology of disease outbreaks, which are described in detail throughout the Foodborne Diseuse Handbook. It is very fortunate for the consumerthat there existsin the food industry a dedicated cadre of scientific specialists who scrutinize all aspects of food production and bring their expertise to bear on the potential hazards they know best. A good sampling of the kinds of work they do is contained in these four new volumes of the Hmdbook. And the benefits of their research are obvious to the scientific specialist who wants to learn even more about food hazards, to the scientific generalist who is curious about everything and who will be delighted to find a good sourceof accurate, up-to-date information, and to consumers who care about what they eat. We are confident that these four volumes will provide competent, trustworthy, and timely information to inquiring readers, no matter what roles they may play in the global campaign to achieve food safety.
Y. H. Hui J. Richard Gorhrrm David Kitts K. D. Murre11 Wui-Kit Nip Merle D. Pierson Syed A. Scrttar R. A. Smith David G. Spoerke, Jr. Peggy S. Stnnjield
Preface
Most people prefer to think of marine environments farthest removed from human settlements as being pristine and pure; however, much of the ocean is under attack on several fronts. One of the foci of this fourth volume of the Foodborne Disease Hundbook is the topic of pollutants, which are being deposited in the oceansin unprecedented quantitiesas raw sewage (often with viable pathogens and parasites), industrial effluents containing toxic or radioactive chemicals, trash and garbage, pesticides in runoff from crop lands, and top soil, one of our most valuable natural resources. Some of these potentially harmful organisms and chemicals enter the food webs of freshwater and marine organisms. Someof these organisms are harvested for human food and have the capacity and tendency to concentrate and sequester in their bodies many of the chemicals and pathogens present in the aquatic environments. The methods of how harmful organisms or hazardous chemicals are detected, analyzed, and identified, and how they can affect human health, are thoroughly reviewed in this volume. In contrast, some marine organisms do not collect toxins from the environment but rather produce their own toxins as a part of normal metabolic processes. When such fish are usedas human food, the result can belife-threatening.Thisvolumediscussesthe species and toxins of importance, analytical methods, and epidemiological aspects of intoxication. The seafood processor was one of the first food industries required to implement the HACCP principles. The development of the seafood HACCP program and its benefits to the consumer are discussed in this volume. A cloud of controversy hovers over the concept of food irradiation. In this volume we present informative facts needed for the reader to come to an enlightened conclusion about the safety of food irradiation. We caution that no matter how successful irradiation might be, mishandling after irradiation treatment makes the product unsafe. We also discuss the continuing need to adhere to HACCP principles and essential sanitary standards that make it possible for HACCP to work. Many, perhaps even most, of the food toxicity issues addressed in this volume are subtle and unknown to most of us. For example, the use of food additives, radioactive isotopes, pesticide chemicals applied to our crops, toxicants occurring naturally in some of our foods, and the therapeutic and growth-promoting drugs fed to domestic animals V
Vi
Preface
are important topics of food toxicology. In addition, plasticizers i n many kinds of vessels used for food storage and handling mayin some instances be a source of toxic chemicals. Most consumers are quite unaware of rat hairs, beetle setae, and fly eggs in their food, and less aware of what effect, if any, such adulterants might have on their health. The editors and contributors to the fourth volume of the Foodborne Disease H L U I ~ book have provided an abundance of facts and supporting explanatory information enabling readers to make confident decisions about their health. Moreover, all sectors of the food industry have the tools needed to apply the sanitary practices and HACCP-driven safeguards that will result in the prevention of foodborne diseases and helpto make available wholesome foods for all consumers around the world. Volume 4 is a composite of current information and policies that enable proper risk-assessment decisions to be made regarding potential food toxicants derived naturally in the environment or through agricultural production and food processing practices.
Y. H . Hui David Kitts Peggy S. Stcrt1jieId
Contents
Itltroduction to the Handbook Pwfrtce Contributors Contents of Other Vnlurnes
...
ill
I’
Xi
...
.v11 l
I. Poison Centers 1.
SeafoodandEnvironmentalToxicantExposures:TheRoleofPoisonCenters David G. Spoerke, Jr.
11.
Seafood Toxins
2.
Fish Toxins Bruce W. Hctlstertd
1
23
3. Other Poisonous Marine Animals Bruce W. Hdstead
51
4. Shellfish Chemical Poisoning L w d m E. Llewellyn
77
5. Pathogens Transmitted by Seafood
109
Russell P. Herwig
6. Laboratory Methodology for Shellfish Toxins Dcwid Kitts
183
Vii
Viii
7.
Contents
Ciguatera Fish Poisoning Yoshitsugi Hokama and Joanne S. M. YoshikLrwa-Ebesu
209
8. Tetrodotoxin Joanne S. M. Yoshikawa-Ebesu, Yoshitsugi Hokama, and Tamao Noguchi
253
9.
287
Epidemiology of Seafood Poisoning b r a E. Fleming, Dolores Katz, Judy A. Bean, and Roberta Hammond
10. The Medical Management of Seafood Poisoning Donna Glcrd Blythe. Eileen Hack, Giavanni Washington, and b r a E. Fleming
31 1
11. The U S . National Shellfish Sanitation Program
32 l
Rebecca A. Reid and Timothy D. Durance 12. HACCP, Seafood, and the U.S. Food and Drug Administration Kim R. Young, Miguel Rodrigues Kamat, and George Perry Hoskin
339
111. Environmental Toxins
13. Toxicology and Risk Assessment Donald J. Ecobichon
347
14. Nutritional Toxicology David Kitts
379
15. Food Additives Laszlo P. Somogyi
447
16. Analysis of Aquatic Contaminants Joe W. Kiceniuk
517
17. Agricultural Chemicals Debra L. Browning and Carl K. Winter
537
18. Radioactivity in Food and Water Hank Kocol
557
19. Food Irradiation Hank Kocol
57 1
20.
Drug Residues in Foods of Animal Origin Austin R. L m g and Jose E. Roybol
579
21.
Migratory Chemicals from Food Containers and Preparation Utensils Yvonne V. Yutrn
599
Contents
ix
22. FoodandHardForeignObjects:AReview J. Richard Gorham
617
23.Food,Filth,andDisease:AReview J. Richard Gor-ham
627
24.
Ides
Food FilthandAnalyticalMethodology:ASynopsis J. Richnrd Gorhnm
639
645
This Page Intentionally Left Blank
Contributors
Judy A. Bean Children's Hospital of Cincinnati, Cincinnati, Ohio,and National Institute of Environmental Health Sciences (NIEHS) Marine and Freshwater Biomedical Sciences Center, University of Miami Rosensteil School of Marine and Atmospheric Sciences, Miami, Florida
Donna Glad Blythe Department of Epidemiology and Public Health, University of Miami School of Medicine, Miami, Florida Debra L. Browning Food Safe Program, Department of Food Science and Technology, University of California-Davis, Davis, California Timothy D. Durance Food, Nutrition, and Health, University of British Columbia, Vancouver, British Columbia, Canada Donald J. Ecobichon Department of Pharmacology and Toxicology, Queen's
Univer-
sity, Kingston, Ontario, Canada
Lora E. Fleming Department of Epidemiology and Public Health, University of Miami School of Medicine, and National Institute of Environmental Health Sciences (NIEHS) MarineandFreshwaterBiomedicalSciencesCenter,University of MiamiRosensteil School of Marine and Atmospheric Sciences, Miami, Florida
J. Richard Gorham
Department of Preventive Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland
Eileen Hack Department of Epidemiology andPublicHealth,University School of Medicine, Miami, Florida Bruce W. Halstead
of Miami
InternationalBiotoxicologicalCenter,WorldLifeResearchInsti-
tute, Colton, California
Roberta Hammond State of Florida Department of Health, Tallahassee, Florida Russell P. Herwig School of Fisheries, University of Washington, Seattle, Washington xi
xii
Contributors
Yoshitsugi Hokama Pathology Department, JohnA. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii George Perry Hoskin Office of Seafood, Division of Science and Applied Technology, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, D.C.
Miguel Rodrigues Kamat Office of Seafood, Division of Programs and Enforcement Policy, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, D.C.
Dolores Katz Department of EpidemiologyandPublicHealth,UniversityofMiami School of Medicine,Miami,andState of FloridaDepartment ofHealth,Tallahassee, Florida
Joe W. Kiceniuk Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada
David Kitts
Food, Nutrition, and Health, University British Columbia, Canada
of British Columbia, Vancouver,
Hank Kocol* HealthPhysicist,Roseville,California Lyndon E. Llewellyn Australian Institute of Marine Science, Townsville, Queensland, Australia
Austin R. Long
Pacific Regional Laboratory Northwest, U.S. Food and Drug Administration, Bothell, Washington
TamaoNoguchi RebeccaA.Reid
Laboratory of Food Hygiene, Nagasaki University, Nagasaki, Japan Department of Fisheries and Oceans, Vancouver, British Columbia,
Canada
Jose E. Roybal AnimalDrugsResearchCenter,
U.S. Food and DrugAdministration,
Denver, Colorado
Laszlo P. Somogyi ConsultingFoodScientist,Kensington,California DavidG. Spoerke, Jr. BristleconeEnterprises,Denver,Colorado Giavanni Washington Department of Epidemiology and Public Health, University Miami School of Medicine, Miami, Florida
of
Carl K. Winter Department of Food Science and Technology, Universityof CaliforniaDavis, Davis, California
Joanne S. M. Yoshikawa-Ebesu Oceanit Test Systems, Inc., Honolulu, Hawaii Kim R. Young Office of Seafood, Divisionof Programs and Enforcement Policy, Center for Food Safety and Applied Nutrition,U.S. Food and Dmg Administration, Washington, D.C. Yvonne V. Yuan School of Nutrition,RyersonPolytechnicUniversity,Toronto,Ontario, Canada
* F o n w r c!ffi/itrtiorz: California Department of Health Services, Roseville, California.
Contents of Other Volumes
VOLUME 1: BACTERIAL PATHOGENS
I. Poison Centers 1.
The Role of U.S. Poison Centers in Bacterial Exposures David G. Spoerke, Jr.
11. Bacterial Pathogens 2 . Bacterial Biota (Flora) in Foods Jut1re.s M. Joy 3. Aerotmtzns hydrophiltr Carlos Aheytcr, Jr., Srmuel A. Palumbo, cltrd Gertrrd N. Steltnrr, Jr.
4. Update: Food Poisoning and Other Diseases Induced Kcrthleetl T. Rtrjkowski cwd Jcrtnes L. Smith
by Bacillus cereus
5. B r u c e l l ~ ~ Shirley M. Hullitrg nrtd Edward J. Youtrg 6.
Campylobncter jejutri Dorz A. Frtrtzco ntzd Charles E. Williatm
7.
Clostritliunr botulirwn Johtr W. Austitl erne1 Karen L. Dodds
8.
Clostridium pelfiingetzs Dorotl1.v M. Wrigley xiii
xiv
Contents of Other Voolumes
9. Eschericlritr coli Mcrrguerite A. Neil, Phillip I. Tarr, David N. Toylor, arld Mtrrcio Wolf 10. Listerirr rrrormytogerres
Cutherirre W. Dortnelly 11.
Bacteriology of Salrnorwllcr Robill C. Anderson crnd Richlrrd L. Ziprirl
12. Salmonellosis i n Animals Dmid J. Nisbet nrrd Richrrrd L. Zipritr
13. Human Salmonellosis: General Medical Aspects Richcrrd L. Zipritr trtrd Michrrel H. Hrme 14. Shigelltr
Allthotry T. Muurelli rrr~dKeith A. Lntrrpel 15.
Strrl~hyloc,occust1ureu.s Scott E. Mcrrtirr, Eric R. Myers, artd Jokrr J. I m d o l o
16. Vibrio cholertre Charles A. Kaysner rrnd June H. Wetherirlgtorl 17.
Vibrio prrrtrhtremolytic~~.s TuuTjyi Clmi t r r d Johrr L. Pace
18.
Vilwio vulnificus Arlc1er.v Dtrlsgntrr-d,Lise H@i,Debi Lirtkous, turd Jcmes D. Oliver
19.
Yersirritr Scott A. Mirlrrick, M i c h e l J. Smith, Steverr D. Wengant, mrd Peter Ferrg
111. Disease Surveillance, Investigation, and Indicator Organisms
20. Surveillance of Foodborne Disease EW[JII C. D. Todd 21.
22.
Investigating Foodborne Disease D d r L. Morse, Grrtllrie S. Birkhecrd, crrrd Jock J. Guwvich Indicator Organisms in Foods Jtrrtlrs
M. Jcry
xv
Contents of Other Volumes
VOLUME 2: VIRUSES, PARASITES, PATHOGENS, AND HACCP
I. Poison Centers 1.
The Role of Poison Centers in the United States Dmid G. Spoerke, Jr.
11. Viruses 2. Hepatitis A and E Viruses There,sa L. Cromearls, Michael 0. Favoro1~,Omnntr V. Naitrcrtr, Mtrrgo1i.s
trtld
Htrrold S.
3. Norwalk Virus and the Small Round Viruses Causing Foodborne Gastroenteritis Hrrzel Appleton
4. Rotavirus Syed A. Strttw, V. Sustrrl Spritrgthorpe, raid Jmorl A. Tetro 5.
Other Foodborne Viruses Syecl A. Sattrrr trtrci Jcrsotr A. Tetra
6. Detection of Human Enteric Viruses in Foods Lec~-A~Irr JtrYkus 7. Medical Management of Foodborne Viral Gastroenteritis and Hepatitis Su:trnne M. Matsui trnd Rtrmsey C. Cheurrg 8. Epidemiology of Foodborne Viral Infections Thomas M. Liithi
9. Environmental Considerations in Preventing the Foodborne Spread of Hepatitis A Syed A. Srrttar a t d Scrbtrh Bidawid 111. Parasites 10. Taeniasis and Cysticercosis Zbigrriew S. Pawlowski m d K. D. MLrrrell
11.
12.
Meatborne Helminth Infections: Trichinellosis William C. Crrnlpbell Fish- and Invertebrate-Borne Helminths
John H . Cross
xvi
Contents of Other Volumes
13. Waterborne and Foodborne Protozoa Rotlrrld Frryer. 14. Medical Management Prrul Pro& 15. Inmunodiagnosis of Infections with Cestodes Bruno Gottsteitl
16. Immunodiagnosis: Nematodes H . Ray Gamble
17. Diagnosis of Toxoplasmosis A I m M.Johnsow rrtld J. P. DuDey 18. Seafood Parasites: Prevention, Inspection, and HACCP Atw M.Adams n t d Debra D. DeVlieger
IV.
HACCP and the Foodservice Industries
19. Foodservice Operations: HACCP Principles 0. Peter Stlyder, Jr.
20. Foodservice Operations: HACCP Control Program 0. Peter Snyder, Jr. It1de.v
VOLUME 3: PLANT TOXICANTS
I. Poison Centers 1.
U.S. Poison Centers for Exposures to Plant and Mushroom Toxins Dovid G. Spoerke, Jr.
11. Selected Plant Toxicants
2. Toxicology of Naturally Occurring Chemicals in Food Ross C. Beier crnd Herbert N. Nigg 3. Poisonous Higher Plants Doreen Grcrce L m g crttd R. A. Smith 4. Alkaloids R . A. Smith
Contents of Other Volumes
5.
Antinutritional Factors Related to Proteins and Amino Acids It-vitl E. Liener
6. Glycosides W d t r r Mujcrk und Miclruel H . Berm
7. Analytical Methodology for Plant Toxicants Alister David Muir 8. Medical Management and Plant Poisoning Robert H. Poppetlgcr 9. Plant Toxicants and Livestock: Prevention and Management Michcrel H. R d p k s 111. Fungal Toxicants 10. Aspergillus
.&$a Kozakiewicz 11.
Clnviceps and Related Fungi Gretchen A. Kuldau and Charles W. Bacotl
12. Fusarium Walter F. 0. Murcrsrrs 13.
Perricilliutu John 1. Pitt
14.
Foodborne Disease and Mycotoxin Epidemiology Sara H d e Hen? and F. Xavier Bosclr
15.
Mycotoxicoses: The Effects of Interactions with Mycotoxins Heather A. Kosllitlsky. Adrietrtre Woytowich, crtrci George G. Kl1rrchatourirrn.s
16.
Analytical Methodology for Mycotoxins James K. Porter
17. Mycotoxin Analysis: Immunological Techniques Futl S. QILI 18.
Mushroom Biology: General Identification Features Drrvid G. Spoerke, Jr.
xvii
xviii
Contents of Other Volumes
19. Identification of MushroomPoisoning(Mycetismus),Epidemiology, and Medical Management David G. Spoerke, Jr. 20. FungiinFolkMedicineandSociety David G. Spoerke, Jr.
Seafood and Environmental Toxicant Exposures: The Role of Poison Centers
Epidemiology 1 AAPCC A. 2 B. Who staffs poison a center'? 3 C. Whattypes of callsarereceived? 4 D. How are calls handled? 5 E. What references are used'! 6 F. How are poisoncentersmonitoredfor quality? 7 G. Professional and public education programs 7 Related H. toxicology organizations 7 I. International affiliations IO J. Toxicology and poison center Web sites IO 11. PoisonInformationCenters intheUnitedStates 10 References 2 l I.
1.
EPIDEMIOLOGY
Epidemiological studies aid treatment facilities in determining risk factors and who becomes exposed, and in establishing the probable outcomes of various treatments. A few organizations have attemptedto gather such information and organizeit into yearly reports. The American Associationof Poison Control Centers (AAPCC) and some federal agencies work toward obtaining epidemiological information, while the AAPCC has an active role in assisting with the treatment of exposures Epidemiological studies assist government and industry in determining package safety, effective treatment measures, conditions of exposure, and frequency of exposure. Studies on seafood and environmental exposures provide information on the type of people most commonly involved in exposures. Are they children, adults at home, outdoorsmen, industrial workers, or blue-collar workers. Studies can also tell us which bacterial species are most commonly involved. What symptoms are seenfirst, what is the onset
Spoerke
2
of symptoms, and are theirany sequalae may also be determined and compared to current norms. A.
AAPCC
1. What Are Poison Centers and the AAPCC? The group in the United States concerned, on a daily basis, with potential poisonings due to household agents, industrial agents, biologics, and food poisoning (including seafood poisoning) is the AAPCC. This is an affliliation of local and regional centers that provides information to health care professionals and the lay public concerning all aspects of poisoning. These centers also refer patients to treatment centers. This group of affiliated centers is often supported by government and private funds, as well as industrial sources. in the late 1950s; the first is thought to have been in Poison centers were started the Chicago area. The idea caught on quickly andat the peak of the movement there were hundreds of centers throughout the United States. Unfortunately there were few or no standards for what could be called a poison center, including the type of staff, hours of operation, or information resources. One center may have hada dedicated staff of doctors, pharmacists, and nurses trained specifically in handling poison cases, while another had nothing but a book on toxicology in the emergency room or hospital library. In 1993 the Health and Safety Code (Sec. 777.002) specified that poison centers provide 24-hour service for public and health care professionals and meet the requirements established by the AAPCC. This action helped the AAPCC standardize the staffs and activities of the various poison centers. The federal government does not fund poison centers, even though for every dollar spent on poison centers there is a savings of $2-$9 in unnecessary expenses (1,2). The federal agency responsible for the Poison Prevention Packaging Act is the U.S. Consumer Product Safety Commission (CPSC). The National Clearinghouse for Poison Control Centers initially collected data on poisonings and information on commercial product ingredients and toxic biological agents. For several years the National Clearinghouse provided product and treatment information to the poison centers. At first most poison centers were fundedby the hospital in which they were located. As the centers grew in size and the numberof calls increased, both city and state governments took on the responsibility of contributing funds. In recent years local governments have found it very difficult to fund such operations and centers havehad to look to private industry for additional funding. Government funding may take several forms, either as a line-item on a state’s budget, as a direct grant, or moneys distributed on a per call basis. Some stateswith fewer residents may contractwith a neighboring state to provide services to its residents. Some states are so populous that more than one center is funded by the state.Industrialfunding also varies,sometimes as a grant,sometimes as paymentfor handling the company’s poison and drug information-related calls,and sometimes as payment for collection of data regarding exposure to the company’s product. Every year the AAPCC reports a summary of all kinds of exposures.
2. Regional Centers The number of listed centers has dropped significantly since its peak of more than 600. Many centers have been combined into regional organizations. These regional poison centers provide poison information, telephone management, and consultation, collect pertinent data, and deliver professional and public education. Cooperation between regional poison
The
of Poison Centers
3
centers and poison treatment facilities is crucial. Regional poison information centers, in cooperation with local hospitals, should determine the treatment capabilitiesof the hospia working relationship with their analytical toxicoltals in the region and identify and have ogy, emergency and critical care, medical transportation, and extracorporeal services. This evaluation should be done for both adults and children. A“region” isusually determined by stateauthorities in conjunctionwith local health agencies and health care providers. Documentation of these state designations must be in writing unless a state chooses (in writing) not to designate any poison center or accepts a designation by other political or health jurisdictions. Regional poison information centers should serve a population base of more than one million people and must receive at least 10,000 human exposure calls per year. The number of certified regional centers in the United States is now less than 50. Certification as a regional center requires the following: Maintain a 24 hourslday, 365 dayslyear service. Provide service to both health care professionals and the public. Have available in the center at all times at least one specialistin poison information. Have on call by telephone at all times a medical director or qualified designee. Readily accessible service by telephone from all areas within the region. Comprehensive poison information resources and comprehensive toxicology information covering both general and specific aspectsof acute and chronic poisoning should be available. A list of on-call poison center specialty consultants. Writtenoperationalguidelines that provideaconsistentapproachtoevaluation, follow-up, and management of toxic exposures should be obtained and maintained. These guidelines must be approved in writing by the medical director of the program. A staff of certified professionals answering the phones (at least one of the persons on the phone has to bea pharmacist or nurse with 2000 hours and 2000 cases of supervised experience). A 24 hourdday physician (board certified) consultation service. An ongoing quality assurance program. Othercriteria,determined bythe AAPCCandestablished with membershipapproval. The regional poison information center must be an institutional lnember ingood standing of the AAPCC. Many hospital emergency rooms still maintain a toxicology reference such as the POISINDEXB system to handle routine exposure cases, but they rely on regional poison centers to handle most of the calls in their area.
B. Who Staffs a Poison Center? The staff of poison centers varies considerably from center to center. The three professional groups most often involved are physicians, nurses, and pharmacists. Who answers the phones is somewhat dependent on thelocallaborpool, moneys available, andthe types of calls being received. Other persons who answer the phone include students in medically related fields, toxicologists, and biologists. Persons responsible for answering the phones are either certifiedby the AAPCC or arein the process of obtaining the certifi-
4
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cation. Passage of an extensive examination on toxicology is required for initial certification, with periodic recertification required. Regardless of who takes the initial call, there is a medical director and other physician backup available. These physicians have specialized training or experience in toxicology and are able to provide in-depth consultations for health care professionals calling the center.
1. Medical Director A poison center medical director should be board certified in medical toxicology or in internal medicine, pediatrics, family medicine, or emergency medicine. The medical director should be ableto demonstrate ongoing interest and expertise in toxicology as evidenced by publications, research, and meeting attendance. The medical director must have a medical staff appointment at a comprehensive poison treatment facility and must be involved in the management of poisoned patients.
2. ManagingDirector The managing director must be a registered nurse, pharmacist, or physician, or hold a degree in a health science discipline. The individual should be certified by the American Board of Medical Toxicology (for physicians) orby the American Board of Applied Toxicology (for nonphysicians). The director must be able to demonstrate ongoing interest and expertise in toxicology.
3. Specialists in PoisonInformation These individuals must be registered nurses, pharmacists, or physicians, or be certified by the AAPCC as a specialist in poison information. Specialists in poison information must complete a training program approved by the nledical director and must be certified by the AAPCC as a specialist in poison information within two examination administrations of their initial eligibility. Specialists not currently certified by the associqt' L Ion must spend an annual average of no less than 16 h o u d w e e k in poison center-related activities. Specialists currently certified by the AAPCC must spend an annual average of no less than 8 hourdweek. Other poison information providers must have sufficient background to understand and interpret standard poison information resources and to transmit that information understandably to both health professionals and the public.
4. Consultants I n addition to physicians specializing in toxicology, most centers also have listsof experts by in many other fields as well. Poison center specialty consultants should be qualified training or experience to provide sophisticated toxicology or patient care information in their area(s) of expertise. In regard to seafoodor environmental toxins, this would include specialists in pesticides, heavy metals, botanical exposures, marine toxins, and hydrocarbons, just to name a few. These experts should be willing to donate their expertise in identifying and handling cases within their specialty. Most poison centers do not have the money to pay a wide variety of consultants.
C. What Types of Calls Are Received? All types of calls are received by poison centers, most of which are handled immediately, while others are referred to more appropriate agencies. Which calls are referred depends
The Role of Poison Centers
5
on the center, its expertise, and the appropriateness of a referral. Below are lists of calls which generally fall into each group Remember, there is considerable variation between poison centers; if there is doubt, call the poison center and they will tell you if your case is more appropriately referred. Poison centers do best on calls regarding acute exposures. Complicated calls regarding exposure to several agents over a long period of time which produce nonspecific symptoms are often referred to other medical specialists, to the toxicologist associated with the center, or to an appropriate government agency. The poison center will often follow-up on these cases to track outcome and the type of service given. Types of calls usually accepted Drug identification Actual acute exposure to a drug or chemical Actual acute exposure to a biological agent (plants, mushrooms, various animals) Information regarding the toxic potential of an agent Possible food poisonings (including seafood poisoning) Exposure to environmental toxins Types of calls often referred Questions regarding treatment of a medical condition (not poisoning) Questions on common bacterial, viral, or parasitic infections General psychiatric questions Proper disposal of household agents such as batteries, bleach, insecticides Use of insecticides (e.g., which insecticide to use, how to use it) unless related to a health issue (e.g., a person allergic to pyrethrins wanting to know which product does not contain pyrethrins)
1. Data Collection Records of all calls/cases handledby the center should bekept in a form that is acceptable as a medical record. The regional poison information center should submit all its human exposure data to the association’s National Data Collection System. The regional poison information center shall tabulate its experience for regional program evaluation on at least an annual basis. N. AAPCC Toxic Exposure Surveillmce Systern (TESS) In 1983theAAPCC formed TESS from the former National Data Collection System. Currently TESS contains nearly 16.2 million human poison exposure cases. Sixty-five poison centers, representing 181 3 million people, participate in the data collection. The information has various uses for both governmental agencies and industry, providing data for product reformulations, repackaging, recall, bans, injury potential, and epidemiology. The summation of each year’s surveillance is published i n the late summer or fall in the Arnericm Jolrrrlal of E~nerge~lcy Mecliciwe.
D.
How Are Calls Handled?
Most poison centers receive requests for information via the telephone. Calls come from both health care professionals and consumers. Only a few requests are received by mail or in person, these are often medicolegal or complex cases. Most centers can be reached as well as by a local number. Busy by a toll-free phone number in the areas they serve, centers have a single number that rings on several lines. Calls are often direct referrals from the 91 1 system.
Spoerke
6
Poison information specialists listen to the caller, recording the history of the case on a standardized form developed by the AAPCC. Basic information such as the agent involved, the amount of the agent, time of ingestion, symptoms, previous treatment, and currentconditionarerecorded, as well as patient information such as sex, age, phone number, who is with the patient, relevant medical history, and sometimes patient address. All information is considered a medical record, and is therefore confidential. The case is evaluated (using various references) as Infonnation only, no patient involved Harmless and not requiring follow-up a follow-up call is given Slightly toxic, no treatment necessary but Potentially toxic, treatment given at home and follow-up given to case resolution Potentially toxic, treatment may or may not be given at home, but it is necessary for the patient to be referred to a medical facility Emergency, an ambulance and/or paramedics are dispatched to the scene. Cases are usually followed until symptoms have resolved. In cases where the patient is referred to a health care facility, the receiving agency is notified. The history is relayed, toxic potential discussed, and suggestion for treatment given.
E. What ReferencesAreUsed? References used also vary from center to center, but virtually all centers use a toxicology system called POISINDEXB which contains lists of products, their ingredients, and suggestions for treatment. The systeln is compiled using medical literature and toxicology specialists throughout the world. Biological products such as plants, insects, mushrooms, and animal bites etc are handled similarly. There is an entry for each individual plant containing a description, the toxic agent present, potential toxic amounts, and so forth. The physician or poison information specialist is then referred to a treatment protocol that may be for a general class of agents; for example, exposure to malathion is referred to a protocol on organophosphate pesticides. An unknown skin irritation or potential infection would deserve a consult with an infectious disease specialist. Questions involving specific agents, such as lead or mercury, are directed to individual treatment protocols. POISINDEXB is available on microfiche, CD-ROM, over a network, or on a mainframe. It is updated every 3-months. Various texts are also used, but much of this infomation is already i n POISINDEXB. It is often difficult to identify some potentially toxic marine animals using a description given over the phone, so often the assistance of a marine biologist is used. If a type of marine food poisoning is involved, the help of an infectious disease consultant and an epidemiologist may be requested. Some poison centers have more experiencewith certain types of poisonings than do others, so often one center will consult another. These are often more complex cases, or cases involving centers in the same region. For example, a poison center in Utah may consult with one in California or Hawaii concerning a lionfish envenomization. A recent trend has been for manufacturers to contract with one poison center to provide poison information services for the whole country. Product information is given
The Role of Poison Centers
7
to only that center and exposures throughout the country can only be handled effectively in that one center.
F. How Are Poison Centers Monitored for Quality? Most poison centers havea system of peer review in place. One person takes a call, another reviews it. Periodic spot reviews are done by supervisory and physician staff. General competence is ensured by certification and recertification via examination of physicians and poison infonnation specialists.
G. Professional and Public Education Programs The regional poison information center provides information on the management of poiat soning to health professionals throughout the region. Public eduction programs aimed educating both children and adults about poison safety and potential dangers should be provided. In thepast, some centers provided stickers or logos suchasOfficer Ugh, Safety Sadie, and Mr. Yuck that could be placed on or near potentially toxic substances. While the intent was to identify potentially toxic substances that children should stay away from, the practice has been much curtailed because in some cases the stickers actually attracted children. Poison prevention week is held each yearin the spring. National attention is focused on the problem of potentially toxic exposures. During this week many centers run special programs for the public. This may include lectures on prevention, potentially toxic agents in the home, potentially toxic biological agents, or general first aid methods. Although an important time for poison centers, public and professional education is a year-round commitment. Physicians are frequently involved in medical toxicology rounds, journal clubs, and lectures by specialty consultants. Health fairs, school programs, and various men’s and women’s clubs are used to educate the public. The extent of these activities is often determined by the amount of funding from government, private organizations, and public donations.
H. Related Toxicology Organizations ACGIH American Conference of Governmentaland IndustrialHygienists Address: Kemper Woods Center; Cincinnati, OH 45240 Phone: 5 13-742-2020 FAX: 5 13-742-3355 ABAT American Board of AppliedToxicology Address: Truman Medical Center, West, 2301 Holmes St., Kansas City, MO 64 108
Phone: 8 16-556-3 1 I 2 FAX: 8 16-88 1-6282 AACT AmericanAssociation of ClinicalToxicologists Address: c/o Medical Toxicology Consultants, Four Columbia Dr., Suite810, Tampa, FL 33606 AAPCC AmericanAssociation of PoisonControlCenters
8
Spoerke
Address: 3201 New Mexico Ave. NW, Washington, DC 20016 Phone: 202-362-72 17 FAX. 202-362-8377 ABEM AmericanBoard of EmergencyMedicine Address: 300 Coolidge Rd., East Lansing, MI 48823 Phone: 5 17-332-4800 FAX. 5 11-332-2234 ACEP American College of Emergency Physicians (Toxicology Section) Address: P.O. Box 6 I99 1 I , Dallas, TX 7526 1-991 1 Phone 800-798- 1822 FAX: 214-580-2816 ACMT American College of Medical Toxicology (formerly ABMT) Address: 777 E. Park Dr., P.O. Box 8820, Harrisburg, PA 17105-8820 Phone: 717-558-7846 FAX: 7 17-558-7841 e-mail:
[email protected] (Linda L. Koval) ACOEM AmericanCollege of OccupationalandEnvironmentalMedicine Address: 55 West Seegers Rd., Arlington Heights, IL 60005 Phone: 708-228-6850 FAX: 708-228- 1856 ACS Association of ClinicalScientists Address: Dept. of Laboratory Medicine, University of Connecticut Medical School, 263 Fannington Ave., Farmington, CT 06030-2225 Phone: 203-679-2328 FAX: 203-679-2328 ACT AmericanCollege of Toxicology Address: 9650 Rockville Pike, Bethesda, MD 20814 Phone: 301 -57 1 - 1 840 FAX: 301-571-1852 AOEC Association of OccupationalandEnvironmentalClinics Address: IO10 Vermont Ave. NW, #513, Washington, DC 20005 Phone: 202-347-4976 FAX: 202-347-4950 e-mail:
[email protected] ASCEPT AustralianSociety of ClinicalandExperimentalPharmacologistsand Toxicologists Address: 145 Macquarie St., Sydney N.S.W 2000 Australia Phone: 61-2-256-5456 FAX: 6 1-2-252-33I O BTS BritishToxicologySociety Address: MJ Tucker,ZenecaPharmaceuticals,22B 1 1 Mareside,Alderley Park, Macclesfield, Cheshire, SKI0 4TG UK Phone: 0428 65 5041 CAPCC CanadianAssociation of PoisonControlCenters Address: Hopital Sainte-Justine, 3 175 Cote Sainte-Catherine, Montreal, Quebec H3T 1 C5, Canada Phone: 5 14-345-4675 FAX: 5 14-345-4822
The Role of Poison Centers
9
CSVVA (CEVAP) Center for the Study of Venoms and Venomous Animals Address: UNESP, Alarneda Santos, N 647, CEP 01419-901, Sa0 Paulo. sp, Brazil Phone 55-0 1 1-252-0233 FAX: 55-01 1-252-0200 EAPCCT EuropeanAssociation of PoisonControlCenters Address: J. Vale, National Poisons Infornlation Centre, P 0 Box 81898 Dep, N-0034 Oslo, Norway Phone: 47-260-8460 HPS HungarianPharrnacologicalSociety Address: Central Research Institute for Chemistry, Hungarian Academy of Sciences, H- 1525 Budapest, P.O. Box 17, Pusztaszeri ut 59-67 Phone: 36- 1- 135-21 12 ISOMT International Society of Occupational Medicine and Toxicology Address:USCSchool of Medicine,222OceanviewAve.,Suite 100, Los Angeles, CA 90057 Phone: 213-365-4000 JSTS JapaneseSociety of ToxicologicalSciences Address: Gakkai Center Building, 4-16, Yayoi 2-chome, Bunkyo-ku, Tokyo 113, Japan Phone: 3-38 12-3093 FAX: 3-3812-3552 SOT Society of Toxicology Address: 1101 14th St., Suite 1100, Washington, DC 20005-5601 Phone: 202-371 - 1393 FAX: 202-37 1- 1090 e-mail:
[email protected] SOTC Society of Toxicology of Canada Address: P.O. Box 517, Beaconsfield, Quebec H9W 5V1, Canada Phone: 5 14-428-2676 FAX: 5 14-482-8648 STP Society of ToxicologicPathologists Address: 875 Kings Hw., Suite 200, Woodbury, NJ 08096-3172 Phone: 609-845-7220 FAX: 609-853-041 1 SSPT Swiss Society of PharmacologyandToxicology Address: Peter Donatsch, Sandoz Pharma AG, Toxicologue 88 1/130, CH4132 Muttenz, Switzerland Phone: 41-61-469-5371 FAX: 41-6 1-469-6565 WFCT WorldFederation of Associations of ClinicalToxicologyCenters and Poison Control Centers Address:CentreAnti-Poisons,HopitalEdonardHerriot, 5 p1 d’Arsonva], 69003 Lyon, France Phone: 33 72 54 80 22 FAX: 33 72 34 55 67
Spoerke
10
1.
InternationalAffiliations
The AAPCC and its members attend various world conferences to learn of toxicology problems and new methods used by other agencies. An especially close relationship has formed between the American and Canadian poison center associations. Once a year the AAPCC and CAPCC hold a joint scientific meeting and invite speakers and other toxicology specialists from throughout the world to attend.
J. Toxicology and Poison Center Web Sites Association of Occupational ctnd Environmental Clinics: This group is dedicated to higher standards of patient-centered, multidisciplinary care emphasizing prevention and total health through information sharing, quality service, and collaborative research. Address:
[email protected] Finger Lrrkes Regional Poison Center. Address:
[email protected] Mediccll/Clinical/Occupntional Toxicology Professionctl Groups A list of primarily U.S. professional groups interested in toxicology. There is a description of each group, addresses, phone numbers, and contact names. Keyword: poison centers, toxicology Address: http://www.pitt.edu/-martint/pages/motoxorg.htm Poison Net A mailing list dedicated to sharing information, problem solving, and networking in the areas of poisoning, poison control centers, hazardous materinot als, and related topics. The list is intended for health care professionals, the lay public. The moderators do not encourage responses to individual poisoning cases from the public. Keywords: poisoning, poison control centers
II. POISON INFORMATION CENTERS IN THE UNITED STATES The Poison Control Center telephone numbers and addresses listed below are thought to be accurate as of the date of publication. Poison Control Center telephone numbers and addresses may change. The address and phone number of the Poison Control Center nearest you should be frequently checked.If the number listed doesnot reach thepoison center, contact the nearest emergency service, such as9 l 1 or your local hospital emergency room. The author disclaims any liability resulting from or relating to any inaccuracies or changes in the phone numbers provided below. This information should NOT be used as a substitute for seeking professional medical diagnosis, treatment, and care. (* Indicates a regional center designated by the American Association of Poison Control Centers.)
11
The Role of Poison Centers ALABAMA
ARKANSAS
Birrningharrr
Little Rock
Regional Poison Control Center* Children's Hospital of Alabama 1600 Seventh Ave., South Birmingham, AL 35233-171 1 800-292-6678 (AL only) 205-933-4050
Arkansas Poison and Drug Information Center University of Arkansas College of Pharmacy 4301 West Markham, Slot 522 Little Rock, AR 77205 800-482-8948 (AR only) 501-661-6161
Tusccrloosu
Alabama Poison Control System, Inc. 408A Paul Bryant Dr. East Tuscaloosa, AL 3540 1 800-462-0800 (AL only) 205-345-0600 ALASKA Arlchorqe
Anchorage Poison Center Providence Hospital P.O. Box 196604 3200 Providence Dr. Anchorage, AK 995 19-6604 800-478-3193 (AK ody) Ftrirbartks
Fairbanks Poison Center Fairbanks Memorial Hospital 1650 Cowles St. Fairbanks, AK 99701 907-456-7 I X2 ARIZONA Pkoetl~s
Samaritan Regional Poison Center'@ Good Samaritan Medical Center 1130 East McDowell Rd., Suite A-5 Phoenix, AZ 85006 602-253-3334 7irc.son
Arizona Poison and Drug Information Center* Arizona Health Sciences Center, Room 1 l56 1501 N. CampbellAve. Tucson, AZ 85724 800-362-0101 (AZ only) 602-626-6016
CALIFORNIA Fresrlo
Frcsno Regional Poison Control Center" Fresno Community Hospital and Medical Center 2823 Fresno St. Fresno, CA 93721 800-346-5922 (CA 011ly) 209-445- 1222 Los Angeles
Los Angeles County University of Southern California Regional Poison Center* 1200 North State. Room I 107 Los Angeles, CA 90033 800-825-2722 2 13-222-3212 Orarlge
University of California lrvine Medical Center Regional Poison Center* 101 The City Dr. South Route 78 Orange, CA 92668-3298 800-544-4404 (CA only) 7 14-634-5988 Richmorld
Chevron Emergency Information Center 15299 San Pablo Ave. P 0. Box 4054 Richmond, CA 94804-0054 800-457-2202 510-233-3737 or 3738
Spoerke
72 S~lc.rtrrnento
Regional Poison Control Center* University of California at Davis Medical Center 2315 Stockton Blvd.. Rm. HSF-124 Sacramento. CA 95817 800-342-3293 (northern CA only) 9 16-734-3692 Scrn Diexo
San Diego Rcgional Poison Center* University of California at San Diego Medical Center 225 Wcst Dickinson St. San Diego, CA 92013-8925 800-876-4766 (CA only) 6 19-543-6000 Son Frt~nci.sc.o
San Francisco Bay Area Poison Center* San Francisco General Hospital 1001 PotreroAve., Rm. IE86 San Francisco, CA 94122 800-523-2222 4 15-476-6600 Son Jose
San Jose Regional Poison Center Santa Clara Valley Medical Center 751SouthBascomAve. San Jose, CA 95128 800-662-9886, 9887 (CA only) 408-299-51 12,51 13, 51 14 COLORADO Den\vr
Rocky Mountain Poison Center* 1010 Yosernite Circle Denver. CO 80230 800-332-3073 (CO only) 303-629- 1 123 CONNECTICUT
DELAWARE Wilnrington
Poison Information Center Medical Center of Delaware Wilmington Hospital 501 West14thSt. Wilmington, DE19899 302-655-3389
DISTRICT OF COLUMBIA Wt~S1Iill~tOll
National Capital Poison Center* Georgetown University Hospital 3800 Reservoir Rd. NW Washington, DC 20007 202-625-3333
FLORIDA Jtrcksonville
Florida Poison Information Center University Medical Center 655 West Eighth St. Jacksonville, FL 32209 904-549-4465 or 764-7667 Tnllohtrssee
Tallahassee Memorial Regional Medical Center 1300 Miccosukk Rd. Tallahassee, FL 32308 904-68 1-541 1 Tu~npo
Tampa Poison Information Center* Tampa General Hospital Davis Islands P.O. Box1289 Tampa, FL 33601 800-282-3171 (FL only) 813-253-4444
~tJ'Llllill,~fOII
Connecticut Poison Control Center University of Connecticut Health Center 263 Farmington Ave. Farmington, CT 06030 800-343-2722 (CT only) 203-679-3456
GEORGIA Atlontcr
Georgia Regional Poison Control Center* Cerady Memorial Hospital
13
The Role of Poison Centers
80 Butler St. SE Box 26066 Atlanta, C A 30335-3801 800-282-5846 (CA only) 404-61 6-9000 Macon
Regional Poison Control Center Medical Center of Central Georgia 777 Hemlock St. Macon, CA 3 I208 912-744-1 146, 1100, 1427 Savannah
Savannah Regional Poison Control Center Memorial Medical Center Inc. 4700 Waters Ave. Savannah, GA 31403 912-355-5228 or 356-5228
Chicago, IL 606 12 800-942-5969 (Northeast IL only) 3 12-942-5969 Normal
Bronlenn Hospital Poison Center Virginia at Franklin Normal, IL 61761 309-454-6666 Springfield
Central and Southern Illinois Poison Resource Center St John’s Hospital 800 East Carpenter St. Springfield, IL 62769 800-252-2022 (IL only) 217-753-3330 Urlmna
HAWAII
Honolulu
Kapiolani Women’s and Children’s Medical Center 1319 Punahou St. Honolulu, HI 96826 800-362-3585, 3586 (HI only) 808-941 -4411
IDAHO Boi.W?
Idaho Poison Center St Alphonsus Regional Medical Center 1055 North Curtis Rd. Boise, ID 83706 800-632-8000 (ID only) 208-378-2707
National Animal Poison Control Center University of Illinois Department of Veterinary Biosciences 2001 South Lincoln Ave., 1220 VMBSB Urbana, IL 61801 800-548-2423 (Subscribers only) 217-333-2053
INDIANA
Indinnapolis
Indiana Poison Center* Methodist Hospital 1701 North Senate Blvd. Indianapolis, IN 46202-1 367 800-382-9097 3 17-929-2323
IOWA ILLINOIS Chiccrgo
Chicago and NE Illinois Regional Poison Control Center Rush Presbyterian-St. Luke’s Medical Center 1653 West Congress Pky.
Des Moines
Variety Club Drug and Poison Information Center Iowa Methodist Medical Center 1200 Pleasant St. Des Moines, IA 50309 800-362-2327 5 15-24 1-6254
Spoerke
14 IOM'U
Cih
University of Iowa Hospitals and Clinics 200 Hawkins Dr. Iowa City, IA 52246 800-272-6477 or 800-362-2327 (IA only) 3 19-356-2922 Cih St. Luke's Poison Center St. Luke's Regional Medical Center 2720 Stone Park Blvd. Sioux City, IA 51 104 800-352-2222 (IA, NE, SD) 712-277-2222
Si0lt.X
KANSAS
Lolii.s\~ille
Kentucky Poison Control Center of Kosair Children's Hospital 315 East Broadway P.0 Box 35070 Louisville, KY 40232 800-722-5725 (KY only) 502-589-8222
LOUISIANA Hou~ncc
Terrebonne General Medical Center Drug and Poison Information Center 936 East Main St. Houma, LA 70360 504-873-4069
Karrscrs C i h
Monroe
Mid America Poison Center Kansas University Medical Center 39th and Rainbow Blvd., Rm. B-400 Kansas City, KS 66160-7231 800-332-6633 (KS only) 9 13-588-6633
Louisiana Drug and Poison Information Center Northeast Louisiana University School of Pharmacy, Sugar Hall Monroe, LA 7 1209-6430 800-256-9822 (LA only) 3 18-362-5393
7opeka
Stormont Vail Regional Medical Center Emergency Department 1500 West 10th Topeka, KS 66604 9 13-354-6100 Wicllitcl
Wesley Medical Center 550 North Hillside Ave Wichita, KS 67214 3 16-688-2222
MAINE
Portlnnd
Maine Poison Control Center Maine Medical Center 22 Bramhall St. Portland, ME 04102 800-442-6305 (ME only) 207-87 1-2950
MARYLAND KENTUCKY Baltinrore
Ft. Tllovra.7
Northern Kentucky Poison Information Center St Luke Hospital 85 North Grand Ave. Ft. Thomas, KY 41075 5 13-872-51 1 1
Maryland Poison Center* University of Maryland School of Pharmacy ' 20 North Pine St. Baltimore, MD 21201 800-492-2414 (MD only) 410-528-7701
75
The Role of Poison Centers MASSACHUSElTS Boston
St. Pa111
Center
Massachusetts Poison Control System* The Children’s Hospital 300 1222 800-222Longwood Ave. Boston, MA 021 15 800-682-9211 (MA only) 617-232-2120 or 735-6607
Minnesota1 Regional Poison Center* Medical St Paul-Ramsey 640 Jackson St. St Paul, MN 55101 (MN only) 612-221-2113
MISSISSIPPI MICHIGAN Adriurr
Bixby Hospital Poison Center Emma L. Bixby Hospital 818 Riverside Ave. Adrian, MI 4922 1 517-263-2412 Detroit
Poison Control Center Children’s Hospital of Michigan 3901 Beaubien Blvd. Detroit, MI 48201 800-462-6642 (outside metropolitan Detroit) 3 13-745-571 1
Juck~ort
University of Mississippi Medical Center 2500 North State St. Jackson, MS 39216 60 1-354-7660 Huttieshrg
Forrest General Hospital 400 S 28th Ave. Hattiesburg, MS 39402 601-288-4235
MISSOURI Kcrrtsus City
Blodgett Regional Poison Center 1840 Wealthy St. S.E. Grand Rapids, MI 49506 800-632-2727 (MI only)
Poison Control Center Children’s Mercy Hospital 2401 Gillharn Rd. Kansas City, MO 64108-9898 8 16-234-3000 or 234-3430
Kolonltrzoo
St. Louis
Grmd Rupids
Bronson Poison Information Center 252 East Lovell St. Kalamazoo, MI 49007 800-442-41 12616 (MI only) 6 16-341 -6409
Regional Poison Center* Cardinal Glennon Children’s Hospital 1465 South Grand Blvd. St. Louis, MO 63104 800-392-9111 (MO only) 800-366-8888 (MO, West IL) 3 14-772-5200
MINNESOTA Minneupolis
Hennepin Regional Poison Center* 701ParkAve. S. Minneapolis, MN 55415 612-347-3144 612-347-3141 (Petline)
MONTANA Derwer
Rocky Mountain Poison and Drug Center Denver, CO 80204 800-525-5042 (MT only)
16
Spoerke
NEBRASKA
NEW MEXICO
Olll(1hLI
Albuquerque
The Poison Center* Children's Memorial Hospital 8301Dodge St. Omaha, NE 681 14 800-955-9119 (WY, NE) 402-390-5400, 5555
New Mexico Poison and Drug Information Center* University of New Mexico Albuquerque, NM 87 13I 800-432-6866 (NM only) 505-843-2551
NEVADA NEW YORK Lcrs Vrp1.s
Hurnana Hospital-Sunrise" 3 l86 Maryland Pky. Las Vcgas, NV 89109 800-446-6I79 (NV only) RPilO
Washoe Medical Center 77 Pringle Way Rcno, NV 89520 702-328-4 144 NEW HAMPSHIRE Lellailol~
New Hampshire Poison Center Dartmouth-Hitchcock Medical Center 1 Medical Center Dr. Lebanon, NH 03756 800-562-8236 (NH only) 603-650-5000 NEW JERSEY
Newcrrk New Jersey Poison Information and Education Systems* 201 LyonsAve. Newark, NJ 071 12 800-962-1253 (NJ only) 20 1 -923-0764 Phi1lip.shur.g
Warren Hospital Poison Control Center 185 Rosberg St. Phillipsburg. NJ 08865 800-962- 1253 008-859-6768
Buffalo
Western New York Poison Control Center Children's Hospital of Buffalo 219 Bryant St. Buffalo, N Y 14222 800-888-7655 ( N Y only) 7 16-878-7654 Mineolu
Long Island Regional Poison Control Center" Winthrop University Hospital 259 First St. Mineola, NY 11501 5 16-542-2323, 2324, 2325 New York C i v
Ncw York City Poison Control Center* 455 First Ave., Rm. 123 New York, NY 10016 2 12-340-4494 2 12-764-7667 Npck Hudson Valley Regional Poison Center Nyack Hospital 160 North Midland Ave. Nyack, NY 10920 800-336-6997 (NY only) 914-353-1000 Rochester
Finger Lakes Regional Poison Control Center University of Rochester Medical Center 601 Eltnwood Ave. Rochester, NY 14642 800-333-0542 (NY only) 7 16-275-515 1
17
The Role of Poison Centers Syrmuse
DAKOTA
Central New York Poison Control Center SUNY Health Science Center 750 E Adams St. Hospital Luke’s St NY 13210 Syracuse, 800-252-5655 Fargo, 3 15-476-4766
NORTH CAROLINA Ashevillc
Western North Carolina Poison Control Center Memorial Mission Hospital 509 Biltmore Ave. Asheville, NC 28801 800-542-4225 (NC only) 704-255-4490 or 258-9907 Chcrrlotte
Carolinas Poison Center Carolinas Medical Center 100 Blythe Blvd. Charlotte, NC 28232-2861 800-848-6946 704-355-4000 Durlzom
Duke Regional Poison Control Center P.O. Box3007 Durham, NC 27710 800-672-1697 (NC only) 919-684-811 1 Greensboro Triad Poison Center Moses H Cone Memorial Hospital 1200 North Elm St. Greensboro, NC 2740 l - 1020 800-953-4001 (NC only) 919-574-8105
NORTH
F0 rg o Poison Dakota North
Center
720 North 4th St. ND 58122 800-732-2200 (ND only) 701-234-5575
OHIO Akron
Akron Regional Poison Center 281 Locust St. Akron, OH 44308 800-362-9922 (OH only) 216-379-8562 C~lrlron
Stark County Poison Control Ccnter Timken Mercy Medical Center 1320 Timken Mercy Dr. NW Canton, OH 44667 800-722-8662 (OH only) 2 16-489- 1304 Cincinrlari
South West Ohio Regional Poison Control System and Cincinnati Drug and Poison Information Center* University of Cincinnati College of Medicine 231 Bethesda Ave., ML #l44 Cincinnati, OH 45267-0144 800-872-51 1 1 (Southwest OH only) 5 13-558-51 1 1 Cleveland
Greater Cleveland Poison Control Center 2074 Abington Rd. Cleveland, OH 44106 2 16-23 1-4455
Hickory
Colundm.7
Catawba Memorial Hospital Poison Control Center 810 Fairgrove Church Rd. SE Hickory, NC 28602 704-322-6649
Central Ohio Poison Center* 700 Children’s Dr. Columbus, OH 43205 800-682-7625 (OH only) 614-228-1323
Spoerke
18 Dayton
West Ohio Regional Poison and Drug Information Center Children’s Medical Center One Children’s Plaza Dayton, OH 45404- 18 15 800-762-0727 (OH only) 5 13-222-2227 Loruin
County Poison Control Center Lorain Community Hospital 3700 Kolbe Rd. Lorain, OH 44053 800-821-8972 (OH only) 216-282-2220
940 NE 13th St. Oklahoma City, OK 73 104 800-522-4611 (OK only) 405-27 1-5454
OREGON Portland
Oregon Poison Center Oregon Health Sciences University 3 181 SW Sam Jackson Park Rd. Portland, OR 97201 800-452-7165 (OR only) 503-494-8968
Sandusky
Firelands Community Hospital Poison Information Center 1101 Decatur St. Sandusky, OH 44870 419-626-7423 Toledo
Poison Information Center of Northwest Ohio Medical College of Ohio Hospital 3000 Arlington Ave. Toledo, OH 49614 800-589-3897 (OH only) 419-381-3897 Yuungsfown
Mahoning Valley Poison Center St Elizabeth Hospital Medical Center 1044 Belmont Ave. Youngstown, OH 44501 800-426-2348 (OH only) 21 6-746-2222 Zanesville
Bethesda Poison Control Center Bethesda Hospital 2951 Maple Ave. Zanesville, OH 43701 800-686-4221 (OH only) 614-454-4221 OKLAHOMA
PENNSYLVANIA Hershey
Central Pennsylvania Poison Center* Milton Hershey Medical Center Pennsylvania State University P.O. Box 850 Hershey, PA 17033 800-521-6110 717-531-611 1 Lancaster
Poison Control Center St. Joseph Hospital and Health Care Center 250 College Ave. Lancaster, PA 17604 7 17-299-4546 Philadelphia
Philadelphia Poison Control Center” One Children’s Center 34th and Civic Center Blvd. Philadelphia, PA 19104 215-386-2100 Pittsburgh
Pittsburgh Poison Center* One Children’s Place 3705 Fifth Ave. at DeSoto St. Pittsburgh, PA 15213 41 2-68 1-6669
Oklahonlu C i h
Williamsport
Oklahoma Poison Control Center Children’s Memorial Hospital
The Williamsport Hospital Poison Control Center
79
The Role of Poison Centers
777 Rural Ave. Williamsport, PA 1770I 717-321-2000
RHODE ISLAND
800 East 21st St. P.O. Box 5045 Sioux Falls, SD 57 117-5045 800-952-0123 (SD only) 800-843-0505 (IA, MN, NE) 605-336-3894
Providence
Rhode Island Poison Center* 593 Eddy St. Providence, RI 02903 40 144-5727
SOUTH CAROLINA Charlotte
Carolinas Poison Center Carolinas Medical Center 1000 Blythe Blvd. Charlotte, NC 28232-2861 800-848-6946 Cohonhia
Palmetto Poison Center University of South Carolina College of Pharmacy Columbia, SC 29208 800-922-11 17 (SC only) 803-765-7359
SOUTH DAKOTA Aberdeen
Poison Control Center St Luke’s Midland Regional Medical Center 305 S. State St. Aberdeen, SD 57401 800-592-1889 (SD, MN, ND, WY) 605-622-5678 Rapid Cif?, Rapid City Regional Poison Control Center 835 Fairmont Blvd. P.O. Box 6000 Rapid City, SD 57709 605-341-3333 Sioux Fcrlls McKennan Poison Center McKennan Hospital
TENNESSEE Knoxville
Knoxville Poison Control Center University of Tennessee Memorial Research Center and Hospital 1924 Alcoa Hwy. Knoxville, TN 37920 615-544-9400 Memphis
Southern Poison Center, Inc. Lebanheur Children’s Medical Center 848 Adams Ave. Memphis, TN 38103-2821 901 -528-6048 Nashville
Middle Tennessee Regional Poison Center, Inc. 501 Oxford House 1161 21st Ave. S, B-IOIVUII Nashville, TN 37232-4632 800-288-9999 (TN only) 615-322-6435
TEXAS Conroe
Montgomery County Poison Information Center Medical Center Hospital 504 Medical Center Blvd. Conroe, TX 77304 409-539-7700 Dallas
North Central Texas Poison Center* Parkland Memorial Hospital 5201 Harry Hines Blvd. P.O. Box 35926 Dallas, TX 75235 800-441-0040 (TX only) 214-590-5000
Spoerke
20
El Paso El Paso Poison Control Center
Thomas General Hospital 4815 Alameda Ave. El Paso, TX 79905 915-533-1244 Gnlveston
Texas State Poison Control Center University of Texas Medical Branch 8th and Mechanic St. Galveston, TX 77550-2780 800-392-8548 (TX only) 7 13-654-1701 (Houston) 409-765-1420 (Galveston) Lubbock
Methodist Hospital Poison Control 3615 19th St. Lubbock, TX 79413 806-793-4366
Box 67 Charlottesville, VA 22901 800-45 1- 1428 (VA only) 804-924-5543 Richmond
Virginia Poison Center Virginia Commonwealth University MCV Station Box 522 Richmond, VA 23298-0522 800-552-6337 (VA only) 804-786-9123
WASHINGTON Washington
P.O. Box 5371 Seattle, WA 98105-0371 800-732-6985 (Within WA) 206-526-2121
UTAH Salt Lake C i h
Utah Poison Control Center* Intermountain Regional Poison Control Center 410 Chipeta Way, Suite 230 Salt Lake City, UT 84108 800-456-7707 (UT only) 801-581-2151
VERMONT Burlington
Vermont Poison Center Medical Center Hospital of Vermont 11 1 Colchester Ave. Burlington, VT 05401 802-658-3456
WEST VIRGINIA Chcrrleston
West Virginia Poison Center* West Virginia University 31 I O MacCorkle Ave. SE Charleston, WV 25304 800-642-3625 (WV only) 304-348-4211 Porkershurg
St. Joseph’s Hospital Center 19th St. and Murdoch Ave. Parkersburg, WV 26 101 304-424-4222
WISCONSIN VIRGINIA Cl~trlorte.sville
Blue Ridge Poison Center* University of Virginia Health Sciences Center
Mndisorr
Regional Poison Control Center University of Wisconsin Hospital 600 Highland Ave. Madison, WI 53792 608-262-3702
21
The Role of Poison Centers Milwaukee
Poison Center of Eastern Wisconsin Children’s Hospital of Wisconsin Poison Wisconsin The West Ave. 9000 Hospital Memorial Children’s Box 1997 P.O. Milwaukee, Dodge8301 W1 53201 414-266-2222
WYOMING Omoha
Center” St.
681 Omaha, NE 14 800-955-91 19 (WY, NE) 402-390-5400. 5555
REFERENCES DL Harrison, JR Draugalis, MK Slack, PC Langly. Cost effectiveness of regional poison control centers.ArchlnternMed156:2601-2608,1996 2. CPSC. CPSC Chairman Ann Brown suggests information technology study to support work of poison ccnters. News release 94-047, March 15, 1994. 1.
This Page Intentionally Left Blank
2 Fish Toxins
I. 11.
111.
1V.
V. VI. VI1. VIIl.
1.
Introduction 23 Toxigcnesis 24 DystrophicationandToxigenesis 24 Icthyosarcotoxic Fish 25 A. Lampreys and hagfish: cyclostome fish 25 B. Sharks,skates,rays,andchimaeras:elasmobranchfish C. Ciguatoxic fish 28 D. Clupeotoxic fish 33 E. Gempylotoxic fish 35 Scombrotoxic F. fish 35 G. Tetrodotoxic 37 fish Icthyootoxic Fish 40 Icthyohcmotoxic Fish 42 Icthyohepatotoxic Fish 43 Icthyoallyeinotoxic(Hallucinogenic)Fish 44 Acknowledgment 45 References 45
26
INTRODUCTION
Fish toxins are of two types: the small, molecular oral biotoxins that are poisonous to eat, and the large molecular venoms that are injected into the body by means of a specialized device known as the venom apparatus. Thus all venoms are poisons, but not all poisons are venoms. In this chapter, only the oral biotoxins are discussed in regard to the names of thetransvectors,theirgeographicaldistribution,clinicalcharacteristics,andabrief statement of the toxin involved. Reports of food poisoningof marine origin are increasing in frequency and outbreaks appear to be spreading geographically. Part of this increase may be credited to heightened awareness, more travel to areas of the world where marine toxicity is endemic, and a greater opportunity for exposure to oral fish toxins, There also has been an increase in
23
Halstead
24
the importation of toxic marine food products into North America, Europe, Russia, Taiwan, Japan, and elsewhere. However, travel and awareness are only two facets of the overall epidemiology of the marine food biotoxication problem. There is evidence that suggests that pollution may be an added factor that needs to be taken into consideration.
II. TOXIGENESIS Toxicity in marine organisms is the result of a progression of biochemical events taking place in the bodyof the target organism. Various combinations of atoms of carbon, hydrogen, oxygen, nitrogen, chlorine, sulfur, and phosphorus are synthesized by the organism into complex biotoxin molecules thatmay have extreme complexity and toxicity. The process by which this is accomplished is referred to as biogenesis, biosynthesis, or, more specifically, as toxigenesis. In actuality, very little is known about the precise chemical processes involved. There is a growing amountof chemical data that suggests that in some instances marine bacteria play a role in toxin biosynthesis. Oral marine biotoxins may develop in the bodies of marine organisms as a result of naturallyoccurringprecursorchemicalagents, or theymay develop as a resultof human-induced chemical pollutants. In either case, the resulting biotoxins are capable of producing serious public health problems. These problems may occur in endemic areas wherever marine biotoxins exist or in far-removed areas to which toxic marine products have been transported. The increase of marine biotoxications worldwide are of grave economic and public health significance.
111.
DYSTROPHICATION AND TOXIGENESIS
There is an aspect of ecotoxicology that appears to have a bearing on the topic of foodborne marine intoxicants, that is, the matter of ocean eutrophication and its resultant dystrophication due to pollution. The process of ocean eutrophication is a phenomenon that has been well documented (1 - 3 ) . Eutrophication is a process of nutrient enrichment involving ocean ecosystems. Dystrophication generally is looked upon as a posteutrophication process in which there is oxygen depletion resulting from the actionof aerobic bacteria upon organic matter accompanied by other poorly defined chemical alterations in the marine environment. These two processes generally are looked upon as an aging activity in a body of water. A vast array of chemical agents, military and industrial pollutants, pesticides, and heavy metals are entering the marine environment and contributing to the eutrophication process. This ocean enrichment process is takingon global proportions and is of growing concern to marine toxicologists. More detailed information on this subject has been published in Ref. 4. Current evidence suggests thatthe combined onslaught of all of these chemical substances entering the ocean environment undoubtedly contributes to the degradative enrichment process. This involves a series of chemical and physical vector forces that presently appearto defy analysis. The ever-increasing chemical contamination of the ocean environment strongly suggests that the growing number of outbreaks of oral intoxications may be related events.
Fish Toxins
25
The bacterial degradation of organic matter by proteolytic bacteria produces a decrease in dissolved oxygen and may increase the growth of sulfate bacteria and the production of hydrogen sulfate and sulfur. Pollutants of various types may upset the phytoplanktoniccycleandaltertheworkofchemicalmediators,resulting ineutrophicationand dystrophication. All of these factors may cause an increasein toxic phytoflagellate blooms and various forms of bacterial toxigenesis. Yasumoto et al. (5,6)and Noguchi etal. (7) reported that the bacterial Psc~1rrlorr1orrcrs and Vibrio species were found in association with toxic pufferfish and toxic phytoflagellates. The investigators concluded that the bacteria were responsible for the production of tetrodotoxin and saxitoxin. Kotaki et al. (8) isolated the bacteriunl Mornellrr species and concluded that it was responsible for the biosynthesis of saxitoxin in cultures of the toxic dinoflagellate P r [ ) t ~ ) ~ o t ~ (Gorryauleu) ~ ~ l l l ~ r . ~ tcrn~crrensisis. The studies cited above provide substantial evidence that microbial organistus are responsible for the production of such toxins as tetrodotoxin, saxitoxin, and some of their congeners. It is possible that the ciguatoxin complex, palytoxin, and probably other marine toxins yet to be identified may also be the products of bacterial activity acting in association with a variety of marine plants and animals (5,6,9-21); Aubert feels that it is difficult to explainthetoxigenesis of phytoplankton by any other means than by bacteria (M. Aubert, personal communication, 1991). Bacterial toxigenesis has now become an area of major epidemiological concernin dealing with outbreaksof organic marine biotoxications. There are various waysof classifying food-borne outbreaks resulting from the ingestion of toxic fishes. Here the outbreaks are arranged phylogenetically accordingto the fish transvectors involved. All of the fish presented in the sections below are members of the phylum Chordata.
IV.
ICHTHYOSARCOTOXIC FISH
Ichthyosarcotoxic fish are those that contain a poison within the flesh of the fish (i.e., the musculature, viscera, skin, or slime) which, when ingested, causes a biotoxication. This category should not be confused with that which causes ichthyootoxism, in which the poison is restricted to the gonads or roe of the fish.
A.
LampreysandHagfish:CyclostomeFish
The cyclostome fish are members of the class Agnatha. The cyclostomes, which include thelampreysand hagfish,are a group of fishlikevertebrateshavinganeel-likeform, cartilaginous or fibrous skeleton, no definite jaws or bony teeth, and a primitive type of cranium. There are no pelvic girdles, paired limbs or true ribs. There are 6-14 pairs of gill pouches opening either directly into the pharynx or into a separate respiratory tube. Only a single nostril is present. Because of their structural simplicity, cyclostomes generally are considered the most primitive of true vertebrates. The hagfish are strictly inhabitants of temperate and subtropical inshore marine waters of the Atlantic and Pacific Oceans. Hagfish are members of the family Myxinidae. The skin of the hagfish is richly supplied with large mucous cells. A large hagfish is said to be capable of filling a twogallon bucket with slime. The slime is reputed to be toxic. Hagfish are rarely eaten as food.
26
Halstead
Representative Species Family: Myxinidae (hagfish) Species: Myxiwe glutinosa Linnaeus. Atlantic hagfish. Length 31 in. (79 cm). Distribution: North Atlantic. Family: Petromyzonidae (lampreys) Species: Petmtnyzon rnarinus Linnaeus. Sea lamprey. Length 33 in. (84 cm). Distribution: Coasts and rivers of both sides of the Atlantic, rivers of the Mediterranean.
Cyclostomc Poisoning Clinicd Chrrrrrcteristics: Poisoning from cyclostomes is rare because they seldom are eaten. The slime is said to be toxic to eat. Symptoms consist of nausea, vomiting, dysenteric diarrhea, tenesmus, abdominal pain, and weakness (22-26). There are no recent accounts of cyclostome poisoning. Treetrttnent:Treatment is symptomatic. See Refs. 27-29. Prevetltion: Most cyclostome poisonings are said to be caused because of failure to deslime the fish. For prevention, some authors claim that if the fresh fish is covered with salt and left in a concentrated brine solution for several hours prior to cooking, the fish is safe to eat (30,31).
B. Sharks, Skates, Rays, and Chimaeras: Elasmobranch Fish The elasmobranch fishes include the sharks, skates, rays, and chimaeras, all of which are members of the class Chondrichthyes. The poisoning is referred to as elasmobranch poisoning when it involves sharks, skates, or rays. When involving chimaeras, also known as the elephantfish or ratfish, the intoxication is known as chimaera poisoning. The sharks, skates, and rays are fishlike vertebrateswith well-developed lower jaws and bony teeth; two pairs of appendages supported by pectoral and pelvic girdles; a cartilaginous skeleton that, while more or less calcified, lacks any true bone; scales, essentially toothlikeinstructure,known as placoidscales;twonostrils,eachsingleandpartially subdivided; and blind olfactory sacs, not opening into the mouth. The posterior end of the vertebral column is either straight or heterocercal. A sympathetic nervous system, pancreas, spleen, and contractile arterial cone are present. There is a series of two, three, or more heart valves and a swim bladder is present. There are five to seven pairs of gills and five to seven gill clefts, each of the latter opening separately to an exterior dorsal fin or fins, and spines, if present, are rigid and not erectile.The skin is with or without dermal denticles, teeth are numerous, and the upper jaw or palatoquadrate cartilage is not fused to the cranium, although it may be attached locally to it. The nostril cartilage is fused to the cranium. At least some of the vertebrae of the trunk region have transverse ribs and the two halves of the pelvic girdle are fused into a single bar. The anus and urogenital canals discharge into a common cloaca and the males are without prepelvic or frontal tenacula.
1, Poisonous Sharks Representrrtive Species Family: Isuridae (mackerel sharks) Species: Crr~-chcrrodoncxrrchrirrs (Linnaeus). Great white shark. Length 20 f t (6 m).
Fish Toxins
27
Distribution: Cosmopolitan: tropical, subtropical, and warm temperate seas worldwide. Family: Carcharhinidae (requiem sharks) Species: Ccrrchcrrhinus me1mopteru.s (Quoy and Gaimard).Black-tip reef shark. Length 6.5 ft (2 m). Distribution: Tropical-Indo Pacific region. Species: Carchnrhitrus amboinet?.sisMuller and Henle. Pig-nosed Shark. Length 82 in. (2.8 m). Distribution: Eastern North Atlantic, Indo-Pacific oceans, South Africa, Madagascar, Gulf of Aden: Indian Ocean, and Australia. 12 f t Species: Cerrcherr11inu.s Ieucas (Valenciennes).Bullheadshark.Length (3.6 m). Distribution: Warm waters of the Atlantic, Pacific, and Indian Oceans. Family: Hexanchidae (cow sharks) Species: Heptranc1riLr.s perlo (Bonnatere). Seven-gilled shark. Length 6.3 ft (2 m). Distribution: Atlantic, Mediterranean, South Africa, and Japan. Family: Dalatidae (sleeper sharks). Species: Sormiosus tnicrocephcrlus (Bloch and Schneider). Greenland shark. Length 6.3 ft (2 m). Distribution: Arctic Atlantic, North Sea. east to the White Sea, and west to the Gulf of St. Lawrence. Family: Sphyrnidae (hammerhead sharks). Species: Sphyrmr ;ygaetrcr (Linnaeus). Scalloped hammerhead shark. Length 14 ft (4 m). Distribution: Tropical to warm temperate belt of the Atlantic and Pacific Oceans.
The elasmobranch form of icthyosarcotoxism is most commonly caused by eating sharkliversandtheflesh of some of the tropical sharks. Skates and rays are seldom involved in food poisoning. Clinicd Characteristics: The ingestion of fresh Greenland shark flesh is toxic to both dogs and man. The symptoms consist of nausea, vomiting, diarrhea, difficulty in walking, convulsions, respiratory distress, and muscular twitching. The local natives gradually build up a tolerance to the poison. The toxicity of Greenland shark poisoning is believed to be due to large amounts of trimethylamine oxide (32). Serious intoxications have resulted from eating the tropical sharks Ctrrchcrroclon curcherrim, C(rrcharhinus tnekrnopterus, Ccwcharhinus amboinensis, Ccrrckrrrhitrus leuc m , He~)trernchinsperlo,and Sphyrnrr zygtrerrcr. The symptoms resulting from the ingestion of shark liver may be severe, developing within 30 minutes after eating. The symptoms consist of nausea, vomiting, diarrhea, abdominal pain, headache, joint aches, tingling about the mouth, and a burning sensation of the tongue, throat, and esophagus. As time progresses, the symptoms involving the nervous system may worsen, resulting in muscular incoordination and difficulty in breathing due to muscular paralysis, followed by coma, and finally death. Ingestion of the flesh of certain species of tropical and arctic sharks may be dangerous to eat, butthe symptoms usually aremild,consistingmainlyof a gastrointestinal upset.
Halstead
28
Trecrtnw1t: Treatment is symptomatic. See ciguatera fish poisoning. See Refs. (2729).
Prevetrtion: Avoid eating the liver of any shark unless it is known for certain to be edible. The livers of tropical and arctic sharks are known to be especially dangerous to eat. The flesh of tropical and arctic sharks is potentially poisonous and should be eaten with caution.
2. Poisonous Chimaeras The chimaeroids-elephantfish or ratfish-differ from the sharks, skates, and raysin that they have only four pairs of gills and four pairs of gill clefts, with only one opening to the exterior on each side of the head. The dorsal fin and spine are erectile. In the adult, the skin is naked, without dermal denticles. Teeth are represented by six pairs of grinding plates; the upper jaw or platorate cartilage is fused with the cranium and the rostral cartiof the lage is articulated to the cranium, not fused. Ribs are lacking and the two halves pelvic girdle separate. There is no cloaca, and the urogenital aperture is distinct from the anus and posterior to it. The males have an erectile prepelvic tenaculum, usually with a frontal tenaculum on the head (33).
Represewtative Species Family: Chimaeridae (Chimaeras) Species: Chirnrrercr ttwt1stro.w Linnaeus. Length 39 in. European chimaera. Distribution: North Atlantic and Mediterranean.
( 1 m).
Clrimcrerrr Poisotlirzg The musculature and visceraof some of the elephantfish and ratfish have been found to be toxic, but the nature of the chimaera poison is unknown. Cliwiccrl Chcrructeristics: The symptomatology of chimaera poisoning in humans is unknown. Treatmetzt: Treatment is symptomatic. Prevention: Chimaeras should not be used for human consumption.
C. Ciguatoxic Fish Ciguatoxic fish cause oneof the most widespread and seriousforms of ichthyosarcotoxism known. More than 400 species of fish are alleged to have transvectored the ciguatoxin complex poisons that serve as the causative toxins in ciguatera fish poisoning. The fish involved are for the most part tropical or warm, temperate zone reef or inshore species found between 35"N latitude and 34"s latitude in the Caribbean and tropical Pacific and Indian Oceans. Occasionally, offshore fish may be involved, but by far the bulk of the outbreaks have occurred in insular areas. Historically, ciguatera-like symptoms have resulted from eating marine turban shells (Turbo picrr) in the Caribbean (25,34,35), and similar outbreaks have been caused by eating coconut crabs in Tahiti and in the Ryukyu Islands (36,37). No freshwater fish have been incriminated. Many of the ciguatoxic fish are valuable food fish that on occasion become toxic within a few hours of ingesting toxic dinoflagellates or algae in association with toxic dinoflagellates. Carnivorous fish may become toxic as a result of ingesting toxic herbivores. Thus ciguatera is a toxic food chain problem. Once a fish becomes poisonous, the
Fish Toxins
29
toxicity within the body of the fish may continue for a period of many years. One of the species of dinoflagellates .that has been incriminated in ciguatera fish poisoning is Gambierdiscus tosicus Adachi and Fukuyo. Several other dinoflagellate species are highly suspect as causative agents of ciguatera fish poisoning, including Ampl~idiniutncnr'tertre Hulbert, Ostreopsis ovtrt~Fukuyo, Prorocentrum C ' O I ~ C L I V U IFukuyo, I? P. limtr (Ehrenberg) Dodge, and P. me.~iccrnu~r~ Tafall (38). There is growing evidence that suggests that the in a symbiotic primary causative agent in this toxicity cycle may be bacteria that live relationship with dinoflagellates or possibly macroalgae (see Sec. 111). Ciguatoxic fish are a group of phylogentically diversified species, most of which are members of theclassOsteichthyes,thebonyfish.Thebiology of thesefishis as diversified in habitat, habits, feeding, and reproduction as it is in morphology. Consequently, it is impossible to present a stereotyped characterization of a ciguatoxic fish. Moreover, you cannot detect a ciguatoxic fish by its appearance. In one part of an island, any given fish species may be edible, whereas on the opposite side of the island or on an adjacent reef, the same species may be deadly poisonous. This is the major problem in ciguatera fish poisoning. The members of the class Osteichthyes are characterized as having a skeleton, in part or all with true bone; the skull has sutures; and the teeth are fused to the bones. The soft fin rays usually are segmented. Nasal openings on each side usually are double and more or less dorsal in position. The biting edge of the upper jaw usually is formed by a functional lung dermal bones, the premaxillae and the maxillae. A swim bladder or usually is present. There is an intestinal spiral valve in only a few lower groups. Internal fertilization is relatively rare, and there is a pelvic copulation device in only one group (phallosthoids). The embryos are not encapsulated in a case (39).
Representdve Species An attempt to list all of the ciguatoxic fish species that have been incriminated to date would not be feasible; consequently, only a small representative group of species is listed below. The fish are arranged in alphabetical order according to their family names and within the family by their generic names. Family: Acanthuridae (surgeonfish) Species: A c c r t h m s lineatus (Linnaeus). Striped surgeonfish. Length 7 in. (18 cm). Distribution: Indo-Pacific. Family: Balistidae (leatherjackets, filefish, triggerfish) Species: AIutera scriljttr (Osbeck). Scribbled filefish. Length 19 in. (50 cm). Distribution: All warm seas. Species: Btrlistoitfes conspicillurn Bloch and Schneider. Clown triggerfish (Fig. 1 1). Length 13.7 in. (35 cm). Distribution: Tropical Pacific from Polynesia to Madagascar, China, Japan. Family: Carangidae (jacks, pompanos) Species: Ctrrtrn.~ hi[)i)os(Linnaeus). Jack, crevalle. Length 29.5 in. (75 cm). Distribution: Tropical Atlantic. Cuvier. Blue jack. Length 25.5 in. (65 cm). Species: ccrrcrnx melatn~~pgus Distribution: Tropical Pacific. Family: Lutjanidae (snappers) 35.5 in. Species: Lutjarzr4.s bohtrr (Forskil).Redtwo-spottedsnapper.Length (90 cm).
30
Halstead
Distribution: Tropical Indo-Pacific. Species: Lutjrrnus gibbus (Forskil). Humpback snapper. Length 15.5 in. (40 cm). Distribution: Tropical Indo-Pacific. 19.5 in. Species: Lutjatzus vaigiensis (Quoy and Gaimard). Redsnapper.Length (50 cm). Distribution: Indo-Pacific. Family: Mugilidae (mullets). Species: CIlelow vcligiensis (Quoy and Gaimard). Mullet. Length 12 in. (30.5 cm). Distribution: Indo-Pacific. Species: Mugil cepkrr1u.s Linnaeus. Mullet. Length 12 in. (30.5 cm). Distribution: Cosmopolitan in warm temperate seas. Family: Mullidae (goatfish, surmullets) Species: Mulloidicl~thysc/ur(jIatrrma (Forskil). Goatfish. Length 13 in. (35 cm). Distribution: Indo-Pacific. Species: Parupet~euschtyser:\ldros (Lacepide). Goatfish. Length 13 in. (33 cm). Distribution: Indo-Pacific, East Africa. Family: Muraenidae (moray eels) Species: Gytnnothortr.u javcrrlicus (Bleeker). Giant brown moray eel. Length 5 ft (1.5 m). Distribution: Indo-Pacific. Species: Gymtlothorc/.r rnelecrgris (Shaw and Nodder). White-mouthed moray eel. Length 39 in. ( I m). Distribution: Indo-Pacific, Japan. Family: Scombridae (tunas, mackerels, albacore) solrrttdri (Cuvier). Wahoo. Length 78 in. (2 m). Species: Acc~t~thocybiun~ Distribution: Circumtropical. Species: Scombemmorus crrvcrllrr (Cuvier). King mackerel. Length 59 in. (1.5 m). Distribution: Tropical Atlantic. Family: Serranidae (sea basses, grouper) Species: Cephnlopholis nrgus (Bloch and Schneider). Peacock grouper. Length 20 in. (51 cm). Distribution: Indo-Pacific. (Forskil). Brown nnarbled grouper. Length 23.5 Species: E~~ine~~halus.fu.scoglrttr~tu,s in. (60 cm). Distribution: Indo-Pacific. Species: Mycterol~erccr vetzertow (Linnaeus).Poisonousgrouper.Length35 in. (90 cm). Distribution: Western tropical Atlantic. Species: Plectroponrrrs olipccrrlthus Bleeker. Blue-lined coral grouper. Length 2 1 in. (55 cm). Distribution: Indo-Pacific. Species: Vrrriola louti (Forskil). Lyretail grouper. Length 23 in. (60 cm). Distribution: Indo-Pacific. Family: Siganidae (rabbitfish). Species: Sig~rnlrslinearus (Valenciennes). Rabbitfish. Length 1I in. (30 ~111). Distribution: Indo-Pacific. Species: Sigrrrrrrs prrellus (Schlegel). Rabbitfish. Length 10 in. (27 cm). Distribution:Indo-Pacific.
Fish Toxins
31
Family: Sphyraenidae (Barracuda) Species: Sphyraetu~hnrrczcudcl (Walbaum). Great barracuda. Length 5.2 ft (1.6 m). Distribution: All warm seas, except eastern Pacific.
Cigutrtern Fish Poisotlitlg Ciguatera fish poisoning results from the ingestionof any of a large variety of shore and reef fish which are usually subtropical or tropical in their distribution. The degree of freshness of the fish has no bearing on its toxicity. The victim becomes poisoned as a result of ingesting a toxin within the flesh of the fish. Ciguatera is not a form of ordinary bacterial food poisoning. Ciguatera fish poisoning involves a complex of poisons: ciguatoxin, molecular formula CI,,,HXhOll,, molecular weight 1 1 12, median lethal dose (LDS,,) 0.45g/kg mouse intraperitoneal (IP) (40-45); maitotoxin, molecular formula ClhlhH2?cS?07J, approximate molecular weight 3396.1, LDcotoxicity 0.13 g/kg mouse IP (46); scaritoxin, molecular formula and toxicity unknown (47). Ciguatoxin and maitotoxin are two of the most toxic marine poisons known. Ciguatoxin acts by increasing the membrane permeability to sodium ions of excitableneurons by openingthevoltage-dependentsodiumchannels(43,48,49).Repeated doses of ciguatoxin to mice on an experimental basis have been found to produce severe ultrastructural morphological changes in the cardiac muscle cells and endothelial lining cells of blood capillaries in the heart. Damage to the capillaries was followed by effusion of serum and erythrocytes into the interstitial spaces of the myocardium. Swelling of the endothelial lining cells of capillaries caused narrowingof the lumen and accumulation of of cardiac blood platelets in capillaries, which resulted in multiple single-cell necrosis muscle cells (50). These experimental findings in mice may possibly explain some of the clinical cardiac findings in individuals that have suffered multiple exposures to ciguatera fish poisoning. The most toxic part of the fish is usually the liver, followed by the intestines, then the testes, ovaries, and the muscle. As noted in Sec. IV.C, the toxin originates in the food web of the fish. There is no evidence of a seasonalincidenceinciguatoxicity,butthespawning season in some of the larger predacious fish may be a more dangerous period than the other seasons of the year. There is no way to detect a ciguatoxic fish by its appearance. Clitliccrl Chcrrcrcteristics: Ciguatera fish poisoning produces a constellation of 175 gastrointestinal, cardiovascular, and neurological symptoms, some of which are pathognomonic for the disease. The onset of the poisoning may occur within minutes and up to 48 hours after the fish is ingested. The initial symptoms generally consist of paresthesias and tingling or numbness of the lips, tongue, and extremities. The neurosensory symptoms may be accompanied by nausea, abdominal pain, vomiting, diarrhea, salivation, general malaise, and muscle and joint pain. The gastrointestinal symptoms are present in about 75% of cases, but usually resolve within 24 hours. There is a neurosensory symptom that is of diagnostic importance in ciguatera poisoning: the reversal of temperature sensation in which cold objects (water, ice, etc.) feel hot, produce a stinging sensation, or are painful upon contact. Warm objects may feel cold. This temperature-reversal sensation appears in more than 89% of cases. Cardiovascular symptoms usuallyconsist of bradycardia and hypotension, which later may change to
32
Halstead
tachycardiaandhypertension.Cardiovascularsymptomsgenerallyresolvewithin 48 hours, but may persist for several weeks. Neurological symptoms of perioral and extremity paresthesias, ataxia, pruritus, mental depression, hysteria, maculopapular skin eruptions, blisters, desquamation, loss of hair and nails, cranial nerve palsy, vertigo, tremors, chills, headache, sweating, dysurea, hiccups, visual blurring, superficial hyperesthesia, motor weakness, respiratory distress, myalgia, arthralgia, temperature reversal, hyporeflexia, metallic taste, loose or painful sensation of the teeth, and extraocular muscle pain Inay be present. Their occurrence and duration vary with the individual. Seldom is fever present. Extreme muscle weakness is a common complaint. Paralysis of the extremities may occur. Physical findings are variable and nonspecific in ciguatera poisoning. The severity of the case varies withthe individual’s sensitivity to the toxin, the toxicity of the fish, and the amount of toxic fish eaten. Recovery varies greatly from case to case, and can be from 48 hours to several days, weeks, months, and, in some cases, years. Fatalities are uncommon, but do occur. Fatalities may be due to cardiovascular collapse or respiratory paralysis. Exposure to the ciguatoxin complex does notconfer immunity. There are no accurate statistics worldwide as to the incidence or mortality rate of ciguatera fish poisoning. There is a gross underreporting of outbreaks even in regions in which ciguatera is endemic. The subject of ciguatera fish poisoning has been under intensive investigation by numerous workers over an extended period of time (25,483 190). Treatment: The treatment of ciguatera fish poisoning is largely symptomatic, but thereare a fewspecificmodalitiesthatmaybeespeciallyhelpful.Theidentity of the fish is of minor value because about 300 different species of tropical reef fish have been incriminated thus far and the amount of toxin in the fish varies greatly from one specimen to the next. The diagnosis of ciguatera fish poisoning is based on the symptomatology presented. Gastric lavage or vomiting induced by sticking ones finger down the throat, or using apomorphine or ipecac, should be done as soon as possible. This should be followed by the administration of a slurry of charcoal to absorb the poison in the intestinal tract. Nausea and vomiting can be controlledby using an antiemetic drug such as prochlorperazine. Hypotension can usually be helped with the useof a pressor drug such as dopamine or dobutamine. Calcium gluconate may also be helpful in treating the hypotension and myocardial insufficiency. Bradycardia may be controlled with the use of atropine. Cool showers and the use of hydroxyzine may be helpful in relieving the pruritus. Intravenous sodium ascorbate (25 g diluted in 250 1111 of normal saline per day for10 days) and vitamin B complex have been ernployed in relieving someof the toxic effectsof ciguatoxin. Mannitol has been found to provide symptornatic relief in many cases (121,130). The fruit of the nono tree (Morinda citrijiolia Linnaeus) has been used for centuries by South Pacific islanders to treat the symptoms of ciguatera fish poisoning ( 2 ) . The juice of this fruit is now sold in the United States and elsewhere throughout the world under the trade name “Noni.” Theusual dosage is 3-4 ounces of the juice per day. The product is nontoxic and should be tried. A variety of other therapies have been employed, but none of them have been shown to be completely effective. See Refs. 27-29. Preverlrion: There is no reliable methodof detecting a ciguatoxic fish by its appearance. However, there are a few basic guidelines that are helpful. Such large, predacious reef fish as snapper, barracuda, grouper, and jacks should be eaten with caution. The larger the fish is the greater is the potentialof ciguatoxication if the fish iscaptured in an endemic
Fish Toxins
33
region. Large fish generally accumulate the toxic products fromthe various trophic levels in the food chain below them. Ciguatera is essentially the end resultof a toxic biochemical magnification problem in which humans are the final recipient of the toxic agents from all of the marine organisms in the trophic levels below them. When catching fish in a suspected ciguatoxic region, it is always advisable to seek the advice of the locals as to the edibility of the fish. The viscera (i.e., liver, gonads, and intestines) of many tropical reef fish are toxic and should not be eaten. Such ordinary cooking procedures as frying, baking, boiling, or drying do not render a fish safe to eat. Ifin doubt concerning the toxicity of the fish, eat only a small amount and wait for a period of several hours before eating additional quantities of the fish. Tropical moray eels frequently are toxic, maybe deadly, and should not be eaten. Offshore fish generally are safer to eat than inshore reef species. Bioassay methods have been used to detect ciguatoxic fish utilizing a variety of test animals,includingbrineshrimp,ants, flies, cats,dogs,mongooses,rats,guineapigs, plants, chickens, frogs, and so on. See Ref. 48 for a review of these bioassay methods. Cats have been found to be extremely sensitive to ciguatoxin. All of these bioassay methods tend to be cumbersome, time consuming, and, in most instances, expensive. Several immunoassay methods have been developed for the detection of ciguatoxic fish. One of thefirst of these techniques utilized a radioimmunoassay method for the detection of minute quantities of antigens and antibodies (91-94). This test was refined further into an enzyme immunoassay method (95) that was found to be easier to run, less expensive, more feasible for screening all sizes and varieties of fish, and could be used to test liver and musculature. A more inexpensive, but still reliable assay is the stick test developed by Hokama (79,96,97) and Hokama et al. (98). The stick test measures ciguatoxin and polyether compounds, including okadaic acid. The stick test has been used commercially to a limited extent in Hawaii. The stick test appears to be the most reliable and practical assay method currently available for the screening of ciguatoxic fish. Unfortunately the stick test is generally not available in most endemic ciguatoxic regions of the world.
D. Clupeotoxic Fish Clupeotoxic fishare members of theorderClupeiformeswhichincludes theherrings, anchovies, and related species. Clupeiform fishes become poisonous after eating toxic dinoflagellates such as Osrreopsis siarnensis Schmidt (99). Undoubtedly other species of dinoflagelllates are also involved but have yet to be incriminated. This fom1 of poisoning is rare and resembles ciguatera, but it is very rapid in its action and has a high mortality rate. The membersof the order Clupeiformes include the families Clupeidae, the herrings; Engraulidae, the anchovies; Elopidae, the tarpons; Albulidae, the bonefish; Pterothrissidae, the deep-sea bonefish; and Alepocephalidae, the deep-sea slickheads. However, the families most commonly incriminated in human clupeotoxications are members of the Clupeidae and Engraulidae. The clupeiform fish are characterized as follows. They are softrayed fish with the anterior vertebrae simple, unmodified, and without auditory ossicles; symplectic bone is present and there are no interclavicles.The recessus lateralis is present, and the infraorbital canal merges with the preopercular canal within a chamber of the neurocranium. Parasphenoid teeth are absent. There is no large foramen on the anterior ceratohyal, the parietals are separated by the supraoccipital, and the opercular bones are
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distinct. The pharyngeal bones are simple above and below,with the lower not falciform. The bones of the jaws are developed; the maxillary is broad, always distinct from the premaxillary, and forms part of the margin of the upper jaw. There are no barbs. The shoulder girdle is well developed and connected withthe cranium by a bony posttemporal. a slit behind the fourth. The air bladder,if present, hasa pneumatic There are four gills with duct. The dorsal and anal fins are without true spines; the ventral fins are abdominal and the adipose fin can be present or absent. This is a large group of fish comprising most of themarinesoft-rayedfish.Mostareplanktonfeederswithnumerous,longgillrakers (39.100).
Representcltive Species Family: Clupeidae (sardines, herrings) Species: Clupmodon thrissa (Linnaeus). Thread herring. Length 9 in. (25 cm). Distribution: Indo-Pacific, China, Japan, Korea. Species: Cluperr sprnttus Linnaeus. Sprat. Length 6 in. ( 1 S cm). Distribution: Northeastern Atlantic, Mediterranean. Species: Hcrrewgulrr ovnlis (Bennett). Sardine. Length 6 in. ( I S cm). Distribution: Indo-Pacific, Red Sea. Species: Opisthot?etncr oglinum (LeSueur). Atlantic thread herring. Length 9 in. (25 cm). Distribution: West Indies. north to Cape Cod. Family: Engraulidae Species: Etrgrcrulis et~crnsicholus(Linnaeus). Anchovy. Length 7 in. (20 cm). Distribution: Eastern Atlantic and Mediterranean. Species: Thrissincr baelmrcr (Forskil). Anchovy. Length 4 in. ( 12 cm). Distribution: Indo-Pacific, Red Sea, enters river mouths.
Clupeotoxications result from eating clupeiform fish such as sardines, herring, and anchovies. Most poisonings have occurred in tropical island areas and were caused by eating fish captured close to shore. It is believed that clupeotoxism is seasonal and most likely to occur during the summer months. Clupeotoxin has been isolated (99), but its molecular structure has not been elucidated. Clirzicul Clrcrrrrc.ter-istics: The symptoms and signs of clupeotoxism are distinct and usually violent. The first indication of an intoxication is a sharp metallic taste that may be present immediately following ingestion of the fish, followed by nausea, dryness of the mouth, vomiting, malaise, abdominal pain, and diarrhea. The gastrointestinal upset may be accompanied by a feeble pulse, tachycardia, chills, cold and clammy skin, vertigo, a drop in blood pressure, cyanosis, and other evidences of vascular collapse. Within a very short period of time, or concurrently, a variety of neurological disturbances ensue, such as nervousness, dilated pupils, violent headaches, numbness, tingling, hypersalivation,musclecramps,respiratorydistress,progressivemuscularparalysis,convulsions, coma, and death. Death may occur in less than IS minutes. Ferguson (101) claimed that the poison was so rapid in its action that natives died while in the very act of eating the yellow-billed sprat (Clulwn tlrrissrr). Pruritus and various typesof skin eruptions, including desqualnation and ulcerations, have been reported in v i c t i m that have survived. There are no accurate statistics available regarding the mortality rateof clupeotoxism, but judg-
Fish Toxins
35
ing from the documented case reports, the fatality rateis very high and the victims generally die within minutes to hours. It is believed that clupeotoxisln in some instances may be related to ciguatera poisoning, but this has not been documented. Some cases appearto exhibit ciguatera-like symptoms. Since clupeoid fish are primarily plankton feeders, it is likely that some of these fish are ingesting highly toxic dinoflagellates. Tretrtnzent: Follow thetreatmentrecommendedforciguaterafishpoisoning.See Refs. 27-29. Prcventiorr: There are no reliable methods of detecting a clupeotoxic fish and preventing intoxication. Outbreaks of this intoxication are rare and there are insufficient data concerning the nature of the poison. No screening methods have been developed for the testing of clupeotoxic fish. Most of the clupeotoxic fish are generally valuable food fish.
E. Gempylotoxic Fish The gempylids, escolars, or pelagic mackerels are a small group of predacious oceanic fish. They have a band-shaped body, large. sharp teeth, and are distinguished from the true mackerels by the complete absence of a later keel or ridge on the caudal peduncle. Two dorsal finsarepresent,thefirst of which is spinous and longer thanthesecond. Gempylids produce an oil that has a pronounced purgative effect.
Representcrtive Species Family: Gempylidae (castor oil fish) Species: Rrrvcttus pr-etiosus Cocco. Castor oil fish. Length 4.2 ft (1.3 m). Distribution: Tropical Atlantic and Indo-Pacific.
Gernpylicl Poisonirrg The gempylid poisoning formof ichthyosarcotoxism is caused by ingesting the flesh or sucking the rich, oily bones of the fish. People suffering from constipation in the South Pacific islands use gempylid fish for its relief. Clinical Clrnrcrcteristics: Ingestion of theoil contained i n theflesh and bones of gempylid fish causes diarrhea, which, although pronounced, is generally without pain or cramping (26,102,103). No other untoward effects have been reported. Gempylotoxisln has also been referred to as gempylid diarrhea. The oil is similar to castor oil, comprised mainly of oleic acid, but it has different pharmacodynamic properties ( 104,105). Treutmerlt: No treatment is required. Prew1tiotl: Avoid eating gempylid fish.
F.Scombrotoxic
Fish
Scombrotoxism,orscombroidpoisoning, is caused by perciform fish of thesuborder Scombroidei, all of which are members of the single family Scombridae, the tunas and related species. One of the members of the order Beloniformes, family Scomberesocidae, the Japanese saury (Cololabis s a i r . c ~ )has also been incriminated in sombroid poisoning. Recently, such otherfish species as mahi-mahi, jack, bluefish, herring, sardines, and anchovies have been reportedto cause scombroid poisoning (87). Scombroid poisoning accounts for about 5% of food-borne poisonings i n the United States. Scombroid fish are characterized by their adaptation for swift locomotion, having
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a sharp profile anteriorly and a slender tail with a widely forked caudal. There is a series of detached finlets on the back behind the second dorsalfin and on the undersurface behind the anal fin, a feature that distinguishes them from most other fish. Scombroids have two dorsal fins, of which the first is composed of spines and the second of soft rays. The fins fit into grooves or depressions on the body, the bones of the head lie flat, and the gill covers fit tightlyagainstthesides. Thescalesare usuallysmall,thin, andmetallicin appearance, offering a minimum of friction in the water. Scombroids are the epitome of grace, form, and speed. Tunas and related species are largely oceanic, migrating great distances through the open seas. Mackerels are largely inhabitantsof littoral waters. Scombroids are distributed widely throughout all temperate and tropical seas, and some occasionally are found in arctic and antarctic waters. They usually swim in large schools. Scombroids generally swim near the surface of the water during spawning season. During the warmer months they approach the shore, but retire to deeper water during the cold months. They feed on to the surface at night. the plankton swimmingin the deeper water during the day, and rise Scombroids are predatory and voracious feeders. In addition to plankton, they also feed on a wide variety of moderate-size fish. Some of the tunas can be found at depths of 200 m or more, whereas other scombroids seldom descend below 40 m . The sauries of the family Scomberesocidae resemble the needlefish, but have short jaws and are identified by a series of five to seven finlets following both the dorsal and anal fins. There are four species and none attain a length much beyond 35 cm. Sauries inhabit temperate and tropical waters. They arevery abundant in some regions and constitute an important foodfisheryin Japan. Sauries feed on planktonandsmall fish. The sauries also have been incriminated in scombroid fish poisoning, but outbreaks have been limited to Japan. Repr~ser~tcrtive Species Family: Scomberesocidae (sauries) Species: Cololabis saircr (Brevoort). Saury. Length 12 in. (30 cm). Distribution: Japan. Family: Scombridae (tunas. nmckerels, albacore) Species: Euthgnm~.s peltrrtis (Linnaeus). Skipjack. Length 19 in. (50 cm). Distribution: Cosmopolitan in warm seas. Species: Scolllber. jcrpotzic~~ Houttuyn. Pacific mackerel. Length 15 in. (40 cm). Distribution: Cosmopolitan in warm seas. Species: Thunnlr.~tl1gtIr~u.s(Linnaeus). Bluefin tuna. Length I O ft (3 m). Distribution: Cosmopolitan in subtropical and temperate seas.
Sconlhroicl (Histtmirle) PoisorlirlR Scombroid poisoning is caused by the improper preservation of scombroid fish or other fish species that results in certain bacteria, mainly species of the family Enterobacteriaceae ( C l o . ~ f r i d i wLrrctobmillus, ~, Prote~rs,Vibrio), acting on histidine in the muscle of the fish convertingit to histamine.The toxicity of histamine is enhanced by the presence of certain potentiators (e.g., cadaverine and putrescine) that act by inhibiting intestinal histamine-metabolizing enzymes. The enzyme inhibition increases the intestinal uptake of unmetabolized histamine (106-108). Histamine ingested by itself generally is much less toxic. Scombroid poisoning is the most common form of ichthyosarcotoxism and occurs
Fish Toxins
37
throughout the world wherever scombroid fish are eaten. Moreover, it is the only form of ichthyosarcotoxism in which bacteria play an active role i n toxin production within the body of the fish. Clirlical Chrrracteristics: The symptoms of acutescombroidpoisoningresemble those of histamine intoxication. The symptoms are characteristic and appear with almost monotonous consistency. Toxic scombroid fish frequently can be detected immediately upon tasting the fish. Victims state that the fish has a sharp or peppery taste. Symptoms usually occur within a few minutes after ingestion of thetoxin and consist of intense headaches, dizziness, throbbing of the carotid and temporal vessels, epigastric pain, burning of the throat, cardiac palpitation, rapid and weak pulse, dryness of the mouth, thirst, inability to swallow, nausea, vomiting, diarrhea, and abdominal pain. Within a short time a generalized erythema and an urticarial eruption may develop, covering the entire body and accompanied by a severe pruritus. The face of the individual becomes swollen and flushed, the eyes become injected, and coryza develops. In severe cases there may be bronchospasm, suffocation, and severe respiratory distress. Various other minor discomforts such as fever, chills, malaise, tremors, metallic taste, and cyanosis of the gums and tongue may occur. There is a danger of shock, and deaths have been reported. However, the acute symptoms generally are transient, lasting only 8-12 hours (48,108-1 1l ) . Treatment: The treatment of scombroid poisoning is largely directed toward relieving the symptoms of the histamine reaction. This form of poisoning is generally selflimiting and fatalities are rare. Minor intoxications can usually be treated with diphenhydramine (Benedryl). Scombroid poisoning may cause respiratory distress, in which case the victim should be taken immediately to an emergency treatment center. Epinephrine is the treatment of choice for respiratory problems in this instance, but it should be used withcaution in olderindividuals with a historycardiacproblems.SeeRefs.(27-29, 92,l 12-1 15). Prevenrion: Scombroid fish and other related species believed to cause this form of ichthyosarcotoxism should be refrigerated promptly or eaten soon after capture. It has been shown that the histamine content in some of the scombroid fish increases from 0.09 mg/100 g of tissue to about 95 mg/ 100 g of tissue when kept at room temperature (20°C25°C) for about10 hours ( 1 16).Toxic scombroid fish cannot always be detectedby appearance or odor. The histamine content in the flesh may be very high with little or no evidence of putrefaction. Scombroid or any other fish having a sharp or peppery taste should be discarded. Scombroid fish with histamine levels greater than 20 mg/ 100 g should be discarded.
G. Tetrodotoxic Fish Tetrodotoxications constitute one of the most violent formsof marine biotoxications. This type of ichthyosarcotoxism commonly is designated as tetrodon or puffer poisoning. The causative transvectors of the poison tetrodotoxin are members of the order Tetraodontiformes, formerly the Plectognathi, which includes the families Tetraodontidae, the puffers; Diodontidae, the porcupinefish; Canthigasteridae, the sharpnosed puffers; and Molidae, the molasor ocean sunfish. The order also includes such other fish families asthe spikefish, filefish, trunkfish, three-toothed fish, and triggerfish, but these are not included in any fish mayalsotransvectany or all of the degree of detail in this chapter. These same ciguatera complex of poisons (e.g., ciguatoxin, maitotoxin, or possibly palytoxin). For
38
Halstead
the 111ost part, this section focuses on the fish families Tetraodontidae, Diodontidae, and Canthigasteridae. Tetrodotoxications are the resultof ingesting a poison known as tetrodotoxin, molecular formula C,,H,,N,O,, molecular weight 319, and LDS,, toxicity in mice is I O pg/kg ( 1 17,118). Tetrodotoxin actsby preventing nerve conductionby an extremely specific and reversible blockage of the inward movement of sodium ions through the cell membrane of an activated neuron (48,119-121). The tetraodontiform fish are characterized by the absence of parietal, nasal, or infraorbital bones, and usually have no lower ribs; the posttenlporal region is present and is siniple and fused with the pteroticof the skull; the hyomandibular and palatine are firmly attached to the skull. Gill openings are restricted. The maxilla is usually firmly united or fused with the premaxilla. Scales are usually modified as shields or plates. The lateral line may be present or absent, and sometimes is multiple. The swim bladder is present except in molids, and there are 16-30 vertebrae. Tetraodontifornles can produce sounds by grinding the jaw teeth or the pharyngeal teeth or by vibrating the swim bladder. The stomach of some of these fish is highly modified to permit inflation to an enormous size. Fish with this ability are commonly called puffers. Inflation is caused by gulping large quantitiesof water into aventral diverticulum of the stomach when the fish is frightened or annoyed. Deflation occurs by expelling the water. If the fish is removed from the water, inflation can occur with air (39,122).Puffers feed mainly on corals and mollusks, but tend to be omnivorous. A wide range of phylogenetically unrelated aquatic organisms are now known to transvect tetrodotoxin, including starfish, gastropod mollusks, the Australian blue-ringed octopus, tropical reef crabs, gobyfish, and a variety of freshwater amphibians. This subject has been reviewed in greater depth elsewhere (48).
Representcrtive Species There are a large numberof fish species incriminated in transvectoring tetrodotoxin, but only a few representative fish are listed below. Family: Canthigasteridae (sharp-nosed puffers) Species: Canthigaster rivuhtus (Temminck and Schlegel). Rivulated Goby. Length 3 in. ( I O cm). Distribution: Indo-Pacific, Japan. Family: Diodontidae (porcupinefish) Species: Chilonzycteru.7 c!jjitziirlis Giinther. Porcupinefish. Length 6 in. ( 1 7 cm). Distribution: Southern California, Galapagos Islands, Hawaii, Japan. Species: Diodorl hystri.r Linnaeus. Porcupinefish. Length 35 in. (90 ctn). Distribution: All warm seas. Family: Tetraodontidae (puffers, blowfish, fugu) Species: Arothron lzisjdus (Linnaeus). White-spotted puffer. Length21 in. (53 cm). Distribution: Indo-Pacific, Panama, Japan, Australia, South Africa, Red Sea. Species: Arothron meleagris (LacCpkde). Guineafowl puffer. Length 13 in. (33 cm). Distribution: West coast of Central America, Indo-Pacific. Species: Fugu Imrddis (Temminck and Schlegel). Fugu. Length 14 in. (35 cm). Distribution: China, Japan. Species: kcgocephcc/u.s luncrris (Bloch and Schneider).Puffer.Length 12 in. (35 Clll).
Fish Toxins
39
Distribution: Indo-Pacific, Red Sea, China, Japan, eastern coast of Africa. Species: Sldlaeroides umul~/tu.s(Jenyns). Puffer. Length 1 I in. (28 cIl1). Distribution: California to Peru, Galapagos Islands. Species: S~h7er.oide.vrnncu1rrtu.s (Bloch and Schneider). Botete. Length 10 in. (25 cm). Distribution: Atlantic coast of the United States south to Guiana. Species: Tettwodorl lineatus Linnaeus. Puffer. Length 18 in. (46 cm). Distribution: Rivers of northern and western Africa.
Tetrorlorr (Puffer.) Poisoilirlg Method ofIntmication: Puffer poisoning or tetrodon poisoning is caused by ingesting the flesh, viscera, or skin of toxic tetraodontiform fishes (i.e., sharp-nosed puffers, puffers, porcupinefish, and other related fish). Certain goby fish, gastropod mollusks, and tropical reef crabs are also capable of transvectoring tetrodotoxin and causing a biotoxication. Puffer fish are more dangerous to eat imnlediately prior to and during the reproductive season, during which time the poison content in the body of the fish increases with the gonadal activity. The skin, liver, ovaries, and intestines arethe most toxic portions of the fish. The tnusculature of the fish usually is safer to eat than other parts of the fish, but at times it also may be toxic. The toxicityof the fish cannot be determinedby its appearance, freshness, or size since even small puffers may contain sufficient poison to be lethal. Puffer and fugu poisoning continuesto be the major causeof fatal food intoxications in Japan, where puffer meatis looked upon as a gourmet delicacy. The sale of toxic puffers is carefully regulated by public health authorities in Japan, butthis has not prevented periodic outbreaks of fatal food poisoning. TheU.S. Food and Drug Administration (FDA) recently has permitted the importation of Japanese puffers for sale in fugu restaurants in the United States (25,87), but this does not guarantee the safety of puffer products. All puffers are potentially toxic unlessthey have been cultivated artificially (123-125). There is evidence that several different strains of marine bacteria may play an important role in the biosynthesis of tetrodotoxin in the body of the fish (5,7). Clinicd CharLlcteri.7tic.s:The onset and symptomatology in puffer poisoning varies greatly depending upon the person and the amount of poison ingested. Initial symptoms usually consist of paresthesias of the lips and tongue, malaise, pallor, dizziness, and ataxia that develop within 10-45 minutes after ingestion of the fish, but may occur as much as 3 hours or niore after ingestion. The paresthesias are described as a tingling or a prickly to the fingers and toes, and gradually develops sensation that may subsequently spread into numbness.In severe cases,the numbness may involve the entire body, which has been described by victims as a floating sensation. Hypersalivation. profuse sweating, extreme weakness, precordial pain, headache, subnormal temperatures, hypotension, and a rapid, weak pulse usually appear early in the succession of symptoms. Nausea, vomiting, diarrhea, and epigastric pain frequently are present. The pupils are constricted during the early part of the intoxication, but later become dilated.As the disorder progresses,the pupillary and corneal reflexes are lost. Shortly after the development of the paresthesias, respiratory symptoms become a prominent part of the clinical picture. Respiratory distress, as noted by an increased rate of respiration, movements of the nostrils, and shallow respiration, generally is observed. Respiratory distress later becomes very pronounced, and the lips, extremities, and body become intensely cyanotic. Petechial hemorrhages involving extensive areas of the body, blistering, and subsequent desquamation may occur. Muscular twitching, tremor, andloss
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of motor coordination become progressively worse and finally terminate in extensive muscular paralysis. The first areas to be involved areusually the throat and larynx, resultingin aphonia, dysphagia, and, later, complete aphagia. The muscles of the extremities become of the patient paralyzed and the patient is unable to move.As the end approaches, the eyes become fixed and glassy, and convulsions may occur. The victim may become comatose, but in most instances the patient remains conscious and the mental faculties remain acute until shortly before death. The fatality rate of puffer poisoning is about 59% in untreated cases. There are no accurate mortality statistics worldwide. Treutmerlt: Early treatment seeks to remove the gastric stable toxin, which is partially inactivated by alkaline solutions. If the victim is seen within 3 hours of ingestion, 1 L 2%sodiumbicarbonate.Thisis gastriclavageshouldbeperformedwithatleast followed with activated charcoal in sorbitol solution. If the victim has difficulty swallowing or breathing, or is not alert, intragastric manipulation should be preceded by endotracheal intubation. Supplemental oxygen and ventilatory assistance should be promptly instituted as respiratory paralysis progresses. The physician should rememberthat the paralyzed victim may be fully conscious and should offer the victim frequent verbal reassurances. Hypotension induced by tetrodotoxin may require the intravenous (IV) administration of crystalloid fluid augmentation. Bradyarrhythmias generally respond to atropine (0.5 mg IV up to 2.0 mg). Severe heart block may require the placement of a temporary transvenous pacemaker. While a minor intoxication may be limited to paresthesias, all victims should be observed for at least 8 hours to detect deterioration, particularly respiratory failure. Under no circumstances should anyone with dysphagia be given liquids by mouth. The fruit of the nono tree (Morindcr citrijola Linnaeus) has been used for centuries by South Pacific islanders to treat the symptoms of ciguatera fish poisoning (2) and may be helpful in the treatment of tetrodon poisoning. The juice of the fruit is now sold in the United States and elsewhere throughout the world under the trade name “Noni.” The usual dosage is 3-4 ounces of the juice per day, or four capsulesof the concentrate. The product is nontoxic and should be tried. See Refs. 27-29. Preverltion: If one follows the old Mosaic sanitary laws in Deuteronomy 14:9- 10eliminate all scaleless fish from the diet-then puffer poisoning will never be a problem. If one is living in Japan and has a desire to eat fugu, he should purchase the fish from a first-class, authorized restaurant with a licensed puffer cook. However, even following this procedure will not absolutely guarantee food safety. Eating puffers, at best, is a game of Russian roulette. In any event, the skin and viscera of the fish should never be eaten. No cooking or drying procedure destroys the poison.
V.
ICHTHYOOTOXIC FISH
Ichthyootoxism is one of the lesser-known forms of fish poisoning. Ichthyootoxic fish constitute a group of fish that produce a poison generally restricted to the gonads. This group of toxic fish does not include the ichthyosarcotoxic puffers because the poison in puffers is distributed widely throughout the body. The musculature and other partsof the body in ichthyootoxic fish generally are safe to eat. Some of these fish are found only in freshwater. There does not appear to be any particular phylogenetic affinity other than the fact that the fish involved are all members of the class Osteichthyes, the true bony
Fish Toxins
41
fish (see Sec. 1v.C for a description of the class Osteichthyes). Most of the intoxications resulting from the ingestion of ichthyootoxic fish occur during the reproductive season, during which the gonadal activity of the fish is at its peak.
Repre.sewtative Species Family: Acipenseridae (sturgeons) Species: Huso huso (Linnaeus). Sturgeon. Length 6 ft (1.8 m). Distribution: Black Sea, Sea of Azov, Caspian Sea, Mediterranean Sea, and rivers that drain into these seas. Family: Lepisosteidae (gars) Species: Lepisosteus tristoechus (Bloch and Schneider). Alligator gar. Length 19 ft (6 m). Distribution: Rivers of Cuba, bays and coastal waters of the Gulf of Mexico. Family: Esocidae (pikes) Species: Esox lucius Linnaeus. Northern pike. Length 48 in. ( I .2 m). Distribution: Freshwaters of Europe, northern Asia, and North America. Family: Cyprinidae (minnows) Species: Brrrbus barbus (Linnaeus). Barbel. Length 35 in. (89 cm). Distribution: Freshwaters of northern and central Europe. Species: Schizothorctx irltermedius McClelland. Snow trout, marinka. Length 18 in. (46 cm). Distribution: Freshwaters of central Asia. Species: Tirlccrtinccz (Linnaeus). Tench. Length 24 in. (64 cm). Distribution: Freshwaters of Europe. Family: Stichaeidae (pricklebacks) Species: Stichaeus grigorjewi Herzenstein. Japanese prickleback. Length 20 in. (51 cm). Distribution: Freshwaters of Japan and Korea. Family: Cottidae (sculpins, cabezon) Species: S~.orl.’aenichtll?,smrrrmorrrtus (Ayres). Cabezon. Length 30 in. (76 cm). Distribution: Pacific coast of North America.
