Advances in Applied Microbiology, Volume 16

ADVANCES IN

Applied Micro biology VOLUME 16

CONTRIBUTORS TO THIS VOLUME

Kenneth L. Applegate Eric Atherton John R. Chipley R. R. Colwell Arnold L. Demain Thomas H. Jukes Kiyoshi Higuchi

R. Kenworthy Robert E. Lennon Johannes Meienhofer William B. Sarles Claude VBzina

ADVANCES IN

Applied Microbiology Edited by D. PERLMAN School of Pharmacy The University of Wisconsin

Madiso n, Wisconsin

VOLUME 16

@

1973

ACADEMIC PRESS, New York and London A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1973,BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS LIST OF CONTRIBUTORS ..............................................................................

ix

Public Health Significance of Feeding Low Levels of Antibiotics to Animals THOMASH. JUKES ................................. I. Introduction .................................. 11. Clinical Studies in Human Medicine ................................................. 111. Toxicological Studies with Some “Feed-Type” Antibiotics ................... IV. Alleged Hazards of Feeding Antibiotics to Animals ............................. V. Discussion ...................................................................................... References ......................................................................................

1 5

11

17 25 28

Intestinal Microbial Flora of the Pig

I. 11. 111. IV. V.

R. KENWORTHY ............... Introduction .............. Metabolic Activities of the Microflora ....................._.... ....................... Significance of Microbial Metabolites in Nutrition and Health ......... Intestinal Microflora and Disease . ...................... ................................ Summary and Conclusions ......... .... ..................................................... References .........

31 33 37 39 49 50

Antimycin A, a Piscicidal Antibiotic ROBERTE. LENNONAND CLAUDE VlhINA

I. Introduction

................................................... .

11.

111. IV . V. VI . Development of Antimycin as a Piscicide ........................................... VII. Applications as a General Piscicide .....

V

56 60 66 67 68 69 83

vi

CONTENTS

VIII. Applications as a Selective Piscicide .................................................. IX. Summary and Conclusions ................................................................ References ......................................................................................

87 91 92

Oc h ratoxins KENNETH L.APPLEGATEAND JOHN R. CHIPLEY

I. Introduction .................................................................................... 11. Fungi Producing Ochratoxins ............................................................ 111. Occurrence of Ochratoxins and Toxin-Producing Organisms IV. Factors Affecting Ochratoxin Production ............................................. V. Structural and Biochemic VI. Extraction and Detection VII. Biological Effects .............................................................................

97 97 98 99 100 102 105 107

Cultivation of Animal Cells i n Chemically Defined Media, A Review

IYOSHI HICUCHI Introduction ......... ......... ........ ...... ...... Preliminary Studies in the Development of Chemically Defined Media ... Cultivation of Mammalian Cells in Chemically Defined Media ............. Nutritional Factors Not Generally Recognized as Required by Mammalian Cells in Vitro ...... V. Growth Factors Associated with Serum Macromolecules ...................... VI. Concluding Remarks ........................................................................ References ..........................................................................

I. 11. 111. IV.

111 112 114 120 130 132

Genetic a n d Phenetic Classification of Bacteria

R. R. COLWELL I. Introduction .......... ........................................ 11. Numerical Taxonom ystematics ......................... 111. Microbial Ecology - Primary Productivity ...........................................

..... .....

167

vii

CONTENTS

Mutation and the Production of Secondary Metabolites

I. 11. 111. IV. V. VI. VII.

ARNOLD L. DEMAIN Introduction .... Increasing Prod Elimination of Undesirable Secondary Products ........ ...................... ... Formation of New Secondary Products ........... Elucidation of Biosynthetic Pathways ................ Future Prospects .............................................................................. Summary.. ......... References ............... ...................................... ...,......................... ...

.

I

177 178 192 193 194 196 197 199

Structure-Activity Relationships in the Actinomycins

I. 11. 111. IV. V. VI. VII.

JOHANNES MEIENHOFER AND ERICATHERTON Abbreviations ............. Introduction ............... Proposed Actinomycin Nomenclature . Occurrence and Preparation Biological Activity ........................... .................................,............, .. Conformation and Mole Problems Ahead .......... Concluding Remarks ......

..,..........................................................................

203 204 207 216 248 269 289 290 29 1

Development of Applied Microbiology at the University of Wisconsin

WILLIAMB. SARLES I. Small Beginnings ....................................................................... 11. The True Start at Wisconsin: H. L. Russell’s Work .............................. 111. Early Expansion ...... ................................................... IV. The Department of Agricultural Bacteriology ... V. The Department of Bacteriology and Its New ............................ VI. Effects of Grants from Federal Agencies for Training Graduate Students and for Research ........ ................... VII. Explanation and Epilogue ..............,..,................................ ....... References .................,.............................. .....................

.

.

AUTHOR INDEX.......................................................................................... SUBJECTINDEX.......................................................................................... CONTENTSOF PREVIOUS VOLUMES..............................................................

30 1 304 307 308 319 320 32 1 322

323 343 345

This Page Intentionally Left Blank

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

KENNETH L . APPLEGATE,Department of Poultry Science, Ohio State University, Columbus, Ohio (97)

ERIC ATHERTON,Children’s Cancer Research Foundation and Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts (203) R. CHIPLEY,Department of Poultry Science, Ohio State University, Columbus, Ohio (97)

JOHN

R. R. COLWELL,ODepartment of Biology, Georgetown University, Washington, D.C. (137) ARNOLDL. DEMAIN,Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts (177) THOMAS H . JUKES, Division of Medical Physics, The University of California, Berkeley, California (1) KIYOSHI HIGUCHI, Microbiological Associates, Inc., Bethesda, Maryland (111)

R. KENWORTHY, Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, England ( 31) ROBERT E . LENNON,Fish Control Laboratory, Bureau of Sport Fisheries and Wildlife, Fish and Wildlife Service, United States Department of the Interior, La Crosse, Wisconsin (55) M E I E N H O F E R , Children’s Cancer Research Foundation and Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts (203)

JOHANNES

W I L L I A MB. SAFUES, Professor Emeritus of Bacteriology, The University of Wisconsin, Madison, Wisconsin (301) CLAUDEV ~ Z I N A Department , of Microbiology, Ayerst Research Laboratories, Montreal, Quebec, Canada (55)

*Present address: Department of Microbiology, University of Maryland, College Park, Maryland.

ix

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ADVANCES IN

Applied Microbiology VOLUME 76

This Page Intentionally Left Blank

Public Health Significance of Feeding Low Levels of Antibiotics to Animals

THOMAS H.

JUKES

Division of Medical Physics, The University of California, Berkeley, California I. Introduction

...............................................................

1

11. Clinical Studies in Human Medicine ............................. 111. Toxicological Studies with Some “Feed-Type”

5

.................................... Antibiotics ..................... IV. Alleged Hazards of Feeding Antibiotics to Animals ... V. Discussion ..................... .................................... References ..................................................................

11

I.

25 28

Introduction

T h e feeding of low levels of antibiotics to farm animals was introduced experimentally in 1949 and commercially in 1950. Chlortetracycline, oxytetracycline, penicillin, and streptomycin were the first to be used, and other antibiotics, especially bacitracin and tylosin, soon followed into use. The term low level originally meant up to 50 gm per ton of feed (about 50 ppm). Levels u p to about 200 ppm are often used, however, and I shall include levels up to 200 ppm in this discussion rather than attempt to exclude all findings with levels above 50 ppm. The initial use of low-level antibiotic feeding was to promote growth of chickens, pigs, and calves. I n 1948, we were looking for sources of vitamin B12that could be added to poultry feeds, because a lack of this vitamin was decisive in limiting growth on diets containing vegetable proteins, such as soybean meal. Our procedure was to test microbial cultures that could be produced by deep-tank fermentation. One such organism is Streptomyces aureofaciens. The results of the first experiment, started December 2, 1948, are shown in Table I. The growth of chicks receiving Aureomycin mash was more rapid than that of chicks receiving a complete diet. The second experiment, started 2 weeks later, is reported in Table 11. The effect of liver extract was due to vitamin B12, and the effect of Streptomyces aureofaciens mash was caused largely by vitamin B12and Aureomycin, now known as chlortetracycline. Very few experiments have been repeated so many times. Penicillin mash was completely ineffective; perhaps the penicillin had decomposed.

1

2

THOMAS H. JUKES

TABLE I EFFECTOF AUREOMYCIN FERMENTATION ON CHICK GROWTHn ____

~~

Days on experiment 11 15 21 25

0 Addition per kilogram of basal diet None None Liver extract, 0.4 ml Liver extract, 0.6 ml Liver extract, 1.0 ml Autoclaved liver extract, 1.0 ml Autoclaved liver extract, 3.0 ml Aureomycin fermentation, 100 ml Aureomycin fermentation, 300 ml Penicillin fermentation, 100 ml Penicillin fermentation, 300m1, a From

No. of survivors at 25 days

Average weights (gm)

39 39 39 39 39 39 39

62 59 66 76 76 74 84

82 74 93 104 113 99 116

100 100 140 156 177 146 182

107 112 175 197 235 192 238

3 1 6 7 10 8 11

39

67

89

125

167

11

39

94

140

215

274

11

39

65

76

Discontinued

39

63

75

Discontinued

T. H. Jukes and E. L. R. Stokstad (unpublished data, 1948). TABLE I1

SECONDEXPERIMENT:EFFECTSO F LIVEREXTRACT (SUPPLYING VITAMINB I ~ ) , AND AUREOMYCIN FERMENTATION ON THE GROWTH OF CHICKS RECEIVING A DIET DEFICIENTI N VITAMIN BI~“.” Days on experiment

Addition per kilogram of basal diet None None Liver extract, 2.0 ml Aureomycin fermentation, 200 ml Aureomycin fermentation, 400 ml

0

11

15

21

25

No. of survivors at 25 days

36 36 36

64 64 76

85 76 101

124 90 160

136 106 212

2 5 12

36

95

135

212

274

11

36

99

139

212

274

11

“From T. H. Jukes and E. L. R. Stokstad (unpublished data, 1948). bDecember, 1948; 12 chicks per group. A preceding experiment indicated that 1.0 ml liver extract (15 antipernicious anemia unitslml) gave maximum growth. The deaths are attributable to vitamin B12 deficiency.

3

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

In spite of the publication of hundreds of reports confirming this finding, the following comment was published in 1968: “The earlier experiments depicting this beneficial effect of antibiotics were not conducted under today’s standards of scientific experimentation, and many critics seriously question whether antibiotics actually do provide the alleged ‘growth stimulation”’ (Smith, 1968). The editor of FDA Papers refused to publish my rebuttal of this statement. In this context, it is of interest to note that Begin (1971) reported that “antibiotics retain their growth-promoting ability over a great many years in which antibiotics have been in more or less continuous use.” He found that the growth response produced in chicks was 7% in 1953 and 10% in 1970, in the same laboratory. From the first, it was evident that the feeding of antibiotics produced resistant microorganisms in the digestive tract because the surviving organisms have to tolerate antibiotics (Table 111). Some typical experiments showed a temporary drop in numbers of intestinal bacteria for the first day or two, followed by a return to levels higher than before. What we were not prepared for was the fact that the changed and resistant flora were in some way beneficial, due either to the presence of certain new bacterial strains or the absence of certain former strains or species. It took us some time to adjust to such a concept, accustomed as everyone was to the idea of resistant organisms being pathogenic. Indeed, the growth effect is so illogical that I recommend others trying the experiment with chicks. Furthermore, sulfonamides, when added to a purified diet for rats, can produce vitamin deficiencies when the vitamins are omitted from TABLE I11 AEROBIC BACTERIAL COUNTS IN RAT FECES~.* Bacterial counts (X lo-’) Hours after start of CTC feeding ~

Aerobes, total Aerobes, CTC resistant

~

Diet

0

12

36

84

144

P C P C

96 134 0.51 0.46

453 136 0.49 2.19

224 186 37.0 7.8

161 195 19.2 22.0

278 360 26.6 70.0

“From Johansson et al. (1953). *P, purified diet; C, commercial diet. CTC resistant medium (agar)had 10 ppm CTC. Conclusion: Significant increase in CTC resistant bacteria within 12 to 36 hours.

4

THOMAS H. JUKES

the diet. The rat obtains certain vitamins from its intestinal flora, and sulfonamides depress the biological synthesis of these vitamins. Our experiments showed that sulfonamides did not produce the growth response in normal chickens that we obtained with chlortetracycline. Another surprise was that antibiotics lessened the requirements of rats and other experimental animals for several vitamins and minerals. The blood levels of riboflavin and calcium in chicks were increased by feeding chlortetracycline. Rats were protected against thiamine (vitamin B,) deficiency by the addition of penicillin or chlortetracycline to a purified deficient diet. Sulfamerazine and streptomycin had the opposite effect. These findings, however, did not explain the “antibiotic growth effect,” which we found with diets that contained more than sufficient quantities of all known vitamins. The responsibilities for introducing a new practice into animal husbandry and a new type of additive into feeds are weighty and considerable. Would the procedure lead to the appearance and spread of resistant strains of harmful bacteria? I expected the question would be answered by the disappearance of the beneficial effects of antibiotic feeds and that, within a year or two, their use would cease as a result of the spread of resistance. The introduction of a biologically active substance into the feeding of domestic animals that are used to produce meat and eggs is a step that calls for a number of points to be examined for the protection of consumers. Among these points are the following:

1. What levels of residues of the additive will be present in the food product that reaches the consumer? Obviously, some level will be present, for, as any chemist will tell you, molecular purity does not exist, and there is no such thing as a “zero level” of a chemical in the tissues of an animal that has received the chemical by mouth, provided that the chemical can pass from the intestines into the blood stream. However, if the compound is readily destroyed by mild heat, it may be reduced by cooking to a level that can have no conceivable biological effect.

2. Is the antibiotic toxic at any level, and, if so at what level? 3. Will the residues in human food produce allergic reactions? 4. Will the use cause problems of resistant pathogens?

5. Will the residues disappear after a reasonable withdrawal period? 6. Will the use in feed impair the usefulness of the antibiotic in clinical medicine, veterinary or human?

5

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

II.

Clinical Studies in Human Medicine

Our experiments were at Lederle Laboratories, where the primary concern was, and is, human medicine rather than feeding livestock. Indeed, it was a long time before we could supply chlortetracycline mash for animal feeds because the antibiotic was needed for patients. However, this was opportune, because it enabled chlortetracycline to be tested directly on human beings for safety before it was used as a feed additive. Also, we had long been interested in tropical diarrhea because of our work with folic acid and sprue, which was started at Lederle. With the collaboration of Lederle’s International Clinical Department, chlortetracycline was provided for infants and children, and experiments were reported in the literature from both tropical and nontropical countries, as summarized in Table 1V. I will touch on some of these briefly. TABLE IV EXPERIMENTS WITH INFANTS AND CHILDRENIN WHICHCHLORTETRACYCLINE AND OXYTETRACYCLINE WERE ADMINISTERED DAILY Duration

of Investigator

Country

Premature infants Robinson (1952)

Israel

Perrini (1951)

Italy

Dosage per day CTC 50 mg/kg body wt. 25 mglkg body wt.

treatment

Number of treated

-

15

-

10

-

47

-

36

Snelling and Johnson

(1952)

Canada

50 mg total

H. L. Cubas (personal communication, 1955)

Peru

40 mg total

Infants and children Macdougall(l957) Lewis et al. (1956) C. H. Carter (personal communication 1953) McVay and Sprunt (1953)

Kenya India USA USA

Jolliffe et al. (1955-1956) Italy Guzman et al. (1958) Guatemala Goff (1955) USA Bulatao-Jayme et al. (1957) Philippines Mackav et al. (1956) Jamaica Loughiin et aZ.(1957-1958) Haiti Litchfield et al. (1957-1958) USA

50 mg 25 mg

2-7 weeks 7 weeks

38 10

1-3 years u p to 20 months 7 months 15-30 months 8-36 months 18 months 2 years 12 months

20 23 181 92 25 140 256 243

5 or 50 mg 6-12 months

2

150 mg 500 mg 20 mg 50 mg 50 mg 50 mg 32 mg OTC 50 mg

1263

6

THOMAS H. JUKES

Robinson (1952), Director of Public Health, Tel Aviv, gave Aureomycin to one of each set of premature twins or triplets. Five of 15 controls died from intercurrent infections; all treated infants survived (Table V). No bacteriology was studied. TABLE V

RESULTS IN ELEVENSETS OF TWINS(CASES1-11) AND Two SETS OF TRIPLETS(CASES 12 AND 13)”.* Babies receiving Aureomycin

Controls

Weight (gm)

Weight (gm)

Case No.

Duration of treatment (days)

BT‘

AT‘

Daily gain

Outcome

BT

1 2 3 4 5 6 7 8 9 10 11 12

26 13 12 28 25 15 19 19 19 20 22 23

1140 1540 1870 1650 1670 2000 1940 1440 2000 2000 1450 2030

2000 1830 2140 2430 2500 2600 2430 1850 2400 2500 1980 2650

31 22 22 28 33 40 25 21.5 21 25 24 27

Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive Alive

13

32

1400 2470

33.5

Alive

1260 1800 1890 1700 1170 2000 2120 1720 2120 2050 1580 1950 1840 1400 1400

AT

Daily gain

1670 16.5 1910 5 2010 10 2500 28 1750 23 1850 -10 2480 19 1900 9.5 2450 17 2550 25 2000 19 2350 17.5 2280 18.5 2150 23 1800 12.5

Outcome Alive Dead Alive Alive Alive Dead Alive Alive Alive Alive Alive Alive Dead Dead Dead

“From Robinson (1952). bWith two exceptions (Cases 4 and 10) all the babies who received Aureomycin gained considerably more weight than the controls. The average daily gain was 29.5 gm in the treated group compared with 18 gm in the controls. Five of 15 controls died from intercurrent infections, whereas all the babies who received a full course of Aureomycin survived. BT, before treatment; AT, after treatment.

Snelling and Johnson (1952), Hospital for Sick Children, Toronto, found that an epidemic of acute nutritional disturbance in 1949, characterized by diarrhea, vomiting, dehydration, and toxemia, was controlled by Aureomycin (Table VI). They said in 1952, “Because of the success of this, as a prophylactic measure we have placed all of the premature babies in our unit on aureomycin, 50 mg daily, since that time.”

7

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

TABLE VI AUREOMYCIN(50 MG DAILYFOR 26 TO 56 DAYS)IN PREVENTION O F CROSS INFECTION IN THE HOSPITAL FOR SICK CHILDREN" Statistic

Aureomycin

No. of cases Deaths % Mortality Nonfatal infections Total morbidity % Morbidity

Controls 48 8 16.6 8 16 33.3

47b 1 2.1 6' 7" 14.9

a From Snelling and Johnson (1952). b T ~ of o these had cross infection gastroenteritis which failed to respond to other antibodies, before aureomycin was started. rOne case was admitted with a second degree bum from a hot water bottle.

Guzman et al. (1958) studied school children in Guatemala and found an initial stimulatory effect of Aureomycin in the rates of growth on both weight and height ( p < 0.01) (Table VII). No side effects were reported. UNADJUSTED

TABLE VII MEANGROWTH RATES OF GUATEMALAN SCHOOL CHILDREN" ~

Treatment

Penicillin Aureom ycin Placebo Penicillin Aureomycin Placebo

No.

Weight (kglmonth)

Height (gm/month)

~~

Starting age

Period One (November 1953-November 1954) 64 0.20 0.42 9.92 92 0.23 0.47 9.27 0.40 9.64 104 0.18 Period Two (December 1954-November 1955) 54 0.17 0.35 9.26 92 0.19 0.38 8.49 0.34 8.65 104 0.15

Treatment frequency

208.06 184.79 -b 207.39 198.99 -b

"Cuzman et al. (1958). bArbitrarily defined as identically zero. Actual frequency of placebo administration did not differ significantly from that for penicillin and Aureomycin.

Loughlin et al. (1957) concluded that "Oxytetracycline might be used safely as a food preservative in underdeveloped countries to improve nutrition." None of the staphylococci or enterococci growing in rectal cultures became increasingly resistant to oxytetracycline.

8

THOMAS H. JUKES

C. H. Carter (personal communication, 1955) gave 150 mg of chlortetracycline daily to 20 mentally retarded children at the Florida Farm Colony. They gained 6.5 lb per year as compared with 1.9 Ib for controls. Unfortunately these experiments were never published. McVay and Sprunt (1953) found no evidence of development of resistance by P-hemolytic streptococci in patients with rheumatic fever (Table VIII). Their study also included 400 geriatric patients as well as children. TABLE VIII CHLORTETRACYCLINE (CTC) IN THE PROPHYLAXIS OF RHEUMATIC FEVER^,* P-Hemolytic streptococci Positive throat swabs after 10.5 months (%) Resistance (total numbers) Increase No change Decrease Respiratory infections (%) Decrease No change Increase

CTC

Placebo

19

30

6 3 11

4 8 12

14 9 0

3 8 1

“From McVay and Sprunt (1953). Authors’ conclusion: “Extensive studies [450 patients] revealed no evidence of the development of significant resistance by Croup A P-hemolytic streptococci to prolonged aureomycin therapy.” “Average treatment, 9 months.

Chlortetracycline and oxytetracycline were both used b y Lewis and co-workers (1956) in a study of Indian children with nutritional deficiencies. The blood analyses are summarized in Table IX. The authors commented favorably upon the possible use of small doses of antibiotics for kwashiorkor. Macdougall (1957) reported that African children with marasmus showed marked improvement during 2-7 weeks of treatment with chlortetracycline, 50 mg daily, as compared with controls (Table X). Bulatao-Jayme et al. (1957) reported that children receiving chlortetracycline, 50 mg daily for 18 months, had fewer cases of illness than controls, as shown in Table XI. Initially, there were 114 children in the experimental group and 108 controls; at 18 months, these numbers were 75 and 65, respectively.

9

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

TABLE IX EFFECT OF 25 MG OF CHLORTETRACYCLINE (CTC) OR OF OXYTETRACYCLINE (OTC) DAILYON BLOODPROTEINS IN INDIAN CHILDREN WITH NUTRITIONAL DEFICIENCIES~ Hemoglobin

Albumin

Globulin

Treatment

No. of cases

Before

After

Before

After

Before

After

Control Antibiotic

7 10

8.9 8.2

9.6 11.0

2.30 2.18

2.89 3.72

3.40 3.38

3.55 3.02

“From Lewis et al. (1956). Authors’ comment: “All of the cases which were treated with small doses of antibiotic recovered and in the series not given antibiotic there were three deaths relatively late in the period of hospitalization. If the administration of small doses of antibiotic can prevent such accidents this would be sufficient reason for their use in the treatment of kwashiorkor.” TABLE X WEIGHTGAINAND GENERALNUTRITIONALSTATUSAT ENDOF TRIALPERIOD IN AUREOMYCINAND PLACEBO GnouPsa’b Treatment: No. of cases: Average daily weight gain (gm): SD: Nutritional status Good Fair Poor Very poor Moribund

Aureomycin 38 45.3 20.01

Placebo 34 14.1 21.95

Before

After

Before

After

0 0 5 12 21

24 14 0 0 0

0 0 2 9 23

7 10 8 3 6

From Macdougall (1957). *Aureomycin group received50 mg ofchlortetracycline daily. Children were “severely undernourished with no chemical evidence of active infection.” The data in this table are adjusted by multiple regression methods for differences in initial age and weight.

In the fifteen studies summarized in Table IV, any reported effects of continuous administration of chlortetracycline and oxytetracycline were beneficial. There were no cases described of infection with resistant pathogens. The results of feeding the tetracycline antibiotics included lowered morbidity and mortality, decreases in infections, and slight to moderate increases in growth as compared with controls. I noted this work with interest, because I hoped that what chlortetracycline did for farm animals it might do for children.

10

THOMAS H. JUKES

TABLE XI OF ILLNESSES AND NUMBER OF SUBJECTS TYPEAND DURATION (FILIPINO CHILDREN, AGES 2-12) AFFECTED DURING 18 MONTHS" Chlortetracycline, 50 mg daily

Type of illness

Cases

Average duration of illness

Cases

Average duration of illness

28 24 24 11

4.6 9.5 7.4 17.5

33 16 47 14

4.6 11.8 10.0 16.4

6

7.3

15

22.9

0 0 6 99

-

7 9 7 148

2 3.4 33

Acute upper respiratory infection Measles Acute conjunctivitis Chickenpox Skin infections and allergy Gastrointestinal infections Undiagnosed fevers Other

All illnesses 'I

From Bulatao-Jayme et

Controls

(11.

6.7 ~

8.66

~

11.22

(1957).

Why were these findings not followed up by more extensive nutritional studies? I believe the reasons included the following: 1. Cost. The work was applicable mostly to impoverished subjects. Antibiotics are far more expensive per gram for human use than in animal feed. 2. Lack of commercial interest. There is no demand for rapid growth in children. 3. Lack of economic motivation by the pharmaceutical industry. 4. Emphasis on use of antibiotics for treatment rather than for continuous dosage. Certainly there were no problems with toxicity or identified resistant disease. There were no reports of salmonellosis. Children grow far more slowly than farm animals. Nevertheless, there is still a good opportunity to use low-level feeding with antibiotics to improve nutrition and reduce diarrhea among children in an impoverished condition, especially in tropical countries. According to Borgstrom (1968) only one-seventh of the world's population has access to water from a faucet, and fecalism is the characteristic hygienic feature of the malnourished, overcrowded world. This sounds like the conditions under which chickens and pigs are reared intensively, and where antibiotic feeding has been useful. I believe that additional large-scale experiments are indicated. Suitable anti-

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

11

biotics can b e purchased in bulk as used in animal feeds for about 2 or 3 cents per gram. Fifty milligrams per day should be tried. Close surveillance of morbidity and resistant pathogens should be included. The small intestines of germfree animals, or animals fed antibiotics, are shorter and thinner than those of apparently healthy (“conventional”) controls (Coates et al., 1955;Elliottand Owen, 1955).Diarrhea in farm animals, especially pigs and calves, is often relieved by feeding antibiotics. The utilization of food by animals is improved by adding low levels of antibiotics to the diet. These observations recall the symptoms of the human disease tropical sprue, which is accompanied by diarrhea, malabsorption, and pathology of the wall of the small intestine. Is the ‘‘conventional’’ animal, living in an unsanitary environment, in a condition that is mildly “spruelike”? Conversely, since feeding antibiotics specifically alleviates the intestinal effects in animals, would the same treatment be useful in sprue? Anderson et al. (1954) and French et al. (1956) found that antibiotics produced almost uniform clinical improvement in sprue. French et al. suggested that antibiotics were effective in tropical sprue through alterations in the intestinal flora, but Sheehy and PerezSantiago (1961) concluded that the mechanism was “more complex than simple elimination of bacterial competition.” These workers found improvement in jejunal morphology and vitamin B 12 absorption in 6, and xylose absorption in 7, of 12 patients treated with tetracycline and chloramphenicol. Prolonging the treatment for 6 months produced intestinal improvement in a higher proportion in studies by Guerra et aE. (1965). They found, that in 8 patients, vitamin BIZabsorption became normal in 6, jejunal morphology nearly normal in all, and xylose absorption normal in all. The treatment was oral tetracycline or oxytetracycline, 1 gm daily for 1 month and 0.5 gm daily for the next 5 months. Klipstein and Falaiye (1969) reported that oral tetracycline, 1 gm daily, produced improvement in 3 weeks in 11 of 12 sprue patients. Prolonging the antibiotic therapy for 6 months led to further improvement. The criteria were remission of megaloblastic anemia, cessation of steatorrhea, increased xylose absorption, and reversal of jejunal abnormalities. The absence of deleterious resistant enterobacteria resulting from prolonged ingestion of tetracycline is clearly inferable in these therapeutic effects of tetracycline on the pathology of the small bowel. 111.

Toxicological Studies with Some ”Feed-Type” Antibiotics

The toxicities of chlortetracycline (OTC), oxytetracycline (CTC), and tylosin have been studied and reported in long-term tests.

12

THOMAS H. JUKES

Lifetime studies with animals are preferred for the toxicological evaluation of a food additive; in such studies, a toxic level must be reached so that an estimate of the range between the effective and toxic dose may be made. Experiments were carried out with rats fed chlortetracycline at 8 different levels ranging up to 5% of the diet (Dessau and Sullivan, 1961). At this top level, most of the rats survived until the experiment was terminated at 25 months; the survivors were healthier than the controls, although their fur and bones were literally yellow with chlortetracycline. Their diet contained about five hundred thousand times the level present in the muscle meat of chickens receiving 2000 ppm of the antibiotic in their feed. Evidence of drug toxicity was seen only in the group receiving 5% (50,000 ppm) of chlortetracycline. Ten of the 40 rats died in the first 6 months, but 29 of the 30 remaining lived until 25 months. Two cases of infection occurred in the 5% group, as compared with 38 cases in the controls. Unfavorable deviations seen only in the 5% group included acute gastrointestinal disturbances, growth depression, fatty material in livers, and testicular atrophy. In the entire population, including all groups, suppurative lung disease was the most common cause of death. This disease, and ear infections, were markedly lower on the higher levels of the antibiotic (Table XII). There were 17 cases of lung lymphoblastoma in groups 1 to 5 and none in groups 6 to 9. TABLE XI1 EFFECTSOF PROLONGED ADMINISTRATION OF CHLORTETRACYCLINE (CTC) ON HATS" Infections Croup Dose level No. of Tumors, No. ofCTC animals Obesity Skin Lungs Ear all types

1 2-5 6-9

None 40 1-100ppm 160 0.05-5% 160

1 4 18

14 68 42

18 85 15

6 41 3

33 114 112

Mortality 0-6 months 1 0

lob

7-24 months 19

73 22

"From Dessau and Sullivan (1961). bAll in 5% dosage group.

Chlortetracycline at a level of 1% in the diet was well tolerated. The daily dose of the antibiotic at this level corresponded to about 0.7 gm per kilogram of body weight daily, equivalent to almost 50 gm per day for a 70-kilogram human being. Normal reproduction took place through two generations on this diet (Hallesy et al., 1964). Twenty successful matings in the second generation produced 165

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

13

live young in the controls and 190 in the antibiotic group. The drug had no effect on weight gain, fertility, or reproductive ability. Aside from the toxic effects of the highest level, chlortetracycline suppressed infections and resulted in apparently better health over a period of two years, which corresponds to most of the lifetime of a rat. The effects of tetracycline and oxytetracycline on prolonged feeding were studied exhaustively by Deichmann and co-workers (1964). These investigators did not use levels higher than 0.1%, but up to this level the antibiotics increased survival time in 11 of the 12 experimental groups. In a second experiment along similar lines, pneumonia occurred more commonly in the control groups than on the higher levels of antibiotics. No signs of toxicity were noted in rats on oxytetracycline or tetracycline at levels of 0.01% and 0.3% for 24 months. The supplemented rats “appeared more vigorous and gained weight more rapidly than the control animals” during the first 18 or 19 months. After this, degenerative changes were encountered in all groups, unrelated to drug intake. No signs of toxicity were found in dogs and pigs receiving levels of OTC and CTC up to 1% for periods of u p to 2 years. The results with tylosin, a macrolide antibiotic, were essentially similar, except that tylosin was well tolerated for 2 years at even higher levels, up to 5% of the diet (Anderson et al., 1966). Survival of rats on the drug-supplemented diet was better than in control groups. Reproduction and lactation were unaffected through three generations on 1% tylosin. In general, these studies have produced much interesting information on geriatric changes in rats, but very little of this is related to drug toxicity. The high incidence of tumors in the control groups (Dessau and Sullivan, 1961) emphasizes the difficulties of detecting tumorigenicity in low levels of chemical additives fed to rats. Longevity can be increased in rats by supplementation with high levels of appropriate antibiotics. The results make it difficult to believe that antibiotic feeds are likely to be harmful to farm animals. Nothing in any of these four reports suggests that continuous and prolonged exposure to antibiotics led to deleterious effects resulting from the emergence of resistant strains of pathogens. These studies in animals are paralleled by extensive experience with prolonged administration of the tetracyclines to human subjects. Hines (1956) reviewed some reports in this field. As an example, h e commented : Perhaps the most remarkable record of sustained antibiotic therapy is that of McVay and Carroll (1952) who treated 2 cases of blastomycosis with 1908 and

14

THOMAS H. JUKES

1042 gm. of chlortetracycline over 21 and 11 month periods respectively. The daily intake varied between 3 and 4 gm. Since these 2 patients received massive amounts of chlortetracycline over a prolonged period of time, comprehensive studies were performed to detect evidence of toxic effects but none were observed. Initially, and every three months thereafter, a liver punch biopsy and a sternal marrow aspiration were performed and the material thus obtained was examined microscopically. A complete peripheral blood study and urinalysis were carried out each week, a blood nonprotein nitrogen was obtained each month, an electrocardiogram every two months, and four determinations of the urinary 17-ketosteroids were carried out during the study. Repeated determinations of bromsulphalein retention and cephalincholesterol flocculation tests were conducted. Two glucose tolerance tests were also found to be within normal limits. Few if any therapeutic agents have ever been administered at this high dose level for so long a period without some evidence of toxic manifestations.

The toxicological studies therefore give no reason for questioning the safety of animal feeds containing the antibiotics described. Also, these studies, in addition to showing a lack of frank toxicity, faiIed to reveal any occurrence of disease resulting from resistant pathogens despite the prolonged exposure of the test animals and human subjects to the antibiotics. The rats fed levels of chlortetracycline ranging from 500 to 10,000 ppm (0.05% to 1%) appeared to benefit in terms of lower morbidity and mortality, although a number of cases of obesity occurred. The bones and teeth of these animals were deeply colored by the antibiotics. Allergic reactions to antibiotics can occur as in the case of numerous other therapeutic agents. Penicillin has been quite prominent in this effect: the only such reactions to antibiotics as reported in food products are from penicillin in milk. A case of dermatitis attributed to this cause was described by Borie and Barrett (1961).The presence in milk usually resulted from mastitis treatment, not from feeding. The use of penicillin for mastitis has been discontinued. The opportunity to study possible occurrence of allergy was greatly increased b y the use of CTC and OTC for delaying spoilage in poultry meat and fresh fish. These foods w&redipped in a weak solution of the antibiotic, and the product was sold with a finite tolerance of the additive higher than would be encountered in meat from animals receiving antibiotic feeds. The dipping process was used in the United States and Canada for several years. No authenticated cases of sensitivity reactions. to CTC or OTC came to light as a result of their use. The practice also gave an opportunity to examine the meat for pathogenic microorganisms, such as Salmonella. In an investigation reported by the Canadian Food and Drug Directorate (Thatcher and

15

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

Loit, 1961), Salmonella was isolated from only one sample of poultry meat dipped in CTC, but 16 isolates of four species were obtained from 16 untreated samples (Table XIII). Four different serotypes TABLE XI11 ISOLATIONOF Salmonella FROM MARKETPOULTRY TREATED AND NONTREATED WITH CHLORTETRACYCLINE (CTC)“

No. of specimens yielding Salmonella (from 90 treated, 80 nontreated chickens) CTC-treated Species

“Fresh”

S. gallinarum S. typhimurium S. oranienburg

0 0 1 0 -

S . indiana Total recovery

No CTC

“Spoiling”

“Fresh”

“Spoiling”

0

1 4

0 0 1

1

0

0 0 0

5 4

-

14

1 2

From Thatcher and Loit (1961).

were recognized, including S. typhimurium. The authors stated that cell isolates grew in broth containing 0.21 ppni of CTC, but none grew at 0.43 ppm. Thatcher and Loit (1961) commented: “No data to indicate an increased health hazard as a result of the use of CTC as a poultry preservative were obtained. No evidence was revealed for modification of hazard due to the presence of staphylococci, enterococci, coliforms, or pathogenic yeasts.” The use of tetracyclines for delaying spoilage of fish and meat relied on the principle that the organisms were psychrophils, predominantly pseudomonads. Spoilage is caused by these organisms in the range between room temperatures and refrigerator temperatures. Growth of coliforms and pathogens, such as staphylococci, at such temperatures is very slow. The studies with tetracyclines in food spoilage are cited here because they gave rise to information on the levels of residues in meat, and in the rate of disappearance of such residues. Many studies have been made in the tissues of animals receiving antibiotics. The tetracyclines have an affinity for bones and teeth, in which they are found after being administered at high levels. They are not retained in soft tissues. The tetracyclines are excreted fairly rapidly in the urine and in consequence are found at higher levels in the kidney than in other tissues. Some antibiotics used in

16

THOMAS H. JUKES

feeds, including tylosin, bacitracin, and penicillin, have not been detected in tissues by the methods available for their assay (Table XIV). Chlortetracycline disappears from muscle after a withdrawal period of 4 or 5 days (Table XV). It is broken down b y cooking into isochlortetracycline, a compound without known biological activity (Shirk et al., 1956-1957). TABLE XIV TISSUELEVELSOF ANTIBIOTICS FOLLOWING FEEDINGa TissuesCand level (ppm) Antibiotic level (ppm)

Time fed (days)

Species

CTCb, 100 CTC, 100 Penicillin, 50

98 98 98

Swine

CTC, 200 CTC, 200 CTC, 2000 CTC, 2000 Lincob, 400 Linco, 400 Tylosin, 50 Tylosin, 1000 Erythromycin, 400 Bacitracin, 1000 OTCb, 220 Penicillin, 200

21

Chickens

5 10

8 108 42

Days of withdrawal

0 5 0 0 4 0 3 0 2 0 3 0 0 0 0

K 0.51 0.07 0

L 0.46 0.04 0

0.62 2.2 0.26 0.03 1.6 12. 0.20 0.02 0.37 0.24 0 0 0 0 Trace 0

0 0 5.6 0

M 0.08 0 0 0.26 0 0.63 0-0.02

0 0

0

0 0 0

0 1.2 0

0 0.3 0

F 0

0 0 0.12 0 0.17 0 0 0 0 0 0 0

-

0

“Chicken data summarized from review by Shor (1971). Swine data from Messersmith et al. (1967). bCTC, chlortetracycline; OTC, oxytetracycline; Linco, lincomycin. ‘K, kidney; L, liver; M, muscle; F, fat.

The next point with respect to safety is: Has the use of antibiotics in animal feed impaired their effectiveness in clinical practice? Fear of such an impairment has been advanced as a reason for banning the use of “clinical” antibiotics for animal feeds (Swann, 1969). The tetracyclines, penicillin, streptomycin, and erythromycin all continue to be used widely in medicine. Bulger and Sherris (1968), for example, found that the majority of strains of Staphylococcus aureus were sensitive to antibiotics except for penicillin. In no case had there been a trend toward more resistance in the preceding 8 years (Table XV). Problems of resistance arise in specific instances, but no evidence exists linking such problems with animal feeds.

17

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

TABLE XV

PERCENTAGE OF STRAINSOF Staphylococcus aureus SENSITIVE TO VARIOUS ANTIBIOTICS a

Percent of strains sensitive Antibiotic

1959

1961

1963

1965

1967

Penicillin G Tetracycline Streptomycin Kanamycin Erythromycin Chloramphenicol

14.9 39.5 14.4 90.4 56.8 71.3

28.9 65.6 51.2 75.8 90.8

29.1 64.2 66.4 94.2 72.9 92.0

30.3 73.4 77.4 94.0 82.6 91.7

33.0 82.8 87.4 95.4 92.0 96.0

167

553

66 1

759

349

Total strains

“Bulger and Sherris (1968).

IV.

Alleged Hazards of Feeding Antibiotics to Animals

The alleged hazards of feeding antibiotics to animals were reviewed and summarized in the Antibiotic Task Force Report (FDA Task Force, 1972) as “guidelines” under the headings of “Human Health Hazard” and “Animal Health Hazard.” These guidelines were also developed under the same headings, but at greater length in Appendices B and C of the Report. I shall refer to this report at some length because of its bearing on the subject of this article. I shall first discuss the guidelines relating to “Animal Health Hazard,” because, if there are hazards from feeding antibiotics, the ill effects should appear first in the farm animals that consume feeds containing antibiotics. Any ill effects will then provide clues to some or all of the human health hazards. Indeed, when antibiotic feeding was first introduced, I expected that if the practice became ineffective or deleterious, this would be discovered in animal husbandry, and antibiotic feeding would be stopped because of lack of usefulness. This would be a safeguard against human health hazards. The Task Force Report lists the assumptions considered in determining guidelines as follows: A. Antimicrobial drug resistant pathogenic bacteria are considered undesirable and harmful to the host animal as well as to those animals of the same species in the immediate environment. B. Currently available information indicates that pathogenic organisms harboring R-factors are equally as virulent as either antibiotic sensitive or resistant organisms.

18

THOMAS H. JUKES

The sentence is confusing because “pathogenic organisms harboring R-factors” are resistant organisms, by definition. Actually there is experimental evidence with Salmonella against this statement, because antibiotic resistance was found to be more readily acquired b y “rough,” nonvirulent strains than by smooth, virulent strains of Salmonella cholerae-suis (Jarolmen and Kemp, 1969a). This finding was ignored in the Task Force Report. C. Bacteria harboring R-factors -both pathogenic and “nonpathogenic” -are considered to be particularly hazardous because of their ability to transfer multiple drug resistance.

Evidently this ability has not led to ineffectiveness of antibiotics for farm animals. Multiple drug resistance is not new, even though its detection is a novelty. D. The use of antibacterial agents in feed at lower levels may induce sensitivity in the aniniai; when the same drug is used for therapy it may cause allergic or hypersensitive reactions which are considered undesirable.

Allergic or hypersensitive reactions are not a problem in farm animals. E. The use of a drug at the optimum dosage is intimately related to the safety as well as the efficacy of the drug.

This statement has no perceptible relevance to the matter under discussion. The section continues with a series of “bacteriological, pharmacological, and epidemiologic guidelines to establish the presence of a possible hazard to animal health; the guidelines are either (1) so far unsubstantiated or unexamined by experimental evidence, (2) unrelated to animal health, (3) already scrutinized b y the FDA, or (4) unrealistic; for example: If there is an increase in the number of multiple-drug-resistant animal pathogens present in animal feeds which contain antibiotics.

Animal pathogens should not be present in significant numbers in properly processed animal feeds. I am not aware of any such hazards arising from common use. Products that might conceivably carry pathogens, such as protein concentrates and bone meal, are routinely sterilized before use. Transmission of pathogens through animal feeds would be a matter of malpractice rather than antibiotic use. The appendix on animal health hazard starts with a preamble that

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

19

recites the effect of low concentrations of antibiotics in selection for the emergence of resistant strains, for example: The effect of drugs given nutritionally has been underestimated in the past, because the concentrations of drugs seemed too small to be significant. For a selection effect, however, nutritional concentrations of 5-100 ppm. in animal feed are far from insignificant. Guinee has shown that concentrations of antibacterial drugs as low as 2 ppm. facilitate the development and persistence of resistant bacteria.

The growth-promoting effect of antibiotics has been reported at levels as low as 1 ppm. Obviously, therefore, such low levels do not seem “too small to be significant.” Resistance of bacteria, in itself, is not harmful. The preamble then states that the use of antibacterial drugs as feed additives “has resulted in almost complete populations of livestock and poultry being subjected to such pressures with the resultant changes of pathogens becoming refractive to the therapeutic agents used.. . Actually, the same therapeutic agents continue to be used. The tetracyclines, penicillin and sulfonamides have not lost their effectiveness in veterinary practice even after more than 20 years of adding low levels to feeds for farm animals. The preamble then quotes two sentences from a report of a Committee on Salmonellosis as follows:

.

9,

A review of the literature on antibiotics in animal feed and on R-factors indicates that as currently practiced, the additives are causing undesirable changes in the balance between host and pathogen. Additional research is often of value, of course, but there is ample data now in the literature to support more rigid control of antibiotics in animal feed and water.

The reference is to a report on salmonellosis. This will be discussed later with respect to the human health hazard. The continued effectiveness of antibiotics at low levels in increasing the growth rate of farm animals is difficult to reconcile with the concept that a hazard to the animals has been occurring by feeding antibiotics. The guidelines for measuring a hazard to human health as a result of incorporating antibacterial drugs in animal feeds are enumerated in the Task Force Report as follows: A. Many diseases of man, particularly those caused by enteric bacilli, are caused by bacteria that are present in the flora of the individual prior to his illness.

20

THOMAS H. JUKES

B. Antibiotic therapy is less successful when the causative bacteria are resistant, as shown by in vitro testing, to the prescribed drug than when they are sensitive.

In uitro testing is not the way to measure the success of antibiotic therapy. C. Antibiotic resistant bacteria, including those harboring R-factors, are as virulent as are antibiotic sensitive bacteria.

This point was discussed above with reference to the question of a hazard to animal health. It is a generalization which needs to be referred to the antibiotic and the bacterial species. Many resistant strains of pathogens are virulent and hazardous. The origin of the resistance is the matter at issue. D. Evidence suggests that the use of certain antibiotics in animals and man promotes an increase in the reservoir of Salmonella through promotion of cross-colonization and infection, prolongation of the carrier state and relapse of disease.

Unsanitary practices, proliferation of Salmonellae in unrefrigerated food, fecal contamination by human beings and animals, including feral rats and pet turtles, as well as farm livestock, all are major factors in salmonellosis (Cherubin et al., 1969, 1970; Cherubin and Winter, 1970; Sato, 1967; Sat0 et al., 1970; Pocurull et al., 1971; Kiser et al., 1971; Dixon, 1965; Winshell et al., 1970). Neu et al. (1971) surveyed the resistance to antibiotics in Salmonella isolates in the northeastern United States in 1965-1969. They found that most isolates with transferable drug resistance had come from municipal hospitals and from children under 10 years of age. They concluded that resistant Salmonella are passed between children in low socioeconomic areas and are not introduced from farm animals or food processing. Salmonellosis was discussed in the Antibiotic Task Force Report, appendix 13, pages 4-8, in which the problem in foods as a result of contamination in slaughterhouses is emphasized. An estimate is made of the incidence of salmonellosis as follows: 23 reported outbreaks in 1970 involved an average of 108 cases per outbreak. T h e mortality is 0.26%. From surveillance in certain areas, “the estimated true number of foodborne outbreaks from any etiology in 1970 was 3,600,” about 13% of which (468) may be Salmonella. Of these 468, 77% (359) would have been caused b y pork, beef, or poultry products, representing 38,772 cases and 100 deaths. The argument is that the number of cases (and deaths) could be reduced by stopping the use of antibiotics in animal feeds because this use increases the shedding of

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

21

SaZmoneZZa by farm animals. The experimental evidence for this is based almost entirely on references to studies with human subjects and laboratory mice (Askeroff and Bennett, 1969; Bohnhoff and Miller, 1962; Dixon, 1965; Rosenstein, 1967), again indicating that clinical medicine may be to blame for a problem that is wrongly being attributed to animal feeds. Indeed, the report contains the following statement: The available data from studies on other animals provides an overwhelming indication that antimicrobial agents in animal feed are ineffective in eradicating Salmonella strains from the gastrointestinal tract of food-producing animals, prolong carriage of infecting strains, and render uninfected animals more susceptible to infection by small inoculae [sic] of Salmonella. These effects, though unconfirmed in food-producing animals, [emphasis added] lead to the conclusion that the practice of incorporating certain “growth promotant” antimicrobial agents in the feed of swine, poultry and cattle has probably enlarged the reservoir of Salmonella in these species beyond that which would pertain in the absence of these drugs. If the present reservoir among foodproducing animals is sufficient to result in at least 38,772 cases and 100 deaths and the likelihood of human infection varies directly with changes in this reservoir, then even a one percent change in the reservoir would be reflected in 3,877 cases and one death in humans each year.

However, the antibiotics used as “growth promotants” in animal feeds are not effective (and not used) against salmonellosis, and obviously cannot be expected to be “effective in eradicating Salmonella strains from the gastrointestinal tract of food-producing animals.” The arguments against antibiotics in animal feeds that are based on the question of salmonellosis do not seem to be related to the main causes of the Salmonella problem: The composition of the human bacterial florae is generally dynamic, and bacteria ingested in foodstuffs may become a part of this resident florae [sic]. The origin of bacteria in foodstuffs is not clearly defined, but bacteria of animal origin infect humans and may cause disease.

