THE FILARIA
World Class Parasites VOLUME 5
Volumes in the World Class Parasites book series are written for researchers, students and scholars who enjoy reading about excellent research on problems of global significance. Each volume focuses on a parasite, or group of parasites, that has a major impact on human health, or agricultural productivity, and against which we have no satisfactory defense. The volumes are intended to supplement more formal texts that cover taxonomy, life cycles, morphology, vector distribution, symptoms and treatment. They integrate vector, pathogen and host biology and celebrate the diversity of approach that comprises modern parasitological research.
Series Editors Samuel J. Black, University of Massachusetts, Amherst, MA, U.S.A. J. Richard Seed, University of North Carolina, Chapel Hill, NC, U.S.A.
THE FILARIA
edited by
Thomas R. Klei Louisiana State University Baton Rouge, LA and T.V. Rajan University of Connecticut Health Center Farmington, CT
KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW
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TABLE OF CONTENTS
Preface
vii
Lymphatic filarial infections: an introduction to the filariae J.W. Kazura
1
Vector-Parasite Interactions in Mosquito-Borne Filariasis L.C. Bartholomay and B.M. Christensen
9
Evolutionary Relationships Among Filarial Nematodes O. Bain
21
Filarial Genomics: Gene Discovery and Gene Expression S.A. Williams and S.J. Laney
31
The Epidemiology of Onchocerciasis and the Long Term Impact of Existing Control Strategies on this Infection P. Fischer and D.W. Büttner
43
The Epidemiology of Filariasis Control E. Michael
59
Host Factors, Parasite Factors, and External Factors Involved in the Pathogenesis of Filarial Infections D.O. Freedman
75
Natural History of Human Filariasis – The Elusive Road B. Ravindran
87
In Utero Exposure to Filarial Antigens and its Influence on Infection Outcomes P.J. Lammie
97
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Immune Effectors Important in Protective Resistance A. Hoerauf
109
Immune Regulation and the Spectrum of Filarial Disease C.L. King
127
Wolbachia Bacterial Endosymbionts M.J. Taylor
143
Approaches to the Control and Elimination of the Clinically Important Filarial Diseases C.D. Mackenzie, M. Malecela, I. Mueller and M.A. Homeida
155
Vaccines For Filarial Infections Paul B. Keiser and Thomas B. Nutman
167
Index
179
PREFACE Filarial nematodes constitute an important group of human pathogens in tropical regions of the world. These parasites have an ancient lineage and have evolved to utilize blood feeding arthropods as intermediate hosts and vectors. The chronic diseases associated with these worms are generally separated into two categories; lymphatic filariasis caused by infections of the lymphatic dwelling parasites Wuchereria bancrofti and Brugia malayi, and onchocerciasis, or river blindness, caused by infections of Onchocerca volvulus. In addition to general morbidity, these infections are associated with the chronic conditions such as recurrent fevers, hydrocoel and elephantiasis caused by lymphatic filariae, and blindness and chronic skin disease cause by O. volvulus. Other filariae such as Loa loa specifically infect humans. A large number of filariae, such as the dog heart worm, Dirofilaria immitis, parasitize domestic and wild animals and it is likely that zoonotic filarial infections may alter the outcome of infections with human parasites. Nonetheless, the focus of the chapters in the book is on the causative agents and manifestations of lymphatic filariasis and onchocerciasis. With the advancement of new technologies, the understanding of the biology and epidemiology of, and host responses to these agents is changing rapidly. These diseases are of a spectral nature, with many infected individuals showing no overt clinical signs, while others suffer the consequences of chronic disease. In the past, explanations for these differences have focused on the polarization of immune responses to these agents into either Th1 or Th2 types. New thoughts are emerging on immune regulation, as well as on nonimmune factors involved in the pathogenesis of these infections. Not surprisingly, our understanding of the host parasite interaction is becoming more complex. For many years, the understanding of filarial infections and the biology of these worms has been hampered by the absence of ideal animal models. New model systems, using murine hosts, have recently been utilized. These include the introduction of nonhuman parasites into susceptible mice and B. malayi or O. volvulus infections of a variety of genetically modified mouse strains. It remains to be seen, however, how easily concepts developed in these systems will be translated into knowledge relevant to human infections. Discussions of host response results from both experimental and field studies are integrated throughout this volume. Although most studies in this field focus on the vertebrae host, exciting new information is emerging on interactions in the mosquito intermediate hosts for Brugia. This is in part due to advances in the ability to genetically manipulate these hosts. The results of these studies may have some impact on control programs in the future. Although known to exist for many years, exciting new observations on the importance of the endosymbiont Wolbachia on parasite development and its potential role in pathogenesis are under way in many laboratories. These studies will likely
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change our understanding on many aspects of filarial infections and may lead to new chemotherapeutic regimes. Molecular technologies are generating genetic information on these parasites at an astonishing rate. The usefulness of these data is just beginning to be realized and this genetic information seemingly has unlimited potential. However, in this regard, long-term efforts to identify parasite proteins, which would be logical targets for vaccine development, have failed in general. It is uncertain if the expanded genomics database and knowledge of vaccinology may change this outcome in the near future. Similarly, long-term efforts to identify a safe and effective anthelmintic, active against the adult parasites, have not been successful. However, recent data on the use of multiple drug treatments may soon change these thoughts in lymphatic filariasis and rational approaches using the growing base of genetic information may also have a significant overall impact. Recently the usefulness of such an adultacide has been questioned. Nonetheless, even in the absence of such a drug, treatment strategies to control onchocerciasis using ivermectin, which is effective against the microfilarial stage of the parasite, have had a major impact in reducing the morbidity of this infection in some regions of Africa. Strategies using combinations of microfilarialcidal drugs have more recently been developed to eliminate the causative agents of lymphatic filariasis. This plan is being implemented through the combined efforts of several international agencies and nongovernment organizations. The utilization of mathematical models offers a unique potential aid in the development, implementation and assessment of such plans. If successful, its impact on human health in endemic regions will be enormous. Questions exist, however, on the potential success of this approach in all regions of the world. Further concerns exist on the potential negative impact of this campaign on the continuation of basic and applied research on these diseases. This book is designed to provide the reader with brief insights and opinions of experts in this field. The chapters cover concepts which in most instances overlap all of the filariae and largely focus on new ideas which are incompletely defined and/or may as yet not be broadly accepted. The summaries, and innovative and provocative thoughts put forward hopefully will stimulate future directions of investigation. For those of us who have studies these parasites and the disease they cause for some years, it is an exciting time. We hope this volume extends this excitement to the reader. Thomas R. Klei and T.V. Rajan, January 24, 2002
LYMPHATIC FILARIAL INFECTIONS: AN INTRODUCTION TO THE FILARIAE
James W. Kazura Professor of Medicine and International Health Division of Geographic Medicine Case Western Reserve University School of Medicine University Hospitals of Cleveland, Cleveland, OH USA.
ABSTRACT: Among the large number of nematode parasites for which humans are the definitive host, lymphatic filariae are among those with the greatest medical and public health significance. In contrast to geohelminths such as Ascaris and Trichuris species that primarily affect children, Wuchereria bancrofti, Brugia malayi, and Brugia pahangi have their most obvious clinical impact during adulthood. Accordingly, lymphatic filariasis significantly decreases the socioeconomic status of affected communities. This is reflected in objective measures such as Disability Adjusted Life Years (DALY) (Coreil et al., 1998; Haddix et al., 2000) This introductory chapter will describe the salient biologic and epidemiological features of human lymphatic-dwelling filariae that have enabled them to be highly successful in maintaining the complex ecologic niche that involves interaction between the definitive vertebrate host and the obligatory mosquito vector. Issues of particular relevance to control strategies and future directions for research will be highlighted. More detailed discussion of protective immunity, the immunology and pathogenesis of disease, prospects for eradication, and filarial biology are presented in other chapters. Keywords: Filariasis, Wuchereria, Brugia.
GENERAL BIOLOGY AND LIFE CYCLE OF HUMAN FILARIAL PARASITES A number of filarial nematode species utilize humans as their definitive host. These include the genera Onchocerca, Mansonella, Loa, Wuchereria, and Brugia. This chapter will provide an overview of lymphatic filariae and the complexity of diseases associated with these parasites as a means of introduction to this group of nematodes. The primary natural host for W. bancrofti, B. malayi, and B. timori is Homo sapiens. Cats and Mongolian jirds (Meriones unguiculatus) may also be infected with Brugia species. Although brugian filariasis may therefore be considered a zoonosis, non-human hosts do not appear to be an important
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reservoir of human filariasis in nature. Some primates have been experimentally infected with W. bancrofti. Human filarial infection is initiated when infective or third-stage larvae (L3) are released from the proboscis of female mosquitoes during the process of blood feeding. Unlike malaria parasites, several genera of mosquitoes may serve as vectors for filariae. In addition to Anopheles species, Culex, Aedes, and Mansonia mosquitoes can transmit W. bancrofti and/or B. malayi. There are marked differences in the competence of various mosquito species to act as vectors, and these properties are under genetic control (Severson et al., 1999). Following deposition on the skin, proteases and other enzymes are secreted by L3, allowing penetration through local connective tissue and migration of the parasite to local lymphatic vessels (Maizels et al, 2001 ). 8 ± 1 days after entry, L3 molt (i.e., shed their cuticle) and fourthstage larvae (L4) appear. These developmental events are critical in the parasite’s life cycle as failure to penetrate and develop in lymph vessels eliminates the possibility to continue transmission. Subsequent development of L4 to adult worms occurs over a period of two to twelve months. Immunogenic molecules secreted during this stage of life cycle may induce and recall allergic-type responses (Selkirk et al, 1993). Sexually mature male and female adult worms residing in afferent lymphatic vessels copulate, and fecund female worms subsequently release embryonic forms (first-stage larvae or L1). These larvae presumably enter the blood stream after passing through from the local lymphatic circulation into the thoracic duct. A morphologic feature of Wuchereria and Brugia L1, commonly referred to as microfilariae, that distinguishes them from other human filariae such as Onchocerca volvulus and Loa loa is the sheath. This surface structure is a remnant of the embryonic eggshell. The median reproductive life span of adult W. bancrofti worms is estimated to be four to six years, whereas microfilariae live for one year or less. The density of microfilariae in the peripheral blood stream is noteworthy in its temporal profile or periodicity. In most areas of the world, microfilariae have a nocturnal periodicity such that large numbers of parasites are present in the peripheral blood at night, coincidental with the peak biting time of the local mosquito vector. During the day, when mosquito vectors are not feeding, microfilariae are not present in peripheral venous blood but are sequestered in deep vascular beds. The molecular basis of microfilarial periodicity and its similarity to biologic ‘clocks’ of other organisms remain obscure. Both innate host and parasite factors are presumably involved as periodicity can be affected by change in the human sleep cycle. Completion of the life cycle takes place in the mosquito, which ingests microfilariae included in the blood meal. Ingested microfilariae exsheath, penetrate the gut of the insect, and molt to form second-stage larvae (L2). The transition to L3 and the capacity to transmit infection to the human host is completed within a period of 10 to 14 days. Of particular significance to the control and pathogenesis of human filariasis is the fact that the host
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adult worm burden depends on the intensity of exposure to L3 (Kazura et al., 1997; Norman et al., 2000; Tisch et al., in press). Thus, transmission has a major impact on the propensity to develop infection and disease both at the individual and population level.
CLINICAL MANIFESTATIONS OF HUMAN FILARIASIS AND THEIR PUBLIC HEALTH SIGNIFICANCE As is the case with other helmithiases, it is important to distinguish between infection and disease. Infection specifically refers to the establishment of worms in the human host whereas disease indicates that the infection has in some manner caused a host pathologic response.. The most widely recognized clinical manifestations of W. bancrofti infection are related to lymphatic dysfunction that is manifest as lymphedema and swelling of the arm or leg (i.e. colloquially referred to as elephantiasis in its most extreme degree), the breast, or male genitalia (e.g., hydrocele, thickening of the vas deferens, and/or skin of the scrotum). Disease due to Brugia infection differs from that to W. bancrofti in that hydroceles generally do not occur in the former. Less common but nevertheless medically important manifestations of human filariasis include chyluria secondary to dysfunction of the lymphatics draining the renal pelvis and tropical (pulmonary) eosinophilia. The latter is most frequently been reported from areas of India and to a lesser extent, Africa. The pentad of nocturnal wheezing, eosinophilia (often greater than 5000 per blood), lack of microfilaremia, elevated IgE, and improvement following administration of the anti-filarial drug diethylcarbamazine typify the latter syndrome (Ottesen and Nutman, 1992). If untreated, tropical eosinophilia may result in the development of restrictive pulmonary disease with interstitial fibrosis. It is important to note that the majority of infected individuals living in endemic areas do not exhibit overt clinical signs of lymphatic filariasis. Even in areas of the world where disease is common, generally less than ten percent of the population at risk have overt lymphedema of the leg. Nevertheless, this apparently asymptomatic state should not be construed as without medical significance. Lymphoscintiographic imaging of the legs and ultrasound examination of the architecture of the lymphatic vessels of the male genitalia indicate that many such cases have abnormal lymphatic function (Dreyer et al., 2000). These subtle manifestations of infection may well compromise the normal defenses of the skin against common bacterial flora and those introduced by minor injuries. In addition, immunologic alterations secondary to chronic filarial infections likely influence immunity to non-helminthic microbes and vaccines (Malhotra et al., 1999; Gopinath et al, 2000). Thus, the impact of filariasis and its control extends beyond specific syndromes attributable to the parasite itself. In addition to the chronic disease manifestations described above, filarial infection may cause acute inflammation of an extremity (acute
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adenolymphangitis) and the male genitalia (e.g., epididymitis, funiculitis). These syndromes generally last two to four weeks and resolve slowly. Their pathogenesis is poorly understood. The propensity to develop acute inflammatory disease is directly related to transmission intensity and may be due to allergic-type responses to antigens secreted by migrating L3 or L4 and secondary bacterial infections (Alexander et al, 1999; King et al., 2001). It is believed that multiple episodes of acute inflammatory disease contribute to the development of chronic lymphatic obstruction of the extremities. A link between acute inflammatory conditions of the scrotal contents and hydrocele formation has not been established.