Ichthyootoxism (Fish Roe Poisoning) Mechanism of Intoxication: Ichthlyootoxism, or fish roe poisoning, results from ingestion of various salt- and freshwater fish of Europe, Asia, and North America, and to a lesser extent, the tropics. The of roemany freshwater and estuarine fish of eastern Europe and Asia are dangerous to eat during their reproductive period,usually March-June. Most ichthyootoxicfisharemembers of thefreshwaterCyprinidaeminnowgenera Burbus, Schizothorclx, and Tinca, found in Europe and Asia, and the genus Srichaeus of the family Stichaeidae, found in Japan and Korea. These fish have caused innumerable intoxications in Europe and Asia. A Pacific North American species of the Cottid genus, Scorpaenichthys, also has produced intoxications. Although cooking is said to destroy most ichthyootoxins, it cannot be relied upon as a completely safe procedure since the poison in some fish appears to be resistant to of ichthyootoxic fish generally are safe to cat during heat. The musculature and other parts the reproductive season. The chemical nature of ichthyootoxins is unknown. For a more comprehensive review of this topic, see Refs. 25 and 48. Cliniccrl Chctracteristics: Symptoms develop soon after ingestion oftheroeand
Halstead
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consist of abdominal pain, nausea, vomiting, diarrhea, headache, fever, bitter taste in the mouth, dryness of the mouth, intense thirst, a sensation of constriction of the chest, cold sweats, irregular pulse, low blood pressure, cyanosis, pupillary dilation, syncope, chills, dysphagia, and tinnitus. In severe cases there may be muscle cramps, paralysis, coma, and death.Barbus roe usually does not cause death, but fatalities have resulted from eating Schizothom.r roe ( 5 3 ) . Treatment: Treatment is symptomatic. There are no known antidotes. Prevention: Avoid eating the roe of any fish during the reproductive season unless you have positive knowledge that the roe is safe to eat. This preventive advice is particularly pertinent to the freshwater and brackish water fish of Europe andAsia and all tropical marine species. Cooking fish roe cannot be relied upon to inactivate ichthyootoxins.
VI.
ICHTHYOHEMOTOXIC FISH
Ichthyohemotoxic fish consist of a variety of different species of eels. All of the fish of this group are membersof the order Anguilliformes (Apodes). The members of this group are characterized by an eel-like body, abdominal pelvic fins (when present), and an air bladder connected with the intestine by a duct. Gill openings are narrow or slitlike. The scales, if present, are cycloid. The dorsal and analfins are very long and usually confluent (1 26). The research on ichthyohemotoxicfish, or fish having toxic blood, reveals that there isverylittleclinical infomlation involving humans. Hemotoxins are largely parenteral poisonsandseldomtoxicwhentaken by mouth. Verylittle is known concerning the chemical nature of these posions.
Representcltive Species Family: Anguillidae (freshwater eels) Species: Anguilla anguilla (Linnaeus). Common European eel. Length 39 in. ( I m). Distribution: Europe, fresh- and saltwater rivers. Family: Congridae (conger eels) Species: Conger conger (Linnaeus). Conger eel. Length 79 in. (2 m). Distribution: Atlantic Ocean, Mediterranean Sea. Family: Muraenidae (moray eels) Species: Muraetza helena (Linnaeus). Moray eel. Length 59 in. (1.5 m). Distribution: Eastern Atlantic and Mediterranean Sea.
1chtk~ohemoto.rism~to.ris~n Mechanism of Intoxication: Ichthyohemotoxins are largely parenteral poisons, although there are a few instances on record in which individuals have become intoxicated due to ingestion of large quantities of the poison by mouth. Most of the ichthyohemotoxic or fish generally are recognized as good food fish. However, ingestion of fresh blood serum from these fish may cause food poisoning. This is a rare form of marine food poisoning. Clinical Characteristics: Very littleis known concerning the symptomatology of ichthyohemotoxism in humans. Fish serum intoxications may be of two types: syste1nic9 a form that results from drinking fresh, uncooked fish blood, and topical. The symptolns of the systemic form consist of diarrhea, bloody stools, nausea, vomiting, hypersalivation,
Fish Toxins
43
skin eruptions, cyanosis, apathy, irregular pulse, weakness, paresthesias, paralysis, respiratorydistress,andpossiblydeath.Forthetopicalform,thereisasevereinflammatory response when raw eel serum accidentally comes in contact with the eye or the tongue. Oral symptoms consist of burning, redness of the mucosa, and hypersalivation. Ocular contact invokes a severe burning sensation and redness of the conjunctivae, lacrimination, and swelling of the eyelids. Eye irritation may persist for several days. Usually recovery is spontaneous. For a more comprehensive discussion of this subject, see Ref. 48. Treatment: Treatment is symptomatic. There are no known specific antidotes. Prevewtiow: Care should be takenin the handlingof eel blood. Raw eel blood should not be ingested. Cooking is said to destroy the toxic properties of eel blood.
VII. ICHTHYOHEPATOTOXIC FISH The livers of certain edible species of fish are sometimes found to be toxic to eat. Most of the outbreaks of ichthyohepatotoxism have occurred in Japan. Nothing is known concerning the chelnical nature of the poisons involved. It is believed that in some instances the intoxications are due to hypervitaminosis A. The fish involved are members of the class Osteichthyes.
Representdve Species Family: Scombridae (tunas, mackerels, albacore) Species: Scornberomorus niphonius (Cuvier and Valenciennes). Japanese mackerel. Length 39 in. ( 1 m). Distribution: Japan, Korea, China. Family: Serranidae (Sea bass, grouper) Species: Stereolepis ischinagi (Hilgendorf). Sea bass. Length 78 in. ( 2 m). Distribution: Japan, Korea.
I c h t h ~ o h e ~ ~ ~ t ~(Fish t ~ ~ Liver i s t r /Poisoning) Symptoms of ichthyohepatotoxism appear within 30 minutes- 12 hours after ingesting the fish liver. The initial symptoms consist of nausea, vomiting, fever, and headache. The headache may be very severe and is said to be intensely aggravated by the slightest movement of the body, head, or eyes. A mild diarrhea may be present, but abdominal pain generally is absent. The face of the victim usually becomes flushed and edematous, and a macular rash having large patchy erythematous areas develops. Within 3-6 days, desquamation appears. Large areas of skin may peel off around the nose, mouth, head, neck, and upper extremities, and gradually extends over the entire body. Epilation may 30 days. Vesicular formation of the oral result. Desquamation may continue for about mucosa and bleeding from the lips may occur. Orbital pain, joint aches, and cardiac palpitation with a rapid pulse may be present. Victims have complained of a slippery sensation on the tip of the tongue. Most of the more acute symptoms disappear in about 3-4 days. Residual symptoms consist of chapping of the lips, stomatitis, and mild hepatic dysfunction. Recovery usually is uneventful. No fatalities have been reported. The liver may be enlarged, but no jaundice has been observed. Treatment: Treatment is symptomatic. There are no specific antidotes. Prevention: Care should be taken in eating fish livers. In general, the liver is one of the most dangerous parts of a fish to eat. If a fish is poisonous, a greater concentration
Halstead
44
of the poison is likelyto be foundin the liver than almost any other part of the fish. Cooking does not destroy the poison. Most outbreaks of ichthyohepatotoxism have resulted from eating fish livers that have been sauted or in soup. The toxicity of of fish liver cannot be determined by its appearance. It is recommended that fish livers be eliminated from the diet unless there is reliable information that it is safe to eat.
VIII. ICHTHYOALLYEINOTOXIC (HALLUCINOGENIC) FISH Ichthyoallyeinotoxism, or hallucinogenic fish poisoning, is caused by ingesting certain types of reef fish known to occur in the tropical Pacific and Indian Oceans. This biotoxication may result from eating either the head or flesh of the fish. The source and chemical nature of the poison is unknown. Most of the fish species incriminated in ichthyoallyeinotoxism also are involved in ciguatera fish poisoning. Whether there is a relationship between these two types of intoxications is not known. All of the hallucinogenic fish are members of the class Osteichthyes.
Representative Species Family: Kyphosidae (sea chubs) Species: Kyphosus citwrmcetIs (Forskil). Sea chub. Length 20 in. (50 cm). Distribution: Indo-Pacific. Family: Mugilidae (mullets) Species: Mugil cephalus (Linnaeus). Common mullet. Length 12 in. (30 cm). Distribution: Cosmopolitan. Family: Mullidae (Goatfish, surmullets) Species: Upetleus arge Jordan and Evermann. Goatfish. Length 12 in. (30 cm). Distribution: Indo-Pacific.
Ichtkgocrllyeinott,.risrn (Hdlucinogenic Fish Poisoning) Mech~rnistnqf Intoxication: Ichthyoallyeinotoxism, or hallucinogenic fish poisoning, is caused by eating the flesh or head of certain species of toxic reef fish, producing hallucinations. The poison reputedly is concentrated in the head of the fish, which is said to be the most dangerous part of the fish to eat. The nature of the poison is unknown. This type of biotoxication is sporadic, uncommon, and completely unpredictable. You cannot detect a hallucinogenic fish by its appearance. The poison is not destroyed by cooking. Clirlicrrl Chnrcrcteristics: The poison affects primarily the central nervous system. 2 hours after ingestion of the fish, continue The symptoms may develop within minutes to for about 24 hours, and then gradually subside. Symptoms consist of dizziness, loss of equilibrium, lack of motor coordination, hallucinations, and mental depression.A common complaint of the victim is that it feels as though “someone is sitting on my chest,” or there is a sensation of a tight constriction around the chest. The conviction that they are going to die or other frightening nightmares are characteristic aspects of the clinical picture. Other complaints consist of itching, burning of the throat, muscular weakness, and, rarely, abdominal distress. No fatalities have been reported. This form of poisoning is generally mild. Treatment: Treatment is symptomatic. No specific antidote is available. Prevention: Caution should be exercised in eating those species of reef fish that
Fish Toxins
45
have been incriminated in ichthyoallyeinotoxism. When possible, natives should be consulted before eating the fish in tropical areas. Hallucinogenic fish cannot be detected by their appearance.
ACKNOWLEDGMENT It is with deep appreciation that I acknowledge the technical assistance Medrano in the preparation of this chapter.
of Leonette C.
REFERENCES 1. ED Goldberg. The Health of the Oceans. Paris: Unesco Press, 1976. 2. BW Halstead. Poisonous and Venomous Marine Animals of the World. Washington, DC: U S . Government Printing Office, 1970. 3. M Aubert. Mediators of microbiological origin and eutrophication phenomenon. Mar Pollut Bull21:24-29,1990. of poisoning from the ingestion of red seaweed 4. BW Halstead, RL Haddock. A fatal outbreak Gmcilurin fsudoe in Guam-A review of the oral marine biotoxicity problem. J Natl Toxins l(l):87-Il5, 1992. of 5. T Yasumoto, A Endo, D Yasumura, H Nagai. M Murata, M Yotsu. Bacterial production tetrodotoxin and its derivatives. In: P. Gopalakrishnakone, CK Tan, eds. Progress in Venom and Toxin Research. Singapore: National University of Singapore, 1987, pp 310-313. 6. T Yasumoto. D Yasumura, M Yotsu, T Michishita, A Endo, Y Kotaki. Bacterial production of tetrodotoxin and anhydrotetrodotoxin. Agric Bioi Chem 50:793-795, 1986. 7. T Noguchi, D-F Hwang, 0 Arakawa, K Diago, S Sato, H Ozaki, N Kawai, M Ito. K Hashimoto. Palytoxin RS the causative agent in the parrotfish poisoning. In: P Gopalakrishnakone and CK Tan, eds. Progress in Venom and Toxin Research. Singapore: National University of Singapore, 1987, pp 325-335. 8. Y Kotaki, Y Oshima, T Yasumoto. Bacterial transformation of paralytic shellfish toxins in coral reef crabs and a marine snail. Bull Jpn SOCSci Fish 51:1009-1013, 1985. 9. T Noguchi, A Uzu. K Koyama, J Maruyama, Y Nagashima. K Hashimoto. Occurrence of tetrodotoxin as the major toxin in a xanthid crab Aterh.crtispo,-idirs. Bull Jpn Soc Sci Fish 4911887-1892,1983. IO. MNV Dimanlig, FJR Taylor. Extracellular bacteria and toxin production in protogonyaulax species. In: DM Anderson, AW White, DG Baden, eds. Toxic Dinoflagellates. New York: Elsevier,1985,pp103-108. ProtogonymI~~x 1 1 . T Ogata, M Kodama, T Ishimaru. Toxin production in the dinoflagellate mnorensis. Toxicon25:923-928,1987. S Sakamoto.UShimidu.Production of paralytic 12. TOgata,MKodama,KKomaru, shellfish toxins by bacteria isolated from toxic dinoflagellates. In: E Graneli, B Sundstrom, LEdler, DMAnderson,eds.ToxicMarincPhytoplankton.NewYork:Elsevier,1989, pp 311-315. 13. M Kodama, T Ogata. S Sato. Bacterial production of saxitoxin. Agric Bid Chem 52: 10751077,1988. 14. M Kodama, T Ogata. Toxification of bivalves by paralytic shellfish toxins [abstract]. Asia Pac J Pharm 3:99-109, 1988. B Sund15. M Kodama. Possible links between bacteria and toxin production. In: E Graneli, strom, L Edler,DM Anderson, eds. Toxic Marine Phytoplankton. New York: Elsevier, 1990, pp 52-61.
46
Halstead
16.TYasumoto.Production ofparalyticshellfishtoxinsbybacteriaisolatedfromtoxic dinoflagellates. In: E Graneli.B Sundstrom, L Edler, and DM Anderson. eds. Toxic Marine Phytoplankton. New York: Elsevier, 1989, pp 3-8. 17. EM Cospcr, C Lee, EJ Carpenter. Novel "brown tide" blooms i n Long Island elnbayments: asearchforthecauses.In:EGraneli.BSundstrom.LEdler, DMAnderson,eds.Toxic Marine Phytoplankton. New York: Elsevier. 1990, pp 17-28. 18. MKodama.TOgata. S Snkamoto.Possibleassociation ofnlarinebacteriawithparalytic shellfish toxicity of bivalves. Mar Ecol Prog Ser 61:203-206. 1990. ofparalytic 19. MKodama. T Ogata, S Sakamoto, S Sato, THonda,TMiwatani.Production shellfish toxins by a bacterium Momrelltr sp. isolated from PI-otogorl?nlrlcr.r fcrrrtcmt~.sis.Toxicon28:707-714,1990. 20. Y Shimizu, S Gupta, H-N Chou. Biosynthesis of red tide toxins. In: S Hall, G Strichartz. eds. Marine Toxins: Origin, Structure, and Molecular Pharmacology. Washington, DC: American Chemical Society. 1990, pp 21-28. 21. TJ Smayda. Novel and nuisance phytoplankton blooms in the sea: evidence for global epidemic. In: E Grancli, B Sundstrom, L Edler, DM Anderson, eds. Toxic Marine Phytoplankton. New York: Elsevier, 1990, pp 29-40. Ics enlpoisonnernents par les hiutres. les moules, 22. A Chevallier. EA Duchesne. Memoire sur les crabs, et par certains poissons de mer et de riviere [in French]. Ann Hyg Pub Paris 45: 387-45 1, 185 1. 73. HCouticre.Poissonsvenimeux et poissonsvcneneux[inFrench].TheseAgreg Carre et Naud,Paris,1899. 24.ENPawlowsky.Fische.Pisces.In:GifttiereUndlhreGiftigkeit [in German]. Jcna: Verlag von Gustav Fischer, 1927. pp 245-247. of theWorld.Washington,DC: 25.BW Halstcad.PoisonousandVcnolnousMarineAnimals U.S. Government Printing Office, 1967. 26.BW Halstead.Marinebiologicalhazards.JOceanTechno12(3):46-49.1968. 27. PS Auerbach. Clinical therapy of marine envenomation and poisoning. In: AT Tu, ed. Handbook of Natural Toxins. Vol. 3. Marine Toxins and Venoms. New York: Marcel Dekker, 1988, pp 493-565. 28. BW Halstead, PS Auerbach. Dangerous Aquatic Animals of the World. Princeton. NJ: Darwin Press, 1992, pp 241-255. 29.BW Halstead,PSAuerbach,DCampbell.AColourAtlas of DangerousMarineAnimals. Boca Raton. FL: CRC Press, 1990, pp182-186. 30. HEngelsen. 0111 Giftfiskoggiftigefisk[inNorwcgian].NordHygTidskriff 3316-325, 1922. 31, OV Linstow. Die Giftthiere und ihre Wirkungauf den Menschen [in German]. Berlin: August Hirschwald, 1894, pp 53-59. 32. U Anthoni, C Christopherson, L Gram. NH Nielsen, P Nielsen. Poisonings from flesh of the Greenland shark Sornirlos1r.s ,,lic,occ.l'ltlc/trs Inay be due to trimethylamine. Toxicon 29(10): 1205-1212,1991. 33. HB Bigelow, IP Farfante, WC Schroeder. Fishes of the Western North Atlantic. New Haven, CT: Sears Foundation for Marine Research, Yale University, 1948. Las masdel ralnomaritimo 34.AParra.Descriptiondediferenrespiezasdehistorianatural. representadas en setents ycinco laminas [in Spanish]. Havana:En la llnprenta de la Capitania General,1787.pp 1-135. 35. BW Halstead.PoisonousandVenolnousMarineAnimalsoftheWorld.Washington,DC: U.S. Government Printing Office. 1965. of toxiccrabs i n 36.YHashinloto, S Konosu,TYasumoto,AInoue,TNoguchi.Occurrence RyukyuandAlnami islands. Toxicon 5:85-90, 1967. 37. R Bagnis. Concerning a fatal case ofciguatera poisoning i n the Tuanlotu Islands. Clin Toxicol 31579-583. 1970.
Fish Toxins
38.
39. 40. 41.
42. 43.
44.
45. 46. 47. 4s
I
49 50 51 52.
47
KA Steidinger,DG Baden. Toxic marine dinoflagellates.In: DL Spector, ed. Dinoflagellates. Orlando, FL: Academic Press, 1984. pp 201 -261. JS Nelson. Fishes of the World. New York: John Wiley and Sons, 1984. M Murata, AM Legrand, Y Ishibashi, T Yasutnoto. Structures of ciguatoxin and its congener. J Am Chem Soc 11 1:8929-8931. 1989. M Murata, AM Legrand, Y Ishibashi, M Fukui, T Yasumoto. Structures and cOnfigUratiot1s of ciguatoxin from the moray eel G?.i,lttotlro~tcsjrr~,trnieus and its likely precursor from the dinoflagellate Garrrhidi.sclr.s toxicrrs. J Am Chetn SOCI12:4380-4386, 1990. T Yasurnoto, M Murata. Polyether toxins involved in seafood poisoning. In: S Hall and G Strichartz, eds. Marine Toxins: Origin, Structure, and Molecular Pharmacology. Washington. DC: American Chemical Society, 1990, pp 120- 132. c Frelin, M Durand-Clement, J-N Bidard, M Lazdunski. The molecular basis Of CigUatoXin action. In:S Hall and G Strichartz, eds. Marine Toxins. Washington, DC: American Chemical Society,1990, pp 192-199. A" Legrand, P Cruchet, R Bagnis, M Murata. Y Ishibashi. T Yasumoto. Chromatographic and spectral evidence for the presence of multiple ciguatera toxins. In: E Graneli. B Sundstrom. L Edler, and DM Anderson, eds. Toxic Marine Phytoplankton. New York: Elsevier, 1990, pp 374-378. CA Rakotoniaina, DM Miller. Assays for ciguatera-type toxins. In: DM Miller,ed. Ciguatera Seafood Toxins. Boca Raton, FL: CRC Press, 1991, pp 73-86. CW Venable, DM Miller. NMR of marine toxins. In: DM Miller.ed. Ciguatera and Seafood Toxins. Boca Raton, FL: CRC Press, 1991, pp 88-101. E Chungue, R Bagnis. N Fusetani, Y Hashimoto. Isolation of two toxins from a parrotfish Sccrnrs Rihbus. Toxicon15:89-93.1977. BW Halstead. Poisonous and Venomous Marine Animals o f the World. 2nd ed. Princeton. NJ:DarwinPress. 1988. MF Capra, J Cameron. Ciguatera i n Australia. In: DM Miller, ed. Ciguatera Seafood Toxins. Boca Raton. FL: CRC Press, 1991. K Terao, E Ito. TYasumoto.Lightandelectronmicroscopicstudies ofthemurineheart after repeated administrations of ciguatoxin or ciguatoxin-4c. Natural Toxins 1: 19-26, 1992. P Martyr.De Orbe Novo, the Eight Decadesof Peter Martyr d' Anghere. 1912 English translationbyFAMacNutt, 2 vols. New York: G. P. Putnam Sons, 1555. W Harmansen. An Account Taken From The Journal of the Voyages of Admiral Wolphart Harmansen to the East lndies [personal journal, in French]. Unpublished manuscript, 1601. pp 1-18.
53. G Knox. Poisonous fishes as means for prevention of cases of poisoning by them. VoennoMedZh 161399-464, 1888. 54. DR Steinbach. Aus dem Schutzgebiete der Marshall-Inseln [in German. with English translation]. In: F Danckelman, ed. Mittheilungen von Forschungsreisenden und Gelehrten aus den Deutschen Schutzgebieten. Berlin: Emst Siegfried Mittler und Sohn, 1895, pp 157-171. special reference to ciguatera in the 55 EW Gudger. Poisonous fishes and fish poisoning with West Indies. Am J Trop Med 10:43-55, 1930. 56. R Matsuo. Study of poisonous fishes at Jaluit Island [in Japanese]. Nanyo Gunto Chihobyo Chosa Ronbunshu 2309-326, 1934. 57. RL Gilman. Review of fish poisoning in the Puerto Rico-Virgin Islands area. US Navy Med Bull40:19-27,1942. 58. Y Hiyama. Poisonous Fish [in Japanese]. Odawara-shi, Juji, Japan: Japan Marine Product Research Laboratory, 1942, pp 1-24. 59. BW Halstead, WM Lively Jr. Poisonous fishes and ichthyosarcotoxism. US Armed Forces Med J 5:157-175, 1954. 60. Y Hashimoto. A note on the poison of a barracuda. Sphyrctenn picudn Bloch and Schneider. BullJpn Soc Sci Fish 21:1153-1157, 1956.
Halstead
48
61. JERandall.Areviewofclguatcra,tropical fish poisoning,with a tentativeexplanation of its cause. Bull Mar Sci Gulf Carib 8:236-267. 1958. 62. AH Banner, S Sasaki,PHelfrich. CB Alcnder.PJ Scheucr. Bioassay o f ciguateratoxin. Nature 189: 229-230,1961. 63. AH Bmncr, P Hclfrich. The Distribution o f Ciguatcra in the Tropical Pacific. An Epidemiological Survey of Fish Poisoning in the Tropical Pacilic. Technical report no. 3. Honolulu: HawaiiMarineLaboratory,1964. 64. MJ Cooper. Ciguatera and other marine poisonings i n the Gilbert Islands. Pac Sci 18:41 1 440, 1964. 65. PJ Schcuer, W Takahashi, J Tsutsumi, T Yoshida. Ciguatoxin: isolation and chemical nature. Science 155: 1267- 1268, 1967. 66. R Bagnis, F Berglund, PS Elias,GJ van Esch, BW Halstead, K Kojima. Problems o f toxicants i n marine food products. Bull World Health Organ 42:69-88, 1970. 67. DP De Sylva, AE Hine. Ciguatera-marine fish poisoning-a possible consequence of thcrmalpollutionintropical seas'? In:MRuivo, ed. MarinePollutionand Sea Life.Surrey. England: Fishing News Books, 1972, pp 594-597. 68.FERussell.Ciguaterapoisoning: ;I report of 35 cases.Toxicon 13:383. 1975. (grouper) tropical fish poisoning. 69. HR Jones, Jr.Acuteataxiaassociatedwithciguate1.a-typc Ann Neurol 7:49 I , 1980. J 281:948, 70. FM Tatnall, HG Smith, PD Welshy, PC Turnhull. Ciguatera poisoning. Br Med 1980.
71. HA Hanno.Ciguaterafishpoisoning in theVirginIsland[letter].JAMA 245:464, 1981. 72. JGMorrisJr.PLewin,NTHargrctt.CWSmith. PA Blake,RSchneider.Clinicalfeatures of ciguatera fish poisoning: a studyofthe disease i n the U.S. Virgin Islands. Arch Intern Med 142:1090. 1982. 73. NC Engleherg. JG Morris Jr. JN Lewis. JP McMillian, RA Polard, PA Blake. Ciguatera fish poisoning: a major cotnmonsource outbreak i n the U.S. Virgin Islands. Ann Intern Med 98: 336, 1983. 74. NW Withers.Ciguaterafishpoisoning.AnnuRevMed 3397-1 1 I , 1982. 75. AM Gelh, DMildran.Ciguaterafishpoisoning. NY State Med J79:1080,1979. 76.RPearce,CHinesJr.TWBurns, CE Clark.Ciguatera(fishpoisoning).SouthMedJ76: 560. 1983. 77. AM Lcgrand.RBagnis.Mode ofactionof ciguateratoxins. I n : EPRagelis, ed. Seafood Toxins. Washington,DC:AmericanChemicalSociety. 1984, pp 217-223. 78. DR Tindall, RW Dickey, RD Carlson, G Morey-Gaincs. Ciguatoxigenic dinoflagellates from the Caribbean Sea. In: EP Ragelis, ed. SeafoodToxins. Washington. DC: American Chemical Society. 1984. pp 225-240. 34:28-31. 79. Y Hokalna. Anupdate o f methodsforciguatoxindetection.SPCNewslett 1985. 80. JL Allsop, L Martin. H Lebris, J Pollard, J Walsh, S Hodgkinson. Les lnanifestations neurologiques de la ciguartera. RevNeurol 142:590, 1986. 8 I . NC Gillespic. RJ Lewis, JH Peam. ATC Bourke, MJ Hohnes, JB Bourke, WJ Shields. Ciguatera i n Australia: occurrence, clinical features, pathophysiology, and management. Med J Aust 145:584, 1986. 82. AMHHo,LM Fraser, EC Todd. Ciguatera poisoning: a reportofthree cases. Ann E m r g Med 15:1225. 1986. 83. RA Bagnis, AM Legrand. Clinical features on 12,890 cases of ciguatera (fish poiSOnillg) in French Polynesia. In: P Gopalakrishnakone and CK Tan, eds. Progress in Venom and Toxic pp 372-384. Research. Singapore: National University of Singapore, 1987. 84. ND Lewis. Ciguatera in the Pacific: incidence and ilnplications for marine resource development. In:EPRagelis. ed. SeafoodToxins.Washington,DC:AmericanChemicalSociety, 1984, pp 289-306.
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ND Lewis.Diseaseanddevelopment:ciguaterafishpoisoning.Soc SCi Med23:983. 1986. 86. p Gopalakrishnakone, CK Tan. Progress in Venom and Toxin Research. Singapore: National University of Singapore, 1987. 1991. 87. FE Ahmed, ed. Seafood Safety. Washington, DC: Natronal Academy Press, 88. GM Calvert. The recognition and management of ciguatera fish poisoning. In: DM Miller. ed. Ciguatera Seafood Toxins. Boca Raton, FL: CRC Press, 1991, pp 1- 1 1. 1991. 89. DM Miller, cd. Ciguatera Seafood Toxins. Boca Raton, FL: CRC Press, FE Russell, NB Egen. Ciguatcric fishes, ciguatoxin (CTX) and ciguatera poisoning. Toxicol 90. 1991. ToxinRev10:37-62, 91. J Laigret, G Bereziat, G Cuzon. J Polonovski. Etude comparative des acides gras extraits de s provenant de zones toxicogeneset non toxicogenes du lagon poissons C t e t l o d l ~ r m striurus de Tahiti. Bull Soc Pathol Exot 66:235-239, 1973. of ciguatoxin. 92. Y Hokama, AH Banner, D Boyland. A radioimmunoassay for the detection Toxicon15:317-325,1977. 93. PF Parc, R Ducousso, S Chanteau, E Chungue, R Bagnis. Problemes poscs par la detection ilnmunologique dc la ciguatoxine dam les tissus pisciaires [in Frcnch]. Med Oceanogr 14: 1-4,1980. 94. S Chanteau, I Lechat, F Parc, R Bagnis. Essai de detectionladeciguatoxine par une methodc immunoenzymatique [in French]. Bull Soc Pathol Exot 74:227-232, 1981. DC Baden,TYasumoto,MNukina, PJ 95. YHokama,LHKimura, MAAbad,LYokochi, Scheuer, Y Shimizu.An enzyme ilnlnunoassay for the detection of ciguatoxin. In: EP Ragelis, ed. Seafood Toxins. Washington, DC: American Chemical Society, 1984, pp 307-320. of ciguatoxin and related 96. Y Hokama. Simplified solid-phase immunobead assay for detection polyethers. J Clin Lab Anal 4:213-217, 1990. 97. Y Hokama. Immunological analysisof low molecular weight marine toxins. J Toxicol Toxin Rev 10:l-35, 1991. 98. Y Hokama, SAA Honda,MN Kohayashi, LK Nakagawa, AY Asahina, JT Miyahara. Monoclonal antibody (MAb) in detection of ciguatoxin (CTX) and related polyethers by the stickenzyme immunoassay (S-EIA) in fish tissues associated with ciguatera poisoning. In: S Natori.KHashimoto, Y Ueno. eds. MycotoxinsandPhycotoxins '88. Amsterdam:Elsevier Science, 1989, pp 303-310. 99. T Yasumoto, M Satake, Y Onuma, J Roux. Toxins involved in ciguatera, clupcotoxism and shark poisoning. Fifth Indo-Pacific Fish Conference, Noumea. New Caledonia, November 1997. 100. DS Jordan, BW Evermann. The Fishes of North and Middle America: A Descriptive Catalogue. Part 1. Washington,DC:SmithsonianInstitution.1896. 101. W Ferguson. On the poisonous fishes of the Caribbean Islands. Trans R SOC Edinb 9:6579, 1823. 102. RT Lowe. A History of the Fishes of Madeira. London: Bernard Quaritch, 1843. 103. A Kramer. Der purgierfisch der gilbcrtinsein [in German]. Globus 79( 12): 181-1 83, 1901, 104. Dl Macht, J Barba-Gose. Pharmacology of Rlr,Ietrlr.spreriosus, or "caster-oil fish." Proc Soc Exp Biol Med 28:772-774, 193 1. 105. Dl Macht, J Barba-Gose. Two new methods for pharmacological comparison of insoluble purgatives. J Am Pharm Assoc 20:556-564, l93 1, 106. SL Taylor, J Hui, DE Lyons. Toxicologyof scombroid poisoning. In: EP Ragelis, ed. Seafood Toxins. Washington, DC: American Chemical Society, 1984, pp 417-430. 107. HA Frank, DH Yoshinaga. Histamine formation in tuna. In: EP Ragelis, ed. Seafood Toxins. Washington, DC: American Chemical Society, 1984, pp 443-451. 1 08. SL Taylor. Histamine food poisoning: toxicology and clinical aspects. CRC Crit Rev Toxicol 17:92,1986. 109. T Kawbata, K Ishizaka, T Miura. Studies on the food poisoning associated with putrefaction 85.
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of marine products. I. Outbreaks of allergylike food poisoning caused by "samma sakuraboshi"(driedseasoned saury) andcannedseasonedmackerel.BullJpnSoc Sci Fish21: 335-340,1955. 1 IO. GM Dack. Food Poisoning, 2nd ed. Chicago: University of Chicago Press, 1956. 1 1 1. HBouder.ACavallo,JBouder.Poissonsveneneuxetichtyosarcotoxisme.BullInst Oceanogr 1-66, 1962. 112. JAWilliamson, PJ Fenner, JW Burnett, JF Filkin.cds.VenomousandPoisonousMarine Animals: Medical and Biological Handbook. Sydney: Universityof New South Wales Press. 1996. 113. JMeier, J, White.eds.ClinicalToxicology ofAnimalVenomsandPoisons.BocaRaton, FL:CRCPress,1995. 114. BW Halstead. Dangerous Marine Animals, 3rd ed. Ccntreville, MD: Cornell Maritime Press. 1995. 115. PSAuerbach, ed. WildernessMedicine.Management of WildernessandEnvironmental Emergencies, 3rd ed. NewYork:Mosby,1995. 116. E Geiger. Histamine content of unprocessed and canned fish. A tentative method of qualitative determination of spoilage. Food Res 9:293-297. 1944. 1 17. K Tsuda, R Tachikawa, K Sakai, C Tammura,S Ikuma, 0 Amakasu, M Kawamura, S Ikutna. O n the structure of tetrodotoxin. Chem Pharmcol Bull 12:642-645, 1964. 1 18. RBWoodward.Thestructureoftetrodotoxin.PureApplChem 9: 49-74,1964. 119. T Namhashi. Mode of aclion of dinoflagellate toxins on nerve membranes. In: VR LoCicero. ed. Proceedings of the First International Conference on Toxic Dinoflagellates. Wakefield, MA: Massachusettes Science and Technology Foundation, 1974, pp 395-402. I n : EP Ragclis, ed. Seafood 120. HS Mosher, FA Fuhrman. Occurrence and origin of tetrodotoxin. Toxins. Washington, DC: American Chemical Society, 1984, pp 333-341. 121. G Strichartz,NCastle.Pharmacologyofmarinetoxins.In: S Halland G Strichartz, eds. Marine Toxins. Washington. DC: American Chemical Society, 1990, pp 2-20. 122. JC Tyler. Osteology, Phylogeny, and Higher Classification of the Fishes o f the Order Plectognathi (Tetraodontiformes). Seattle: U.S. Department of Commerce. 1980. 123. TMatsui, S Hamada, S Konosu. [inJapanese].Bull Jpn Soc SciFish 47535, 1981. 124.TMatsui, S Hamada, K Yamamori.[inJapanese].BullJpnSocSciFish 48:l 179. 1982. 125. T Matsui, S Hamada,C Shimizu. [in Japanese]. Bull Jpn Soc SciFish 48:253, 1982. 126. LS Berg. Classification of Fishes Both Recent and Fossil. Ann Arbor, MI: J. W. Edwards, 1947.
3 Other Poisonous Marine Animals
1. 11.
Ill.
Introduction S I lnvertebratcs S 1 A. Poisonous marine Protista (Protozoa) S1 B. Poisonous cnidarians (Coelenterata) 53 C. Poisonous echinodcrms (sea cucumbers, sea urchins) D. Poisonous mollusks 56 63 E. Poisonousarthropods: crabs andlobsters Vertebrates 66 Poisonous marine turtles B. Poisonous marine tnammals Acknowledgtnent 72 References 72
A.
1.
54
67 68
INTRODUCTION
Although toxic marine fishes, paralytic shellfish poisoning, diarrhetic shellfish poisoning, brevitoxic (neurotoxic) shellfish poisoning, and amnesic shellfish poisoning probably account for the bulk of the foodborne intoxications caused by marine organisms, there are a variety of other toxic marine organisms capableof causing severe oral intoxications and even fatalities. This chapter deals with someof these lesser-known marine biotoxications. The topics discussed have been arranged phylogenetically.
II. INVERTEBRATES
A.
Poisonous Marine Protista (Protozoa)
The phylum Protista, or Protozoa, consists of single-celled microscopic organisms, most of which are free livingand inhabit an aquatic environment. Afew live i n the body fluids of other animals. Most protistans liveas independent cells, but some are grouped as colonies. Marine protistans poisonous to man are largely members of the class Mastigophora of the order Dinoflagellata. Since dinoflagellates exhibit both animal and plant characteris51
52
Halstead
tics (i.e., motility and chlorophyll), theyare claimed by bothzoologistsandbotanists. Hence they sometimes are referred to as plant-animals. They also are designated as phytoplankton. They abound in neritic waters and i n the high seas, ranging from the tropics to polar oceans. Dinoflagellates form an important part of the ocean plankton as synthetic producers of carbohydrates, proteins, and fats. During their periodic maxima, they may cause yellow, brown, green, black, red, or milky local discolorations of the sea. The “blooming” of thesetoxic plankton in excessive numbers frequently causes the mass mortality of the fish and other animals livingin the region. Phytoplankton blooms often areassociated with weatherdisturbancesorweatheralterationsthatbringabout changes in water masses or upwellings. Conditionsmost favorable for the growth of dinoflagellates are found more oftenin coastal waters than far offshore. Dinoflagellate blooms can causeseriouseconomiclosses i n a regionbecause of theirtoxicityandthemass mortalities of fish they cause. Toxic dinoflagellates play a major role as transvectors of poisons that are ingested by a variety of mollusks, causing paralytic, brevitoxic (neurotoxic), and diarrhetic shellfish to.ricus (Adachi and Fukuyo) serves as a poisoning.Thedinoflagellate Gtrmhie~~cli.scu.s transvector of theciguatoxincomplexinciguaterafishpoisoning. In addition,several other species of dinoflagellates are suspected as causative agents in ciguatera fish poisoning; they are A~nphidiniumcarterere (Hulbert), Ostreopsis ovuttr (Fukuyo), Prorocerltrurn conccrvum (Fukuyo), P. l i m r (Ehrenberg), and P. tuexicmrm (Tafall) (1).
Family: Pfiesteriaceae (dinoflagellate). Species: Pfiestericr piscicidn (Steidinger, Burkholder, et al.) Pfiesteria dinoflagellate. Distribution: Maryland, south along the southeastern coast of the United States.
Over the past 20 years there has been an increase in toxic dinoflagellate blooms worldwide which has resulted in mass mortalities of shellfish and finned fish capable of adversely affecting human health. One of the worst of these toxic blooms was caused by a new species of dinoflagellate known as Pfiesteritr pisciciclum, first reported by Burkholder et al. (2) in 1992. It was later determined by Steidinger et al. (3) that this dinoflagellate was a new species, a new genus, and a new family of the order Dinamoebales. This dinoflagellate is unique because of its polymorphic multiphasic life cycle. The dinoflagellate ranges in size from 5 to 250 pm. Moreover, the dinoflagellate produces potent toxin(s), the molecular structure of which has not been elucidated. It is estimated that this dinoflagellate has produced a mass mortality of more than one billion invertebrates and fish along the Atlantic coast involving the Pamlico and Neuse estuaries in North Carolina and the lower shores of Maryland. The cause of the toxic dinoflagellate bloom was due directly to excessive pollution by hog, chicken, and other untreated wastes that were being dumped into the rivers. The dinoflagellate requires fresh finfish or their excreta for excystment and the release of its potent neurotoxin(s). Mechnrrism of Intoxiccrtiorz: Human exposure to Pjiestericc may be in the form of an aerosol or water containing the toxic dinoflagellate or contact with the bodies of fish or shellfish that have been killed by Pfiesteria. Fish that have been killed by Pfiesteria have skin lesions over their bodies and should not be handled.
Other Poisonous Marine Animals
53
Cli~itwIC I ~ ~ ~ r ~ ~ ~ t eContact r i . s t i (with ~ ~ :Pjesteriu toxin may result in paresthesias of the extremities and circumoral area, joint and muscle aches, headaches, itching, nausea, vomiting,abdominalpain,memoryloss,disorientation,sweating,respiratorydistress, emotional changes, and skin lesions (4-6). Twtrtmetrt: The treatment is symptomatic. There is no known antidote. Pretvntiotl: Avoid handling any dead or dying fish having skin lesions in water contaminated by Pfiestcviel. Do not swim in estuaries or coastal waters contaminated by Pje.ster.it/.Persons working with cultures of Pjiesterin should be properly covered with protective clothing and masks to avoid contact with either water or air contaminated with Pje.steri(r toxin. Contaminated fish should not be eaten.
B. Poisonous Cnidarians (Coelenterata) Cnidarians or coelenterates are best known for their stinging abilities. However, i n this chapter, only their toxicity is discussed as it relates to biotoxications by ingestion. Cnidarians. or coelenterates, are simple metazoans having primary radial, biradial, or radiobilateral symmetry. They are composed essentially of two epithelial layers and an internal cavity, the gastrovascular cavity or coelenteron, which opens only throughthe mouth. Another dominant characteristicof the group is the presence of tentacles equipped with stinging nematocysts. The group is characterized further by showing a remarkable degree of polymorphism, having an alternation of generations with a sexual and asexual phase, as well as a specialization of individual polyps as in the siphonophores. A single species may present a variety of forms of either the sessile polyp or the free-swimming medusoid type. For at least a part of their life span, most coelenterates are attached or sedentary. The phylum Cnidaria (Coelenterata) includes three classes: Hydrozoa, the hydroids; Scyphozoa, the jellyfish; and Anthozoa, the sea anemones, corals, and alcyonarians. Apparently hydroids are not used as food. Jellyfish commonly are eaten in Japan and elsewhere, and there have been no reported cases of poisonings. The nematocysts of hydroids and jellyfish contain proteinaceous toxins that are inactivated by heating and gastric juices. However, oral biotoxications have resulted from the ingestion of sea anemones in the Philippines, New Guinea, and Samoa. Palytoxin, which is produced by anthozoan Ptrlyfhoa species. is not discussed in this section because palythoans generally are not considered to be a foodstuff. However, palytoxin has been found to be transvectored by fish and other organisms (7,8).
Represmttrtive Species Family: Actiniidae (sea anemones) Species: Phy.sobr~crchiacInu~~Itr.si(Kent). Lulnane (Samoa). Diameter 2 in. ( 5 cm). Distribution: Indo-Pacific. Family: Actinodiscidae (sea anemones) Species: Rhotfrrctis howc~si(Kent). Matalelei (Samoa). Diameter 4 in. (30 cm). Distribution:Indo-Pacific. Family: Stoichactiidae (sea anemones) Species: Rcrdicrr~tl~u.s p t r u r n o t e r r s i s (Dana). Matamala samasama (Samoa). Diameter 3 in. (8 cm). Distribution:Indo-Pacific.
Halstead
54
Sea Anemone Poisoning Intoxications resulting from the ingestion of poisonous sea anemones in Samoa and other parts of the tropical IndoPacific region have been reported by Farber and Lerke (9) and Martin (lo), and are discussed by Halstead ( 1 1.12) and Hashimoto (13). The nature of the poison is unknown. Mechanistn of Intoxication: Sea anemones commonly are eaten in Samoa and elsewhere in the tropical Indo-Pacific region,but they generally are cooked. Rhodncris howesi (Matalelei) andPhysobmchia douglasi(Lumane) generally are consideredto be poisonous when raw, but safe to eat when cooked. Rarlionthus pnurnotensis (matamala samasama) and some of the other members of this genus are considered to be poisonous to eat raw or cooked. Small children are frequent victims of sea anemone poisoning intheIndoPacific region. Clinical Chorcrcmiytics: The initial symptoms of sea anemone poisoning consist of acute gastritis with nausea, vomiting, abdominal pain, cyanosis, and prostration. Shortly after ingestion of the sea anemones, the victim may become comatose, which may last a period of 36 hours or more. During this comatose period, the superficial reflexes may be absent. The blood pressure and pulse remain normal. Pulmonary edema has been reported. The patient may go into profound shock, and death usually ensues. Trecrtment: The treatment is symptomatic. There are no specific antidotes. Prevention: The safest procedure for prevention is to avoid eating sea anemones.
C.
Poisonous Echinoderms (Sea Cucumbers, Sea Urchins)
Echinoderms include the starfish, sea urchins, and sea cucumbers. They are all members of the phylum Echinodermata. The members of this group are characterizedby their radial symmetry, a body with usually five radii around an oral-aboral axis, calcareous platesthat form a more or less rigid skeleton, or plates and spicules embedded in the body wall. Spines and pedicellariae are presentin the asteroids and echinoids,but are absent in some of the others. The coelom is complex and includes a water vascular system with tube feet. The digestive tract may or may not include an anus. The sexes usually are separate. Echinoderms, with the exception of a few planktonic holothurians, are all benthic and all are marine. The phylum Echinodermata is divided into four classes: Asteroidea, the starfish; Ophiuroidea, the brittle stars; Holothuroidea, the sea cucumbers; and Echinoidea, the sea urchins, heart urchins, and sand dollars. Some species of starfish are reported to be toxic, but little is known concerning the nature of the poisons or their effects on humans. Poisonings have resulted from eating sea cucumbers and the ovaries of sea urchins.
1. Poisonous Sea Cucumbers Sea cucumbers are free-living echinoderms having an elongate, wormlike or sausagelike body, without free arms but with a series of tentacles circling the mouth, located at the anterior end of the body. The intestinal tract is long and looped, terminating in an anus at the posterior end. The skeleton consists of variable-size, irregularly arranged plates embedded in the skin. Tube feet are present but are not situated in a furrow. I n some species of sea cucumber, a number of white,pink,orredtubulesareattached to the common stem of the respiratory trees. These are the so-called organs of Cuvier. or Cuvierian tubules. If the sea cucumber is irritated, the Cuvierian tubules are discharged through the anus. Upon contact with the water, they swell and elongate into sticky slender threads
Other Poisonous Marine Animals
55
that serve to entangle the predator. Only part of the tubules are emitted at any one time, and the expelled tubules are soon replaced by new ones. In some species, the organs of Cuvier are quite toxic, containing large concentrations of holothurin. Holothurians are sluggish creatures, generally moving over the bottom of the sea by means of rhythmic contractions of the body. The tube feet are used principally as organs of attachment. Sea cucumbers are not important i t e m of food for fish, but they are used as food by many Pacific islanders and Asians. Sea cucumbers are sold commercially under the names of “trepang” and “beche-de-mer.” They are prepared by boiling them, causing them to eviscerate, shorten, and thicken. After thorough drying, the trepang is ready for marketing. Trepang is used for flavoring soups and stews.
Represewt~rtiveSpccirs Family: Holothuridae (sea cucumbers) Species: Holotkuriu cwgus (Jaeger). Sea cucumber. Length 12 in. (30 cm). Distribution:Indo-Pacific. Species: Holothuria tl~b1do.w(Gmelin). Sea cucumber. Length 10 in. (25 cm). Distribution: Mediterranean and adjacent Atlantic Ocean.
Scrr C~vnr1x.rPoisorling Very little information is available on the clinical effects resulting from the ingestion of poisonous sea cucumbers. Mechnisrn of I~rto.vicrrtion:Intoxications have been reported from eating poisonous sea cucumbers (14,15). The causative toxins in seacucumbersarecalledholothurins, of sugars, steroid which involve a complex of saponins. Saponins are complex compounds moieties, or triterpenoid moieties and are characterized by forming a durable foam when their water solutions are shaken. See Refs. 12, 13. 16, and 17 for a more detailed discussion of the chemistry of holothurins. Clirlictrl Clltrmcteristics: Reported symptoms of dermal contact with sea cucumber poison are burning pain, redness, anda violent inflammatory reaction. Liquid ejected from the visceral cavityof some species, when contacting the eye, may cause blindness. Nothing appears to be known concerning the symptoms resulting from the ingestion of poisonous sea cucumbers, but fatalities have been reported ( 18-20). Treatrncnt: Treatment is symptomatic. Pharmacological studies suggestthat anticholinesterase agents may be effective in the event of ingestion of holothurin (21). Prevention: Check with the locals prior to eating sea cucumbers. This is particularly important in tropical regions.
2. Poisonous Sea Urchins Sea urchins are free-living echinoderms having globular, egg-shaped, ora flattened body. The viscera are enclosed within a hard shell, or test, formed by regularly arranged plates carrying spines articulating with tubercles onthetest.Betweenthespinesaresituated three-jawed pedicellariae, which are of interest to the venonlologist and have been described at great length elsewhere ( 1 l , 12). Tube feet are arranged in 10 meridian series rather than in furrows. A double porein the testcorresponds to each tube foot. The intestine is long and coiled, and an anus is present. The gonads are attached by mesenteries to the inner aboral surface of the test. The mouth, situatedon the lower surface, turns downward and is surrounded by five strong teeth incorporated i n a complex structure called “Aristotle’s lantern.” Sea urchins move by means of spines on the oral side of the test.
56
Halstead
Represerztc/tive Species Family: Echinidae (sea urchins) Species: P~rracet1trotu.slividus (Lamarck). European sea urchin. Diameter of test 3 in. (7 cm). Distribution: Atlantic coast of Europe, Azores, West Africa. Family: Toxopneustidae (sea urchins) Species: Tri/meusres g r d l l a (Linnaeus). Sea urchin. Diameter of test 4 in. (10 cm). Distribution: Indo-Pacific, Japan, East Africa, Australia. Secr Urchin Poisonitlg
Several species of European and Indo-Pacific echinoids serve as commercial food sources. Only the gonads are eaten, either raw or cooked. During the reproductive season, generally the spring and summer, the ovariesof certain species of sea urchins are reported to develop toxic products that are injurious to man (8,1 1 -13,22-25). Mecllcrnisrn o f Itltoxiccrtiorl: Sea urchin poisoning is the result of ingesting toxic sea urchin gonads. The chemical nature of the poison is unknown. Cliniccrl Characteristics: The symptoms of sea urchin poisoning consist of general epigastric distress, nausea, diarrhea, vomiting, severe migrainelike headaches, and swellto be an allergic type of reaction in some ing of the lips and mouth. There is believed cases. Ttwtmerzt: Treatment is symptomatic. There is no known antidote. Prevewtior1: Care should be taken when eating the ova of known toxic species of sea urchins during the reproductive season. It is especially important to contact the local inhabitants as to the edibility of sea urchin eggs, especially in tropical regions.
D. Poisonous Mollusks Mollusks have been incriminated in a number of types of oral food intoxications aside from paralytic shellfish poisoning, diarrhetic shellfish poisoning, and amnesic shellfish poisoning. Mollusks are members of the phylum Mollusca. Mollusks are unsegmented invertebrates with a soft body and usually secreting a calcareous shell. A muscular foot is present that may be modified to serve various functions. Covering at least a portion of the body is soft skin, the mantle, the outer surfaceof which secretes the shell. Respiration is by means of gills or a modified primitive pulmonary sac. Jaws are present in some species. In four of the five classes, food is obtained by the use of a rasplike device called a radula. In the cone shells and a few others, the radula ribbon is lost and the teeth are modified into hollow, harpoonlike structures that may contain venom. The phylum Molluscais generally divided into five classes: Amphineura, the chitins; Scaphopoda, the tooth shells; Gastropoda, the snails and slugs; Pelecypoda, the bivalves (scallops, oysters, clams); and Cephalopoda, the octopuses, squids, and cuttlefish. Most of the toxic species of mollusks are gastropods, pelecypods, and, rarely, cephalopods.
1. Poisonous Gastropods: Abalone Abalone are gastropod mollusks that have a rough, horny coating that quite frequently is hidden by a thick cover of algae and other growth. The spire of the shell is flattened and the epipoda is bordered with a fringe and tentacles that project around the margin of the shell. Along the margin of the shell in older specimens, there is a single row of holes through which feelers may project and from which water passing over the gills is discharged. The living mollusk projects its head out from under the edge of the shell in the
Other Poisonous Marine Animals
57
area where therow of holes terminate. The tip of the broad muscular foot is pointed backward from under the spiral. The lining of the shell is pearly iridescent. Abalone shell has been used extensively as a source of mother-of-pearl i n the manufacture of buttons. ornaments, and trinkets. The muscular foot is used i n the preparation of soups, chowders, and as steaks. The toxic substance foundin the viscera of abalone is believed to originate through their food web, namely, certain species of seaweeds belonging to the genus L)r~.srrraresticr (26).
Represmtrrtitv Species Family: Haliotidae (abalone) Species: Hrrliotis di.sc~u.s(Reeve). Abalone. Length 6 in. (15 cm). Distribution: Japan.
Abnlorle Viscer-lr Poisorrirrg Abalone poisoning has been reported fro111 eating Hrrliotis di.scw.s and H. siebolcli in Japan (27). Mechcrrrisrrr of 1rlto.riccrtiotr: Poisonings from abalone are theresult of eating the viscera of the mollusk. The custom of eating the entire mollusk is practiced in Oriental countries. Elsewhere, only the muscular foot (abalone steak) is eaten. The tnuscular foot generally is safe. Although only two species of Japanese abalone, H r r l i o t i s discus (Reeve) and H. sieboldi (Reeve), have been incriminated, the viscera of other species of abalone are suspect. Clirriccrl Clrrrmc.tr.ri.stics: The symptoms of abaloneviscerapoisoninghavebeen described as a sudden onset of a burning or stinging sensation over the entire body, followed by an urticarial rash, itching, erythema, pain in the face and extremities, edema. and subsequent development of skin ulceration. It has been observed that the skin lesions are limited to those parts of the body exposed to sunlight, and there is a distinct boundary between covered and exposed parts of the body (26). Tretrtmerrt: Treatment is symptomatic. Pre\wtion: Do not eat the viscera of the abalone. Only the muscular foot or steak should be eaten.
2. Poisonous Gastropods: Turban Shells The turban shells are members of the gastropod mollusk family Turbinidae. They
are related to the top,or trochus, shells of the Trochidae. As their name implies, they generally are conical or top-shaped, spiral, and have a pearly luster on the inner surface of the shell. The turban shells are largely algae feeders. Some of the turban shells have bee11 found to be toxic. Rel~r-e.~~~rltrrtitl~~ Species
Family: Turbinidae (turban shells) Species: Turho crrgyro.sfor,lu.s (Linnaeus). Turban shell. Dialneter 3 in. (8 ~ 1 1 1 ) . Distribution: Indo-Pacific. Species: Tur-hoIr1rrrwwrrrfus (Linnaeus). Turban shell. Dialneter 8 in. (20 cm). Distribution: Indo-Pacific.
T ~ r l ~Shell r t ~ Poisorrirlg Human outbreaks have beenreportedfromeatingturbanshellstaken at M;lrcus Island, the Marianas Islands, and the Western Pacific (12,13.28). Several toxic sLIbstallces
Halstead
58
have been isolated from 7'. trrggrostottlus (Linnaeus), butthe chemical nature of these compounds hasnot been fully determined.A poison has been isolated from T. rwI-tt1oI-utu,s (Linnaeus) thatisbelieved to beidentical to ciguatoxin (29). Saxitoxinhas also been found in the gut of this turban shell. Mechmism of Itltosicertion: The toxic substances apparently are foundin the midgut gland and the gut of theturbanshell and apparently are obtained by feeding on toxic algae. Ingestion of the whole mollusk may cause poisonings in humans. Ciguatoxin and saxitoxin are also believed to occur in these mollusks. See Chapter 2 for further information on ciguatoxin and saxitoxin. Clillicd ChNrcrL.teI-iSt1'c.s: The synlptoms of turban shell poisoning consist of gastrointestinal upset, nausea, vomiting, diarrhea, fatigue, temperature-reversal sensation, and pruritus. In general, the symptoms resemble those of ciguatera fish poisoning (28). TI-ecrttnerlt:Treatment is symptomatic. See Chapter 2, Sec. 1V.C on ciguatera fish. PI-evention: The turban shells are believedto be safe to eatif the viscera are removed. However, since ciguatoxin and saxitoxin Inay be present, to prevent poisoning one should be extremely cautious when eating these shellfish. It is advisable to check with the local inhabitants concerning their edibility.
3. Poisonous Gastropods: Whelks The whelks area large and aggressive familyof carnivorous gastropod mollusksthat range from tropical to polar seas. They have a vertical distribution that ranges from the littoral zone to great depths. Their shells come in a variety of shapes, sizes, and colors. Whelks commonly are observed clambering about on rocks or plowing their way through mud, sand, or gravel, with the muscular foot largely buried in the bottom. When at rest, the mollusk retracts the foot and thereby closes the aperture with a horny operculum. Most whelks are more scavengers than active predators, feeding on dead fish and other scraps, but they tend to shun anything in an advanced state of decay. Some of the whelks feed on live mollusks, and at times may cause considerable damage to oyster beds. Whelk poison is said to be concentrated i n their salivary glands.
Represeutdve Species Family: Buccinidae (whelks, ivory shells) Species: Bcrbylnrlirr jrrpotIiccr (Reeve). Japanese ivory shell. Length 3 in. (7 cm). Distribution: Japan. Species: Neptrrneer to~tiglrrr(Linnaeus). European whelk. Length 4 in. (10 cm). Distribution: Northern Europe. Species: Nepturlerr inteI-scwlpttr (Sowerby). Whelk. Length 6 in. ( I S cm). Distribution: Japan.
Whelk Poisotlitlg Several outbreaksof poisonings fromthe ingestion of the Japanese ivory shell (Btrbylotlicr jcrponi~tr)have been reported in Niigata, Japan (12,13,30,3 1). Human intoxications have been reported by Kanna and Hirai (32) for Neptunea itrtersarlptcr in Japan. Fange has reported finding the toxic substance tetrarnine i n the salivary glands of N. crllticqLdcr from Sweden (33-3). No clinical data were presented in the Swedish reports. M e c j ~ c r j l ic!fActiotl: .~~ Whelk poison is believed to be restricted to the salivary glands of the mollusk. Intoxication results when these glands are ingested in whole shellfish in the raw, cooked, or canned state.In B. jqmliccr, the poison was found to be concentrated in the lnidgllt gland. The poison found in B. jtrporlictr is called surugatoxin and has been
Other Poisonous Marine Animals
59
found to be a potent mydriatic, ganglion blocker, and hypotensive agent (36). Its molecular formula is C22Hl,,BrN,0,,, nlolecular weight 660 (13). Paralytic shellfish poison also has been detected in the digestive glands of N . decemcostlrta (Say) taken in eastern Canada (37). Clirlicrrl Cl~crmcterisfics: The toxic substance present in poisonous whelks istetramine. Tetramine is an autonomic ganglionic blocking agent. Intoxication from tetramine may result in nausea, vomiting, anorexia, weakness, fatigue, faintness, dizziness, thirst, dysuria, mydriasis, aphasia, numbness, photophobia, impaired visual accommodation, and dryness of the mouth. Trecrtrnent: Treatment is largely symptomatic. Preverltiorl: Poisonous whelks are said to be safe to eat if the salivary glands are removed.
4. Poisonous Bivalve Mollusks The pelecypods, or bivalves, have their bodies laterally compressed and are surrounded entirely by the lateral mantle folds, which are greatly enlarged and covered bya longitudinally divided shell. They lack a proboscis and a radula. The byssus gland, which is found at the base of the foot, secretes sticky threads that harden and serve as mooring lines to the substratum. The byssus threads of mussels are a good example of this attachment mechanism. The foot in bivalves is used as a burrowing organ. The mantle cavity lies on both sides of the foot and contains a pair of modified and enlarged gills, or ctenidia, also known as branchia. The stomach is associated with a crystalline style. The nervous system is simple and concentrated in the posterior portion of the body. Most bivalves are dioecious, and fertilization takes place in the water or in the mantle cavity. Pelecypods are largely filter feeders. In addition to paralytic, amnesic, brevitoxic (neurotoxic), and diarrhetic shellfish poisoning, there are other forms of bivalve shellfish intoxications, including callistin, venerupin, giant tridacna clam, and scallop poisoning.
5. Poisonous Bivalve Mollusks: Callistin Shellfish The genus Ctrllista belong to the family Veneridae, a group of bivalve mollusks of the class Pelecypoda. The members of this genus have an ovate shell that is acunlinate on the back and rounded at the front. The surface of the shell in Ccdlista brevi.si~~hotrrrtrr is marked by rough growth lines and is covered with a smooth and polished periostracum, under which light purplish-brown rays are seen on the yellowish background in young specimens. Members of the genus Cullisfa have been incriminated in human intoxications.
Repre.sewtative Species Family: Veneridae (Venus clams) Species: Ccrllisftr hrevisiphotlcrtrr (Carpenter). Japanese callista. Length in. 6 (15 cm). Distribution: Japan.
The history of callistin shellfish poisoning appears to be of recent origin. The first report of an outbreak of poisoning was by Asano et al. (38) and was caused by eating C. brevisil,hnntrttr taken in the vicinityof Mori, Hokkaido, Japanin 1950. There wasa second report of an occurrence in the same region in 1953. The Mori Health Ccnter subsequently banned the sale of this shellfish.
Halstead
60
Mechcrnistn ofPoi.soning: Callistin shellfish poisoning results from eating the ovaries of the Ctrllistn bivalve, which contains a high concentration of choline. The shellfish are said to be toxic only during the spawning season, May-September. Clirricrrl Charcrcteristics: The onset of symptoms generally occurs within 1 hour after ingestion of the toxic shellfish. The chief symptoms are itching, flushing of the face, urticaria, a sensation of constriction in the chest, epigastric and abdominal pain, nausea, vomiting, dyspnea, cough, asthmatic manifestations, hoarseness, paralysis or numbness of the throat, mouth, and tongue, thirst, hypersalivation, drop in blood pressure, increase in pulse rate, leucocytosis, sweating, chills, and fever.In general, this biotoxication resembles a severe allergic reaction. No fatalities have been reported. Treatnrent: Treatment is symptomatic.. Antihistamines have been recommended. Pretrntiow:The callistin shellfish are saidto be safe to eat if the ovaries are removed. The ovaries can be identified by smearing a small amount of the whitish- or yellowishcolored gonads ona glass slide and observing the material undera microscope. The female ovary can be identified by the white, granular-appearing ova. The male gonadal substance appears as a milky paste. Cooking does not destroy the toxic principalof callistin shellfish poison. The freshness of the shellfish is not a factor in the occurrence of this disease.
6. Poisonous Bivalve Mollusks: Venerupin Shellfish Venerupin shellfish poisoning was named after the bivalves Dosinicl jcrporliccr and Tq’es serniclecusscrtr~,which are members of the pelecypod family Veneridae. In addition, the Japanese oyster (Crtmostrecr g i p s ) , a bivalve of thefamilyOstreidae,hasalsobeen incriminated as a causative shellfish species. The toxicity of the bivalves is believed to be due to the transvectoring of one or several toxic speciesof dinoflagellates of the genus Prorocerrtrum. One of the agents is thought to be P. rrltrric.le-lebouric~e(12).
RL~l~resenttrtitle Species Family: Ostreidae (oysters) Species: Crcrssostrecr gigas (Thunberg). Oyster. Length 25 in. (25 cm). Distribution: Japan, British Columbia, southern California. Family: Veneridae (Venus clams) Species: Dosinitr jcporriccr (Reeve). Japanese dosinia. Length 3 in. (7 cm). Distribution: Japan. Species: Ttrpes semidecussata (Reeve). Japanese littleneck. Length 2 in. ( 5 cm). Distribution: Japan, British Columbia south to California.
Vcrrerrrpir~Slrellfislr Poisorrirrg Outbreaks of venerupin shellfish poisoning were first reported i n 1957 near Niigata, Japan, and later in the Kanagawa and Shizuoka Prefectures, Japan, during the months of January-April ( 1 3,39). There is no record of this type of shellfish poisioning occurring elsewhere, despite the fact that the species of shellfish involved are found in other parts of Japan and have been introduced into the United States. There donot appear to be any recent outbreaks reported. M t 4 ~ a r r i s mc!fItrtc).~ictrtion:Venerupin poison appears to be transvectored by a toxic species of dinoflagellate of the genus Prorocentrur?~,which is ingested by the shellfish and concentrated in the digestive gland or “liver” of the mollusk. The chemical nature of the poison is unknown. Clinicer/ Cl?nmctPri,stic.s:The symptoms of venerupin, or asari, shellfish poisoning
Other Poisonous Marine Animals
61
usually develop within48 hours of ingesting the shellfish.The initial symptoms are nausea, gastricpain,vomiting,constipation,headache,andmalaise.Bodytemperatureremains normal. Within 36 hours, additional symptoms such as nervousness, hematemesis, and bleeding from the mucous membranes of the nose, mouth, and gums develop. Halitosis is a dominant part of the clinical picture. Jaundice, petechial hemorrhages, and ecchymoses of the skin generally are present, particularly about the chest, neck, and upper portion of the arms and legs. Leucocytosis, anemia, retardation of blood clotting time, and evidence of liver dysfunction have been noted. The liver generally is enlarged, but painless. In fatal cases, the victim becomes extremely excitable, delirious, then comatose. There is no evidence of paralysis or other neurotoxic effects as in paralytic shellfish poisoning. The low mortality rate has been credited to early diagnosis and prompt medical care.In severe cases, the victim usually dies within 1 week. Recovery is extremely slow and the victim remains in a weakened condition for an extended period of time. The fatality rate is about
33%. Trecltment: Treatment is symptomatic, buttheuse of IV glucose, vitamins B, C, and D, and insulin have been recommended. Prevetltim: ShellfishtakenintheShizuokaandKanagawaPrefectures,Japan, should not be eaten during the months of January-April. Ordinary cooking procedures do not destroy the poison. Toxic shellfish cannot be detected by their appearance. The only certain method to determine the safety of the shellfish is to prepare tissue extracts and test them on laboratory animals.
7. Poisonous Bivalve Mollusks: Tridacna Clams Some species of giant c l a m of the family Tridacnidae have shells nlore than 39 in. (1 m.) in length. Tridacna clams are recognized by their massive shells and large irregular teeth set on a broad hinge plate; they can be found burrowed or wedged into coralon the bottom of tropical seas. The shape and color of the shell often blends in with the coral rock, but the giant clam is detected quickly by the brilliant coloration of its siphon edges and mantle. Two species of the giant tridacna clams have been incriminated in French Polynesia in human intoxications, which clinically resemble ciguatera fish poisoning. Representntive Species Family: Tridacnidae (tridacna clams) Species: Triductra gigas (Linnaeus). Tridacna clam. Length 54 in. (137 cm). Distribution: Indo-Pacific. Species: Tridclctlo tmrima (Roding). Tridacna clam. Length 14 in. (35 cm). Distribution: Indo-Pacific.
TridLtcm Shelljsh Poisowing Tridacna shellfish poisoning clinically resembles ciguatera fish poisoning (see Chapter 2 . Sec. 1V.C). The symptoms consist of gastrointestinal, vasomotor, and various disturbances, including a loss of motor coordination. Bagnis (40) provides the most complete account of tridacna shellfish poisoning, which involved33 people and a numberof domestic animals that had eaten TridLroln nrnxirncr at Bora-Bora, Society Island, French Polynesia. The poison involved was believed to be of the ciguatoxin complex. Trecrtment: Treatment is symptomatic (see Chapter 2, Sec. 1V.C). Prrvention: It is advisable to check withthelocalinhabitantsprior toingesting tridacna clams.
62
Halstead
8. Poisonous Bivalve Mollusks: Scallops Representative Species Family: Pectinidae (scallops) Species: Patinopectin yessoensis (Jay). Scallop. Length 6.3 in. (160 mm). Distribution: Northern Japan.
Sccrllop Poisoning Yasumoto et al. (41 ), Murata et al. (42), and Okada and Niwa (43) reported biotoxications in 1985 resulting from the eating of toxic scallops (Prrtinopectin yessoensi.s) harvested from northeast Japan. They isolated two toxins called pectenotoxin (PTXI and PTXI-5). PTXI was found to be a major component of dinophysotoxin (DTX) isolated from the mussel Mytilis eclulis taken from the northeast coast of Japan (41). Dinophysotoxin derives its name because of its association with the dinoflagellate Dinophysis forti;. Dinophysotoxin has also been found in toxic mussels found in Bantry Bay, Ireland (43). Another toxin, yessotoxin (YTX), has also been isolated from P. yessoensis by Murata et al. (42). Mechcrnism sf’ Action: Scallop poisoning is caused by eating poisonous scallops containing pectinotoxin or yessotoxin. Clinicnl Cl~trr.ncteri.stics:The symptoms generally consist of nausea, vomiting, and diarrhea. Neurological disturbances are generally not present. Trecrtnrent: The treatment is symptomatic. There is no known antidote. Prevention: Check with the local public health authorities if there is any question as to their edibility.
9. Poisonous Cephalopods Cephalopods are nlollusks of the class Cephalopoda that have bilateral symmetry and a well-developed head containinga circumoral crown of mobile appendages bearing suckers and/or hooks (exceptin Nrrutilus). The mouth has chitinous, beaklike jaws and a chitinous tonguelike radular band of teeth. The shell is variously modified, reduced, or absent and is enveloped by the mantle; an external shell occurs only in Nmrtilus, which is restricted to the Indo-Pacific area. They are soft-bodied animals, with their primary skeletal features consisting of a cranium-a mantle/fin support comprised of a cuttlebone or gladius. The central nervous system is highly developed and is associated with well-organized eyes. A funnel or siphon tube expels water from the mantle or body cavity. The coloration of cephalopods is variable, depending upon the species and habitat. Most cephalopods have numerous chromatophores and iridocytes i n the skin to accommodate rapid changes in colors and patterns. The size of cephalopods varies greatly, from less than 1 in. (2.5 cm) to 70 ft (20 m) in length and weighing over 1 ton. Locomotion is achieved by drawing water into the mantle cavityand expelling it i n a jetlike manner through the funnel. Octopi are sometimes observed crawling alongthe bottom of the sea on their a r m . Cephalopods are largely predatory and feed carnivorously on crustaceans, bivalves, and fish. Human intoxications resulting from the ingestion of octopus and squid have been reported. Repwsentrrtil>e Species Family: Ommastrephidae (squid) Species: Ommnstrephes slotrni p~rc$c~u,s(Steenstrup). Pacific squid. Length 12 in. (30 cm) Distribution: Pacific Ocean. Family: Octopodidae (octopi)
Other Poisonous Marine Animals
63
Species: Octopus vulgcrris (Lamarck). Octopus. Length 32 in. (80 cm). Distribution: Warm temperature and tropical waters, all oceans.
Cepkolnpod Poisorritrg Intoxications resulting from the ingestion of cephalopods generally are rare. However, a series of 779 outbreaks involving 2974 persons occurred in Japan from 1952 to 1955 caused by the ingestion of squid and octopus. Fatalities were reported (44). Apparently there have been no recent outbreaks reported. Meckrrrrisnr of 1rrto.ricrrtiorr:The nature of the poison involved in cephalopod poisonings is unknown. It is unknown whether bacteria may have playeda role in these cephalopod outbreaks. ClitriccrlCIr(1rcrcteristic.s: Symptoms did not develop in the Japanese outbreaks of cephalopod poisoning until about 10-20 hours after ingestion of the squid or octopus. The symptoms consisted of nausea, vomiting, abdominal pain, diarrhea, low-grade fever, headache, chills, weakness, dehydration, paralysis, and convulsions. Neurological sympa toms were not a dominant part of the clinical picture. Recovery was generally within period of 48 hours. In most instances, cephalopod poisoning resembles severe gastroenteritis, but death may occur. Trecrtrtrerrt: Treatment is symptomatic. There are no known specific antidotes. Prevetrtiorl: TheJapaneseoutbreaks of cephalopodposioningoccurredwithout warning and there was no evidence in the appearance or taste of the cephalopods. Both of the species of squid and octopus incriminatedin the Japanese outbreaks are eaten commonly throughout the world without any ill effects.
E. Poisonous Arthropods: Crabs and Lobsters The phylum Arthropodais the largest single group within the animal kingdom, containing more than 800,000 species. Arthropods are characterizedas having a body usually divided into a head, thorax, and abdomenof like os unlike somites, which are variously fusedand with each segment bearing a pair of jointed appendages. The exoskeleton is chitinous and is molted at intervals. The digestive tract is complete and divided into fore, mid-, and dorsally. The hindgut.The body spaces serve as ahemocoel,and theheartislocated phylum Arthropoda is divided intoa large numberof classes, but only twoof them, Merostomata, which includes the horseshoe crabs, and Crustacea, which includes the lobsters, crayfish, and crabs, are pertinent to toxicologists.
1. Poisonous Horseshoe Crabs The class Merostomata includes the horseshoe crabs or king crabs, which are characterized by an arched cephalothorax, a horseshoe-shaped carapace, a wide unsegmented abdomen possessing three-jointed chelicerae, pedipalpi, and six-jointed legs. They inhabit coastal waters with sandy or muddy bottoms at depths of less than 6 fathoms. Horseshoe crabs are scavengers and feedon polychaete worms, mollusks,a variety of small marine animals, and T d r y i e u s can be and algae. The Asiatic members of the genera Ctr~citroscorpir~s very toxic to eat.
Rcpreserrttltive Species Family: Xiphosuridae (horseshoe crabs, king crabs) Species: Crrrcirroscor1~ilr.srotrrrrrlictrudrr(Latreille). Asiatic horseshoe crab. Length 13 in. ( 3 3 cm).
Halstead
64
Distribution and habitat: Philippines, Indonesia, Malaysia. Species: Tdlypleu.7 gigas (Muller). Asiatic horseshoe crab. Length 19 in. (50 cm). Distribution: Torres Straits, Vietnam, east coast of Bay of Bengal.
Horseshoe Crcrh Poisoning Human intoxications from eating Asiatic horseshoe crabs have been reported by Smith ( 4 3 , Soegiri (46), Waterman (8,47,48), and Banner and Stephens (49). Mostof the reported outbreaks have occurredi n Thailand, but they probably occur elsewherein Southeast Asia wherever these horseshoe crabs are endemic. Horseshoe crab poisoning is referred to as mimi poisoning in Thailand. Mrchmism of Intmicrrtion: Asiatic horseshoe crab poisoning is caused by eating the unlaid green eggs, flesh, or viscera during the reproductive season. Despite their periodic toxicity, the large masses of unlaid green eggs are highly prized by Asiatic peoples (47,48). The poison in Asiatic horseshoe crabs is believed to be chemically identical to saxitoxin (50). Cliniccl/ ChLlrac.teristic,s:The onset of symptoms in Asiatic horseshoe crab poisoning usually occurs within 30 minutes of ingestion of the poison. The initial symptoms consist of nausea, vomiting, abdominal cramps, headache, dizziness, slow pulse rate, decreased body temperature, aphonia, cardiac palpitation, numbness of the lips, parethesias of the lower extremities, and generalized weakness. More-severe symptoms may occur in rapid succession: aphonia, sensation of heat in the mouth, throat, and stomach, inability to lift the arms and legs, generalized muscle paralysis, trismus, hypersalivation, drowsiness, and loss of consciousness. The mortality rate is unknown but is said to be very high. Death, when it occurs, takes place within a period of 16 hours. Trecrtmetzt: Treatment is symptomatic. Prevention: Although Asiatic horseshoe crabs commonly are eaten in many parts of Southeast Asia, they should be avoided during the reproductive season.
2. Poisonous Tropical Reef Crabs The class Crustacea includes the lobsters, crayfish, and crabs. Most of the known toxic reef crabs are members of the crustacean family Xanthidae. The xanthid crabs have a carapace that is transversely oval, hexagonal, or subquadrate, rarely subcircular, and almost always broader than long. The front of the carapace tends to be broad and is never produced in the form of a rostrum. The legs of the crab are of the ambulatory type rather than the swimming type. The family Xanthidae is found throughout the tropical littoral, inhabiting coral reefs. Most of the family feeds on a variety of foods, including algae. Some of the xanthid crabs have been foundto be extremely toxic and have caused human fatalities. Representative Species Family: Xanthidae (mud or reef crabs) Species: Atergutis yoridus (Linnaeus). Reef crab. Width of carapace 2 in. (5 cm). Distribution: Indo-Pacific. Species: Corpilius rntrculntlts (Linnaeus). Spottedreef crab. Width of carapace 4 in. (10 cm). Distribution:Indo-Pacific. Species: Demanin toxiccl (Garth). Poisonous reef crab. Width of carapace 2 in. ( 5 cm).
Other Poisonous Marine Animals
Distribution: Indo-Pacific. ?tl Reef crab. Width of carapace 2 in. Species: PlotypodiL/ ~ 1 y ( 1 1 1 ~ ~ 0 1 7 (Ruppell). cm). Distribution: Indo-Pacific. Species: Zozytmrs aeneus (Linnaeus). Width of carapace 3 in. (7 cm). Distribution: Indo-Pacific.
65
(5
Tropicul Reef Cmb Poisoning Reports of human biotoxications from tropical reef crabs have appeared at infrequent intervals. Most of these intoxications have occurred in the tropical Indo-Pacific region. References 2, 13, and 5 1-65 contain reports on poisonings by tropical reef crabs. Mechrrrlisrn of Itmxiccrtion: The poison contained in Zozymus aeneus, according to Refs. 8 and 66-68, appeared to be chemically identical to saxitoxin. Yasumoto et al. (697 I ) found saxitoxin, neosaxitoxin,and gonyautoxins to be present in various other species of tropical reef crabs including Nemanthias impressus (Lamarck), Actaeodes tornerltosus (H.MilneEdwards), Eriphicr scahricwltr (Dana), Pi1ur~111u.s vespertillo (Fabricius), Schi:.ophrys asperu (H. Milne Edwards), andThnlarnita species, and Halstead and Schantz (72) found themin Perctwn plani,s.sirnum(Herbst). Yasurnuraet al. (73) found tetrodotoxin in the reef crab Zozymus c m c u s . Clirlicnl Chcrrcrcteristics: The symptoms of tropical reef crab poisoning consist of paresthesias, muscular paralysis, aphasia, nausea, vomiting, and collapse. Death may occur within 2 hours to several days (66). The symptoms may resemble those of paralytic shellfish poisoning or tetrodotoxism (see Chapter 2, Sec. 1V.G.). Treatment: Treatment is symptomatic. Prevention: The eating of tropical reef crab is at best an extremely hazardous game of Russian roulette. Tropical reef crabs should not be eaten.
3. PoisonousCoconut Crabs Coconut or robber crabs (Birgus lntro) are land hennit crabsthat are equipped with powerful pincers capable of cutting coconuts from palm trees and opening them. Coconut crabs are capable of climbing palm trees and can do so with considerable agility. Under most circumstances, coconut crabs are eaten and looked upon as a great delicacy, but under certain conditions they may be toxic and may cause fatalities.
Representcltive Species Family: Coenobitidae (coconut crabs) Species: Birgus lntro (Linnaeus). Coconut crab. Length of carapace 5 in. (13 cm). Distribution: Indo-Pacific.
Coconut Crnb Poisonirlg Human biotoxications from coconut crabs have occurred from time to time in the Tuamotu and the Ryukyu Islands in the Indo-Pacific region (8,66,74). Mechanism ofI7?ro.rimrion: Coconut crabs commonly are eaten throughoutthe IndoPacific region, but under certain circumstances they may become extremely poisonous. It is believed that these crabs become toxic by feeding on the roots of certain toxic terrestrial plants. Native islanders in the Tuamotus have suggestedthat one of these toxic plants may be the “Piratea” [Ceoclosrmbrcrcul$ercr (Forster)], a member of the family Hernandiaceae, found in swampyareas on variousPacificatolls (74).Hashimoto(13)reported
Halstead
66
several other poisonous plants that the coconut crab may be feeding on in the Ryukyus, such as Diospyros tt~rrritimrr(Blume Bijdr) of the family Ebenaceae, Herncmdiu .so~~or(r (Linnaeus) of the family Hernandiaceae, and Aloccrsicr mtrcrorrhizc~(Linnaeus) Schott of the family Araceae. Clirlicrrl CI1omcteristics: The symptoms of coconut crab poisoning consistof a violent gastrointesintal upset, headache, chills, joint aches, extreme exhaustion, and muscular weakness. Deaths have been reported. Treatment: Treatment is symptomatic. Prevention: If there is the slightest question concerning the edibility of the coconut crab, avoid eating it. Unfortunately these crabs generally are considered edible anda great delicacy, and reliable local information is difficult to obtain to prevent poisoning. 4. Poisonous Lobsters The term lobster as used in the vernacular sense may include any member of the families Homaridae, the true lobsters; Palinuridae, the spiny lobsters; Scyllaridae, the slipper or shovel lobsters; and the deep-sea lobsters,all of which are members of the arthropod class Crustacea. The true and spiny lobsters are of commercial importance. Lobsters have a rigid, segmented exoskeleton, andfive pairs of legs, one or more pairsof which are modified into pincers or chelae, with the chela on one side usually larger than on the other. The eyes are on movable stalks, and there are two pairs of long antennae. Several pairs of swimmerets are on the elongated abdomen. A flipperlike tail is used for swimming; flexure of the tail and abdomen are used to propel the animal backward. All lobsters are marine and bottom dwelling. Lobsters are nocturnal in habit and scavenge for dead animals, fish, invertebrates, and seaweed. A number of poorly documented accounts have indicated that someof the spiny lobsters in French Polynesia have caused human intoxications. The exact species involved have not been documented (8.74).
Representrrtive Species Family: Palinuridae (spiny lobsters) Species: Paliwurus species. Spiny lobster. Length variable, about Distribution: Indo-Pacific.
12 in. (30 cm).
Lobster Poisorlirlg Lobster poisoning generally is considered to be a rare occurrence and apparently nothing is kllown concerning the nature of the poisons involved or the clinical characteristics of the intoxications they produce. It is assumed that the poisons involved originate through the food chain of the lobster, similar to the toxic tropical reef crabs which inhabit the same reef environment and have similar eating habits. Clirlictrl Cl7arcrcteristics: The clinical characteristics are unknown. Trmment: Treatment is symptomatic. prel~ptltiotl:Data on prevention of lobster poisoning are extremely meager. The best policy is to check with the local inhabitants.
111.
VERTEBRATES
Vertebrate fish poisonings are covered in Chapter 2, so this discussion is limited to poisonous marine vertebrates other than fish, that is, marine reptiles and mammals.
Other Poisonous Marine Animals
A.
67
PoisonousMarineTurtles
Marine turtles are reptiles of the order Chelonia (Testudinata) and are characterized by a broad body encased in a bony shell comprised of a rounded dorsal carapace and a flat ventral plastron that are joined at the sides and coveredby polygonal laminae (i.e., Scut% scales, or leathery skin). The jaws are edentulous and equipped with horny sheaths. The quadrate bone is united to the skull. The ribs are fused to the shell, and the sternum is absent. All turtles, tortoises, and terrapins are oviparous in their reproduction. There are five species of marine turtles that have been reported as poisonous to man.
Represetrtcrtive Species Family: Chelonidae (marine turtles). Species: Ccrretrcrcvrrettrr gigcrs (Deraniyagala). Loggerhead turtle. Length of carapace 47 in. (1.2 m). Distribution: Tropical Pacific and Indian Oceans. Species: Cl?e/orlinmyclcrs (Linnaeus). Green sea turtle. Length of carapace 47 in. (1.2 m). Distribution: All tropical and subtropical oceans. Species: Et-etmockelys irnhricntrr (Linnaeus). Hawksbill turtle. Length of carapace 35 in. (89 cm). Distribution: All tropical and subtropical oceans. Family: Derlnochelidae (leatherback turtles) Species: Dertnochelys coricrcea (Linnaeus). Leatherback turtle. Length of carapace up to 9 ft (2.7 m). Weight of about 1500 Ib (700 kg). This is the largest of all the turtles. Distribution: Largely circumtropical. Family: Trionychidae (soft-shelled turtles) Species: Peloche/ys Dibrotri (Owen). Soft-shelled turtle. Lengthof carapace 6 ft (1.8 m). Distribution: Rivers and coastal areas of Southeast Asia.
Moritre Turtle Poisonins (Cllelotlito~~icrrtio~~) Marine turtles have been reported as poisonous to eat at sporadic intervals over the years. Unfortunately very little specific information is available concerning manyof these outbreaks. References 8,12,52,66,75-82 report on turtle poisoning (chelonitoxications). Mechcrrrisnl o f htoxiccrtiotr : The origin of turtle poison (chelonitoxin) is unknown, of the opinion that the poison but most investigators who have studied the problem are originates in the food chain of the turtle. Most marine turtles appear to be omnivorous, and feed on marine algae, among other things. It is believed that they become poisonous in humans by feeding as a result of feeding on toxic algae. Turtle poisoning may be caused on the flesh, fat, viscera, or blood of various species of tropical sea turtles. The chemical nature of chelonitoxin is unknown. Clitlicd Clrnrcrcteristics: The symptoms of chelonitoxication appear to vary with the amount of turtle ingested and the individual. Symptom develop within a few hours to several days after ingestion. The initial symptoms consist of nausea, vomiting, diarrhea, of the exepigastric pain, tightness of the chest, pallor, tachycardia, sweating, coldness tremities, and vertigo. Frequently there is reported an acute stomatitis, consisting of a dry, burning sensation of the lips, tongue, and lining of the mouth, and thirst. Swallowing
68
Halstead
becomes very difficult, and hypersalivation may be pronounced. The tongue develops a white coating, and the breath becomes very foul. The tongue later may develop pinheadsize, reddened pustules. The oral symptoms may be slow to develop, buttheyusually become very severe after several days. The lingual pustules may persist for several months or may break down into ulcers. Some victims develop a severe hepatomegaly with right upper quadrant tenderness. The conjunctivae become icteric. Headaches and a feeling of “heaviness of the head” are reported. Deep reflexes may be diminished. Somnolence is one of the pronounced symptoms present in severe intoxications, and is usually indicative of an unfavorable prognosis. At first the victim is difficult to awaken, then becomes comatose, which is followed rapidly by death. The symptoms presented are typicalof hepatorenal disease. The clinical characteristics of marine turtle poisoning are discussed in Refs. 12,77,78,8I , and 83-85. Trentment: Treatment is symptomatic. There is no known antidote. Prevention: There are no reliable external characteristics indicativeof a toxic marine turtle to aid in prevention of poisoning. Natives frequently will test suspect turtle meat by feeding samples of it to dogs or cats. It is advisable to seek the advice of the local inhabitants before eating marine turtles.
B. PoisonousMarine Mammals Mamnals are characterized by bodies that are covered with hair and skin containing various types of glands. The skull possesses two occipital condyles. The jaws usually have differentiatedteeththatarecontained in sockets. The limbs are adapted variously for walking, climbing, burrowing, swimming, or flying. The feet have claws, nails, or hoofs. The heart is four-chambered, with only a left aortic arch. The lungs are large and elastic. There is a diaphragm between the thoracic and abdominal cavities. The male has a penis and fertilization is internal. The eggs are small or minute and usually are retained in a uterus for development. The females have mammary glands that secrete milk to nourish the young. Body temperature is regulated. Several orders of marine lnamnals have been found to be toxic to eat: the order Cetacea, which includes whales, dolphins, and porpoises; Pinnipedia, which includes walruses and seals; and Carnivora, which includes the polar bear.
1. Poisonous Cetaceans There are only four species of cetaceans (whales, dolphins, and porpoises) that have been reported as toxic to man: the sei whale (Brrlcrenoptercr borealis), white whale (Delphinq>terus leucrrs), sperm whale (Physeter crrtodorl), and Southeast Asiatic porpoise (Neophoerrem phocnenoide). Members of the family Bulnenopteridrre are the finback whales, which includes the sei whale. Some of the members of this family are among the largest of living animals. The largest member of the group, the blue or sulphur-bottom whale [Sibbddus 1nmculu.s (Linnaeus)], attains a length of 98 ft (30 m ) and a weight of 110 tons ( 1 12.500 kg). This is one of the two families of baleen whales i n which the embryonic teeth are replaced by baleen plates in the adult animals. Finback whales frequently are called rorquals, which refers to a whale having folds or pleats. The rorquals are equipped with longitudinal furrows, usually 10 to 100 in number, that are present on the throat and chest. These furrows increase the capacity of the mouth when it is opened. The members of this family are the
Other Poisonous Marine Animals
69
fastest swimmers of the baleen whales. Their food consists largely of euphausiid shrimp, copepods,amphipods,andotherzooplankton.Whalesareconsideredamongthemost healthy of all living mammals since evidence of pathology is seldom observed. The liver of the sei whale is considered to be toxic to eat. The family Monodontidae, or white whales, consists of only two species, the white whale (Dell,hirlcrl,ter-lrs leuccrs) and the beaked narwhal (Momdon monocerosj. They inhabit arctic seas and sometimes ascend rivers. Eating white whale has produced fatalities (86,87). There is no information available concerning the toxicity of the narwhal; apparently they are used for human food at times, but their meat is usually fed to sled dogs. White whales have a body shape similar to members of the Delphinidae. The snout is blunt and there is no beak. There are no external grooves on the throat. White whales usuallylive in schools, sometimes consisting of more than 100 individuals. They feed mainly on benthic organisms, cephalopods, crustaceans, and fish. White whales are of economic importance and are hunted mainly for their skins, which are sold as "porpoise leather." The family Physeteridae, or the sperm whales, inhabitsall oceans. The sperm whale (Physeter-cntodon) attains a large size of up to 65 ft (20 m ) and 55 tons (55,880 kg) in weight. The sperm whale is said to be the only cetacean with a gullet large enough to swallow a human. The characteristic featuresof the sperm whale areits tremendous barrelshaped head and the underslung jaw. The sperm whale feeds on squid, cuttlefish, fish, and elasmobranches. Theoil and fesh of the sperm whale have been reported to be toxic, but the documentation of this is poor (88). The family Delphinidae includes the dolphins and porpoises. They inhabit all oceans and the estuaries of many large rivers; some species may ascend rivers for great distances. Some species seem to prefer warm coastal waters; none of them are found in the polar regions. The term dolphin generally refersto small cetaceans having a beaklike snout and a slender streamlined body, whereas the term porpoise refers to small cetaceans having a blunt snout and a rather stout, stocky body. Their food consists of fish, crustaceans, and cuttlefish. The Asiatic porpoise( N e o l h ) c m n a phocaenoicle.~)ascends estuaries and rivers has been reported as toxic (89-91). for more than 1000 miles (1600 km). Neophocaer~~~
Family: Balaenopteridae (sei whales) Species: Bcrlcrenoptertr borecrlis (Lesson). Sei whale. Length 60 ft (1 8 m). Distribution: Atlantic Ocean, south to the Gulf of Mexico; Pacific Ocean, Bering Sea south to Baja California. Family: Monodontidae (white whales) Species: Delphitlnpter-us 1errccr.s (Pallas). White whale. Length l8 ft (5.5 m). Distribution: Arctic and subarctic seas. Family: Physeteridae (sperm whales) Species: Physeter- ctrtodon (Linnaeus). Sperm whale. Length 60 ft ( 1 8 m). Distribution: Polar, temperate, and tropical seas. Family: Delphinidae (dolphins, killer whales, and pilot whales) Species: Asiatic porpoise [Neophoc-amrrphocvenoides (Cuvierj]. Length 5 f t (1.5 m) for Asiatic porpoise. Distribution: Coastal areas, estuaries, rivers, and lakes of China.
Halstead
Some cetaceans havebeen reported as poisonous to eat including Baltrenoptercr borealis (92), Delphinrryterus 1eucrr.s (86,87), Physeter. catorlot1 (88), and Neophoccretm pkocmrtoides (89-91,93). The subject of poisonous cetaceans hasbeen reviewed by Halstead ( 12.8 1). Mechanism of Intoxication: Sei whale. The liver ofthesei whale (Bcrlcrettopter(t borealis) is reported to be toxic to cat. 1euccr.y) is White whale. The viscera and meat of the white whale (Del~hinrrl~terus in fatalities. reported to be toxic and may result Sperm whale. The oil and meat of the sperm whale (Physeter ccrrodon) may be poisonous in some localities. Asiatic porpoise. The liver, other viscera, and muscle of the Asiatic porpoise (Neophourettcr phocrrettoides) are reported to be poisonous and may cause death. The nature of the poisons has not been determined, but it is suspected that in some instances vitamin A may be the causative agent. Clitticcl1 CIlctracteristics: Seiwhalepoisoning.Theonset of symptomsbegins within 24 hours after ingesting the liver and consists of severe occipital headaches, neck pain, flushing of the face, nausea, vomiting, abdominal pain, diarrhea, fever, chills, photophobia, tearing, and erratic blood pressure. After several days, the victim's lips become dryanddesquamationdevelopsaroundthemouth,graduallyspreadingto the cheeks, forehead,andneck.Thedesquamationusuallydoes not involvetheentirebody.Although jaundice is not reported, there is evidence of liver impairment. Acute symptoms generally subside within 2 days, but the desquamation may continue for a longer period of time. White whale poisoning. Ingestion of the flesh of the white whale may cause death, but little appears to be known concerning the clinical characteristics. Sperm whale poisoning. No information is available on the clinical characteristics of sperm whale poisoning. Asiatic porpoise poisoning. The symptoms of Asiatic porpoise poisoning consist of abdominal pain, nausea, vomiting, bloating, swelling and numbness of the tongue, loss of vision, cyanosis, numbness of various areas of the body, hypersalivation, greenish tinge to the saliva, and muscle paralysis. Death may be rapid, and the fatality rate is said to be very high. Treatment: The treatment of cetacean poisoning is symptomatic. The nature of the poison is unknown. Prelwttion: T o prevent cetacean poisoning, the liver of the sei whale should never be eaten. The flesh of the white whale should never be eaten. The oil and flesh of the sperm whale reportedly are toxic in some areas and should be eaten only with extreme caution. The Asiatic porpoise viscera and flesh should not be eaten.
2. PoisonousWalrusesand Seals Walruses and seals are members of the mammalian order Pinnipedia. The pinnipeds are a group of marine mammals havinga spindle-shaped body and limbs modified into flippers for aquatic locomotion. The toes are included into webs, and the tail is very short. The males usually are larger than the females. The livers of walruses and certain species of seals may at times be poisonous.
Other Poisonous Marine Animals
71
Family: Odobenidae (walruses) Species: 0doI7en~1.srostncrrus (Linnaeus). Walrus. Length 1 1 f t (3.5 m). Distribution: Arctic Ocean, northeast coast of Siberia, northwest coast of Alaska, north to northwest of Greenland, Ellesmere Island. Family: Phocidae (seals) Species: EriSncrthus bnrbotus (Erxleben). Bearded seal. Length 9 ft (2.7 111). Distribution: Inhabits the edge of the ice along the coasts and islandsof arctic North Atnerica and northern Eurasia. Species: N(@10ccr ciwerctr (Peron). Australian sea lion. Length 8 ft (2.4 m). Distribution: South and southwest coasts of Australia. Species: Prrstr hi.s~,irr'tr(Schreber). Ringed seal. Length 4.5 ft (1.4 111). Distribution: Circumboreal, near the edge of the ice, to the North Pole.
Wdrm and S e d Poisot1ing Walruses (Od0berlrrs ro.smrru.s)have been reportedto have toxic livers(94). Several reports have also appeared on the toxicity of the liver of the bearded seal (Erignathus barbatus) (95-100). The Australian sea lion (Neophoca cinerea) hasbeen reported to have poisonous flesh and allegedly has caused produced deaths in humans and dogs (101,102). Mechmism of Itltoxicntiott: Walrus and seal poisoningis usually causedby ingesting the livers of the rogue bull walrus (Odobetlus) or the bearded seal (Erignr~thus),or the flesh of the Australian sea lion (Neophoca).Intoxications are believed to be caused by an excessive intake in vitamin A, which is present in the liver. However, in the case of the Australian sea lion, the nature of the poison is unknown. Clitliccrl Clr~rrcrcteristics:The symptoms of walrus and seal poisoning are said to be sirnilar to that of polar bear poisoning (see Sec. III.B.3). Tretrtnrent: The treatment of walrus and seal poisoning is similar to that for polar bear poisoning (see Sec. III.B.3). Preverltion: There is no reliable method of detecting a toxic walrus or seal by visual examination. Avoid eating walrus and seal livers and the flesh of Australian sea lions.
3. PoisonousPolar Bears Polar bears [Tllcllrlrctos tntrrifitnus (Phipps)] are marine carnivoresof the class Mammalia thatarecharacterizedbyhavingfourorfivetoes;claws;mobilelimbs;completeand separate radii and ulnas, tibias and fibulas; small incisors; and canines that are slender fangs. The only marine carnivore toxic to man is the polar bear. Most rnanmologists are of the opinion that there is only a single species. Relwesertttrtive Species Family: Ursidae (bears) Species: Tlln1arcto.s rnnritirnus (Phipps). Polar bear. Length 8 ft (2.5 m). Distribution: Arctic, circumpolar.
Polar Berrr Poisoning The biogenesis of the poison present in the polar bear has not been established, but it is believed that the toxin is due to the presence of excessive quantities of vitamin A.
72
Halstead
Meckcrnisnr cfb~toxic~rrion: Polar bear poisoning is caused by eating the liver or kidneys, which apparently concentrate large quantities of vitamin A. Clirzicd ChcrrLlc,reristics:The symptoms of polar bear poisoning usually begin about 2-5 hours after ingesting either the kidneys or the liver. The predominant symptoms are intense throbbing or dull frontal headaches, nausea, vomiting, abdominal pain, dizziness, drowsiness, irritability, weakness, muscle cramps, visual disturbances, and collapse. The headaches may become intense during the first 8 hours and may cause insomnia since they are aggravated by lying down. Gradually the headache lessens in severity and may disappear by the following day. Numerous cases have been cited i n which desquamation occurred on various parts of the body, particularly the face, arms, legs, and feet. Tonic a case in which photosensitiand clonic convulsions may be present. Sutton (103) reported zation was a prominent symptom. If fatalities do occur from these intoxications, they are rare. The amount of liver ingested appears to have a direct bearing on the severity of the symptoms. Eskimos believethat ingestion of polar bear liver may result in depigmentation of theskin.However,investigationfails to substantiatethisbelief (104). The clinical characteristics of polar bear poisoning are discussed in Refs. 12,96,99,103, and 105-1 IO. Trear~nent:Treatment of polar bear poisoningis symptomatic. Emetics and laxatives promptly administered sometimes are useful in relieving the severity of the symptoms. The clinical manifestations gradually disappear after ingestion of the toxic meat has been discontinued. Prevelltion: There is no reliable method of detecting toxic polar bear liveror kidneys by visual examination. The age of the bear seems to have no bearing on the edibility of the meat. The liver from cubs has been known to cause intoxication ( 1 1 I ) . In general, it is best to discard polar bear liver or kidneys. If these parts are eaten,they should be eaten in amounts of less than a 0.5 Ib (230 g).
ACKNOWLEDGMENT It is with deep appreciation that I acknowledge the technical assistance Medrano i n the preparation of this chapter.
of Leonette C.
REFERENCES 1.
2. 3.
4.
5. 6.
7.
KA Steidinger, DG Baden. Toxic marine dinoflagellates. I n : DL Spector, ed., Dinoflagellates. Orlando: Academic Press, 1984, pp 201-261. JMBurkholder.EJNoga.CJHobbs,HBGlasgow.New“phantom”dinoflagellateisthe causative agent of major estuarine fish kills. Nature 358:407-410, 1992. KA Steidinger, JM Burkholder, HB Glasgow, CW Hobbs, J K Garrett, EW Truby, EJ Noga. Gen. Et. Sp. Nov. (Pfiesteriaceae. Fam. Nov.), a newtoxic SA Smith. P’esreritr piscicid~~ dinoflagellatc with a complex life cycle. J Phycol 32:1.57-164, 1997. HB Glasgow, JM Burkholder. DE Schmecel, PA Tester. PA Rublee. Insiduous effects of a toxic dinoflagellate on fish survival and human health. J Toxicol Environ Health 46:SOl522, 199.5. DL Matuszak, JL Taylor, C Dickson, GC Benjamin. Toxic pfiesteria-surveillance for hunun disease in Maryland. Md Med J 47(3):144-147, 1998. BHilcman.Pfiesteriahealthconcernsrealized.ChcrnEngNews 75:14-15, 1997. BW Halstead, RL Haddock. A fatal outbreakof poisoning from the ingestion of red seaweed
Other Poisonous Marine Animals
8.
9.
IO. II.
12.
13.
14. 15.
16. 17. 18.
19.
20. 21.
22.
23. 24. 25.
26. 27. 28
29. 30. 31.
32.
73
~ r l ( c ; / ~ t.s[&e ( r ; ~ ~in Guan-areviewofthcoralmarinebiotoxicityproblem. J Natural Toxins l(l):87-l15, 1992. BW H&tcad. Dangerous marine animals, 3rd ed. Centrevilk, MD: Cornell Maritime Press, 1995. LFarbcr,PLerke.Studiesonthetoxicpropcrty of Rkodrrctis /rowe.sii (Matalnalu). In: I KeeganandWVMacfarlane,eds.VenomousandPoisonousAnimalsandNoxiousPlants o f the Pacific Region. Ncw York: Pergamon Press, 1963, pp 67-74. Rlrodactis kowe.sii (Coelenterata). Pac EJ Martin. Obscrvations on the toxic sea anemonc, Sci14(4):403-407,1960. BW Halstead. Poisonous and Venomous Marine Animals of the World. Washington. DC: U.S. Government Printing Office, 1965. BW Halstead. Poisonous and Venomous Marine Animals of the World, 2nd ed. Princeton, NJ:DarwinPress,1988. Y Hashimoto. Marine Toxins and Other Bioactive Marine Metabolites. Tokyo: Japan Scientific Socictics Press, 1979. T Yarnanouchi. On the poisonous substances containcd in holothurians. Pub1 Set0 Mar Biol Lab 4(2-3):183-203, 1955. RFNigrelli. S Jakowska. Effects of holothurin, a steroid saponin from the Bahamian sea cucumber (Actirro/Jygrro p s s i z i ) , on various biological systems. Ann NY Acad Sci 90:884892,1960. PJ Scheuer. Chemistry of Marine Natural Products. New York: Academic Press. 1973. PL Scheuer. Chemical and biological perspcctives contents. In:PJ Scheuer, ed. Marine Natural Products. NewYork:AcademicPress,1978,pp1-299. JB Clcland. Injurics and diseasesof man in Australia attributable to animals (cxcept insccts). J Trop Med Hyg 16:25-47, I91 3. ACastellaniand AJ Chalmers.Venomousanimals (continued): Piscesandamphibia.In: 203Manual of TropicalMedicine.3rded.NewYork:Wm.WochandCo.,1919,pp 241. S Frankel, C Jellinek. Ber essbare Holothurien [in German]. Biochem Z 185:389-391, 1927. SL Friess. Some pharmacological activities of the sea cucumber neurotoxin. Arner lnst Bio SciBull13(2):41,1963. M. Phisalix. Animal venilneux et venins (2 vols.) [in French]. Paris: Masson et Cie, 1922. EN Pawlowsky. Fische. Pisces [in German]. In: Gifttiere Und lhre Giftigkeit. Jena: Verlag von Gustav Fischer, 1927, pp 245-247. KV Earle. Pathological effects of two West Indian echinoderms. Trans R SOCTrop Med Hyg 33:447-452,1940. E Kaiser, H Michl. Die Biocheme der tierschen Gifte [in German]. Vienna: Franz Deuticke Wien,1958. Y Hashimoto, K Noito, J Tsutsurni. Photoscnsitization of animals by viscera of abalones, Hdiotis spp. Bull Jpn Soc Sci Fish 26: 12 16- 122 I , 1960. Y Hashirnoto, J Tsutsumi. Isolation of a photodynamic agent from the liver of abalone, Hdiotis discrrs /rccrlrrai. Bull Jpn Soc Sci Fish 272359-866, 1961. Y Hashimoto,TOkaichi,LD Dang, TNoguchi.Glenodinine,anichthyotoxicsubstance produced by a dinoflagellate, Peridini~mrpolonicurtr. Bull Jpn SOCSci Fish 34:524, 1968. T Yasumoto, Y Kotaki. Occurrence of saxitoxin in a green turban shcll. Bull Jpn Soc Sci Fish 43:207-21 1. 1977. C Sckiguchi,M Yokoyama, E Nishino. T Yonetani, M Shimpo. (NSA) [in Japanese]. Niigata Eiken Gyoho 95: 1, 1960. C Ji-Shcng. Marine toxins and toxicosis in China. In: A. Tu, ed. Tosin-Related D i s e ~ t , s c ~ . New Delhi: Oxford, 1993. pp 237-267. K Kanna, M Hirai. On the Toxicityof Nepturren irrterscdpta Sowerby. Lecture at the annual meting of the Japan Society of Fisheries, Tokyo.
74
Halstead
RFange. An acetylcholine-like salivary poison inthemarinegastropod Ne/7tlt1letl clrltiylta. Nature180(4578):196-197,1957. 34.RFange.Paperchromatographyandbiologicaleffectsofextracts of thesalivarygland of Neprrrnen nrrriqua (Gastropoda). Acta Zoo1 39:39-46, 1958. 35.RFange.Thesalivarygland of Nq7tur1ea cmtiyua. AnnNYAcad Sci90:689-694,1960. 36. S Hall, G Strichartz, E Moczydlowski, A Ravindran, PB Reichardt. The saxitoxins: sources, chemistry, and pharmacology. In:S Hall, G Strichartz, eds. MarineToxins: Origin. Structure, and Molecular Pharmacology, Washington, DC: American Chemical Society, 1990. pp 2965. in easternCanada.Fish 37. A Prakash, JC Medcof,ADTennant.Paralyticshellfishpoisoning Res Board Canada Bull 171:1-87. 197 1 . 38. M Asano, F Takayanagi, Y Furchuhara. Studies on the toxic substances in marine animals. 11. Shellfish poisoning from Ca//isttr hrer,isi/7/?or~~~r~~ Carpenter, occurred inthevicinity o f Mori, Kayabe County [in Japanese]. Sci Pap Hokkaido Fish Sci Inst (7):26-36. 1950. 39. T Akiba. Food poisoning due to oyster and baby clam in Japan, and toxicological effects of the toxic substance. Unpublished manuscript. 40. R Bagnis. A propos de quelques cas d’intoxications par des mollusques due genre “bnitier,” dans une ile de la socit [in French]. Bull Soc Pathol Exot 60:580-592, 1967. 41. T Yasumoto, M Murata. Y Oshima, M Sano, GK Matsumoto. J Clardy. Dimhetic shellfish toxins. Tetrahedron 41:1019, 1985. of yessotoxin. A novel 42. M Murata, M Kumagi, JS Lee, T Yasumoto. Isolation and structure polyether compound implicated in diarrhetic shellfish poisoning. Tetrahedron Lett 28:5869, 1986. in foodpoisoning.JToxicolToxicRev17: 43.K Okada, MNiwa.Marinetoxinsilnplicated 373-384,1998. 44. T Kawabata, BW Halstead, TF Judefind. A report of a series of recent outbreaks of unusual cephalopod and fish intoxications in Japan. Am J Trop Med Hyg 6:935-939, 1957. 45. HM Smith.Apoisonoushorseshoecrab.JSiamSocNatHist9:143-145,1933. 46. DR Soegiri. A case ofMimipoisoning[inDutch].In:CBonneandGMStreef.eds.Geneeskundig Tijdschrift voor Nederlandsch-Indie. Batavia: G. Kolff and Co., 1936, pp880-883. 47.THWaterman.PoisonousPacifichorseshoecrabs.Unpublished,1953. 48. TH Waterman.XiphosurafromXuong-Ha. AmSci41:301-302, 1953. 49. AH Banner, BJ Stephens. A note on the toxicity of the horseshoe crab in the Gulf of Thailand. Nat Hist Bull Siam SOC 21(3-4):197-203, 1966. 50. N Fusetani, H Endo. K Hashimoto, M Kodama. Occurrence and properties oftoxinsinthe horseshoe crab Curcinoscorpius rotundicauda. Toxicon Suppl 3: 165- 168, 1983. 51. AH Banner, JE Randall. Preliminary report on a marine survey of Onotoa, Gilbert Islands. AtollResBull13:43-62,1952. 52. MJ Cooper. Ciguatera and other marine poisonings i n the Gilbert Islands. Pac Sci 18:41 1440, 1964. 53. D Guinot. Les crabes comestibles de I’Indo-Pacifique [in French]. Paris: Editions de la Fondation Singer-Polignac, 1967. 54. A Inoue, T Noguchi, S Konosu, Y Hashimoto. A new toxic crab, Arerh.trti.s.porirllr.s.Toxicon 6.1 19-123, 1968. 55. S Konosu, Y Hashimoto. Occurrence of toxic crabs in the Ryukyu and Amami Islands and similarity of the crab toxin to saxitoxin. S Pac Comm Tech Rep Seminar: 1-6. 1968. 56. Y Hashimoto, S Knosu, A Inoue, T Saisho.S Miyake. Screening of toxic crabs in the Ryukyu andAmami Islands. Bull Jpn Soc Sci Fish 35:83, 1969. 57. S Konosu, A Inoue, T Noguchi, Y Hashimoto. A further examination of the toxicity of three species of xanthid crab. Bull Jpn Soc Sci Fish 35:88, 1969. 58. ACAlcala,BW Halstead.Humanfatalityduetoingestionofthe crab Dermrrria sp. inthe Philippines. Clin Toxicol 3:609-61 l , 1970.
33.
Other Poisonous Marine Animals
75
59. JP Erhardt, P Niaussat. De leventuelle toxicite du decapode brachyoure C~roililrs~ O ~ I L ' C ' . Y I I S Forskl, etude d'excmplaires provenant de I'atoll de Clipperton [in French]. Cah Pac (14): 114-1 15. 1970. 60. Y Hashimoto, S Konosu, M Shibota, K Watanabc. Toxicity of a turban-shell i n the Pacific. Bull Jpn Soc Sci Fish 36: I 163- 1 17 l . 61. S Konosu, T Noguchi, Y Hashimoto. Toxicity of a xanthidcrab Zosirtrfts oerrelrs and several other species i n the Pacific. Bull Jpn Soc Sci Fish 36:715-719, 1970. 62. GE Mote, BW Halstead, Y Hashimoto. Occurrence of toxic crabs in the Palau Islands. Clin Toxicol 3597-607. 1970. 63. YF Tch, JE Gardiner. Toxin from the coral reef crab Lop/toro,7!.rttlr.s pictor. Phartnacol Res Comm 2 2 5 1-256, 1970. 64. JS Garth. Derrrnrrin fo.uica. a new species of poisonous crab from the Philippines. Micronesia 7( 1-2): 179- 183. l97 1. 65. BW Halstead, KW Cox. A fatal caseof poisoning by the red-spotted crab Cmpi/i~tsr r r c t c h t l r . s (Linneaus) in Mauritius. R Soc Arts Sci Mauritius 4(2):27-30, 1973. in 66. Y Hashimoto, S Konosu, T Yasumoto, A Inoue, T Noguchi. Occurrence of toxic crabs Ryukyu and Amami islands. Toxicon 523-90, 1967. 67. S Konosu, A Inoue. T Noguchi, Y Hashimoto. Comparison of crab toxin with saxitoxin and tetrodotoxin. Toxicon 6.1 13-1 17, 1968. 68. T Noguchi, S Konosu, Y Hashimoto. Identity of the crab toxin with saxitoxin. Toxicon 7: 325-326,1969. 69. T Yasumoto, Y Oshima, T Konta. Analysis of paralytic shellfish toxins of xanthid crabs i n Okinawa. Bull Jpn SOCSci Fish 47:957-959, 1981. 70. T Yasutnoto, Y Oshima, M Tajiri, Y Kotari. Paralytic shellfish toxins in previously unrecorded species of coral reef crabs. Bull Jpn Soc Sci Fish 49:633-636. 1983. in 71. T Yasumoto, D Yasumura, Y Ohizutni, M Takahashi. AC Alcala, LC Alcala. Polytoxin two species of Xanthid crabs from the Philippines. Agric Biol Chem 50:163-167, 1986. 72. BW Halstead. ET Schantz. Paralytic Shellfish Poisoning. Rome: World Health Organization, 1984. 73. D Yasumura, Y Oshima. T Yasumoto. AC Alcala. LC Alcala. Tetrodotoxin and paralytic shellfish toxins in Philippine crabs. Agric Biol Chem 50593-598. 1986. 74. R Bagnis. F Berglund, PS Elias, GJ van Esch, BW Halstead, K Kojima. Problems of toxicants in marine food products. Bull World Health Organ 42:69-88, 1970. 75. EH Taylor. Amphibians and Turtles of the Philippine Islands [English edition]. Manila: Bureau of Printing, 1921, pp 162-163. 76 A Loveridge. Reptiles of the Pacific World. New York: Macmillan, 1945. 77 J Bierdrager. A caseof turtle poisoning in New Guinea [in Dutch]. Geneesk Tijdschr Nederlandsch-Indie 76( 18): 1933-1944, 1936. 78 P Deraniyagala. The Tetrapod Reptiles of Ceylon. London: Dula and Co., 1939. 79. A Carr. So Excellent A Fishe. Garden City, NY: Natural History Press, 1967. 80. BW Halstead, WM Lively Jr. Poisonous fishes and ichthyosarcotoxism. Pac Air Forces Conmand Surg Newslett l ( I): 1 -S, 1959. 81. BW Halstcad. Poisonous and Venomous Marine Animals of the World. Washington, DC: U S . Government Printing Office, 1970. 82. dcR Champetier, RN Rasolofonirivia, N Razafimahefa. JD Rakotoson, D Raboson. Intoxications by marine animal venoms in Madagascar (icthyosarcotoxism and chelonitoxism): recent epidetniological data. Bull Soc Pathol Exot 90286-290, 1997. 83. M Kinugasa, W Suzuki. An etiological investigation of a mass outbreak of food poisoning in the Shinchiku caused by eating a sea turtle caught along the coast at the town Koryu. district, Taiwan [English translation from Japanese]. Taiwan lgohkai Zassi 39(74):66-74. 1940. 84. T Romcyn, GT Haneveld. Vergiftigingcn door het eten van schildpadvless (Eretrtroc./te/y,s
76
Halstead irdwicccre) op Nedcrlands Nieuw-Guinea. Nederiandsch Tijschrift voor Genceskunde 1 156- I 159, 1956.