Bacteria in foodstuffs can originate from many sources. Walton and Lewis (1971) have discussed the role of food handlers and contamination from abattoirs in England. It is difficult to pinpoint antibiotics in feeds as aggravating problems caused by bacteria in foods. There is also a possibility that bacteria in foods might be decreased in numbers by the use of antibiotics. Williams Smith (1970) found that E. coli of animal origin did not become a part of the resident flora of a human subject. In 1959 and 1960, descriptions of the resistance transfer factor were published in Japan (Akiba et al., 1960). This factor is carried by an episome that occurs in Enterobacteriaceae and is transferred from cell to cell. The distinctive feature of the episome is its content of a seg-

22

THOMAS II. JUKES

ment of DNA carrying the information for its transfer to another bacterial cell through the cell wall. Genes for resistance to antibiotics and sulfonamides readily become integrated with the episome, and multiple drug resistance was found in Japan to be transferred in vitro from E . coli to Shigella, a causative organism for dysentery (Akiba et al., 1960; Watanabe and Fukasawa, 1961). This observation was viewed against the fact that multiple drug resistance in Shigella had first been detected in 1955 in Japan, in a dysentery patient who had just returned from Hong Kong. The earlier studies were reviewed by Watanabe (1963). The fact that the resistance could be transferred from the ubiquitous E . coli to a pathogen showed the need for research in the field. An increase in transferable resistance in cultures of virulent Salmonella typhimurium was noted in Great Britain during 1964 and 1965 (Anderson, 1968). This was predominantly associated with the spread of a multiple-resistant strain, identifiable as phage type 29. It was first isolated from calves. The infection peaked in 1965. Most of the infected calves had been kept under intensive conditions, and their case histories went back to contacts with calves brought from a single dealer (Anderson, 1968) who had purchased newborn calves throughout the country. These were transported immediately to his premises, and given “shotgun” therapy. The failure to feed these calves with colostrum has been mentioned as contributing to the ease with which they developed infections. The multiple-resistant S. typhimurium infection which appeared at this location was then distributed widely by reshipment of the infected calves to many points. The infection produced a high mortality rate in calves, sometimes over 50% (Anderson, 1968). Unlike most salmonellae, S. typhimurium cases causes infection in both man and domestic animals. Six human deaths were attributed to this outbreak. It was suggested that transferable tetracycline-resistance in this strain may have originated from “a single tetracycline-resistant organism which was able to start a focus of infection in a herd of calves.” This outbreak had wide repercussions in Great Britain and aroused much speculation in the press. This interest was reflected in the “Swann Report” (Swann, 1969). This report concluded that the outbreaks of infection, including human cases, due to s. typhimurium phage type 29, included cases resulting from multiple-resistant organisms which “acquired their resistance through the use of antibiotics in animals.” Whether this was the case it is not possible to decide. The explosive nature of the incident, and its subsequent recession is suggestive of an unusual genetic event, coupled with ex-

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

23

ceptionally opportune conditions for dispersal of the strain throughout the country. The incident was not attributable to feeds containing antibiotics; the use of these for calves is not permitted in Great Britain. The possibility for the recurrent emergence of such a virulent strain coupled with circumstances favoring its rapid spread must be weighed. The sudden nature of the outbreak, followed b y subsidence, provokes inquiry into why such incidents do not occur frequently (although the obvious malpractice in calf management was evidently one of the reasons for this epidemic). The following additional questions are pertinent: (1)Why are antibiotic feeds still effective even under conditions of widespread use? ( 2 ) Is multiple, transferable resistance in intestinal enterobacteria, such as E . coli, more common in human beings living in agricultural surroundings where antibiotics are widely used, than in urban areas? (3) How readily is resistance transferred in vivo as compared with in uitro? ( 4 ) How persistent are resistant strains of enterobacteria, especially E . coli and Salmonella? And how do immunogenicity and virulence correlate with resistance? (5)Is resistance typical of hospital environments? Of these questions, the most paradoxical problem is posed b y the effectiveness of antibiotic feeds under conditions that should favor the spread of all types of resistance. When the usual “feed” antibiotics are given to animals, intestinal bacteria become predominantly antibiotic tolerant or resistant (Jukes, 1955). Perhaps these bacteria tend to “overgrow” some of the pathogens. Nonpathogenic “rough” strains of Salmonella were found to acquire a resistance transfer factor more readily than “smooth” pathogenic strains (Jarolmen, 1971). This perhaps suggests a difference in the cell walls as playing a part in the transfer of the episome. Resistant rough strains are predominantly less virulent and strongly immunogenic (Jarolmen and Kemp, 1969a; Watanabe and Watanabe, 1969). The incidence of infectious resistance in gram-negative cultures from hospital patients in an agricultural area (Carroll, Iowa) and in an industrial area (Willimantic, Connecticut) was studied by Jarolmen et al. (1973). Several hundred cultures from each location were examined over a period of more than a year. T h e industrial area had a slight but insignificant “edge” in percentage of isolates resistant. In Connecticut, an average of 32.5% resistant cultures was found in 326 isolates; in Iowa, 28.2% in 290 isolates (Table XVI). Using smaller samples, and working in Germany, Wiedemann and Knothe (1971) reported that the incidence of R-factor-positive enterobacteria in city dwellers was the same as in farm workers. Employees in feed mills

24

THOMAS H. JUICES

PATTERNS

OF

TABLE XVI ANTIBIOTICRESISTANCE- 1971' Percent of isolates resistant

Escherichia coli

Enterobacteria

Klebsielln

Proteus

Corm.* Iowa'. Conn. Iowa Conn. Iowa Conn. (233)d (145) (27) (43) (26) (41) (40)

Antibiotic

20 27 3 3 83 25 7"" 26

Ampicillin Dihydrostreptomycin Neomycin Kanamycin Erythromycin Tetracycline Chloramphenicol Sulfonamides ~

15 30 8" 6 96"" 34 1 21 ~~

78 26 7 4 100 32 11" 19""

79 14 0

2 91 13 0 0

81 31 15"" 8 91 36 12 31

73 12 0 0 97 19 2 12

13 8 3 0 100 86 18 35

Iowa (61)

23 11 0 2 95 93 21 33

~~

"From Jarolmen et al. (1972). bWindham Community Hospital, Willimantic, Connecticut. CSt.Anthony Hospital, Carroll, Iowa. dTotal number given in parentheses. "Significant at .05 level. "*Significant at .01 level.

who handled antibiotic feeds had a lower incidence. Moorhouse (1971) found no significant difference with respect to resistant enterobacteria between infants in families with and without contacts with livestock in Ireland. Transfer of resistance in vivo has been reported to be a rare occurrence (JaroImen and Kemp, 1969b). However, if infected specimens containing Salmonella, particularly intestinal, were incubated in nutrient broth before plating, R-factor transfer readily occurred, apparently in vitro. Williams Smith (1969) studied the colonization of the alimentary tract of a human being by E . coli strains of animal and human origin. The animal strains were poorer colonizers than the human strains. The amount of transfer of R-factors was small, even when very large numbers of donor cells were given, and the resistant organisms did not persist in the tract for long. Williams Smith has also reported an interesting study on incidence of E . coli containing Rfactors in river water. The principal source was insufficiently treated human sewage in predominantly urban areas (Williams Smith, 1970). Regnier and Park (1972), in discussing Williams Smith's findings, concluded that the contamination of sea water at public beaches could be b y E. coli of both human and farm animal origins. They concluded

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

25

that “the probability of contracting a serious disease by bathing in sewage-polluted water is so small as to be epidemologically undemonstrable.” Discussing the possibility that R+ E . coli may become established in the gut as a result of such bathing, the authors comment that “this possibility seems slight, because large doses are required for transfer to occur, and not particularly important, because most people already carry R+ E . coli.” They conclude that there is no public health risk unless bathing waters are “visibly contaminated with faeces.” Their conclusions do not place much weight on the resistance transfer factor as a public health hazard. The Swann Committee noted that most cases of Salmonella infection, except typhoid and paratyphoid, seen in EngIand and Wales came from infected food, and direct transmission is seen mainly “within hospitals or other institutions or within the family.” Their report also stated that they believe most cases of infection with Salmonella come directly or indirectly from farm animals, and that the majority of these travel by indirect routes such as contaminated meats or utensils. The report noted that infection might pass directly from animals to man, especially to farmers and veterinarians. V.

Discussion

No deleterious effects on public health in the United States that are attributable by experimental evidence to the use of antibiotics in animal feeds have been reported in more than 20 years of such use. Even when tetracyclines were used in the United States and Canada in a dipping process for poultry meat and fish, no deleterious effects on consumers were reported. Such poultry and fish contained levels of tetracyclines far higher than any encountered in animals receiving antibiotics in feeds. The use of antibiotic feeds has increased the production of food for human beings. This may be expressed in terms of economic value; for example, Bird (1969) estimated that “antibiotics have saved 3 million tons of broiler feed with a value of $240 million solely by their effect on feed conversion.” However, the result is that the meat supply for people is more abundant and therefore cheaper, and this has some beneficial effects on public health by increasing the intake of protein of high biological value. The theoretical, arguments recently raised against feeding antibiotics are so portentous that the introduction of the practice for the first time in 1972 would probably not have been countenanced. But antibiotics were introduced into animal feeds in 1950, and the arguments against the practice remain in the area of theory. Surely twenty

26

THOMAS H. JUKES

years would have been long enough to allow clear-cut deleterious effects on public health to emerge, if such effects were ever going to appear. The principal arguments were as follows: From the standpoint of public health, the use of antibiotics in feeds for farm animals was questioned b y the Antibiotic Task Force principally for the following reasons: (1) dissemination of resistance (a) through episomal transfer factor from commensal enterobacteria in animals to pathogenic recipients in animals or man, or, (b) directly by increasing the reservoir of resistant pathogens in animals, and transmission of these to man via food; (2) increased shedding of Salmonella caused by feeding antibiotics to animals; (3) ingestion of antibiotic residues in human food, producing bacteriological and pharmacological hazards. Category (1) represents an effect that could be quantitatively only a fraction of the more direct effect produced by the clinical use of antibiotics in medical practice and by human-to-human contact, and (lb) is primarily a problem of sanitary handling of the food supply to protect the consumer against contaminated food, whether or not the pathogens in it are resistant. Category (2) also is a matter of routine sanitary food practice to reduce the incidence of salmonellosis. The general improvement of sanitation is a more logical approach than an attempt to reduce percentagewise the Salmonella count in contaminating materials which should not be there in the first place. In any case, the increased shedding of Salmonella following antibiotic treatment has been shown for human beings and laboratory mice, but not for farm animals. Category (3) does not seem to constitute a hazard in the light of controlled experiments in which antibiotics were fed for prolonged periods to human subjects, especially to children. A difference between 1950 and 1972 is that today there is a highly favorable climate for obtaining bans on technological practices in agriculture. Because of an abundant food supply and an agricultural surplus, it has become fashionable to mount attacks on real or fancied contaminants in food products resulting from the use of chemicals. Such attacks are favorably received b y many members of the general public and, in consequence, by their legislative representatives. Witness the introduction in Senate and House (HR14941) of bills to authorize definitions for “organically grown food which has not been treated with preservatives, hormones, antibiotics or synthetic additives of any kind,” that each farm or establishment engaged in producing organically grown or processed food shall be registered with the Secretary of Agriculture, who shall inspect each such place annually. The proposed legislation reflects a loss of confidence in the food supply, and this makes it easy to arouse distrust in any and all

FEEDING OF LOW LEVELS OF ANTIBIOTICS TO ANIMALS

27

procedures in technological agriculture. Some of the factors leading to this loss of confidence may be as follows, 1. The rejection of “establishment” scientific values by the counterculture. 2. The success of the book Silent Spring, with its theme that foods contain poisonous residues placed there by industry; e.g., Chapter 11, referring to the food supply, is entitled, “Beyond the Dreams of the Borgias.” 3. T h e flood of imitations of Silent Spring. 4. The increase in sensitivity of analytical methods to the point where many or most biological materials can be shown to contain residues of substances commonly regarded as poisonous. These refined methods also show the presence of traces of pesticides, hormones, etc., and publicizing these residues has caused widespread distrust in scientific agriculture. 5 . The “Delaney Clause” in the Food Additives Amendment. The clause automatically places a ban on any additive that gives a positive test as a carcinogen, without permitting evaluation of whether the additive has a “threshold level” below which it might not be biologically active. This interpretation makes the public suspicious of any residue at any level. 6. The activities of reporters and television commentators, who are able to command attention with alarming stories about residues in foods. 7. The existence of a cult of food quackery whose high priests have moved into the intellectual vacuum caused by rejection of established values. 8. The urbanization of the American population so that most people are completely ignorant of the chemical technology that is necessary for agriculture. 9. The reiteration of the theme that capitalism in general, and as typified by “agri-business,” is contrary to the welfare of the consumer. These add up to a conviction that banning the use of chemicals of almost any type in agriculture is a “good” cause. I conclude that the responsibility for the spread of resistance in human clinical medicine rests primarily with those who are using antibiotics in clinical practice, not with the feed industry. All the evidence in the United States points to hospitals and other human clinical contacts as being responsible for the spread of resistance, no matter whether episomal or chromosomal. T h e continued efficacy of antibiotics in animal feeds shows clearly that there is no significant resistance problem at the level of the farm. This efficacy has been

28

THOMAS H. JUKES

abundantly documented in the relevant scientific literature and by numerous unpublished reports. A recommendation of the FDA Task Force states that “the feeding of antibacterial agents would logically seem to give rise to a human health hazard” (page 10). Claude Bernard (1865) said, “If an idea presents itself to us, we must not reject it simply because it does not agree with the logical deductions of a reigning theory.” I n summary, the use of antibiotic feeds for farm animals is of benefit to the consumer. There is no evidence that, as practiced in Canada and the United States it leads to the accumulation of significant residues in meat. Nor is there evidence for the spread of transferable resistance from farms into microorganisms that cause public health problems to the human population. The fact that antibiotic feeding of farm animals continues to be effective shows that this practice in the vast majority of cases is not causing pathogenic resistance among the animals that are exposed to it. REFERENCES Akiba, T., Koyoma, K., Ishiki, Y., Kimura, S., and Fukushima, T. (1960). Nippon Iji Shimpo 1866; 46 (cited by Watanabe, 1963). Anderson, E. S. (1968). Annu. Reu. Microbiol. 23, 131. Anderson, C. M., Frazer, A. C., French, J. M., Hawkins, C. F., Ross, C. A. C., and Sammons, H. G. (1954). Gastroenterologia 81,98. Anderson, R. C., Worth, H. M., Small, R. M., and Harris, P. N. (1966). Cosmet. Toxicol. 4,l. Askerkoff, B., and Bennett, J. V. (1969).N . Engl. Med. J . 281, 3. Begin, J. J. (1971). Poultry Sci. 50, 1496. Bernard, C. (1865). “An Introduction to the Study of Experimental Medicine.” AbelardShuman, New York. Bird, H. R. (1969).Nat.Acad. Sci.-Nut. Res. Counc., Publ. 1679,31. Bohnhoff, M., and Miller, C. P. (1962).J . Infec. Dis.111, 117. Borgstrom, G. A. (1968). Congr. Med. Women’s Int. Ass., l l t h , 1968 pp. 1-24. Borie, P., and Barrett, J. (1961). Brit. Med. J . 2, 1267. Bulatao-Jayme, J., Intengan, C. L., Quiogue, E. S., Martinez, E. O., and Pascual, C. R. (1957). Nutr. News 9, 34. Bulger, R. J., and Sherris, J. C. (1968). Ann. Intern. Med. 69, 1099. Cherubin, C. E., and Winter, J. (1970). Amer. J. Med. Sci. 260, 34. Cherubin, C. E., Fodor, T., Denmark, L., Master, C., Fuerst, H. T., and Winter, J. (1969). Amer. J . Epidemiol. 90, 112. Cherubin, C. E., Szmuness, M., and Winter, J. (1970). Intersci. Conf. Antimicrob. Ag. Chemother., loth, 1970 p. 67. Coates, M. E., Davies, M. K., and Kon, S. K. (1955).Brit.J.Nutr. 9,110. Deichmann, W. B., Bernal, E., Anderson, W. A. D., Keplinger, M., Landeen, K., Macdonald, W., McMahon, R., and Stebbins, R. (1964).Ind. Med. Surg. 33,787. Dessau, F. I., and Sullivan, W. J. (1961).Toxicol.Appl. Pharmacol. 3,654. Dixon, J. M. S. (1965). Brit. Med. J . 2, 1343. Elliott, R. F., and Owen, B. (1955). Cited in Jukes (1955), p. 26.

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FDA Task Force. (1972). “Report to the Commissioner of the Food and Drug Administration on the Use of Antibiotics in Animal Feeds,” (FDA) 72-6008. U.S. Food and Drug Administration, Roshville, Maryland. French, J. M., Caddie, R., and Smith, N. M. (1956). Quart.J. Med. 25, 333. Goff, C. W. (1955). Clin. Orthop. 6,95. Guerra, R., Wheby, M. S., and Bayless, T. M. (1965).Ann. Intern. Med. 63,619. Guzman, M. A., Scrimshaw, N. S., and Monroe, R. J. (1958). Amer. J. Clin. Nutr. 6,430. Hallesy, D. W., and Hine, C. H. (1964).Toxicol.Appl. Pharmacol. 6,9. Hines, L. R. (1956). Antibiot. Chemother. (Washington, D.C.) 6,623. Jarolmen, H. (1971).Ann.N.Y. Acad. Sci. 182,72. Jarolmen, H., and Kemp, G. A. (1969a).J . Bacteriol. 97, 962. Jarolmen, H., and Kemp, G. A. (1969b).J . Bacteriol. 99, 487. Jarolmen, H., Langworth, B. F., and Kemp, G. A. (1973). In press. Johansson, K. R., Peterson, Y. E., and Dick, E. C. (1953).J.Nutr. 49,135. Jolliffe, N., Frontall, G . , Maggioni, G . , Corbo, S., and Lanciano, 0. (1955-1956). Antibiot. Annu. p. 19. Jukes, T. H. (1955). “Antibiotics in Nutrition,” Antibiotics Monograph No. 4, pp. 1-128. Med. Encycl., New York. Kemp, G. A. (1972).J . Anim. Sci. 35, 463. Kiser, J. S., Gale, G . O., and Kemp, G. A. (1971). CRC Critical Reu. Toxicol. p. 55. Klipstein, F. A., and Falaiye, J. M. (1969). Medicine 48, 475. Lewis, R. A., Bhagat, M. P., Waghe, M. A., Kulkarni, B. S., and Sotoskar, R. S. (1956). Amer. J . Trop. Med. H y g . 5, 483. LitchfieId, H. R., Turin, R., and Zion, L. (1957-1958), Antibiot. Annu. pp. 102-106. Loughlin, E. H., Alcindor, L., and Joseph, A. A. (1957). Antibiot. Annu. pp. 95-106. Macdougall, L. G. (1957).J . Trop. Pediat. 3, 74. Mackay, I. F. S., Patrick, S. J., Stafford, D., and Cleveland, F. S. (1956).J.Nutr. 59,155. McVay, L. V., and Carroll, D. S. (1952). Amer. J . Med. 12, 289. McVay, L. V., and Sprunt, D. H. (1953). N . Engl. J. Med. 249,387. Messersmith, R. E., Sass, B., Berger, H., and Gale, G. 0. (1967).J. Amer. Vet Med. Ass. 151, 719. Moorhouse, E. (1971). Ann. N.Y. Acad. Sci. 182, 65. Neu, H. C., Winshell, E. B., and Winter, J. (1971). N.Y. StateJ. Med. 71, 1196. Perrini, F. (1951). Boll. SOC.Ital. Biol. Sper. 27, 1151. Pocurull, D., Gaines, W., Stuart, A., and Mercer, D. H. (1971).Appl. Microbiol.21,358. Regnier, A. P., and Park, R. W. A. (1972). Nature (London) 239, 408. Robinson, P. (1952). Lancet 1,52. Rosenstein, B. J. (1967).J . Pediat. 70, 1. Sato, G. (1967).J . Infec. Dis. 117, 71. Sato, G., Miyame, T., and Miura, S. (1970).Jap.J. Vet. Res. 18,47. Shirk, R. J., Whitehill, A. R., and Hines, L. R. (1956-1957). Antibiot. Annu. pp. 843-848. Shor, A. L. (1971). “Tissue Residue Levels in Poultry Products, a Summary.” American Cyanamid Co., Princeton, New Jersey. Sheehy, T. W., and Perez-Santiago, E. (1961). Gastroenterology 41, 208. Smith, D. (1968). FDA (Food Drug Admin.) Pap. 2, p. 78. Snelling, C. E., and Johnson, R. (1952). Can. Med. Ass. J. 66, 6. Swann, M. M., chairman. (1969). “Report of Joint Committee on the Use of Antibiotics in Animal Husbandry and Veterinary Medicine.” Her Majesty’s Stationery Office, London. Thatcher, F. S., and Loit, A. (1961). Appl. Microbiol. 9, 39. Walton, J. R., and Lewis, L. E. (1971). Lancet 2, 255.

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Watanabe, T. (1963). Bacteriol. Rev. 27, 87. Watanabe, T., and Fukasawa, T. (1961). J . Bacteriol. 81, 669. Watanabe, T., and Watanabe, M. (1969).Med. Biol. (Tokyo) 79, 139. Wiedemann, B., and Knothe, H. (1971).Ann. N.Y. Acad. Sci. 182, 380. Williams, Smith, H. (1969). Lancet 1, 1174. Williams, Smith, H. (1970). Nature (London) 228, 1286. Winshell, E. B., Cherubin, C., Winter, J., and Neu, H. C. (1970). Antimicrob. Ag. Chemother. p. 86.

Intestinal Microbial Flora of the Pig

R. KENWORTHY Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford, England I. Introduction ....................................... A. General ................................................................ B. Composition of the Intestinal Microflora 11. Metabolic Activities of the Microflora ............................ A. Fermentation of Carbohydrate ...... B. Catabolism of Amino Acids ..................................... C. Catabolism of Other Compo D. Synthesis of Vitamins .......... .......................... 111. Significance of Microbial Metabolites in Nutrition and Health .................................................... A. Products of Carbohydrate Fermentation B. Amino Acid, Urea, and Choline Catabolism .............. IV. Intestinal Microflora and Disease .................................. A. Escherichia coli and Gastroenteric Disease in Pigs ...................................................... B. Swine Dysentery ....... C. Clostridium welchii ( p D. Salmonellosis ........................................................ V. Summary and Conclusions References ..................................................................

31 31 32 33 33 35 36 36 37 37 39 39 39 46 47 48 49 50

1. Introduction

A.

GENERAL

Despite the theory, widely accepted in the late nineteenth century and championed b y Metchnikoff, that noxious products of microbial metabolism in the intestine led to “autointoxication” and thence to many disease states, the significance of the gut microflora in mammalian physiology and nutrition became apparent only with the advent of broad spectrum antibiotics and the availability of germfree animals. Not surprisingly then, interest in the intestinal microflora of animals, including that of the pig, was sporadic for the first 50 years of this century. Heinick (1903), Pfeiler (1929), and Kosarev (1940) attempted identification of the porcine intestinal microflora, but the major stimulus to further investigations was the observation of Stokstad and Jukes (1950a,b) and Jukes et al. (1950) that the active growth-promoting factor in crude residues from Streptomyces aureofaciens was not vitamin BIZ but Aureomycin.

32

R. KENWORTHY

B. COMPOSITION OF THE INTESTINAL MICROFLORA For the next 5 years, research activity into the intestinal microflora of the pig was intensified in an effort to improve the economics of production (see reviews by Braude et al., 1953; Stokstad, 1954). Inevitably much of the early work was either incomplete or contradictory, but by the end of the decade it was established that the major components of the microflora were lactobacilli and streptococci (Quinn et al., 1953; Briggs et al., 1954; Wilbur et al., 1960) and that moderate changes in diet and housing conditions had no influence on microfloral composition (Briggs et al., 1954; Larson and Hill, 1955). 1 . The “Normal” Microflora Subsequent work by Smith and Crabb (1961), Pesti (1962), Kenworthy and Crabb (1963), Smith and Jones (1963),and Uchida et al. (1965) substantiated that the lactobacilli are the stable, dominant members of the microflora, and established that to date the basic, “normal” flora of the conventional pig under standard conditions of husbandry can be considered to comprise lactobacilli, streptococci, Bacteroides spp., and Escherichia coli; these groups have been recovered from all levels of the intestinal tract. To this flora Clostridium welchii (perfringens) can be added, particularly while the animal is sucking (Smith and Crabb, 1961; Kenworthy and Crabb, 1963; Van der Heyde and Hendrickx, 1964); and veillonellae (Smith and Crabb, 1961; Kenworthy and Crabb, 1963; Fuller and Lev, 1964) and yeasts (Wilbur et al., 1960;Van Uden and Do Carmo Sousa, 1962; Kenworthy and Crabb, 1963; Koch, 1964) can justifiably be included after the piglet passes the age of about 14 days. Further data relating to specific identifications within the major groups is provided in Table I. Finally it should be stated that a systematic overall attempt to recover and quantify strict anaerobes has not yet been described; it is possible that the results of such an investigation could add to the “normal” composition referred to above, especially insofar as populations of the large intestine are concerned. 2. Organisms Isolated Relatively Infrequently Other groups reported with less frequency are Enterobacter cloacae, Proteus spp., Providence spp., Pasteurella pseudotuberculosis and Actinobacillus spp. (Dickinson and Mocquot, 1961), Klebsiella spp. (Dickinson and Mocquot, 1961; Kenworthy, 1967), Citrobacter spp. (Kenworthy, 1967), Salmonella spp. (see Heard et al., 1969; Smith, 1969), Peptostreptococcus elsdenii (Alexander and Davies,

INTESTINAL MICROBIAL FLORA OF THE PIG

TABLE I “NORMAL” MICROFLORAOF THE PIC - SPECIFIC ~

Genus

Species

Lacto bacillus

acidophilus fermenti breuis cello biosis plantarum salivarius jugurti bovis equinus faecium faecalis or liquefaciens coli

Streptococcus

Escherichia

33

IDENTIFICATION ~~~

Reference

Fewins et al. (1957), Fuller et al. (1960), Kenworthy (1967)

Raibaud and Caulet (1957) Fuller et al. (1960) Raibaud and Caulet (1957), Smith and Jones (1963) Bartley and Slanetz (1960), Fuller et al. (1960), Kjellander (1960), Dickinson and Mocquot (1961), Smith and Crabb (1961), Pesti (19621, Kenworthy and Crabb (1963), Uchida et al. (1965)

1963; Fuller and Lev, 1964; Giesecke et aZ., 1970; Aalback, 1972), Clostridium butyricum (Baker et al., 1950), Sphaerophorus spp., (Fuller and Lev, 1964; Aalback, 1972), Vibrio coli (von Balmoos, 1950; McNutt, 1953; Deas, 1960), and certain spirochaetes which will be discussed at greater length in the section relating to disease. 3. E$ect of Diet on the Composition of the Microflora It is worthy of comment that the generally comparable results (quantitative and qualitative) of the more recent workers on pig intestinal microbiology confirm earlier observations that relatively minor differences in dietary formulations are unlikely to affect the flora. When differences between dietary formulations (Wilbur et al., 1960; Kellogg et aZ., 1964; Smith, 1965) or housing conditions (Kenworthy, 1967; Hill and Kenworthy, 1970) become more gross, however, alterations in the intestinal microflora are likely to occur. II. Metabolic Activities of the Microflora

A.

FERMENTATION OF CARBOHYDRATE

Digestion and utilization of carbohydrate is a complex topic, and interpretation of the literature is fraught with difficulties related to the ages of the pigs studied, dietary formulations and the treatment of

34

R. KENWORTHY

complex carbohydrates prior to inclusion in the feed. These issues are not considered appropriate for lengthy discussion here, but insofar a2 they exercise an influence on the microbial flora they justify some comment.

1 . Fermentation of Cellulose Fermentation of cellulose has been discussed or referred to by Mangold (1934), Baker (1939), Trautmann and Kirchof (1940), Trautmann and Asher (1941), Vartiovaara and Roine (19421, Vartiovaara et al. (1944), Baker and Harriss (1947), Vartiovaara (1949), Phillipson (1947), Woodman and Evans (1947a,b), Forbes and Hamilton (1952), Freeman et al. (1970), and Farrell and Johnson (1970). The general conclusion from this literature is that cellulose digestion in the pig is essentially a bacterial process carried out in the cecum and large intestine. The microorganism mainly responsible was described by Trautmann and Asher (1942) as a large, more or less spherical bacterium readily stained with iodine and probably belonging to the coccaceae.” Additionally, Vartiovaara and Roine (1942) cultured bacteria from the cecum of a sow anaerobically on a medium containing cellulose as the only form of carbohydrate and isolated from subcultures 2 morphological forms. One was a gram-negative rod; the other, predominating form, was an anaerobic mesophilic, nonsporing gram-positive coccus occurring in pairs or short chains. Attempts to isolate the organisms in pure culture were not successful; butyric and acetic acids were the main fermentation products. “

2. Fermentation of Starch Baker and Nasr (1947) reported that raw potato starch, as distinct from maize starch, for example, was metabolized by microorganisms in the cecum and large bowel, mainly by C. butyricum. Further studies of starch fermentation were recorded at some length by Baker et al. (1950); these authors concluded that C. butyricum provides the main source of starch-hydrolyzing enzyme in the cecal contents of pigs and that lactobacilli, enterococci, yeasts, and coliforms do not play a leading part, but further metabolize the carbohydrate products of starch decomposition. Additionally, Baker et at?. (1950) commented that the general microbial population of the cecum showed an increase in fermentative activity upon the arrival of a new substrate; an observation very pertinent to the later studies of Porter and Kenworthy (1969) and I. R. Hill et al. (1970), and to the mechanisms of initiation of gastroenteric disease (see later).

INTESTINAL MICROBIAL FLORA OF THE PIG

35

3. Fermentation of Glucose and Pyruvate Michel (1961) has described in vitro studies of the breakdown of glucose and pyruvate by the intestinal flora taken from different levels of the alimentary tract. Glucose was fermented to organic acids. The flora of the stomach produced lactic acid throughout fermentation: the flora of the small intestine produced lactic acid during the latent and early exponential phases of growth, after which volatile acids were formed . At the end of fermentation the quantity of volatile acids expressed as acetic acid was 8.0 gm/liter, corresponding to the utilization of 50% of the available glucose. Pyruvate was rapidly decarboxylated, the reaction products being acetic and lactic acids and C O z ; in the presence of NH3 synthesis of amino acids occurred, and these were again catabolized.

B. CATABOLISM O F AMINO ACIDS 1 . In Vitro Studies Much of the information available on catabolism of amino acids by the intestinal microflora has stemmed from an interest in the growthpromoting properties of antibiotics and other growth-promoting compounds such as copper sulfate (see below). A major contribution has been made by Michel (1956, 1962), who reported that the microbial flora always showed considerable in vitro catabolic activity toward amino acids, with simultaneous deamination and decarboxylation. The activity varied considerably according to the anatomical site from which the flora was isolated and with the season of the year during which the isolations were made. Deamination was generally more rapid than decarboxylation except insofar as catabolism of the dicarboxylic acids alanine and arginine were concerned. T h e reaction products were thus an organic acid, NH3, an amine, and C O z ;furthermore when sulfur-containing amino acids were the substrate, the first products were COz and NH3, and then some H2S was evolved. The nonnatural isomers of essential amino acids were not catabolized. Of the strains of microorganisms isolated and examined biochemically by Michel(l956, 1962), only homofermentative lactobacilli failed to produce NH3 from arginine. The most consistently active organism was Streptococcus faecalis, and a wide range of activity was observed in the gram-negative, lactose-negative bacilli; but heterofermentative lactobacilli, Proteus, various cocci, E . coli, various gram-positive bacilli, and yeasts are all listed as having closely similar activities.

36

R. KENWORTHY

Interested in the effects of dietary change on the microflora, I. R. Hill et aE. (1970) also carried out in vitro studies of amino acid catabolism by organisms in gut content removed within 48 hours after weaning. Increased deaminative and decarboxylative activity occurred compared with material from the intestines of unweaned controls. Also, confirming the findings of Michel (1962), deamination and decarboxylation proceeded simultaneously; further, active decarboxylation continued at pH values up to 7.5, thereby throwing into question the relevance of intestinal pH values to microbial activity in the gut, and the validity of extrapolating from observations made in pure culture.

2. In Vivo Studies In vivo studies of amine production in the intestine of pigs have been carried out by Larson and Hill (1960) and Porter and Kenworthy (1969). The former authors reported readily demonstrable amounts of amines in the ileal contents of pigs from 4 days to 14 weeks old; agmatine, putrescine, cadaverine, histamine, ethanolamine, tyramine, and tryptamine were all identified by paper chromatography. Porter and Kenworthy (1969) observed that the catabolic activity of the normal flora increased after a dietary change was made, and elevated levels of amines were found when estimated directly in intestinal content or indirectly by measuring output of urinary heterocyclic amines. The predominant diamines were cadaverine and putrescine. C. CATABOLISM OF OTHERCOMPOUNDS Choline and urea are catabolized in vitro by the microflora of the pig (Michel, 1961). From choline, trimethylamine is produced, but as with amino acid catabolism there are considerable variations between isolates from different parts of the tract, and between pigs. The urease activity of the flora was found to be very high. Reiser and Mott (1971) studied the effects of the microflora on serum cholesterol levels and fecal bile acids in germfree pigs which were allowed to become infected. A dramatic decrease in cholesterol level occurred after infection; this could be correlated with development of a flora which metabolized primary bile acids to secondary bile acids.

D. SYNTHESISOF VITAMINS Synthesis of components of the vitamin B complex by the intestinal flora is known to occur in many species of animals (Kon, 1945). Nasr (1950) reported that C. butyricum can synthesize folic acid, riboflavin, nicotinic acid, aneurin, pyridoxine and pantothenic acid when p -

INTESTINAL MICROBIAL FLORA OF THE PIG

37

aminobenzoic acid and biotin are added to the medium. Further, Nasr (1949)and Baker et al. (1950)demonstrated that members of the vitamin B complex could be absorbed and utilized to a limited extent under experimental conditions of vitamin B deficiency, and with a diet rich in potato starch. These observations accord with those discussed at length by Kon (1945)relative mainly to small rodents. There is also considerable evidence that vitamin BIZ is synthesized in the alimentary tract by the microflora. Noer (1950)showed that a Bacillus subtilis and a corynebacterium isolated from pig intestinal mucosa could synthesize vitamin BIZin uitro, and Barber et al. (1953) demonstrated that synthesis of vitamin BIZ occurs between the duodenum and cecum of the pig. However, Michel(l962) while confirming these observations, found that only a very small amount of the vitamin was in the free state. Furthermore the flora synthesizes analogs of vitamin Blz which cannot be directly utilized by the animal anyway (Wijmenga et al., 1950; Folkers and Wolf, 1954). 111. Significance of Microbial Metabolites in Nutrition and Health

A.

PRODUCTS OF CARBOHYDRATE

FERMENTATION

The pig traditionally consumes a diet rich in carbohydrate, and therefore, as would be expected from the nature of the microflora described and the in oitro studies of Michel (1961),organic acids are found in all regions of the alimentary tract. The amount and type of acid are related to the region of the tract, the diet (Friend et al., 1963), and the age of the animal under study. Thus, lactic acid is found in decreasing amounts from stomach to large bowel, and in the stomach of the young piglet the concentration of acid is related to the time of feeding (K. J. Hill et al., 1970).Volatile fatty acids (VFA’s) occur mainly in the cecum and colon (Elsden et al., 1946; Barcroft et al., 1944;Alexander and Davies, 1963;Friend et al., 1963). 1 . Lactic Acid The nutritive value of lactic acid per se is uncertain; indeed there is no direct evidence that it is absorbed in the pig (Cranwell, 1968). MBllgard (1946)has presented evidence that lactic acid facilitated the absorption of calcium, and that pigs fed cultures of lactic acid bacteria grew better than controls. It should be stated, however, that Braude (1964)was unable to confirm these observations, and the present author has yet to see any convincing evidence, from his own experiments or elsewhere, that lactic acid supplied either directly or by

38

R. KENWORTHY

attempted inoculation of lactic acid bacteria produces reproducible beneficial effects either on growth rates or health. On the evidence to date, it seems likely that much of the lactic acid produced in the gut is further metabolized b y the microflora and may in this way provide some energy to the host. 2. Volatile Fatty Acids According to some authors there seems littIe doubt that the products of microbial fermentation of cellulose in the cecum and colon of the pig can provide an energy source for the animal (Vartiovaara et al., 1944; Vartiovaara, 1949; Woodman and Evans, 1947a; Forbes and Hamilton, 1952; Farrell and Johnson, 1970). However, Freeman et al. (1970) found that the level of fiber in the ration had no influence on blood acetate levels, and that blood concentrations of acetate in fed and fasted pigs were not substantially different; they therefore concluded that endogenously produced acetate was quantitatively of more nutritional significance to the animal than acetate absorbed from the alimentary tract. These findings are largely supported by those of Farrell and Johnson (1970), who measured production rates of VFA’s in the cecae of pigs given diets containing 26% or 8% cellulose. Only 2.7% and 1.9% of the apparent digestible energy of the 26% and 8% cellulose diets, respectively, came from VFA’s, and it was concluded that the cecum plays only a small role in the breakdown of feed substances. It is possible to reconcile these views, however, since Nordfelt (1954) showed that digestibility of crude fiber increases as the pig increases in body weight, and the increase is most marked when pigs below 100 kg body weight are compared with mature animals of 180 kg and above. The pigs studied by Freeman et al. (1970) were only 6-12 weeks old and would therefore probably not be much in excess of 35-40 kg body weight, and those used by Farrell and Johnson (1970) weighed about 33 kg. Also, variations in results could follow from differences in sources of fiber employed, level in the diet, character of the nonfibrous portion of the ration and plane of nutrition (Forbes and Hamilton, 1952). Barcroft et al. (1944) and Barcroft (1945) have shown that the chief sites of absorption of VFA’s in the pig are the cecum and colon, but there is apparently some absorption from the stomach and small intestine. Friend et al. (1964) studied the VFA content of pig blood and, making a number of assumptions, calculated that acetic, propionic, and butyric acids could contribute between 15 and 28% of the maintenance energy requirements of a 30-kg pig; these data now need to be evaluated in the light of the findings of Freeman et al. (1970) and Farrell and Johnson (1970), referred to above.

INTESTINAL MICROBIAL FLORA OF T H E PIG

B. AMINO ACID, UREA,

AND

39

CHOLINE CATABOLISM

From the studies of Michel and FranGois (1955), FranGois and Michel(1960), and Larson and Hill (1960),the effects of catabolism of amino acids, urea, and choline should be considered mainly deleterious. This conclusion is based on the growth-promoting effects of certain compounds, mainly antibiotics, and evidence that in their presence the catabolic activity of the flora toward amino acids is reduced. Also, additional support can be found when the remaining literature relative to use of antibiotics as growth promoters is considered. There is much evidence, for example, that although antibiotics given with the feed reach the cecum and large intestine, they do not cause dramatic changes in the composition of the microflora (Braude et at., 1953). In the pig some studies have shown that penicillin increased the total bacterial count (Bridges et al., 1951; Dyer et al., 1952; Kline et al., 1952) whereas others have shown little or no effect of antibiotics on anaerobic, coliform, lactobacillary, or yeast cell counts (Bridges et al., 1951, 1952; Brown and Luther, 1950; Cuff et al., 1950; Wahlstrom et al., 1950, 1952; Barber et al., 1953). In only a relatively few experiments were numbers reduced (Noland et al., 1951; Larson and Carpenter, 1952; Sieburth et al., 1951). Additionally Smith and Jones (1963) were unable to show any qualitative or quantitative effect on the intestinal microflora of the pig during administration of copper at levels sufficient to stimulate growth:Finally, antibiotics have a greater effect under subclinical disease or stress conditions (Warner and Hanson, 1950), and there is increased metabolic activity by the normal flora with greater production of toxic amines, under a condition of stress such as that imposed by weaning (Porter and Kenworthy, 1969). However, it cannot be concluded that amino acid catabolism b y intestinal microbes is necessarily entirely negative to the host. Some absorbed ammonia may be metabolically useful in the synthesis of nonessential amino acids, providing the necessary carbohydrate is consumed; furthermore, one of the most important defense mechanisms of the body is the production of ammonia for neutralization of acids, and formation of ammonium salts which spare the fixed base (Na, K, Ca) necessary for the alkali reserve and the bones. IV. Intestinal Microflora and Disease

A. Escherichia coli AND GASTROENTERIC DISEASEIN PIGS Disorders of the alimentary tract associated with E . coli are identified clinically as gastroenteritis and bowel edema. T h e former is further subclassified into neonatal diarrhea, and postweaning scours,

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which generally affects pigs 3-6 weeks old, depending on the age at which they are weaned. The various disease syndromes, their epidemiology, pathogenesis, and the characteristics of the E . coli strains involved have been discussed at length b y Sojka (1965).The issues of bacteriological interest largely center around endotoxin and enterotoxin production and modes of action, the antigenic formulation of pathogenic serotypes, and the significance of the K88 antigen. There is no evidence that colicine production plays any significant part in E . coli-associated enteric disease of pigs (Tadd and Hurst, 1961; Salajka and Sarmanova, 1971).

1 . Neonatal Diarrhea Enteric disease associated with E . coli infection in newborn piglets has been reviewed b y Sojka (1971) and Kohler (1972). Comment here will therefore attempt only to bring out the essential points of bacteriological interest and controversy, and will relate mainly to the K88 antigen and enterotoxin. a. K88 Antigen

Muschel (1960) and Glynn and Howard (1970) showed that complement resistance in E . coli is due to the presence of adequate amounts of K antigen, and the latter authors also demonstrated that some K antigens are more effective, weight for weight, than others. The K antigens appear to inhibit agglutination and hemolysis of red cells and, by analogy, bacteriolysis and bactericidal activity, b y impeding the attachment of antibody to the target cell. Howard and Glynn (1971)also found that strains of E . coli with sufficient K antigen to resist killing by complement were poorly phagocytosed, and suggested that the effects of K antigen on phagocytosis and complement killing or lysis could all be explained by their ability to impair antibody binding. There is unanimity that b y far the majority of strains of E. coli associated with neonatal disease possess the K88 antigen (Sojka, 1965, 1971; Wittig, 1965a,b; Kaszubkiewicz et al., 1967; Truczynski et al., 1966; Beh, 1971; Gyles e t al., 1971) and much attention has been focused on it, and its significance in pathogenesis. The antigen occurs as a fur of fine filaments and confers upon the organism adhesive properties (Stirm et al., 1967); the pathological significance of these findings may lie in the potential of K88-containing strains to become closely associated with the intestinal epithelium thereby permitting establishment and proliferation of the organism in the small intestine (Smith and Linggood, 1971). Thus,

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if the K88 antigen is one with high complement resistance (Glynn and Howard, 1970), and if the properties of adhesion and hemagglutination postulated b y Stirm et al. (1967) are related to a hypothetical ability of microorganisms to associate closely with mammalian intestinal epithelium, an immunological/physical basis is provided to explain some facets of neonatal diarrhea in conventionally reared piglets, and reproduction of disease in colostrum-deprived animals (Smith and Linggood, 1971). However, it should be recalled that there are strains of E . coli associated with neonatal diarrhea, which do not possess the K88 antigen (Gyles et al., 1971; Sojka, 1971). Furthermore, the concept of total dependence on K88 antigen for establishment and proliferation of E . coli strains in the upper small intestine has been challenged by the work of Miniats and Gyles (1972), and Kenworthy (1973). Miniats and Gyles (1972) found that in gnotobiotic pigs the ability to colonize the upper small intestine was not necessarily related to presence of the K88 antigen, and concluded that there must be other effector mechanisms. Kenworthy (1973) conducting balance and digestibility studies on gnotobiotic pigs before and during infection with an enteropathogen observed that in the first 24 hr after infection a gut stasis occurred, followed by a great increase in volume of fecal output. If the stasis affected the small intestine, the need to postulate an adhesive mechanism to account for proliferation of motile microorganisms in the upper region of this organ under the experimental conditions described would be obviated. Finally, if K antigens are important in pathogenesis, and survival depends upon stimulation of relevant immunological defense mechanisms, these mechanisms have not yet been adequately defined. There is a rapid reduction in mortalities in initially germfree pigs as the age of infection increases from birth to 4-6 days (Kohler and Bohl, 1966) or from birth to 3 weeks, after which mortality is relatively low (R. Kenworthy, unpublished), despite low levels of circulating antibody and immunoglobulin up to 5 days postinfection (Porter and Kenworthy, 1970).

b. Enterotoxin The other main subject of bacteriological interest in neonatal diarrhea of pigs is that of enterotoxin. The first detailed investigations regarding E . coli enterotoxin and its significance in baby ptgs were reported by Smith and Halls (1967), and since that time the literature has become quite voluminous (see review by Kohler, 1971). Enterotoxin production has been identified

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with the presence of a transmissible plasmid (Smith and Halls, 1968a) which according to Smith and Linggood (1971) appears to play no part in proliferation of the organisms in the small intestine, but does contribute to the diarrhea in the presence of the K88 plasmid. The contradictory observations of Miniats and Gyles (1972) on this issue have already been referred to. The enterotoxin has been isolated as heat-stable (ST) and heatlabile (LT) fractions (Smith and Halls, 1967; Gyles and Barnum, 1969), which are considered to be essentially two forms of the same enterotoxin (Smith and Gyles, 1970). The heat-labile toxin is antigenic and was produced by all the enteropathogenic strains examined by Smith and Gyles (1970); according to Trusczynski and Pilaszek (1970), LT enterotoxin is also pyrogenic for pigs. The heat-stable toxin is nonantigenic and is found only in those strains that naturally possess, or have possessed, K88 antigen (Smith and Gyles, 1970). However, until the physical and chemical characteristics of enterotoxin are better defined, some doubts must remain relative to interpretation of its biological activities. Trusczcynski and Ciosek (cited in Trusczcynski and Pilaszek, 1970) stated that the enterotoxin obtained by the method of Smith and Halls (1967) probably contained a small amount of endotoxin, and Truszczynski and Pilaszek (1970) have commented that it is not yet possible to compare directly endotoxin and enterotoxin because purified enterotoxin free of endotoxin has not yet been prepared. Similarly Jacks et al. (1972) failed to separate enterotoxin and endotoxin components from 08 :K87, K88a,b:H19. There are many other anomalies which also need to be resolved before the full role of enterotoxin in neonatal diarrheic disease of pigs can be finally evaluated. For example, Truszczynski and Pilaszek (1969) recorded that enterotoxins derived from strains belonging to the same serotype produced positive or negative reactions in ligated segments of the same pig. Also 0139:K82 was more frequently positive in their hands than was 08:K87 (B) K88a,b (L), in contrast to the observations of Smith and Halls (1967). Smith and Halls (1967) also commented on pig-to-pig variation in response to enterotoxic preparations; furthermore, some animals were unreactive to cell-free extracts of cultures which had proved satisfactory when injected into the ligated intestine as live organisms. Additionally, Stevens (1971) found that pigs are highly sensitive to enterotoxin during the first 3 days of life, and also in the immediate post weaning period, although unweaned litter mates are very resistant. Finally, Moon and Whipp (1970) found three patterns of response to E . coli in the ligated intestine of pigs. The response to nonenteropathogenic strains was

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INTESTINAL MICROBIAL FLORA OF T H E PIG

negative at all ages, to some enteropathogenic strains positive at all ages and to certain other strains positive in pigs less than 2 weeks old, intermediate in pigs 2-6 weeks old, and negative in pigs more than 6 weeks old. 2. Postweaning Diarrhea Associated with Proliferation of Pathogenic Serotypes of E . coli This subject has received only a small amount of attention compared with that applied to neonatal diarrhea. Bacteriologically it is associated with proliferation of certain strains of E . coli throughout the intestinal tract within a few days of a dietary change being made (Buxton and Thomlinson, 1961; Kenworthy and Crabb, 1963; Miniats and Roe, 1968). The proliferating coliforms are most commonly hemolytic on blood agar plates, but not necessarily so (Sojka et al., 1960; Thomlinson, 1963), and are usually limited to one serotype at any particular time. The serotypes most commonly isolated by various authors are shown in Table 11. It is interesting that the K88 TABLE I1 SEROTYPESOF Escherichia coli COMMONLY ISOLATEDFROM WEANEDPIGS WITH ENTERlC

0138:K81 (B) 0139:K82 (B) 0141:K87 (B) (Richards and Fraser, 196 1)

08:K87 (B) K88 (L) 0138:K81 (B) 0134:K82 (B) 0141:K85 (B) 0141:K85 (B)K88 (L) 0141:K87 (B) (Nielsen et al., 1968)

DISEASE

0138:K81 (B) 0141:K81 (B) 0141:K85a,b (B)-K85a,c(B) 0141:K85a,b (B) 0141:K85a,c (B) (Sojka, 1965,1971)

08:K87, K88a,c 0116 KV 17, K88a,c 0138:K91 0147:K89, K88a,c (Gyles et al., 1971)

antigen does not figure so prominently in these older animals as in the neonate. Furthermore, the pathogenicity of 0141:K85a,c which lacks the K88 antigen, in postweaning diarrhea has been clearly related to its ability to adhere to, and proliferate in, the anterior small intestine (Smith and Halls, 1968b). Thus the significance attached to the K88 antigen (Smith and Linggood, 1971) relative to these characteristics must be kept in perspective. Additionally, with regard to pathogenesis and the significance of enterotoxin, 0139:K82 does not appear to be a consistent producer of the latter (Smith and Halls, 1967). Kenworthy and Allen (1966) examined the significance of the coliform in postweaning diarrhea, and concluded that the role of the organisms in pathogenesis was secondary to the disturbance in

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physiological function created b y the change in diet. There seems to be no justification for retaining the terms “nutritional scours” and “bacteriological scours” in relation to this syndrome (Kenworthy, 1967), especially in view of later studies showing increased metabolic activity of the normal microflora after dietary change (Porter and Kenworthy, 1969). The E . coli metabolites which exacerbate postweaning diarrhea need clarification both in terms of their relative significance in the syndrome and of their mode of action. The metabolites that have attracted attention are endotoxin (Sojka et al., 1957; Buxton and Thomlinson, 1961), enterotoxin, largely by extrapolation from cholera in man and disease in the neonatal pig and lacking any experimental evidence in support, and to a much lesser extent toxic products following decarboxylative and deaminative activity of microorganisms on amino acids (Porter and Kenworthy, 1969; I. R. Hill et al., 1970). The inflammatory response recorded by Kenworthy (1970) and Drees and Waxler (1970) following infection of germfree pigs with E . coli is certainly in accord with the pathology of the naturally occurring disease as described by Richards and Fraser (1961) and Thomlinson and Buxton (1962) and indicates participation of endotoxin and/or other agents which pass the epithelial barrier. However, according to Kenworthy (1973), the clinical signs and histopathology of the experimental syndrome produced by infection of gnotobiotic pigs were more reminiscent of a complex-mediated hypersensitivity than anaphylaxis, in contrast to the hypothesis of Buxton and Thomlinson (1961). Comment on participation of products of amino acid catabolism is largely speculative and circumstantial at the present time.