EPIDEMIOLOGY OF HUMAN LYMPHATIC FILARIASIS W. bancrofti is endemic throughout subtropical and tropical areas of Africa, Asia, islands of the Western and South Pacific, and selected areas of the Caribbean Sea and South America. B. malayi infection is more limited in its distribution. It is found only in Asia, particularly India (e.g., Kerala), Indonesia, and the Philippines. B. timori is confined to the Timor Islands. It is estimated that approximately 120 million persons in the world are infected with W. bancrofti and B. malayi, with many more at risk (Michael et al., 1996). The majority of cases are in India and Africa. Historically, W. bancrofti was endemic in many regions of the Americas, including the southern United States. Coincidental with improved sanitation and decreases in the abundance of the mosquito vector, the distribution became more limited. Foci currently remain in Haiti and coastal equatorial Brazil. Ironically, bancroftian filariasis is spreading in other areas of the world, particularly Africa and South Asia. This is in large measure due to migration of human populations from rural to urban areas of developing countries and the propagation of breeding sites for ‘urban’ culicine mosquitoes (e.g., standing water that collects in empty cans and other human refuge that is disposed of improperly). Intensive use of anti-filarial chemotherapy is reported to have eliminated filariasis from most areas of China (Xu et al., 1997). There are several epidemiologic features of filariasis that provide insight into the pathogenesis of lymphatic disease and control strategies. First, studies from multiple endemic areas indicate there is an age-related increase in the rates of infection (determined by microfilaremia or the presence of circulating W. bancrofti antigen) and lymphatic pathology. This implies that repeated exposure to L3 and/or cumulative adult worm burden correlate with the propensity to develop disease. Second, despite the age-related increase in infection and worm burden, only a minority of infected individuals has overt disease manifestations such as elephantiasis. This heterogeneity in disease pattern may be due to individual differences in immune responses, the intensity or pattern of exposure to L3, secondary bacterial infections, and polymorphisms of genes not directly involved in antigen-specific immunity ( de Almeida et al., 1996; Dreyer et al., 2000; King et al., 2001). Third, at a
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population level, there is a direct relationship between transmission intensity and the prevalence of lymphedema of the extremity but not hydrocele (Kazura et al., 1997; Tisch et al., in press). This suggests that the pathogenesis of the two conditions differ. Finally, infection burdens measured by the density of microfilaremia or level of filarial antigenemia tend to increase up to the age of approximately 30 years and then plateau. It is not clear if this ‘leveling off’ in worm burden represents acquisition of partial resistance against ‘new’ incoming L3 (e.g., concomitant immunity) or an inherent limitation of the number of worms that can be established in the host.
CONTROL OF HUMAN FILARIASIS Over the past several years there has been a resurgence of interest in the possibility that human filariasis can be eliminated as a public health problem or even eradicated by mass distribution of annual or semi-annual single dose therapy with safe and inexpensive anti-filarial medications such as diethlycarbamazine (DEC), ivermectin, and albendazole alone or in various combinations (Dean, 2000; Horton et al., 2000). This goal is remarkable in view of earlier pessimism that drugs such as DEC could not be delivered in a manner that led to decreases in infection load. It was previously believed that the drug did not kill adult worms and that it had to be given daily over a period of 10 to 14 days. It is now clear that DEC kills a portion of adult W. bancrofti and that single doses are as effective as multiple doses in decreasing microfilaremia (Dreyer et al., 1998; Freedman et al., 2001). Single annual doses of DEC alone or in combination with ivermectin have been shown to reduce microfilaremia, transmission, and reverse hydroceles in rural areas where anopheline mosquitoes are the major vectors of W. bancrofti (Meyrowitsch et al., 1996; Bockarie et al., 1998). Similar studies using albendazole with ivermectin are underway in areas of Africa where DEC cannot be used because onchocerciasis is endemic. DEC is contraindicated in this situation because it may lead to an acute decrease in visual acuity when O. volvulus microfilariae in the cornea are killed. The enthusiasm generated by publicity surrounding the goal of controlling lymphatic filariasis on a global scale poses several challenges to both basic and applied research. First, despite recent reports that single dose chemotherapy decreases microfilaremia and transmission potential by more than 90 percent compared with pre-treatment levels, there is as yet no convincing evidence that transmission of W. bancrofti can be eliminated completely. Thus, it is not known how long mass chemotherapy should be administered. Based on the assumption that the median reproductive life-span of adult W. bancrofti is approximately five years, it is believed that at least four to six annual drug treatments will be required. However, it appears that even repeated doses of anti-filarial medications such as DEC do not ‘cure’ infection as documented by conversion of circulating antigen-positive to negative status or by ultrasound detection of living worms in the scrotal contents (our unpublished data, 20,21). These findings underscore the need
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for continuing development of newer compounds that more effectively kill adult parasites. Second, the enthusiasm generated by global programs for eradication should not be taken as an indication to abandon research on the basic biology of filariasis and vaccine development. On the contrary, the recent ‘rediscovery’ of Wolbachia organisms as filarial endosymbionts – observations that largely resulted from the filarial genome project – is a good example of how basic research may quickly be translated to the field situation. Wolbachia are highly susceptible to tetracycline, and the drug has been shown to decrease the reproductive potential and viability of Onchocerca female worms (Taylor, 2000; Hoerauf et al., 2001). In the context of vaccine development, the recent generation of sensitive assays to detect circulating antigens has enabled more precise classification of worm loads than was previously possible. Thus, there are now improved methods to classify persons in endemic areas according to their infection and a clearer definition of individuals who are ‘putatively immune’ than was possible when microfilaremia was the only objective measure of this parameter (Chanteau et al, 1994; Weil et al., 1996). Third, valid endpoints to assess the progress and success of filarial control programs remain to be established. Several have been proposed, such as detection of filarial infection in pools of mosquito vectors by polymerase chain reaction, antigenemia (especially in children), and community disease rates. Finally, unlike the case for bancroftian filariasis, anti-filarial medications such as DEC appear to be relatively less effective and lead to more severe side effects in brugian than bancroftian filariasis. In addition, assays specific for circulating antigens of B. malayi are not available. Thus, monitoring of the efficacy of control efforts against this parasite are limited to detection of microfilaremia. Development of drugs effective against B. malayi and detection of circulating antigen assays for this parasite are thus needed.
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Haddix, A. C., and A, Kestler. 2000. Lymphatic filariasis: economic aspects of the disease and programmes for its elimination. Transactions of the Royal Society of Tropical Medicine and Hygiene 94:592-3. Coreil, J., G. Mayard, J. Louis-Charles, and D. Addiss 1998. Filarial elephantiasis among Haitian women: social context and behavioural factors in treatment. Trop Med Int Health 3:467-73. Severson, D.W., D. Zaitlin, and V.A. Kassner. 1999. Targeted identification of markers linked to malaria and filaroid nematode parasite resistance genes in the mosquito Aedes aegytpii. Genet Res 73:217-24. Maizels, R. M., N. Gomez-Escobar, W. F.Gregory, J. Murray , and X. Zang. 2001. Immune evasion genes from filarial nematodes. International Journal of Parasitology 31:889-98. Selkirk, M. E., W. F. Gregory, R. E. Jenkins , and R. M. Maizels. 1993. Localization, turnover, and conservation of gp15/400 in different stages of Brugia malayi. Parasitology 107:449-57. Kazura, J. W., M. Bockarie, N. Alexander, R. Perry, F. Bockarie, H. Dagoro, Z. Dimber, P. Hyun, and M. P. Alpers. 1997. Transmission intensity and its relationship to infection
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and disease due to Wuchereria bancrofti in Papua New Guinea. Journal of Infectious Diseases 176:242-6. Tisch, D., F. E. Hazlett, M. P. Alpers, W. Kastens, M. Bockarie, and J. W. Kazura. Epidemiologic and ecologic determinants of filarial antigenemia in a Wuchereria bancrofti endemic area of Papua New Guinea. Journal of Infectious Diseases (in press). Norman, R. A., M. S. Chan, A. Srividya, S. P. Pani, K. D. Ramaiah, P. Vanamil, E. Michael, P. K. Das, and D. A. Bundy. 2000. EPIFIL: the development of an agestructured model for describing the transmission dynamics and control of lymphatic filariasis. Epidemiol Infect 124:529-41. Ottesen, E. A., and T. B. Nutman. 1992. Tropical pulmonary eosinophilia. Ann Rev Med 43:417-24. Dreyer, G., J. Noroes, J. Figueredo-Silva, and W. F. Piessens. 2000. Pathogenesis of lymphatic disease in bancroftian filariasis: a clinical perspective. Parasitology Today 16:544-8. Malhotra, I., P. Mungai, A. Wamachi, J. Kioko, J. H. Ouma , J. W. Kazura, and C. L. King. 1999. Helminth- and Bacillus Calmette Guerin-induced immunity in children sensitized in utero to filariasis and schistosomiasis. Journal of Immunology 162:6843-8. Gopinath, R., M. Ostrowski, Justement, A. S. Fauci, and T. B. Nutman. 2000. Filarial infections increase susceptibility to human immunodeficiency virus infection in peripheral blood mononuclear cells in vitro. Journal of Infectious Diseases 182:1804-8. Alexander, N. D., R. T. Perry, Z. B. Dimber, P. J. Hyun, M. P. Alpers, and J. W. Kazura. 1999. Acute disease episodes in a Wuchereria bancrofti endemic area of Papua New Guinea. American Journal of Tropical Medicine and Hygiene 61:319-24. King, C. L., M. Connelly, M. P. Alpers, M. Bockarie, and J. W. Kazura. 2001. Transmission intensity determines lymphocyte responsiveness and cytokine bias in human lymphatic filariasis. Journal of Immunology 166:7427-36. Michael, E., D. A. Bundy, and B. T. Grenfell. 1996. Re-assessing the global prevalence and distribution of lymphatic filariasis. Parasitology 112:409-28. Xu, B., Z. Cui, Y. Zhang, J. Chang, Q. Zhao, Q. Huang, and X. Lin. 1997. Studies on the transmission potential of filariasis in controlled areas of Henan Province. China Med Journal 110:807-10. de Almeida, A. B., M. C. Maia e Silva, M. A. Maciel, and D. O. Freedman. 1996. The presence or absence of active infecdion, not clinical status, is most closely associated with cytokine responses in lymphatic filariasis. Journal of Infectious Diseases 173:1453-9. Dean, M. 2000. At last, the fight against lymphatic filariasis begins. Lancet 355:385. Horton, J., C. Witt, E. A. Ottesen and 19 coauthors. 2000. An analysis of the safety of the single dose, two drug regimens used in programmes to eliminate lymphatic filariasis. Parasitology 121 (supplement): 147-60. Dreyer, G., D. Addiss, A. Santos, J. Figueredo-Silva, and J. Noroes. 1998. Direct assessment in vivo of the efficacy of combined single-dose ivermectin and diethylcarbamazine against adult Wuchereria bancrofti. Transactions of the Royal Society of Tropical Medicine and Hygiene 92:219-22. Freedman, D.O., D. A. Plier, A. B. de Almeida, A. L. de Oliveira, J. Miranda , and C. Braga. 2001. Effect of aggressive prolonged diethylcarbamazine therapy on circulating antigen levels in bancroftian filariasis. Tropical Med International Health 6:37-41. Meyrowitsch, D. W. , P. E. Simonsen, and W. H. Makunde. 1996. Mass DEC chemotherapy for control of bancroftian filariasis: comparative efficacy of four strategies two years after treatment. Transactions of the Royal Society of Tropical Medicine and Hygiene 90:423-8. Bockarie, M. J., N. D. Alexander, P. Hyun, Z. Dimber, F. Bockarie, E. Ibam, M. P. Alpers, and J. W. Kazura. 1998. Randomised community-based trial of annual singledose diethylcarbamazine with or without ivermectin against Wuchereria bancrofti in human beings and mosquitoes. Lancet 351:1662-8.
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24. Taylor, M. J. 2000. Wolbachia bacteria of filarial nematodes in the pathogenesis of disease and as a target for control. Transactions of the Royal Society of Tropical Medicine and Hygiene 94:596-8. 25. Hoerauf, A., S. Mand, O. Adjei, B. Fleischer, and D. W. Buttner. 2001. Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet 357:1415-6. 26. Chanteau, S., J. P. Moulia-Pelat, P. Glaziou, N. L. Nguyen, P. Luquiaud, P. Plichart, C. Plichart, P. M. Martin, and J. L. Cartel. 1994. Og4C3 circulating antigen: a marker of infection and adult worm burden in Wuchereria bancrofti filariasis. Journal of Infectious Diseases 170:247-50. 27. Weil, G. J., R. M. Ramzy, R, Chandrashekar, A. M. Gad , R. C. Lowrie Jr, and R. Faris. 1996. Parasite antigenemia without microfilaremia in bancroftian filariasis. American Journal of Tropical Medicine and Hygiene 55:333-337.
VECTOR-PARASITE INTERACTIONS MOSQUITO-BORNE FILARIASIS
IN
L.C. Bartholomay and B.M. Christensen Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1656 Linden Drive, Madison WI 53706
ABSTRACT Transmission of the causative agents of lymphatic filariasis is dependent on the availability of susceptible mosquito hosts. Mosquito susceptibility is a heritable trait, but genes that control susceptibility have not yet been identified. Likewise, nothing is known about gene expression in filarial worms as it relates to adaptation of the parasite to the mosquito host environment. Mosquitoes are equipped with several means to prevent the establishment of developing parasites. Each environment that worms encounter within the mosquito, including the mouthparts, midgut, hemolymph, and thoracic muscles, can be an obstacle to further development. Of particular interest in present research efforts are genes and gene products expressed in these tissues that could be manipulated to alter susceptible phenotypes to a refractory state. The technology to manipulate mosquito genomes now exists, and has the potential to generate refractory populations that could be used in integrated control strategies. Keywords: mosquito, vector competence, Brugia malayi, Wuchereria bancrofti, susceptibility, genetics.