85
86. 87. 88.
89. 90. 91. 92. 93. 94. 95.
96. 97. 98.
99. 100.
101.
102. 103.
104. 105.
106. 107. 108.
109. 1 IO.
111.
100:
VK Pillai. MB Nair, K Ravindranathan, CS Pitchumoni. Food poisoning due to turtle flesh. J Assoc Phys India 10(4):181-187, 1962. V Stefansson. My Lifc with thc Eskimo. New York: Macmillan, 1924, pp 32-35. V Stefansson. Arctic Manual. New York: Macmillan, 1944, pp 98-283. Y Sahashi. Nurtritive value of sperm whale oil and finback whale oil. Sci Papers Inst Phy Chem Res 20(416):245-253, 1933. DJ Macgowan. Porpoise poison. Med Rcp Imp Marit Customs China (27):12. 1884. DJ Macgowan. Poisonous fish in China. Bull US Fish Comm 6: 130- 13 I , 1887. BE Read. Chinese Materia Medica: Fish Drugs. Peking: Peking Natural History Bulletin, 1939 M Mizuta, T Ito, T Murakami, M Mizobe. Mass poisoning from the liver of Sawara and Iwashikujira [English translation from Japanese]. Jpn Med J 1710:27-34, 1957. DJMacgowan.Poisonousfishandfish-poisoninginChina.ChineseRecMissionJ 17(2): 45-49,1886. FH Fay. Carnivorous walrus and some arctic zoonoses. Arctic 13: 111-122, 1960. J Lindhard. Sundhedsforholdene paa "Danmark-Expeditionen" [in Danish]. Hospitalstidcnde 325355347, 1910. A Krogh, M. Krogh. A study of the diet and metabolism of Eskimos[in Norwegian]. Meddel om Gronland 51:11-52, 1913. EO Jordan. Food Poisoning and Food-Borne Infection. Chicago: University of Chicago Press, 1931,pp55-64,1931. K Rodahl, T Moore. The vitamin A content and toxicity of bear and seal liver. Biochem J 37:166-168,1943. K Rodahl. Toxicity of polar bear liver. Nature 164(4169):530-531, 1949. R Rausch. The toxicity of polar bear liver. Unpublished, 1956. WH Leigh. Reconnoitering voyages and travels with adventuresin the new colonies of South Australia. In: Reconnoitering Voyages and Travels with Adventures in the New Colonies of South Australia, Cornhill, London: Smith, Elder, and Co. 1839, p 164. JB Cleland. Injuries from animals. Med J Aust 2(22):491-492, 1942. RL Sutton. Is polar bear liver poisonous? JAMA 118: 1026, 1942. V Stefansson. The Friendly Arctic: The Story of Five Years in Polar Regions. New York: Macmillan,1921. H Khl. Kann die Liber der Fleischfresser gifting sein'? In: R. Ostertag-Stuttgart, ed. Zeitschrift fur Fleisch-und Milchhygiene. Berlin: Verlagsbuchhandlung von Richard Schoetz, 1929; pp 45-49. 0 Boje.Toxin i n thefleshoftheGreenlandshark[inNorwegian]Mcddelom Granland 125(5):1-16,1939. JK Doutt. Toxicity of polar bear liver. J Mammal 21:356-357. 1940. RL Sutton. Is polar bear liver poisonous? JAMA 118: 1026. 1942. W Beckcr, C Klotzsche. Die hypervitaminose A [in German]. Arztl Woch 24545-550, 1955. H Jcghers, H Marraro. Hypervitaminosis A: its broadening spectrum. Am J Clin Nutr 6:335339,1958. EK Kane. Arctic Explorations: The Second Grinnell Expedition in Searchof Sir John Franklin, 1853, '54, '55. Philadelphia: Childs and Peterson, 1856.
4 Shellfish Chemical Poisoning
I. Introduction 78 11. Paralytic Shellfish Poisoning 79 A. Causativc toxin and its source 80 B. Molecular mechanism of action 81 C. Toxin uptake 82 D. Toxin metabolism, transport, and elimination 82 E. Treatment 83 111. Diarrhetic Shellfish Poisoning 84 A. DSP-causing agents and their sources 84 B. Known molecular pharmacology 85 C. Cellularimpactsofokadaicacidactions 85 D. Organandtissueeffects of DSTs 86 E. Toxinuptakemetabolism,transport,andelimination F. Treatment 86 IV. Neurotoxic Shellfish Poisoning 87 A.Thecausativetoxinsandtheirorigins 87 B. Molecular pharmacology of the toxin 87 C. Toxin uptake, metabolism, and distribution 89 D. Toxin elimination 89 E. Whole animal effects 90 F. Treatment for NSP 90 V. Amnesic Shellfish Poisoning 90 A. Causative toxin and its origin 91 B. Molecular target of domoic acid 92 C. Domoicaciduptakebythegut 92 D. Toxin distribution and elimination 92 E. Pathology 93 F. Treatments for ASP 93 VI.MinorShellfishToxins 94 A. Cyclized, peptidic hepatotoxins 94 B. Tetrodotoxin 95 of Toxins 95 VII.CO-Occurrence
86
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VIII.
ShellfishResponse t o Toxins 96
IX. Effect on Toxins by ShellfishPreparations07 X. Concluding Remarks 97 Acknowledgment 98
References 98
1.
INTRODUCTION
Molluscan shellfish have long been a gastronomic treat for humans, a delight that has tragically for some, ended in poisoning and even death. The cause of these poisoning’s can be twofold. First, poor food hygiene may lead to bacterial spoilage of the shellfish, resulting in human illness such as botulism( 1 ), a type of poisoning not unique to shellfish. The second cause, which rarely occurs i n other food sources and is the subject of this chapter, results from the shellfish sequestering toxic compounds. Many shellfish gain sustenance by sieving the water column and feeding upon the minute organisms therein. At times the organisms comprisingthe shellfish’s diet are toxic, so as well as nutrients, shellfish consume compounds that are seemingly harmless to them (2.3) but are toxic to those organisms further up the food chain. These toxic microorganisms are present in the environment as a matter of course, but are usually in numbers too low to present a problem. Their populations do on occasion bloom, intoxicating exposed shellfish to levels harmful tothe consumer. These toxins can remain withinthetissues of theshellfish for days, weeks, or months after exposure of the shellfish to the toxic microalgae. Therearefourmajor shellfish poisoningsyndromescaused by bioaccumulated toxins: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP). amnesic shellfishpoisoning(ASP),andneurotoxic shellfish poisoning(NSP).Themicroalgal blooms thatgiverise to toxicshellfishareincreasing i n frequencyandimpactona globalscale (4). This has beenattributedtofactorssuchasglobalwarming (5,6) and increasedhumanimpactoncoastalwaters,includingagriculturalfertilizerrunoffand wastewater discharge (7,8). Globalization of the toxic shellfish problem may also be due, i n part, to the deposition of toxic microalgae from the ballast water of international shipping into areas where it lacks competitors and thus is able to flourish (9). Evidence of this global increase is the occurrence of toxic algal blooms and associated human intoxications in regions with no recorded history of such events. For instance, New Zealand experienced an NSP episode in 1993 (10) that was the first ever occurrence of this malady outside of the Americas. The public health impact of shellfish poisoning in naive regions is magnified because the local health authorities are inexperienced at recognizing the event unfolding before them, thus delaying implementation of suitable countermeasures. The shellfish that primarily cause human intoxication are the bivalve molluscs. This class of molluscs includes clams and oysters and is characterized by a two-piece hinged shell that protects the soft body. Some of the species reported i n shellfish poisonings are listedinTable 1. Thetoxinsaregenerallyaccumulated i n thedigestivegland,which counts among its functions excretion of digestive enzymes and some nutrient absorption, and in the siphon, the tubular organ that draws water into the animal’s body cavity and gut. Many adult bivalves attach to a solid surface or live in the sediment. This makes them ideal for farming as the main requirements for their growth are a suitable substrate
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Shellfish Chemical Poisoning
Table 1 ShellfishSpeciesConfirmed to HaveCausedHuman Intoxications and the Syndromes Observed
Species
Shellfish poisoning syndrome
Example references
NSP PS P PSP ASP DSP DSP DSP DSP DSP PSP PSP PSP PSP PS P PSP ASP
10 11 12 13 14.15 16 17 15 15 18 19 11 20 21
22 23
and high-quality water from whichto feed. However, farmingcan also exacerbate shellfish toxicity as it concentrates the shellfish population, magnifying the toxic effectof an algal bloom. Rapid international freight and improved shipping technology mean that shellfish can be exported to many countries from a single region. Thus it is possible, that a shellfish poisoning event may occur far from where the product originated. As an example, the Galician Rios in Spain supplies approximately 40% of all European shellfish (24). Shellfish 200 people being hospitalized all over intoxication i n this one region led to more than Europe (25). For these reasons, monitoring shellfish toxicity has become a responsibility incumbent upon industries and government in virtually every region of the world. These programs ensure that shellfish bound for export and domestic consumption, and, at times, those imported. are either toxin free or contain toxin levels far below that which will cause human illness (26,27). These efforts have dramatically decreasedthe possibility of people being poisoned, but the safeguards arenot absolute. Public health authorities, other health professionals. government authorities, and shellfish farmers should therefore be aware of the various shellfish toxins, their effects, and etiology.
II. PARALYTIC SHELLFISH POISONING Paralytic shellfish poisoning (PSP) is the longest studied shellfish poisoning syndrome, with the toxic principle being recognized early this century (28,29). Human intoxications PSP is an are now global (Figure l ) , and i n some countries i n the Asia-Pacific region, almost annual concern (57). As the name suggests, paralysis is a prominent symptom of PSP. In theearlystages of theintoxication,victimstypicallyexperiencetinglingand numbness of the mouth, tongue, face, and extremities. Accompanying these effects may
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;9
\
V
Fig. 1 Regions of the world from where shellfish to havc caused human intoxication havc originated. (Amnesic shellfish poisoning A;diarrhetic shellfish poisoning m; neurotoxic shellfish poisoning V; paralytic shellfish poisoning ). (16,20.22,23,30-56).
be nausea and vomiting. Hypertension is usually evident in PSP victims (S8,59).In severe cases, the patient will exhibit advanced neurological dysfunction suchas ataxia, weakness, dizziness, a sense of dissociation, followedby complete paralysis. Thereis a compounding effect of central nervous system depression, rendering the diaphragm nonfunctional (60), and death mayresult from cardiorespiratory failure. Mortality rates from PSP have reached 40% (22).
A.
Causative Toxin and Its Source
PSP is caused by saxitoxin (STX; C,,,H17N704; molecular weight 299) and its chemical relatives (Figure 2); this family of toxins will be referred to here as the paralytic shellfish to guinea pigs at only S pg/kg when toxins (PSTs). STX is highly toxic, being lethal injected intramuscularly (61) and mice when injected intraperitoneally (29). PSTs are tricyclic molecules with the 1,2,3- and 7,8,9-guanidino groups of STX possessing ~ K , Jof 1 1.3 and 8.2, respectively (62-64). Thus, at physiological pH, the 1,2,3- guanidino carries a positive charge, whereas the 7,8,9-guanidino group is partially deprotonated (65). This polar nature of STX makes it readily soluble in water and lower alcohols but insoluble in organic solvents. It is extremely stable in solution at neutral and acidic pHs, even at high temperatures. However, alkaline exposure will oxidize the toxin to an inactive derivative (66). Both marine and freshwater microalgae produce PSTs. In freshwater, blue-green to freshalgae (Anlrbtrerltr circirdis) producePSTsandcantransfertheirtoxins water shellfish (Altrthyrilr conclollr) (69), although no reports exist of PST intoxication via thisroute.MarineshellfishsequesterPSTsfrommotilemarinemicroorganismscalled dinoflagellates, particularly Ale.randrium catenelllr, A. n~ir~lrtut?~, A. oster!feldii, A . tcrmcrreme, Gymnoclitliw?? ccrtencrturn, and Pyrodit~iuml~trharner~sew r . cor?IpressmI. At this
81
Shellfish Chemical Poisoning
STX neoSTX
B1 Gonyautoxin 2 Gonyautoxin 3 decarbamoylSTX
RI
R4 RZ
R3
H OH H H H
H H H H
H H H
H H
OSOS
OSOY
H H
H H H
H
soy
’ In this derivative, a proton replaces of all the structure beyond the wavy line, including R+ Fig. 2 Thc structure of saxitoxin and o f
naturally
occurring chemical variants (62,63,67,68).
time there is a debate concerning the possible production influence on PST production (70,7 I).
of PSTs by bacteria or their
B. Molecular Mechanism of Action PSTs prevent sodium ions from passing through the voltage-sensitive sodium channel (VSSC), binding to the channel with nanomolar affinity (72,73). This large protein, of approximately 260 kDa, spans cell membranes mainly in nerve and muscle. Depolarization of the cell membrane initiates a conformational change in the VSSC, opening a pore that selectively transports sodium ions into the cell. The VSSC is a multisubunit protein, with the largest subunit andthat which contains the pore, the a-subunit, containing four internal of these sequence repeats, it is hypothesized amino acid sequence repeats. Within each that six transmembrane a-helices assemble around the central ion transporting pore (74). Depending on the tissue and VSSC isoform, other smaller proteins, the P-subunits, attach to the a-subunit and affect channel properties such as the speed of ion conduction and channel activation and inactivation rates (75,76). To date, VSSCs have only been detected in animals, occurring in all vertebrates and in most invertebrate phyla, including molluscs (77), jellyfish (781, and flatworms (79). PSTs bind at the opening of the pore that allows the ions to pass through the VSSC (80,81). Whether ion passage is hindered by physical occlusion of the pore, or by an allosteric modulation of the pore’s structure making it unable to conduct ions, is still not fully resolved. Modified STX can havea dramatically altered ability to bind to the VSSC. For instance, the sulfated PST, B 1 (Figure 2), has an affinity 400 times less than STX for the VSSC rat skeletal muscle (73) and neoSTX (Figure 2) is four fold better than STX at binding to the same channel. Crucial also to the ability of STX to bind to the VSSC
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is the charge state of the 7,8,9-guanidino group, with its deprotonation preventing it from ligating to the channel (65). Mammals also express several VSSC isoforms to which STX can bind with only micromolar affinity. They exist in lnatnmalian sensory neurons (82), cardiac muscle(83). skeletal nluscle in the early stages of development (84), and experimentally denervated skeletal muscle (85). Differences in VSSC amino acid sequences underlie this diversity in toxin sensitivity, and mutation of a single amino acid can convert a toxin-insensitive VSSC to a channel that is easily blocked by STX (86).
C. ToxinUptake STX uptake from the gut appears to be quite efficient since no toxin is eliminated i n the feces of test animals (58,87,88). Absorption of drugs and toxins from the mammalian gut mainly occurs by passive processes and not via the specific active transporters for nutrients (89). Passive uptake across the gut epithelium favors nonionized and lipophilic molecules that readily penetrate cell membranes.It is curious thenthat the cationic STX is efficiently absorbed. Of importance for ionizable compounds the likePSTs, the pH of the gut environment affects toxin uptake by altering the charge state of the toxin. For example, since most absorption occurs in the alkaline intestine, in this part of the gut STX will lose a proton from the 7,8,9-guanidiniunl, making it less polar and therefore approaching a state more amenable to diffusion across the lipid bilayer. Also, the overall charge state in some PSTs can be modified by the presence of anionic sulfate groups, which can negate the positive charges provided by the guanidino groups. Passive absorption of charged, hydrophilic compounds occursin the gut by diffusion through the tight intercellular junctions of the epithelium (89). This process, called paracellular diffusion, is restricted to molecules of approximately 5 2 0 0 Da and is unaffected by pH. This exclusion limitisnotabsolute andmaybeaffected by agentsthatsequester calcium and magnesium. The complex biological matrix in which the toxin is ingested may contain natural ion chelating agents, making it easier for PSTs to enter the bloodstream via this route. Although paracellular diffusion is much less efficient than uptake through the epithelial cells, it may be a significant route of toxin absorption, as STX is not much greater than the size exclusion limit, even if not affected by ion chelators. Also, those PSTs significantly smaller than the parent STX molecule, for example, decarbamoylSTX (Figure 2; molecular weight 256), would be better able to pass through the tight junctions.
D. Toxin Metabolism,Transport,andElimination The first point where PSTs may be modified after ingestion is in the gut itself. Gastric acid can convert small amounts of the less toxic sulfated PSTs to more potent compounds (90). This may prove significant if a very large amount of these sulfonated toxins is ingested and critical amounts of the highly toxic STX or neoSTX are produced. Once in the blood, STX is returned to physiological pH, and thereforeto a predorninantly doubly charged and highly toxic state. With STX again being quite polar, there is little possibility of it crossing the blood-brain barrier, which is best traversedby lipophilic agents. This is borne out by a recent study where no toxin reached the brain of cats that received 2.7 pg/kg STX (87). Curiously though, at higher doses ( I O pg/kg) in this same study, STX did enter the brain. This dose dependence of STX’s ability to invade the
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brain may explain earlier reports of STX exerting central effects (60.91,92). One other explanation for this unexpected observation may be a process where charged drugs, to which STX can be equated, binds to plasma proteins (89), some of which may be actively taken across the blood-brainbarrier, carrying STX along with them. PST’s effects on central systems can also be achieved upon regions of the brain not encapsulated by the impermeant blood-brain barrier, such as the circumventricular region. Toxin is eliminated predominantly via the urine, with no elimination of PSTs via the feces (58,87,88). Excretion of PSTs occurs rapidly,no matter how the toxin is administered. For instance, the bulk of IV administered saxitoxinol, a chemically reduced STX, and STX was urinated by rats within hours of injection (66,88). In humans, the half-life after oral ingestion of toxic shellfish is approximately I O hours (58). Metabolic conversion of PSTs while passing through mammals has been little studied, especially in humans. Examination of the serum and urine of human PSP victims revealed a significant increase in Cl compared to its relative gonyautoxin-2, which differ only in an additional sulfate on the C type PST (58). This sulfation of gonyautoxin-2 in humans contrasts with rats, where there was no metabolism of STX or saxitoxinol during their-passage into the rat’s urine (66,88).
E. Treatment Victims should undergo immediate gastric evacuation to remove toxic contents unyet digested and absorbed. This can be achieved not only orally, but also by enemas to clear the intestines. This will also decrease the impact of any conversion of lesser toxic PSTs in the gut. Artificial ventilation for victims of severe PSP is the only recommended treatment to date. Symptomatic relief should accompany all other efforts. The dominant strategy for developing a medicinal treatment has been to use antibodies developed to STX, a carrier protein to make an antigenic epitope, that created against toxin conjugated to may sequester PSTs and out-compete the sodium channel to bind the STX (93-96). If the antibody has a significantly lower affinity than the sodium channel for STX, which was indeed the case with one antibody that possessed a micromolar affinity for the toxin (95). then it must compete for thetoxin by weight of numbers. This would requires administration of large amounts of the antibody. Another limitation of this approach is that the antibodies may be quite specific to single PSTs such as STX; other PSTs to which the victim was exposed maybe able to bypass the antibody and still act. A mixtureof antibodiesmay be necessary if this approach is to succeed. Despite these potential problems, there has been some successi n animal modelswhen treated with STX antibodies, although they have not been tested on PST mixtures (93-96). Since it seems that STX can cross the blood-brain barrier at high doses, antibodies will be ineffective in inhibiting this pool of toxin since they cannot cross the blood-brain barrier. Surprisingly, in guinea pigs, the potassium channel blocker 4-aminopyridine (4-AP) effectively counteracted the effectsof lethal IV doses of STX (61). This drug reverted the loss of blood pressure and enhanced neuromuscular transmission to allow the diaphragm to again function. Large dosesof 4-AP were necessary, however, and may cause serious side effects such as seizures and convulsions. If administered in a hospital, steps can be taken to ameliorate these side effects. A clue to an alternative drug strategy is present in a recent study on cats (87). Cats given lethal doses of STX were kept alive forthe duration of the experiments by continuous administration of the adrenergic agonist, dobutamine, in conjunction with mechanical
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ventilation. In these animals, because breathing was assisted, cardiac rather than respiratory arrest was the cause of death. Dobutanline increases cardiac contractile strength and volume, and providing it along with artificial ventilation, as was done with the cats, may be a suitable emergency medical strategy for PSP victims (87).
111.
DlARRHETlC SHELLFISH POISONING
The firstreport of what became known as diarrhetic shellfish poisoning (DSP) was in 1978 in the Tohoku district in Japan (IS). Since then reports of DSP have emerged from every continent except Africa and Australia (Figure1 ). DSP has never resultedin a human fatality, despite the causative toxins being lethal to mice when injected intraperitoneally (LDi0 of 192 pg/kg) (97). Diarrhea is the major symptom exhibited by victims as well as other gastrointestinal upsets such as vomiting, nausea, and abdominal cramps. These (IS). symptoms may become so severe as to incapacitate the patient
A.
DSP-Causing Agents and Their Sources
The major causative toxin for DSP is okadaic acid (OA) and its relatives, the dinophysistoxins (Figure 3). To be consistent throughout this chapter, the suite of DSP-causing toxinswillbereferredto asdiarrheticshellfishtoxins(DSTs).Okadaicacidderivesits name from the organism from which it was first isolated, the sponge Herlichondricr okcrh i , andwashypothesizedtobeproduced by microorganisms(97).Okadaicacid (C4,HtjXOl3; molecular weight 804) isa complex lipophilic polyether, readily soluble in many organic solvents (99) and sensitive to degradation by acid or base exposure (48). DSP isa somewhatconfusingsyndrome inthatseveralstructurallydistinctpolyether toxin families, the yessotoxins and pectenotoxins, often occur i n shellfish alongside OA and the DTXs and are also referred to as DSTs. Only dinophysistoxin and OA are truly diarrheagenic (IOO), and will be focused upon here, but the other toxins confuse toxicity monitoring results because of their effects on mice used i n the regulatory bioassay. Pectenotoxin is clearly an hepatotoxin (IOO), whereas how yessotoxin mediates its toxicity is still unclear (101); experimental animals injected with it die from cardiac failure (102).
H acid Okadaic Dinophysistoxin-l Dinophysistoxin-2
H H H
CH3 CH3
CH3 CH3 H
Fig. 3 Structure of' thepolyether diarrhetic shellfish toxins, okadaic acid and dino1Jhysistoxins (48,97.98).
Shellfish Chemical Poisoning
B.
85
Known Molecular Pharmacology
Okadaic acid is a very potent inhibitor of several classes of serinekhreonine phosphatase, acting at nanomolar and even picornolar concentrations (107-109). These enzymes are a variable subunit that then multisubunit proteins targeted to their substrate protein by forms a heterodimer with a structurally invariant subunit which catalyses the dephosphorylation of serine and threonine residues ( 1 IO). Serine-threonine phosphatases are subclassified depending upon their substrate specificity, their requirement for cofactors such as to several inhibitory peptides and divalent cations and calmodulin, and their sensitivity okadaic acid itself. OA-sensitive subclasses are PP-l and PP-2A, whereas PP-2C is unaffected by OA,andmicromolaramounts ofthetoxin arerequiredtoinhibitPP-2B ( l 0 9 , l I l , I 12). It is the catalytic subunits of PP-l and PP-2A that are affected by OA, mainly by noncompetitive inhibition (1 13,l 14). These two isoforms are highly homologous, explaining the similarity in their OA sensitivity. The homology is not complete and the differences i n amino acid sequence cause subtle differences in their affinity for OA. A sequence of four amino acids containing two acidic residues from rabbit PP-l can be a replaced by the corresponding amino acids from rabbit PP-2A, which contain instead cysteine and a basic amino acid, arginine. This increased the toxin sensitivity of the chimeric mutant so that it approached that of PP-2A from which the arginine-cysteine sequence was derived ( 1 15). The carboxylic acid of OA is essential to its activity, as demonstrated by methyl esterification, a modification which abolishes OA's abilityto inhibit its target phosphatases ( I 16,l 17). Also, oxidation of the 27-OH greatly reduced its abilityto inhibit PP-2A. From molecular modeling, this portion of molecule is not involved in bonding to the enzyme, but is more likely to be involved in binding the phosphate substrates (1 18). Apart from its acute toxicity, chronic exposure to OA may have a genotoxic effect. At low concentrations. OA forms DNA adducts in mammalian cell lines, between 2 and 100 adducts per 10' bases, a phenomenon not observed at higher concentrations of 2 10 nM ( 1 19). Regular dietary exposure to OA levels that may not elicit DSP may therefore have a teratogenic effect.
C. Cellular Impacts of Okadaic Acid Actions In those cells that take up DSTs, the toxins cause a buildup of phosphorylated proteins. Phosphatases are counterbalanced within the cell by protein kinases,which introduce phosphate groups onto proteins. Since the phosphorylation state of an enzyme, or receptor, often governs whether it is active or not, this interplay between kinases and phosphatases regulates cell function and manyof the cascades which underlie cellular processes. Further, of a kinases and phosphatases can act upon each other. For example, dephosphorylation kinase renders it inactive and phosphorylation of a phosphatase may switch the enzyme into action ( I 1 I ). In the presence of OA, protein kinases continueto function, but in those pathways containing PP- I and PP-2A, which are inhibited, phosphorylated proteins will become overabundant. Thus enzymic cascades and membrane receptors may continue to
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function long beyond their optimal time period, or may not switch on again until the OA is eliminated. Because OA has an effect on such a crucial enzyme, it may result in many effects within the same cell. This, of course, depends upon the efficiency of toxin uptake by the cell and the metabolic status of the cell when the toxin is absorbed, which would govern which subunit the PP-1 and PP-2A catalytic subunits would be dimerized to at the time. Multiple effects can be shown with vero cells, a monkey kidney cell line, where OA inhibited protein, DNA, and RNA synthesis, all at nanomolar concentrations ( 1 20). Certain cell types are also more sensitive to OA than others. For example, in culture, neuronal cells are affected at subnanomolar concentrations, whereas astrocytes and fibroblasts (121) require much more toxin for an effect to be realized. Another case in point would be rat hepatocytes, which require micromolar concentrations of OA for cytotoxicity to be observed ( 122). The role of celltype and cell susceptibility is also demonstrated by the differences between cellular and in vitro effects. Protein synthesis in a cell line was inhibited by SO nM OA, whereas in in vitro studies, generic protein synthesis using a rabbit reticulocyte lysate system was almost 10,000-fold more sensitive to OA, being inhibited by SO% at a concentration of 6.5 pM (120).
D. Organ and Tissue Effects of DSTs DSTsproducetwomajorphysiologicalresponses totheirmolecularactions: an acute effect-diarrhea-and a potentially chronic effect-tumor promotion. Intraperitoneal injection of DTX- 1 desquamates intestinal epithelia (100,123). This, in conjunction with induction of excessive fluid secretion in the intestine ( 124), underlies many of the gastrointestinal effects seen in DSP victims. DSTs alone do not induce tumor formation, but when applied to the skin of mice after a tumor-initiating substance, dimethylbenzanthracene, virtually all test animals developed skin tumors of varying malignancy ( 125,126).
E. Toxin Uptake Metabolism, Transport, and Elimination The sensitivity of these toxins to acid and base exposure would be expected to impact upon the stability of the DSTs while in the human gut before absorption, but the presence of DST degradation products in fecal material has never been reported. If not degraded to a form chemically different from the parent DST, the lipophilicity of the toxins would allow them to passively diffuse through the gut epithelium and toxin uptake would be expected to be quite efficient. Oral administration of tritiated OA to mice resulted in its In pregnant rats, 7% of distribution to liver (2%), kidney (2%), and blood (6%) (127). administered OA crossed the placenta and contaminated the fetal pups (127). Pregnant victims of DSP must receive special care because if DST canalso cross the human placenta it would be concentrated in a single fetus. OA was eliminated in urine, with almost 8% of the administered tritiated OA being detected in the urine within 24 hours (127). Curiously, OA has a secondary effect within the gut of increasing epithelial permeability via paracellular diffusion (128), which may increase uptakeof DSTs themselves or of hydrophilic compounds accompanying these toxins.
F. Treatment DSP is rarely life threatening and usual treatment is to make the patient as comfortable as possible for the durationof the intoxication. Symptomatic treatment for severe diarrhea,
Shellfish
87
such as fluid replacement, should be employed. Because epithelial destruction by the DSTs contributes to 111uch of the diarrhetic effect, administration of classic antidiarrheals is unlikely to provide any relief.
IV. NEUROTOXIC SHELLFISH POISONING The Gulf of Mexico has a long history of toxic microalgae blooms that cause massive fish kills and respiratory irritation in humans. It was eventually recognized that the toxic to induce what agent from these blooms could also be transmitted to humans via shellfish NSP wasrestrictedfor becameknownasneurotoxicshellfishpoisoning(NSP)(129). manyyearstotheAmericasuntiltheearly1990s,whencaseswerereportedinNew Zealand and Australia (Figure l ) . Victims of this syndrome exhibit many of the same symptoms as people who suffer fromthe fish-derived seafood poisoning syndrome. ciguatera. Typical symptoms are tingling in the face, throat, and digits. dizziness, fever, chills, muscle pains, abdominal cramping, nausea, diarrhea, vomiting, headache, reduced heart rate, and pupil dilation (50). There have been no recorded deaths from NSP, although the causative toxin is fatal to test mammals ( 1 30- 132) when administered by various routes, including orally.
A. The Causative Toxins and Their Origins Like the PSTs and DSTs, the compounds that cause NSP originate from dinoflagellates. Inthis case, the culprit alga is Ptychodiscus hruvis (formerly known as Gyrrrnodiniunr b r e w ) ,which gives it name to the toxins, the brevetoxins. Many naming conventions have appeared over the years with regard to brevetoxins. but here we will refer to the toxins as PbTxs, an abbreviation derived from their taxonomic origin. PbTxs are lipophilic, 10to I I-ring polyether compounds (Figure 4) which can be divided into two classes. Type 1 brevetoxins contain l I hexameric rings except for two 7-membered and one 8-membered ring. Type 2 brevetoxins possess only IO rings with one more 8-membered ring than type l PbTxs and an unusual 9-membered ring. They also differ from type 1 PbTxs inthat they have a terminal pentameric ring ( 133). Decomposition of both types of brevetoxins is accelerated in aqueous solutions with pHs greater than 10 and less than 2 ( 135). When dry, however, PbTxs are extraordinarily stable with PbTx-2 and -3 being stable in this state to 300°C ( 1 3 5 ) . I n rats, PbTx-2 and -3 possess LDS,,s of 60 and 200 pg/kg via the intravenous route ( 130,131 ). Similar LD,,, values are obtained when administered intraperitoneally (131). but the toxin’s effectiveness is markedly reduced when ingested, with the respective oral LD,,,s being S20 and 6600 yg/kg ( 13 1 ).
B. Molecular Pharmacology of the Toxin PbTxs bind to the voltage-sensitive sodiunl channel described i n detail in Sec. 11. It binds, however, to a different region of the VSSC than that via which the PSTs act and elicits a very different effect. The VSSC binding site for PbTxs involves both the first and fourth of the four homologous domains within the a-subunit ( 136,137), and quite likely part of one of the extracellular loops of the channel ( 138). Theaffinity of the toxins for theVSSC is nanomolar ( 139,140).and unlike with the PSTs, toxin-insensitive channel isoforms have
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Me
H0 R
0
Fig. 4 Brevetoxins type 1 (A) and 2 (B) and some of the chemical variations that nature (133,134).
may occur in
yet to be reported and mutations of the channel which affect brevetoxin binding have not been reported. PbTxs shift the activation voltage of the VSSC to more negative potentials (141144). This puts the channel nearer the triggering threshold where the change in potential difference across the cell membrane initiates the seriesof conformational changesto open and allow conduction of sodium ions. Inactivation of ion conduction is also believed by some to be slowed by brevetoxins (132,141). The end result of either of these two effects, or their combined effect, is to lengthen the mean open time for the VSSC, allowing a higher overall number of sodium ions to enter the cell. This hyperexcitability of cells reliant upon action potentials can lead directly to cellular malfunction. The additional sodium loadmay also overwhelm theprocesses,such as the N a + / K ' transporter,that maintain the concentration gradient of sodium across the cell membrane, providing the
Shellfish
89
electromotive force for action potentials. PbTx affected VSSCs also display subconductance states; that is, the rate of ion flow through the channel is reduced (141,145). The VSSC might not be the only receptor affected by the brevetoxins, because other smallpopulations of brevetoxinbindingsiteshavebeenobserved(138,139,146,147). These other binding sites were not identified, but the candidates include the nicotinic receptor-ionophore complexof the neuromuscular junction( 148)and the aryl hydrocarbon receptor, which is a ligand-activated transcription factor (149).
C. Toxin Uptake,Metabolism,andDistribution The lipophilic PbTxs easily pass through the gut epithelia into the bloodstream. Oral administration of PbTx-3 to rats resulted in almost all of the toxin being absorbed ( 1 SO). and within hours SO% of the toxin had accumulated equally in the liver and stomach. The ingested toxin was also readily distributed to the intestine (14%), heart (8%').and kidneys (695). Of the other tissues tested-spleen, lung, fat, muscle, plasma, testes, brain, and skin-no more than 4% of toxin appeared. Although IV administration of PbTx-3 results in a different pattern of tissue distribution in rats to that after ingestion, what is revealed is that uptake of brevetoxin is incredibly fast, with it disappearing from the blood within 1 minute of injection (IS1). Within 1 hour of the IV PbTx-3 injection, almost 70% of the 20% in the liver, and the remainder radiolabeled toxin appeared in the skeletal muscle, in the intestinal tract ( 1S 1 ). Orally ingested compounds first pass through the liver for detoxification, but in some cases, the compounds may attack the liver. In the case of the brevetoxins,thisisindeedthecase,wherePbTx-3affectedmouseliverefficiency by ( 152). enhancing sodium entry into liver cells, thereby inhibiting oxygen consumption Therefore the large anlount of PbTx that resides in the liver after its uptake could result in significant and possibly long-term liver dysfunction in NSP victims. The lipophilic nature of the brevetoxins would allow them to not only easily cross the gut epithelium, but also the brain barrier. This supposition is supported by a study with the chemically and pharmacologically related toxin ciguatoxin, which binds to the same sitc on the VSSC as the brevetoxins and elicits the same effects (153), which can induce many effects in the brains of mice (154). Curiously though, with PbTx-3, very little toxin reached the brain of rats, getting to no more than approximately 1% of h e toxin dose at any one time ( 150, IS l ) . PbTxs are vulnerable to metabolic conversion in mammals. This was demonstrated after rats were orally dosed with radioactive PbTx-3 and fecal extracts contained several radioactive compounds apart from the parent toxin, indicating some modification of the toxin had occurred during its movement through the body ( IS I ) .
D. Toxin Elimination PbTxs exit the lnamnalian body by both urination and defecation. In experimental m a n mals, the route of'toxin administration can affect which of the two routes will predominate. After IV administration of radiolabeled brevetoxin, 75% of the toxin was eliminated via the feces, whereas only 14% exited via the urine and the remainder was still in the rat's body after 6 days ( 15 I ) . After oral dosing with PbTx-3, however, some of the toxin is not absorbed, passing directly through the gut to be eliminated with the feces. Equivalent amounts of the toxin were eliminated in both the urine and feces, but because feces in-
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cludes unabsorbed toxin, it was suggested thaturinaryeliminationmight be dominant over the fecal route (150). The symptoms produced by the toxin may therefore accelerate in test mammals (135) and its elimination by increasing defecation and urination rates humans.
E. WholeAnimal Effects Intravenous injection of PbTx into anesthetized dogs caused apnea, bradycardia. uncontrolled spontaneous skeletal muscle twitching, and tonic contractions. Heart rate and blood pressure immediately dropped upon injection, but recovered quickly. Doses greater than 80 pg/kg causedrespiratory arrest in dogs ( 1 SS). This short-lasting bradycardia, hypotension, and respiratory inhibition is known as the Bezold-Jarish refex and also occurs in anesthetized cats given PbTx (156j.In awake rats, the respiratory rate is depressed, which the animal compensates for by increasing breath volume (157). A substantial decrease in core and peripheral body temperatures can occur in mammals given a brevetoxin (157). Thisobservation, in conjunction withtheneurologicaldysfunctionsuchasataxiaand simultaneousseizures of limbpairs,indicatesbrainandspinalchordinvolvement (156,157).
F. Treatmentfor NSP As with PSTs, antibodies to PbTxs have been investigated as a means of treating NSP victims. Pretreatment of rats with goat polyclonal antibodies directed toward PbTx-3 prevented any NSP symptoms i n rats after injection with PbTx-2 (130,157). Immediate injection of PbTx antibodies after injection with PbTxalsoprotectedthe animals from the toxin’s effects (130), except for some subtle emanations of toxicity such as mild ataxia. This was somewhat surprising since brevetoxin disappears from the blood so quickly and wouldnotbe exposed to the antibody. Brevetoxin is a reversible toxin ( I 5 8 ) , andan antibody may manage to sequester the toxin during the periods that it is dissociated from the receptor and before it reassociates. This is unlikely to be a suitable route for treating NSP victims, since quitea long time is likelyto occur between intoxication and realization by the victim that they may need medical attention.
V.
AMNESIC SHELLFISH POISONING
In 1987. on Prince Edward Island in Canada,an unusual shellfish poisoning event unfolded involving the blue mussel(Mytilus erlulis), a shellfish implicated in many different poisoning syndromes (TableI ) (46). Of the symptoms manifestedin the victims, themost curious was the effect on memory, an effect that lasted days or longer, which led to the syndrome being dubbed amnesic shellfish poisoning (ASP). Almost 200 people were affected and unfortunately, 3 of the victims died (159). Of interest, approximately 50,000 people are believed to have consumed mussels from the same batch that caused the poisoning (160). There have been few ASP events since (Figure l ) . Other symptoms experienced by ASP victims include nausea, vomiting, headache, diarrhea, and abdominal cramps. Neurological symptoms may follow, such as confusion, memory loss, and disorientation (161 ). I n severe cases, seizures, followed by coma and death may occur.
Shellfish
A.
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Poisoning
Causative Toxin and Its Origin
Because of the unusual nature of the s y m p t o m and the number of people affected in the first ASP event, Canadian authorities mobilized a substantial scientific team to ascertain whether the cause of the intoxication was anthropogenic or natural. They discovered that domoic acid (DA) (Figure3 , a known neuroexcitatory toxin, was the cause of the poisonings and had been accumulated in the shellfish from a diatom Pseurlo-t/it:schia (previously Nit:sc.hitr) ~nrrrget~s forma rtrultiseries ( 13,166). DA derives its name from the first organChotlclricr nrmtrttr, locally known ism in which it was found, the macroscopic red alga, as domoi (163). It was originally purified as the antihelminth in a long-used traditional medicine (163). This use, coupledwith the observation that approximately 50,000 people ingested DA-contaminated shellfish in Canada in 1987, giving a morbidity rateof approximately 0.4% and a mortality rate of 0.006%, indicates that DA is not very toxic compared to other shellfish toxins. Other DA-producing diatoms include Pseuno-rritcsc,hia seritrtcr, P. rtrultiseries, P. trustrnlis, P. psc.uek)cleliccrtis.sirtItr, P.clPlic.trti.ssitnci, and P. tur&ldcr (167-17 I). Of concern is an outbreak of DA poisoning in seabirds in California in 1991, where the vector was not shellfish but anchovies ( I7 1 ), a widely eaten organism not normally subjected to toxicity monitoring. Thus a different pathway exists that may lead to an ASP-like outbreak. Domoic acid (C,,H:,NO,,; molecular weight 31 I ) is a tricarboxylic acid that differs very little from the glutamate receptor agonist kainic acid, both being cyclized analogues
2 C O O H
'PCOOH
A
B
C
D
E
F
Fig. 5 Domoicacid ( A ) andthevariousanaloguesknown to date withregions o f isomerism depicted with the circular ;~rrows ( 162- 165). Note that not a l l of these compounds occur i n shellfish but arc specific to thc macroscopic red algae ( 164). Kainic acid, the ncurotoxin that defines the subset o f glutanmtc receptors activated by domoic acid and its analogues, is depicted in F.
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of L-glutamate (Figure 5 ) . DA is only weakly toxic to mammals, having an LDS,)in mice (intraperitoneally) of 3.6 mg/kg (172).It is a polar molecule, insolublein organic solvents, but soluble in water (-8 g/L) and slightly soluble in methanol (173). This polarity arises from three carboxylic acids and an amino group in the pentameric ring, which have pK,s of 2.1, 3.7, 5.0, and 9.8, respectively (163,174). The ionic state of DA affects its overall toxicity, with intraperitoneal DA injection into mice in an acidic vehicle significantly less toxic than at physiological pH( 175).
B. Molecular Target of Domoic Acid Domoic acid was recognized many years ago as a potent amino acid neuroexcitant (176), mainly through its structural similarity to kainic acid, a compound used to delineate a subclass of ionotropic glutamate receptors (IgluRs). IgluRs are ligand gated, neuronal ion channels, which respond to L-glutamate by opening and allowing the passage of cations. IgluRs trigger many intracellular cascades either directly by their introduction of cations or indirectly by the second messengers produced by these cascades, such as cyclic AMP or reactive oxygen species (177,178). IgluRs have been subdivided depending upon their sensitivity to three compounds: NMDA, AMPA, and kainate. It is primarily the kainate IgluRs that are sensitive to DA, although there is some evidence that AMPA IgluRs may also respond to the toxin (179). From the amino acid sequences of IgluRs cloned to date, hydrophobicity plots predict there are four transmembrane regions with both the carboxy and amino tails of the subunits being intracellular. The molecular diversity of the IgluRs arises not just from their differences in amino acid sequences, but also from the fact that they are made up of five, sometimes different, subunits which combine to surround the central ion-conducting pore (180,181). It is possible that domoic acid may affect other kainate-sensitive glutamate systems. For example, kainateis a nontransported competitive inhibitorof an excitatory amino acid transporter, EAAT2 ( 1 82). SinceDA can access the kainate binding site in kainate IgluRs, it is possible that it may also inhibit this enzyme.
C. Domoic Acid Uptake by the Gut The hydrophilicity and ionic properties of DA make it difficult for it to cross cell membranes in the gut epithelium. Evidence for the poor uptake efficiencyof DA by the human gut is its low morbidity rate. In fact, orally administered DA in both rats and mice was virtually completely eliminated in the feces and none of it made it to the urine (172). Since it possesses both basic and acidic pK,s, it can exist in charged states in both the stomach and intestines. For instance, in the stomach, a variety of charge forms from deprotonation of the various carboxylic acids can exist, whereas in the small intestine, all 3carboxylics will be charged while a pool of DA also bearing a positive charge on the imino will develop. The most likely route then for DA uptake is paracellular diffusion, an inefficient and size-selective method.
D. Toxin Distribution and Elimination Despite DA’s poor ability to cross the gut epithelium, it obviously can enter the human bloodstream and elicit drastic effects in ASP victims. It does not have much time to do this though, as it has been shown in monkeys that IV DA disappears from the blood very
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quickly, with 60-90% of the injected dose appearingin the urine within 6 hours of injection (183). Once DA has successfully entered the bloodstream it returns to physiological pH and all of the carboxylic acids will be charged, significantly hinderingit from crossing the blood-brain barrier, althoughnot absolutely (160). However, someof the human brain lacks the blood-brain barrier (184), exposing it to absorbed DA. After attacking these brain regions there may be a flow-on effect, whereby trauma in these exposed regions of the brain triggers excessive glutamate release within the brain and causes general excitotoxicity.Kainate-receptoractivationleads to activation of other IgluRs, with NMDAreceptor activation being the major cause of cellular death in cultured cerebellar granule neurons (185). DA that does enter the blood is primarily eliminated from mammals in their urine ( 1 86). Injection of radiolabeled DA into rats resulted in virtually the entire toxin dose being urinated within several hours of injection. Little metabolism of DA occurs during its time in the body because, in this same study, all of the radiolabel in the urine was associated with the parent domoic acid.
E. Pathology Kainate IgluRs are a prominent receptor family in the human brain, and since the bloodbrain barrier does not enclose all of the human brain,it is vulnerable to DA attack resulting in lesions and other neuropathologies (161,184,187). In mice, high doses were necessary to inflict observable damage upon these unprotected regions of the brain, the so-called circumventricular region. Lesions were generally localized but did extend a short way into neighboring regions of the brain ( 1 84), which may reflect the flow-on effect of DA neurotoxicity discussed above.Some animal models may be more susceptible to this effect. For instance, rats receiving an intraperitoneal injection of DA suffered significant lesions in most areas of the central nervous system (CNS), including cerebrum, cortex, hippocam( 1 88). As alluded to pus, hypothalamus, olfactory system and septum, and even the eye above, small amounts of DA can permeate the blood-brain barrier, albeit very slowly, and when it does, it can inflict additional neurological lesions apart from the circumventricular region (160).
F. Treatmentsfor ASP Symptomatic treatment and life support are the only recommended treatment. There are clues in some studies, however, of medicinal strategies that may be employed. The most direct strategy is the use of a kainate IgluR antagonist, CNQX, which ameliorated DA’s action on retinal tissue (1 89). And because kainate receptor activation by DA can elicit neuronal damage by their activation of the NMDA class of IgluRs, it is not surprising that the competitive NMDA receptor antagonist, D( -)-2-amino-5-phosphonopentanoic acid, and non-NMDA receptor antagonist, NBQX, greatly reduced domoate toxicity by almost 80% (185). Dextromethorphan is also another NMDA IgluR receptor antagonist that showed promise in mice as a DA antidote (190). Alternative pharmaceutical strategies may arise from inhibition of systems peripheral to the IgluRs. Pretreatment of rats with diazepam, a benzodiazepine sedative and anesthetic, can reduce DA-induced convulsions in rats at 5 mg/kg but achieved little else at this dose (191). Its efficacy if administered after DA intoxication was not explored. Kynurenic acid, a tryptophan metabolite found in the mammalian brain, protected mice
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from DA toxicity for several hours, even when administered some time after the onset of motor seizures (192). Gastric lesions, which appear after DA administration in mice, were also significantly reduced by kynurenic acid (193). Administration of tryptophan and the organic acid transport blocker, probenecid, augmented the protective effects of kynurenic acid (190). Activation of the 5-HT1A subclass of serotonin receptors in rat hippocampi by the drug 8-hydroxy-dipropylaninotetralinnegated most of the effects of DA that was injected directly into the hippocampus (194).
VI. MINOR SHELLFISH TOXINS Several peptidic hepatotoxins, previously considered to occur only in freshwater microalgae. have now been found in marine shellfish and phytoplankton. They have not, as yet, been implicated in any shellfish poisoning, but we should be wary of their potential to causehumanintoxication.Also, the long-knownmarine toxintetrodotoxin,which has caused human intoxications from gastropod nlollusc ingestion rather than bivalve molluscs, has been found in a dinoflagellate known to be a source of PSTs. This raises the possibility that i t may toxify bivalve shellfish. In this section, these two toxins will be briefly summrtl-ized.
A.
Cyclized, Peptidic Hepatotoxins
Microcystins are cyclic heptapeptides from species
of thefreshwaterblue-grcenalgae
Microcystis (Figure 6), long known as hepatotoxins (197). The amino acid composition of the microcystin may vary, but the presence of the novel hydrophobic amino acid, 3amino-9-methoxy- IO-phenyl-2,6,8 trimethyl deca, 4,6 dienoic acid (ADDA)is essential to its bioactivity. Like okadaic acid, microcystins are potent inhibitors of the serine/threonine protein phosphatases, binding to the same site on the enzymes as OA (198j, and also act as tunlorpromoters (199). Althoughacutehumanintoxication bythesepeptidesafter eating marine shellfish has not been reported, they have been detected in the blue mussel
Me 0
Fig. 6 The structure of the cyclic heptapcptide toxin. microcystin (195,196). Thc side chain of the essential and unique amino acid. ADDA is to thc left of the molecule.
95
Shellfish Chemical Poisoning
(Mytilus edulis) (200), a shellfish responsible to date for ASP, DSP, and PSP (Table l), and other commercial marine bivalves (201). We must be wary of their presence in the marine environment because chronic exposure to subacute doses of microcystins may impact upon human health, especially their potential role in cancer development.
B. Tetrodotoxin Tetrodotoxin (TTX) (Figure 7), is the famous pufferfish poison, highly prized in the fugu tradition in Japan. Human intoxication and death has occurred after consuming mollusks as in all of infested with this toxin. although the mollusks involved were not bivalves, the shellfish poisoning syndromes described above, but were gastropods. Seventeen Taiwanese in 1994 were poisoned after eating samples of Nrrs.sari~r.scustus and N. conoiddis, with one elderly victim dying, although most were probably from complicationsthat arose after the initial effects of the poisoning had abated (205). TTX competes with a comparable affinity for the same binding site on the VSSC as the PSTs, acting in the same manner as PSTs. Despite these similarities in their molecular pharmto elicit the same effects acology, TTX is structurally dissimilar to the PSTs (Figure 7), but like the PSTs, they possess a guanidino group (PK,~8.8) essential to its toxicity (202-204). The possibility of TTX intoxication from bivalve shellfish is of concern because of a recent report that thedinoflagellate Alexmdriurn n l i m t w r l , a dinoflagellateknown to havecausedPSP intoxicationviabivalveshellfish,mayalsoproduce TTX(206).Thisdiscoverywas made due to the fact that toxic individuals of the commercial edible scallop, Patirzopecten yessoerlsis, contained TTX along with PSTs. Thus TTX may abecause of PSP and analysis of culprit samples is necessary to confirm that PSTs and not TTX were the causative toxin.
VII.
CO-OCCURRENCE OF TOXINS
From Table 1, it can be seen that some shellfish, such as Mytilus edulis, are responsible for more than one type of shellfish poisoning. The conditions that initiate and maintain a bloom for one toxic microalgae may be just as suitable for other toxic species and so a bloom may contain several speciesof toxin-producing organisms. Also, slow detoxification of one toxin from a shellfish may overlap with a bloom of another toxic species, adding to the shellfish’s toxin load. Thus it is possible that people may experience more than one type of intoxication, making the diagnosis of such an event far more difficult
pK, = 8.8 Fig. 7 The neurotoxin,tetrodotoxin (202-204)
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and the treatment even more problematic. In fact, both diarrhetic and paralytic shellfish toxins have co-occurred in mussels from Spanish waters (24). A potential complication of the co-occurrence of toxins is the effect by okadaic acid on paracellular diffusion. As described above, this process is the means by which molecules traverse the gut epithelia through the tight junction barrier, allowing small hydrophilic molecules not amenable to passing through cellular barriers to enter the human system. Okadaic acid has been found to increase this permeability ( 1 28) which may not only affect its own uptake, but also of hydrophilic toxins such as the PSTs or domoic acid with which it may co-occur.
VIII. SHELLFISH RESPONSE TO THE TOXINS Many of the factors that govern the ability of a toxin to be taken up across the human gut also influence the ability of shellfish to accumulate the toxin. Domoic acid, which is poorly taken up across the gut epithelium, is also very poorly accumulated by shellfish (207). Lipophilic compounds like the brevetoxins, however, which are retained in the fat tissue of mammals for long periods (151), may be expectedto be accumulated effectively and retained by shellfish for long periods after a toxic algal bloom. This is indeed the case, with detectible levels of brevetoxin remaining in shellfish some months after the toxic dinoflagellate Prychodiscus brevis had disappeared (208). Poisonous shellfish do detoxify when no longer exposed to the intoxicating organism, with the rateof detoxification being species dependent and influenced by salinity, water temperature, and the size of the shellfish (207,209-21 1). Different life stages of toxifying microalgae can produce differentamounts oftoxin.Gametes,zygotes,and thefirstfew stages of vegetatively growing cellsof the DA-producing diatomP.seud~-tzit,-.schie/ purrpws f. rwltiser'ies do not produce domoic acid (212). The age structure o f a toxic algae bloom influences the amount of toxin a shellfish is exposed to. What effect then. do these toxins have on the shellfish themselves. By definition, shellfish that accumulate these toxins must be resistant to some degree, as the compounds often reside in the animals' tissue for long periods of time. Shellfish do possess the receptors that are inhibited by some of these toxins. The mollusk Aplysicr is known to have kainate-sensitive glutamate receptors (213) and a VSSC (77). Resistance may arise from the possession of insensitive isoforms of the enzymes and receptors targetedby the toxins. As has been discussed, simple mutations of the VSSC can result in much reduced PST sensitivity, and a naturally occurring mutation may underlie the apparent resistance of shellfish nerves to PSTs (2,3). Similarly, a single point mutation in a serine-threonine protein phosphatase can cause a 50-fold decrease in the sensitivity of this enzyme to OA (214). The toxin sensitivity of these receptors i n toxin-accumulating species of shellfish has yet to be elucidated. Alternatively, physical separation of the toxin away from its active site, such as in the digestive gland, may confer resistance. Depuration rates of particular toxins are complicated by their metabolic conversion within the shellfish which may modify their chemistry. Incubation of sulfated PSTs with homogenates of different tissues of the scallop (Plmqm~retrnrrrRe1lcrnicv.s) and littleneck clam (Prororhcrca stcrmitwa) desulfated the toxins, converting them to the most potent PST, saxitoxin (215,216). This transformation would remove a negative charge from the molecule, changing its overall charge state and therefore its pharmacokinetics in the shellfish.
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IX. EFFECT ON TOXINS BY SHELLFISH PREPARATIONS The means of preparing shellfish may impact on the amount and state of the toxins that are ingested by potential victims. Raw shellfish, which are commonly consumed, would obviously bethe most dangerous way to eat any toxic shellfish. Some of the shellfish toxins are heat stable and water soluble, such as the PSTs and domoic acid. One would expect therefore that boiling of shellfish in water, as in the making of soup, would extract the toxin. Although this possibility has not been studied with shellfish, anecdotal evidence comesfromJapanesecaseswhereparticularcrabswhichcanaccumulatePSTswere cooked as part of miso soup and consumersof the broth died from PSP (217). In contrast, research with the hepatopancreas of the lobster (Hotntrrus arner-iccrnus), which can accumulate PSPs, showed that boiling removed very little of the PSTs present in the samples (218). Steaming, however, which caused a loss of tissue water, did significantly reduce the toxicity. It may be that the toxin is biochemically anchored in the tissue and not easily extracted by cooking, withthis anchorbeingpresentonly in somespecies. A similar scenario can be raised with lipophilic shellfish toxins and their possible extraction from shellfish tissues into cooking oils.
X.
CONCLUDING REMARKS
It must be remembered that the toxins described herin are naturally occuring compounds, unlikely to have evolved for the purpose of killing humans. Rather,they have some natural function, or are the physiological resultof the organism’s metabolism,not yet fully understood. Their occurrencc in nature is probably underestimated, only being detected after our attention has been attracted by some event such as a human intoxication. efficacy Individual variation in the healthof shellfish consumers can impact upon the of shellfish toxins in humans. For instance, people who suffer stomach and intestinal ailments that affect gut pH may experience different pharmacokinetics with regard to the toxins, possibly improving the efficiency of toxin uptake. Medication given to counteract these ailments, such as drugs to inhibit acid production, could have the opposite effect. Also, the existenceof an ulcer may provide a direct method for toxin entry into the bloodstream for toxins such as domoic acid which are taken up poorly across the gut wall. The occurrence of shellfish poisoning may also be lower than the reality. Mild poias sonings may notbe recognized by victims as poisonings, but rather self-diagnosed generic food poisoning or as an allergic response. The general community is often more aware of these two ailments than shellfish poisoning, and unless they present themselves to a shellfish toxin-aware health professional. the intoxication may never be identified. The adage thatthe world is getting smaller due to modern technology is as true with the food we eat as with anything else. Shellfish have increasingly become a staple of the global diet. Toxins transmitted from the environmentto the consumer are a danger faced today by many people in many countries, not just those which traditionally have a seafood diet. A shellfish poisoning eventnot only affects the victims and their loved ones, but can have an economic impact due to closure of the shellfish industry and a flow-on effect to the whole seafood industry due to reduced consumer confidence in general seafood safety. Steps have been taken in many countries to ensure that their shellfish are safe. These include monitoring of water supplies for harmful microorganisms and direct
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toxicity testing of selected shellfish. These monitoring studies have upper limits that are usually far below the real acute toxic dose, but little is known about the effects of chronic consumption of some of thesetoxins.Increasedsensitivity of analyticalmethodsand expansion of surveys to other members of the marine and aquatic environment may extend our knowledge of the actual taxonomic distribution of these toxins and fully measure how exposed humans are to these compounds. In due course, it will also reveal the role the toxins play in the physiology and ecology of the organisms.
ACKNOWLEDGMENTS This is contribution number 987 of the Australian Institute of Marine Science.
REFERENCES 1.
2.
3.
4.
5.
6. 7.
X.
9.
10.
I I. 12.
13
IN Ashie, JP Smith. BK Simpson. Spoilage and shelf-life extensionof fresh fish and shellfish. Crit Rev Food Sci Nutr 36537-121, 1996. innerves BMTwnrog. T Hidaka, H Yalnaguchi. Resistance to tetrodotoxin and saxitoxin 10: of bivalve molluscs. A possible correlation with paralytic shellfish poisoning. Toxicon 273-278,1972. RC Kvitek, MK Beitler. Relative insensitivity of butter clam neurons to saxitoxin: a preadaptation for sequestering paralytic shellfish poisoning toxins as a chemicnl defense. Mar Ecol Prog Ser 69:47-54, 1991. GM Hallegraeff. A review of harmful algal blooms and their apparent global increase. Phycologia 32:79-99, 1993. JA Patz, PR Epstein, TA Burke. JM Balhus. Global climatechange and emerging infectious diseases. JAMA 275:217-223, 1996. PA Tester. Harmful lnarinc phytoplankton and shellfish toxicity. Potential consequences of climate change. Ann N Y Acad Sci 740:69-76, 1994. CWY Lam, KC Ho. Red tides in Tolo Harbour. Hong Kong. In: DM Anderson, T Ncn1oto, eds. Red Tides: Biology, Environmental Science and Toxicology. New York:Elsevier. 1989, pp 49-52. TJ Smnyda. Novel and nuisance phytoplankton blooms i n the sexevidence for a global epidemic. In: E Graneli, B Sundstrom, L Edlcr. DM Anderson. eds. Toxic Marine Phytoplankton. New York:Elsevier, 1990. pp 29-40. GM Hallegraeff, CJ Bolch. Transport of diatom and dinoflagellate resting spores via ship’s ballast water: implications for plankton biogeography and aquaculture. J Plankton Res 14: 1067-1084, 1992. H Ishida, N Muramatsu, H Nukaya. T Kosuge. K Tsuji. Study on neurotoxic shellfish poisoning involving the oyster, Crmwstren gigcts. in New Zealand. Toxicon 34:1050- 1053, 1996. I Karunasagar, Y Oshimo, T Yasumoto. A toxiu profile for shellfish involved in an outbreak of paralytic shellfish poisoning in India. Toxicon 28:868-870, 1990. 0 Arakawa, T Noguchi, D Hwang,D Chang. J Jeon, K Hashimoto. Paralytic shellfish poisoning bythemussel MTrihrs eddis i n Korea. In: T Okaichi, DM Anderson, T Ncmoto, e d ~ . Red Tides: Biology, Environmental Science and Toxicology. New York: Elsevier. 1989. pp 407-4 10. JLC Wright. RK Boyd, ASW de Freitas, M Falk, RA Foxall. WD Janlicson, MV Laycock. AW McCulloch, AG McInnes. P Odense, VP Pathak, MA Quilliam, MA Ragan. PG Sink
Shellfish Chemical Poisoning
14.
15.
16.
17.
18. 19.
20.
99
P Thibault, JA Walter, M Gilgan, DJ Richard, D Dewar. Identification of domoic acid, a neuroexcitatory amino acid,in toxic mussels from eastern Prince Edward Island. Can J Chem 67:48 1-489, 1989. M Kumagai, T Yanagi, M Murata. T Yasumoto, M Kat, P Lassus, JA Rodriguez-Vazquez. Okadaic acid a s the causative toxin of diarrhetic shellfish poisoning i n Europe. Agric Biol Chem50:2853-2857,1986. T Yasumoto, Y Oshima, M Yamaguchi. Occurrence of a new type of shellfish poisoning in the Tohoku district. Nippon Suisan Gakkaishi 44: 1249-1255, 1978. G Lelnbeyc,TYasumoto,JZhao,RFernandez.DSPoutbreak in ChileanFiords.In:TJ Smayda, Y Shimizu,eds. Toxic Phytoplankton Blooms in the Sea. New York: Elsevier, 1993, pp 525-529. L Boni, A Milandri, R Polctti, M Pompe. DSP cases along the coast of Emilia-Rornagna (northwestern Adriatic Sea). In: TJ Smayda, Y Shimizu, eds. Toxic Phytoplankton Blooms. New York: Elsevier, 1993. pp 475-481. RE Long,JCSargent,KHammer.Paralyticshellfishpoisoning: a case reportandserial clcctrophysiologic observations. Neurology 401:3 10-3 12, 1990. I Karunasagar, B Joseph, KK Philipose, I Karunasagar. Another outbreak o f PSP in India. HarmfulAlgaeNews l7:l, 19Y7. RQ Gacutan, MY Tabbu, EJ Aujero, F Icatlo Jr. Paralytic shellfish poisoning due to Pyrodirti w r l hoIImItvI.se var. c w r r y m s s o in Mati, Davao Oriental. Philippines. Mar Biol 87:223-227, 1985.
21.
22.
23.
24.
25.
26. 27. 28. 29. 30. 31.
32. 33.
D Hwang, T Noguchi. Y Nagashima.1 Liao, K Hashimoto. Occurrence of paralytic shellfish poisons in the purple clam Solrctellino tliphos (bivalve). Nippon Suisan Gakkaishi 53:623626.1987. F Rosalcs-Loessener, E De Porras, MW Dix. Toxic shcllfish poisoning in Guatemala. In: T Okaichi. DMAnderson,TNemoto,eds.Red Tides: Biology. Environlncntal Science and Toxicology. New York: Elsevier, 1989. pp 113- I 16. KW Kizer. Domoic acid poisoning. West J Med 16159-60. 1994. A Gag-Martinez, JA Rodriguez-Vazquez, P Thibault, MA Quillianl. Simultaneous occurrence of diarrhctic and paralytic shellfish poisoning toxins in Spanish mussels in 1993. Nat Toxins 4:72-79, 1996. J Luthy. Epidemic paralytic poisoning in western Europe, 1976. In: DL Taylor, HH Seliger. eds. Toxic Dinoflagellate Blooms. Amsterdam: Elsevier. 1979, pp 321-326. HH Huss. Development and use of the HACCP concept i n fish processing.Int J Food Microbiol15:33-44,1992. DL Park. Surveillance programmes for Inanaging risks from naturally occurring toxicants. FoodAddit Contan 12:361-371, 1995. in the common sandcrab. Science H Sonuncr.The Occurrence of the paralytic shellfish poison 76574-575. 1932. H Sonuner. KF Mcyer. Paralytic shellfish poisoning. Arch Pathol 24:560-598, 1937. CT Tan, EJ Lee. Paralytic shellfish poisoning i n Singapore. Ann Acad Med Singapore 15: 77-79.1986. D Montebruno. Paralytic shellfish poisoning in Chile. Mcd Sci Law 33:243-246, 1993. MEE Popkiss, DA Horstman. D Harpur. Paralytic shellfish poisoning. a report of 17 cases in Capetown. S AfrMcd J 55:1017-1023, 1Y7Y. BC Rodrigue, RA Etzel, S Hall. E De Porras, OH Velasquez, RV Tauxe, E Kilbournc. PA Blake. Lethal paralytic shellfish poisoning i n Guatemala. Am J Trop Med Hyg 42:267-27 1, 1990.
34. E Todd. G Avery, CA Grant. An outbreak of severe paralytic shellfish poisoning in British Columbia. Can Conunun Dis Rep 19:99- 102, 1993. 35 EWC Todd, T Kuiper-Goodman. W Watson-Wright, MW Gilgan, S Stephen. J M:llT, S pleas:incc.MA Quilliam. H Klix, HALuu. CFB Holmes. Rece11t illnesses from seafood toxins
7 00
36. 37 38. 39.
40. 41. 42.
43. 44. 45. 46. 47.
48. 49. 50. 51.
52. 53.
54.
55.
56. 57. 58.
Lle wellyn in Canada: paralytic, amnesic and diarrhetic shellfish poisoning. In: TJ Smayda, Y Shimizu, eds. Toxic Phytoplankton Blooms i n the Sea. New York: Elsevier, 1993, pp 335-339. BD Gcssner, JP Middaugh. Paralytic shellfish poisoning in Alaska: a 20-year retrospective analysis. Am JEpidemiol141:766-770,1995. DM Anderson, JJ Sullivan, B Reguera. Paralytic shellfish poisoning in northwest Spain: the toxicity of the dinoflagelatte Gyr?~r~orlinium c m w m m . Toxicon 27:665-674, 1989. LA Proenca, L Rorig. Mussel production and toxic algal blooms in Santa Catarina State, southern Brazil. Harmful Algae News 12/13:5. 1995. TJ Laila. First report of Gyrmodirriurn caterltrturlr from Atlantic Morocco. Harmful Algae News12/13:7,1995. E Macdonald. Dirwphysis bloom in West Scotland. Harmful Algae News 93, 1994. N Lundholm, J Skov. Psr~rdorzif,.sc.hinpse~cdor/c~/ic~ari.ssir,ltr i n Scandinavian coastal waters. HarmfulAlgaeNews5:4,1993. G Lcmbeye. Major PSP outbreak in Chile. 1991-1992. Harmful Algae News 2: 1-2, 1992. FH Chang, DM Anderson, DM Kulis, DC Till. Toxin production of A/csodriur,r tniuufuur (Dinophyceae) from the Bay of Plenty, New Zealand. Toxicon 35393-409. 1997. Anonymous. Shellfish poisoning: boy dies and 13 treated. Harmful Algae News 8:6, 1994. Anonymous. Paralytic shellfish poisoning: New Brunswick. Canada. Morbid Mortal Wkly Rep 25347-348, 1976. MA Quilliam, JL Wright. The amnesic shellfish poisoning myslery. Anal Chem 61:1053AI060A, 1989. JM Hughes, MA Horwitz, MH Merson, WH Barker, EJ Gangarosa. Foodborne disease outbreaks of chemicaletiologyintheUnitedStates,1970-1974.Am J Epidenliol 105233244,1977. T Yasumoto, M Murata, Y Oshima, M Sano, GK Matsumoto, J Clardy. Diarrhetic shellfish toxins. Tetrahedron 41:1019-1025, 1985. M Kat. Diarrhetic shellfish poisoning in the Netherlands related to the dinoflagellate D i m p/y.si.s otwrtrirrtrfo. Antonie Van Leeuwenhoek 49:417-427, 1983. PD Morris. DS Campbell, TJ Taylor, JI Freeman. Clinlcal and epidemiological features of neurotoxic shellfish poisoning in North Carolina. Am J Public Health 81:471-474, 1991. Q Yuzao, Z Ziaping, H Ying, L Songhui, Z Congju. Occurrence of red tides on the coasts Sea. New of China. In: TJ Smayda, Y Shimizu, eds. Toxic Phytoplankton Blooms in the York: Elsevier, 1993, pp 43-46. Q Adnan. PSP and red tide status in Indonesia. In: TJ Smayda,Y Shimizu, eds. Toxic Phytoplankton Blooms in the Sea. New York: Elsevier. 1993, pp 199-202. t Gyrfmr/i~liun~ c'crtcJrltrtur?f ALBarbera-Sanchez, S Hall,EFcrraz-Reyes. A / e . ~ r ~ m / r i r wsp.. and PSP in Venezuela. In: TJ Smayda, Y Shimizu. cds. Toxic Phytoplankton Blooms in the Sea. NewYork: Elsevier, 1993. pp 281-285. D Mcdina, G Inocentc. C Lopez. PSP in bivalve molluscs along the Uraguayall co21st. In: TJ Smayda, Y Shimizu, eds. Toxic Phytoplankton Bloonls i n thc Sea. New York: Elsevier, 1993, pp 425-428. Y Fukuyo, K Yoshida, T Ogata, T Ishimaru, M Kodama. P Pholpunthin, S Wisessang, V Phanichyakam, T Piyakarnchana. Suspected causative dinoflagellates of paralytic shellfish poisoning in the Gulf of Thailand. In: T Okaichi. DM Anderson, T Nemoto, eds. Red Tides: Biology, Environnxntal Science and Toxicology. New York: Elsevier, 1989, pp 403-406. GM Hallegraeff, C Sumner. Toxic planktonblooms affect shellfish f a r m . Aust Fish 45(December):15-18,1986. RA Corrales. JL Maclean. Impacts of harmfuld g on~seafarming in the Asia-Pacific areas. J Appl Phytol 7:151-162, 1995. BD Gcssner. P Bell, GJ Doucette, E Moczydlowski, MA Poli, FVan Dolah, S Hall. Hypertcnand identification of toxin in human urine and serum following a cluster of musselassociated paralytic shellfish poisoning outbreaks. Toxicon 35:7 1 1-722, 1997.
Shellfish Chemical Poisoning
101
59. JPK McCollum, RCM Pearson, HR Ingham, PC Wood, HA Dewar. An cpidcmic of mussel poisoning in north-castEngland.Lancet1968:767-770,1968.
60. HL Borison, WJ Culp, SF Gonsalves, LE McCarthy. Central respiratory and circulatory depression caused by intravascular saxitoxin. Br J Pharmacol 68301-309, 1980. 61. FT Chang. RM Bauer,BJBenton, SA Keller, BR Capacio.4-aminopyridineantagonizes saxitoxin-andtetrodotoxin-induccdcardiorespiratorydepression.Toxicon34:671-690. 1996. 62. EJ Schantz, VE Ghazarossian, HK Schnoes, FM Strong,JP Springer, JO Pczzanite, J Clardy. The structurc of saxitoxin. J Am Chem Soc 97:1238-1239, 1975. 63. Y Shimizu, C Hsu, WE Fallon, Y Oshima, I Miura, K Nakanishi, K. Structure of neosaxitoxin. J Am Chcm Soc 100:6791-6793, 1978. 64. RS Rogers, H Rapaport. The pKa's of saxitoxin. J Am Chem SOC102:7335-7339, 1980. 65. G Strichartz. Structural determinants of the affinity of saxitoxin for neuronal sodium channels. Elcctrophysiological studies011frog peripheral nervc. J GenPhysiol84:281-305. 1984. 66. RC Stafford, HB Hines. Urinary elimination of saxitoxin after intravenous injection. Toxicon 33:1501-1510, 1995. 67. T Harada. Y Oshima, T Yasumoto. Structures of two paralytic shellfish toxins, gonyautoxins V andVI,isolatedfrom a tropical dinoflagellate, Pyrodirlium bakamer~sevar. cwnprcwa. Agric Biol Chem 46: 1861- 1864, 1982. 68. T Noguchi, Y Ueda, K Hashimoto, H Seto. Isolation and characterization of gonyautoxin1 from the toxic digestive gland of scallop Pntinopecten yessoensis. Nippon Suisan Gakkaishi 47:1227-1231, 1981. 69. AP Negri, GJ Jones. Bioaccumulation of paralytic shellfish poisoning (PSP) toxins from the cyanohacterium Ambaerlcc rircimlis by the freshwater mussel Ahthyria c m r l o k c . Toxicon 33:667-678.1995. 70. S Gallacher, KJ Flynn, JM Franco, EE Brueggemann, HB Hines. Evidence for production o f paralyticshellfishtoxins bybacteria associatedwith A / ~ . V ~ I ~ spp. I ~ U(Dinophyta) III in culture. Appl Environ Microbiol 63239-245, 1997. 71. ES Silva. Intracellular bacteria: the origin of dinoflagellate toxicity. J Environ Pathol Toxicol Oncol 10: 124- 128, 1990. 72. JB Wcigele, RL Barchi. Analysis of saxitoxin binding in isolated rat synaptosomes using a rapid filtration assay. FEBS Lett 91310-314. 1978. 73. X Guo, AUehara, A Ravindran,SHBryant. S Hall,EMoczydlowski.Kineticbasisfor i n sodium channels of canine heart and denervated insensitlvity to tetrodotoxin and saxitoxin rat skeletal muscle. Biochemistry 26:7546-7556, 1987. of voltage-gatedionchannels.Natunvis74. HTcrlau, W Stuhmer.Structureandfunction senschaftcn85:437-444,1998. 75. SC Cannon, AI McClatchey, JF Gusclla. Modification of the Na' current conducted by the rat skeletal muscle alpha subunit by coexpression with a human brain heta subunit. Pflugers Arch 423: 155- 157, 1993. 76. N Makita, PB Bennett, AL Gcorge. Molecular determinants of beta1 subunit-induced gating modulation in voltage-dependent Na' channels. J Neurosci 16:71 17-7127, 1996. of the 77. JRDyer,WLJohnston. VF Castellucci, RJDunn.Cloningandtissuedistribution Ap!\sia Na' channel alpha-subunit cDNA. DNA Cell Biol 16:347-356, 1997. 78. JD Spafford, AN Spencer, WJ Gallin.A putative voltage-gatcd sodium channel alpha subunit (PpSCNI ) from the hydrozoan jellyfish, Po/yorchi.s penicilllltus: structural comparisons and evolutionary considerations. Biochem Biophys Res Commun 244:772-780, 1998. 79. MC Jeziorski, RM Grcenberg, PA Anderson. Cloning of a putative voltage-gated sodium channel from the turbellarian flatworm Bdellolrru cnndirirr.Parasitology 115:289-296, 1997. 80. J Satin, JT Limbcris, JW Kyle, RB Rogart, HA Fozzard. The saxitoxin/tetrodotoxin binding site on cloned rat brain IIa Na channels is in the transmembrane electric field. Biophys J 67: 1007-1014.1994.
102
Llewellyn
GM Lipkind. HA Fozzard. A structural model of the tetrodotoxin and saxitoxin binding site of the Na' channel. Biophys J 66: 1- 13, 1994. 82. AN Akopian,LSivilotti,JNWood.Atetrodotoxin-resistantvoltage-gatedsodiumchannel expressed by sensory neurons. Nature 379257-262, 1996. 83. LL Cribbs, J Satin, HA Fozzard, RB Rogart. Functional expression of the rat heart I Na' channel isoform. Demonstration of properties characteristic of native cardiac Na channels. FEBS Lett 275: 195-200, 1990. 84. MMWhite,LQ Chen, RKleinfield,RGKallen,RL Barchi. SkM2, a Na' channel cDNA clone from denervated skeletal muscle, encodes a tetrodotoxin-insensitive Na' channel. Mol Pharlnacol39:604-608,1991. 8 5 . RC Kallen,ZHSheng,JYang,LQ Chen, RB Rogart,RLBarchi.Primarystructureand expression of a sodium channel characteristicof denervated and immature rat skeletal muscle. Neuron 4:233-242, 1990. 86. J Satin, JW Kyle, M Chen, P Bell, LL Cribbs, HA Fozzard, RB Rogart. A mutant of TTXresistant cardiac sodium channels with TTX-sensitive properties. Science 256: 1202-1 205, 1992. 87. D Andrinolo, LF Michea, N Lagos. Toxic effects, pharmacokinetics and clearance of saxitoxin, a component of paralylic shellfish poison (PSP), in cats. Toxicon 37:447-464, 1999. 88. HB Hines, SM Naseern, RW Wannemacher. ['H]-saxitoxinol nletabolis~nand elimination in the rat. Toxicon 3 1 :905-908, 1993. 89. AG Gilman, TW Rall, AS Nies, P Taylor. The Pharmacological Basis of Therapeutics, 8th ed. NewYork:PergamonPress,1990. 90. T Harada, Y Oshima, T Yasumoto. Assessment of potential activation of gonyautoxin V in the stomach of mice and rats. Toxicon 22476-478, 1984. 91. FT Chang, BJ Benton, RA Lenz, BR Capacio. Central and peripheral cardio-respiratory effects of saxitoxin (STX) in urethane-anaesthetized guinea-pigs. Toxicon3 1:645-664, 1993. of saxitoxin in the cerebrospi92. HL Borison,LE McCarthy. Respiratory and circulatory effects nal fluid. Br J Pharmacol 61:679-689. 1977. 93. BJ Benton, VR Rivera, JF Hewetson, FCChang. Reversal of saxitoxin-induced cardiorespiratory failure by a burro-raised alpha-STX antibody and oxygen therapy. Toxicol Appl Pharmacol 124139-5 I , 1994. 94. SR Davio. Neutralization of saxitoxin by anti-saxitoxin rabbit serum. Toxicon 23:669-675, 1985. 95. R1 Huot, DL Armstrong, TC Chanh. In vitro and in situ inhibition of the sodium channel blocker saxitoxin by monoclonal antibodies. J Toxicol Environ Health 27:381-393, 1989. 96. B Kaufman, DC Wright, WR Ballou, D Monheit. Protection against tetrodotoxin and saxitoxin intoxication by a cross-protective rabbit anti-tetrodotoxin antiserutn. Toxicon 29:58l 587, 1991. V Endcn,JClardy,YGopichand, F 97. KTachibana,PScheuer.YTsukitani,HKikuchi, Schmitz. Okadaic acid, a cytotoxic polyether from two marine sponges of the genus Hrrlic.hondricr. J Am Chem Soc 103:2469-2471, 1981. 98. M Murata, M Shimitami, H Sugitani, Y Oshima, T Yasumoto. Isolation and structural ehcidation of the causative toxin of the diarrhetic shellfish poisoning. Nippon Suisan Gakkaishi 48:549-552,1982. 99. MA Quilliam, JLC Wright. Methods fordianhetic shellfish poisons. In GM Hallegraeff, DM Anderson,ADCernbella. eds. Manual on HarmfulMarineMicroalgae.Paris:UNESCO, 1995, pp 95-1 1 I . on experimental marine 100. K Terao, E 110, T Yanagi, T Yasumoto. Histopathological studies toxin poisoning. I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-l and pectenotoxin-l. Toxicon 24: I141 - I 151, 1986. of yessotoxin. Nat Toxins 5: 101. H Ogino, M Kumagai, T Yasumoto. Toxicologic evaluation 255-259,1997.
81.
Shellfish
Poisoning
103
102. K Terao, E110, M Oarad:\, M Murata. T Yasumoto. Histopathological studies on experimental marine toxin poisonit1g-5. The effects i n mice of yessotoxin isolated from Ptrtitl~)/~e~Tetl yes,soc~t~.si.sand of a dcsulfated derivative. Toxicon 28: 1095-1 104, 1990. 103. T Y:lsumoto. y Oshima, W Sugawara, Y Fukuyo, H Oguri, T Igarishi, N Fujita. Identification
104. 105.
106.
107. 108.
109.
1 IO.
I II. 112.
113.
114. 115.
116. 117.
118.
119.
120
121
122.
of I);no/,lly.s;.s,fi,r.tii as the causative organismof diarrhetic shellfish poisoning. Nippon Suisan Gakkaishi 46: 1405- 13 1 1, 1980. y Murakatni. Y Oshima, T Yasulnoto. Idcntificatlon of okadaic acid as a toxic component of :l marine dinoflagellate P r o r ~ ~ t w ~ t rlinrtr. t m Nippon Suisan Gakkaishi 48:69-72, 1982. T Hu, J Marr, ASW Defreitas, MA Quilliam, JA Walter, JLC Wright. S Pleasance. New diol esters (of okadaic acid) isolated from cultures of the dinoflagellates froroccnfrum h r r r and frorocerltrlrrtr cotwnww. J Nat Prod 55: 1631 - 1637, 1992. JS Lee, T Igarashi, S Fraga, E Dahl, P Hovgaard, T Yasumoto. Determination of diarrhetic shellfish toxins in various dinoflagellate species. J Appl Phycol 1: 147-152, 1989. A Tnkai, M Murata. K Torigoe, M Isobe, G Mieskes, T Yasumoto. Inhibitory effect of okadaic acid derivatives on protein phosphatases. Biochetn J 284:539-544, 1992. YM Li, JE Casida. Cantharidin-binding protein: identification as protein phosphatase 2A. Proc Natl Acad Sci USA 89: 1 1867- I 1870, 1992. TA Haystead, AT Sim, D Carling, RC Honnor, Y Tsukitani, P Cohen, DG Hardie. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature337:78-81. 1989. J Goldberg, HB Huang. YG Kwon, P Grcengard. AC Nairn, J Kuriyan. Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-I . Nature 376:745753.1995. P Cohen. The structure and regulation of protein phosphatases. Ann Rev Biochetn 58:453508.1989. CW Laidley. E Cohen, JE Casida. Protein phosphatase in neuroblastoma cells: ['Hlcantharidin binding site in relation to cytotoxicity. J Phartndcol Exp Ther 280:1152-1158, 1997. C Bialojan. A Takai. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J 256:283-290, 1988. A Takai. G Mieskes. Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phos1991. phatase activity of protein phosphatases. Biochem J 275:233-239, Z Zhang, S Zhao, F Long, L Zhang. G Bai,H S h i m , M Nagao, EYC Lee. A mutant of protein phosphatase-l that exhibits altered toxin sensitivity. J Biol Chem 269:16997- 17000, 1994. CFB Holmes, HA Luu, F Carrier. FJ Schmitz. Inhibition of protein phosphatases- 1 and-2A with acanthifolicin. Comparison with diarrhetic shellfish toxins and identification of a region on okadaic acid important for phosphatase inhibition. FEBS Lett 270:216-218. 1990. S Nishiwaki.HFujiki,MSuganuma,HFuruya-Suguri, R Matsushima,Ylida,MOjika, Yamada. D Uemura, T Yasumoto. Structure-activity relationship within a series of okadaic acid derivatives. Carcinogenesis 1 1 : 1837- 1841, 1990. K Sasaki, M Murata, T Yasumoto, G Mieskes, A Takai. Affinity of okadaic acid to type-l and type-2A protein phosphatases is nlarkedly reduced by oxidation of its 27-hydroxyl group. Biochem J 298259-262, 1994. V Fessard, Y Grosse. A Pfohl-Leszkowicz, S Puiseux-Dao. Okadaic acid treatment induces DNA adduct formation in BHK2 1 C 13 fibroblasts and HESV keratinocytes. Mutat Res 361 : 133-141.1996. WG Matias, M Bonini, EE Creppy. Inhihition of protein synthesis in a cell-free system and vero cells by okadaic acid. a diarrhetic shellfish toxin. J Toxicol Environ Health 48:309317, 1996. a potent MT Fernandez, V Zitko, S Gascon, A Novelli. The marine toxin okadaic acid is neurotoxin for cultured cerebellar neurons. Life Sci 49:PL157-PL162, 1991. T Aune, T Yasumoto, E Engeland. Light and scanning electron microscopic studies on effects
104
Llewellyn
ofmarinealgaltoxinstowardfreshlyprepared hepatocytes. J Toxicol Environ Health 34: 1-9,1991. 123. E Ito, K Tcrao. Injury and recovery process of intestine caused by okadaic acid and related compounds. Nat Toxins 2 3 7 1-377, 1994. 124. L Edebo, S Lange, XP Li, S Allenmark. Toxic mussels and okadaic acid induce rapid hypersecretion in the rat small intestine. APMIS 96:1029-1035, 1988. 125. M Suganuma, H Fujiki, H Suguri, S Yoshizawa, M Hirota, M Nakayasu, M Ojika, K Wakamatsu, K Yamada, T Sugimura. Okadaic acid: an additional non-phorbol-l2-tetradecanoate13-acetate-type tumour promoter. Proc Natl Acad Sci USA 85: 1768-1771, 1988. 126. H Fujiki, M Suganuma, H Suguri, S Yoshizawa, K Takagi, N Uda, K Wakamatsu,K Yamada, M Murata, T Yasumoto, T Sugimura. Diarrhetic shellfish toxin, dinophysistoxin-I , is a potcnt tumor promoter on mouse skin. Jpn J Cancer Res 79: 1089-1093, 1988. 127. WC Matias, EE Creppy. Transplacental passage of [‘HI-okadaic acid in pregnant mice measured by radioactivity and high-performance liquid chromatography. Hum Exp Toxicol 15: 226-230,1996. 128.JTripuraneni,AKoutsouris,LPestic,PDeLanerolle,GHecht.Thetoxinofdiarrhetic shcllfishpoisoning,okadaicacid,increasesintestinalepithelialparacellularpermeability. Gastroenterology 1 12:100- 1 IO. 1997. 129. SMRay,DVAldrich. Gyrnnodiriiunr breve: inductionofshellfishpoisoninginchicks. Science148:1748-17.50,1965. 130. CB Templeton, MA Poli. R Solow. Prophylactic and therapeutic use ofan anti-brevctoxin (PbTx-2) antibody in conscious rats. Toxicon 27: 1389-1395, 1989. 13 1. DC Baden, TJ Mende. Toxicity of two toxins from the Florida red tide marine dinoflagellate, Phckodiscus brevis. Toxicon 20:457-461, 1982. 132.REGawley,KSRein,MKinoshita, DC Baden.Binding of brevetoxinsandciguatoxinto the voltage-sensitive sodium channel and conformational analysis of brcvetoxin B. Toxicon 301780-78.5,1992. 133. Y Lin, M Risk, SM Ray, D van Engen, J Clardy, J Golik, JC James, K Nakanishi. Isolation and structure of brevetoxin B from “red tide” dinoflagellate Phchodiscus hrevis (Gyrnrtodinitmr breve). J Am Chem Soc 103:6773-6776, 1981. 134.KNakanishi.Thechcmistryofbrevetoxins: a review.Toxicon23:473-479,1985. 135. DC Baden.Brevetoxins:uniquepolyetherdinoflagellatetoxins.FASEB J 3:1807-1817, 1989. 136. MA Poli, TJ Mende, DC Baden. Brevetoxins, unique activators of voltage-sensitive sodium channels,bindtospecificsitesinratbrainsynaptosomes.MolPharmacol30:129-135, 1986. 137. VL Trainer, DC Baden, WA Catterall. Identificationof peptide components of the brevetoxin receptor site of rat brain sodium channels. J Biol Chem 269: 19904-19909, 1994. 138. VL Trainer, WJ Thomsen, WA Catterall, DC Baden. Photoaffinity labelling of the brevetoxin receptor on sodium channels in rat brain synaptosomes. Mol Pharmacol 40:988-994, 1991. 139. RA Edwards,VLTrainer, DC Baden.Brevetoxinsbindtomultipleclassesofsitesinrat brain synaptosomes. Brain Res Mol Brain Res 14:64-70. 1992. 140. VL Trainer, E Moreau, D Guedin, DC Baden, WA Catterall. Neurotoxin binding and allosteric modulation at receptor sites 2 and 5 on purified and reconstituted rat brain sodium channels. J Biol Chem 268:17114-17119, 1993. 141. G Jeglitsch, K Rein, DC Baden, DJ Adams. Brevetoxin-3 (PbTx-3) and its derivatives modulate single tetrodotoxin-scnsitive sodium channels i n rat sensory neurons. J Pharmacol Exp Ther 284516-525, 1998. 142. WD Atchison, VS Luke, T Narahashi, SM Vogel. Nerve membrane sodium channels as the target site of brevetoxins at neuromuscular junctions. Br J Pharmacol 189:731-738, 1986. 143. JM Huang, CH Wu, DC Baden. Depolarizing action of a red-tide dinoflagellate brevetoxin on axonal membranes. J Pharmacol Exp Ther 229:615-621, 1984.
Shellfish Chemical Poisoning
105
147. PL Whitney, J A Delgado, DG Baden. Complex behavior of marine animal tissue extracts ill thc competitive binding assay of brevctoxins with rat brain synaptosomes. Nat Toxins 5 : 193-200, 1997. 148. JP Gallagher, P Shinnick-Gallagher. Effectsof crude brevetoxin on membranc potential and spontaneous or evoked end-platcpotentials in rathemidiaphragm.Toxicon23:489-496, 1985. Rein, DGBaden,PJWalsh, DE Hinton, K Tullis,MSDenison.Bre149. BSW&burll,KS vetoxin-6 (PbTx-6). a nonaromatic marine neurotoxin, is a ligand of the aryl hydrocarbon receptor. Arch Biochem Biophys 343: 149- 156, 1997. 150. M Cattet, JR Geraci. Distribution and elimination of ingested brevetoxin (PbTx-3) in rats. Toxicon31:1483-1486,1993. 151. MA poli, CB Templeton. WL Thompson, JF Hewetson. Distribution and elimination of brevetoxin PbTx-3 in rats. Toxicon 28:903-910, 1990. (PbTx-3) inhibits oxygen consump152. FA Rodriguez, N Escobales, C Maldonado. Brevetoxin-3 tion and increasesNa' content in mouse liver slices througha tetrodotoxin-sensitive pathway. Toxicon 32:1385- 1395, 1994. 153. A Lombet, JN Bidard, M Lazdunski. Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltage-dependent Na' channel. FEBS Lett 219:355-359, 1987. of 154. YG Peng, TB Taylor, RE Finch,PDMoeller.JSRamsdell.Neuroexcitatoryactions ciguatoxinonbrainregionsassociatedwiththermoregulation.Neuroreport6:305-309. 1995. 155. GL Johnson, JJ Spikes, S Ellis. Cardiovascular effects of brevetoxins i n dogs. Toxicon 23: 505-515. 1985. 156. HL Borison, LE McCarthy, S Ellis. Neurological analysis of rcspiratory, cardiovascular and neuromuscular effects of brcvetoxin in cats. Toxicon 23517-524, 1985. 157. CB Templeton, MA Poli, RD LcClaire. Cardiorespiratory effects of brevetoxin (PbTx-2) in conscious, tethered rats. Toxicon 27: 1043- 1049, 1989. 158. T Yuhi, A Wada, R Yamamoto, M Urabc, H Niina, F Izumi, T Yanagita. Characterization of ['Hlbrevetoxin binding to voltage-dependent sodium channels in adrenal medullary cells. ArchPharmacol 350:209-212, 1994. 159. TM Perl, L Bedard, T Kosatsky, JC Hockin, ECD Todd, RS Remis. An outbreak of toxic encephalopathy caused by eating mussels contaminated with domoic acid. Eng J Med 322: 1775- 1780, 1990. 160. E Preston, I Hynie. Transfer constants for blood-brain barrier permeation of the ncuroexcitatory shellfish toxin, domoic acid. Can J Neurol Sci 18:39-44, 1991. 161. MJ Teitelbaum, R Zatorre, S Carpenter, D Gendron, AC Evans, A Gjedde, NR Cashman. Neurologic scquclac of domoic acid intoxication due to the ingestion of contaminated musscls. N Eng J Med 322:1781-1787, 1990. 162. JLC Wright, M Falk, AG McInnes, JA Walter. Identification of isodomoic acid D and two new geometrical isomers of domoic acid in toxic mussels. Can J Chem 68:22-25, 1990. 163. TTakemoto, K Daigo.Constituents of C/wtrrlrirr nrrtrnta. Chem PharmBull 6578-580, 1958. 164. M Macda, T Kodama,T Tanaka, H Yoshizumi, T Takemoto,K Nomoto. T Fujita. Structures of isodomoic acids A, B and C, novel insecticidal amino acids from the red alga Chondrin nr'nrrrfn.Chem Pharm Bull 34:4892-4895, 1986.
106
L le wellyn
165.LZaman, 0 Arakawa,A Shimosu, Y Onoue, S Nishio, Y Shida, TNoguchi. Two new isomers of domoic acid from a red alga, Clzmdrier arrncctcc. Toxicon 35:205-212, 1997. 166. SS Bates,JCBird, ASWDeFreitas,RFoxall,MGilgan, LA Hanic,GRJohnson, AW McCulloch, P Odense, R Pocklington, MA Quilliam, PG Sim, JC Smith. DV Subba Rao, Nir:.sc/ricr purlgerls as the primary source ECD Todd, JA Walter, JLC Wright. Pennate diatom of domoic acid, a toxin in shellfish from eastern Prince Edward Island, Canada. Can J Fish Aqaut Sci 46:1203-1221, 1989. 167. DV Subba-Rao, MA Quilliam, R Pocklington. Dornoic acid-a neurotoxic amino acid produced by the marine diatom Nit3chirr p~cngc’nsin culture. Can J Fish Aquat Sci 45:20762077.1988. 168. DL Garrison, SM Conrad, PP Eilers, EM Waldron. Confirmation of domoic acid production by Pseudonitz.shia ctusfr.cr/is (Bacillariophyceae) cultures. J Phycol 28:604-607, 1992. 169.MCVillac, DL Roelke, FP Chavez, LA Cifuentes. CA Fryxell. P.srrr~lor~ir~sc~hin trrrstrcrlis Frenguelli and related species from the west coast of the USA: occurrence and domoic acid production. J Shellfish Res 12:457-465, 1993. Nitzschicr psercr/oc/e~/ic~rrtissir,,N-asource of 170. JL Martin, K Haya, LE Burridge, DJ Wildish. domoic acid in the Bay of Fundy, eastern Canada. Mar Ecol Prog Scr 67:177-182, 1990. 171. L Fritz, MA Quilliam, JLC Wright. An outbreak of domoic acid poisoning attributed to the uustr.cr1i.s. J Phycol 28:439-442, 1992. pennate diatom P.seLrd~)~lir~.s~/liu 172.FIverson.JTruelove,ENera,LTryphonas,JCampbell,ELok.Domoicacidpoisoning and mussel-associated intoxication: preliminary investigations into the response of mice and rats to toxic mussel extract. Food Chem Toxicol 27:377-384. 1989. 173. M Falk, PF Seto, JA Walter. Solubility of domoic acid in water and in non-aqueous solvents. Can J Chem 69: 1740- 1744, 1991. of domoicacid.Can J Chem67: 174.MFalk,JAWalter,PWWiseman.Ultravioletspectrum 1421-1425,1989. 175. MS Nijjar, MS Madhyastha. Effect of pH on domoic acid toxicity in mice. Mol Cell Biochem 167:179-185,1997. 176.TJBiscoe,RHEvans, PM Headley,MMartin.JCWatkins.Domoicandquisqualicacids 255: 166-167, aspotentaminoacidexcitantsoffrogandratspinalneurones.Nature 1975. 177. EK Michaelis. Molecular biology of glutamate receptors inthe central nervous systeln and their role in excitotoxicity, oxidative stress and aging. Prog Neurobiol 54:369-415. 1998. 178. S Ozawa, H Kamiya, K Tsuzunki.Glutamatereceptorsinthemammalianccntralnervous sytem. Prog Neurobiol 54581-618, 1998. 179. JA Larm, PM Beart, NS Cheung. Neurotoxin domoic acid produces cytotoxicity via kainateand AMPA-sensitive receptors in cultured cortical neurones. NeurochemIn1 3 1 :677-682, 1997. 180. WWisden,PH Seeburg. Mammalian ionotropic glutamate receptors. Curr Opin Neurobiol 3:291-298,1993. 18 1. S Nakanishi. Molecular diversity of glutamate receptors and impkations for brain function. Science258:597-603,1992. 182. RJ Vandenberg, JL Arriza, SG Amwa, MP Kavanaugh. Constitutive ion fuxes and subslrate 1. 1995. binding domains of human glutamate transporters. J Biol Chem 270:17668-1777 183. J Truelove, F Ivcrson. Serum domoic acid clearance and clinical observations in the CynOmOlgus monkey and Sprague-Dawley rat following a single i.v. dose. Bull Environ Contaln Taxicol 52:479-486, 1994. 184. JE Bruni,RBose,CPinsky.GBGlavin.Circumventricularorgalloriginofdomoicacidinduced neuropathology and toxicology. Brain Res Bull 26:419-424, 1091. 185. FW Berman, TF Murray. Domoic acid neurotoxicity in cultured cerebellar granule neurons is mediated predominantly by NMDA receptors that are activated as a consequence of exitatory amino acid release. J Neurochem 69:693-703, 1997.
Shellfish
Poisoning
107
~ ,Hierlihy. Renal clearance of domoic acid in the rat. Food Chcm Toxicol 10: 186. CA S U Z USL 701-706. 1993. 187. L Tryp[lollas, F [verson. Neuropkithologyof excitatory neurotoxins: the domoic acid model. Toxicol Pathol 18: 165- 169. 1990. 188. L Tryphonas. J Truelove, F Iverson. Acute parenteral neurotoxicity of domoic acid i n c p o 1990. lnolgus monkeys. Toxicol Pathol 18:297-303. to excitatory amino acidsand excite189. CD Zcevalk, WJ Nicklas. Nitric oxide in retina: relation toxicity. Exp Eye Res 58:343-350, 1994. 190. R Base, C Pinsky, GB Glavin.Sensitive murine model and putative antidotes for behavioural toxicosis from contaminated mussel extracts. Can Dis Weekly Rep 16:91-98. 1990. 191. S NakajiIna, JL Potvin. Neural and behavioural effects of domoic acid, an amnesic shellfish toxin, i n the rat. Can J Psycho1 46:569-581, 1992. 192. C Pinsky, GB Glavin, R Bosc. Kynurenic acid protects against neurotoxicity and lethality of toxicextractsfromcontaminatedAtlanticcoastmussels.ProgNeuropsychophannacol BiolPsychiatry 13595-598, 1989. 193. GB Glavin, R Base, C Pinsky. Kynurenic acid protects against gastroduodenal ulcerationi n mice injected with extracts from poisonous Atlantic shellfish. Prog Neut.opsychopharmaco1 BiolPsychiatry 13569-572, 1989. 194. SK Shartna, K Dakshinamurti.Suppressionofdomoicacidinducedseizuresby%(OH)DPAT. J Neural Transm 93:87-98, 1993. 195. DP Boles, AA Tuinman, PL Wessels, CC Viljoen, H Kruger, DH Williams, S Santikarn, RJ Smith, SJ Hammond. The structure of cyanoginosin-LA, a cyclic heptapeptide toxin from the cyanobacterium Micrmysris nerugirtosa. J Chcm Soc Perkin Trans 1:231 1-2318, 1984. S Santikarn, RJ Smith. JCJ Bru-na, DH 196. DP Botes, PL Wesscls, H Kruger, MTC Runnegar. Williams. Structural studies on cyanoginosins-LR,-YR, -YA, and -YM, peptide toxins from cyanobacterium M i c w c w i s ~ e r t c g i ? ~J .Chem ~ ~ . Soc Perkin Trans 1 :2747-2748, 1985. 197. WW Carmichael. Thc toxins of cyanobacteria. Sci Am 270:78-86, 1994. 198. C MacKintosh, KA Bcattie, S Klumpp, P Cohen, CA Codd. Cyanobacterial rnicrocystirl-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both manmnls and higher plants. FEBS Lett 264: 187-192, 1990. 199. R Nishiwaki-Mntsushima, S Nishiwaki, T Ohta, S Yoshizawn, M Suganuma, K Harada, MF Watanabe, H Fujiki. Structure-function relationships of microcystins, liver tumor promoters. in interaction with protein phosphatase. Jpn J Cancer Res 82:993-996, 1991. 200. DE Williams, SC Dawe. ML Kent, RJ Andersen, M Craig, CFB Holmes. Bioaccumulation and clearance of microcystins from salt water mussels, Mytilus ed~tlis,and in vivo evidence for covalently bound microcystins in mussel tissues. Toxicon 35: 1617-1625, 1997. RJ Andersen, CF Holmes. Identification 201. DZX Chen, MP Boland, MA Smillie, H Klix, C Ptak, of protein phosphatase inhibitorsofthe microcystin class in the marine environment. Toxicon 31:1407-1414, 1993. 202. K Tsuda, S Ikutna, M Kawamura, R Tachikawa, K Sakai. Tetrodotoxin. VII. On the structure of tetrodotoxin and its derivatives. Chem Pharrn Bull 12: 1357-1 374, 1964. 203 T Goto, Y Kishi, S Takahashi. Y Hiratn. Tetrodotoxin. Tetrahedron 21:2059-2088, 1965. 204 RB Woodward. The structure of tetrodotoxin. Pure Appl Chem 9:49-74, 1964. 205. C Yang, K Han, T Lin, W Tsai, J Dcng. An outbreak of tetrodotoxin poisoning following gastropod mollusc consumption. Hum Exp Toxicol 14446-450, 1995. 206. M Kodama,S Sato, S Sakamoto, T Ogata. Occurrenceof tetrodotoxin in Alesnndrilrnr fctr)tNrerrse, a causativedinoflagellateofparalyticshellfishpoisoning.Toxicon34:1101-1105, 1996. 207. GD Wohlgeschaffen, KH Mann, DV Subba Rao, R Pocklington. Dynamics of the phycotoxin dolnoic acid: accumulation and excretion i n two comnercially important bivalves, J Appl Phycol 4297-310, 1992. 208. H Ishida, N Muralnatsu, T Kosuge, K Tsuji. Studyof neurotoxic shellfish poisoning involving
108
209.
210. 21 1.
212.
213. 214. 215. 216. 217. 218.
Llewellyn
New Zealand shellfish,CrcrssosrrecI gigrts. In: T Yasumoto, Y Oshima,Y Fukuyo, eds. Harmful and Toxic Algal Blooms. Paris: UNESCO, 1996, pp 491-494. J Blanco, A Morono, J Franco, MI Reyero. PSP detoxification kinetics in the mussel Mytilus gefllul”nvirlcicr1is. One- and two-compartment models and the effect of some environmental variables. Mar Ecol Prog Ser 158:165-175, 1997. W Silvert, DV Subba-Rao. Dynamic model of the flux of domoicacid, a neurotoxin, through a Mxtilus edulis population. Can J Fish Aquat Sci 49:400-405. 1992. W Silvert, AD Cembella. Dynamic modelling of phycotoxin kinetics in the blue mussel, Mytilus rdulis, with implications for other marine invertebrates. Can J Fish Aquat Sci 52: 521-531,1995. DV Subba Rao, ASW De Freitas, MA Quilliam, R Pocklington, SS Bates. Rates of production of domoic acid, a neurotoxin amino acid in the pennate marine diatom Nir:.schier purtgerls. In: E Graneli, B Sundstrom.L Edler, DM Anderson, eds. Toxic Marine Phytoplankton. New York: Elsevier, 1990, pp 413-417. LE Trudeau, VF Castellucci. Excitatory amino acid neurotransmission at sensory-motor and interneuronal synapses of AplysicI ccrlifornica. J Neurophysiol 70: 1221- 1230, 1993. L Zhang, Z Zhang, F Long, EYC Lee. Tyrosine-272 is involved in the inhibition of protein 1, 1996. phosphatase-l by multiple toxins. Biochemistry 35: 1606-161 in YShimizu,MYoshioka.Transformationofparalyticshellfishtoxinsasdemonstrated scallop homogenates. Science 21 2547-549, 1981. JJ Sullivan, WT Iwaoka, J Liston. Enzymatic transformation of PSP toxins in the littleneck Biochem Biophys Res Commun 114:465-472, 1983. clam (Prorothncrr srcu~rir~ecr). Y Hashimoto. Marine toxins and other bioactive marine metabolites. Tokyo: Japan Scientific Societies Press, 1979, pp 53-55. JF Lawrence, M Maher, W Watson-Wright. Effectof cooking on the concentrationof toxins associated with paralytic shellfish poisonin lobster hepatopancreas. Toxicon 32:57-64, 1994.
5 Pathogens Transmitted by Seafood"
I. Introduction 109 11. BacterialandViralPathogensAssociatedwithRawandUnderprocessedShellfish
121
Bacterial and Viral Pathogens Primarily Associated with Improper Processing or Handling of Seafood 159 of Seafood-BornePathogens 164 IV. Development ofRapidMethodsforDetcction 165 V. ConclusionsandRecommendations VI. Additional Sources of Information: World Wide Web Sites for Pathogens Associated with Seafoods 17 1 References 172 111.
1. A.
INTRODUCTION Overview of Seafood-Borne Disease
Risks and hazards are associated with all forms of human activities. While humans may choose to minimize or eliminate some risks by changing or eliminating a certain type of behavior or activity, all humans must consume food and water to live. In some regions of the world, the consumptionof food and watercan be, unfortunately, routinely associated with exposure to pathogenic organisms. Inhabitants of developed countries generally assume that their supply of food and water is safe, and government regulatory and public health agencies are charged with the task of ensuring the safety of these items. In many regions, however, infectious disease is a wayof life, responsible for the deathsof millions of people each year in developing countries. Part of this infectious disease is associated with the transmission of pathogenic microorganisms in the food and water that is consumed. The World Health Organization( I ) reported that in 1997, of a global total of 52.2 million deaths, 17.3 million or one-third were due to infectious and parasitic diseases.
* This chapter is dedicated to the educational and scientific contributions of John Liston and Jack R. Matches, two former faculty members and microbiologists in the School of Fisheries, University of Washington. 109
110
Hetwig Other and unknown causes 9%
Infectious and parasitic diseases 43%
9%
Perinatal and maternal causes 10%
Fig. 1 Major causes of death in developing countries (1).
The leading causes of death from infectious diseases were acute lower respiratory infections (3.7 million), tuberculosis (2.9 million), diarrhea (2.5 million), H I V / A T D S (2.3 million), and malaria (1.5-2.7 million). Diarrheal and parasitic diseases may be associated with the consumption of contaminated water and food. Major differences in the causes of human death exist between developing and developed countries (Figs. 1 and 2). Infectious and parasitic diseases cause43% of the deaths in developing countries and approximately 1% of the deaths in developed countries. An excellent choice of food for protein and other essential nutrients is seafood. In many regions of the world seafood and other aquatic products are a major portion of the diet, while in other regions of the world seafood is viewed as a delicacy or something
Diseases of the circulatory system 46% Fig. 2 Major causes of deathin developed countries (1).
Pathogens Transmitted by Seafood
111
unusual. Besides supporting the healthy livesof millions of people, seafood is also capable of pathogenic of supporting the growth of or acting as a vehicle for the transmission organisms. The principalbiologicalagentsthat cause seafood-borne disease are bacteria, viruses, and parasites. The major human diseases caused by fish-borne parasites are trematodiasis, cestodiasis, and nematodiasis. Worldwide, but primarily in developing countries, trematodes are the most important food safety hazard linked to fish and fishery products. The trematodes infecting the greatest number of people are species of the genera Clonorchis, Opisthorchis, and Parvrgonirnus (2). The World Health Organization (2) estimates that tens of millions of people are affectedby fish-borne trematodes. Most of these people live in developing countries. Parasites area minor problem for seafood consumersin most developed countries. The focus of this chapter is on the bacteria and viruses associated with aquatic foods that are pathogenic to humans, although the reader should understand that parasitic infections are themajor problem associated with the consumption of seafood in many regions of the world. Bivalve mollusks present a much greater risk of infecting humans with bacterial and viral pathogens than do crustaceansandfinfish.Thegreatestnumberofseafoodassociated disease outbreaks and cases in developed countries are causedby the consumption of raw or insufficiently cooked bivalve mollusks that were harvested from waters contaminated with human sewage or waters wherea fraction of the indigenous microflora are pathogenic to humans. These two types of contamination are associated with enteric bacteria and viruses (Escherichiu coli, Salmonella, hepatitis A, Norwalk-like viruses) and bacteria naturally found in surface water and sediments (Vibrio vuln$cu.s, V. pcrrclhaetnolyticu.7, V. cholerae, Aercmo11c1.s hydrophila, Clostridiutn Dotdinurn). Another group of pathogenic microorganisms (suchas Stclphvlococcus aureus) are primarily introduced into fishery products by humans who handle or process seafoods after harvesting.
B. Consumption of Seafood and the Rise in Aquaculture 1. Increasing Consumption of Seafood In recent years the supply of fish has continued to increase steadily and in 1995 the total world production of finfish, crustaceans, and mollusks from capture fisheries and aquaculture reached 1 12.9 million metric tons. Much of the increase in annual aquatic production is attributable to aquaculture. For cultured finfish and shellfish, the annual contribution to total finfish and shellfish rose from 11.7% in 1989 to 18.5% i n 1995. For food fish, more than one-quarter of the total world supply was derived from aquaculture. Aquaculture is one of the fastest growing food-producing sectors, providing an acceptable supplement and substitute for wild fish and plants. The relative importance and role of aquaculture in different countries varies widely. I n 1995, for example, more than 60% of the total aquatic production in China came from aquaculture. This was nearly twice that seen in France, India, Republic of Korea, and the Philippines. A considerably lower contribution was reported in Thailand (13%), Norway (9%). and the United States (7%) (3).
2. The Safety of Aquaculture Products There are increasing concerns related to the safetyof food products from aquaculture and studies are now focusing on these concerns. Are there greater hazards associated with aquaculture products versus wild-caught products? Are different pathogens associated with
Herwig
112
Table 1 Prevalence of Snlrrrorlrllrr i n AquaculturalSpecies,’
Country Product Tilapia Catfish Catfish Eel
Prawns
Reference % Positive Africa South U.S.
us. Japan Philippines
55 3 21 26
187 188 1x9 190
16
191
aquacultural products? The greatest amountof aquaculture is done i n developing countries i n tropical and subtropical regions of the world, yet most of the scientific literature and research related to seafood safety has been performed i n “Western” countries with seafood captured or raised in temperate waters. Historically, cultured fish have not been considered important vectors of human pathogens. This situation may be changing, partly due to increasing animal densities as a consequence of a rapidly growing industry and partly due to increasing awareness by health care providers of pathogens in aquatic species that may result in human illness (4). Not surprisingly, bacteria that are known to be pathogenic to humaw have been isolated from locations where aquaculture products are produced and from products that have been sent to the marketplace. Table 1 lists the results from several studiesas summarized by D’ Aoust (5) that indicate the prevalence of Srrlnlowello i n different aquacultural species, including tilapia, catfish, eel, and prawns. A more recent review (6) summarized the association of pathogenic bacteria with cultured fish (Table 2). Although evidence has been published showing the presence of a variety of pathogenic organisms in culturedfishandshellfish,whetherthere is a proportionately
Striped bass hybrid
Pathogens Transmitted by Seafood
113
larger number of cases or outbreaks of seafood-borne disease associated with cultured animals has not been established. More research is required in this emerging sector of seafood production.
C. Cases and Outbreaks: Statistics and Government Agencies to Oversee Seafood Safety 1. United States-Developingan Interest in Shellfish Sanitation and the Food Safety Initiative Many i n the public may perceive food safety issues as something completely new, but several federal, state, and local agencies have been concerned with these issues for many years. Regarding seafood safety, there is a lengthy history of government involvement i n the safety of shellfish. Most of the pathogenic bacteria and viruses associated with seafood-borne disease are associated with the consumption of bivalve mollusks. The early history of shellfish-borne disease and the establishment of shellfish sanitation regulations in the United States is summarized i n the manual of operations for the National Shellfish Sanitation Program (7). While the exact cause or microbiology of disease was not known at the time, public health controls on shellfish became a national concern in the United States in the late 19th and early 20th centuries. At that time, public health officials noted a large number of illnesses associated with the consumption of raw oysters, clams, and mussels. Shellfish-associated disease outbreaks were also recorded in Europe. During the winter of 1924-1925, widespread outbreaks of typhoid fever occurred in New York, Chicago, and Washington, D.C. These outbreaks were traced to oysters that had been contaminated with sewage. Local and state public health officials and the shellfish industry became so alarmed that they requested that the surgeon general of the United States Public Health Service develop the controls necessary to ensure a safe supply of shellfish. The surgeon general called a conference in 1925 of representatives from state and municipal health authorities, state conservation commissions, the Bureau of Chemistry [later to become the Food and Drug Administration (FDA)], the Bureau of Commercial Fisheries [now called the National Marine Fisheries Service (NMFS)], and the shellfish industry. TheNationalShellfishSanitationProgram(NSSP)developedfromtheprinciplesand shellfish controls formulated at this conference. To strengthen the mission of the NSSP. in 1982officialsfrom22statesformedtheInterstateShellfishSanitationConference (ISSC). The ISSC allows state regulatory officials to establish uniform guidelines and to exchange information about the sources of safe shellfish. The ISSC adopted the NSSP manual of operations and has established procedures that enable it to adopt changes i n the manual (7). The Centers for Disease Control and Prevention (CDC) is largely responsible for collecting and tabulating data related to prevention and control of disease, injury, and disability in the United States. Alerts and information about current foodborne disease outbreaks and cases are disseminated in a publicationcalled Mnrbidify and Morralip Weekly. The most recent publications that summarize the numbers and causes for foodborne and seafood-related disease outbreaks are the “Summary of Notifiable Diseases, United States 1997” (8) and “Surveillance for Foodborne-Disease Outbreaks-United States, 1988-1992” (9). Data related to shellfish-borne disease was previously collected by the FDA’s Northeast Technical Services Unit (NETSU). The NETSU data were used
114
HeMg
and cited in previous reviews (10,ll). While the NETSU considers reports of outbreaks (two or more persons who become ill after consumption of a common food) and individu cases, the CDC database usually includes only outbreaks. In addition, the CDC data are collected exclusively through the voluntary submission of outbreak forms from state public health departments. The NETSU data are considered more inclusive and precise than the CDC data for shellfish-borne disease (10). In 1994, Rippey(1 1) published a review that summarized the infectious diseases associated with molluscan shellfish consumption. The data presented were related to reports presented through 1990, and as thorough as he could be, Rippey admitted that his data represented only a small portion of the actual number of cases that occur annually because of the weakness of the reporting system and the nature of most foodborne disease. Unfortunately, data collection related to shellfish-born disease is no longer being performed by NETSU, and no other agency in the United States has formally begun to collect this data in a similar manner. The result of these weaknesses is that the data for the number of cases of seafood-borne illness in the United States are most likely poorer and less representative than that collected 10 years ago. With these caveats in mind, data reported by the CDC suggest that among the dif ent kinds of food that are tabulated, there are a significant number of foodborne-disease outbreaks (25%) and a smaller fractionof cases that are associated with seafood (Figs.3 and 4). For the purposes of this review, seafood is considered “shellfish” and “other fish” in the categories that are listed in the CDC data. The data suggest that seafood has fewer cases associated with each outbreak compared to other typesof foods. From 1988 to 1992, 6095 foodborne disease outbreaks representing 77,373 cases were reported to the CDC. Seventy-five deaths were reported to be caused by foodborne disease (9). The outbreaks reported include only a fraction of the cases of foodborne disease that occur each year. For the average of 15,500 cases and 14 deaths reported each year by the CDC’s surveillance system, there are an estimated 6 million cases that actually occur each year (12). The etiology was not determined for 59% of the reported outbreaks. Most of the outbreaks that had an unknown etiology had an incubation period of 15 hours or more, suggesting an infectious agent. Manyof these outbreaks may have been caused by viruses (9). The capability of testing serum for antibodies to foodborne viruses is not widely available.