3. Bowel Edema Edema disease in pigs was first described by Shanks (1938) and received a considerable amount of attention until the focus was transferred to diarrhea of the neonate. Edema disease is characterized by (a) association with the same serotypes of E . coEi as are commonly isolated from postweaning diarrhea (Sojka, 1965); (b) apparently no diarrhea - on the contrary constipation frequently occurs (although this claim is questioned by some investigators); and (c) evidence of neurological disturbance. Opinions expressed relative to etiology are numerous and have been reviewed b y Sojka (1965), but there is little doubt that E . coEi is involved, probably through one or more of its toxic principles. Reproduction of the disease following intravenous injection of crude

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preparations from organisms disintegrated by various means has been claimed by many workers including Erskine et al. (1957), Timoney (1957), Rastegaeva (1957, 1958), Terpstra and Akkermans (1959), Jones and Smith (1964), and Pickrell et al. (1969).It is worthy of comment too that Jones and Smith (1964) claimed reproduction of the disease by intravenous administration of material from strains of E . coli other than the specific serological types recovered from naturally occurring disease. Erskine et al. (1956), Hess and Suter (1958), and Bouckaert et al. (1960) failed to reproduce edema disease by oral administration of live organisms, in contrast with Gregory (1958), Smith and Halls (1968b), and PickrelI et al. (1969). However, Smith and Halls (1968b) were successful with only one strain of pig. Sojka et al. (1957) suggested that the disease was an enterotoxemia, but added that an equally essential factor in pathogenesis must be the presence of an environment that can precipitate the process. Additionally, Schimmelpfennig (1970) has presented evidence that the toxic factor isolated from the gut in edema disease is identical with E . coli neurotoxin. In contrast, Hess (1956),Lemcke e t aE. (1957), Erdos et al. (1957), Ohshima and Miura (1961),and Buxton and Thomlinson (1961) have proffered theories that the disease was an anaphylactic phenomenon possibly related, according to the latter authors, to absorption of endotoxin. At the present time none of these hypotheses have been supported b y other than circumstantial evidence. However, the contrasting views do serve to emphasize the lack of controlled, experimental data available on the coliformassociated diseases of pigs, largely owing to lack of reproducible models. The otherwise detailed pathology of Thomlinson and Buxton (1962) did not include studies of blood vessels or nervous tissue, but there was good correlation between their findings on field cases and experimental models. Other authors (Richards and Fraser, 1961; Jones and Smith, 1964) did not find the widely disseminated lesions described by Thomlinson and Buxton (1962), but Kurtz et al. (1969) have described a generalized necrotic arteritis as the prominent lesion in 106 pigs affected with subacute and chronic edema disease. It seems entirely feasible that, in the graded states of this syndrome, endotoxic shock with its associated pathology could represent an acute manifestation involving perhaps IgE, and that the more commonly occurring, less acute states rely for their symptomatology on a complex-mediated hypersensitivity. Clearly there is much to be learned about the physiological biochemical response of the intestine to “stress” situations before the true significance of intestinal E . coli in enteric disease states can be evaluated.

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B. SWINE DYSENTERY This disease was first described by Whiting et at. (1921), but the etiology had not been systematically investigated until relatively recently (see review by Harris and Glock, 1971). The disease is contagious and infectious, and the organisms of greatest interest have been Vibrio coli and members of the Treponemataceae.

1 . Vibrio coli This organism has been associated with swine dysentery for many years, and James and Doyle (1947), Roberts (1956), Truszczynski (1957), Davis (1961), and Lussier (1962) have reported reproduction of the disease by experimental exposure to pure cultures of the organisms. The success rate was not convincing, however, usually not exceeding 50% of those exposed, and the induced diarrhea was often of short duration only. Also there have been reports of failure to reproduce the disease using pure cultures of V . coli (Gorrie, 1946; Boley et al., 1951; Deas, 1960; Manninger et al., 1960; Terpstra et al., 1968) despite success in establishing the organisms in the gut (Andress et al., 1968; Andress and Barnum, 1968).

2 . Treponemata I n 1968 Terpstra et al. using fluorescent methods identified a spirochetelike organism in smears of the intestinal contents of diseased pigs, and since that time much attention has been devoted both to verifying the presence of these organisms and attempting to grow them in pure culture. Vallejo (1969) suggested that they were probably Borrelia, and Todd et al. (1970) stated that they had isolated the organisms in culture but had no evidence as to their etiological role in swine dysentery. In the same year Taylor (1970) described six different morphological forms of spirochetes in the feces of pigs and outlined a method for recovery and subculture, but he was not successful in obtaining pure cultures. In 1971 Harris and Glock reviewed swine dysentery and reported isolation of Treponema in pure culture using the strictly anaerobic techniques of Hungate (1950); the same authors also recovered Borrelia, but like Taylor (1970) were unable to obtain pure cultures of this genus. Because of the difficulty in differentiation between Borrelia and Treponema b y standard bacteriological methods at the present time, much emphasis has been laid upon their ultrastructural appearance (see Listgarten and Socransky, 1965). Harris et al. (1971) described the isolation and propagation in pure culture of 4 strains of small spirochete and 4 strains of large spirochete. The small spirochete had

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one or two axial fibrils originating from each end of the protoplasmic cylinder, was 0.24-0.30 p in diameter, and resembled the immobile type (b) described by Taylor (1970). The large spirochete had 7-9 axial fibrils originating from each end, and the cell diameter was 0.29-0.38 p; this organism resembled the type (d) of Taylor (1970) and Taylor and Blakemore (1971). Harris and Kinyon (1972) have classified the large spirochete as Treponema hyodysenteriae, and Harris et al. (1972) reported that this organism is structurally similar to that which invades the early lesion of swine dysentery. Moreover, T . hyodysenteriae alone or in combination with V. coli reproduces clinical swine dysentery, whereas V. coli alone does not. Glock (1972) has studied the pathogenesis of swine dysentery and consistently found the large spirochete invading the goblet and epithelial cells of the colonic mucosa. It thus appears that T . hyodysenteriae is the organism primarily associated with swine dysentery, but as Harris and Glock (1971) have pointed out, whether or not the spirochete is capable of invading the gut epithelium without preconditioning by other microbial factors is open to conjecture; and this latter proviso may also require extension in terms of the host’s contribution. Indeed in some respects the disease is similar to the coliform-associated diarrheas; for example, if Taylor (1970) and Harris and Kinyon (1972) are discussing the same organism, it is not demonstrable except just prior to onset of clinical signs, it apparently disappears after recovery, and can be found again only when relapses occur. Indicating that it is probably present in low numbers normally and its proliferation is triggered b y some host response.

C . Clostridium welchii (perfringens) ENTERITIS There have been reports from many parts of the world describing a necrotizing enteritis in baby pigs associated with proliferation of C. welchii type C in the intestine (Field and Gibson, 1955; SzentIvanji and Szabo, 1955; Barnes and Moon, 1964; Meszaros and Pesti, 1965; Matthias et al., 1965), and the subject has been reviewed by Bergeland (1970). Field and Goodwin (1959) reproduced the disease easily in newborn piglets by dosing them with whole cultures of C. welchii type C , and by feeding bacteria-free toxin from type C cultures. However, suspensions of washed organisms were largely ineffectual. It is particularly interesting relative to microbially associated gastroenteric disease in general that when the disease was reproduced by

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administration of cell-free toxin, large numbers of type C organisms were recoverable from the intestine, yet from normal healthy animals recovery of type A only is reported. Once again a characteristic pattern relating to gut enteropathogens emerges - namely, that the pathogens are present at all times but require a hostlgut response for proliferation and production of clinical symptoms. Although most reports of C. welchii enteritis have incriminated type C, Thorne (1971)has stated that all 100 strains of C. perfringens isolated by him from different types of enteritis (hemorrhagic, necrotizing, ulcerating) belonged to type A.

D. SALMONELLOSIS The term salmonellosis is frequently used to refer simply to the presence of salmonellae in any particular animal species; it is a fine point whether such terminology is valid, since the suffix “osis” may connote departure from normality; as most species of farm animals in particular, and probably most wild animals in general, have their own endemic salmonella populations, the inevitable question arises as to what is normal? The term salmonellosis is therefore used here to indicate an association between large numbers of salmonellae and clinical disease, and in this context the main species of significance to the pig are S. cholerae-suis and S. typhimurium. Moreover, since the latter organism is a common pathogen to a wide range of animal species with no special peculiarities relating to the pig, it will not be considered further here. The pig is the natural host of S. cholerae-suis, which may be associated with an acute, subacute, or chronic disease variously known as paratyphoid, swine typhoid, pig typhus, or necrotic enteritis. The organism is known to be widespread geographically (see Field, 1959), but recoveries from healthy animals are either extremely low (Lutje, 1938, 1939) or quite negative (Heard et al., 1969). It is generally accepted that S. cholerae-suis is one of the more difficult of the salmonellae to isolate from infected feces, and Smith (1952) expressed the view that some of the liquid enrichment media are too toxic to support the growth of the organism. However, Heard et al. (1969) examined the effects of various media on recoveries from feces experimentally inoculated with graded levels of S . cholerae-suis and, in addition, heavily contaminated with Pseudomonas aeruginosa and Proteus spp. Selenite F broth and deoxycholate citrate agar were of little value, but enrichment in tetrathionate broth followed by plating on brilliant green agar (BGA) or brilliant green phenol red agar (BGPR) gave good results. Despite these findings, no S . cholerae-

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suis were isolated from a 2-year survey which included 3127 fecal examinations and represented 13,355 pigs on farms, in markets, and in abattoirs. The authors concluded that perhaps the incidence of symptomless excretors was not as high as it had been in the past, but the situation may in fact be more complicated than this interpretation implies. S. cholerae-suis is characterized by its invasiveness, and, as with many salmonellae, the mesenteric lymph nodes are the predilection sites; furthermore, there is much evidence to show that the organism is more likely to assume a pathogenic role and, by inference, proliferate in the gut, thereby rendering identification and isolation easier, if the resistance of the host is lowered by intercurrent disease or poor environmental conditions (Edwards et al., 1948; Hutyra et al., 1949; Field, 1959). Since Heard et al. (1969) only introduced tetrathionate/BGA or BGPR media at a time in the survey which appear to exclude at least 7, and possibly 9, of their 11 “situations” investigated they may have missed several “stress” situations. With regard to other salmonellae isolated from pigs, it may be assumed that any of them could become associated with a disease state in the host animal. It is now 15 years since Buxton (1957) observed that the development of a fatal Salmonella bacteremia was dependent upon a variety of factors concerning the age and general well-being of the host, and that other factors relating to the microenvironment of the parasite still remain to be explained. It is particularly pertinent to complete this section with reference to these comments because they still apply and are equally relevant to all the bacterially associated enteric diseases discussed. It cannot be overemphasized that the physiological and biochemical changes in gut function which must precede the proliferation of pathogenic microorganisms are as important in pathogenesis as are the bacterial strains or serotypes recovered from the gut during a clinical disease state. V. Summary a n d Conclusions The “normal” microflora of the pig comprises lactobacilli, streptococci, Escherichia coZi, and Bacteroides spp, and the dominant, stable members are lactobacilli. Clostridium welchii (perfringens) can be added to these groups when the animal is sucking, and veillonallae and yeasts can be included when the piglet passes the age of about 14 days. There is some evidence that fermentation of carbohydrate may contribute to the nutrition of the animals; this is most convincing in relation to digestion of cellulose in older animals, but VFA’s produced

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in the alimentary tract of the young pig may provide a proportion of the maintenance energy requirements. Catabolism of amino acids by the gut flora is a process mainly deleterious to the host, but there could be some beneficial contributions from the production of NH3 toward synthesis of nonessential amino acids and preservation of fixed base. The principal enteric diseases associated with bacteria are colibacillosis, swine dysentery, C. welchii toxemia, and salmonellosis. These diseases are similar in their pathogenesis to the extent that they require a host response to predisposing “stress” factors, and there is evidence that the organisms involved are normal inhabitants of the alimentary tract or its immediately associated structures, such as mesenteric lymph nodes. ACKNOWLEDGMENT I t is a pleasure to acknowledge the helpful criticism provided by my colleagues Drs. E. F. Annison, A. C. Baird-Parker, and P. Porter and Mr. W. D. Allen during preparation of this script.

REFERENCES Aalback, B. (1972). Actu Vet. Scand. 13, 228. Alexander, F., and Davies, E. M. (1963).J . Comp. Pathol. 73, 1. Andress, C. E., and Barnum, D. A. (1968). Can. J. Comp. Med. 32, 529. Andress, C. A,, Barnum, D. A., and Thompson, R. G . (1968).Can.J.Comp. Med. 32,522. Baker, F. (1939). Sci. Progr. (London) 34,287. Baker, F., and Harriss, S . T. (1947). Nutr. Abstr. Rev. 17, 3. Baker, F., and Nasr, H. (1947).J . Roy. Microsc. Soc. [3] 67, 27. Baker, F., Nasr, H., Morrice, F., and Bruce, J. (1950).J . Pathol. Bacteriol. 62,617. Barber, R. S., Braude, R., Kon, S. K., and Mitchell, K. G. (1953). Brit. J . Nutr. 7, 306. Barcroft, J. (1945). Proc. Nutr. Soc. 3, 247. Barcroft, J., McAnally, R. A., and Phillipson, A. T. (1944).J . E x p . Biol. 20, 120. Barnes, D. M., and Moon, H. W. (1964).J . Amer. Vet. Med. Ass. 144, 1391. Bartley, C. H., and Slanetz, L. W. (1960). Amer. J . Pub. Health Nut. Health 50, 1545. Beh, K. J. H. (1971). Aust. Vet. J . 47, 379. Bergeland, M. E. (1970). In “Swine Health: Common Diseases Affecting Baby Pigs. A Research Review” (A. D. Leman, ed.), p. 41. College of Veterinary Medicine, University of Illinois, Urbana. Boley, L. E., Woods, G. T., Roth, R. D., and Graham, R. (1951). Cornell Vet. 41, 231. Bouckaert, J. H., Oyaert, W., and Sierens, R. (1960). Deut. Tieraerztl. Wochenschr. 67, 349. Braude, R. (1964).See Charlet-Lery (1965),discussion, p. 283. Braude, R., Kon, S. K., and Porter, J. W. G. (1953).Nutr. Abstr. Rev. 23,473. Bridges, J. H., Dyer, I. A., and Burkhart, W. C. (1951).J.Anim. Sci. 10, 1040. Bridges, J. H., Dyer, I. A,, and Burkhart, W. C. (1952).J . Anim. Sci. 11,474. Briggs, C . A. E., Willingale, J. M., Braude, R., and Mitchell, K. G. (1954). Vet. Rec. 66, 241.

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Brown, J. H., and Luther, H. G. (1950).J . Anim. Sci. 9, 650. Buxton, A. (1957). “Salmonellosis in Animals. A Review.” Commonwealth Agricultural Bureaux, Bucks, England. Buxton, A., and Thomlinson, J. R. (1961). Res. Vet. Sci. 2, 73. Charlet-Lery, G. (1965). In “Energy Metabolism” (K. L. Blaxter, ed.), p. 279. Academic Press, New York. Cranwell, P. D. (1968). Nutr. Abstr. Reu. 38, 26. Cuff, P. W., Maddock, H. M., Speer, V. C., and Catron, D. V. (1950).J.Anim. Sci. 9,653. Davis, J. W. (1961).J. Amer. Vet. Med. Ass. 138, 471. Deas, D. W. (1960). Vet. Rec. 72, 65. Dickinson, A. B., and Mocquot, G. (1961).J. Appl. Bacteriol. 24, 252. Drees, D. T., and Waxler, G. L. (1970). Amer. J . Vet. Res. 31, 1147. Dyer, I. A,, Harrison, J. T., Nicholson, W. S., and Cullinson, A. E. (1952).J . Anim. Sci. 11, 465. Edwards, P. R., Bruner, D. W., and Moran, A. B. (1948). Ky., Agr. Erp. Sta., Bull. 525. Elsden, S. R., Hitchcock, M. W. S., Marshall, R. A., and Phillipson, A. T. (1946).J.Erp. Biol. 22, 191. Erdos, J., Hirt, G., and Szabo, I. (1957). Acta Vet. (Budapest) 7, 67. Erskine, R. G., Lloyd, M. K., and Sojka, W. J. (1956). Cited by Sojka (1965). Erskine, R. G., Sojka, W. J., and Lloyd, M. K. (1957). Vet. Rec. 69, 301. Farrell, D. J., and Johnson, K. A. (1970). Anim. Prod. 14, 209. Fewins, B. G., Newland, L. G. M., and Briggs, C. A. E. (1957).J . Appl. Bacteriol. 20, 234. Field, H. I. (1959). In “Infectious Diseases of Animals. Diseases Due to Bacteria” (A. W. Stableforth and I. A. Galloway, eds.), Vol. 2, pp. 540-556. Butterworths, London. Field, H. I., and Gibson, E. A. (1955). Vet. Rec. 67, 31. Field, H. I., and Goodwin, R. F. W. (1959).J.Hyg. 57, 81. Folkers, K., and Wolf, D. G. (1954). Vitam. Horm. (New York) 12, 1. Forbes, R. M., and Hamilton, T. S. (1952).J . Anim. Sci. 11, 480. Frangois, A., and Michel, M. (1960). Extr. Cah. Coll. Med. 1960 No. 12. Freeman, C. P., Noakes, D. E., and Annison, E. F. (1970). Brit. J . Nutr. 24,705. Friend, D. W., Cunningham, H. M., and Nicholson, J. W. G. (1963). Can. J . Anim. Sci. 43, 156. Friend, D. W., Nicholson, J. W. G., and Cunningham, H. M., (1964). Can. J. Anim. Sci. 44, 303. Fuller, R., and Lev, M. (1964).J. Appl. Bacteriol. 27, 434. Fuller, R., Newland, L. G. M., Briggs, C. A. E., Braude, R., and Mitchell, K. G. (1960). J . Appl. Bacteriol. 23, 195. Giesecke, D., Wiesmayr, S., and Ledinek, M. (1970).J . Gen. Microbiol. 64, 123. Clock, R. D. (1972). Proc. Congr. lnt. Pig Vet. SOC., 2nd, 1972 p. 62. Glynn, A. A., and Howard, C. J. (1970). Immunology 18, 331. Gorrie, C. J. R. (1946).Aust. Vet.J . 22, 135. Gregory, D. W. (1958). Vet. Med. 53, 77. Gyles, C. L., and Barnum, D. A. (1969).J . lnfec. Dis. 120, 419. Gyles, C. L., Stevens, J. B., and Craven, J. A. (1971). Can. J. Comp. Med. 35, 258. Harris, D. L., and Clock, R. D. (1971). lowa State Vet. 1, 4. Harris, D. L., and Kinyon, J. M. (1972).PTOC.Congr. lnt. Pig Vet. SOC., 2nd, 1972 p. 72. Harris, D. L., Kinyon, J. M., Mullin, M. T., and Clock, R. D. (1971).Can.J. Comp. Med. 36, 74. Harris, D. L., Clock, R. D., Christensen, C. R., and Kinyon, J. M. (1972). PTOC.Congr. lnt. Pig Vet. SOC., 2nd, 1972 p. 73.

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Antimycin A, a Piscicidal Antibiotic’ ROBERT E. LENNON Fish Control Laboratory, Bureau of Sport Fisheries and Wildlife, Fish and Wildlife Service, United States Department of the Interior, La Crosse, Wisconsin AND

CLAUDEVBZINA Department of Microbiology, Ayerst Research Laboratories, Montreal, Quebec, Canada I. Introduction .............................................................. A. Use of Toxicants in Fishery Management ............... B. Desirable Characteristics of a Piscicide ...,..............

A. Chemical Structure C. TotaI Synthesi

............ .............. ...........................................

111. Fermentation ... ....................... IV. Biosynthesis ....................

...................

B. I4C-Labeled Precursors.. ... V. Mechanism of Action ............. VI. Development of Antimycin as a Piscicide ..................... A. Laboratory Trials ................................................. B. Field Trials ......................................................... C. Detoxification or Removal of Antimycin in Water ............................................ D. Degradation of Antimycin in Water .......... E. Formulation of Antimycin ................... F. Registration ....... VII. Applications as a Ge A. Lakes and Ponds ....................................... B. Rivers and Streams ........................... C. Marine and Brackish Waters .............. VIII. Applications as a Selective Piscicide ........... A. Lakes and Ponds ......... B. Rivers and Strea IX. Summary and Conclusions.. .................... ...................... References ..........,............. . . . . . . . I . . . . . .

56 56 59 60 60 63 64 65 66 67 67 68 68 69 69 75 78 78 80 83 83 83 85 87 87 87 90 91 92

‘The terms “antimycin” and “antimycin A” are used as synonyms in the present review.

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I. Introduction Antimycin A is one of the oldest antifungal antibiotics. The discoverers (Leben and Keitt, 1948) found it to be very active against a variety of saprophytic and pathogenic fungi and had good hopes of combating successfully mycotic infections of man, animals, and plants. Their hopes vanished when the substance was found to be very toxic to animals. If antimycin A were discovered today, it would undoubtedly have the same destiny as the many toxic microbial metabolites that are isolated every year. For Professor Frank M. Strong and his collaborators at the University of Wisconsin the interest in this novel substance was strengthened by their finding (Ahmad et al., 1950) that antimycin A was a strong inhibitor of aerobic respiration; they elucidated its structure in 1961 (Van Tamelen et al., 1961). In the meantime, the antibiotic had become an important biochemical tool, and contributed importantly to the advance in our knowledge of respiration. Impetus was given to their studies when Derse and Strong (1963) recognized that antimycin A was more toxic to fish by several orders of magnitude than to all other forms of life, and proposed its application in fish management. This finding instigated an extensive development program at the Fish Control Laboratories in La Crosse, Wisconsin, to evaluate the piscicidal properties of the antibiotic. At the same time, high-producing strains and improved fermentation conditions were developed at Ayerst Research Laboratories, Montreal. It is the first facet of the program that will be emphasized here. Antimycin A was first reviewed by Strong (1956). The chemistry, the toxicity to various forms of life, and the mechanism of respiration inhibition were covered in the excellent review by Rieske (1967),who very recently updated this information (Rieske, 1973). The teleocidal properties of antimycin A were reviewed briefly by Vezina (1967),and a preliminary account of fermentation was recently given by the same author (VCzina, 1971). A. USE OF TOXICANTS IN FISHERY MANAGEMENT Many substances that enter streams, lakes, and seas as pollutants are detrimentally piscicidal because the death of exposed fish is an unwanted and uncontrolled result. In contrast, in this review of antimycin as a piscicide we will discuss the development and application of a substance to achieve beneficial kills of fish. But first, we should answer the frequently asked question, “Why should anyone want to kill fish?” The answer includes the following objectives: for food, for the control of a pest situation, or for needed improvements in the management and production of food or game fishes.

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Man’s use of fish toxicants dates back thousands of years. Native South American and Asians have long employed rotenone-bearing plants to stun and kill wild fish in streams and ponds for collection and use as food. Chinese farmers have practiced fish culture in small ponds for centuries, and they found it necessary at times to apply saponin-bearing plants to the water to control competing or predaceous fishes. The American Indian collected fish in streams by stunning them with extracts of walnut. In modern societies, however, toxicants are not employed in the capture of food fishes. The long history of killing fish with natural poisons contributed to the adoption of toxicants as aids in modern fishery management. Titcomb (1914) recorded what is thought to be the first attempt to eliminate competing and predaceous fishes from a trout lake in Vermont. H e employed copper sulfate as the toxicant, but the results were not wholly satisfactory. The use of toxicants in fisheries increased rapidly in the 1930’s and 1940’s, and Prevost (1960) stated that the reclamation of waters b y poisoning fish had become the best management tool available. The fish problems amenable to correction by toxicants are too numerous to be discussed other than sketchily here. Freshwater and marine fishes are frequently involved in pest situations that involve man’s safety and welfare (Lennon, 1970). Fish toxicants afford relief in many pest situations, but controls are either lacking, inadequate, or impractical for other pest situations. Rotenone has been employed, for example, in new hydroelectric impoundments in South America to control large populations of piranhas that threatened injury or death to humans and livestock (Fontenele, 1963).Some of the finest food and game fishes may serve as intermediate hosts for dangerous parasites of man and domestic animals (Bauer, 1961; Van Duijn, 1962), and fish toxicants can be applied in some circumstances to eliminate infected fish. The control of the highly destructive sea lamprey in the Great Lakes depends largely on the use of a selective toxicant (Applegate et al., 1961; Baldwin, 1968). The toxicant is applied in many of the American and Canadian streams tributary to lakes Superior, Michigan, Huron, and Ontario to kill the larval offspring of sea lamprey spawning. The lamprey suppression project has been called the largest aquatic control measure ever attempted (Bardach, 1964). The exotic carp in the United States is the principal target of fish toxicants. This fish occurs in the 48 contiguous states and has achieved overwhelming dominance in many rivers and lakes. The carp grows fast and large, and its range is expanding because the fish is exceedingly wary and adaptable. Its environmental and economic impacts are enormous and adverse (Sigler, 1958). By its abundance, large size,

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and rooting feeding habit, it disrupts the bottom and causes troublesome turbidities in once-clear rivers and lakes, displaces valued food and game fishes, destroys fish spawning beds, and wrecks aquatic vegetation important to waterfowl as food and shelter. Without doubt, much effort will be expended in years to come to bring populations of carp under control, and suitable toxicants may continue to play an important role in the control. Pet fishes and cultured bait fish have caused pest problems when permitted to escape into wild waters. Wild goldfish are often as undesirable as carp because of abundance and habits. The walking catfish in Florida also is an example of an unfortunate escape (Idyll, 1969), and bringing the species under control with toxicants or other measures may be difficult. The purposeful stocking of game fish in foreign waters may give rise to unforeseen problems. T h e introduction of the largemouth bass into Cuba resulted in losses of cyprinodont fishes that are important in malaria control (Rivero, 1936). In Lake Atitlan, Guatemala, the introduced largemouth bass preyed to a damaging degree on the economically important, freshwater crab and on the young of the rare and flightless giant pied-billed grebe (Powers and Bowes, 1967). Antimycin was used to eradicate the bass in a grebe sanctuary adjacent to Lake Atitlan, but no attempts have been made to control the bass in the main lake. Many pest fish situations are associated with the rapid expansion of pond-fish culture and shrimp culture throughout the world. Competing, predaceous, or disease-carrying fishes may gain access to culture ponds and damage or destroy production. Wild sunfishes and minnows may become serious competitors in ponds devoted to intensive culture of channel catfish in southern United States, and it is fortunate that antimycin can be used as a selective toxicant for the offending species (Burress and Luhning, 1969a). Toxicants also have been used to eliminate predaceous fish from shrimp-culture ponds in the United States and Asia. Native or introduced populations of game fish in streams and lakes may become seriously unbalanced because of unwise stocking, selective or unequal fishing pressures, or characteristics of the habitat which favor a less desirable species over a more desirable species. Yellow perch, for example, may be detrimentally competitive with brook trout in small lakes or become abundant but worthlessly stunted in cold, soft-water lakes (Zilliox and Pfeiffer, 1956; Riel, 1965; Radonski, 1967). The sport-fishing value of many farm ponds and lakes may be damaged by great excesses of stunted bluegills that result from

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59

insufficient predation, fishing pressure, or food. Toxicants are widely used to correct this problem (Emig, 1966; Burress and Luhning, 1969b). Large, cannibalistic trout or other predaceous fish may require elimination from ponds and lakes that are managed intensively for trout fishing. Many such waters are treated regularly with fish toxicants at intervals of 3 to 10 years (Hooper et al., 1964; Stroud and Martin, 1968). I n short, fish toxicants and techniques for using them in management of waters are in process of evolving, and they offer promise for the solution of many fishery problems.

B. DESIRABLECHARACTERISTICS OF A PISCICIDE Today, a piscicide must possess characteristics that are both desirble to fishery managers and acceptable to regulatory agencies. The fishery managers want a piscicide to be: 1. Safe to human handlers and field workers. 2. Sufficiently powerful to be effective in small quantities against target fishes, thus reducing logistic problems on large waters or remote waters. 3. Available formulations specifically designed to reach target animals in clear or turbid, shallow or deep, weed-choked or weed-free, thermally stratified or unstratified, and flowing or standing waters. 4. Effective against target animals in warm or cold, soft or hard, acid or alkaline, and fresh or marine waters. 5. Selective or relatively selective against target animals by nature of the biological activity of the compound or by application techniques. 6. Nonrepellent to target animals. 7. Irreversible in effect once a dose-threshold is attained. 8. Sufficiently persistent in water to obtain lethal exposure of target animals. 9. Harmless or relatively so to nontarget aquatic life, especially those plants and animals that serve importantly as fish food. 10. Safe to wildlife, livestock, and humans who may use the treated water for drinking and bathing. 11. Capable of being detoxified rapidly and completely in water by administration of a safe and economical agent. 12. Economical to purchase and apply. 13. Relatively stable in storage. In addition, fishery agencies join with regulatory agencies, e.g., U.S. Environmental Protection Agency, US. Food and Drug Administration, and state departments of health and agriculture, in requiring that a piscicide be: 1. Safe in short- and long-term exposures to manufacturers, dis-

60

ROBERT E. LENNON AND CLAUDE V$ZINA

tributors, users, and nontarget life. Evidence is necessary that the piscicide causes no carcinogenic, teratologic, mutagenic, reproductive, or other adverse effects. 2. Degradable to harmless fractions in water under a wide variety of limnological conditions. Managers must have the capability to detect and measure the breakdown process and the disposition of residues in the environment. 3. Nonpersistent in water, bottom soil, and nontarget plants and animals. 4. Not subject to biomagnification in food-chain organisms. 5. Free of compromising influences on subsequent use of water for irrigation, livestock watering, domestic purposes, and recreation. 6. Registered for specific fishery uses by appropriate federal and state regulatory agencies. It is unlikely that any one compound or single formulation thereof can suffice to correct the wide variety of fishery problems and fulfill the safety requirements listed above. Of the fish toxicants known to fishery managers, antimycin comes closest by far in meeting needs and requirements. II. Chemistry

A.

CHEMICAL STRUCTURE

Antimycin A was isolated b y Dunshee et al. (1949) as an apparently homogeneous antibiotic, but subsequent studies by Lockwood et al. (1954) revealed its complexity: four different components were detected in bioautographs (paper chromatography and yeast) and designated as antimycin A,, AP, AS, and A, in the order of their increasing Rfvalues. Harada et al. (1958) were the first to isolate fractions A, and AS in pure form, and one year later Liu and Strong (1959) further obtained pure fraction Al. Subsequent results obtained from alkaline degradation studies by Strong and his collaborators led to the complete structure elucidation (Dickie et al., 1963; Van Tamelen et al., 1961); the structure was confirmed by Birch et al. (1961). The structure of antimycin A1 and A3 is shown in Fig. 1. The molecule consists of a 3-formamidosalicylic acid residue linked through an amide group to a dilactone ring which bears an acyl side chain and an n-butyl or n-hexyl side chain. Further studies on the separation of the complex b y use of the countercurrent distribution technique were conducted by Kluepfel et al. (1970): the four major components Al, Az , AS, and A4 were isolated in gram quantities, and three additional fractions Ao, A,, and & were detected and designated in the order of

61

ANTIMYCIN A, A PISCICIDAL ANTIBIOTIC

Component n-Alkyl FH3

Acyl

A,

n-Hexyl

Isovaleryl

A3

n-Butyl

Isovaleryl

0

/

OHCNH H

V n-Alkyl

&

O-Acyl

FIG.1.

Structure of antimycin A, and AS, according to Dickie et al. (1963).

their increasing polarity. For the first time, fraction A4 was isolated in pure, crystalline form; components An, As, and A, represented less than 1% of the complex. All seven components, Ao to &, were submitted to pyrolysis and the pyrolyzates separated and collected by gas-liquid chromatography (Schilling et al., 1970). Three peaks were observed and the corresponding pyrolysis products were submitted to mass spectrometry; on the basis of their mass number, infrared, ultraviolet and nuclear magnetic resonance spectra, and microchemical analysis, their structures were elucidated. All three peaks of pyrolysis products were derived from the nonaromatic moiety of the molecule; no significant radioactivity could be detected in the products isolated after pyrolysis of antimycin A, 14C-labeled in its aromatic moiety or threonine residue. These results of Schilling et al. (1970) confirmed the structure proposed by Van Tamelen et al. (1961) for components A1 and At, and established the structure of Az and Ad; the structure of An, As, and A,, which were not available in pure form, was also proposed. The components of antimycin A are listed in Fig. 2. The pyrolysis-GLC method of Schilling et a2. (1970) is a relatively rapid method for the semiquantitative analysis of the components present in the antimycin A complex. Endo and Yonehara (1970) applied similar techniques to the analysis of the blastmycin complex Component

A,, d

:

F\O HHO I C , 0

OHCNH

CH 0

o=c n- Alkyl

H

3

C V O-Acyl

A, A2 A3

4 A5 A6

n-Alkyl

(a) n-Hexyl

Acyl

(b) n-Butyl (c) Octyl (d) n-Hexyl Heptyl

Hexanoyl Heptanoyl Butyryl Isovaleryl

n-Hexyl n-Butyl n-Butyl Ethyl Ethyl

Butyryl Isovaleryl Butyryl Isovaleryl Butyryl

FIG.2. Structure of antimycin & to &, according to Schilling et al. (1970).

62

ROBERT E. LENNON AND CLAUDE VBZINA

(which mainly consists of antimycin As). They confirmed that antimycin A, was the main component, and detected three minor components, one of which is antimycin A4. Components of antimycin A have the same biological activity. Liu and Strong (1959) previously observed that antimycin A3 was much more active than A1 and A,, when tested against Saccharomyces cerevisiae Y-30 in the cylinder plate assay; however, Tappel (1960) reported that all three fractions have identical activity as electrontransport inhibitors. More recently, Kluepfel et al. (1970) found that the four major components A1 to A4 exhibited the same fungicidal activity against Saccharomyces cerevisiae Y-30; the higher activity A, reported before was found to be only apparent, and could be attributed to its higher diffusion coefficient in agar. The teleocidal activity was also the same for the four major components. The proportion of individual components in the antimycin A complex is a characteristic of the producing streptomycete. Kluepfel et al. (1970) determined the centesimal composition of the complex produced by Streptomyces sp. B-265 and several high-producing strains derived from it: mutations to increased production did not necessarily lead to a change of composition, but, in the mutants examined, the change, when observed, was in favor of A,. The structure-activity relationships of antimycin A and its derivatives, prepared by Dickie et al. (1963) and Van Tamelen et aE. (1961), were discussed by Rieske (1967), who concluded that the substituted dilactone ring, the phenolic hydroxyl group and an N-carbonyl group individually and collectively are indispensable for inhibitory activity of antimycin A. The mechanism of inhibition is determined by the aromatic nucleus and its substituted groups, whereas the dilactone ring and its substituted alkyl groups, in addition to conferring lipid solubility, would provide better fit between antimycin A and its binding site on the enzyme (Rieske, 1967). Tappel (1960) proposed also that antimycin A inhibits respiration because of its capacity to chelate with metal ions. Recently, Neft and Farley (1971) contributed information on the role of the formamido group of antimycin A on its activity against several enzymes of submitochondrial particles: the formamido group in position ortho to the phenolic hydroxyl could be replaced by pnitro or p-formamido groups without loss of activity. According to Neft and Farley (1971), the main role of the formamido group would be to decrease the p& of the phenolic hydroxyl through its electronwithdrawing action toward the phenolic ring (also see Rieske, 1973).

63

ANTIMYCIN A, A PISCICIDAL ANTIBIOTIC

B. DEGRADATION The degradation of antimycin A is illustrated in Fig. 3. This diagram is derived from the initial studies conducted under mild alkaline conditions by Strong and his collaborators (see especially Van Tamelen et al., 1961). The mechanism of degradation has been studied as a means of elucidating the structure of the antibiotic and is of

q C O N H

23

OH H,C

H,CH(CH.JZ

R

0

NHCHO

Antimycin A, : R = n-hexyl: CzsH40Nz0, Antimycin A, : R = n-butyl : CZ6H3,N20,

.

COOH

OH

CH,

I

I I

1 R' O

NHCHO

n

:

Antimycin lactone C14H2404 : R = OOCCH,CH(CH,),

Blactmycic acid CIZH,,N,O,

R' = n-C,H, CI,HZ*O* R = OOCCH,CH(CH,),

I

c

COOH I CONH-CH

I

P

O

H

HCoH AH,

$.

HCOOH F o r m i c acid

R' = n-C,H,,

+

Hydroxy lactone

L

CllHZOH

3

R = OH

NHZ

R' = n-C,H,,

Antimycic acid

+

C11H14N205

-n- Hexyllevulinic acid

(Y

C1LH2W03

3-b i n 0 salicylic acid C7H7NO3

Glycine C2H5N02

Methylethylacetic acid ' ~5Hl002

FIG.3. Alkaline degradation of antimycin A. Diagram drawn from data of Dickie et al. (1963) and Van Tamelen et al. (1961).

64

ROBERT E. LENNON AND CLAUDE V ~ Z I N A

interest in conjunction with the use of antimycin A as a piscicidal antibiotic. Derse and Strong (1963) recognized in antimycin A the characteristic of being rapidly degraded in natural waters, a sine qua non condition for a toxic agent in fish management. Hussain (1969) has recently determined the kinetics and mechanism of hydrolysis of antimycin A,, in neutral and alkaline solutions, to obtain a better understanding of the rate of degradation in natural waters. H e observed, by spectrophotometric measurements, that at neutral and alkaline pH values the overall degradation follows consecutive first-order kinetics: Antimycin A1 -% blastmycic acid

+ antimycin lactone -% fatty acids

kr and kz when determined separately were also found to follow firstorder kinetics. The pH dependency of each rate constant was studied. The apparent activation energy for klwas determined fluorometrically at two different hydroxyl ion concentrations and found to be 18 kcal/ mole, including 12 kcal/mole for the heat of ionization of water. T h e net activation energy, 6 kcal/mole, was much lower than that for the hydrolysis of y-valerolactone and y-butyrolactone, and has been anticipated by Van Tamelen et al. (1961).

C. TOTALSYNTHESIS Kinoshita and Umezawa (1969, 1970) have recently reported the total synthesis of dehexyldeisovaleryloxyantimycin A, (alkyl and acyl side chains replaced by hydrogens); the synthetic compound showed a minimum inhibitory concentration of 0.1 pglml against Piricularia oryxae; the MIC values against various species of Corticium, Gloeosporium, Glomerella, and Leptosphearia varied between 50 and 100 pg/ml. Similar activities were observed when a diasteromeric mixture of blastmycin (antimycin A3) obtained by total synthesis (Kinoshita et aZ., 1969) was tested against the same microorganisms. Kinoshita et al. (1972) also established the absolute configuration of antimycin lactones as 2(R), 3(R), 4(S), from which they could determine the absolute structure of antimycin A. Rieske (1973) recently confirmed that dehexyldeisovaleryloxyantimycin A was as potent as antimycin A, in inhibiting electron transport. Further investigation of Kinoshita and his co-workers (cited.by Rieske, 1973) involved the synthesis of antimycin A analogs containing a 15-member ring (instead of the natural 9-member ring). The 15s epimer was much more active than the 15R epimer in inhibiting Piricularia oryzae and electron transport. Rieske (1973)concluded that the specificity of action depends much more on the configuration of the dilactone than on its lipid solubility.

65

ANTIMYCIN A, A PISCICIDAL ANTIBIOTIC

D. MICROBIALAND ENZYMATIC TRANSFORMATIONS A large number of microorganisms, mainly aspergilli and streptomycetes, were tested b y Singh and Rakhit (1971) for their ability to transform antimycin A. Aspergillus ochraceus AY F-219 was found to be the most effective organism; conidia were more active than the growing or resting mycelium in transforming the antibiotic. Conidia were suspended in 0.02 M phosphate buffer, p H 8, to a concentration of 1 x 109/ml; antimycin A in acetone was added to a final concentration of 1mg/ml; glucose was also added (1mglml) as an energy source. The mixture was incubated on a rotary shaker for 72 hours at 28°C. Untransformed antimycin A and transformation products were isolated and characterized. A mixture of compounds (111) and (IV) (Fig. 4) was obtained through reactions C and B, respectively. The transformation products had no antibacterial activity and showed about 2% of the activity afforded by antimycin A against Saccharomyces cerevisiae Y-30; the products also had only a weak effect on respiration of S. cerevisiae and Candida albicans. These results confirm previous conclusions on the necessity of an intact dilactone ring for activity.

00 OH

OH

and/or

(11)

~

~

R

HH

0

NHCHO

~

(W)

0

(DI)

4. Transformation of antimycin A by conidia of Aspergillus ochraceus. From Singh and Rakhit (1971). FIG.

Singh et al. (1972) used purified enzymes in an attempt to cleave the ester or amide bond in antimycin A. Trypsin, chymotrypsin, pancreatin, peptidase (from hog intestine), carboxypeptidases A and B, wheat germ, pancreatic lipase, and a crude preparation of Escherichia coli penicillin acylase were inactive, but deacylation was observed with hog kidney acylase. The reaction was carried out with 500 mg of the

o

~

66

ROBERT E. LENNON A N D CLAUDE V ~ Z I N A

enzyme and 1 gm of antimycin A in 1 liter of 0.05 M phosphate, p H 8.0; the mixture was incubated aerobically at 37°C for 17 hours. One polar product was isolated and characterized as compound (V) (Fig. 5 ) on the basis of nuclear magnetic resonance spectroscopy and pyrolysis-gas-liquid chromatography (Schilling et al., 1970). Deacylantimycin A had no antibacterial activity, and only 10-15% of the fungicidal (S. cerevisiae Y-30) activity of antimycin A; it affected only weakly the respiration of S. cerevisiae and C . albicans. Although deacylantimycin A can be a useful substrate for the preparation of semisynthetic antimycins, no compound has been isolated so far which would have altered antimicrobial spectrum or decreased toxicity.

O-Acyl

O-H

(V)

(1)

FIG.5. Deacylation of antimycin A by hog kidney acylase. From Singh et al. (1972).

Ill. Fermentation The original strain of Leben and Keitt (1948) was designated as Streptomyces sp. NRRL 2288: it produced the four major components A,-A, of antimcyin A complex; another isolate was later reported (Lockwood et al., 1954) which produced a similar complex. The Japanese workers (Harada and Tanaka, 1956) isolated antipiricullin A which proved to be very similar to the antimycin A complex; the producing organism was designated as s. kitazawaensis. The same year, Sakagami et al. (1956) reported the isolation of virosin, another antimycin A-like antibiotic, produced by a variant of S. olivochromogenus. Watanabe et al. (1957) then isolated blastmycin which is also a complex, the main component of which was found to be antimycin AS; S. blastmyceticus was the producing organism. More recently, another complex was reported by Schmidt-Kastner (1963) and designated as phyllomycin which also comprises antimycin A fractions; this complex was produced by S. umbrosus. Camiener et al. (1960) were the first to recognize that several strains of S. antibioticus could simultaneously produce antimycin A

ANTIMYCIN A, A PISCICIDAL ANTIBIOTIC

67

and actinomycins, and made extensive taxonomic studies of this group of antimycin A-actinomycin producers. Karasawa et al. (1959) conducted similar studies on blastmycin-producing strains. It is a well known fact that antimycin A-producing streptomycetes are commonly recognized in antibiotic screening programs directed to the isolation of antifungal antibiotics. When Derse and Strong (1963) reported antimycin A as a useful tool in fish management, a strain improvement program was conducted: spontaneous and induced variability in Streptomyces sp. NRRL 2288 was exploited in isolating high-producing strains; through four steps of ultraviolet irradiation and selection, a strain was isolated which in suitable media yielded ca. 5 gm of antimycin A per liter. An account of this work was given by Vkzina (1967). When antimycin A fermentation was scaled up in aeratedagitated fermenters, it was found necessary to adjust p H to 6.0 and feed the carbon source continuously for maximum yields and minimum fermentation time (Vhzina, 1971). IV. B iosy nt hesis

A. GROWTHEXPERIMENTS

To our knowledge the first report on the biosynthesis of antimycin A is that of Birch et al. (1962); using S . kitazawaensis in a complete medium they obtained yields of about 15 pglml. Kannan et al. (1968) used a complete medium and devised a synthetic medium for S . antibioticus NRRL 2838, an actinomycin and antimycin A producer. Although the yields were low (- 2 pg/ml in synthetic medium), they found that tryptophan was well assimilated, but not required for growth. There was no absolute relationship between growth and antimycin A production. Working with the same organism hehAEek et al. (1968a) could isolate from culture broths a new microbial metabolite, N-formylaminosalicyclic acid, which they proposed as a precursor of antimycin A. RehAEek and h a r c (1968) described the isolation and characterization of that metabolite. RamanKutty et al. (1969) studied the role of phosphate on the formation of antimycin A in a synthetic medium: addition of 5 gm per liter of medium led to maximum yields when effected at 18 hours of incubation of s. antibioticus NRRL 2838. The role of phosphate was found to lie in the conversion of poly-P-hydroxybutyrate into readily assimilable source of carbon. Neft and Farley (1972a) devised a synthetic medium for Streptomyces sp. AY- B-265. They observed that iron was very inhibitory and could not be added to the medium for maximum yields. They confirmed the enhancing effect of tryptophan on antimycin A production.

ROBERT E. LENNON AND CLAUDE V ~ Z I N A

68

B. 14C-LABELEDPRECURSORS Birch et al. (1962) added formic-I4C,acetic-l-14C,and pyruvic-2-I4C acids to a complete medium inoculated with S. kitazawaensis. They observed that acetic acid was well incorporated into moieties A, B, C and D (Fig. 6), whereas pyruvic acid was incorporated into moieties B and C only. They concluded that moiety A originates through the acetate-malonate pathway, and moiety B results from pyruvate directly; they also deduced from their studies that moiety C (the acyl fragment) is from the amino acids valine, leucine, and isoleucine, and that moiety D (threonine) originates from the aspartate pathway. They speculated that fragment E (salicyclic nucleus) is from shikimic acid, and moiety F comes from C1-unit transfer mechanisms; however, formic acid-14C was not incorporated.