INTRODUCTION As we strive to eliminate lymphatic filariasis, it is important to remember that mosquitoes are obligate intermediate hosts, essential for the maintenance and transmission of filarial worms. Increasing our understanding of the genetic interplay between mosquito and parasite has the potential to reveal new strategies for disrupting transmission and enhance the efficacy of antihelminthic treatments as a control strategy. These types of studies in vector biology have been facilitated by (1) the development of the Mongolian gerbil, Meriones unguiculatus, as a laboratory host for Brugia species (Ash and Riley, 1970), and (2) the selection of a strain of Aedes aegypti susceptible to B. malayi and B. pahangi, therefore providing a model mosquito for subsequent studies (Macdonald, 1962a). The result has been a wealth of information on the genetic basis for susceptibility and numerous studies on the biological and biochemical relationships between parasite and
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mosquito that contribute to a compatible (susceptible) or incompatible (refractory) association. A primary emphasis of these studies in recent years has been an understanding how mosquitoes mount defense responses capable of killing ingested filarial worms. With the development of viral transducing vectors and transformation technology for mosquitoes, it is possible to address specific questions on the influence of mosquito gene expression on parasite development. In the foreseeable future, defense responses could be manipulated to generate a filarial worm refractory mosquito population to be used in integrated control strategies.
PARASITE DEVELOPMENT IN THE MOSQUITO Microfilariae (mf) of Wuchereria bancrofti and B. malayi circulate in the blood of vertebrate hosts, often with a periodicity corresponding to peak feeding times for vector species, and are ingested when a female mosquito takes a blood meal. Development within the mosquito is dependent upon the morphological, physiological and biochemical compatibility of the mosquito vector for the parasite. In susceptible mosquito species, mf travel through the mouthparts and foregut to the mosquito midgut with a blood meal. Within hours, mf traverse the single cell layer of the midgut epithelium to enter the hemolymph (Figure 1). The mf can exsheath during their tenure in the midgut (Chen and Shih, 1988). In other cases, the sheath is damaged during the migration across the midgut, facilitating exsheathment in the hemocoel (Christensen and Sutherland, 1984). Parasites that remain in the midgut then make their way to the thoracic musculature and differentiate into sausageshaped first larval stage (L1) intracellularly (Figure 1). After molting twice, L3s migrate to the head tissues and proboscis to be transmitted during a subsequent blood feeding. Unlike malaria parasites that are injected with saliva into a host, infective-stage filarial worms actively break out of the proboscis within a drop of hemolymph and must find and enter the puncture wound made by the mosquito, hair follicles, or other abrasions, making transmission highly inefficient. Under optimal temperature conditions, the
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developmental process takes approximately 10–12 days, during which time parasites increase in size 4–6 times. Filarial worm development in the mosquito is not a benign process. Worms inflict damage on their mosquito hosts as they traverse the midgut, and while developing in the thoracic muscles, impairing movement and flight. Consequently, developing parasites disable or even kill their mosquito hosts. Despite being pathogenic to mosquitoes, it is unlikely that filarial worms constitute a selective pressure for refractoriness because, even in highly endemic areas, the prevalence of infection rarely exceeds 1% of the total mosquito population.
MODELS AND MOSQUITO MANIPULATIONS Laboratory studies of the interaction between mosquitoes and filarial worms commonly utilize the Aedes aegypti-Brugia species model. Techniques for doing so have been described in detail by Townson (1997). MOSQUITOES Natural vectors of filarial parasites primarily include mosquitoes in the genera Aedes, Anopheles, Culex, and Mansonia, but much of the research done on the genetics of susceptibility of mosquitoes to filarial parasites utilized selected strains of Ae. aegypti. Although not a common natural vector of filarial parasites, Ae. aegypti is easily reared in the laboratory and both classic and molecular maker linkage maps of the genome exist (Munstermann, 1990, Severson et al., 1993, Antolin et al., 1996,). Armigeres subalbatus has been the subject of intense research on the immune response of mosquitoes to filarial worms because this mosquito is naturally refractory to B. malayi by virtue of a strong melanotic encapsulation response, but is susceptible to B. pahangi (Yamamoto et al., 1985). Several routes of introduction are possible to infect mosquitoes for experimental purposes in the laboratory: (1) mosquitoes can feed directly on infected experimental hosts, (2) parasites can be presented to mosquitoes through an artificial membrane on a glass feeding apparatus, (3) parasites can be inoculated directly into the hemocoel using pulled glass capillary needles (allowing an assessment of parasite development in the absence of a blood meal), and (4) parasites can be injected into the midgut through the hindgut using pulled capillary needles, allowing for the assessment of parasite development in the absence of exposure to the cibarial armature and salivary gland secretions. EXPERIMENTAL HOSTS B. malayi and B. pahangi are easily maintained in gerbils, and can also be maintained in cats and dogs. Unfortunately, a convenient animal model for W. bancrofti does not exist; therefore, experimental infections of mosquitoes require a source of infected human blood. In laboratories where a source of blood from infected individuals is unavailable, cryopreserved mf in blood can provide a source of parasites (see Bartholomay et al., 2001).
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GENETICS OF SUSCEPTIBILITY Only a few species of mosquitoes are capable of supporting the development of filarial worms, and susceptibility can vary between strains within species. Macdonald (1962a), using Ae. aegypti and B. malayi as model organisms, was the first to demonstrate that susceptibility to filarial worms was a heritable trait. The primary locus controlling susceptibility, designated (filarial susceptibility, B. malayi), is sex-linked and recessive; consequently, a refractory strain with the dominant (F,F) or heterozygous (F, genotype is readily selected from the same parental strain (Macdonald, 1962b). The same locus also influences Ae. aegypti susceptibility to subperiodic and periodic strains of both B. malayi and W. bancrofti but not Dirofilaria species, which exhibit a different developmental strategy (Macdonald and Ramachandran, 1965). The molecular genetic basis for susceptibility has recently been reviewed by Beerntsen et al. (2000) and Severson et al (2001). Using restriction fragment length polymorphism (RFLP) markers, Severson et al. (1994) identified a quantitative trait locus (QTL) (fsbl) that influenced susceptibility in a recessive manner (the gene) and a second QTL (fsb2) that seems to affect fsbl in an additive manner. Furthermore, the intensity of infection is influenced by another gene identified as QTL idb[2,LF181], which is linked to fsb2, and influences ingestion ability and midgut penetration (Beerntsen et al., 1995). The challenge now is to identify the genes associated with the above loci. The search could be facilitated if the Ae. aegypti genome were to be sequenced. The applicability of mechanisms controlling susceptibility in Ae. aegypti to natural vector-pathogen interactions is questionable. For example, Cx. pipiens pipiens, a natural susceptible vector for W. bancrofti, was recently selected for increased susceptibility (Farid et al., 2000). Susceptibility in this population was reduced when selection pressure was not provided, and it was not possible, even with pair-wise mating strategies, to select a refractory strain. These findings suggest that the genetic basis of susceptibility in this vector species is quite different and more complex than that of Ae. aegypti. Furthermore, strains of Cx. pipiens that are susceptible to W. bancrofti are refractory to B. malayi (unpublished). Unfortunately, efforts to understand the genetic basis of susceptibility have largely ignored the role of parasite genetic polymorphisms in the filarial worm-mosquito association—a role that is obviously not trivial. Evidence suggests that different strains of parasites are differentially infective to the same mosquito species (Wharton, 1962), and Laurence and Pester (1967) clearly illustrated that one can use selection strategies with a filarial worm to increase its ability to infect a particular mosquito species.
BARRIERS TO PARASITE DEVELOPMENT
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Within the mosquito, each of the organs and tissues that filarial parasites encounter potentially serve as barriers to further development. These barriers affect the compatibility of the vector-pathogen association (vector competence). Blood containing mf moves through the proboscis by pumping action created by the cibarial and pharyngeal pumps. In some mosquito species, the pumps are lined with denticulate structures and spines that can fatally damage passing mf (see McGreevy et al., 1978). In most cases, mf that reach the midgut unscathed rapidly traverse its single epithelial layer to arrive in the hemocoel; thus, the peritrophic matrix, which is not completely formed for at least 12 hours after a blood meal, is not a barrier to development of filarial parasites as it can be for malaria parasites. But the midgut environment can affect vector competence for filarial worms. Coagulation of the blood meal can hinder the migration across the midgut because parasites are immobilized. Consequently, the anti-coagulant potency of mosquito saliva in the blood meal can influence vector competence. Cx. pipiens pipiens, while susceptible to W. bancrofti, is completely refractory to B. malayi, mf of which are killed by unknown factors in the midgut. However, B. malayi mf injected into the hemocoel (bypassing the midgut) develop to the infectious stage (unpublished). In this system, therefore, refractoriness resides in the midgut lumen only, but the factors responsible for the observed effect are unknown. Once in the hemocoel, mf are immersed in the hemolymph, which consists of plasma and blood cells called hemocytes. By unknown means, filarial worms are sometimes recognized as foreign and elicit a hemocytemediated immune response known as melanotic encapsulation. Although the means of recognition are not understood, this response can be highly specific, i.e., in Ar. subalbatus, ingested B. pahangi develop but B. malayi are killed by a strong melanotic encapsulation response (Yamamoto et al., 1995). Alternatively, B. pahangi may be capable of suppressing or evading the immune response (Beerntsen et al., 1989). The mf sheath may function in the recognition process. Exsheathed B. pahangi mf intrathoracically inoculated into Ae. aegypti elicit significantly reduced melanization response in comparison to sheathed mf (Sutherland et al., 1984), and Chen and Laurence (1985) showed that cast sheaths in the hemocoel can be encapsulated. Melanization of these cast sheaths may function to divert the immune response from exsheathed mf (Agudelo-Silva and Spielman, 1985). Unlike the mechanism of recognition, the biological and biochemical processes leading to melanotic encapsulation have largely been elucidated and characterized (Figure 2). The process begins when melanotic materials are deposited on a filarial worm, which becomes encased in a dark and hardened capsule. Melanized capsules may prevent nutrient uptake by the parasite (Chen and Chen, 1995) or subject parasites to reactive oxygen species (Nappi and Ottavani, 2000), ultimately killing the parasite. The pathway of melanin biosynthesis involves a complex cascade of reactions beginning with tyrosine and ending in the polymerization of the capsule (Figure 1). The biochemistry
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of these reactions has been elucidated (reviewed in Paskewitz and Christensen, 1996, and Beerntsen et al., 2000), revealing the necessity of phenol oxidase (PO) at several junctures of the pathway. The importance of a specific PO in melanin biosynthesis was verified using a dsSIN virus constructed with an antisense RNA targeted to the copper-binding region of Ar. subalbatus As-proPO-I Ar. subalbatus mosquitoes infected with this virus had reduced PO activity in the hemolymph and the melanotic encapsulation response was almost completely inhibited (Shiao et al., 2001). Recently, an additional enzyme that converts phenylalanine to tyrosine, phenylalanine hydroxylase (PAH), has been cloned from Ae. aegypti and Ar. subalbatus, and its transcription is upregulated in Ae. aegypti mosquitoes that are actively melanizing mf (unpublished). PAH likely functions to provide additional tyrosine, the rate-limiting substrate, for melanin biosynthesis. This reaction requires the cofactor tetrahydrobiopterin that is generated by dihydropterin reductase (DHPR). DHPR has been identified in an expressed sequence tag (EST) library from B. malayi-infected Ar. subalbatus (unpublished).
In addition to melanotic encapsulation, melanin biosynthesis is critical for egg chorion tanning. Thus, following a blood meal containing pathogens, the processes of egg development and melanotic encapsulation must compete for resources. Ar. subalbutus infected with B. malayi elicit a strong melanization response and are compromised in terms of egg development. By melanotically encapsulating parasites these mosquitoes, though less fit reproductively, avoid the damage inflicted by developing
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worms (Ferdig et al., 1993). The observed fitness cost of melanotic encapsulation must be considered if attempts are made to engineer mosquitoes to enhance the melanotic encapsulation response. In addition to the hemocyte-mediated immune response, an induced humoral response can adversely affect the development of filarial worms. Mosquitoes rapidly respond to bacterial insult with a potent array of antimicrobial peptides, such as defensins and cecropins, produced by and exported primarily from the fat body (reviewed for Ae. aegypti by Lowenberger, 2001). Our understanding of the regulation of antimicrobial peptide genes was recently reviewed by Hoffman et al. (1999) and, though fascinating for its analogies to vertebrate innate immunity, will not be discussed here. Lowenberger et al. (1996) demonstrated that the humoral immune response, (induced by intrathoracic inoculation of bacteria into Ae. aegypti and followed by exposure to B. malayi) resulted in significantly reduced intensity and prevalence of infection. Synthetic cecropins from another insect were found to have adverse affects on parasite motility in vitro and on development in vivo in Ae. aegypti when the peptide and worms were co-injected (Chalk et al., 1995). Purified defensin from Ae. aegypti coinjected with B. pahangi similarly affected parasite development (Albuquerque and Ham, 1996). Using viral transducing vectors and transformation technologies (discussed below), studies are underway to target the specific effects of defensin and cecropin on developing parasites. Parasites that avoid cell-mediated and humoral responses and reach the thoracic muscles may still fail to develop. Host gene expression, or lack thereof, responsible for this phenomenon has not been identified.