6,095 outblreah
Fig. 3 Distribution of foodborne disease outbreaks in the United States, 1988-1992 (9).
115
Pathogens Transmitted by Seafood
Shellfish
Other Fish
1Yo
2%
Other Foods 97%
Fig. 4 Distribution of foodborne disease cases in the United
States, 1988-1992 (9).
Shellfish-borne and “otherfish” diseases represented 1% and 2%of the total number of cases, but 2% and 23% of the outbreaks, respectively. In general, seafood-borne disease outbreaks are characterized by a small number of cases. On the other hand, an “outbreak” represented by only a single case is not reported in the CDC data. The responsible pathogen is not identified in more than half of the foodborne disease outbreaks that are reported to the CDC. During this 5-year period there were no cases associated with “other fish” that were attributable to Vibrio cholerae, V. parahuemolyticus, and V. vulniJcus. Examining outbreaks that were associated with shellfish during this same period, these three pathogens together caused one to four outbreaks (9). In 1988, a multistate hepatitis A outbreak with 61 cases was caused by eating raw oysters (13). An outbreak of V. cholerae occurred in Guam, an island in the U.S. Pacific territory, was caused by eating contaminated reef fish. In this single outbreak, 26 people became ill and 1 died. In 1991, there were two outbreaks of V. chokrae caused by tainted food imported to the United States. One outbreak with two cases was attributed to crabs that were imported illegally from Ecuador (14). Most of the disease outbreaks and cases associated with seafood, as reported by the CDC (9), are caused by chemical agents such as scombrotoxin, ciguatoxin, and paralytic shellfish poisoning. These seafood-borne disease agents are discussed elsewhere in this book. During the past 3 years there has been growing public concern about the safety of the food supply in the United States. Much of this concern was ignited by outbreaks caused by the emerging pathogen E. coli 0157:H7. In May 1997, a report called “Food Safety from Farm to Table: National Food-Safety Initiative” was presented to President Clinton. This report presented an intergovernmental agency strategy to prevent foodborne disease and included plans to develop elements of an improved foodborne disease surveillance system. The first component of the new system is the Active Foodborne Disease Surveillance System, known as FoodNet, a collaborative effort between the CDC, FDA, and the U.S. Department of Agriculture (USDA), and selected counties in seven states (California, York, and Oregon)participating in Connecticut, Georgia, Maryland, Minnesota, New CDC’s Emerging Infections Program. FoodNet is designed to conduct population-based
116
Herwig
active surveillance of seven bacterial foodborne pathogens (Saln~onella, ShipAlcr, C m 7 p ~ lobacter, E. coli 0157:H7, Listeria, Yersirlia, and Vibrio),and to determinethe magnitude of diarrheal illnesses and the proportion of these illnesses that are attributable to foods. The population of these seven FoodNet sites i n 1997 was 20.3 million people (7.7% of the U.S. population). The objectives of FoodNet are to (a) describe the epidemiology of new and emerging bacterial, parasitic, and viral foodborne diseasesof national importance, (b)morepreciselydeterminethefrequency andseverity of foodbornediseases in the United States, and (c) determine the proportion of foodborne disease caused by eating specific foods. To addresstheseobjectives,FoodNetconductsactivesurveillanceand related studies: a population survey, a physician survey, and a case-control study of E. coli 0157:H7 infections ( 1 516). A positive outcome from the initiation of FoodNet is perhaps a more realistic estimate about the incidence of foodborne diarrheal disease in the United States. Data from FoodNet suggest that the incidence of diarrheal illness in the United States is about 1.4 episodes/person/year, or some 370 million episodes each year. If only 25% of diarrheal disease is food related, the burden of foodborne diarrheal disease in the United States far exceeds current estimates (16). In theUnited States, four federal agencies play major roles in carrying out food safety regulatory activities: the FDA, the Food Safety and Inspection Service (FSIS) of the USDA, the Environmental Protection Agency (EPA), and the NMFS. Seafood safety is under the jurisdiction of the FDA, EPA, and NMFS. The FDA has jurisdiction over domestic and imported seafoods thatare marketed in interstate commerce. The FDA’s Center for Food Safety and Applied Nutrition (CFSAN) seeks to ensure that seafoods are safe, sanitary, nutritious, wholesome, and honestly labeled. The CFSAN also has control over seafood processing plants. The EPA establishes tolerances for pesticide residues in seafoods, and is responsible for protecting against other environmental chemical and microbial contaminants in water that might threaten the safety of seafoods. The NMFS conducts a voluntary seafood inspection and grading program that is primarily a food quality activity (16). The seafood industry is in the early stages of transitioning to hazard analysis critical control point (HACCP) programs. It is generally accepted in the food science community that the use of HACCP programs i n all aspects of food production, processing, and distribution is proactive and an excellent approach toward food safety. In 1995, the FDA issued itsfinalruleon HACCP programs for seafood. Written HACCP plans for seafood are now required, and must be specific for each processor and type of seafood. In response to the need to train members of the seafood industry i n HACCP procedures, the National Seafood HACCP Alliance for Training and Education was created. Thisand other organizations provide HACCP training courses and model plans that can be used as templates for developing specific plans ( I 6).
2. Asia: The Importance of Seafood in Their Diets The people of Asian countries consume a larger portion of fish and fishery products in theirdietsthancitizensinnlostWesterncountries,thereforeseafood-bornediseaseis generally a much larger percentage of the overall problems associated with foodborne disease. I n 1996, Lee et al. (17) published an epidemiological study of food poisoning in Korea and Japan. From 1981 to 1990, the most commonly incriminated vehicles of foodborne illness in Korea were fish and other seafood (31.8%). meat and animal products (25.0%), grainsandvegetablesincludingmushrooms(17.5%),andcompoundfoods
Pathogens Transmitted by Seafood
117
( 1 8.3%). During the same time,in Japan the most comnlon vehicles were seafood ( 2 I .7%), meat and animal products (3.6%),and graimand vegetables (14.6%). Amongthe seafoods, the major causes of food poisoning in Korea were shellfish (9.3% of total) and puffer fish (5.0%).In Japan, shellfish and puffcr fish accounted for 6.9% and 2.8%, respectively, of the total number of disease outbreaks. In Korea, of the 115 deaths from food poisoning from 1981 to 1990, 42.6% were caused by seafoods, 13.1%J by meat and animal products, and 33.1% by grains, vegetables, and mushrooms. In Japan, of the 106 deaths from food poisoning during the same period, 62.3% were caused by seafoods and 22.5% by grains, vegetables, and mushrooms. No fatalities were attributed to meat and animal products. In Korea, 58.6% of the outbreaks of food poisoning from 1981 to 1990 were due to bacteria, 18.6% were due to toxic compounds, and 23.3% were due to unknown causes. In Japan, 61.3% of food poisoning cases were due to bacteria, 23.5% were due to toxic compounds,and 15.2% weredue to unknowncauses.Thefollowing bacterialspecies were incriminated in Korea: Vibrio spp. (37.6%), Strlrrrotrellcr spp. (23. I %), other species (17. I %), S t t r ~ ~ l r ~ l o ~ ~ (14.9%), o c c u s and E. coli (6.8%). In Japan, the bacterial foodborne problemswere Vibrio spp.(47.3%). Strrl,l,ylococclrs spp.(24.8%), Salmowellcr spp. (14.8%), and other species(9.6%).With data such as these,it is understandable why there needs to be research related to seafood safety in these countries (17).
D. Microbial Classification and Identification of Pathogens 1, Prokaryotes (Bacteria and Archaea) A revolution in describing the taxonomy of microorganisms has occurred over the past 20 years, largely initiated by the contributions by Carl Woese and his coworkers (182 1 ). The taxonomy of microorganisms is now based on the phylogenetics. or evolution, of organisms rather than on interpretation of the results of phenotypic properties. The phylogeny of microorganisms can be inferred by finding the changes that have occurred i n molecules that act as chronometers of evolutionary history over thousands of years. The 16s rRNA (ribosomal RNA) and 30s rRNA are the molecules of choice today. Since the fossil record for microorganismsis extremely poor and virtually nonexistent, the relationship between microorganismsis now determinedby sequencing representative molecules. After the sequences are obtained, the phylogenetic relationship is suggested by performingintensivecalculations withtheaid of personalormainframe computers. A variety of algorithms have been developed to create phylogenetic trees, also known as "trees of life." The work by Woese and others clearly illustrate that there are three major groups or domains of organisms, called the Btrcteritr, Arclraea, and Errkatptr. The natnes A ~ ~ J ~and I CErrkrrryr YI were formerly known as Archaebacteriaand Eukaryotes, in use and found in the literature today. Besides respectively, and these older names are still observing the three major trunks that can be determined by sequencing the representative molecular chronometers, these three domains of life have major differences in their cellular biochemistry and genetic organization. The 16s rRNA sequences and phylogenetics for many of the bacterial food pathogens have been determined and are available fromvariety a of electronic database servers. One of the best resources for phylogenetic analysisand 16s rRNA sequences is the Ribosomal Database Project (RDP) (22) located at Michigan State University. The URL for their web site is www.cllle.lllsu.edt1/RDP/.
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118
Of interest, most of the seafood-borne bacterial pathogens cluster into two phylogenetic groups and arenot widely distributed throughout the "tree of life." No human microbial pathogens are found in the Archaea. Bacterial pathogens associated with seafood are members of the y (gamma) subdivision of the Proteobnctericr and the gram-positive bacteria (Fig. 5). Besides the basic scientific curiosity about the phylogenetic position of seafood-borne pathogenic bacteria, this information can also be used to develop molecular probes and polymerase chain reaction (PCR) primers. Protocols developed for the phylogenetic analysis of microorganisms are used to examine the composition or identity of organi s m present on a food or environmental sample without havingto culture or grow organisms on media. y SubdivisionProteobucteriu Vibrio group Vibrio cholerue subgroup Vibriocholerae Vibrio vulnificus Vibriofisheri assemblage Vibrioparahaemolyticus Aerornonus group Aerornonas hydrophilu subgroup Aeromonas hydrophila
Enterics and relatives Eschericl~iu-Salmor~ell~~ group Escherichiacoli
Salmonellaspecies Yersiniu group Yersiniaenterocolitica Plesiomonas shigelloides Catnpylobacter and relatives Canlpylobacter fetus subgroup Campylobacter jejuni
Gram positive phylum Clostrium and relatives Clostridium botulinum subgroup Clostridiumbotulinum Bacillus-Lactobacillus-Streptococcu~. subdivision
group
StUphY~OCOCCUS
Staphylococcus aureus Listeriu-Bruc/lot}fri~~ Group Listeria monocytogenes
Streptococci Streptococcusiniae
Fig. 5 Phylogeneticposition of pathogenicbacteriaassociatedwith sedood. No111ellclalure for the lnajor groups and subgroups are thc t e r m used by the Ribosomal Database Project ( 2 2 ) .
Pathogens Transmitted by Seafood
119
The discovery of foodborne bacterial pathogens was made possible by the ability to culturetheorganismsonbacteriologicalmedia.Today, therapididentificationand differentiation of many foodborne pathogens is possible because of molecular genetic and immunologic methods. Nevertheless, for the development of theserapid tools, a pure culture of the pathogenic organism was required, meaningthat the culture was most likely initially isolated, characterized, and grown on bacteriological media in the laboratory. In other words, if the pathogenic organism has not been cultured, it has not been identified or implicated as causing seafood-borne illness.
2. Viruses In addition to the bacteria that may cause disease, animal viruses that can cause human illness are associated with seafood. Compared to bacteria, viruses are small, ranging from 25 to 75 nm, and are therefore not observable under the light microscope. While animal viruses come in a variety of shapes, mostof those associated with food are spherical. The of DNA or RNA, but most of the genetic material of animal viruses may be composed food viruses contain RNA, usually single stranded. Virus particles do not have any metabolism of their own. but require living animal host cells for replication. to host by direct or indirect transmission. Direct transmission, Viruses pass from host also known as contact transmission,is probably the most common way that a virus particle passes from one host to another. For example, direct transmission of viruses that cause gastrointestinal disease occurs through an anal-oral route, usually by hands that are contaminated with fecal material containing the infectious virus. Indirect transmission of viruses may occur by (a) vectors, which are intermediate animals within which the viruses are transported; and Inay multiply; (b) fomites, inanimate objects on which the viruses (c) vehicles, foods and water which may transport the viruses. Viruses that have indirect modes of transmission are required to be more stable and durable compared to those that can only be transmitted directly. Viruses are classified using a different scheme compared to other microorganisms and other higher forms of life. A “tree of life” that consolidates and organizes all of the different viruses does not exist.The primary viruses associated with seafoods are members of two viral families, the Picortuviridue and the Crdiciviriche. The Picortmviridcre are snlall single-stranded RNA viruses comprising some of the important pathogens of humans and animals. These viruses are small, having an icosahedral structure, and their nucleic acid consists of positive sense, linear, single-stranded RNA. The Cdiciviridw is also a positive sense, linear, single-stranded family of viruses. Fig. 6 shows the taxonomic groups of viruses associated with foods using the taxonomic nomenclature as described by the International Committee on the Taxonomy of Viruses (23). Six kindsof viruses causing infectionin humans are associatedwith the consumption of seafood: hepatitis A, human caliciviruses (Norwalk virus and Norwalk-like virus), hepatitis E, astrovirus, group A rotavirus,and human adenovirus. Human poliovirus in seafoods is primarily of interest for historic reasons and since this virus is routinely used in laboratory experiments with seafood. The morphologies of human viruses and the detection of these viruses is often performed with theaid of an electron microscope. Samples containing virus particles are stained with a solution containing an electron-dense compound that creates a “negativestain” image that can be observed under an electron microscope. Fig. 7 displays the electron micrographs of representative viruses that may cause seafood-borne disease.
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Positive Stranded Single StrandedRNA Viruses Family Picornaviridae Genus Enterovirus human poliovirus Genus Hepatovirus hepatitis A (HAV) Family Caliciviridae Genus Calicivirus human caliciviruses, alsoknownas small round structured viruses (SRSV) Norwalk virus Norwalk-like viruses, including Hawaii strain, Snow Mountain strain, Taunton strain, Southampton strain Hepatitis E Family Astroviridae Genus Astrovirus Small Round Viruses (SRV) (probable classification) Double Stranded RNA Viruses Family Reoviridae Genus Rotavirus group A rotavirus Double Stranded DNA Viruses Family Adenoviridae Genus Mastadenovirus human adenovirus Fig. 6 Viruses and virus families associated with food. Viruses that are most frequently associated with seafood are in boldface print. Taxonomic nomenclature as described by the Sixth Report by the International Conunittee on Taxonomy of Viruses (23).
While hepatitis A, the Norwalk viruses, and other viral pathogens are thought to cause many outbreaks and cases of gastroenteritis and hepatitis in humans, the good news for food lnicrobiologists and the seafood industry is that there are many human infectious viruses that are not known to be transmitted by seafoods to humans. These include human immunodeficiency virus (HIV), herpesvirus, Hantavirus, rabies virus, rhinoviruses causing the common cold, and the agent of bovine spongiforrn encephalopathy. The last disease agent is not a virus, but a prion.
E. Summary of Clinical Presentations of the Diseases Table 3 summarizes the symptoms,infectivedose,incubationperiod,andduration of seafood-borne diseases. The time intervals listed for the incubation period and the duration of disease are approximations and vary between individuals, episodes, pathogenic strains,
ransmitted Pathogens
h
by Seafood
727
Fig. 7 Electronmicrographs of representative viruses associated wlth seafood (A) calicvirus, (B) astrovirus, (C) human rotavirus, (D) human adenovirus. Micrographs were electronically retrieved from the Universal Virus Database approved by the International Committee on Taxonomy of Viruses. This site is maintained at the Australian National Universlty.
and the numberof bacteria or virus particles consumed. In this table, pathogenic organisms are separated from each other based on the types of symptoms that are commonly observed. For some of the pathogens the infective dose is based on feeding trials with human volunteers and/or the number of organisms that were enumerated on the incriminating or may item of food. Most of the foodborne pathogens cause a diarrheal disease that may not be accompanied by vomiting and nausea, but some seafood-borne pathogens may cause septicemia or neurologic effects that may lead to death. Additional details about the individual seafood-borne pathogens are provided below.
II. BACTERIAL AND VIRAL PATHOGENS ASSOCIATED WITH RAW AND UNDERPROCESSED SHELLFISH A.
Bacteria
In developed countries, most of the problems associated with seafood are caused by the consumption of raw, underprocessed, or mishandled shellfish, particularly bivalve mollusks. These invertebrates concentrate microorganisms that arein surrounding waters by a filter-feeding process. Someof the pathogenic organisms that are concentrated are indigenous to the aquatic environment, while other pathogens are introduced from terrestrial sources or by the fecal pollution of humans or other warm-blooded animals. After fish
Table 3 Symptoms, Infective Dose, Incubation Period. and Duration of Seafood-Borne Diseasesa Organism
Symptoms
Infective dose
Upper ~~a.stroiritestirin1 tract syniptoins (riatisea, \vmiirirrg) occur Jirsr or predomiriute Sraph~lococci~.s nurect.s Nausea. vomiting, diarrhea. Less than I mg of toxin will abdominal pain produce symptoms.
Incubation period or onset time to symptoms 1-6 hour, mean 2-4 hours
Loic,er ,qa.rrstroiritesfinof rmcr symprotns (abdominal cramps, diarrhea) occiir first or predominate 1- 10 cells 8-72 hours Snlmoriella spp. Abdominal pain, diarrhea, vomiting, nausea Some serotypes are highly in- Variable. depending on seroEscherickia coli Abdominal pain. diarrhea. fective for infants and nausea; some strains may type young children. E. coli is a cause watery or bloody dinormal inhabitant in the arrhea. gut of humans and other mammals. 18-36 hours Cunipylohucter jejLoii Small, perhaps less than 200 Abdominal pain. diarrhea. vomiting. nausea cells 24-48 hours Yersiiiia enterocolitico Fever and abdominal pain are Unknown. but thought to be the primary symptoms; fregreater than 10' cells quently have diarrhea and/ or vomiting; may imitate flu and acute appendicitis. Vihrio cholerue (01 8-96 hours 10" cells with production of Abdominal pain. diarrhea, and 0 1 3 9 ) cholera toxin (CT). Much vomiting, nausea. "rice walower number if stomach ter" stool acid is neutralized 18-36 hours Suspected to be 10"-10' cells Vihrio cholerue (non1. Abdominal pain. diarrhea. vomiting. nausea 01 and non-0139) 2. Extraintestinal infection such as septicemia. wound infections. ear infections 3-76 hours Diarrhea, abdominal pain. Vihrio parahuenrolyti10-10- Kanagawa positive cells CllS vomiting, nausea
Duration of disease
6
2-3 days
5 days Variable. depending on serotype
7-10 days 3-21 days
Varies between individuals, mild, watery diarrhea to acute diarrhea Diarrhea may be quite severe lasting 1 week
X Illness usually mild, with duration of 3-5 days
2
aF
L
v.
e,
U2
0
Pathogens Transmitted by Seafood
-d 0 0
d
c
a
I
r-
-
e,
0
D
124
3
S .M
Henvig
3
S .-
Pathogens Transmitted by Seafood
Hewig
126
and shellfish are harvested, pathogenic microorganisms may also be introduced when the food is processed and transported. y-Proteobacteria and gram-positive A variety of bacteria, including members of the organisms, are associated with shellfish-borne disease in humans. Table 4 lists the Gram’s stain reaction, cellular morphology, major phenotypic characteristics, and the natural hab tats where these pathogenic bacteria are normally found. For many of these organisms, more thorough and detailed reviews about the individual genera and species have been published. In the following sections I will summarize the major features of each of the pathogenic organisms and briefly outline protocols that are routinely used to detect and enumerate these pathogens. Additional details can be found in the citations listed.
1. Vibrio Species Of the several species that comprise the genus Vibrio, three species areof primary concern in seafood-borne disease:V. cholerae, V. parahaemolyticus,and V. vulnijicus. To a much lesser extent, some additional speciesof Vibrio have been suggested to cause human disease such as wound infections and gastroenteritis, includingV. alginolyticus, V. carchariae, V . cincinnatiensis, V. damsela, V. jluvialis, V. fumissii, V. hollisne, V. metschnikovii, and V. mimicus (24). Fig. 8 shows the distribution of cases caused by Vibrio species in the United States from 1989 to 1998 (M. Glatzer, personal communication). During this Vibrio species were attributed same period89%of the deaths caused by seafood-associated to V. vuln$cus (Fig. 9) (M. Glatzer, personal communication). (Since the database was established primarily to follow the number of cases associatedV.with vulnijicus, the number of deaths and cases associated with the other Vibrio species should be examined with caution.) All species of the genus Vibrio are gram-negative rods that are often motile with either peritrichous or single polar flagella. They are facultative anaerobes with both fermentative and respiratory metabolisms. In cultural studies, members of the genus Vibrio are frequently isolated from estuarine water, sediments, and biota. Many strains are chitinolytic, capable of hydrolyzing chitin, a biopolymer composed of monomers of N-acetyld-glucosamine (25). Chitin is found in the exoskeleton of arthropods and in certain fungi. In aquatic environments, strains of Vibrio have been foundin close association with crusta-
V. flwralis V V. mrmrcus
-
3%
alginolyticus 1%
5%
V. vulnificus
55%
parahaemolytlcus 16%
Fig. 8 Cases of Vibrio species causing seafood-borne illness in the United States, 1989-1998 m. Glatzer, personal communication).
127
Pathogens Transmitted by Seafood V. parahaernolyticus -
V. cholerae 6%
!io/.
V. vulnificus
89%
Fig. 9 Deaths caused by seafood-associated Vibrio species in the United States, 1989-1998 (M. Glatzer, personal communication).
ceans. These include crabs, shrimp, and lobsters, seafoods that are of great importance for commercial and recreational harvesters. The reported incidence of potential pathogenic species of Vibrio in fish and shellfish is relatively high. Unlike many of the other pathogenic bacteria listed in this chapter, pathogenic and nonpathogenic strains of the genus Vibrio are autochthonous microorganisms in freshwater, estuarine, and the marine environment. U.
Vibrio cholerue
Disease. Vibrio cholerue is the causative agent responsible for the disease called cholera and in the most severe form is sometimes called cholera gravis. Cholera has killed many people and it continues to be a problem in many developing countries. Outbreaks, epidemics, and pandemics of cholera have been recorded throughout history. Since 1817, the world has been affected by seven pandemics of cholera (26). Specific distinctions in the genus are made based on the production of cholera enterotoxin, also known as cholera toxin (CT), serogroup, and the potential for epidemic spread. The basis of serotyping V. cholerue is the lipopolysaccharide somatic (0)antigen. Until recently, the distinction was simple, V. cholerue 0 1 that produced CT were associated with the epidemics and all other strains were nonpathogenic or occasional pathogens. Now, two serogroups-01 and 0139-are associated with epidemic disease, but not all strains of these serogroups produce CT. These strains do not produce cholera, and are not pathogenic (24). V. cholerue strains of the 0 1 serogroup that produceCT have long been associated with pandemic cholera. CT-negative V. cholerae strains have been occasionally isolated in cases of diarrhea or extraintestinal infections. This serogroup can be further divided into two serogroups called Ogawa and Inaba. V. cholerue 0 1 can also be divided into two biotypes, classical and El Tor. The causative agents of the first four pandemics are not known, but those of the fifth and sixth pandemics are due to the classical biotype of V. cholerue 0 1 . For the past 20 years the classical biotype has only been isolated in Bangladesh. The causative agent of the present, seventh pandemic cholera, the El Tor biotype of V. cholerue, began in 1961 in Sulavesi, Indonesia. The seventh pandemic extended across Asia into the Middle East during the 1960s and through Africa, southern Europe, and the Pacific Islands during the 1970s.This pandemic reached South America
128
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in January 199 I . Within a year a majority of countries in South and Central America were affected (26).Until the emergence of the 0139 serogroup, all isolates that were identified as V. cholerere on the basis of biochemical tests, but were negative for 01 serology were referred to as “non-01.” By late 1992,a new cholera epidemic was caused by the emergence of a new serotype, V. cholerrre 0139,also known as V. cholerrre Bengel (24,26). V. cholercw 0139, which emerged in theBay of Bengalarea i n 1992 hassince been detected in 10 countries. This new strain continues to be confined to southeast Asia (Bangladesh, China, India, Indonesia, Malaysia, Myanmar, Nepal, Pakistan, Sri Lanka, Thailand). Upuntil 1994,imported cases were reported in Estonia, Germany. Hong Kong, Japan, Korea, Singapore, Switzerland, Thailand, and the United States (27).V. cholerrre 0139 appears to be a hybrid of the 01 and non-Ol strains. The clinical presentation and modes of transmission of V. cholercre 0139 are the same as V. cholerere 0 I . In important virulence characteristics, V. cholerne 0 1 39 is indistinguishable from V. cholercrc 01 El Tor strains (28). However, this organism does not produce the 0 1 lipopolysaccharide and has a polysaccharide capsule that is similar to non-Ol strains (24). Cholera remains a global threatand one of the key indicators of social development. While the diseaseno longer poses a threat to countries with a minimum standardof healthy living, it remains a challenge in countries where accessto safe drinking water and adequate sanitationcannotbeassured.Almosteverydevelopingcountryisnowfacingeither a cholera outbreak or the threat of an epidemic (27). In India, cholera is estimated to be responsible for the deaths of 20 million people during this century. Since1991,the cholera pandemicin South America hasbeen responsible for the deaths of 10,000people. Death from cholera is caused by extreme dehydration and loss of electrolytes. An individual with cholera may lose up to 20 L of liquidlday and the tnortality rate may be 30-50% if an infected person is untreated (26). Three major types of V. cholerrre are recognized and are differentiated based 011 serology: 01, 0139,and non-OI/non-0139. The 0 1 serotypeisdivided into classical (nonhemolytic) or El Tor (hemolytic) types. V. c h o l e r m 01 causes cholera, which may be a mild case of diarrhea or a life-threatening disorder. The more recently discovered serotype, 0139,is very similar to 01. Non-Ol V. cholerere causes a less severe disease, usually a gastroenteritis or soft-tissue infection and septicemia. Most strains of non-01/ non-0 139 V. cholertre are non pathogenic (24,29). The symptoms associated with V. cholerne 01 range from a mild, watery diarrhea to an acute diarrhea with rice water stools. The mechanisms for pathogenesis have been well described and include two important aspects, colonization of the small intestine and of intracellular cyclic adenoproduction of cholera toxin. The toxin causes increased levels sine monophosphate (CAMP) and the secretion of water and electrolytes (Na‘, K - , Cl-, bicarbonate) into the lumen of the small intestine (26).
Ecology. Cholera is a disease that is primarily waterborne, but food that has contacted contaminated water may also carry organisms of V. c~l~olcrrre 01. The food that has been most frequently implicated in outbreaks is seafood, both molluscan shellfish and crustaceans. Estrada-Garcia and Mintz (30)showed that seafood was the most commonly implicated vehicle in foodborne cholera outbreaks around the world, with more than 12 outbreaks associated with seafood since 1961 (Table 5 ) . The primary reservoir for V. (Aolerrre is infected humans. Short-tertn carriage of this pathogen by humans is important in transmission of the disease. Persons with acute cholera excrete 107-10s V. cholerrre cells per gram of stool. People who are excreting
nd
Pathogens Transmitted by Seafood
129
Table 5 SeafoodsImplicatedinCholeraTransmissiond
Location Scverlrh pcrrlderrlic
1973 1974 I974 1977 1978 1982 1994
Raw shellfish Raw and undercooked shellfish Salted raw fish Raw salted fish and clams Steamed prawns Cooked squid "Samba1 sotong" Rawfish
198 199 200 20 1 35 36 202
1991 1991 1991 1992
Raw seafood ceviche Cooked crab Cooked crab Shrimp and fish
203 14 204 205
Louisiana Louisiana
1978 1986
34 206
Colorado
1988
Cooked crab Cooked crab and shrimp, raw shrimp and crab Raw oysters
Italy Portugal Guam Gilbert Islands Singapore Singaporc Italy L n r i r ~Arrlericrr epirlenlic
Ecuador Ncw Jersey (Ecuador) New York (Ecuador) California (Pcru) U.S. Gldf Cotr.st
207
large volumes of diarrhea may easily release a trillion viable and pathogenic cells in I O L of diarrhea. Asymptomatic carriers may live in the same household as an individual suffering from acute illness. In various studies 4-22% of individuals may be asymptomatic carriers. Although there is evidence that V. cholerne can survive and multiply in the environment (31), the rates of isolation of CT-producing V. cholera 0 1 from the environment correlate primarily with the degree of sewage contamination (29). Several reports have illustrated the close association between Vibrio species and crustacean zooplankton. While most research related to public health has focused on the shellfish that humans purchase and consume, smaller planktonic animals may be causing very significant problems for humans. Huq et al. (32) described a simple filtration method to remove plankton-associated V. cholercre in raw water supplies in developing countries. In laboratory experiments, the bulk of the plankton was removed with a filter constructed from either nylon net or sari material, the latter being inexpensive and readily available in Bangladeshi villages. Seafood may be contaminated if harvested from water polluted by sewage or from environments where V. cholerne 0 1 occurs naturally independentof human fecal contamination. In artificially contaminated oysters and clams, V. ckolerne 0 1 survived for more than 3 weeks when refrigerated (33). Crabs boiled for less than 10 minutes or steamed for less than 30 minutes may still harbor viable V. choler-ne 0 1 organisms (34). In the United States, the first cholera outbreak associated with seafood occurred in Louisiana. Eleven cases of cholera were associated with the consumptionof home-cooked crabs that were either boiled or steamed. Secondary contamination of seafood has been reported. Steamed prawns (35) and cooked squid left for at least 4 hours at room temperature (36) were implicated in two cholera outbreaks in Singapore. In businesses that maintain fish live in aquaria for human consumption, the aquaria
130
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water may contain pathogenic organisms such as V. choler.cre. Kam et al. (37) described the investigation of 12 cholera cases in Hong Kong. Microbiological investigations demonstrated that contaminated seawater in fish tanks that were used for keeping seafood alive was the most likely vehicleof transmission. The source of seawaterin some facilities Inay be contaminated. Restaurant owners in Asia who serve seafood often receive seawater for their holding tanks from “water vehicles.” The operators of these vehicles Inay draw seawater from polluted harbors or typhoon shelters, protected anchoring areas that provide shelter for fishing junks and other small vessels during monsoons (38). Estrada-Garcia and Mintz (30) listed several factors that make seafood a possible also important for the other Vibrio vehicle for cholera transmission. These factors are species that are pathogenic to humans: In many parts of the world seafood is consumed raw or undercooked. Seafood may be contaminated with V. cholcrcre in the aquatic environment. V. cholercre persists for many weeks in seafood, even if refrigerated (39) and multiplies rapidly if introduced onto cooked seafood (40). Therefore secondary contamination may be a problem. V. cholernc adheres to chitin, and chitin-absorbed bacteria seem to be more resistant to variations in pH and heat than free-living bacteria (41). V. cholercle colonize the surfaceof copepods and may concentratein the gastrointestinal tracts of animals that ingest copepods (42). MicrobiologicalProcedures.Traditionalmicrobiologicalproceduresfor
V. chol-
erne and other pathogenic Vibrio strains are well established and have changed little in
recent years. Most species of Vibrio grow well at alkaline pH and a key step in the protocols istheinitial enrichment in alkaline peptone water. Enrichments are streaked onto thiosulfate citrate bile salt (TCBS) agar and typical colonies are tested with a series of biochemical and physiological tests. Fig. 10 summarizes the protocol for V. cholercre (43). The key confirmation for the identificationof V. cholercre 0 1 is agglutination in polyvalent antisera. Antiserum against 0 1 3 9 is now available.
b.
Vibrio vulniJcus
Disease. Vibrio vultlificus isthe mostseriouspathogenic Vibrio species in the United States, responsible for 95% of the seafood-related deaths. A disease caused by V. vultzificus was first described by Roland in 1970 (44) who detailed the case of endotoxic syndrome and leg gangrene acquired in New England coast water. The etiologic agent was misclassified as V. parnhaernolvticus (44). Investigators later examined their collections of lactose-positive vibrio strains and a new species of Vibrio, V.
[email protected], was declared in 1979 because the pathogen could produce cutaneous lesions (vulnificus, meaning wounds in Latin) (45,46). In the state of Florida, this organism is the leading cause of foodborne deaths. While most of the cases that have been documented are from the southeast region of the United States,V. vulngcus was recently found in the waters and shellfishin Europe. In 1996, Dalsgaard et al. (47) reported the first clinical and epidemiological data about a series of V. drzificus infections in northern Europe. Arias et al. (48) presented the first report of the detection of V. vult$ficus naturally present in seawater and edible shellfish along the Spanish Mediterranean coast. This pathogenic bacterium may be Inore widespread in temperate and tropical waters than previously thought. v . \)uln.$cu.y manifests itself in three fornls of human disease, with two forms of seafood-borne disease having a very high rate of mortality. First, v. v u i t l i f i c w lnay cause
131
Pathogens Transmitted by Seafood
Vibrio cholerae Oysters
Oysters
Weigh 25 g sample and add 225 ml of APW Homogenize for 2 min
Weigh 50 g sample and add450 ml Alkaline Peptone Water (APW) Homogenize for 2 min
Split homogenatein half
250 m1 anddilutions
Than Foods Other
Prepare serial dilutionsof homogenate 250 ml anddilutions
t
Incubate homogenate and APW dilutions 6-8 h, 16-24 h at 35-37°C
Incubate homogenateand APW dilutions 6-8 h, 16-24 h at 42°C
Streak all enrichments onto Thiosulfate Citrate Bile Salts (TCBS) agar (and, optionally, Modified Cellobiose PolymyxinB Colistin (mCPC) agar) TCBS is incubated 18-24h at 3537°C; mCPC is incubated 18-24 h at 39-40°C
t
Pick typical colonies onto 1%Tryptone + 1% NaCl (T,N,) or Trypticase Soy agar (TSA) + 1.5% NaCl Incubate 12-24h at 35-37°C
I
t
Perform preliminary biochemicaland physiological tests Triple Sugar Iron (TSI) Kligler Iron Agar (KIA) Arginine Glucose Slant (AGS) 1% Tryptone + 3% NaCl TIN^), 1% Tryptone + 0% NaCl (TINo) Gelatin Agar (GA), Gelatin Salt (GS) Hugh-Leifson glucose broth Oxidase Gram stain
t
Confirm V. cholerae 01, V. cholerae 0139, V. cholerae non-01, V. cholerae non-0139, and V. mimicus using serological and biochemical tests
Fig. 10
Enrichmcnt.isolation,andidentificationprotocol
for Vihrio cholerrre (43).
Henvig
132 Table 6 Characteristics Among Patients with
Vibrio vulnificus Infections in the United States,
1988-1996O
Gastroenteritis septicemia Wound Characteristic Primary Median age, years (range) Males (9%) Fever Diarrhea Abdominal cramps Nausea Vomiting Shock Localized cellulitis Bullous lesions Hosuitalized
infection 54 (24-92) 89 91 58 53 59 54 64
-
49 97
35 (0-84) 57 57 100 84 71 68 0
0 65
59 (4-91) 88 88
-b
30 91
89
' Data presented by Shapm et al. (49). Characteristlc not associated with syndrome.
a primary septicemia often resulting from the consumption of raw oysters. The mortality rate for this form is about 60%. Second,V. vulnijicus may cause wound infections on the skin. This disease may be causedby organisms present in seawater andlor shellfish, and has a mortality rate of 20-25%. The third form of the disease is a gastroenteritis that rarely causes death. Table 6 summarizes the characteristics among patients with V. vulnacus infections in the United States from 1988 to 1996 (49). V. vuZn$cus septicemia is nearly always associated with the consumption of raw oysters. Previous reports have shown the prevalence of raw oyster consumption in the general United States population to be around 17%, with a prevalence reaching as high as 32% in coastal states such as Florida(50). Data clearly indicate the hazards associated with the consumption of raw shellfish, particularly oysters, harvested from the Gulf Coast of the United States. Oysters causingV. vulnijkus disease have been harvestedin Louisiana, Florida, and Texas (Fig. 11). Although many of the shellfish-associated cases of V. Unknown
Fig. 11 States of harvest for shellfish associated withVibrio vulnificuscases (M. Glatzer,Personal communication).
Pathogens Transmlfted by Seafood Unknown
133
9 states
Anzcna
2%
New York 2%
Florida
38%
Alabama
Texas
9%
11%
Fig. 12 States of consumption of shellfish for cases associated with Vibrio vuln$cus personal communication).
(M.Glatzer,
vulnijicus occurred in these harvest states, there were also a significant number of cases as California, and states that in states where there are major population centers, such receive shipmentsof shellfish from the Gulf (Fig.12). These data also illustrate the complexity of the problems that are faced by federal and state public health officials in dealing with the interstate transport of raw shellfish distributedto retail and food service establishments. Nearly three-quarters of the cases of V. vulnifcus disease are associated with the consumption of shellfish in restaurants (Fig. 13). The common symptoms are fever, chills, nausea, and hypotension (low blood pressure). Symptoms associated with gastroenteritis are often present, but are less common. An unusual symptom that also occurs is the development of secondary lesions that are often found on the extremities. These lesions may develop into necrotizing fasciitis or vasculitis that may require the amputation of limbs or surgery. Fortunately infection with V. vulnijicus is not a serious problem for healthy people; nearly all serious and deadly Unknown Miscellaneous
Party
4%
4%
5%
qestaurant 72% Fig. 13 Location of shellfish consumption for cases associated with Vibrio vuln$cus (M. Glatzer, personal communication).
134
Herwig
infections occur in people with an underlying disease that causes an elevated level of iron of patients in the blood or in those who are immunosuppressed. About three-quarters with raw oyster-associated V. vulnificus primary septicemia have preexisting liver disease ( 5 1S 2 ) . In Florida, the reported rateof raw oyster-associated V. vuln$cu.s primary septicemia is 60 times greater among personswith liver disease thanin those without liver disease (53). Liver or blood-related diseases may predispose an individual to serious V. vulrl$cr~ septicemia. In recentyears,additionalwork has indicated that patients who are immunosuppressed are also susceptible to V. vuln$cus and V. ckolerue infections. Raw oyster-associated V. vulnificus septicemia has been reported in at least one patient with HIV infection (54) and a fatal case of V. cholerae non-01 sepsis was described i n a 51-year-old patient who was undergoing chemotherapy for leukemia and reported consuming raw oysters 48 hours before he became ill ( 5 5 ) . A report (56) describing the acquisition of V. vulr~ijcus septicemia from wounds in two patients with solid organ transplants supports the notion that immunosuppressed persons are at increased risk for severe Vibrio infections. Some patients with underlying liver diseases continue to consume raw oysters even after they are informed about the risks. Tayloral. et (57) studied the raw shellfish consumption practices of patients with liver disease being seen in the outpatient gastroenterology clinic at Walter Reed Army Medical Center. One-fifth of patients with known liver disease, even when previously informed about the risks, reported eating raw shellfish. In 199 I , California was the first state to require restaurants and other establishments that serve or sell Gulf Coast oysters to warn prospective customers about the possible risks. Florida and Louisiana also use these warning regulations. In October 1995, the ISSC rejected an FDA proposal to ban the sale of raw oysters obtained from the Gulf of Mexico during warm-weather months. Instead, oyster harvesters are now required to refrigerate oysters within 6 hours after harvesting from this region. The U.S. government has received pressure from national consumer groups to set a standard requiring the Gulf Coast shellfish industry to eliminate pathogenic bacteria, such as V. ~ ~ u l n ~ j i cfrom r ~ s ,oysters. The consumer groups notethat technologies are available that can eliminate this organism. A mild heat pasteurization technology is offered by AmeriPure Oyster Companies of Empire Louisiana. Another technology that appears to be promising, a high-pressure treatment, is also being developed (58).
Ecology. V. vulnificw is a normal inhabitant of the estuarine environment, having of the United States, the Gulf of Mexico, and in estuarine been isolated on both coasts environments around the world. The ecology of V. \~uh$cu.sis very similar to V. pcrrcrhrremolvticus. As filter-feeding shellfish, oysters concentrate the number of microorganisms found in the water column in their gut. For example, one study found a nearly 10,000-fold increase in concentration, from 7 colony forming units (cfu)of V. vuln[jicu.s in seawater to 10" cfu/g of oyster. As found with other marine vibrios, there is no correlation between the presence of V. vulrlificus and fecal coliforms. Therefore, measuring thelevel of E. coli or fecal coliforms is not a good monitoring method for V. vulrlificus. Some of the highest concentrations of V. vulniJcus have been found in the intestines of finfish (59). V. vulnificus also undergoes seasonal population variations correlated with temperature. The data of Motes et al. (60) clearly show the relationship between temperature and the levels of V. vulr1ificu.s in oysters. During warm-weather months,when more than 85% of the shellfish-associated V. vuln$cus occur, most probable number (MPN) counts were usually 103-10'/g of oyster meat. During cold-weather months,when infections have not
Pathogens Transmitted by Seafood
135
been reported, MPN counts were less than IO/g. Gulf Coast data suggest that the number of V. wtln[ficus organisms i n oysters is strongly correlated with water temperature until the temperature reaches 26”C, above which there appears to be no additional increase in the number of bacteria. Seasonal temperature change explains most of the variability in the Vibrio levels in the Gulf. Salinity explains an additional 10% of the variability in these levels and also explains the differences between sites. Shapiro et al. (49) summarized data that was collected by the Gulf Coast Vibrio Surveillance System, which includes public health agencies in the states of Florida, Alabama, Louisiana, and Texas. Using data collected by the CDC, they performed oyster trace-backs and examined the temperature of the place of harvest. For primary septicemia infections caused by V. vulrz(ficus, they found that all of the implicated oysters were harvested in the Gulf of Mexico when water temperatures were greater than 22°C. Among the 72 traced cases between 1988 and 1995, only 3 infections occurred in persons who consumed oysters harvested in waters with temperatures less than 20°C. While water temperature can serve to predict infections by V. vuh~[ficus,Shapiro et al. (49) did not prove that higher temperature is the cause of the increased incidence of V. vuln$cus infections. Unfortunately for the oyster industry in the Gulf, their data support the strong association between “summer harvesting” and illness, but summer harvesting represents 6 months of the year (May-October). Since 1972, oyster harvesting during this period has increased from 15% to 40% of the total annual production. A simple response to this problem, such as a closure of the harvest from May to October, would resultin a severe economic impact to the Gulf Coast oyster industry. Methods to reduceV. vuln$cus in live oysters after harvest include depuration(also called relaying), irradiation, and heat treatment. Gamma irradiation, although effective in reducing V. vuln$clrs in oysters in doses of 1.O- 1.5 kGy, also increases oyster mortality during storage. Also, this process is not approved by the FDA. Conventional depuration of oysters with an indigenous microflora of V. vulw$cus has been unsuccessful (61,62) and if conducted at temperatures higher than 21°C may actually increase the number of V. vuln$cus in oysters. Motes and DePaola (63) evaluated suspension relaying of Gulf Coast oysters to offshore waters that are normally free of V. vuln$cus. They observed a decline of V. v~rlr~$cus in relayed oystersthat was suggested tobe associated with exposure to high-salinity environments essentially devoid of V. v u l ~ z $ c ~ ~ s . Besides using methods to reduce the populations of potentially pathogenic v. vu/r~$cus cells in oysters, procedures can be used to tninilnize the increase in v. vuln(fifi~us numbers after harvest. Reducing the time oysters remain outside of refrigeration can decrease consumer exposure to high numbers of V. vulrz$cus; the oysters must be cooled immediately after harvest to eliminate postharvest growth of V. vuln$cus. Under typical Gulf Coast industry practices, oysters are held on the deck of harvest vessels without refrigeration or icing until the vessel docks. The NSSP does not require shellstock to be refrigerated on harvest vessels. By the summer of 1995 most Gulf Coast states had regulations on the time shellstock can remain outside refrigeration that are more restrictive than those set forth in the NSSP manual (64).
MicrobiologicalProcedures. Theenrichment,enumeration,andisolation procedures for V. vuln$c~u are very similar to those used for V. parcrhaemolyticus. Many studies attempt to perform enumerations for both Vibrio species using the same samples. Mostprotocolsbeginwith an enrichment step in alkalinepeptonewater,followedby plating onto a selective and differential medium called TCBS agar. Similarto V. cholerae,
Herwig
136
V. vu1rlijicu.s can grow under alkaline conditions. Inrecentyears, however, a modified cellobiose polymyxin B colistin (mCPC) agar has replaced the use of TCBS for isolating V. vulnificus. Suspected colonies are examined in a variety of biochemical tests. Fig. 14 outlines the enrichment and identification procedures for V. vulrzijicus and V. purcrhnetno-
lyricus (43). Some V. vultz@xs researchers believe that the environment contains a very diverse population of V. vultlificw strains and that only a certain subset appears to be associated with human disease(65,66).To examinethe diversity that exists within the species, molecular methods have been used including restriction fragment length polymorphism (RFLP) and ribotyping. Tamplin etal. (65) described RFLP and biotype profiles for environmental
Vibrio vuinificus
Vibrio parahaemolyticus
Weigh 50 g of sample and add 200 ml of Phosphate Buffer Saline(PBS)
Weigh 50 g of sample and add 200 ml of 2-3% NaCl solutionor Phosphate Buffer Saline (PES)
Prepare serial hutions in PBS
PreDare serial dilutions in saline or PBS
Inoculate Alkaline PeptoneWater (APW) MPN Incubate 12-16 h at 3537°C
Inoculate APW or Alkaline Peptone Salt broth (APS) MPN Incubate 16-18 hat 3537°C
t
Streak enrichments onto Modified Cellobiose Polymyxin B Colistin (mCPC)agar Incubate 18-24 hat 39-40°C
+
Streak enrichments onto Thiosulfate Citrate Bile Salts (TCBS) agar Incubate 18-24 hat 35-37°C
Pick typical colonies onto l%Tryptone1% NaCl (TINl agar) or Trypticase Soy agar + 1% NaCI, and Gelatin agar (GA) or Gelatin Salt agar (GS) Incubate 12-24 hat 35-37°C
+
Perform prelimmary biochemical testing to differentiate species Gram stain Oxidase Motility Arginine Gelatin Salt (AGS) Triple Sugar Iron (TSI) 0/129 vibriostat sensitivity ONPG test
Perform additional physiological, biochemical, serological, and pathogenicity tests
Fig. 14 f?ruiyticlls
Pathogens Transmitted by Seafood
137
and clinical V. vlrlw$c.u.s strains. They found high levels of variation in RFLP profiles among 53 clinical and 78 environmental isolates as described by pulsed-field gel electrophoresis. In contrast, ribotype profiles showed greater similarity. Jackson et al. (66) presented evidence that V. vuln;fic~rsinfections result in the proliferation of a single pathogenic strain, not a mixture of strains, and that high-risk individuals may be susceptible torelativelylowconcentrations of V. r~uln~ficus, as low as 10' cells/g of oyster if the pathogenic strain is present. Characterization of V . 1~1rl11$crrs strains has led to subdivision of the species into two biotypes which are now suggested to be basedon serological properties and host range, rather than the more variable biochemical properties. Biotype 1 strains are pathogenic for humans, exhibit several immunologically distinct lipopolysaccharide(LPS) types, and are indole positive (67). Biotype 2 strains appear to be virulent for both humans and eels and express a conmon LPS type, and the majority of biotype 2 strains are indole negative (67,68).
c'. Vihrio ~~nr~r11tremolytic'rl.s Disease. The firstreport of a V. pnr~rhaernolyticusfoodborneoutbreakwas in 1950. This organism was first isolated in 1953 as the causative agent for food poisoning i n Osaka, Japan (69). In some regions of the world V. parnkaernolyticw is the leading cause of gastroenteritis associated with food. In 1994, 102 outbreaks of foodborne disease involving 4726 cases were reported to the Taiwan Department of Health. Of these outbreaks, 72.5% were caused by bacterial pathogens with V. partrknemolyticus responsible for 56.7%, Stcll'h?.lococc.lrs mre1r.s 20.3'76, Bcrcilllrs cereu.s 14.9%, and Salmonella spp. other than S. typhi and S. pcrrcrtyphi 8.1% (70). V. ~'c~r~~hrrenrol~ticus has been the leading high proportion (54%) of the cause of foodborne illness i n Taiwan for many years. A large outbreaks, defined as having more than 50 cases, was associated with commercial lunch-boxes suppliedto elementary and juniorhigh schools. In Japan, V. pczr~rkezernolyticus is the leading cause of foodborne disease, with as much as 70% of the bacterial foodborne disease i n the 1960s being caused by this organism. Most of these outbreaks were associated with the consumption of seafood. Whereas most Japanese outbreaks involve fish, in the United States outbreaks primarily involve crab, shrimp, lobster, and oyster. The largest outbreak caused by V. p r o hrlenlo1yticu.s occurred in the summer of 1978 and affected 1133 of 1700 people attending a dinner in Port Allen, Louisiana, in which boiled shrimp was served. The shrimp were boiled in the morning then repacked i n the wooden crates in which they had been stored before they were cooked. The boiled shrimp were stored in an unrefrigerated truck until they were served approximately 8 hours later. In the last 2 years, two major outbreaks caused by V. pcrrtrhrrenro1~ticu.shave occurred in the United States. Thereis some concern among regulatory officials and within the industry about whether the recent outbreaks are an anomaly or the start of a new trend. During July-August 1997, the largest outbreak i n North America in recent years of confirmed V. ~~nrcrhaemo1~~ticu.s infections occurred (7 1). Illness in 209 people was associatedwith eating raw oysters harvested from California, Oregon, and Washington in the United States and British Columbia in Canada. One person died from the outbreak. During this outbreak, most ill people had no underlying illness. There was a suggestion that a slightly higher than normal water temperature contributed to this outbreak. Mean Pacific coast sea surface temperatures recorded by the U.S. Navy ranged from 12°C to 19°C from May through September. These temperatures were I"C-5"Chigherthanthe temperaturesfor the sameperiod in1996.Thelargest
138
Henvig
previous outbreak of V. PLrrah~lerno1yticu.sinfections reported in North America occurred in 1982 and resulted in 10 culture-confirmed cases (71). In 1998, an even larger V. purerIlnernolyticus foodborne outbreak occurred along the Gulf Coast. This outbreak caused illness in more than 400 people (M. B. Glatzer, personal communication). The symptoms associated with V. ~~arrrhtret~~ol~ticr~,s are primarily diarrhea and abdominal cramps with fever, nausea, and vomitingto a lesser extent. The infectious doseis thought to be large, somewhere between lo5and IO’ cells. Pathogenic V. ~~~rrahnemolyticus strains, which cause acute gastroenteritis after consumption of raw or partially cooked seafoods, have been known to produce thermostable direct hemolysin (TDH), also known as the Kanagawa phenomenon, or TDH-related hemolysin (TRH), orboth TDH and TRH (72,73). Most clinical isolates (96.5% of 2720 strains) have been Kanagawa phenomenon positive, whereas 99% of 650 environmental and food cultures have been found to be Kanagawa phenomenon negative (74,75). Suthienkul et al. (76) used PCR to analyze genes encoding TDH (tdl1) and TRH (tr.11) in 489 clinical strains and found that 81 % of the 489 strains were TDH’TRH-, 5.5% were TDH-TRH’, 2% were TDH-TRH’, and 11.5% were TDH-TRH-. Hence the investigators thought that identification of V. parahnemol~ti~us, irrespective of its virulence factors, is necessary for public health (77). V. ~~a~~rhnemolyticus is not a reportable disease in all states. During the 1997 outbreak,publichealthofficials in Washington,California,andBritishColumbiabecame aware of the outbreak through routine reporting. An editorial note in Morbidity mcl Mortality Weekly (71) suggested that all states should consider making V. ~~trr~rh~retrrolyticus and other vibrioses reportable.
Ecology. V. ~~arc~haerrrolyticus is a naturallyoccurringbacteriuminestuaries around the world. It is found in sediments, plankton, and in a variety of fish and shellfish. Pioneering work performed in the 1970s in Rita Colwell’s laboratory clearly documented the ecology of this human pathogen in the Chesapeake Bay (78). V. ~~arahaemolyticus was found to have a strong association with zooplankton during the warm summer months, and appeared to “overwinter” in the sediments during the cooler periods of the year. Most Vibrio species are chitinolytic, and thus are involved with the biodegradation of crustacean exoskeletons and other forms of chitin (79). Several studies have not found a correlation between the number of V. pLrrCrhClern[)lyti(.u,~ and fecal coliforms in environmental samples (61$0). V. yrrrcrhc~emolyticus has a remarkably rapid growth time, as short as 8 or 9 minutes at 37°C in bacteriological media. Even in seafoods, generation times of 12-18 minutes havebeenreported(24).Withsuch a shortgenerationtime,thisspeciescangrowto remarkably high concentrations i n a relatively short time. Recently another type of shellfish has been implicated as a vehicle in seafood-borne V. f,crr~rhnemolyticusdisease. Bean et al. (81 ) demonstrated that the consumption of cooked crayfish was associated with V. pcrrcrhaemolyticus infection. Crayfish are commercially harvested along the Gulf Coast of the United States. Of interest, no crayfish consumption was reported in V. vuln$cus infections. Crayfish have been described as “nouvelle cuisine” and are becoming an increasingly popular seafood item in many areas outside the Gulf Coast region. MicrobiologicalProcedures. Procedures verysimilartothoseusedfor V. vulr1ificu.s are used for V. parahaemolyticus (Fig. 14) (43). Sometimes additional amounts of NaCl are included in the diluent or enrichment broths used for V. ~~~rrrhaemolyticus. Isolates of V. pcrrahcretnolyticus can be serotyped according to their somatic (0)and capsular
Pathogens Transmitted by Seafood
139
(K) antigens, based on a scheme developed by Sakazaki et al. (82) who exanlined 2720 strains. Presently there are 120 antigens and 59 K antigens. Although many environmental and some clinical isolates cannot be typed bythe K antigen, the majority of clinical stains have recognized 0 types. While the antigenic characteristic is helpful in identifying and monitoring different strains, there appearsto be no correlation between serotype and virulence. Not all strains of V. / ~ r ~ ~ r r l ~ r ~ e t ~cause r ~ ) / yhuman t i c ~ ( . ~disease.I n many environmental studies pathogenic strainsof V. pcr~rrhcrenrolytit~us are never orrarely found. To enumerate or identify pathogenic strains, special attention is paid to finding strains that produce a hemolysin, called TDH (thermostable direct hemolysin) or Kanagawa hemolysin. A special blood agar called Wagatsuma agar (83) can be used to detect the production of this special hemolysin. but i n recent years a gene probe has been used to detect tdh genes. Primer sets for specific amplification of the TDH gene fragment have been described, and by using a preenrichment step i n alkaline peptone water for 8 hours, I O cells can be detected (84). Kaysner et al. (85) concluded that the urease-positive biotype is a useful marker for identifying potentially pathogenic strainsof V. ptrrnhtrernolyticlrs isolated from mollusks grown in the Pacific Northwest. This phenotype may only be useful for a preliminaryscreen,since all TDH-positivestrainswereureasepositive,however, notall urease-positivestrainsproducedTDH.Theassociation of positiveureaseactivityand TDH. has not been observed, however,in strains isolated from other regionsof the world (86). Chen and Chang (86a) described a rapid method for detecting V. /,rr~rrhaemo/yticrrs in oysters by immunofluorescencemicroscopy.Antibodieswerepreparedagainsttwo outer membrane proteins of V. pcrmhrretrrolyticus and an indirect staining method was employed. It was suggestedthat this could beused as a rapid screen ofV. ~~tr~crhLlerr~o!\‘ricu.s in oysters, with a presumptive positive result obtainable within 24 hours. Some problems remained, however, with cross-reactions between closely related but generally nonpathogenic organisms, such as V. crlgir~olyticrrs.
2 . Salmonella (1. Diserrse. Species of Soltwtrelltr are gram-negative, facultative anaerobic, nonspore-forming, peritrichously flagellated rods that fernlent glucose and utilize citrate as a sole sourceof carbon. Some species arenot motile. S c h r o t w l l t r spp. are very closely related ~ ~ ~ r rspecies e. of Srrlrrronellcr are to E. coli and are lnenlbers of the E l l t e ~ o b t r c ~ t e r - i ~ ~Several recognized and hundreds of different serotypes have been identified. The nomenclature of the Salmonella group has changed over the years and is based on a taxonomic scheme that uses biochemical and serologic infomiation and on the principles of numerical taxonomy and DNA/DNA homology (87). Manywould argue that all of the different “species” and strains are actually a single species with numerous subspecies. The three species that are generally recognized on a biochemical basis include S. typhi, S. cholertresui.~,and S. erlteritidis (88). Theserotypes are identified according to the Kauffnlann-White antigenic scheme and are segregated based on their somatic (O), capsular (Vi), and flagellar (H) profiles. Serologic tests are complex and labor-intensive techniques involving the agglutination of surface antigens with Salmonella-specific antibodies. Before 1950 typhoid fever, the disease caused byS. phi, was the primary infection caused by Salttrorwlltr in Western countries. This disease was the most frequent form of water-borne disease, and thousands of people died of typhoid fever during the early part
140
Hewig
of this century. Most of the outbreaks occurring earlier this century were related to S. hphi in the consumption of raw mollusks. At present, nontyphoid Srrltnonella spp. are one of the leading causes of bacterial foodborne disease in the United States and in many other non-Asian countries. Scrlrnorrell~ is associated with shellfish that is harvested from contaminated water or with seafoods that may have come in contact with other meats, poultry, or dairy products during preparation. The acute symptoms of the disease, called salmonellosis, are nausea, vomiting, ab1-2 days. dominalcramps,diarrhea,fever,andheadaches.Acutesymptomsmaylast Some people may suffer more chronic symptoms including arthritis-like symptoms that may follow 3-4 weeks after the onset of the acute symptoms. The infectious dose may be low, withas few as 15 cells causing the disease. As with other gastrointestinal diseases, the severity of the infection depends on the health and age of the person and strain differences within the genus.All age groups are susceptible, but symptoms are most severe in the elderly, infants, and the infirm. AIDS patients frequently suffer salmonellosis (estimated at 20-fold more than the general population) and suffer from recurrent episodes. The disease is caused by penetration and passage of Srrlrnonella organisms into the epithelium of the small intestine where inflammation occurs. There is evidence that an enterotoxin may be produced (87). Strlnwnella is among the leading causes of foodborne illness in the United States. It has been estimated that 2-4 million cases of salmonellosis occur in the United States each year. The CDC estimates that 75% of S. enterifidis outbreaks are associated with the consumption of raw or inadequately cooked Grade A eggs (87).
h. Ecology. Salmonellu arewidelydistributed amonganimals,particularlydomesticatedpoultryandswine,and in the environment.Environmentalsourcesinclude water,soil,insects,factorysurfaces,kitchensurfaces,animalfeces,andwildanimals. Scdrnor~ellaare primarily a problem associated with raw meats, poultry, and eggs, but they are also associated with milk and dairy products, fish, shrimp, frog legs, sauces and salad dressing, and manufactured foods that have ingredients that include the above.SLrlmonella species can be readily isolated from fish from aquaculture farms. Table 1 lists the prevalence of S d r n o r w l l c r in aquaculture farms in South Africa, the United States, Japan, and the Philippines ( 5 ) . There is evidence that Scrlrnonelkr is frequently isolated from these operations. Many aquaculture farms may be using antibiotics to safeguard the health of farmed fish and shellfish. c. Microhiologictrl Procedures. Traditionalmethodsfortheenrichment,isolation, and identification of Sdrnonella often include several steps or transfers and may take more than a week to perform. Usually, a preenrichment step in lactose broth precedes enrichment steps in tetrathionate broth and Rappaport-Vassiliadis (RV) broth. These steps are followed by plating on selective media that may include bismuth sulfite agar, xylose lysine deoxycholate agar, and hektoen enteric agar, biochemical screening, and serotyping. Fig. 15 describestheprotocolrecommended by the FDA.Recentstudiesindicate that modified semisolid Rappaport-Vassiliadis (MSRV) broth is very effective at yielding results that are better than or equal to those obtained with RV broth (89).In the last 10 years several research laboratories and commercial firms have described more rapid screening methods (90). Several of these methods have been recognized and approved by international agencies.
147
Pathogens Transmitted by Seafood Weigh 25 g of tissue Add 225 ml Lactose broth and blendfor 2 minutes
+
i
Transfer to wide-mouth, screw cap jar (500 ml and let stand for 60 min at room temperature
*
Mix well, adjust pH, if necessary to 6.8 0.2 Add up to 2.25 ml of surfactant (Tergitol Anionic 7 or Triton X - l 00)
+
Loosen jar lids and incbbate 24 k 2 h at 35°C
Transfer 0.1 ml of mixture
Rappaport-Vassiliadis (RV) broth Incubate 245 2 h at 42 rt 0.2%
Tetithionate (TT) broth Incubate 24 rt 2 h at 43f 0.2%
+
Transfer loopful from TT and RV broths onto: Bismuth Sulfite (6s) agar Xylose Lysine Deoxycholate (XLD) agar Hektoen Enteric (HE) agar Incubate 24 f 2 h at 35°C
/
Pick 2 or more colonies from each selective agar Pick typical colonies onBS agar at 24 rt 2 h, 48 f 2 h Transfer to Triple Sugar Iron (TSI) agar, Lysine Ironagar Incubate 24 k 2 h at 35°C
+
Apply biochemical and serological tests to presumptive Salmonella from TSI agar Urease test Polyvalent flagellar(H) test Lysine decarboxylase broth Phenol red dulcitol broth KCN broth Malonate broth Indole test Polyvalent somatic (0) test Phenol red lactose broth Phenol red sucrose broth Methyl Red-Voges Prokauer tests Simmons citrate
Fig. 15
Enrichmentandisolationprocedurefor
Salnronella
species
(90).
Herwig
142
3.
Escherichia coli
(1. Disecrse. The bacterial species E. coli has been described and known for many years. The organism is a common inhabitant of the facultative anaerobic microflora found in the intestinal tracts of humans and other warm-blooded animals. E. coli is a gramnegative, motile rod, capable of fermenting lactose and other sugars at 35°C. This organism is a member of the family of bacteria known as the Enterobacteriaceae. Isolates can be serologically differentiated on the basis of three major surface antigens, the somatic (O), flagellar (H), and capsule (K) antigens. Most of the strains found i n humans and in contaminated water samples are avirulent. Only certain strains of E. coli are pathogenic to humans and these strains are referred to as theenterovirulent E. coli (EEC) group. Four classes of E. coli are generally recognized: (a) enterotoxigenic E. coli (ETEC), (b) enteropathogenic E. coli (EPEC), (c) enterohemorrhagic E. coli (EHEC). primarily of the serotype 0157:H7, and (d) enteroinvasive E. coli (EIEC) (91). In addition to these four classes, diffuse-adhering E. coli (DAEC) and enteroaggregative E. c d i (EaggEC) strains have been recently recognized and associated with diarrhea in children in certain parts of the world (92). ETEC causes gastroenteritis andis sometimes called travelers’ disease. The infective dose is IO’ or more cells, possibly less for infants. This E. coli class is not a problem in countries with high sanitary standards. Humans are the principal reservoir of ETEC that cause human disease. EPEC causes watery or bloody diarrhea primarily i n infants, with few cases reported for adults. The most common foods associated with this disease are raw beef and chicken, and infant formula prepared with contaminated water. Humans are an important reservoir for EPEC. EHEC, primarily associated with serotype 0157:H7, causes hemorrhagic colitis, a disease that can be particularly severe for infants and young children. The vehicle generally associatedwith this disease is raw or undercooked ground beef. Cattle appear to be the natural reservoir for E. coli 0157:H7. EIEC causes bacillary dysentery, a mild form of dysentery, where there is the appearance of blood and mucous i n stools. The infective dose for EIEC is thought to be low and it is associated with food contaminated with human feces, since humans are a major reservoir.
h. Ecology. E. coli andothercloselyrelatedentericbacteriaarenonnalinhabitants of the guts of humans and other warm-blooded animals. Pathogenic strains of E. c d i may be a problem in shellfish and fish that are harvested from water containing human and other fecal pollution. Even though there are few reported cases of E. coli causing shellfish-borne disease, E.coli is a useful organism for monitoring the environment and shellfish for fecal contamination.
c. Mic.rohiolo~~iic.LI1 Procedures. Proceduresfor the enumeration of E. coli and fecal coliforms have beenusedformanyyears. The bacteriological quality of surface water, drinking water, and shellfish growing water is generally determined and regulated based on the presence and levels of fecal coliform and E. coli. The term “fecal coliform” is described by an operational definition, that is, by the ability to ferment and produce gas in particular media, at an elevated temperature, within a specific time. Classical enumeration procedures for fecal coliforms include the inoculation of a series of broth tubes for initial enrichnlent, followedby the transfer of positive tubes into more selective media. Using a series of dilutions of broth tubes results in the calculation of an MPN based on the number of positive tubes overa series of three dilutions. Conventional MPN procedures for total and fecal coliforms and E. coli require several days for incubation of the inoculated
Pathogens Transmitted by Seafood
143
media and aliquots from positive tubes from one type of medium to another at very specific times. Fig. 16 describes the conventional MPN procedure beginning with a presumptive MPNs (91). To test using lauryl sulfate tryptose broth and brilliant green lactose broth perform the fecal coliform test, small aliquots from positive lauryl sulfate tryptose tubes are transferred toEC broth tubes. Positive tubes from this step are streaked onto a differential medium and later presumptive colonies of E. coli are examined in a series of biochemical tests (Fig. 16). For the enumeration of fecal coliforms in shellfish growing waters A-
Weigh 50 g tissue, add450 ml phosphate-buffered dilution water Blend for 2 minutes
t
Prepare declmal dilutionsof blended material
Inoculate Lauryl Sulfate Tryptose(LST) MPN tubes Incubate for 48 ? 2 h at 35°C Examine after 24 rt 2 h and again at 48 k 2 h
+
Tubes are scored positiveif gas is present Transfer loopful from positive tubesto Brilliant Green Lactose Bile (BGLB) tubes
t
Calculate coliform MPN based on positve BGLB tubes, those with gas
EC Broth Method for Fecal Coliforms and Confirmed Test for E. coli Transfer loopful from positive LST tubes from the PresumptiveTest to EC tubes Incubate 48 rt 2 h at 45.5"C Examine after24 rt 2 h and again at 48 rt 2 h
+ +
Tubes are scored positive if gas is present
Streak loopful from positivetube to Levine's Eosin Methylene Blue (L-EMB) agal Incubate 18-24 hat 35°C
Transfer suspected E. coli colonies to Plate Count agar slants Perform biochemicaltests Gram stam Indole production Voges-Proskauer (VP)-reactive compounds Methyl red-reactwe compounds Citrate utilization Gas lrom lactose
Calculate E. coli MPN based on propbrtionof EC tubes that contain E. coli
144
Herwig
+ + +
Collect water sample
Prepare decimal dilutions
Inoculate A - l MPN tubes Incubate 3 h at 35"C, followed by 21 h at 44.5%
Tubes are scored positiveif gas is present
Fig. 17 MPN protocolusing A - l medium for shellfish growing waters (91).
1 brothcanbeused. Water samples are incubated at 35°C for 3 hours before theyare moved into a 44.5"C water bath incubator (Fig. 17). A newer variation of the conventional protocol incorporates the use of a fluorogenic substrate, 4-methylumbelliferyl-~-D-glucuronide (MUG), thatwillfluoresce under UV light when the enzyme P-glucuronidase cleaveslnethylutnbelliferonefromthesubstrate(Fig. 18). Achromogenicsubstrate 5 bron~o-4-chloro-3-indoyl-~-D-glucuronide (S-GLUC or BCIG) can also be used. These substrates have been incorporated into several selective media for the Enterobacteriaceae for rapid detection of E. coli (93). Chromogenic and fluorogenic substrates are used in several commercially available systems, such as those with the trade name Petrifilm, for the enumeration of coliforms and E. coli (94). At the present time there are no simple procedures available for the direct cultivation of EPEC, ETEC, and EIEC from foods. Weigh 25 - 50 g shellfish meat and add phosphate-buffered dilution water Blend for 2 minutes
PreDare serial dilutionsof blended material
Incubate Lauryl Sulfate Tryptose(LST) tubes as in Conventional MPN Procedure
I
t
Transfer loopful from positive LST tubes to EC-MUG broth tubes Incubate 24 h at 44.5 f 0.2%
t
Determine fluorescencein tubes All tubes that fluoresceare positive Calculate E. coli MPN
Fig. 18 EC-MUG method for dctermining E. coli MPN i n shellfishnle:l(s (91 ).
Pathogens Transmitted by Seafood
145
Since Shiga toxin-producing E. coli (STEC), especially serotype 0157:H7, arewell recognized as in1portallt food pathogens, several selective media have been described for these in detail in regulatory manuals organisnls (89). The conventional procedures are described such as the Bacteriological Analytical Manutrl (9 l), Oflcial Metl1od.s of Anc4wi.s (?fAoAC IilterilatiollLl[(95), and Recornmended Procedures for the Ex-nminarion of Sea W m r and Shellfish (96).
4.
Campylobacter jejuni
(1. Diseclse. Ctlinpglobacter jejuni is a gram-negative,curved,andmotilerod.It is a nlicroaerophilic organism requiring reduced concentrations of atmospheric oxygen for growth. Because of its microaerophilic requirements, optimum conditions for growth are achieved in the laboratory when 3-5% oxygen and 2-10% carbon dioxide are Provided. Before 1972 and after techniques were developed to culture this Organism from i primarily associated with abortion and enteritis feces, it was thought that C. j e j ~ n was in sheep and cattle. In recent years, however, C. jejuni is believed by some to bethe leading cause of bacterial diarrheal disease in the United States, causing more cases than S c h n o n e l k ~and Slligellrr combined (97,98). C. jejuni andothercloselyrelatedspecies,forexample, C. lari, C. coli, and C. }~?'ointestir~e~lis, cause diarrhea that may be watery or sticky and may contain blood and fecal leukocytes. It is believed that 99% of cases are caused byC. jejuni. Other symptoms that are often present include fever, abdominal pain, nausea, headaches, and muscle pain. The illness usually occurs 2-5 days after ingestion of contaminated food or water and than usually lasts 7-10 days. The infective dose is considered to be small, perhaps less 200 organisms (99). The pathogenic mechanisms for C. jejuni are still not completely understood. Death from C. jejuni is extremely rare, but there are reports of abortion induced by the organism. Some investigators suspect that C. jejuni is the leading cause of gastroenteritis in the world (100).
b. E c o l ~ ~ q y .C. jejuni isnotusually found in healthyhumans, but it is found in healthy farm animals, birds, and flies. In foods, C. jejuni is frequently associated with raw chicken; raw milk is also a source. C. jejuni is sometimes found in surface waters, such as streams and ponds. Shellfish have been implicated as a vehicle in human cases of C. jejunienteritidis (101,102).In 1993,69% of investigated mussels and 27%of oysters traded in the Netherlands were found to be contaminated with Ccrtnpylobacterspp. (1 03). Initial and later testing suggestedthat most of the isolates were C. lari. C. lari is a thermophilic species that was first isolated from gulls and subsequently from other avian species, dogs, cats, and chickens. Throughout the world, C. jejuni is the most significant known agent of human gastroenteritis. C. lari is isolated much less frequently from clinical samples, but is also recognized as a causative agent of gastroenteritis in humans (104). Studying the distribution of Cnrnpylobacter along the West Coast of the United States over an 8-year period, Abeyta et al. ( I 02,105) indicated that Camnpylobacter are well distributed in shellfish growing waters. In temperate aquatic environments, Campglobacter may exhibit a seasonality in its population numbers. Wilson and Moore (106) reported that few Cmlpylobacter were recovered between May and August; most were foundin the cooler winter months (November-March). Less than 6% of the shellfish examined between May and August contained C~rmpylobacter.Thermophilic Campylobocter spp. were detected in 42% of the shellfish. The greatest percentageof these (57%) were urease-positive thermophilic campylobacters
Herwig
146
(UPTC), atypical types that do not appear to be closely associated with domestic or farm animals or man. Their significance in foodborne human diseaseis probably minimal. These atypical Ccrmpvlobncters have not been commonly reported elsewhere. This may be the result of misidentification as C. coli.
c. Microbiologicc~lProcedures. Cc~mpylobcrc~ter areusuallypresentin highnumbers in the diarrheal stools of individuals, but isolation requires antibiotic media and a special microaerophilic atmosphere. Crrmpylobcrcter was difficult to culture until protocols for using the appropriate gas atmospheres were developed for enrichment and isolation of this organism. Ccrmpylobc~cterwill grow only under microaerophilic (microaerobic) conditions which can be supplied using special gas mixtures purchased from gas vendors or by using special “gas-paks” that generate the proper atmosphere within an anaerobe jar. More recently Abeyta et al. (105) reported that selected enrichment broths supplemented with the commercially available enzyme Oxyrase were evaluated for recovery of Ce~mpylobercterunder nornlal atmospheric conditions from shellfish. Results indicated that the Oxyrase enzyme was useful. Growth of the organism is not very fast, and isolation of C. jejurzi from food may take several days to a week. Water samples can be tested for the presence of Ce~mpylobc~ctrrby filtering samples through a 0.45 pm positively charged filter and adding the filter to an enrichment broth, a Bolton broth withantibiotics.Afterincubation in the enrichment broth, aliquots are streaked onto selective medium and incubated under microaerophilic conditions. Presumptive Cclmpylobacter colonies are tested with a variety of physiological and biochemical tests. Shellfish and other fishery products are examined in a similar fashion, except that the samples are placed in a stomacher or blendedto prepare a homogenate. Fig. 19 shows the suggested protocols for testing for Ccrmpylobcrcrer in water and shellfish samples as suggested by Hunt et al. (97). 5. Listeria monocytogenes cl. Disease. Listericl monocytogerws is a gram-positiverod,motile by means of peritrichousflagella.Theorganism is not a spore former. Some studies suggest thata significant number of humans Inay be intestinal carriersof this organism. It has been found in a variety of mammals, both domesticated and wild, a number of different bird species, and in some species of fish and shellfish (107,108). Lennon et al.(109) described a perinatal outbreak in Auckland, New Zealand, and hypothesizedbut did not prove an association with raw fish or shellfish. Schlech (1 IO) provided an extensive review on listeriosis and suggested that there has been a significant increase in the incidence of the disease. Listeriosis is an atypical foodborne pathogenof public health concern because of the severity and the nongastroenteric nature of the disease. While a relatively unco1nn1on disease, the high mortality rate suggests the need for continued vigilance and study. Listeriosis is clinically defined when the organism is isolated from blood, cerebrospinal fluid, or from otherwise sterile sites. The symptoms and outcome of listeriosis can be very serious and include septicemia, meningitis, encephalitis, and intrauterine or cervical infections in pregnant women which may result in spontaneous abortion or stillbirth. The onset of these disorders is usually preceded by influenza-like symptoms such as nausea, vomiting, and diarrhea. The onset time of serious forms of listeriosis is unknown but may range from a few days to 3 weeks
(107,108). The infective dose for
L. mo~zoc~ytogenes is unknown but is believed to vary with
147
Pathogens Transmitted by Seafood Water Samples Filter 2 - 4 liters through 0.45 pm positively-charged filters
t
Add filter to 100 m1 of Bolton broth with antibiotics Incubate 3 h at 30"C, followed by 2 h at 37°C under microaerobic conditions
+ +
Incubate, with shaking28 h at 42 C, without shaking 48 h at 42°C
I
t
Streak enrichmentsonto Abeyta-Hunt-Bark agar or Modified Campylobacter Blood-Free Selective agar base (CCDA) using anerobic jars under microaerobic conditions at 37-42°C
Test presumptive Campylobacter colonies with physiological and blochemical tests Hippurate hydrolysis Antibiotic sensitivities Nitrate reduction H2S from cysteine
Triple Sugar Iron (TSI) Temperature range of growth 3.5% NaCl
Glucose utilization MacConkey agar Glycine
Shellfish Samples Measure 100-200 g shell liquor and meat Blend at low speedor stomach for 60 seconds
Remove 25 ghomogeGate for sample analysis Add to 225 Bolton broth(1:l 0 homogenate)
t
+
Remove 25 g of i:l 0 homogenate Add to 225 Bolton broth (1:lOO homogenate)
Incubate 4 h at 37°C - for samples producedor processed c10 days previously OR Incubate 3 h at 30°C, followed by 2 h at 37°C - for frozen samplesor samples produced or processed 2 10 days previously
t
Incubate, with shaking28 h at 42°C: without shaking 48 h at 42°C
Streak enrichments onto Abeyta-Hunt-Bark agar'or Modified Campylobacter Blood-Free Selective agar base (CCDA) using anerobic jars under microaerobic conditions at 37-42°C
Test presumptive Campylobacter colonies with physiological and biochemical tests
Fig. 19 Protocols for examining water and shellfish salnples for Cunrpylohactcr species (97)
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148
the strain and the susceptibility of the human. Fewer than 1000 organisms can cause the disease.Ingested L. tnonocytogenes invades thegastrointestinalepithelium,entering monocytes, macrophages, or polymorphic leukocytes. The presence of L. rrrorrocytoSerlc’s in phagocytic cells permits access to the human brain and probably allows for migration tothe fetus in pregnant women. The pathogenesis of L. rrror~ocyrogerrescenters on its ability to survive and multiply in phagocytic host cells (107,108).
b. Ecology. L. monoc.ytogerles differs in many aspects from most other foodborne pathogens because it is ubiquitous and is resistant to diverse environmental (and food processing) conditions such as low pH and high NaCl concentrations. This pathogen is microaerobic and psychrotrophic. Two properties that contribute to its widespread distriit bution in the environment are its ability to survive for long periods of time, although is not a spore former, and its ability to grow at low temperatures. L. ttrorlocyroserlc’.s is widely distributed in a number of mammalian, avian, fish, and shellfish species (107,108). The organism appears to be widely distributed in freshwater and less so in bottom sediments. Colburnet al. (1 1 1) conducted a study to determine the incidence of Listeritr species in freshwater tributaries draining into Jumboldt-Arcta Bay in California. Lister-icr species were detected in 8 1% of freshwater samples and30% of sediment samples. Whether these organisms are indigenous to freshwater or are the result of runoff from livestock found in the vicinity of the rivers is uncertain. The organism is very hardy and resists the effects of freezing, drying, and heat. L. tnowocytogetzes has been associated with raw milk, cheeses (primarily soft varieties), ice cream, raw vegetables, fermented raw-meat sausages, poultry, raw meats, and raw and smoked fish. Seafoods, particularly ready-to-eat forms, have been frequently reported to be contaminated with L. rtrotloc~togewes( 1 12-1 14). The organism has the abilityto grow in temperatures as low as 3OC, permitting growth i n refrigerated foods. This characteristic may be a particular problem in ready-to-eat foods, such as cold smoked salmon and cooked fish products, since these products are not further heat processed before they are consumed. A relativelyhigh incidence of the organism (6-369’0) has been found in these products ( 1 13).
c. Microbiological Procedures. The traditional methods of analyzing food for L. monocytogenes are complex and time consuming, requiring a total time of 5-10 days. Protocols ( 1 15) are outlined in Fig. 20. Sample processing begins withan enrichment step using trypticase soy broth which is supplemented with yeast extract, phosphate salts, and pyruvic acid. Selective agents (acriflavin, nalidixic acid, and cyclohexamide) are added to the enrichment. Aliquots from the enrichment are streaked onto selective media and then presumptive L. rr7orlocytogerle.s colonies are examined in several biochemical and physiologicaltests.Finally,serologic,mousepathogenicity,andtheChristie,Atkins, Munch-Peterson (CAMP) testare performed. In recentyears,specificnonradiolabeled DNA probes have been developed which allow for simpler and faster confirmation of suspect isolates. Agersborg et al. (1 16) detected L. nrot1ocytoRene.s in seafood products using PCR and primers for fragments of the lysteriolysin 0 (/dy) gene and forthe invasionassociated protein gene (icrp). They were able to detect one to five L. trrorlocytogerlescells in S g of product in 55 hours.
6.
Yersinia enterocolitica
N. Disease. Thegenus Yersinicr hasthreepathogenicspeciesforhumans: Y. ellterocolitic.cr, a common intestinal pathogen in humans; Y. pseuclotuberculnsis, an intestinal pathogen in rodents which occasionally infects humans; and Y. pestis, the infectious agent
149
Pathogens Transmitted by Seafood Weigh 25 g of sample and add 225 ml of Enrichment Broth (EB) [Trypticase Soy Broth with Yeast Extract (TSBYE) supplemented with monopotassium phosphate, disodium phosphate, pyruvic acid]
I
+
Blend or stomach. Transfer to 500 ml flask Incubate 4 h at 30°C
+
Add selective agents acriflavin, nalidixic acid,and cycloheximide, and incubate additional44 h for a total of 2 days at 30°C
At 24 and 48 h, streak EB culture ontoboth Oxford Medium (OXA) and Lithium Chloride Phenylethanol Moxalactum (LPM)agar or LPM + esculin/Fe3+ agars. PALCAM agar may be substituted for LPM agars Incubate OXA andPALCAM media for 24-48 h at 35°C Incubate LPM mediafor 24-48 h at 30°C
Streak typical Listeria colonies from OXA and LPM (or PALCAM) onto TrypticaseSoy Agar with Yeast Extract (TSAYE)
+ +
Perform biochemical and physiological tests Cellular morphology and motility Catalase Gram stain Carbohydrate testing Hemolytic activity
Perform serological, mouse pathogenicity,Christie-Atkins-Munch-Peterson (CAMP) test
of bubonic and black plague. As members of the Enterobacteriaceae, Yersini(l are small, rod-shaped, gramncgative. oxidase-negative, facultative anaerobes that ferment glucose. Y. cwter’ocdificlr is often isolated from clinical specimens such as wounds, feces, sputum, and lymph nodes. It is not considered part of the normal human microflora. Y. entewcolirictr is a heterogeneous species, being separated into several subgroups according to biochemical activity and somatic (0)lipopolysaccharide antigens ( 1 17- 119). Yersiniosis in humans is characterized by synlptoms of gastroenteritis with diarrhea and/or vomiting. Most systematic infections occur in children, especially those less than 5 years old. Fever and abdominal pains are the hallmarks of this disease, with the organism causing physicians to misdiagnose appendicitis. Crohn’s disease, and mesenteric lymphadenitis. Most symptomatic infections result in self-limiting diarrhea. Yersiniosis may also give rise to a variety of autoimmune complications including reactive arthritis, erythema nodosum, iridocylitis, Homerulonephritis, carditis, and thyroiditis. These diseases have been reported to follow acute infection ( 1 19). This outcome is infrequent before the age of I O years and
750
Herwig
occurs most often in Scandinavian countries. Yersiniosis is a rare disease in the United States, but appears to be a more common disease in northern Europe, Scandinavia, and Japan. b. Ecology. Y. etlterocoliticcl hasbeenfound in a variety of environmentsand has been isolated from the intestinal tracts of many different mammalian species, as well as from birds, frogs, fish, flies, fleas, crabs, and oysters ( 1 19). Y. etlterocoliticn can be found in meats, raw milk, fish, and oysters. This organism is also commonly found in a variety of terrestrial and freshwater habitats, including soil, vegetation, lakes, rivers, wells, and streams. Pigs are the only animal species from which Y. enterocoliticcr of biovar 4 serovar 0 3 , the variety most commonly associated with human disease, has been isolated with any degree of frequency. Individual isolates of Y. erlterocditicxr from pigs and humans appear identical with each other in terms of serovar, biovar. RFLP of chromosomal and plasmid DNA, and virulence determinants ( 1 17). Y. enterocoliticrr, including pathogenic strains, can survive for extended periodsin the environment, such as in soil, vegetain rivers than tion, streams, lakes, wells, and spring water (120). Its survival was lower i n soils; Chao et al. ( 120) suggest that the presence of protozoans in the aquatic environment are responsible for their enhanced decline. The relatively low optimum temperature for growth may account for the higher incidence of yersiniosis in temperate regions of the world and the tendency for infections to peak during late autumn and winter where Y. crlterocoliticrr is endemic (121 ). c. Mic.robiologicer1 Procedures. If highconcentrations of Y. erlteroc-oliticcr are 36-48 hours;however, suspectedthen thisspeciescanbepresumptivelyidentifiedin confirmation or enrichment of samples for Y. enterorditiea may add another 2-3 weeks to the procedure. Fig. 2 1 summarizes the protocol ( 1 18) for the enrichment and isolation of Y. enterocoliticcr. Samples for enrichment are placedin peptone sorbitol bile broth and incubated for 10 days at 10°C. This step takes advantageof the ability of Y. erlterocoliticcr to grow at lower temperatures. Following the enrichment step, samples are streaked onto selective agars and presumptive colonies are tested through a series of differential media, physiological, and biochemical tests. Some investigators have recommended the use of two enrichment media, suggesting thatnosingle procedure will recover all pathogenic serotypes. The second enrichment method uses irgasan ticarcillin chlorate broth followed by plating on Snlrnot~ellrr-Shi~~e/ler agar (89).If high countsof this organism are suspected, aliquots of the homogenized sample can be directly plated on selective media and later colonies can be probed with a virulence gene probe. Determination of pathogenic strains requires additional steps and may include an autoagglutination test, the low calcium response Congo red agarose virulence test, and the crystal violet binding test. Other tests include DNA colony hybridization with probesto detect virulence factor genes. and intraperitoneal infection of adult mice pretreated with iron dextran and desferrioxamine B. Screening isolates for invasive potential usingan in vitro HeLa cell assay is alsoan effective procedure for determining pathogenic strains.
7 . Plesiomonas shigelloides cr. Disetrse. P1esionIoncr.s.skigelloitle.s is a motile,oxidase-positive,catalase-positive, facultative anaerobic, gram-negative rod that has been isolated from freshwater, freshwater fish, and shellfish and from many types of domesticated and wild animals. The genus are consists ofa single species. Manyof the phenotypic properties found in P. .shi,~e/loidr.s shared with the genera Vibrio and Aerornonas. For example, P. .shic~e//oicfe.es is Silnilar to
751
Pathogens Transmitted by Seafood Weigh 25 g of sample, add 225 ml Peptone SorbitolBile Broth (PSBB) Homogenize 30 seconds
t
Incubate 250 ml of homogenate for 10 days at 10°C OR
1
If high counts of Yersiniasuspected, spread-plate0.1 r n l of homogenized sample onto Macconkey agar or Celfsulodin-lrgasan-Novobiocin(CIN) agar, and transfer 1.O ml of homogenate to 9.0 ml solution of 0.5% KOH in 0.5% saline and spread-plate onto MacconkeyAgar and CIN Agar Incubate for 24 h at 30°C
Perform colony hybridization withYersinia virulence gene probe Transfer 0.1 PSBB of enrichment to 1.0 ml solution of 0.5% KOH in 0.5% saline and mix Transfer 0.1 ml enrichment to 1.O ml to 0.5% saline Streak from each saline tube onto Macconkey agar and CIN agar Incubate 24 h at 30°C
4-J
Pick presumptive Yersinia colonies and inohlate Lysine Arginine Iron (LAI)agar slants, Christensen's Urea agar,and Bile EsculinAgar Incubate 48 h at room temperature
t
Cultures yielding typical reactions onLA1 agar are streaked onto Anaerobic Egg Yolk (AEY) agar Incubate at room temperature
Perform gram stain, biochemical,and other physiological tests Lysine, arginme. ornithine decarboxylase Phenylalanine deaminase Motility at 2226°C and 3537°C Acids from: mannitol, sorbitol, cellobiose, adonitol. Inositol, sucrose, rhamnose, raflinose, meliblose. salicin, trehalose, xylose Simmons citrate Indole Voges-Proskauer test Llpase beta-D-glucosldase test Pyrazinamidase
152
Herwig
Vibrio spp. in its susceptibility to the vibriostatic agent O/ 129. Like Aerornontrs species, P. shigelloides does not have a requirement for sodium and is unable to grow in 6% NaC1. Humans and other animals may be carriersof the organism, showing no overt sylnptoms of disease. Much of the earlier information about this pathogen came from outside of the United States, but in recent years studies from the United States are supporting the role of this organism as a pathogen that causes diarrhea. Many of these cases are associated with the consumption of raw bivalve mollusks or with foreign travel (122,123). p . shigel1oide.s may cause a mild self-limiting gastroenteritis with fever, chills, abdominal pain, nausea, diarrhea, and vomiting with symptoms beginning 20-24 hours after consumption of the contaminated food or water. The infection may cause diarrhea for 12 days in healthy adults. There may be high fever and chills, and protracted dysenteric symptoms in infants and children under 15 years of age. The infectious dose is presumed to be quite high and is thought to be more than one million organisms. In healthy people, gastrointestinal illness caused by P. skigelloides infection may be so mild that infected individuals do not seek medical treatment. consequently the number of cases associated with this organism is assumed to be vastly underreported (124,125). A cluster of cases occurred in North Carolina in November 1980 following an oyster roast. Thirty-six of 150 people who had eaten roasted oysters experienced nausea, chills, fever, vomiting, diarrhea, and abdominal pain beginning 2 days after the roast. The averag duration of the symptoms was 2 days. P. skigelloides was recovered from oyster samples and patient stools (126). b. Ecology. P. shigelloides is a gram-negativerod thathas beenisolatedfrom freshwater, freshwater fish, and shellfish and from many types of domesticated and wild aninlals ( 123,127-130). Most human infections caused by this organism are suspected to be waterborne. The organism may be found in unsanitary water that is used for drinking water, recreational water, or water used in food processing. Studies indicate a seasonal effect from environmental sources, with an increase i n the reported casesof diarrhea during the warmer months ( 127,128).
c.MicrobiologicalProcedures. P. s1~igelloide.scan beculturedonInany of the media used for isolation and enrichment ofthe enteric bacteria. This speciesis tolerant of bile salts and brilliant green. Because lactose is generally slowly fermented, P. shigelloides appears as lactose negative on solid media. Because of a lack of competitiveness, enrichment techniques may be of limited usefulness (13 l). Procedures for the enrichment and isolation of P. shigelloides are not standardized and have not been published by the FDA ( 132). Koburger and Wei (124) suggesteda protocol where samples are diluted and plated onto Inositol brilliant green bile salt agar and PL agar. One to 10 g of sample are added to 90 m 1 of (etrathionate broth. Following a 24-hour incubation period, suspected colonies are examined in differential media and the enrichment broth is streaked onto the isolation media. Fig. 22 outlines the procedure (124). P. sl~igelloidescan be differentiated from Aeromonas spp. using a series of biochemical tests.
8. Aeromonas hyrophila and Other Aeromonas Species
N. Disease. Aerotnonu h y h p k i l n andother Aerornonas speciescanoften be isolated from food and the environment. CertainAerolnonus species are human pathogens that may cause gastroenteritis. Among the suspected foods are prefrozen or inadequately cooked seafood and oysters (133). Based on hybridization studies, the taxonomy and speci-
Pathogens Transmitted by Seafood
153
Add 10 g of sample to 90 ml Tetrathionite Broth Incubate at 40°C for 24 h
+
Streak onto duplicate platesof two of the following selective media Incubate at 35°C for 24 h Macconkey agar Billiant Green Lactose Bileagar PL agar Salmonella shigella agar Inositol Brilliant GreenBile Salts agar
+ +
Pick three typical coloniesfrom each of the selective media into Triple Sugar Iron agar and Inositol Gelatin deeps Incubate at 35°C for 24 h
Perfom oxidase test and gram stain from TSI slant
ation of the genus A P I Y ) I ) I Ounderwent II~ revision in 1980s. The taxonomy of the genus is still evolving and still confusing. Since DNA-DNA hybridization is not routinely employed in most clinical laboratories. most still rely on a series of phenotypic tests to differentiate Aeronlo/m species. The species name A. hydrophilrr has been broadly used i n the older literature to refer to the whole group of mesophilic aeromonads, but it should he more narrowly definedby the phenotypic characterizationof Popoff or DNA-DNA hybridization. If the speciation of a strain is in question, or if an isolate has not been thoroughly Aer.otnmm sp. or aeromocharacterized, then investigators should refer to the isolate as nads. Based on phenotypic testing and definitions, the generally accepted speciesof Aeromo)uI.s associated with diarrhea in humans are A. hydrophiltr, A. ( ~ r v i wA., \ ~ c ~ l n t r ibiovar i sobricl and biovar lvrorrii ( 134,135). Two types of gastroenteritis have been associated with A. hydrophila, a cholera-like illness with “rice water” diarrhea, and a dysenteric of illness characterized by loose stools containing blood and tnucus. The infective dose the organism is unknown, but SCUBA divers who have ingested small amounts of watcr have become ill and A. hydrophilu has been isolated from their stools ( 1 36).
b. E ~ o l o ~ y y .AcJromo1russpp. are commonly associated withfish and seafood and arewidelydistributed in the aquatic environment (137). Most of the published studies have dealt with A. /ryhophi/u, a bacterial species that is present in all freshwater environments and in estuaries. Some strains are capable of causing disease in fish and amphibians as well as humans. who may acquire infections through open wounds or by ingestion of organisms in food orwater. A. /rydrop/riI~rhas beenfrequentlyisolatedfromfishand shellfish. It has also been found in market samples of red meats and poultry. There are
154
Herwig Prepare dilutionsor use 25 gof original samplefor enrichments For dilutions, weigh 25 g and blend with 225 ml of 0.1% peptone water
+
For enrichments and MPN procedures, inoculate sample into Alkaline PhosphateWater or Tryptic Soy Broth Containing30 mg/L ampicillin Incubate at 28°C for 24 h
Streak enrichmentsor MPN tubes onto Starch Ampicillin (SA) agar or Bile Salts Brilliant Green Starch (BBGS) agar Incubate at 28°C for 18 to 24 h
+ + +
Flood the surface of the agar plates with Lugol's iodine solution Count typical colonies that have surrounding zones of starch hydrolysis
Pick typical colonies and streak onto nutrient agar that does not contain carbohydrate, such as Nutrient agar, TrypticSoy agar Streak DNase testagar
Perform gram stain, catalase test, resistance to 0/129 vibriostatic agent Inoculate Kaper's medium and observe reactions
relatively few published cases in which foodborne gastroenteritis.
Aerotnolm species have been associated with
c. Mictnbiological Procedures. Protocolsfor the enrichmentandisolation of pathogenic species of Aerovronm have not been officially accepted by the FDA i n the United States. Media for isolation of aeromonads usually exploit a resistance to ampicillin. Palumbo et al.(138) described a protocol for the isolation and identificationof Aerorwrltrs species from food (Fig. 23). Food samples can be blended and diluted i n peptone water and spread onto a surfaceof starch ampicillin agar or bile salts brilliant green starch agar. The latter medium is suggested by investigators who formulated it for samples i n which a large number of Proteus spp. might be encountered. Typical colonies on these media are identified after incubating by flooding the surface with an iodine solution. If low numbers of A e r o ~ n ~spp. ~ ~are m expected, food samples can be inoculated into enrichment broths or a seriesof MPN tubes with alkaline peptone water or tryptic soy broth containing ampicillin. After 24 hours, aliquots from the enrichments are streaked on the plating media. Suspected colonies from the plating media canbe tested for additional phenotypic proper-
Pathogens Transmitted by Seafood
155
ties to identify whether A. hyiro~philaor other Aewtw11m species were isolated. Species identification is confirmed by performing a series of biochemical tests (125,138).
B. Viruses There are four major categories of viruses that cause gastroenteritis in humans: rotavirus, enteric adenovirus, calicivirus (that includes Norwalk virus and its relatives). and astrovirus. Hepatitis virus is a foodborne virus whose symptoms and signs are associated with liver disorders. Raw shellfish havebeen implicated in outbreaks of foodborne viral gastroenteritis in the United States, Europe, and Australia (139-143). Outbreaks have occurred following the consulnption of shellfish harvested from waters contaminated with human sewage (143,144). Mostof the viruses associated with seafood-borne disease are calicivirus and hepatitis A. Table 7 lists the structural features and diseases caused by viruses associated with foodborne disease. Unlike most bacteria, viruses are not easily inactivated by normal cooking procedures. Shellfish tissue may "protect" the virus and individuals may become ill after consuming cooked shellfish that are contaminated with enteric viruses. To study the extent of the hazard presented by oysters contaminated with virus, DiGirolamo et al. (145) contaminated samples of whole and shucked Pacific oysters with IO' plaque-fonning units of poliovirus/ml and heat processed the oysters in four ways: stewing, frying, baking, and steaming. Results indicated that a significant portion of virus in oysters withstood these methods of processing. The survival rate varied from 7% to 10%. Cooking experiments performed with contaminated mussels revealed that S minutes after the opening of the mussels' valves, rotaviruses and hepatitis A virus could still be recovered in the steamed shellfish (146). While depuration or relaying shellfish may be a satisfactory method for removing pathogenic bacteria from these animals, such methods must be used with caution for the removal of human virus. Power and Collins (147) studied the elimination of poliovirus, E. coli, and a coliphage from the common mussel (Myrillrs cdulis). In their experiments. the relative rates of elimination during depuration were E. c w l i > coliphage > poliovirus. It was concluded that E. coli cannot beareliableindicator of viruseliminationfrom mussels. A dramatic experiment that showed the potential danger of relying on indicator bacteria and depuration was performed in Australia. Following the widespread outbreaks of oyster-associated gastroenteritis in Australia in 1978. several programs were introduced to minimize the occurrence of further outbreaks. One program included the depuration of oysters and the use of human volunteers, as an interim measure, to test samples before sale to the public. After eating Georges River oysters that were thought to be safe. S2 volunteers became ill and Norwalk virus was foundto be the cause of the infection. Depuration, as carried out in pollution-free water for 7 days, was therefore concluded to be an unsatisfactory method ( 148).
1. Hepatitis A Virus (1. Disctrw. HepatitisAvirus (HAV) is also referred toas Heptovirus A. and i n older literature as infectious hepatitis. Five kinds of hepatitis are recognized: A, B. C, D. and E. Hepatitis A and E are of concern with food, but type E is not found inNorth America. HAV is classified with the enterovirus group of the Picornaviridae family. Like other enteric viruses, HAV is transmitted by the fecal-oral route. The most common form
Herwig
156
Table 7 Structural Features and Symptoms of Human Viruses Associated with Seafoods,’
Virus Virus most ,frequently trssociaterl with seafood:
Hepatitis A
Calicivirus [includes Nonvalk virus and other small round structured viruses (SRSV)]
diameter, 27virus, round nm icosahedral symmetry, not enveloped, single stranded RNA, genome length of 7500 nth 35-39 nm diameter. round virus, icosahedral symmetry, not enveloped, one or two structural proteins, single stranded RNA, genome length of 7700 nt
Virus less ,frequently associated with seufood: 80 diameter, nm double-
Human adenovirus
Human poliovirus
shelled wheel-like capsid, icosahedralsymmetry.11 segments of doublestranded RNA, genome length of 18,500 nt 27-30 nm diameter, round surface and pointed star with 6 points, polyhedral symmetry, several structural proteins, single stranded RNA, genome length of 6800-7900 nt 75 nm diameter, not enveloped, fastidious growth in culture, double-stranded DNA, helper or satellite virions may be present, genome length is 30000360000 nt 28-30 nm diameter, round virus, icosahedral symmetry, not enveloped, single stranded RNA, genome length of 7400 nt
Structure and disease symptoms from sunlrnarles present (208). ” n t = nucleotides.
Epidemics of acute diarrhea and vomiting in older children and adults, often food or water borne
Major cause of severe dehy-
Rotavirus (group A)
Human astrovirus
Fever, malaise, nausea, anorexia, abdominal discomfort, followed by jaundice
111
drating diarrhea in infants and young children
Watery endemic diarrhea of children, in day care ccnters, some disease outbreaks, role in HIV-related diarrhea
Endemic diarrhea of infants and young children
Cause of polio in humans
Murphey et al. (23) and Blacklow and Herrmann
Pathogens Transmitted by Seafood
157
of transmission is person to person by fecal contamination. The incubation period for HAV ranges from 10 to 50 days with a mean of approximately 30 days, depending upon the number of virus particles consumed. The infectious dose is thought to be 10-100 particles. The virus infects the liver producing a debilitating, low-mortality disease that sometimes includes jaundice. The infected person is characterized by a sudden onset of fever, malaise, nausea, anorexia, and abdominal discomfort, followed by several days of jaundice. Death from HAV isveryrare. Most adultshaveanimmunity to HAV that provides lifelong protection against reinfection (23,149,150).
b. Ecology. HAV is an entericvirusthatiscloselyassociatedwithinfectedhumans and fecal contamination. Less than 10% of HAV cases are food associated or water associated. With foods, it is most frequently associated with bivalve mollusks, but it may also be found in foods associated with a large amount of handling, such as salads. In the later association, contamination may occur during preparation by a food handler who is infected with HAV. In the United States, between 5000 and 35,000 cases of food poisoning caused by HAV are estimated to occur each year. One of the more noteworthy outbreaks associated with seafood includes an outbreak in Sweden in 1955. Oysters held in a harbor awaiting sale around Christmas were contaminated with fecal pollution from a toilet that was over theharbor.Oystersfromtheharborwereeatenrawand629peoplebecame ill. This outbreak was the first report of HAV transmission by shellfish. In 1988 an HAV outbreak occurred in Shanghai, China. Clams taken from water that was contaminated with human sewage were eaten raw, resulting in nearly 300,000 cases of HAV infection (126). c. Microbiologiccrl Procedures m c l Detectiorl. Thediagnosis of an HAVinfection can be performed by detecting antibodies to HAV in human serum (126).
2. Human Calicivirus (Norwalk and Norwalk-Like Viruses) (1. Disetrse. Thehumancaliciviruses,whichincludeNorwalk-likevirusesorthe Norwalk family of viruses, are called small, round, structured viruses (SRSV) i n Europe. These viruses are 35-39 nm in diameter and are a serologically related group of viruses. Norwalk viruses are classified in the Caliciviridae family, and are positive sense, linear, single-stranded RNA viruses. Norwalk viruses cause gastroenteritis, typically with diarrhea and vomiting, with an incubation period of 24-48 hours and a duration of infection lasting from 24 to 60 hours. The disease caused by Norwalk viruses is often referred to as viral gastroenteritis. The infectious dose is thought to be low. The attack rate for Norwalk viruses is particularly high; often more than 50% of individuals who consulne contaminated food become ill. Humans appear to be susceptible to repeated infectionwith the same strain since the virus does not elicit a strong immunological response (23,149.150).
b. Ecology. Some investigatorssuspectthatNorwalkvirusesmaybeamongthe leading agents causing foodborne disease. Water is the most common source of outbreaks, but raw or insufficiently cooked bivalve mollusks have also been recognized as vehicles for transmission. Food outbreaks are often associated with the consumption rawofshellfish. In 1978 morethan 2000 people who ate oysters were infected in New South Wales, Australia. Some groups who consumed oysters showed attack rates of 85% (140). In December 1994 and January 1995, Florida experienced the largest outbreak of oyster-associated gastroenteritis ever reported. The largest shellfish and trout harvest for the Apalachicola Bay fishing industry occurs for the New Year’s holiday, when oyster
158
Herwig
roasts are a tradition. Of 223 oyster eaters, 58% became ill, compared with 3% of nonoyster eaters. Most oyster eaters ate only cooked (grilled, stewed, or fried) oysters. Oyster eaters who reported eating thoroughly cooked oysters were as likely to become ill cornpared to those who ate raw oysters. The outbreak demonstratedthat oysters cooked to the point where consumers consider them done or overdone werestill capable of transmitting enough virus to cause disease in a substantial proportion of people. The outbreak may have resulted from overboard dumpingof feces during a cornmunity outbreak of diarrheal illness. McDonnell et al. (15 1) suggested that using fecal coliform monitoring to define water quality is inadequate. Contamination of oysters with SRSV occurred during a time when routine water quality monitoring indicators were in acceptable ranges both in Florida and Texas. c. Mic~rohiologicdProceclrrres N I I ~Detectiorl. Norwalkvirusescanbedetected using serologic methods, but in recent years gene probes and PCR amplification methods have become increasingly more popular. While the DNA molecular procedures are theoretically very sensitive and rapid, nluch of the research effort has been directed toward developingmore efficientextractionproceduresandreducingtheproblemsassociated with inhibitory compounds that are present i n complex food matrices. I n 1996. Atmar et al. (152) described a multicenter, collaborative trial that was performed to evaluate the reliability and reproducibility of a reverse transcription PCR method ( 153) for the detection of Norwalk virus in shellfish tissues. The sensitivity and specificity of the assays were 85% and 9170, respectively, when results were detcrmined by visual inspection of ethidium bromide-stained agarose gels. The test sensitivity and specificity improved to 87% and 100%afterconfirmation by hybridizationwith a ctioxigenin-labeled,virusspecific probe.
3. Group A Rotavirus CL Disctrsc.. Rotaviruses are members of the Reoviridae family. They have a genome consisting of l l double-stranded RNA segments surrounded by a distinctive twolayered protein capsid. Particles are 80 nm in diameter. Six serologic groups have been identified, three of which (A, B, and C ) infect humans. Rotaviruses cause acute gastroenteritis. The most widesprcad group A rotavirus is also known as infantile diarrhea, winter diarrhea. acute nonbacterial infectious gastroenteritis, and acute viral gastroenteritis. Rotavirus infection is the most common cause of dehydrating diarrhea in children in the United States. Rotavirus, the most important cause of pediatric gastroenteritis in the United States, is responsible for an estimated one-third of all hospitalizations for diarrhea in children less than 5 years of age (154). Rotavirus gastroenteritis is a mild to severe disease characterized by vomiting. watery diarrhea, and low-grade fever. The infective dose is presumed to be low, from 10 to 100 virus particles. Since a person with a rotavirus diarrhea excretes large numbers of virus particles ( IOs-lO"' particles/ml of feces), infectious doses can easily be acquired from contaminated hands or utensils ( 154,155).
h. Ecology. Rotavirusesarctransmitted by thefecal-oralroute.Person-to-person contact is thought to be the most common means of transmission. Rotaviruses are very stable in the environment ( 154,155). These viruses do not appear to be the most frequent virus type associated with seafood.
Pathogens Transmitted by Seafood
159
c. Microbiolo,yiccrl Procedures ~rrrdDetection. Specificdiagnosis of the disease is made by identification of the virus in the patient's stool. Enzyme immunoassay is the test most widely used to screen clinical specimens, and several commercial kits are availableforgroupArotavirus.Electronmicroscopyandpolyacrylamidegelsareusedin some laboratories. More recently a reverse transcription PCR (RT-PCR) method has been developed to detect all three groups of human rotavirus (156-158).
4. Other Viruses:HumanAstrovirus,HumanAdenovirus, and Poliovirus Other viruses may also be found associated with shellfish, including human astrovirus, human adenovirus, and poliovirus. These viruses have notbeenreported as being frequently associated with seafood.
111.
BACTERIAL AND VIRAL PATHOGENS PRIMARILY ASSOCIATED WITH IMPROPER PROCESSING OR HANDLING OF SEAFOOD
A.
Bacteria
1.
Clostridium botulinum
(7. Diserrsc~. Foodbornebotulism, as distinguishedfromwoundbotulism andinfant botulism, is an extremely severe form of food poisoning caused by the ingestion of foods containing a potent neurotoxin that is formed during growth of Clostridium botulir u m . The toxin is heat sensitive and can be destroyed if food is heated to 80°C for 10 minutes or longer. The incidence of the disease is low, but the mortality rate is very high if the infected patient is not treated quickly. Approximately 10-30 outbreaks are reported in the United States each year, and these are mostly associated with inadequately processed home-canned foods. Some cases of botulism may go undiagnosed because symptoms are transient or mild or are misdiagnosedas Guillain-Barr6 syndrome. Seafood products have been involved in botulism outbreaks ( 126,159,160). C. b o t r r l i r r u r r r is a gram-positive, obligately anaerobic, spore-forming rod that may produce a neurotoxin. Seven types of C. botulirzrrrn (A, B, C, D, E, F, and G) are recognized, based on the antigenic nature of the toxin produced. Types A, B, E, and F cause human botulism, and typeE is mostly associated with seafoods in the United States. Different types of C. botrrlitrum are found in different areas of the world. Overall, type E is infrequcntly found around the world, but predominates in northern regions and in most temperate aquatic environments. This type is found in high numbers around the Great Lakes and in the coastal areas of Washington and Alaska. Along the Pacific coast of the United States, however, the prevalent type shifts from E to A and B below latitude 36"N. In Europe, type B predominates in the aquatic environments around the United Kingdom, while in other aquatic environments, type E is most frequently found. Surveys of Asia report high levels of type E spores around the Caspian Sea and in the northern areas of Japan. I n tropicalareas of Asia,typesCandDarefoundathigherlevelsinaquatic environments thantype E. Nanogram quantities of botulinum toxin can cause illness. Onset of symptoms in foodborne botulinum usually occur 18-36 hours after ingestion of the toxic food, although this can vary from 4 hours to 8 days. Early signs of intoxication
160
Herwig
include marked lassitude, weakness, and vertigo, usually followed by double vision and progressive difficulty in speaking and swallowing. Difficulty in breathing, muscle weakness, abdominal distention, and constipation may also occur ( 126,159,160). Storage of fresh and processed fish using modified atmospheric packaging (MAP) has gained widespread application in Europe. The technology has been approached more cautiously in North America because of the risk associated with C. borulinum E. The conditions often encountered in MAP fish products are conducive to the growth andtoxin production of C. borulinurrr E evenat temperatures as low as 33°C (161 ). Mild temperature abuse conditions of MAP fish products are of additional concern. Furthermore, the restricted growth of normal spoilage organisms may enhance the growth of C. horulitrum. These possibilities were shown by Eklund ( 162) in sallnon inoculated with C. b ~ t u l i t ~ ~ t t ~ spores and stored under MAP conditions. While spoilage was delayed for 10 days, toxin production occurred earlier. Whether toxin production precedes or follows spoilage is debated by some, but there is little question that with mild temperature abuse, botulinum toxin can be detected prior to organoleptic deterioration. To ensure the safety of MAP fish, strict temperature control at low temperatures (
h. Ecology. Vegetative organisms and spores of C. botulir~utr~ arewidelydistributed in nature. The organism requires an anaerobic environment for growth,but the spores are resistant to a wide variety of environmental perturbations. Organisms and spores have of streams, lakes, and been found in forest soils, cultivated soils, the bottom sediments coastal waters, in the intestinal tracts of fish and mammals, and in the gills and viscera of crabs and other shellfish. Since C. botulirlutn is so widely distributed i n nature, the food industry and food consumers have learned to take measures to prevent the growth of C. botulit7um cells from spores and toxin production. Two major mechanisms are used to prevent foodborne botulism: (a) maintaining the pH at less than 4.6 and (h) utilizing a heat treatment during food processing to kill C. boruliturrrr spores and vegetative cells. Smoked fish, both hot and cold smoked (e.g., Kapchunka) have caused outbreaksof type E botulism (126,159,160). In 1987, eight cases of type E botulism occurred, two in New York City and six in Israel. All eight patients had consumed Kapchunka, an uneviscerated, salted, air-dried, whole whitefish. The product was made in New York and transported by individuals to Israel. All eight patients developed symptoms within 36 hours of consuming the Kapchunka. One patient died, two required breathing assistance, three were treated with antitoxin, and three recovered spontaneously. The Kapchunka contained high levels of botulinum type E toxin, despite the high levels of salt that should have prevented the growth of the organisms. One possible explanation was that the uneviscerated fish contained low levels of salt when air dried at room temperature, became toxic, and then were rebrined. of Kapchunka Regulations were publishedto prohibit the processing, distribution, and sale and Kapchunka-type products in the United States (164).
c. Microbiologiccrl ~ n Detection d Plncec1ure.s. Examiningsuspectfoodsforbotulinum toxin involves using procedures to detect and identify the toxin. The most widely accepted method is to inject extracts of the food into passively immunized mice, called the mouse neutralization test. This animal assay takes 48 hours to complete. The severe and sometimes fatal outcome caused by C. botulinunr toxins requires rapid identification of the neurotoxin so treatment can begin and public health agencies can minimize the spread of the illness. Current animal tests and other bioassays may take days to complete, too long a time when the seriousness of the consequences are considered. Following this
Pathogens Transmitted by Seafood
161
assay, suspect food can be cultured in an enrichment medium for the detection and isolation of C. botulinurn. The microbiological procedure takes at least 7 days to complete. Fig. 24 outlines a protocol (159) for screening smoked fish for viable vegetative cells or spores of C. botuliwum.