OH&

FIG.6. Antimycin A moieties according to their origin. Redrawn from Birch et al. (1962).

RehiEek et al. (1968a) fed tryptophan-2-14C and tryptophan-5-14C to cultures of S. antibiotics NRRL 2838 and found high incorporation of these precursors into formylaminosalicylic acid and fragment E of antimycin A, thus proving the hypothesis of Birch et al. (1962) that this fragment is formed through the shikimate pathway. Neft and Farley (1972b), using Streptomyces sp. AY B-265, corroborated the results of RehaEek et al. (1968a) and found that tryptophan was better incorporated than phenylalanine and shikimate into moiety E of antimicin A. However, incorporation of labeled tryptophan was not diluted out by unlabeled tryptophan, and tryptophan addition to the medium did not enhance yields of the antibiotic. Incorporation of 2-ring-tryptophan-I4C demonstrated that carbon-2 of the indole ring was incorporated into the formamidocarbonyl (moiety F) of antimycin A.

V. Mechanism of Action The first attempt to uncover the mechanism of action of antimycin A was made by Ahmad et al. (1950), who observed that the antibiotic

ANTIMYCIN A, A PISCICIDAL ANTIBIOTIC

69

was a potent inhibitor of succinoxidase (2 x lo-* M ) . Potter and Reif (1952) extended these studies to several enzymatic systems and concluded that it specifically blocks the passage of electrons at a site located between cytochrome b and cytochrome c. When components of the mitochondria1 respiratory system became known, the antimycin A site could be identified with more precision: Rieske and Zaugg (1962) found that only complex I11 is affected by antimycin A, and concluded that the antimycin A-sensitive site is a primary linkage between cytochromes b and c. The interested reader is referred to the two excellent reviews by Rieske (1967, 1973) for a complete account of present knowledge. In his second review, Rieske (1973) discussed in detail the toxicity of antimycin A for various forms of life, and the diversity of its effects which stems directly from a single, highly specific interaction with the respiratory chain. He concluded that antimycin A has become an indispensable tool for probing the relationships between structure and function in the cytochromes b-cl segment of the respiratory chain. It is noteworthy that most bacteria, including the antimycin Aproducing streptomycetes (Rehakek et al., 1968b), are insensitive to antimycin A, even though they have a full complement of cytochromes a, b, and c. They may have no antimycin A-sensitive site or alternate electron transfer pathways. At the other extreme, fish is more sensitive than any other form of life by several orders of magnitude. As a gilled animal, fish is separated from its aquatic environment by a membrane only one cell layer thick; this may account for the higher toxicity of the antibiotic against fish as compared with mammals.

VI. Development of Antimycin as a Piscicide A. LABORATORY TRIALS Derse and Strong (1963) were the first to suggest the potentials of antimycin as a fish toxicant. Their U.S. Patent No. 3,152,953 on METHOD O F KILLING FISH WITH ANTIMYCIN was based on observations that fish are much more sensitive than higher animals to antimycin (Strong and Derse, 1964). At that time, Derse and Strong at the Wisconsin Alumni Research Foundation and the University of Wisconsin respectively invited the Fish Control Laboratory at La Crosse, Wisconsin to participate in further research on antimycin as a piscicide. Walker et al. (1964) related the results of trials of antimycin against 24 species of fish in indoor trials and 25 species of fish in outdoor trials. They learned that the range of sensitivity among

70

ROBERT E. LENNON AND CLAUDE

VBZINA

various species of freshwater fish is great. Gizzard shad perished from 24-hour exposures to 0.04 ppb of pure antimycin at 22°C whereas 40 ppb were required to kill black bullhead at the same temperature. The fish most sensitive to antimycin included trouts, perches, and herrings, with all specimens succumbing to less than 1ppb in 24-hour bioassays at 12°C. Fish of intermediate sensitivity included pikes, sunfishes, suckers, sticklebacks, minnows, and carps that perished as a result of 24-hour exposures to 1.6 ppb of antimycin at 12°C. The most resistant fish in laboratory trials included goldfish, channel catfish, black bullhead, and yellow bullhead in indoor bioassays and the same species plus gars and bowfins in outdoor trials. Some resistant specimens survived 24-hour exposures to antimycin at 20-100 ppb at 12°C. Antimycin also was bioassayed against selected invertebrates in the laboratory. Water fleas (Daphnia magna) survived 24-hour exposures at 12°C to 1 ppb of the toxicant, but perished in 100 ppb. Crayfish survived 96-hour exposures to 10 ppb at 12°C. Damselfly nymphs required 48-hour exposures to 500 ppb at 12°C for lethal effects. Tiger salamanders survived 80 ppb but were killed by 600 ppb in 96 hours at 12°C. Bull frog tadpoles were resistant to 24-hour exposures to 20 ppb of antimycin at 12"C, but died at 40 ppb. There were no grossly toxic effects of antimycin on plankton, bottom fauna, or aquatic plants in outdoor pools in midsummer and early fall. The concentrations of toxicant applied ranged up to 20 ppb. Further tests in hatchery ponds at 10 ppb showed no effects on plankton, bottom fauna, and aquatic plants that could be attributed to the toxicant. Concurrently, Loeb (1964) demonstrated that antimycin was lethal to large carp when force-fed at doses of less than 1 mg of toxicant per kilogram of body weight over a period of 2 days at 18.3"C. Poison baits were palatable to carp when fed at a 1%level in 200-mg baits that were composed of equal amounts of flour and fish pellet meal. Carp were killed consistently by doses of 3 milligrams per kilogram of body weight at 18.3"C within 1-4 days after ingestion. Loeb also killed all carp, yellow perch, and unidentified minnows in a 0.1hectare pond by application of 1.9 gm of antimycin, or approximately 3 ppb. After 96 hours, the pond was drained, and brown bullheads were the only fish remaining alive. Crayfish, frogs, and newts present appeared unaffected by the toxicant. The results obtained b y Walker et al. (1964), Loeb (1964), and Berger (1967) confirmed that antimycin has potential as a piscicide. The chemical is effective in very small quantities against fish, in-

ANTIMYCIN A, A PISCICIDAL ANTIBIOTIC

71

cluding the carp, which frequently is a target species; it is odorless, colorless, and nonrepellent in water; it degrades within 24-96 hours; and it is relatively harmless in fish-killing concentrations to plankton, macro- and microinvertebrates, and aquatic plants. On the basis of these results, the Wisconsin Alumni Research Foundation and the Fish Control Laboratory decided to attempt development of antimycin as a piscicide. The decision was made, however, in knowledge that extensive and strict new requirements imposed by law - never before applied to a fish toxicant-would have to be met. The new laws administered by the U.S. Food and Drug Administration and having bearing on piscicides include: Food Additives Amendment of 1958, Color Additive Amendment of 1960, Federal Hazardous Substances Act of 1961, and the Kefauver-Harris Drug Amendments of 1962. The Pesticide Regulation Division (formerly in the U.S. Department of Agriculture but now in the Environmental Protection Agency) extended its control of pesticides to include fish toxicants under the 1959 Amendment to the Federal Insecticide, Fungicide, and Rodenticide Act of 1947. Accordingly, representatives of WARF and the Fish Control Laboratory consulted repeatedly with the regulatory agencies on studies needed during development of antimycin as a piscicide. The aquatic studies that contributed to U.S. and Canadian registration of antimycin as a piscicide in 1966 were conducted by Berger et al. (1969) at the Fish Control Laboratory. Solutions of crystalline antimycin in acetone exhibited good shelf life, as did a formulation of antimycin on sand (1% antimycin, 24% Carbowax, and 75% sand). The investigators found a further advantage that antimycin can be detoxified by potassium permanganate. Their preliminary tests revealed that 300 ppb of potassium permanganate detoxified 5 ppb of antimycin within 6 hours to a point where fingerling bluegills could survive if added to the medium after the 6 hours. The Laboratory trials included 31 species of fish of various sizes and life stages. The bioassay waters varied in temperature, hardness, pH, and turbidity. As in earlier trials (Walker et al., 1964), trouts were the more sensitive to the toxicant. Twenty-four of the 31 species succumbed to 5 ppb or less, and only black bullheads survived exposures to 25 ppb. The exposure of fertilized fish eggs disclosed a further advantage of antimycin. T h e eggs of rainbow trout, northern pike, goldfish, carp, white sucker, and channel catfish perished after short and long exposures to the toxicant. All fertilized goldfish eggs, for example, were killed by 2-hour exposures to 7.5 and 10 ppb at 17"C, and all goldfish and carp eggs died when exposed to 2.5 ppb

72

ROBERT E. LENNON AND CLAUDE V ~ Z I N A

throughout the 7-day incubation period. Concurrently, Valentine (1966) was finding that antimycin kills the fertilized eggs of zebra fish. Berger et al. (1969) also determined that the newly hatched fry of bowfin, rainbow trout, channel catfish, and largemouth bass were generally more sensitive than fertilized eggs to antimycin. On the other hand, juvenile and adult fish of 10 species tended to be more resistant than fry or fingerlings to the toxicant. A summary table (Table I) indicates the order of antimycin toxicity to the 31 species of fish, including various life stages and different water qualities. Twenty-five of the 31 species are killed by 10 ppb or less of antimycin; shortnose gar, bowfin, and channel catfish require up to 25 ppb for kills; and white catfish, flathead catfish, and black bullhead require up to 200 ppb. Thus, the range of sensitivity among freshwater fish to antimycin is wide, and there is obvious potential for selective application of the toxicant. TABLE I TOXICITY OF ANTIMYCIN TO 31 FISHES, INCLUDING VARIOUS LIFE STAGES AT DIFFERENT WATER TEMPERATURES AND QUALITIES"

ORDER OF

1.0 ppb Rainbow trout Brown trout Brook trout Lake trout Walleye Yellow perch

5.0 ppb (continued) Pumpkinseed Green sunfish Fathead minnow Northern redbelly dace Brook stickleback Largemouth bass

5.0 ppb

7.5-10.0

White sucker Smallmouth bass Freshwater drum Black crappie Bigmouth buffalo Quillback Spotted sucker Northern pike Carp Longear sunfish B1u e gi 11 Redear sunfish

ppb Goldfish

25 ppb Shortnose gar Bowfin Channel catfish

200 ppb White catfish Flathead catfish Black bullhead

"From Berger et al. (1969).

Other investigators in the U.S. Fish and Wildlife Service extended the list of fish tested in 1966 to include spot, longnose killifish, sea catfish, sheepshead minnow, white amur, and tilapia. Of them, only

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the sheepshead minnow could be considered among the more resistant species. The tests afforded the first evidence that antimycin is effective against fish in marine water. Whereas most of the bioassays run by Berger et al. (1969) were in deionized water reconstituted to 42 ppm in total hardness, 30 ppm in total alkalinity, 7.40 in pH, and saturated with 0 2 at 12"C, some definitive trials were conducted in waters of different hardness, pH, and temperature. The bioassays of antimycin against fingerling rainbow trout in waters of 20, 48, 90, 180, 360, and 400 ppm in total hardness disclosed that the toxicant is slightly less effective on fish in hard water. The findings were confirmed later by 96-hour trials against rainbow trout, goldfish, and bluegill at 20 and 400 ppm in total hardness. I n contrast, the effectiveness of antimycin against fish is influenced significantly by the pH of the medium. In trials against goldfish at 12"C, the 96-hour 100% effective concentrations (ECloo,) were 0.20 ppb at pH 5 , l . l O ppb at pH 8, and 60 ppb at pH 10. Further tests against rainbow trout and carp confirmed that the activity of antimycin against fish is reduced at higher pH levels. With respect to temperature, fish are more susceptible to antimycin at warmer temperatures. The exposure of 22 species of freshwater fish to the toxicant at 7", 12", 17",and 22°C in the laboratory yielded 2-fold to 5-fold differences between ECloots.Cold weather trials of antimycin at 5 ppb killed juvenile to adult fish of sensitive and resistant species at 3.4-5.5"C, and the conclusion was that the mortality is retarded rather than reduced by cold. I n trials under 6 inches of ice at pH 8.5 to 9.0, 1 ppb of antimycin caused no significant mortalities among eight species of fish, and 5 ppb killed only the more sensitive species. A higher concentration of toxicant or more favorable pH may have resulted in greater mortality of exposed fish. Because the turbidity of water may influence the performance of a fish toxicant, antimycin was tested against fish in the presence of 1000 and 5000 ppm of clay in suspension. At the higher turbidity, the toxicant was only slightly less toxic than in nonturbid controls in 24- to 96-hour bioassays. Further tests with 5 ppb of antimycin in outdoor pools at 3.4 to 5.5"C killed all fish within 15 days in clear water and within 18 days in turbid water. The toxicity appeared to be retarded b y turbidity to a lesser extent than by temperature. Berger et al. (1969) also injected antimycin into the body cavities of fish, and the specimens died. The behavior of injected fish is similar to that of fish immersed into solutions of antimycin. Moreover, the order of toxicity in injected fish is similar to that in immersed fish. Rainbow trout were the most sensitive to injections, carp were intermediate, and black bullheads were least sensitive.

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The same investigators subjected a candidate field formulation to trials in a Plexiglas column 1 foot in diameter and 8 feet high. The antimycin in Carbowax was coated on 40-mesh sand grains in such a way to release the toxicant uniformly into the water within the first 5 feet of depth from the surface. When applied at the surface of the 8-foot column of water, the release of antimycin was uniform within the first 5 feet, but not complete. Samples siphoned off at depths of 1-5 feet were toxic to rainbow trout; a sample at 6 feet of depth killed 12 of 16 trout; a sample at 7 feet was nontoxic; but a sample from the bottom at 8 feet killed all trout. Following improvements and field trials of the formulation, it was registered in 1966 as a toxicant for fishery uses under the trade name FINTROL-5. Berger and Hogan (1966) demonstrated that antimycin can be used for selective removal of scale fishes in the presence of channel catfish. The eggs of goldfish and carp are four to eight times more sensitive than channel catfish eggs to antimycin; largemouth bass fry are 100 times more sensitive than channel catfish fry to the toxicant; and fingerling-size goldfish, carp, fathead minnow, green sunfish, bluegill, and largemouth bass are many times more sensitive than fingerling channel catfish. Callaham and Huish (1968) further explored the selective potentials of antimycin in trials against golden shiner, bluegill, redear sunfish, and largemouth bass. They demonstrated that the piscicide can be applied selectively against certain species in a multispecies complex of fishes or used selectively against certain sizes of fish within a species. Other investigations of antimycin-fish relations in the laboratory were focused on specific questions that pertain to use of a toxicant in fishery management. Marking (1969) tested the compatibility of rhodamine B and fluorescein sodium with antimycin because these fluorescent dyes are often employed to trace the movement of water and toxicants during stream reclamations. Both dyes at field-use concentrations are nontoxic to fish, and they have no significant influence on the activity of antimycin against fish. Howland (1969) recognized the possibility that both antimycin and rotenone may be employed in a lake-stream reclamation project, e.g., one toxicant in the lake and the other in streams tributary to the lake. He tested the interaction of antimycin and rotenone in fish bioassays and noted a slight additive effect. He concluded that the two toxicants are compatible in water and no nullifying interaction occurs. Because earlier investigations indicated the sensitivity of antimycin to pH, Marking (1970)and Marking and Dawson (1972) plotted the half-life of antimycin’s biological activity against fish in waters

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having 40-44 ppm total hardness, 12"C,and pH's 6-10. At pH 6, the half-life is 310 hours, but at pH 9, 9.5, and 10, the half-lives are 8.7, 4.6, and 1.5 hours, respectively. Marking demonstrated, however, that the antimycin may not decompose to a significant extent within a few hours, but rather the high p H inhibits its killing power. For example, the killing power is inactivated rapidly at p H 9, but it is restored almost completely if the bioassay medium is rebuffered down to pH 7 . In contrast, the ion content of the water has little influence on the half-life of antimycin except for the effect of ions on pH. Gilderhus (1972) conducted bioassays in flowing water to define the lengths of exposure necessary to a given concentration of antimycin to produce 100% mortality of target fishes. H e termed this length of exposure the Effective Contact Time (ECT) and determined that the ECT for 5 ppb of antimycin against carp in hard water at p H 7.5-8.0 is 6 hours at lTC, 3 hours at 17"C,and 2 hours at 22°C. The ECT for 5 ppb of antimycin against white sucker is 6 hours at 12"C, 2 hours at lTC, and 1 hour at 22°C. Green sunfish are more resistant and require 11 hours of exposure at 12"C,4 hours at lTC, and 1 hour at 22°C. Doubling the concentration of toxicant shortens the ECT by only 38% whereas increasing temperature 5°C shortens the ECT by 62%. On the basis of these results, Gilderhus emphasized employment of streamside bioassays to determine appropriate concentration of toxicant and ECT's for target fishes prior to reclamation of a stream. The laboratory trials discussed above were accompanied by an extensive series of field trials against fish in a wide variety of water types.

B. FIELDTRIALS

The first field trials of antimycin as a piscicide were made in small ponds at the Delafield, Wisconsin Warmwater Fisheries Research Station in 1963 (Walker et al., 1964). The water exceeded 200 ppm in total hardness, and the temperature ranged from 15" to 21°C during the trials. The toxicant was applied at 10 ppb against 18 species of fish in one pond and against 19 species in the other. Northern pike were the first fish to exhibit distress by surfacing in a narcotic state. Rainbow trout, white sucker, carp, walleye, and sunfishes followed in order with the same symptoms as northern pike, and most specimens were dead. within 48 hours. Longnose gar, bowfin, black bullhead, yellow bullhead, and brown bullhead were not greatly affected, and 70% of them were recaptured alive when the ponds were drained after 20 days. Monitoring disclosed that the antimycin degraded with-

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in 72 hours after application, and fish introduced to the water after 72 hours survived until the ponds were drained. The pretreatment volume of plankton in the first pond was 0.018 cm3/liter, and the posttreatment volume was 0.047 cm3/liter; the pretreatment plankton in the second pond was 0.0035 cm3/liter, and the posttreatment volume was 0.039 cm3/liter. None of the minor changes was attributed to the toxicant. There were no observable changes in submersed and emergent, aquatic plants in the ponds during the experiments. Preand posttreatment samples of bottom fauna were examined, and the authors concluded that there were no significant changes in the species composition or numerical abundance of the 15 taxonomic groups present. No harmful effects of the piscicide could be detected in the frogs, salamanders, and turtles present. Gilderhus et al. (1969) reported the results of field trials in ponds and lakes in Arkansas, Nebraska, New Hampshire, New York, Wisconsin, and Wyoming in 1964-1966, and in streams in Wisconsin in 1965-1967. Their conclusions included: antimycin is effective as a piscicide in standing and flowing, and soft and hard waters; the effective concentration for most fish, including prime target species, is 10 ppb or less in waters below pH 8.5; fish eggs are killed b y concentrations used to control free-swimming fish; the effects in fish are irreversible; the liquid or sand formulations of antimycin do not repel fish; the concentration to be employed in a reclamation should be determined by on-site bioassays against target species in target water; and the piscicide can be detoxified by 1 ppm or less of potassium permanganate. They also noted that antimycin degrades rapidly, especially under alkaline conditions, and most waters can be restocked with fish within 2 weeks after treatment. The authors had abundant opportunity to observe the effects of the piscicide on nontarget life. In general, there were no discernible effects among aquatic invertebrates, but autumn pulses of rotifers, water fleas, and copepods in ponds at Cape Vincent, New York, were drastically reduced by concurrent exposures to antimycin and the season’s first killing frosts. On another occasion, in Wyoming, 10 ppb of antimycin caused no harm to freshwater shrimp in a trout lake, but higher concentrations applied in spring tributaries to the lake did reduce the shrimp numbers. The other invertebrates exposed to the toxicant without harm during the trials included crayfish, mayflies, damselflies, water boatmen, backswimmers, and midges. No evidence of mortality or harm was detected among mole salamanders, frogs, turtles, water snake, herons, puddle ducks, diving ducks, gulls, and terns that were present during treatments. Many herons and ducks

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were observed eating antimycin-killed fish, but without harm. Thus, the specificity of antimycin to fish was further affirmed. The trials by Gilderhus et al. (1969) also demonstrated how antimycin can be applied in streams and lakes, how overdosing or underdosing can be avoided, and how chemical and physical factors in the receiving waters may influence the performance of a toxicant. After these trials, Burress (1968,1970a) experimented with controlling sunfish populations, tilapia, and walking catfish in small ponds; Radonski (1967) suggested the potentials of antimycin for selective control of yellow perch; Burress and Luhning (1969a,b) developed applications of antimycin to obtain selective removal of scale fishes from channel catfish ponds; selective thinning of sunfish populations in ponds; and Burress (1970b) determined the sensitivity of the new exotic white amur to antimycin. Callaham (1968) and Callaham and Huish (1969) observed the effects of 5-ppb applications of antimycin on plankton and benthic animals in small, softwater ponds in Georgia. They noted severe reductions among the planktonic Rototaria, Cladocera, and Copepoda in two of three ponds, but no great influences of the piscicide on the benthic Tendipededidae, Ceratopogonidae, and Culicidae. The two ponds that suffered reductions in plankton were apparently overdosed with antimycin at 5 ppb because total hardness ranged from 5 to 8 ppm, pH from 6.4 to 7.3, and temperature from 19”to 22”C, i.e., conditions very favorable to the toxicant. The third pond had a total hardness of 15 ppm, pH 8.2, and temperature of l T C , and the antimycin had no significant effect on the zooplankton. As noted earlier, the toxic activity of antimycin is reduced in higher pH water and to a lesser extent by cold temperature. The results obtained by Callaham and Huish including a reduction of plankton in two ponds but not in another pond lend emphasis to the recommendation by Berger et al. (1969): “The concentration of toxicant to be used in a reclamation should be determined by preliminary bioassays on site against target fish in target water.” Burress ( 1 9 7 0 ~ )conducted a 2-year study on methods and equipment for on-site bioassays of toxicants against target and nontarget animals. H e determined that 75-gallon, 3-mil polyethylene bags serve well as bioassay vessels when suspended in target water. The loadings of organisms in vessels are light, and bioassays of 72-96 hours in duration are feasible. A series of bags containing test concentrations and organisms can be protected from depredation or accidental puncture by surrounding the immediate area in the water with a stout, anchored seine.

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c.

DETOXIFICATION OR REMOVALOF ANTIMYCIN

IN

WATER

Walker (1966) tested the potentials of potassium permanganate for detoxifying fish-killing concentrations of antimycin. He determined that 0.5 ppm of potassium permanganate deactivates 0.5 to 5 ppb of antimycin in water within 15 minutes to a degree safe for rainbow trout. Less than 0.5 ppm of potassium permanganate worked more slowly, and 1 ppm worked faster but was in itself toxic to rainbow trout. Walker (1967) discussed additional experiments in detoxifying antimycin with potassium permanganate. Using simulated stream conditions, he observed that the deactivation of antimycin b y the permanganate was more rapid in soft water and in warmer water. In static tests, the permanganate was significantly more toxic to fish at lower temperatures and less effective on antimycin than at higher temperatures. A field trial involved application of 2 ppm of potassium permanganate to deactivate 7.5 ppb of antimycin in a mill pond at 5°C. The water which began to spill from the pond 5 hours later was nontoxic to rainbow trout and fathead minnows in live cages at 100 and 500 yards downstream from the dam. Loeb and Engstrom-Heg (1970) mentioned that antimycin can be detoxified with either potassium permanganate or chlorine. Lennon (1973) reported on experiments to reduce or eliminate toxic wastes in laboratory effluents and noted that activated charcoal is highly effective in removing antimycin from water. Solutions of antimycin that contained up to 100 times the lethal concentration for rainbow trout were rendered nontoxic after passing through a 6-inch column of activated charcoal. Gilderhus et al. (1969) described the continuous application of 1 ppm of potassium permanganate for 40 hours in the outlet stream of a spring-fed lake to detoxify 10 ppb of antimycin. The application was successful in preventing mortality of fish downstream. Slifer (1970) prepared formulas and discussed equipment necessary for administering potassium permanganate in streams to detoxify antimycin. D. DEGRADATION OF ANTIMYCIN I N WATER

The earliest paper on the potential of antimycin as a piscicide contained mention that the compound degrades rapidly in water (Derse and Strong, 1963). The authors considered the lack of persistence an advantage and stated that 10 ppb of antimycin in water degrades within 7 days. The rate of degradation is accelerated in the presence of light, high alkalinity, and warm temperatures. The process of breakdown under alkaline conditions was discussed further b y Walker et al. (1964). Berger and Hogan (1966) conducted bioassays to determine whether the products of naturally degraded antimycin

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are harmful to fish. Fingerling bluegills survived a 2-month exposure in pools where 10 ppb of antimycin had degraded before the fish were stocked. The review of antimycin trials in the laboratory by Berger et al. (1969) contains data on the natural degradation of the compound in waters of various qualities and temperatures. Fingerling fish of species most sensitive to antimycin were used to detect the rate of degradation. The survival of fingerling bluegills for at least 96 hours, for example, indicated the degradation of 10 pyb of antimycin within 144 hours in outdoor pools with water at 252 ppm in total hardness, pH 8.4-8.6, and 15.5-17.2"C. T h e rate of degradation is relatively slow in cold water. Ten parts per billion antimycin took 13 days in clear water and 17 days in turbid water to degrade in pools at 3.4-5.5"C, total hardness of 260-320 ppm, and p H 7.4-8.2. These degradation rates, however, cannot be considered as absolutes because the metabolic rates of the indicator fish can be assumed to be lower in the very cold water. Lee et al. (1971) employed yeast assays to detect the degradation of antimycin in water. They demonstrated that the half-lives of antimycin in water at various pH's ranges from 5 hours at p H 7 to 6 minutes at p H 10. These half-lives are very brief when compared with the half-lives determined by Marking and Dawson (1972) in fish bioassays. The latter ranged from 120 hours at p H 7.5 to 1.5 hours at pH 10. The Marking-Dawson experiments involved exposure of six species of fish of different but known sensitivity to antimycin to solutions of the piscicide that had been allowed to degrade for selected periods of time before the fish were added. Concurrently, the same species of fish were exposed to fresh solutions of antimycin with known concentrations. The differences in LC50's for fish in the aged and fresh solutions reflects the degradation that has taken place in the aged solutions. Fluorometric studies by these authors (L. L. Marking and V. K. Dawson, unpublished) have confirmed the halflives observed in bioassays with fish. Lee et al. (1971) found that water hardness and alkalinity have no effect on the degradation of antimycin. On the other hand, light does have an effect, but less than pH, on the degradation of antimycin. Yeast assays showed that the half-life of the piscicide in aqueous solution in sunlight and in open shade is less than 20 minutes. Additional studies on the photolytic sensitivity of antimycin have been made at the Fish Control Laboratories, U.S. Fish and Wildlife Service (Lennon, 1972). Solutions of antimycin were exposed to ultraviolet light for 0, 1, and 3 hours prior to bioassay, and the 96-hour LCSO'S for fingerling chinook salmon were 0.056, 0.360, and 4.00 pg/liter,

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respectively. The depth of water also influences the rate of degradation by ultraviolet shielding. The experiments by Lee et al. (1971) included yeast and fish assays of antimycin in the presence of soil and lake sediments. Concentrations of suspended solids from 0.025 to 1.25% by weight in assay media had no effect on the toxicity of antimycin to yeast or fish. Marking and Dawson (1972) also observed the effects of temperature on the rate of antimcyin degradation. They showed that there is an effect and listed half-lives of 120 hours at 12"C, 93 hours at 17"C, and 68 hours at 22°C in water at 42-44 ppm total hardness and p H 7.5. The effect of temperature on the rate of antimycin degradation, however, is less than that exerted by pH. The degradation of antimycin in treated lakes and streams is detected b y submerging a series of live cages containing one or more species of sensitive fish. Each cage contains at least 10 fish, and the first cages are placed in the target water prior to initiation of treatment. The dead fish in each cage are counted and removed each 24 hours, and cages of fresh fish are put in place at that time. When all fish in cages have survived for 48 hours in the target lake, the time of their introduction into the water is listed as the end point of degradation. In a stream, the survival of fish in the live cages may detect either the degradation of the toxicant or its passage beyond the target site. Gilderhus et al. (1969) reported that fish-killing concentrations of antimycin persisted for more than 2 weeks in small, trout ponds having 10 ppm of total hardness, pH 7.0-7.2, and 12-13°C. Ten parts per billion of the piscicide lasted only 6 days in a small lake having 223 ppm of total hardness, p H 7.8, and 13°C. In another lake having 204 ppm of total hardness, pH 9.3, and 16.6"C, an application of 7.5 ppb of antimycin was deactivated or degraded so rapidly that few of the target fish were killed. Berger et aE. (1969) applied antimycin under thick ice cover in small pools. At p H 8.5 to 9, the 5 ppb of toxicant degraded to a point harmless to fingerling brown trout within 120 hours after application.

E. FORMULATION OF ANTIMYCIN A fishery manager has a much more difficult task in reaching a target fish with a piscicide than an agriculturist has in attacking an insect pest with an insecticide. The receiving water poses more obstacles to a piscicide application than is generally appreciated by managers and formulators. Furthermore, the long history of pesticide uses on fields and forests has conditioned us to think in terms of surface applications of certain concentrations per unit area, such as

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gallons or pounds per acre. T h e formulators and users of piscicides must keep in mind that a body of water is a complex, three-dimensional system, and seldom are two bodies of water alike in most respects. A body of water has surface area, but it also has depth which may average shallow, deep, or very deep. Furthermore, an expression of average depth may be misleading unless the topography of the bottom of the stream or lake is known and considered. Lennon et al. (1970) described a stream as much more complex than a lake in physical, chemical, biological, and political characteristics. Many of the characteristics, singly or in combination, may influence the selection or performance of piscicide formulations. A lake may be a sheltered and relatively static body with little or no inflow and outflow, or a dynamic body well mixed by wind-driven currents or by large inflows and outflows. Lakes also may be chemically and thermally stratified in summer and winter to extents that affect the performance of piscicide formulations. There are also mechanical obstacles to be considered by formulators and users of piscicides, and they include: the surface film of the water, overstory of terrestrial vegetation, beds of emergent and emersed aquatic vegetation, debris, adverse currents, adverse turbidity, difficult topography of surrounding land, and difficult access to the target water. A further point to be considered by formulators and users of piscicides is the fact that a target fish is seldom found throughout a body of water. Ideally, a formulation should be tailored or applied against a target fish in his ecological niche and thus avoid rendering the whole body of water toxic. Once research had demonstrated such advantages of antimycin as specificity to fish, toxic in extremely small quantities to fish, irreversible toxicity, nonrepellency to fish, and rapid degradation in water, studies were begun to develop field formulations that would meet target needs and yet preserve the desirable features of the parent compound. The first formulations used in laboratory trials consisted of crystalline antimycin dissolved in quantities of acetone that in themselves are nontoxic to fish (Derse and Strong, 1963; Walker et al., 1964; Berger et al., 1969). Stock solutions of antimycin in acetone remain stable and piscicidal for 2 years or more in cool, dark storage. The acetone formulations are satisfactory for laboratory bioassays and small-scale field experiments where the solutions can be injected into the water. The volatility of the solvent limits effective use of these formulations as sprays administered from boats or aircraft. T h e escape of solvent into the air during spraying may leave insoluble particles of antimycin on the surface film of the receiving water.

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Walker et al. (1964) experimented briefly with a formulation of antimycin that contained an emulsifiable concentrate, and it appeared to evoke a more rapid response in fish than acetone-antimycin. Developmental efforts were focused soon, however, on a dry, granular formulation that would not adhere to overstory vegetation or debris, would penetrate the surface film of water readily, and would release the toxicant within a certain depth from the surface of water. The first dry, heavier-than-water formuIation consists of antimycin in Carbowax coated on 40-mesh sand (Lennon, 1966). It was registered in the United States and Canada in 1966 as FINTROL-5. It contains 1%of antimycin, 24% of Carbowax, and 75% of sand by weight. It releases the antimycin into the water within the first 5 feet of depth from the surface. It is easily and uniformly spread over the surface of water by means of hand-powered or motor-powered, grass-seed spreaders from shore, boats, or aircraft (Lennon et aZ., 1967). T h e development and registration of FINTROL-15 followed. It is similar to FINTROL-5 except that it has 5% of antimycin and is designed to release the piscicide within the first 15 feet of depth from the surface. Progress has been made toward the development of a FINTROL-30 for treatment of the first 30 feet of depth. Because many fishery managers prefer or are equipped to dispense a liquid piscicide, especially in streams, FINTROL-CONCENTRATE was registered in 1969. It contains 10% of antimycin, a surfactant, and acetone, and is proving useful when injected into streams and shallow lakes. Hacker (1969) added ethyl alcohol as an antifreeze to the formulation and applied it successfully under the ice in midwinter to rid streams and small ponds of carp. The metering of precise amounts of a toxicant into a stream over a period of hours poses many problems involving equipment, the integrity of toxicant solutions, and manpower to service the apparatus and prevent interference. Ayerst Laboratories, Inc. plans to solve many stream-treatment problems b y developing a FINTROL-BAR that can be suspended inside a short length of stovepipe or plastic tubing, anchored below the surface of a stream, to release its antimycin uniformly over a certain period of hours. A FINTROL-BAR weighs approximately 250 gm and contains 35 gm of antimycin. A candidate bar dissolves in about 7 hours at 10°C and in about 5 hours at 15°C. Depending on the volume of stream flow, a fraction of a bar or one or more bars would be placed in the stream to release the desired concentration of antimycin in parts per billion for a desired amount of time.

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F. REGISTRATION Registrations of the FINTROL formulations of antimycin as piscicides have been obtained by Ayerst Laboratories, Inc., New York, New York in the United States and Canada. Much of the research leading to registrations was accomplished by Ayerst Laboratories, Inc., the Wisconsin Alumni Research Foundation, Madison, and the U.S. Department of the Interior’s Fish Control Laboratories at La Crosse, Wisconsin and Warm Springs, Georgia. The registrations permit use of the FINTROL formulations in freshwater streams and lakes and in marine waters against problem fishes. Methods and concentrations for target fish in waters of various pH’s and temperatures are specified. Degradation of the piscicide is usually complete within 2-7 days, and a bioassay method for confirming the degradation is described in package inserts. Detoxification with potassium permanganate is outlined. Antimycin-treated water must not be used for drinking by man or animals, or for crop irrigation, until fingerling fish of sensitive species (rainbow trout, bluegill, or suitable marine fish) survive 48 hr of exposure in live cages in the treated water. Fish killed by the piscicide must not be eaten by humans or livestock. The cautions specify keeping FINTROL formulations out of the reach of children, pets, livestock, and wildlife. Users should avoid inhalation of dust or contact with skin. The use of protective gloves and safety glasses is necessary. If FINTROL comes in contact with skin or eyes, flush repeatedly with water immediately. Prior to use in any private waters, the directors of state or provincial conservation departments should be contacted to determine whether a permit is required and to obtain any special instructions that may apply to the particular geographical area. VII. Applications as a General Piscicide

A. LAKESAND PONDS

The reclamation of fish production, farm, and ranch ponds with toxicants has become an established and economical practice in at least 29 countries, including the United States and Canada (Lennon et al., 1970). Total and partial reclamations of lakes and reservoirs up to hundreds of hectares in surface area have become effective management practices within the past 2 decades. Streams are often treated with toxicants in conjunction with lake reclamations, and the treatment of large river systems is now occurring on a larger scale. Forty-

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nine states in the United States and at least 7 of 11 provinces in Canada have used or are using fish toxicants in the management of waters. Inasmuch as the practice of reclamation is common and registered piscicides are marketed, the reports on reclamations are more often administrative job-completion reports of limited availability rather than published papers. Selected papers and job reports are cited here to demonstrate the variety of circumstances under which antimycin is applied to waters to correct probIems in fisheries. Powers and Bowes (1967) applied 9.3 ppb of antimycin in the FINTROL-5 formulation in shallow waters of the Giant Grebe Refuge at Lake Atitlan, Guatemala. The purpose was to eliminate the exotic largemouth bass that were preying to a serious extent on chicks of the rare and flightless, giant pied-billed grebe. The treatment was considered a success; largemouth bass up to 5.3 lb were removed; and fingerling fish of native species were restocked 10 days later in the treated waters to serve as food for the grebes. There were no adverse effects detected in nontarget, aquatic life. Northern pike, minnows, white sucker, pumpkinseed, and yellow perch were targets of a 5-ppb application of antimycin in FINTROL-5, FINTROL-30, and FINTROL-CONCENTRATE formulations in 44acre Beauty Lake in Quebec (Chamberland, 1966). The oligotrophic lake has a maximum depth of 42 feet; the FINTROL-5 was applied by seed-spreaders over the shallow portions; FINTROL-30 was applied similarly over deep water; and FINTROL-CONCENTRATE was used in small tributaries to the lake. The rapid release of toxicant in the deeper portions of the lake was demonstrated by the surfacing and kill of American smelt within 2 hours of application. The treatment was successful in eliminating target fishes. The FINTROL-30 formulation overcame the thermal stratification of water. Detoxification of the antimycin was complete within 2 weeks, and the lake was restocked with trout later. Observations made during 96 hours after treatment disclosed no adverse effects on plankton, aquatic insects, leeches, turtles, and aquatic plants. Powers and Schneberger (1967) discussed the potentials of antimycin for control of carp in Wisconsin. They cited applications of the piscicide that caused kills of enormous quantities of carp and rapid clearing of the once-turbid waters. Sayre (1969) employed FINTROL5 to rid a series of gold dredge ponds in Oregon of predaceous and competing fishes in a large, trout-restoration project. The toxicant was distributed by a helicopter over 250 surface acres of exposed pond water within 6 square miles of dredge tailing area. There were about 4 square miles of moving ground water through the dredged

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area, and many target fishes existed in caverns and channels between ponds. The treatment eliminated the target species from the ponds and underground connecting waters. Potassium permanganate was used with success to detoxify some effluent waters. An application of 5 ppb of antimycin in a high altitude, softwater lake in Idaho was too high a concentration (Rabe and Wissmar, 1969). The target cutthroat trout were in distress and dying within 1.5 hours, and the kill was complete within 24 hours. Thirteen days later, no living crustaceans could be found in the lake. A new survey 2 years later disclosed the presence of crustaceans and no change in the diversity of forms. Hacker (1971) summarized the control of carp with antimycin in the upper Fox River region in Wisconsin. The project involved 17 lakes with 4126 surface acres, 37 ponds, 18,000 acres of marshes, and 234 miles of streams. The estimated kill included 1.5 million pounds of rough fish, principally carp. The elimination of carp was estimated to be nearly 100%. Most of the antimycin was distributed by helicopter, and the author concluded that the sand-based formulations are superior in lake and slough applications, whereas the liquid formulation is best suited to streams. Montgomery (1972) discussed a novel application of FINTROLCONCENTRATE into small lakes in Georgia to obtain complete kills of fish. Sufficient antimycin in the liquid formulation was applied by means of a venturi-type boat bailer into the water to produce a 1-ppb concentration in the entire oxygenated water volume. The application per lake, however, was made along the entire margin of the lake in one pass, The toxicant diffused throughout the water and produced the desired kills of fish. Obtaining the correct concentration of antimycin proved to be critically important. T h e 1-ppb concentration killed all fish in one lake, but less than 1 ppb produced only a partial kill in another lake. The perimeter application of antimycin in the small lakes was easy, quick, effective, and economical. B. RIVERS AND STREAMS Antimycin in the FINTROL-CONCENTRATE and FINTROL-BAR formulations is well suited to the reclamation of flowing water because it is effective in cold water, only brief exposures are required to kill fish, it does not repel fish, and it is subject to detoxification by potassium permanganate. In contrast, rotenone-based piscicides do not work well in cold water, and they repel fish. Lennon and Berger (1970) reported the results of early trials of antimycin against target fishes in Wisconsin streams. Carp, for example, make no attempt

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to flee from a bolt of antimycin moving downstream, killing concentrations can be applied and maintained by drip or spray stations within practical limitations of time and economy, and potassium permanganate solution can be introduced into the water over a period of time to detoxify the antimycin quickly. Sayre (1969) discussed the application of antimycin in a 20-mile portion of a river system in Oregon. Hacker (1969) reported success in applying FINTROL-CONCENTRATE under the ice to kill carp in the Beaver Dam River system in Wisconsin. Again, Hacker (1971) summarized carp control by antimycin in 234 miles of streams in the Fox River system in Wisconsin and pointed out that the liquid formulation of the toxicant is suited to use in streams. Slifer (1970) developed formulas and suggested techniques for use of antimycin in streams. Gilderhus (1972) gave the practice of stream reclamation a boost by defining the effective contact times (concentration duration of exposure) necessary to produce kills of target fish with antimycin and rotenone in flowing systems. Five- to 12-inch carp, for example, require at least 6 hours of exposure to 5 ppb of antimycin at 12"C, 3 hours at l T C , and 2 hours at 22°C for kills. Green sunfish and largemouth bass require 11 and 13 hours, respectively, of exposure to 5 ppb at 12°C for kills. Thus, maintaining concentration of toxicant and an adequate duration of exposure are essential to successful reclamation of flowing waters. The new FINTROL-BAR formulation of antimycin was given a major test in 13.5 miles of Fish Creek in Michigan in June, 1972 (Smith, 1972). The bars were suspended inside 1-foot sections of 4-inch plastic pipe, and the pipes were staked below the surface of the stream. The FINTROL-BARS yielded a 12-ppb concentration of antimycin over an 8-hour period and largely eliminated the target white sucker, northern pike, bluegill, black crappie, and other species. Only one live fish-a white sucker-was found during electrofishing surveys 5 days later. Observers noted that fish made no attempt to avoid the bolt of antimycin moving downstream, and there was no damage to mayflies and crayfish in the stream. The Wisconsin Department of Natural Resources completed a DRAFT Environmental Impact Statement in June, 1972, regarding its intent to conduct a fishery rehabilitation of the Rock River in Dane, Dodge, Columbia, Fond du Lac, Green Lake, Jefferson, Rock, Washington, Walworth, and Waukesha counties in Wisconsin. The 4500square mile area lies in southeastern Wisconsin and encompasses the 2802 miles of streams and 100,400 acres of marshes that are designated for treatment with antimycin in some portions and with roten-

+

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one in others to eradicate the very abundant carp and other rough fishes. The waters at present have negligible sport fish and wildlife value, but once reclaimed, they will have enormous value. Unattractive turbidities will be reduced by elimination of the carp, and clear water will become attractive for general recreation. The total cost of this enormous undertaking will approach $5 million, but the benefits to sport fishing, waterfowl, other wildlife, and water-related public recreation in the populous southeastern part of the State are expected to exceed costs by far.

c. MARINEAND BRACKISHWATERS Cooperating with the Fish Control Laboratories, the National Marine Fishery Services Laboratory at Gulf Breeze, Florida, subjected antimycin to indoor and outdoor bioassays against selected saltwater fishes (Lowe, 1966; Berger et al., 1969). The results established that the piscicide is effective against marine fishes, and the concentrations needed are comparable to those employed against freshwater fishes. FINTROL-5 proved very effective in eradicating competitor fishes from shrimp-oyster culture ponds in Louisiana (Huner, 1968). The antimycin at 5 ppb had no adverse effects on the oysters, brown shrimp, and white shrimp under culture, nor on other invertebrates, such as oyster drill snails and glass shrimp. Finucane (1969) applied antimycin at 7 ppb in a marine impoundment in Florida and observed the results among fish and invertebrates. Fish sensitivity varied among families and species. Thirty-eight species were susceptible and several species were resistant to the concentration of toxicant tested. Lancelets were highly susceptible, blue crabs showed some sensitivity, but the wide variety of zooplankton and invertebrates present appeared to be unaffected by the piscicide. At p H 8.1 and 3O.l0C, the antimycin degraded in 5 days to an extent that the water was safe for sensitive fishes. Later, Finucane (1970) used 12 ppb of antimycin in the FINTROL-5 and FINTROL-15 formulations to reduce or eliminate predaceous fishes from a saltwater impoundment prior to the initiation of experiments in mass production of pompano. VIII. Applications a s a Selective Piscicide

A. LAKESAND PONDS

The greatest advantage of antimycin lies in its adaptability to selective control of target fishes. At this time when environmental concerns require that controls applied in pest situations be as selective as possible to the offending organisms, the selective potentials of antimycin

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in the FINTROL formulations loom importantly in fishery management (Ligler, 1969). Walker et d.(1964) documented the wide range of sensitivity to antimycin among freshwater fishes, and Berger and Hogan (1966) followed up by pointing out possibilities for selective removal of competing scale fishes from channel catfish ponds. Attempts to achieve selective control of problem fishes by use of antimycin followed rapidly in various parts of the country. Callaham and Huish (1968)evaluated antimycin as a selective toxicant for bluegill in small largemouth bass ponds in Georgia. Radonski (1967) demonstrated that troublesome yellow perch could be removed from a Wisconsin lake by a concentration of antimycin that was harmless to more desirable, resident fishes. Burress and Luhning (1969a,b) worked widely in the South to show that antimycin can be employed for eradication of competing scale fishes in channel catfish culture, and for selective thinning of sunfish populations in ponds. Pfeiffer and Ellis (1968) obtained a selective kill of gizzard shad with antimycin in a Kentucky lake. Stinauer (1968) found that 2 ppb of antimycin drastically reduced or eliminated gizzard shad, carp, bluegill, longear sunfish, and white crappie without serious harm to largemouth bass in strip mine ponds in Illinois. Avault and Radonski (1968) further defined uses of antimycin to remove trash fish from catfish ponds. Foye (1968) eradicated yellow perch and five cyprinids from a trout lake in Maine with a 0.5-ppb concentration of antimycin that caused only a partial kill of brook trout and redbelly dace and no kill of white sucker and burbot. Kafia (1969) observed that blue catfish in culture ponds fed heavily but without harm on scale fishes killed b y selective application of antimycin in Illinois ponds to produce selective kills of trash fish. H e concluded that the t6xicant is an excellent tool for fishery management. The above-mentioned literature deals with selective-kill concentrations of antimycin applied throughout a body of water. By virtue of its nonrepellency to fish, antimycin can be applied to a portion of a body of water to achieve control of target fish residing there. Thus, toxification of the entire body of water is avoided, efforts and costs are less, and potential impacts on the environment are reduced. In a Wisconsin lake, for example, 4 ppb of antimycin were applied in a bay that contained a large number of spawning carp, and an adjacent bay served as an untreated control (Gilderhus et aZ., 1969). Ninety-six hours later, great numbers of dead eggs and no fry were observed in the treated bay, but live eggs and fry were numerous in the untreated bay. An application of 50 ppb of antimycin against a spawning congregation of carp in the bay of an Iowa lake did not repel the fish, and many were killed (Lennon and Berger, 1970).