NEW TECHNOLOGIES FOR MOSQUITO GENOME MANIPULATION The recent development of viral expression vectors and mosquito transformation techniques provide the requisite tools to assess the role of specific genes involved in mosquito immunity to filarial worms. Using these technologies, it is possible to express or silence genes of interest in vivo to assess their effects on vector competence. This may ultimately result in development of transgenic mosquitoes, refractory to parasites, to be used in integrated control strategies. SINDBIS VIRUS Although several viral expression vectors exist for gene expression in mosquitoes, the most widely used is the Sindbis (SIN) virus expression system. Sindbis (Togaviridae: Alphavirus) is a positive sense RNA arbovirus naturally transmitted by Culex species to avian hosts. The infection is noncytopathic to the mosquito and thus SIN viruses have been developed as vectors both for expressing, and silencing genes (in an antisense manner). These SIN vectors can either be injected directly into the hemolymph
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(TE/3'2J virus) to replicate in salivary glands, neural and respiratory tissue, or be ingested by larvae or adult mosquitoes (MRE/3'2J virus) to specifically infect midgut cells, avoiding the necessity to inject mosquitoes (a process that can potentially turn on genes involved in wound healing); therefore, these expression vectors can be used to express or silence genes of interest in vivo in a tissue specific manner. SIN expression systems have proven to be powerful tools to test the effects of genes involved in the mosquito response to filarial worms. In fact, SIN vectors have been used to express endogenous defensin in Ae. aegypti (Cheng et al., submitted), to effectively block the transmission of other arboviruses (reviewed in Atkinson et al., 2001) and the avian malaria parasite, Plasmodium gallinaceum (Capurro et al., 2000), and to silence a critical enzyme in the melanin biosynthetic pathway (Shiao et al., 2001). This expression system, reviewed in Atkinson et al. (2001), is limited to transient, non-heritable expression of a gene of interest in infected individual mosquitoes. TRANSFORMATION It is now possible to permanently introduce a gene of interest into mosquito germlines to manipulate insect genotypes and assess resulting phenotypes using transposable element-based systems. Four transposable elements from insects have been used to generate transformant An. stephensi (Minos), Ae. aegypti (Hermes, Mosl, and Piggybac) and Cx. pipiens quinquefasciatus (Hermes) (reviewed by Atkinson et al., 2001). Green fluorescent protein (GFP) is an effective molecular marker for screening transformants. Promoters for genes such as vitellogenin (Vg) (reviewed by Raikhel, 1992), and actin can be used to express genes in a tissue specific manner under temporal control factors. For example, the Vg promoter is triggered by a blood meal to express genes in the fat body for export into the hemolymph. The potential power of transformation and its relevance for research relating to mosquito-filarial worm interactions is illustrated by the transformation of Cx. pipiens quinquefasciatus with the promoter for Drosophila actin 88 (Act88F) driving expression of GFP (Allen, Christensen, Atkinson, unpublished data); this promoter controls a gene specifically expressed in indirect flight muscles in the thorax, and therefore could be used to drive expression of genes that may have adverse effects on developing parasites in a tissue specific manner (Figure 3). Furthermore, a transformed line of Ae. aegypti was generated using a construct containing the Hermes transposable element, Vg promoter, and coding region for Ae. aegypti defensin A, thus demonstrating that transgenic mosquitoes can be engineered to express potential antiparasite genes following a blood meal (Kokoza et al., 2000).
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CONCLUSIONS Distinct parallels exist between filarial worm-mosquito and filarial worm- human host relationships. Developing worms inflict pathology on their hosts, resulting in an immune response, which worms are capable of evading and/or suppressing. The tools are rapidly becoming available to answer specific questions regarding the molecular crosstalk that occurs during the interaction between parasite and host resulting in the above-mentioned phenomena. Using functional genomics, whole genome sequences for parasite and host will undoubtedly reveal unexplored aspects of this complex interaction. Therefore, our understanding of the relationship of mosquito and parasite may provide new insight into that of human and parasite, and vice versa. Furthermore, exploring this interaction may lead to new understandings of and, consequently, novel strategies to prevent transmission of lymphatic filariasis.
ACKNOWLEDGEMENTS We thank R. E. Hammad, H.A. Farid, M. L Allen and P.W. Atkinson for kindly providing figures, and Julián F. Hillyer for technical assistance. This work was supported by grants from the National Institutes of Health (AI 19769 and AI 46032).
REFERENCES Agudelo-Silva, F., and A. Spielman. 1985. Penetration of the mosquito midgut wall by sheathed microfilariae. Journal of Invertebrate Pathology 45: 117-119. Albuquerque, C.M.R., and P.J. Ham. 1996. In vivo effect of a natural Aedes aegypti defensin on Brugia pahangi development. Medical and Veterinary Entomology 10: 397-399. Antolin, M.F., C.F. Bosio, J. Cotton, W. Sweeney, M.R. Strand, and W.C. Black IV. 1996. Intensive linkage mapping in a wasp (Bracon hebetor) and a mosquito (Aedes aegypti) with single-strand conformation polymorphism analysis of random amplified polymorphic DNA markers. Genetics 143: 1727-1738. Ash. L.R., and J.M. Riley. 1970. Development of Brugia pahangi in the jird, Meriones
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unguiculatus, with notes on infections in other rodents. Journal of Parasitology 56: 962968. Atkinson, P.W., A.C. Pinkerton, and D.A. O'Brochta. 2001. Genetic transformation systems in insects. Annual Review of Entomology 46: 317-346. Bartholomay, L.C., E. El Kordy, H.A. Farid, and B.M. Christensen. 2001. A practical technique for the cryopreservation of Dirofilaria immitis, Brugia malayi, and Wuchereria bancrofti microfilariae. American Journal of Tropical Medicine and Hygiene 65: 162-3. Beerntsen, B.T., S. Luckhart, and B.M. Christensen. 1989. Brugia malayi and Brugia pahangi: inherent difference in immune activation in the mosquitoes Armigeres subalbatus and Aedes aegypti. Journal of Parasitology 75: 76-81. , D.W. Severson, J.A. Klinkhammer, V.A. Kassner, and B.M. Christensen. 1995. Aedes aegpti: a quantitative trait locus (QTL) influencing filarial worm intensity is linked to QTL for susceptibility to other mosquito-borne pathogens. Experimental Parasitology 81: 355-362. , A.A. James, and B.M Christensen. 2000. Genetics of Mosquito Vector Competence. Microbiology and Molecular Biology Reviews 64: 115-137. Capurro, M.L., J. Coleman, B.T. Beerntsen, K.M. Myles, K.E. Olson, E. Rocha, A.U. Kretli, and A.A. James. 2000. Virus-expressed, recombinant single-chain antibody blocks sporozoite infection of salivary glannds in Plasmodium gallinaceum-infected Aedes aegypti. American Journal of Tropical Medicine and Hygiene 62: 427-433. Chalk, R., H. Townson, and P.J. Ham. 1995. Brugia pahangi: the effects of cecropins on microfilariae in vitro and in Aedes aegypti. Experimental Parasitology 80: 40-406. Chen, C.C. and B.R. Laurence. 1985. The encapsulation of the sheaths of microfilariae of Brugia pahangi in the hemocoel of mosquitoes. Journal of Parasitology 71: 834-836. and C.M. Shih. 1988. Exsheathment of microfilariae of Brugia pahangi in the susceptible and refractory strains of Aedes aegypti. Annals of Tropical Medicine and Parasitology 82: 201-206. and C.S. Chen. 1995. Brugia pahangi: Effects of melanization on the uptake of nutrients by microfilariae in vitro. Experimental Parasitology 81: 72-78. Cheng, L.L., L.C. Bartholomay, K.E. Olson, C.A. Lowenberger, S. Higgs, B.J. Beaty, and B.M. Christensen. 2001. Characterization of an endogenous gene expressed in Aedes aegypti using an orally infectious recombinant Sindbis virus (Submitted). Christensen, B.M., and D.R Sutherland. 1984 Brugia pahangi: Exsheathment and midgut penetration in Aedes aegypti. Transactions of the American Microscopical Society 4: 423433. Farid, H.A., R.E. Hammad, S.A. Kamal, and B.M. Christensen. 2000. Selection of a strain of Culex pipiens highly susceptible to Wuchereria bancrofti. Egyptian Journal of Biology 2: 125-131. Ferdig, M.T., B.T. Beerntsen, F.J. Spray, J.Y. Li, and B.M. Christensen. 1993. Reproductive costs associated with resistance in a mosquito-filarial worm system. American Journal of Tropical Medicine and Hygiene 49: 756-762. Johnson, J . K . , J.Y. Li, and B.M. Christensen. 2001. Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedes aegypti. Insect Biochemistry and Molecular Biology. (In Press). Hoffman, J.A., F.C. Kafatos, C.A. Janeway, and R.A.B. Ezekowitz. 1999. Phylogenetic perspectves in innate immunity. Science 284: 1313-1317. Kokoza,V., A. Ahmed, W-L Cho, N. Jasinkiene, A.A. James, and A. Raikhel. 2000. Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti. Proceedings of the National Academy of Sciences 97: 9144-9149. Laurence, B.R. and F.R.N. Pester. 1967. Adaptiation of the Filarial worm Brugia patei to a new mosquito host, Aedes togoi. Journal of Helminthology 41: 365-392. Lowenberger, C.A., M.T. Ferdig, P. Bulet, S. Khalili, J.A. Hoffman, and B.M. Christensen. 1996. Aedes aegypti: Induced antimicrobial proteins reduce the establishment and development of Brugia malayi. Experimental Parasitology 83: 191201.
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Innate immune response of Aedes aegypti. 2001. Insect Biochemistry and Molecular Biology 31: 219-229. Macdonald, W.W. 1962a. The selection of a strain of Aedes aegypti susceptible to infection with semi-periodic Brugia malayi. Annals of Tropical Medicine and Parasitology 56: 368-372. 1962b. The genetic basis of susceptibiliy to infection with semi-periodic Brugia malayi in Aedes aegypti. Annals of Tropical Medicine and Parasitology 56: 372-382. and C.P Ramachandran. 1965. The influence of the gene (filarial susceptibility, Brugia malayi) on the susceptibility of Aedes aegypti to seven strains of Brugia, Wuchereria, and Dirofilaria. Annals of Tropical Medicine and Parasitology 59: 64-73. McGreevy, P.B., J.H. Bryan, P. Oothuman, and N. Kolstrup. 1978. The lethal effects of the cibarial and pharyngeal armatures of mosquitoes on microfilariae. Transactions of the Royal Society of Tropical Medicine and Hygiene 72: 361-368. Munstermann, L.E. 1990. Linkage map of the yellow fever mosquito, Aedes aegypti, In Genetic maps: Locus maps of complex genomes, vol. 5. SJ. O’Brian (ed.) Cold Spring Harbor Laboratory, Cold Spriring Harbor, N.Y. p. 3179-3183. Nappi, A.J., and E. Ottaviani. 2000. Cytotoxicity and cytotoxic molecules in invertebrates. Bioessays 22: 469-480. Paskewitz, S.M., and B.M. Christensen. 1996. Immune Responses of Vectors. In the Biology of Disease Vectors. Beaty, B.J., and W.C. Marquardt (eds.) University Press of Colorodo, Niwot, Colorado, p 371-392. Raikhel, A.S. 1992. Vitellogenesis in Mosquitoes. Advances in Disease Vector Research 9: 139. Severson, D.W., A. Mori, Y. Zhang, and B.M. Christensen. 1993. Linkage map for Aedes aegypti using restriction fragment length polymorphisms. Journal of Heredity 84: 241247. and 1994. Chromosomal mapping of two loci affecting filarial worm susceptibility in Aedes aegypti. Insect Molecular Biology 3: 67-72. S.E. Brown, and D.L. Knudson. 2001. Genetic and physical mapping in mosquitoes: molecular approaches. Annual Review of Entomology 46: 183-219. Shiao, S.H., S. Higgs, Z. Adelman, B.M. Christensen, H.S. Liu, and C.C. Chen. 2001. Effect of prophenoloxidase expression knockout on the melanization of filarial worms. Insect Molecular Biology 10: 315-321. Sutherland, D.R., B.M. Christensen, and K.F. Forton. 1984. Defense reactions of mosquitoes to filarial worms: role of the microfilarial sheath in the response of mosquitoes to inoculated Brugia pahangi microfilariae. Journal of Invertebrate Pathology 44: 275-281. Townson, H. 1997. Infection of mosquitoes with filaria. In The Molecular Biology of Disease Vectors. Crampton, J.M., Beard, C.B., and C. Louis (eds.) Chapman and Hall, London, UK. p. 101-111. Yamamoto, H., N. Kobayashi, N. Ogura, H. Tsuruoka, and Y. Chigusa. 1985. Studies on filariasis VI: The encapsulation of Brugia malayi and B. pahangi larvae in the mosquito, Armigeres subalbatus. Japanese Journal of Sanitary Zoology 36: 1-6. Wharton, R.H. 1962. Studies on filariasis VI: The biology of Mansonia mosquitoes in relation to the transmission of filariasis in Malaya. Bulletin of the Institute of Medical Research, Federated Malay States 11.
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EVOLUTIONARY RELATIONSHIPS FILARIAL NEMATODES
AMONG
Odile Bain Parasitologie comparée et Modéles expérimentaux, associé à 1’INSERM (U 445) Institut de Sysématique, FR 1541 CNRS Ecole Partique des Hautes Etudes, Muséum National d’Historie Naturelle 61 rue Buffon, 75231 Paris cedex 05
ABSTRACT Due to their highly evolved biology characterized by specialized eggs and the microfilariae, which migrate in host lymph or blood and are transmitted by hematophagous arthropods, the filariid Onchocercidae were considered recent nematodes. Currently, their origin is thought to be remote, hidden in the Secondary era, with the first representatives in crocodiles and transmitted by culicids (150 M years). But the main expansion occurred during the Tertiary, synchronously with bird and mammal diversification. Among the 80 genera of Onchocercidae, a few are parasites of humans: Brugia-Wuchereria, Mansonella, Onchocerca and Loa. This list does not, however, include all the agents of zoonoses. The human filariae result from two evolutionary processes: either they have evolved from parasites of primates or humans have been infected by the capture of filariae parasitic in zoologically unrelated groups. Keywords: Onchocercidae, reptiles, birds, mammals, Gondwana, Jurassic, crocodiles, Tertiary, humans, zoonoses.