2. Staphylococcus aureus (1. Disease. Srcrl,h),lococcus clweus foodpoisoning is caused bythemicrobial production of a toxin. The onsetof symptoms is usually very rapid and acute, but depends on the amount of toxin that is consumed and the susceptibility of the individual to the toxin. The symptoms include nausea, vomiting, retching, and abdominal cramps. Recovery generally takes 2 days, but severe cases may take longer. Death from staphylococcal food poisoning is very rare. The infective doseis less than 1 pg of toxin, a level that is achieved when S. nureu.7 populations exceed 1 Os/g ( 132,165,166). Staphylococcal food poisoning is not particularly associated with seafood or fisheries products, except when seafood isan ingredient in the preparationof salads. It is not associated with raw shellfish. Foods that require considerable handling and are maintained at warm temperatures are frequently involved in staphylococcal food poisoning (165,166).
b. Ecology. Humans and other warm-blooded animals are the primary reservoirs for S. clweus. This organism is normally present in the nasal passages, throats, hair, and skin of more than 50% of individuals. Foods that are maintained below 7°C or above 60°C will not support the growth of S. nureus (132,165,166). C. Microbiologicd Procedures. S. ( I U ~ ~ MareS gram-positivecocci that occur in pairs, short chains, or grape-like clusters. Some strains producea heat-stable protein toxin thatcausesillness in humans.Fig. 25describes a directplatingmethodandan MPN method for the enumeration of S. [rureu.s in food samples (165).
Place smoked fishin water-tight plastic bag andadd Trypticase Peptone GlucoseYeast Extract (TPGY) broth Remove air from bag and seal Incubate 5 days at 28°C
Remove 20 ml of enrichment and centrifuge Adjust pH if necessary I
Add 5% solution of trypsin to culture broth
t
Prepare dilutionsof trypsinized and nontrypsinized broth and Inject mice Observe mice for botulism and record observations Death of mice is presumptive evidence
+
Confirm botulism toxin using mice protected with toxin antisera
Fig. 24 Protocol for testing smoked fish for C/o.stridium hotulinunl and production of toxin (159).
162
Herwig Direct Plate Count Method Weigh 50 g of sample and add 450 ml of Phosphate Buffer Saline (PBS) Blend for 2 minutes
+ +
Using 1 ml of homogenate, inoculate 3 platesof Baird-Parker agar by spreadplating Incubate 45-48 hat 35°C
Count colonies tybicalfor S. aureus
Select more than one colony of each type and test for coagulase
t
Perform additional biochemical tests Catalase Glucose, anaerobic utilization Mannitol, anaerobie utilization Lysostaphm sensitivity Thermostable nuclease production
MPN Method
+ + +
Prepare sampleas above
Using blended sample, inoculate MPN tubes containing Trypticase Soy Broth (TSB) + 10% NaCl and 1% sodium pyruvate Incubate 48 k 2 h at 35°C
Transfer loopful from each turbid tube to Baird-Parker agar Incubate 48 h at 35°C
Select colonies typicalfor S. aureus test for coagulase and perform additional biochemical tests
3. Streptococcus iniae
N. Disease. Thegenus Str'eytoL'oc'c'u.7 is comprised of gram-positive,microaerophilic cocci that are not motile and occur in chains and pairs (25). S. iwiw is a pathogen for aquatic animals, but it was recently reported to cause serious disease in humans. The pathogen is associated with live finfish culture and aquaria. Frequently, in seafood markets
Pathogens Transmitted by Seafood
163
and restaurants in Asia or in Asian markets locatedin Western countries,finfish are maintained live in aquaria. Humans that contact these fish may be exposed to S. itliere. h. Ecology. S. itliere was firstreportedin1976 to causesubcutaneousabscesses in Amazonfreshwaterdolphins at aquariums in San Francisco and New York. Later, meningoencephalitis outbreaks caused by streptococci have been recognized in a variety of cultured finfish, including tilapia, yellowtail, rainbow trout, and coho salmon. S. itliae may colonize the surfaceof fish or cause invasive disease associated with 30-50% mortality rates (167-169). S. itliere is a bacterium recently linked to intensive recirculation tank systems in the United States. A virulent strain has been isolated from humans: this infected several customers who purchased live Tilupia sp. and presumably became infected through an open wound while cleaning the fish. While most of the problem are associated with skin lesions, systemic infections have been reported in Canada (167,168). During the winter of 1995-1996 in the Toronto area, a cluster of four cases of invasive S. itliae infection occurred in peoplewho hadrecentlyhandledfresh,wholefishfrom a farm.Results of surveillance identified a total of nine patients with invasive S. itzie~einfection (cellulitis of the hand in eight and endocarditis in one). All the patients had handled live or freshly killed fish, and eight had percutaneaus injuries. Six of the nine fish were tilapia, which are commonly used in Asian cooking. All of the patients were of Asian descent and all the patients reported preparing whole, raw fish. Eight patients recalled injuring their hands by puncturing the skin with the dorsal fins, a fish bone, or a knife used to clean the fish. Fish were purchased from markets where they were maintained live and were killed and gutted when purchased. The head, tail, and fins were left on the fish carcass. Cellulitis developed within 16-24 hours after the injuries.All of the patients responded to antibiotics within 2-4 days. The investigators concluded that S. irzine can produce invasive infection after skin injuries during the handlingof fresh fish grown by aquaculture. Using molecular typing methods, the authors identified a clone of S. itline that causes invasive disease in both humans and fish ( 167- 169). This organism may be an emerging pathogen since it was only recently identified as a pathogen in fish produced by the aquaculture industry.
c. Microhiologicd Procwlures. Routineexamination of patients,fish,andfishrearing ponds and aquaria for S. irlicre is not performed. Since the genus Streptococcus is of routine clinical importance, protocols for the identification of S. itlirre can be easily performed. Characteristics used to identify streptococci as S. itliere are that they are phemolytic ontrypticasesoyagarwith 5%) sheep's blood; they are not groupable with Lancefield groups A-V antiserum; they are susceptible to vancomycin; they do not produce gas, are nonmotile, are positive for pyrolidonyl arlamidase and leucine aminopeptidase; and they producc negative results on bile esculin, Voges-Proskauer, and hippurate tests. Not surprising for an environmental organism, most strains grow at 10°C but not at 4S"C, and they do not grow in 6.5% NaCl. Surface swabs from fish can be inoculated onto colistin-nalidixic acid blood agar and incubated at35°C in 5% CO: for 18-24 hours ( 158,169). Public health agencies and officials need to be aware of S. itzicre, a pathogen emerging in the past few years. B. Viruses Viral pathogens described in a previous chapter section may also be associated with seafood that has been improperly processed or handled. These pathogens primarily include
Henvig
164
hepatitis A, human calicivirus, and group A rotavirus. Viral pathogens may be introduced by fecal contamination of the seafood from an infected food handler or from processing waterorfoodingredients thatare contaminated withfecalmaterial. In addition,viral pathogens that are present on raw products may not be inactivated if the contaminated seafood receives insufficient cooking.
W. DEVELOPMENT OF RAPID METHODS FOR DETECTION OF SEAFOOD-BORNE PATHOGENS Much has been published and discussed about the development of rapid methods for the detection of pathogenic microorganisms. While a “dip stick,” “early pregnancy” style prototype kit is desired by regulatory agencies and food processors, the science for this technology is not presently at a stage for use on most pathogens, but recent work appears to be very promising. Improvements in sensitivity and speed of analysis have occurred. Most of theimprovementsandresearchefforts inrecentyearshavefocusedonPCR technologies. Hill (170) and Scheu et al. (17 1 ) provided extensive reviews on PCR and its applications for detectionof foodborne pathogens. Muchofthe difficulty in implementing PCR for the analysis of food samples lies in the problems associated with preparation and purification of DNA templates from food matrices. If most of these problems can be resolved, PCR technologies offer an investigator the opportunity to quickly identify specific pathogenic species or strains and the presence of specific genes associated with pathogenesis. DNA canbe extracted from food samples and thereis no need to perform enrichmentsteps.Hill (170) also listsmanyPCRprimers,amplificationconditions,and references for detecting a wide variety of foodborne pathogens. Limitations of the PCR method have been discussed in previous reviews ( 170,171) and articles. Two major limitations are the differentiation of living and nonliving cells, and the inhibitionof the PCR by food. PCR protocols maynot differentiate between viable and nonviable microorganisms. Amplification methods only indicate whether the target sequence of DNA is present, and not whether the organisms are alive or dead. DNA from dead microorganisms may leadto false-positive results. One way to minimize the detection of dead cells is a preenrichment step prior to the PCR analysis. This step increases the sensitivity of the test and restricts detection to culturable cells (172). The presence of intact RNA as a sign of active metabolism mayalso be a valuable indicator of the viability of the microorganisms (171). Messenger RNA (mRNA) can be detected as the template for be difficult to RT-PCR. Cells need to be rapidly extracted, howcver, and intact mRNA may isolate. Ribosomal RNA (rRNA) maybe a better target for analysis because of its higher stability. For most investigators, working with RNA is more difficult than working with DNA. A large problem in using PCR methods with foods is the apparent presence of PCR inhibitorsinfoods (171). False-negativeresultscanoccurforvariousreasons: (a) the presence of substances that chelate divalent magnesium cations, ions that are necessary for DNA polymerase, (h) degradationof nucleic acid targets or primcrs through nucleases, (c) substances that directly inhibit DNA polymerase. To reduce false-negative results it is very important for the investigatorto include the appropriate controls when developing and using PCR protocols. While most publications have described the detection of single pathogenic species
Pathogens Transmitted by Seafood
165
or strains, detection of several pathogens may be possible with a single test. For example, Wang et al. (173) showed the development of a protocol using universal culture medium and the same PCR conditions for detecting more thana dozen foodborne bacterial species in foods. Internal probe hybridization and nested PCR procedures were used as confirmation. Short enrichment culture procedures without DNA isolation may be the best approach because they are easy to perform and have a high sensitivity. Table 8 lists a variety of PCR systems that have been developed for the detection in seafood products. In several investigations, seafood of bacterial and viral pathogens was among the different foods for which the molecular method was developed. A more comprehensive table that was published by Scheu et al. (171) lists seafoods and other types of foods. For many of the listed protocols the detection limit is extremely low, between 1 and 100 cells/g. While PCR methods are very attractive because of their sensitivity and speed, especiallywhen compared to traditional enrichment and cultural methods, these methods may not be practical for routine analyses performed by the quality control laboratories present in seafood companies and other small agencies. Several of the protocols use radioactive probes and require the skills of laboratory personnel specially trained in molecular techniques such as PCR, gel electrophoresis, and restriction digests of DNA.
V.
CONCLUSIONS AND RECOMMENDATIONS
A.
E. colias anIndicator Organism
Routine monitoring for fecal coliforms or E. c ~ o l iare inadequate for protecting the public againstmostseafood-bornebacterialandviralpathogens.Depuration of contaminated shellfish in clean water may rcmove bacterial contaminants, butmaynot be a reliable method for the elimination of viral contamination (106). Several authors have suggested that routine screening of bivalves for the presence of health-significant enteric viruses before public consumption may help to prevent disease outbreaks among shellfish consumers. Most current regulations concerning the contamination of shellfish and their growing waters are based solely on bacteriological standards, primarily E. coli or fecal coliforms. This aspect is of concern because many bacterial organisms failto provide a reliable indication of the virological quality of the bivalve shellfish and their waters (146).
B. Consuming Raw Seafood The consumption of raw animal protein is inherently an activity with potential risk and consumers should be informed about the types of seafood and processing procedures where the risks maybe unacceptable. If people refused to consume raw animal protein and regulatory agenciesprohibitedthesale of suchproducts in foodserviceestablishments,the number of cases and outbreaks associated with infectious bacteria would be greatly reduced. Many people, particularly those with liver or other blood disorders, become seriously ill and diewhen they consume raw oysters. In the United States, three states (Florida, California, and Louisiana) require warning labels or signs where raw oysters are sold. Fig. 26 shows the warning labels that are required in two states. Should this labeling be required i n all restaurants and stores that sell raw oysters? Some would argue that widespread warnings would make little difference. The long and active campaign against cigarette smoking serves as a vivid example. Klontz et al. (174) suggested that the consumer
Table 8
PCR Systems for the Detection of Bacterial and Viral Pathogens in Seafood Productsq
Organism Bacterial pathogetis Clostridium hotuliriuni A3.E
E. coli 0157
E. coli enteroinvasive Listeria nionocyrogeries L. monocytogerzes L. monocytogetzes
Type of sample Milk, meat juice. canned tuna, mushrooms. sausage Raw hamburger. raw milk, raw oysters, bean sprouts Seafood. greens, dairy products 250 different food types Cold smoked salmon Meat products, vegetables. seafood
Amplified gene
Detection system
Detection limit
Reference
Botulinum neurotoxin genes
Gel
4 cfu/g following 48hour enrichment
209
sltl, sltII (vertoxin 1 and 2 genes)
Immunoinagnetic separation. multiplex PCR. gel Restriction enzyme digestion, gel Gel Nested PCR, gel
1 - 10 cells/g
210
10’ cfu/ml
21 1
100 cfu/ml 100 cfu/g
212 213
Gel
1 cfu/g following nonse-
214
lective and selective enrichments 2 x 10’to 2 x IO’cfu/g
215
40 cells
216
I - 10 cells/g after 3hours enrichment
217
Less than 40 cells/g
218
EIEC virulence plasmid Listeriolysin 0-gene prfA (transcription activat-
ing protein) h(vA (IisA. literiolysin 0 gene), iap (invasion associated protein) 16s rDNA
Salnioriella
Beef, fish, pork samples
Salnionella typlii
Salrnoriella spp.
Crabmeat salad, fried chicken. peppersteak, ratatouille salad, smoked mussels Oysters
himA
Salniotiella spp.
Oysters
1iti.s
5 s rDNA-23s rDNA spacer region
gene
Gel and “P-labeled oligonucleotide probe Gel
Gel and Southern blot. dot blot, radioactive ohgonucleotide probe Gel and radioactive oligonucleotide probe
X
8.s
Gel, Southern blot, fluorescein labeled oligonucleotide probe Multiplex PCR h/yA amplicon as internal control, gel nested PCR. gel Gel and radioactive oligonucleotide Nested PCR. gel Restriction endonuclease digestion of amplicons. gel
Vihrio cliolerue
Oysters. crabmeat, shrimp. lettuce
Cholera toxin operon
V. cholerae
Raw oysters
l i l y 4 hemolysin gene ctx cholera enterotoxin
Vihrio puruhaeriiolyticics
Shellfish
Cloned DNA fragment
Fish. water Oysters
2 3 s rDNA Cytol ysin
Oysters and clams
5‘ noncoding end
RT-PCR. gel
Oysters
RT-PCR, gel
Oysters, shellfish
Pan-enterovirus HAV capsid Polymerase gene
Oysters, clams
Polymerase gene
Shellfish
Polymerase gene
Virol pcifhogens Enteroviruses. polioviruses Enteroviruses. poliovirus 1, hepatitis A virus Enteroviruses, polio virus I, HAV, Nonvalk virus Nonvalk virus
Round structured viruses
&Tablemodified after Scheu et al. (171).
RT-PCR, restriction endonuclease digestion, gel RT-PCR, restriction endonuclease digestion, gel RT-PCR, gel, Southern blot, internal oligonucleotide probe
10 cfulg following an 8hour enrichment
219
30 cfulg 3 cfulg after 6-hours enrichment
220
9 cfu/g after 3-hours enrichment 12- 120 cells/PCR 10’ cfulg after 24-hours enrichment
221
2 pfulg 0 pfu/polio virus/PCR 295 pfu HAVIPCR OL-lO” pfulml
48,222 223
224 225.226 227
228 229
168
Henvig
Florida(FloridaAdministrativeCode
10D-3.091 ( 6 ) a.)
“Consumer information: There is risk associated with consuming raw oysters or any raw animal protein. If you have chronic illnessof the liver, stomachor blood, or have immune disorders, you are at greater riskof serious illness from raw oysters, and should eat oysters fully cooked. If unsure of your risk, consult a physician.”
California(CaliforniaCodeofRegulationsTitle
17)
“Eating raw oysters may cause severe illness and even death in persons who have liver disease (for example alcoholic cirrhosis), canceror other chronic illnesses that weaken the immune system. If you eat raw oysters and become ill, you should seek immediate medical attention. If you are unsure if you are at risk, you should consult your physician.” Fig. 26 Warning labels for raw oysters from Florida and California.
of raw oysters is, by nature, a “risk-taker.” Performing a survey in Florida, they found that the prevalence for eating raw oysters was higher for men18-49 years old. The prevalence was even higher for persons who reported being cigarette smokers or acute or chronic alcohol drinkers.
C. Transport of Seafood Fresh, raw, and potentially contaminated seafood is often transported long distances from the location of harvest or processing. Physicians, emergency room, and other medical personnel who work in these distant areas need to be aware of seafood-borne disease. Several isolated cases have been reported where individuals who consumed contaminated seafood in “nonseaside” communities have died from V. vulnijcus. For example, a 61year-old man with adult-onset noninsulin-dependent diabetes died after consuming raw oysters in Louisville, Kentucky that were harvested in Florida ( 175). In Oklahoma, another man with underlying liver disease and a history of eating raw oysters died within15 hours of arriving at the hospital of V. vuln$cus sepsis (176). For V. vuln$cus infections, early treatmentwithantibioticshasbeenshown to improvesurvivalinsepticemicpatients ( 177,178), so prompt recognition of the infection by physicians is critical to reduce mortality.
D.
V, parahaemolyticus and V. vulnificus as Notifiable Diseases
In the United States, federal and state public health agencies need to consider adding V. I’““ahaernol~,ricu.~ and V. vuln$cus to the list of infectious diseases that are notifiable. In 1997,52 infectious diseases were designated notifiable as at the national level in theUnited States (8). Of these, only a few are associated with food, including botulism, cholera, cryptosporidiosis, E. coli 0157:H7, hepatitis A. salmonellosis,shigellosis,andtyphoid fever. Somewhat ironic is the fact that, in the United States during the past I O years, the
Pathogens Transmitted by Seafood
169
number of deaths caused by V. w l t ~ j f i c x exceeded s the number caused by foodborne botulism, yet V. \wln(fifiCv.sis not notifiable to the CDC. Also, reporting of nationally notifiable diseases to the CDC by individual state governments is voluntary, not mandatory. Reporting is only mandated at the state and local levels. Thus the list of diseases that are notifiable varies slightly between states. The FoodNet program in the United States is a good start, since all Vibrio species are reported, but only a limited number of states and counties are participating in this new program.
E. International Trade in Seafood and Public Health Many species of raw and processed fish and shellfish are being imported and exported from many different countries around the world. Regulatory agencies face a very difficult challenge in attempting to keep all of the imported, along with domestically produced seafood products, safe for human consumption. Consumer education programs need to inform the public about the potential risks and the procedures that can be followed in the home and in retail establishments to reduce or minimize possible seafood disease risks. The United States imports more than 50% of the total consumed seafood from 172 countries, originating from about 6000 foreign firms (179). In December 1997, the FDA required ail seafood processors to adopt a quality assurance program based on the HACCP concept. These measures arealso being applied to seafood importers. HACCP focuses on preventing hazards ratherthan relying on spot-checks and the random sampling of products at the endof the production line. Under HACCP each food processor and importer prepares a plan for identifying and monitoring the steps of their operation that may contribute hazards to human health. While the seafood industry, regulatory agencies, seafood technologists, and scientists embrace the principals and objectives of HACCP, the effectiveness of these measures i n reducing the incidence of seafood-borne pathogenswill probably not be known for several years. The dauntingtask faced by the FDA was highlighted in April 1998 when the U.S. General Accounting Office (GAO) released a report entitled “Food Safety: Federal Efforts to Ensure the Safety of Imported Foods are Inconsistent and Unreliable.” The GAO report stated that the FDA inspects less than 2% of all food imports, including seafood imports. With such a low level of inspection, the proactive approach of HACCP may be the only logical way to keep seafood, and other food commodities, as safe as reasonably possible for human consumption.
F. The Rise of Aquacultured Seafood Freshwater and marine aquacultured products are becoming increasingly important in the production of seafood. Studies may need to be performed to see if new bacterial and viral pathogens are associatedwith these products.To date,no one has foundthat these products al. are inherently more hazardous thanwildfish and shellfish. For example, Pullela et ( I 80) examined the nature and number of indicator and pathogenic microbes in fish reared using recirculating and nonrecirculating freshwater systems. L. monocyfogenes, Y. enterocolitictr, E. coli 0 157:H7, and Salmonella spp. were not isolated. C.botulinunr, however, was isolated from all the aquacultured fish sampled except pacu and tilapia grown in a recirculating aquaculture system.
170
Herwig
G. Importance of Environmental Quality For the survival and success of their industry, fish and shellfish harvesters and processors have a stake in water and sediment quality. This chapter focused on microbial pathogens that are associated with seafood. Many of these pathogens are introduced by pollution into fish and shellfish in the aquatic environment. The better the environmental quality where the seafood is harvested, whether in an aquaculture farm, an intertidal beach, or in the open sea, the less likely that microbial or chemical contaminants will cause problems for the seafood consumer.
H. Passive Transport and Introduction of Pathogenic Organisms The transport of pathogenic microorganisms from one part of the world to another in the ballast water and sewage of large ships is alarming and probably requires consideration. Most of the attention by the popular press in recent years has focused on the transport of exotic species of invertebrates and plants, suchas zebra mussels and European green crabs, from one part of the world to another. McCarthy and Khambaty ( 18 1) recovered toxigenic V. clzolerne 01, serotype Inaba, biotype El Tor from ballast, bilge, and sewage water from five cargo ships docked in ports of the U.S. Gulf of Mexico. Four of these ships had taken on ballast water in cholera-infected countries; the fifthtookonballastina noninfected country. Isolates examined by pulsed-field gel electrophoresis were indistinguishable from the Latin American epidemic strain, C6707. On the basis of their findings, the FDA recommended that the U.S. Coast Guard issue an advisory to shipping agents and captains requesting that ballast waters be exchanged on the high seas before entry of ships into U.S. ports. More recently, following the largest V. p c l m h n e m n l ~ t i coutbreak ~~.~ in North America in many years, in which more than 400 cases were reported, the FDA began an investigation of shellfish contamination causedby ballast water discharged from ships. This suspicion was spurred by the fact that the serotype isolated(03:K6) is common in Japan, India, and Taiwan, but had never before been detected in Texas waters (182).
1.
Development of Test Kits
The development of rapid and relatively simple test kits for seafood-borne pathogens, biotoxins, and hazardous chemicalsis an area of research that needsthe support of governments, the research community, andthe seafood industry. T o help importers monitor shipments for HACCP compliance, several companies have developed rapid test kits. For example, Neogen offers akit that tests histamine levels using an enzyme-linked immunosorbent assay (ELISA) and yields visual color results in 1 hour (183). Colwell and her associates at the University of Maryland have developed protocols and kits for the rapid detection of V. cholerne 01 and V. cholerne 0139 (184-1 86), and a kit is now commercially available (Cholera SMART).
J.
Relative Risks Associated with Consumption of Seafood
Although a varietyof foodborne diseases are associated withfish and shellfish, the number of human fatalities that occur each year, particularly in developed countries, from the
171
Pathogens Transmitted by Seafood
Table 9 WorldWideWebSitcswithInfortnationaboutPathogensAssociated with Seafoods
URL
Site Description ~~
U.S. Food and Drug Administration Seafood Information and Resources. Center for Food Safety and Applied Nutrition Thc Bad Bug Book The National Food Safety Database Centers for Disease Control and Prevention Emerging Infectious Diseases. A peer-reviewed journal that is available on the Internet, produced by the National Center for Infectious Diseases, CDC FoodNet Interstate Shellfish Sanitation Commission Seafood Network Infortnation Center (SeafoodNIC), Sea Grant Extension Program, University of California, Davis National Scafood HAACP Alliance for Training and Education
~
~~
~~~~
www.fda.gov vm.cfsan.fda.gov/seafood 1 .html
vm.cfsan.fda.gov/-mow/intro.httnl
www.foodsafety.org www.cdc.gov www.cdc.gov/ncidod/EID
www.cdc.gov/ncidod/dbmd/foodnet
www.issc.org seafood.ucdavis.edu
seafood.ucdavis.edu/haccp/ha.htnl
consumption of fish and shellfish is extremely low compared to the mortalities caused by other infectious and noncommunicable diseases. Other routine activities performed daily by humans around the world are much more hazardous than consuming seafood. Large differences certainly exist between developed and developing countries. For example, in developed countries infectious and parasitic diseases account for 1% of the deaths each year. In developing countries this figure is much larger, 43%. Some of these infectious disease agents may be associated with seafood.
VI.
ADDITIONAL SOURCES OF INFORMATION: WORLD WIDE WEB SITES FOR PATHOGENS ASSOCIATED WITH SEAFOODS
Several agencies in the United States provide large amounts of information for the consumer or educator interested in seafood safety and the characteristics of seafood-borne pathogens. Much of this information is freely available on the Internet or by contacting the agencies by telephone or mail. Table 9 lists the URL for several of the sites related to seafood safety.
ACKNOWLEDGMENTS I would like to acknowledge the assistance of Robyn Estes and Jennifer McLarnan, who searched the University of Washington libraries for the nun1erous articles that were re-
Herwig
172
quired to prepare this review. Unpublished data about the number of cases of shellfishborne disease caused by Vibrio species were kindly provided by Marc Glatzer, shellfish specialist at the U.S. Food and Drug Administration, Tallahassee, Florida.
REFERENCES 1.
2.
3. 4. 5.
6.
7.
8.
9. IO. 11.
12.
13.
14. 15. 16.
17. 18.
19. 20.
World Health Organization. The world health report 1998: Life in the 21st century-a vision for all. Geneva. Switzerland: World Health Organization, 1998. World Health Organization. Control of food borne trematode infections. WHO Study Group, Manila, Philippines, 18-26 October 1993. Geneva, Switzerland: World Health Organization. 1995. KJ Rana. Trends in global production, 1984-1995. Rome: FAO Inland Water Resources and Aquaculture Service, 1997. KJ Greenlces, J Machado, T Bell, SF Sundlof. Food borne microbial pathogens of cultured aquatic species. Vet Clin N Am Food Anim Prac 14:101-112, 1998. J-Y D’ Aoust. Snlrnonellu and the international food trade. Int J Food Microbiol 24: I 1-3 1. 1994. CF Fernandes, GJ Flick, JL Silva, TA McCaskey. Comparison of quality in aquacultured fresh catfish fillets 11. Pathogens E. coli 0 I57:H7, Cumpylobacter, Vibrio, Plesiontonus, and Klebsiella. J Food Protect 60: 1182-1 188, 1997. Program and Enforcement Branch. Office of Seafood. Center for Food Safety and Applied Nutrition.Nationalshellfishsanitationprogrammanualofoperations.Washington,D.C.: U.S. Food and Drug Administration, 1995. Centers for Disease Control and Prevention. Summary of notifiable diseases, United States, 1997. MMWR Mobid Mort Wkly Rep 46:l-90, 1998. Centers for Disease Control and Prevention. Surveillance for foodborne-disease outbreaksUnited States, 1988-1992. MMWR Mobid Mort Wkly Rep 45:l-66, 1996. human health, RJ Wittman, GJ Flick. Microbial contaminationof shellfish: prevalence, risk to and control strategies. Annu Rev Public Health 16: 123-140, 1995. SR Rippey. Infectious diseases associated with molluscan shellfish consumption. Clin Microbiol Rev 7:419-425, 1994. Council for Agricultural Science and Technology. Foodborne pathogens: risks and consequences. Task Force Report no. 122. Ames, IA: Council for Agricultural Science and Technology,1994. JC Desenclos, KC Klontz, MH Wilder, OV Nainan, HS Margolis, RA Gunn. A multistate outbreak of hepatitis A caused by the consumption of raw oysters. Am J Public Health 81: 1268- 1272, 1991. L Finelli, D Swerdlow, K Mertz, H Ragazzoni, K Spitalny. Outbreak of cholera associated with crab brought from an area with epidemic disease. J Infect Dis 166:1433-1435, 1992. S Yang. FoodNet and Enter-net: emerging surveillance programs for foodborne diseases. Emerg Infect Dis 4:457-458, 1998. National Research Council, Committee to Ensure Safe Food from Production to Consumption. Ensuring safe food from production to consumption. Washington, D.C.: National Academy Press, 1998. W-C Lee, T Sakai, M-J Lee, M Hamakawa, S-M Lee, I-M Lee. An epidemiological study of food poisoning in Korea and Japan. Int J Food Microbiol 29:2-3, 1996. GJ Olsen, CR Woese, R Overbeek. The winds of (evolutionary) change: breathingnew life into microbiology. J Bacteriol 176: 1-6. 1994. GJ Olsen, CR Woese. Ribosomal RNA: a key to phylogeny. FASEB J 7.1 13-123, 1993. CR Woese. Bacterial evolution. Microbiol Rcv 51:221-271, 1987.
Pathogens Transmitted by Seafood
21.
22.
23.
24. 25.
26. 27. 28. 29. 30. 31.
32.
33.
34.
35.
36. 37. 38.
39. 40. 41. 42.
43.
44.
173
GE Fox, E Stackebrandt, RB Hespell, J Gibson. J Maniloff, TA Dyer, RS Wolfc. WE Balch. RS Tanner, LJ Magmm. LB Zablen, R Blakemore, R Gupta. L Bonen, BJ Lewis. DA Stahl, KR Luehrsen, KN Chcn, CR Woese. The phylogeny of prokaryotes. Science 209:457-463, 1980. BL Maidak. GJ Olsen, N Larsen. R Overbeek. MJ McCaughey, CR Woese. The RDP (ribosotnal database project). Nucleic Acids Res 25:109-11 I , 1997. FA Murphcy, CM Fauquet. DHL Bishop, SA Ghabrial, AW Jarvis, GP Martelli. MA Mayo. MD Summers, eds. Sixth report of the International Conmittcc on Taxonomy of Vituses. vol. IO. New York:SpringerVerlag,1995. JD Oliver, JB Kaper. Vihrio species. I n : MP Doyle, LR Beuchat, TJ Montvillc, cds. Food microbiology: fundamentals and frontiers. Washington, D C : ASM Press, 1997, pp. 228-264. JG Holt,NR Krieg, PHA Sneath, JT Staley. ST Williams. eds. Bergey’s manual of determinative bacteriology. Baltimore: Williams & Wilkins.1994. MJ Albert, M Neira. Y Motarjemi. The role of food i n the epidemiology of cholera. World Health Stat Q 50:l1 1- 1 1 8, 1997. World Health Organization. Cholera i n 1997.WklyEpidetniolRec73:201-208.1998. MJAlbert. Vihrio cholertre. JClinMicrobiol32:2345-2349,1994. JB Kaper, JG Morris Jr. MM Levine. Cholera. Clin Microbiol Rev 8:48-86. 1995. J EpiT Estrada-Garcia, ED Mintz. Cholera: foodborne transmission and its prevention. Eur detniol12:461-469,1996. RR Colwell, A Huq.Environmentalreservoirof Vibrio chlerrre. The causative agent of cholera. AnnNYAcadSci15:44-54, 1994. RR Colwell. A simple filtration AHuq,B Xu, MARChowdhury,MSIslam,RMontilla, method to remove plankton-associated Vihrio cholercre i n raw water supplies in developing countries. Appl Environ Microbiol 622508-25 12, 1996. J A DePaolo. Vibrio c.holercte in marine food and environmental waters: literature review. Food Sci 46:66-70, 1981. PA Blake, DT Allegra, JD Snydcr, TJ Barrett, L McFarland, CT Caraway, JC Fceley, JP Craig, JV Lee, ND h h r . RAFeldman.Cholera-a possible endemic focus in theUnited States. N Engl J Med 302:305-309. 1980. i n Singapore, Ann KT Goh. Surveillance of food poisoning and other food-bornc diseases AcadMedSingapore16577-582.1987. KT Goh, S Lam, S Kumarapathy, JL Tan. A common source foodbornc outbreak of cholera in Singapore. Int JEpidemiol13:210-215,1984. K-M Kam, T-H Leung, Y-YP Ho, NK-Y Ho, TAP Saw. Outbreak of Vibrio ckolercre 0 1 in Hong Kong related to contaminated fish tank water. Public Health 109:389-395, 1995. SH Lee, ST Lai, JY Lai, NK Leung. Resurgence of cholera in Hong Kong. Epidetniol Infect 117:43-49,1996. in tural East WM McCorlnack, MS Islam, M Fahimuddin, WH Mosley. Endemic cholera Pakistan. Am JEpidetniol89:393-404.1969. JL Kolvin, D Roberts. Studies on the growth of Vihrio cholerne biotype eltor and biotype classical in foods. J Hyg (Cambr) 89:243-252, 1982. DR Nalin, V Daya, A Reid, MM Lcvine, L Cisneros. Adsorption and growth of Vibrio c . / r o / erne on chitin. Infect Immun 25:768-770, 1979. AHuq, EB Small, PAWest,MIHuq,RRahman,RRColwell.Ecologicalrelationships between Vihrio cholertre and planktonic crustacean copepods. Appl Environ Microbiol 45: 275-283,1983. E Elliot, CA Kaynser, L Jackson. MLTamplin. Vihrio chlercre, V. ptrrtrh~rer,lo!\ticIrs, V. v~r/r~$crt.s, and other Vihrio spp. In: Bacteriological analytical manual. 8th ed. Rev. A. Gaithersburg, MD:AOAC International, 1998, pp. 9.01-9.27. infection FP Roland. Leg gangrene and endotoxin shock due Vihriopcrrn/~~er,lo!\.rierr.s-at~ to acquired in New England coastal waters. N Engl J Med 282: 1306, 1970.
174
Herwig
PA Blake, MH Merson, RE Weaver, DC Hollis, PC Heublein. Disease caused by a marine Vibrio. Clinical characteristics and epidemiology. N Engl J Med 3OO:l-5. 1979. 46. JJ Farmer. V i h i o ( “Ber~ec.keo”)v d r t i f i w s . the bacterium associated with sepsis, septicaemia, and the sea. Lancet 2:903, 1979. 47. ADalsgaard,NFritnodt-Moller.BBruun.LHoi, JL Larsen.Clinicalmanifestationsand molecular epidemiology of Vihrio wt/rl;fic.rr,s infections i n Denmark. Eur J Clin Microbiol InfectDis15:227-232,1996. 48 CR Arias, R Aznar, MJ Pujalte, E Carny. A comparison of strategies for the detection and recovery of Vibrio vdnijicus from marine samples of the western Mediterranean coast. Syst Appl Microhiol 21 :128- 134, 1998. 49. RL Shapiro. S Altekruse, L Hutwagner, R Bishop, R Hammond. S Wilson, B Ray, S Thompson, RV Tauxe, PM Griffin. The role of Gulf Coast oysters harvested in warmer months in Vibrio 1wln~ficu.sinfections in the United States. 1988-1996. J Infect Dis 178:752-759, 1998. 50. KC Klontz.JCADesenclos.L W o k , S Hoecherl,CRoberts, RA Gunn.Therawoyster eater-risk taker? Use of the Behavioral Risk Factor Surveillance System. Epidemiology 2: 437-440, 199 1. 51. JG Morris Jr, RE Black. Cholera and other vibrioses in the United States. N Engl J Med
45.
52.
53. 54.
55. 56.
57. 58. 59.
60.
312343-350, 1985. KC Klontz, S Lieb, M Schreiber, HT Janowski, LM Baldy, RA Gum. Syndronles of Vihrio w l r l i j i c x t s infections. Clinical and epidemiologic features in Florida cases. 198 1 - 1987. Ann
InternMed109318-323.1988. JC Desenclos, KC Klontz, LE Wolfe, S Hoecherl. The risk of Vibrio illness in the Florida raw oyster eating population, 198 1 - 1988. Am J Epidcmiol 134290-297, 199 1. KP Chin, MALowe,MJ Tong, ALKoehler. Vibrio w/rt(ficu.s infectionafterrawoyster ingestion in a patient with liver disease and acquired immune deficiency syndrome-related complex. Gastroenterology 92:796-799, 1987. KC Klontz. Fatalities associated with Vibrio parcrht/rrrro!\.rit.Irs and Vibrio cholercrc~non-0 1 infections inFlorida(1981to1988).SouthMed J 83500-502. 1990. A Ali, MR Mehra, DD Stapleton. SA Kemlnerly, K Ramireddy. FW Smart, S Augustine. H 0 Ventura. Vihrio v d r l i f i f i c w sepsis i n solidorgantransplantation: a medicalnemesis.J 1995. Heart Lung Transplant 14598-600. MD Taylor, 0 Squire, KC Klontz. Raw shellfish consumption and liver disease. Risk factor for septicemia. Infect Dis Clin Prac 6562-564, 1997. 29. 1998. Center for Science in the Public Interest. Press release. June A DePaola, GM Capers. D Alexander. Densities of Vihrio I w l r l i j i t w s i n the intestines of fish from the U.S. Gulf Coast. Appl Environ Microbiol 60:984-988, 1994. ML Motes, A DePaola, DW Cook, JE Veazey, JC Hunsuckcr. WE Garthright, RJ Blodgett. SJ Chirtel. Influence of water temperature and salinity on Vibrio 1 w / r l ( f i c l t . s i n northern Gulf and Atlantic Coast oysters (Crtrssostrecr lirgirlicu). Appl Environ Microhiol 64: 1459-1465, 1998.
61.
SH Jones, TL Howell, KR O’Neill. Differential elimination of indicator bacteria and pathogenic Vihrio sp. from eastern oysters (Crnssostren virgit~iccr,Gtnelin. 1791) i n a colntnercial controlled puification facility i n Maine. J Shellfish Res 10: 105- 1 12, 199 I . 62. ML Tamplin, GM Capers. Persistence of Vihrio w/rtificlr.s i n tissues of Gulf Coast oysters, Crmsostrea virginicu, exposed to seawater disinfected with UV light. Appl Environ Microbioi 58: 1506- 15 IO. 1992. 63. ML Motes, A DePaola. Offshore suspension relaying to reduce levels of Vihrio v d r i i f i c u s i n oysters (Crcrssostrrtr virgir~ico).Appl Environ Microhiol 62:3875-3877, 1996. 64. DW Cook. Refrigeration of oyster shellstock: conditions which minimize the outgrowth of Vihrio w / n i f i c u . s . J Food Protect 60349-352, 1997. 65. ML Tamplin, JK Jackson. C Buchrieser, RL Murphrce, KM Portier. V Gangar. LC Miller,
Pathogens Transmitted by Seafood
66.
67.
68. 69. 70. 71.
72.
73. 74.
75.
76.
77.
78.
79. 80. 81
82.
83.
84.
85.
175
C w Kaspar. Pulsed-ficld gel electrophoresis and rihotype profilcs of clinical and environmental Vibrio vrtlnifiurs isolates. Appl Environ Microhiol 62:3572-3580, 1996. JK Jackson, RL Murphree, ML Tamplin. Evidence that mortality from Vihrio \7u/r~ificlr.s infection results from single strains among heterogeneous populations in shellfish. J Clin Microhiol35:2098-2101.1997. EG Biosca, JD Oliver, C Amaro. Phenotypic characterization of Vibrio vu/rl$clt.s biotype 2, lipopolysaccharide-based homogeneous0 serogroup within Vibrio \ w / r z i f c l t s . Appl Environ Microhiol62:918-927,1996. C Amaro, EG Biosca.Vibrio \ h i f i c u s biotype 2, pathogenic for eels, isalso an opportunistic pathogen for humans. Appl Environ Microhiol 62: 1454-1457, 1996. T Fujino, D Okuno,S Nakada, K Oyana, RM Fukai,T Ueho. On the bacteriological examina1953. tion of shirasu-food poisoning. Med J Osaka Univ 4:447-450, T-M Pan. C-S Chiou, S-Y Hsu, H-C Huang, T-K Wang, S-I Chiu, H-L Yea, C-L Lee. Food borne disease outbreaks i n Taiwan, 1994. J Formos Med Assoc 95:417-420, 1996. Centers for Disease Control. Outbreakof Vibrio pm'crhaer?ro/~ri~,tr.s infections associated with eating raw oystcrs-Pacific Northwest, 1997. MMWR Morh Mortal Wkly Rep 47:457-462. 1998. YMiyamoto.Y Ohara, TNikkawa, S Yamai, T Kato,YYamada,MOhashi.Simplified purification and hiophysicochemical characteristicsof Kanagawa phenomenon-associated hemolysin of Vibrio /~etr"hoerr~o!\ri~lr.s. Infect lmnun 28:567-576, 1980. M Nishibuchi, JB Kaper. Nucleotide sequence of the thermostable direct helnolysin gene of Vibrio ptrrur/lcrc,rrro!\tiett.s. J Bacteriol 162:558-564, 1985. T Honda, YX Ni, T Miwatani. Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio pcrrc'huen~o/~ric~t.s and related 1988. to the therrnostahle direct hemolysin. Infect llnmun 56:961-965, M Nishibuchi, M Ishibashi, Y Takeda, JB Kaper. Detection of the thermostable direct hemolysin gene and related DNA sequences in Vibrio por~r/lcrnrlo/~ticus and other vibrio species by the DNA colony hybridization test. Infect Irnmun 49:481-486, 1985. 0 Suthienkul, M Ishibashi, Tlida, N Nettip. S Supavej, B Eampokalap, M Makino, T Honda. Ureaseproductioncorrelateswithposscssionofthe rrh gene in Vibrio panrhcrertro!\rit.Irs strains isolated i n Thailand. J Infect Dis 172:1405-1408, 1995. K Venkateswaran, T Kurusu. M Satake, S Shinoda. Comparison of a fluorogenic assay with aconventionalmethodforrapiddetectionof Vihrio /,e~rahrrerrro!\'tie~t.s in seafoods.Appl Environ Microbiol 62:35 16-3520, 1996. and related halophilic vibrios. SW Joseph, RR Colwell, JB Kaper. Vihrio /,ctrtrhcterrro/~?ie~,s Crit Rev Microhiol 10:77-124, 1982. T Kaneko, RR Colwcll. Adsorption of Vihrio /"rrer/ltrc.rrro/~ric~t.s onto chitin and copepods. ApplMicrohiol29:269-274,1975. A DePaola,LH Hopkins, JT Peeler, B Wentz, RM McPhearson. Incidenceof Vibrio pcrrcrhrrc,rrlo!\'ficus i n U.S. coastal waters and oysters. Appl Environ Microhiol 56:2299-2302, 1990. NH Bean, EK Maloney, ME Potter, P Korazemo, B Ray, JP Taylor, S Seigler. J Snowdcn. Crayfish: a newly recognized vehicle for vibrio infections. Epidemiol Infect 121:269-273, 1998. R Sakazaki, S Iwanami, K Tamura. Studies on the enteropathogenic, facultatively halophilic bacterium, Vibrio p"r~rhoetrro/~ficlr.s. 11. Serological characteristics. Jpn J Med Sci Biol 2 I : 313-324,1968. S Wagatsuma. A medium for the test of the hemolytic activity of Vibrio parerhrr~~rr~o!\tic~ts. Media Circle 13: 159- 16l , 1968. I Karunasagar, G Sugumar, PJA Reilly. Rapid polylnerase chain reaction method for detection of Kanagawa positive Vibrio p~rr~h~rcrrro!\.ri~~tr.s in seafoods. Int J Food Microbiol 31: 1-3, 1996. CA Kaysner, C Aheyta Jr.PA Trost, JH Wetherington, KC Jinneman, WE Hill, MM Wekell.
176
86.
86a.
87. 88.
89. 90.
91.
92.
93.
94.
95. 96. 97. 98.
99.
100. 101.
102.
103.
104.
Herwig
Urea hydrolysis can predict the potential pathogenicity of Vibrio /'ara/rtrer,ro/yticrts strains isolated in the Pacific Northwest. Appl Environ Microbiol 60:3020-3022, 1994. R Osawa. T Okitsu, H Morozumi, S Yamai. Occurrence of urease-positive Vibrio p r r n t h r r e rtro!\.ticw.s in Kanagawa,Japan,withspecificreference to presence of thermostabledirect hemolysin (TDH) and the TDH-related-hemolysin genes. Appl Environ Microbial 62:725727, 1996. HC Chen, TC Chang. Rapid detection ofVibrio /'err(rhrre,rro!\ticrr.s i n oysters by itnmunolluorcscence microscopy. In1 J Food Microbiol 2931 1-3 19, 1996. J-Y D'Aoust. Sa/r~ronel/er species. In:MP Doyle, LR Beuchat,TJ Montville, cds. Food microbiology: fundamentals and frontiers. Washington, D.C.: ASM Press. 1997, pp. 129-158. WH Ewing. The notnenclature ofSnhmmdln, its usage, and definitions for the three species. Can J Microbiol 18: 1629-1637, 1972. E deBoer.Updateonmediaforisolationof E~rter[)btrc.ter-irre,erre from foods. I n t J Food Microbiol45:43-53.1998. WH Andrews. CA June, P Shcrrod, TS Hatnmack,RM Amaguana. Scr/lwne//rr. In: Bacteriologicalanalyticalmanual.8th ed. Rev.A.Gaithersburg,MD:AOACInternational,1988, pp. 5.01-5.20. AD Hitchins.PFeng, WD Watkins,SRRippey. LA Chandler. Esc~heric~hirrcoli andthe coliform bacteria. In: Bacteriological analytical manual. 8th ed. Rev. A. Gaithersburg, MD: AOAC International, 1998, pp. 4.01 -4.29. MP Doyle. T Zhao, J Meng. S Shaohua. Escherichia coli 0157:H7. In: MP Doyle. LR Beuchat, TJ Montville, eds. Food microbiology: fundatncntals and frontiers. Washington, D.C.: ASMPress. 1997,pp.171-191. EW Frampton, L Restaino. Methods for E.scherichiLr coli identification in food, water and clinical samples based on beta-glucuronidase detection. J Appl Bacteriol 74:223-233. 1993. K Vcnkateswaran, A Murakoshi, M Satake. Comparison of cotntnercially available kits with standard mcthods for the detection of coliforms and E.scheric.hitrcoli i n foods. Appl Environ Microbiol62:2236-2243,1996. AOAC International. Official methods of analysis of AOAC International. Arlington, VA: AOACInternational,1995. Atnerican Public Health Association. Recommended procedures for the examination of sea water and shellfish. 4th ed. New York: American Public Health Association, 1970. I n : Bacteriological analytical manual. 8th ed. JM Hunt. C Abeyta. T Tran. Crirr~y?\'/ohocter. Rev. A. Gaithersburg, MD: AOAC International, 1988, pp. 7.01-7.24. I Nacharnkin. Ccrr~rpy/?\.lohtrc~ter j e j w i . In: MP Doyle, LR Beuchat. TJ Montville. eds. Food microbiology:fundamentalsandfrontiers.Washington, DC.: ASMPress. 1997,pp.159170. RC Reinhard, TJ McAdam, GJ Flick, RE Croonenberghs, RF Wittman, AA Diallo, C FernanL > / / ~ I , preltnrodes. Analysis of Crrlrrpylobacterjejuni, Crrnrpylohrrcter co/i, S ~ I / I I I ~ I I K/ebsic~//rr n i r w , and E.scheric.hia c d i 0 157:H7 in fresh hand-picked blue crab (Co//inecte.ss c r p i d r t s ) meat. J Food Protect 59:803-807, 1996. RJ Gilbert. Food-borne infections and intoxications: recent problems and ncw organisms. Br F o ~ dJ 90:71-73, 1988. gastroenteritis associated MR Griffin, E Dalley, M Fitzpatrick, SH Austin. Crrr~r/~y/ohtrcter with raw clams. J Med Soc N J 80:607-609. 1983. C Abeyta Jr, FG Deeter, CA Kaysner, RF Stott, MMWekell. Crrrnp~lobucterjejurri in a Washington State shellfish growing bed associated with illness. J Food Protect 56323-325, 1993. JS Vliegcnthart. Aanwcaigheid en betekenis van Catnpylobacter in schelpdieren. DeWare(n) Chemicus 24: 115-1 17, 1994. B Mishu, CM Patton, RV Tauxe. Clinical and epidemiologic features of non-jejrrni, non-cdi Canrpylobaeter species. In: I Namchatnkin. MJ Blaser, LS Tompkins, eds. CrrnrpvIohtrctc,r
Pathogens Transmitted by Seafood
177
jejuni: current status and future trends. Washington, D.C.: American Society for Microbiol105.
106. 107. 108.
109. 110. 111.
112. 113. 114.
115. 116.
117. 118.
119. 120. 121.
122. 123. 124.
125.
126.
ogy.1992,pp.31-41. C Abeyta Jr. PA Trost, DH Bark, JM Hunt, CA Kaysner, BJ Tenge, MM Wekell. The use of bacterial membrane fractions for the detection of Canlpyloharter species in shellfish. J Rapid Meth Automat Microbiol 5:223-247, 1997. IG Wilson, JE Moore. Presence of Salmonella spp and Can~pylohocterspp in shellfish. EpidemiolInfectI16:147-153,1996. JM Farber, PI Peterkin. Listeria monocytogenes, a food-borne pathogen. Microbiol Rev 55: 476-51 I , 1991. J Rocourt,PCossart. Listericr nronoc.~togene.s.In:MPDoyle, LR Beuchat,TJMontville, eds. Food microbiology: fundamentals and frontiers. Washington, D.C.: ASM Press, 1997, pp. 337-352. S Tonkin, N Yeates, R Stamp, D Lennon, B Lewis, C Mantell, D Becroft, B Dove, K Farmer, K Mickleson. Epidemic perinatal listeriosis. Pediatr Infect Dis 3:30-34, 1984. WF Schlech 111. Lowbury Lecture. Listeriosis: epidemiology. virulence and the significance of contaminated foodstuffs. J Hosp Infect 19:211-224, 1991. KG Colburn, CA Kaysner, C Abeyta Jr, MM Wekell. Listeria species in a California coast estuarine environment. Appl Environ Microbiol 56:2007-2011, 1990. R Dillon, T Patel,S Ratnam. Occurrence ofListerin in hot and cold smoked seafood products. Int J Food Microbiol 22:73-77, 1994. PK Ben-Embarek. Presence, detection and growth of Listeria monocytogenes in seafoods: a review. Int J Food Microbiol 23:17-34, 1994. SD Weagant,PN Sado, KG Colburn, JD Torkelson, FA Stanley, MH Krane, SC Shields, CF Thayer. The incidence of Listeria species in frozen seafood products. J Food Protect 5 1 : 655-657,1988. AD Hitchins. Listeritr rt,onoc:\.togene.s. In: Bacteriological analytical manual. 8th ed. Rev. A. 10.01-10.13. Gaithersburg, MD: AOAC International, 1998, pp. A Agersborg, R Dahl, I Martinez. Sample preparation and DNA extraction procedures for polymerase chain reaction identification of Listeria rnonocytogenes in seafoods. Int J Food Microbiol35:275-280,1997. G Kapperud. Yersirlia enterocolitica. Int J Food Microbiol 12:53-65, 1991. SD Weagant, P Fcng, JT Stanfield. Yersinicr enterocolitica and Yersinin pse~tdotuherc.~closis. In: Bacteriologicalanalyticalmanual.8th ed. Rev. A. Gaithersburg,MD:AOACInternational, 1998, pp. 8.01-8.13. TL Cover, RC Aber. Yer.sinin e~lteroco1itic.u.N Engl J Med 6:16-24, 1989. WL Chao, RJ Ding, RS Chen. Survival of Yer.sinia enterocolitica in the environment. Can J Microbiol34:753-756,1988. RM Robins-Browne. Yersinicc enterocoliticn. In: MP Doyle, LR Beuchat, TJ Montville, eds. Foodmicrobiology:fundamentalsandfrontiers.Washington,D.C.:ASMPress,1997, pp.192-215. WA Rutah, FA Sarubi Jr, CS Finch, JN McCormack,GE Steinkraus. Oyster-associated outbreak of diarrhoeal disease possibly causedby Plesionmnas shigelloides. Lancet 1:739, 1982. SD Holmberg, IK Wachsmuth, FW Hickman-Brenner, PA Blake, JJ Farmer 111. P1e.siomonu.s enteric infections in the United States. Ann Intern Med 105:690-694, 1986. JA Koburger, C-I Wei. Plesionroncrs .shigelloides. In: C Vanderzant, DF Splittstoesser, eds. Compendium of methods for the microbiological examination of foods. 3rd ed. Washington, D.C.: American Public Health Association, 1992, pp. 517-522. SM Kirov. Aeromonns and Plesiornonm species. In: MP Doyle, LR Beuchat, TJ Montville, eds. Food microbiology: fundamentals and frontiers. Washington, D.C.: ASM Press, 1997, pp. 265-287. Food and Drug Administration. Bad bug book. Foodborne pathogenic microorganisms and natural toxins handbook. Retrieved via the Internet at vm.cfsan.fda.gov/-mow, 1992.
178
Herwig
Ple.sion/otlu.s shigel127. T Arai, N Ikejima,T Itoh, S Sakai, T Shimada, R Sakazaki. A survcy of loides fromaquaticenvironments,domesticanimals,petsandhumans.JHyg (Lond) 84: 203-21 1, 1980. 128. T Tsukamoto, Y Kinoshita, T Shimada, R Sakazaki. Two epidemics of diarrhoeal disease possibly caused by Plesiotwnus shigelloirles. J Hyg (Lond) 80:275-280, 1978. Edrvurdsiellu turrla and Plesiortror?c/sshige//oides 129. LR Van Damme, J Vandepitte. Isolation of from mammals and birds in Zaire. Rev Elev Med Vet Pays Trop 37: 145- 15 I , 1984. 130. DK Winsor, AP Bloebaum,JJ Mathewson. Gram-negative, aerobic, enteric pathogens among intestinal microflora of wild turkey vultures (Cothartes m m ) in west central Texas. Appl Environ Microbiol 42: 1 123- I 124, 198I . .shige//oitle.s from oysters using 131. SM Freund, JA Koburger, C1 Wei. Isolation of P/esiorrro~~u.s tetrathionate broth enrichment. J Food Protect 51:925-929, 1988. 132. Food and Drug Administration. Bacteriological analytical manual. 8th ed. Rev. A. Gaithersburg, MD: AOAC International, 1998. 133. SM Kirov. The public health significance of Aerorrror~trsspp. i n foods. Int J Food Microbiol 20: 179- 198, 1993. Aeronwr~us:anexpanding 134. JMJanda, SL Abbott.Evolvingconceptsregardingthegenus panorama of species, disease presentations, and unanswered questions. Clin Infect Dis 27: 332-344,1998. 135. JM Janda. Recent advances in the study of the taxonomy, pathogenicity, and infectious synhomes associated with thc genus Aeronronas. Clin Microbiol Rev 4:397-410, 1991. 136. RJ Seidler, DA Allen, H Lockman, RR Colwell, SW Joseph, OP Daily. Isolation, enumeration, and characterization of Aerornoms from polluted waters encountered in diving opera1980. tions. Appl Environ Microbiol 39:1010-1018, Aeromonns species in fish, fish-eggs, shrimp 137. ML Hanninen, P Oivanen, V Hirvela-Koski. and freshwater. Int J Food Microbiol 34:17-26, 1997. 138. S Palumbo, C Abeyta, G Stelma Jr. Aerornonus hydrophilu group. In: C Vanderzant, DF Splittstoesser, eds. Compendium of methods for the microbiological examination of foods. 3rd ed. Washington, D.C.: American Public Health Association, 1992, pp. 497-5 15. 139. ON Gill, WD Cubitt,DA McSwiggan, BM Watney, CL Bartlett. Epidemic of gastroenteritis caused by oysters contaminated with small round structured viruses. Br Med J 287.15321534,1983. 140. CS Grohmann, HB Greenberg, BM Welch, AM Murphy. Oyster-associated gastroentcritis in Australia: the detection of Norwalk virus and its antibody by immune electrcn microscopy and radioimmunoassay. J Med Virol 6: 1 1-19, 1980. 141. H Appleton. Foodborne viruses. Lancet 336: 1362- 1364, 1990. 142. RA Gunn, HT Janowski, S Lieb, EC Prather, HB Greenberg. Norwalk virus gastroenteritis following raw oyster consumption. Am J Epidemiol 115:348-351, 1982. 143. DLMorse,JJGuzewich, JP Hanrahan,RStricof,M Shayegani, RDeibel. JC Grabau, NANowak, JE Hernmann, G Cukor, NR Blacklow.Widespreadoutbreaks of clam- and oyster-associated gastroenteritis. Role of Norwalk virus. N Engl J Med 314:678-681, 1986. 144. CW Hedberg, MT Osterholm. Outbreaks of food-borne and waterborne viral gastroenteritis. Clin Microbiol Rev 6:199-210, 1993. 145. RDiGirolamo,JListon,JRMatches.Survival ofvirus i n chilled, frozen, andprocessed oysters. Appl Microbiol 2058-63, 1970. 146. FX Abad, RM Pinto, R Gajardo, A Bosch. Viruses in mussels: public health implications and depuration. J Food Protect 60:677-681, 1997. 147. UF Power, JK Collins. Differential depuration of poliovirus, E.scherichiu coli, and a coliphage by the common mussel, Mytihrs edulis. Appl Environ Microbiol 55: 1386-1390, 1989. 148. CS Grohmann, AM Murphy, PJ Christopher, E Auty, HB Greenberg. Norwalk virus gastroenteritis in volunteers consuming depurated oysters. Aust J Exp Biol Mcd Sci 5 9 2 19-228, 1981.
Pathogens Transmitted by Seafood
179
149. AZ Kapikian. Overview of viral gastroenteritis. Arch Virol Suppl 12:7-19, 1996. 150. DO Cliver. Foodborne viruses. In: MP Doyle, LR Beuchat, TJ Montville, eds. Food microbiology: fundamentals and frontiers. Washington, D.C.: ASM Press, 1997, pp. 437-446. 151. S McDonnell, KB Kirkland. WC Hlady, C Aristeguieta, RS Hopkins, SS Monroe, RI Glass. Failure of cooking to prevent shellfish-associated viral gastroenteritis. Arch Intern Med 157: I 1 1 - 1 16, 1997. 152. RL Atmar, FH Neill, CM Woodley, R Manger,CS Fout, W Burkhardt, L Leja, ER McGovern, F Le Guyader, TG Metcalf, MK Estes. Collaborative evaluation of a method for the detection of Norwalkvirus in shellfish tissues by PCR. Appl Environ Microbiol 62:254258, 1996. 153. RL Atmar, FH Neill, JL Romalde, G-F Le, CM Woodley, TG Metcalf, MK Estes. Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR. Appl Environ Microbiol61:3014-3018,1995. S Jin, JC Smith, PA Woods, MJ Clarke, MSHo,JR 154. RI Glass, PEKilgore.RCHoltnan, Gentsch. The epidemiology of rotavirus diarrhea in the United States: surveillance and estimates of disease burden. J Infect Dis 174(suppl I):S5-Sll, 1996. of poliovirus recovery techniques 155. B Mignotte, D Terver, L Schwartzbrod. Comparative study from mussel tissues. Mar Pollut Bull 342375479. 1997. 156. AB Dix, LA Jaykus. Virion concentration method for the detection of human enteric viruses in extracts of hard-shelled clams. J Food Protect 61:458-465, 1998. 157. E Dubois, F Le Guyader, L Haugarreau, H Kopecka, M Cornier, M Pommepuy. Molecular epidemiological survey of rotaviruses in sewage by reverse transcriptase seminested PCR and restriction fragment length polymorphism assay. Appl EnvironMicrobiol63: 1794- 1800, 1997. J Luthy,PHubner.Seminested RT-PCRsystemsforsmallround 158. DHafliger,MGilgen, structured viruses and detection of enteric viruses in seafood. Int J Food Microbiol 37:2736, 1997. 159. HM Solomon, R Lilly Jr. Clostridium hotulinum. In: Bacteriological analytical manual. 8th cd. Rev. A. Gaithersburg, MD: AOAC International, 1998, pp. 17.01-17.10. 160. KLDodd,JWAustin.Clostridiumbotulinum.In: MP Doyle, LR Beuchat, TJ Montville, eds. Food microbiology: fundamentals and frontiers. Washington, D.C.: ASM Press, 1997, pp.288-304. 161. G Hobbs. C. / w r u / i n u t n and its importance in fishery products. Adv Food Res 22: 135- 185, 1976. Clostridium 162. MW Eklund. Effects of CO,-modified atmospheres and vacuum packaging on boru/inurrr and spoilage organisms of fishery products. Proceedings of the First National Conference on Seafood Packaging and Shipping, Washington, D.C., 1982, pp. 298-330. ofbotuli163. LS Post. DA Lee, MM Solbergm, D Furgang, J Specchio, C Graham. Development nal toxin and sensory deterioration during storageof vacuum and modified atmosphere packaged fish fillets. J Food Sci 50:990-998, 1985. of type E botulism associ164. Centers for Disease Control and Prevention. International outbreak ated with ungutted, salted whitefish. MMWR Morb Mort Wkly Rep 36:812-813, 1987. 165. RW Bennett, CA Lancette. Sttr/’h?./ococ,c.usaureus. In: Bacteriological analytical manual. 8th ed. Rev. A. Gaithersburg, MD: AOAC International, 1998, pp. 12.01-12.05. 166. LM Jablonski, CA Bohach. Strr/’/l)’/oc.oc.c.ltswreus. In: MP Doyle, LR Beuchat, TJ Montville, eds. Food microbiology: fundatnentals and frontiers. Washington, D.C.: ASM Press, 1997, pp. 353-375. Srr-epel,lococ.cwsjn&167. Centers for Disease Control and Prevention. Invasive infection with Ontario, 1995-1996. MMWR MorbMortalWklyRep 5550-653, 1996. 168. M Weinstein, D Low, A McGeer, B Willey, D Rose, M Coulter, P Wyper, A Borczyk, M Lovgren,RFacklam.Invasiveinfectiondueto Streptoc.oc.c.u.s in&: a new or previously unrecognized disease-Ontario. 1995- 1996. Can Cotnmun Dis Rep 22: 12913 1, 1996.
180
Herwig
169. MR Weinstein, M Litt, DA Kertesz, P Wyper, D Rose, M Coulter. A McGeer, R Facklam, C Ostach, BM Willey,ABorczyk, DE Low.Invasiveinfectionsduetoafishpathogen, Streptococ~cusinine. S. irline Study Group. N Engl J Med 337589-594, 1997. 170. WE Hill. The polymerase chain reaction: applications for the detection of foodborne pathogens. Crit Rev Food Sci Nutr 36:l-2, 1996. 171. PM Scheu, K Berghof, U Stahl. Detection of pathogenic and spoilage micro-organisms in food with the polymerase chain reaction. Food Microbiol 15: 13-3 I , 1998. 172. KL Josephson,CP Gerba, IL Pepper. Polymerase chain reaction detection of nonviable bactcrial pathogens. Appl Environ Microbiol 59:35 13-35 15. 1995. 173. R-F Wang, W-W Cao, CE Cerniglia. A universal protocol for PCR detection of 13 species of foodborne pathogens in foods. J Appl Microbiol 83:727-736, 1997. 174. KC Klontz, JC Desenclos, LE Wolfe, SA Hoecherl, C Roberts, RA Gunn. The raw oyster consumer-a risk taker? Use of the Behavioral Risk Factor Surveillance System. Epidemiology 21437-440, 199 1. 175. MB Ali, MJ Raff. Primary Vihrio r l t / n ; j i c u . s sepsis in Kentucky. South Med J 83356-357. 1990. 176. LD Rotz, DP Buckley, DP Fine. Overwhelming sepsis withVibrio ~ t / r ~ ~ ' $ c . u .as :coastal pathogen in Oklahoma. J Okla State Med Assoc 89349-352, 1996. 177. RJ Hally, RA Rubin, HS Fraimow, ML Hoffman-Terry. Fatal Vihrio porohemo!\tic~t.ssepticemia in a patient with cirrhosis. A case report and review of the literature. Dig Dis Sci 40: 1257-1260,1995. 178. M Tsuzuki, T Ino, F Maruyama, M Okamoto, K Ezaki. M Hirano. Vihrio w/rl;jicu.s scpticemia in a patient with severe aplastic anemia. Int J Hematol 67:175-178, 1998. 179. ES Garrett, ML Jahncke, JM Tennyson. Microbiological hazards and emerging food-safety issues associated with seafoods. J Food Protect 60:140Y-1415, 1997. 180. S Pullela, CF Fernandes, GJ Flick, CS Libey, SA Smith, CW Coale. Indicative and pathogenic microbiological quality of aquaculturcd finfish grown in different production system. J Food Protect 61:205-210, 1998. of epidemic Vibrio e/?o/ercteby 181. SA McCarthy, FM Khambaty. International dissemination cargo shipballastandothernonpotablewaters.ApplEnvironMicrobiol60:2597-2601, 1994. 182. J Briley. Texas oyster sites will be cleared if FDA finds no more evidence of Vihrio. Food Chcm News 40:25-26, 1998. 183. Anonymous.Newsystemforseafoodsafety.EnvironHealthPerspect106:A475-A478. 1998. 184. JA Hasan, A Huq, GB Nair, S Garg, AK Mukhopadhyay, L Loomis, D Bernstein. RR colwell. Development and testing of monoclonal antibody-based rapid immunodiagnostic test kits for directdetection of Vihrio chlercre 0139 synonymBengal. J ClinMicrobiol 33: 2935-2939,1995. 185. JA Hasan, D Bernstein, A Huq, LLoomis,ML Tamplin, RRColwell.CholeraDFA:an improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vihrio cholerne 0 1 . FEMS Microbiol Lett 120: 143-148, 1994. 186. JA Hasan, A Huq, ML Tamplin, RJ Siebeling, RR Colwell. A novel kit for rapid detection of Vihrio cholrrue 01. J Clin Microbiol 32:249-252, 1994. 187. DR Mackenzie, DJ Livingstone. Salmonellae in fish and food. S Afr Med J 42:999-1003, 1968. the channel PL Poelma, A Romero. Bacteriological survey of 188. WH Andrews, DR Wilson, catfish (Icrolunts purlemus) at the retail level. J Food Sci 42:359-363, 1977. 189. LE Wyatt, R Nickelson, C Vanderzant. Occurrence and control of Sa/nlone//a in freshwater catfish. J Food Sci 44:1067-1073, 1979. //rr of eel cUltUrc pollds. Nippon 190. K S&&], S Kobayashi, R Kawanishi.S n / r ~ ~ o r ~ econtamination Suisan Gakkaishi 55:675-679, 1989.
Pathogens Transmitted by Seafood 191.
192.
193. 194. 195.
196. 197.
181
PJA Reilly, DR Twiddy. Str/rtlorlel/rr and Vibr-ioC / l ~ J / f > r m ’in brackish water cultured tropical prawns. lnt J Food Microbiol 16:293-301, 1992. JR MacMillan, T Santucci. Seasonal trends in intcstinal bacteral floraof farn-raised channel 1990. catlish. J Aquat Anim Health 2:217-222, LE Wyatt, R Nickelson, C Vanderzant. Edrc*ctrc/.sic~/lrrtcwllct in freshwater catfish and their environment. Appl Environ Microbiol 38:710-714, 1979. C-K Leung, Y-W Huang, OC Pancorbo. Bacterial pathogens and indicators in catfish and pond environment. J Food Protect 55:424-427, 1992. PW Lowery, LM MacFarland. HK Threefoot. Vihrio h o l l i s c t e septicemia after consumption of catfish. J Infect Dis 154:730, 1986. PC Nedoluha. D Wcsthoff. Microbiological flora of aquacultured hybrid striped bass. J Food Protect 56:1054-1060, 1993. Plesionror~ct.~ ,shige/loid~~.s isolated HSugita.TNakamura,YDcguchi.Identificationof Irom freshwater fish with the microplate hybridization method. J Food Protect 56:949-953. 1993.
198.
199. 200.
201. 202. 203.
204.
205.
206.
207. 208. 209.
210.
21 I . 212. 213
WB Baine. M Mazzotti. D Greco, E Izzo, A Zampieri. G Angioni, G-M Di, EJ Gangarosa, F Pocchiari. Epidemiology of cholera in ltaly in 1973. Lancet 21370-1374. 1974. PA Blake, ML Roscnberg. JB Costa. PS Ferreira, CL Guimaraes, EJ Gangarosa. Cholera in Portugal.1974. I. Modes of transmission. Am J Epidemiol105:337-343,1977. MH Merson. WT Martin, JP Craig, GK Morris, PA Blake, GF Craun, JC Feeley, JC Camaofnoncho. EJ Gangarosa. Cholera on Guam, 1974: epidemiologic findings and isolation toxinogenic strains. Am J Epidemiol 105:349-36 I . 1977. RCMclntyre,TTira.TFlood, PABlake.Modesoftransmissionofcholera in anewly infected population on an atoll: implications for control measures. LancetI :3l 1-3 14. 1979. World Health Organization. Cholera, Italy. Wkly Epidemiol Rec 69:332, 1994. JT Wcher. ED Mintz, R Canizarcs, A Scrniglia, I Gomez, R Sempetegui, A D’Avila, KD Grcene, ND Puhr, DN Carneron, et a l . Epidemic cholera in Ecuador: multidrug-resistance and transmission by water and seafood. Epidemiol Infect 112: 1-1 I , 1994. 1991. MMWR Morb Mortal Wkly Rep Centers for Disease Control. Cholera-New York, 40:516-518, 1991. P-J Eberhart, RE Besser, MP Tormey, D Koo, D Feikin. MR Araneta, J Wells, L Kilman. GW Rutherford. PM Griffin, R Baron, L Mascola.An outbreak of cholera from food served on an international aircraft. Epidemiol lnfcct 1 l6:9- 13, 1996. PWLowry,ATPavin.LMMcFarland,BHPeltier,TJBarrett,HBBradford,JMQuan.J Lynch,JBMathison,RA Gum, PABlake.Cholera in Louisiana.Wideningspectrumof seafood vehicles. Arch Intern Med149:2079-2084. 1989. Centers for Disease Control. Toxigenic Vihrin rholcroe 0 1 infection acquired in Colorado. MMWR Morb Mortal Wkly Rep 38:19-20. 1989. NR Blacklow, JE Hernnann. Astrovirus gastroenteritis. Trans Am Clin Climatol Assoc 106: 58-66, 1994; discussion106:66-68, 1994. EA Szabo, JMPemberton. AM Gibson, MJ Eyles, PM Desmarchelier.Polymerasechain reaction for detection of C/o.stridim h o t l t l i u l r r r r types A, B and E in food, soil and infant faeces. J Appl Bacteriol 76539-545, 1994. KC Jinneman, PA Trost, WE Hill,SD Weagant, JL Bryant, CA Kaysner. MM Wekell. Comparison of template preparation methods from foods for amplification of E.scherirhitr coli 0157 Shiga-like toxins type I1 and I1 DNA by multiplex polymerase chain reaction. J Food Protect58:722-726, 1995. MR Andersen, CJ Omiecinski. Direct extraction of bacterial plasmids from food for polymerase chain reaction amplification. Appl Environ Microbiol 58:4080-4082, 1992. NS Bansal. Development of a polymerase chain reaction assay for the detection of Lisrerirl rrrorloc:\roaerre.s in foods. Lett Appl Microbiol 22:353-356, 1996. MC Simon. D1 Grey. N Cook, LA Jaykus, L-R De, MS Sobsey. DNA extraction and PCR
182
214.
215.
216. 217. 218. 219. 220.
221.
222. 223.
224.
225. 226. 227. 228. 229.
Herwig
methods for the detection of Listeria morm-ytogerres in cold-smoked salmon. Appl Environ Microbiol 622322424, 1996. C Niederhauser, C Hofelein, J Luthy, U Kaufmann, HP Buhler, U Candrian. Comparison of “Gen-Probe”DNAprobeandPCRfordetection of Listericc nronocytogenes innaturally contaminated soft cheese and semi-soft cheese. Res Microbiol 144:47-54, 1993. K Wernars, CJ Heuvelman, T Chakraborty, SH Notermans. Use of the polymerase chain reaction for direct detection of Listeria rrlonocytogews in soft cheese. J Appl Bacteriol 70: 121-126,1991. Q Zhu, CK Lim, YN Chan. Detection of Saln~or~ella tyyhi by polymerase chain reaction. J Appl Bacteriol 80:244-25 1, 1996. AK Bej, MH Mahbubani, MJ Boyce, RM Atlas. Detection of Scrlrmnella spp. in oysters by PCR. Appl Environ Microbiol 60368-373. 1994. DD Jones, R Law, AK Bej. Detection of Seclrrwnelltr spp. in oysters using polymerase chain reaction (PCR) and gene probes. J Food Sci 58:1191-1197, 1993. WH Koch, WL Payne, BA Wentz, TA Cebula. Rapid polymerase chain reaction method for detection of Vibrio cholerne in foods. Appl Environ Microbiol 59:556-560, 1993. YH Shangkuan, YS Show, TM Wang. Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae andtobiotype Vibrio choleme 01. JApplBacteriol79:264-273, 1995. CY Lee, SF Pan, CH Chen. Sequence of a cloned pR72H fragment and its use for detcction Iof Vibrio parcthaenlolyticus in shellfish with the PCR. Appl Environ Microbiol 61:131 1317,1995. CR Arias, E Garay, R Aznar. NestedPCR method for rapid and sensitive detectionof Vibrio wlnijicus in fish, sediments, and water. Appl Environ Microbiol 61:3476-3478, 1995. WE Hill, SP Keasler, MW Trucksess, P Feng, CA Kaysner, KA Lampel. Polymerase chain reaction identification of Vibrio vulnijicus in artificially contaminated oysters. Appl Environ Microbiol 57:707-711. 1991. of a method for detectionof enteroviruses DN Lees, K Henshilwood, WJ Dore. Development as amodel.ApplEnvironMicrobiol 602999-3005, inshellfishbyPCRwithpoliovirus 1994. LA Jaykus, R De Leon, MS Sobsey. Application of RT-PCR for the detcction of enteric viruses in oysters. Water Sci Techno1 27:49-53, 1993. H Chung, LA Jaykus, MD Sobsey. Detection of human enteric viruses in oysters by if! vivo and in vitro amplification of nucleic acids. Appl Environ Microbiol 62:3772-3778, 1996. RL Atmar, TG Metcalf, FH Neill, MK Estes. Detection of enteric viruses in oysters by using the polymerase chain reaction. Appl Environ Microbiol 59:631-635, 1993. V Gouvea, N Santos, M do Carmo Timenetsky, MK Estcs. Identification of Norwalk virus in artificially seeded shellfish and selected foods. J Virol Meth 48:177-187, 1994. C1 Gallimore, DW Brown.Detectionofsmallround DN Lees, K Henshilwood, J Green, structuredviruses inshellfishbyreversetranscription-PCR.ApplEnvironMicrobiol61: 4418-4424,1995.
6 Laboratory Methodology for Shellfish Toxins
Introduction I83 11. Methods of PSP Testing185 A. Bioassay methods 185 B. Chemical methods 190 C. Immunological assays 192 111. Domoic Acid Testing 194 A. Bioassays 195 B. High-performance liquid chromatography (HPLC) C. Capillary electrophoresis 197 Gas chromatography D. 197 E. Radioassays 197 F. Enzyme-linked immunosorbent assay (ELISA) 197 IV. Conclusions 201 References20 1 I.
1.
196
INTRODUCTION
Paralytic shellfish poisoning (PSP) occurs following the consumption of shellfish that have been exposed to toxic marine dinoflagellates and contain a potent source of neurotoxins, called paralytic shellfish toxins (PSTs) ( l ) . PSTs consist of at least 18 different toxins that can be divided into subclasses containing hydrogen atom (e.g., saxitoxin) or hydroxyl (neosaxitoxin-relatedtoxins)groupsontheR1position.Gonyautoxinstoxins (GTXs), are characterized by 1 l-hydroxy-saxitoxin sulfate derivatives (Fig. 1). Dinoflagellates are important ocean plankton that contribute to the foundation of the marine food chain as a primary source of carbohydrates, proteins, and lipids. The organisms are microscopic, single-cell photosynthetic algae, which during periodic maxima produce local discolorations of coastal waters. Shellfish poisonings are difficult to control because of the unpredictable and sporadic occurrence of the dinoflagellates which produce PSTs. The initial 183
184
Kitts
NH
Toxin
H H H
H OH H H H H H
H H
Saxitoxin Hc-N-OH'-saxitoxin H dc2saxitoxin saxitoxin doc3 Saxitoxin ethanoic acid OH Neosaxitoxin H c-N-OH-neosaxitoxin OH H dc OHneosaxitoxin OCONH2 GonyautoxinH 1 gonyautoxin-l OH dc H Gonyautoxin-2 gonyautoxin-2 H dc H Gonyautoxin-3 gonyautoxin-3 H dc OHGonyautoxin-4 gonyautoxin-4 OH dc H Goilyautoxin-5 OH Gonyautoxin-6 Cl c2
c3 c 4
RI
R2
H
CHzCOOH OCONH2 H
R3
I4
H3
OH
oso;
oso; OCONHZ oso; oso; OH OCONH2 oso; oso; OH
oso3' H H H H H
H H OH OH
oso;
H
oso3-
OH OCONH2 OH
oso; H
oso; H
OCONHSOY
ocoNHso; OCoNHso3' 0coNHso; OCONHSOi 0coNHso;
IC-N-OH:carbamoyl-N-hydroxy 2dc:decarbamoyl 'doc:deoxycarbamoyl Fig. 1 Saxitoxinandrelated PSP isomersassociatedwithparalyticshellfishpoisoning.
relationship between PSTs and the presence of plankton from the genus Gntzy1ulnx was reported by Sommer et al. (2). In fact, toxic dinoflagellate blooms occur sporadically in large numbers from as many as 20 species of dinoflagellates throughout the world, with the greatest frequencies occurring in the north and south temperate zones (3). Outbreaks of PSP occur in both protected and unprotected waters in which conditions arc prone to an upwelling of nutrient-rich water often associated with organic loadingof seawater from run off or sewage disposal (4), or unique changesin salinity, temperature, light, and water turbulence. Seasonal outbreaks of red tide vary in coastal regions, as evidenced by the
Laboratory Methodology for Shellfish Toxins
185
reported outbreaks between mid-May and late October alongthe Pacific Coast,in contrast to the period of Inid-July to late September in Atlantic Canada (5).Dinoflagellates represent a rich source of food for bivalve shellfish and changes in water conditions or the presence of predators,suchasdiatoms, will change thedinoflagellatepopulation.The principle group of shellfish associated with PSP outbreaks has been the bivalve mollusks, such as clams, mussels, scallops, and oysters. These species accumulate PSTs from poison011s dinoflagellates in the hepatopancreas and are then metabolized, excreted, or released within several weeks. One exception is the Alaskan butter clam (Srrsidorrlus Lqi,qtrrltc’/r.s), which mobilizes PSTs from the hepatopancreas to the siphon, where they can remain for of becolnillg several years (6). Shellfish become toxic with PSTs only after a few days exposed to a red tide bloom or extremely high concentrationsof poisonous dinoflagellates i n cohabitat waters. Other intertidal organisms such as gastropods, periwinkles. whelks, and certain crab specieshave also been foundto contain PSP (7-9). The blooms of Gor~yt r c r k r . ~species are often referred to as red tide b l o o m due to the color produced from a xanthophyll derived from light-harvesting antenna ( I O ) and the fact that the bloolns are 90-95% monospecific tothisspecies of dinoflagellatethatreachasninnyas 20.00030,000 organisndml of seawater. The distribution of PSP in shellfishisvariableand I n the case with red depends on the species of bivalve and the specific tissue involved. tide blooms, the correct environmental conditions produce a generation of algae that produce domoic acid poisoning, also referredto as amnesic shellfish poisoning (ASP). Subsequently, shellfish and finfish which feed by filtering seawater concentrate the algae and accumulate domoic acid toxin, principally i n hepatopancreatic tissue. As is the case with other sources of seafood toxins, the principle agent is confined to the viscera. Shellfish poisoning represents both a serious public health concern as well as having potentially devastating economic consequences where shellfish harvesting, tourism, recreation, and restaurants are important localindustries.Thistypeofpoisoningcannotbe completely controlled or prevented and the absence of an antidote has resulted in temporary incapacitating illness and even death. Although the number of mortalities attributed to PSP following consumption of contaminated shellfish is relatively low, the control of shellfish poisoning is largely the result of extensive monitoring procedures principally controlled by government health bodies. Since there is very little information on the predictors of when and where sourcesof the toxins may occur, PSP and ASP monitoring are imperative to ensure a safe and viable industry. This chapter describes the different laboratory methods that have been used to detect shellfish poisons and estimate toxicity.
II. METHODS OF PSP TESTING
A.
Bioassay Methods
1. Mouse Bioassay (Mouse Lethality Technique) The standard tnouse bioassay, initially developed by S o n m e r and Meyer ( l ) , is currently the official method for measuring shellfish toxicity ( 1 1). The procedure measures total toxicity of a shellfish sample by challenging, with intraperitoneal injection, white Swiss mice of standard size ( 1 8-22 g) and sex (mal?) with an acid extract of homogenized shellfish tissue (Table I ) . The mouse bioassay, which was adopted in 1965 as an AOAC method in Canada. remains the accepted procedure for the assessment of PSP toxicity. Time to death is recorded for 3-5 mice to screen samples and in 10-12 animals to assay specific samples expected to contain PSTs. The survival time of mice administered acidic
Kitts
186
Table 1 ProceduralInformation for BioassayandChemicalPSTDctection Prmcipal
Assay
Reference
Houselly Chlcken emhryo
Elcctrophoresis Thin layer chromatography HPLC
HA-ISP-MS
Total toxlclty (mean mousc death tllnc) LD,,, in housclly Total toxlcity ( % mortality) Morphology or vlability o f N2A cell enzymatic metabolism Separatm on cellulose acetate strips TLC wlth lluorescent detcctlon (KOH + H@:) HPLC-cyanocolurnn separation; perlodate Ruorcsccnt derivative Ion spray nuss spectrometry Capillary electrophorcSIS with UV detection
CE-UV
STX
saxitoxin;Neo
=
Limitations
=
neosaxitoxin: GTX
Sensitivlty 30 pg/ 100 g shelllish 0 . 2 pg PSP/mI 20 pg/100 g shelllish 0.11 pg STX 3.0 pmole/ml STX 2 pg STX 2 pg/100 g tissue 0.4 pg/ 100 g tissue No quantltation
0.5 ng STX, GTX-2. GTX-3. 8.0 ng NCO.GTX- 1-4
0.1 pM STX 5.0 pM STX, 5.6 I.IM Nco-STX
=
CV = -+ 20%intcrlah varlation = 60-70% Microtechnique 96-hour Incubation time Effects o f extraction procedure unknown
2.13.1618.2 1 23 24 3 1-36
S5
Volume limitations
45
Nonuniform Iluoresccnce intensities for all PSTs limited t o laboratory environment Spcc~alizcdequipment
50-54
60-6 1
56
gonyautoxlns.
shellfish extracts is indicative of the presence and the toxicity of PSTs in the sample, which in turn is expressed in mouse units. One mouse unit is defined, as the amount of poison that will kill a 20 g mouse in 15 minutes with symptoms of paralysis or respiratory failure. The use of the mouse unit (MU) as a measure of toxicity was derived initially before the availability of a reference saxitoxin standard and was calculated from the following expression: log dose (MU) = 145/r - 0.2 where MU
=
mouse units, and
t = time in minutes.
MU/ Shellfish quarantines are put in place when toxicity from PSTs reaches 400 100 g shellfish. Human illness has been reported at 600 MU/100 g shellfish and death caused by PSP can occur at 3000 MU (12 ) . Schantz et al. ( 13) were successful in preparinga highly purified source of saxitoxin from the digestive glands of toxic mussels (Myrilus ctrlifrrtrimlus) and also from the siphons of toxic Alaska butter clams (Sc1.ridoma.sgigcrnteus). The reference saxitoxin standard was determined to elicit a toxicity of 5500 -+ 500 MU/mg and was subsequently used as the reference standard for the official mouse lethality bioassay. One mouse unit was determined to be equivalent to 0.18 pg saxitoxin dihydrochloride poison required to kill a mouse in approximately 10-20 minutes. For example, a value greater than 70-80
Laboratory Methodology for Shellfish Toxins
187
pg PST/100 g fresh shellfish is considered unfit for human consumption in Canada. Under permit for canning purposes, shellfish may be taken from areas with PST levels of 80160 pg/100 g tissue, since the canning process may reduce the toxin content by 90% when analyzed by the AOAC method (14). The lethal PST dose for humans is I O mg of toxin from consumption of shellfish ( 15). This value is obtained from mouse units calcuto five mice are referred to lated to the nearest 5 seconds. Mean time to death for three in Sommer’s table, and the total toxicity from the lethality time is standardized against a known saxitoxin standard solution. Such factors as strain and sex of the mice, route of administration. and pH or presence of ions such as sodium have been evaluated on the acute median lethal dose (LD,,,) of saxitoxin (16,17). Examplesof calculated toxicity from three variable mouse death times are shown below: Example I : Average weight of mice (g) = 24.97 = I . l 1 MU Death time (min) = 4.27 = 2.32 MU Corrected mouse units
= 1.1 1
Toxicity = 2.575 MU
* 0.18 pg poison/MU
4:
2.32 = 2.575 MU Q
100 g meat
= 92.7 yg PST/ IO0 g meat
Example 2: Average weight of mice (g) = 24.50 = 1.10 MU Death time (min) = 7.906 = 1.25 MU Correctedmouseunits
= 1.10
Toxicity = 1.375 MU
x.
:C
1.25 = 1.375 MU
0. I8 pg poison/MU
:k
100 g meat
= 49.5 yg PST/100 g meat
Example 3: Average weight of mice (g) = 24.30 = 1 . I O MU Death time (min) = 1.82 = 9.50 MU Corrected mouse units
= 1.82 * 9.50 = 10.45 MU
Toxicity = 10.45 MU
:k
= 376.2
0.18 pg poison/MU
Q
100 g meat
yg PST/ 100 g meat
Female mice are more susceptible than male mice at higher doses of toxin, with the LDS,) per mouse being directly related to the average body weight of the test animal. Acute LD,(, values determined for oral (263 pg/kg), intraperitoneal (10 pg/kg), and intravenous (3.4 pg/kg) ingestion demonstrated the relative toxicity of PST for different routes of exposure. Increases in pH above 4.0, or the addition of sodium ions,will reduce intraperitoneal toxicity of saxitoxin, but has less effect on the oral or intravenous toxicity. Conversely, boiling extracts at low pH levels( 1.S-4.0) for up to I O minutes can have no effect on mowe toxicity (14). The volumeof injection medium containing the toxic lnaterial was that the shown to have no effect on the toxicity. It was concluded from these findings median time to death of mice exposed to low concentrations of PST as a criterion of toxicity was not reliable at pH values above 4, or in the presence of sodium ions above a 0.1 M concentration following intraperitoneal exposure. Further studies by Park et al. ( 1 8) have confirmed the importance of the final pH of the sample extract, especially when
188
Kitts
samples are stored for various times. Storage of standard solutions for as long as 4 weeks at 4°C and pH 6.0 produced a SO% decrease in toxicity, even after adjustment to pH 2 prior to testing. The limit of sensitivity for the mouse bioassay is approximately 30 pg/ 100 g shellfish tissue, with an accuracy of approximately 20% of the maximum allowable content (e.g., 80 pg saxitoxin equivalents/ 100 g sample) (19), a level set by both North American and European communities (20). Other researchers have claimed that the lnouse bioassay may underestimate marginally toxicshellfish by as much as 60% (21), while interassay variation in sample analysis can reach 2 2 0 % (22). The errorin toxicity estimation depends on the mice being within the 18-20 g weight range, and the subjectivity of estimating time to death. Moreover, the mouse bioassay is limited to accurately deternlining the potential toxicity of shellfish extracts containing sulfamate toxins, since the conditions of sample preparation are not sufficiently acidic to ensure complete hydrolysis of the toxin complex to a corresponding carbamate state. In addition to the technical limitations. the mouse bioassay requires large mouse colonies to supply mice for testing with a relatively uniform body weight. The lethality procedure is relatively expensive becauseof the need to maintain a specific mouse colony and therefore it is not suitable for use in field conditions. The method, albeit the only true indicator of total toxicity, represents an unacceptable procedure for animal welfare groups. which has resulted in further pressure for its replacement with a more humane method for reliably monitoring PST in large numbers of contaminated shellfish samples.
2. Other Bioassays There have been many attemptsto develop alternative bioassay methods for PST measurement and screening, including houseflies (23), chicken embryos, brine shrimp and selected microbial assays (24), selected microbial bioassays, and tissue biosensors (25-27). The housefly and chicken embryo assays have noteworthy limitations concerning sensitivity, relatively long incubation periods, and specialized techniques which preclude their use as alternativestothemousebioassay.Aradiolabeldisplacementassaywasproposed by Davio and Fontelo (28), which was based on the affinity of saxitoxin and other paralytic toxins to compete for nerve membranes. Samples were mixed with tritiated saxitoxin and incubated witha suspension of rat brain membranes. Following incubation, the mernbranes were washed and radjoactivity was determined. The concentration of PST in the sample was inversely proportional to the radioactivity recorded by liquid scintillation counting. This assay was reported to be very sensitive ( 1 000 times that of the mouse bioassay); however, the method did not measure total toxicity and there was a problenl finding shellfish samples sufficiently free of PSTs, to be run as negative controls. Similar solid-phase, radioreceptor binding assays have been developed for estimating cumulative PST toxicity (29,30). Good agreement i n estimating saxitoxin equivalents over a wide concentration range (0.5 pM-0.1 mM) of saxitoxinusingboththemousebioassay ( r = 0.946) and HPLC ( r = 0.878) has been reported with dinoflagellate toxins (30). Furthermore, this radioreceptor assay also correlated very well (0.967) with HPLC results on PST-contaminated clams, mussels, and oysters over a concentration range of 40-8000 pg STX/100 g shellfish. A tissue biosensor procedure designed to rapidly (e.g., S min) measure small quantities of PSTs (e.g., S fg STX) has also been reported (26). Thebiosensor, composed of a sodium electrode which covers a frog bladder membrane, measures the affinity of various shellfish toxins to block sodium channels. The active transfer of sodium across a frog bladder from the internal to external side of the membrane is detected by a sodium electrode within a flow cell (25,27). There is little information available concerning the
Laboratory Methodology for Shellfish Toxins
189
influence of marine conditions (e.g., salt concentration, temperature) on the perfornxmce of this procedure. An extension of the tissue culture methodology concept involving rapidly growing mouse neuroblastoma cells was initially developed by Kogure et al. ( 3 1 ). Similar strategies have been developed using bacterial cultures (32). The channel blocking potential of saxitoxin and related toxins is used to antagonize the toxic effects of added known agents (e.g., ouabain and veratridine) which stimulate sodium influx and depolarize the action potential on excitable membranes. The effect of the presence of PSTs enables nerve cell or bacterial growth to continuein culture. Evaluation of both the morphology and viability of neuro-2A (N2A) cells was reportedto be simple, inexpensive, and more sensitive than chromatography methods for saxitoxin and gonyautoxin (31). Jellett et al. (33,34) automated this method by using a microplate reader to measure absorbances of crystal violet from vital stained neuroblastonla cells exposed to both pure saxitoxin standard and acidic shellfish extract. The extract was processed through an LC-l8 SPE (Supelco) cartridge. Detectionlimitswere as low as 10ngsaxitoxinequivalentsandcorrelatedverywell with the mouse bioassay over a wide range of toxin concentrations (0-5500 pg/IOO g). Subsequent studies by Manger et al. ( 3 5 ) employed cultured neuroblastoma cells (N2A, mouse cells) using a microtiter plate format; however, this protocol relied on enzymatic colorimetric end-point measurements to assess PST toxicity. The neuroblastoma cells me(3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazoli~~1~~) tabolizethereducingagentMTT when positively correlated with the concentration of saxitoxin standard in a concentration range of 0-0.6 ng. Moreover, these researchersalso demonstrated that this bioassay would work well with tissue extracts collected from crab viscera and produced results i n close agreement with the standard mouse bioassay (36). A detailed comparison of the sodium channel cytotoxicity assay with the traditional mouse bioassay for PST detection in shellfish has been reported (37).
3. Macroscopic and Biotechnology Methods Toxin-producing dinoflagellates can be monitored using a light microscope and, therefore, in contrast to the mouse bioassay. may provide an early warning signal of impending toxic blooms. This allows cultured shellfish to be harvested or salmon ranchesto be moved away prior to an infestation of toxic algae (38). PST production is a stable characteristic in selected strains of dinoflagellates; however, training is required to distinguish toxic from nontoxic strains under light microscopy. A specific oligonucleotide gene probe was developed to recognize regions of DNA that are unique to any given organism or group of related organisms. It was postulated that using DNA probesto recognize toxic dinoflagellates could provide a rapid and cost-effective early warning method for the presence of impending red tide blooms. Specific DNA probes were also considered useful to serve as molecular tags for tracking the movement of toxic blooms, or for studying the dynamics of phytoplankton growth and succession in nlarine waters. Specific molecular weight soluble proteins have been identified in both crabs and clams (8,39,40) and correspond to estimates of toxicity using the mouse bioassay or fluorescence detection. The induction of a specific 79 kDa, protein in crabs exposed to saxitoxin injections was used as the basis for ELISA and Western blotting methods to screen clam and crab tissue for PSTs (41). Finally, Fairey et al. (42) published a gene manipulation procedure whereby a c,fOs luciferase reporter gene construct was cloned in an N2A mouse neuroblastoma cell line to enable a direct cytotoxicity measurement, utilizing a luciferase catalyzed light gen-
790
Kifts
eration as an end-point measurement. This procedure enabled sensitive detection of saxitoxin over a range of 1-10 ng/ml. As is the case with other cell cytotoxicity or receptor binding assays, this method does not provide information on the identity of the chemical toxin and therefore would work well in parallel with HPLC methods.
B. Chemical Methods A chemical approach to monitoring PSTs in shellfish was first developed by Bates and Rapoport (43) and Bates et al. (44) using a fluorometricmethod.Thisprocedure also provided the basis for several subsequent chemical assays, including thin-layer chromatography (TLC) ( 4 3 , low-pressure liquid column, and HPLC methods. The method used an oxidation step with hydrogen peroxide under alkaline conditions to produce a fluorescent derivative of saxitoxin, 8-an~ino-6-hydroxymethyl-2-i1~~i1~opurine-~(2H)-propionic acid. Although based on the separation of toxins on a weak cation exchange resin eluted with sulfuric acid, a major limitation of this method is that only saxitoxin is measured, and while some workers havehad success withthe method, the results can underestimate toxin content by as much as 65% compared to the mouse bioassay (46). The reason for the underestimation of toxicity is due to the fact that other PSTs which are less likely eluted from the weak ion-exchange column dueto the high polar nature have limited florescence. Moreover, only saxitoxin is measured relative to the known standard. For example, the carbamate and sulfocarbamoyl PSTs are two of the most predominant isomer sources in some shellfish; however, while the carbamate toxins the are most toxic, the sulfocarbamoyl toxins are the least toxic (47). Thus a situation for underestimating PSTs occurs if there are other PSTs present in samples other than saxitoxin which exhibit specific toxicities. The finding by Sullivan et al. (48) that metabolic transformation of PST occurs in the littleneck clan1 demonstrates the potential for multiple fornx of PST. Halstead (3) reported that the success of the batch fluorometric method based on the strong adsorbability of saxitoxin on acidic ion-exchange columns is due to the fact that saxitoxin was the primary saxitoxinpresentincontaminatedshellfish. It is important tonote,however,thatthe method of Bates and Rapoport distinguishes between acceptable and unacceptable shellfish with some false-positive but no false-negative results (49). A number of researchers have demonstrated that PST can be derivatized to yield fluorescent compounds, although the degree of fluorescence is extremely variable (5052). The N- 1 hydroxy toxins, which include neosaxitoxin and GTX- 1 and -4, have lower fluorescence(Fig.2a), yet displayhighspecifictoxicities(Fig.2b).Otherprocedures (54), capillary electrophoresis ( 5 5 ) , and HPLC including TLC (45.53), cellulose (50,52,54,56) are based on the measurement of fluorescence. The HPLC method offers in the sample and isInore the potential for identifying individual PST isomers present sellsitive than theInOuse bioassay. HPLC procedures represent sensitive and accurate techniques which are suitable for handling a large number of samples for routine monitoring. Sullivanet al. (57) reported a method that involvedpreparation of an acidextractof shellfish followed by ion interaction chromatography using a polystyrene divinyl benzene resin column. A binary gradient HPLC method witha postcolumn oxidation derivatization and fluorescence detection enabled the detection of nanogram quantities of the N-1 hydroxyl toxins. I n general, the detection chemistry for this method is based on the oxidation of all toxins with periodate under alkaline conditions and detection of fluorescent products with two chromatographic separations. Good correlation between the HPLC method and the InOuse bioassay requires correction for the contribution of each PST toward the total
191
Laboratory Methodology for Shellfish Toxins 2 r
Type of Toxin 1.2
B
Type of Toxin
Fig. 2 Relative fluorescence (A) and toxicity (B) of different PSP. (From Ref. 54.)
toxicity (58). A semiautomated method employing thefluorescenceresponseusingan autoanalyzer system was also successful with good accuracy (CV = 9.5%) for samples 260 pg/lOO g and sensitivity 10 pg/IOO g toxin for screening samples (59). This procedure was proposed to be coordinated with the mouse bioassay for initial screening of samples performed by the semiautomated method and subsequent confirmationof samples total in a range of 60-250 pgI100 g shellfish meat tested using the mouse bioassay for
792
Kitts
toxicity. Further advancements in lowering detection limits for low molecular weight marinetoxins,includingsaxitoxin,havebeenreportedusingionspray(60),electrospray (61), capillary electrophoresis mass spectrometry (62,63). The two quanidinium groups on the saxitoxin molecule provide the charged sites for monitoring detection of saxitoxin at a fragment ion spectrum of 300 m/:. Principal ions for detection arett1/: 300 (saxitoxin), m/:316 (neosaxitoxin), m/: 380 (gonyautoxin-5), and m / z 396 (gonyautoxin-6). Using atmospheric pressure ionization and combined liquid chromatography-mass spectrometry analysis, a detection of 30 pg saxitoxin has been achieved, which represents a saxitoxin concentration detection limit of 0.1 FM/ 1 p1 injection (compared to 0.0 14 pM and 20 pM injection for HPLC) (57). The detectionlimit for saxitoxin using electrospray ionizationis similar to ion spray when flow injection analysis is used (61). Moreover, there is very little difference in the PST spectrum between the ion spray and electrospray methods.
C. Immunological Assays Immunological methods that have shown promise for monitoring PSTs include radioimmunoassay (64) and direct (65) and indirect (66-68) ELISA assays (Table 2). The direct ELISA developed by Usleber et al. (69) has been used in dipstick (70,71) and immunofiltration (69) formats. The small molecular size common to all PSTs requires that appropriate conjugates are used to covalently attach haptenic components to functional groups of toxins for generating appropriate immunogens required for generating specific antibodies. The coupling of amine functional groups of bovine serum albumin to purified PST, derived from clam siphon glands using formaldehyde, was first reported by Johnson et al. (72) and used by Carlson et al. (64) and Chu and Fan (66) in developing a radioimmunoassay format. The high specificity for saxitoxin and low cross-reaction with NI-hydroxylated toxins (2-10% cross-reactivity with neosaxitoxin) using this approach resulted in a potential severe underestimation of toxin content in PST-contaminated shellfish where toxicity was in fact attributed to PSTs otherthan saxitoxin. Similar limitations were present with very specific polyclonal (66-68) and monoclonal (73,74) ELISA assays. Antisaxitoxin sera generated using a glutaraldehyde-reacted saxitoxin-polyalanine carrier produced some cross-reactivity with neosaxitoxin, GTX-2 and -3, and mixtures of N-21 sulfocarbamoyl derivatives in both contaminated shellfish and dinoflagellate organisms that were fourfold more sensitive than the mouse bioassay and twofold more sensitive than HPLC (67). Immunological assays using the ELISA format have reported STX detection limits of about 3-4 ng/g of tissue in mussels and clams (69). Efforts to overcome the limitation of specificity of PST antisera in the faceof diverse numbers of PST isomers and variability i n PST composition of different shellfish due to metabolic interconversions were made by Chu et al. (68), with the generation of antibodies to neosaxitoxin instead of saxitoxin. This polyclonal antiserahad a relatively greater cross-reactivity with saxitoxin (up to 50% depending on the ELISA format). Despite the improvements using greater cross-reacting antibodies derived from neosaxitoxin. the fact still remains that ELISAs are limited by the underestimation of some toxic samples (e.g., dc-GTX-1-4 are present in significant quantities). Other researchers have used a glucose oxidase conjugate to facilitate measuring very small quantities of neosaxitoxin standard by employing periodate coupling and have reported success at generating specific antibodies for both neosaxitoxin and GTX1 and -4, with cross-reactivity of 3.4%, 3.4%, 0.1%, and 0.04% for saxitoxin, GTX-1 and -3, descarbamoylsaxitoxin, and N-sulfocarbamoylsaxitoxin, respectively (75). As a consequence of cross-reactivity with different PST analogues, detection limits ranged from
x
Laboratory Methodology for Shellfish Toxins e,
V
C
E
2
2
E n
Y
W
U
C
U
6
2
e,
W
.d
U
.-
e,
W
3
Q
8
m
a
M
E
3
m
.
x
.-> .-m U
e,
m
.d
m
U
G
5
e,
-a .3
‘C a
C M 0 C
2 E W
Kitfs
194
17 to 22 pg/ml for both neosaxitoxin and GTX, 0.58 to 1.4 ng/ml for saxitoxin, 1.45 ng/ m1 for GTX-2 and -3, and 12 to 61 ng/ml for dcSTX and C l and 2, respectively. Partialsuccess inaccuratelymeasuringPSTsinbothspecificPSTmixturesand PST-contaminated shellfish from different species has been accomplished on solid-phase systems using specific STX or neoSTX antibodies with either STX-HRP or neoSTX-HRP conjugate markers (76,77). In conclusion, a requirement for an ELISA method that will respond to all PSTs including the C group of toxins remains a primary challenge in diagnostic test development.
111.
DOMOIC ACID TESTING
Domoic acid is a neurotoxin that has been detected in various seafoods including mussels, razor clams, and anchovies (78-81). In addition to domoic acid, a number of cis-trrrns isomers, collectively referred to as isodomoic acids, have been identified (Fig. 3) (82), althoughtoxicitiesarerelativelylower (81). Domoicacid is arare,watersoluble but natural, amino acid that is structurally related to kainic acid, a potent excitotoxin that produces its effects via an interaction at specific glutamic acid receptors (83). The toxin, which acts as an agonist to glutamate, expresses neurotoxicity through an interaction at kainate receptors (84). The toxin is produced by at least two speciesof red algae, Chondrin urrnot(1 (85)and Alsidiurn cor-allium (86),and isa secondmetabolite of the diatom Nirzschin pungens (87). N. purlgens is widely distributed in coastal waters of the Atlantic, Pacific and Indian Oceans. However, not all strains have been demonstrated to produce domoic acid (88). Grimmelt et al. (80) reported that more than 90% of total domoic acid was recovered in mussel hepatopancreas tissue, related to the content of cholorphyll a, a useful marker for algae biomass. Domoic acid cannot be destroyed by cooking or freezing, but reports
A
B FH3
Fig. 3 Do~noicacid (A) and the diastereoisomer of dornoic acid (B).
Laboratory Methodology for Shellfish Toxins
195
have disclosed that it is possible to wash out the toxin by flushing contaminated mussels with uncontaminated water for 18 days (80). The governmentof Canada has a safetylevel for domoic acid setat 20 ppm in wet mussel tissue. The consumptionof mussels containing less than this amount is unlikely to pose a health hazard (89). Domoic acid,a potent agonist of glutamatergic kainate receptors located in the dorsal hippocampus of the brain (84) produces symptoms of gastrointestinal and neurologic disorders (e.g., temporary or permanent memory loss). The intoxication syndrome is known as amnesic shellfish poisoning (ASP) and includes symptoms of vomiting, diarrhea, and/ or abdominal cramps within 24 hours, memory loss, confusion and disorientation, seizures, coma, and cranial nerve palsies within 48 hours (90). As with PSTs, there is no antidote for treatment of domoic acid poisoning. Initial physical symptomsof domoic acid poisoning occur within 0.5-24 hours, depending on the severity of the intoxication (91). Physical symptoms include vomiting, diarrhea, and headache, with more severe intoxications causing excessive bronchial secretions, difficulty in breathing, coma, permanent memory loss, and even death. The first record of foodborne illness attributed to domoic acid in North America occurred in a Canadian maritime provincein 1987, when numerous people became ill and three elderly people died from amnesic shellfish poisoning (92). Until 1987 the only shellfish poisoning reported in the maritime area with regularity was PSP. Leftover mussels were assayed using the mouse bioassay for PST; however, test mice displayed involuntary scratching of their shoulders with their hind legs which was not typical of PSP symptoms (90). The source of the outbreak was traced to three river estuaries on Prince Edward Island. Wild and cultivated mussel samples were taken for laboratory analysis for potential toxic agents, including bacteria, viruses, and chemical residues. No bacterial or viral pathogen, heavy metals, polychlorinated biphenyls, or pesticides were detected in the samples to (79). The domoic acid concentrations in the leftover mussel samples ranged from 31 128 mg/ 100 g of mussel tissue. Estimates of total domoic acid ingested by the patients ranged from 60 to 290 mg (79). Illnesses reported in past years in this area were traced to consumption of wild shellfish that had been harvested in toxic areas where warning notices had been posted. The outbreak of domoic acid poisoning occurred on Prince Edward Island in November 1987. Histologic studies of brain samples collected from those killed by the poisoning revealed neuronal necrosis or cell loss and astrocytosis, particularly in the hippocampusandtheamygdaloidnucleus(93).Subsequent tothe dolnoicacid outbreak in eastern Canada were two outbreaks reported on the West Coast of the United States. In September 1991, a high incidence of sick and dying pelicans in Monterey Bay was traced to domoic acid-contaminated anchovies. A similar outbreak occurred in November 1991 on the Long Beach peninsula in Washington State with razor clams. Most of the domoic acid appeared to reside in the gut lumen of mussels, with small alnounts found in the intracellular compartments (94). Meat of small mussels contained relatively more toxin per unit weight than large mussels because of their relatively large digestive glands.
A.
Bioassays
The original method used for domoic acid detection was based on the mouse bioassay for PST (95). The overall behavioral signs are quite distinct from the paralysis associated with PSP, but yield compatible standard curves (95). Mice injected intraperitoneally with is similar an acid extract derived from boiled, whole mussels for domoic acid detection
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Kitts
to the PST assay but produces an atypical response that is characterized by scratching, convulsions, and death. I n previous studies that used the mouse bioassay, mice were observedfor4hoursafterintraperitonealextractinjectionforbehavioralresponses (scratching) oruntil death. Onlyrarely does the animal die morethan 3 hours postinjection. False positives can occur in mice that show decreased locomotor activity and recovery to normal I hour after injection (80). There is some biological variability in the toxicity estimates of domoic acid when using onset of scratching or even time to death in different mice. In contrast to PST, mouse weight is not a critical factor in estimating the toxic effects of domoic acid-contaminated extracts (80). In mussels the method produces a noeffect level of 24 ppm and 94 ppm for death in mice when performed intraperitonally. An LDio of 83.5 pg domoic acid/mouse ( 5 mg/kg) has been reported for intraperitoneal injections (80). When administered orally, domoic acid doses between 35 and 70 mg/kg (or ppm) body weight are required to produce toxic symptoms inboth mice andrats. Besides havinga degree of similarity with the human clinical situation, the mouse bioassay is rapid for sample screening. However, becauseof the low sensitivity of this test (40 pg/ g drained wet tissue), below the current legal limit (20 pg/g tissue) for edible tissue, this procedure is not used.
B. High-Performance Liquid Chromatography (HPLC) Due to the limitations of the mouse bioassay, shellfish extracts are routinely analyzed for domoic acid using HPLC methods. In addition to the standard AOAC method for sample extraction of shellfish material used in the conventional mouse bioassay (95,96), which recovers only 70-80% domoic acid, an additional extraction method was developed which includes a cleanup procedure prior to HPLC analysis using a C l 8 solid-phase extraction. Two isocratic mobile phases including a 10% acetonitrile with 0.1%1trifluoroacetic acid (92) phase and a 12% acetonitrile phase containing 2% orthophosphoric acid (97) were developed, with a conmon UV-detection wavelength step (242 nm). Rapid analysis (S minutes) and excellent sensitivity(0.5 pg/g of toxin i n shellfish tissue) enable these methods to meet the demands of high sample volume turnover in laboratories as well as being below the tolerance limit of 20 pg/g of tissue set by regulatory agencies. Two certified reference materials for domoic acid quantitation consist of an instrument calibration solution (DACS- 1 ) released in May 1989 and a mussel tissue reference material obtained from Mstilus ecldis, released in August 1989 from the Marine Analytical Chemistry Standards Program.Thesestandardizedextractshave been of greatassistanceinprovidingboth quality control for domoic acid analysis in different laboratories and confirmation ofsymptoms of intoxification in mouse bioassays (98). The extraction and cleanup procedure for tissue domoic acid determinations has also been reported (99). Due to the complex differences in sample matrix for crab viscera, anchovies, and even porpoise stomach contents, the problem of extraneous and unidentifiable peaks has been reported (81). A relatively simple NaCl sample cleanup and extraction procedure overcomes this limitation, yielding a detection limit of approximately 0.5 ppnl (81). Other methods have used a fluorenylmethoxycarbonyl derivative of domoic acid for monitoring trace amounts of domoic acid in seawater and phytoplankton and have reached minimum detection limits of 15 pg/ml (100). HPLC methodologies have also been applied to urine and feces from monkeys and blood serum from humansin a study reported by Lawrence (101). Although the procedure was not appropriate for urine and blood samples that required additional cleanup before analysis, it did work well for domoic acid analysis i n feces at concentrations greater than 1 mg/kg ( 1 ppm). Blanchard and Tasker( 102) also usedH P I X for donloic acid determina-
Laboratory Methodology for Shellfish Toxins
197
tion in the serum of rats, pigs, and rainbow trout, with a detection limit of 0.20 ppm in serum. The assay demonstrates a high degree of reproducibility and reasonable sensitivity. of HPLC/mass spectrometry methods are extremely sensitive for the quantitation domoic acid (60,103-105). The ion spray mass spectrum is characterized by a prominent peak resulting from protonated molecules at tn/z 312. Detection limits as low as 100 ng/ g of shellfish have been reported using reverse-phase liquid chromatography coupled with ion spray mass spectrometry.
C. Capillary Electrophoresis Mussel tissue homogenates extractedwith water and processed further usingan octadecylsilica solid-phase extraction cartridge prior to capillary electrophoresis (CE) has proved successful. Using mussel extracts spiked with domoic acid at 3.91-500 ppm, electropherograms showed a strong linear relationship between peak area and domoic acid concentration (106). For domoic acid determination i n contaminated tissue, the treated extract was mixed with a known amount of domoic acid or with water. Domoic acid concentration was calculated from the difference in peak areas of the electropherograms. The detection limit for domoic acid inwettissue was I O ppm. Removal of lipids from the shellfish extract facilitates electroendonosis and shortens analysis time. A modification of the HPLC procedure using capillary electrophoresis has also been reported by Pleasance et al. (62).
D.