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The advantages of FINTROL-5 and FINTROL-15 in selective control were made evident in the elimination of a population of heavily parasitized minnows from a Minnesota trout lake (Lennon and Berger, 1970). The lake is 80 feet in depth and is managed for rainbow trout. By late summer in 1967, the lake was stratified in three, thermally distinct layers; the target minnows resided in the uppermost layer of warm water approximately 15 feet deep; the nontarget rainbow trout resided in the colder water of the thermocline, below 15feet in depth. FINTROL-5 was applied along the shoreline, and FINTROL-15 was distributed over deeper water. The antimycin released within the 15 feet of the warm, surface layer eliminated the diseased minnows; the toxicant was prevented from diffusing into deeper water by the temperature barrier between the epilimnion and thermocline; and rainbow trout residing in the thermocline largely escaped harm. The minor kill of trout observed was among specimens that moved into the epilimnion to feed. Schorfhaar and Frankenberger (1970) partially treated two, large and deep lakes in Michigan to reduce overpopulations of white sucker, rock bass, pumpkinseed, bluegill, and yellow perch and improve conditions for trouts, whitefish, smallmouth bass, and largemouth bass. In one lake, the shoreline band of water out to the 20-foot contour was treated with 1 ppb of antimycin in FINTROL-CONCENTRATE formulation; in the other lake, the toxicant was applied over the entire surface to provide a 1-ppb concentration down to 20 feet of depth; thus, only small fractions of the total water volumes received killing concentrations of the piscicides, but these fractions of the lakes contained most of the target fishes. Yellow perch had completed spawning in Ottawa Lake, and large areas of the bottom at 10-15 feet of depth were covered with egg masses. Following treatment, the eggs and fry were dead in all the masses checked. The kill of fish was calculated to be 40 lbs per acre in Ottawa Lake and 30 Ibs per acre in Golden Lake, and the dead consisted largely of target species. Nontarget game fish were not seriously affected. The smallmouth bass and largemouth bass were more resistant to antimycin in the colder, harder waters of Ottawa Lake than in the warmer, softer waters of Golden Lake. The authors considered the treatments successful and economical. Bonham (1972a-c; Smith, 1972b) discussed ways to reduce overabundant populations of bluegill in four Michigan lakes by means of partial treatments with antimycin. The results ranged from poor to excellent. The low concentrations of toxicant, the high p H of the water, and dilution of the narrow bands of treated water by wind-driven currents contributed to poor results in Murray Lake and Long Lake. At 0.5 ppb, antimycin in the uppermost, 5-foot layer of water in Cowden Lake,

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having a maximum depth of 50 feet, killed 35% of the target panfish, and the treatment was considered to be a moderate success. A concentration of 0.7 ppb of antimycin distributed throughout Pine Lake at pH 8.1 and 22.8"C achieved the desired 85-90% reduction of bluegill. Observers estimated that 10,900 lbs of bluegill and 1200 lbs of pumpkinseed were killed. There was no harm to the nontarget largemouth bass in any of the lakes. Many bass, in fact, continued spawning activities in shallow water during the course of antimycin treatments. Greater success may have been obtained if on-site bioassays were used on each lake prior to treatment. The treatment of a 100-acre reservoir in Missouri with 1 ppb of antimycin at p H 7.5-8.0 and 15.5"C caused a 94% reduction of the target bluegill, 97% reduction of target redear, and a desired but unspecified reduction of green sunfish (Goddard, 1972). The kill of nontarget largemouth bass amounted to 2.4 lbs per acre, and it consisted mostly of small fish that averaged 0.5 gm in weight. None of the nontarget channel catfish was killed. The project, designed to improve fishing for panfish and bass, was considered a success. Several of the authors cited above pointed out the unique advantages of antimycin for selective control of certain target fishes. On the basis of their experiences and recommendations, an increase in the use of antimycin for selective control of troublesome fish may be expected.

B. RIVERS AND

STREAMS

The selective removal of target fish from streams b y means of antimycin is largely untested. Stream reclamation efforts to date have been focused on removal or reduction of all fish within treated areas. Motivation for development of selective applications, however, is afforded b y the established practice of killing sea lamprey larvae in Great Lakes tributaries with lampricides that cause little or no harm to native fishes (Baldwin, 1968). In addition, MacPhee and Ruelle (1969) have developed Squoxin as a selective toxicant for squawfishes that prey heavily on young salmon in Pacific Coast streams. The advantage of treating massive but temporary aggregations of target fish with a nonrepelling piscicide was illustrated in Idaho (Anonymous, 1969). Nineteen ppb of antimycin in FINTROLCONCENTRATE formulation were metered for 2 hr into a portion of river where large numbers of carp had congregated. Approximately one-half million, 2-year old carp were killed in a 2-acre area. Only four trout were lost. Successive congregations of gizzard shad in the inlet to a channel catfish lake in Arkansas were annihilated by metering small quantities of liquid antimycin for a few minutes into the

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flowing water (Lennon and Berger, 1970). Killing three congregations within a period of 5 days sharply reduced the number of gizzard shad in the lake. There was no loss of channel catfish, and the selective treatments were considered to b e very effective and economical. Brynildson (1970) attempted to eliminate carp and other rough fish from a channel catfish stream in Wisconsin. The antimycin killed large numbers of carp, white sucker, and green sunfish in the stream without harm to the channel catfish. The turbidity in the water disappeared after removal of the carp. The benefits of the reclamation were expected to be short-lived, however, because some large backwaters were not treated and the carp in them escaped exposure to the piscicide. Opportunities for selective control of fish in streams will occur where there is a substantial difference between the sensitivities of target and nontarget fish to antimycin, or where spawning or temperature-oriented congregations of target fish occur in densities that displace or discourage the presence of nontarget fish. It is reasonable to expect that antimycin will become more widely used in streams because of its effectiveness on fish, nonrepellency, rapid degradation, and ease of detoxification with potassium permanganate. IX. Summary a n d Conclusions Antimycin is extremely and quite specifically toxic to fishes. Comprehensive bioassays of the antibiotic in the laboratory against a variety of freshwater and saltwater fishes in waters of various qualities and temperatures delineated the range of fish sensitivities and the relative safety to other aquatic animals. The most sensitive fishes are killed by antimycin in parts per trillion, whereas the least sensitive fishes succumb to the compound in parts per billion. This range of responses indicated possibilities that antimycin may be effective as a selective toxicant for certain target fishes. Following the laboratory studies, trials of antimycin were made in hard water and soft water, and warm water and cold water lakes and streams. The results demonstrate that antimycin is: effective in very small quantities against fish; irreversibly toxic to fish once a dose-threshold is reached; nonrepellent to fish; relatively harmless in fish-killing concentrations to other aquatic animals and to aquatic plants; nonpersistent in the aquatic environment; subject to natural degradation within hours to days in natural waters; and subject to detoxification by potassium permanganate. High p H and low temperature do affect the performance of antimycin against fish. The compound is less effective and degrades more

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rapidly at high pH than at low pH, and compensations must be made in accordance with the pH of receiving waters. Although the toxicity is greater at warm temperatures, the piscicide can be employed effectively and practically at near freezing temperatures and under ice cover. Because antimycin possesses most of the attributes desired or required in a piscicide, formulations were developed to suit use-patterns in lakes and streams. FINTROL-5, -15, and -30 are sand-based formulations and designed to release antimycin into the water within the first 5, 15, and 30 feet of water from the surface down, respectively. FINTROL-CONCENTRATE is a liquid formulation designed for injection into or spraying on lakes and streams. FINTROL-BAR is a solid formulation that is suspended in a stream to release its antimycin into the flowing water uniformly over a certain period of hours. Registrations have been obtained on some formulations in the United States and Canada, and petitions for registration of others are in progress. Antimycin has been used operationally as a general piscicide by fishery managers in the United States and Canada with good results, and the use is increasing as advantages of the compound become more widely known. The use of antimycin as a selective toxicant against certain target fishes, however, is growing even more rapidly. A body of water may be treated as a whole with a concentration of the piscicide that kills very sensitive target fish but leaves more resistant fishes unharmed. Or, only those portions of a lake or stream that harbor target fish may be treated with the nonrepelling piscicide. The latter is an advantage that is new and promising in fishery management. REFERENCES Ahmad, K., Schneider, H. G., and Strong, F. M. (1950). Arch. Biochem. 28, 281. Anonymous. (1969). Amer. Fish. U . S . Trout News 14, 18. Applegate, V. C., Howell, J. H., Moffett, J. W., Johnson, B. G . H., and Smith, M. A. (1961). G t . Lakes Fish. Comm., Tech. R e p . 1, 1. Avault, J. W., Jr., and Radonski, G . C. (1968). Proc. Southeast. Ass. Game Fish Comm. 21, 472. Baldwin, N. S. (1968). Limnos 1, 20. Bardach, J. E. (1964). “Downstream: A Natural History of the River.” Harper, New York. Bauer, 0. N. (1961). In “Parasitology of Fishes” (V. A. Dogie1 et al., eds.), pp. 320-334. Oliver & Boyd, Edinburgh. Berger, B. L. (1967). Proc. Southeast. Ass. Game Fish Comm. 19, 300. Berger, B. L., and Hogan, J . W. (1966). Bur. Sport Fish W i l d . R e s . Publ. 17, 75. Berger, B. L., Lennon, R. E., and Hogan, J. W. (1969). Invest. Fish Contr. 26,l.

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Birch, A. J., Cameron, D. W., Harada, Y., and Rickards, R. W. (1961). J. Chem. SOC., London p. 889. Birch, A. J., Cameron, D. W., Harada, Y., and Rickards, R. W. (1962). J. Chem. SOC., London p. 303. Bonham, M. D. (1972a). “Partial Chemical Treatment of Long Lake, Kent County, with Fintrol-5.” Michigan Department of Natural Resources, Fisheries Division, Grand Rapids, Michigan. Bonham, M. D. (1972b). “Partial Chemical Reclamation of Pine Lake, Kent County, with Fintrol Concentrate.” Michigan Department of Natural Resources, Fisheries Division, Grand Rapids, Michigan. Bonham, M. D. (1972~).“Partial Chemical Reclamation of Murray Lake, Kent County, with Fintrol-5.” Michigan Department of Natural Resources, Fisheries Division, Grand Rapids, Michigan. Brynildson, C. (1970). “Selective Chemical Fish Eradication of Mill Creek, Richland County,” Manage. Rep. No. 32. Wisconsin Department of Natural Resources, Bureau of Fish Management, Madison, Wisconsin. Burress, R. M. (1968). Bur. Sport Fish. Wildl. Res. Publ. 64, 116. Burress, R. M. (1970a). Bur. Sport Fish. WiEdl. Res. Publ. 88, 36. Burress, R. M. (1970b). Bur. Sport Fish. Wildl. Res. Publ. 106, 63. Burress, R. M. (1970~).Bur. Sport Fish. Wildl. Res. Publ. 106, 62. Burress, R. M., and Luhning, C. W. (1969a). Inuest. Fish Contr. 25, 1. Burress, R. M., and Luhning, C. W. (1969b). Invest. Fish Contr. 28, 1. Callaham, M. A. (1968). Ph.D. Thesis, University of Georgia, Athens. Callaham, M. A., and Huish, M. T. (1968). Proc. Southeast. Ass. Game Fish Comm. 21, 476. Callaham, M. A., and Huish, M. T. (1969). Proc. Southeast. Ass. Game Fish Comm. 22, 255. Camiener, G. W., Dietz, A., Argoudelis, A. D., Whitfield, G. B., DeVries, W. H., Large, C. M., and Smith, C. G. (1960). Antimicrob. Ag. Annu. p. 494. Chamberland, E. (1966). “Experimental Poisoning of Beauty Lake (cty Gatineau, P. Que.) with ‘FINTROL,”’ Proj. Rep. Bureau Biologique du Quebec enrg. Drummondville, Quebec. Derse, P. H., and Strong, F. M. (1963). Nature (London) 200, 600. Dickie, J. P., Loomans, M. E., Farley, T. M., and Strong, F. M. (1963).J. Med. Chem. 6, 424. Dunshee, B. R., Leben, C., Keitt, G. W., and Strong, F. M. (1949).J. Amer. Chem. SOC. 71, 2436. Emig, J . W. (1966). In “Inland Fisheries Management” (A. Calhoun, ed.), pp. 375-392. California Department of Fish and Game, Sacramento. Endo, T., and Yonehara, H. (1970). J. Antibiot. 23,91. Finucane, J. H. (1969). Trans. Amer. Fish. SOC. 98,288. Finucane, J. H. (1970). Amer. Fish Farmer 1, 5. Fontenele, 0. (1963). Commer. Fish. Rev. 25, 46. Foye, R. E. (1968). Progr. Fish Cult. 30, 216. Gilderhus, P. A. (1972). J . Fish. Res. Ed. Can. 29, 199. Gilderhus, P. A., Berger, B. L., and Lennon, R. E. (1969). Inuest. Fish Contr. 27, 1 Goddard, J. A. (1972). “Little Prairie Lake: Memorandum.” Missouri Department of Conservation, Jefferson City, Missouri. Hacker, V. A. (1969). “The Winter Chemical Treatment of Streams with Antimycin.”

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Wisconsin Department of Natural Resources, Oshkosh, Wisconsin. Hacker, V. A. (1971). Wis.Conseru. Bull. 36, 3. Harada, Y., and Tanaka, S. (1956).J . Antibiot., Ser. A 9, 113. Harada, Y., Uzu, K., and Asai, M. (1958).J . Antibiot., Ser. A 11, 32. Hooper, F. F., Williams, J. E., Patriarche, M. H., Kent, F., and Schneider, J . C. (1964). “Status of Lake and Stream Rehabilitation in the United States and Canada with Recommendations for Michigan Waters,” Rep. No. 1688, p. 1. Michigan Department of Conservation. Howland, R. M. (1969). Progr. Fish Cult. 31, 33. Huner, J. V. (1968). Proc. La. Acad. Sci. 31, 58. Hussain, A. (1969).J . Pharm. Sci. 58, 316. Idyll, C. P. (1969). Nut. Geogr. Mag. 135, 864. Kafia, J. (1969). Farm Pond Harvest 3, 7. Kannan, L. V., Kozova, J., and RehaEek, Z. (1968). Foliu Microbiol. (Prague) 13, 1. Karasawa, K., Tanaka, N., Yonehara, H., and Umezawa, H. (1959).J.Gen. Appl. Microbiol. 5, 13. Kinoshita, M., and Umezawa, S. (1969). Bull. Chem. Soc. Jap. 42,854. Kinoshita, M., and Umezawa, S. (1970). Bull. Chem. Soc. Jap. 43, 897. Kinoshita, M., Wada, M., and Umezawa, S. (1969).J . Antibiot. 22, 580. Kinoshita, M., Aburaki, S., and Umezawa, S. (1972). J . Antibiot. 25, 373. Kluepfel, D., Sehgal, S. N., and Vezina, C. (1970).J . Antibiot. 23, 75. Leben, C., and Keitt, G. W. (1948). Phytopathology 38, 899. Lee, T. H., Derse, P. H., and Morton, S. D. (1971). Trans. Amer. Fish. Soc. 100, 13. Lennon, R. E. (1966). Wis. Consero. Bull. 31, 4. Lennon, R. E. (1970). In “Principles in Plant and Animal Pest Control” (C. E. Palm, Chrm.), Vol. 5, pp. 6-41. National Academy of Sciences, Washington, D.C. Lennon, R. E. (1972). “The Fish Control Laboratories: Annual Report for 1971.” U.S. Fish Control Laboratory, La Crosse, Wisconsin. Lennon, R. E. (1973). “The Fish Control Laboratories: Annual Report for 1972.” U.S. Fish Control Laboratory, La Crosse, Wisconsin. Lennon, R. E., and Berger, B. L. (1970). Inuest. Fish Contr. 40, 1. Lennon, R. E., Berger, B. L., and Gilderhus, P. A. (1967). Progr. Fish Cult. 29, 110. Lennon, R. E., Hiinn, J. B., Schnick, R. A., and Burress, R. M. (1970). F A 0 Fish. Tech. Pup. 100, 1. Ligler, W. (1969). Farm Pond Haruest 3, 1. Liu, W.-C., and Strong, F. M. (1959).J . Amer. Chem. Soc. 81,4387. Lockwood, J. L., Leben, C., and Keitt, G. W. (1954). Phytopathology 14, 438. Loeb, H. A. (1964). N . Y. Fish Game J . 11, 160. Loeb, H. A., and Engstrom-Heg, R. (1970). N . Y. Fish News 32, 1. Lowe, J . I. (1966). “Quarterly Program Progress Report.” U.S. Bureau of Commercial Fisheries, Biological Laboratory, Gulf Breeze, Florida. MacPhee, C., and Ruelle, R. (1969). Trans. Amer. Fish. Soc. 98, 676. Marking, L. L. (1969). Progr. Fish Cult. 31, 139. Marking, L. L. (1970). Bur. Sport Fish. Wildl. Res. Publ. 88, 26. Marking, L. L., and Dawson, V. K. (1972). Trans. Amer. Fish. Soc. 101, 100. Montgomery, A. B. (1972). “Annual Project Report 1972, Fort Benning, Georgia.” US BSFW, Division of Fishery Services, Atlanta, Georgia. Neft, N., and Farley, T. M. (1971).Fed. Proc., Fed. Amer. Soc. E x p . B i d . 30,1190 (abstr.). Neft, N., and Farley, T. M. (1972a). Antimicrob. Ag. Chemother. 1, 274. Neft, N., and Farley, T. M. (1972b).J . Antibiot. 25, 298.

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Pfeiffer, P. W., and Ellis, B. F. (1968). “Annual Report, Division of Fisheries: Small Lake Studies.” Kentucky Department of Fish and Wildlife Resources, Division of Fisheries, Frankfort. Kentucky. Potter, V. R., and Reif, A. E. (1952).J . Biol. Chem. 194, 287. Powers, J. E., and Bowes, A. L. (1967). Trans. Amer. Fish. Soc. 96, 210. Powers, J. E., and Schneberger, E. (1967).Wis. Conseru. Bull. 32, 14. PrCvost, G. (1960). Can. Fish Cult. 28, 13. Rabe, F. W., and Wissmar, R. C. (1969). Progr. Fish Cult. 31, 163. Radonski, G. (1967). Wis. Conseru. Bull. 32, 15. RamanKutty, M., Kannan, L. V., and Rehicek, Z. (1969). Indian J . Biochem. 6,230. Rehifek, Z., and Svarc, S. (1968). Chem. Ind. (London) p. 1523. Rehifek, Z., Kannan, L. V., RamanKutty, M., and Puza, M. (1968a).Hindustan Antibiot. Bull. 10, 280. RehaEek, Z., RamanKutty, M., and Kozovi, J. (1968b). Appt. Microbiol. 16, 29. Riel, A. D. (1965). Progr. Fish Cult. 27, 37. Rieske, J. S. (1967). In “Antibiotics” (D. Gottlieb and P. Shaw, eds.), Vol. I, pp. 542584. Springer-Verlag, Berlin and New York. Rieske, J. S. (1973). (Personal Communication). Rieske, J. S., and Zaugg, W. S. (1962).Biochem. Biophys. Res. Commun. 8,421. Rivero, L. H. (1936). Trans. Amer. Fish. Soc. 66, 367. Sakagami, Y., Takeuchi, S., Sakai, H., and Takashima, M. (1956).J.Antibiot., Ser. A 9 , l . Sayre, R. (1969). “Powder River Rehabilitation - Use of Fintrol-5 for Fish Eradication.” Oregon State Game Commission, Fishery Division, Portland, Oregon. Schilling, G . , Berti, D., and Kluepfel, D. (1970).J . Antibiot. 23, 81. Schmidt-Kastner, G. (1963).Justus Liebigs Ann. Chem. 668, 122. Schorfhaar, R., and Frankenberger, L. (1970). “Costs and Methods Involved in Partial Chemical Eradication Projects Using Antimycin A,” Michigan Department of Conservation, Fisheries Division, Lansing, Michigan. Sigler, W. F. (1958). Utah, Agr. E x p . Sta., Bull. 405, 1. Singh, K., and Rakhit, S. (1971).J . Antibiot. 24, 704. Singh, K., Schilling, G., Rakhit, S . , and Vbzina, C. (1972).J . Antibiot. 25, 141. Slifer, G. E. (1970). “Stream Reclamation Techniques,” Manage. Rep. No. 33, p. 1. Wisconsin Department of Natural Resources, Madison, Wisconsin. Smith, D. W. (1972a).“Chemical Reclamation of Fish Creek, Montcalm County, Using Fintrol Bars (Antimycin).” Michigan Department of Natural Resources, Fisheries Diviqion. Gfiind Rapids, Michigan. Smith, D. W. (1972b). “Partial Chemical Reclamation of Cowden Lake, Montcalm County, with Antimycin.” Michigan Department of Natural Resources, Fisheries Division, Grand Rapids. Michigan. Stinauer, R. (1968). “Antimycin A Trials in Strip Mine Waters.” Illinois Department of Conservation, Havana, Illinois. Strong, F. M. (1956). In “Topics in Microbial Chemistry,” pp. 1-43. Wiley, New York. Strong, F. M., and Derse, P. H. (1964). U.S. Patent 3,152,953. Stroud, R. H., and Martin, R. G. (1968). “Fish Conservation Highlights 1963-1967.” Sport Fishing Institute, Washington, D.C. Tappel, A. L. (1960). Biochem. Pharmacol. 3, 289. Titcomb, J. W. (1914). Trans. Amer. Fish. S O C . 44, 20. Valentine, J. J. (1966). M.S. Thesis, University of Connecticut, Storrs. Van Duijn, C., Jr. (1962). In “Fish as Food” ( G . Borgstrom, ed.), Vol. 2, pp. 573-593. Academic Press, New York.

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Van Tamelen, E. E., Dickie, J. P., Loomans, M. E., Dewey, R. S., and Strong, F. M. (1961).J. Amer. Chem. Soc. 83, 1639. VBzina, C. (1967).Antimicrob. Ag. Chemother. p. 757. VBzina, C. (1971). Pure Appl. Chem. 28, 681. Walker, C. R. (1966). Bur. Sport Fish. W i l d . Res. Publ. 17, 79. Walker, C. R. (1967). Bur. Sport Fish. W i l d . Res. Publ. 39, 173. Walker, C. R., Lennon, R. E., and Berger, B. L. (1964). Inuest. Fish Contr. 2, 1. Watanabe, K., Tanaka, T., Fukuhara, K., Miyairi, N., Yonehara, H., and Umezawa, H. (1957).J. Antibiot., Ser. A 10, 39. Zilliox, R. G., and Pfeifler, M. (1956). N.Y. Fish GarneJ.3, 167.

Oc hratoxins KENNETH

L. APPLEGATE

AND

JOHNR. CHIPLEY

Department of Poultry Science, Ohio State University, Columbus, Ohio I. Introduction

.... ....................

.............. IV. V. VI. VII.

Factors Affecting Ochratoxin Production ........................ Structural and Biochemical Properties ........................... Extraction and Detection ............................................. Biological Effects ............. ...............

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97 97 98 99 100 102 105 107

I. Introduction Mycotoxicoses, the diseases caused by the consumption of any one of many fungal toxins (Bamburg et al., 1970), have been receiving increased attention in recent years. Specifically, diseases caused by ingestion of ochratoxins which are a group of acutely toxic and highly carcinogenic mold metabolites produced by strains of aspergilli and penicillia are currently being reported. The ochratoxins form a group of naturally occurring heterocyclic compounds that differ chemically through chloro and ethyl ester moieties and are frequently produced on foods and feedstuffs destined for human and animal consumption. The pathological potential of the ochratoxin compounds coupled with the ubiquity of the toxinproducing organisms heightens their significance when related to public health. The present article is not intended by the authors to be a comprehensive review of all research concerned with ochratoxins. Rather, it is an attempt to familiarize the reader with the more pertinent information regarding the chemical and physical properties of these compounds. II. Fungi Producing Ochratoxins Ochratoxin is a metabolite produced by species of Aspergillus (Scott, 1965; Van der Merwe et al., 1965; Nesheim, 1967; Lai et al., 1968; Hesseltine et al., 1972) and Penicillium (Townsend et al., 1966; Carlton et al., 1968; Van Walbeek et al., 1969; Carlton and Tuite 1969, 1970a; Scott et al., 1970a; Budiarso et al., 1971a). Hesseltine 97

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et al. (1972), using 44 representative strains of the nine species composing the A . ochraceus group, reported that ochratoxins A and B were found in some strains of A . sulphureus, A . sclerotiorum, A . alliaceus, A . melleus, A . ochraceus, A . ostianus, and A. petrakii. No ochratoxins were produced by A . elegans or any of the six strains of A. auricomus. Five of the 9 strains of A . sclerotiorum and 5 of 11strains of A . ochraceus were negative, while all 7 of the A . alliaceus strains tested were positive. The highest yields of ochratoxins A and B were obtained from A . ochraceus NRRL-3174 and NRRL-3519. Similar findings were reported by Scott (1965). He established the toxinproducing potential of 46 strains of 22 species, comprising Aspergillus, Fusarium, Paecilomyces, and Penicillium by the biosensitivity of ducklings, rats, and mice following ingestion of moldy grains. T h e presence of ochratoxin A and subsequent isolation of two strains of Penicillium viridicatum from red spring wheat were also reported b y Scott et al. (1970a). Lai et al. (1968) reported that among the 34 cultures of eight species within the Aspergillus ochraceus group, cultures of A . sulphureus and A . ochraceus and two cultures of A. melleus produced significant amounts of ochratoxin A when grown on soybeans, wheat, and liquid medium. Fourteen isolates of A. ochraceus were screened by Nesheim in 1967. He reported that of the 14 isolates, 4 produced ochratoxin A following inoculation on wheat, soybeans, and liquid medium. Toxicosis in mice and swine fed corn molded with various Penicillium species has been reported b y Carlton et at., 1968; Carlton and Tuite, 1969, 1970a,b. Van Walbeek et al. (1968) reported that an A . ochraceus isolated from brewery hops and a Penicillium sp. isolated from packaged ham produced both ochratoxin A and small amounts of aflatoxin. 111. Occurrence of Ochratoxins a n d Toxin-Producing Organisms Aspergilli and penicillia species are widely distributed in nature and have been isolated from soils, decaying vegetation, animal feeds, and from foodstuffs intended for human consumption (Van der Merwe et al., 1965; Shotwell et al., 1969a,b; Engelbrecht and Purchase, 1969; Scott et al., 1970a,b; Trenk et al., 1971). According to Campbell (1967), A. ochraceus is covered by United States Patent No. 1,313,209 for the organism’s ability to induce desirable flavor changes in coffee during fermentation of the beans. Van Walbeek et al. (1968) reported that of the 128 fungi isolated from 74 food samples obtained from household and retail stores located in Canada, 16 isolates were found to produce ochratoxins. Similarly, Scott et al. (1970a) reported the presence of ochratoxin A and isolation

OCHRATOXINS

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of P . viridicatum from red spring wheat which was destined to be feed for livestock. Christensen et al. (1967) isolated various strains of fungi from black and red pepper, among which were colonies of A. ochraceus which, when grown for 8-10 days on moist autoclaved corn, proved to be toxinogenic to white rats and 2-day-old Pekin ducklings. Japanese investigators (Yamazaki et al., 1970) have reported significant yields of ochratoxin from moldy rice contaminated with two strains of A. ochraceus. These fungi also produced large amounts of ochratoxin A when grown in nutrient solutions containing 1% L-phenylalanine and 2% yeast extract. Similarly, Trenk et al. (1971) observed the development of A. ochraceus on rice, corn, and wheat bran with subsequent productions of ochratoxins A and B. A. ochraceus has reportedly been isolated from moldy pecan meats (Doupnik and Bell, 1971). The A. ochraceus isolates were considered by Doupnik and Bell to be part of the 10 most prevalent fungal isolates from pecan meats screened for toxinogenicity according to chick sensitivity. IV.

Factors Affecting Ochratoxin Production

Schindler and Nesheim (1970) reported on moisture conditions that influence ochratoxin A production in shredded wheat. They reported that water levels of 40-70 ml per flask of 100 gm of wheat with an incubation period of 19-21 days at ambient temperatures would give maximum toxin production. Christensen (1962) demonstrated that wheat stored at between 20 and 25°C with a moisture content of from 15.0 to 15.5% is susceptible to invasion by A. ochraceus, while wheat with moisture levels of 16.0% and greater is highly susceptible to mold deterioration. H e further stated that a difference of less than 1% in moisture content of wheat greatly influences the rate at which A. ochraceus will invade wheat grains. Townsend et al. (1966) have reported that a Penicillium species produced significant levels of rubratoxin A and B when grown on a Modified Raulin Thom medium. Czapek-solutions having 1% corn steep and 3% glucose supplement also appear to be an excellent synthetic medium for the production of various toxogenic substances by the organism P . viridicatum (Budiarso et al., 1971a). Davis et al. (1969) reported that A. ochraceus NRRL-3174 produced 27 mg of ochratoxin A per 100 ml of “semisynthetic” medium, consisting of 4% sucrose and 2% yeast extract. The toxin was extracted from the cultures following 12 days of stationary incubation at 25°C. They also reported that trace amounts of ochratoxin B were pro-

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duced in a 1% yeast medium, while larger amounts of ochratoxin B would be obtained if 16% and 32% sucrose media were used. Average yields of 39-65 mg of ochratoxin A per liter of substrate were obtained from NRRL-3174 cultures developing in 4 liters of a 4% sucrose and 2% yeast extract nutrient solution after 7 days at 25°C (Davis et al., 1972). Attempts to produce ochratoxin in submerged cultures on conventional liquid media, such as those of Czapek, Raulin-Thom, and glucose-ammonium nitrate, were not successful (Brian et al., 1961). Ferreira (1967) reported the production of large quantities of ochratoxin A by A. ochraceus in 10-liter Marubishi fermenters. The carbon and nitrogen sources of choice were sucrose and glutamate, respectively. The maximum yield of ochratoxin A observed in shake-flasks and that found in 10-liter fermentations did not differ significantly and amounted to approximately 100 mg per liter of medium. Using the synthetic basal medium of Adyes and Mateles (1965), Lai et al. (1970) reported that trace elements are required by A. melleus and A. ochraceus, but not by A. sulphureus, for growth and ochratoxin A production. They further stated that the composition of the medium affected the synthesis of the toxin more than the growth of the mycelium. Trace elements of Fe3+,Zn2+,Cu2+,B3+, and Mo6+ were not needed for growth and ochratoxin production by A. sulphureus. They were required, however, in some combinations for development of A. melleus NRRL-3519 and A. ochraceus NRRL3174. A. sutphureus NRRL-4077 produced the maximum amount of ochratoxin A within 8-10 days from glucose and sucrose (200-210 mg of toxin/liter of medium), with mannose, galactose, and xylose producing 140, 135, and 70 mg of toxin per liter of substrate, respectively.

V.

Structural and Biochemical Properties

The molecular formula of ochratoxin A has been established by Van der Merwe et al. (1965) as 7-carboxy-5-chloro-8-hydroxy-3,4-dihydro3R-methylisocoumarin, linked from its 7-carboxy group to L-P-phenylalanine by an amide bond; ochratoxins B and C are the dechloro and ethyl ester derivatives, respectively, of ochratoxin A (Fig. 1). Synthesis of ochratoxin A was accomplished by Steyn and Holzapfel (1967) using an initial preparation of 7-carboxy-5-chloro-3,4-dihydro8-hydroxy-3-methylisocoumarin, which could then be linked from its 7-carboxy group to L-P-phenylalanine by coupling an acid azide to L-P-phenylalanine (Fig. 2). Steyn (1971) has recently published a review article concerning the in vitro and in vivo aspects of the ochratoxins as well as the synthesis of both ochratoxin A and B per se.

0

101

OCHRATOXINS

?OoH

CH,-CH-NH

-C

;w

c H3

C1

H

H

H

Ochratoxin A

Ochratoxin B

0 ll C-O-CH,-CH,

OH

CH,-CH-NH-C 0

CH3 Cl

n

Ochratoxin C

4-Hydroxymellein

FIG. 1. Structural f o r m u l a s of ochratoxins A, B, and C and 4 - h y d r o x y m e l l e i n .

Cole et ul. (1971)reported the extraction of the chemical compound, 4-hydroxymellein, from cultures of A. ochruceus (Fig. 1).Th’IS compound is similar to the dihydroisocoumarin moiety of the ochratoxins and it has been suggested by these authors that 4-hydroxymellein is a possible biosynthetic precursor of the ochratoxins per se.

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KENNETH L. APPLEGATE AND JOHN R. CHIPLEY

n

Chloroisocouniarin

/

1.-

Phenylalanine

/-

Ochratoxin A

FIG.2. Synthesis of ochratoxin A.

Searcy et al. (1969) have demonstrated b y use of phenylalanine-lI4C and sodium acetate-2-14C that phenylalanine is incorporated unaltered into the phenylalanine moiety of the ochratoxin A molecule, while the isocoumarin moiety is mostly derived via acetate condensation. Wei et al. (1971), using a synthetic basal medium similar to that of Ferreira (1967), except containing 0.75% N a 3 U , demonstrated that during the biosynthesis of ochratoxin A by A. ochraceus, 70% of the 36Clwas incorporated in the isocoumarin moiety. VI.

Extraction and Detection

Steyn and Van der Merwe (1966) recommended 12 hours of a Soxhlet-type extraction for toxic substances from media with chloroform-methanol (1: 1). Folhwing the Soxhlet extraction, the chloroform-methanol is diluted 50% with chloroform, extracted with 0.1 M aqueous sodium bicarbonate, and the ochratoxins were removed by further chloroform extractions following acidification of the chloroform-methanol solution with 2 M hydrochloric acid. The ochratoxin extracts are resuspended in chloroform after being previously airdried, spotted on thin-layer chromatographic (TLC) plates and developed in tanks containing benzene-acetic acid (3 : 1).According to these authors, the above methodology can be used to detect ochratoxin A in maize at levels of 0.1 ppm.

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A TLC screening procedure for the detection of 18 mycotoxins has been reported by Scott et at. (1970b). The procedure consists of spotting unknown samples, along with known standards, on TLC plates coated with Asorbosil 5 silica gel, developing the plates in a solvent system consisting of toluene-ethyl acetate-90% formic acid (6 :3 : 1) and benzene-methanol-acetic acid (34 : 2 : 1).The plates are viewed under ultraviolet (UV) light preceding and following heat activation and spraying of plates with P-anisaldehyde-methanol-glacial acetic acid-sulfuric acid (0.5:85 : 10 : 5 v/v/v). Eppley (1968)reported on a screening test for zearolenone, aflatoxin, and ochratoxin. The procedure incorporates a water-chloroform extraction with sequential elution of the mycotoxins from a silica gel column. Scott and Hand (1967) described a method for the detection and semiquantitative estimation of ochratoxin A in flour and other cereal products. The sample is extracted with aqueous-methanol and n-hexane and the toxin is partitioned on a Celite column. Ochratoxin A is separated from contaminating compounds by thin-layer chromatography. By their procedure, 0.01 p g of ochratoxin A can be detected in 10 pl of final sample extract, corresponding to a detection limit of about 25 pglkg of cereal foodstuff. Scott et al. (1971) also reported on a simple and rapid method for purifying ochratoxin A from extracts of P. viridicatum without the aid of TLC plates. The methodology requires absorption of chloroform extracts of the fungus onto chromatographic columns composed of silica gel impregnated with 5% oxalic acid dihydrate. Ochratoxin A is eluted from the columns b y chloroform-acetone (99 : 1 or 98 :2 ) washes. Further purification of the toxin is accomplished via extraction of its column fractions with 0.5 M sodium bicarbonate, then acidification of its aqueous phase, with reextraction of toxins with chloroform. Similarly, Stoloff et al. (1971) reported on a multimycotoxin screening method for aflatoxins, ochratoxins, zearalenone, sterigmatocystin, and patulin. The method is based on selective extraction of the sample with acetonitrile-water, defatting with isooctane, and removal of water-soluble components by transfer of the mycotoxins to chloroform. The individual toxins are separated by thin-layer chromatography. The method has been applied to corn, barley, oats, and wheat. Pitout (1969) has recommended a spectrophotometric method for the assay of ochratoxin A via carboxypeptidase A. The test requires that ochratoxin A be hydrolyzed by HC1 or carboxypeptidase A. Hydrolysis by either of these methods will result in a change in the absorption spectra from 380 nm to 330 nm. As a result of this shift, Pitout (1969) has stated that the spectrophotometric method of analy-

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KENNETH L. APPLEGATE AND JOHN R. CHIPLEY

sis of ochratoxin A is more sensitive than is the colorimetric ninhydrin method. The rates of hydrolysis of ochratoxin A and B in vitro by bovine carboxypeptidase A and enzymes extracted from rat liver, small intestine, and large intestine have been studied by Doster and Sinnhuber (1972).They reported the V,,, values for the hydrolysis of ochratoxin A and B by carboxypeptidase to be 5.15x lo-' and 4.35 x mole/liter per minute, respectively. Incubation of the extracts of rat tissue with equimolar amounts of ochratoxin A or B indicated that ochratoxin B was hydrolyzed 6-7 times faster than ochratoxin A. The inhibitory effects of ochratoxin A on in vitro bovine carboxypeptidase A has been reported by Pitout and Nel(l969).Their findings indicate that the inhibitory action of ochratoxin A resembles that of certain dipeptides. An inhibition constant of 14.2 mM was observed and a binding energy of ochratoxin A to the enzyme was estimated to b e 2.5 kcal per mole. They further suggested that the toxin has an influence both on carbohydrate and protein metabolism. Similarly, Chu and Butz (1970) reported a spectrophotofluorodensitometer method for measurement of ochratoxin A on TLC plates. The lower detection limits of this method were 0.5-1.0ng of toxin per spot. Their report also included a method for extraction and primary treatment of ochratoxin A from cereal products with a resultant sensitivity of from 10 to 50 pglkg of product. Chu (1970)also reported the fluorescence emission spectra of ochratoxin A and B on silica gel to be 340 nm and 325 nm, respectively, the emission maxima for both toxins being 475 nm. The investigation of excitation maxima and emission maxima for ochratoxins A, B, and C has been studied b y Trenk and Chu (1971).They reported that the excitation maxima of these three toxins shifted from 380 nm to a longer wavelength of 390 nm following exposure of these toxins to ammonia. The emission maxima of the toxins were also increased from 435 to 445 nm after similar ammonia treatments. Vorster (1969)reported on a rapid technique for screening of cereals and groundnuts for aflatoxins, ochratoxins, and sterigmatocystin. T h e procedure is based on the subjective evaluation of thin-layer chromatograms of the extract, with a replication accuracy of ~ 2 0 % . Nesheim (1969)used hot chloroform to extract ochratoxins from A. ochraceus cultures grown on moist shredded wheat breakfast cereals. Purification of the toxins following primary chloroform extractions are accomplished via aqueous bicarbonate. Ochratoxins A and B are reportedly separated by gradient silica-gel column chromatography using an acetic acid-benzene eluent; the fractions are analyzed by TLC densitometry with final purification of ochratoxins A and B by crystallization of the toxins from benzene and methanol, respectively.

OCHRATOXINS

VII.

105

Biological Effects

Broce et al. (1970) reported a 24-hour bioassay for ochratoxins A and B using Bacillus cereus mycoides LSU. Their calculated coefficient of variation was 5.9%, with a test sensitivity of 1.5 and 3.0 p g of ochratoxins A and B, respectively. Using short-term experiments with ducklings, Theron et al. (1966) reported that ochratoxin A caused a mild fatty infiltration of the liver when administered to the ducklings orally at doses of 100 pg. Weanling male rats given comparable doses of the toxin developed widespread hyaline degeneration of the liver cells with focal necrosis. According to Englebrecht and Purchase (1969), exposure of African green monkey kidney epithelial cells to ochratoxins resulted in enlarged nucleoli, a decrease in normal mitosis, and prophase and metaphase blocks after 24 hours of toxin exposure. The accumulation of glycogen in rat liver following ingestion of ochratoxin A has been studied by Pitout (1968). His investigations have indicated that interactions of ochratoxin with nucleic acids or their derivatives does not occur. However, it was demonstrated that the toxin does inhibit the phosphorylase enzyme system. No inhibitory effect of ochratoxin A on the enzymes glucomutase, glucose-6phosphate dehydrogenase, phosphorylase a or hexokinase were reported by Pitout; he did, however, postulate that the inhibitory effects that do occur might be due to competition of the toxin with 3’,5’-cyclic AMP for its enzyme phosphorylase b kinase. Chu (1971) has studied the interactions of ochratoxin A with serum albumin from bovine. Using spectrophotometric analysis, Chu reported that the absorption maximum of ochratoxin A (395 nm) shifts to a longer wavelength of 400 nm as a result of interactions of the ochratoxin A with various protein fractions of the bovine serum. Moore and Truelove (1970) reported that ochratoxin A and one of its breakdown products, dihydroisocoumarin, severely inhibited coupled respiration when applied at low concentration to rat liver mitochondria. Buckelew et al. (1972) have demonstrated that a number of toxic fungal metabolites which possess an alpha and beta unsaturated carbonyl system or which act as uncoupling agents of oxidative phosphorylation may be detected by the use of Bacillus megaterium spores. Toxicosis in mice fed corn molded with various Penicillium species has been reported by Carlton et al. (1968; Carlton and Tuite, 1970a). The pathological symptoms included hydropic degeneration, hypertrophy of hepatocytes, vaculation and cytoplasmic alterations of various body cells. The toxigenic properties of metabolites produced by Penicillium species grown on corn and fed to swine, guinea pigs, and

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rats have also been investigated by Carlton and Tuite (1969, 1970b). At necropsy, focal pale areas of necrosis, hepatic lesions, and bile duct hyperplasia were noted in test animals. Rats intubated daily with 500 pg of ochratoxin A, or fed daily diets of barley inoculated with Penicillium viridicatum and containing 250 p g of the toxin, developed anorexia and had similar pathological changes in the kidneys. It was further stated that a large part of the intubated toxin could not be accounted for from analysis of urine and feces, and little accumulation of the toxin was observed in the liver or kidneys (Van Walbeek et at.,

1971). Purchase and Van der Watt (1971) reported tumorigenic responses, using a long-term subcutaneous testing of rats with ochratoxin A, although they believed the tumorigenic responses to be in part the result of the carrier solvent, sunflowerseed oil. They further stated that administration of ochratoxin A orally to rats did not result in carcinogenic responses. Budiarso et al. (1971b) investigated the hepatorenal damage incurred in mice as a result of ingestion of P . viridicatumcontaminated rice. Their findings indicated that diets approaching an LD5o level were those diets containing 10 and 15% concentrations of the Penicillium-contaminated rice. Mice with ages varying from 2 through 18 weeks were studied. Budiarso et al. stated that ochratoxin resistance is not acquired with age since carcinogenic responses were manifested as hepatic and renal lesions in older mice after 8, 10, 14, and 18 weeks of growth. The sensitivity of mice to fungal mats and rice molded with isolates of Penicillium and Aspergillus has been investigated b y Carlton et al. (1972). Pathological signs appeared within 4 to 6 days and were exemplified b y renal and hepatic lesions. The histopathologic alterations were dominated b y a necrotizing cholangitis accompanied by dissimenated focal hepatic cell necrosis, with periductal edema and fibrosis. The toxicity of the hydrolyzate of ochratoxin A, isocoumarin carboxylic acid, was found to be much lower (LD50100 p g per egg) than that of ochratoxin A (LD50 16.96 p g per egg) as reported b y Yamazaki et al. (1971). Isolates of Aspergillus ochraceus obtained from peanuts (Doupnik and Peckham, 1970) were highly toxic to chicks, causing death of all birds during the first week of exposure. Postmortem examinations revealed small hemorrhages in the proventriculi of birds dying between days 2 and 5. Emaciation, dehydration, and dry gizzard linings were observed throughout the investigation. The principal microscopic findings were extensive hepatic injury consisting of either fatty changes or necrotic foci, along with suppression of bone marrow activity and depletion of lymphoid elements.

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The acute toxicity of ochratoxin A and C and dihydroisocoumarin to day-old New Hampshire Leghorn chicks has also been studied (Chu and Chang, 1971). The LD,, was reported to be 166 and 216 p g per assay for ochratoxin A and C, respectively, while no toxic effects were reportedly observed in chicks that had been fed upward of 500 p g of the substance dihydroisocoumarin. Peckham et al. (1971) reported that chicks given daily oral doses of 100 p g of ochratoxin A died after 2 days, and single subcutaneous doses of 400 p g of ochratoxin A were also lethal. The 7-day average oral lethal dose for chicks was estimated to be 1890 pg of ochratoxin A, while chicks given daily oral doses of 100 pg of ochratoxin B survived for 10 days. Sublethal doses of combined ochratoxins A and B resulted in growth suppression, visceral gout being the principal gross finding. Feeding of ochratoxins at concentrations of 1, 2, and 4 ppm to White Leghorn pullets from 14 weeks to 1 year of age resulted in delayed sexual maturity and reduced or terminated egg production (Choudhury et al., 1971). These authors further reported that feeding graded levels of ochratoxin to layers reduced egg hatchability and depressed for the first 2 weeks of life the performance of the progeny. Still et al. (1971) have suggested that ochratoxins are in part responsible for bovine abortion, fetal resorption, and deaths in many cases of domestic animals where specific causes are unknown. REFERENCES Adyes, J., and Mateles, R. I. (1965). Biochim. Biophys. Acta 86, 418. Bamburg, J. R., Strong, F. M., and Smalley, E. B. (1970).J. Agr. Food Chem. 17,443. Brian, P. W., Dawkins, A. W., Grove, J . F., Hemming, H. G., Lowe, D., and Norris, L. F. (1961).J. E x p . Bot. 12, 1. Broce, D., Grodner, R. M., Killebrew, R. L., and Bonner, F. L. (1970).1.Ass. Ofic. Anal. Chem. 53,616. Buckelew, A. R., Jr., Chakravarti, A., Burge, W. R., Thomas, V. M., Jr., and Ikawa, M. (1972).J. Agr. Food Chem. 20,431. Budiarso, I. T., Carlton, W. W., and Tuite, J. (1971a).Toxicol. Appl. Pharmacol. 20,194. Budiarso, I. T., Carlton, W. W., and Tuite, J. (1971b).Toxicol. Appl. Pharmacol. 20,357. Campbell, T. C. (1967). Va. J . Sci. 18, 67. Carlton, W. W., and Tuite, J. (1969). Toxicol.Appl. Pharmacol. 14, 636. Carlton, W. W., and Tuite, J. (1970a). Toxicol.Appl. Pharmacol. 17, 289. Carlton, W. W., and Tuite, J. (1970b). Toxicol. Appl. Pharmacol. 16, 345. Carlton, W. W., Tuite, J., and Mislivec, P. (1968). Toxicol. Appl. Pharmacol. 13, 372. Carlton, W. W., Tuite, J., and Caldwell, R. W. (1972).Toxicol. Appl. Pharmacol. 21,130. Choudhury, H., Carlson, C. W., and Semenuik, G. (1971). Poultry Sci. 50, 1855. Christensen, C. M. (1962). Cereal Chem. 39, 100. Christensen, C. M., Fanse, H. A., Nelson, G. H., Bates, F., and Mirocha, C. J. (1967). Appl. Microbiol. 15, 622. Chu, F. S. (1970).J. Ass. Ofic. Anal. Chem. 53,696.

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Chu, F. S. (1971).Arch. Biochem. Biophys. 147, 359. Chu, F. S., and Butz, M. E. (1970).J . Ass. Ofic. Anal. Chem. 53, 1253. Chu, F. S., and Chang, C. C. (1971).J. Ass. Ofic. Anal. Chem. 54, 1032. Cole, R. J., Moore, J. H., Davis, N. D., Kirksey, J. W., and Diener, U. L. (1971).J. Agr. Food Chem. 19,909. Davis, N. D., Searcy, J. W., and Diener, U. L. (1969). Appl. Microbiol. 17, 742. Davis, N. D., Sansing, G. A,, Ellenburg, T. V., and Diener, U. L. (1972). Appl. Microbiol. 23, 433. Doster, R. C., and Sinnhuber, R. 0. (1972). Food Cosmet. 10, 389. Doupnik, B., Jr., and Bell, D. K. (1971). Appl. Microbiol. 21, 1104. Doupnik, B., Jr., and Peckham, J. C. (1970). Appl. Microbiol. 19, 594. Engelbrecht, J. C., and Purchase, I. F. (1969). Afr. Med. J. 43,524. Eppley, R. M. (1968).J. Ass. Ofic. Anal. Chem. 51, 74. Ferreira, N. P. (1967). In “Biochemistry of Some Microbial Toxins” (J. R. Mateles, ed.), p. 157. MIT Press, Cambridge, Massachusetts. Hesseltine, C. W., Vandergraft, E. E., Fennell, D. I., Smith, M. L., and Shotwell, 0. L. (1972). Mycologia 64, 539. Lai, M., Semeniuk, G., and Hesseltine, C. W. (1968).J. Pathol. Bacteriol. 58, 1056. Lai, M., Semeniuk, G., and Hesseltine, C. W. (1970). Appl. Microbiol. 19, 542. Moore, J. H., and Truelove, B. (1970). Science 168, 1102. Nesheim, S. (1967).J. Ass. Ofic. Anal. Chem. 50, 370. Nesheim, S . (1969).J . Ass. Ofic. Anal. Chem. 52, 975. Peckham, J. C., Doupnik, B., Jr.. and Jones, 0. H., Jr. (1971). Appl. Microbiol. 21,492. Pitout, M. J. (1968). Toxicol. Appl. Pharmacol. 13, 299. Pitout, M. J. (1969). Biochem. Pharmacol. 18, 1829. Pitout, M. J., and Nel, W. (1969). Biochem. Pharmacol. 18, 1837. Purchase, I. F., and Van der Watt, J. J. (1971). Food Cosmet. Toxicol. 9, 681. Schindler, A. F., and Nesheim, S. (1970). J . Ass. Ofic. Anal. Chem. 53,89. Scott, D. B. (1965). Mycopathol. Mycol. Appl. 25, 213. Scott, P. M., and Hand, T. B. (1967).J . Ass. Ofic. Anal. Chem. 50, 366. Scott, P. M., Van WaIbeek, W., Harwig, J., and Fennell, D. I. (1970a). Can.J . Plant Sci. 50, 583. Scott, P. M., Lawrence, J. W., and Van Walbeek, W. (1970b). Appl. Microbiol. 20,839. Scott, P. M., Kennedy, B., and Van Walbeek, W. (1971). J. Ass. Ofic. Anal. Chem. 54, 1445. Searcy, J. W., Davis, N. D., and Diener, U. L. (1969). A p p l . Microbiol. 18, 622. Shotwell, 0. L., Hesseltine, C. W., and Goulden, M. L. (1969a). J . Ass. O@c. Anal. Chem. 52,82. Shotwell, 0. L., Hesseltine, C. W., and Goulden, M. L. (1969b). Appl. Microbiol. 17, 765. Steyn, P. S. (1971). In “Microbial Toxins” (S. Kadis, A. Ciegler, and S. J. Ajl, eds.), Vol. VI, p. 179. Academic Press, New York. Steyn, P. S., and Holzapfel, C. W. (1967). Tetrahedron 23, 4449. Steyn, P. S., and Van der Menve, K. J. (1966). Nature (London) 23, 418. Still, P. E., Macklin, A. W., Ribelin, W. E., and Smalley, E. B. (1971).Nature (London) 234,563. Stoloff, L., Nesheim, S., Yin, L., Rodricks, J. V., Stack, M., and Campbell, A. D. (1971). J. Ass. Ofic. Anal. Chern. 54,91. Theron, J. J., Van der Merwe, K. J., Leibenberg, N., Joubert, H. J. B., and Nel, W. (1966).J. Pathol. Bacteriol. 91,521.