INTRODUCTION Filarial worms occupy a numerically minute place in the immense phylum of nematodes. However, their highly specialized biology and the fact that a few of them parasitize humans give them a particular interest. The word filaria evokes long and thin tissue-dwelling worms and, in this broad sense, is applied to two groups distinguished by their biology. In one, embryonated eggs with a thick shell are expelled to the outside, and ingested by coprophagous or omnivorous insects. Thus, the cycle is identical to that of the spirurid ancestors. Examples are mainly in birds (filariae from air sacs, orbital cavities) and also in mammals (Filaria, in (Anderson 2000)). In the other group, the only one to be considered, e.g. the family Onchocercidae (Figure 1), the constraint of being restricted to the host’s tissues without any direct communication with the exterior has resulted in an original adaptation: the embryo has become mobile, and so can migrate in the
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circulating fluids, lymph or blood, of the host to places favorable for its ingestion by hematophagous arthropod vectors such as lice, fleas, various diptera (mosquitoes, simuliids, ceratopogonids, tabanids), and also ticks and other acarians. Within the vector filarial development commences and continues as far as the third larval stage, which is a resistant phase in all phasmidian nematodes, whether they are free living or parasitic (Chabaud 1954). Development continues in the definitive host, after the infective larvae have been deposited on or inoculated into it. Retracing the history of Onchocercidae is not possible due to the lack of sound data, but some clues can be obtained by conjoint analyses of the parasites morphology and biology, host range, the host’s distribution and paleontological data. The Onchocercidae were considered to be recent nematodes, due to their highly evolved life cycle. Their origin is now thought to be remote, hidden in the Secondary era. The sub-family Oswaldofilariinae, which is exclusively parasitic in reptiles, is very interesting from this point of view. It comprises a morphologically primitive genus, Oswaldofilaria, two species of which are parasites of crocodiles. They are astonishingly similar although one is found in South America and the other in southern Africa. The other Oswaldofilariinae are parasites of Lacertilia (Iguanidae, Tejiidae, Agamidae, Gekkonidae and Scincidae). Their diversity is great and, in each geographical region, there is a corresponding unique
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genus. But their geographical distribution includes only the ancient southern continents: Australia, South America, the Ethiopian region, and India but not the rest of Asia. Taken together, these data suggest that crocodiles harbored the first Oswaldofilariinae during the Gondwana epoch and that their expansion occurred as this continent was breaking up, favoring the isolation of newly-evolved genera. This can be precisely dated to the end of the Jurassic, about 150 M years ago (Chabaud and Bain 1994). Several life cycles have been elucidated in this sub-family, including that of an Oswaldofilaria from a crocodile. Mosquitoes are intermediate hosts for all species which seems to be the ancestral behavior. At this epoch of the Secondary era, the hematophagous Culicidae had already separated from their sister group, the Chaoboridae (oral communication of Pr L. Matile, MNHN). Some filariae thus survived the great late Jurassic crisis. However, their main diversification occurred in the Tertiary, contemporary with the appearance and explosive radiation of the two groups of vertebrates that they predominantly parasitize, birds and mammals, which made available new niches (Figure 2). The hosts of the Onchocercidae, being all terrestrial vertebrates, do not differ greatly one from the other, and the universality of the biochemical pathways has made possible the transfer of filarial parasites from one host group to another. Birds and mammals have in general different filarial genera but captures between them have occurred (Bartlett and Greiner 1986). During this vast Tertiary period, geographical isolation and exchanges between continents played an important role in the establishment of the filarial faunas. For example, in South America which was isolated during most of the Tertiary, one may distinguish three successive stages in the history of the Onchocercinae parasitic in mammals, represented mainly by the Dipetalonema line (Bain, Baker et al. 1982). First, the earliest paleoendemic fauna were composed of genera parasitic in marsupials and edentates. This can probably be dated to the end of the Secondary or the beginning of Tertiary periods, as can be their hosts. Second, some genera presenting rather greater affinities with Ethiopian forms are parasitic in two groups of exclusively South American mammals, the caviomorph rodents and the platyrhine monkeys. No paleontological traces of these hosts have been found in South America before the Oligocene; their similarities with the two homologous African groups suggest that they arrived from this region at the end of the Eocene, thanks to an intercontinental bridge (Hoffstetter 1982; Thomas 1992). This hypothesis, which remains debatable for the rodents only (Hartenberger 1998), is strongly supported in the two groups of hosts by the similarities of their intestinal nematodes, trichostrongylids on one hand (Durette-Desset 1971) and oxyurids on the other (Quentin 1973).
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The similarities are too great to be explained by convergences. The filariae, like the intestinal nematodes, were probably introduced with their hosts during this early part of the Tertiary. Finally, during the Pleistocene (3 M years ago), when the two American continents were united, new fauna infiltrated with the influx of mammals from the Nearctic regions. Some species of widespread genera, such as Brugia and Cercopithifilaria, are representative of this modern fauna. This is also the case with the species of Litomosoides which diversified in the neotropical murids, whereas the line seems to have originated from parasites of bats in the Eocene. This recent expansion could explain why the unique filarial species, L. sigmodontis, which develops in laboratory mice, belongs to the genus Litomosoides. The few onchocercids parasitizing humans must be considered within the context of this vast historical background which covers at least 60 M years. They are a heterogeneous group, the result of two distinct evolutionary processes: (i) some parasites of humans have evolved from those of the hostgroup to which they belong, the primates; and (ii) humans have also been infected by capture of filariae which are fundamentally parasites of a zoologically unrelated host-group.
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BRUGIA-WUCHERERIA These two genera have more morphological and biological similarities than divergences (reduced buccal capsule, but an esophagus with a glandular part; sheathed microfilariae; an infective stage with a long tail and three lappets, Culicid vectors and lymphatic location). Molecular analyses of these filariae (Xie, Bain et al. 1994) and their endosymbiotic bacteria Wolbachia (Bandi, Anderson et al. 1998; Casiraghi, Anderson et al. 2001) confirm these conclusions. Thus, it seems more advisable to treat these two genera as a whole. They have a dozen species (Sonin 1975; Eberhard 1984). One species is parasitic in Tupaidae, a family of Asiatic insectivores which is a sister group of primates; the microfilaria of this species, B. tupaiae, does not possess the specialized caudal nuclei typical of the genus Brugia, and thus it resembles Wuchereria spp. The following four species are parasites of primates: W. bancrofti, in humans from the Oriental Pacific area, which has spread recently through the tropical belt; W. kalimantani, restricted to Indonesian leaf-monkeys and apparently derived from the human parasite; B. malayi, in humans from India to Korea and monkeys in South-East Asia, and B. timori, in humans but restricted to two small Indonesian islands. Three species are known from lagomorphs (hares) in the Indian subcontinent, Russia and North America. The other species are parasites of carnivores in the Old and New Worlds. The host range is thus at first sight incongruous but these mammals have in common the fact that they are all ancient - they appeared at the end of the Secondary era (Tupaidae) or at the beginning of the Tertiary, during the Paleocene. At this remote time, the expansion of these mammals generated a great diversity of new niches, which permitted the radiation of the nascent parasitic lineage. As it has been emphasized for other nematodes of vertebrates (Chabaud 1981), the date of appearance of host-groups is often more important than their zoological affinities. The human filariae Wuchereria bancrofti, B. malayi and B. timori thus belong to a group with an ancient origin, which seems to have diversified in South-East Asia. The geographically restricted B. timori may represent a parasite of local wild primates at least in the past. B. malayi presents a special problem; it is currently accepted that this species has a broad host range, being parasitic also in carnivores and even in very specialized hosts such as pholidotes (pangolins). This diverse host range has been proven in the case of domestic carnivores (Buckley and Edeson 1956), but the presence of B. malayi in pangolins requires confirmation by a morphological study, which apparently has not been done.
MANSONELLA This genus is very complex and is not yet completely understood despite the significant progress resulting from the work of Orihel and Eberhard (1984). All representatives of the genus have total regression of the
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buccal capsule, a simplified esophagus reduced to a thin tube, and four lappets on the caudal extremity of the adult female and the infective larva. The 25 species of the genus, however, form several morphological groups, which constitute as many sub-genera (Eberhard and Orihel 1984). Sandnema is morphologically the most primitive; it comprises two species parasitic respectively in an insectivore and a cercopithecid monkey in Asia. Tupainema is monospecific and a parasite of tupaids. The sub-genus Mansonella is parasitic in carnivores in the Holarctic region (Procyon and Ursus), and in sciurids and humans in South America. The sub-genus Tetrapetalonema is represented by 13 species parasitic in platyrhine monkeys in South America. Lastly, the sub-genus Esslingeria, distinguished from the previous one by the arrangement of the head papillae, is parasitic in anthropoid monkeys and humans in Africa, as well as caviomorph rodents in South America. The vast lineage of Mansonella thus has a remote origin, attested by its insectivorous, primate and carnivorous hosts, and the point of dispersion seems to have been in the Asiatic region, where the most primitive forms exist. Subsequently, two continental migration routes were apparently used: towards Africa through the Arabian peninsula, and towards North America through the Behring Strait. The South American species could have migrated either during the Pleistocene for North America (sub-genus Mansonella), or from Africa at the end of the Eocene (Tetrapetalonema, Esslingeria of caviomorphs). The three species parasitic in humans belong to two sub-genera, the histories of which differ. M. (E.) perstans and M. (E.) streptocerca are parasites which are common in African anthropoids but these harbour more species, 6 in total (Bain, Moisson et al. 1995). Thus a small group diversified in these primates whose radiation may be dated, like that of their hosts, to the Oligocene (Coppens 1984). Very recently, M. (E.) perstans was introduced into South America by human migration; the two South American species of Esslingeria in caviomorph rodents cannot explain this human parasitism because they are more closely related to Tupainema. In contrast, M. (M.) ozzardi (of which the adults are well known thanks to (Orihel and Eberhard 1982)) probably represents a capture, originating from parasites of carnivores or sciurids. This conclusion is based on the remarkable similarity of the right spicule, a character which seems to be able to enlighten several points in the Mansonella line.
LOA LOA This is the only species of the genus; it is restricted to the Congolese forest region and is a parasite of humans and cercopithecids (Duke and Wijers 1958), and sometimes anthropoids. In humans, occult filariasis is frequent, and independent of the intensity of transmission (Fain 1978). In cercopithecids, the worms are larger and more fertile (Duke and Wijers 1958),
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suggesting that these monkeys were the original hosts of L. loa. The birthplace of these primates is the Arabo-African continent (Thomas and Senut 1999).
ONCHOCERCA VOLVULUS The genus Onchocerca is represented by about thirty species. Only one is parasitic in humans (and sometimes in anthropoids), whereas all the others are parasites of ungulates. They are spread throughout the Asiatic, Holarctic and Ethiopian regions, Australia having only two recently imported species. The most primitive form of the Onchocerca line is a parasite of an African equid and has a limited radiation, comprising three derived species, which occurred in these hosts. Camelids and suids also have their species, but it is in cervids and bovids, the expansion of which is dated to the Miocene, that the greatest number of species is known. Among those parasitic in bovids, there is a limited onchocercal line, the adults of which inhabit spherical, well defined nodules, like the human parasite O. volvulus. These nodular species also share a double adaptive character: the hypodermal lateral chords are hypertrophied whereas the musculature is very reduced (Bain 1981b). Two nodular species have been described in bovines from the Asiatic region, but they are better known in Australia. One is dermal and the other, O. gibsoni, is located in the aponeuroses. In Africa there is a vicariant species in these two habitats, of which one, the dermal species, is O. ochengi. This species is the closest morphologically to O. volvulus and it is likely that the human filaria resulted from capture of this bovine parasite, or of a common ancestor. The vector for O. volvulus and O. ochengi is the same (Denke and Bain 1978) and the morphology of the infective larva is almost identical (Wahl and Schibel 1998). These factors make the calculation of the Annual Transmission Potential impossible. Onchocerciasis is a disease having an African origin and only recently was it imported into the New World. There the foci are more fragmented and transmission is more easily interrupted because a high proportion of ingested microfilariae are destroyed by the pharyngeal teeth of the local simuliid vectors. The diversity of origin of the human filariae demonstrates the impressive plasticity of these nematodes and the present-day filarial zoonoses probably reflect what happened in the past. Their animal reservoirs are diverse: 1) carnivores for dirofilariases, sometimes with microfilaraemia being able to develop in humans (Nozais, Bain et al. 1995), 2) ungulates such as cattle, horses and even wild boar (Uni, Bain et al. 2001) (Takaoka, Bain et al. 2001) as for onchocerciases; and 3) African monkeys for Mansonella rodhaini (cf (Richard-Lenoble, Kombila et al. 1988) and Meningonema perruzzi (cf (Boussinesq, Bain et al. 1995)). In forest areas, reservoirs were sometimes not identified (Microfilaria vauceli Galliard and Brygoo, 1955, Microfilaria semiclarum Fain, 1974) and the occurrence of other species parasitic in humans is unpredictable.
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ACKNOWLEDGMENTS Many thanks to S. Babayan, PhD student of my team who prepared the figures, and to Pr. J. Baker who patiently corrected my English.