Gas Chromatography
Attention has also been given to developing a gas chromatographic/mass spectrometry method employing a two-step reactionof domoic acid to N-trifluoroacetyl-0-?er!-butyldinlethylsilyl prior to analysis (107), or derivatization of domoic acid with dimethylfornlamide dimethyl acetal to produce a volatile derivative of domoic acid, namely, N-formyl0-methyl domoic acid,in a single reaction step (108). Confirmation of domoic acid using the several diagnostic ions i n the mass spectrum was reported to be sufficiently below regulatory levels of 20-30 ppm for dornoic acidin razor clams, blue mussels, and Dungeness crabs (108). As with the HPLC/mass spectrometry methods, which require sophisticatedequipment,gaschromatographymethods atthepresenttimearealsolimited to access to official analytical laboratories.
E. Radioassays The generation of domoic acid antisera was first reported by Newsome et al. (109) using a rabbit polyclonal antidomoic acid serum in a tritiated radioimmunoassay format. Radioimmunoassays are highly sensitive and accurate, but the cost of radiotracers and radioactive waste disposal is high. Furthermore, radioimmunoassays are not applicable to field tests because of the possibility of radioactive contamination. In addition to radioimmunoassay, a competitive radioreceptor assay employing a recombinant kainate glutamate re(5 nM) and varying concentrations of domoic acid has ceptor with tritiated kainic acid been developed ( 1 IO). The detection limit for domoic acid using the cloned receptor was approximately 1 nM, with an ICso of 3.4 nM (1.02 ng/ml).
F. Enzyme-Linked lmmunosorbent Assay (ELISA) Newsome et al. (109) first raised antiserum against domoic acid by conjugating domoic acid to BSA using ethyl dimethylaminopropyl carbodiimide hydrochloride (EDC) reagent. The antibodies raised i n rabbits were also formatted for ELISA. No cross-reactivity was found with glutamic acid, aspartic acid, kainic acid, and saxitoxin. The ELISA method
Kitts
198
proved to be more sensitive than radioimmunoassay for domoic acid quantitation i n rat urine (40 ng/ml-2.6 yg/ml), but it producedinconsistentresultswithvariousratand monkey antiserum dilutions. Subsequent studies compared antisera obtained i n mice with domoic acid immunogen conjugates derived from ovalbumin (OVA), keyhole limpet hemocyanin (KLH), and BSA carrier proteins using either EDC or N-hydroxysuccinimide
1.OE-2 1.OE-3 1.OE-4 1.OE-5 1.OE-6 1.OE-7 1.OE-6 1.OE-9
Dilution of Anti-OVA-DA
0.W
0.04
0.08
0.12
KLH-DA (ug/rnL)
Fig. 4 (A) Different dilutions and affinity absorbance readings of anti-OVA-Dornoic acid. -X = RI-antisera; -C = R2-antisera. (B) Influence of differentcoatinglevelsof KLH-Dornoic acid antisera on affinity of detection. '- = RI-antisera; -f = R2-antisera. ~
Laboratory Methodology for Shellfish Toxins
199
reactions ( 1 1 l ) . Both conjugation efficiencies as well as serum titers were superior with ovalbumin and KLH, with OVA displaying the highest affinity for free domoic acid in a competitive ELISA. The antiserum did not cross-react with kainic acid, glutamic acid, aspartic acid, or saxitoxin, and when formatted for a competitive ELISA produced lower limits of accurate determinations of 0.2 pg/ml domoic acid in urine, 0.25 pg/ml domoic acid i n plasma, and 10 pg/ml domoic acid in milk. It was concluded that the concentration
S
50
0
100
150
200
250
Free Domoic Acld (nglml)
% Inhibition = 12.1286 log (Dornolc Acid] + 3.78298
r = 0.9929
1
10
100
1“
Domoic Acid (nglmL)
Fig. 5
(A) Performance of different antisera in competitive ELISA for domoic acid using KLHDomoicacidcoating of 0.025 pg/ml (a) and 0.050 pglml (b). f = R1-antisera; t f = R2antisera. (B) Standard curvc for competitive ELISA for domoic acid.
Kitfs
200
of domoic acid in body fluids of individuals with ASP could be determined directly using this method. The anti-OVA domoic acid was also used for domoic acid determination in mussel extracts(1 12). Spike-recovery experiments showed that domoic acid concentration in both aqueous and acid mussel extracts could be measured to within 8% of the actual value using competitive ELISA. The ELISA yielded a domoic acid detection limit of 0.5 pg/g of extract and correlated(r = 0.96) with the standardHPLC method. The competitive ELISA also detected domoic acid for several samples that were undetected by HPLC. Due to the limited amount of serum expected to be recovered from mice, domoic acid antibodies can be raised in rabbits and the ELISA for domoic acid can be standardized according to the methods reported previously (111,112). The anti-domoic acid titers are made in buffers resulting in reductions in absorbances as the dilution of titers increases (Fig. 4a). In the example given herein, with rabbit antisera, the optimal domoic acid antisera dilution waslo-'. Determining the optimal coating level of antisera on the microtiter plate is also essentialfor obtaining optimal absorbance readingsof toxin, which can vary depending on individual antisera (Fig. 4b). In this example, the coating level for antisera obtained for rabbit 1 (R-l) was approximately 0.025 lg/ml, compared to 0.05 pg/ml for rabbit 2 (R-2). Using the KLH conjugate, tests with plates coated with KLH alone should be confirmed for a negative control, thus providing absorbance readings of 0.00 and confirming that the domoic acid antisera derived using this conjugate does not react with the KLH protein carrier. The reactivity of the antisera to the antigen is also especially important in a competitive ELISA and will vary depending on the coating concentration of KLH-domoic acid antisera (Fig. 5a). In the present example, inhibition was less at the 0.05 pg/ml coating level than at 0.025 pg/ml. Both coating levels demonstrate that the more free domoic acid available, the greater the reduction in absorbance and thus the more competitive the reaction. Anti-domoic acid obtained fromR-2 was more sensitive,
n
80
M
2 70 v
z2
60
U
8
.W
50
e 40 0
0
U
4
GtJ
30 20
U
8 10 0
l
2
3
4
5 6 7 SampleNumber
8
9
1
0
Fig. 6 Comparison of domoic acid determinations using HPLC,Mouse antibody ELISA andRabbit antibody ELISA. W = HPLC; = Mouse ELISA; 0 = Rabbit ELISA.
Laboratory Methodology for Shellfish Toxins
201
hence a small change in the concentration of free domoic acid results in a large change in the percent inhibition. A typical domoic acid ELISA standard curve using the aforementionedcoatingconcentrationandantiseratiterisshown i n Fig.5b.Oncomparingthe different estimates of dotnoic acid concentration in sea mussel extracts by HPLC and the two ELISA methods, it can be seen that both the mouse and rabbit ELISA procedures are in good agreement with the HPLC method (Fig. 6). In most cases, as reported earlier (1 l?), ELISA results for domoic acid are higherthan HPLC measurements, but the rabbit ELISA correlates well with the HPLC results ( r = 0.964). The rabbit antisera, however, gives relatively higher readings for domoic acid than the mouse antisera, although good agreementexistsbetweenthetwoprocedures ( v = 0.987). Againthehigherreadings obtained using ELISA compared to HPLC may be due to the presence of domoic acid isomers that cross-react with either antisera; possibly Inoreso than with the rabbit antisera.
IV.
CONCLUSIONS
The persistent and yet unpredictable nature of shellfish contamination with PSTs or domoic acid poisons has resulted in an absolute requirement for routine testing of shellfish harvested for human consumption. With the development of reliable, practical, and costeffective monitoring procedures performed by both government laboratories and shellfish processors, the current problems of monitoring vast areas of coastline would be reduced, and a sound quality control standard for shellfish poisons in processed shellfish could be established. With the greater testing made possible by alternative methodsto the traditional mouse assay comes an enhancement of the process of risk assessment which translates into increased public confidence in the safety of the product. These important changes i n quality control assessment and public confidence could also demand greater market value of shellfish resources. It is a fact that the public reacts adversely to marine products in general when there is news of a PSP or ASP outbreak. Adverse publicity, although specific to the bivalve mollusk industry, can also have an impact on the fish industry as well. More efficient and practical monitoring methods for contaminated shellfish could further reduce the risk of food poisoning. Finally improvements in routine monitoring of seafood toxins in shellfish by more efficient and reliable methods could also contribute to a greater utilization of the entire shellfishery without overharvesting of selected areas designated as safe.
REFERENCES 1. H Sotnmer. K Mcyer. Paralytic shellfish poisoning. Arch Pathol 24560-598. 1937. 2. H Sommer, WF Whedon. CA Kofoid, R Stohler. Relation of paralytic shellfish poison to certain plankton organisms of the genus Gonyrtlox. Arch Pathol 24:537-559, 1937. 3. BW Halstead. Seafood toxins. EP Ragelis, ed. American Chemical Society Symposium Series 262. Sea Food Toxins. Washington, D.C.: Food and Drug Administration, 1984, pp. 3751, 333-344. 4. A Prakash. Dinoflagellate bloorns-an overview. I n : VR Lo Ciccro, ed. Toxic dinoflagellate blooms. Wakefield.MA:MassachusettsScienceandTechnologyFoundation,1974, pp. 2-6. 5 . J Gibbard, J Naubert.Paralyticshellfishpoisoning intheCanadianAtlanticCoast. Am J PublicHealth 38:550-553, 1948.
202
Kitts
6. EJ Schantz. Studies on shellfish poisons. J Agric Food Chcm 17:413-416, 1969. 7. JC Medcof. The St. Lawrence rough whelk fishery and its paralytic shellfish poisoning problem. Publication no. 1201. Ottawa: Fisheries Research Board of Canada, 1972. 8. KG Barber, DD Kitts, PM Townsley. Response of the shore crab Hemigmpsus oregonesis to saxitoxin injections. Bull Environ Toxicol 40: 190- 197, 1988. 9. DS Smith, DD Kitts.Proteinbiomarkersforparalyticshellfishtoxins.In:MFingerman, R Nagabhushanam, M-F Thompson, eds. Recent advances in marine biotechnology. Vol. 2, Environmentalmarinebiotechnology.NewDelhi:OxfordPVTPublishing,1998.pp. 131.
IO.
11. 12. 13.
14. 15.
16. 17. 18.
19.
20.
21. 22.
23.
24. 25.
26.
BB Prezelin, SA Randall. Photosynthetic characteristics and organization of chlorophyll i n marine dinoflagellates. Proc Natl Acad Sci USA 75: 1801- 1804, 1978. Association of Official Analytical Chemists. Official methods of analysis. 15th ed. Vol. 2. Washington, DC.: 1990,pp.881-882. RF Bond, JC Medcof. Epidemic shellfish poisoning i n New Brunswick. Can Med Assoc 79: 1-14,1957. EJ Schantz, JD Mold, DW Stanger, J Shavel. FJ Riel, JP Bowden, JM Lynch, RS Wyler, B Riegel, H Sommer. Paralytic shellfish poison. VI. A procedure for the isolation and purification of the poison from toxic clam and mussel tissues. J Am Chern SOC795230-5235, 1957. DB Quayle, N Bourne. The clam fisheries of British Columbia. Bulletin 179. Ottawa: Fisheries Research Board of Canada, 1972. CM Yentsch. Accumulation of algal botoxins in mussels. RA Lutz, ed. In: Mussel culture and harvest: a North American perspective. Amsterdam: Elsevier Scientific, 1980, pp. 223245. NR Stephenson, HI Edwards, BF MacDonald, L1 Pugsley. Biological assay of the toxin frotn shellfish. Can J Biochem Physiol 33:849-857, 1956. CS Wiberg, NR Stephenson. Toxicologic studies on paralytic shellfish poison. Toxicol Appl Pharmacol2:607-615,1960. DL Park, WN Adam, SL Graham, RC Jackson. Variability of mouse bioassay for determination of paralytic shellfish poisoning toxins. J Assoc Anal Chem 69547-550, 1986. JW Hurst, R Selvin, JJ Sullivan, CM Ycntsch, RRL Guillard. Intercomparison of various assay methods for the detection of shellfish toxins. In: DM Anderson, A White, DC Baden, eds. Toxic dinoflagellates. Proceedingof the Third International Conferenceon Toxic Dinoflagellates. New York: Elsevier, 1985, pp. 427-432. European Communities. Richtlinie des Rates votn 15. Juli 1991 zur Festlegung von Hygiene(9 1/492/EWG). Amtsblatt evorschriften zur erzeugung und vermarktung lebender Muscheln der Europaischen Gemeinschaften L 286: I - 14, 1991. EF McFarren. Report on collaborative studies of the bioassay for paralytic shellfish poison. J Assoc Agric Chem 42:263-271, 1959. YShimizu.Developments in thestudyofparalyticshellfishpoisons.In:DLTaylor, HH Seliger, ed. Toxicdinoflagellateblooms.Amsterdam: Elsevier/North Holland,1979, pp. 321 -326. MR Ross, A Siger, BC Abbott. The house fly: an acceptable subject for paralytic shellfish DC Baden, eds. Toxicdinoflagellates. New toxinbioassay.In:DMAnderson,AWhite, York: Elsevier, 1985, pp. 433-437. OL Park, I Aguirre-Flores, WF Scott, EAlterman.Evaluationofchickenembryobrine shrimp and bacterial assays for saxitoxin. J Toxicol Environ Health 18589-594, 1986. B Cheun, S Takagi, T Hayasi, Y Nagashima,E Watana. Determination ofNa' channel blockToxins ers in paralytic shellfish toxins and pufferfish toxins with a tissue biosensor. J Nat 71109-1 13, 1988. B Cheun, H Endo, T Hayashi, Y Nagashima, E Watanabe. Development of an ultra high sensitive tissue biosensor for determination of swellfish poisoning tetrodotoxin. Biosens Bioelectron12:1185-1190.1996.
Laboratory Methodology for Shellfish Toxins
203
27. B Chcun, M Loughran. T Hayasi, Y Nagashima, E Watanahe. Use of a channel biosensor for the assay of paralytic shellfish toxins. Toxicology 36: 137 1-1377. 1998. 28. SR Davio, p Fontelo. A competitive displacement assay to detect saxitoxin and tetrodotoxin. AnalBiochern141:199-204,1984. 29. MR Vicytes, AG Cahado, A Alfonso, MC Louzano, AM Botana. LM Totana. Solid phase radioreceptor assay for PST. Anal Biochem 21 1 :87-93. 1993. 30. GJ Doucettc,MM Logan. JS Ransdcll. FM Van Dohah. Development and prclirninary validation of :l microtitre plate-based receptor binding assay for PSP toxin. Toxicon 35:625-636. 1997. for tetrodotoxin, 31. K Kogure,MLTamplin,USimidu.RRColwell.Atissuecultureassay saxitoxin and related toxins. Toxicon 26: 191-197, 19x8. TH Birkbeck. A tissue culture assay for direct detection of sodium 32. S Gall:acher. channel blocking toxins i n bacterial culture supernatcs. FEMS Microbiol Lett 71: 101- 107. 1992. 33. JF Jellet, LJ Marks, JE Stewart, ML Dorey, W Watson-Wright. JF Lawrence. Paralytic shellfish poison (saxitoxin family) bioassays: automated endpoint determination and standardization of the in vitro tissue culture bioassay. and comparison with the standard mouse bioassay. Toxicon 30: 1 143- I 156. 1992. 34. JF Jellet, JE Stewart, MV Lycock. Toxicological evaluationof saxitoxin. neosaxitoxin, gonyautoxin 11, gonyautoxin II plus 111 and decarbamoylsaxitoxin with the n m s e neuroblastoma cell bioassay. Toxicol In vitro 957-65, 1995. 35. RL Manger, LS Leja. SY Lee, JM Hungerford, MM Wekell. Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitive sodium channels: semi-automated assay for saxitoxins. brevetoxins and ciguatoxins. Anal Biochem 214: 190- 194, 1993. 36. RL Manger. LS Leja, SY Lee, JM Hungerford, MM Wekell. Assessment of marine toxins by cell bioassay. In: F Shahidi, Y Jones. DD Kitts. eds. Seafood safety, processing and biotechnology. Lancaster, PA: Technomic Publishing, 1997, pp. 1 1- 16. PJ Lake. Comparison of mouse bioassay and other sodium channel cytotoxicity 37. P TNIU~II, assays for detecting paralytic shellfish poisoning toxins in shellfish extracts. J AOAC l n t 79: 1 130-1 133, 1996. 38. CJ Bird, CA Murphy, MA Ragan, EL Rice.
RR Gutell. Towards an 18s ribosomal RNA gene phylogeny of the red algae (Rhodophyta). Aquatech conference, Halifax, Nova Scotia, 1992, pp. 25. 39. DD Kitts. DS Smith. T Owen.An enzyme-linked immunosorhent assayto detect the presence of paralytic shellfish poison using an induced crab protein marker. Food Agric Immunol 3: 49-56, l99 I . 40. DD Kitts, DS Smith, MK Beitler. J Liston. Presence of paralytic shellfish poisoning toxins andsolubleproteins in toxicbutter clams (SmidorrIus gigcrrltelrs). BiochemBiophysRes Commun 18451 1-517. 1992. 41. DD Kitts, DS Smith. Cross-reacting antigens in the butter clam (Su.t-it/oruu.vRigtlrltelrs). Toxicon30:967-978, 1992. 42. ER Fairey.JSEdmunds, JS Ramsdell. A cell based assay for brevetoxins, saxitoxins and ciguotoxins using a stably expressed c-fos-luciferase reporter gene. Anal Biochem25 I : 129132, 1997. 43. HA Bates, H Rapoport. A chemical assay for saxitoxin. the paralytic shellfish poison.J Agric
FoodChem23:237-239,1985. HABates. R Kostriken,HRapoport.Achctnicalassay for saxitoxin.improvementsand modifications. J Agric Food Chcm 26252-254. 1978. 45. LJ Buckley. M Ikawa, JJ Sasner. Isolntion of Gonytrdtrs torttarrn.sis toxins from soft shell clams ( M y(wcrmri~I)and a thin-layer chromatographic-Ruorelnctric method for their detection. J Agric Food Chcm 24: 107- I 1 I . 1976. 46 RJ Bose, JE Reid. Evidence for the heterogeneity of paralytic shellfish toxin i n clam and
44.
204
47. 48. 49. 50. 51.
52.
53. 54. 55. 56. 57. 58. 59.
60. 61. 62.
63.
64.
65.
66. 67.
Kitts
mussel samples gathered near Prince Rupert, British Columbia. In: DL Taylor, HH Seliger, cds. Toxic dinoflagellate blooms. New York: Elscvicr, 1979, pp. 399-402. S Hall, PB Reichardt. Cryptic paralytic shellfish toxins. In: EP Ragelis, ed. Seafood toxins. Washington, D.C.: Atncrican Chemical Society, 1984, pp. 1 13-1 24. JJ Sullivan, WT Iwaoka, J Liston. Enzymatic transformation of PSP toxins in the littleneck clam (Prorothcrur srominecl). Biochem Biophys Res Cotntnun 114:465-472, 1983. SS Hall, PB Rcichardt, RA Neve. Toxins cxtracted from an Alaskan isolateof Protogoqwclrrr sp. Biochem Biophys Res Commun 97:649-653, 1980. JJ Sullivan, WT Iwaoka. High pressurc liquid chromatographic determinationo r toxins associated with paralytic shellfish poisoning. J Assoc Anal Chem 66:297-303, 1983. JF Lawrence. C Menard. Liquid chromatographic determination of paralytic shellfish poisons in shellfish after prechrotnatographic oxidation. J Assoc Anal Chem 74:1006-1012, 1991. JF Lawrence. C Menard, CF Charbonncau. SS Hall. A study of ten toxins associated with paralytic shellfish poison using chromatographic oxidation and liquid chromatography with fluorescence detection. J Assoc Anal Chcm 74:404-409. 1991. of gonyaulax toxins and other NH Shoptaugh, LJ Buckley, M Ikawa, JJ Sasner. Detection guanidine compounds on thin-layer silica gel chromatograms. Toxicon 16:509-513, 1978. JJ Sullivan, MG Simon, WT Iwaoka. Comparison ofHPLCandmousebioassaymethods for determining PSP toxins in shellfish. J Food Sci 48: 13 12- I3 15, 1983. WE Fallon, Y Shimizu. Electrophoretic analysis of paralytic shcllfish toxins. J Environ Sci HcalthA12:455-464,1977. P Thibault, S Pleasance, MV Laycock. Analysis of paralytic shcllfish poisons by capillary electrophoresis. J Chromatogr 542483-501, 1991. JJ Sullivan, MM Wekell, LL Kentala. Application of HPLC for the determination of PSP toxins i n shellfish. J Food Sci 52:26-29. 1985. JJ Sullivan, WT Iwaoka, J Liston. Enzymatic transformation of PSP toxins in the littleneck clam (Prororhcrcrr stumimrr). Biochctn Biophys Res Cotntnun 1 14:465-472, 1983. J Jonas-Davies, JJ Sullivan,LL Kcntala, J Liston, WT Iwaoka, L Wu. Semiautomated method for the analysis of PSP toxins in shellfish. J Food Sci 49: 1506-1509, 1516, 1984. MA Quilliurn, BA Thomson, GJ Scott, KWM Siu. Ion-spray mass spectrometry of marine neurotoxins. Rapid Cotntnun Mass Spectrom 3:145-150, 1989. HB Hincs. Electrospray ionization of sclcctcd low-molecular-weight natural biotoxins. Biol Mass Spectrotn 22:243-246, 1993. S Pleasance. P Thibault, J Kelly. Comparison of liquid-junction and coaxial interfaces for capillary electrophoresis-mass spectrometry with application to compounds of concern to the aquaculture industry. J Chromatogr 591:325-339, 1992. S Pleasance, SW Ayer, MVLaycock,PThibault.Ionspraymassspectrometryofmarine toxins. 111. Analysis of paralytic shellfish poisoning toxins by flow-injection analysis, liquid chromatography/mass spectrometry and capillary electrophoresis/tnass spectrometry. Rapid Cotnmun Mass Spectrotn 6:14-24, 1992. RE Carlson, ML Lever, BW Lee, PE Guire. Development of immunoassays for paralytic shellfishpoisoning:aradioimmunoassayforsaxitoxin.In:RagelisE, ed. Seafoodtoxins. Washington, D.C.: American Chcmical Society, 1984, pp. 18 1- 191. E Usleber, E Schneider, G Terplan. Direct enzyme immunoassay i n microtitration plate and test strip format for the detection of saxitoxin in shellfish. Lett Appl Microbiol 13:275-277, 1991. in shellfish. FS Chu, TSL Fan. Indirect cnzymc-linked immunosorbcnt assay for saxitoxin J Assoc Anal Chcrn 68:13-16, 1985. A Cembella, Y Parent, D Jones, G Lambourcau. Specificity and cross-reactivity of an adsorption-inhibition enzyme-linked immunoassay for the detection of paralytic shellfish toxins. In: E Graneli, B Sundstrom, L Edler,DM Anderson, eds. Toxic marine phytoplankton. New York: Elsevier Science. 1989, pp. 339-344.
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205
68. FS Chu,X Huang. S Hall. Production and characterization of antibodies agatnst neosaxitoxin. J AOAC Int 75:341-345, 1992. 69. E Usleber, E Schneider, G Terplan. Direct enzyme imnunoassay in microtitration plate and test strip format for the detection of saxitoxin in shellfish. Lett Appl Microbiol 13:275-277, 1991. of 70. E Schneider. E Usleber, G Terplan. Test strip enzyme immunoassay for the detection saxitoxin. Food Agric lmtnunol 3:103-104, 1991. 71. E Usleber, E Schneidcr, G Tcrplan. MVLaycock. Two formats of enzyme immunoassay for the detectionof saxitoxin and other paralytic shellfish poisoning toxins. Food AdditContam12:405-423,1995. 72. HM Johnson, PA Frcy, RAngelotti, JE Campbell, KH Lcwis. Haptenic properties of paralytic shellfish poison conjugated to proteins by formaldehyde treatment. Proc Soc Exp Biol Med 117:425-430,1964. 73. RC Huot, DL Armstrong. TC Chanh. In vitro and in situ inhibition of the sodium blocker saxitoxin by lnonoclonal antibodies. J Toxicol Environ Health 27:381-394, 1989. 74. R Hack, V Rcnz, E Martlbauer, G Terplan. A monoclonal antibody to saxitoxin. Food Agric Ilntnunol2:47-48,1990. 75. C Burk, E Usleber, R Dietrich, E Martlbauer. Production and characterization of antibodies against neosaxitoxin utilizing a novel immunogen synthesis procedure. Food Agric Immunol 713155322.1995. 76. H Huang,K-HHsu,FS Chu. Direct cotnpetitive enzyme-linked inmunosorbent assay for saxitoxin and neosaxitoxin. J Agric Food Chetn 44: 1029-1035, 1996. in naturally 77. FS Chu, KH Hsu, X Huang,RBarrctt,CAllison.ScreeningofPSPtoxins occurring sarnplcs with three different direct competitive ELISA. J Agric Food Chem 44: 4043-4047,1996. EW Dyer, DJ Embree, M Falk. 78. CJ Bird, RK Boyd, D Brewer, CA Craft, ASW deFreitas. MC Flack, R Foxall, C Gillis, M Greenwell, WR Hardstaff, WD Jamieson. MV Laycock, P Leblanc, NI Lewis, AW McCulloch, GK McCully. M McInemey-Northcott, AG Mclnnes. JL McLachlan. P Odensc, D O’Neil, VP Pathak, MA Quilliam, MA Ragan, PF Seto, PG Sim, D Tappen. P Thibault, JA Walter, JLC Wright, AM Backman, AR Taylor, D Dewar. M Gilgan, DJC Richard. Identification of domoic acid as the toxic agent responsible for the P.E.I. contaminated mussel incident. Technical report no. 56. Atlantic Research Laboratory. 1988, pp. 86. 79. JLC Wright, RK Boyd, ASW deFrcitas, M Falk, RA Foxall, WD Jamieson, MV Laycock, AW McCulloch, AG McInnes, P Odense, V Pathak, MA Quilliam, MA Ragan. PG SinP Thibault, JA Walter, M Gilgan, DJA Richard,DDewar.Identification o f domoicacid, a ncuroexcitatory amino acid, in toxic mussels from eastern P.E.I. Can J Chetn 67: 48 1-490, 1989. S Wager, GR Johnson,JF Amend. Relationship 80. B Grimmelt, MS Nijjar. J Brown, N MacNair. between dolnoic acid levels in the blue mussel (Mytilu.7 c d d i s ) and toxicity in mice. Toxicon 28:501-508.1990. 81. CL Hatfield, JC Wekell, EJ Gnuglitz, HJ Barnctt. Salt clean-up procedure for the determination of dotnoic acid by HPLC. Nat Toxins 2:206-21 I , 1994. 82. JLC Wright, M Falk, AG Mclnnes, JA Wnltcr. Identification of isodomoic acid D and two geometrical isomers of domoic acid i n toxic mussels. Can J Chem 68:22-25, 1990. 83. JT Slevin. JF Collins, JT Coyle. Analoguc interactions with the brain receptor labeled by [‘H] kainic acid. Brain Res 265:169-172, 1983. 84. GDebonnel,LBeauchesnc,CDCMontigny.Domoicacid,thealleged“musseltoxin,” might produce its neurotoxic effect through kainate receptor activation: an electrophysiological study in the rat dorsal hippocampus. Can J Physiol Pharlnacol 6729-33. 1989. 85. K Daigo. Studies on the constituents of Clzor~clr-itrnnrratcc. 11. Isolation of an antihelnlintical constituent. Yakugaku Zasshi 79353-356, 1958.
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86. G Impellizzeria, S Mangiafico, G Oriente. M Piattelli. S Sciuto, E Fattorusso. S Magno, C Santacroce, D Sica. Amino acids and low molecular weight carbohydrates of some marine redalgae.Phytochemistry14:1549-1557,1975. 87. D Subba Rao, MA Quilliam. R Pocklington. Domoic acida neurotoxic amino acid produced bythemarinediatom Nitzchicc pungetts in culture. Can JFishAquatSci45:2076-2079, 1988. 88. MA Quilliam, JLC Wright. The amnesic shellfish poisoning mystery. Anal Chem 61: 1053A1989. 1054A.1056A.1058A-l060A, 89. HBS Coacher. Outbreak of mussel poisoning i n Canada. Identification and determination of a new toxin in Prince Edward Island mussels. Food Lab Ncwslett 1332-33, 1988. An outbreak of toxic encepha90. T Perl, L Bedard. T Kosatsky, J Hockin. EDC Todd. R Retnis. lopathy caused by eating mussels contaminated with dotnoic acid. N EnglJ Med 32217751780,1990. 91. EDC Todd. Domoic acid and amnesic shellfish poisoning-a review. J Food Protect 56:6983, 1993. 92. MA Quilliam, PG Sim, AW McCulloch. High performance liquid chromatographyofdomoic J EnvironAnal acid,amarineneurotoxin.withapplicationtoshellfishandplankton.Int Chem 36: 139- 154, 1989. 93. J Teitelbaum, R Zatorre. S Carpenter, D Gendron. A Evans, A Gjedde, N Cashman. Neurologic sequelae of dotnoic acid intoxication due to the ingestion of contaminated mussels. N Engl J Med 3221781-1787, 1990. 94. 1 Novaczek, MS Madhyastha. RF Ablett. A Donald, G Johnson, MS Nijjar.Sims. DE Depuration of domoic acid from live blue mussels (Mytillts rchrlis). Can J Fish Aquat Sci 49:3 12318. 1992. 95. AOAC.Officialmethodsofanalysis.Arlington.VA:AssociationofOfficialAnalytical Chemists,1984. L Tryphonas, J C:unpbell, E Lok. Domoic acid poisoning 96. F Iverson, J Truelove, E Vera, and mussel-associated intoxication: preliminary investigations into the response of mice and rats to toxic mussel extract. Food Chem Toxicol 273777384, 1989. 97. JF Lawrence, CF Charbonneau, C Menard, MA Quilliam, PC Sim. Liquid chromatographic determination of dotnoic acidi n shellfish products using the AOAC paralytic shellfish poison extraction procedure. J Chromatogr 462349-356, 1989. 98. WR Harstaff,WDJamieson. JE Milky, MA Quilliam. PG Sim. Referencematerialsfor dotnoic acid, a marine neurotoxin. J Anal Chem 338520-525. 1990. 99. MA Quilliam. MXie,WR Hardstaff.Arapidextractionandclean-upprocedureforthe determination of dotnoic acid in tissue samples. Tcchnical report 64. Ottawa: National Research Council of Canada, Institute for Marinc Biosciences, I99 I . 100. R Pocklington. JE Milky, SS Bates, CJ Bird. ASW deFreitas, MA Quilliam. Trace deternmination of donloic acid in seawater and phytoplankton by high performance liquid chromatography of the fluorenyllnethoxycarbonyl (FMOC) derivative. Int J Environ Anal Chem 38:351368.1990. 101. J Lawrence. Determination of domoic acid i n seafoods and in biological tissues and fluids. CanDisWklyRep16(supplIE):27-31, 1990. 102. J Blanchard. R Tasker. High-performance liquid chrotnatographic assay for domoic acid in serum of different species. J Chrotnatogr 526546-549. 1990. 103. MA Quilliam, BA Thomson, GJ Scott. KWM Siu. Ion-spray mass spectrometry of marine neurotoxins. Rapid Colnmun Mass Spectrom 3: 145-150, 1989. 104. P Thibault. J Walter. Identification of domoic acid, a neuroexcitatory amino acid. in toxic mussels from eastern Prince Edward Island. Can J Chem 67:481-490, 1989. D Lewis. Comparison ofUV absorption and electrospray 105. JF Lawrence, BP-Y Lau, C Cleroux. mass spectrometry for the high performance liquid chromatographic determination of dolnoic 659:1 19-126. 1994. acid in shellfish and biological samples. J Chromatogr
Laboratory Methodology for Shellfish Toxins
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106. AL Nguycn, JHT Luong, C Masson. Capillary electrophoresis for detection and quantitation of domoic acid in mussels. Anal Lett 23: 1621 -1634, 1990. 107. S Pleasance, M Xie, Y LeBlanc, MA Quillium. Analysis of domoic acid and related compoundsby mass spectrometryandgaschrotnatography/massspectrometryasN-trifluoroacetyl-0-silyl derivatives. BiomedEnvironMassSpectrom19:420-427,1990. 108. SW Hadley, SK Braun, MM Wekrll. Confirmation of domoic acidas an N-formyl-0-methyl derivative in shellfish tissues by gas chrotnatography/tnass spectrometry. I n : F Shahidi. Y Jones, DD Kitts, eds. Seafood safety. processing and biotechnology. Lancaster, PA: Technomic Publishing, 1997, pp. 25-32. 109. H Newsome. J Truelove, L Hierlihy, P Collins. Determination of domoic acid in serum and urine by immunochemical analysis. Bull Environ Contam Toxicol 47329-334, 1990. 1 IO. FM Van Dolah, JF Doughtie, DR Hampson, JS Ramsdell. High capacity receptor assay for domoic acid: use of a recombinant glutamate receptor produced in a baculovirus expression system. In: Forbes JR. ed. Proceedings of the 4th Canadian Workshop on Harmful Marine Algae. Can Tech Rep Fish Aquat Sci 2016:63-64, 1994. I 1 1. DS Smith, DD Kitts. A competitive enzyme-linked immunoassay for dotnoic acid determination in human body fluids. Food Chetn Toxicol 32: 1147-1 154, 1995. 112. DS Smith, DD Kitts. Enzyme immunoassay for the determination of domoic acid in mussel extracts. J Agric Food Chem 433677371, 1995.
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7 Ciguatera Fish Poisoning Yoshitsugi Hokama Universih of Hawaii, Honolulu, Huwaii
Joanne S. M. Yoshikawa-Ebesu Oceanir l e s t System,
Honoluluv Hnnwii
I. Introduction 210 11. History 210 111. Biology2 1 1 IV. Chemistry213 V. Pharmacology216 VI. Epidemiology 2 18
VII.
VIII. IX.
X.
A. Geographic distribution 2 18 Incidence B. 21 8 C. Fish associated with ciguatera poisoning 219 1 DetectionMethods22 A. Bioassay 221 B. vitro Inbioassay 223 C. In vitro cell assay 225 D. Immunoassay 226 Membrane E. immunobead assay 227 Chemical F. methods 235 G. Summary 235 Clinical 235 Pathology 237 A. Human 237 B. Animal studies 238 Therapy 242 A. Earlier treatment 242 Recent B.treatment 242 C. Summary 244 References 244
209
210
1.
Hokama and Yoshikawa-Ebesu
INTRODUCTION
This chapter covers one of the common foodborne diseases associated with consumption of fish from coral reefs and the near-shore environment of the oceans in the tropics and in some instances the semitropics to depths down to 60 m. The region is circumglobal between the Tropic of Cancer and the Tropic of Capricorn. Seafood poisoning encompasses a broad spectrum initiated by a variety of marine toxins. Some of the better known seafood poisonings include paralytic shellfish poisoning (PSP); scombroid poisoning; pufferfish poisoning; amnesic shellfish poisoning (ASP); ciguatera poisoning; and diarrhetic shellfish poisoning (DSP). PSP is associated with dinoflagellates producing saxitoxins and gonyautoxins, which accumulate in shellfish during heavy dinoflagellate blooms at certain timesof the year ( 1,2). Scombroid poisoning results from the spoilageof fish tissues due to bacterial contamination and subsequent production of the enzyme decarboxylase, which converts amino acids to amines (e.g., histidine to histamine), thus inducing clinical changes mimicking histamine-like attacks. This poisoning isassociatedwithpoor or inadequate fishpreservationandstorageprocesses (3,4). Pufferfish poisoning is associated with tetrodotoxin (TTX) found in fish of the order Tetraodontoidea and in certain crustacea (5,6), and is probablyof bacterial origin (7,8). Tetrodotoxin is also found in the newt and certain speciesof octopus. ASP is caused on St. Andrew‘s by domoic acid and was first reported in a mussel poisoning outbreak Island, Canada, in late 1987 (9). The neuroexcitotoxin (domoic acid) produced by Pseudonitizches was identified as the causative factor. DSP causes severe diarrhea in patiem consuming shellfish or fish containing okadaic acid or its analogs (IO, 1 I ) . The emphasis in this chapter concerns the seafood poisoning known as ciguatera.It is becoming a major problem as fish consumption in the United States increases and the fishing industries of endemic areas (tropics, semitropics, and temperate zones) increase their export and distribution of fish worldwide.
II. HISTORY Fish poisoning probably dates back to antiquity. It was cited in Homer’s Odyssey (800 B.c.) and was observed during the time of Alexander the Great (356-323 B.c.), whose armies were forbiddento eat fish in order to avoid the accompanying sickness and malaise that could threaten his conquests (12 ) . Ciguatera poisoning was described as early as 1606 in the South Pacific island chain called New Hebrides (13). A similar outbreak there and in nearby New Caledonia was reported by the famous English navigator Captain James Cookin 1774 (14). He described the clinical symptomsof his sick crew, symptoms that coincide with the clinical manifestations described today for ciguatera poisoning (l5,16). In addition, viscera from the same fishes given to Cook’s crew were also given to pigs, causing their deaths (14). The term ciguateru originated in the Caribbean to designate intoxication induced by the ingestion of the marine snail, Turbo livonn pica (called cigucr), described bya Cuban ichthyologist. Today the term is widely accepted to denote a particular type of fish poisoning resulting from the ingestion of certain fish (primarily reef fishes) encountered around islands in the Caribbean, the Pacific, and elsewherein the tropics. Current information points to at least one of the many polyether toxins, such as okadaic acid, palytoxin, or maitotoxin, which are structurally closely associated with ciguatoxin, as also being
Ciguatera Fish Poisoning
21 1
Table 1 ClinicalSymptomsAssociatedwithCiguateraFishPoisoning
Category or system Digestive
Neurological
Cardiovascular
General
Nausea, often followed by vomiting; diarrhea; painful defecation; abdominal pain and cramps. Symptoms generally abate after 24 hours, leaving an asthenic and dehydrated patient. Dysesthesia, principally with sensitivity to cold, temperature reversal; paresthesia, painful tingling of the palms of the hands and soles of the feet on contact with cold water; superficial hyperesthesia, with sensation of burning and electrical discharge: often mydriasis is present; patellar and achilles reflexes sometimes diminished. Neurological symptoms generally persist for 1 week. It is not unusual to see contact dysesthesia lasting a month. Pulse slow (35-50 beatdmin), often irregular pulse, low arterial pressure (heart sounds distant); EKG may show dysrhythmia from sinus bradycardia (slow heart beat) to bursts of supraventricular or ventricular extra systoles (rapid systolic heart beat). Ventricular tachycardia, excessively rapid action o f thc heart, may also occur. There may also be a 1st A-V (atricular-ventricular) block. Cardiovascular disorders usually disappear in 48-72 hours and may be mistaken for a heart attack. Toxins from carnivorous fish tend to cause cardiovascular problems. Asthenia, making it difficult to walk, keeping the patient in bed for several days; arthralgia, especially of knee, ankle, shoulder, and elbow; dorsolumbar stiffness. myalgia, especially leg muscles; headache; marked and constant chilliness, but no problems of thermal regulation; lipothynlia and dizziness; itching 2-3 days after onset and may persist for many days; oliguria, sometimes during the first 48 hours.
associated with ciguatera ( 1 1,I7,18). The characteristic clinical symptoms are described in Table l and are discussed further in the clinical section. Since the 1970s, muchprogresshasbeenmade in ciguateraresearchbyseveral investigators (17-27), encompassing all phases of the problem. These areas, covered in this chapter, include biology; chemistry, including the closely related polyethers; pharmacology; clinical; epidemiology; immunological analysis, or the testing and detection of the major toxin ciguatoxin and its related polyether toxins, and other biological testing methods for assessing ciguatoxin and related polyethers; pathology; and therapy.
111.
BIOLOGY
Fish poisoning outbreaks occurred throughout the tropical regions of the Pacific before, during, and after World War 11. It became a serious problem for the military during the war because outbreaks of ciguatera occurred among the troops stationedin various areas endemic for ciguatera poisoning, including Midway, Wake, Guam, Johnson Island, French Polynesia, New Caledonia, the Marshalls, and the Marianas. Other tropical areas of the world with ciguatera include the Caribbean, Gulf of Mexico, and Indian Ocean. In the in postwar years, investigators from the University of Hawaii, Japan, and Tahiti went search of the etiological factor causing ciguatera poisoning.
212
Hokama and Yoshikawa-Ebesu
Banner et al. (28) were pioneers in the study of the ecology and biology of ciguatera fish poisoning. Randall (29), in his investigations of toxic fish, showed that all fish of the same species in the same area were not necessarily carriers of the toxin, and hence the toxin was not produced by the fish species. Therefore the toxin in the fish must be derived from their food source. This was the beginning of the development of the food chain concept shown i n Fig. 1. The sequence of this concept follows the scheme: dinoflagellates or other sources (bacteria, invertebrates, macroalgae) are consumed by herbivores or omnivores, which in turn are eaten by carnivores that store the toxins. Humans and other mammals develop ciguatera poisoning as a result of eating the contaminated fish. Theetiology of ciguaterapoisoningwasestablishedwhenYasumotoet al. ( I O ) discovered a dinoflagellate resembling a Diplopstrlis sp. as the initiator of the food chain disease. Later the organism was correctly identified as Gumhetdiscus toxicus, a new species of the family Dinophyceae (30). The species was named after its origin of discovery, the French Polynesian Islandsof Gambier in the South Pacific (10.30). Studies have shown the food chain concept to be correct. Campbell et al.(3 1) showed an outbreak of ciguatera poisoning in a species of herbivore (Ctenock~wtus sp., kole). Examinationsof gut smears stained with anti-CTX fluorescence dye showed that 95% of the gut contents contained numerous G. toxicus. All tissues tested positive for ciguatoxin in the stick enzyme immunoassay (S-EIA). Yasumoto et al. ( 1 0) demonstrated G. tosicus in the gut of herbivores. More recently, chemically characterized congeners of ciguatoxin were found i n moray eel tissues (32). The precursor for these two oxidized congeners appears to be C T X K , characterized and obtained from G. toxicus (33,34). G. toxicus, a benthic organism collected from the natural environment yielded both ciguatoxin and maitotoxin, but when culturedi n the laboratory, ciguatoxin production was essentially nil. Maitotoxin, a water-soluble, polyhydroxy-containing compound and one of the most potent toxins (0.125 pg/kg LD,,,,in mouse), was obtained in moderate yields (35). The inability of G. toxicus to produce ciguatoxin in culture may be because of its need for a symbiotic relationship with red-brown alga in nature. G. toxicus was found i n large blooms at certain timesof the year. Among the macroalgae associated withG. toxicus were Tur-hinnria sp., Jcrrzirr sp., Sypridicr sp., andBpopsis sp. (19,36-38). G. rosicus adherence to macroalgae and release for movement in calm water appears to be regulated by a substance(s) produced by the macroalgae (39). Other organisms of significant interest
GAMBIEKDISCUS TOXICUS
Fig. 1 Transmission path of ciguatera toxin from the lnarinc dinoflagellate through herbivorous and carnivorous fishes to man.
Gnl,lbie,.rli.sc.lr.s tosicws
Ciguatera Fish Poisoning
213
Fig. 2 Life cycle of G. toxicus. A) Motile, free-swimmingG. toxicus; B) Precyst: immobile, dark, dense intracellular pigment, initial secretion of mucoid sheath; C ) Cyst: adherence to petri dish bottom, thick mucoid sheath, dark centrally located pigment; D) Exocyst: emergence from thick mucoid wall; E) Division of exocyst; about 20 divisions per exocyst; and F) Conditions unknown, 4-6 months. Prorocentrurn species and coral(Hulichoninvolved in ciguatera poisoning (4,40) include dria sp.) producing okadaic acid and its congeners, and recently Ostreopsis sp. elaborating palytoxin (41,42), which was originally discovered in Pulythou (43). The geographic distribution ofG. toxicus is circumglobal and primarily in the tropics. It has been found in the Pacific and Caribbean by numerous investigators(44).Growth (40,000 of G. toxicus in nature is subject to changes in the environment. Heavy growth cells/L of seawater) in the environment is spotty: in some areas, growth is nonexistent and in other areas growth is lowto moderate (100-1000 cells/L). The proper amount of sunlight, temperature(24OC 2 4"C), salinity (28-40 ppm),and appropriate nutrients are necessary for growth. Salinityof less than 2.8% generally showed no growth; that is, the presence of freshwater is deleterious for G. toxicus growth. No cyclic pattern for growth was observed in a 2-year, monthly collection of G. toxicus from Waianae Boat Harbor, Hawaii (45). In this study, the physical parameters (dissolved oxygen, salinity, pH, temperature, and water) remained constant throughout the study. The only significant finding was the lack of growth of the associated macroalga(Bryopsis sp.), which led to difficulty in finding G. toxicus during this period of no algal growth. The asexual life cycle of G. toxicus was examined in vivo and in vitro (45). The life ofcycle G. toxicus from Waianae Boat Harbor is shown in Fig. 2. It was initially cultured successfully by Yasumoto et al. (37). Since then, numerous laboratories have grownG. toxicus for biological and chemical studies. A comprehensive compilation of G. toxicus and other dinoflagellate literature is presented by Brusle (44).
W.
CHEMISTRY
The isolation, purification, and determination of the structure of the causative factor associated with ciguatera poisoning have constituted a tedious and long-term study initiated by Professor P. J. Scheuer et al. in Hawaii (23-25,46,47). The major obstacle in structural
214
Hokama and Yoshikawa-Ebesu
determination was the source of the natural material in fish, which was scarce even in the most toxic fish, G!'rnnothorclxju\,anicus (one moray eel liver yields about 1 btg purified ciguatoxin). Nevertheless, following the first isolation and characterization in 1967 (23), purified ciguatoxin (0.45 pg/kg LD5[,intraperitoneally in mouse) was obtained and the structure determined (23-26,28). It is a polyether of lipid nature consisting of several hydroxyl groups, solublein organic solvents (chloroform, diethyl ether, methanol, ethanol, 2-propanol,andacetone),withamolecularweight of 11 11.7 5 0.3 and an empirical fornlula of C,,H,,NO,, (47). Thepolyether nature of CTX was noted following the demonstration by Yasumoto et al. (48) that a compound (toxin PII) isolated from Prorocentrutn limL/ had all the biological and TLC (thin layer chromatography) characteristics of ciguatoxin. However, this compound was later identified as okadaic acid, a polyether that was earlier isolated from a sponge ( / f d i c h o n d r iokadui) ~~ by Scheuer's group ( 17). Brevetoxin (49) and many monocarboxylic antibiotics (50) isolated from Streptomyc~sbelong to this group of polyethers. The polyether antibiotics all possess ionophoretic properties affecting thepemleability of variouselectrolytesthroughthecellmembranes.Notsurprisingly, many of the dinoflagellate toxins (brevetoxin, okadaic acid, maitotoxin, and ciguatoxin) appear to have this same effect on various electrolytes (Na', K ' , Gal-), although the precise mechanisms have not all been ascertained. In the late 1980s and early 1990s, the structures of ciguatoxin from moray eel tissues andG. toxicus cultures were determined and reported by Yasumoto and Murata(51). The initial report was presented in 1990 (52). Fig. 3 shows the structure of CTXlB (from morayeel tissues) and CTX4B (from G. to.ricu.s). The yield from 125 kgof Gymr~othorrrxjmonicus viscera was 0.35 mg of pure CTX (molecular weight 11 11.5843 t 0.0053, formula C , , ~ ~ H ~ ~Several ~ O ~ c ~thousand ). liters of G.
37
1 R1 =
( f m Gabbierdiscus toxicus)
R2 = H Fig. 3 Structures of ciguatoxin from G . tosicus (CTX 4B) andlnoray eel liver (CTX1).
Ciguatera Fish Poisoning
215
to.ricus culture yielded 0.74 pg of pure CTX4B (the less polar congener of CTX), with a molecular weightof 1061S 8 4 and the formula C,,H,,O,, (33,5 1,52). Prior to the isolation and identification of CTX4B from cultured G. toxicus, gambiertoxin was only isolated from collecting wild, natural-growing G. toxicus. Yasumoto et al. (33,5 1,52) have reported more than 19 ciguatoxins that are associated with either ciguatera poisoning in fish or G. toxicus from the Pacific. The one major and 10 minor ciguatoxins (polyether isolates) from G. toxicus are referred to as gambiertoxins, while the three major and numerous minor ciguatoxins from three species of fish are called ciguatoxins. Table 2, taken from Legrand et al. (33) and partially updated by Yasumoto's group (personal communication), shows the number of ciguatoxin congeners characterized to date. Other polyether compounds isolated from G. toxicus are of significance. Gambieric acids isolated from the culture medium have shown potent antifungal properties (54) and G. toxicus culture medium weak toxic properties. The drawback is that the yields from Table 2 Ciguatoxins from Various Specics of Fishand G.
Code no.
MW
IA IB(CTX)
1111
1c 2A 1 2A2 2A3 2B 1 2B2 2c
I023
G. tosicm
tosicus
Scnrus Lutjonus 6ol1nr gihhtts (flesh) (viscera) (viscera) 0 e
1057 1095 1057 1061 1095 1061
e
3c 3C
3D
"4A *4B(GT-4B) 4C C-CTX- 1 C-CTX-2
(Caribbean) 0 e e
e
e e
e
e
e e e e
1077 l085
e
0 e
1080**
e
1062"'@ 1023 94 1 I095 1023 l06 1 1023 1061
e
106l
Crrnrrln latus
e
1095
3A 3B 3C 3C
Gymothoros jovor1icu.s
e
e
e e e
e e
e
e
e
e
e
0 1 142 +*l142
e
e ~
+ Carlhhean C-CTX: From Cumrl.r Intrlts, MW I 142 Da, clcctrophyslological studies (neuromuscular and cardiac tlssue) rcsumhle Pacilic CTX: sonic differences detected (53). 0 : M L I J Ot ~o x ~fraction (from Ref. 3 2 ) . * : Stcreo isomcr ** : Two recent CTX congners Isolatcd from moray eel tissucs ( 3 2 ) .
Hokama and Yoshikawa-Ebesu
216
are poor: yields of these toxins are very limited and generally only in the low microgram quantities. Nevertheless, if they could be synthesized in the laboratory, their use against fungal diseases would be of great significance. Lewis et al.( 5 5 ) isolated, purified, and determined ciguatoxin structures from moray eel, confirming Murata et al.'s (52) findings in 1990. Allof these chemical studies demonstrated the diversity of ciguatoxins that contribute to the clinical entity called ciguatera poisoning. Also contributing to these variabilities are the genetic and ethnic makeups of the individuals consuming ciguatoxin-contaminated fishes. Thus the diverse clinical findings in ciguatera poisoning appear to be compatible with the variation of chemical structures described in the study of ciguatoxin chemistry in the marine environment (33,51). Furthermore, this isolation, purification, and structural determination of ciguatoxin was a major breakthrough in ciguatera research, making possible the potential synthesisof ciguatoxin and the preparation of epitopes for better immunological assay developments. Two ciguatoxins were recently isolated in the Caribbean from horse-eye jack (Cmm x latus), a species associated with ciguatera poisoning(53). These two ciguatoxins were considered diastereomers and differed fromCTXlB, the major toxin isolated from moray eel viscera (52). Whether these toxins are related to the numerous minor ciguatoxins of Gyrnnot1mu.r sp. and L U ~ ~ U Isp. I Uremains S to be determined. Caribbean ciguatoxin was found to have similar biological toxicity in the mouse. At this date, it is much too early to concludethat Caribbean ciguatoxin differs significantly from Pacific ciguatoxin. Immunologically, studies with sheep anti-CTX and antibody prepared with ciguatoxin coupled to human serum albumin reactedvery well with tissues of Lutjrrrzus sp., a species involved in a large outbreak in the Caribbean (56). Recently,two newciguatoxinanalogs,2J-dihydroxyCTX3Cand 5 l-hydroxy C T X X , were isolated from moray eel (G. J a \ w ~ i c u sby ) Satake et a1 (32). The precursor CTX3C is a minor analog originating as a component of G. tmicus (34). Synthesis ofCTX1Bis presently being attempted by two laboratories in Japan, with one laboratory starting at the west sphere and the other at the east sphere of the C T X l B molecule (see Fig. 3). Both synthetic spheres (the west consisting of the A, B, and C rings; and the east consistingof J, K, L, and M rings) have been tested with the monoclonal antibody to ciguatoxin (MAb-CTX). Only the east rings J, K, L, and M have shown activity with the stick-enzyme immunoassay (S-EIA) procedure (57).
V.
PHARMACOLOGY
In an earlier pharmacologic study (an assessment of ciguatoxin with rabbit intestine suspended in tyrode solution) the resulting contraction of the muscle was shown to be greater when the blood was initially primed with ciguatoxin, prior to the addition of acetylcholine plus blood with glucose (58). Since no information of the ciguatoxin preparation was given, the data appears questionable in light of today's information on the chemistry of ciguatoxin. Subsequently, Rayner (59) in 1972, with partially purified ciguatoxin preparation, showed that the physiological effect of ciguatoxin was not due to an anticholinesterase action. Instead, ciguatoxin evolves its action through the voltage-dependent sodium channel (60,61). Ciguatoxin is a novel type of Na- channel toxin (60,61). The primary receptor site of ciguatoxin action is the fifth domain of the Na' channel, similar in this
Ciguatera Fish Poisoning
21 7
respect to the primary receptor site of brevetoxin (61). Ciguatoxin and brevetoxin (PbTx) share a common receptor site on the neuronal voltage-dependent Na’ channel (62), although ciguatoxin has a 20-50 times greater affinity for the Na- channel than brevetoxin. Whereas the varied clinical symptoms of ciguatera would suggest involvement of different toxins (15,64), ciguatoxin alone exerts its action on many tissue target sites. I n nervous tissues, ciguatoxin causes a tetrodotoxin-sensitive increase in sodium ion permeability and depolarization of the resting membrane (59). Depending on the magnitude of thedepolarization,theconsequence can be anincreaseinexcitability of theneuronal membrane or a depolarizing type of conduction block at high concentrations. Lower doses of ciguatoxin have marked effects on both the respiratory and cardiovascular systems. Although ciguatoxin has some neuromuscular blocking properties, the respiratory arrest induced by a lethal dose results mainly from depression of the central respiratory center (65).On the cardiovascular system, the effect is often biphasic: hypotension with bradycardia followedby hypertension and tachycardia (66). The hypotension and accompanying bradycardia are readily antagonized by the anticholinergic agents atropine, hexamethonium, and hemicholiniutn (67), whereas the tachycardia and hypertension are mediated by the sympathetic nervous system and suppressed by the adrenergic blockers propranolol and reserpine (67). The effects of ciguatoxin on smooth muscles are quite complex and depend on the predominant autonomic innervation and the postsynaptic receptor function. For example, on isolated guinea pig tissues, ciguatoxin enhances the inhibition of the taenia caecum (68) and excitation of the vas deferens (69). These effects are associated with norepinephrine release and antagonized by guanethidine, propranolol, reserpine, and tetrodotoxin. In contrast, ciguatoxin induces a sustained contraction of the ileum that is potentiated by eserine and completely blocked by atropine (70). Similar effects on excitation and inhibitionare observed i n other tissues. Ciguatoxin contracts the thoracic aortic strip of the rabbit (67) and relaxes the spontaneously contracting rat jejunum (71). Many of these effects of ciguatoxin can best be explained by the depolarization of nerve terminals and the resultant release of lleurotransmitters. Recent examinationof various crude methanol-soluble extracts of a herbivore (Cterloclltretus stripsrrs) and a carnivore (Serioltr clurtrerili) demonstrated that the pharmacologic use of guinea pig tissues (atrium and taenia caecum) in vitro was of great value in the characterization of ciguatoxin-like or maitotoxin-like toxins. C. srrigosus flesh extracts showed maitotoxin characteristics, whereas S. clunlcrili extracts showed ciguatoxin-like activity in the atrium and taenia caecum studies (72). I n electrophysiological studies with rats and human paticnts affected with ciguatera poisoning, Cameron et al (73) obtained findings indirectly suggesting that ciguatoxin acts on tnammalian nerves by prolonging sodium channel actions. The paradoxical sensory discomfort experiences in paresthesia and dysesthesia are most likely a result of an exaggerated and intense nerve depolarization occurring i n peripheral small A delta myelinated and C-polymodel nociceptor fibers (73,74). Other experimental studies of positive-CTX extracts in animals included (a) inhibition of the accunlulation of y-aminobutyric acid and dopamine (60); (b) prolonged sodium channel activation in rats and humans (73,75); (c) increased frequency of depolarization (75,76);(d) effect on myelinatednerve fibers (75);(e) release of norepinephrine (68,75,77); (f) release of intracellular calcium (78); (g) causeof nodal swelling (76); (h) release of acetylcholine (79) from NG 105- 15 neuroblastoma and glioma hybrid cells; (i)
Hokama and Yoshikawa-Ebesu
218
synaptic vesicle depletion (80); (j) fragmented nerve terminal, frog motor nerve terminals (80); and (k) positive inotropic effects in vitro in nmnlnalian atrial tissue assays (81-83). With the exception of the use of pure ciguatoxin preparations, results of crude ciguatoxin preparations used in many of the earlier pharmacologic analyses are questionable because of contaminating compounds.
VI.
EPIDEMIOLOGY
A.
Geographic Distribution
As indicated in the introduction, ciguatera poisoning is found worldwide between latitudes 35"N and 35"s circumglobally. The major endemic areas where most studies have been done are the Caribbean and the Pacific islands, including the lands adjacent to the Indian Ocean and the Gulf of Mexico. Archipelagos of the tropics considered to be safe from ciguatera poisoning are probably nonexistent. The travels of ships today and the periodic destructive hurricanes in the Pacific and Caribbean have contributed to the distribution and increase of the toxic dinoflagellate G. foxicws (84,85). For example, the presence of G. toxicus i n the state of Hawaii may be attributed to troop transports and warships coming from endemic areasof the Pacific to Pearl Harbor, which may have carried cysts or dinoflagellates on the bottoms and sides of ships and inballastwaterreleasedfromships during World War 11. Two major hurricanes in the state of Hawaii, Iwa (1982) and Iniki ( 1991), have further contributed to ciguatera. The increase i n knowledge of ciguatera i n recent years has also heightened awareness of ciguatera.
B. Incidence Ciguatera incidences have been reported throughout the world. The most extensively studied area has been French Polynesia by Bagnis and Legrand (85). In French Polynesia, the culprit involved, G. toxicus, was discovered by Yasurnoto et al. (22). Ciguatera poisoning has been recognized inmost of these epidemiological surveys, in which it is believed that only 20% of the total cases have been reported. In the Pacific, the epidemiology of ciguatera poisoning has received considerable attention becauseof the efforts of the South Pacific Commission (SPC). The countries compiled in Table 3 comprise what is probably the most studied area for the incidence of ciguatera poisoning. The Federated Statesof Micronesia, Palau, Puerto Rico, and Hawaii share relatively low incidencesof toxicity from fish in contrastto French Polynesia. In parts of the Central Pacific, the incidences of ciguatera poisoning are generally higher than i n Hawaii, but much lower than the French archipelagos (90). The incidencesof ciguatera poisoning in the region of the Indian Ocean are relatively low and nonexistent on some islands, such as Madagascar, Comores, Ceylon, and Indonesia.Arecentincident of interest, however, occurred in Madagascar, where 98 of 500 people died after eating the flesh of a shark. Clinical symptoms were not definitive for ciguatera poisoning, and with limited tissues, two new liposoluble compounds, designated carchatoxin A and B, were isolated (91,92). Reunion Island appeared to be unfavorable for toxic algae growth, but its rate of intoxication for ciguatera poisoning is the highest with 13.4 cases/100,000. These incidences may be due to imported fishes (93).
ses/
Ciguatera Fish Poisoning
279
Table 3 Numhcr of Incidences i n French Polynesia, the Micronesian Islands, and Other Arcas
Country 4.8 Federated States 1982-1987 of Micronesia 1982234.9 Marshall Islands 19871982-74.8 Northern Mariana Islands Palau 1960Polynesia French 807 Australia 100 Society S00 Tuamotu I700 Marqucsas 4300 Gambier 7.8 State of Hawaii RicoPuerto
x-l 1
SPC (86) l987
1984
SPC (86) SPC (86) SPC (86) Danielson Danielson and Bagnis 1985 ( 8 8 )
Health 1970-1995 Department of I996
(87)
(unpuhlished data) et Dcmotta al. (89)
I n the Caribbean, ciguatera poisoning varies with the islands involved. The incidence of ciguatera poisoning is low to moderate in Puerto Rico (89), high in St. Barthelemy (94) and high in the Virgin Islands(95). Ciguatera poisoning toxicities were also relatively high in the Dominican Republic and Cuba. Countries such as the United States and Canada have reported sporadic cases of ciguatera poisoning. In the United States, the major states reporting ciguatera poisoning are Hawaii and Florida. Other reports have resulted from importation of fish from endemic areas such as the Caribbean, and in some instances, from the Pacific. Ciguatera outbreaks also occur among tourists returning from endemic areas.
C. Fish Associatedwith Ciguatera Poisoning Ciguateric fish are generally restricted to species feeding on algaeor detritus around tropical reefs (96),especially surgeon fish (Ac,trrlt/rrrritltre)and parrotfish (Smridcre).Carnivores such as snappers(Lrr{jcr/riclcre).jacks (Ctlrcrngitltre),wrasses (Orbrickre),groupers, sea bass, rock cod (Serrcuridtre), moray eels (Murcre/ridcre), triggerfish (Bcrlisricicre),and barracudas (Sl)h!.rcrPnicitre) feed on the herbivores (97). For example, an outbreak on the island of Kauai occurred when a large bloom of G. r o x i u r s among the green algaBrycpvis contaminated the surgeon fish, Ctcwocheretrrs strigo.s~rs,causing an outbreak of ciguatera poisoning following consumption of C. srripsus. G. t0.ricv.s was found in 95% of the gut contents of the surgeon fish when examined with sheep anti-CTX fluorescent-labeled antibody(3 I ). The fishreportedlyinvolved in ciguaterapoisoningarelisted in Table 4.These fishes represent the major causesof ciguatera poisoning in particular regionsof the tropics. In a l l of the regions reported, the families of fish presented are similar. These include moray eels, snappers. groupers, wrasses, barracuda, amberjacks, jacks, surgeonfish, and triggerfish, and prilnarily represent tropical reef fish.
ion
Hokama and Yoshikawa-Ebesu
220 Table 4
FishesIlnplicated in CiguateraPoisoningfromVariousRegions
Geographical American Samoa
Australia
Caribbean (Virgin Islands) Florida
Vcrnoux and Lejeune ( 100) Lawrence et al. (101)
French Polynesia
Hawaii
Ciguatera Fish Poisoning
VII. A.
22 1
DETECTION METHODS
Bioassay
it was imperative to have an assay with specificity, sensitivity, and simplicity of application. Various invertebrate and vertebrate animals have been used for bioassays (28). Later, when extraction procedures were developed and the toxins were shown to be lipids, chemical procedures using fluorescent markers were utilizedfortheisolationandpurification of thetoxins.Purification of ciguatoxinswas achieved i n the late 1960s and late 1980s, enabling the development of immunological procedures (105,106). Banner et al. (28) examined a nurnber of animal species, from the lower forms to mammals. Some of the phyla examined included Protozoa, Ciliates, Arthropoda (CrustaceaandInsecta),Mollusca,Echinodermata,Chordata,Amphibia,Reptilia,Aves,and Mammalia. These animals were fed pieces of raw flesh by voluntary or force feeding, and in some cases were fed residues of alcoholic extracts (28). In feeding experiments with crayfish, significant physiological changes were observed, ranging from paralysis to death. The Africanland snail gave questionable results. The oral feedingof raw and dried toxic O r r j a n u s bohtrr showed variable reactions i n the mouse. The 36 otherspecies that were used demonstratednoresponses. Inlight of the present knowledge of the chemistry and concentration of ciguatoxin in fish tissue, thc results obtained by Banner et al. (28) were to be expected. Oral administration of raw or dried toxic fish flesh to mamn1als requires a larger dose of toxins over long periods to demonstrate any effect. Based on the mouse bioassay for PSP. Banner et al. (28) turned to intraperitoneal injection of toxic fish extracts in mice with some success, but still adhering to the fact that nlan becomes intoxicated via the oral route, they turned to the mongoose (Herpestc~s rnur7go) for the feeding assay. The mongoose can be fed up to 15% of its body weight of raw fish, dried fish, or oil extracts of fish incorporated in raw eggs. Unlike cats. 111011gooses retain all of the food consumcd. Cats tendedto regurgitate fish tissue, so the accuracy of the amounts eaten was questionable. The clinical response to toxic fish following consumption by the mongoose showed similarities to that of humans. The responses were classified into five stages for a single feeding followed for48 hours: no reaction, 0; slight weakness andflexing of the forelimbs, 1 ; slight motor ataxia, pronounced fiexionof the forelimbs and weaknessof the hind limbs, 2; moderate motor ataxia, weakness and partial paralysis of limbs and muscles, 3; acute motor ataxia and extreme weakness, limited movement, or coma, 4; death, 5. All responses 3,4, and 5 were solnetimes accomwere accompanied by loose stools (diarrhea); responses panied by extensive salivation and trembling. Although in the acute cases symptoms were neurological, no histological changes were observedin postmortem examination with light microscopy. Postmortem examination revealed some animals with gastric ulcers, but this phenomenon was also noted earlier in natural, wild, untreated mongooses. Animals usedinciguaterapoisoningbioassaysincludecat,mongoose,chicken, mouse, mosquito, and fish. Cats (Fdi.sctrtrrs) have been shown to be highly sensitive to toxic fishes, and kittens have been used as animal models in bacterial food poisoning. However, for humane reasons, the use of cats has been discouraged in the United States. Nevertheless, in earlier studies, cats were used in other countries (Tahiti, Japan) because of their sensitivity to toxic fish, which permitted the use of small fish samples without extraction procedures. Hypersalivation i n cats was one of the significant physiological 111 the study of ciguatera poisoning,
222
Hokama and Yoshikawa-Ebesu
reactions to ciguateric fish. However, regurgitation of the fish satnples caused difficulties i n obtaining accurate results. In addition, Inaintaining proper and clean housing facilities was a problem (28,107,108). Chicks (Gallus g ~ l l u ssp., G. donresticus) were used as an assay method for ciguatera poisoning assessment by administration of fish extracts via intravenous, intraperitoneal, or intratnuscular methods (58,109). Administration of extracts by intramuscular injection in hens demonstrated pathomorphological changes in the nervous system(58). Oral administration of toxic fish tissues or extracts to chicks induced hypersalivation, loss of weight, decrease in rectal temperature, and acute motor ataxia. Chicks appeared to be two IO five times more sensitive to toxic fish than mice. Chicks have proved to be satisfactory animal models for both feeding and injection assays for fish poisoning. They are readily available, easy to manage, relatively small in size and unlikely to regurgitate. They are highly sensitive to fish toxins and the effects of the toxin are cumulative. However, some negative aspects of using chicks have been pointed out by several investigators, including those using the chick assay (109). The effects of crude ciguatera toxin extracts fed to red snapper (Lufjcr1rr4.sh l r r r r ) were first determined in 1960 ( 1 10).In this study it was demonstrated that the toxin accumulated and was retained in the tissues of the snapper with no adverse effect to the fish. However, fish fed ciguatoxin and maitotoxin showed behavioral changes. Maitotoxin was G. to.vic~us-led fish the probable cause of the behavioral pattern, since experiments with showed patholnorphological changes i n the liver of S~rr.cr~ru.s cwbrillrr ( 1 1 1 ). Feedin, experiments withvariousfishspeciesweredifficulttoassesssincethedinoflagellate G. to.ric~4.scan produce several toxins in addition to CTX4B and CTX4C, maitotoxin and severalminorpolyethertoxins (33). Consequently, toattributetissuechangesonlyto ciguatoxin alone may be misleading. The results could also be due to the species of fish used for the particular assay: the use of freshwater and nontoxic reef fishes (not exposed to G. t o s i c u s in nature) could contribute to nlisleading results. More recent studies using fish and dinoflagellate extracts have demonstrated that fish are sensitive to IP administration but less sensitive than mouse. Mosquitoes (Aedes L w g y 7 t i i ) havebeen usedextensively i n FrenchPolynesia to assess crude ciguatera fish extract for toxicity ( 1 12). Crude fish extracts are injected into the intrathoracic cavity and the mosquito LD5,,of the fish toxicity expressed as grams of fish/mosquito. Significant correlations between toxicity in mosquito, mouse, and cat, and toxicity in man have been shown (108). Not considering the extraction preparation for each fish, the mosquito test is time consuming, but cost saving. It was considered the most reliable bioassay in French Polynesia, but unfortunately, as with all bioassays, unless pure material is tested, the test has no specificity, and the effects of CTXl alone cannot be demonstrated i n a crude mixture of unrelated toxins. Invertebrates have been used in assay systems for qualitative and semiquantitative assessment of fish toxicity. One advantage of their use is that their testing only requires small amounts of materials. The invertebratesutilized by some investigators have included brine shrimp, crayfish, flies, and mosquitoes (28,l 12- 1 16). I n these procedures, the technique is important to the success of using these assay organisms. Mice have been the major animal used to assess ciguatera poisoning. Because oral feeding in both mice and rats showed no promise, investigators (28) use the intraperitoneal route i n the nlouse assay. I n their purification studies, Banner et al. (28) showed that the active toxin was in the diethyl ether fraction and not the polar, water-soluble fraction in
Ciguatera Fish Poisoning
223
the intraperitoneal mouse assay. Most investigators now use the standardized procedure l/t), where t = hours. One for mouse toxicity with the formulation: MU = 2.3 log (1 mouse unit (MU) is the concentration of toxic extract injected intraperitoneally which kills a 20 g mouse in 24 hours. It has been found that with highly purified moray eel CTXIB, 1 MU = 7-8 ng/mouse. With crude fish extracts, the procedure (with modifications within each laboratory) is as follows: Swiss Webster mice weighing 20-25 g are utilized to assess the toxicity of fish extracts. One hundred milligrams of crude fish extract are resuspended i n 1 m l of 1% Tween 60 in saline and injected intraperitoneally into mice. Symptoms displayed by the nlouse are observed at 30 minutes, 1, 2, 4, 6, 8, 24, and 48 hours after injection and rated on a scale of 0-5 according to toxicity (Table 5 ) . The estimation of ciguatoxin by mouse units from known cases of ciguatera fish obtained from the Hawaii Department of Health (HDOH) ranges from 7 to 9 ng (1 17). The mouse test, like the other bioassays presented a lack of specificity, since any toxic factor in fish extracts to which the mouse is sensitive will cause death.
+
B. In Vitro Bioassay The in vitro bioassay to assess marine toxins has been of great interest in the past decade because of its sensitivity and use of minimal amounts of material. However, these test systems require tissue culture techniques and special physiological instrumentation ( 1 IS, 119). Nevertheless, these assays give some measure of specificity, since the actions are at the sites of the sodium channel of neuroblastoma cells or guinea pig atrial tissue (82). For the guinea pig atrial assay (82), 100 mg of each fish extract is resuspended in 1 1111 of Krebs-carbonate solution. One hundred microliters of the suspension is tested on the guinea pig atria. Subsequent inotropic and chronotropic actions are noted, in addition to the extract's pharmacological responseto tetrodotoxin (TTX), verapamil, and the adrenergic blockers (propranolol and phentolamine). The inhibitors TTX and verapamil, and p1 of a IO" M the adrenergic blockers are given after the inotropic responses at 12.5 concentration (68).
Table 5
MouseToxicityAssayScoring
ToxicityDescription 0 1
of visible clinicalsymptoms
in mouse after extract injection
No ill effect 15-60 minutes: muscle contraction in lower back area (flexion), increased respiration.
immobile (inactive), recovery as I , but recover in 2-3 hours, pilo-erection 3 Rccoverin12-24hours:same as 2,musclecontraction,paralysis in extremities (usually hind legs), rapid and irregular breathing, immobile, closed eyes, pilo-erection, slight cyanosis (tail) Symptoms 4 as in 3, but death within 24-18 hours,' 5 Symptoms of 3 and 4, death in lessthan 6 hours
Same 2
.' 1 MU. death withln 24 hours In 20 g mouse; contains 7-9 ng ofciguatoxln in the sample o f 100 mg of crude
cxtract/mousc injectcd (IP) (estimated from HDOH contirmcd clguatcric fish extracts).
Hokama and Yoshikawa-Ebesu
224
The guinea pig atrial in vitro assay, in spite of the specialized techniques involved (including dissection of the atria, left and right, from the heart), is an excellent means of studying marine toxins, especially crude marine toxin extracts. Once inotropic standard patterns are obtained for each pure toxin and the effects of various chemical inhibitors are ascertained, the inotropic patterns of crude fish extracts with inhibitors can be compared with the standardized patterns of the pure samples. Thus CTXlB and other ciguatoxin-like toxins (okadaic acid and brevetoxin) show similar inhibition with tetrodotoxin. That is, ciguatoxin, okadaic acid, and brevetoxin can be readily distinguished from palytoxin and maitotoxin. Sodium channel effector toxins can be distinguished from calcium channel effectors by use of appropriate inhibitors to the inotropic response. Figure4 representsclassicalinotropicpatterns of pureciguatoxin,okadaicacid,palytoxin,andbrevetoxin with or without TTX inhibition.
l
TTX
TTX
Ver
Rinse
Rinse
Fig. 4 Inotropic pattcrns for PTX, CTX. OA and PbTx-3 in the guinea pig atrial assay. a) Pure PTX (240 ng): blocked by verapalnil (ver) but not by tetrodotoxin (TTX). b) Crude CTX (3.2 ng): blocked by TTX. c) Crude OA (1 pg): blocked by TTX. d) Pure PbTx-3 (20 ng): blocked by TTX.
Ciguatera Fish Poisoning
225
C. In Vitro Cell Assay
1. Neuroblastoma Assay Directed cytotoxicity to sodium channels of neuroblastoma cells has been established for purified ciguatoxins, brevetoxins, saxitotoxin, and crude seafood extracts, including finfish as well as from thePacific ( 1 19). Mouseneuroblastoma extracts from the Caribbean (neuro-2a, ATCC) were grown in RPM1 complete medium supplemented with 10% heatinactivated fetal bovine serum, 2 mM glutamine, l mM sodium pyruvate, 50 pg/ml streptomycin, and 50 U/ml penicillin. Actively growing cultures were maintained at 37" C in a humidified 5 % CO: atmosphere. For bioassay, 96-well plates were seeded with 1 X 10' neuroblastoma cells in 200 p1 growth medium and incubated for 24 hours. Culture wells then receive 10 p1 each of the test sample, 10 mM ouabain, and 1 mM veratridine. Toxin samples were tested in replicates of four at various concentrations of the sample. A minimum of 15 wells were processed as ouabadveratridine controls and 5 wells were evaluated as untreated controls (cells only). Purified brevetoxin (PbTx-l) was included as a positive control of sodium channel enhancing activity in conjunction with ciguatoxic finfishextractassays.Nonspecificcellactivitywasassessed by addition of samples and PbTx-l, but no ouabain and veratridine. Cultures were then incubated either 4 or 22 hours for detection of sodium channel enhancing activity or 24-48 hours to assess sodium channel inhibitory activity. MTT (3[4,5-din~ethylthiazol-2-yl]-2.5-diphenyl-tetrazolium) was used to assess cytotoxicity by replacing the overlying medium with 60 p1 of a 1 :6 dilution of MTT stock ( 5 mg/ml PBS, pH 7.4) in complete culture medium. Plates were then incubated for 15 minutes at 37°C or until sufficient formazan deposits were observed in untreated control wells.The overlying 100 p1 of DMSO andtheplatesreadat medium was then replaced in each well with 570 nm in an ELISA plate reader. This procedure measures the viable active cells via mitochondrial dehydrogenase reduction of the colorless MTT to formazan to give a purplish color read at 570 nm. The IDSo for PbTx-l was detected at 250 pg and the ID5,, for purified ciguatoxins were detected at 1 pg for CTXl and 3 pg for CTX3C at 7 hours incubation. CTXl was isolated from moray eel livers and CTX3C fromG. toxicus culture as a minor component. This cell bioassay isof value for marinetoxin studies and may replace mouse assays when the use of live animals for experimental studies becomes prohibited. Measures of specificity and sensitivity for sodium channel activator and inhibitor compounds were excellent. In this respect, it was very similar to the guinea pig experiments, but appeared to have a greater sensitivity. 2. Other Cells In addition to neuroblastoma cells, HeLa cells and fibroblastic malnmalian kidlley cells have been used to evaluate ciguatoxicity (120,121). 3. Radiolabeled Binding Assay Since ciguatoxin and brevetoxin sharea common receptor in the sodium channel (domain S), although with a difference in affinity (CTX1 > PbTx), with the use of labeled brevetoxin UH-PbTx-B), ciguatoxin can be quantitated by competitive binding assay with sodiumchannel-containingproteins.Thishasbeenshown by severalinvestigators (1 18,122) using isolated rat brain synaptosome with 3H-PbTx-B and the CTX3C congener of CTXlB isolated from G. tosicus ( 1 18). This procedure requiresa small amount of fish
226
Hokama and Yoshikawa-Ebesu
extract and the method is rapid, simple, and has a high sensitivity. Although the method of the is useful for research, it may be impractical for large-scale fish screening because method’s use of a beta counter and radiolabeled compounds.
D. Immunoassay 1. Radioimmunoassay The first successful detection of a low nlolecular weight polyether toxin, ciguatoxin, directly from contaminatedfish tissues was reported in 1977 using radioimmunoassay (RIA) with a sheep antibody prepared with purified moray eel ciguatoxin conjugated to human serum albumin (HSA) as carrier (26). Subsequently the RIA was used to screen 5529 Seriolo dutnerili (kahala), a species associated with ciguatera outbreaks in Hawaii from 1979 through part of 1981 (123). The 15% of fishtestingborderline or positivewere discarded and the remaining 85% of fish scoring negative were sold commercially with no false negatives. Although the RIA procedure proved effective, its complexity and cost suggested seeking alternative immunological ciguatera screening methods.
2. Enzyme Immunoassay An enzyme immunoassay (EIA) was subsequently established using sheep anticiguatoxin (anti-CTX) coupled to horseradish peroxidase, based on the same principle used in the of expensive isoRIA (26). This method, although economically feasible because the use to be tedious. Consequently this tope counting instruments was eliminated, was found EIA method was abandoned and was followedby the stick-enzyme immunoassay (S-EIA) employing sheep anti-CTX and monoclonal antibody to ciguatoxin (MAb-CTX) coupled to horseradish peroxidase. This method has been used extensively for surveys of ciguateraendemic areas and clinical confirmation of documented ciguatera poisoning for the Hawaii Department of Health ( 1 24- 129).
3. lmmunobead Assay The application of colored polystyrene particles coated with MAb-CTX as markers for direct detection of ciguatoxin adsorbed onto organic correction fluid-coated bamboo paddles was instituted in 1990 (130). In this solid-phase imlnunobead assay (SPIA), the organic correction fluid coat bound both polar and nonpolar components onto the bamboo paddle sticks. Numerous documented ciguateric fish from the HDOH and reef fishes surveyed in Hawaii were assessed using this procedure (1 3 1,132).
4. Summary The compilation of data of HDOH fish from ciguatera poisoning-implicated fish tested by RIA, S-EIA, and SPIA is shown in Table 6. The data include both the fish implicated in ciguatera poisoning as compiled by the HDOH and the routinely tested Hawaiian reef fishes generally associated with ciguatera poisoning in Hawaii. The major species tested included Ctenochuetus strigosus (kole) andSeriolrr dumerili (kahala). Ciguatera poisoning in patients was clinically documented by physicians or epidemiologists from the Department of Health. Of the 176 ciguatera-implicatedfish tested with these assays, 171 (97.2%) were found to be borderline or positive, while 5 (2.8%) were found to be negative (false negative). These procedures demonstrated a high degree of sensitivity with both sheep anti-CTX and MAb-CTX. On the other hand, the examination of Hawaiian reef fish of unknown toxicity demonstrated a fair to moderate degree of specificity with the MAb-
Ciguatera Fish Poisoning
227
Table 6 Compilation of the Assessments of Test Procedures Prior to the
MLA
Results
delinel Number used Procedure Radioimmunoassay (RIA)
Stick-enzyme 16 immunoassay (S-EIA)
Antibody of fish Reference Negative Positive
Sheep anti-CTX
Sheep anti-CTX
HDOH 46 Routine samples S. dlollerili 5596 Other Species 766 HDOH
824 (14.7%) 17 (2.2%) 16
2 (4.4%)
154
4112 (85.3%) 749 (97.8%) 0
( 100%)
Routinc samples various species 574 HDOH MAb-CTX 83 Routine samples
c. strigosis 712 S. durwrili
168
44
(95.6%)
168 Other species 3539
Solid-phase HDOH MAb-CTX inmunobcad assay (SPIA)
31
Routine samples various species 482
212 (36.9%) 81 (96.8%)
362 (63.1%) 2 (2.4%)
392 (55%) 82 (49%) 1720 (48.6%) 30 (96.8%)
320 (45%) 86 (51%) 1819 (51.4%)
26 1 (54.1qJ)
22 1 (45.9%)
1
(3.2%)
Both State of Hawaii Department o f Health (DOH) implicated toxic tishcs and routine samples of reef fishes arc included. The MIA data are prcscntcd 111 detail i n Tdbles 7 and 8.
CTX in the S-EIA and SPIA procedures showing 45.0-5 1.4% for the unknown fish. This is due in part to nonspecific binding to the coated bamboo stick. This moderatespecificity has limited the commercial use of these tests. However, following extraction, 70% of the borderline and positive extracts of unknown fish tested in the mouse and the guinea pig assays provedto be toxic. The sheep anti-CTX proved to give the best results in specificity (85.3-97.8%) and sensitivity (95.6%) in the RIA procedure in assessment of S. dutnerili and other reef fishes. The lack of a higher specificity suggested a search for a better solidphase medium for binding ciguatoxin and related polyethers.
E. Membrane lmmunobead Assay The membrane inmunobead assay (MIA) technique is based on the same immunological principles used to develop the SPIA ( 130), using a monoclonal antibody to purified moray
Hokama and Yoshikawa-Ebesu
228
eel ciguatoxin (CTX-I) (13 1,132), colored polystyrene beads, and a hydrophobic membrane laminated onto a solid plastic support. The membrane only bindsto lipids and generally not to polar or water-soluble compounds, unlike the liquid paper-coated sticks. The polyether toxins bind to the hydrophobic polyvinylidene fluoride (PVDF) melnbrane and are then detected with the MAb-CTX coated onto colored polystyrene beads. The intensity of the color on the membrane correlates to the concentration of toxin.
1. Preparation of Monoclonal Antibody to Purified
Ciguatoxin (MAb-CTX) Highly purified ciguatoxin was obtained through the courtesy of Professor P. J. Scheuer of the University of Hawaii Department of Chemistry. The toxin was isolated from LW)doiltis (G?.mrlothor.rrx)jrrvmicus (moray eel) livers and consisted of two interchangeable isomers (24).
2. Coupling of CTX1to Human Serum Albumin Purified ciguatoxin ( I pg) was coupled to human serum albumin (HSA)( 1 mg) in 7.5 t n l of 0.05 M phosphate buffered saline (PBS) by the carbodiimide procedure (133). Following exhaustive dialysisof the protein conjugate against salineat 4"C, the conjugate was precipitated with cold acetone in a ratio of 4: 1 (acetone:conjugate). The precipitate was centrifuged, rinsed twice with cold acetone, suspended in saline, and then used for the innnunization of the BALB/c mice. After pervaporation, the acetone phase was analyzed for free ciguatoxin. These steps were critical for the immunization process (133).
3. Immunization of BALB/c Mice Three BALB/c mice, 10 weeks of age, were injected intraperitoneally once a week for 3 consecutive weeks with 0.1 1111 of the CTX/HSA conjugate. A booster was given in the sixth week, 3 days prior to sacrificing the aniruals. Each nlouse received a total of 80100 ng CTX/80-100 pg HSA and 0.2 1111Freund's complete adjuvant (FCA) subcutaneously for each third injection of 0.1 ml of the conjugate.
4. Fusion Step The nonimlnunoglobulin synthesizing mouse myeloma cells selected for fusion were those previously reported by Kearney et al. (134), designated PBX63-Ag8.656B. These cells were grown in Dulbecco's modified eagles medium (DMEM) supplemented with 10% fetal calf serum. Myeloma cells in the logarithmic growth phase were used for fusion. The fusion of the sensitized cells from the BALB/C mice with the myeloma cells was doneby the method of Kennet etal. ( 135-137). These procedures included hybridoma selection, media preparation, and limiting dilution methods. The original active hybridonla containing reactive anti-CTX was designated 5C8. Analysis of 5C8 with commercial immunofluorescence-labeled goat anti-mouseimmunoglobulinisotypes(SigmaChemical Co., St. Louis, MO) demonstratedthe following hybrid cells: IgM,85%; IgG,, 55%; IgC?,,, 14%;IgG2h,3%: and possibly IgG3.This suggested a mixture of clones since the original myeloma produced no immunoglobulins. Nevertheless, 5C8 supernatants and ammonium sulfate fractions(50% precipitates) were used successfully in numerous studies from 1987 to 1994 using a variety of test procedures (124-132). Throughout theyearsfrom1987 to 1996,subculturing of 5C8 causedgradually diminishing activity of the MAb-CTX. Subsequent ELISA isotyping of different lots of SC8 (50% anlmonium sulfate fractions) showed the loss of the IgM, IgG,,,, and IgG2,, isotypes and the appearance of IgG+ The first recloning of 5C8 resulted in six viable,
Ciguatera Fish Poisoning
229
moderately active clones out of 100 plated. The clone designated 2E6 was further cloned by limiting dilutions resulting i n 1 1 (6%) clones with good activity. All of the good and moderately active clones were frozen and stored at -80°C. One of the highly active clones, designated 1C6, has been further cultured and used. Purification by gel affinity protein G column and isotyping in the ELISA procedure demonstrated equal amounts of IgG, and IgG; in the clone IC6. The analyses for sensitivity and specificity were taken from Morgan et al. (138): Sensitivity =
positive (HDOH) fish implicated in ciguatera detected by MIA X 100 total number of positive fish implicated in ciguatera ~
Specificity =
fish presumably without ciguatera toxins, negative in MIA X 100 total number of fish tested, presumed to be negative for ciguatera
5. lmmunobeadPreparation The clone designated 1C6 was selected and its cultured supernatants collected and diluted 1 :20 in saline in a final suspension of 0.1 % colored polystyrene beads. The 1 :20 dilution of antibody was selected as the optimal antibody concentration following titrationof each batch of monoclone IC6 cultured supernatants with the end point determined as approxinlately I : 100 dilution. The 1 :20 dilution circumvents the ‘‘hook effect” ( 139) and appears to be able to detect ciguatoxin at levels as low as 80 pg/g of fish (80 ppt). Sensitivity can be enhanced by increasing the MAb-CTX concentration. Polystyrene beads of blue (0.314 pm in diameter) and red (0.124 pm in diameter) colors were used i n a ratio of 3 : 1. These bead sizes were found to be satisfactory in suspension since larger-size beads, 20.8 pm in diameter, tended to settle readily to the bottom of the vial. The use of a single color of beads was satisfactory,but the combination of two different bead colors gave a deeper hue. Polyvinylidene fluoride membrane was laminated onto a plastic support stick (Fig. 5). Other materials and chemicals used in the MIA include test tubes (8 mm inside diameter 75 mm length), a punch biopsy tool or sharp razor to obtain fish samples, methanol for soaking fish samples, a suspension of 0.1% colored microbeads (0.3 14 pm and 0.124 pm diameter) coated with MAb-CTX. Standard control for positives consisted of pooled crude extracts of HDOH implicated fish from clinically diagnosed ciguatera poisonings. The of Hawaiian reef fishes implicated inciguatera presence of ciguatera in these crude extracts poisoning was previously examined by Hokalna et al. ( 1 30) and Manger et al. ( 1 19) in the neuroblastoma cell bioassay. In addition, the presence of ciguatera was previously
80 mm (plastic suppon)
T 7
~
1
1-1
+OSmrn 20 mm
(membrane) Fig. 5 MembranestickforMembraneImlnuunobeadAssay (MIA) withdimensionsgiven.The membrane portion of the stick is directly laminated onto the longer plasic support.
Hokama and Yoshikawa-Ebesu
230
established at 4 ng/ 12.5 mg of crude extract by itnmunoinhibition of the MAb-CTX with highly purified C T X l B (140).
6. Membrane lmmunobead Procedure The concept of the MIA is similar to that of the solid-phase immunobead assay (SPIA): colored immunobeads will adhere to ciguatoxin (antigen) on the membrane portion of the plastic stick previously exposed to the antigen; positive samples will show visible color changes, while negative ones will not (Fig. 6). A fish tissue sample (approximately S 2 3 mg) cut with a punch biopsy tool or razor blade is placed along with 0.5 ml of methanol and a membrane stick into a test tube. After soaking for 20 minutes, the stick is removed from the test tube and thoroughly air dried for at least 20 minutes. The completely dried membrane stick is immersed into 0.5 ml of latex immunobead suspension, removed after 10 min, rinsed in saline or water, shaken to remove excess liquid,air dried, and the membrane observed for color. The result is scored as negative, no distinct color on the membrane; weakly positive, light bluepurple color on the membrane, with or without a darker band at the meniscus level; or strongly positive, membrane is colored and has more than one dark band at the meniscus level. All fish with results reading weakly positive and higher should not be eaten. Positive control samples at both high and low concentrations of toxins consisted of pooled extracts of implicated HDOH ciguateric fish at7.0 mg/ml and 3.0 mg/ml of methanol containing residual ciguatoxin of 2.24 ng and 0.96 ng ciguatoxin/ml of methanol, respectively. Negative controls were blank membranes used directly or soakedin methanol anddriedthoroughlybeforeimmersingintotheimmunobeadsolution.Thesequential steps of the MIA procedure are presented in Fig. 6. During the development of this method, several synthetic membranes were examined and the hydrophobic membrane proved to be the best, since less than 3% of negative controls showed nonspecific binding of the immunobeads. The data in Table 7 represent an assessment of fish recently involved in clinically documented ciguatera poisoning cases using the MIA. Of 13 fish samples examined, 10 were found to be positive and 2 borderline, containing approximately 60-160 pg ciguatoxin or related toxin per gram of extract. This gave a sensitivity of 92.3%, with 7.7% false negatives. A Sphgmerza bcrrr-clcudcl sample from the island of Hawaii gave an initial negative response in the MIA. However, further examination of the extract of this fish by Membrane stick
I
L
Methanol Tissue sample Step 1
Step 2
Fig. 6 MembraneImmunobeadAssay
Colored bead with MAb -CTX suspension
Negative
Step 3
(MIA) procedure.
Weak Pos. Strong Pos. Step 4
231
Ciguatera Fish Poisoning
Table 7 Membrane lmnunobead Assay of Ciguatera-Implicated Fish Samples, Hawaii Department of Health Species
Remarks
Source Oahu Oahu Kauai Big Island Oahu Kauai Kauai Oahu Maui
unidentified) (species Snapper Barbedos (via Red fish (species unidentified) Barracuda (species unidentified) Crpkalopko1i.s q q u s (mi)
MIA result sample +
+
+ -.I
+
+ + +
+ t
New Jersey) Texas DOH Texas DOH Big Island
+ + ?h
Cooked Cooked sample Cooked samplc Cooked sample Tissue Tissue Tissue Cooked sample Cooked sample sample Cooked Tissue Tissue sample Cooked
-.
Results are shown as +. positlvc; 5 . borderline; and negative. Thls sample was extracted and tested with the MIA and reacted weakly at 7.0 nlglml in laethanol and negative at 3.5 mg/ml. Mouse toxicity results (dead In 7 minutes after intraperltoncal injection) suggest presence of PTX- or MTX-like toxms. The guinea pig atrium analysis suggested the presence of low levels of CTX-like compounds i n this sample (sec Fig. 7). h One patient ate the samespec~es for three consecutive meals: it is uncertain if the same or three different tishcs were consumed. Three others ate only one meal of roi with no toxicity reported. A roi from the same catch was tested and found t o be negative.
mouse toxicity and guinea pig atrium analyses demonstrated that it contained a potent toxin unlike ciguatoxin. I n comparison with a classical ciguatoxin pattern showing strong inhibition by tetrodotoxin (TTX), the S. barrncudcl pattern showed only a slight tetrodotoxin effect (Fig. 7). Nevertheless, the extract at 7.0 mg/ml showed a weak MIA response and a 100 mg dose killed a mouse in 7 minutes. Still, the patient who consumed part of this fish demonstrated classic ciguatera symptoms (141). Taken together, these data suggested the presence of palytoxin, maitotoxin. or as yet undetermined toxins in this barracuda, along with low levels of ciguatoxin-like compounds. The analysis of various fish species with the MIA is presented in Table 8. This table includes the species names, sources, total number of samples, and MIA results directly from fish tissues. The Cmmx sp. (jack or papio) from Hawaii shows a higher percentage of borderline and positive results (23%) than those from Kwajalein and Kosrae (0% and lo%, respectively). In Hawaii, the Crrmnx sp. is one of the major targetsof sportfishermen and is the leading cause of ciguatera poisoning in nearly all of the islands of Hawaii. In summary, of the 154 samples tested with the MIA, 132 (85.7%) were negative, 14 (9.1%) wereborderline,and 8 (5.2%) were positive. The specificity (85.7%) shown iswithin acceptable levels for biological assays of this nature. All negative fishes were consumed without any reports of ciguatera poisoning; in other words, no false negatives were recorded. Figure 8 represents the titration of the standard pooled HDOH-positive extract in the MIA. The data show MIA results of the extracts tested at different concentrations and
232
DOH-poritiw extract 10 rng (b)
Hokama and Yoshikawa-Ebesu
Tetrodotoxin V e r a p d
Rinse
Fig. 7 Inotropic patterns from guinea pig atrium assays for (a) S. burrcrcudn and (h) DOH-positive (containing CTX) extracts with addition of tetrodotoxin, 10 ' M (12.5 p]), verapamil, 10 ' M (12.5 pl), and rinse, respectively. The difference in the TTX block is clearly demonstrated i n the CTX containing extract (h).
Membrane Immunobead Assay of Routine Catches of Fish from Hawaii and Pacific Islands
Table 8
MIA results
Total Species
Ncgative Borderline Positive fish Source
C~YULY sp. (pepiolulua) Curunx sp. (papio) Curmx sp. (papio)
Ctenochaer~rstrigosus (kole) Mugil cephml11.s(mullet) Mulloidichthys aurifluuztnu (weke) Serioln rlumeriti (kahala) Aphureusfitrcn (wahanui) Mu1loirlichthy.s pflugcvi (weke ula) Rodianrts bilinulrrt~rs(a'awa) A p i o r ~virescens (uku) Ccphalopholis arg~ts(roi) S ~ ~ J ~ Nhelleri ~ I I L(barracuda) I Prirrc~or~thu.~~ meeki (aweoweo) A c ~ n r ~ t h u trio.steg~t.s r~s (manini)
Hawaii Kwajalein Kosrae Midway Hawaii Hawaii Hawaii Hawaii Hawaii Hawaii Maui Oahu Oahu Oahu Oahu
Total number (%) Perccntage values arc shown
Other
26 82 20 3 3
3 3
2 (7.6) 4 (5.0) 0 (0.0) 0 0
5
1
4
2 3 2
1
0 1
2 2 1
1 1 1
154 In
parcntheses
20 (77.0) 78 (95.0) 18 (90.0)
2 0
2
2 2
0 0 0 1 0 0
0 0 1
1 132 (86.0)
14 (9.0)
4 (15.4) 0 (0.0)
2 (2.0) 0 0 0 1 0 0
0 0 1
0 0 0
8 (5.0)
233
Ciguatera
3.0+ 2.0+
2.0C
1.6+
1.6+
l.O+
ff
-- -
Fig. 8 Examples of DOH mixedciguatericfishextracts from 0.1 through 10.0 mgextractin 1 ml of methanol (in duplicate), tested in the MIA. The numbers in the parentheses are calculated concentrations of CTX in the crude extracts. The -, t,and numbers represent relative degree of activity.
+
the approximate calculated ciguatoxin concentration. The minimal detection point was 0.08 ng or 80 pg (80 ppt) ciguatoxin in the crude HDOH mixture. This new MIA procedure was compared with previously developed immunological (142). The S-EIA used test procedures which used correction fluid-coated bamboo sticks bamboo skewers with a correction fluid coat and MAb-CTX conjugated to HRP with appropriate substrate as the detection system. The SPIA used a correction fluid-coated bamboo paddle and MAb-CTX adhered to colored polystyrene beads to detect ciguatoxins. The MIA system consists of a solid-phase hydrophobic synthetic membrane laminated onto a solid plastic support. The sensitized membrane is dipped into an aqueous suspension of mixed polystyrene beads of two different colors and diameters bound to MAb-CTX and the intensity of the resulting color on the membrane assessed. This new procedure was used to assess clinically implicated HDOH ciguatera fish samples, a variety of routine fish samples from endemic reef areas of Hawaii, a number and of Caranx sp. from Kwajalein and Kosrae. The results presented in the tables show good detection of HDOH ciguatera-implicated fish, with one exception: direct examinationof the S. barracuda tissue showed no activity in the MIA, but the extract was highly toxic in the mouse assay and showed response in the MIA with the extract at a higher dose (7 mg). Analysis of the extract in the guinea pig assay suggested its major toxic component to be other than ciguatoxin (Fig. 7), as stated earlier. Comparison of Carum sp. from Hawaii, Kwajalein, and Kosrae showed that those from Hawaii had a higher toxicity level (23%) than the samples from Kwajalein (5%) and Kosrae (10%). In part, this may be attributed to the fact that the Caram sp., from Hawaii were obtained from several areas on different islands, while the Kwajalein and Kosrae samples were from one area on a single island. Other groups of fish samples from Hawaii were too small to properly evaluate. Nonetheless, the overall sampling by the MIA shows reasonable ciguatoxin detection levels, with 86% negative, 9% borderline, and 5% positive. The variability dueto nonspecific bindingof the immunobeads to the membrane is minimized by the use of the hydrophobic membrane, with only3% of 179 blank membranes tested showing a borderline reaction. The sensitivity achieved with the small num-
234
Hokama and Yoshikawa-Ebesu
ber of HDOH toxic samples was 92.3%, while the specificity indicated for unknown reef fishes was 85.7%. These values were within the acceptable ranges for a biological test system. The sensitivity of the older procedures (RIA, S-EIA, SPIA) ranged between 95.6% and 97.2% and the specificity was close to 50%. This low specificity suggested nonspecific binding of the antibody conjugate and the antibody bead to the liquid paper on the stick. In part, the antibody bead tended to bind to the bamboo paddles nonspecifically in 22 ( 1 0.6%) of the 207 blank sticks examined. As mentioned, the MIA blanks showed only 2.8% nonspecific binding. The low specificity with liquid paper-coated bamboo paddles needs further studyif these procedures are to be used for commercial ventures. However, the SPIA has been useful in preventing ciguatera in recreational fishermen in Hawaii (132). With the exception of okadaic acid, cross-reaction studies of MAb-CTX with other polyethers are limited (140,143). As indicated by Landsteiner (144), MAb-CTX reacted best with ciguatoxin, its homologous antigen (epitope) at the 1 ng level, while 5 ng of okadaic acid was necessary for comparable activity. Other related polyethers, including brevetoxin, palytoxin, maitotoxin, and congeners of okadaic acid, require levels of 2 5 0 ng to give comparable reactions with MAb-CTX in the S-EIA assay. In the development of the MIA, several factors critical to obtaining accurate and repeatable results were noted. First, the membrane portion of the membrane stick must not be touched, since this can cause false-positive reactions. Second, the membrane stick must be soaked in the methanol/fish sample suspension forat least 20 minutes for optimal results. Third, both the stick and the test tube must be completely dried before adding the latex immunobead suspensionto the test tube; failure to doso can also cause false-positive reactions.Lastly, to preventfalse-positiveresults,themembranestickshouldnotbe soaked in the latex immunobead suspension for longer than I O minutes. If these points are followed, the MIA procedure should prove to be simple and reliable for the detection of ciguatoxin and related polyethers in fish. Preliminary studies suggestedthat the binding sites of ciguatoxin to sodium channel receptors and the monoclonal antibody to ciguatoxin were on opposite sites of the ciguatoxin. Evidence for this interpretation was based on immunological analysis with epithelial extracts of pig small intestine (sodium channels) and MAb-CTX in the guinea pig atrium assay. Ciguatoxin addedto the physiobath containing guineapig atrium induces an inotropic (increase in the atrium contraction, and thus the amplitude of the heartbeat) response. Preaddition of varying concentrations of pig intestine extract or MAb-CTX into the physionot the latter bath inhibits the inotropic effect in the former (pig intestine extract) but (MAb-CTX). Further, immunological studies employingthe S-EIA demonstrated that the west sphere synthetic tricyclic rings A, B, and C did not react with MAb-CTX, but the east sphere synthetic tetracyclic rings K, L, M, and N reacted with MAb-CTX (57). Furthermore, computer molecular modeling for ciguatoxin suggests the reactive siteof ciguatoxin on the fifth domain of the sodium channel is the west sphere, which is similar to brevetoxin in its reaction with the synaptosome of the rat brain tissue (122). Modified immunoassays for polyether toxins have been developed, including radioimmunoassay for brevetoxin (1 18,122). However, to date, no extensive testing or evaluato show sensitivity, but tion of this immunoassay has been established. They appeared specificity has not been evaluated and the procedures were generally not applicable for simple screening of toxic fish.
Ciguatera Fish Poisoning
235
F. Chemical Methods Analyses for polyether marine toxins including palytoxin, tetrodotoxin, okadaic acid,brevetoxin, alld ciguatoxin have useda variety of chemical methods. Thin layer chromatography and high-performance liquid chromatography (HPLC) have been explored. Chemical techniques have been utilized for the analysis of okadaic acid and other polyethers in extracts of fish ( 1 18,145,146). Fluorometric analysis has been developed for assessing tetrodotoxin (okadaic acid, DSP poisoning) and ciguatoxin ( 1 18).
G. Summary Various assays have been developed to identify the toxin of interest in natural marine products, in the purification process, and for screening purposes to maintain wholesome and healthy products for consumption. This has been the case for shellfish and finfish some products. Thus the review and comments addressed in this section were dedicatedto detection.Thebasicgoalsare to achievesimplicity,sensitivity,and specificity i n anassay system to be universally applicable for home, field, and laboratory use in ciguatera poisoning. Most of the bioassays using animals or cell systems tend to be nonspecific. However, for ciguatoxin,use of sodium channel targets suchas synaptosomes and cell bioassay with neuroblastoma indicate some level of specificityandsensitivity. The immunochemical assay appears to be the most promising for practical use cornmercially in screening for ciguatera toxic fish because of its simplicity, practicability, and most of all, its specificity and sensitivity.
VIII. CLINICAL Consumption of toxic fish associated with ciguatera poisoning results in the appearance of clinical symptoms within 10 minutes to 12 hours, but on occasion 24 hours, after consumption (mean 4-6 hours). The gastrointestinal symptoms are characteristic of many other food poisonings, but ciguatera poisoning can be differentiated by unique features affecting the neurologic system. Table I summarizes the many clinical symptoms associated with ciguatera poisoning (15,147). The major toxin implicated has been designated ciguatoxin (23), although the complex symptoms and peculiarities among patients from different tropical and subtropical areas (Caribbean and Pacific) suggest multiple toxins (15,64). For example, Bagnis and Legrand (85) and Bagnis et al. (102), in examining numerous cases from the SouthPacific, showed subtle differences in certain clinical symptoms between Melanesians and Polynesians. Pruritis, ataxia, abdominal pain, and weakness were more commonly reported by Melanesians than by Polynesians. Whether these differences were due to variability in toxins (sourceof fish) or the susceptibility of different ethnicities to ciguatoxin remains unclear (85,102). Diagnosis was based on clinical presentation with paresthesia and dysesthesia, considered a clinical hallmark of ciguatera poisoning, especially in the South Pacific. This essentially differentiates ciguatera from other formsof food poisoning ormild gastroenteritis (64,85,102). In Hawaii, the use of immunological tests including the MIA has aided in the confirmation of ciguatera poisoning by examination of the remaining contaminated fish tissues.
236
Hokama and Yoshikawa-Ebesu
Reports of ciguatera poisoning from the Caribbean (including Florida) and the Pacific (Hawaii, French Polynesia, American Samoa, and Australia) all generally show variations of the clinical symptoms includedin Table 1. These variations can change from area to area. The neurological symptoms, especially dysesthesia and paresthesia, the were major clinical diagnostic hallmarksof ciguatera poisoning followingconsumption of toxic fishes, although these symptoms can also occur in shellfish poisoning and tetrodotoxin intoxication (147). Ciguatera poisoning caused by Scarvs gibbus from the Gatnbier Islands showed initial clinical symptoms characteristic of the ciguatera syndrome. However, after 5-10 days, a second phase appeared, characterized by an ataxic gait, dysmetria, asthenia, loss of static and dynamic equilibrium, and muscle tremor, which was induced by an effect on the cerebellum. This second phase set in for about 7 days and required about 4 weeks for complete recovery (1 5). This variation was probably due to another polyether toxin, isolated and named scaritoxin (148,149). Recently scaritoxin (Table 2) has been identified as a congener of ciguatoxin and named CTX3B (Molecular Weight 1077). Palytoxin has been implicated by Japanese investigators following consumption of a parrotfish (Y/,sisccrru.sovifrom) ( 1 SO). One patient died 4 days after consuming the fish and had symptoms not unlike ciguatera poisoning (150). Earlier (1983), Yasumoto et al. (15 I ) reported the presence of palytoxin in two species of crabs (Loph0zozymrr.s pictor and Detmllicr crlcalcri) associated with lethal poisoning of humans in the Philippines. Palytoxin toxicity has also been reported in Hawaii from smoked Decapterus mac" rosoIm imported from the Philippines ( 152). I n laboratory analysis of toxicity induced by several species, low levels of palytoxin were demonstrated in 4of the 14 fish examined (141). It seems that the term ciguatertr encompasses fish poisoning associated with a group of lipid polyether compounds,of which ciguatoxin appears to be one of the most hazardous and probably the most prominent i n carnivores (23). A study of two outbreaks in Hawaii i n 1985 involving a herbivore (Ctet~ochtretus strigosus) and a carnivore (Seriola d u m r i l i ) showed clinical similarities and differences (64). C. strigosus induced only gastrointestinal and neurological symptoms i n all 16 patients with no hospitalization. Seven of the 14 patients who had eaten S. durnerili were hospitalized and 7 had cardiovascular symptoms in addition to gastrointestinal and neurological problems. A similar observation was reported by Bagnis (15) in Tahiti. This observation of ciguatera poisoning by two fish species of different eating habits suggests the following: (a) there is more than one toxin, (b) changes in the toxin occurred during the passage from herbivore to carnivore, and (c) there were no differences between toxins, but poisoning was a dose-response effect because C. strigosus was small and the viscera (liver, roe, etc.) was not consumed. Other factors worth noting in ciguatera poisoning include (a) multiple poisoning of an individual causes an enhancement of clinical severity and greater sensitivity to the toxin in subsequent poisonings; (b) the poisoning is rarely lethal ( 14,16,85,98,101,102) when the flesh is consumed, and lethality (less than 1%) occurs when the most toxic parts of the fish are eaten (liver, viscera, organs, roe, etc.); and (c) susceptibility to the toxin(s) may vary among individuals. Mammals susceptible to ciguatera poisoning include dogs, cats, dolphins, California sea lions, Hawaiian monk seals, and domestic pigs. Therefore care should be taken in feeding these animals fish from endemic areas. Transmission of toxin via ciguateric mother's milk during breast-feeding has been demonstrated for ciguatera poisoning, along with placental transfer of the toxin, which has resulted in abortions during the first trimesterof pregnancy (85,102). Sexual transmis-
Ciguatera Fish Poisoning
237
sion via seminal fluid from an intoxicated husband to wife has also been reported ( 153). Thereforeciguatoxinanditscongenerscan be transmittedthroughbreast-feeding,the placenta, and body fluids (seminal fluid) from a ciguateric patient to a healthy individual. This is probably made possible by the ability of ciguatera to bind normal serum and tissue proteins, one of which may be albumin. Table 4 summarizes the fish most commonly implicated in ciguatera poisoning i n the Caribbean, including Florida, and the Pacific (Hawaii, French Polynesia, American Samoa, and Australia). Most of the fish listed are carnivores. Part of the variation in toxicity relative to fish between areas may be attributed to ethnic variations in taste for various fish species; another contributing factor could be the variation due to the nature of secondary prevailing toxins. In all studies reported, the cyclic effect of a seasonal pattern of intoxication was inconsistent. I n American Samoa, toxicity has occurred all year round. Toxicityin Florida occurs frequentlyin the spring and silnnner months. In Hawaii, no cyclic pattern of toxicity has been shown. A close examination of 14 fishes consisting of eight species implicated in ciguatera poisoning in 1994 withrespect to clinical symptoms, immunological tests of fish, and guinea pig atrium (sodium channel effect) and mouse bioassays has been reported (141). It was concluded fromthe comparison of the clinical symptomsin the patients and analyses of the fish consumed by the patients i n the immunological test, guinea pig atrium, and mouse toxicity assays that there was a diversity of toxins in the ciguateric fish examined. Potentially, in any given fish there was probably more than one toxin causing the ciguatera symptoms. Diversity of toxins or congeners has been previously suggested by investigators in ciguatera poisoning studies (15,64,85,102,104,14I.l52.154~. Based on the mosquito, mouse, and MIAtests of the consumed ciguatericfish, it has been estimated that illness occurs when man consumes at least23-230 ng ofciguatoxin in a meal. This was referred to as the pathogenic dose (PD) by Bagnis et al. (85,102). The wide range of toxicity suggests variability in individual susceptibility and the effect of previous exposures to very low concentrations of ciguatoxin or congeners (cumulative effect). Others have indicated the minimal level of toxic fish consumption to be 50-100 pg/g of fish (155,156).
IX.
PATHOLOGY
A.
Human
Pathological findings in postmortem studies of humans following death from ciguatera poisoning are relatively scarce. This is due, in part, to the fact that deaths from ciguatera poisoning are very rare (less than 1 % worldwide). Most deaths reported were probably due to palytoxin, particularly to intoxication from eating crustaceans. The crabs Dernclrlitr wqnaudi, D. cdcnltri, and hphozozqr~ru.spicfor from the Philippines and Singapore have been implicated in toxicities and death, but these involved palytoxin as the causative factor. Postmortem examinations of human tissues generally showed little or no histologic tissue changes with routine hematoxylin and eosin (H & E) staining. Evidence of morphologic tissue changes detectable either by light or electron microscopy in human deaths due to ciguatera poisonings is very rare (44). However, three reports in the literature demonstrated these findings: (a) histologic lesions in nerve fibers with striking edemain the vacuoles of Schwann cell cytoplasms and vesicular degeneration of myelin ( 1 57); and (b) muscle biopsy on a ciguatera poisoning victim revealed fiber-
238
Hokama and Yoshikawa-Ebesu
splitting degeneration and necrosisof the liver ( 158,159). No electron microscopy findings have been reported in human fatalities as a consequence of ciguatera poisoning.