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Trenk, H. L., and Chu, F. S. (1971).J. Ass. Ofic.Anal. Chem. 54, 1307. Trenk, H. L., Butz, M. E., and Chu, F. S . (1971).Appl. Microbiol. 21, 1032. Townsend, R. J., Ross, M. O., and Peck, H. M. (1966). J . Pharm. Pharmacol. 18,471. Van der Merwe, K. J., Steyn, P. S., and Fourie, L. (1965).J . Chem. SOC. (London)p. 7083. Van Walbeek, W., Scott, P. M., and Thatcher, F. S. (1968). Can.J . Microbiol. 14, 131. Van Walbeek, W., Scott, P. M., Hanvig, J., and Lawrence, J. W. (1969). Can. J. Microbiol. 15, 1281. Van Walbeek, W., Moodie, C. A., Scott, P. M., Harwit, J., and Grice, H. C. (1971). Toricol. Appl. Pharmacol. 20, 439. Vorster, L. J. (1969). Analyst 94, 136. Wei, R., Strong, F. M., and Smalley, E. B. (1971). Appl. Microbiol. 22, 276. Yamazaki, M., Maebayashi, Y., and Miyaki, K. (1970). Appl. Microbiol. 20,452. Yamazaki, M., Suzuki, S., Sakakibara, Y., and Miyaki, K. (1971). Jap.J . Med. Sci. Biol. 24, 245.

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Cultivation of Animal Cells i n Chemically Defined Media, A Review IYOSHI HIGUCHI Microbiological Associates, Znc. Bethesda, Maryland

I. Introduction ............................................................... 11. Preliminary Studies in the Development of ............... Chemically Defined Media. 111. Cultivation of Mammalian C Defined Media ............................................................ A. Propagation of Mouse Fibroblast Cells (Strain L) B. Cultivation of Cell Lines of Other Mammalian Species ................................................................. C. Cultivation of Cells in Suspension in Defined Media .................................................................. IV. Nutritional Factors Not Generally Recognized as Required by Mammalian Cells in A. Vitamins and Cofactors ........................................... B. Hormones. .................... C. Lipids .................................................................. D. Trace Metals ......................................................... E. Miscellaneous Nutrients and Other Substances ......... V. Growth Factors Associated with Serum Macromolecules .... ... VI. Concluding Remarks ................................................... References ..................................................................

I.

111 112 114 114 115 117 120 120 123 125 126 128 130 132 132

Introduction

This report is primarily a survey of work done in the development of chemically defined media for the cultivation of mammalian cells. However, much nutritional work undertaken in complex media is relevant to the present topic and therefore will comprise a major area covered in this review. Moreover, because of the enormous significance of the role of animal sera in most cell culture media, the results of various studies dealing with attempts to identify and isolate the active macromolecular constituents of serum will be examined. The information collected in this review is presented with the hope that it will contribute to the development of improved formulations of chemically defined media. It is evident from a survey of the literature that, although remarkable progress has been made, there remain serious deficiencies in currently available chemically defined cell culture media. The growth-supporting properties of all known chemically defined cell culture media can be substantially improved by additions of complex I11

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substances such as whole animal serum. To my knowledge, no chemically defined medium permits prolonged proliferation of primary and low-passage animal cell cultures. This deficiency is also seen in the failure of defined media to permit proliferation of cells of normal karyology such as the diploid human cell strain WI-38. It is therefore quite clear that much remains to be done to improve the formulation of chemically defined culture media. Waymouth’s earlier review (1965) on the present subject will undoubtedly be helpful to some readers because of its more detailed discussion of many aspects of the present subject. A somewhat more recent review by Swim (1967) also provides thoughtful comment on the current knowledge of the nutrition of cultured mammalian cells. II.

Preliminary Studies in the Development of Chemically Defined Media

Discussion of the history of the subject will necessarily be brief owing to space limitation; however, at least a short review covering earlier accomplishments is required in order that various aspects of animal cell culture in chemically defined medium can be placed in proper perspective. Probably the work of White (1946) entitled “Cultivation of Animal Tissues in Vitro in Nutrients of Known Constitution” should be recognized as the earliest detailed account of attempts to develop a defined culture medium. H e employed a nutrient mixture consisting of amino acids, vitamins, a carbohydrate, inorganic ions, and hormones, such as insulin and thyroxine. White also considered physicochemical factors, such as osmolarity, pH, and gas atmosphere, in his investigations. Skeletal cells from 8-day chick embryos were maintained in good condition for 58 days, and chick heart cells retained ability to beat for 44 days through repeated renewal of nutrients. In a later paper (1949), White described improvements in his medium that permitted chick heart cells to beat for as long as 80 days. He stated at that time that the goal of his research was not necessarily to obtain rapid cell proliferation, but to maintain tissue cells in healthy functional condition over long periods. This approach was not taken by most other workers whose efforts in the development of chemically defined medium will be reviewed. Most reports on the formulation of chemically defined culture media deal with the degree of rapid cell proliferation obtainable in the medium under consideration. Almost concurrent with the work described above, Morgan e t a2. (1950) described a defined medium designated as Mixture 199. The

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objective of the authors was to develop a chemically defined medium permitting cell multiplication in the absence of complex substances, such as serum, embryo extracts, and protein digests. Their medium contained some 68 defined compounds and supported survival of chick embryo tissues for periods averaging 4-6 weeks; however, no substantial proliferation of cells was obtained. It is pertinent to note that all of the above early workers employed cell cultures derived from primary explants. It was only later that it was recognized that primary cell types are the least likely candidates for continued proliferation in chemically defined medium. Thus, when investigators began to employ a strain of transformed continuously passaged mouse fibroblast cells (strain L), isolated originally b y Earle et al. (1943) and subsequently cloned by Sanford et al. (1948), that a new era opened in the cultivation of animal cells in chemically defined medium. Almost all the early successes in animal cell cultivation achieved in chemically defined media were obtained with this strain of mouse cell. Before proceeding to a review of defined media developed for the cultivation of various cell types, it is pertinent to describe at this point the contributions made by Eagle and his co-workers. Prior to their studies, most attempts at formulation of defined media were either based on preparation of mixtures containing a large variety of compounds that were conceivably useful to cell nutrition, or based on simulations of various biological fluids. Westfall and co-workers (1954a,b, 1955) made important contributions in the latter approach by quantitation of the amino acid and keto acid contents of ultrafiltrates of sera and embryo extracts. Eagle and co-workers, on the other hand, sought to establish the minimal essential nutritional requirements for growth of several species of mammalian cell lines. They showed that besides serum proteins, 13 amino acids (Eagle, 1955a,b; Eagle et al., 1955) 8 vitamins (Eagle, 1955c; Eagle et al., 1957), 6 inorganic ions (Eagle, 1956a), and glucose or some other carbohydrate (Eagle et al., 1958) were required for growth of cells. It must be remembered that dialyzed serum was an essential component of their basal medium; therefore, the above list of essential nutrients were designated as the minimal requirements for growth. The work of HaEand Swim (1957a,b) and Swim and Parker (1958a,b) on the amino acid and vitamin requirements of cells of both human and nonhuman species also contributed greatly to our knowledge of the basic nutritional requirements of mammalian cells in vitro. The above studies made with media containing dialyzed serum aided significantly in the development of truly chemically defined cell media.

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Ill.

Cultivation of Mammalian Cells in Chemically Defined Media

A. PROPAGATION OF MOUSE FIBROBLAST CELLS (STRAINL) Soon after the development of medium 199 b y Morgan et al. (1950), a modification of this medium containing 58 compounds was described by Healy et al. (1954) in which continuous proliferation of L cells was obtained in the absence of serum or any other undefined constituent. A 5- to &fold increase per week in cell population was obtained. A further modification by Healy et al. (1955) resulted in a medium containing 62 compounds that included a mixture of amino acids, vitamins (both water and fat soluble), inorganic salts, glucose, 5 deoxynucleosides, glucuronate, cocarboxylase, flavin adenine dinucleotide, and uridine triphosphate. This new defined medium yielded 9- to 10-fold increases of L cells per week. Another successful medium for the propagation of L cells was described by Evans e t al. (1956a,b). This medium, designated NCTC 109, contained 25 amino acids, 18 vitamins, 6 coenzymes, 5 nucleic acid precursors, 6 inorganic salts, Tween 80, ethanol, phenol red, glucose, acetate, glucuronate plus its lactone, and glutathione. Cell yields in this medium amounted to as high as a 13-fold increase of L cells per week. Defined media described so far have been notable for their complexity in containing substances such as coenzymes, fat-soluble vitamins, and a variety of nucleic acid precursors. Waymouth (1959), however, devised a much simpler medium designated MB 752/1 containing only 40 compounds. Rapid proliferation of L cells (7.5to 8.5-fold increases in 8 days) was reported. Waymouth’s work indicated that strain L cells were uniquely adaptable to cultivation in a rather simple medium. Further evidence that earlier formulations of defined culture medium for culture of L cells were unnecessarily complex was presented by Sanford et al. (1963), who showed that many of the constituents of NCTC 109, such as coenzymes, glutathione, ascorbic acid, certain nucleic acid precursors, glucuronate, Tween 80, and fat-soluble vitamins, were not required for growth of strain L mouse cells. Merchant and Hellman (1962) also showed that L cells can be grown (serum free) in Eagle’s minimal essential medium with doubled concentrations of vitamins and amino acids. Nagle and co-workers (1963) described a chemically defined medium of even simpler composition containing only 34 compounds that produced excellent growth of L cells. The growth rate in this medium was estimated to be as great as a 15-fold increase in cell numbers per week. These workers employed the suspension cell culture method

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in their studies. Their medium also supported growth of a number of other cell lines of diverse animal species; however, further discussion of this work will be presented in later sections of this review. It should be pointed out that all media described so far were employed under procedures requiring periodic renewals of medium during the growth cycle. This meant that 3-4 volumes of fresh medium were employed during a growth cycle for each culture. It would appear that certain constituents of the medium were limiting for continued growth of L cells in each of the above media. An alternative explanation for requirement for periodic renewal of medium would be an accumulation of toxic products. Higuchi (1970a) investigated the growth responses of L cells to graded levels of amino acids and other nutrients and devised a formulation that resulted in very high cell yields (> 5 x lo6 cells per milliliter) under conditions where no renewal of medium was necessary. When daily renewals of medium were made, cell yields approaching 30 x lo6 per milliliter were attained. This work was also conducted in a cell suspension system in vented shake flasks to provide adequate aeration. Birch and Pirt (1970) also described a defined medium for growth of mouse fibroblast cells (strain LS) in which yields averaging 3.3 x lo6 cells per milliliter were obtained without renewal of medium. It would appear from these results that the deficiencies in earlier culture procedures that led to requirements for frequent culture medium renewals were due to depletion of certain essential nutrients and the need to obtain adequate gas exchange. No serious accumulation of toxic metabolic products other than COz was evident in the L cell systems. Higuchi (1970a) reported, however, that production of ammonium ions in his medium approached near inhibitory levels.

B. CULTIVATION O F CELL LINESOF OTHER MAMMALIANSPECIES Following the successful cultivation of mouse fibroblast cell (strain L) in various defined media, intensive efforts were made to propagate cell lines derived from other animal species. Evans and co-workers (1959) succeeded in obtaining growth of a strain of monkey kidney cells (strain LLC-MK2) in defined medium NCTC 109. The first reported successful long-term cultivation of a human cell line in a chemically defined medium was obtained by Holmes (1959). H e cultured a line of human liver origin (Chang) in a medium similar to that of Healy et al., 1955 after approximately 120 days of “adaptation.” Bakken et al. (1961) also reported the cultivation of a strain of human cells in medium NCTC 109. Again, Evans et al. (1964) described successful propagation of cells of 4 mammalian species

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(mouse, rhesus monkey, Chinese hamster, and man) in medium NCTC 109 or its modifications. The human cell line of cancer origin (HEp-2) was also adapted to growth in medium NCTC 109 (Den Beste et al., 1966). It was characteristic of most of the above work that rather prolonged periods of “adaptation” to defined media were required. Serum concentrations were gradually reduced over periods of many months before adaptation occurred. Growth of monkey kidney cells in defined medium NCTC 109 (Evans et aZ., 1959), however, occurred without prolonged adaptation. Growth rates obtained for these cell cultures in defined media were rather low. Procedures for their cultivation describe a 2 for 1 split of cultures at weekly intervals, thus indicating a rather slow rate of growth when compared to the much higher rates of growth obtainable with mouse cells (strain L). In considerable contrast to the rather poor growth rates of cell lines other than strain L obtained in earlier defined culture media, Nagle et al. (1963) demonstrated good rates of growth of cultures derived from 6 mammalian species in their medium previously described in this review. Estimated rates of increase per week (by examination of their graphical data) were as follows: for L cells, 15-fold; for cat kidney cells, 15-fold; for HeLa cells, 10-fold; monkey kidney cells, 7-fold; rat spleen cells, 3.5-fold; and guinea pig kidney, 10-fold. It is probable that the presence of insulin in the medium was a significant factor responsible for the superior growth rates of a variety of cell lines. Blaker et al. (1971) also grew HeLa cells in suspension in a defined medium. The presence of insulin in their medium served to replace the growth-promoting activity of serum. HeLa cells attained populations of 1.6 to 2.4 x lo6 cells per milliliter in their defined medium. The work of Ham (1965) in which single cell platings of near-diploid Chinese hamster cells and mouse L cells were achieved in defined medium is of great interest. I n earlier work (Ham, 1962, 1963a,b, 1964), a variety of defined substances were shown to be stimulatory for clonal growth of mammalian cells in a basal medium containing two purified serum protein fractions. Ham was able to eventually replace the protein fractions with putrescine and linoleic acid (1965). More detail on this work will be presented in a later section. Quite recently, cultures of a human leukemic cell line (RPMI 4265) has been successfully propagated serum free in synthetic RPMI medium 1640 fortified with asparagine, serine, and 0.24% methyl cellulose (Buhl and Regan, 1972). Takaoka and Katsuta (1971) reported on the long-term cultivation of a number of mammalian cell strains in a simple chemically defined medium. Their medium contained no lipids, protein or any unusual

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substance. Of the 10 cell lines adapted to the defined medium, 7 were of rat origin, 1 from the cynomolgous monkey kidney, 1 from strain HeLa, and 1 from the mouse fibroblast strain L. Generally, long periods of adaptation involving selection of variants under condition of several month’s incubation without medium renewal were employed to obtain “adapted” cell cultures. The growth rates observed for their most rapidly growing cell lines as judged by data provided in the graphs, indicated approximately a 10-fold increase of cells per week. It was reported that only 10 of 20 cells lines tested could be “adapted” to growth in their defined medium. Higuchi and Robinson (1973)have recently described a chemically defined medium that includes among its constituents hormones (insulin, hydrocortisone, and thyroxine), lipids (oleate, lecithin, and cholesterol), and protamine sulfate, in addition to the other usual components (Table I). This medium permitted growth in monolayer culture of virtually all continuous cell lines tested. The list of cell lines grown (Table 11) included 7 cell lines of human origin, 3 cell lines of nonhuman primate origin, and 5 lines of rodent origin. Most of these cell lines required no prolonged periods of adaptation in order to grow in the defined medium. Many grew rapidly upon initial transfer from serum-containing growth medium. These results indicated that continuous cell lines of heteroploid karyology characteristically do not require complex unidentified serum proteins for unlimited proliferation. The recent work of Lasfargues et al. (1972) would also suggest a similar conclusion.

c.

CULTIVATION OF

CELLS IN SUSPENSION IN DEFINEDMEDIA

This topic deserves special treatment although there will be duplication of some material presented in earlier sections of this review. The advantages associated with suspension culture methods as contrasted to static cell cultivation are well known. It is pertinent, however, to note that a criterion for distinguishing cells of normal diploid types from transformed cells is the inability of normal cells to grow in suspension even in serum medium (Hayflick, 1967). Early attempts to propagate mammalian cells in serum-free media in suspension were usually disappointing. Cell fragility and accelerated diffusion of important intracellular metabolites may have prevented success in serum-free systems. In 1960, however, Kuchler et al. made the significant observation that methyl cellulose protected cells in agitated suspension culture in serum-free medium. Subsequently, Bryant et al. (1961) successfully cultivated the mouse fibroblast (strain L) in suspension in medium NCTC 109 fortified with methyl cellulose. Bakken et al. (1961) also were able to obtain growth

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TABLE I CULTURES“ CHEMICALLY DEFINEDMEDIUM FOR MONOLAYER

Component L-Amino acids Arginine.HC1 Asparagine.HZ0 Cysteine.HC1.H20 Glutamine Histidine.HC1.HtO Isoleucine Leucine Lysine.HC1 Methionine Phen ylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Vitamins Biotin Choline chloride Folic acid Inositol Nicotinamide Ca-panthothenate Pyridoxal .HCl Riboflavin Thiamine.HC1 Vitamin B 1 p

Concentration (mg/liter)

32 150 22 198 63 33 26 28 15 33 115

105 12 6 46 35

0.002 10.0 1 .0 2.0 0.1 0.2 0.05 0.10 0.20 0.002

Component Inorganic salts and buffers NaCl KCI NaH2P04.H20 NaHCO:, CaCl2.2H2O MgCle’6HZO FeNH4(S04)2.12H20 ZnS04.7H20 HEPES (buffer) Miscellaneous Glucose Na-pyruvate Gluconolactone Phenol red Ethanol Methyl cellulose (15 CPS) Protamine sulfate Oleic acid Lecithin Cholesterol Hydrocortisone L-Thyroxine EDTA Insulin (LENTE)

Concentration (mg/liter)

7400 400 100 1140 265 275 4.8 0.28

2980

1800 110 178 10 360 600 2.0 0.5 1.0 5x

M

M M M 0.064 unitslml

2.5 x

“This medium was designed for use without serum in static cultures of heteroploid cell lines in plastic culture flasks. From Higuchi and Robinson (1973) with permission of the Tissue Culture Association Inc.

in suspension of a line of human skin cells in medium NCTC 109 containing methyl cellulose. Bryant et al. (1961) also recommended that treatment of surfaces of glass culture vessels with silicone was beneficial for cultures in suspension. However, the growth rates obtained in both the above work were rather low. The L cells grown by Bryant et al. (1961) required at least 4.5 days per generation; and the human skin cells of Bakken et al. (1961) were subcultured at only a 1:2 ratio once per week. Merchant and Hellman (1962) also succeeded in culturing L cells in suspension in a defined medium containing

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TABLE I1 CELLLINESGROWNIN CHEMICALLY DEFINED MEDIUM'^ Yields * Cell lines Human origin HeLa (cervical carcinoma) KB (carcinoma of nasopharynx) HEp-2 (carcinoma of larynx) Chang’s liver MA 160 (prostate) Heart (Girardi) FL (amnion) Nonhuman primate origin Vero (African green monkey kidney) BSC-1 (Cercopithecus monkey kidney) LLC-MK, (Rhesus monkey kidney) Rodent species origin L929 (mouse fibroblast) L M (variant of L929) AKR (mouse embryo of AKR strain) LLC-RK, (rabbit kidney) BHK21 (baby hamster kidney)

( 106/ml)

1.7 1.1 1.4 0.7 1.4 0.9 1.5 0.9 1.1 0.9

1.3 1.8 0.8 0.6 0.8

“Data from Higuchi and Robinson (1973) with permission of the Tissue Culture Association Inc. ”The yields represent viable cell counts made usually after 1 week’s incubation after inoculation of less than lo5 cells per milliliter of medium (Table I) per flask. Cultures were refed 3-4 times during the growth cycle. The rabbit cell (LLC-RK1), however, grew very slowly and required almost 1 week for each cell doubling.

methyl cellulose. A cell doubling time of approximately 2.5 days was reported by these workers. In contrast to the rather low rates of growth described above, Nagle et at. (1963)obtained much improved rates of growth in their defined medium described earlier in this review. Cell lines derived from 6 mammalian species inoculated with 1 to 2 x lo5 cells per milliliter produced yields ranging from 2 to 3 x lo6 cells per milliliter after periods of incubation ranging from 7 to 11 days. Bryant (1966) reviewed the work done on cultivation of mammalian cells in suspension in defined media and discussed the limitations of the then available systems. T h e significant role of methyl cellulose in permitting propagation of mammalian cells as suspension cultures was emphasized. Higuchi (1970a) investigated the factors that limited yields and growth rates of L cells in suspension culture. His improved suspension medium produced yields in excess

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of 5 x 106 cells per milliliter and represented a 25-fold increase per week. More importantly, no renewal of medium was required during the entire growth cycle. The improvements consisted of a better balanced composition of amino acids and other nutrients, incorporation of iron and zinc ions, and a system for adequate aeration of suspension cultures. Birch and Pirt (1970) also reported attaining high yields of L cells in suspension in an improved defined medium. Their yields averaged 3.3 x lo6 cells per milliliter and were attained also without renewal of medium during growth. Blaker et al. (1971) described the successful propagation of HeLa cells in suspension in a modification of the defined medium previously described b y Birch and Pirt (1970) for culturing L cells. Yields as high as 2.35 x loficells per milliliter were obtained. Insulin (0.2 U ml) served to replace the serum supplement normally required by HeLa cells. Insulin was also employed by Nagle et al. (1963) in serum-free cultures of HeLa cells in suspension. Studies on the role of physicochemical factors on the growth of L cells in suspension in a defined medium were described recently (Taylor et al., 1971). The workers instrumented spinner culture vessels to monitor variables such as pH, p 0 2 , $ 0 2 , and oxidationreduction potential of the culture medium. An oxygen tension value corresponding to equilibrium with 9% oxygen in the gas phase was shown to be optimal. Other data obtained by these workers (G. W. Taylor, J. P. Kondig, S. C. Nagle, and K. Higuchi, unpublished) showed a relatively broad p H optimum (6.8-7.2) and an optimal pC02 value of between 2.5 and 5% CO, in the gas phase. Undoubtedly, better control of these physicochemical factors will permit improved growth and survival of many types of tissue cells in vitro. IV.

Nutritional Factors N o t G e n e r a l l y Recognized as Required by M a m m a l i a n Cells i n Vitro

This section will be devoted to a review of reports of growthstimulatory effects obtained with well defined substances that have not generally been recognized as required for optimal nutrition of mammalian cells in vitro. It is hoped that this information will be helpful in future design of improved formulations of chemically defined media. A.

VITAMINS AND COFACTORS

The nutritional activities of cofactor forms of the vitamins were examined by Eagle (1956b) and by Swim and Parker (1958a). They observed that the free vitamins were generally equivalent or even

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superior to the coenzyme forms except in the case of folic acid. Leucovorin (citrovorum factor, folinic acid) definitely was superior to folic acid. This was also shown by Fischer and Welch (1957), who reported that a mouse leukemia cell (strain L-5178) required unusually high levels of folic acid, whereas much less than one hundredth as much of citrovorum factor would permit good growth of cells. Neuman and Tytell (1960) observed similar results with the Walker carcinosarcoma cell. Ham (1962) also made the observation that folinic acid was superior to folic acid for clonal growth of the Chinese hamster diploid cell, whereas no difference was seen in tests with HeLa cells. Swim and Parker (1958a) noted that flavin adenine dinucleotide (FAD) was slightly but significantly more active than riboflavin in the proliferation of human uterine fibroblast cells (U-12-79). The above findings suggest that cells that are difficult to cultivate in defined medium containing only the free forms of the vitamins may be helped by the presence of more complex cofactors. Of course, this possibility had been considered b y many early workers in the design of chemically defined media (Morgan et al., 1950; Healy et al., 1954; Evans et al., 1956a). A review of the biochemistry of cultured mammalian cells made by Levintow and Eagle (1961) made no mention of requirements for vitamin BIZ or biotin. Subsequently, Sanford et al. (1963) reported evidence indicating a requirement for biotin by a substrain of L cells in a defined medium. Ham (1967) described experiments in which biotin (3X M ) was essential for successful cloning of a strain of hamster cells. Haggerty and Sat0 (1969) showed that in Waymouth’s medium MB 752/1, omission of biotin resulted in cessation of growth of L cells. Quantitative data on the amount of biotin required for growth of mouse fibroblast strain L and human cell strain HeLa were obtained by Higuchi (1969). Approximately 2 ng of biotin per milliliter was required in a chemically defined medium for optimal growth of both cell lines. Repeated passages in biotin-free medium were required to obtain cells that showed vitamin deficiency and failed to survive unless supplied biotin. A stimulatory effect of vitamin B12 on the growth of L cells was described by Evans et al. (1956a,b). Their medium, NCTC 109, contained an unusually high level (10 pglml) of vitamin Blz plus a variety of purine and pyrimidine nucleosides. Waymouth (1959) also reported that vitamin B12improved the growth of L cells in her defined medium MB 752/1. In subsequent studies made to firmly establish the requirement for vitamin Blz b y L cells, Sanford et al. (1963) and Sanford and Dupree (1964) came to the conclusion that although a variant substrain of L cells showed a requirement for B l z ,the parent

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KIYOSHI HIGUCHI

L ceIl did not require the vitamin. Waymouth (1965)cited earlier work on B12requirements and stated that vitamin BIZ was probably not an essential nutrient for cultured cells but that it was stimulatory for growth. However, Price et al. (1967) showed that a neoplastic C3H mouse cell strain required vitamin B12. No quantitative data were presented. Subsequently, work by Higuchi (1969) showed that vitamin B12was required not only by strain L mouse cells, but also by human cell (strain HeLa). Exceedingly low levels of BIZ (10100 pg/ml) were sufficient for maximal growth of both cell lines. The presence of purine and pyrimidine nucleosides did not replace the B l z requirement. Data from the work on the B1, response of HeLa cells are presented in Fig. 1. There have been several recent reports describing the relationship between vitamin BIZ requirement and purine and pyrimidine nucleosides in the culture medium. Price et al. (1970) and Rotherham et al. (1970) reported that BIZ is dispensable in the nutrition of C3H neoplastic mouse cells when nucleic

0

I

I

I

0.1

1.0

10

Id0

FIG. 1. Growth response of HeLa cells to vitamin B l r . Inoculum cells were grown in B12-free medium for several passages until vitamin BIZ deficiency was indicated by poorer growth as compared to control cultures. Media containing graded levels of vitamin Blz were inoculated with 25,000 cells per milliliter (10 /.~gof cellular protein). Cultures were refed 3 times during 8 days of incubation; then assayed for cell protein yields. From Higuchi and Robinson (1973) with permission of the Tissue Culture Association Inc.

ANIMAL CELLS IN CHEMICALLY DEFINED MEDIA

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acid precursors are present. Participation of folic acid in the above relationship was also described. Another very interesting report of Mangum and North (1968) described a procedure for producing a BIZrequirement in human HEp-2 cells by replacement of methionine with homocysteine in the growth medium. T h e amount of B 12 used to obtain growth in their system was 0.1 pglml; this is at least 1000-fold the level required by L and HeLa cells in a methionine-containing medium (Higuchi, 1969). Ascorbic acid has been employed in a number of formulations of defined media even though little or no direct evidence for its requirement had been established. For example, Mohberg and Johnson (1963) observed that ascorbic acid was extremely labile in incubated medium; moreover its omission from Waymouth’s medium MB 752/1 did not impair growth of L cells. However, the recent report by Park et al. (1971) described a specific requirement for ascorbic acid for colony formation by mouse plasmacytoma cells. Levene and Bates (1970) had also shown a role for ascorbic acid for growth and synthesis of collagen in the 3T6 strain of mouse cell cultures. B. HORMONES Lieberman and Ove (1959a) demonstrated the remarkable growthfactor activity of insulin in a serum-free culture system. Approximately 0.04 unit/ml was required to obtain growth of HeLa and appendix cells approaching growth obtained in a medium containing 10% serum. Higuchi (1963) and Tribble and Higuchi (1963) also showed significant growth-promoting effects of insulin in serum-free media. More recently, Higuchi and Robinson (1973) presented data showing that as little as 10 punits of insulin per milliliter produced enhancement of growth of the HEp-2 strain of human cancer cells. Data are illustrated in Fig. 2. Paul and Pearson (1960) concluded from their studies that the action of insulin on cell cultures could be generally attributed to its enhancement of cell glycolysis; however, current ideas on the role of insulin involve more complex functions for the hormone (Horowitz and Metzenberg, 1965; Gross, 1968; Illiano and Cuatrecasas, 1972). Recently Blaker et al. (1971)reported that insulin served to replace the serum requirement for the growth of HeLa cells in suspension culture. Nagle et al. (1963) had earlier employed insulin for the propagation of HeLa cells in a chemically defined medium. Glucocorticoid steroid hormones have been studied for effects on cell cultures by numerous workers. These substances were generally found to be inhibitory to cultured cells in vitro (Grossfeld and Ragan,

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KIYOSHI HIGUCHI

4Y

>

0

. .

0.01

0.10

1.o

10

100

INSULIN, M ILLIUNITS/ ML

FIG. 2. Growth response of HEp-2 cells to insulin (Lente, Eli Lilly & Co.). Media containing graded levels of insulin were inoculated with 70,000 cells per milliliter (20 pg of cell protein) and incubated at 36°C for 6 days. Media were renewed twice during the growth cycle prior to harvesting for assay of cell protein yields. From Higuchi and Robinson (1973) with permission of The Tissue Culture Association Inc.

1954; Holden and Adams, 1957; Kline et al., 1957; Wellings and Moon, 1961; Pihl and Eker, 1965; Ruhmann and Berliner, 1965). However, there is the report by Arpels et al. (1964) that hydrocortisone prompted longer viability in cell cultures. Berliner and Ruhmann (1966) also reported enhancement of growth of cultured cells by certain of the steroids. Higuchi and Robinson (1973) found that hydrocortisone (lo+ M ) was beneficial for cultivation of certain cell lines in a chemically defined medium. The reported growth stimulatory effects of the glycocorticoid steroids may be related to the ability of these compounds to induce enzyme production in a variety of cultured cells. The following enzymes have been induced in vitro by steroids: alkaline phosphatase (Cox and McLeod, 1961; Griffin and Cox, 1966; Griffin and Ber, 1969); invertase (Hijmans and McCarty,

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1966); tyrosine transaminase (Pitot et al., 1964; Thompson et al., 1966; Gerschenson et al., 1970); and glutamine synthetase (Barnes et al., 1971). White (1946) included L-thyroxine in his early chemically defined medium, but reported no significant effects of this hormone. Lieberman and Ove (1959a) incorporated triiodo-L-thyronine (1.5 x M) in their medium but described its effect as small and inconsistent. However, Siegel and Tobias (1966) and Bartfeld and Siegel (1968) reported significant enhancement of growth of cells in vitro by Lthyroxine at concentrations ranging from lop7 to lop6 M . Higuchi and Robinson (1973) also reported L-thyroxine (2.5 X low8M ) to promote growth of mammalian cells in their chemically defined medium. A recent report by Froelich and Rachmeler (1972) on the effects of cyclic adenosine 3’,5’-monophosphate (CAMP) on cultured human diploid cells strongly indicated that this substance was involved in the phenomenon of contact inhibition of growth. Because of the postulated role of CAMP as an intracellular mediator of multiple hormones (Sutherland et al., 1968), the activity of this substance in cells cultured in vitro is of special interest to those concerned with the study of hormonal action. Klein and Makman (1971) have shown that the protein kinases isolated from 3 cell lines (Chang’s human liver, HeLa, and 3T6 mouse embryo cells) were stimulated 2- to 3-fold by M CAMP.

C. LIPIDS Sat0 et al. (1957) reported that growth of isolated mammalian cells (HeLa) required cholesterol as a nutrient in a medium containing highly dialyzed serum. On the other hand, Holmes and co-workers (1969) found that in a medium containing serum freed of lipoproteins, cholesterol was not required by heteroploid cell lines such as HeLa, KB, and Holmes’ human lung cells, but cholesterol (1pg/ml) was required by primary diploid human fibroblast cells. Higuchi (1970b) reported that a continuous cell line of porcine kidney origin required cholesterol for growth in a chemically defined medium. Data are presented in Fig. 3. Fatty acids have been demonstrated to be essential nutrients for certain cultured cells. Ham (1963b) showed that linoleic acid was required for clonal growth of mammalian cells when albumin was omitted from his basal medium. Dubin et al. (1965) found linoleic acid to promote growth of chick embryo macrophages. Further evidence for the important role of fatty acids in cultured cells was provided by Harary and co-workers (1967). Normal mitochondria1 function in heart and HeLa cells required linoleate and arachidonate.

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FIG.3. Growth response of a porcine kidney cell line to cholesterol. Media containing graded levels of cholesterol were inoculated with 62,000 cells per milliliter (approximately 20 p g of cellular protein) and incubated for 1 week. Cultures were refed twice prior to harvesting for assay of celI protein yields. From Higuchi (unpublished data).

HeLa cells responded to 2.5 x M linoleate b y enhanced growth. Palmitate maintained beating of heart cells. Jenkin and Anderson (1970) found that oleic acid (10-20 pg/ml) in a serum-free medium containing 0.2% bovine albumin replaced calf serum for growth of the LLC-MKs strain of monkey kidney cells. Further studies b y Jenkin et al. (1970) revealed that among various isomers of cisoctadecenoic acids tested, only those acids with the double bond in the central region were stimulatory for growth. Higuchi and Robinson (1973) found that a combination of lipids (cholesterol, oleic acid, and lecithin) was required for growth of human heart cells (Girardi) and the BSC-1 strain of monkey kidney cells in a defined medium. All three compounds were required for optimal growth of cells. An excellent reference material concerning the role of lipids in cultured mammalian cells is the Wistar Institute Symposium edited by Rothblatt and Kritchevsky (1967).

D. TRACEMETALS The major inorganic ions required for growth of mammalian cells in vitro have been known for some time. Shooter and Gey (1952) showed that calcium, magnesium, and potassium ions were required for outgrowth of rat tissue cells. Eagle (1956a) showed the salt requirements of cultured mammalian cells to be satisfied by Na+, K+,

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CaZ+,Mg2+, C1-, and phosphate. Wyatt (1961), after ion-exchange treatment of serum and use of specially purified inorganic salts, did not report any additional requirements than those reported by Eagle. However, an additional inorganic ion, bicarbonate (or CO,) was shown to be essential for animal cells in vitro by Geyer and Chang (1958). They employed rigorous methods to remove or reduce metabolic and ambient COZ in order to demonstrate the requirement. Swim and Parker ( 1 9 5 8 ~also ) found that CO, was essential for six cell lines. Further studies by Geyer and Neimark (1958), Gwatkin and Siminovitch (1960), and Chang et al. (1961) showed that COz served as a precursor for synthesis of oxaloacetate and nucleic acid intermediates; and that COz could be substituted by a combination of oxaloacetate plus purine and pyrimidine nucleosides. Recently, Birch and Pirt (1971) reported on the mineral requirements of mouse L cells grown in suspension in a defined medium. They showed that cultures of strain L fibroblasts yielded 2.4 x lo5 cells per microgram of K, 2.2 X lo5 cells per microgram of P, and almost lo6 cells per microgram of Mg. No requirement for zinc was observed. Omission of Ca'+ in their medium resulted in poor reproducibility of growth. Unpublished data of the reviewer (K. Higuchi), however, showed that Ca'+ was essential for growth of L cells in a defined medium. Almost a linear response in cell yields was observed if cell numbers were plotted against the logarithm of calcium concentration in the range between 10+ and M calcium. For example, cell yields were 2.5 x lo6, 3.5 x lo6,4.4X loG,and 5.3 x lo6 cells per milliliter for calcium concentrations of and M respectively. Only limited knowledge of the trace metal requirements of mammalian cells has been available. Neuman and Tytell (1961b) showed that iron replaced the lactalysate requirement for clonal growth of Walker carcinosarcoma cells. Ham (1962) reported that iron was a requirement for clonal growth of Chinese hamster ovary cells, but not for HeLa cells. He also reported that copper M ) and zinc M ) in combination with lipoic acid (Ham, 1963a) frequently improved the growth of the hamster ovary cells. Theoretical considerations alone dictate that trace elements would be required by mammalian cells; however, substantial contamination in even socalled chemically defined media has delayed progress in the field. Thomas and Johnson (1967) demonstrated absolute requirements for both iron (0.6-1.4 p M ) and zinc (0.6 p M ) for growth of L cells in a chemically defined medium. Treatment of Waymouth's medium MB 752/1 with a chelating resin (Dowex A-1) resulted in a metaldeficient medium. Addition of manganese (0.1 p M ) was toxic, but the

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toxicity was reversible by increasing the iron concentration. Copper (0.4 p M ) was beneficial in iron-deficient medium because it tended to reduce the iron requirement. Higuchi (1970a) also showed that both iron (4 p M ) and zinc (1p M ) were essential for optimal growth of mouse fibroblast cells in a defined medium. No special treatment of the medium was required to demonstrate the cation requirements because of the exceptionally high cell yields (5 X 106/ml)obtained without renewal of the culture medium during the entire growth cycle. E. MISCELLANEOUSNUTRIENTSAND OTHER SUBSTANCES The so-called nonessential amino acids are alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine. These have been described as dispensable for most mammalian cells cultured in oitm. However, a number of cell types has been shown to require one or more of these amino acids for growth. Other cell lines were found to require these amino acids under certain culture conditions. For example, in a Bs-deficient medium 6 “nonessential” amino acids were required for growth of strain U-12-79 human fibroblasts (Swim and Parker, 1958a). Lockart and Eagle (1959) found that serine was essential for clonal growth of strain HeLa. Serine was required by a strain of rabbit fibroblasts ( H a 8 and Swim, 1957a). Serine was also required by a substrain of L cells (Higuchi, 1970a; Nagle and Brown, 1971). Asparagine was essential for growth of Walker carcinosarcoma 256 cell (Neuman and McCoy, 1956), the Jensen sarcoma cell (McCoy et al., 1959), and the mouse leukemia cell (Haley et al., 1961). Proline and serine were needed for optimal clonal growth of the CHD-3 cell and human cell lines (Ham, 1963a). In the glutaminefree medium of Nagle and Brown (1971), alanine was shown to be required for optimal growth of L cells. Keto acids have long been shown to enhance growth of animal cells in culture. Neuman and McCoy (1958) showed growth-promoting activities of pyruvate, oxaloacetate, and a-ketoglutarate for isolated Walker carcinosarcoma cells. Pyruvate was stimulatory for growth of chick embryo cells (Neuman and Tytell, 1961a). Neuman and Tytell (1958) found in studies with the Walker carcinosarcoma 256 cell that certain purines such as hypoxanthine and related compounds (inosine, adenine, etc.) significantly improved clonal growth of cell cultures. Guanine and its derivatives and pyrimidines did not produce growth stimulation. Fisher et al. (1959) showed that hypoxanthine promoted clonal growth of HeLa Ss cells. Hakala and Taylor (1959) showed that the folic acid requirement of several cell lines were replaceable by a combination of glycine (but not serine), a purine, and

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thymidine. Ham (1962) reported that both thymidine and hypoxanthine were required for optimal clonal growth of diploid Chinese hamster cells. Cells cultured from humans afflicted with a deficiency in hypoxanthine-guanine phosphoribosyltransferase activity (LeschNyhan syndrome) showed a requirement for purine for growth (Felix and Demars, 1969). Ham (1964) showed that putrescine or a related diamine was required for clonal growth of a strain of Chinese hamster cells in a medium containing serum albumin but no fetuin. Putrescine, spermidine, and spermine were all effective at M concentration. More recently Pohjanpelto and Raina (1972) identified putrescine as a growth factor for a human fibroblast culture. They found that only a very narrow range of putrescine concentration of approximately 2.5 X l o + M was effective; therefore it was suggested that there was a potential regulatory role for putrescine in cellular growth. In this connection, the work of Alarcon et al. (1961) and Alarcon (1964) indicating that putrescine was the growth regulatory substance in normal striated muscle tissue are of considerable interest. A recent book by Cohen (1971) provides a comprehensive treatment of the role of polyamines in a wide variety of prokaryotic and eukaryotic cells and suggests an important role for these substances in the regulation of RNA synthesis. Lieberman and Ove (1958a) found that salmine sulfate (protamine) promoted attachment of cells to glass in a serum-free medium, but it was not effective in promoting cell growth. However, Neuman and Tytell (1961a) showed salmine to significantly enhance growth of chick embryo cells. Higuchi (1963) also found that protamine sulfate promoted growth of HeLa cells in a serum-free lactalbumin hydrolyzate medium. More recently, Higuchi and Robinson (1973) found that protamine sulfate (2 pglml) promoted growth of two cell lines in a chemically defined serum-free medium. Its mode of action, other than enhancement of cell attachment, is unknown. Amos (1961) had described enhancement by protamine of RNA uptake by chick embryo cells. The highly charged polycationic nature of protamine may permit interaction with cell membrane as well as with other anionic substances in the medium. Catalase has been shown to be a requirement for survival of mammalian cells when small numbers of cells were present (Lieberman and Ove, 195813). Lipoic acid was reported to be beneficial for clonal growth of the CHD-3 strain of Chinese hamster cells (Ham, 1963a). A variety of nonprotein polymers have been tested in attempts to replace the essential macromolecular factors provided by serum. Among the substances tested, methyl cellulose has been extremely

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useful especially in suspension cultures in chemically defined medium (Kuchler et al., 1960; Bryant, 1966; Buhl and Regan, 1972). Katsuta and co-workers (1959) found that alginic acid, dextran, and polyvinylpyrrolidone (70,000 MW) were increasingly effective, in that order, in substituting for serum protein requirement of rat ascites cell cultures. Healy and Parker (196613) found that dextran (100,000200,000 MW) substituted for one of the a-globulin factors for growth of mouse embryo cells. Birch and Pirt (1970) compared the effectiveness of methyl cellulose and polyvinylpyrrolidone for use in suspension culture of L cells and reported that methyl cellulose yielded superior results.

V.

Growth Factors Associated with Serum Macromolecules

The many drawbacks associated with use of animal serum in tissue culture have been discussed recently (Fedoroff et al., 1971). However, no entirely satisfactory substitute for serum is available. There is hope, however, that work on the isolation of active proteins from serum will eventually permit the development of a serum-free medium that will be equal to serum-containing media. Moreover, if the nutritional and other functional properties of isolated serum macromolecules are elucidated, it may be possible to replace proteins with synthetic substitutes. Sanford et al. (1955) prepared four fractions from serum by alcohol precipitation, and found growth-stimulatory activity for L cells in all fractions. Lieberman and Ove (1957, 1958a) isolated a serum glycoprotein that promoted growth of two human cell lines (Appendix and HeLa). Serum albumin was required in addition to the glycoprotein factor to obtain cell growth. Fisher et al. (1958) also described a protein factor which they identified as fetuin, a glycoprotein present in high concentration in fetal sera. Subsequently, Fisher et al. (1959) reported that two purified serum proteins, fetuin and albumin, replaced dialyzed calf serum for clonal growth of HeLa S, cells. Lieberman and Ove (1959b) on the other hand, presented evidence that the glycoprotein factor isolated b y them was not fetuin. Holmes and Wolfe (1961) employed curtain electrophoresis to separate several bovine serum fractions that stimulated growth of human cell lines in a defined medium. An albumin fraction and an a-globulin fraction were active. Marr et al. (1962) also fractionated fetal calf serum and isolated two active components, fetuin and another glycoprotein (amacroglobulin), with growth activity for HeLa cells. Ham (1962) employed a serum-free medium containing fetuin and serum albumin to obtain clonal growth of diploid Chinese hamster cells. In sub-

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sequent work, Ham (1963a,b, 1964, 1965) showed that the two serum protein fractions could be replaced by a combination of linoleic acid and putrescine. Michl (1964) and Michl and Svobodova (1965, 1966) also reported on the growth-promoting activity of an a-globulin fraction of serum. Holmes (1967) isolated a purified a-protein fraction from human serum that differed from fetuin and permitted immediate growth of cell lines that normally required months to adapt to serumfree medium. The recent work of Jainchill and Todaro (1970) suggesting that there is a factor in the y-globulin fraction of serum essential for growth of 3T3 mouse cells complicates our current knowledge of the nature of serum growth factors. Another very interesting work is that of Holley and Kiernan (1968) in which a substance purified 2900-fold from human urine replaced a serum growth factor. This work may have great implication in future studies on the role of serum in cell nutrition in vitro. Tozer and Pirt (1964) employed electrophoresis and gel filtration to fractionate macromolecular constituents of calf serum. They concluded that a fraction probably composed of both a- and pmacroglobulin components possessed growth-factor activity for mouse fibroblasts (strain L929). Their work showed the presence not only of growth-stimulatory factors, but also of dialyzable inhibitory substances in serum. DeLuca et al. (1966) also found that certain preparations obtained from plasma and serum were cytotoxic. Otsuka (1972) described a growth inhibitor for BHK21 cells present in calf serum. The complexity of the problem of serum factors affecting cells in uitm was recently emphasized by Paul et al. (1971), who presented evidence that normal rat serum contained four different factors essential for growth and survival of 3T3 mouse fibroblasts and SV40 virustransformed mouse cells. Eagle (1960) showed that the growth-stimulatory activity of serum proteins was made dialyzable b y digestion with Viokase. H e suggested that the primary role of serum proteins in cell nutrition is to provide low molecular weight nutrients that are initially bound to proteins. DeLuca et al. (1966) also made proteolytic digest of serum proteins and showed that a fraction of approximate molecular weight of 5000 was stimulatory for growth of a Syrian hamster carcinoma cell, but not for the mouse cell (strain L929). Pierson and Temin (1972) recently described the partial purification of a small protein factor (4000-5000 MW) from calf serum that was stimulatory for growth of chick embryo cells. The substance was not insulin and possessed antitryptic activity. Schaer and Schindler (1967) also prepared proteolytic digests of serum albumin that were stimulatory for growth in cells in uitro.

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Healy and Parker (1966a,b), unlike many earlier workers, employed low-passage mouse embryo cells to assess the growth-stimulatory factors in serum. They found that two fractions, an a-glycoprotein and an a-macroglobulin, were stimulatory for growth of mouse embryo cells in an improved chemically defined basal medium. The activity of the a-macroglobulin could be substituted for b y dextran of 100,000200,000 molecular weight. The use of low-passage mouse embryo cells in these studies is to be commended, particularly in view of the demonstrated ability of most heteroploid cells to grow in media free of serum protein factors. Shodell and Rubin (1970) examined the temporal sequence of cellular activities upon stimulation of growth of chick embryo cells by serum. Their results indicated that the effects of serum were initially manifested during the G I phase of the cell cycle. VI.