REFERENCES Anderson R. C. 2000. Nematode parasites of vertebrates. Their development and transmission. 2nd Edition, CABI Publishing, New York, 650 pp. Anderson R. C, Bain O. 1976. Keys to genera of the order Spirurida. Part 3. Diplotriaenoidea, Aproctoidea and Filarioidea. In: Commowealth Institute of Helminthology Keys to the Nematodes Parasites of Vertebrates, No. 3 (Edited by Anderson R. C., Chabaud A. G. & Willmott S.), 59-116. Bain O. 198la. Filariids and their evolution. p. 167, in: Evolution of Helminths (Workshop Proc., EMOP 3), Parasitology, 82, 161-174. Bain O. 1981b. Le genre Onchocerca: hypothèses sur son évolution et clé dichotomique des espèces. Annales de Parasitologie Humaine et Comparée, 56, 503-526. Bain O., Baker M., Chabaud A.G. 1982. Nouvelles données sur la lignée Dipetalonema (Filarioidea, Nematoda). Annales de Parasitologie Humaine et Comparée, 57, 593-620. Bain O., Moisson P., Huerre M., Landsoud-Soukate J., Tutin C. 1995. Filariae from a wild gorilla in Gabon with description of a new species of Mansonella . Parasite, 2, 315-322. Bandi C., Anderson T. J. C., Genchi C., Blaxter M. L. 1998. Phylogeny of Wolbachia in filarial nematodes. Proceedings of the Royal Society, London, B, 265, 2407-2413. Bartlett C. M., Greiner E. C. 1986. A revision of Pelecitus Railliet and Henry, 1910 (Filarioidea, Dirofilariinae) and evidence for the “capture” by mammals of filarioids from birds. Bulletin du Muséum d’Histoire Naturelle 4è sér. 8, section A,B, no. 1, 47-99. Boussinesq M., Bain O., Chabaud A. G., Gardon-Wendal N., Kamgno J., Chippaux J. P. 1995. A new filariid zoonosis of the cerebrospinal fluid of a man probably caused by Meningonema peruzzi, a parasite of the central nervous system of Cercopithecidae. Parasite, 2,173-176. Buckley J.J.C. & Edeson, J.F.B. 1956. On the adult morphology of Wuchereria sp. (malayi ?) from a monkey (Macaca irus) and from cats in Malaya, and on Wuchereria pahangi n. sp. from a dog and a cat. Journal of Helminthology, 30, 1-20. Casiraghi M., Anderson T. J. C., Bandi C., Bazzochi C., Genchi C. 2001. A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology, 122, 93-103. Chabaud A. G. 1954. Sur le cycle évolutif des spirurides et des nématodes ayant une biologie comparable. Valeur systématique des caractères biologiques (suite). Annales de Parasitologie Humaine et Comparée, 29, 206-249. Chabaud A. G. 1974. Keys to subclasses, orders and superfamilies. In : Commowealth Institute of Helminthology Keys to the Nematodes Parasites of Vertebrates, No. 1 (Edited by Anderson R. C., Chabaud A. G. & Willmott S.), pp 6-17. Chabaud A. G. 1981. Host range and evolution of nematode parasites of vertebrates. In: Evolution of Helminths (Workshop Proc., EMOP 3), Parasitology, 82, 169-170. Chabaud A. G., Bain O. 1994. The evolutionary expansion of the Spirurida. International Journal for Parasitology, 24, 1179-1201. Coppens Y. 1984. Hominoides, Hominidés et Hommes. La Vie des Sciences, Comptes-Rendus, série générale, 1, 459-486. Denke A.M., Bain O. 1978. Données sur le cycle d'Onchocerca ochengi chez Simulium damnosum s. l. au Togo. Annales de Parasitologie. Humaine et Comparée, 53, 757-760.
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Duke B. O. L., Wijers D. J. B. 1958. Studies on loiasis in monkeys. I. The relationship between human and simian Loa in the rain forest of the British Cameroon. Annals of Tropical Medicine and Parasitology, 52, 158-175. Durette-Desset M.-C . 1971. Essai de classification des Nématodes Heligmosomes. Corrélations avec la paléontologie des hôtes. Mémoires de Muséum National d’Histoire Naturelle, nlle sér., Sér. A Zool., 49, 1-126. Eberhard, M. L. 1984. Brugia lepori sp. n. (Filarioidea: Onchocercidae) from rabbits (Syhilagus aquaticus, S. floridanus) in Louisiana. Journal of Parasitology, 70, 576-579. Eberhard, M. L., Orihel, T. C. 1984. The genus Mansonella (syn. Tetrapetalonema) : a new classification. . Annales de Parasitologie Humaine et Comparée, 59, 484-496. Fain A. 1978. Les problèmes actuels de la loase. Bulletin de l’Organisation Mondiale de la Santé, 56, 155-167. Hartenberger J.-L. 1998.Description de la radiation des Rodentia (Mammalia) du P a l é o è n e supérieur au Miocène ; incidences phylogénétiques. Comptes-rendus de l’Académie des Sciences, Paris, Sciences de la Terre et des Planètes., 326, 439-444. Hoffstetter R. 1982. Introduction sur les hôtes. I.-Phylogénie des Mammifères : méthodes d’étude, résultats, problèmes. In: Symposium sur la Spécificité parasitaire des Parasites de Vertébrés, 13-17 avril 1981, Mémoires du Muséum National d’Histoire Naturelle, sér. A, pp. 13-20. Nozais J. P., Bain O., Gentilini M. 1995. Un cas de Dirofilariose sous-cutanée à Dirofilaria (Nochtiella) repens avec microfilarémie en provenance de Corse. Bulletin de la Société de Pathologie Exotique, 87, 183-185. Orihel T. C., Eberhard, M.J. 1982. Mansonella ozzzardi: a redescription with comments on its taxonomic relationships. American Journal of Tropical Medicine and Hygiene, 31, 1142-1147. Quentin J.-C. 1973. Affinités entre les oxyures parasites de rongeurs Hystricidés, Erethizontidae, et Dinomyidae. Intérêt paléobiogéogaphique. Comptes Rendus de 1’Académie des Sciences, Paris, 276, sér. D, 2015-2017. Richard-Lenoble D., Kombila M., Bain O., Chandenier J., Mariotte O. 1988. Filariasis in Gabon: human infections with Microfilaria rodhaini. American Journal of Tropical Medicine and Hygiene, 39, 91-92. Sonin M. D. 1975. Filariasis of Animals and Man and the Diseases Caused by Them. Fundamentals of Nematology, 24, pp 396 (in Russian). Takaoka H., Bain O., Uni S., Korenaga M., Tada K., Ichikawa H., Otsuka Y., Eshita Y. 2001: Human infection with an Onchocerca dewittei japonica, a parasite from wild boar in Oita, Japan. Parasite, 8, 261-263. Thomas H. 1992. Crise climatique et évènements géodynamiques. Leur rôle dans l’évolution des primates anthropoides. Bibliothèque d’Orientation Mentha, Paris, 92 pp. Thomas H., Senut B. 1999. Les primates ancêtres de l’homme. Ed. Artcom, Paris, 181 pp. Uni S., Bain O., Takaoka H., Miyashita M., Suzuki Y. 2001. Onchocerca dewittei japonica n. subsp., a common parasite from wild boar in Kyushu Island, Japan. Parasite, 8, 215-222. Wahl G., Schibel J. M. 1998. Onchocerca ochengi: morphological identification of the L3 in wild Simulium damnosum s. 1. verified by DNA probes. Parasitology, 116, 337-348. Xie H., Bain O., Williams S. A. 1994. Molecular phylogenetic studies on filarial parasites based on 5S ribosomal spacer sequences. Parasite, 1, 141-151.
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FILARIAL GENOMICS: GENE DISCOVERY AND GENE EXPRESSION
S.A. Williams1,2 and S.J. Laney1 1Department
of Biological Sciences, Clark Science Center, Smith College, Northampton, MA 01063, USA and 2Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003, USA
ABSTRACT A project to study the genome of the lymphatic filarial parasite Brugia malayi was initiated in 1995. This project has been funded by the World Health Organization and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) with the ultimate objective of identifying new vaccine candidates and drug targets for filariasis. Because fewer than 60 Brugia genes had been cloned by the end of 1994, it was determined that the first goal of the project would be the identification of thousands of new genes. These genes have been identified by randomly selecting clones for DNA sequence analysis (ESTs) from cDNA libraries that have been constructed from all life cycle stages of B. malayi. To date, over 22,000 Brugia ESTs have been entered into the National Center for Biotechnology Information’s dbEST database and about 8000 new genes have been identified (estimated to be about 40% of the complete set of B. malayi genes). In addition to new gene discovery, the 22,000 ESTs can be used to identify genes that are most highly expressed at each stage of development. Such analyses can provide insights into the biology of the organism and can suggest new molecules for study as drug targets and vaccine candidates.
INTRODUCTION Parasite genome projects have been initiated as a new approach to the study of those species that are among the most important human pathogens in tropical regions of the world (Williams and Kemp, 1996). Because conventional genetic approaches (the ability to do crosses and map genes using classical techniques) are either very difficult or impossible for many of these organisms, the application of molecular biology has been critical to the study of their genomes. The World Health Organization and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) launched an initiative in 1994 to study the genomes of five parasites critical to the future of public health in the tropics including those that cause lymphatic filariasis (Unnasch, 1994). The long term goal of these genome initiatives has been to collect data that will foster understanding of
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important biological problems such as parasite drug resistance, pathogenesis, and virulence; and to assist in the identification of new targets for chemotherapy and for the development of vaccines (Williams and Kemp, 1996; Williams and Johnston, 1999; Degrave et al. 2001). For more than 10 years (approximately 1982-1994), standard molecular cloning techniques were applied to the study of filarial parasites (particularly Brugia malayi and Onchocerca volvulus) but few genes were cloned and identified. By the end of 1994, only 60 Brugia genes had been submitted to GenBank. Two important reasons for this lack of progress were: 1) most of these parasites are very difficult to collect and many cannot be maintained in the laboratory and 2) no high quality B. malayi cDNA libraries were available. One common strategy for cloning parasite genes of immunological interest had been to screen cDNA expression libraries with sera from infected patients. Important genes were identified in this way, but the pace was slow and many genes were cloned multiple times in many laboratories. It was clear that a new approach for studying the filarial genome was needed to make rapid progress. The genome project approach represented a complete departure from the way parasite genes had been studied in the past. Genome projects are typically not directed at the identification of individual genes, but instead at the identification, cloning and sequencing of all the organism’s genes. At the first meeting of the Filarial Genome Project (1994), B. malayi was selected as the organism to be studied (Unnasch, 1994). This parasite was chosen over the other two medically important species of filarial parasites, Wuchereria bancrofti and O. volvulus, primarily because of the ready availability of all stages of the life cycle. In addition, molecular phylogenetic studies of filarial parasites had shown that all three of these species are closely related (Xie et al. 1994). Thus, much of the molecular data obtained on one of the species would be applicable to the other two. The genome of B. malayi is about the same size as that of the free-living nematode Caenorhabditis elegans. The genome is AT rich (70%) and there are five pairs of chromosomes (Williams et al. 2000). 10% of the Brugia genome consists of a single repetitive sequence, the HhaI repeat. This repeat is 322 bp in length and is organized in long tandem arrays (McReynolds et al. 1986; Williams et al. 1988). The number of protein-coding genes in C. elegans is estimated to be 19,000 (The C. elegans Genome Sequencing Consortium, 1998) and the number of genes in Brugia is expected to be similar.
cDNA LIBRARIES, ESTS AND CLUSTER ANALYSIS Since fewer than 60 Brugia genes had been cloned by 1994, it was decided that the primary goal of the project would be the identification of at least 5000 new genes. These new genes would help elucidate the biology of
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the organism and would aid in the identification of new vaccine candidates and drug targets. The plan was to identify these new genes by randomly selecting clones for DNA sequence analysis from new cDNA libraries that were to be constructed from all life cycle stages of B. malayi. The construction of many cDNA libraries from all of the life cycle stages would insure that a high proportion of the expressed genes of B. malayi would be represented. Such expressed sequence tag (EST) analysis is a rapid way to identify gene sequences, since no effort is made to completely sequence each cDNA clone. Twelve Brugia cDNA libraries have been constructed in the bacteriophage lambda cloning vector ZAPII (Stratagene, La Jolla, CA) representing the following developmental stages: microfilaria (MF), second stage larva (L2), third stage larva (L3), molting L3 larva (L3M), fourth stage larva (L4), young adult (YA), adult male (AM) and adult female (AF) (Table 1). Unidirectional cloning was chosen to facilitate the use of these libraries in EST analyses. Seven of the Brugia cDNA libraries were constructed using conventional techniques (Williams and Johnston, 1999), three were constructed using subtraction techniques (Diatchenko et al. 1996), and three were constructed using PCR and the SL1 spliced leader sequence (Scott and Yenbutr, 1995). The Brugia cDNA libraries (and cDNA libraries from O. volvulus and W. bancrofti) and all individual cDNA clones are available from the Filarial Genome Project Resource Center at Smith College (contact S.A. Williams at
[email protected]). As of February 2002, 22,439 ESTs containing over 10 million base pairs of Brugia sequence data have been submitted to the National Center for Biotechnology Information’s dbEST database (http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html). Since cDNA clones are selected randomly for sequencing and since duplication (especially of highly expressed genes) is unavoidable, these expressed sequence tags represent about 8000 new Brugia genes. The number of genes represented by the EST data set is determined using a clustering algorithm that groups ESTs derived from the same gene into clusters. The analysis used for this paper was performed at the Institute for Genome Research (TIGR) using CAP3 and algorithms developed at TIGR (Quackenbush, et al. 2001). This cluster analysis is available on the web as one of the TIGR gene indices (http://www.tigr.org/tdb/bmgi/). Although the TIGR clustering was used for this paper, another excellent clustering is available through NEMBASE from Mark Blaxter's laboratory at the University of Edinburgh (http://nema.cap.ed.ac.uk/nematodeESTs/nembase.html) (Blaxter et al. 1999).
USE OF THE EST DATA TO STUDY GENE EXPRESSION Analysis of the large Brugia EST database is providing important information concerning the relative abundance of different mRNAs in different life cycle stages of the parasite. This use of EST data to provide a deeper
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understanding of gene expression in the organism is important since it is very difficult to obtain sufficient parasite material for Northern blot analyses or even quantitative RT-PCR studies. An accurate analysis of the EST data set for the study of gene expression requires that only randomly selected cDNA clones be included. This means including only those clones that are selected at random for sequencing from cDNA libraries made by conventional methods. To this end, data for the following analyses does not include ESTs from the SL (spliced leader) cDNA libraries (Table 1). These libraries were constructed from very small amounts of RNA using PCR and the SL sequence to amplify small amounts of mRNA isolated from difficult to obtain stages of the parasite. These libraries are biased by the PCR and by the use of the SL sequence to favor the amplification of short mRNAs and those that contain the SL sequence at their 5’ end. In addition, libraries made by subtraction methods to eliminate common cDNA clones were also not included in this analysis. Also, some cDNA clones were selected for sequencing by hybridizing gridded libraries with labeled DNA from abundant ESTs. The clones that hybridized with these abundant clones were NOT selected for sequencing. Thus, any clones selected by one of these subtractive hybridization techniques were also not included in this analysis. The only clones included in this analysis were those selected randomly from conventionally constructed cDNA libraries (Table 1).
A large number of the most abundant clusters (those with the most ESTs) belong to two classes: ribosomal RNA genes and mitochondrial genes. Of the clusters containing at least 24 ESTs (70 total clusters), five are ribosomal RNA genes and 14 are mitochondrial genes (data not shown). These rRNA and mitochondrial genes are not included in the analyses in this paper because they are not nuclear genes encoding proteins (Table 1). Applying the above criteria, the entire EST data set was reanalyzed using the most recent TIGR clustering: only 14,904 out of the 24,439 ESTs were used.