B. Animal Studies 1. Ciguatoxin and Brevetoxin Experimental animals administered whole fish tissues, crude organic solvent extracts, and purified ciguatoxin presented with clinical symptoms characterized i n human intoxication with ciguatera poisoning. Wild mongoose given toxic moray eel flesh was noted to have increases in ulcers of the stomach (28). However, i n nature mongoose has been found to develop stomach ulcers due tostressfulenvironments (28). Experimental mice models show few light microscope changes with routine H & E staining. but definite pathologic changes can be seen with the electron microscope i n target organs of mice administered pure ciguatoxin ( 1 60). Repeatedintraperitonealandoraladministrations of ciguatoxin 1B(CTXIB), CTX4C, and PbTx-3 derived from L I I ~ ~ N bI oI hI cI wS and CTX4C purified from G. roxicus to male ICR miceat a dose of 0.1 pglkg for 15 days resulted in marked swelling of cardiac cells and endothelial lining cells of blood capillaries in the heart. A single dose showed no discernible pathologic changes in the heart. Damage to the capillaries was followed by prominent effusion of serum and erythrocytesinto the interstitial spacesof the myocardium. Swelling of the endothelial lining cells of the capillaries caused narrowing of the lumen and accumulation of blood platelets in the capillaries, which caused multiple necroses of single-cell cardiac muscle cells. Myocytes and capillaries appeared normal after 1 month of the treatment with toxins. The effusion in the interstitial spaces resulted i n the formation of bundles of dense collagen, which persisted for 14 months. Diffuse interstitial fibrosis was prominent in the septum and ventricles, with bilateral ventricular hypertrophy. A single dose of 0.1 pg CTXI/kg intraperitoneally resulted in severe acute heart injuries, followed by diffuse myocardial fibrosis ( 160,161 ). It is well established that the west sphere of ciguatoxin acts on the membrane of cells, with the receptor being the fifth domainof the sodium channel, similarto brevetoxin (162). However, ciguatoxin has been shownto have a greater affinity for the receptor than brevetoxin. Both compounds depolarize nerve terminals through their actions on sodium channels, which potentiate the membrane and cause the influx of sodium ions into the cells. It has been suggested that this increase in the sodium influx creates the pathological patterns of the targeted cells when examined by light or electron microscopy ( 160,161 ). Nevertheless, surprisingly, damage by acute levelsof PbTx (200 pg/kg) showed less tissue pathology than CTX at a sample dose of 0.35 pg CTXI /kg. CTX1-treated animals showed marked edema in the heart muscles as well as the nerve tissues. Whether given in shortterm successive or long-term intermittent doses, the effect of CTXl on the heart muscles was similar to the single dose of 0.35 pg CTX 1 /kg. by stimulation of the motor neurons Acute dosesof brevetoxin showed tonic seizures and death with a short opisthotonosis. Pathologic changes in the heart, kidney, and liver by PbTx-3 were less severethan those of ciguatoxicosis. Table 9 summarizesthe comparison of the toxic effects of pure ciguatoxin and PbTx-3 i n ICR mice (161). The effects of CTXI (0.35 pg/kg) on the cardiac tissues at a lethal dose were much more severe than PbTx-3 at a larger lethal dose (200 pglkg), though both toxins are sodium channel ago-
Ciguatera Fish Poisoning
(relative intensity) muscle Cardiac cells Blood capillaries (endothelial lining cells) Thrombosis(heart.liver.penis) Cunlulative effect heart in
239
31 3+ 3+
2+
+ +/-
nists. The affinity for the sodium channel, however, is much greater for CTX I than PbTx3. This physiological difference may account for the variance in the cardiac tissue pathology. Mice given high (death in 65 minutes), moderate (deathin 2 hours 43 minutes), and low doses (death i n 7 hours 15 minutes) of ciguatoxin all demonstrated changes in the small and large intestines. Small intestines showed degeneration of nuclei and swelling (edema) of the lamina propiain the high-dose (CTXI) administration, while the moderatedose CTXl mice showed degeneration of the villi tips. Stripping of brush borders of the villi was observed i n the small intestines with the low-dose CTXl treatments. The lowdose CTXl also caused epithelial surface degeneration, sloughing into the lumen, and increased mucin production. These findings were demonstrated by light microscopy (163). Eighty percent of a mouse unit of semipure CTX 1 B from Gytnt~othormj a w r n i c m s given per mouse induced diarrhea within 10 minutes after intraperitoneal injection and lasted for 30 minutes. The changes were shown i n the large intestines. CTX 1 B induced accelerated mucus secretion and peristalsisi n the colon and stimulated defecation resulting in prominent diarrhea. Large quantities of mucus were secreted in the colon from mature as well as immature goblet cells and epithelial cell damage was observed in the upper portion of the large intestines, but not the lower half. The morphologic changes of the upper large intestine induced by CTX I B were similar to those seen with cholera toxin ( I 64,165). Diarrhea appears to be induced in mice by CTX 1B in doses between I and 7 ng/mu. Mice given less than I ng and greater than 7 ng intraperitoneal injections showed no diarrhea. Mice given oral doses at any levels showed no diarrhea. It appears that variable patterns of diarrhea after intraperitoneal injection of ciguatoxin crude extracts may be attributable to the C T X l B concentration in the crude extract. The pathology induced after administration of ciguatoxin in experimental animals can be summarized as follows: (a) acute poisoning by CTXlB or CTX4C showed damage in the heart, nledulla of the adrenal glands, autonomic nerves, penis, and small and large intestines (163,164);(b) atropine suppresses symptomsof diarrhea, but had no effecton the
240
Hokama and Yoshikawa-Ebesu
injury to cardiac muscle; (c) reserpine aggravatedthe clinical symptoms and pathological findings; and (d) guanethidine and S-hydroxy dopamine and bilateral adrenalectomy had no significant effects on ciguatoxicosis.
2. Dinophysistoxin-l and Pectenotoxin-l Pathological tissue changes occurred in experimental mice administered other related polyether marine toxins. Sequential intrastructural changes were studiedi n the gastrointestinal system of the BALB/c strain of mouse following intraperitoneal administration of dinophysistoxin- 1 (DTX- 1 ) and pectenotoxin- 1 (PTX- 1 ), causative agents of DSP, DTX- 1, a diarrheagenic toxin, produced severe mucosal injuries in the small intestine within an hour after the injectionof the toxin. The injuriesin the small intestines included extravasation of villi vessels, degeneration of absorptive epithelium, and desquamation of the degenerated epithelium from the lamina propia. PTX-I, on the other hand, a nondiarrheagenic toxin from DSP-causing mussels, demonstrated no pathologic changes in the small intestine, but induced pathologic changesin the liver within an hour after intraperitoneal administration. Numerous nonfatty vacuoles appeared in hepatocytes around the periportal regions of the hepatocyte lobules. These vacuoles originatedfrom invaginated plasma membranesof the hepatocyte, as shown by electron microscopy with colloidal iron ( 166).These studies were carried out with suckling BALB/c mice of either gender given various intraperitoneal doses of DTX- 1 and PTX- I .
3. Maitotoxin Maitotoxin (MTX), one of the toxins implicated in ciguatera poisoning and probably the most toxic substance isolated from cultured G. toxicus, induced severe pathomorphologic changes i n the stomach andheart,andatrophy of lymphoidtissueswithreduction of circulating lymphocytes after release of cortisol from the adrenal gland. A reduction of IgM and an increase in calcium in the adrenal glands were also noted in mice. In the gastric nwcosa, multiple lesions were seen with a marked increase in total calcium level 1-24 hours after intraperitoneal administration of 200 or 400 ng/kg of MTX. There was no close temporal association between accumulation of calcium and the morphologic appearance of dead cells in the heart and thymus. After administration of 200-400 ng/kg MTX, marked swelling was seen the in endothelial lining cellsof blood capillaries between cardiac muscle fibers within 30 minutes, followed by cell death of the cardiac muscle cells. MTX at 2200 nglkg also resulted in necrosis of the lymphocytes in the cortex of the thymus at 4 hours and the medulla at 8 hours after administration. Mice pretreated with CoCI,, a calcium channel inhibitor, and/or adrenalectomized mice showed no discernible changes i n the lymphoid tissue after repeated administrations of MTX. It was concluded that MTX initially stimulated calcium influx into the adrenal glands, which induces the release of cortisol into the circulation. The increase of cortisol i n serum results intheacuteinvolution of the thymus and lymphoidtissues,andthus reduction in Ig synthesis ( 167.168).
4. Palytoxin The effects of palytoxin (PTX) on young male ICR mice were examined with ouabain and several cations, including potassium, and the tissues examined by light and electron Illicroscopes for changes. The target organs of PTX were the heart, kidneys, pancreas,
24 1
Ciguatera Fish Poisoning
slnall intestine, and liver. Pathomorphologic changes of the heart induced by PTX were silllilar to those of a lethal dose of KCI. However, no pathologic changes were shown in the kidney for mice treated with KCI. Similar kidney damage in the pro xi ma^ as well as the distal convoluted urinary tubules in mice given 5 mg/kg ouabain were shown, while the heart of Illice given ouabain i n the same dose demonstrated no discernible pathologic changes. Ouabain given together with palytoxin had no effect on the damage induced i n the heart and kidney by palytoxin at 8 mg/kg. It was concluded that palytoxin caused a syt11pto111 col11pIex of hyperkalemia and induced profound inhibition of Na-/K -ATPase ( 169.170). Table 10 presents the LDS,,of mouse toxicity for various known pure nlarine toxins studied. The decreasing order of toxicity i n mouse is as follows: '
MTX
> PTX > CTX > TTX > PbTX > OA > PbTx-B.
5. Toxicity in Fish Generally fish associated with ciguatera poisotlitlg are not affected by consulllption of G. fo.xicll,y, whether in herbivores or in consumption of herbivores by carnivores. For example, toxic fish fed to carnivores caused no toxicity or pathologic changes, but carnivores accumulated the toxin in their tissues ( 1 IO). However, abnormalities occur when fish not associated with ciguateric transmission (freshwater fishes, for example) are fed either G. f o x f u r s or ciguatera-contaminated flesh. Why ciguateric fish are not affected remains to be determined; whether it is metabolic (ability to assimilate toxins into tissues), or a buildup of tolerance with constant exposure to the toxins remains to be studied (44). I n studies prior to availability of pure toxins, pathomorphologic changes induced by administration i n experimental animals were difficult to assess because of the potential mixture of toxins i n crude extract. A case in point is the finding of anticholinesterase activity in fish extracts associated with ciguatera poisoning (52). Present studies with purified ciguatoxin, maitotoxin, palytoxin, and the okadaic acid group of toxins have given a better account of pathomorphologic changes and the clinical symptoms from observations in both the anirnal studies and the symptoms observed in the clinical setting.
6. Summary Thestudies in mice modelswith
pure marinetoxinscarried out by Terao et al. ( 16 1,162,164- 169) probablyreflect the pathology in humans, where studies are very lim-
Table 10 ComparativePotency o f MarineNatural Toxins in Mice
Rcl'erence toxin
L D , , (pg/kg)
Molecular
i n mice (IP)
weight
Source
Hokama and Yoshikawa-Ebesu
242
ited. I n humans, the major target organs affected by marine toxins are the gastrointestinal tract, nervous system, and muscles, explaining the symptomology seen for ciguatera fish poisoning (Table 1 ).
X.
THERAPY
A.
Earlier Treatment
Treatment of ciguatera poisoning can be separated into two periods. I n the first period, limited information about the mode of action, chemistry of the toxins, and definite patterns of s y m p t o m were known for ciguatera poisoning. A thesis by Chanfour ( 17 I ) presents the treatment for ciguatera poisoning in French Polynesia. which was quite similar to the therapy devised by Russel (147). Essentially, treatment was based on the action of the drugs on the s y m p t o m observed. Thus, in humans, drips of atropine at 0.5 nlg every 4 hours were used i n a continuous, multiple-electrolyte solution (147). Atropine appeared to have an effect on the gastrointestinal and cardiovascular symptoms and signs. However, atropine had no significant effect on the skeletal muscle symptoms and showed only a slight improvement i n the neurological findings following injection of atropine. Cyanotic and respiratory diseased patients were placed on oxygen. Patients were maintained on high electrolyte solutions and intravenous daily doses of calcium gluconate, a vitamin B complex, vitamin C, and time-disintegratillg capsules including phenobarbital, hyoscyamine sulfate, atropine sulfate. and hyoscine hydrobromide. These drugs were used i n an attempt to control the presunled pharmacologic action of fish poisoning toxins. These treatments were confined to patients seen within 48 hours of the fish poisoning (147). In cases seen 3-14 days after poisoning, the most severe complaints were sensory disturbances. Atropine had no effect on these patients; that is, no detnonstrable improvements were seen. These patients did best with a high protein diet and administration of intravenous vitamin B complex, calcium gluconate, and oral vitamin C. Extremes i n temperature were avoided because they caused recurrenceof painful pruritus, paresthesia, and nausea ( 147). The use of pyridine-2-aldoxin~e methiodide (2-PAM) based on the anticholinesterase concept of ciguatoxin action produced no improvement i n the patients’ conditions. This was demonstrated in earlier reports ( 172). In 1975, one of the best courses of treatment for ciguatoxin, as recommended by Russel (147) was atropine to control the gastrointestinal corlplaints and cardiovascular disturbances in the early phases of ciguatera poisoning, and intravenous high electrolytes, calcium gluconate, and vitamin B complex, coupled with a high protein diet and vitamin C. Thisappeared togivethemostsatisfactoryresultsfollowingtheacuteperiodand shortened the duration of illness. For pruritus, analgesic cream was used because steroid creams were of no value. For insomnia, diazepam I O mg was rccommcnded because steroids were ineffective.
B.
Recent Treatment
Tocainide has been used with some success i n three patients with long-term, chronic ci( 173). Amitripguatera, i n which neurological symptoms have persisted over several years tyline was also given to these same patients prior to tocainide with no effect (173). At
Ciguatera Fish Poisoning
243
this tilne, 110 significant large-scale studies on the treatment of chronic ciguatera poisoni% with tocainide or aI11itriptyline have been reported. In part, studies of chrOIlic ciguatera a sufficient patient pOpLllapoisoning are limited i n the United States due to the lack of tion. A more recent preliminary study suggested the useof antioxidant therapy for chronic ciguatera poisoning ( I 74). It was suggested that long-term antioxidant therapy might have 21 role i n the nlanagement of chronic ciguatera poisoning. Vitamin E has been shown to provide protection from various chemicals thought to initiate free radical-induced illjury by blocking the deleterious effect of free radicals i n the cellular membrane (175). The suggested therapy for chronic ciguatera poisoning was vitaminE 1000 IU/day for 1 week. followed by vitamin E 2000 IU/day for 2-8 weeks, vitamin C 500 mg/day, and betacarotene 100 mg/day. Amitriptyline (an antisuppressant drug) givenat a low doseo f 2 5 mg to an individual in improvementandeventualrecoveryafterseveral withciguaterapoisoningresulted weeks (176). However, amitriptyline had no significant effect in studies carried out on the Marshall Islands (177). In the western Pacific, traditional remedies prepared from numerous plants have been used both in oral treatment and topical applications for rashes, pruritus, and other skinailmentsassociatedwithciguaterapoisoning.Extracts of leaves.bark.roots,and fish poisoning. Although some fruits of various plants have been traditionally used for plant extracts alleviate someof the symptoms of ciguatera poisoning, none of the hundreds of plants examined was specific for the treatment of ciguatera poisoning ( 178). The most significant clinical discovery in the treatment of ciguatera poisoning occurred serendipitously, when two coma patients in the Marshall Islands (Jaluit Atoll) were treated with mannitol for edema of the brain. Both patients were suspected of ciguatera poisoning.Afewminutesafterintravenousinfusion of mannitol,bothpatientsawoke from their comas (177). Subsequently, all patients with severe ciguatera poisoning have routinely been treated with intravenous infusion of mannitol at the Majuro Hospital in the Marshall Islands. In thefirst JAMA report (177), 24 patientswithacuteciguaterapoisoningwerc treated with intravenous mannitol and each patient's condition improved dramatically.All patients exhibited marked lessening of the neurologic and muscular dysfunction within n1inutes of the mannitol infusion. Relief from gastrointestinal symptoms took longer. Two patients in coma and one i n shock responded within minutes with full recovery after mannitol infusion. These observations were empirical anduncontrolled.Nevertheless,later studies i n Australia and the United States (179,180) have verified and repeated the studies reported in 1988 ( 177). Mannitol is presently used i n severe ciguatera poisoning in most emergency units whereciguaterapoisoningoccurs(Hawaii,Florida,PuertoRico,Australia).Mannitol treatnlent is most effective when given within the first 24-72 hours after ciguatera poisoning, and after 4 weeks following intoxication, mannitol may be of no help ( 177,179,180). The exact mode of mannitcl action i n acute ciguatera poisoning is not clear, but it may be attributable to its pharmacological actions.In addition to being a potent diuretic, mannitolcan causedecreasingcellularedema,reestablishingionicbalanceanddecreasing plasma pH; mannitol is a scavenger of selected chemical structures including hydroxyl group and free radicals ( I 8 1 ). A major effect maybe the interference or removal of bound ciguatoxin at the receptor sites of sodium channels of nerve and muscle tissues ( 1 82). Thus mannitol acts best during the initial period of acute ciguatoxin poisoning.
Hokama and Yoshikawa-Ebesu
244
C. Summary As seen by recent approaches to treatment, nontoxic drugs which would have a protective effect on the sodium channelof the nerves and muscles would probablybe the choice for alleviation of the clinical symptoms associated with ciguatera poisoning.
REFERENCES I.
2.
3.
4. 5.
6. 7.
8. 9.
10. 11.
12. 13. 14.
15.
16.
17.
18. 19.
Y Shinlim. Shellfish aquaculture and paralytic shellfish poisoning. In: Aquaculture: Public Health Regulatory and Management Aspects. Proceedings of the 6th U.S. Food and Drug Administration Science Symposium in Aquaculture. College Station, TX: Texas A&M Uni1982, p. 38. versity Sea Grant College Program, Y Oshima. LJ Buckely, M Alam, Y Shimizu. Heterogeneity of paralytic shellfish poisons: three new toxins from cultured Gortyortl~r.~ t r t r r r u w t s i s cells, Mytr t r w t n l - i c c and Strxic1orrlrt.s g i g t r r l f c w s . Cotnp Biochctn Physiol 5 7 3 l , 1977. SL Taylor, JY Hui, DE Lyons. Toxicology of scombroid poisoning. In:EP Ragelis. cd. Sea1984, p. 417. food Toxins. ACS Symposium Series 262. WR Lange. Scombroid poisoning. Am Family Pract 37:163, 1988. HS Mosher, FA Fuhrman. Occurrence and origin of tetrodotoxin. In: EP Ragclis, ed. Seafood Toxins. ACS Symposium Series 262, 1984, p. 334. FA Fuhrman. Tetrodotoxin. tarichatoxin, and chiriquitoxin: historical perspectives. AnnN YAcadSci479: 1, 1986. M Yotsu, T Yamazaki, Y Meguro, A Endo, M Murata, H Naoki, T Yasumoto. Production of tetrodotoxin and its derivatives by P.sc,rt~~~r,rorltr.s sp. isolated from the skin of pufferfish. Toxicon 2 5 2 5 5 , 1987. T Yasumoto, H Nagai, D Yasumura, T Michishita, A Endo, M Yotsu, Y Kotaki. Interspecies distribution and possible origin of tetrodotoxin. Ann N Y Acad Sci 479:44, 1986. TM Pcrl, L Bedard, T Kosatsky, JC Hockin, ECD Todd, RS Remis. An outbreak of toxic J Mcd 322: encephalopathy caused by eating mussels contaminated with domoic acid. N Eng 1775.1990. T Yasumoto. Y Oshttna, Y Murakatni, I Nakajitna, R Bagnis, Y Fukuyo. Toxicity of benthic dinotlagellatcs. Bull Jpn Soc Fish 46327, 1980. Y Murakami, Y Oshima, T Yasumoto. Identification of okadaic acid as a toxic conqxment of a marinc dinoflagellate Prorocentrutn lima. Bull Jpn Soc Fish 48:69. 1982. BWHalstead.Poisonousandvetlotnousmarineanilnals of theworld.Washington,D.C.: U.S. GovernmentPrintingOfficc1970, p. 1006. P Hclfrich. Fish poisoning in Hawaii. Hawaii Med J 22361, 1963. J Cook. A voyage towards the South Pole and around the world. 3rd cd. J Cook, T Furneaux, W Hodoes, eds. London: W. Strahatl and T. Cadell, 1977. R Bagnis. Clinical aspects of ciguatcra (fish poisoning) in French Polynesia. Hawaii Med J 22361, 1968. NC Englebcrg,JGMorris,JLewis. JP Mcmillan, RA Pollard, PABlake.Ciguaterafish poisoning: a major common-source outbreak i n the U.S. Virgin Islands. Ann Intern Mcd 98: 336, 1983. K Tachibana. PJ Scheuer, Y Tsukitani, H Kikuchi, D Van Engen, J Clardy, Y Gopichand, F Shimtz. Okadaic acid, an cytotoxic polyether from two marine sponges of the genus Halichondria. J Am Chem Soc 1032469, 1981. T Yasumoto, M Murata. Marine toxins. Chetn Rev 93:1897, 1983. T Yasumoto, U Raj, R Bagnis. Seafood poisoningi n tropical regions. Tohoku, Japan: Laboratory of Food Science Hygicnc, 1984.
Ciguatera Fish Poisoning 20.
21. 22.
23. 24. 25. 26.
27.
28. 29.
30.
31.
32.
33.
34.
35. 36.
37.
38.
39. 40. 41.
42.
245
AHB:unner. The biological origin and transmission of ciguatoxin. In: HJ Hum. CE La11cc, e&. Bioactivc products from the sea. NewYork:Marcel Dckkcr, 1974, p. 15. T Yasun1oto, I Nakajirna, R Bagnis, R Adachi. Finding of a dinoflageihte as a likely culprit of clguatera. Bull Jpn Soc Sei Fish 43: 1021. 1977. T Ynsumoto. A Inoue, R Bagnis. Ecological survey of ;I toxic dinoflagellate associated with ciguatera. In: FJR Taylor. HH Seliger, cds. Toxic dinoflagellate blooms. New York: Elsevier, 1979, p. 221. pJ Scheuer, W Takahashi, J Tsutsunli. T Yoshida. Ciguatoxin: isolation and chemical nature. Scicnce 155: 1267, 1967. M Nukina, IM Koyanagi, PJ Scheuer. Two interchangeable forms of ciguatoxin. Toxicon 22: 169, 1984. K Tachibana. Structural studies i n morine toxins. PhD dissertation, University of Hawaii, Honolulu.HA.1980. Y Hokama, AH Banner, D Boyland. A radioinmunoassay for the detection of ciguatoxin. Toxicon 15317. 1977. YHokama, W Takenaka, KL Nishimura,JSMEbcsu.Asimple nwnbranc inmunobead assay for detecting ciguatoxin and related polyethers from human ciguatera intoxication and natural reef fishes. J AOAC I n t 84727. 1998. AH Banner. PJ Scheuer. S Sasaki. P Helfrich, CB A l d e r . Observations on ciguatern type 1960. toxin in fish. Ann N Y Acad Sci 90:770, JE R;undall. A review o f ciguatera tropical fish poisoning, with a tentative exploration of its cause. Bull Mar Sci Gulf Carib 8:236. 1958. R Adachi. Y Fukuyo. The thecal structure of a nmine loxic dinoflagellate Grrrrrbiediscrrs /o.uicrr.s gen. et sp. nov. collected in a ciguatera-endelnic area. Bull Jpn Soc Sei Fish 4557, 1979. B Campbell, K Nakagawa, MN Kobayashi, Y Hokama. Grtrrrhic.rrli.scrr.s tosicrrs in gut content of the surgeon fish Cterroc./rtretrrs sfrigoms (herbivorc) and its relationship to toxicity. Toxicon 25:1125, 1987. M Satakr, M Fukui. AM Legrand, P Cruchct, T Yasumoto. Isolation and structures of new ciguatoxin analogs. 2.3-dihydroxy CTX3C and 5 I-hydroxy CTX3C. accumulated i n tropical rccf lish. Tetrahedron Lett 39:1197. 1998. AM Legrand, M Fukui, P Cruchet, Y Ishibashi, T Yasumoto. Characterization ofciguatoxins from differcnt fish species and wild Grrrrrhic~~t/isc~~r.s to.uic~r,s.In: TR Toteson ed. Proceedings 25. of the 3rd International Conference on Ciguatera Fish Poisoning, Puerto Rico, 1992, p. M Satakc, M Murata. T Yasumoto. Thc structure of CTX3C, a ciguatoxin cogencr isolated from cultured Gambierdiscus toxicus. Tetrahedron Lett 34: 1975, 1993. T Yasumoto, R Bagnis, JP Vernoux. Toxicity of surgeon fishes. 11-properties of thc principle water-soluble toxin. Bull Jpn Soc Sci Fish 42359. 1976. Gnrrlhierclisc,rr.s YShimizu,HShimizu, PJ Scheuer,YHokama,MOyama,JJMiyahara. /o.uic.~r.s;I ciguatera causing dinoflagcllatc from Hawaii. Bull Jpn Soc Sci Fish 48:Xl I. 1982. T Yasumoto, A Inouc, R Bagnis. Ecological survey of a toxic dinoflagellate associated with ciguatern. In: FJR Taylor, HH Scliger, eds. Toxic dinotlagcllatcblooms. Ncw York: Elsevier, 1979. p. 221. D Ichinotsubo. AY Asahina, E Titus, S Chun, TLWP Hong, JL Shirai, Y Hokama. Survey of ciguatera fish poisoning in west Hawaii. Mem Queens1 Mus 34513, 1994. BH Sakamoto. Studies on Grrtrlhie~~~/i.sc,rrs fosicu,~.PhD dissertation, Univcrsity ofHawaii. Honolulu,HA,1998. T Yasumoto, N Seino, Y Murakami. M Murata. Toxins produced by bcnthic dinollngellatcs. BiolBull172:128.1987. M Usami, M Satake. M Ishida. A h u e , Y Kan. T Yasumoto. Palytoxin analogs from the dinollagcllatc O.strc,o/~.si.ssicrrrrcwsis. J Am Chenl Soc I 17:5389, 1995. M Usumi. M Satakc, T Yasumoto, A h u e . The dinoflagellate Ostrcwpsis .sinrr~err,si,sa s a
246 Yoshikawa-Ebesu
43. 44.
45.
46.
47.
and
Hokama
probable source of palytoxin found i n fish and crabs. In: Y Hokama. PJ Schcuer, Y Yasumoto. eds. Proceedings of the International Symposium on Ciguatera and Marine Natural Products, Honolulu, Hawaii, 1995, p . 285. RE Moore, PJ Scheuer. Palytoxin: a new murine toxin from coelcnteratc. Science 172:49S, 1971. I Brusle. Ciguatera fish poisoning.a review. Sanitary and economic aspects. Paris: INSERM, 1997. Y Hokama, JSM Ehesu, DA Ascunsion. H Nagai. Growth and cyclic studiesof Gorrlhierrlis('11s fo.vic.~t.s i n the natural environment and in culture. In: T Yasumoto. Y Oshima, Y Fukuyo, cds. Harmful and toxic algal blooms. New York: Intergovernmental Oceanographic Colnn1ission of UNESCO, 1996, 313. K Tachibana. M Nukina. YG John. PJ Schcuer. Recent developmentsin the molecular stmclure of ciguatoxin. Biol Bull 172:122, 1987. M Nukina,KTachibana, PJ Scheuer.Recent developlnents in ciguateraresearch.In: JB Harris,ed.Naturaltoxins,animal,plant and microbial.Oxford:Clarcndon Press, 1986.
p. 46. 48. T Yasurnoto, K Fujimoto, Y Oshima, A Inouc, T Ochi, R Adachi, Y Fukuyo. Ecological distributional studies OII a toxic dinoflagellate responsible for ciguatera.Tokyo, Japan: Ministry of Education. 1980, p. I . 49.
50. 51.
52.
53. 54.
55.
YY Lin. M Rish, SM Ray. D Van Engcn, J Clardy. J Golik,JC JU~IKS, K Nakanishi. Isolation and structure o f brevetoxin B from the "red tide" dinoflagellate Ptychorliscus brevis (Gyrrlr l o d i r l i r r r t r Drcvc). J Am Chem Soc 103:6773, 1981. JW Westley. The polyetherantibiotics:monocarboxylicacidionophores. AnnRepMed Chem 10246. 1975. T Yasumoto, M Murata. Marine toxins. Chem Rev 93: 1897. 1993. M Murata. AM Legrand. Y Ishibashi, M Fukui, T Yasumoto. Structures and configurations of ciguatoxin from the moray eel Gyrrrrlofhormj m m i c x s and its likely precursor from the dinollagellatc Gtrrrrbierdiscms to.1-icxs. J Am Chem Soc 1 124380, 1990. o f Carihbeanciguatoxins from the JP Vernoux.RJLewis.Isolationandcharacterization horse-eye jack (Crrrmv Irrtrts). Toxicon 3S:889. 1997. H Nagai, Y Mikami, K Yazawu. T Gonoi, T Yasumoto. Biological activities of novel polyether antifungals, ganlbieric acids A and B from a marine dinoflagellate Grrrrrhic,r-rli.scrrsm v i c w . J Antihiot 46:520. 1993. RJ Lewis. M Sellin, MA Poll, RS Norton,JK Maclcod. MM Shell. Purification and charactcrization of ciguatoxins f r o m moray eel (Lycod~rrtis. / t ~ v r r ~ i c r r . s ,Murtrerlitlrrc,). 29: 1 1 15. 1991.
56. JN Lewis. 0 Caines. CLE Christian, R Schneider. Ciguatera fish poisoning-St. Croix. Virgin Islands of the United States. MMWR 30:138. 198 I . 57. M Sasaki, M h u e . M Murata, K Tachibana. Synthesisof a tetracyclic frag!ncnto f ciguatoxin toward preparation o f an artificial antigen for ciguatoxin immunoassay. In: Y Hokama, PJ Scheuer, T Yasumoto, eds. Proceedings of the International Symposium on Ciguatera and 229. Marine Natural Products. Honolulu, HA: Asian Pacific Research Foundation. 1995, p. 58. KM Li. Ciguatera fish poison: a cholinesterase inhibitor. Science 147:1580. 1965. S9. MD Rayner. Mode of action of ciguatoxin. Fed Proc 31 : I 139. 1972. 60. JN Bidard. HPM Vijverbcrg, C Frenlin, E Chungue. AM Legrand, R Bagnis. M Lazocunski. Ciguatoxin is a novel type of Na' channel toxin. J Biol Chen~259:8353. 1984. 61. A Lambert, J N Bitlard, M Lazdunski. Ciguatoxin and brevetoxins share a comiion rcceptor site on the ncuronal voltage-dependent Na' channcl. FEBS Lett 219355, 1987. of thevoltagesensitive Na+ channel. a receptor for 62. WACatteraIl.Molecularproperties multiple toxins. 4th International Conference on Ciguatera Poisoning. Tahiti. 1992, p. 28. 63. Reference tleleted.
Ciguatera Fish Poisoning 64.
65.
66. 67. 68. 69.
70. 71. 72.
73. 74. 75. 76.
77. 78.
79.
80. 81.
82.
83. 84.
85.
247
y H&al11:1, JT Miyahara. Ciguatera poisoning: clinicaland inmunological aspects. J Toxicol ToxinRev 5:25. 1986. KKCheIIg.KMLi.YHMQuiIlctillis. The mechanismofrespiratoryfailureinciguatera poisoning. J Pathol 97:89, 1969. KM Li. A note o n ciguatera [ish poisoning and action o f its proposed antidotes. Hawaii MCd J 24:358. 1965. AM Legrand, M Galonnicr, RA Bagnis. Studies on the mode of ciguatera toxins. Toxicon 2 0 2 1 l , 1982. JT MiyahWa, MM Oya111a. Y Hokama. The mechanism of cardiotonic action of ciguatoxin. Proceedings of the 5th International Coral Reef Congress, Tahiti. 4:449. 1985. y Ohizumi. S Shiha(;l, K Tachihana. Mode of the excitatory and inhibiting actionso f ciguatoxin in the guinea pig vas deferens. J Pharlnacol Exp Ther 217:474, 1981. RJ Lewis. R Endea11. Mock of action o f ciguatoxin from spanish mackerel. S~~~r,rbcrf~rrroroIrs ~,~)rt/rrfc,r,s~~r/;, J Pharmacol Exp Ther 228:756. DM Millcr. RW Dickey, DR Tindall. Lipid-extracted toxins from a dinoilagelhte. G r d ) i c ~ r t/;,vc,Lt,v r o . ~ ; c , l t . ~ .111: EP Ragelis. cd. Seafood toxins.ACSSymposiumSeries 262, 1984. p. 241. JT Miyahara. J E Kurihara. LK Nakagawa, MN Kobayashi. Y Hokama. The sitnilarity of toxin in C‘terloc,/rtrc,trr.s srrigosus and cultured Gtrrtfbic,~r/isc~/ts to.ric/ts [abstract]. 1st Asia-Pacific Congress on Animal Plant and Microhial Toxins. Singapore, 1987, p. 00x0. J Cameron. AE Flowers, MF Capra. Electrophysiological studies on ciguatera poisoning in tnen ( p a r t 11). Neurol Sci 101:93, 1991. J Cameron. AE Flowers. MF Capra. Effects of ciguatoxin on nerve excitability in rats (part I ) . J NeurolSci101:87, 1991. JA Brock. P Jopling. EM McLachlan, R Lewis. Effects of ciguatoxin-l on electrical activity recorded intracellularly from rat tail artery in vitro. Men1 Queens1 Mus 34:454, 1993. E Benoit. P Juzans. AM Legrand. J Molgo. Nodal swelling produced by ciguatoxin-induced selective activation of sodium channels in myelinated nerve fibers. Neuroscience 7 1: I 12 I. 1996. YOhizumi, Y Ishida, S Shihata. Mode of theciguatoxin-inducedsupersensitivity inthe guinea pig vas deferens. J Pharmacol Exp Thcr 221:745, 1982. J Molgo. T Shimaharo. AM Legrand. Ciguatoxin extracted poisonous morayeel cause sodium-dependent calcium mobilization in NG IOX- 15 t~eurohlastolnaxglioma hybrid cells. NeurosciLett 1%: 117, 1993. J Molgo. YM Gaudry-Talarrllain, AM Legrand, N Mnulian. Ciguatoxin extracted from poitriggers acetylcholine release from Torpedo chosonous Inoray eels C;?.rrrr/otl/ort~.\-;~/l,~/~/ic,/t,s linergic synaptosomes via reversed Na’-Ca” exchanges. Neurosci Lett 160:65, 1993. J Molgo, JX Cotnella. AM Legrand. Ciguatoxin enhances quantal transmitter release from frog nerve tcrminals. Br J Pharlnacol 99595. 1990. H Ohshika. Marine toxins from the Pacific IX. Some effects of ciguatoxin on isolated mum malian atria. Toxicon 9:337, 1971. JT Miyaharu. CK Akau, T Yasumoto. Effects of ciguatoxin and maitotoxin o n the isolated guinea pig atria. Res Cotnmun Chem Pathol Phnrmacol 25:77. 1979. RJ Lewis. AW Wong Hoy. Comparative action of three major ciguatoxins on guinea pig atria and ilea. Toxicon 31:437, 1993. G Rigby, GM Hallcgraeff. G A hallast water controls to tninitnize the translocation-What progress have we made ;und where :re we gomg? In: T Yasurnoto, Y Oshima. and Y Fukuyo cds. HNIII~LI~ toxic d g a l hlootns. New York: lntcrgovernrnental Oceanographic Commission of UNESCO. 1996, p. 201. R A Bagnis.AM Legrand. Clinical features in 12.890 cases of ciguatera fish poisoning in French Polynesia. I n : PGopol:lkrisnakove, CK Tan, etls. Progress in venom and toxin rescnrch. Kent Ridge. Singapore: National University o f Singapore. 1987, p . 372.
Hokama and Yoshikawa-Ebesu
248
South Pacific Commission. Coordination of SPC worki n ciguatcra SPC/WP29. South Pacific Commission,1990. p. 3. 87. MT Danielson, B Danielson. The Mangaravca story: Grcenpcace v. Gmnbicrdiscus. Pacific IslandMonthDec: 29-30, 1985. 88. R Bagnis, J Benett, M Barsinas, M Chehret. G Jacquet. 1 Lechat, Y Mitermite, PH Perolat, S Ro11geras. Epidemiology of ciguatcra in French Polynesia from 1964- 1984. Proceedings 4:475, 1985. of the 5th International Coral Reef Congress, Tahiti, 89. E Dcmotta. G Feliu, A Izquierdo. Identification and epidemiological analysis of ciguatcra cases in Puerto Rico. Mar Fish Rev 48:14, 1996. 90. ND Lewis. Epidemiology and impact of ciguatera i n the Pacific: ;I review. Mar Fish Rev
86.
48:6. 1986.
91. GC Habcnnehl, HC Krebs, P Rasoanaivo. A Ramialihariso. Scvcrc ciguatera poisoning in Madagascar: a case report. Toxicon 32: 1539, 1994. 92. P Boisicr, G Ranaivoson, N Rasolofonirina,BAlldriamahefnzafy. J Roux. S Chantau,M Satake. T Yasumoto.Fatal mass poisoning i n Madagascarfollowingingestion of shark (Qo.c.hN,.hirlrlrs l e u c m ) : clinical and epidemiological aspectsand isolation of toxins. Toxicon 33:1359, 1995. 93. JP Quod. Ciguatera in the Indian Ocean: an overview. Proceedings of the 4th International Conference on Ciguatera Fish Poisoning. Tahiti. 1992, p. 37. 94. P Bourdeau. Epidelniologie de la ciguatcra aux antillcs:plateau de Saint-Barthcmlellly, SaintMartin et Anguilla. Etude in 1985 et 19x6. RapportIFREMER-EVAN" 8413303, 1986. p. 304. 95. JG Morris, P Lewin,NT Hargrctt, CW Smith.PA Blake, R Schneider. Ciguatera fish poisoning: epidemiology of the disease on St. Thomas. U.S. Virgin Islands, Am J Trop Mcd Hyg 3 1 5 7 4 , 1982. 96. JE Randall. A survey of ciguatera at Enewctak and Bikini Marshall Islands, wlth notes on the systemic and food habits of ciguatoxic fishes. Fish Bull US 78:201, 1980. 97. JE Randall. Ciguatera: tropical fish poisoning. Sea Frontiers 7: 130. 1961. 98. JM Dawson. Fish poisoning in American Samoa. Hawaii Med J 36:239. 1977. 99. NC Gillespie, RJ Lewis, JH Pearn, ATC Bourke,MJ Holmes, JB Bourke. WJ Shields. Cigua[era in Australia: occurrence, clinical feature, pathophysiology and managemcnt. Med J Aust 145584. 1986. Men1Queens1Mus 34:631, 100. JP Vernoux. J Lejeune. Ciguatcra in the French West Indies. 1994. in Miami. J 101. DN Lawrence. M Enriqucz, RM Lumish, A Macco. Ciguatera fish poisoning AmMedAssoc 244:254. 1980. 102. RA Bagnis, T Kuberski, S Langier. Clinical observations on 3009 cases of ciguatcra (fish poisoning) in the South Pacific. Am J Trop Med Hyg 28: 1067. 1979. 103. BS Anderson, JK Sims, N Wicbcnga, M Sugi. The epidemiology of ciguatera fish poisoning in Hawaii, 1975-1981. Hawaii Med J 42:326. 1983. 2:44. 1988. I 04. Y Hokama. Ciguatcra fish poisoning. J Clin Lab Anal inmunoassay stick test for the dctcctlon of ciguatoxin 105. Y Hokama. A rapid simplified enzyme and related polyethers from fish tissues. Toxicon 23:939, 1985. of ciguatoxin and related 106. Y Hokama. Simplified solid-phase immunobead assay for detection polyethers. J Clin Lab Anal 4:213, 1990. some biological and chemical aspects. 107. DW Hessel. Marine biotoxins: 1. Ciguatera poison: Ann N Y Acad Sci 90:788, 1960. 108. R Bngnis, S Chanteau, E Chungue, JH Droillct, I Lechat, AM Legr2111d.A Pompon. C Prieur. U Roux, C Tctaria. Conqmrison of the cat bioassay, the lnousc bioassay. and the lnosquito
bioassay t o detect ciguatoxicity in fish. Proceedings of the 5th International Coral Rccf Congress, Tahiti. 1985, 4491.
Ciguatera fish Poisoning
249
109. I IO. 111.
I 12.
113. 114.
1 IS. 116.
117.
118.
119.
120. 121.
122.
123.
124 125.
126.
127.
128. clonal antibody (MAb) in detection of ciguatoxin (CTX) and related polyethers by the stick
Hokama and Yoshikawa-Ebesu
250
enzyme immunoassay (S-EIA) in fish tissues associated with ciguatera poisoning. I n : S Natori,KHashimoto.Y Ueno. eds. Mycotoxinsandphycotoxins. 88. Anlsterdam:Elsevicr Science. 1989, p. 399. 129. Y Hokama, AY Asahina, TWP Hong, ES Shang, JT Miyahara. Evaluation of the stick enzyme inmunoassay in Ctrrtrr~.~ sp. and Serioltr dtrrrerili associated with ciguatera. J Clin Lab Anid 4:363, 1990. 130. YHokama.Simplifiedsolid-phaseimmunoheadassay for thedetection of ciguatoxinand related polycthcrs. J Clin Lab Anal 4213, 1990. 13 1 . CA Canal, AY Asahina, Y Hokama, JT Miyahara. Characterizationof toxin(s) in Myrjpri,sri.s sp. by immunological. mouse toxicity and guinea pig assays. J Clin Lah Anal 7:41. 1993. 137. YHokama.AYAsahina, ES Shong, TWP Hong,JLRShirai.Evaluation of theHawaiian reef fishes with the solid-phase imnunobead assay. J Clin Lab Anal 7:26, 1993. 133. Y Hokama. l~nn~unological approaches to understanding marine toxins. In: CE Kimhle, ed. Aquaculture, 2nd ed. Silver Springs, MD: Zarcon Press, 1985. p. 80. 134. JF Kcarney, A Radbrush, B Liesegang, K Rajewsky. A new marine myeloma cell line that has lost inmunoglobulin expression. but permits the construction o l antibody-secreting hyhrid cell lines. J Imnunol 123:1.548, 1978. 135. RH Kcnnet. TJ Mckearn, KB Bechtol, eds. Monoclonal antibodies. New York: Plenum Press. 1980.
136. G Kohler, C Milstein.Continuouscultures
of fused cells secreting antihody of predefined specilicity.Nature256:494,1975. 137. M Schreier. G Kohler. H Heingartner, C Berek, M Trucco, L Forni. Hybridoma techniques. Cold Spring Harhor. NY: Cold Spring Harbor Laboratory Press, 1980. 138. RF Morgan,JRHebel.RJMcCarter.Astudyguide to epidemiologyandhiostatistic. 3rd ed. Gaithersherg. MD: Aspen Publications. 1990. 139. MJ Khosravi. Shifting the “hook effect” i n one-step immunometric assays. Clin Chem 36: 169, 1990. 140. Y Hokama, TWP Hong, M Isobe, Y Ichikawa. T Yasumoto. Cross rcactivity of highly purified okadaic acid (OA) synthetic spirochetal east-sphere of OA and ciguatoxin. J Clin Lab Anal 654, 1992. 141, Y Hokama, JSM Ehesu, K Nishimula. S Oishi, B Mizuo, M Stiles. B Sakamoto, W Takcnaka, H Nagai. Human intoxication from Hawaiian reef fishes associated with diverse marine toxins. J Nat Toxins 5:235, 1996. 142. Y Hokama, KL Nishimura. W Takcnaka, JSM Ebesu. Simplified solid-phase membrane immunohead assay (MIA) with monoclonal anti-ciguatoxin antibody (MAhCTX) for detection of ciguatoxin. J Nat Toxins 7.1, 1998. 143. Y Hokama, LH Kimura. MA Abad, L Yokochi, PJ Scheuer, MNukima. T Yasu111oto,DG Baden, Y Shimizu. An enzyme imnunoassay for the detection of ciguatoxin and coqctitive inhibition by related natural polyether toxins. I n : EP Ragelis. ed. Seafood toxins. ACS Symposium Series 262. Washington, D.C.: American Chemical Society, 1984, p. 307. 144. K L:ln&teiner, ed. The specificity of serological reactions. Cambridge, MA: Harvartl UnivcrsityPress. 194.5. 145. RWDickey. SC Bohzin, DJ F a u l k ~ ~ c FA r , Bencsnth,DAndrzejewski.Idcntilicationof okadaicacid from CaribbeandinoflagellateProrocentrumconcaviunl.Toxicon28:37 I, 1990. 146. RJ Lewis. 11n111unological,biochemical, and chemical features of ciguatoxin-imp~icatiollsfor detection of ciguateric fish. Mem Quccnsl Mus 34:.541, 1994. 147. FE Russell.Ciguaterapoisoning: a report of 3.5 cases. Toxicon 13:383-385, 1975. 148. YGJoh.PJ Scheuer. The chemical nature of scaritoxin. Mar Fish Rcv 48:19, 1986. 149. E Chu11gLIe.R Bagnis, NFusetani.YHashimoto.Isolation of twotoxins from parrotfish Scarus gibhus.Toxicon15:89,1977. 150. T Noguchi. DF Hwang, 0 Arakawa, K Daigo. S Sato. H Ozaki. N Kawai. M Ito, K Hashi-
Ciguatera Fish Poisoning
151.
152.
153.
154. 155.
156.
157. 158.
159. 160.
161.
162. 163.
164.
165.
166.
167.
168.
169.
251
lnoto. Ellytoxin 21s the causitivc agent i n the parrotfish poisoning. In: P Gopalah.tshnakotic. CK Tan, e&. Progress i n venotn and toxin research. Kent Ridge, Singapore. National Univcrsity of Singapore, 1987, p. 325. T Y:lsul11oto, D Yatnamura, Y Ohizutni, M Takahashi. AC Alcala, LC Alcala. Palytoxitl in two species of Philippine crabs. In: T Yasumoto. ed. Studies on tropical fish and shellfish infected by dinoflagellates. Tohoku University, Tohoku. Japan, 1983, p. 45. A KodaI11a, Y Hokama, T Yasumoto. M Fukui,SJ Manca, N Sutherland. Clinicaland Iaborato17 findings ilnplicating palytoxin as a cause of ciguatera poisoning due t o Dcwrpferlrs r r 1 w cro.somo (mackerel). Toxicon 27: 105I , I989. WR Langc. KM Lipkin. GC Yang. Can ciguatera bea sexually transmitted disease’?J Toxicol Clin Toxicol 27:193. 1989. R Endean, JK Griffith, Robins, SA Monks.Multiplctoxins in a specimen o f thenarrowbarred Spanish mackerel S~,orrrhc,rorrlorIrs mrnrllc’rsm. Toxicon 3 1: 195, 1993. Y Hokama, JSM Ebesu, W Takenaka, K Nishimura. R Burke, PF Sullivan. A simple mcmbrnne it~~tnunobead assay (MIA) for detection of ciguatoxin and related polyethers from ciguatera intoxication and natural reed fish. J AOAC Int 81:727, 1998. H Labrousse. S Pauilloc, E Jehl-Martinez. AM L e g t ~ t ~S~ Avrameas. d. Techniques de detection de l a ciguatoxine irl I i , - o el irl vim. Oceanis 18: 189, 1992. JL Allsop. L Martini, H Lebrus,J Pollard. J Walsh, SMS Hodgkin. Les manifestationsneurologiqucs de la ciguatera. RevNcurol 142590, 1986. R Bagnis. M Rouanet. A propos d’un cas tnortuldc ‘ciguatera’ d a m les ides Tuamo Fr. Rev McdVet 121:459, 1970. R Bagnis. Concerninga fatal case of ciguatera poisoning in the Tuarnotu Islands. Clin Toxicol 3579. 1970. K Tcrao, E Ito, T Yasumoto. Light and electron microscopic studies o f themurineheart after repeated administrations of ciguatoxin or ciguatoxin-4C. Nat Toxins 1: 19, 1992. K Terao. E Ito, M Ohkusu, T Yasutnoto. Pathonmrphological aspects of poisonings induced by marine toxins acting o n sodium channel i n mice. In: Y Hokama. PJ Scheuer. T Yasumoto. eds. Proceedings of the International Symposium o n Ciguatera and Marine Natural Products. Honolulu, HI: Asian Pacilic Research Foundation, 1995, p. 263. K Terao, E Ito, M Ohkusu. T Yasumoto. Pathological changes in murine hearts induced by intermittent administration o f ciguatoxin. Men1 Queens1 Mus 34:62 l , 1994. IF Coombc, MF Capra, AE Flowers. J Cameron. Pathologicalchanges t o mammalian gut following administration of ciguatoxin. In: P Gopalakrishnakone and CK T a n , eds. Progress i n venotn and toxin research. National Universityof Singapore, Kent Ridge. Singapore. 1987.
p. 405. E Ito. TYasumoto. K Terao.Morphologicalobservationsofdiarrhea 111 micecaused by experimental ciguatoxicosis. Toxicon 34: 1 1 I , 1996. K Tcrao, E Ito. M Oarada, Y Ishihashi,AM Legrand, T Yasumoto. Light and electronmicroscopic studies of pnthological changes induced in miceby ciguatoxin poisoning. Toxicon 29:633, 1991. K Tcrao. E I t o , T Yanagi. T Yasutnoto. Histopathological studies on experimental marine toxin poisoning. I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pcctenotoxin-1. Toxicon 24: 1 l4 I , 1986. K Tcrao, E Ito, Y Sakatnnki. K Igayashi, A Yokoyama, TYasumoto. Histopathological studies o f expcritnental marine toxin poisoning. 11. The acute effects of maitotoxin on the stomach. heart, and lymphoid tissues i n mice and rats. Toxicon 26395. 19XX. K Terao.E Ito, Y Kakinama. K Igarashi. M Kohayashi. Y Ohizutni, T Yasumoto. Histopathological studies on experimental marine toxin poisoning. IV. Pathogenesis of cxperilnental
maitotoxin poisoning. Toxicon 27979. 1989. K Terao. E Ito. T Yasumoto. Light ancl electron microscopic observations of experilncntal 1992. palytoxin poisoning i n mice. Bull Soc Pathol Exot 55:494,
252
Hokama and Yoshikawa-Ebesu
170. E Ito, M Ohkusu, T Yasumoto. Intestinal injuries caused by experimental palytoxicosis in mice.Toxicon 34343. 1996. 171. B Chanfour. L’intoxication ciguateriquc en Polynesie Francaise. Universitc Louis Pasteur. Doctorat en Medicine Diplome D’ETAT, 198X. 172. MM Okihiro, JP Kecnan,ACIvy. Jr. Ciguaterapoisoningwithcholinesteraseinhibition. HawaiiMed J 24:354, 1965. 173. WR Lange, SD Kreider, MMD Hattwick, J Hobbs. Potential benefitof tocainide in the treatment of ciguatera. Report in three cases. Am J Med 84: 1087, 1988. 174. WR Lange, CS Contorcggi, RI Herring,JL Cardent. Proceedings of International Symposium on Ciguatera and MarineNatural Products. Honolulu, HI: Asian Pacific Research Foundation. 1994, p. 273. 175. JG Bieri, L Corash, US Hubbard. Medical use of vitamin E. N Engl J Mcd 308: 1063, 1993. 176. PB Bowman. Amitriptyline and ciguatcra. Med J Aust 40:802. 1984. 177. N Palafox, L Jain, A Pinano, T Gulick. R Williams, 1 Schatz. Succcssful treatment of ciguatera fish poisoning with intravenous mannitol. JAMA 250:2740, 1988. in western Pacific 178. G Bourdy, P Cabalion, P Amade, D Laurent. Traditional remedies used for the trcatn1ent of ciguatera poisoning. J Ethnopharlnacol 36: 163, 1992. 179. J Pearn, R Lewis, T Ruff, M Tail, J Quinn, N Gillespie. Ciguatcra and mannitol: experience with a new treatment regimen. Med J Aust 151:77, 1989. 180. DG Blythe, DP DeSylva,LE Fleming, RA Ayyar. DG Baden, K Shrank. Clinical experience with IV mannitol in the treatment of ciguatoxin. Bull Soc Pathol Exot 85:425. 1992. 1x1. TF Huang,LLHuang. Effect of mannitol,N-2-mercaptopropio1lylglycerineandsodium nitropriussideinEEGrecoveryfollowingcerebralischemiaandreperfusionin rat. Clin J Physiol 33: 121. 1x2. Y Hokama, JT Miyahara. In vitro effect of 6-carbon monosaccharides on the sodium channel response to crude fish extracts containing ciguatoxin. J Nat Toxins 3:35. 1994.
Tetrodotoxin
I. 11.
Introduction 254 SourceofToxin 255 A. Distribution 255 B. Tetrodotoxin-bearing organisms 255 C. Transmission o f tetrodotoxinthrough the foodweb D. Bacterial origin tetrodotoxin of 257 E. Major sources of human intoxication 260
257
111. Detection Methods 263 A. Mouse bioassay 263 B. High-pcrformnnce liquid chromatography 263 C. Mass spectrometry 263 spectrometry 264 D. Gas chrornatogmphy-mass E. Thin-layer mass spectrometry (TLC-MS) andliquid 264 chromntography-Inass spectrometry F. Thin-layer chromalography 265 G. Capillary isotachophoresis 266 H. IR spectrolnetry 266 I. ' H-Nuclearmagneticresonance (NMR) spectrometry268 J. Electrophoresis 269 K. Cytotoxicity assays 269 L. Immunoassays 269 IV. Clinical 269 A. Symptoms 269 B. Trentlnent 270 C. Incidences 271 V. Toxicology 276 VI. Pharn1acological Actions 276
253
Yoshikawa-Ebesu et al.
254
Use of Tetrodotoxin as a PharmacologicalTool277 A. Organismal level 277 B. Molecularlevel 277 VIlI. Summary 281 References 282 VII.
1.
INTRODUCTION
Tetrodotoxin (TTX) is one of the most potent nonproteinaceous toxins known, responsible for numerous fish poisonings. This toxin is one of' the oldest known toxins, recorded as early as 2700 B.C. in Chinese literature describing the toxicity of puffers and around 2500 B.C. in Egyptian history in hieroglyphs depicting the fatal poisoning of a pharaoh who had eaten one of these fishes (1,2). In the late 188Os, Yoshizumi Tahara named this toxin after the order Tetraodontiformes,sincemany of its memberscarrythetoxin. In additiontotheTetraodontidae (puffer, balloon fish, globe fish, swell fish, fugu, toad fish, and blowfish), two other families, the Canthigasteridae (sharpback puffers) and Diodontidae (spiny puffers or porcupinefish) also include members associated with tetrodotoxin. Tetrodotoxin poisoning cases due to ingestion of puffer have frequently occurred in the Orient, especially in Japan where these fish are a traditional food. The preferred forms of this delicacy are slices of raw flesh (sashimi) and slightly cooked liver (kimo). Japanese people are aware of the puffers' toxicity and have devised ways to reduce tetrodotoxin in the liver. Judging from the statistics in Japan, the number of deaths due to puffer poisoning has steadily declined, from morethan 100 cases (with morethan 50% mortality) every year before 1959, to between 10 and 100 cases (with more than 25% mortality)
Tetrodotoxin ('IXX)
acidTetrodonic
255
every year between 1960 and 1981,to between 1 and I O cases (with morethan 6% mortality)everyyearsince1982 ( 3 ) . Thisdecline in incidence is probablydue toincreased government regulations. Tetrodotoxin is a water-soluble heterocyclic guanidine whose chemical structure has been characterized (Fig. l ) . Tetrodotoxin only has one guanidium moiety. As can be seen in Fig. 1, many derivatives of tetrodotoxin have been found, although their toxicitiesvary widely. First isolated in 1964 from puffer of the family Tetraodontidae, tetrodotoxin has since been found in diverse organisms, including marine fish and an octopus as well as some terrestrial amphibians such as newts and frogs (see Ref. 4). Tetrodotoxin has been found in several bacteria species, including Shewcmella crlgu and Vibrio spp., and is now believed to be of bacterial origin (5-8).
II. SOURCE OFTOXIN
A.
Distribution
Various toxic tetraodontid species are globally distributed (9,lO). However, thesefish are consumed only in certain parts of the world. Areas that have reported tetrodotoxin poisoning cases due to consumption of toxicpufferfishinclude Japan, Taiwan, China, Hong Kong, Thailand, Korea, Singapore, Malaysia, Australia, the United States, Kiribati, Papua New Guinea, and Fiji. Other organisms found to possess tetrodotoxin are listed in Table 1. Like puffers, these animals are found worldwide: Japan (goby, blue-ringed octopus, various gastropods, starfish,xanthidcrabs),Taiwan(goby,variousgastropods,xanthidcrabs),Philippines (goby), Costa Rica and Panama (Atelopus frogs),Australia(blue-ringedoctopus),the United States (newts), and Thailand (horseshoe crab).
B. Tetrodotoxin-Bearing Organisms Puffers were long believed to be the exclusive source of tetrodotoxin (Fig. 1) (9,lO). The distribution of tetrodotoxin in these fish is mainly in the ovaries (eggs), liver, and skin, though the pattern varies to some degree among species. The origin of tetrodotoxin was often debated to be either endogenous or exogenous. In 1964, however, this toxin was unexpectedly detected in the California newt (Tnrichn torosa) (see Ref. 1 l), thus settling the controversy. In this organism, tetrodotoxin was concentrated in the skin, ovary, muscle, and blood. Since then, tetrodotoxin has been found in a variety of animals (Table l). Other newt species including T. rivularis, T. granulosa, T. vulgcrris, T. cristcrtus, T. nlpestris, T. mmoratus, Notophthalmus viridescens, Cyopsis pyrrhogrrster, and C. erlsicaudcr were also discovered to possess tetrodotoxin in almost the same anatomical distribution as T. torosa. In 197 I , tetrodotoxin was foundin the skin, viscera,and muscle of a goby (Yongeicht l y s crirliger) inhabiting the Amami and Ryukyu Islands, Japan. This goby is also distributed in the Philippines and Taiwan, whereit has been implicated in several human poisoning cases. In addition, tetrodotoxin was detected in the skin of Atelopus frogs inhabiting Costa Rica and Panama. In 1978, tetrodotoxin was isolated from the postsalivary gland of the blue-ringed octopus (Hnl~7aloc~hlerenn mcrcrdoscr) which mainly inhabits northern Australia. Human tetrodotoxin cases are occasionally reported in this area due to envenomation by H. nlucu-
256
Yoshikawa-Ebesuet al.
Table 1 Distribution of Tetrodotoxin inAnimals (9)
Animals
Parts
1. Platyhelminthes:
Turbellaria:
Whole body
2. Nemertinea:
3. Mollusca:
Gastropoda:
Cephalopoda:
Whole body Whole body Whole body Digestive gland Digestive gland Digestive gland Digcslive g h d Digestive gland Whole body Digestive gland Digestive gland Postsalivary g h ~ d
4. Annelida:
Polychaeta: 5 . Arthropoda:
Whole body Whole body Whole body EEE
6. Chactognatha:
Head Head 7. Echinodermata:
X. Vertebrata;
Pisces: Amphibia:
Whole body Whole body Whole body Skin, liver, ovary Skin, viscera, gonad Skin, egg, ovary. muscle, blood Skin, egg, ovary Skin, egg, ovary, muscle, blood Skin, egg, ovary. muscle. blocd Skin Skin
lostr. This octopusalso occasionally appears in middle to south Japan. The octopus secretes tetrodotoxin from the posterior salivary gland to paralyze its prey. In 1979, a serious poisoning incident dueto ingestion of the trumpet shell (Chtrmtria .scmliae),whose muscle is widely consumed, occurred in Shimizu, Shizuoka, Japan. The causative agent was isolated from the digestive gland and identified as tetrodotoxin ( 12). Two similar food poisoning incidents followed in Wakayama and in Miyazaki, Japan, in 1982 and 1987, respectively. This toxic carnivorous trumpet shell is widely distributed in middle to south Japan. Other carnivorous gastropods suchas the frog shell or ootrtrr’utoDor’rr (Tutujcr lissostorncl), hatmnushiro,yc~i(Zeuxis suquijorewsis), and trrcrrepi (Niotha clnthmtcr) in Japan and Niotlrtl lineclttr (13) in Taiwan also possess tetrodotoxin i n their
Tetrodotoxin
257
digestive glands. In addition, in 19S0, tetrodotoxin was detected in the ivory shell(Berbylorritr , j c r p o r ~ i c c z ) collected , i n Fukui, Japan. Mass food poisonings due to ingestion of the eggs of the horseshoe crab sporadically occur i n Thailand, where during early 1996 they caused about 20 deaths. Tetrodotoxin in the eggs was later determined to be the cause of death. Xanthid crabs (Ater;~crfi.s,pnr.inlr.s, Zo.sitr~rr.scrcr~tw),flatworms (Plcrrrot~)nrspp.), and ribbon worms (Nemertinea) have also been shown to possess tetrodotoxin. Presently, distribution of tetrodotoxin is known in only a limited number of organisms (Table l). In many of these animals, including puffers, newts, flatworms, and horseshoe crabs, tetrodotoxin accumulatesi n the ovaries and skin, where it may play a defensive role against predators. In other animals, however, tetrodotoxin maybe used as an offensive substance to immobilize and capture prey by secretion or envenomation of the toxin from the proboscis in ribbon worms or salivary glands in the blue-ringed octopus.
C.
Transmission of Tetrodotoxin Through the Food Web
Tetrodotoxin in puffers seems to come directly from their food, such as toxic gastropods ( / ? c t r ? ~ r r r l r r . s / ? j ~ o ~ ~flatworms, ~ri), and starfish (9,lO). Tetrodotoxin commonly accumulates in the eggs of pufiers, presumably transmitted from the female, since thetoxingenerally disappears from the larvae about 1 week after fertilization. In the wild, the toxin usually reappears in the larvae i n about 2 weeks, probably through ingestion of toxic organisms. Indeed, when larval puffers initially possessing tetrodotoxin during the first week after fertilization are cultured and fed a tetrodotoxin-free diet, they do not become toxic (14). Thus the tetrodotoxin toxification mechanism in puffers is likely to occur through the food web, though intestinal bacteriain some species of these fish reportedly produce negligible amounts of the toxin. Puffers, however, seem inclined to seek tetrodotoxin in their diets. Ingested toxin is initially accumulated in the liver, followed by the skin and other tissues. Fish, except those possessing tetrodotoxin, such as puffers and the tropical goby Y0rrgcit.hrhys t.r.inigc~r.,do not accumulate tetrodotoxin even when toxin-containing diets are fed to them at sublethal doses. Other tetrodotoxin-bearing animals such as gastropods and starfish probably BCCLImulate their toxins through the food web as well. In the trumpet shell (Chttmrricrsaulirrc), tetrodotoxin is ingested through its prey. During elucidation of the tetrodotoxin transmission mechanism for this animal, starfish such as Astwpectetr polyacarrfhus.A. ltrtc'S I ~ / I I O . S t I . S , and A. .sco~~trr.i~r.s on which the gastropod feeds were determined to be links in the food web leading to tetrodotoxin toxitication (IS). These toxic starfish are widely distributed in Japan. Tetrodotoxin-containing starfish probably derive the toxin through their diets. The ivory shell (B~l~ylorricr j ~ r p o r ~ ilikely c ~ ) becomes toxified by ingesting toxic puffers that fishermen discard in the ocean. Tetrodotoxin toxification mechanisms through food web links have already been established by model experiments (15). Fromthesestudies,thetoxificationmethodis hypothesizedasillustrated in Fig. 2. Theinitialsource of tetrodotoxinfound in most animals, however. is belicved to be marine bacteria. '
D. Bacterial Origin of Tetrodotoxin Since tetrodotoxin is distributed i n a variety of invertebrates and vertebrates and there is a wide individual variation in toxin content, even among members of the same species,
258
Yoshikawa-Ebesu et al.
bacter'a 1
Pseudomonas SD., etc. I
-
\
U
absorbed on and precipitated Wlth v
planktonic carcass, etc. Tropical
Ribbonworm Arrowworm Xanthid crab
sediment
1
I Puffetfish
l
Large gastropods
Small gastropods
Fig. 2 Assumedmechanism of toxification of tetrodotoxin-bearing animals (9)
the origin of tetrodotoxin was deduced to be a universal organism such as a microbe. Using instrumental high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) analyses for tetrodotoxin, an intestinal bacteria isolated from a toxic xanthid crab (Atergatis jl0ridu.s) collected from Shimoda, Shizuoka, Japan, was discoveredto produce tetrodotoxin (5). This bacterium was alsonoted as analogous to Vibrio.fischeri. Soon after, V. aigit~oiyticusand several other Vibrio spp. isolated from the intestines of the puffer Tcrkififirgusrlyrleri, the starfish Astropcterr po1~trc~rrrrtilu.s. the blue-ringed octopus H(rpcr1ochl~errrrt ~ r r r ~ ~ d oand s ~ rthe , horseshoe crab Ccrrc~irloscorp i u s rnturrdicaudrr were also demonstrated to produce tetrodotoxins using similar analytical techniques. Simidu etal. (7) showedthat 12 of 24 type culture strainsof marine bacteria tested clearly produced tetrodotoxin or related substances (Table 2). Yasumoto et al. (6) and Matsui et al. (8) ascertained that A1teronroncr.s tetmorlotris isolated from a calcareous alga (Jcmia sp.) and She~vcrwellcrputrefaciem isolated from a puffer (T. rliphokles) produced tetrodotoxin and anhydrotetrodotoxin, respectively. Typical tetrodotoxin production by Vibrio group VI11 ( 16) isolated from the intestines of Atergatisjoridus was demonstrated through instrumental analyses for tetrodotoxin as well as cell toxicity assays. An appreciable amount of tetrodotoxin (30 MU/flask) was produced by Vibrio group VI11 strain (Table 3). The tetrodotoxin fraction showed two peaks (tetrodotoxin and anhydrotetrodotoxin) in HPLC analysis, and in GC-MS analysis for the toxin ( C , base) exhibited Inass fragment ions at t r d : 407 (molecular peak), m/: 392 (base peak),and m / z 376, all of which are specificto trimethylsilylated C, base derived from authentic tetrodotoxin (Figs. 3 and 4). It is recognized fromthe above evidencethat many bacteria can produce tetrodotoxin and/or its derivatives. Tetrodotoxin productivity in vitro, however, seems largely dependent upon culture conditions. The optilnal culture conditions for maximumtoxin yield by tetrodotoxin-producingbacteriaremainunknown.Suchbacteriamayproduceminimal
Table 2
Analysis of Tetrodotoxin in BacterialCells (7) HPLC AnhydroTetrodotoxin tetrodotoxin GC-MS
Bacterial strains used
+
+ + + + t
t
+
t
+
+. clearly detected; =
cultivated
111
2 . dil‘licult t o detect; n fl.eshwatcr medium.
-. not detected
Table 3
Analyses for TetrodotoxinandRelatedSubstancesinthe Extracts of Sevcral Vilwio Groups of Bacteria Isolatcd from Intestines of A. jloridus Specimens Collected from Kojitna in Ishigaki Island, Okinawa (16)
Anhydro-Vihrio group
Lethal potency (MU)
HPLC Tetrodotoxin tetrodotoxin GC-MS
+
260
I
0
Yoshikawa-Ebesu et al.
.
.
-
.
I
.
10
I
20
Retention time (min) Fig. 3 HPLC of tetrodotoxinfractionfrom Vihrio group VI11 isolated from a Kojimaspecimen (upper) and o f authentic tetrodotoxin (lower) (16).
amounts of tetrodotoxin in the intestines of fish and invertebrates. In addition, based on the observationsthat trumpet shells can become highlytoxified by ingesting toxic starfish, and that the debris of small gastropods are often found in the digestive ducts of toxic puffers, and that cultured puffers (nontoxic) can become toxified by feeding on the liver of toxic puffers, it is presumed that tetrodotoxins in carnivorous organisms able to accumulate the toxin could originate from tetrodotoxin-producing bacteria.
E. MajorSources of Human Intoxication 1. Puffers Toxic puffers are limited to the family of Tetraodontidae ( 1 7 ) . The toxicity of Japanese puffer species was extensively surveyedby Tani ( 1 7 ) (Table 4). In this study, wide individual, regional, and seasonal variationsof toxicity were recognized. Especially large regional variations in toxicity were reported in the muscle of hignt~jiig~r (Trrk
26 l
I
1
9
l1
13
Retention time (min)
Fig. 4
15
250 350
400
300 Mass (m/z)
450
100
200 400 300 Mass (mlz)
GC-MS of tetrodotoxin fraction of Vibrio group VI11 from a Kojima specimen
(16). GC,
left; MS, right.
f l ~ r v i c f u , ~mrrshifirgu ), (T. e.\-crcurws),tak$~rgrr (T. ohlongus),firtcrtsuhoshi~~gu (Ltrgocephn/us lrrnuris), serlrlirfugu (L. sceler~tus), rnoyof1grr (Arothrotz stellatus), strzcrr~rrrr~itirg~r (A. hispitlus), and okirltrwtfl&u (Chelorldorr ptrtocLr)were later added to the list of toxic species (3,9). For a more complete list of toxic tetraodontid species, refer to Ref. 18. Tetrodotoxication most commonly occurs in Japan, where puffer or fugu is considered a delicacy. Eating fugu either raw or cooked can be hazardous since tetrodotoxin is heat stable. One of the main reasons connoisseurs eat fugu is to experience the slight oral tingling sensation and even feelings of euphoria that occur with consumption due to trace amounts of tetrodotoxin. In Japan, chefs must be specially trained and licensed in order to prepare and serve fugu. Sake or rice wine infused with fugu testes is also believed to be an aphrodisiac ( 1 1). The toxicity of puffer is related to their spawning season, with the highest toxicity levels occurring between March and June for Japanese puffers. Consequently, i n Japan, fuguiseatenprimarily between October and March. The toxinis concentrated in the ovaries, liver, and often, the skin, making the female puffer more toxic than the males, especially during spawning season. The parts of the puffer considered less toxic are the musculature, fins, andtestes.Althoughthemuscletissuemaybetoxic inat least two puffer species, Fugrr p ~ r r d d i sand L . ~ I ~ O C ~ ~ ~ lunnris ~ I I M S (see Ref. 19), no fatal cases of tetrodotoxication have yet been reported due to eating the meat of the puffer as sashimi, or sliced rawfish, i n Japan (18). Onthe other hand, the dishes known as chiri, fillets partially cooked in a stew of skins, livers, intestines, or testes, and kirno. partially cooked (9,10,18). livers. have been associated with many fatal intoxications
2. Goby Although tetrodotoxin-bearing fish were originally thought to be limited to puffers, tetrodotoxin has also been found i n two gobiid species, the Indo-Pacific goby(Gohius criniger)
Yoshikawa-Ebesu et al.
262
Table 4 ToxicityofJapanesePuffers,Surveyed
Family
Species
Ovary
by T a n i (17)
Testes Blood Intestine Muscle Skin Liver
0 0 0 0 0 0
o e o o o o 0 . o X
0 0 0
0 0 0 0
X
0
8
X
o
x
X
o 0
x 0
X
X
o
x
X
X
X
X
X
X
X
X
x
x
X
X
-
o
o
0
X
-
X
X
X
X
-
X
-
x
x
X
X
-
X
X
-
X
X
-
X
X
x x x
X
X
x x x
X
X
X
X
-
X
X
X
0 0 0
0
0 0 0 8 0 0 0 0 X
0 Strongly toxic, lethal at less than IO g. 0 Moderately toxic, not lethal at less than I O g. 0 Weakly toxic. no^ lethal at less than I O 0 g. X Negative. not lethal at less than 1000 g. - N o data availahle.
( 1 8-20) and a subtropical goby (Yongeichthys cr-itliger.) inhabiting the Amami Islands, theRyukyuIslands, thePhilippines,andTaiwan (21). In thelattergoby,the toxin is distributed in the skin, viscera, and muscle (21). Although the origin of tetrodotoxin i n Y. cr-itfigerremains unknown, these fish are scavengers and are probablytoxified through the food web by feeding on the tissues of dead toxic puffers (Chelonodonpcltoccr), whose habitat is similar to that of the goby.
3. Other Sources of Tetrodotoxin Through human toxication cases, tetrodotoxin has been discovered in several species of gastropodmollusks,including N~rsscrriusccrstus (Gould) and N . cor1oickrli.s (Deshayes) ( 19,22), N . linecrtrr, N. clcrthr-crta,TutufLI lissostonrtr, and Zerr.ris suquijor-er1si.s ( I 3 ) , and Cl~ar-oniasaulicre ( 1 2). Another type of tetrodotoxication can occur through envenomation by the blueringedoctopus (Hcrplochlcrenn mcrculosa) (23). Excessivehandling of the animalhas been the cause behind most of these cases.
Tetrodotoxin
111.
263
DETECTION METHODS
The official method for tetrodotoxin determination has been the mouse assay. With the development of more advanced technology, however, additional methods are being devised that are more rapid, sensitive and specific than this assay (24-27).
A.
Mouse bioassay
Twenty-gram mice are injected intraperitoneally with toxin or unknown samples. Toxicity is expressed i n mouse units (MU), where I MU is the amount of tetrodotoxin that can kill a 20 g male mouse of ddY strain in 30 minutes (28,29). One MU is equivalent to 220 ng of tetrodotoxin (28). Although this bioassay is easy, inexpensive, and relatively quick, its sensitivity and specificity are limited.
B.
High-Performance Liquid Chromatography
This technique will probably soon replace the mouse assay for experimental tetrodotoxin analysis to obtain more consistent toxicity data. Several attempts (24,251 have been made to determine tetrodotoxin type using HPLC. An example of one of these methods follows: Analysis for tetrodotoxin is perfomled using reverse-phase HPLC on a YMC-Pack AM314 column (6 mm inside diameter X 300 mm), two constant flow pumps for eluent and reagent, and a reaction coil (Teflon tube, 3 mm inside diameter X 10 m). The column is developed with a mixture of 2 mM sodium I-heptanesulfonic acid and 1% methanol in 0.05 M potassiunl phosphate buffer (pH 7.0) at a flow rate of 1 ml/min (24). The eluate is mixed with an equal volume of 3 N NaOH and heated in a reaction coil at IOO'C. The intensity of fluorcscence is measured at SOS nm with 380 nm excitation. Retention time o f tetrodotoxin is determined using authentic tetrodotoxin solution in diluted acetic acid (IS pg of tetrodotoxin/ml). A calibration curve of thetoxinis derived fromthe dose relationship between tetrodotoxin at concentrations of 0.1 -1 pg/IO p1 and its resulting fluorescent intensity (peak area). From this curve, tetrodotoxin in unknown samples can be determined. An example of such a curve is illustrated in Fig. S. In this case, more than 0.03 pg of tetrodotoxinll0 p1 can be detected.
C.
Mass Spectrometry
The nlass spectrum of tetrodotoxin can be directly measwed by fast atom bombardment 0. I mg of tetrodosecondary ion mass spectrometry (FAB-SIM). For this technique, about toxin and glycerol as matrix are placed on the sample stage of a mass spectrometer, mixed well, and introduced into the ion chamber of the spectrometer (FAB). Both positive and negative mass spectra of tetrodotoxin are then measured (26,30,31). As shown in Fig.6,tetrodotoxinexhibits (M + H ) ' and(M + H - H,O)' ion peaks at t t d : 320 and tn/: 302, respectively, in the positive mass spectrum, and an (M H ) - peak at W / : 318 in the negative. This technique may beused to identify pure samples of tetrodotoxin from mass number 319.
264
Yoshikawa-Ebesu et al.
-
0
15
30
Retention time (min)
Fig. 5 HPLC chron~atogra~n of authentic tetrodotoxins: ( a ) tetrodotoxin;(h) 4-cpitetrodotoxin:(C) anhydrotetlodotoxin (16).
D. Gas Chromatography-Mass Spectrometry Gas chromatography-mass spectrometry ( 12) is recommended to detect tetrodotoxin in contaminated or very dilute samples that are difficult to purify for tetrodotoxin. In this type of analysis, tetrodotoxin and its derivatives are decomposed to 2-amino-6-hydroxymethyl-8-hydroxyquil~azoline(C, base) withalkalihydrolysis and then converted with to GC-MS trimethylsilane (TMS) to TMS derivatives. These derivatives are submitted analysis as follows: A I O nil flask containing 0.2 m l of tetrodotoxin solution (25 MU, equivalent to about S pg tetrodotoxin) and 0.2 rill of 1 .S N NaOH is heated at 80°C-90°C for 30 minutesto derive a C, base from the toxin. After being cooled to room temperature, the reaction mixture is adjusted to pH 3-5 with 10% HCI and extracted three times with 0.5 1111 of 1-butanol. The CCJbase in the residue is converted to the TMS derivative in the presence of N,O-his (trimethylsilyl) acetamide, trimethylchlorosilane, and pyridine (2:1 : I). Each example of GC-MS selected ion monitoring (SIM) (m/: 407, W / : 392, m/: 376) and the resulting mass spectra are illustrated in Fig. 4. Each peak of SIM ( m / : 407, m/: 392, / ] I / : 376) typical of those of Cq base which appears at a retention time of 14.0 minutes is evidenced by spectrometry. Sharp fragment ions appear at HI/:407 [M'; C, base-(TMS)?], m/: 392 ( M ' - CH,), and / T I / : 376 [ M ' - H - (CH,)?].
E. Thin-Layer Mass Spectrometry (TLC-MS) and Liquid Chromatography-Mass Spectrometry Both TLC-MS (32) and LC-MS (30) are promising detection methods for tetrodotoxin. The latter methodis presently being developed.An example of each procedure is described below.
265
Tetrodotoxin ("10)
100-
-
185(2G+H)'
50. (M+H)' 320
f l
l00
c
200
300
400
500
200
300
400
500 m/z
Fig. 6 Positiveandnegativefastatombombardmcnt Cij) (27).
(FAB) lmss spectra (MS) of tetrodotoxin
For TLC-MS, the TLC plate used is an LHP-K high-performance precoated plate (Whatman, Clifton, NJ). The solvent system is pyridine :ethyl acetate: acetic acid: water ( 15 :5 : 3 :4). For MS, a FAB-MS apparatus, a JEOL DX-303 mass spectrometer with a JEOL DA-S000 data system,is used. An exampleof an analysis of tetrodotoxin, tetrodonic acid, and anhydrotetrodotoxin with this system is shown in Fig. 7 (32). For LC-MS, HPLC and MS are combined to increase measurement accuracy. In this example, the HPLC apparatus used is an Hitachi L-6200 with an ODs-3 column. The solvent system is 50% CHiCN with an ionization level of ESI. Fig. 8 depicts an analysis of tetrodotoxin derivatives using this method (30).
F. Thin-LayerChromatography The TLC technique (26) is conducted on a 5 cm X 20 cmsilica gel precoated plate (Merck) with a solvent system of pyridine: ethyl acetate: acetic acid: water (75 :25 : 15 : 30), 3-butano1 : acetic acid: water(2: 1 : l), or 1-butanol :acetic acid :water (60:1 S : 25). Tetrodotoxin
266
Yoshikawa-Ebesu et al.
100 7
50 302
0
0.2
0.4
0.6
0.8
1.0
100
200
300
1
412
400
Rf
500
m/z
Fig. 7 TLC-MS of tetrodotoxin and derivatives (32): (left) TLC; (right) MS.
is visualized as a pink spot with the Weber reagent or as a yellow fluorescent spot under a UV lamp (365 nm) after spraying with 10% KOH followed by heating at 100°C for 10 minutes. The Rf values of tetrodotoxin are 0.70 with pyridine:ethyl acetate: acetic acid: water (75 : 25 : 15 :30), 0.45 with 3-butanol :acetic acid : water (2 : 1 : 1 ), and 0.2I with 1 -butanol : acetic acid: water (60:15 : 25).
G. Capillary lsotachophoresis When tetrodotoxin (2.1 pg) is applied to an isotachophoresis apparatus, a zone appears at the position with a potential unit value of 0.32. The analytical time is less than 20 minutes. This method enables quantitation of tetrodotoxin content in crude extracts of puffer. The detection sensitivity for tetrodotoxin using capillary isotachophoresis (26) is about 0.2-5 pg, which is equivalent to that of the mouse assay. This procedure, however, eliminates the necessity for using live animals.
H. IR Spectrometry In this method, tetrodotoxin in a KBr pellet is analyzed by a spectrometer (26). The resulting spectrum is complex (3350, 3240, 1670, 1612, 1075lcm) but helpful for identification of the toxin from various sources.
267
Tetrodotoxin
TIME(M1N.)
-
a)
5 .O
G
10.0 l
. 75
TIC
J
G
320 * 5.0 SCAN NO. 200
,
100
: 1250
SAMPLE NO.
SCAN NO. 67-59 TIME(MIN) : 4.2
, -
100
1
276
-
-
60
320
153
249
185 I
I
I
I
I
I
I
1
II
100
1
'
"
l
!
l
,
l
I
l
l
l
l
l
l
l
l
l
l
l
300
200
0
-
400 TIME(M1N.)
1.0
5
3.0
2.0
5.0
4.0
6.0
1633 23
- "IC
f--" 1
I
l
I
I
1
I
50 SAMPLE NO. : 1246
100
304 * 30.0
I
SCAN NO.
SCAN NO. : 78 TIME(M1N) : 4.9
100
-
-
133
100
304
-
158
-
0
-
235
; 100
I
I
I
I
!
~
.
.
.
.
,
.
,
1
'
200
,
,
1
i
l
.
,
,
~
.
l
.
.
.
,
l
0
:
300
400
Yoshikawa-Ebesu et al.
268
H (9.9)
5.49
4.25
3.96 4.02
4 4a 5 7 8 9 11
Tetrodotoxin
2.34 (9.5) 4.08 4.29 4.02 4.04
Newt toxin
5.48 (9.5) 2.33 (9.5) 4.23 4.06 4.27 3.94 4.00
Chemical shifts are expressed as ppm. Coupling constants in parentheses are given as Hz. Fig. 9
1.
'H-NMR spcct~u~n of ncwttoxinanditsdata
on tetrodotoxin.
H-Nuclear Magnetic Resonance (NMR) Spectrometry
This procedure can be used to determine the chemical structure of pure tetrodotoxin and related compounds. One milligram of authentic crystalline tetrodotoxin or purified newt toxin is dissolved in 0.3 1nl of 3% CD,COOD in DzO and submitted to analysis at 400 MHz using a JEOL GSX-400 spectrometer with tetramethylsilane (Me,Si) as the external standard ( 1 2 ) . Fig. 9 shows an example of an analysis using this method.
Tetrodotoxin
J.
269
Electrophoresis
One microliter of tetrodotoxin solution ( I O MU or 2 pg tetrodotoxin) is applied to a 5 cm X 18 cm cellulose strip (Chemetron) with 0.08 M Tris buffer (pH 8.7) and electrophoresed at 0.8 mA/cm for 20 minutes. For detection of tetrodotoxin the strip is treated as described for TLC. In this type of electrophoresis (26), tetrodotoxin moves toward the cathode with a mobility clearly smaller than that of the saxitoxin standard, which exhibits a blue fluorescent spot under UV light after being treated with alkali.
K. Cytotoxicity Assays An assay based on the cytotoxicity of tetrodotoxin was first developed in 1988 (33). In this method the mouse neuroblastoma cell line neuro-2A (ATCC, CCL 13 I ) was used to quantitatively measure sodium channel blocking toxins such as tetrodotoxin and saxitoxin based on their ability to protect the cells from the cytotoxic effects of veratridine and ouabain. Since cytotoxicity was evaluated by microscopic examination of the morphological differences between living and dead cells, this procedure was time consuming and subjective. An improvement to this method was recently made usinga water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliun~, monosodium salt (WST-I), which is an indicator of living cells (34).
L. Immunoassays Several immunoassays have been developed for tetrodotoxin. These methods(35-37) had low sensitivity or required considerable time to yield results (38). Recently an enzyme immunoassay (EIA) was developed to quantitatively measure tetrodotoxin. This method israpid as well as highlysensitive to tetrodotoxinatconcentrations as low as 2-100 ng/ml (38). Rather than using an antibody raised against tetrodonic acid or tetrodotoxin conjugated to keyhole limpet hemocyanin as in previous assays, this assay uses an antibody against tetrodotoxin conjugated to bovine serum albumin to increase the sensitivity and specificity for this toxin. Moreover, this EIA can yield results in 30 minutes after 60 minutes of preparation (38).
IV. CLINICAL Clinical cases of tetrodotoxin poisoning are diagnosed based on the symptoms and history of exposure to the toxin.
A.
Symptoms
Tetrodotoxication is characterized by a myriad of symptoms, due almost entirely to the sodium conductance blocking actionof the toxin. The type, severity, andvariety of symptoms depend on the amount of toxin ingested. There are four main stages or degrees of tetrodotoxication based on the severity and evolvement of the poisoning (see Table 7, p. 273). Depending upon the amountof toxin ingested, symptoms usually appearwithin 1045 minutes of exposure, though some cases have reported being asymptomatic until as
270
al.
Yoshikawa-Ebesu et
much as 3-6 hours after exposure. Oral paresthesia is usually the initial symptom, and gradually spreads to the extremities and trunk. Other early sylnptorns include taste disturbance, dizziness, headache, diaphoresis, and pupillary constriction. These may or may not be accompanied by gastrointestinal symptomsof salivation, hypersalivation, nausea, vomiting,hyperemesis.hematemesis,hypermotility,diarrhea.andabdominalpain.These symptoms are characteristicof first degree tetrodotoxicationi n a system devisedby Fukuda and Tani (39). Second degree tetrodotoxicationis characterized by additional neuromuscular symptoms, such as advanced general paresthesia, paralysisof phalanges and extremities, pupillarydilation,and loss of pupillaryandcornealreflexes.Respiratorydistressmayalso begin. In third degreetetrodotoxication,patientsexperienceincreasedneuromuscular symptoms. Paresthesia of the larynx may lead to dysphagia and aphagia. Other neuromuscular symptoms include dysarthria. lethargy, muscular incoordination, and ataxia; sensations of floating due to numbness; cranial nerve palsies and muscular fasciculations. Cardiovascularandpulmonarysymptoms of hypotension or, morerarely,hypertension; vasomotor blockade; cardiac arrhythmias including sinus bradycardia, asystole, tachycardia, andatrioventricularnodeconductionabnormalities;cyanosis,pallor,anddyspnea may also occur during this stage of intoxication. Dermatologic symptoms of exfoliative dermatitis, petechiae, and blistering are also often observed. Fourth degree tetrodotoxication involves respiratory failure, extreme hypotension, seizures, and loss of deep tendon and spinal reflexes. Although some patients may exhibit impaired mentalfacultiesandmayevenbecomecomatose,mostpatientsremainfully conscious and alert until inmediately before death occurs. Since death usually occurs in 6-24hours, if thepatientsurvives past 24 hours the prognosis for recovery is good. Otherwise, death is caused by progressive ascending paralysis involving the respiratory nluscles. I n addition to the symptoms just described, several unusual symptoms have been reported in a few cases. These include hypertension (22,40) and cranial diabetes insipidus (4 I ). I n the patients who experienced dramatic increases in blood pressure in response to tetrodotoxin. Deng et al. (40)noted they all had preexisting hypertension and sensitivityto sympathetic stimulation. The case involving cranial diabetes insipidus is described below.
B. Treatment At present, there are no known antidotes or antitoxins to tetrodotoxin, SO treatment of symptolns is supportive. Diagnosis is based on the clinical symptoms and history of consumption of toxic organisms. To reduce exposure to unabsorbed tetrodotoxin, emetics lnay be administered if vomiting has not already occurred. In addition, gastric lavage, especially with 2 % sodium bicarbonate, followed by activated charcoal is recomniended (42). Fluid and electrolyte replacement therapy may be usedto reduce resulting fluid loss. Atropine may also be given to counteract hypotension and bradycardia (42). In cases of respiratory difficulty or failure, oxygen and other ventilatory support, including endotracheal intubation. is often necessary. Several researchers report that administration of the anticholinesterases edrophoIlium and neostigmine enhance the recovery of motor power and markedly reduced paresKao (48). thesia ;llldnL1mbness (43-47). Although these reports are contrary to those of
Tetrodotoxin
271
Chew et al. (46,47) suggest that tetrodotoxin causes a competitive reversible block at the motor end-plate as well as at the motor axon and muscle membrane. The effectiveness of the anticholinesterases can thus be explained by their action in increasing the quantal release of acetylcholine at the neuromuscular junction, thereby reversing the tetrodotoxin blockage (47).
Incidences C. 1. Japan In Japan, many puffer poisoning cases occur every year, resulting in numerous deaths with approximately 7% mortality over the past 10 years (Table 5) (3). Japanese people know that puffer, especially its liver (kirno) is very toxic, but more than a few kirtro fans dare to ingest the liver, believing that the toxin has been eliminated fromit using their own “special” detoxification method. Consequently most puffer poisoning cases are caused by ingestion of toxic livers. Since the Japanese Ministry of Health and Welfare published a guideline of ediblepuffers in 1983,withupdates i n 1993and1995(Table6) (3). the number of puffer poisonings i n Japan seems to have diminished (Table 5). These guidelines only prohibit puffer livers to be served in restaurants. Consequently many tetrodotoxin poisonings still occur due to consumption of homemade puffer liver dishes from fish usually caught by a family member. Tetrodotoxin poisonings due to the ingestion of digestive glands of the edible trumpet shell (Cl~rrro~~irr strulirre) occurred in 1979 i n Shimizu, Shizuoka Prefecture (12). in 1982 in Wakayama, and in 1987 in Miyazaki, Japan. Typical examples of tetrodotoxin poisoning cases are as follows: Poisorlirlg Due t o the Liver of‘ the Plrfler “Korrlorlfirgu” (Trrkifiqu poecilonA 48-year-old man in NagasakiCity,NagasakiPrefecture,atemore thanfour slightly cooked livers (kimo) along with some flesh of wild T. poecilonotus i n the evening in October 1996. The fish had been caught earlier i n the day. Thirty to 60 minutes after in his hands and ingestion of the puffer tissues, the man began to suffer from numbness (1.
orus).
Table 5
PufferPoisoningCases Number
Year
of cases
1987
35
1988 1989
26
1990
31 33
1991
29
1992 l993 1994
33
1995
l996 Total
28 16 30 21 382
Number o f patients
S2 46 45
(1987-1996) inJapan ( 3 ) Mortality Number of deaths 4 S
5
(%)
1.7 10.9 11.1
55
1
I .8
45 57 44
3 4 4
6.7 7.0
23
I
42
2
4.3 4.8
34
3
488
32
9.1
8.8 6.6
272
et
EdiblePufferSpeciesAvailable Welfare) (3)
Table 6
Yoshikawa-Ebesu
a/.
i n Japan(Ministry of Healthand
Edible part Male Family
Species
* Applicable t o this species caught
Muscle gonad Skin
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
-
0 0 0 0
-
0 -
0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
-
-
0 0 0 0
0 0 0 0
-
-
0 0 0 -
i n Ar~akcSea and Tachihana Bay.
limbs, followed by cyanosis and respiratory failure during the next 60 minutes. Although he was immediately hospitalized, the patient died during the following hour. Toxicity scores of the puffer livers and flesh were 715-4260 MU/g (equivalent to 0.14-0.85 mg of tetrodotoxin/g)andless than 5 MU/g (equivalentto 0.01 n1g of tetrodotoxin/g), respectively. In this case, the cause of death was detemined to be tetrodotoxin in thewild 7‘. poecilonofus liver, and the victim died due to ingestion of a total toxicity score of more than 10,000 MU (about 2 mg tetrodotoxin), equivalent to the LDS,, of tetrodotoxin for man.
b. Poisotling Due t o the Liver of tlw Pufer “Kurosrrbclfirgu (Lqocepholus gloveri). A 59-year-old woman cooked and consumed 3.5 livers (kimo) (estimated at 300 g) of fresh wild puffer, L. gloveri. Fifty minutes after ingestion she complainedof numbness of her lips and the tip of her tongue, followed by numbness of her fingers. After vomiting once, the victim was immediately hospitalized. Five hours after ingestion the victim devel”
Tetrodotoxin
273
Table 7 The Four Main Degrees of Tetrodotoxication and Associated Symptoms
Degree FirstNrurotnuscularsymptoms(paresthesia of lips,tongueandpharynx;tastedisturbance; dizziness: headache; diaphoresis; pupillary constriction); gastrointestinal symptoms (salivation, hypersalivation, nausea, vomiting, hyperemesis. hematemesis. hypertnotility, diarrhea, abdominal pain) SecondAdditionalncuromuscularsytnptonls(advancedgeneralparesthesia;paralysis of phalanges and cxtremities; pupillary dilatation, reflex changes) ThirdIncreasedneuromuscularsymptoms(dysarthrta;dysphagia,aphagia;lethargy;incoordination, ataxia; floating sensation; cranial nerve palsies; ~nuscularfasciculations). cardiovasculnrlpulmonary symptoms (hypotension or hypertension; vasomotor blockadc: cardiac arrhythmias including sinus bradycardia, asystole, tachycardia, and atriovcntricular node conduction abnormalities; cyanosis: pallor; dyspnea). dermatologic sylnptollls (exfoliativc dermatitis, petechiae, blistering) loss o f FourthRespiratoryfailure,impairedmentalfaculties,extremehypotension,seizures, deep tendon and spinal reflexes
oped dyspnea, mydriasis, and paralysis of the extremities. Artificial respiration through an endotracheal tube was then administered. Nine hours after ingestion, arrhythmia began and was successfully treated with antiarrhythmic drugs; the patient recovered soon after. The toxicity score of the leftover wild puffer livers was 3.5 MU/g (equivalent to 0.0007 mg tetrodotoxin/g). The total amount of toxin ingested by the patient was estimated to be 900-1000 MU (or 0.18-0.2 mg of tetrodotoxin), which may be near the minimum dose necessary to cause tetrodotoxin symptoms. Although L. glover-i isbelieved to be nontoxic, specimens from Taiwan occasionally show weak toxicity (42 MU/g; 0.008 mg of tetrodotoxin/g) in the liver (3 1).
c. Poi.soni~r~g Due tothe Flesh of “Dokrr.snbr!fugu (L. lunaris lunnris). Specimens of L. 1urltrri.s 1uncrri.s (49), a puffer similar to the nontoxic species “sabafugu” (L. luncrris .sprrdicus)were caught in the Vietnam Sea in September 1959 and the heads, skin, and viscera removed. The flesh with tails were quickly frozen. Five men ( 1 7-27 years old) each ate one pieceof the fried L. lunar-is lulraris flesh at noon i n October 1959. All five men developed signs and s y m p t o m typical of tetrodotoxin poisoning 1 hour after ingestion and were hospitalized within 3 hours of ingestion. Three of the men died within 3.5-6.5 hours following ingestion. The causative fish was initially thought to be the nontoxic “sabafugu” (L. lurrrrr-is .s/~rrdicrrs). Since the flesh was toxic, however, classification of the fish was reinvestigated, resulting in the identification of a new species, L. lurttrr-is 1unar.i.s. I ’
cl. Poisorrir~gDue to the Digestive Glarld of n Trumpet Shell (Char-onin sauliae). In December 1979 a 41-year-old man ate a boiled digestive gland (approximately 60 g) of “boshubora” or trumpet shell (C. scrulitre) which he caught in the Shilnizu Sea. Thirty minutes after ingestion, he began to develop paralysisof his lips and limbs, followed by cyanosis and respiratory paralysis. He was immediately hospitalized and received artificial respiration. Although he remained unconscious for 2 days, he recovered fully in 5 days.
274
Yoshikawa-Ebesu et al.
The leftover digestive gland of the C. sarrlicrc. showed a toxicity score of 17.000 in the bioassay for tetrodotoxin. Thus the victim MU (equivalent to 3.7 mg of tetrodotoxin) may have ingested about 10,000 MU (2 mg of tetrodotoxin), the approximate LD5,,of the toxin for man. Tetrodotoxin was also identified as the causative agent i n samples from subsequent similar tetrodotoxin poisoning cases in Wakayama i n 1982 and in Miyazaki in1987using TLC,electrophoresis,andinstrumentalanalyses with IR. 'H-NMR. and GC-MS.
2. United States Incidences of tetrodotoxication are rare i n the United States. As i n Japan, the main Sollrce of this poisoning is puffer. Only 10 known fatalities from the consumption of puffer have occurred in the United States, 3 of them between 1950 aid l990 (50,5 1). Four other deaths occurred in Hawaii between 1903 and 1925 (before statehood), all due to consumption of Awthron sp. (52). An additional case occurred i n Hawaii in 1986 clue to consumption of the liver of Diodoll hystri.x (42). Although the patient did not present t o a hospital until 24 hours after exposure, he recovered within 1 week. More recently, in 1996, there were three cases of tetrodotoxin poisoning in California. Three men consumed 0.25- I .5 oz. of puffer brought from Japan as a prepackaged, ready-to-eat product (53). The two men who had eaten only 0.25 oz. each of the puffer began to experience oral paresthesia and dizziness within 10-20 minutes, while the other man who had eaten 1.5 oz. of puffer began to experience these symptoms within 2-3 minutes. All three men experienced additional typical tetrodotoxin poisoning symptoms, including anxiety, feelings of doom, headache, difficulty speaking, chest tightness. facial flushing, shaking, nausea, vomiting, weakness, and collapse. After hospitalization, all were treated with intravenous hydration, gastric lavage, and activated charcoal. All victims recovered within 24 hours (53). There have been two other cases of tetrodotoxication i n the United States. one of them fatal, due to swallowing the Oregon rough-skinned newt (Ttrrichcr grrrrlulostr) (54). In both cases, the victims may have been under the influence of alcohol, and swallowed the newts as pranks. Consistent with the presence of tetrodotoxin in this species of newt, the two individuals involved experienced the classic signs and symptomsof tetrodotoxicatlon.
3. Elsewhere Although not as common as i n Japan, puffer poisoning occurs in other Asian and Pacific countriesincludingTaiwan,HongKong,Thailand,Singapore.Malaysia,Kiribati,Fiji, Australia, and Papua New Guinea. Unfortunately their records may not always be as complete as those in Japan. In Taiwan, for example, tetrodotoxin poisoning is not a reportable disease, and many physicians are unfamiliarwith the symptomatology of the disease ( 18). Nonetheless, a study of the Taiwan Poison Control Center data from 1988 to 1995 showed a significant number of tetrodotoxin poisonings: a total of 20 incidents involving 52 patients ( 1 8). A detailed listing of these incidents including dates and locations of occurrences, sources of toxin, number of deaths, gender distribution, age of victims. and laboratory confirmation are shown in Table 8. In Singapore, the ingestion of SpAcreroitles rmcrrltrtrrs and Arothrorf stelltrtrrs have resulted in several tetrodotoxin poisonings (41,46,47). Althoughthe patients in these cases experienced typical tetrodotoxication symptoms, no fatalities were reported among them. An unusual symptom was noted in an additional tetrodotoxication case in Singapore
Table 8
Demographic Data of Tetrodotoxin Poisoning in Taiwan, 1988-1995 (18)
Date of occurrence
Location of occurrence:
Mar 1988 Mar 1988 Apr 1988 Jun 1988
Northwest Northwest Midwest Northeast
Whole puffer Flesh of puffer (L. luritrris) Whole puffer Roe of puffer (L. lirriciris)
Jan 1989
Northwest
May 1989 Jul 1989 May 1990 .Tun 1991 Dec 1991 Dec 1992 Jan 1993 Jan 1994 Apr 1994 Apr 1994 May 1994
Northwest Midwest Southeast Midwest Midwest Southeast Midwest Midwest Southwest Midwest Southwest
Decapitated puffer and liver of puffer Roe of puffer Whole puffer Whole puffer Flesh of puffer Whole puffer Whole puffer Whole puffer Roe of puffer Roe of puffer Whole puffer Gastropod mollusks ( N . e m firs and N. corioiddis)
Jul 1994 Dec 1994 Apr 1995 Oct 1995
Northeast Northwest Midwest Midwest
Source of toxin
Whole Whole Whole Whole
No. of patients (deaths) 4 ingested, 1 was poisoned (0) 5 ingested. 2 were poisoned ( 1). 5 (0) 2 (1)
2 P
:
0
.Age (years) 60 36 9, U" (4) 3-60 62
2 (0) l(1) 3 (1) 1 (0)
I(1) 3 (1) 1 (0) 4 (0) 2 (0)'
5 ingested. 3 poisoned ( 0 ) 26 poisoned, I7 reported ( 1 )
fish of Gobirrs criniger puffer puffer puffer
The location of occurrence was dclincd according to different parts of the Taiwan island M/F/U denotes male. female. and unknown sex. respectively. ' Only m e case was reported t o the Poison Control Center-Taiwan with adequate clinical information ,'The symbol "U" denotes unknown age. _I
Gender distribution (M/F/U)
9 im-2 38 53-59. U ( I ) 57 53 5-40 38 25-29. U ( I ) 50 15-71 12-7 I
32-34 19 50 56
Toxin assay None 120 MU/g (inusclej None 1200 MU/g (roe). 45 MU/g (muscle) None I100 MU/g (roe) None None None None 150 MU/g (muscle) None 150 MU/g (roe) None None 150 MU/g ( N . cu.sriis), 13 MU/g ( N . C O I I O I ddS) None None None None
z.3 J
Yoshikawa-Ebesu et al.
276
involving the consumption of Ar-orhr-orz r-eficwlar-is (41). In this case, a 24-year-old man developed nausea and abdominal discomfort after eating this fish. Soon after hospitalization, he became apneic and lost consciousness. After successful cardiopulmonary resuscitation, he remained in a deep c o m and was placed on mechanical ventilation. One noteworthy feature of this case is the first reported occurrence of cranial diabetes insipidus due to tetrodotoxin poisoning. Tambyah et al. (41) suggest that tetrodotoxin may have blockedsodiumchannels i n the axons of thenlagnocellularneurons of the neurohypophysis, thus inhibiting the release of vasopressin and resulting i n diabetes insipidus. Since cranial diabetes insipidus is indicative of extensive irreversible brain damage and impending death, even more unusual was the patient’s complete physical and mental recovery within 50 hours. Reports of tetrodotoxication from other countries are sporadic. These include eight cases (one fatality) from unidentified puffer species in Hong Kong (5556); several cases Ltrgoceph(llu.7 Irrgocephalus (Linnaeus) in Kiribati (for(two fatalities) from the puffer merly the Gilbert Islands) (57); four cases (two fatalities) from Diodorl h y t r - i s in Papua New Guinea (58); five cases (one fatality) from the same puffer species in Fiji (43); four cases (one fatality)in Australia, two from Amhl~r-/?~r~chnre.~ r-ichei,one from Sphcrer-oides gluher- (44,59), and one from Termcferm glaher- (60); seven nonfatal cases i n Thailand, six from Terr-croclorl frrrlgi (61) and one from an unknown puffer species (62); and four cases (one fatality) from an unknown puffer species i n Malaysia (63).
V.
TOXICOLOGY
Many animals as well as humans are sensitive to tetrodotoxin. I n humans, the LD?,,of tetrodotoxin i n man (50 kg) is approximately 10,000 MU, equivalent to 2 mg of tetrodotoxin. Early studies examined the toxic nature of tetrodotoxin in cats, dogs, pigs, chickens, pigeons, sparrows, doves, rabbits, mice, guinea pigs, rats, snakes, frogs, turtles, dragonflies, newts, puffers, and various freshwater and marine fish (for a detailed review, see Refs. 19,481.
VI.
PHARMACOLOGICAL ACTIONS
I n the intact animal, tetrodotoxin has toxic effects on the neuromuscular, cardiovascular, and respiratory systems, probably all due to its voltage-insensitive blocking effect on sodium channels. Tetrodotoxin can induce local anesthetic, emetic, hypothermic, hypotenall of sive, vascular smooth muscle relaxant, and respiratory depressant symptoms (64), which reflect the progressive paralysis of the excitability mechanismof nerve and muscle. Tetrodotoxin also possesses a direct relaxant effect on vascular smooth muscle that may be responsible for producing hypotension i n vivo (65). For an extensive historical review on the pharmacology and toxicology of tetrodotoxin, see Ref. 19. Additional effects of tetrodotoxin have been noted i n various tissues, including pacinian corpuscles, and cardiac and neural tissues. In Pacinian corpuscles, tetrodotoxin may block the sodium channel action potentialas well as reduce the mechanoreceptor potential (see Ref. 66). In rat heart mitochondria, tetrodotoxin decreases in vitro calcium uptake, inhibiting respiration and ATP (adenosine triphosphate) generation capacity at high doses;
Tetrodotoxin
277
this effect is probably due to blockade of the sodium-calcium uniporter by the toxin (67). In embryonic chick heart cell aggregates. tetrodotoxin blocks spontaneous activity in a dose-dependent manner and reduces action potential duration, but has little effect on interbeat interval (see Ref. 68). Neurophysiological studies have been conducted in at least two different human tetrodotoxication cases. I n both cases, although the conduction velocity and amplitude of both muscle and sensory nerve action potentials were decreased, neither temporal dispersion nor focal conduction block occurred. This phenomenon seemed diffuse, occurring in most or all fibers in affected nerve trunks and resulting in synchronous slowingof impulse conduction (58,641. Recent animal studies have shown 4-arninopyridine can reverse saxitoxin and tetrodotoxin-induced cardiorespiratory depression (69). Since this compound is a potent potassium channel blocker, however,it can have serious side effects and even toxicity at higher doses, making its use as a tetrodotoxin antidote unfeasible without further studies.
VII. A.
USE OF TETRODOTOXIN AS A PHARMACOLOGICAL TOOL Organismal Level
Due to its sodium channel blocking effect, tetrodotoxin possesses anticonvulsant and neuroprotective activity in certain situations. In some in vitro studies, low concentrations of tetrodotoxin can have anticonvulsant-like effects similarto those of phenytoin or carbamato duplicate this effect. During pathologizepine (70,71 ); in vivo studies have been unable cal conditions such as seizures or ischemia, sodium influx from voltage-dependent sodium channels may cause a cascade of undesirable effects: opening of calcium channels and calcium-dependent glutamate release, opening of potassium channels, and relief from magi n vitro and in nesium-dependent block of NMDA-type glutamate receptors (72). Both vivo studies have demonstrated that selective sodium channel blockers such as tetrodotoxin can block the initial sodium influx and thus prevent most of these detrimental effects that may contribute to cellular damage during ischemia.In vitro models examined include rat hippocampal slices, optic nerves, neuronal cultures, and spinal explant cultures; in vivo models include gerbil and rat forebrain ischemia, isolatedrat head, and rat c l d’lac < r arrest (see Ref. 72). The toxicityof naturally occurring sodium channel blockers such as tetrodotoxin precludes their therapeutic usefulness (73). Nonetheless, they have been instrumental i n further elucidation of the damaging nature of such phenomenon as ischemia and in the subsequent development of preventive measures against these conditions.
B. Molecular Level Neurotoxins, especially tetrodotoxin. have long been used as tools to study sodium channels in excitable membranes because of their specificity for these channels. The unique action of tetrodotoxin on blocking action potentials by selective inhibition of the membrane sodium conductance upon depolarization wasfirst discovered in lobster giant axons (74). This action was found to be exerted at nanomolar concentrations, to occur without affecting the kinetics of the transient conductance (potassium) increase, to block the tran-
Yoshikawa-Ebesu et al.
278
sient channel without impairing the gating mechanism, and to be effective only when tetrodotoxin was applied to the outside of the squid axon and nodal lnenlbranes (see Ref. 65). Thus tetrodotoxin must act on the outer surface of the sodium channel. In addition, it was noted that action potentials not based on sodium ion fluxcs were insensitive to tetrodotoxin. These included calcium-based action potentialsof various animal nluscles (see Ref. 75). Subsequent studies showed that tetrodotoxin had no effect on neurotranslnitter (both acetylcholine and norepinephrine) release from various nervous tissues (65), contirming the direct effect of tetrodotoxin on sodium channels. The original mechanism of action of tctrodotoxin was thought to be via the guanidinium group on the toxin “plugging” the sodium channel pore and physically preventing the passage of sodium ions through the pore (76). This was based on the fact that the guanidinium moietics were critical for the pharmacological actions of the toxin, and that free guanidinium ions could pass through sodium channels. The elucidationof the chernical structure of tetrodotoxin, however, altered this idea. One of thc first hypotheses of tetrodotoxin binding to sodium channels is illustrated in Fig. 10. In this model, Hille (77) proposed that the tnain binding site on both tetrodotoxin and saxitoxin, a toxin with similar actions to tetrodotoxin, for the sodium channel receptor wasthe unusually electropositive. homologous hydrated ketone group on carbon 1 0 of tetrodotoxin (designatedby the circled X i n Fig. 10). Further hydrogen bonding and electrostatic attraction between the guanidinium group and the negative charge of the selectivity filter were helievcd to stabilize the complex. Moreovcr, this model helped to explain why even minor moclifcations in the structure of either tetrodotoxin or saxitoxin caused drastic reductions in biological activity; for example,the low biological activityof anhydro, alkyl. and deoxy derivatives of tetrodo-
Tetrodotoxin
Tetrodotoxin
279
toxin can be accounted for by the loss of hydrogen bonds between the modified toxin and sodium channels (75,771. Most analogues of tetrodotoxin, including nortetrodotoxin. are relatively inactive against sodium channels. A methoxamine product of nortetrodotoxin, however, was discovered to be about one-third as active as tetrodotoxin (78). Previously the guanidinium moiety and the cationic head at the carbon 2 position were thought to be most important in the binding of tetrodotoxin t o the sodium channel (see Fig. 1). This finding suggested the importance of the 1 I end of the tetrodotoxin molecule i n sodium channel binding. Although all action potentials that are blocked by tetrodotoxin are based on sodium currents, the converse is n o t necessarily true. Certain types of excitable tissues with action potentials due to sodium currents are insensitive to tetrodotoxin. For example, the nerve Iron1 the newt Ttrrichtr torostr is at least 30,000 times more resistant to tetrodotoxin than the frog nerve, and the nerve of the Atlantic puffer (Spheroides nrtrcwltrtrrs) is 1000 times n1ore resistant to tetrodotoxin than frog nerve(6.5).Tetrodotoxin-resistant sodium channels also have much lower c o t l d ~ ~ c t than a ~ ~thcir ~ e ~toxin-sensitive counterparts (79).
I extracellular
intracellular
v
coo-
Fig. l l Sodium chnnncl structure. including regions o f the protein that have been associated with various functions. T, the TTX-binding site: H, the crucial portion of the inactivation gate: +. the and is necessary for activation gating; P. phosphorylacharged portion that sensed menlhrnnc voltage tion sitcs along an extensive cytosolic loop that is absent in skeletal muscle and ecl electroplax: G. sitcs of extracellular glycosylation. (Inset) An expanded view o f the domain IV-S6 region, crucial for the binding o f local anesthetics and phcnyloin. Numbering of amino acid residues begins a t the amino ( N H ? )terminus and continues sequentially through approximately I800 amino acids toward the carhoxyl (COOH) terminus (72).
280
Yoshikawa-Ebesu et al.
Other tetrodotoxin-resistant sodium-based action potentials are found in nerves of puffers, octopus, mollusks, denervated mammalian muscle, etnbryonic and newborn rat by Baker muscle, and rat skeletal muscle L6 Inyotubes (see Refs. 75.80). Experiments and Rubinson (81) with carbodiimide-nucleophile treated nerves showed that the group in the sodium channel involved in the sensitivity of the action potential to tetrodotoxin can, under appropriate conditions, be dissociated from the ability of the channelsto generate action potentials. Thus sensitivity to tetrodotoxin can be abolished while retaining the physiological function of the sodium channel. Tetrodotoxin has been used to demonstrate that sodium channels sensitive and resistant to thistoxin have different physiological and pharmacological properties (see Ref. 80). In rat dorsal root ganglia, both types of channels are present in immature as well as (82,83). adult neurons, suggesting that they both may be essential to sensory integration Indeed, distinct types of sodium channels may be involved i n conveying different types of sensory information (80). For example, tetrodotoxin-resistant sodium channels may be more important in nociception than tetrodotoxin-sensitive ones (84,85), especially under chronic neuropathic pain conditions (86). Further insight into the interactions between tetrodotoxin and sodiunl channels, as well as the differences between tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels, has been accomplished with the use of molecular biological methods. I n particular, the S5-S6 region of each domain is thought to contain two short segments, SS1 and SS2, that may partly span the membrane as a hairpin (Fig. 1 1 ). Various point mutations in this region have been made in an attempt to map the site of tetrodotoxin and saxitoxin block of the sodium channel. A single point mutation of the rat sodium channel I1 at position 387, replacing glutamic acid with glutamine. was found to reduce its sensitivity to tetrodotoxin and saxitoxin by more than three orders of magnitude (87). This mutation effectively rendered the rat sodium channelI1 insensitive to concentrations of tetrodotoxin up to I O FM, while hardly affecting the macroscopic current properties. The glutamic acid residue at position 387 is conserved in all sodium channels with known sequence and is located between segments SS and S6 of domain I. In the present sodium channel model, this would place glutanlic acid 387 on the extracellular side of the membrane, probably in close proximity to the channel opening (87). The results of this study are consistent with this location, since the binding site for tetrodotoxin is known to be located on the extracellular side (77). Other point mutationsof the rat sodium channel I1 have shown similar results. Mutaat equivations involving two groupsof predominantly negatively charged residue. located lent positions i n the seven-amino acid SS2 segment of the four repeated domains, strongly reduce tetrodotoxin and saxitoxin sensitivity. Mutations of adjacent residues, howevcr, have little or no effect on sensitivity to these toxins (88). These researchers have found that all mutations reducing net negative charge in the amino acid clusters i n segment SS2 markedly diminish single-channel conductance. and have suggested that these two clusters of residues form ring structures which form part of the extracellular pore opening and/ or the pore wall of the sodium channel (88). Molecular techniques have also been used to discover the structural determinants that confer tetrodotoxin sensitivity or resistance of certain sodium channels.AS mentioned, sodill111 channels in brain and innervated skeletal muscles are tetrodotoxin-sensitive and respond to nanomolar concentrations of tetrodotoxin. On the other hand, those which are tetrodotoxin-resistant, such as sodium channels in denervated skeletal tmsclc and RH1
Tetrodotoxin
281
sodium channels in heart, are only blocked by micromolar tetrodotoxin concentrations. RH1 sodium channels are also more sensitive to cadmium than are tetrodotoxin-sensitive sodium channels. Two of the seven amino acidsin segment SS2of domain I differ between RH1 and the tetrodotoxin-sensitive sodium channel isoforms (see Ref. 89). A point mutation of one of these two amino acids, cysteine at position 374 to either phenylalanine or tyrosine, in anRH1 mutant, confers three propertiesof tetrodotoxin-sensitive sodium channels: (a) a 730-fold increase in tetrodotoxin sensitivity; (b) a 28-fold decreased sensitivity to cadmium;and(c)alteredadditionalblock by tetrodotoxinuponrepetitivestimulation (89). Similar homologous mutations have also resulted in reduced tetrodotoxin affinity: an aromatic tyrosine or phenylalanine substituted for cysteine at position 374 i n tetrodotoxinsensitive, voltage-gated sodium channels (90); cysteine substituted for tyrosine at position 401 of the skeletal muscle isoform (p 1) of the rat muscle sodium channel (91 ); and serine changed to phenylalanine at position 376 in sensory neuron specific voltage-gated sodium channels (92). Apparently then, the aromatic residue at position 374 is critical for highaffinity tetrodotoxin binding. The magnitude of the increased tetrodotoxin binding affinity due to a point mutation at this position in RH1 sodium channels suggeststhat this interaction may be through an ionized hydrogen bond (89). Furthermore, the importance of the two amino acid clusters in tetrodotoxin and sodium channel binding proposed by Terlau et al. (88j may be limited to secondary electrostatic interactions (89). The salient features of sodium channels havebeen collectively analyzed to generate a potential model for the tetrodotoxin binding site of the sodium channels of both cardiac and skeletal muscle (93). I n general, this model fits well with most studies. For example, modifications of the hydroxyls at carbons 9 and I O of tetrodotoxin (to tetrodonic acid) result in a totally ineffective molecule (see Ref. 65). Another feature of this model was revealed when the toxin binding site was analyzed with the tetrodotoxin removed: the binding site consisted of a structure resembling a funnel that terminated in a narrowed region. The dimensions and chemical properties of this region werc consistent with those of the hypothesized selectivity filter of Hille (77,94) (Fig. 10). This novel model of thetetrodotoxinandsaxitoxinsodiumchannelbindingsite provides a basis for the differential binding of these toxins to various channel types. This model does not, however, consider interactions of the tetrodotoxin/saxitoxin binding site with other portions of the sodium channel protein. Thus, further experiments and analyses are needed to confirm the actual structure of this macromolecule.