Concluding Remarks

The present review of the literature on the development of chemically defined media for culturing animal cells has pointed out the following: (1) Heteroploid cell types are generally capable of continuous growth in some form of chemically defined medium. (2) Normal diploid cells are incapable of continued proliferation in any chemically defined medium currently available. (3) These are undefined factors present in serum that are essential for growth of normal diploid cells. (4)Serum growth factors represent multiple substances, some of which may be replaceable by synthetic polymers such as methyl cellulose, polyvinylpyrrolidone, and dextran; or by low molecular weight substances. Current knowledge of mammalian cell nutrition does not appear to be adequate to permit formulation of a medium that allows cultivation of normal diploid cells in a truly chemically defined medium. Future workers in the development of chemically defined media must therefore seek to elucidate the role of the serum factor(s) essential for growth of normal diploid cells. Availability of a suitable substitute for serum would be extremely valuable, both scientifically and in applied medicine. Consequently, more intensive research in this area is urgently needed. REFERENCES Alarcon, R. A. (1964). Arch. Biochem. Biophys. 106, 240-242. Alarcon, R. A., Foley, C . E., and Modest, E. J. (1961). Arch. Biochem. Biophys. 94, 540-541. Amos, H. (1961). Biochem. Biophys. Res. Commun. 5 , 1-4.

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Katsuta, H., Takaoka, T., Hosaka, S., Hibino, M., Otsuki, I., Hattori, K., Suzuki, S., and Mitamura, K. (1959).Jap.J . E x p . Med. 29, 45-70. Klein, M. I., and Makman, M. H. (1971). Science 172, 863-864. Kline, I., Leighton, J., Belkin, M., and Orr, H. C. (1957). Cancer Res. 17, 780-784. Kuchler, R. J., Marlowe, M. L., and Merchant, D. J. (1960).Exp. Cell Res. 20,428-437. Lasfargues, E. Y., Coutinho, W. G., and Lasfargues, J. C. (1972). In Vitro 7, 264-265 (abstr.). Levene, C. I., and Bates, C. J. (1970). J . Cell Sci. 7, 671-682. Levintow, L., and Eagle, H. (1961).Annu. Rev. Biochem. 30, 605-640. Lieberman, I., and Ove, P. (1957). Biochim. Biophys. Acta. 25, 449-450. Lieberman, I., and Ove, P. (1958a). J. Biol. Chem. 233,637-642. Lieberman, I., and Ove, P. (1958b).J . E x p . Med. 108,631-637. Lieberman, I., and Ove, P. (1959a). J . Biol. Chem. 234, 2754-2758. Lieberman, I., and Ove, P. (195913). Science 129,43-44. Lockart, R. Z., and Eagle, H. (1959). Science 129, 252-254. McCoy, T. A., Maxwell, M., and Kruse, P. F. (1959). Cancer Res. 19,591-595. Mangum, J. H., and North, J. A. (1968). Biochem. Biophys. Res. Commun. 32,105-110. Marr, A. G. M., Owen, J. A,, and Wilson, G. S. (1962). Biochim. Biophys. Actu 63,276285. Merchant, D. J., and Hellman, K. B. (1962). Proc. Soc. E x p . Biol. Med. 110, 194-198. Michl, J. (1964). Nature (London) 202, 1133-1134. Michl, J., and Svobodova, J. (1965). Biochim. Biophys. Acta 97, 168-169. Michl, J., and Svobodova, J. (1966). E x p . Cell Res. 42, 205-206. Mohberg, J., and Johnson, M. J. (1963).J. Nut. Cancer Inst. 31, 603-610. Morgan, J. F., Morton, H. J., and Parker, R. C. (1950).PTOC.Soc. E x p . Biol. Med. 73,l-8. Nagle, S. C., and Brown, B. L. (1971). J . Cell Physiol. 77, 259-264. Nagle, S. C., Tribble, H. R., Anderson, R. E., and Gary, N. D. (1963). PTOC.SOC. E x p . Biol. Med. 112, 340-344. Neuman, R. E., and McCoy, T. A. (1956). Science 124, 124-125. Neuman, R. E., and McCoy, T. A. (1958). Proc. SOC. E x p . B i d . Med. 98, 303-306. Neuman, R. E., and Tytell, A. A. (1958). Exp. Cell Res. 15, 637-639. Neuman, R. E., and Tytell, A. A. (1960). Proc. S O C . E x p . B i d . Med. 103, 763-767. Neuman, R. E., and Tytell, A. A. (1961b). Proc. Soc. E x p . Biol. Med. 107, 876-880. Neuman, R. E., and Tytell, A. A. (1961a). Proc. Soc. E r p . Biol. Med. 106, 857-862. Otsuka, H. (1972). J . Cell Sci. 10, 137-152. Park, C. H., Bergsagel, D. E., and McCulloch, E. A. (1971). Science 174, 720-722. Paul, D., Lipton, A., and Klinger, I. (1971). Proc. Nat. Acad. Sci. U.S. 68, 645-648. Paul, J., and Pearson, E. S. (1960).J . Endocrinol. 21, 287-294. Pierson, R. W., and Temin, H. (1972).J . Cell Physiol. 79, 319-330. Pihl, A., and Eker, P. (1965). Biochem. Pharmacol. 14, 1065-1072. Pitot, H. C., Peraino, C., Morse, P. A,, and Potter, V. R. (1964). Nut. Cancer Inst., Monogr. 13, 229-242. Pohjanpelto, P., and Raina, A. (1972). Nature (London), New Biol. 235, 247-249. Price, F. M., Rotherham, J., and Evans, V. J. (1967).J. Nat. Cancer Inst. 39, 529-538. Price, F. M., Rotherham, J., and Evans, V. J . (1970). J. Nut. Cancer Inst. 44, 85-90. Rothblatt, G. H., and Kritchevsky, D., eds. (1967). “Lipid Metabolism in Tissue Culture Cells,” Wistar Inst. Symp. Monogr. No. 6. Wistar Inst. Anat. B i d , Philadelphia, Pennsylvania. Rotherham, J., Price, F. M., and Evans, V. J . (1970). J . N a t . Cancer Inst. 44, 91-97. Ruhmann, A. G., and Berliner, D. L. (1965). Endocrinology 76,916-927.

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Sanford, K. K., and Dupree, L. T. (1964).Ann. N.Y. Acad. Sci. 112, 823-830. Sanford, K. K., Earle, W. R., and Likely, G. D. (1948).J . Nut. Cancer Inst. 9, 229-246. Sanford, K. K., Westfall, B. B., Fioramonti, M. C., McQuilkin, W. T., Bryant, J . C., Peppers, E. V., Evans, V. J., and Earle, W. R. (1955).J.Nut. Cancerlnst. 16,789-802. Sanford, K. K., Vupree, L. T., and Covalesky, A. B. (1963).Exp. Cell Res. 31,345-375. Sato, G., Fisher, H. W., and Puck, T. T. (1957).Science 126, 961-964. Schaer, J. C., and Schindler, R‘. (1967).Biochim. Biophys. Acta 147, 154-161. Shodell, M., and Rubin, H. (1970). In Vitro 6, 66-74. Shooter, R. A,, and Gey, G . 0. (1952).Brit. J . E x p . Pathol. 33, 98-103. Siege], E., and Tobias, C. A. (1966).Nature (London) 212, 1318-1321. Sutherland, E. W., Robison, G. A., and Butcher, R. W. (1968). Circulation 37,279-306. Swim, H. E. (1967). Lipid Metab. Tissue Cult. Cells, Symp., 1966 Wistar Inst. Syrnp., Monogr. No. 6, pp. 1-14. Swim, H. E., and Parker, R. F. (1958a).Arch. Biochem. Biophys. 78,46-53. Swim, H. E., and Parker, R. F. (195813).Can.]. Biochem. Physiol. 36,861-868. Swim, H. E., and Parker, R. F. (1958~). J. Biochem. Biophys. Cytol. 4, 525-528. Takaoka, T., and Katsuta, H. (1971).E x p . Cell Res. 67, 295-304. Taylor, G. W., Kondig, J. P., Nagle, S. C., and Higuchi, K. (1971).Appl. Microbiol. 21, 928-933. Thomas, J. A,, and Johnson, M. J. (1967).J . Nat. Cancer Inst. 39, 337-345. Thompson, E. B., Tomkins, G . M., and Curran, G. F. (1966).Proc. Nat. Acad. Sci. U.S. 56, 296-303. Tribble, H. R., and Higuchi, K. (1963).J.Znfec. Dis. 112,221-225. Tozer, B. T., and Pirt, S. J. (1964).Nature (London)201,375-378. Waymouth, C . (1959).J.Nut. Cancer Inst. 22,1003-1016. Waymouth, C. (1965). In “Cells and Tissues in Culture” (E. N. Wilmer, ed.), Vol. 1, pp. 99-141. Academic Press, New York. Wellings, S. R., and Moon, H. D. (1961).Lab. Invest. 10, 539-547. Westfall, B. B., Peppers, E. V., Sanford, K. K., and Earle, W. R. (1954a).J. Nat. Cancer Inst. 15, 27-35. Westfall, B. B., Peppers, E. V., and Earle, W. R. (1954b).J . Nat. Cancer Inst. 15, 433438. Westfall, B. B., Peppers, E. V., and Earle, W. R. (1955).J . Nat. Cancer Inst. 16, 337347. White, P. R. (1946).Growth 10, 231-289. White, P. R. (1949).J . Cell. Comp. Physiol. 34, 221-241. Wyatt, H. V. (1961).E x p . Cell Res. 23,97-107.

Genetic and Phenetic Classification of Bacteria

R. R. COLWELL' Department of Biology, Georgetown University, Washington, D . C .

I. Introduction ...... 11. Numerical Taxonomy in Bacterial Systematics ................ A. Micrococcus/Staphylococcus .................... B. Ruminococcus ......... ..... ............... C. Streptococcus .............. . D. Lactobacillus ........................................ E. Corynebacterium ................................. ... F. Propionibacterizcm ........ ............... G . Brevibacterium ....... ....................................... H. Pseudomonas ............... I. Thiobacillus ........................................ J. Arthrobacter ........................................ K. RhizobiumlChromobacteriumlAgrobacterium L. HalobacteriumlHalococcus ............ ............... M. Aeroinonas ............................................. N. Enterobacteria ............. .. P. Vibrio ..............................

s. Bacteroides ........................................ .... T. Nocasdia .......... U. Mycobacterium ......................................... Productivity ............... 111. Microbial EcologyIV. Summary ..................................................... ............... References ........................ ..

137 139 139 143 144 146 148 149 150 151 153 154 155 157 157 158 159 160 161 163 164 164 165 167 168 169

I. Introduction

Each year since 1960 the number of papers appearing in the literature on the theory and application of those multivariate biometric analyses known as numerical taxonomy (NT) in microbiology has doubled. Since the publications of Sneath (1957a,b), more than 250 papers have appeared on NT of bacteria, in addition to those dealing with viruses, yeasts, and fungi. An abbreviated review of the NT of bacteria is provided, with treatment also of results of molecular taxonomic studies. The methods and theory of molecular taxonomy, especially measurement of deoxyribonucleic acid (DNA) base com'Present address: Department of Microbiology, University of Maryland, College Park, Maryland 20742.

137

138

R. R. COLWELL

position and nucleic acid hybridization, have been reviewed competently elsewhere (Gillespie and Spiegelman, 1965; Brenner et al., 1969; De Ley, 1969, 1970; Mandel et al., 1970; Brenner and Falkow, 1971; Staley and Colwell, 1973). The results of molecular taxonomic studies provide valuable information and are largely correlative with NT analyses. Some shortcomings of taxonomy in microbiology should be mentioned. Tests are not uniformly applied to all genera of bacteria. For reasons not always understood, tests applied to one genus may not even be considered in another taxonomic group. Good discriminatory tests are not available for all genera, and tests considered to be “classical” for some genera are of no value and should not be used at all. Some tests do not test what they purport to test, or are subject to unacceptably large experimental error. Direct comparison of data from one laboratory to another is not now done, although it is hardly a difficult task for the computer. Since interest and activity have been directed toward establishing national and international microbiological data repositories (Rogosa et al., 1971), standardized testing, coding, and computer data storage methods are needed. Methods for testing vary widely, as evidenced by the volume of literature abstracted by Skerman (1970). Twenty or thirty variations of a single method exist, with the result that taxonomic conclusions vary considerably. New methods for determining characters are also needed. Some effort has been made to update and revise older test methods and to introduce new characters (De Ley, 1962; Hill et al., 1965; Stanier et al., 1966; Schaefler and Malamy, 1969; Schaefler et al., 1969). Regulatory enzymes (Cohen et al., 1969; Feist and Hegeman, 1969) and comparative allostery (Jensen, 1970; Jensen and Stenmark, 1970) are good sources of reliable taxonomic characters. Thus, a limitation of taxonomy of the bacteria lies in the methods for testing and coding taxonomic characters; there is no functional limit in multivariate computational procedures and software. With the methods available, only a fraction of the bacterial genome is measured, either phenetically (De Ley, 1962, 1969) or b y molecular genetic criteria (Brenner, 1970; Brenner and Falkow, 1971). Relatedness among bacterial strains, i.e., nucleic acid binding, offers useful taxonomic information but carries interpretive problems. Divergence in specific genetic or physical regions of the bacterial chromosome, conservation of ribosomal RNA genes and transfer RNA genes, difference in genome size, and the complications introduced into assessment of genetic relatedness by extrachromosomal elements make interpretations of molecular genetic data for taxonomy or phylogeny

GENETIC AND PHENETIC CLASSIFICATION OF BACTERIA

139

very difficult. An effective approach to microbial taxonomy at present appears to be polyphasic taxonomy, which employs data from all levels, molecular to ecological (Colwell, 1968). Construction of a natural taxonomy (Heslop-Harrison, 1962) for the bacteria may not be achieved otherwise. Despite limitations, remarkable success has been obtained in applying numerical taxonomic methods to the bacteria. Molecular genetic data have reinforced taxonomic conclusions drawn from numerical analysis. The following sections cite contributions of NT to bacterial systematics. II. Numerical Taxonomy i n Bacterial Systematics

A. MicrococcuslS taphylococcus In one of the first NT studies of bacteria, Hill (1959) analyzed data for staphylococcus strains provisionally classified according to the scheme of Shaw et al. (1951), including Staphylococcus aureus, S. saprophyticus, S. lactis, S. roseus, and S. afermentans. Two main clusters resulting from the analysis were regarded as separate genera. Three groups sorted out: (a) S. aureus; (b) S. saprophyticus (the group commonly referred to as S. epidermidis or the coagulase-negative S. albus group); and (c) S. roseus. Groups (a) and (b) were found to be more closely related to each other than to group (c). S. roseus was redefined as Micrococcus roseus, and S. lactis and S. afermentans were not found to comprise natural groupings (Hill, 1959; Heslop-Harrison, 1962). In 1960, Pohja published on micrococci isolated from fermented meat. Most of the meat micrococci were identified as S. lactis (Shaw et al., 1951). Three distinct subtypes of S. lactis were recognized, and the relationship of S. lactis to S. saprophyticus was found to be somewhat diffuse. Pohja and Gyllenberg (1962) extended the study of micrococci of fermented meat origin, expanding the treatment of the data to include other multivariate analytic methods (Sneath, 1957a,b). Halophilic cocci were found to be distinctly separable from other strains, but, not being a single homogeneous cluster, probably represent more than one species. Nonhaltolerant types of S. lactis were also heterogeneous. A classification of Staphylococcus spp. by means of principal component analysis was proposed by Hill et al. (1965). Good agreement between deoxyribonucleic acid (DNA) base composition and NT of the gram-positive cocci was observed by several workers (Rovira, 1965; Silvestri and Hill, 1965; Rosypal et al., 1966; BohiEek et al., 1967; BohaEek and Kocur, 1970; Raj and Colwell, 1971) (see Table I). DNA data for S. aureus suggest it to be more

TABLE I OVERALL BASE COMPOSITION, EXPRESSED AS PERCENTGUANINE+CYTOSINE (G C ) , OF DEOXYRIBONUCLEIC ACID (DNA) OF BACTERIAL GENERAAND SPECIES"

+

% G+C

Organism

27-34 29-30 29-35 30-39 32-35 31-37 32-37 3 1-43 30-40 33 32-38

Fusobacterium/Sphaerophorus Sarcina (anaerobic) Campylobacter Staphylococcus S . uureus S. saprophyticus S . lactis S porosarcinu C ytophagalSporocytophagu Flavobacterium aquatile Lactobacillus L. jugerti L. helveticus L. salivarius L. bulgaricus L. acidophilus Strep tococcus S . faeculis var. zymogenes S. faecalis var. Ziquefaciens

r\

32-38

33-42 34-38

i

S. sanguis s. uberis S . salivarius S. viridans S . sanguis S . cremoris S. duruns S . innominatus S . bovis S . faecium

34-4 1

i

38-40

I

1

38-42 40 41 35-4 1 38-42 35-40 38 41-51 39-47 39-4 1 41-43 41-47 41-47 (41-43, 44-45, 46-47)* 43-52

S.pyogenes Pediococcus Aerococcus Pasteurella multocida Listeriu monocytogenes BacteroideslRistella1/Eggerthella Acinetobac ter A. calco-aceticus A . unitratus A. citroalcaligenes A . lwofii Lactobacillus L. buchneri L. viridescens L. brevis

t

43-48 43-47 140

TABLE I (Continued) ~

~~

~

%

Organism

L. plantarum L. casei L. lactis L. leichmannii L. delbrueckii L. fermenti L.cellobiosus , Vibrio V. parahaemolyticus V. cholerae Pseudomonas piscicida Pasteuretta (Yersinia) Saprospira grandis S pirillurn Escherichia Erwinia E . aroideae E. carotouora E. atroseptica Fergusonia shigelloides Plesiomonas Serratia Aeroinonas A. punctata A. formicans Alcaligenes faecalis Bijido bac terium Corynebacterium C. michiganensis C. fasciens Thiobacillus T. thio-oridans T. concretiuorus T. neapolitanus T. ferro-oxidans T. thioparus T. thiocyano-oridans T . denitrijicans T. noveltus T. trautweinii Planococcus (marine) Micrococcus (halophilic) M . mucitaginosus M . morrhuae Pseudomonas P . bathycetes P.fluorescens P. aeruginosa

c+c

44-46 46-47

1

49-52

40-50 44-46 46-48 45 46-48 46-48 49-6 1 51

1

51-53 51-53 52 56-58 57-63 57 59 59 57-64 47-60 54 55 51-68

t t

5 1-52 56-57

62-68

1

39-5 1 56-61 56-59 57-61 57-66 57 61 64 14 1

142

R. R. COLWELL

TABLE I (Continued) Organism

% G+C

Xanthomonas Arthrobac ter A . globiformis Rhizobium R . leguminosarum R. meliloti R. japonicum Halo bacterium Propionibacterium P . shermanii P . freudenreichii P. arubinosum Rrevibacterium linens MycobacteriumlNocardia M . tuberculosis M . kansasii M . phlei M . scrofulaceum M . mariunum M . smegmatis Micrococcus M . denitrijicans M . conglomeratus M . varians M . luteus M . roseus M . (Staphylococcus)roseus M . (Staphylococcus)ufermenturi,\ Bordetella bronchiseptica Cellulomonas biazotea

62-63 59-74 65

1

t

59-63

1

67

63-66 66-68 66-70 67 67 70 63 64-70 64-65 64-67

67-68 66-75 66-67 68-70 68-70

t

73-75 70 75

“The values, rounded to the nearest percent, are taken from references cited in the text. *See text, Section II,Q, on Acinetobacter.

homogeneous in DNA base composition than S. Zactis (Auletta and Kennedy, 1966; Rosypal and Rosypalova, 1966; Garrity et al., 1969). However, the ranges overlap. Micrococci from precooked frozen seafoods and marine sources were separated into two clusters, corresponding to the genera S t a p h y Zococcus and Mic~ococcus(Evans et aZ., 1955; Colwell and Raj, 1962). DNA base compositions confirmed N T findings. DNA base composition analyses and N T of micrococci and staphylococci were done b y Rosypal et al. (1966), who divided these

GENETIC AND PHENETIC CLASSIFICATION OF BACTERIA

143

organisms, on the basis of moles percent guanine plus cytosine (G C) content, into several groups. Strains with high phenotypic similarity demonstrated similar DNA base composition. Rosypal et al. (1966) concluded that two distinct genera were discernible, Micrococcus, with a G C range of 66.3-73.3%, and Staphylococcus, with 30.7-36.4%. Auletta and Kennedy (1966) examined catalase-positive, grampositive to gram variable strains of the genus Micrococcus. Two groupings by G C content were found: (1) the range 30-39% included all named staphylococci except S. roseus and S. afermentans, several micrococci, Gafiya tetragena and G. homari; (2) the range 59-72% included the remaining micrococci, the sarcinae, S. roseus, S. afermentans, and an atypical strain of G a . y a , ATCC 6007. Interestingly, some strains of staphylococci with G + C in the range 30-39% were incapable of forming acid from dextrose under anaerobic conditions, thus representing an intermediate group within Staphylococcus, distinguishable from S . aureus and S. epidermidis (Auletta and Kennedy, 1966). The wider range of 30.7-40% reported in the literature for S. aureus is probably due to the variety of methods and calculations employed by different investigators, rather than to a real variation in base composition (Garrity et al., 1969). The data of Mortensen and Kocur (1967), correlating DNA base composition and acid production from glucose, are in good agreement with the conclusions of NT studies and the DNA analyses of Auletta and Kennedy (1966) and Garrity et a2. (1969). In a survey of several bacterial taxa, Focht and Lockhart (1965) found no difference between Ga$kya and Staphylococcus on the basis of NT, and the DNA base composition reported for Ga$kya (Auletta and Kennedy, 1966) confirmed the synonymy of these genera. Compilation of available DNA base composition data by Bohidek and Kocur (1970; BohGek et d . , 1969) clarified several points. Attempts to identify the groups of gram-positive cocci and to determine species within the groups on the basis of a few selected characteristics, such as fermentation of carbohydrates (Gibson, 1967; Mortensen, 1970), lysostaphin susceptibility (Klesius and Schuhardt, 1968), novobiocin resistance (Hirsch and Grinsted, 1954), or other single, i.e., monothetic characters (Lockhart and Hartman, 1963), have not been successful.

+

+

+

B. Ruminococcus Ruminococci, R. albus and R . flavefaciens, isolated from rumen contents of fistulated sheep were studied by Jarvis and Annison

144

R. R. COLWELL

( 1967). That the two species were “natural groups,” i.e., genetically independent or ecologically distinguishable, was tested by applying the methods of NT. Results of the numerical analysis showed two main clusters corresponding to the existing two species, as well as two intermediate groups. All strains classified as R. albus, except one, were included in a single cluster, the one exception falling into a cluster intermediate to R. flauefuciens. The second main cluster represented R. fluuefaciens strains. The classification produced agreed closely with that derived by classical methods. The advantage of numerical classification was that intermediate strains could be assigned to appropriate species and the degree of relationship between members of the same group could be assessed objectively.

C. Streptococcus Numerical classification of streptococci was first done by Colobert and Blondeau (1962), who examined strains of S. faecalis. The streptococci were found to group into two large, clearly delineated groups (Colobert and Blondeau, 1962). Within each, two subgroups could be discerned. One of the major clusters was identified as S. faecalis, including S. faecalis var. liquefaciens and S. faecalis var. zymogenes. S . faecalis was divided into S. faecalis proprium and S. faecium. Other subclusters of enterococci were identified as S. durans and S. innominatus. Factor analysis provided a means for separation of S . faecalis strains into three groups: (1) S. faecalis proprium; (2) S. faecium, and ( 3 ) S. durans (Defayolle and Colobert, 1962). Thus, biotypes were identified as proprium, faecium, durans, tiquefaciens, innominatus, etc., by Colobert and Blondeau (1962) and Defayolle and Colobert (1962). Comparing type R factor analytic results with type Q (Michener and Sokal, 1957) and the classical taxonomic approach, the investigators concluded that the results of the studies were compatible. Strains of Streptococcus belonging to the fecal, pyogenic, and viridans groups were studied by Raj and Colwell (1966). Results of the computations revealed a large, homogeneous group containing the enterococcus strains and a discrete cluster of other streptococci belonging to the pyogenic and viridans groups. Phenetic clusters within the major enterococcal group comprised S. faecalis and its varieties, the unclassified enterococcus strains, S. bovis, S. durans, and S. faecium. The S. faecalis species cluster, demonstrating an overall intergroup mean similarity value (S) of 83.5%, comprised three discernible subgroupings: S. faecalis (typical or “proprium”),

GENETIC AND PHENETIC CLASSIFICATION O F BACTERIA

145

S. fuecalis var. liquefuciens; and S. fuecalis var. zymogenes. The subgroupings were considered functional biotypes. The pyogenic and viridans strains studied by Raj and Colwell (1966) clustered separately from the S. bovis strains, which appeared to be more closely related to the enterococcus group. S. bovis demonstrated cross-relationships (significantly high S values) with the unclassified group of enterococci. Thus, the classified group constituted one of the “evolution buds” of S. faeculis hypothesized by Colobert and Blondeau (1962) and named S. innominatus. Raj and Colwell (1966) considered S. durans and S. faecium to be separate species clusters and these were treated as natural groupings. A more inclusive study of Streptococcus spp. was undertaken by Colman (1968), who examined a wide representation of streptococcal strains, including streptococci, aerococci, leuconostocs, and a pediococcus strain. Three independent clustering algorithms were used to analyze the taxonomic data. Colman (1968) concluded that S. agalactiue (Lancefield group B) and S. salivarius were species with distinctive properties. On the other hand, S. fuecalis and S. faecium strains formed a single phenon at 85% S, a result in disagreement with observations of Colobert and Blondeau (1962) and Raj and Colwell(1966),who found these species separate but within the enterococci cluster. Kandler et al. (1968)found all S. faecutis strains to contain a lysinealanine type of cross-linkage in the murein and all strains of S. faecium, a lysine-aspartic acid type of murein. By starch gel electrophoresis of glucose-6-phosphate dehydrogenase and glyceraldehyde3-phosphate dehydrogenase, Williams and Bowden (1968) were able to allocate 36 strains of enterococci to two major divisions: (a) S. fuecalis and (b) S. faecium, with S. durans a variant of S. faecium. Weissman et al. (1966), in examining relationships among Lancefield groups and serotypes of streptococci by nucleic acid homology (DNA/ RNA), found Lancefield group H to be more closely related to groups A and C than to either group D or F. The homology data provided evidence for the proposal that Lancefield groups A, C, H, and F have a closer relationship to each other than to D, and are consistent with and thus confirming the major groupings of streptococci obtained by numerical taxonomy. The aerococci, gram-positive, aerobic, nonmotile cocci, were observed b y Colman (1968) to form a discrete cluster, as did S. pyogenes (Lancefield group A) and other clusters of unclassified streptococci. Lack of DNA reassociation between Aerococcus spp. and S . fueculis was observed b y Schultes and Evans (1970), supporting a taxonomic separation of aerococci and streptococci.

146

R. R. COLWELL

The viridans-like streptococci of man may contain three recognizable divisions in addition to the well-established species S. salivarius. S. agalactiae, S. pneumoniae, S. pyogenes, S. salivarius, and S. faecalis are the only well-documented streptococcal species isolated from man. The three population nodes found b y Colman (1968) among the viridans-like streptococci were suggested to represent centers of aggregated species. Reference strains of Aerococcus, Corynebacterium, Diplococcus, Leuconostoc, Staphylococcus, Streptococcus, and strains of streptococci isolated from various sites within the oral cavity were included in an NT study by Carlsson (1968). The oral streptococci were found to cluster together. The healthy mouth flora was concluded to comprise a rather restricted group within Streptococcus. An NT study of gram-positive catalase-negative cocci from frozen snap bean and whole-kernel corn was carried out by Splittstoesser et al. (1968). All but two of the vegetable isolates clustered at 68% S, the lowest linkage value for the analysis. One group of strains was readily recognized as being heterofermentative cocci, Leuconostoc mesenteroides, and a subgroup of dextran-negative leuconostocs, as L. paramesenteroides. Also included with the homofermentative cocci were S. lactis and S. uberis. A fifth cluster was identified as S . faecalis. DNA base composition data for the leuconostocs and streptococci showed, in general, that the G C value fell in the range 36-42% (Suzuki and Kitahara, 1964; Rosypal and Rosypalova, 1966; Colwell et al., 1967).

+

D. Lactobacillus The classical subdivisions of Lactobacillus are the subgenera Thermobacterium, S treptobacterium, and Betabacterium (OrlaJensen, 1919, 1943) (see Table 11). Only a few reports of numerical taxonomic studies of the lactobacilli are available, and these are limited b y the range of strains studied and number of tests employed. Using paper chromatography, Cheeseman and Berridge (1959) examined amino acids and peptides found in lactobacilli. The chromatographic patterns were compared b y computer. The analysis did not permit species separation but indicated a generic level of similarity for the strains. A cluster analysis of lactobacilli was prepared b y Davis (1964), who detected three groups approximating the classification of OrlaJensen (1919, 1943), i.e., Thermobacterium, Streptobacterium, and Betabacterium. Hauser and Smith (1964) carried out an NT analysis of lactobacilli isolated from Canadian cheddar cheese. Most of the lactobacilli

147

GENETIC AND PHENETIC CLASSIFICATION OF BACTERIA

OUTLINEOF Subgenera and species Thermobacterium L. helveticus L. jugerti L. bulgaricus L. lactis L. acidophilus L. leichmannii L. delbrueckii L. salivarium Streptobacterium L. plantarium L. casei Beta bacterium L. fermenti L. buchneri L. brevis L. cellobiosus L. viridescens

THE

TABLE I1 CLASSIFICATION OF THE GENUSLACTOBACILLUS" Vitaminb requirements

Serology'

% G +Cd

Cell wall' composition

32-35

Lys-Asp Lys-Asp Lys-Asp Lys-Asp Lys-Asp Lys-Asp Lys-Asp Lys-Asp

Pyridoxal, folic acid

44-46 46-47

DAP LYS-Asp

Riboflavin Folic acid Riboflavin

52-53 44-46 43-47 51-54 36-43

Lys-Asp Lys-Asp LYSAsp Orn-Asp Lys-Ser-Ala

Riboflavin, Riboflavin, Riboflavin Riboflavin Riboflavin, Folic acid Riboflavin Riboflavin,

pyridoxal pyridoxal

folic acid

38-39 37-40 38-51 49-51 32-39 50-51 50

folic acid

"From Gyllenberg and Eklund (1968). bData from Rogosa and Sharpe (1959). 'Data from Sharpe (1962). dData from Miller et al. (1970). 'Data from Kaiidler (1967).

isolated from cheddar cheese resex-led either L. plantarum, L. casei, or intermediate forms. The families Lactobacillaceae and Propionibacteriaceae were compared in an NT study of 82 strains of streptococci, lactobacilli, and propionibacteria (Seyfried, 1968). A clear-cut separation at the 70% S level occurred for the streptococci, propionibacteria, and lactobacilli. The genus Streptococcus was found to form a distinct cluster with four subgroups: S. faecalis, and its varieties, liquefaciens and zymogenes, and S. durans and S. faecium. The pyogenic streptococci, S . pyogenes, S. zooepidemicus, and S. agalactiae comprised the second subgroup, and S. Zactis and S. cremoris, the third. The S. citrophilus and S. diacetilactis subgroup was separated from the above at 69% S (Seyfried, 1968) (see above, Section II,C, on Streptococcus). The lactobacilli examined by Seyfried (1968) clustered at 67% S, with subgroup saltations identifying the subgenera Thermobacterium, Streptobacterium, and Betabacterium. The groupings were confirmed by DNA base composition data (Table I).

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Evidence indicated L. bijidus and organisms related to it should be collected in the genus Bijidobacterium (Orla-Jensen, 1919). The numerical analysis of Seyfried (1968) showed reasonably good agreement with taxonomic conclusions based on DNA analyses.

E. Corynebacterium The genus Corynebacterium comprises a large and heterogeneous group of gram-positive organisms within the order Actinomycetales. One of the earliest numerical taxonomic studies of the genus was undertaken b y Moore and Davis (1963). Oral isolates were sorted into nine groups on the basis of overall similarities. Four of the groups showed similarities to C. flavidum, C. renale, and C . diphtheriae. Three groups resembled C. ovis, one group C . bovis, and three C . equi and C . hofmanii. Moore and Davis (1963) concluded that two main types of corynebacteria exist, comprising saccharolytic and nonsaccharol ytic strains, Relationship of plant pathogens to other coryneforms was investigated b y da Silva and Holt (1965). Four distinct clusters were observed with the largest, delimited b y a phenon level of 82%, containing five of the phytopathogenic species, Corynebacterium poinsettiae, C. tritici, C . sepedonicum, C . insidiosum, and C . michiganense. Other clusters observed were: Arthrobacter globiformis and Brevibacterium linens (83% S); Corynebacterium flaccumfaciens, Cellulomonas biazotea; Cellulomonas jimi and a bean wilt bacterium; and Corynebacterium fasciens. The following conclusions emerged: (a) phytopathogens differed sufficiently from the type species of the genus Corynebacterium diphtheriae to be excluded from the genus Corynebacterium; (b) Corynebacterium fasciens should be excluded from the Corynebacteriaceae; ( c ) Brevibacterium linens and Arthrobacter globiformis were sufficiently similar to warrant a new combination, A. linens; and (d) A . tumescens should be excluded from the genus Arthrobacter. The Listeria monocytogenes, Cellulomonas biaxotea, and C . $mi groups were considered to be acceptable species. Harrington (1966) examined strains of Corynebacterium, Mycobacterium, Nocardia, Arthrobacter, and Jensenia. Harrington also observed the plant pathogenic Corynebacterium spp. to be distinct from animal parasites of the genus. For the Corynebacterium diphtheriae strains, a range of similarity values from 87% for the “gravis t m i t i s ” pairing, to 51% for the intermedium pairing, was found. Harrington (1966) chose to reclassify C. ulcerans as a variety of C. diphtheriae. Further, C . pyogenes was excluded from the genus and Harrington (1966) concurred with Sneath and Cowan (1958) in

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grouping C . pyogenes with Streptococcus. Harrington (1966) acted upon the suggestion of Cummins (1962) that the genera Corynebacterium, Mycobacterium, and Nocardia be merged. From numerical analysis of data obtained for coryneform bacteria, Davis and Newton (1969) did not find Harrington’s (1966) recommendation of a common genus for Corynebacterium, Mycobacterium, and Nocardia tenable. C . equi, which clustered with the mycobacteria in an earlier study (Harrington, 1966), did not form a homogeneous group (Davis and Newton, 1969). Division into animal and plant corynebacteria (da Silva and Holt, 1965) was indicated. Also, since Brevibacterium linens clustered closely with Arthrobacter globiformis and other groupings of BrevibacteriumlArthrobacter were noted, many of the strains currently placed in Brevibacterium were reassigned to Arthrobacter. DNA base composition analyses of B . linens and A. globiformis corroborate the synonymy of these species (Rosypal and Rosypalova, 1966; Werner, 1967) (see Table I). Kurthia xopfii was considered better placed with the ListerialErysipelothrixI Microbacterium thermosphactum complex, with probable relationship to animal corynebacteria also noted. Isolates from peas, beans, and corn were compared with cultures of Corynebacterium, Microbacterium, and Arthrobacter by Splittstoesser et al. (1967). Five groups were recognized among the vegetable isolates. Most of the named species within the three genera included in the study differed as much from each other as they did from the vegetable isolates. An NT study of Corynebacterium pyogenes isolated from disease processes in animals showed C. pyogenes to be a “good species” (Roberts, 1968), with the isolates related at high S values (>75%). Unfortunately, no named species, i.e., type culture strains of Corynebacterium or Streptococcus, were included in the study; if this had been done, the relationship of Corynebacterium pyogenes to Streptococcus spp., questioned by the studies of Cummins (1962), Sneath and Cowan (1958), and Harrington (1966), might have been clarified.

F. Propionibacterium Compared with other genera of bacteria, the taxonomy of the propionic acid bacteria rests on a firm foundation provided by the distinguished pioneering work of van Niel (1928). Van Niel recognized eight species: P . pentosaceum, P . freudenreichii, P . shermanii, P . jensenii, P . technicum, P . peterssonii, P . thonii, and P . rubrum. Werkman and Brown in 1933 described P . arabinosum, P . zeae, and P . rafinosaceum. The first NT study of the propionic acid bacteria was

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that of Antila and Gyllenberg (1963), who examined strains of propionic acid bacteria isolated from milk and cheese. P. peterssonii and P. jensenii were grouped together, and P. arabinosum was divided into two subgroups, but otherwise the numerical results were not different from the classical scheme for these bacteria. The study of Antila and Gyllenberg (1963) provided good evidence for separation of the species P. casei. Further analysis revealed that P . freudenreichii, P. shermanii, and the small subgroup of P. arabinosum were fused at a high level of similarity. Four major groups of propionibacteria were obtained: (a) P. freudenreichii, P. shermanii, P. casei, and P. arabinosum; (b) P . technicum and P . pentosaceum; (c) P. peterssonii (and P. jensenii) and P . rafinosaceum, and (d) P. thonii and other red-pigmented types included in the study. Eight Propionibacterium species were collected and examined by Malik et al. (1968). It was concluded that four groups were represented by the strains examined, interrelating at a matching coefficient of 68%: P . freudenreichii and P. shermanii; P. rubrum, P. jensenii, and P. peterssonii; brownish red pigmented P. thoenii [sic]; and P. arabinosum, P. peterssonii, and a single strain of P. shermanii. Malik and his co-workers (1968) concluded, as did Antila and Gyllenberg (1963), that the two species P . shermanii and P. freudenreichii should be consolidated.

G. Brevibacterium

The small, gram-positive, nonsporulating, motile bacteria, producing yellow- or orange-pigmented colonies on nutrient agar and possessing the capability of utilizing glucose, have long been difficult to identify and classify. Chatelain and Second (1966) examined named strains of Flavobacterium, Corynebacterium, Brevibacterium, and Listeria spp., as well as unknown isolates of human origin, i.e., spinal fluid, blood, pus, etc., or contaminated serum or transfusion plasma. Five distinct groupings of strains were noted: (a) Brevibacterium linens and Corynebacterium pseudodiphtheriticum (>80% S); (b) B . ammoniagenes and B. stationis (>go% S); (c) a Flavobacterium and Corynebacterium spp. cluster (>70% S); (d)Listeria denitrijicans (> 80% S), and Listeria monocytogenes (strains of which were found to be very nearly identical). Peripheral strains, which remained unclustered, included B. helvolum, C. zerosis, C . fermentans, a Brevibacterium sp., and C. aquaticum. The two major branches of the groupings separated at 60% S; one branch was composed of Brevibacterium and nonmotile Corynebacterium, the other of motile coryneforms and Listeria spp. Phenon 3 contained motile strains

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producing a yellow or orange pigment and all were placed in the genus Brevibacterium, either B . fermentans or B . oxydans. The taxonomy of the brevibacteria is far from satisfactory. The type species is uncertain; therefore, the genus Brevibacterium is illegitimate. The relationship of these bacteria to the coryneforms, i.e., Corynebacterium, Microbacterium, and Arthrobacter, is not entirely clear. Fiedler et al. (1970) concluded that the variation of murein types found in the strains of Brevibacterium match the variation found in corynebacteria and arthrobacter. Thus, it has been recommended that Brevibacterium be abolished, since the organisms placed in this genus have characteristics of Corynebacterium and other coryneforms.

H. Pseudomonas Species of the genus Pseudomonas were found to be distinct, as a group, from the Enterobacteriaceae, Bacillaceae, and Micrococcaceae (Colwell and Liston, 1961a; Colwell et al., 1965). A cluster of pigmented strains showing high similarity to P . aeruginosa was noted by Colwell and Liston (1961a). Four groups were observed: a marine group, including P . elongata; a cluster of strains including P . fragi and Vibrio (Pseudomonas) percolans; a grouping of strains including P . ovalis, Vibrio (Pseudomonas)tyrogenus, P . denitrificans, P . fluorescens ATCC 11251; and a large cluster of strains identified as the “fluorescent group.” The fluorescent group was further subdivided into psychrophilic strains resembling P . aeruginosa, mesophilic P . aeruginosa, and fluorescent pseudomonads. A lower generic level of 60% S and species level of 75% S was suggested (Colwell and Liston, 1961a). Pseudomonas was found to be clearly distinct from the other genera examined and Aeromonas strains, other than A . formicans, were more closely related to members of the Enterobacteriaceae than to Pseudomonas. A study of fluorescent pseudomonads was carried out by Rhodes (1961),who observed that aeromonads separated as a group and most of the isolates were grouped as P . jluorescens at an S value of 2 80%. P . aeruginosa was found to be closely related to P . jluorescens and, on the basis of average overall S values, a neotype strain of P. fluorescens was selected. Based on examination of strains representing 46 different species of Pseudomonas and six species of Aeromonas, a proposal for the taxonomy of the genus Pseudomonas was made by Lysenko (1961). Strains were sorted into two main groups.

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COLWELL

A taxonomic comparison of Xanthomonas and Pseudomonas slip. by Colwell and Liston (1961b)divided Xanthomonas species into two related groups. Pseudomonas solanacearum strains were observed to form a group distinct from Xanthomonas and representing a true taxonomic species (intra, S = 75%). Relatedness among several species of Pseudomonas and Xanthomonas was measured by De Ley and Friedman (1965) and De Ley et al. (1966a),- using DNA homology (De Ley and Schell, 1963). Twenty-eight Xanthomonas spp. showed homology with X . pelargonii of 3 75% S . Labeled DNA from P. fluorescens hybridized with Xanthomonas DNA to the same extent as with other pseudomonads. These data confirmed the conclusions drawn by Colwell and Liston (1961b) on the basis of numerical analysis, namely, that Xanthomonas spp. are very closely related to Pseudomonas spp., and host specificity within the genus Xanthomonas was not sufficient evidence for species demarcation. In a quantitative approach to the study of bacterial species, Liston et al. (1963)presented the concept of a hypothetical median organism, using P. aeruginosa as the model species. Groups isolated b y Gyllenberg et al. (1963a) from market milk were identified as P . fluorescens. One of the groups was concluded to belong to the “psychrotrophic aeruginosa group” of Colwell and Liston (1961a). Three other groups were identified as P . fragi. The “psychrotrophic aeruginosa” group was found to lie closer to P . fragi than to the pigmented P . fluorescens group. Gyllenberg et al. (196313) was able to distinguish two groups of market milk, samples of good keeping quality and samples of poor quality. Tests for the occurrence of pseudomonads, particularly the “psychrotrophic P . aeruginosa” type, was found to be a reliable test for distinguishing between the groups of milk samples. Marine bacteria have been subjected to phenetic analysis and a predominant portion of the natural flora of marine animals, seawater, and sediment have been shown to be Pseudomonas spp. (Colwell and Gochnauer, 1968). Characterization and identification of marine bacteria were studied b y Colwell et al. (1967), who established a computer “file” on marine forms. Bacterial populations sampled at stations at several depths, from 2 to 5000 meters, in Antarctic waters, were sampled. Pseudomonas spp. formed a major portion of the bacterial genera represented. A numerical analysis of bacteria isolated from Antarctic and tropical seawaters was carried out by Pfister and Burkholder (1965), who pointed out that numerical taxonomic techniques can be carried out

GENETIC AND PHENETIC CLASSIFICATION OF BACTERIA

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on board oceanographic vessels equipped with high-speed computers. On-board analyses enable investigators to remain at sea for long periods and to make systematic appraisals of marine microbial populations from various geographic locations. Bacteria from deep-sea sediments collected at 9400-10,400 meters in the Philippine and Marianas trenches of the Pacific Ocean, were grouped into five phenetic clusters, four of which were identified as Pseudomonas spp. Similar isozyme patterns and DNA base compositions were found for strains identified as Pseudomonas bathycetes. Pseudomonas piscicida, the pigmented and icthyotoxic bacterium, was found to be related to P . geniculata, P . atlantica, and P . rubescens (Hansen et al., 1965). However, mean DNA base compositions of P . piscicida was 44.5&0.7%G + C (Mandel et al., 1965). The relatively low % G C content for the P . piscicida strains was somewhat disconcerting, since the overall DNA base composition range for Pseudomonas spp. is 15-20% higher (see Table I). This discrepancy between the phenetic and molecular genetic data remains to be clarified. Phytopathogenic pseudomonads, traditionally described on the basis of host range, symptomatology, and morphological and physiological tests, were examined by Sands et al. (1970). Groupings obtained were: dihydrolase-negative, phytopathogenic fluorescent pseudomonads, fluorescent saprophytes, and nonfluorescent pseudomonads. A series of phenotypes with relative areas of homogeneity was observed.

+

I. Thiobacillus A taxonomic survey of the thiobacilli from a variety of sources, i.e., soil, estuarine mud, biological filters, activated sludge and minewater, was undertaken by Hutchinson et al. (1965, 1966, 1967). Four groups were clearly differentiated, corresponding to T . trautweinii, T. novellus, T . thioparus, and T . neapolitanus (Hutchinson et al., 1965).Within the T . trautweinii group was included a strain tentatively identified as Pseudomonas jluorescens and a strain similar to P . stutzeri. Both organisms were characterized by the ability to grow as faculative autotrophs, oxidizing thiosulfate to polythionates with a corresponding increase in the pH value of the medium. T . trautweinnii was excluded from the genus Thiobacillus. S values from comparison of duplicated strains ranged from 88 to 100, with a mean of 95.7, suggesting that differences between strains and errors of testing can seriously affect the S values. An S value of 97% between two strains derived from division of a single

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strain immediately prior to testing was observed. On the other hand, classification of thiobacilli b y overall similarity was found to be independent of tests used. Two groupings of strains, corresponding to T. thio-oridans and T . ferro-oxidans, were obtained. Named strains of T . thio-oxidans and T. concretivorus fell into the T . thio-oxidans group. There were no major differences among the named species of the acidophilic iron oxidizers, and it was concluded that the Ferrobacillus ferrooxidans should be renamed ThiobacilEus ferro-oxidans, since they were all capable of oxidizing sulfur and thiosulfate as well as ferrous iron. The taxonomy of the anaerobic thiobacilli has been studied by Hutchinson et al. (1967) using newly isolated strains, comparing these with other species of the genus grown under aerobic and anaerobic conditions. T. denitrijicans was found to be a valid species. Jackson et al. (1968) found that the ordering of strains on the basis of S values (Hutchinson et al., 1965,1966,1967)followed that of DNA base composition. The unequivocal separation of T . thiopurus and T . neapolitanus was confirmed, as was the observation that T. thiooxidans and T . ferro-oxidans were clearly separable (Table I).

J. Arthrobacter The range of similarity values for Arthrobacter spp. suggests a diversity among the strains. However, Bhat (1967; Mullakhanbhai and Bhat, 1967) considered Arthrobacter, as a genus, to be homogeneous. A two-stage principal component analysis of Arthrobacter and arthrobacter-coryneform soil isolates, as well as cultures of Brevibacterium linens, Nocurdia cellulans, Corynebacterium michiganense, and Jensenia canicruria, was done by Skyring and Quadling (1969a, 1970). Observations included separation of A . tumescens from A . globiformis and, as concluded by other investigators (Cummins, 1962), the grouping of A . globiformis, A. pascens, A. aurescens, and A. ramosus (A. ureafaciens and A. atrocyaneus were not included in the cluster). Also observed was the phenotypic and G t C content similarity of B . linens and the arthrobacters. Data were provided which supported the conclusion that the phytopathogenic coryneform C . michiganense is significantly different from the type species of the genus Corynebacterium. The soil arthrobacters did not fall into discrete clusters without intermediate forms but did, rather, suggest a “quasicontinuum with discernible foci of greater relative density, characterizable in terms of nutritional and other attributes” (Skyring and Quadling, 1969b).