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2,556 ESTs were eliminated because they came from SL cDNA libraries or because they were obtained by subtraction methods designed to eliminate highly expressed cDNAs. 3,356 ESTs were excluded because they represented rRNA or mitochondrial genes. Finally, 3,623 ESTs were excluded due to low quality or short sequences. Table 2 shows the 52 most abundant clusters represented in this refined data set (those with at least 24 ESTs and excluding rRNA and mitochondrial genes). Analysis of the data contained in Table 2 shows that there are many interesting genes that warrant further study. The most abundant cluster in Table 2 is rbp-1/TC1784 (1.09% of the total data set). It shows similarity to an RNA binding protein, although its function is unknown. It is the most highly expressed gene in the Mf stage (3.21%) and is also highly expressed in all of the other life cycle stages. The second most highly expressed gene based on this cluster analysis is another of unknown function (aaf-1 for abundant adult female transcript/TC1819). The ESTs from this gene represent 0.91 % of the total data set indicating that this gene is expressed at very high levels. Adding to the interest in this gene is the fact that it is expressed only in mature adult worms and primarily in adult females. In fact, it is by far the most highly expressed gene seen in the adult female (AF) stage (4.01%). In adult males (AM) the cluster is represented at 0.35% making it the fourteenth most abundant in adult males (data not shown). No ESTs of this cluster were obtained from the MF, L3, L3M, L4 or young adult (YA) cDNA libraries. Other genes in Table 2 show fascinating expression patterns. For example, the sixth most abundant cluster in the data set (aad-1 for abundant adult/TC1736) is one that has homology to a predicted C. elegans protein (T25D3.2) that shows weak similarity to a human melanoma antigen. Based on the EST data, this gene is expressed primarily in adult males and females, although a few ESTs are found in the L3 and YA data sets. In contrast, the cluster representing the gene alt-2a (TC3577) is expressed only in L3 larvae. This gene is the seventh most abundant in the total data set and is the most abundant in the L3 data set. Another related gene (alt-1a/TC1812) is also expressed only in L3 larvae. Other genes with interesting expression patterns include a serine protease inhibitor (serpin; spn-2/TC1863) that appears to be expressed only in microfilariae and perhaps in developing MF in adult females, another serpin (spn-1/TC1849) that is expressed only in L3, a gene with similarity to vespid allergen antigen (vaa-1/TC1805) that is highly abundant in L3, a major sperm protein (msp-2/TC1838) found exclusively in AM, and a novel (TC1896) that is seen only in AM. If one examines only the ESTs from the molting L3 stage (L3M), the 19 most abundant clusters (those with at least 5 ESTs) show some very interesting expression patterns. As can be seen in Table 3, seven of the 19 genes most highly expressed in the L3M stage are novel with no informative similarity to any genes in the databases. Of these seven, four appear to be
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uniquely expressed in the L3M stage (alt-2b/TCl797, TC1790, TC1901, and TC2162), while the other three have a few ESTs in other stages. What are these genes and what critical function do they encode for the parasite as it molts from an L3 to an L4 larva? These genes are of particular interest because the L3M is the first molt in the human host and the parasite is thought to perhaps be most vulnerable at this stage of development. Six of the 19 most abundant L3M clusters are collagens which is not surprising since these parasites must synthesize a new cuticle for the molt from L3 to L4 . One of these collagens is found exclusively in the L3M stage (TC1858). In addition, four of the 19 most abundant clusters encode two actin homologues (act-2/TCl84l and act-4/TC1840), a troponin C homologue (tpc-1/TC1833), and a myosin heavy chain homologue (myh-1/TC1924) indicating the active synthesis of muscle tissue during this transitional stage. Many other genes have been identified in our preliminary analyses that appear to be stage-specific. Stage-specific genes are first identified by their frequent occurrence in the EST data set of one stage of development but not others. To further test stage-specificity, gene-specific primers are designed for the gene of interest and used in PCR reactions to test all of the cDNA libraries. If the PCR test indicates that cDNA clones for that gene are found in only one stage of development, then the gene is classified as being stage-specific. Many clones identified in the EST project have been selected for further investigation by members of the Filarial Genome Project and other laboratories (Williams, 1999). Since over 300 clones have been requested, the list of genes currently under investigation is much larger than can be referenced here. All of the EST clones are freely available from the Filarial Genome Project Resource Center (curated by Steven A. Williams at Smith College) and can be obtained by contacting
[email protected].
GENOMIC LIBRARIES AND GENOME MAPPING For genome mapping and large-scale genome sequencing, BAC (bacterial artificial chromosome) libraries have been constructed (Sun et al. 2001). The BAC libraries have been gridded onto high-density nylon filters and have been screened with many different gene probes to validate that they contain a reasonable representation of the B. malayi genome. Genomic libraries are essential reagents for many purposes: analysis of filarial gene organization, obtaining the 5' ends of partial cDNA sequences, examining the intron-exon structure of genes-of-interest, defining promoter and other regulatory regions, and mapping in preparation for genome sequencing. The BAC library is currently being used as a resource by the filarial research community to isolate and sequence genomic copies of genes identified in the EST analysis of cDNA libraries. Aliquots of BAC clones of interest can be requested from the Filarial Genome Project Resource Center (
[email protected]).
37
38
39
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The map will be a valuable tool for research into the biology of Brugia, especially when it is compared to the genome map and sequence of C. elegans (The C. elegans Genome Sequencing Consortium, 1998). Many genes in C. elegans are organized into operons from which polycistronic pre-mRNAs are transcribed and subsequently resolved by trans-splicing into monocistronic mature mRNAs (Zorio et al. 1994). If this organization is found in Brugia, then the mechanism of trans-splicing could be a specific anti-filarial drug target.
FILARIAL PARASITES HAVE THREE GENOMES: NUCLEAR, MITOCHONDRIAL AND BACTERIAL When DNA is isolated from Brugia parasites (and from most other filarial parasites), three different genomes are recovered. The nuclear genome contains most of the genes and has been the major focus of the Filarial Genome Project. However, in the course of sequencing 24,000 ESTs, “contaminating” sequences from two other genomes were obtained. Sufficient numbers of ESTs containing mitochondrial sequences were recovered such that most of the mitochondrial genome has now been sequenced using a PCR-based strategy (M. Blaxter, personal communication). Unexpectedly, ESTs were also obtained from a third genome. These ESTs were found to most closely match sequences from alpha-proteobacteria, particularly those of the Rickettsia branch. Further analysis indicated that these sequences were most closely related to Wolbachia species, a group of Rickettsia-like bacteria that are common endosymbionts of arthropods and nematodes (Werren, 1997). Intracellular bacteria in filarial parasites were first described based on electron microscopy studies (McLaren et al. 1975), but the identification of these bacteria as Wolbachia was not made until the EST sequences found by the Filarial Genome Project were analyzed. Various antibiotics have now been shown to have antifilarial effects by reducing the viability of adults and microfilariae and by abrogating the development of embryonic forms in utero (Townson et al. 2000). Tetracycline has been shown to prevent the molting of L3 to L4 larvae in culture (Smith and Rajan, 2000) and preliminary clinical data indicate that doxycycline may be useful in the treatment of filarial infections (Hoerauf et al. 2000). A consortium has been established to sequence the genome of four Wolbachia species from insects and nematodes and one of the four chosen is the Wolbachia of B. malayi (Slatko et al. 1999). Recent studies indicate that lipopolysaccharide-like molecules from the Wolbachia endobacteria may be involved in mediating the inflammatory responses induced by filarial nematodes in their mammalian host (Taylor et al. 2000; Brattig et al. 2000). The Wolbachia genome warrants detailed analysis and may supply additional anti-filarial drug targets and vaccine candidates.
FUTURE DIRECTIONS The Filarial Genome Project has recently reached a milestone in completing the EST phase of the research effort. Current and future research
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efforts are now being directed at genome mapping, genome sequencing, and the arraying of all 8000 known B. malayi genes on "gene chips" to facilitate global gene expression studies. The complete sequencing of the Wolbachia endosymbiont and the mitochondrial genome are also high priorities. The Institute for Genome Research (TIGR) with assistance from the Filarial Genome Project has recently initiated large-scale sequencing of B. malayi genomic DNA(http://www.tigr.org/tdb/e2kl/bmal/). A continuing goal of the Filarial Genome Project is to continue to contribute resources that will be useful in understanding the biology of filarial parasites and in the development of new antifilarial drugs and/or a vaccine against lymphatic filariasis.
ACKNOWLEDGEMENTS The authors wish to acknowledge all members of the Filarial Genome Project including the members of the following laboratories: Steven A. Williams (Smith College), Mark L. Blaxter (University of Edinburgh), Reda Ramzy (Ain Shams University, Cairo, Egypt), Barton Slatko (New England Biolabs), Tania Supali (University of Indonesia), and Alan L. Scott (Johns Hopkins University). The authors wish to give special thanks to Michelle Lizotte-Waniewski for construction of most of the conventional cDNA libraries and to John Quackenbush and Geo Pertea of TIGR for their contributions to the organization and analysis of the EST clusters.
REFERENCES Blaxter, M.L., D.B. Guiliano, A.L. Scott, and S.A. Williams. 1997. A unified nomenclature for filarial parasites. Parasitology Today 13: 416-417. Blaxter, M.L., M. Aslett, D. Guiliano, and J. Daub. 1999. Parasitic helminth genomics. Parasitology 118: S39-S51. Brattig, N.W., U. Rathjens, M. Ernst, F. Geisinger, A. Renz, and F.W. Tischendorf. 2000. Lipopolysaccharide-like molecules derived from Wolbachia endobacteria of the filaria Onchocerca volvulus are candidate mediators in the sequence of inflammatory and antiinflammatory responses of human monocytes. Microbes and Infection 2: 1147-57. Degrave, W.M., S. Melville, A. Ivens, and M. Aslett. 2001. Parasite genome initiatives. International Journal of Parasitology 31: 531-5. Diatchenko, L., Y.F. Lau, A.P. Campbell, A. Chenchik, F. Moqadam, B. Huang, S. Lukyanov, K. Lukyanov, N. Gurskaya, E.D. Sverdlov, and P.D. Siebert. 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proceedings of the National Academy of Sciences, USA 11: 6025-30. Hoerauf, A., L. Volkmann, C. Hamelmann, O. Adjei, I.B. Autenrieth, B. Fleischer, and D.W. Buttner. 2000. Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis. Lancet 355:1242-3. McLaren, D.J., M.J. Worms, B.R. Laurence, and M.G. Simpson. 1975. Micro-organisms in filarial larvae (Nematoda). Transactions of the Royal Society of Tropical Medicine and Hygiene 69: 509-14. McReynolds, L.A., S.M. DeSimone, and S.A. Williams. 1986. Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal
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parasite Brugia pahangi. Proceedings of the National Academy of Sciences, USA 83: 797801. Quackenbush, J., J. Cho, D. Lee, F. Liang, I. Holt, S. Karamycheva, B. Parvizi, G. Pertea, R. Sultana, and J. White. 2001. The TIGR gene indices: analysis of gene transcript sequences in highly sampled eukaryotic species. Nucleic Acids Research 29: 159-164. Scott, A.L. and P. Yenbutr. 1995. Molecular cloning of a serine protease inhibitor from Brugia malayi. Infection and Immunity 63: 1745-53. Slatko, B.E., S. O’Neill, A.L. Scott, J.L. Werren, and M.L. Blaxter. 1999. The Wolbachia Genome Consortium Meeting Summary. Microbial and Comparative Genomics 4: 161-5. Smith, H.L., and T.V. Rajan. 2000. Tetracycline inhibits development of the infective-stage larvae of filarial nematodes in vitro. Experimental Parasitology 95:265-70. Sun, L.V., J.M. Foster, G. Tzertzinis, M. Ono, C. Bandi, B.E. Slatko, and S.L. O’Neill. 2001. Determination of Wolbachia genome size by pulsed-field gel electrophoresis. Journal of Bacteriology 183: 2219-25. Taylor, M.J., H.F. Cross, and K. Bilo. 2000. Inflammatory responses induced by the filarial nematode Brugia malayi are mediated by lipopolysaccharide-like activity from endosymbiotic Wolbachia bacteria. Journal of Experimental Medicine 191: 1429-36. The C. elegans Genome Sequencing Consortium. 1998. Genome sequence of the nematode C. elegans. A platform for investigating biology. Science 282: 2012-18. Townson, S., D. Hutton, J. Siemienska, L. Hollick, T. Scanlon, S.K. Tagboto, and M.J. Taylor. 2000. Antibiotics and Wolbachia in filarial nematodes: antifilarial activity of rifampicin, oxytetracycline and chloramphenicol against Onchocerca gutturosa, Onchocerca lienalis and Brugia pahangi. Annals of Tropical Medicine and Parasitology 94: 801-16. Unnasch, T.R. 1994. The filarial genome project. Parasitology Today 10: 415-6. Werren, J.H. 1997. Biology of Wolbachia. Annual Reviews of Entomology 42: 587-609. Williams, S.A., S.M. DeSimone, and L.A. McReynolds. 1988. Species-specific oligonucleotide probes for the identification of human filarial parasites. Molecular and Biochemical Parasitology 28: 163-169. Williams, S.A. and D. J. Kemp, 1996. Parasite genome projects. In Encyclopedia of Molecular Biology and Molecular Medicine, R.A. Meyers, (ed.). VCH Publishers, Inc., New York, p. 306-312. Williams, S.A. 1999. Deep within the filarial genome: progress of the Filarial Genome Project. Parasitology Today 15: 219-24. Williams, S.A. and D.A. Johnston. 1999. Helminth genome analysis: the current status of the filarial and schistosome genome projects. Parasitology 118: S19-S38. Williams, S.A., M.R. Lizotte-Waniewski, J. Foster, D. Guiliano, J. Daub, A.L. Scott, B. Slatko, and M.L. Blaxter. 2000. The filarial genome project: analysis of the nuclear, mitochondrial and endosymbiont genomes of Brugia malayi. International Journal of Parasitology 30: 4119. Xie, H., O. Bain, and S.A. Williams. 1994. Molecular phylogenetic studies on filarial parasites based on 5S ribosomal spacer sequences. Parasite 1: 141-151. Zorio, D.A.R., N.N. Cheng, T. Blumenthal, and J. Spieth. 1994. Operons represent a common form of chromosomal organization in C. elegans. Nature 372: 270-2.