VIII. SUMMARY One of thebest ways to prevent tetrodotoxin poisoning is, of course, not to eat toxic puffer. but many find the delicacy too delicious to resist. Fortunately, both the number of incidences and mortality rate for tetrodotoxication have declined in Japan, where this type of poisoning occurs most often. Key factors i n this reduction are probably increased governmental regulations and the availability of net-cultured nontoxic puffers. Unfortunately, supportive treatment remains the only remedy for tetrodotoxication. Ovcr the past few decades, many tetrodotoxin poisonings have been reported due to the ingestion of seemingly unrelated organisms. Thc discovery of tetrodotoxin in a
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diverse array of animals, both marine and terrestrial, has led to the hypothesis of a bacterial source of the toxin. Presently the toxicity scoreof tetrodotoxin is officially measured by the mouse assay (4). but improved HPLC analysis for the toxin is being developed to replace it due to the increased accuracy, time savings, and ethical concerns of this method. Many instrumental analyses (7,8), including'H-NMR,C'7"NMR. IR, GC-MS, TLC-MS. and LC-MS, are also presently available to identify tetrodotoxin.In addition, cytotoxicity and immunological assays for tetrodotoxin may also be employed for mass screening of samples. Although tetrodotoxin is a potent neurotoxin with an LD,,, of 2 n1g for man, in some areas it is also an important analgesic for the severe pains of rheumatism, neuralgia, and cancer. Furthermore, this toxin is a significant biochemical and pharmacological reagent a s a sodium ion channel blocker (10).
REFERENCES 1. BW Halstead. Poisonous fishes. Public Health Rep 73:302. 1958. 3. PJ Scheucr. Toxins from fish and other marine organisms. Adv Food Res 18:141, 1970. 3. Pufferfishes available in Japan. a n illustrated guide t o their identification. Tokyo: Veterinary Sanitation Division. Environmental Bureau, Ministry of Health and Welfare, 1994. 4. T Yasumoto, M Murata. Marine toxins. Chcm Rev 93: 1897. 1993. 5. T Noguchi, JK Jeon, 0 Arakawa, H Sugita, Y Deguchi, Y Shida. K Hashimoto. Occurrence o f tetrodotoxin and anhydro-tetrodotoxin in Vibrio sp. isolated from the intestines of a xanthid crab. Atcrgatis floridus. J Biochem 99:3 1 1. 1986. 6. T Yasumoto, D Yasumura. M Yotsu, T Michishita. A Endo, Y Kotaki. Bacterial production o f tetrodotoxin and anhydrotctrodotoxin. Agric Bin1 Chcm 50:793, 1986. 7. U Sitnidu. T Noguchi, DF Hwang. Y Shida. K Hashimoto. Marine bacteria which produce tetrodotoxin. Appl Environ Microbiol 53: 1714, 1987. X. T Matsui, S Taketsugu. K Kodama, A Ishii, K Yamamori, C Shimizu. Production of tctrodotoxin by the intestinal bacteria o f a puffer fish Z > k ( f i r p / r l i p h o h / o . s . Nippon Suisan Gakkaishi 55:2199. 1989. o f toxic fishes related to food hygiene. 9. T Noguchi. T Abc, K Hashimoto. A pictorial handbook
Tokyo: Midori-shobo.1988. T Noguchi. Recent studies on nlarinc toxins. Jpn J Toxicol Environ Health 39:81, 1993. 1 1 . FA Fuhrman. Tetrodotoxin. Sci An1 217:61. 1967. 12. H Narita. T Noguchi, J Maruyama, Y Ueda, K Hashitnoto, Y Watanabe, K Hida. Occurrence of tetrodotoxin in a trumpet, "boshubora" C'hnrorrirr snrtlitrc,. Bull Jpn Soc Sci Fish 47:983. IO.
19x1. 13.
14.
15.
16.
17
DF Hwmg, LC Lin, S S Jeng. Occurrence o f tetrodotoxin-rclated toxins in the gastropod triolusk Niothrr thfhrtrttr from Taiwan. Nippon Suisan Gakkaishi 58:63, 1992. T Saito, J Mnruyama, S Kanoh, JK Jeon. T Noguchi. T Harada, 0 Murata, K Hashimoto. Toxicity of cultured pufferfish ancl its resistibility against tetrodotoxin. Nippon Suisan Gakk :us. ,h'L 50:1573, 1984. T Noguchi. Food chain-associated toxification of tetrodotoxin-bearing animals. In: K. Hashim o t o , ed. Recent advances in tetrodotoxin research. Suisangaku Series 70. Tokyo: Kouseishakouseikaku,1988, p. 85. K Hashitnoto, T Noguchi, S Watabe. New aspectso f tetrodotoxin. In: AE Pohland. VR Dowell. JL Richard, eds. Microbial toxins i n foods and feeds, cellular and nwlecular action. New York: Plenum. 1990, p. 575. 1 Tani. Toxicological studies on Japanese puffertishes (in Japanese]. Tokyo: Teikoku-shobo. 1945.
Tetrodotoxin
283
18. BW Hnlstead. Poisonous and vcnonwus marine nnitnals o f the world. 2nd ed. Princeton. NJ:
DarwinPress. 1988. 19. CC Yang. SC Liao. JF Dcng. Tetrodotoxin poisoning in Taiwan: an analysis o f poison ccn1CI' data. Vet Hum Toxicol 38:282, 1996. a case 20. VH Fmg. SY Chow.Electrophysiologicalstudiesonacutetetrodotoxinpoisoning: report. Chin Med J (Taipei) 58:299, 1996. 21. T Noguchi. Y Hashitnoto. Isolation of tetrodotoxin from a goby Go/7irr.sc,r-ir~igc)r-.Toxicon 1 1: 305, 1973. 22. CC Yang, KC Han. TJ Lin. An outbreak of tctrodotoxin poisoning following gastropod m o l lusk consumption. Hum Exp Toxicol 14:446. 1995. 23. PW Gage, AF Dulhunty. Effects o f toxin from the blue ringed octopus (Hte/'Lc/och/rcc,rrcc r r t w l lostr).
I n : DF Martin, GM Padilln, eds. Marinc Pharmacognosy. New York: Academic Press.
1973. p. 85.
Y Nagashima. J Maruyama. T Noguchi. K Hashitnoto. Analysis o f paralytic shellfish poison and tetrodotoxin by ion-pairing high performance liquid chromatography. Nippon SuisunGakkaishi 53:819, 1987. 25. M Yotsu. A Endo. T Yasutnoto. An improved tetrodotoxin analyzer. Agric Biol Chctn 53:
24.
893. 1989. 26. Y Onouc, T Noguchi. K Hashitnoto. Tetrodotoxin determination methods. In: EP Ragclis. ed. Seafood toxins. Washington. D.C.: American Chemical Society, 1984, p . 345. K Hashimoto. Fast atom bombardmcnt- and secondary 27. J Maruyama, T Noguchi. S M;I~SLIIXI~:~. ion-mass spectrometry o f paralytic shclllish poisons and tetrodotoxin. Agric Biol Chcm 48: 2783. 1984. 28. Standard methodso f analysis in food safety regulation. chemistry. Tokyo: Japan Public Health Association, I99 l , p . 296. 29. T Kawabatu. Assay method for tetrodotoxin. In: Food hygicnc cxatnination manual. vol. 11. [ i n Japanese] Tokyo: Japan Food Hygiene Association, 1978. p. 232. 30. Y Shida. 0 Arnknwa, Y Onoue. T Noguchi. LC/MS of marine toxin-l. Proceedings of the 46th AnnunlConference on Mass Spectrometry. Tokyo: Japan Mass Spectrometty Society. 1998, p . 137. 31. DF Hwang, T Noguchi. 0 Arakawa. T Abe. K Hashitnoto. Toxicological studies on several species of puffer in Taiwan. Nippon Suisan Gakkaishi 54:2001, 1988. 32. Y Nagashima, S Nishio, T Noguchi,0 Arakawa, S Kanoh, K Hashitnoto. Detectionof tetrodo-
toxin by thin-layer chrorn~~toglaphy/f~lst atombotnhardmenttnassspectrometry.AnalBiochem 175:258. 1988. 33. K Kogurc, ML Tamplinc. U Sitnitlu. RR Colwcll. Atissue culture assay for tetrodotoxin. saxitoxin and related toxins. Toxicon 26: I9 1. 1988. 34, K Hamasaki. K Kogure. K Ohwada. A biological method for the quantitative measurement of tetrodotoxin (TTX): tissue culturc bioassay i n combination with ;I water-soluble tetrazolium salt. Toxicon 34:490. 1996. 35 S Watabe, Y Sata, M Nakaya. K Hashitnoto, AEnomoto. S Kuninogawa. K Yamnuchi. Monoclonal antihody raised against tctrodonic acid. a derivative of tetrodotoxin. Toxicon 27:265. 1989. 36. K Matsumura, S Fukiya. Indirect competitive enzyme immunoassay for tetrodotoxin using a biotin-avidin system. J AOAC Int 75:883, 1992. 37. K Mntsutnura. A monoclonal antibody against tetrodotoxin that reacts to the active group for toxicity. Eur J Phartnacol 293:41. 1995. 38. KKawatsu.Y Hamano. T Yoda. Y Tcrano. T Shihata. Rapid and highly sensitive cnzylne immunoassayforquantitativedctcrtnination o f tetrodotoxin.Jpn J Med SciBiol S O : 133, 1997, 39. T Fukuda. I Tani. Rccortls o f puffer poisonings, rcpt. 3 [ i n Japonesc]. Nippon Igaku Oyohi Kenko Hoken 3258:7, 1941.
Yoshikawa-Ebesu et al.
284
40. JF Dcng. RL Tominack, HM Chung, WJ Tsai. Hypertension a s an unusual feature in an outbreak of tetrodotoxin poisoning. Clin Toxicol 29:7 l , 1991. 41. PATambyah.KPHui,PGopalakrishnakone,NKChin,TBChan.Central-nervous-system effects of tetrodotoxin poisoning. Lancet 343:538, 1994. 42. JK Sims, DC Ostman. Pufferfish poisoning: emergency diagnosis and management of mild human tetrodotoxication. Ann Emerg Med 1.5: 1094, 1986. 43. M Sorokin. Puffer fish poisoning. Mcd J Aust 1:957, 1973. 44. TA Torda, ESinclair, DB Ulyatt.Pufferfish(tetrodotoxin)poisoning:clinicalrecordand suggested management. Med J Aust 1599, 1973. 45. TC Tan. Puffer fish poisoning. Singapore Faln Physician 639, 1980. 46. SK Chew, LS Chew,KW Wang, PK Mah, BY Tan. Anticholinesterase drugs in the treatment of tetrodotoxin poisoning. Lancet 2:108, 1984. KW Wang, PK Mah, BY Tan. Puffer fish (tetrodotoxin) poisoning: clinical 47. SK Chew, CH Goh. report and role of anti-cholinesterase drugs in therapy. Singapore Med J 24:168, 1983. 48. CY Kao. Tetrodotoxin, saxitoxin and their signilicancc in the study of excitation phenomena. Pharlnacol Rev 18:997, 1966. (Ltr~ot.e/’h~hh r .c~r r i s 49. 0 Tabeta,HKumagai.Morphologicaldifferencebetweensabafugu spfrdiclts) and doku-sabafugu (L. hrrlcrris Izrr~crris) with special reference to the food intoxication occurred in Kita-Kyushu City, 1959. J Food Hyg Soc Jpn 21:405, 1980. 50. CS Rcddy, AW Hayes. Food-borne toxicants. I n : AW Hayes. ed. Principles and methods of toxicology. 2nd ed. New York: Raven, 1989, p. 67-1 IO. 51. WR Lange. Puffer fish poisoning. Am Fam Physician 42: 1029. 1990. 22361. 1963. 52. P Helfrich. Fish poisoning in Hawaii. Hawaii Med J 53. Centers for Disease Control. Tetrodotoxin poisoning associated with eating puffer fish trans45:389, 1996. ported from Japan-California, 1996. MMWR 54. SG Bradley,LJ Klika. A fatal poisoning from the Oregon rough-skinned newt(Trrrichtrg m r w losa). JAMA 246:247, 1981. 55. K Sun. J Wat, P So. Puffer fish poisoning. Anaesth Intensive Care 22307, 1994. 56. FL Lau. CK Wong, SH Yip. Puffer fish poisoning. J Accid Emerg Med 12:214, 1995. 57. MJ Cooper. Ciguatera and other marine poisonings in the Gilbert Islands. Pacific Sci 18:41 1, 1964.
58. AJ Trevett. B Mavo, DA Warrell. Tetrodotoxic poisoning from ingestion of a porcupine fish (Diotlorr hy.srri.v) in Papua New Guinea: nerve conduction studies. Am J Trop Med Hyg 56: 30.1997. 2673, 1951. 59. C Duncan. A case of toadfish poisoning. Med J Aust 60. J Tibballs. Severe tetrodotoxic fish poisoning. Anaesth Intensive Care 16:215, 1988. 61. S Laobhripatr. K Limpakarnjanarat, 0 Sangwonloy. S Sudhasancya,BAnuchatvorakul, S Leelasitorn, K Saitanu. Food poisoningdue to consumption of the freshwater puffer Tetrclotlorl $mgi in Thailand. Toxicon 28: 1372, 1990. J Med Assoc 62. J Kanchanapongkul. W Tantraphon. Pelagic paralysis from puffer fish poisoning. Thai76:285. 1993. 4031, 198.5. 63. PCW Lyn. Puffer fish poisoning: four case reports. Med J Malaysia 64. K Oda, K Araki, T Totoki. H Shibasaki. Nerve conduction study ofhuman tetrodotoxication. Neurology39:743, 1989. 65. T Narahashi. Chemicals as tools in the study of excitable membranes. Physiol Rev 54:813, 1974. 66. J Bell. S Bolanowski, MH Holmes. The structure and functionofpacinian corpuscles: a review. Prog Ncurobiol 42:79, 1994. I n : M Thompson, R 67. AK Agarwal. Effect of Inarine toxins on heart mitochondrial function. Sarojini.andRNagahhushanam,eds.Bioactivecompoundsfrommarineorganisms.New Delhi, India: Oxford, 1991, p. 14.5. 68. JR Clay. AS Kristof. J Shenasa, RM Brochu. A Shricr. A review of the effects of three cadi-
Tetrodotoxin
69.
70. 71.
72. 73.
74. 75. 76. 77. 78. 79. 80. 81.
82. 83.
x4. 85.
86.
87. 88. 89.
285
oactive agents on theelectricalactivity from embryonicchickheart cell aggregates: TTx, ACh, and E-4031. Prog Biophys Mol Biol 62:185, 1994. FCT Chang, DL Spriggs,BJ Benton. SA Keller. BR Capaciao. 4-aminopyridine reverses saxitoxin (STX)- and tetrodotoxin (TTX)-induced cardiorespiratory depression in chronically instrumented guinea pigs. Fund Am Appl Toxicol 38:75, 1997. MA Burack, SF Stasheff, WA Wilson. Selective suppression inofvitro electrographic seizures by low-dose tetrodotoxin: a novel anticonvulsant effect. Epilepsy Res 22: 1 15, 1995. CP Taylor, PR Patrylo, ML Weber. JL Bloomquist, FE Dudck. Na’ channel blockers reduce prolonged seizure-like activity in low-[Ca”],, solutions. Epilepsia 37(suppl 5):21 (Abstracts), 1996. CP Taylor, LS Narasimhan. Sodium channels and therapy of central nervous system discases. AdvPharlnacol 3947. 1997. SM Goldin, K Subbarao, R Shartna. AG Knapp. JB Fischer, D Daly, GJ Durant, NL Reddy, LY Hu, S Magar.MEPcrlman,J Chen, SHGraham, WF Holt.DBerlove,LDMargolin. Neuroprotectiveuse-dependentblockersofNa-and Ca” channelscontrollingpresynaptic release of glutamate. Ann NY Acad SCI765229, 1995. T Narahashi, JW Moore, WR Scott. Tetrodotoxin blockage of sodium conductance increase in lobster giant axon. J Gen Physiol 47:965. 1964. JM Ritchie, RB Rogart. The binding of saxitoxin and tetrodotoxin to excitable tissue. Rev Physiol Biochem Phartnacol 79:1, 1977. CY Kao, A Nishiyama. Actions of saxitoxin on peripheral neuromuscular systems. J Physiol (Lond) 180:50,1965. a structural hypothesis. Biophys J 15: B Hille. The receptor for tctrodotoxin and saxitoxin: 615, 1975. RY Tsien, DPL Green, SR Levinson. B Rudy, JKR Sanders. A pharnwmlogically active derivative of tetrodotoxin. Proc R Soc B I91 :555, 1975. ML Roy, E Reuveny, T Narahashi. Single-channel analysis of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channelsin rat dorsal root ganglion neurons. Brain Res65034 I , 1994. S Yoshida. Tetrodotoxin-resistr\nt sodium channels. Cell Mol Neurobiol 14:227, 1994. PF Baker, KA Rubinson. Chemical modification of crab nerves can make them insensitive to the loco1 anaesthetics tetrodotoxin and saxitoxin. Nature 257:4 12, 1975. N Ogata, H Tatebayashi. Ontogenic development of the TTX-sensitive and TTX-insensitive Na’ channels in neurons of the rat dorsal root ganglia. Dev Brain Res 65:93, 1992. ML Roy, T Narahashi. Differential properties of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons. J Neurosci 12:2104. 1992. JB Arbuckle. RJ Docherty. Expression of tetrodotoxin-resistant sodium channelsin capsaicinsensitive dorsal root ganglion neurons of adult rats. Neurosci Lett 185:70, 1995. AN Akopian, L Sivilotti, JN Wood. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 379:257, 1996. SD Novakovic.ETzoumaka.JGMcGivern.MHaraguchi.LSangameswnran, KR Gogas, RM Eglen. JC Huntcr. Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions. J Ncurosci IX:2174. 1998. M Noda, H Suzuki, S Nunla, W Stuhmer. A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II. FEBS Lett 259:213. 1989. H Terlau, SHHeincrnann,WStuhmer,MPusch,FConti,KImoto. S Numa. Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel 11. FEBS Lett 29393, 1991. J Satin. JW Kyle, M Chcn, P Bell. LL Cribbs. HA Fozzard, RB Rogart. A mutant of tetrodotoxin-resistant cardiac sodium channels with tetrodotoxin-sensitive properties. Science 256:
1202, 1992. 90. J Satin. JT Limberis, JW Kyle, RB Rogart, HA Fozzard. The soxitoxin/tetrodotoxin binding site on cloncd rat brain Ila Na channels is in the transmembrane electric ficld. Biophys J 67:
1007, 1994.
286 91.
Yoshikawa-Ebesu et al. 1 Favre. E Moczydlowski, L Schild. Specificity for block by saxitoxin and divalent cations a t
a residue which determines sensitivity to sodium channel subtypcs to guanidinium toxins. J GenPhysiol 106:203. 1995. 92. L Sivilotti, K Okuse, AN Akopian, S Moss, JN Wood. A single serine residue confers tetrodotoxin inscnsitivity on the rat sensory-neuron-specific sodium channel SNS. FEBS Lctt 409: 49, 1997. 93. GM Lipkind, HA Fozzard. A structural model of the tetrodotoxin and saxitoxin binding site of the Na' channcl. Biophys J 66:1, 1994. 94. B Hille. Ionic channels of nerve: qucstions for theoretical chcmists. Biosystems 8:195, 1997.
9 Epidemiology of Seafood Poisoning
I. Introduction 288 11. Epidemiologic Overview 288 Epidemiology A. 288 B. Epidemiology of seafood poisoning 289 Surveillance C. 290 111. EpidemiologyofSeafoodPoisoning291 1V. EpidemiologyofSeafoodPathogens292 Bacteria A. 292 B. Viruses 296 C. Parasites 297 D. Allergies 297 Toxins E. 298 V. Prevention 303 Environmental A. 303 B. Education 304 VI. Conclusions 304 References 306
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INTRODUCTION
Seafood poisoning accounts for a largeandgrowingproportion of all food poisoning incidents. In the United States, fish, shellfish, and other marine organisms are responsible for at least 1 in 6 food poisoning outbreaks with a known etiology, and for 1.5% of the deaths associated with these particular outbreaks. The proportion of foodborne illnesses attributable to seafood, based on reporting for 1988-1992, has markedly increased over the previous decade, when seafood consumption was associated with 10% of foodborne illnesses with an identified etiology (1-4). In other parts of the world, the impact of seafood poisoning is even greater. In the period 1971-1990, seafood was the single most important vehicle in food poisoning outbreaks in Korea (32%) and Japan (22%), where seafoodwasresponsiblefor 43% and 62%, respectively, of outbreak-relatedfatalities (.5,6).As with general food poisoning, seafood poisonings share the following epidemiologic characteristics: ingestion as the primary route of exposure, a wide variety of etiologies (bacterial, viral, parasitic, and toxin), significant underreporting, and an apparently increasing incidence in human populations. This chapter reviews the general epidemiologic principles and issues relatedto seafood-borne disease, explores reasons for the apparently increasing incidence of seafood poisoning, and summarizes the epidemiology of bacterial, viral, parasitic, and toxin-related seafood illnesses.
II.
EPIDEMIOLOGIC OVERVIEW
A.
Epidemiology
Laboratory scientists establish causation by manipulating the laboratory environment. Epidemiologists most often establish causation by observing the natural environment, specifically by assessing naturally occurring patterns of disease in populations. Often, surveillance information provides the first evidence of an unusual pattern. Epidemiologists then design studies to identify differences between ill people and well people that could explain why the disease occurred. T o prove epidemiologic causation, the following are important: an appropriate time to the agent under study), strengthof associasequence (i.e., the disease follows exposure tion (people with the exposure are more likely than those without the exposure to get disease: the greater this increased risk, the greater the evidence for causation). evidence of a dose-response relationship (the probability of illness increases as the exposure increases), biologic plausibility, consistency with other studies, and laboratory evidence (such as toxicologic) that supports the causal relationship (7-1.5). For a seafood poisoning outbreak, it is important to show that the persons who became ill were infected with the suspect organism and had consumed the appropriate seafood vehicle before they became ill: that the attack rate (percentill) was higher among those who consumed the seafood than among those who abstained, and was highest among those who ate the most seafood; and that as the seafood vehicle was contaminated with the specific pathogen or toxin. Of note, with many foodborne diseases, seafood-borne poisonings often appear as disease clusters (i.e., unusual increasesin morbidity or mortality in time and space), since seafoodis often shared among families and friends as well as through commercial venues such as restaurants (13,161.
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B. Epidemiology of Seafood Poisoning Key epidemiologic activities in establishing causation are (a) identifying a target population for study, (h) formulating a case definition, (c) tneasuring exposures of interest, and (d) comparing exposures or outcomesi n subgroups within the target population (see Fig. 1). Ideally the target population is at high risk for both exposure and disease, and thus has a high incidence of disease. In the case of seafood poisonings, a target population would consume seafood contaminated with the agent under investigation. In acute outbreaks, the target population is clear: the group within which the outbreak is occurring. In order to nleasure the amountof disease causedby a specific exposure in the target population, a case definition is required. The case definition may be based on historical a consensus or developed for the purposes of a particular investigation. For example, classic case definition for fish-associated scombroid seafood illness would be the acute onset of gastrointestinal symptoms and upper torso skin rash (both eliminated by antihistamines) within minutes to hours of eating fish. To the extent possible, the case definition should also incorporate some objective measurements such as biomarkers to reduce misdiagnosis. The case definition should also have clinical relevance so that it can be used in the diagnosis and reporting of disease. An exposure isany agent(chemical,biological,etc.)orcharacteristic (a human behavior, a particular genotype) suspected of causing or increasing the risk of disease. In the epidemiology of seafood poisoning, exposures usually are physical agents, such as chemicals or bacteria. Measures of these exposures and their surrogates, physiologic effects, are known as biomarkers (73). Biolnarkers have been divided into markersof exposure and markers of effect. Biotnarkers of exposure are the actual levels of the toxins or their metabolites in body fluids and seafood, such as methyl mercury in the hair of a person who consumes contaminated fish, or identification of a specific virus in the stool of persons suffering from seafood-associated gastroenteritis (17,18). Markers of effect are indicators of subclinical physiologic change, such as conduction changes in the peripheral
Establish the presence o f an outbreak. Confirmthediagnosis. Develop a CLISK definition. Have informal conversations with affected persons to generate hypotheses. S. Develop a questionnaire. 6. Administer the questionnaire to well and ill persons. 7. Look for associations between exposllres and ihless: cakUlatK n lncasure of effect (risk ratio or odds ratio). X. Based on #7. identify a vehicle or transvector. 9. Test the suspected vehicle for the organism.
I. 2. 3. 4.
Tips:
Avoid testing foods a t random before a pathogenic agent is idcntifed in patients and an association between illness and specific food(s) is established. Always collect sanlples of stool/vomitusfromill persons. Keep some stool samples fresh, unpreservcd, unfrozen, so they can be tested for viruses. (Refs. 12-14. 16. 21)
Fig. 1 Steps intheinvesLigation
of n seafood-borneoutbreak
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nerves of persons suffering from the fish toxin disease ciguatcra (19.20). I n addition to biomarkers,epidemiologistsuseotherIneasurements of exposure such as information about food consumption and symptoms associated with a seafood poisoning outbreak collected through questionnaires (21). The final step in an epidemiologic investigation is the calculation of a measure of effect that indicates how much more likely exposed people in the target population are to developdiseasecompared totheunexposedpeople inthetargetpopulation. These measures usually are the risk ratio or the odds ratio, both of which can be interpreted to be the attack rate (or incidence of disease)in the exposed population divided by the attack rate in the unexposed population.
C. Surveillance Surveillance is the ongoing and systematic collection, analysis, interpretation, and dissemination of health-related data. This information isused to plan, implement. and evaluate public health interventions ( 1 1-15,21). With regard to foodborne disease, surveillance permits the early detection of new or newly recognized diseases; early identification of outbreaks so they can be contained (see Figs. 1 and 2): the identification of specific food sources of infection; and the conduct of studies to develop new ways to keep foods clean. Surveillance for seafood poisoning involves two groups of activities: collection of information about human illnesses, and monitoring of marine habitats for evidence of contamination, Data on human illnesses come primarily from traditional passive surveillance programs based on state laws that require clinicians and laboratories to report cases of specific diseases (e.g., paralytic shellfish poisoning, ciguatera, Vibrio infections, tetro-
On July 30, 1987. an outbreak of an acute neurologic illness occurred in Champcrico (population 689 1 ) on the coast of Guatemala ( 2 2 ) . Patients con1plained of headache. numbness o f face and extremities,difficultywalking,dizziness,andother synlptonls. Twenty-sixof 187 cases died o f respiratory arrest within hours of onset. To establish the characteristics and sizeof the outbreak. initially a “case” was broadly defined onset betwccn July 23 as an acute illness in a person with at least two neurologic symptoms with and August 7. Subsequently for the formal epidemiologic study. the case definition was narrowed to an acute illness with headache, numbness o f two or n~oreareas o f the body, and a t least two 0 1 five specific symptoms. Nineteen households, each with sick and well persons in it, were interviewed with a food questionnaire. Colnparison ofCoods eaten at lunch on July 30 showed that clams were associated with the illness. The shellfish were gathered at local beaches by individual families. Fifty-six of S7 patients reported eating clams, compared to S of41 controls. The odds ratio for clam soup was 38.5 [95% confidenceinterval (CI) = 4.6 325.01, signifying that cases werealmost 40 tinm more likely compared with well family members to have eaten clam soup. Soup and clanls were tcstcd using a mouse bioassay and found to contain saxitoxin [the chemical agent of paralytic shcllfish g of clan1 meat; the lethal dose was calculated :IS poisoning (PS€‘)) at levels up to 30,000 1~.U/100 11.000-35,000 pU.Toxin was also isolated from the stomach contents of a fatal casc. at three samplingsitcs along After this epidenlicof PSP, shellfish surveillance was established the Pacific coast. There was also increased awareness anlong thc fishernlen, consuI11ers. and health care providers of the potential seafood consumption. ~
Fig. 2 Seafood-borneoutbreakcasestudy
Epidemiology of Seafood Poisoning
29 1
dotoxin poisoning)to health departments ( 1,23). Health departmentsuse these case reports to monitor long-term trends in disease incidence (i.e., the number of new cases of disease i n a defined population during a specific time period) and to identify disease clusters that may signal outbreaks. Marine habitat surveillance involves monitoring of shellfish beds for paralytic shellfish poisoning (PSP), infectious organisms such as Vibrio species, and toxins (16,241. One example of surveillance techniques at work was the January 1995 closure of oyster beds in Florida’s Apalachicola Bay after studies traced multiple disease clusters of Norwalk viralgastroenteritis to theconsumption of oysters from the bay. Subsequent investigation revealed an earlier outbreak among oyster harvesters, who may have spread the infection to oyster eatersby dumping raw sewage from their boats into the bay, where the oyster beds became infected (25,26). Although the exact temperature and length of time of cooking were uncertain, studies conducted during this investigation also raised doubts about the usefulness of cooking as the sole means of preventing infection (27).
111.
EPIDEMIOLOGY OF SEAFOOD POISONING
One factor in the apparent increasing incidence of seafood poisoning is the worldwide increase in seafood consumption (28). In the United States, the average annual per capita consumption of commercial seafood increased from 5.7 to 6.7 kg from 1980 to 1992. In Canada, total per capita seafood consumption increased from 6.6 to 7. I kg from 1980 to 1990 (29,30). This consumption is not evenly distributed among populations. Degner et al. (28) studied a random sample of 8000 Florida households by telephone survey and found that the avcrage annual seafood consumption was 7.6 kg (with finfish consumption of 6.0 kg) 33% of 1071 peoplesurveyedfromMiami(30). However, in thel-weekrecall,only Dade County consumed marine tinfish (31). Different ethnic subpopulations also have different seafood consumption and preparation patterns. For example, many Southeast Asian and South American groups traditionally eat raw fish, while northern Europeans consume fish preserved by pickling. These cultural differences have important implications for prevention and control. Health warnings that are not culturally targeted and i n the appropriate language may not reach these ethnic subpopulations. For example, locally caught fish contaminated by heavy metals such as mercury are often consumed by ethnic subpopulations that may not have been informed about the dangers (14,lS). In addition, many populations, including ethnic subpopulations, believe that the consumptionof raw seafood is healthier and/or has aphrodisiac properties (as with raw oysters or fugdpufferfish) ( 3 2 3 ) . Seafood poisoning also varies etiologically by vehicle or transvector. In the United States during the period 1983-1992,fish was the transvector for scombroid and ciguatoxin, while shellfish was the transvector for Norwalk-like viruses, paralytic shellfish poisoning, Vibrio species, and hepatitis A virus ( 1 J ) . Seafood poisoning has certain transmission mechanisms in common with all foodborne illness. One is improper food handling. I n 1992, the Centers for Disease Control and Prevention (CDC) recorded 240 foodborne outbreaks witha known etiology (51% of total outbreaks reported). For those outbreaks, the contributing factors included ilnproper holding temperatures (62%), inadequate cooking (29%). contaminated equipment ( 18%), food from unsafe sources (7%), and poor personal hygiene (29%) (1). For seafood, as for other types of food, the relative importance of restaurant and
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other commercial establishments versus private food sourcesin the epidemiology of food poisoning is unclear. Twenty percent of the seafood consumed is derived from recreational and/or subsistence fishingthatisoutside most public health controls (2). Yet the true contribution of this food source to food poisoning is unknown because outbreaks involving these sources are less likely to be reported than restaurant outbreaks. A further global problem with all foodborne illness outbreaks is that,in nonendemic areas, medical personnel often do not recognize the illness or know of reporting requirements, further clouding the true epidemiologic picture due to significant under-reporting (1,2,16,32,33). As with other foodborne pathogens, the internationalization of the food supplymore than 50% of the commercial seafood eaten in the United States is imported-has greatly facilitated the transmission of seafood pathogens. National and international commerce allows forthe rapid and wide geographic spreadof contaminated seafood. Examples range from choleraon international airplanes to Norwalk-like gastroenteritis in five different states associated with the same contaminated collection site (17,27,34-40). The pathogenic organisms themselves can cross national and international boundaries, as seen with the spread of bacterial and dinoflagellate species through the dumping of ballast and bilge 3) (34,39,40). water from international shipping (see Fig. of public These new transmission dynamics have had a major impact on the practice a public health standpoint, the tracing and recall of contamhealth and epidemiology. From inated seafood is much more difficult when the product is widely distributed, and therefore the potential for prolonged outbreaks is much greater (1 7). Epidelniologically it is much more difficult to study human disease when the target populations become diffuse. In addition to the transmission mechanisms it shares with other foodborne illnesses, seafood poisoning has unique modes of transmission related to the marine environment. Discharge of treated and untreated human fecal material, other sewage, and industrial wastes into estuaries and coastal watersis extremely common, even in developed nations. The coastal waters of the United States receive more than 3 X 10" L of municipal sewage each day. Viruses can settle out of the water column and attach to sands, clays, aquatic life forms, and sediments, accumulating in the loose layer over the compact bottom sediment. This layer can be easily resuspended after storms, dredging, or even boating; tidal currents can then transport these resuspended viruses to distant waters (41). Shellfish, especially the filter feeding bivalve mollusks (oysters, scallops, mussels, clams, cockles), live in estuarine areas and obtain their food by filtering large amounts of water. A wide variety of organisms and toxins pathogenic to humans can accutnulate in the shellfish alimentary tract, especially if the filtered water is contaminated by sewage or chemical pollutants. After contamination it can take hours to weeks of filtering in uncontaminated water before these pathogenic organisms are removed. Since the alimentary tract of these bivalves fonns the major edible portion for humans, these mollusks can serve as extremely effective vehicles fora wide range of organisms and toxins pathogenic to humans (30,42,43). The other commonly consumed group of shellfish is the crustaceans (e.g., shrimp, crab, and lobsters). Although not filter feeders, they can acquire contamination from contaminated water.
IV. EPIDEMIOLOGY OF SEAFOOD PATHOGENS
A.
Bacteria
Traditionally the most frequently reported seafood poisoning outbreaks (Table I ) have been associated with bacteria. Although increased sophisticationof laboratory testing now
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implicates viral origins, new bacteria-associated seafood poisonings are appearing due to the increasing worldwide consumption of seafood by sensitive subpopulations.
1. Vibrios Vibrios are natural inhabitantsof marine environments. Pathogenic marineVibrio species, and V.wltl$cus, most commonlycause especially V. cholercre, V.parcrktrer~lol~~tic~rs, disease when they are ingested in raw shellfish and, less commonly, i n fish (44,45). Many Vibrio species are not associated with fecal contamination, so the use of fecal colifornl counts as clean water indicators does not ensure that water or seafood beds are free of pathogenic vibrios (3). Cholera, caused by the toxin-producing V. cholerae, can be a severe infection with mortality rates as high as 50%, but is more commonlyan asymptomatic infection. Seafood is the most common vehicle of outbreaks. The infection can be acquired through fecal contamination of food by a person who is ill or an asymptomatic carrier, through seafood harvested in a sewage contaminated area, or through seafood gathered i n natural environmentalreservoirs of V. cholerrre. Nevertheless,epidemiologicstudieshaveshownthat unhygienic food handling in the home or on the street are the most common sources of seafood-borne cholera (34,46). Cholera has the potential for rapid international spread, as evidenced by the seven pandemics since 1817, including the recent Peruvian epidemic. In a particularly dramatic example of the potential for the spread of disease through international travel, 75 people developed cholera after taking an airplane flight from Latin America to California during which they consumed a cold seafood salad plate from Lima, Peru (35-37). In another example of international spread, shellfish beds in the United States have been contaminated of ships arriving from Latin with V. cholercre transported in the bilge and ballast water America (34,40). V. pcrrahaemo1~tieu.sis associated with the consumption of inadequately cooked or refrigerated crustaceans and fish (32). In Japan, upto 50% of reported foodborne outbreaks have been caused by V. ~~crrcrIrhnen~o1~ticu.s; in Guam, the risk of laboratory confirmed V. ptrrrrhtretnol~tie~~.s food poisoning was highly associatedwith recent seafood consumption, especially fish (OR = 37.59, C1 8.30-220.2). In the United States, V. vuln~ficusis rare outside the coastal states of the Gulf of Mexico, but is relatively common in these states; Florida recorded 141 cases during the period 1981- 1993. This organism can be associated with mortality as high as 50% in persons who are immunocompromised or have liver disease; the risk of oyster-related V. ~ulw$cus infection is 80 times greater i n adults with a history of liver disease or who of infection from eating oysters is have chronic alcoholism (2,26,43,45-48). The risk highest in the months April through October, when more than 90% of raw oyster-associated V. vulw$cus infections occur (49). Florida and other states require restaurants that serve raw oysters to post warnings about the dangers of consunling raw oysters. Although not directly seafood consumption,V. vuln$cu.s also can cause wound infectionsin persons who go wading or swimmingin contaminated waters. In Florida, during the period 19811993, one-third of the 141 V. w l n @ x . s infections were wound infections (49). 2. Listeria In New Zealand and elsewhere, Listcrirr ~lowoc~togetles, found on the external surfaces of fresh and frozenfish as well as in the processing plant environment, has been implicated in several seafood-borne outbreaks of listeriosis. These outbreaks have included perinatal infections associated with transmission from infected mothers, who may be asymptomatic
Table 1 Reported Seafood Poisoning Outbreaks by Etiology Etiology
Seafood transvector
Clinical presentation
Reference
Mollusks Mollusks, crustaceans, fish Mollusks Mollusks Shellfish. seafood
Septicemia Gastroenteritis, septicemia (at risk immunocompromised, liver disease) Gastroenteritis Gastroenteritis Gastroenteritis (at risk inimunocompromised)
27.43. 108 43, 108 29. 43. 52
Shellfish Shellfish Shellfish. seafood Seafood
Gastroenteritis Gastroenteritis Gastroenteritis Listeriosis
43 43 42. 43. 55 50, 51
Mollusks Shellfish
Hepatitis Gastroenteritis
42. 53. 62, 63, 108 17. 18, 26. 27. 53. 60. 101, 108
Shellfish Shellfish
Gastroenteritis Gastroenteritis
43 43
Fish (raw) Herring, cod. whiting. haddock. salmon Fish
Abdominal discomfort. eosinophilia. allergy
32. 43. 64-66, 70
Abdominal discomfort, eosinophilia. allergy. eosinophilic meningitis
66
Bacterial Sulnioriellu (typhi. purutyphi) Vihrio fcholerue. purcrhcreniolyticit.s. niimicus, hollisue. j m i a l i s , vultiij?cirs) Sli ig ellu Cuniphylobucter Aeromotius hydrophila, veronii sohriu, cuviue Bacillus cereus EdLcw-dsiellu turdu E. coli (including enterotoxigenic) Listeriu nioriocyrogeries Viral Hepatitis A Small round structured viruses, Nonvalklike viruses (Nonvalk. cockle. snow mountain. calicivirus) Rotavirus Astrovirus Parasitic Airisrikis
Giiut hosfonia
29. 43, 57, I08 35, 42-44, 48, 53. 68. 108
z! 3. 3 0
m
M
22
W
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E
W
m
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(50,Sl). A wide range of seafood transvectors have been associated with these reported listeriosis outbreaks, from raw fish and shellfish to ready-to-eat (but insufficiently processed) cooked shrimp and crabmeat, and smoked mussels.
3. Salmonella Strltnonelltr and Aeromonrrs species are frequent contaminants of raw fish and shellfish, especially if the seafood has had prolonged exposure to elevated temperatures. The risk of transmission is higher for shellfish than for fish because shellfish often are refrigerated for many days without obvious spoilage andthen eaten raw (29,52). As with Vibrio infections, persons who are elderly, immunocompromised, or very young are at greater risk I C I S , enteropathofor more severe gastroenteritis after infection with A ~ ~ O I I I O Iespecially genic strains. 4. Other A wide spectrum of bacterial species has been cultured from shellfish and fish without definitive etiologic connections to seafood poisoning outbreaks ( 5 1-55). Some of these organisms, like Arrornonas, are part of the nonnal flora of the seafood. Others are more likely introduced by unhygienic hutnan handling; enterotoxigenic Esclwrichitl coli, the conmon pathogen of travelers' diarrhea, and Stc~~?ltr'loc.occu.s rwreus have both been detected on seafood (52-56).
5. Future Threats Internationaltradeinwild-harvestedseafood,andincreasinglyinaquacultureseafood products, has facilitated the introduction of pathogens into new geographic areas, as well as into seafood and hunlan communities (30). For example, Strlmonellcr crgoncr was first introduced to Europe following the importation of Peruvian fish meal, with subsequent rapid spread into other food products, producingan increase in human outbreaks. Furthermore, the intensive use of antibiotics in the aquaculture industry, leading to antibiotic accurnulation in seafood tissues, increases the potential for the development of multiply resistant bacteria (57,58).
B. Viruses As discussed above, shellfish harvested in waters contanlinated with raw or inadequately treated sewage are extremely efficient transvectors of seafood pathogens because the shellfish filter the water as they feed, concentrating the pathogens (1,2,29,59). Paradoxically refrigeration increases the pathogens' geographic range, permitting transportof apparently healthy shellfish to many geographic areas, thus extending and prolonging outbreaks. Inadequate procedures for tracing and recallof contaminated seafood also serve to extend outbreaks ( 1 7,18,27).
1. Small Round Structured (Norwalk-Like) Viruses In theperiod1978-1987,approximatelyone-third of seafoodoutbreaks intheUnited States and two-thirds of all cases were associated with consumption of raw molluscan shellfish,accordingto CDC and FDA records (2). In themajority of thesecases,the pathogens were snlall round structured viruses (SRSVs) (26,29,43). SRSVs,orNorwalk-likeviruses,areclassifiedascalicivirusesandarecoInmon causes of outbreaks of gastrointestinal illness in the United States. Because the infectious dose is small, cooking theshellfishdoesnotreliablyeliminatetherisk of contracting
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gastroenteritis. For example,i n an outbreak of Norwalk-like gastroenteritis in North Carolina following consumption of oysters at a church supper, the attack rate (56%) did not differ based on whether the oysters were eaten raw or steamed for at least 12 minutes. Outbreaks of Norwalk-likegastroenteritishavebeenreportedaftergrilling,stewing, steaming, and fryingthe shellfish (26.27.60). Furthermore, routine water quality tests from the areas of contaminated shellfish collection were within normal limits, demonstrating that fecal coliform monitoring is inadequate protection for SRSV contamination (27,61).
2. Hepatitis A The proportion of hepatitis A cases in the United States that are attributable to foodborne or waterborne outbreaks is small (7.5% in the period 1983-1989) (43). Nevertheless, the most common causeof these outbreaksis the consumptionof raw or inadequately prepared shellfish taken from sewage-contaminated waters (32,42,62,63). As with other seafoodrelated pathogens, suchas V. clwlerae, seafood poisoning with hepatitis A has been associated with a significant geographic spread due to seafood export patterns. For example, an outbreak of hepatitis A after consumption of raw oysters from Florida resulted in cases in five different states, including Hawaii (38). C.
Parasites
Consumption of raw and inadequately cooked seafood, especially in certain ethnic subpopulations, is associated with parasitic infections, particularly with anisakids and cestodes (232). Correct food preparation and handling will eliminate the majorityof these diseases.
1. Anasikiasis Anasikiasis is a rare nematode (roundwoml) infection acquired through the consumption of rawfish(especiallycod,herring,mackerel,andsalmon)andcephalopods(such as squid). Traditionally associated with Asian cultures, because of changing consumption patternsanasikiasisisnowreported in Europeand the UnitedStates,although rarely (64,65). Although often asymptomatic, patients can presentwith eosinophilia, abdominal discomfort, invasion, or hemorrhage dueto burrowing of the womi, as well as granulomatous and frank allergic reactions (32,43,65). A similar disease associated with the helminth Gntrrhostomn has been reported in Southeast Asia and the Middle East.
2. Diphyllobothriasis Diphyllobothriasis or fish tapeworm disease was traditionally associated with gefilte fish preparation by Jewish women. Although often asymptomatic. megaloblastic anemia (secondary to vitamin B I Zdeficiency) and eosinophilia are classic findings. Approximately 10% of people in Scandinavia are reportedly infected with Diphyllobothrium. Although usually associated with freshwater fish consumption, diphyllobothriasis has been reported with the ingestion of raw Pacific salmon (32,66).
D. Allergies Seafood allergies include true food allergies (i.e., type I IgE-mediated hypersensitivity) and food intolerance (i.e., nonimmunologic hypersensitivity) (67,68). It is estimated that 3% of 3-year-old Finnish children are allergic to fish, and the prevalence of fish allergy is close to 1 in 1000 in the general Norwegian population (67). In a study in Spain of 3034 persons over 14 years old seen as outpatients in an allergy unit, skin testing and/or
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RAST testing found 30 (0.98%) persons with a possible food allergy, with 14.9% related to seafood (especially shellfish) and 4.2% to fish. Symptoms ranged from acute cutaneous disorders to gastrointestinal complaints to respiratory symptoms (69). In South Africa, among 105 persons with suspected fish allergy, adverse reactions were reported with shellfish [prawns (46.7%), crayfish (43.8%), abalone (35.2%), and black mussels(33.3%)] and withfish [hake(24.8%),yellowtail(21.9%).salmon(15.2%),andmackerel(15.2%)]; these reports were confirmed by double-blind, placebo-controlled food challenge (67). Allergic reactions, ranging from urticaria to anaphylaxis, have been reported after consumption of seafood with parasitic or certain natural toxin (i.e., scombroid) contamination. Allergic reactions after seafood ingestion have been reported even with negative skin or exposure test to fish or shellfish; these are often associated with parasitic infestation and specific allergic reaction rather than seafood-associated allergy. In particular, chronic intermittent allergic reaction type I and/or I11 with eosinophilia have been reported after the consumption of Aniscrkis-parasitizedfish. The ingestion of even safely cooked but to allergicreactionswithIgE-mediatedsensitization Anisakis-parasitizedfishhaslead (64,65,70).
E. Toxins The marine environment contains a variety of chemicals, both natural and man-made,that can be acutely and/or chronically toxic to humans if ingested in large enough quantities. Unfortunately the marine organismsthat humans prefer as food tend to concentrate toxins through a process known as bioaccumulation. Many of the fish that humans eat are at the top of the marine food chain; that is, they are predators (salmon) or even predators of predators (sharks). Toxins from the smaller organisms these predators eat accumulate in the predators’ tissues (particularly if the toxins are lipophilic), and often reach levels high enough to cause acute or chronic disease in humans. The largest and oldest fish of each species contain the most toxin, and also tendto be the most desired by seafood consumers. Molluscan shellfish, also prized as a food source, are filter feeders, concentrating natural and chemical toxins indiscriminately (2,43,7 1,72). Often the toxins that marine organisms accumulatehave no obvioushealtheffects on thefish andshellfish,thustheyremain available for capture for human consumption, and they appear healthy and appetizing to the consumer. Among the diseases caused by natural toxins, only scombroid, botulism, ciguatera, PSP, and tetrodotoxin (fugu) are required to be reported to the CDC (1). Individual states mayrequirereporting of other diseases (26). Therefore, as withallthe seafood-borne diseases, under-reporting is a major epidemiologic issue.
1. Bacteria The seafood diseases caused by bacterial toxins generally are associated with improper food preparation, handling, and storage. Scombroid poisoning is probably the most commonly reported fish-associated illness in the United States (1,3,32,67,68,73). It is often mistakenly diagnosed as “fish allergy” because the symptoms resemble an IgE-mediated reaction and respond rapidly to antihistamines. Scombroid poisoning actually is caused by bacterial overgrowth associated with inadequate fish storage, especially of fish i n the Scotnbroidae family (i.e., tuna, mackerel, and jacks)as well as mahi-mahi or dolphinfish, bluefish, and sardines. The surface bacteria (e.g., halophilic Vibrio spp., Proteus, Klebsiella, Enrerobacter) decarboxylatethehistidinepresent naturallyin dark-meat fish to
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produce high levels of histamine, optimally when the temperature is between 20°C and 30°C. The phenomenon occurs with consumption of fresh and canned fish. The bacteria Clostridium botulinutn type E is prevalent in both freshwater and marine environments. The bacteria produce botulism toxin E on smoked fish, fish eggs, and uneviscerated and salted whitefish. The majority of the cases are associated with inadequate cannillg procedures. Although smoking or light cooking may not kill the bacterial spores, the toxin is heat labile. The toxin produces an acute gastroenteritis followed by cranial nerve dysfunction and symmetrical descending weakness. Although rare, this disease is OccasionalIy seen among ethnic minority communities and with home canned fish (2,32,33,68). Stcll,hy/ococcu.sL I U ~ P U elaborates S an enterotoxin on improperly stored seafood, especially if the fish is garnished with cream sauces or mayonnaise (63).
2. Marine The marine toxin diseases are caused by a myriad of natural toxins produced by minute organisms such as dinoflagellates and diatoms. These phytoplankton are part of the base of the marine food web and are ubiquitous in the marine world (74). Humans are most often exposed through consumption of fish and shellfish that accumulate these toxins. The toxins, small nonpeptides, are some of the most powerful natural substances known; ciguatoxin is toxic to humans in a total body dose of 70 ng. Because these toxins are tasteless, odorless, andheat and acid stable, normal screening and food preparation procedures will not prevent intoxication if the fish or shellfish is contaminated (39,75). The marine toxin diseases are categorized into two groups based on their primary transvectors. Shellfish harbor the toxins that produce paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), diarrheic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). Fish carry the toxins responsible for ciguatera poisoning and tetrodotoxin (fugu or puffer) poisoning. The shellfish-associated diseases generally occur in association with algal blooms or “red tides,” which may be characterized by patches of discolored water and dead or dying fish. The fish-associated diseases are more localized to specific reef areas (ciguatera poisoning) and fish (fugu poisoning). Underdiagnosis and underreporting, especially in endemic areas, make it difficult to know the true worldwide incidence of the marine toxin diseases. For example, it is believed that ciguatera affects at least 50,000-100,000 people per year who live in or visit tropical and subtropical areas, but there is significant underreportingof this relatively common marine toxin disease in endemic areas (16,39). The primary target of marine toxins is the neurologic system, although affected individuals usually present witha wide rangeof symptoms, resultingin a confusing clinical picture. Gastrointestinal symptoms begin minutes to hours after eating contaminated seafood. In the case of PSP, fugu, and ciguatera, accompanying acute respiratory distress may be fatal within hours. Ciguatera and ASP may also produce debilitating chronic neurologic symptoms lasting monthsto years. Chronic disease (neurologic, immunologic, etc) associated with the marine toxins is an area of active scientific research. I n the past, these illnesses have been highly localized to endemic island and coastal communities. With increasing worldwide seafood consumption and trade,as well as international tourism, these diseases are expanding beyond their traditional geographic boundof diagnosis and treatment of disease in aries. One side effect has been the high costs traditionally nonendemic areas. In Canada, with an estimated 1000 casedyear related to tourism and food importation, the average medical cost per case of ciguatera was $2470 in 1990 (76,77).
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In addition to increased worldwide seafood consumption, other anthropogenic factors may have helped spread the dinoflagellates and their toxins. Human-assisted transportation of the dinoflagellates or their cysts occursin spat cultivation (young bivalve shellfish sold conimercially to global markets for aquaculture) and dumping of ship ballast water. In response, new international regulations require ships to purge ballast water i n the open ocean prior to docking (40). Human-generated environmental changes, such as reef destruction and eutrophication, also may help explain the apparent increase in human marine toxin disease, as well as the increase in red tides reported worldwide. Global climate change, which some suggest are linked to human activities, also may help explain the apparent global increase in algal blooms as well as the appearance of new marine toxin diseases like Pfiesteria (30,75,78-80). There is even new research connecting red tides to cholera outbreaks since copepods carrying V. cholercre feed on marine algal blooms; thus these algal blooms can lead to cholera disselnination and outbreaks associated with increasingly frequent monsoon flooding (79). cr. Parrrlyic Shel@sh Poisoning. ClassicPSPsymptomsincludeparesthesias of the mouth and extremities, frequently accompanied by gastrointestinal symptoms, within minutes to hours after eating. The disease occurs worldwide. Although often associated with a red tide or algal bloom, significant epidemics of PSP can occur in humans in the absence of a known red tide, as discussed in Fig. 2 with the Guatenlalan PSP epidemic of 1987 ( 2 2 ) . The dinoflagellates associated with PSP produce at least 12 toxins that are heat and acid stable; saxitoxin was the first characterized and the best understood (39). In a population-based study in Alaska after five local outbreaks of PSP, 70% of the 170 people interviewed had eaten shellfish gathered from the same area and 13% reported symptoms consistent with at least one episode of PSP (81). The case-fatality rate was about 8.5-9.5% in two large series; the 1987 Guatemalan outbreak on the Pacific coast had a case fatality rate of 14% overall, but 50% in young children, who may be more sensitive to PSP toxins. Access to emergency medical services in acute cases is crucial to survival. Chronic health effects have not been studied (22).
b. Neurotoxic ShellJsh Poisoning. Red tides that occur offtheFloridacoastare associated with two distinct clinical syndromes, depending on the route of exposure. Ingestion of contaminated shellfish (and less commonly contaminatedfish) causes gastroenteritis and neurologic symptoms similar to but less severe than those associated with PSP. Inhalation of toxins, predominantly brevetoxin, from the sea spray associated with the red tide and accompanying fish kills causes an upper respiratory syndrome in humans and other mammals (39). The classic causative organism, Gytnrlodinium breve, is a dinoflagellate restricted to the Gulf of Mexico and Caribbean waters, although similar species occur throughout the world (82). NSP was first identified by Walker in 1844 on the west coast of Florida. Since then, NSP has been reported from the Gulf of Mexico (including the coasts of Texas, Alabama, and Mississippi), the east coastof Florida, and the North Carolina coast. Recent prolonged NSP red tides in the Gulf of Mexico have been associated with die-offs of endangered manatees and dolphins, as well as respiratory problems in humans (26,83). Fish feed harvested from red tide-contaminated areas also has been blamedfor fish kills in the aquaculture industry. c. Diarrheic ShellJsh Poisottitlg. The first cases of DSP were reported from the Netherlands in the 196Os, followed by similar reports in the late 1970s from Japan. Since with the peak season from then, Inore than 1300 cases have been reported from Japan,
Epidemiology of Seafood Poisoning
30 1
April to September. Other outbreaks have been reported in Europe and South America as well as the Far East (39). i ~ . dinoflagThe causative organisms are the marine dinoflagellates D i t ~ o p h y ~These ellates are widely distributed, but do not always form redtides. They produce atleast nine different toxins consisting of okadaic acid and its derivatives. Because okadaic acid is a potent animal carcinogen, the issue of chronic disease from DSP poisoning needs to be addressed in humans (39,84).
d. Anulesic Shellfish Poisonir~g. First recognized in an outbreak onPrinceEdward Island, Canada, i n 1987, this syndrome produces nausea, vomiting, severe headache, abdominal cramps, and diarrhea within 15 minutes-38 hours of eating mussels, accompanied in about 25% of cases by acute lnemory loss. The 1987 outbreak, associated with consumption of mussels, produced 153 cases and 4 deaths. Some persons with ASP develop apparently permanent neurologic deficits, especially dementia. The toxin responsible is domoic acid, elaboratedby the pennate diatomNit:.schicr prngnc’s. It has been suggested that the index bloomof the diatom may havebeen related to fertilizer runoff from extensive identified as a continuing tobacco farming in the area (39,85). The organism was later problem among seabirds, sea lions, and shellfish in Washington, Oregon, and California. e. Cigrrcrtero Poisoniwg. Ciguatera isthemost conmon foodborne illnesscaused by a marine toxin. Its most distinguishing symptom is temperature reversal (hot coffee tastes cold, ice cream tastes hot). It is caused by the consumption of reef fish that have toxicus and other reef-dwelling dinofed on organisms contaminated with Grrmbic~rdiscrr,s flagellates. Ciguatera has been reported since ancient times and occurs i n tropical and subtropical areas around the world, with epicenters i n the Caribbean and the Indo-Pacific islands. It is not associated with red tides. Ciguatera is another of the seafood-related diseases that have spread geographically because of tourism and national and international commerce (39,75). For example, a single amberjack from a dealer in Key West (Florida), sold to two restaurants and two grocery stores,resultedin at least 20 ciguateracases i n at least two states (86). An estimated 10,000-50,000 people/year who live in or visittropical and subtropical areas develop ciguatera poisoning. The CDC and others estimate that only 2-10% of cases are actually reported i n the United States. Human impact on the environment may also play a role in the spread of ciguatera. Disturbances of coral reefs by military and other human activities stinlulate rapid recolonization by G. to.ricus, which appears to grow better after disturbance than under normal conditions (75). Ciguatera has had a measurable social and economic impact in endemic regions. Because the toxin is so widespread among reef fish, and because it cannot be detected by smell or taste or destroyed by cooking, populations in several endemic areas have abandoned local fish as a food source. Lewis (87) found that ciguatera in the South Pacific caused depression of both the local and export fishing industries and tourism, and had an indirect affect on human health due to avoidance of fresh fish consumption.
J: Pufet:fish PoisorlinR/FuRu. Pufferfish poisoning ortetrodotoxinintoxication (fugu) produces symptoms similarto PSP, with case fatality rates as high as 60%. Tetrodotoxin poisoning is found worldwide, associated predominantly with the ingestion of pufferfish. Fugu poisoning was known to the ancient Chinese as early as 2800 B.C. Unlike diseases caused by marine toxins, fugu poisoning is not due to dinoflagel-
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lates. The toxin, tetrodotoxin, is found i n the order of fish known as Tetraodontiformes, especially in the family Tetraodontidae (puffers). Marine bacteria colonize thegutand skin mucosal layers of the pufferfish following infection, and produce persistent levels of in gonads, liver.and.toalesser extent, muscle tetrodotoxinwhichthefishsequesters (39,75). The most toxic pufferfish are found along the coasts of China and Japan, where they are considered a delicacy and are eaten only after preparation by specially trained chefs. In Japan, where the seafood is known as fugu, 60 poisonings were reported in the period 1974-1979, with 20 deaths. About 50%;of fatalfood poisonings i n Japan each year are due to eating fugu; cases are more conlmon among men than women, probably related to eating habits. Cases have been reported in Europe associated with the mislabeling of imported fish, and elsewhere in the world due to ignorance of the toxic potential of thepuffers.Thus, fuguisavery circumscribedpublic health problem withahigh mortality. Chronic health effects have not been studied (39). g. Otlwr Ncrturd Mtrrinr To.rins. Numerous other types of poisoning are associated with the consumption of a wide variety of fish and shellfish. However, at present these poisonings have very restricted geographic and race-ethnic population distributions. These include a pseudoallergic reaction after eating Japanese callista during the spawning season to hallucinatoly fish poisoning after eating nmllet i n the tropical Pacific and Indian Oceans to hypervitaminosis A and neurologic disorders after consuming shark contaminated with unknown toxins (68,73,75). In addition, consumption of edible red algae, agorlori, i n Japan has been associated with acute foodbome illness and death (88). As with theotherseafooddiseases,internationalseafoodcommerce.aswellastourism,may change the geographic and population profiles of these illnesses in the future.
3. Chemical Toxins Humans, through their industrial activities, liberate mercury and other heavy metals from the earth and send them into the seas. where they bioaccun~ulate throughthe food chain and come back to humans in the form of contaminated seafood. Man-made chemicals and chemical by-products such as polychlorodibenzo-p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs), manyof them lipophilic and nondegradable, follow the same pathways and are consumed by humans. Infants and fetuses are at particular risk because many of (72.89). these substances are incorporated into breast milk and cross the placenta Evaluating the impact of these toxins requires a thorough understanding of the ecotoxicology of pollutants. For example, arsenic bioconcentrates i n shellfish. Nevertheless. years of research have revealed that little of the organic arsenic accumulated by humans from the consumption of seafood is converted into toxic inorganic arsenic. Thereforc, seafood containing arsenic represents a low risk to hurnan consumers (90,91). Traditionally it was believed that many chemical toxins contaminated the marine environment through single-source polluters such as specific industries. One extreme example of single-source pollution was the epidemic of. chronic neurologic disease that occurred when the people of Minimata, Japan, consumed fish contaminated with a methylmercury effluent from a local chloralkali plant (92). Other exanlples include highly visible marine oil spills that can force closure of shellfish beds and fish spawning areas for years. For example, after the Braer oil spill i n theShetlands,thesediment of thetraditional herring spawning beds rose from “background oil levels” o f 5 0 ppb to 100-350 ppb (93).
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However, i n recent years, more global sources of pollution have been recognized. For example. many freshwater and marine fish have become contaminated with methylmercury not from industrial point sources but viaa complex exposure pathway originating withglobalburning of fossilfuelthatproducesinorganicmercury as a by-product (72,94,95). Atmospheric and oceanic currents then spread this mercury to distant sites. Persistent contaminants, such as heavy metals and organochlorines, have been found i n the Arctic food chain and i n biological sampling of Arctic natives (72). Even radioactive materialscanbebioconcentrated in seafood.Radioactivewastedumping.atmospheric fallout, leaks from spent reactors, and runoff from contaminated rivers have lead to measurable radioactivity in seafood found in some Arctic regions (96). Pesticide residues increasingly are being found in seafood. This is probably due to agricultural runoff and the lipophilic properties of many pesticides that cause them to bioaccumulate in both the seafood and the hunlan consumer. In 1993, in the FDA residue monitoring program, 15.8% of 444 imported fish/shellfish samples analyzed for pesticide residues were above regulatory levels; by comparison, only 3.3% of 2261 imported fruits were above regulatory levels during the same sampling time period (97). Long-term exposure to relatively low levels of these chemical contaminants through seafood ingestion may be important in the etiology of immune diseases and cancer. Multiple myeloma, a relatively rare and deadly form of B-cell neoplasm, has been associated with the consumption of fish contaminated with dioxins (possible immunogens) in Baltic Sea fishermen and Alaskan natives (98). Recent research points to the increased sensitivity of children, infants, and fetuses to heavy metals and pesticides, especially to bioaccumuto the dramatic lowering of these allowable residue lated neurotoxins, which may lead levels in seafood and other foods i n the future (89.99).
V.
PREVENTION
Primary disease prevention, the goal of public health, reduces the incidence of disease by preventing human infection ( 1 1 - 13,23). Secondary andtertiary prevention seek to reduce, respectively, the duration of and complications from disease. Of the three approaches, primary prevention is almost always the most effective, from both economic and public health standpoints, although it may not always be possible. Primary prevention of seafood poisoning has two foci: the environment in which the seafood grows and is processed, and the people who prepare and eat the seafood.
A.
Environmental
After a large outbreak of typhoid fever was traced to contaminated shellfish, i n 1925 the U.S. Public Health Service established the Interstate Shellfish Sanitation Program (ISSP) to ensure that contaminated shellfish donot reach the retail market ( 1 7,42). Recommendations to prevent shellfish contamination included sewage control, limiting shellfish harvests to areas with clean water based on fecal coliform counts, and tagging all boxes of shellfish to indicate harvest location and date. Because fecal coliform counts arenot sensitive to the presence of many Vibrio species and Norwalk-like viruses, this has led to a shift from wastewater-associated bacterial outbreaks to outbreaks involving wastewater-
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derived viral pathogens and bacteria of environmental origin (100-102). More recently, in the wake of a 1998 Texas outbreak of V. ~ ~ t r ~ t r k c r e r ~ r o l ~the t i c uISSP s , has adopted new standards that call for the sampling of shellfish meat from areas associated with illnesses caused by V. y~r~.crh~renlol~ticus. The recent passage of the National Food Safety Initiative is a significant primary prevention intervention that includes the following seafood safety initiatives ( 103). Required seafood testing for pathogens and toxins as soon as it has left the hands of the fisherman. Increased personnel, epidemiologic, and laboratory resources devoted to surveillance programs. Implementation of a Hazard Analysis and Critical Control Point (HACCP) approach to seafood processing. HACCP rules require food industries to design and implement preventive measures that identify points where contamination is likely to occur and to implement controls to prevent it. Epidemiologic evaluation will be very important i n the future to determine the implementation and efficacy of these programs.
B. Education In surveys in Florida and elsewhere, from 17 to 25% of theadultpopulationreported eating raw clams and oysters in the preceding year (49,103).These data suggest the need for targeted food safety messages to educate consumers about the dangers of seafoodborne illnesses, especially sensitive subpopulations such as the immunosuppressed, the elderly, and children. The health care providersof these sensitive subpopulations need to be aware of the potential dangers of seafood consumption. I n addition, food processors andfoodservice workers need information about proper food handling procedures for each step of the process from the marine environment to the consumer’s plate. Seafood testing information, as well as food handling and storage procedures. needs to be conlmunicated back tothefishermen and the seafoodindustry to seek out and providesafer seafood. Finally, decreasing global pollutionwill protect not only one seafood supply, but also the crucial habitats.
VI.
CONCLUSIONS
Seafood-borne poisonings, both acute and chronic, are under-reported, but are probably increasing in incidenceandgeographicspread. I n particular,therehasbeenverylittle research into chronic disease associated with seafood poisoning. Furthermore, subpopulations, such as ethnic and the illlmunosuppressed, may be at increased risk for seafood diseases. Although primary prevention is the most desirable, the continual emergence of new seafood-borne diseases signifiesthat active disease surveillance will always be necessary. Finally, although required in the United States by the Food Safety Act, reliance on the testing of food will never be enough, since such testing must always be targeted and it is impossible to test for new or different pathogens and toxins in all seafood prior to human consumption. Therefore, education with regard to seafood safety (particularly issues suchas the consumption of raw seafood andthe importance of sanitation) will remain important (3,38).
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The education of health care providers and public health officials in traditionally nonendemic areas is necessary concerning the diagnosis andthe importance of official reporting of imported "exotic" or emerging seafood diseases (37). Groups that should be targeted include international travelers (both workers and tourists), recent emigrants, certain ethnic subpopulations, and imlllunosuppressed individuals. I n addition, education and cooperation of seafood industry workers concerning the risks of seafood-borne poisonings as wellasprimaryand secondary prevention programs are necessary for these programs to function effectively ( I 1-13.104). There should also be cooperative exploraas seafood tion and education about new and inexpensive protection technologies, such irradiation, between international and national health agencies, and the seafood industry (4,105). Given the high reported prevalence of disease with multiple etiologies associated with the consumption of raw or inadequately cooked seafood, thorough cooking of all seafood is a basic recommendation (53,62). This is particularly true for persons who are immunocompro~nised and/or suffering from liver disease (2,50,106), although not a coniplete panacea (for example, some viruses have high virulence, and many toxins cannot be removed by cooking) (27.60). Furthermore, it is critical that the necessary time and temperature studies be done with seafood to determine the degree of cooking that will denature a virus such as SRSV. It is also important to conmunicate the dangers of raw seafood consumption to ethnic subpopulations using culturally sensitive and appropriate language (33,94,107). In addition. laboratory and epidemiologic evaluation of possibly protective culturally acceptable practices, such as adding acidic substances (i.e., lemon juice in the Latin American marinated seafood dish of ceviche against Vibrio cholera) should alsobe explored (14,15,34.35). Alternative technologies such as the use of naturally occurring bacteria in seafood with antagonistic activity toward pathogens such as Lister-icr are being tested (51). Good sanitation. including facilities for human waste disposal and for proper hand washing with soap, are important to protect food and water supplies. Furthermore. education should be focused on the dangers of human fecal waste near drinking, agricultural, or aquacultural water supplies to prevent the contamination of irrigation and runoff water (34). Educational efforts targeting food handlers, food handling, and preparation to both prevent fecal contamination and maintain food integrity without spoilageor bacterial overgrowth should be undertaken (34.46,53). Even food storage should be emphasized; for example. i n Hong Kong, cholera may have been spread through the consumption of fish kept i n tanks with contaminated water (44). As seen i n several seafood-borne outbreaks, national and international collllnerce can lead to the rapid, wide, and prolonged spread of contaminated seafood with multiple resultant disease outbreaks ( I , 17,27.34,35,37,38). Fecal colifornl counts are agood initial screen for water contamination by fecal material, but are inadequate in the case of some bacteria, and even more for many pathogenic viruses and both natural and chelllical toxins. New laboratory techniques such as polymerase chain reaction (PCR) will lead to better diagnosis, tracing, and ultinmtely prevention of seafood poisonings (3,17,18). Todd and Harwig (29) recommend a risk analysis approach for seafoodthat has the following characteristics: widely consumed, international sources (both feral and aquaculture), associated with emerging diseases, and potentially produced under unsanitary conditions. They emphasize the importance of integrating this risk assessment approach into the HACCP system to more accurately determine the hazards and control of seafood processes. They particularly recommend focusing on potentially unsafe preparation and con-
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sumption practices (such as raw or smoked seafood), since these processes are known to increase the riskof disease. Additional recommendations include economic data collection and presentation to evaluate both the impact and cost-effectiveness of intervention programs, as wellas of present seafood practices (15,29,5 1,57). Thus, epidemiology will necessarily play an active role in evaluating the impact of seafood poisoning and future preventions and interventions.
REFERENCES Centers for Disease Control. Surveillance for foodhorne disease outhrcnks-United States, 1988-1992. MMWR 4.5:SSI-S, 1996. 2. FE Ahtned. Issues in fishery products safety i n the United States. Environ Toxin Water Qual I.
8:141-1S2. 1993. 3. EK Lipp, JB Rose. The role of seafood i n foodhorne diseases in the United Stateso f Atnelica. RevSciTech 16:620-640. 1997. 4. Anonymous. Irradiation for the prevention of foodborne disease. Bull Pan Am Health Organ 29:279-28 I . 199.5. S. W-C Lee, T Sakai. M-J Lee, M Hamakawa, S-M Lee, I-M Lee. An epidemiologic study of food poisoning in Korea and Japan. lnt J Food Microbiol 29: I 141- I 148. 1996. 6. TYK Chan. Shellfish borne illnesses: a Hong Kong perspcctive. Trop Geogr Mecl 47305307, 199s. 7. BS H u h , JD Griffith, TC Wilcosky, eds. Biological markers i n epidemiology. New York: OxfordUniversityPress, 1990. 8. PA Schulte. Methodologic issues i n the use of biologic markers in epidemiologic research.
Am JEpidemiol12:1006-1016,1987. 9. JL Kelscy. WD Thompson, AS Evans. Methods OxfordUniversityPress. 1996.
i n observational epidemiology. New York:
Press. 1992. 10. RR Monson. Occupational epidemiology. Boca Raton, FL: CRC 1 I . World Health Organization. Environtnental health criteria 27: environmental cpidemiology. Geneva: World Health Organization. 1983. 12. T Beaglehole, R Bonita. J Kjellstrom. Basic epidemiology. Geneva: World Health Organization, 1993. 13. Centers for Disease Control. Guidelines for investigating clusters of health events. MMWR. 39: 1-23, 1990. 14. E Todd. Surveillance of foodborne disease. I n : YH Hui. JR Gorhaln, KD Murrcll. DOCliver. eds. Foodborne disease handbook. New York: Marcel Dekker, 1994, pp. 46 1--.536. FK Kaferstein. Food safety. I n : JA Herzstein, WB Bunn. I S . Y Motarjemi. G Moy, A Reilly, LE Fleming, M Harrington, J Jeyaratnam, IR Gardncr. International occupational and environmentalmedicine. St. Louis: Mosby. 1998, pp. 602-619. 16. LEFleming, JA Bcan, DCBaden.Epidemiology oftoxic marine phytoplankton.In:GM Hallegraeff, DAN Anderson, AD Cetnbclla, eds. UNESCO-IOC manual 011 harmfLtl tnaritw phytoplankton #33. Paris: UNESCO, 199.5. 17. SF Dowell. C Groves, KB Kirkland, et al. A multi-state outbreak of oyster-associated gastro-
enteritis: inlplications for inter-state tracing of contaminated shellfish. J Infect Dis 17: 14971503. 199s.
18.
19
M Sugieda, K Nakajima, S Nakajitnn. Outbreaks of Norwalk-like virus-associated gastroenteritis traced to shellfish: co-existence o f two genotypes i n one specimen. Epidctniol Infect 1 16:339-346. 1996. DR Ayyar, WJ Mullaly. Ciguatera: clinical and clcctrophysiologic observations. Neurology 26354, 1977.
Epidemiology of Seafood Poisoning
307
20. G S(>zzi.P Marotta, D Aldcghi,G Tredichi, L Calvi. Polyneuropathy SeColldWy to ci&uatoXill poisoning. Ita1 JNeurolSci 9:491-49S. 1988. 21. DL Morse, GS Birkhead, JJ Gusewich. In: YH Hui, JR Gorham, KD Murrell, DO Cliver. c&. Foodborne disease handbook. NewYork: Marcel Dekker, 1994, pp. 547-603. 22. DC Rodrigue, RA Etzel, S Hall, et a l . Lethal parolytic shellfish poisoning in Guatelnaln. Am J Tr017MedHyg 42:267-271. 1990. 23. DN L:IwrcIlce, MB Enriqucz. R M Lumish, A Maceo. Ciguatera poisoning i n Miami. J Ani Med ASSOC244(3):254-258, 1080. 21. 1llterllation;llL.ahor Organization World Health Organimtion. Aquatic (nlarine and freshwater) biotoxins.EnvironmentalHealthCriteria37.Geneva:WorldHealthOrganization, 1984.
25. C'c11ters for Disease Control. Multistate outbreak of viral gastroenteritis associated withCOIIsumption of oysters----ApalachicoIa Bay, Florida. December 1994-January 1995. MMWR
44:37-39, 1995. RSHopkinS. S Heber.RHamn1ond. Water related disease i n Florida:continuingthreats require vigilance. J FI Mcd Assoc 84:441-415, 1997. 27. S McDonnell. KB Kirkland. WG Hlady. et a l . Failure of cooking to prevent shelllish-associated viral gastroenteritis. Arch Intern Med 157: 1 1 1- 1 16, 1997. 28. R Degner. C Adams. S Moss, S Mack. Per capita fish and shellfish consumption i n Florida. 1994. Industry Report 94-2. Gainesville. FL: Florida Agricultural Market Research Center, 29. ECD Todd. J Harwig. Microbial risk analysis o f food i n Canada. J Food Protect (suppl): IO--
26.
18. 1996.
30. 31.
32. 33. 34.
35. 36.
37. 38. 39.
40.
41
42 43
ECD Todd. Emerging discases associated with seafood toxins and other water-borne agents. Ann N Y AcadSci740:77-94, 1994. K Porticr. U Yonghwan. R Degner, S Mack. Statistical analysis of Florida per capita fish and shellfish consumption data. Industry Report 9s-l. Gainesvillc, FL: Florida Agricultul.al Market Research Center, 1995. J Eastaugh, S Shephard. Infectiousand toxic syndromes from lish and shellfish consumption. ArchInternMed149:1735-1740, 1989. MR Watters. Organic neurotoxins in seafoods. Clin Ncurol Neurosurg 97: 119-124, 19%. MJ Albert, M Neira, Y Motarjemi. The role o f fwd i n the epidemiology o f cholera. World Hedth Stat Q SO:I 1 I - 1 I 8, 1997. RV Tauxe, ED Mintz, RE Quick. Epidemic cholerai n the new world: translating lield epide1: 141- 146, 1995. miology into new prevention strategies. Emerging Infect Dis Centers lor Disease Control (CDC). Cholera associated with an international airline fight, 19'92. MMWK 166:1433-1435. 1992. J Ebcrhart-Phillips, RE Besser, MP Tormey, et a l . An outbreak of cholera from food served on an international aircraft. Epidemiol Infect 1 16:9-13. 1996. JCA Dcsenclos. KC Klontz. MH Wilder. et a l . A multi-state outbreak of hepatitis A caused by the consumption o f raw oysters. An1 J Public Health 8 1 : 1268- 1272. 199 I , D Bxden, LE Flcrning. JA Bcan. Marine toxins. I n : FA deWolff, ed. Handbook of clinical neurology: intoxicationsof the nervous system. Part11. Natural toxins and drugs. Amstertlam: Elsevier. 1995, pp. 1 1 1 -175. GM Halleg~~eff, CJ Bolch. Transport o f diatom and dinollagellate resting spores in ships' ballast water: implications for plankton biogeography and aquaculture. J Plankton Res 14: 1067-1084, 1992. VCRan.TGMetcnlf, JL Melnick.Hunxulviruses in sediments. sludges. and sails. Bull World Health Organ 64: 1 -- 14, 1986. J A Hughes. MH Merson, EJ Gangarosa. The safety of eating shellfish. JAMA 237: 19801981, 1977. G Fang, V Araujo, RL Gucrrant. Enteric infections associated with exposure t o animals and anind products. Infect Dis Clin N An1 5:681-701, 1991.
308
Fleming et al.
44. SH Lee, ST Lai, JY Lai, NK Leung. Resurgence of cholera
111
Hong Kong. Epidetniol Infect
1 17:43-49, 1996.
45. GR Matte, MH Matte, MIZ Satto. PS Sanchez, IG Rivera, MT Martins. Potentially pathogenic Vibrtos associated with mussels froma tropical region on the Atlantic coast of Brazil. J AppIBacteriol77:281-287.1994. 46. JT Weber, ED Mintz, R Canizares. et al. Epidemic cholera in Ecuador: multidrug resistance and transmission by water and seafood. Epidetniol Infect 112: 1-1 I , 1994. 47. M Landgraf, KBP Lcme. ML Garcia Moreno. Occurrence of emerging pathogenic Vihrio spp. in seafood consutncd in Sao Paulo City, Brazil. Rev Microbiol 27:126-130, 1996. 48. RL Haddock. AF Cabanero. The origin of non-outbreak Vihrio parahaer,/o/~tic/t.s infections in Guam. Trop Gcogr Med 46:42-43, 1994. 49. WC Hlady,KCKlonrz. The epidemiology of Vibrio infections in Florida. 1981-1993. J Infect Dis 173: I 176- 1 183, 1996. of Listeritr rr,orloc:\to,~erre.s in ready-to-catseafood SO. SAMcCarthy.Incidcnccandsurvival products. J Food Protect 603722-3726, 1997. SI. PK Ben Embarek. Presence, detection and growth of L/sterit/ rl,orfo~!.togerfe.s in seafoods: a review. Int J Food Microbiol 23:17-34, 1994. 52. S Merino, X Rubires, S Knochel,JMTotnas.Etncrgingpathogens: Aeromm1.s spp. Int J Food Microbiol 28: 157- 168, 1995. 53. HC Chen. Seafood microorganisms and seafood safety. J Food Drug Anal 3:133-144. 1995. 54. JJ Florcs Abuxapqui, G de J Suarez Hoil. MR Heredia Navarrcte, MA Puc Franco, J Franco Monsreal. Calidad tnicrobiologica de10s alimentos marinos enl a ciudad de Merida, Yucatan. VetMex27:319-324.1996. E c d i and SIU/J/Iu/treus 5s. AM Romero Ayulo, R Abreu Machado, VA Scussel. Enterotoxigenic i n fish and seafood from the southern region of Brazil. Int J Food Microbiol 24:171-178, 1994. 56. M Samadpour. JE Ongerth, J Liston, et al. Occurrence of Shiga-like toxin producing Escher i c h coli in retail fresh seafood, beef. lamb, pork, and poultry from grocery stores in Seattle, Washington. Appl Environ Microbiol 60: 1038-1040. 1994. 57. JY D’Aoust. Salmonella and the international food trade. Int J Food Microbiol 24:l 1-31. 1994. 58. ED Park, DV Lightner, DL Park. Antimicrobialsin shrimp aquaculture in the United States: regulatory status and safety concerns. Rev Environ Contatn Toxicol 138: 1-20, 1094. 59. DO Cliver. Epidemiology of foodborne viruses. In: YH Hui, JR Gorhaln, KD Murrell, DO Cliver, eds. Foodborne disease handbook. NewYork:MarcelDekker,1994.pp.159-175. 60. KB Kirkland. RA Meriwcther, JK Leks, WR MacKenzie. Steaming oysters does not prevent Norwalk-like gastroenteritis. Public Health Rep 1 1 1527-530, 1996. 61. RA Gunn. HT Janowski. S Lieb, EC Prather, HB Greenberg. Norwalk virus gastroenteritis following raw oyster consumption. Am J Epidctniol 115:348-351, 1982. con62. A Melc. MC Rastelli, ON Gill. et al. Recurrent epidemic hepatitis A associated with sumption of raw shellfish. probably controlled through public health measures. Am J Epidctniol130:540-545, 1989. 63. L Sagliocca, A Mele, L Ferrigno, C L d . Case control study of risk factors for hepatitis A: Naples 1990- 1991. Ita1 J Gastrocntcrol 27: I8 1- 184. 64. A Moreno Ancillo, MT Caballero, R Cabanas cl al. Allergic reactions to A/~rt.srrki.s.sirry/e.iparasitizing seafood. Ann Allergy Asthma Imtnunol 79946-250, 1997. 65. P Bouree, A Paugatn,JC Petithory. Anisakidosis: report of 25 cases and review of the literature. Comp Itntnunol Microbiol Infect Dis 18:75-84. 1995. 66. JK Griffiths, GT Keusch. Eating yuppie cuisine. In: D Schlossberg. ed. Infections of leisure. New York: Springer Verlag, 1994, pp. 294-315. 67. C Zinn. A Lopata. M Visser, PC Potter. The spectrum of allergy to South African bony fish (Teleosti). S Afr Mcd J 87: 146- 151. 1997.
Epidemiology of Seafood Poisoning 68.
309
AM Saavedra-Delgado.DD Mctcalfe. Seafood toxins. Clin Rev Allergy llnlnunol I 1241-
260. 1993. A Joral, F Villas. J Gartnendia, 0 Villarcnl. Adverse reactions to food in adults. J lnvcslig Allcrgol Clin Immunol 5:47-49. 1995. 70. A Montoro, MJ Perteguer, T Chivato. R Laguna,C Cuellar. Recidivous acute urticaria caused by Al~istrkis, s i ~ I ~ p / cAllergy ~ . ~ , 52:985-991, 1997. 69.
71. 0 Roots. Polychlorinated biphenyls and chlororganic pesticides, assesstnet11 of health risk 6(3/4):124-135. associated with the consumption of seafood. Proc Estonian Acad Sci Ecol 1996.
72. p Ayotte. E Dewailly. S Bruneau, S Careau. A Vezina. Arctic air pollutionand hutnan health: 1995. What effects should be expected? Sci Total Environ 160/161:529-537. and shellfish.Adv Food Nutr 73. DD Rawles, GF Flick, RE Martin. Biogenic amines in fish Res 39:329-365, 1996. 74. DA Anderson. Red tides. Sci Aln 271:62-68, 1994. 75. BW Halstead. Other poisonous marine animals. In: YH Hui. JR Gorham, KD Murrcll, DO 1994. pp. 497Clivcr, eds. Foodborne diseasehandbook.NewYork:MarcclDekker, 528. 16. ECD Todd. How ciguatera affects Canadians. In: TR Tostcson, ed. Proceedings of the Third International Conference, Puerto Rico. 1990. pp. 181-196. 17. WR Lange, FR Snyder. PJFudnla. Travel and ciguatera fish poisoning.Arch Intern Med 152:2049-2053, 1992.
78. R Viviani. Eutrophication, marine biotoxins, human health. Sci Total Environ
(suppl):631-
662.
79. PR Epstcin. TE Ford. RR Colwell. Marine ecosystems. In: PR Epstein, D Sharp, eds. Health and climate change. London: LancetLtd, 1994, pp.14-17. XO. PA Tester. Harmful marine phytoplankton and shellfish toxicity. Ann NY Acad Sci 740:6977, 1994. 81. BD Gessncr. M Schloss. A population based study of paralytic shellfish poisoning in Alaska. Alaska Med 38(2):54-58, 1996. 82. LEFleming.DGBaden.Neurotoxicshellfishpoisoning:publichealthandhutnanhealth effects. White paper for the Proceedings of the Texas Conference on Neurotoxic Shellfish Poisoning, Corpus Christi, Texas, 1998. S Wright. Brevetoxicosis 111 tnanatecs (Tri83. GD Bossart, DG Bnden. R Ewing, B Roberts. c ~ h c d r r t s/trmtrtu.s / t r t i r m t r i . s ) from the 1996 epizootic: gross, histopathologic and immunohistochemical fcaturcs. Toxicol Pathol 26:276-282. 1998. 84. J Landsberg. Neoplasia and biotoxins in bivalves: Is there a connection? J Shellfish Res 15: 203-230. 1996.
85. TM Perl, L Bedard. T Kosatsky, JC Hockin. ECD Todd, R Retnis. Encephalopathy caused bycontatnitxatcd mussels. N Engl J Med 322:1775-1780, 1990. 86. R Hatnmond. Ciguatera: ;I case study of a foodborne outbreak. Fla J Environ Health 137: 7-9, 1992. 87. ND Lewis. Diseaseand development: ciguatera lish poisoning. Soc Sci Med (Aust) 23:983993, 1986. 88. T Noguchi. T Matsui. K Miyazawa, et al. Poisoning by the red alga “opornori” (Crcrc,i/trrirc \vr.roclr.str)on the Nojima coast. Yokohatna. Kanagawo Prefecture. Japan. Toxicon 32: 15331538, 1994. 89. P Grandjean, P Weihc. LL Needham.et al. Relation of a seafood diet t o tnercury, selenium. arsenic, and polychlorinated biphenyls and other organochlorine concentrations inhuman milk. Environ Res 7 I :29-38, 1995. 90 JM Neff. Ecotoxicology of arsenic in the marine enviromnent. Environ Taxicol Chetn 16: 9 17-927, 1997. 91 SP Lall. Macro and trxe clerncnts in fish and shellfish. I n : IJ Jeon. WG Ilkins. eds. Analyzing
Fleming et al.
310
92.
93. 94. 95.
food for nutrition labeling and dangerouscontamin~utlts.New York: Marcel Dckker. 1995. pp. 187-213. M Hamda.Minamatadisease:methyltncrcurypoisoning i n Japancaused by environmental pollution. Crit Rev Toxicol 25:1-24, 1995. J Goodlad. Effects of the Braer oil spill on the Shetland seafood industry. Sci Total Environ 186:127-133.1996. LE Fleming, S Watkins, R Kadcrtnan, et al. Mercury exposure in humans through food cot1sutnption from the Everglades of Florida. Water Air Soil Pollut 80:41-48, 1995. R Buzina, P Stcgnar. K Buzina-Suboticancc. M Horvat, I Petric. TMM Farley. Dietary mercury intake and human exposure in an Adriatic population. Sci Total Environ 170:199-208, 199s.
96. TG Sayzkina, I1 Kryshev. Current and potential doses from Arctic scafood consutnption. Sci TotalEnviron202:57-65. 1997. 1993. J Assoc 97. Anonymous.FoodandDrugAdministrationpesticideresiduemonitoring: Offic Anal Chcm Int 77(5):161A-185A. 1994. 9X. G Schwartz.Multiplemyeloma:clusters,cluesanddioxins.CanccrEpidetniolBiotnarkcrs Prev6149-56,1997. 99. National Research Council. Pesticides in the dicts of infants and children. Washington. D.C.: NationalAcademyPress,1993. 100. SR Rippey. Infectious diseases associated with molluscan shellfish consumption. Clin Microhiol Rev 7:419-425. 1994. 101. A Stollc, B Sperner. Viral infections transmitted hy food of anitnal origin: the present situationintheEuropeanUnion.ArchVirolSuppl 13:219-228. 1997. 102. CP Gerha, SM Goyal, RI LaBelle, et al. Failure of indicator bacteria to reflect the occurrcncc of enteroviruses in marine waters. Am J Public Health 69: 1 116-1 I 19. 1979. 103. FoodandDrugAdministration.May1997.Wcbsitc: vm.cfsan.frla.gov/-.d~ls/i~sre~)ort.htt~~l. 104. ELBaker,ed.Surveillance i n occupationalhealth and safety.Washington.D.C.:American PublicHealthAssociation,19x9. 105. PLoaharanu.Irradiation as a cold pasteurizationprocess o f I'ood. VetParasitol64:71-82. 1996. 106. D Hellcr. ON Gill. E Raynhatn. et al. An outbreak of gastrointestinal illness associated with consumption of raw depurated oysters. Br Met1 J 292:1726-1727. 19x6. 107. pJ Shubat, KA Raatz, RA Olson. Fishconsutnptionadvisories and outreuch programs for southeast Asian immigrants. Toxicol Ind. Health 12:427-434, 1996. lox. pH Rheinstein. KC Klontz.Shellfish borne illnesses. An1 h111 Physician 47: 1837-1840. 1993. 109. M Wittner. JW Turner. G J q u e t t e . LRAsh. MP Salgo, HB Tanowilz.Eustrongylidinsis. 21 p:ur;lsitic infection acquired hy eating sushi. N Engl J Med 320: 1124-1 126. 1989.
10 The Medical Management of Seafood Poisoning
I. 11.
Introduction 31 I Fish-RelatedPoisonings A. B. C. D.
Botulism
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312
Scombroid poisoning 312 Tetrodotoxin or pufferfish poisoning Ciguatera 313 Ill. Shcllfiah-Related Poisonings 315 A. Vibrios 315 B. Shellfish toxins 316 IV. Conclusion 3 17 References 3 1 X
1.
3 13
INTRODUCTION
Since seafood is now universally accepted a s a nutritious, high-protein, low-fat red meat substitute, it is being increasingly consumed ( 1 ). Activities associated with tourism and travel,aswellasever-increasinginternationaltrade,facilitateconsumerexposuretoa widearray of seafoodproducts,bothfreshandfrozenfromdifferentmarinesources. Withcontemporaryconsumerhabitsinvolvingmoretwo-incomefamilies,greaterfrequency of eating out in restaurants is also seen. Many patrons who might not routinely cook fish at home, enjoy eating it when it is well-prepared at a restaurant. It is therefore
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unexpected when a seemingly healthy and probably expensive dinner results in food poisoning. In this chapter, the medical management of acute seafood poisoning is approached by separatingseafoodintofish-relatedpoisoningandshellfish-relatedpoisoning.Fishrelated poisonings include botulism, scombroid poisoning, fugu poisoning, and ciguatera. Shellfish-related poisonings include Vibrio, and the four distinct shellfish poisonings (paralytic, neurotoxic, diarrheic, amnesic). Fish-related poisoning, secondary to bacterial toxins or parasites, is well known to be associated with inadequate refrigeration and/or improper preparation. Howcver, toxinmediated seafood poisoning can occur in a perfectly prepared and healthy fish. Shellfishrelated poisoning, especially bacterial and viral,is known to be associated with inappropriate harvesting, handling, or storage. Shellfish poisoning can also be caused by extrinsic toxins produced by algal blooms. Diagnosis of seafood poisoning is, by necessity, associated with a history of recent seafood ingestion. Because most seafood poisonings are self-limited, they are usually dismissed and not diagnosed or reported. Sharingof food oftcn leads to small disease clusters ill and the or epidemics of seafood poisonings. However, if more than one person gets cluster is reported to the public health authorities, a more accurate etiology can then be gleaned through epidemiological investigation. Before asking the epidemiological questions, however, the poisoning sufferer mustbe treated. This chapter dealswith the medical treatment of seafood-borne poisonings.
II. FISH-RELATED POISONINGS A.
Botulism
Type E botulism is associated with air-dried, and both hot and cold smoked fish.Clostricli m b o t d i r r w ? r is responsible for type E botulism (2,3). Pressure cooker temperaturcs are highaltitudeswithlowboilingpoints.Unrequired to killthisbacteriaespeciallyat dercooking can result in acute gastroenteritis, bilateral sixth cranial involvement with diplopia and ptosis, progressive descending symmetrical flaccid paralysis, and respiratory arrest. Treatment is ventilatory support, intravenous hydration. gastric lavage, cathartics, and enemas. Fortunately, a trivalent antitoxin E is available and should be used i n all suspected type E botulism victims. All sinlilarly canned and smoked fish should be safely discarded. Survivors usually recover completely.
B. Scombroid Poisoning Scombroid poisoning is a pseudoallergic intoxication also known as "histaminc food poisoning." It resembles an acute allergic reaction and results from the ingestion of spoiled fish from the Scombridae family (including tuna, mackerel, jack, and bonito). These fish have a high content of dark meat that is rich in histidinc. Unlike thc othcr fish-related toxins, scombroid is caused by inadequate refrigeration, allowing bacterial growth which can transform histidine into histamine.Histamineis not affected by hcat or any other method of preparation. Scombroid poisoning is, however, entirely preventable if the fish is itntnediately and properly refrigerated. Signsandsymptomsbeginminutesafteringestion.Theyincludedramaticskin flushing, throbbing headache, oral burning, abdominal cramps, nausea, and diarrhea. Hives
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and swelling of the tongue are less common. Victims report that the fish tasted peppery or metallic (4).The disease course is self-limited and symptoms abate i n less than a day if left untreated. Antihistamines and H: blockers, shorten the course and ameliorate the symptoms. Angioedema and bronchospasms require epinephrine.
C. Tetrodotoxin or Pufferfish Poisoning Famed as one of the most potent nonprotein poisons i n nature, tetrodotoxin is naturally found i n pufferfish, from which the coveted “fugu” is prepared. Long considered a delicacy i n Japan, fugu is said to produce exhilaration, perioral tingling, a floating sensation, and a feeling of overall warmth (S). It is also considered to be an aphrodisiac. Fugu chefs are specially trained to take particular care in cutting away the more toxic fish parts such as the viscera and gonads. Tetrodotoxinfunctions by blockingvoltage-dependentsodiutnchannels in nerve cell membranes. This blockage causes perioral paresthesia. nausea, and dizziness. The more rapid the onset of the symptoms, the poorer the prognosis (5,6).More severe symptoms include weakness, generalized numbness,loss of coordination, slurrcd speech, bradycardia, hypotension, and respiratory paralysis. Subsequent death results from respiratory arrest. There is no antitoxin at present. Gastrointestinal decontamination is recommended and supportive respiratory therapy is life saving. Survivors reportedly have spontaneous and complete recovery.
D. Ciguatera Ciguatera was first recorded by Caribbean explorers i n IS 1 1 . It is the most colnnion cause of tropical fish poisonings (4,6-8). It is barely recognized in the medical literature, and remains misdiagnosed, inadequately treated, and underreported. Ciguatera is caused by the ingestion of ciguatoxic fish. These fish are contanlinated with ciguatoxins that arc produced by the marine dinoflagellate Gcrrrrbirr.ili.sc,rr.s to.ric~r4.s(8).Ciguatera has been associated with the consumption of reef fish including barracuda, grouper, snapper, jack, tropical mackerel, morays, parrotfish. surgeonfish, hogfish, sea bass, and wrasse.
1. Acute Phase Ciguatera is classically described as an acute illness. Gastrointestinal symptoms typically begin 3-6 hours after the ingestion of the fish (7,9,lO). The gastrointestinal symptoms are overlapped or followed by a myriad of neurological symptoms. Cardiovascular symptoms are less comnlon and rarely severe. Ciguatera is seldom fatal. The gastrointestinal symptoms of ciguatera are typical of any food poisoning: nausea, vomiting, cramps, abdominal pain, and watery diarrhea. These symptoms are generallyself-limited i n aday or two. The neurological symptoms include intense pruritus, paresthesia, and dyesthesia. They usually begin several hours after the ingestion of the toxic fish and overlap the gastrointestinal phase. Occasionally neurological symptoms do not begin until 3 or 4 days later, well after the gastrointestinal symptoms have subsided. Patients report a myriad of highly unusual complaints, including tap water tasting carbonated, bugs crawling on their skin, a sensation of their teeth falling out, cold feeling hot. and air conditioning as intolerable. Chills are a frequent complaint. but actual temperature elevation is not seen. Urination can burn and sexual intercourse can be painful. Arthralgias, weakness, and easy fatigability are cotnlnon complaints which can last for years. Alcohol
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ingestion is known to aggravate all of these symptoms. Paralysis, especially facial nerve palsies and coma, have been reported ( 7 3 ) . Cardiovascular events include bradycardia and hypovolemic hypotension. Death is rare and usually attributed to hypovolemic shock. Individual variation in symptom response is the rule even when all affected eat the same source of contaminated fish. The diagnosis of ciguatera remains clinical. There are no currently available clinical diagnostic tests for ciguatera in humans.
2. Chronic Phase I n chronic ciguatera, the gastrointestinal and cardiovascular events have subsided, but the neurological symptoms persist. These neurological symptoms are usually diminished in intensity, but can be exacerbated, with patients describing "good days and bad days." Patients can usually identify triggering events such as the ingestion of alcohol, nuts, or fish (1 1 - 15). Physical exercise is exhausting, and libido and joie devivre are diminished. Chronic fatigue, arthralgias, and myalgias are particularly frustrating. Despair can be overwhelming. Reassurance that almost all ciguatera is eventually self-limited is a most welcome prognosis.
3. Treatment The treatment of ciguatera is prilnarily supportive therapy. Severe volume depletion and hypotensive shock require urgent treatment with an intravenous normal saline or lactated ringers solution. Bradycardia is usually mild, with normal sinus rhythm at 50-60 beats/ min, andusually does notrequiretreatmentwithatropine.Gastricdecontamination is probably futile, as symptoms typically begin several hours after the ingested meal. Antiemetics and antidiarrheics should be avoided. Neither antibiotics nor corticosteroids have been shown to help (6,7,12,16). Antioxidants and B-complex vitamins seem harmless,but are of questionable value. Antihistamines and H z blockers may decrease pruritus. Nonsteroidal anti-inflammatory drugs help chronic arthralgias, myalgias, and dental pain. Calcium channel blockers are used for palpitations caused by subsequent tachycardias, which are self-limited. Intravenous (IV) mannitol therapy has been shown to bring a dramatic improvement if administered early (1 l): results have been less impressive if given more than 48-72 hours after ingestion of the toxic fish (1 2- 14,16). In hyperosmolar concentrations, decreased cerebral edema and peripheral nerve edema occur. Clinical reports from controlled studies indicate that a 20% mannitol IV infusion over 30-120 minutes at a dose of 1 g/ kg body weight is safe and well tolerated in the hydrated patient, and seemingly effective if given early enough (8). Less dramatic, symptomatic improvement has been reported when IV mannitol therapy has been administered as late as 1-7 weeks following the onset of ciguatera in uncontrolled studies (1214). The delay in therapy is the result of the typical delay in the diagnosis of ciguatera. Chronicciguatera, in general, does not respond to IV mannitol therapy ( 12.13). Chronic treatment is symptomatic. Antihistamines, H2 blockers, analgesics, and nonsteroidal anti-inflammatory drugs may help to relieve the paresthesia, dysesthesia, and arthralgia ( 1 l,l2). Amitryptiline and fluoxetine have been reported to be beneficial for the chronic fatigue, insomnia, and clinical depression ( 1 7,18). There is no antitoxin for ciguatera, and local folklore and native homeopathic remedies have questionable efficacy. Most ciguatera victims recover spontaneously within a few weeks (19).
Medical Management of Seafood Poisoning
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SHELLFISH-RELATED POISONINGS
A.
Vibrios
315
1. Exposure Infectionbythepathogenic Vibrio species generally occurs following theingestion of contaminated water and food, especially raw or insufficiently cooked shellfish. Vibrios are natural inhabitants of tidal rivers and bays that have moderate salinity. Marine shellfish and plankton are the main reservoirs for Vibrio species. Of these Vibrio species. V. cholerne, V. / ) ~ ~ ~ C I I I C I ~ I and ~ I ( ~V.I ~wltriji(~ans ~ ~ ~ ~ ~ ~ . ~have , been implicated most frequently in seafood poisonings (20-28). V. vulrrjjicwrrs can result in septicemia with a high mortality rate. The othcr Vibrio species are associated with gastroenteritis of varying severity (211.
2.
Clinical Symptoms
Vibrio cholor~tr The Vibrio species responsible for “epidemic” cholera are V. cholertr serogroup 01 biotype E l Tor strain and the recently identified V. cholern serogroup 01 39 biotype Bengal. No major outbreaks of this disease have occurred i n the United States since 191 1. Casesreported in theUnitedStates have beenassociatedwithtravelersfromSouth America during the most recent pandemic, or from illegally smuggled crustaceans that were held at improper temperatures (21-24,291. Epidemiologic studies have shown that unhygienic food handling is the most commonsource of seafood-bornecholera CI.
(20,24,25,29,30). Acute diarrheal illness occurring 24-48 hours after the ingestion of contaminated shellfish or fish is conmon to both subgroups. Diarrhea. abdominal pain or cramps, and nausea are generally reported, with fever, vomiting, and headache in about SO% of cases. I n severe cases, stool volume can exceed 250 n N k g in the first 24 hours (31). Coniplications from the effectsof fecal volume and electrolyte depletion include hypovolemic shock and acute renal failure. With early rehydration these complications can be avoided and the illness resolved in a few days. However, untreated, cholera can result in death in a matter of hours. V. cholarrr serogroup non-01 is primarily caused by the consumption of raw, improperly cooked or stored shellfish. Diarrhea is usually less severe than in epidemic cholera. Abdominal cramps, nausea, vomiting, and fever are conmon symptoms whichusually persist for about a week (29,3 1,32). b. Vibrio ~~t~rc~htrerllol~tit.us V. ~ ~ ~ ~ r r r l r c r ~infections ~ r ~ ~ o lare ~ ftypically i ~ ~ ~ ~ linked .~ to the ingestion of undercooked or improperly handled seafood. In Japan, up to SO% of foodborne disease outbreaks have been associated with this Vibrio species (27). Generally, symptoms include watery diarrhea, accompanied by abdominal cramps, nausea, and vomiting. Less colmnon are fever, chills, and dysentery, along with bloody, mucoid stools. The incubation period for this illness is between 4 hours and 4 days, with symptoms present for approximately 3 days.
Vibrio ~ ~ u l r r ~ f i c ~ t r r l s V. w h $ u r r r . s is thc most important cause of severe Vibrio infections in the United States, but is rare outside the Gulf of Mexico coastal states. Typically patients with V. c‘.
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Blythe et al.
~ w l n i f i c ~ infections ~.s are men over the age of 40 who are immunocon~proll~ised or have cirrhosis, hemochromatosis, or diabetes mellitus. Most patientshave ingested raw oysters from coastal states between May and September within 2 days of onset of symptoms. approaches SO%, with most deaths due touncontrolled Mortality i n theUnitedStates sepsis ( 2 6 , 2 8 3 ) . In a reported case series from Japan, mortality was 68%, and the authors noted that the mortality rate appeared to increase with greater delay between the onset of illness and the initiation of the antibiotic treatment (34). Vibrio can be confirmed by the identification of the causative organism from the diarrheic stool of infected individuals. Laboratory isolation of the organism to differentiate species of Vibrio is possible.
3. Treatment Rapid and adequate replacementof fluids and electrolytes is necessary for allVibrio infections. A fluid, administered orally, containing an Na*content of 90 mmol/L, 20 mmol/L of K*, 80 mmol/L Cl-, 10 mmol/L citrate, and 110 mmol/L glucose has been recommended by the World Health Organization (3 1). This treatment is safe for infants if it is alternated with the consumption of sodium-free fluids such as breastmilk or water. If patients are severely dehydrated, intravenous fluid replacement is required. The use of antibiotics for the treatment of cholera is controversial. It can have the effect of shortening the duration and volume of fluid loss, and clearing the organism from the stool. Multidrug resistance of cholera has been noted in recent studies (30,3 1 3 ) . Tetracycline, ciprofloxacin, chloramphenicol, and erthromycin have been used. Tetracyclineisconsideredthetreatment of choicefor V . \wlniJicu.s infections (30,31,34) in combination with gentamicin. I n addition to antibiotics, surgical debridement and management of shock, coagulopathies, and renal and respiratory failure are critical. Most casesof V. I"/r"/zcrfnrol~ticu.sare self-limited, and require neither antimicrobial treatment nor hospital care. Severe cases should be treated with fluid replacement and antibiotics.
B. Shellfish Toxins There are four distinct shellfish poisoning syndromes caused by the marine toxins: paralytic (PSP), neurotoxic (NSP), diarrheic (DSP), and amnesic (ASP) shellfish poisoning. The toxins responsible for shellfish poisonings are produced by microscopic algae known as diatoms and dinoflagellates (9). Shellfish are filter feeders and they accumulate many local toxins, someof which are heatand acid stable. Toxins implicated in disease outbreaks are often associated with harmful algal blooms, such as red tides (6).
1. Paralytic Shellfish Poisoning Saxitoxin is the major toxin responsible for PSP. It is produced by the dinoflagellates Gorryrrrlas ctrtclltr and G. ttrmtrr.etrsi.s.Saxitoxin manifests toxicity by interfering with the voltage-dependent sodium channels i n nerve cell membranes. This mechanism is similar to that of tetrodotoxin, and quite possibly explains the similar clinical presentations. Of all the shellfish poisonings, PSP and ASP produce the most severe illness. Patients experience paresthesia of the mouth, face, and limbs, frequently associated with nausea, vomiting, and diarrhea. Most deaths occur within the first few hours after ingestion and are attributable to respiratory paralysis. Prompt respiratory therapy is imperative for survival because there isno antidote presently available. Mortality from this disease ranges
Medical Management of Seafood Poisoning
31 7
from 1% to 12% (36-38). PSP usually lasts about 3 days; those surviving past 24 hours have a reportedly excellent prognosis.
2. Amnesic Shellfish Poisoning ASP is caused by domoic acid (DA) which is produced by the diatom Nif:.schitr p r t g e r r s . Domoic acid acts as a potent excitatory neurotransmitter which affects the motor axons. Like the other shellfish poisonings, ASP is associated with gastroenteritis. A unique toxic encephalopathy caused by eating mussels contanlinated with dolnoic acid was reported in Canada. Cognitive dysfunction, loss of short-term memory, confusion, disorientation, seizures, and coma presented in an outbreak of ASP hours after eating mussels harvested off Prince Edward Island (36). For some individuals, the cognitive dysfunction and memory loss were permanent, and deaths were also reported. Treatment is symptomatic.
3. Neurotoxic Shellfish Poisoning The culpable agent i n NSP, brevetoxin. is produced by the dinoflagellate Ptyhotliscw brevis, responsible for the Florida red tides. Brevetoxins open the sodium channels on nerve membranes, which inducesan influx of sodium. NSP presentswith acute gastrointestinalandneurologicalsymptoms.Vomiting and diarrheaoccursil~lultancneously with paresthesia, temperature reversal, vertigo, pupil dilation, throat tightness, and a choking sensation. Symptoms begin about 3 hours after ingestion of toxicshcllfish and lastno more than a few days (6).NSP is milder than PSP and is reportedly self-limited. Treatment is symptomatic.
4. Diarrheic Shellfish Poisoning DSP is caused by okadaic acid (OA), whichis claborated by the dinoflagellates Diwplrysis and Pr.oroccvtrrwu. Okadaic acid induces diarrhea by stimulating the phosphorylation that controls sodium secretion i n the gut (6). This poisoning is common in Japan. An ephemeral illness, gastroenteritis occurs 12 hours after ingestion and persists for about 24 hours. There have bccn no neurological manifestations or deaths reported. Antiemetics and antidiarrheics should be avoidcd. DSP is reportedly self-limited with the elimination of the toxin.
W. CONCLUSION From the medical and public health point of view, the best treatment for seafood poisoning is the primary prevention of illness. Therefore, primary prevention should focus on the harvesting, transport. storagc. and preparation of seafood. Nevertheless, even with proactive measures, seafood poisoning cannot be completely prevented, especially for sensitive subpopulations consuming raw or inadequately cooked seafood. Botulism from undercooked canned or smokedfish, is usually thc result of inexperience. Fortunately, antibotulism toxin E is life saving, provided that emergency medical care is promptly available. Fugu poisoning can turn a thrill-seeking adventure meal into a self-destructive and hardly glamorous near-death experience for those risk takers fortunate enough to survive it. The risk of poisoning from tetrodotoxin is closely related to precautions taken in the preparation of the puffertish. Scombroid poisoning requires surface bacterial growth and is prevented by prompt to only a few days. and continuous refrigeration. Even refrigerator shelf life is limited
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Fish should be frozen early if consumption cannot be ensured within 2 days. Scombroid poisoning is easily preventable. Ciguatera is not so easily prevented. Highly prized, usually healthy and delicious coral reef fish can occasionally be toxic. The large predator fish, barracuda, is typically the most toxic and should be avoided. The sale of barracuda is, in fact, banned i n Miami, Florida. Barracuda is still considered a delicacy in most of the Caribbean, especially by the natives. Unfortunately, there is no reliable field test currently available to analyze fish for ciguatoxins before they are sold to the consun1cr. There are also no clinical diagnostic tests for ciguatera. Leftover fish canbe tested for toxicity, however, there is rarely sufficient fish remaining from the meal for toxin analysis. The true incidence of ciguatera is unknown and only speculative. An antitoxin is not available, but if correct diagnosis can be made quickly, IV nwlnitol treatnlent is helpful. Shellfishharvestingiscloselymonitoredtoavoidacute Vibrio gastroenteritis. V. 1v~h(ficm.s canalso cause a toxic septicemia with gangrene and death, especially when in these highassociated with liver disease. Raw shellfish should be avoided altogether risk consutners, and not just in the “month’s without R’s.” Fortunately the four shellfish toxicities-PSP, NSP. DSP, and ASP-are less common. Their descriptivenames-paralytic, neurotoxic, diarrheic, and amnesic-are nlenacing indicators of their potent toxins. Unfortunately no antitoxins are available.
REFERENCES 1.
2.
3. 4.
S. 6.
RL Dcgner. CM Adams. SC Moss. SK Mack. Per capita fish and shellfish consumption in Florida. Industry Report 94-2. Gainesvillc: Florida Agricultural Research Center, 1994. H Badhey, DJ Cleri, RF D’Amato, et al. Two fatal cases of type E adult food borne hotuolism with early symptoms and terminal neurological sign. J Clin Microhiol 23:616-61 X. 1986. TL Bott. JS Deffncr. EMcCoy,EM Foster. C/o.stridiIm b o t c t l i u m r type E i n fish from the Great Lakes. J Bacteriol 91:919-924, 1966. LE Fleming. DC Blythe, DC Baden. Ciguatera fish poisoning. Shoreland‘sTravel Med Month l(6):1-4. 1997. AR Mills, R Passmore, R Pelagic. Paralysis. Lancet 1:161-164. 1988. DC Baden. LE Flcnling, JB Bean. Marine toxins. Handbook Clin NeuroI 2 l(65):14 1 - 175. 1995.
7. LR Juranovic, DL Park. Foodhornc toxins o f marinc origin: ciguatera. Rev Environ Contam Toxicol I 17:s 1-94. 199I. X. R Bngnis. Ciguatern fish poisoning. In: ID Falconer. cd. Algal toxins in seafood and drinking water. New York: Academic Press, 1993, 105- I 15. 9. C Frennette, JD Maclean. TW Gyorkos. A large coInmon-source outhreak of ciguatera fish poisonings. J Infect Dis 158:I 12X- 1