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K. RhizobiumlChromobacteriumlAgrobacterium Lange (1961) demonstrated that cross-inoculation group separations of the rhizobia for species were not satisfactory. Distinctly different host groupings were apparent from data collected during his study of nodule bacteria associated with indigenous legumes of southwestern Australia. As pointed out by Lange, the economic importance attached to agronomic legumes and the resulting disproportionate attention paid to these resulted in a skewed classification of the nodule bacteria. Graham (1964) undertook an extension of Lange's work and reported on a numerical taxonomic study of the rhizobia. Bacillus polymyxa, Chromobacterium violaceum, and Beijerinckia indica were found to show low affinities with root-nodule bacteria. Agrobacterium radiobacter and A. tumefaciens grouped with fast-growing Rhizobium, especially with R. meliloti. All comparisons between the two Agrobacterium species exceeded 80% S, and values greater than 90% were found to be common. A. radiobacter, A. tumefaciens, and R. meliloti showed similarities greater than 70% S with R . leguminosarum, R. phaseoli, and R . trifolii. Strains of R . leguminosarum, R . trifolii, and R . phaseoli grouped together with similarity values in the range 80-97%. Slow-growing rhizobia formed a group with similarities in the range 70-95%. Graham (1964) observed a distinct division among the Rhixobium spp. and suggested: (a) consolidation of R . trifolii, R. leguminosarum, and R . phaseoli into a single species, R . leguminosarum; (b) consolidation of A. radiobacter and A . tumefaciens; and (c) inclusion of the latter as synonyms of R . radiobacter in the genus Rhizobium. Also suggested was the creation of a new genus Phytomyxa to contain strains of slow-growing root nodule bacteria. In an attempt to distinguish between bacterial populations of different rhizospheres, Brisbane and Rovira (1961; Rovira, 1965) classified isolates according to their similarities with each other. About three-fourths of the rhizosphere isolates belonged to a single group related to the genera Rhizobium and Agrobacterium, which were linked together as a single genus by Graham (1964). The remainder of the isolates formed several small groups apparently not related to any of the known cultures. Similarity coefficient and clustering methods were compared by 't Mannetje (1967a). Some of the newer clustering techniques were applied by 't Mannetje (196713) to the data of Graham. His major conclusions were in agreement with Graham (1964). A numerical analysis of the family Rhizobiaceae was undertaken by Moffett and Colwell (1968). Relationships among the strains

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representing the genera Rhizobium, Agrobacterium, Chromobacterium, Pseudomonas, Enterobacter, Escherichia, Vibrio, and Proteus were examined on the basis of all available evidence, i.e., computed similarities, nucleic acid data, and isozyme similarities. The results of the analyses supported separation of Chromobacteriurn from other genera of the Rhizobiaceae. In fact, there was no evidence for retaining the family as presently composed (Breed et al., 1957). The root nodule bacteria were thought to be better placed in the family Pseudomonadaceae, a conclusion also reached by De Ley et al. (Heberlein et al., 1967; De Ley, 1969) from DNA homology studies. The major separation of the Rhizobium species observed by Moffett and Colwell (1968) was into the slow-growers and the fast-growers. The numerical analysis further showed results supporting the consolidation of Rhizobium (i.e., the fast-growers) into two or more species, R . meliloti and R . leguminosarum being retained as separate species. The computed data suggested that A. gypsophilae and A. pseudotsugae be removed from Rhixobium and transferred to separate generic groups. De Ley et al. (196613) questioned the relationship of A. pseudotsugae to other agrobacteria from the viewpoint of DNA base composition. The taxonomic conclusions, cited b y Moffett and Colwell (1968; Colwell et al., 1968), were in good agreement with those of Heberlein et al. (1967), who did DNA/DNA hybridization experiments with strains of Agrobacterium, Rhixobium, and Chromobacterium spp. Sneath (1956, 1960) observed that Chromobacterium strains formed two clusters, corresponding to the mesophilic ( C . violaceum) and psychrophilic ( C . lividum) strains. Moffett and Colwell (1968) confirmed the separation of Chromobacterium into two groups. Goodall (1966) reexamined Sneath’s data and found essentially the same groupings. Bacterial soil isolates have been examined by techniques designed to achieve classification to the generic level (Rovira, 1965; Gyllenberg and Rauramaa, 1966; Bowie et al., 1969; Skyring and Quadling, 1969b). Skyring and Quadling (1969a,b) analyzed binary-encoded descriptions of cultures, mostly of soil origin. A two-stage principal component procedure was used. A difference was detected in the kinds of isolates predominant in flax rhizosphere samples when compared to isolates derived from the corresponding control soil populations. The control soil sets of isolates were predominantly arthrobacter-coryneform-like organisms, and the rhizosphere isolates were pseudomonas-like. Condensation of considerable ecological

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and taxonomic data in a useful and revealing form was achieved (Skyring and Quadling, 1969b).

L. HalobacteriumlHalococcus Halobacteria require a high concentration of salt for growth and have been known for nearly a hundred years. A set of moderate and extreme halophiles was studied, and two major groupings were obtained: a group of rodlike extreme halophiles and a less homogeneous but taxonomically distinct cluster of halophilic cocci (Colwell and Gibbons, 1973). A single species relationship of strains within the clusters was noted, and two species were suggested: Halobacterium salinarium and HaEococcus morrhuae. From a comparison of halophiles with a set of halotolerant streptococci, staphylococci, and micrococci, it was concluded that the moderately halophilic bacteria are taxonomically distinct from the extremely halophilic forms. Genetic relatedness among various strains of halophilic bacteria was assessed by DNA-DNA and DNA-RNA reassociation studies. All the strains of extremely halophilic rods were closely related, and the extent of divergence of base sequence was found to be similar for the major and minor DNA components (Moore and McCarthy, 1969a,b). The degree of relatedness of the extreme halophiles of the genus Halobacterium to a reference strain ranged from 54% homology for H . halobium to 71 and 7875, respectively, for H . cutirubrum and H . salinarium, confirming species synonymy detected in other studies, including NT (Colwell and Gibbons, 1973). Experiments with ribosomal RNA were also carried out by Moore and McCarthy (196913). Relationship between the extremely halophilic rods and cocci and a more distant relationship to moderate halophiles was revealed. The genetic relatedness measured by DNA/RNA hybridizations is remarkably paralleled by the phenetic relationships measured by NT. M. Aeromonas In a study of gram-negative bacteria carried out by Colwell and Mandel(1964), A . formicans and A . punctata strains were considered to be intermediate between the genera Pseudomonas and Escherichia (Table I). The taxonomy of Aeromonas strains was examined in depth by Smith (1963)and Eddy and Carpenter (1964). Two discrete groups of Aeromonas strains were found: pigmented and nonpigmented “Bacterium salmonicida” and Aeromonas. Although it was concluded that A . salmonicida should be placed in a separate genus (Smith, 1963), inspection of the S value triangular matrix showed inter-S values to lie predominantly in the 6 0 4 0 % range, a finding which

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indicates that A . salmonicida and Aeromonas spp. could be retained within a single genus. Eddy and Carpenter (1964) came to this conclusion after inspecting results of an NT study of aeromonads and related strains. Eddy and Carpenter (1964) also concluded that A . punctata, A . formicans, A . salmonicida, and Vibrio comma were closely related and that C27 strains should be excluded from the genus Aeromonas and transferred to a proposed new taxon, Plesiomonas shigelloides. The other strains of the study, A . punctata, A. formicans, Photobacterium spp., and V . comma, were observed to be separate phenons at S values between 88 and 90%.

N. ENTEROBACTERIA Many investigators share the opinion that the enterobacteria have been “overgenerated” with little or no real phenotypic evidence for the number of taxonomic distinctions made among the Enterobacteriaceae. Kreig and Lockhart (1966) studied a relatively small, but representative, sample of strains and observed a single large cluster comprising the genera Enterobacter (Aerobacter), Escherichia, Salmonella, Arizona, Citrobacter, and Shigella. Krieg and Lockhart (1966) concluded that many of the clinically significant members of the Enterobacteriaceae occurred as a single cluster, with a spectrum of related organisms. Lockhart and Holt (1964) reported an analysis of bacteriological data where Kauffman’s (1961) descriptions of Salmonella serotypes served as Operational Taxonomic Units (OTU’s) (Sokal and Sneath, 1963). Unfortunately, the physiological data given b y Kauffman for each type was not based on characters of a single type strain but represented a summary of reactions. No significant groupings came out of the analysis (Lockhart and Holt, 1964). The genus Serratia was studied by Colwell and Mandel (1965; Colwell et al., 1964), who presented N T and DNA base composition data for Serratia marcescens, including the varieties S . kiliensis, S . plymuthica, and S . marinorubra. Overall group homogeneity was noted, and a single species, S . marcescens, was recognized. Direct comparison of S values with DNA base compositions permitted exclusion of four strains from the species group and detection of the relationship of these prodigiosinproducing strains with Enterobacter spp. Bascomb et al. (1971), in addition to revising Klebsiella, gave evidence for a “Serratia biotype 11,” supported by Grimont (1972). A numerical study of the genus Erwinia was done by Lockhart and Koenig (1965), who concluded that Erwinia carotovora, E . aroideae,

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E . atroseptica, and E . ananas should be placed in a single species, separate from E . amylovora. E . nimipressuralis and E . chrysanthemi were suggested to be distinct species. Strains of E . amylovora were found not to be very similar to one another or to other members of the Enterobacteriaceae. Correlation of DNA base composition data for Erwinia spp. (Starr and Mandel, 1969)with the diagram of relationships given by Lockhart and Koenig (1965) is good (Table I). Molecular relationships among members of the Enterobacteriaceae have been reviewed by Brenner and Falkow (1971). In terms of polynucleotide sequence relatedness, the species Escherichia coli is described as a group of strains with DNA of >90% relatedness. Nucleotide sequence relatedness of other genera of the family to E . coli has been measured and all enterobacteria were found to exhibit 50% or less reaction with E . coli DNA.

0. Pasteurella Talbot and Sneath (1960) examined strains of Pasteurella septica, most of which were from human lesions. Cat strains showed a high degree of similarity, and P . septica appeared to be distantly related to P . pestis and P . pseudotuberculosis. P . septica is associated with infections of the upper respiratory tract. A separation of P . septica strains from P . pseudotuberculosis and P . pestis was observed. Smith and Thal (1965) extended the work of Talbot and Sneath (1960) to cover all listed species of Pasteurella. Their data clearly showed P . pneumotropica, haemolytica var. ureae, multocida, and haemolytica were a separate group from the P . “X,” pestis, and pseudotuberculosis strains. The proposal of van Loghem (1944-1945, 1946) to accommodate P . pestis and P . pseudotuberculosis in a separate genus, Yersinia, was adopted by Smith and Thal (1965), who further proposed that Yersinia be classified within the family Enterobacteraceae. Pasteurella “X” was also assigned to Yersinia as “Yersinia X.” Of the oxidase-positive group of Pasteurella strains, P . multocida and P . haemolytica var. ureae, renamed P . ureae, were considered to be species. Two types of P . haemolytica, A and T, were noted but separation into different species was not considered (Smith and Thal, 1965). Janssen and Surgalla (1968) described a new species, P . piscicida, pathogenic for white perch (Roccus americanus). A numerical study by Allen and Pelczar (1967) of cultures isolated from healthy white perch revealed no P. septica (multocida) strains in the normal commensal flora of fish. The groups isolated from white perch were

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Bacillus, Achromobacter, Pseudomonas, Aeromonas, Enterobacter, Micrococcus, and an unidentified pseudomonad group. P. Vibrio I n 1963, Sebald and Veron published a classification of Vibrio and related species based on percent G + C composition of DNA, and VCron (1966a,b) subsequently did a numerical taxonomic study of the genus. The phena observed were: Campylobacter, Spirillum, Pseudomonas sp., P. terrigena, Moraxella lwofii, Plesiomonas shigelloides, P. jluorescens, Vibrio ichthyodermis, Vibrio sp., Aeromonas punctata, Aeromonas sp., A. salmonicida, Vibrio costicolus, and V . metchnikovii. A further grouping into two major clusters was noted, one containing Spirillum, Camp ylobacter, Pseudomonas terrigena, Moraxella lwofii, and Plesiomonas shigelloides; and the other Vibrio, Aeromonas punctata, and A . salmonicida. When VBron (1966b) compared the percentage of S groupings with the percentage of G f C clusters, a significant correlation was noted, and the phena Spirillum, Vibrio, and A. punctata, were found to be homogeneous. Veron concluded that the family Spirillaceae should include the genera Spirillum and Campylobacter, and the family Vibrionaceae, the genera Vibrio, Aeromonas, and Plesiomonas (Table I ) . Fujino et al. (1953) reported on food poisoning caused by a saltrequiring bacterium found on seafish. Identified by Fujino as Pasteurella parahaemolytica n. sp., later examined and named Pseudomonas enteritis by Takikawa (1958), and renamed Uceanomonas by Miyamoto et al. (1961), the organism was finally classified as Vibrio parahaemolyticus by Sakazaki et al. (1963). Zen Yoji et al. (1965) applied computer techniques to the classification of these halophilic vibrios. Two species were recognized. An extensive numerical taxonomic study of the genus Vibrio was done b y Colwell and co-workers. Colwell and Yuter (1965)reported correlation of phenetic data (S a 83%) with overall DNA base composition for V. cholerae and El Tor vibrios. The median organism as defined by Liston et al. (1963)was found to correspond to the mean strain in the range of overall DNA composition values. Isozymes, DNA base composition, and NT of Vibrio cholerae and Vibrio parahaemolyticus were examined (Adeyemo et al., 1967; Colwell et al., 1968), and a polyphasic taxonomy of the genus Vibrio was proposed b y Colwell (1970). Data for Vibrio cholerae and the variety El Tor, V . parahaemolyticus, V . marinus, and other Vibrio spp. were processed (Colwell, 1970); results of numerical analyses showed low relationships of Vibrio spp.

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to Pseudomonas and Spirillum spp. ( s65% S). Measurement of DNA/ DNA and DNAIRNA homologies, both inter- and intraspecies, for the genus Vibrio was done (Citarella and Colwell, 1970), and a direct correlation of the numerical data and the data from studies of DNA/ RNA and DNA/DNA heteroduplex formation was observed. Numerical taxonomic studies of Vibrio spp. proved useful for identification of the commensal bacterial flora of Chesapeake Bay invertebrate animals. Bacterial cultures isolated from samples of animals, water, and sediment were identified and classified b y computer. The predominant microbial groups and their enzymatic capabilities, as well as total number of viable bacteria per sample, were determined (Lovelace et al., 1968).

Q. AchromobacterlAcinetobacter/Moraxella/Neisseria As pointed out by Jones and Sneath (1970), recent work suggests at least five main phenetic groups among the gram-negative, aerobic, nonmotile cocci or coccoid rods: (i) Moraxella lacunata ( M . lacunata, M . bovis, and M . nonliquefaciens); (ii) M . lwofii; (iii) Neisseria catarrhalis ( N . catarrhalis, N . caviae, and N . ovis); (iv) Acinetobacter anitratum, and (v) Neisseria meningitidis ( N . meningitidis, N . gonorrhoeae, and other species). M . osloensis, M . kingii, and M . phenylpyruvica appear to be distinct, but other strains, vaguely defined as Achromobacter or Acinetobacter, are of uncertain taxonomy. Mima and Herellea also are in a somewhat confused taxonomic state. In a preliminary study of gram-negative coccobacilli, Thornley (1960) demonstrated that pigmented and nonpigmented Pseudomonas strains from poultry and from culture collections could be grouped together and separated completely from Achromobacter isolates. There were three subgroups within the Achromobacter group of isolates, each of which was fairly homogeneous and possessed low intergroup similarities. Pinter and Bende (1967) examined relationships among bacterial strains isolated from clinical material and identified as Acinetobacter lwofii and Acinetobacter anitratus. Results showed mean intragroup S values of 81.9% and 87.3% for A. lwofii and A. anitratus, respectively, with intergroup S-value of 71.9%. The Alcaligenes faecalis and Bordetella bronchiseptica strains formed a homogeneous group, whereas Pseudomonas aeruginosa strains were well separated, as was Aeromonas liquefaciens. Oxidase-positive species of gram-negative diplobacteria have been classified in the genus Moraxella (Baumann et al., 1968a), with oxidase-negative strains assigned to Achromobacter, Acinetobacter,

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Mima-Herellea, Moraxella lwofii, Bacterium anitratum, etc. Pinter and Bende (1968)chose to investigate oxidase-negative gram-negative diplobacteria. Two groups were observed. Thornley (1967, 1968), in a masterful presentation of data for the gram-negative or gram-variable, nonmotile coccoid rods, examined relationships of these isolates with other genera. All but 10 of the 195 strains were found to group at 72.5% similarity. Thornley’s study did not include strains of the oxidase positive Moraxella species. Baumann et al. (1968a) subjected nutritional and physiological data for strains of oxidase-negative moraxellas to computer analysis. Two major clusters were observed, both of which were placed in the genus Acinetobacter: A . calco-aceticus and A . citroalcaligenes. The G C content of the genus was found to be in the range 39-47%, with A . calco-aceticus, 39-41 moles %, and A . citroalcaligenes, 41-47 moles % G+C. Strains of Acinetobacter studied by Pinter and Bende (1968), gramnegative, nonmotile, nonflagellated coccoids or cocco-bacilli, were subjected to DNA base composition analysis by De Ley (1968). Numerical taxonomic and DNA base composition data were compared directly. Pinter and d e Ley (1969) reexamined data for Acinetobacter strains, eliminating characters with identical responses. Only distinctive properties were thus scored. Acinetobacter anitratus and two A . lwofii groups were discerned from the combined NT and DNA base composition analyses. A . anitratus appeared identical to the A . anitratus of Thornley (1968) and the A . calco-aceticus var. anitratus of Baumann et al. (1968a,b). A . lwofii was identical to Acinetobacter sp. of Thornley (1968) and to groups Acinetobacter sp. and Acinetobacter lwofii of Baumann et al. (196813). No real cutoff points among the Acinetobacter could be determined, since minor groupings of strains were found by Pinter and De Ley (1969) to link the glucidolytic anitratus group of 42% G + C with the less biochemically active lwoffii group of 47% G + C . Strains of bacteria selected by the Subcommittee on Moraxella and Allied Bacteria of the International Committee on Nomenclature of Bacteria (International Association of Microbiological Societies) were tested for nucleic acid homologies by Johnson et al. (1970).All oxidase-negative Moraxella strains had measurable homologies in their DNA and the homology groups correlated closely with the phenetic groupings of Baumann et al. (1968b; Bovre et al., 1969). The oxidase negative, Acinetobacter spp., showed little if any detectable DNA homology with the oxidase positive, Moraxella, strains.

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Significant rRNA homology between the two groups was observed and was considered to be suggestive of distant kinship.

R. CytophagalFlavobacteriumlFlexibacter Marine isolates identified as Flavobacterium or Cytophaga were studied by Hayes (1962). Two clearly defined groups were observed from a sorted S value matrix, and three smaller, less obvious, clusters were also discernible. Floodgate and Hayes (1963) found it difficult to classify the phenons as Flavobacterium or Cytophaga. The largest group was classified as Flavobacterium when the type species of the genus, F . aquatile ATCC 11947, was found to be similar. However, F . aquatile has since been reclassified as Cytophaga. The other group was identified as Cytophaga (Floodgate and Hayes, 1963). De Ley and van Muylem (1963) determined DNA base compositions of the Floodgate and Hayes (1963) strains and found the strains demonstrating swarming to possess a 3 3 5 4 0 . 6 % G C. From the available data, the majority of the strains examined by Floodgate and Hayes (1963) appear to be Cytophaga spp. The other group of strains, separated on the basis of numerical taxonomy from the swarmers, possessed an overall G + C of 63-64% (De Ley and van Muylem, 1963) and were identified as Flavobacterium spp. Data for flexibacteria strains were analyzed by Colwell (1969). Three major subclusters were detected. The phenons closely approximated groupings formed on the basis of overall DNA compositions. Characters such as cellulose digestion correlated with other characters of the groups formed, but none was found to be a suitable key character for a monothetic taxonomy (Lockhart and Hartman, 1963) since variation in frequency of occurrence was observed (Colwell, 1969). Good correlation, in general, with taxonomic decisions made on the basis of classical taxonomy was noted, but positioning of individual strains and allocation of certain strains to generic rank, viz., Herpetosiphon spp., Microscilla spp., and Flexbacter dorothea was not justified on the basis of the NT study. Gram-negative, nonsporing, rod-shaped bacteria predominant in cannery environments, and designated “flavobacteria” were examined by Bean and Everton (1969). The majority of the isolates were identified as Flavobacterium spp. (Breed et al., 1957), the remainder being classified as Xanthomonas, Bacillus, and Corynebacterium spp. and Enterobacteriaceae. The strains did not form a very homogeneous taxonomic group and probably comprised a wide variety of genera. Heterotrophic bacteria growing in association with continuous culture of Chlorella sorokiniana were examined b y Litchfield et al.

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(1969). Five major clusters were identified: Pseudomonas, Acinetobacter, Flauobacterium, and Bacillus (one strain). S. Bacteroides Barnes and Goldberg (1968) carried out a numerical study of the family Bacteroidaceae. Results provided five phena: (i) Eggerthella clostridiformis and Bacteroides necrophorus strain NCTC 7155; (ii) Bacteroides and Bacteroides fragilis (Sphaerophorus necrophorus ATCC 12290 also showed high similarity to these strains); (iii) Sphaerophorus spp; (iv) a group of isolates from chickens considered to be a separate cluster; and (v) an additional group of chicken isolates subsequently shown to be gram-positive, hence doubtful members of the family Bacteroidaceae. Two fusobacteria strains, Fusobacterium polymorphum and F . biacutus, showed little similarity to each other or to the five phena of the analysis. A second analysis provided similar results, with the exception that a phenon was discerned which included Sphaerophorus, Fusobacterium, and Bacteroides melaninogenicus spp., clustering at 75% S. The close relationship observed between Fusobacterium and Sphaerophorus was supported by the DNA base composition data of Sebald (1962), who suggested uniting the genera.

T. Nocardia Nocardia were examined in one of the earliest numerical taxonomic studies done on bacteria (Cerbon, 1967). Three main branches were observed among the Nocardia strains: (I) Nocardia asteroides with three subgroups, one of which included Actinomyces pasteuroides, A, corneus, N . leishmanii, N . gypsoides, N . transualensis, as well as N . asteroides; and (11) N . corallina, N . globerula, N . pretorian, N . rhodnii, and N . phenotolerans. Branch I1 was related to Branch I at an S level of 47-56% and to Branch 111 ( N . brasiliensis strains) at 39%. Branch I11 constituted a cluster of highly metabolically active strains. Strains intermediate between Branches I and I1 formed a cluster, comprising N . polychromogenes, N . blackwelli, and N . asteroides. I n an extended analysis of the genus Nocardia, Tsukamura (1969) examined strains identified by other investigators as nocardiae. Tsukamura (1969) concluded from his analysis that the species N . asteroides was divisible into two species, Nocardia asteroides and Nocardia farcinica.

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U. Mycobacterium The nonpigmented, rapidly growing mycobacteria were studied by Bojalil and Cerbon (1960,1961). The only well-defined species identified in the group of strains studied was Mycobacterium fortuitum. Further study of the rapidly growing mycobacteria carried out by Cerbon and Bojalil (1961) suggested that the three species M . smegmatis, M . phlei, and M . fortuitum constituted natural groups, with M . smegmatis and M . phlei much more closely related to each other than to M . fortuitum. Mycobacteria (photochromogens, scotochromogens, nonphotochromogens, and rapid growers) were examined by Bojalil et al. (1962). Three major branches of strain clusters related at S 2 60% were found: (i) highly metabolically active strains, with two well-defined species, M . smegmatis and M . phlei and M . peregrinum sp. nov.; (ii) rapid growers, M . fortuitum, M . piscium, M . marinum, and M . thamnopheos, and also M . acapulcensis sp. nov., M . runyonii sp. nov., and M . jlavescens sp. nov.; (iii) slow growers, M . kansasii (photochromogens), M . avium (nonphotochromogens), M . marianum (scotochromogens), and M . gordonae sp. nov. (scotochromogens). A study of corynebacteria and related strains (Harrington, 1966) yielded results in general agreement with those of Cerbon and Bojalil (1961), who found M. smegmatis and M . phlei on the same branch of their dendogram, the branch dividing in the latter study, done by Harrington (1966), at the 60% level into two groups. Two new species of Mycobacterium, M . chitae and M . novum, were described by Tsukamura (1967a). A statistical approach to the definition of Mycobacterium species has been presented by Tsukamura (1967b). Tsukamura and Mizuno (1968) used the hypothetical mean organism (HMO), based on the hypothetical median organism of Liston et al. (1963), to derive strain clusters of mycobacteria. Tsukamura and Mizuno (1968) suggested that the genus Mycobacterium could be divided into subgenera. Numerical taxonomy analyses of mycobacteria have dealt predominantly with the rapid-growing mycobacteria. Slow-growers have generally been less amenable to taxonomic analysis, and this dilemma was pointed out by Wayne (1964), who also cited the attention of the tuberculosis investigators” as being fixed on “virulence” tests. The problem of growth rate was attacked by Wayne (1967), who presented some useful solutions. Using selected characters and employing the hypothetical median organism (Liston et al., 1963), Wayne concluded that M . avium and “Battey” strains of Runyon’s mycobacterial Group “

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I11 (Runyon, 1965) are a single species. Group I1 scotochromogenic mycobacteria were divided into M . aquae and M . scrofulaceum. The most extensive analysis of the mycobacteria carried out to date is that of Bogdanescu and Racotta (1967). Five clusters were observed, with mean matching indices above 80%: I (human and bovine mycobacteria); I1 (“atypical” mycobacteria belonging to Run yon groups I, 11, and 111); I11 (mycobacteria related to M . fortuitum); IV (scotochromogenic mycobacteria with greater metabolic abilities); V ( N . smegmatis). By using overall similarities, the groups were ordered, with the extreme position of M . smegmatis noted, as suggested earlier by Bojalil et al. (1962). DNA base composition data obtained for 14 mycobacterial species or varieties (Wayne and Gross, 1968) showed separation of the genera Corynebacterium and Mycobacterium, whereas a complete overlap between Nocardia and Mycobacterium was demonstrated. Thus, the proposal (Harrington, 1966) to lump corynebacteria, mycobacteria, and nocardias into a single genus found no support from the DNA base composition data (Table I). Wayne and Gross (1968) observed a bimodal distribution of frequencies of G C values for Mycobacterium spp., but the bimodality did not separate mycobacteria on the basis of growth rate, i.e., slow growers and fast growers. The hypothesis that M . tuberculosis is not centrally placed within the genus Mycobacterium, but is, rather, a peripheral species (Bogdanescu and Racotta, 1967) was strengthened by the DNA data. M . smegmatis, considered by many investigators to be in an extreme position within the genus (Bojalil et al., 1962), was shown to have an overall DNA base composition value near the upper limit for the genus (Wayne and Gross, 1968). Wayne et al. (1971) and Kubica et al. (1972) provide a new departure in the use of NT through a cooperative study of the mycobacteria. Gross and Wayne (1970) measured nucleic acid homology among eight species of mycobacteria and compared clustering patterns based on % S from NT analyses (Wayne, 1967) with hybridization data. The sequence of phenetic divergence of the mycobacterial species, ranging from M . tuberculosis at one extreme to the rapidly growing species at the other, i.e., M . smegmatis and M . phlei, was found to reflect degree of genetic divergence as well. When the phenetic similarity (% S) of M . tuberculosis to each of the test organisms was computed against the elution temperature (&) of the strain pair complexes, correlation coefficients of 0.85 and 0.84, respectively, were obtained. Using M . kansusii as the reference system, correlation coefficients of 0.87 and 0.86, respectively, were obtained. These compare favorably

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with similar computations done by Colwell(l970)for the genus Vibrio (correlation coefficient of 0.91). The good correlations obtained between the genetic and phenetic similarity measurements are reassuring, considering the fact that the standard deviation for the similarity value (% S ) is probably in the order of 5-lo%, and only a small proportion of the genes are involved in estimation of phenetic resemblance. Furthermore, the D N A hybridization data probably include errors in estimation of genetic relatedness (Gross and Wayne, 1970) due to differences in genome size, complete divergence of large segments of the genomes, occasional mismatched base pairs in otherwise similar genome segments, or possible presence of satellite DNA in one or both strains of a pair.

Ill. Microbial Ecology- Primary Productivity Many NT studies of bacteria were initiated primarily to determine what kinds of bacteria were present in a given environment. The role of bacteria in mineralization of organic matter and primary productivity cannot be measured precisely at present by the techniques available. Studies dealing with bacteria in marine ecological systems usually have enumerated total viable populations by direct or plate count methods, coupled in some instances with identification and classification of dominant microbial types in the culturable populations. Dominant types of bacteria found on sea fish have been identified and classified, at least to generic level, by NT and molecular genetic analysis. Pfister and Burkholder (1965) applied NT to bacteriological analyses of seawater, with the objective of identifying and classifying potential “biological indicators” for water masses. A collection of bacterial isolates from the Indian Ocean were similarly examined by other investigators (Johnson et al., 1970). A most severe handicap has been the limited usefulness of “type” cultures. Named cultures and type species resident in culture collections have rarely represented the dominant species found in nature. Where phena have included named or type species, the latter have not been central in the species clusters. Identification and classification of species resident in a given habitat thus remains a problem. That type cultures are so often “nontypical” probably derives from type cultures being designated for reasons of chronology, history, or personal preference, but rarely, if ever, because the organism demonstrates a central tendency in a sample of a large number of strains tested for a wide variety of characters. The bias of medical microbiology has also influenced microbial ecology since nearly all tests

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used in identification and classification of bacteria were originally designed for identifying and classifying species pathogenic for man. The intensively studied bacterial species, therefore, are human pathogens, but these species are found to be peripheral species, or “specialized” species. Ironically, evolution of pathogenic forms will most probably be deduced through comparative study of nonpathogenic species from natural environments. Model studies of ecosystems have usually perfunctorily included the bacteria. Since bacteria and other microorganisms are now recognized as significant links in food chains, attention is being drawn to definition and quantification of the role of bacteria in energy conversion. For example, an extensive analysis of 28 environmental parameters measured in Lake Maggiore was carried out by Goldman et al. (1968) and represents an approach to systems analysis that will be more commonly applied to ecological problems. Computation of correlation coefficients for variables in the aquatic system was done. Bacterial plate counts were found to correlate with total phytoplankton biomass and with total zooplankton (Gerletti and MelchiorriSantolini, 1968). Types of bacteria and their metabolic capabilities were not examined, and in future studies, this information will need to be known to understand and/or predict the role of bacteria in a given ecosystem. As an example to illustrate this point, two areas of Chesapeake Bay have been monitored for several years (Lovelace et al., 1968). Total viable, aerobic, heterotrophic counts for both areas, one of which is commercially productive for shellfish and the other no longer successfully harvested, reveal identical populations on a quantitative basis. However, from NT analyses carried out on pure cultures of bacteria isolated from samples taken from the two areas at given time intervals over a 5-year period, it was discovered that the distribution of taxonomic groups was not identical in the two areas. At the site in Chesapeake Bay where severe mortalities of the oyster population were observed, a dominance of Vibrio spp. was detected in the water column, sediment and shellfish. Vibrio spp. isolated from the diseased shellfish populations have since been shown to be pathogenic (Tubiash et aZ., 1970; Kaneko and Colwell, 1973). The potential of numerical taxonomy and molecular genetic methods in microbial ecology is only just beginning to be tapped. IV. Summary Numerical taxonomy, coupled with molecular genetic studies, has been successfully applied to microbial systematics. The tradition of quantitative experimentation, lack of fossil record with few or no

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phylogenetic constructs, and acceptance of a dynamic taxonomy, shifting to accommodate new information, characterizes microbial systematics and probably accounts for the general acceptance of numerical taxonomy by microbiologists. The combination of phenetic and genetic methods in microbial taxonomy provides a powerful tool for developing an objective, reproducible, and information-rich taxonomy for the bacteria. DNA base composition and base sequence measurements obtained by DNA/DNA hybridizations have, in nearly every instance, confirmed relationships established by measurement of the expressed phenotype, using the methods of numerical taxonomy. New methods are needed, however, to improve the resolution of relatedness based on phenotypic expression. Precise measurement of the gene product can be done, for example, by amino acid sequencing of proteins, which, although feasible, is not yet a routine laboratory procedure. Isozyme analysis and determination of allosteric regulation of branch-chain pathways approach the taxonomic precision of amino acid sequencing of enzyme proteins, and offer promise. In addition to new test methods and standardization of tests, methods for calibrating similarity coefficients are needed to improve taxonomic resolution. Also, hierarchical ranking should be based on “similarity equivalents,” i.e., with reference to a “standard table of similarity values” based on number of strains studied, number of tests coded, statistic used, etc. The contributions of numerical taxonomy to microbial systematics in the past decade has been substantial. Alpha taxonomy in microbiology is being replaced by numerical and molecular genetic methodology. The most exciting applications of numerical taxonomy are to come, and no doubt these will be in the newly developing field of microbial ecology. ACKNOWLEDGMENTS The author acknowledges support received from the National Science Foundation (Grant GB 18274), Sea Grant Project GH-91, and Contract N00014-71-A-0220-0006 between the office of Naval Research, Department of the Navy, and Georgetown University. Thanks are extended to D. J. Brenner and S. Falkow for providing the author with a preprint of their manuscript. The author is grateful to M. Rogosa and L. R. Hill for reading the manuscript.

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Allen, N., and Pelczar, M. J. (1967).Chesapeake Sci. 8, 135-154. Antila, M., and Gyllenberg, H. (1963).Milchwissenschaft 18, 398-400. Auletta, A. E., and Kennedy, E. R. (1966).J . Bacteriol. 92, 28-34. Barnes, E. M., and Goldberg, H. S. (1968).J . Gen. Microbiol. 51, 313-324. Bascomb, S., Lapage, S. P., Willcox, W. R., and Curtis, M. A. (1971).J . Gen. Microbiol. 66, 279-295. Baumann, P., Doudoroll, M., and Stanier, R. Y. (1968a).J . Bacteriol. 95, 58-142. Baumann, P., DoudoroR, M., and Stanier, R. Y. (1968b).J. Bacteriol. 95, 1520-1541. Bean, P. G., and Everton, J. R. (1969).J . Appl. Bacteriol. 32, 51-59. Bhat, J. V. (1967). Rep. Conf. Taxon. Bacteria, Czech. Coil. Microorg. Fac. Sci. Univ. PurkynB, Brno, Publ. 40,219-222. Bogdanescu, V., and Racotta, R. (1967).J . Gen. Microbiol. 48, 111-126. Bohacek, J., and Kocur, M. (1970). Rep. Conf. Taxon. Bacteria, Czech. Coll. Microorg., Fac. Sci. Uniu. Purkyni., Brno, Publ. 47, 205-216. Bohieek, J., Kocur, M., and Martinec, T. (1967).J . Gen. Microbiol. 46, 369-376. Bohaeek, J., Kocur, M., and Martinec, T. (1969).Antonie van Leeuwenhoek; J. Microbiol. Serol. 35, 185-188. Bojalil, L. F., and Cerbon, J. (1960).Amer. Rev. Resp. Dis. 81, 382. Bojalil, L. F., and Cerbon, J. (1961).J. Bacteriol. 81, 338-345. Bojalil, L. F., Cerbon, J., and Trujillo, A. (1962).J . Gen. MicrobioZ. 28, 333-346. Bovre, K, Fiandt, M., and Szybalski, W. (1969).Can. J . Microbiol. 15, 335-338. Bowie, I. S., Loutit, M. W., and Loutit, J. S. (1969).Can. J . Microbiol. 15, 297-302. Breed, R. S., Murray, E. G . D., and Smith, N. R., eds. (1957).“Bergey’s Manual of Determinative Bacteriology,” 7th ed. Williams & Wilkins, Baltimore, Maryland. Brenner, D. J. (1970).Develop. Ind. Microbiol. 11, 139-153. Brenner, D. J., and Falkow, S. (1971).Aduan. Genet. 16, 81-118. Brenner, D. J., Fanning, G . R., Johnson, K E., Citarella, R. V., and Falkow, S. (1969). J . Bacteriol. 98, 637-650. Brisbane, P. G . , and Rovira, A. D, (1961)./. Gen. Microbiol. 26, 379-392. Carlsson, J. (1968).Odontol. Revy 19, 137-160. Cerhon, J. (1967).Rev. Latinoamer. Microbiol. Parasitol. 9, 65-68. Cerbon, J., and Bojalil, L. F. (1961).J. Gen. Microbiol. 25, 7-15. Chatelain, R., and Second, L. (1966).Ann. Inst. Pasteur, Paris 111, 630-644. Cheeseman, G. C., and Berridge, N. J. (1959).J . Appl. Bacteriol. 22, 307-316. Citarella, R. V., and Colwell, R. R. (1970).J . Bacteriol. 104, 434-442. Cohen, G. N., Stanier, R. Y., and LeBras, C . (1969).J . Bacteriol. 99, 791-801. Colman, G. (1968).J . Gen. Microbiol. 50, 149-158. Colobert, L., and Blondeau, H. (1962).Ann. Inst. Pasteur, Paris 103,345-362. Colwell, R. R. (1970). “Culture Collections of Microorganisms” (H. Iizuka and T. Hasegawa, eds.), pp. 421-436. University Park Press, Baltimore, Maryland. Colwell, R. R. (1969).J . Gen. Microbiol. 58, 207-215. Colwell, R. R. (1970).J . Bacteriol. 104, 410-433. Colwell, R. R., and Gibbons, N. E. (1973).AntonieuanLeewenhoek;]. Microbiol. Serol. (in press). Colwell, R. R., and Gochnauer, M. B. (1968).Proc. S y m p . Microbiol. Inst. A p p l . Microbiol. Univ. Tokyo, IOth, 1968 pp. 113-136. Colwell, R. R., and Liston, J. (1961a).J . Bacteriol. 82, 1-14. Colwell, R. R., and Liston, J. (1961b).J. Bacteriol. 82, 913-919. Colwell, R. R., and Mandel, M. (1964).J . Bacteriol. 87, 1412-1422. Colwell, R. R., and Mandel, M. (1965).J . Bacteriol. 89, 454-461.

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Mutation a n d the Production of Secondary Metabolites'

ARNOLDL. DEMAIN Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts

. I. Introduction ..__. 11. A. Auxotrophic Mut B. Mutants Resistant to Toxic End Products.................. C. Mutants Resistant to Tox or to Toxic Precursors ... D. Mutants Directly Selecte ................. E. Revertants of Nonproducing Cultures ...................... F. Additional Possibilities. .......................................... 111. Elimination of Undesirable Secondary IV. Formation of New Secondary Products V. Elucidation of Biosynthetic Pathways ............................ VI. Future Prospects ......................................................... VII. Summary .................... References .........

177 178 183 189 190 190 192 192 192 193 194 196 197 199

I. Introduction Microbially produced secondary metabolites are a group of products which are extremely important to the health and nutrition of our society. As a group which includes antibiotics, toxins, and plant growth factors, they have tremendous economic importance. Secondary metabolites are usually produced during the idiophase (a phase subsequent to the growth phase or trophophase) of development, have no general function in growth processes (although they may contribute to survival of a particular producing organism), are produced b y certain restricted taxonomic groups of organisms, and are usually formed as a mixture of closely related members of a chemical family (Weinberg, 1970). Since the production of secondary metabolites appears to be affected b y the same regulatory mechanisms that control primary metabolism (Demain, 1971), i.e., induction, feedback regulation, and catabolite regulation, and because each of these mechanisms is genetically determined, it is understandable why mutation procedures have had such a major effect on the production of secondary metabolites. Indeed, it is the chief factor responsible 'Contribution No. 2050 of the Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.

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for the hundred- to thousandfold increases obtained in production of antibiotics from the time of their initial discovery to the present. An additional characteristic common to all secondary metabolites is the ease with which the microbial producers of these compounds lose their ability to form the product, i.e., “strain degeneration.” Thus, mutation is of importance for secondary product formation in the negative, as well as in the positive, direction. In this review, I shall examine some of the ways in which microbial mutants have been used to increase the production of secondary metabolites, to eliminate undesirable products, to produce new secondary products, and to elucidate secondary biosynthetic pathways. Knowledge of the genetics of secondary metabolism and the use of genetic recombination to improve production of these products have been desirable goals for many years but have not yet borne fruit and will not be considered in the succeeding pages. There is no doubt that recombination processes will some day take their proper place in industrial microbiology, but progress is slow because (a) organisms with well-known genetics are, generally speaking, not commercially important, and (b) there has been a lack of a large-scale industrial commitment to the genetic study of commercial strains. In addition to Sermonti’s excellent text (Sermonti, 1969), the following papers are recommended for a discussion of recombination in industrial microbiology: Alikhanian (1962), Calam (1964), Jones (1966), Bradley (1966), and Elander (1969). Of particular interest to the use of recombination is the demonstration by Holt and Macdonald (1968) that breeding for increased antibiotic yield is possible. Of further significance are their efforts to map the genes of penicillin biosynthesis. Another topic of importance to those interested in secondary metab. olism is the genetics of sporulation in Bacillus, since spores and especially the compounds which are specifically formed during sporulation are indeed secondary products. However, the field is really in its infancy, and it will be several years before the genetics of sporulation are well understood. In view of the fact that sporulation will not be discussed in this paper, the reader is referred to the review of Schaeffer (1969) for an excellent analysis of the sporulation process.

11. Increasing Production of Secondary Metabolites Increases in broth potencies of penicillin G from 1941 to 1969 are shown in Fig. 1. Each point represents the highest yield quoted in the literature during each year. Since this type of information is usually proprietary in nature, it would b e expected that a curve drawn from industrial yields would have a greater slope, but its shape would

179

MUTATION AND SECONDARY METABOLITES

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FIG.1. Maximum literature values for ability to produce penicillin from 1941 to 1969 (Demain, 1971).

probably be similar. The data are reported in micrograms per milliliter and can be converted to units per milliliter by multiplying by 1.67. The steep part of the curve (1941-1946) is mainly due to advances in the art of submerged fermentation. It can be seen that between 1946 and 1969 there was a slower, but still exponential, increase in penicillin yields. A similar situation is seen in Fig. 2, which describes the advances in potencies of streptomycin broths. These dramatic improvements essentially resulted from procedures involving mutagenesis followed by the testing of random survivors and of morphological (and color) mutants. Most of the penicillin-producing mutants are descendants of the Wisconsin strain, Penicillium chrysogenum Q-176, which was made available to industry in late 1945 (Stauffer and Backus, 1954).

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FIG.2. Maximum literature values for ability to produce streptomycin from 1945 to 1967 (Demain, 1971).

The most common method used to obtain high-yielding mutants is that of treating a population with a mutagenic agent until a certain “desired” kill is obtained, plating out the survivors and testing each resulting colony or a randomly selected group of colonies for product formation in flasks. It is tedious, laborious, and intellectually unsatisfying-but it has worked in the past. Another common procedure is to select the obvious morphological mutants (including those with changes in color) for testing in flasks. Although these procedures are responsible for the dramatic increase in yields in the past, many complaints are heard in the fermentation industry that it is becoming more and more difficult to obtain sGperior cultures. Workers in the fermentation industry are dismal about the continuation of the exponential rise in productivity using classical methods. It is my feeling that as

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yields of secondary products reach the 10-15 gm/liter range, new methods of selection are needed. The exponential nature of strain selection (Fig. 1) requires that a new mutant of P . chrysogenum in 1969 had to produce 1500 pglml more penicillin than its parent to show 15% superiority, whereas in 1946 only 100 pg/ml were required. Perhaps this is asking too much of the mutational process, and we should be satisfied with smaller percentage increases. However, setting a goal of 5% increase, for example, introduces other problems, such as the difficulty of detecting such small increases in the face of the known variability of fermentations. Selection of morphological mutants for testing was a popular early procedure because of its ease and convenience. A good part of the success of the University of Wisconsin program for development of superior penicillin producers was due to selection based on morphological change (Stauffer and Backus, 1954). Studies on morphological mutations in the organisms producing cycloheximide, nystatin (Spiiek et al., 1965a), and tetracyclines (Blumauerova et uZ., 1969a) indicate a definite qualitative connection between colonial morphology and production of secondary products. T h e question of increased production was not considered in these studies; however, Alikhanian et al. (1959) reported that morphological mutants of Streptomyces rimosus contained a lower percentage of superior oxytetracycline producers than did survivors with unchanged morphology. On the other hand, they found that their most outstanding high-producing mutants differed in morphology from their less productive parents. Indeed, the two key mutants of their stain selection program were morphological mutants. In certain cases, morphological mutation has also been shown to be related to the overproduction of primary metabolites. Roth et uZ. (1966) found that histidine regulatory mutants of Salmonella typhimuriurn picked b y virtue of resistance to triazolealanine, formed colonies which were wrinkled instead of smooth. In every case examined, the wrinkled phenotype was associated with derepressed levels of the histidine biosynthetic enzymes. I n many such mutants histidine is overproduced to the extent that it is excreted into the medium (Roth and Ames, 1966). Differences in colonial morphology have also been noted between strains which are “stringent” or “relaxed’’ with respect to amino acid control of ribonucleic acid synthesis (Edlin and Broda, 1968). Similarly it has been reported that a high proportion of conidial color mutants of AspergiEZus niger are superior producers of citric acid (Ilczuk, 1968). What do we know about the enzymatic basis of morphological

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mutants? Certainly nothing in the case of microorganisms which produce valuable secondary metabolites. We must turn to Neurospora crassa for some information on the basic biochemical nature of morphological mutations. In this species, the morphological change in the colonial mutant col-2 (increased branching leading to a dense, compact colony) is due to mutation in the structural gene for glucose6-phosphate dehydrogenase causing a decrease in affinity for both glucose 6-phosphate and NADP (Brody and Tatum, 1966). This structural change results in a 6- to &fold increase in the internal concentration of glucose 6-phosphate, a 5-fold increase in fructose 6-phosphate, a 2- to 3-fold increase in glucose l-phosphate, and a 50% decrease in the use of the pentose phosphate pathway (Brody and Tatum, 1967a,b). T h e resultant deficiency of NADPH is thought to be one of the major reasons for the altered morphology (Lechner and Fuscaldo, 1968). Two other distinctive types of morphological mutants, balloon and frost, also suffer from defective glucose-6-phosphate dehydrogenases (Scott and Tatum, 1969). A fourth type of colonial mutation, ragged, is due to a 90% deficiency of phosphoglucomutase and a decreased affinity of this enzyme for glucose l-phosphate and glucose 1,6-diphosphate (Brody, 1969). In all four types of morphological mutants, changes also occur in the amount of various carbohydrate polymers in the cell wall of N . crassa. It is thus clear that a single structural change in an enzyme can result in pleiotropic effects on steady-state levels of intermediate metabolites and of coenzymes, on metabolic pathways, on cell wall composition, and on morphology. Since so many antibiotics involve sugar phosphates and NADPHE in their biosynthetic paths, it is no wonder that product formation is affected, both positively and negatively, by morphological mutation. In all mutation programs, the ideal cellular form for mutagenesis is the uninucleate spore. In some antibiotic-producing species, however, the organism sporulates very poorly or not at all. In such cases, involving filamentous forms, hyphal bits are usually employed, but successful mutation is difficult owing to their multinucleate nature. In other cases, the original culture sporulates well, but as the family of higher producers is developed, sporulation becomes less and less abundant and mutation becomes more and more difficult. The recent report by Esposito et al. (1972) is very encouraging in this regard. These workers found that the nonsporogenous Streptomyces mediterranei, the producer of rifamycin, could be mutated to an abundantly sporulating form. Although the success achieved in increasing yields by mutation followed by random or morphological screening is impressive, we

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must sadly acknowledge the fact that we are completely ignorant of the basic enzymatic or regulatory changes brought about in the cell as a result of these rare “successful” mutations. If this information were known, we could devise rational screens to select only those survivors which have been mutated at such sites. It is clearly time to start examining the biochemical changes evoked in superior mutants. In the absence of such pertinent data, the next best approach is to examine the production abilities of small segments of the mutagenized population to determine whether certain groups of survivors are more likely to contain superior cultures than the overall population. Although the literature is practically devoid of such comparisons on a real scientific basis, there are bits and pieces scattered among the published works on secondary metabolites which are worthy of discussion.

A. AUXOTROPHIC MUTANTS AND REVERTANTS On the surface, it wouid appear that isolation of auxotrophic mutants would be a time-consuming and worthless method for selection of superior producers of secondary products. After all, these cultures are going to be placed in a complex fermentation medium which will supply their nutritional requirements; thus, the auxotrophs would be expected to behave just like their wild-type parents. However, this reasoning appears to be erroneous. Alikhanian et al. (1959) studied the tetracycline-producing ability of 53 auxotrophic mutants of S. rimosus, the parental cultures being capable of producing 1200-3200 pglml oxytetracycline in a corn steep medium. All the auxotrophs produced less than their parents, the vast majority making less than 25% of parental potencies. Whereas most of the auxotrophs did not show increased antibiotic production when the growth requirement was added to the complex medium, one group of 10 mutants responded dramatically to the supplementation. Yields were increased from 4-28% of the parenta