THE EPIDEMIOLOGY OF ONCHOCERCIASIS AND THE LONG TERM IMPACT OF EXISTING CONTROL STRATEGIES ON THIS INFECTION
Peter Fischer and Dietrich W. Büttner Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany
ABSTRACT Onchocerciasis is a disease of great public health importance in Africa. Due to the long life span of the adult worms and the absence of a safe macrofilaricidal drug, long-lasting control programs are needed. Although onchocerciasis was successfully controlled in West Africa by vector control, the current strategy is the interruption of transmission by community-based ivermectin treatment. This microfilaricidal drug can reduce morbidity significantly, but cannot clear infection in most parts of Africa. The long term impact of ivermectin in control programs is dependent on persistent elimination of microfilariae from their human reservoir. Supportive measures are required to ensure the success of the current control efforts. Keywords: River blindness, onchocerciasis, public health importance, ivermectin, control programs
LIFE CYCLE AND DISTRIBUTION The filarial parasite Onchocerca volvulus, the agent of onchocerciasis, is a public health problem in many parts of tropical Africa and to a lesser degree in Latin America. The adult worms live in subcutaneous nodules, onchocercomas. During their reproductive life span of about 9-11 years, female worms produce millions of microfilariae. These larvae migrate in the skin and can be taken up by a susceptible blackfly vector during its blood meal. In the vector, the first stage larva penetrates the midgut wall and migrates to the flight muscles where it molts twice. The third stage larva migrates to the head of the blackfly, becomes infective and can be transmitted via the blood meal. Man is the only natural vertebrate host of this parasite. The development within the vector takes about 1-2 weeks, while the development of the parasite to the adult stage in the human being takes one year or longer, at which time a female worm mates and produces microfilariae. It was estimated in 1993 that 17.7 million people were infected with O. volvulus. About 99% of the infections can be found in sub-Saharan Africa (WHO 1995). The highest prevalence of infections is in the Democratic
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Republic of Congo, Nigeria, Cameroon and Uganda, with about 10 million people infected. Historically, onchocerciasis had the highest prevalence in West Africa, but due to the success of the Onchocerciasis Control Programme (OCP), the prevalence has dropped considerably in that area. Onchocerciasis foci in Latin America are usually small. About 1.6 million persons are assumed to be at risk, while only about 130,000 persons are estimated to be infected in Mexico, Guatemala, Ecuador, Venezuela and in a few foci in Brazil and Colombia (Espinel 1998). Similarly, only about 30,000 people are assumed to be infected in the Yemen. The distribution of O. volvulus is largely dependent on the presence of suitable breeding sites for its Simulium vector. In some endemic areas, almost all adults are infected, whereas only a few kilometers further away from the vector breeding site the prevalence of onchocerciasis drops to zero. This is especially true if the local vector species has a short flight range as for example Simulium neavei and S. yahense. However, certain species of the S. damnosum complex can drift for more than 100 km. This has great implications for reinvasion in vector controlled areas. In Liberia, the savanna species of the S .damnosum complex can seasonally invade far distant forest areas and become a biting nuisance (Garms, et al. 1991). These blackflies may transmit both the local forest O. volvulus parasites or, if carrying savanna parasites, those as well since vector-parasite transmission complexes play no fixed role in the epidemiology of onchocerciasis (Toe et al. 1997). The prevalence of infection within a population is dependent on exposure to Simulium bites. Prior to onchocerciasis control, almost all adults above the age of 19 years were infected in hyperendemic areas in Burkina Faso, Liberia and Uganda (Fig. 1).
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In Uganda more than 40% of these adults had palpable nodules (Fischer et al. 1993). It takes about 8 to 10 years of residence in such a highly endemic area until a plateau of prevalence of microfilaria carriers is reached, and 2 to 4 years longer before a plateau of palpable onchocercomas can be observed (Fig. 2).
Within a population in a hyperendemic area, putatively immune individuals are also seen. These persons are exposed for a long period of time (e.g. 17 years) to transmission and are negative for the infection clinically, parasitologically and by PCR-based tests even if assessed multiple times over many years. In addition, specific antibody responses indicate contact with infective larvae. The prevalence of putative immune individuals in hyperendemic areas is usually less than 5% of the total population. It cannot be excluded that putatively immune persons present a group of individuals with a lower susceptibility to O. volvulus and that protective immunity is not absolute, but rather a function of transmission pressure. Prevalence of O. volvulus infection is subjected to natural variation. Although there are seasonal changes of transmission rates in many areas almost no seasonal variation in prevalence of human infection is seen because of the long duration of development and the long life span of the worms. However, environmental changes like deforestation can lead to a decline of forest dwelling vector species, followed by a decline of prevalence (Fischer et al. 1997a). A rapid increase of the human population may diminish the vectorfilarial parasite contact and may dilute the force of transmission considerably. A similar effect can be observed if the vector species is to a certain degree zoophilic and the population of its animal hosts is increasing. It has been shown that O. volvulus and its closest relative, the S. damnosum s.l. transmitted cattle parasite Onchocerca ochengi, share many antigens.
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Therefore, due to the immunological cross-reactions between both species, O.ochengi can also induce an immune response in humans that might lead to a zooprophylactic effect (Wahl et al. 1998). Since immunological crossreactions are common among filarial parasites, other human or even animal parasites inoculated by man-biting vectors may induce some degree of protective immunity against O. volvulus infection.
CLINICAL SIGNS AND PUBLIC HEALTH RELEVANCE The most impressive clinical signs of onchocerciasis are eye diseases leading to the common name river blindness. The intensity of infection is related to the level of transmission and, therefore, in most cases eye pathology depends on the worm load. In 1993 it was estimated that about 270,000 people were blind and 500,000 more were severely disabled due to onchocercal eye diseases (WHO 1995). Whereas onchocercal blindness is typically a sign of a long lasting infection, onchodermatitis is often found in children and young adults (Fischer et al. 1993). In addition, the hyperreactive or sowda form of onchocerciasis, originally described from the Yemen, is also found in other countries. The clinical picture of sowda is linked to a mild to severe, often asymmetric papular dermatitis, enlarged local lymph nodes, low microfilaria densities and a strong antifilarial antibody response (Brattig et al. 1994). During recent years, several studies have highlighted the psychological and socioeconomic impact of onchocercal skin disease. A multicountry study of the WHO revealed that affected persons spend US $ 20 more each year (about 15% of their annual income) on health-related expenditures and more time at health care facilities and that affected children are more likely to drop out of school (WHO 1995). In Nigeria, farmers with onchocercal skin disease have about one third less farm land under cultivation than those without the disease (Oladepo et al. 1997). Onchocercal skin disease can lead to serious stigmatization, with profound social and economic consequences. An increased prevalence of epilepsy is reported from areas hyperendemic for onchocerciasis in Uganda and an association is suspected (Kaiser et al. 1996). Although there is still no evidence for a causal relationship between O. volvulus infection and epilepsy, it is noteworthy and requires further research. Since the middle of the last century an endemic form of dwarfism, the so-called “Nakalanga” syndrome, is known to occur in onchocerciasis foci in Uganda. Many of the affected individuals are also epileptics. This form of endemic dwarfism is still observed in Uganda (Kipp, et al. 1996). The progress of onchocerciasis control may help to elucidate the relationship between onchocerciasis, epilepsy and the “Nakalanga” syndrome, since successful intervention may affect all three. In onchocerciasis, high microfilaria densities can lead to immunosuppression to both parasite and non-parasite antigens. This has important implications for the efficacy of vaccination and it has been shown
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that onchocerciasis patients may have a diminished cell-mediated immune response to tetanus toxoid vaccinations (Cooper et al. 1998). Cellular hyporesponsiveness is caused by high microfilaria densities and immunoreactivity can be restored by the microfilaricidal drug ivermectin (Soboslay et al. 1992). Therefore, ivermectin can help indirectly to increase the efficacy of vaccinations. Heavy O. volvulus infection may lead to increased susceptibility to other infectious agents like HIV. In vitro studies on peripheral blood mononuclear cells from filariasis patients indicate that filarial infections may increase the susceptibility to HIV-1 (Gopinath et al. 2000). However, no epidemiologic association between HIV-1 infection and onchocerciasis has been observed (Fischer et al. 1995). In many cases, O. volvulus infection causes no obvious clinical signs, but high microfilaria densities may lead to a diminished life expectancy. Persons infected with O. volvulus show an eosinophilia, which is induced by the microfilariae and not by live adult worms (Wildenburg, et al. 1995). The long-term consequences of hypereosinophilia in persons with onchocerciasis are still not known. It cannot be excluded that even in persons with generalized onchocerciasis and with no clinical signs of eye and skin diseases, parasites cause irreversible pathological damage.
PARASITE STRAINS The clinical picture of onchocerciasis is variable in different geographical regions and often differs between ecological zones within a region. Prior to the OCP, a high prevalence of blindness was seen notably in the West African savanna. The prevalence of blindness and severe eye diseases sometimes reached 10% or more, especially if young, unaffected adults moved out and left the blind, older people behind. In the forest areas in West Africa, severe eye disease due to O. volvulus infection is rare. Therefore, it has been hypothesized that a distinct strain of O. volvulus different from that in the forest areas in the West African savanna is responsible for the high prevalence of blinding. Morphometrical and biochemical differences have been described between parasites from these areas. Most impressive is the discovery that forest and savanna parasites can be differentiated by specific DNA sequences. These non-coding and fast evolving molecular markers are organized in a 150 bp long tandem repeat (O150) with about 4000 copies per haploid genome. In West Africa, the sequence differences in this repeat correlate well with the epidemiologic patterns of blindness (Zimmerman et al. 1992). In Eastern Africa different strains of O. volvulus have been reported from western Uganda and the Abu Hamet focus in the Sudan (Fischer et al. 1996a, Higazi et al. 2001). Based on the O-150 classification, it was assumed that the parasites in Brazil and Guatemala are indistinguishable from savanna strains in West Africa, although they may have been introduced with slaves originated from both forest and savanna areas (Zimmerman et al. 1994). Onchocercal blindness is rare in Brazil, but was more frequently observed in
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Guatemala and Mexico, with more onchocercomas in the head. It appears reasonable to conclude that several different strains of O. volvulus occur throughout its large distribution area, but strain differences are not sufficient to explain all the geographical variation of the disease. The human host, biting habits of the vector or environmental factors may also influence the clinical picture of onchocerciasis. Since all strains of O. volvulus have public health importance, strain classification appears to lose importance. However, with advanced intervention, intraspecific molecular markers may gain practical importance again. They may be helpful to identify the origin of parasites if introduced in control areas or as population markers if drug resistance develops.
IDENTIFICATION OF ENDEMIC MODELING OF EPIDEMIOLOGY
AREAS
AND
The classical way of identification of O. volvulus infected persons is the parasitological demonstration of microfilariae in the skin. Modern and more sensitive methods include the detection of DNA (O-150) by PCR in skin snips or even skin scratches (Fischer et al. 1996b, Toe et al. 1998). Many studies are aimed at the development of a sensitive, non-invasive field method for the detection of O. volvulus infection. A Mazzotti test, which uses a topical diethylcarbamazine (DEC) lotion to induce a skin reaction in microfilaria positive persons can be applied to detect onchocerciasis recrudescence (Toe et al. 2000). Serological assays require blood collection and are often not specific for O. volvulus. However, specific recombinant antigens have been described and a rapid-format antibody card test for the diagnosis of onchocerciasis in the field has been developed (Weil et al. 2000). An advantage of antibody assays is that some of them are able to detect prepatent infection. A disadvantage is that antibody response is often long lasting and the short-term effects of intervention efforts cannot be detected. For epidemiological purposes, the identification of endemic areas by palpation of selected high risk age groups (e.g. men over 19 years of age) for onchocercomas is suitable and can be used for the rapid epidemiological mapping of onchocerciasis (REMO, Whitworth et al. 1999). There is a good correlation between the prevalence of adult nodule carriers and the prevalence of microfilaria carriers (WHO 1998). For the selection of villages for REMO detailed geographical information is important and a framework of indicator villages has to be selected around potential vector breeding sites. REMO data can be analyzed by Geographical Information System (GIS) software to obtain maps of onchocerciasis distribution. Several models have been developed to describe the epidemiology of onchocerciasis and to estimate the effect of intervention programs. The computer program ONCHOSIM was developed to study the epidemiology and effect of control measures of onchocerciasis mainly in the savanna areas of West Africa (Plaisier et al. 1990). Another program, called SIMON, was
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developed based on data collected from onchocerciasis areas in the forest of Sierra Leone (Davies 1993). Both programs use the community microfilarial load as parameter which describes the geometric mean microfilarial load of adults in a community who are at least 20 years old, including non-infected individuals. The main value of these computer programs lies in the prediction of the outcome of intervention procedures in different epidemiological settings.
CONTROL OF IVERMECTIN
ONCHOCERCIASIS
PRIOR
TO
The surgical removal of onchocercomas by nodulectomy was used in Central America to control O. volvulus infection. This procedure reduced the microfilarial loads significantly. Due to its high cost, nodulectomy could not be applied for large scale control of onchocerciasis in Africa. The drug suramin has not only micro- but more importantly also macrofilaricidal action and was used in Venezuela and Sudan for broad scale treatment of onchocerciasis patients. The treatment regime involves repeated intravenous injections and the drug is too toxic to be used for mass treatment (Chijioke et al. 1998). In O. volvulus infection diethylcarbamazine (DEC) rapidly kills microfilariae, but does no harm to the adult worms. However, in heavily infected persons it causes serious side effects, sometimes leading to increased eye damage (WHO 1987). Therefore, DEC is not recommended any longer for the treatment of onchocerciasis. Vector control was the essential strategy to control onchocerciasis for many years. The most important program was the OCP, established in 1974 in the savanna of West Africa. Over time, the OCP was extended to an area of about in 11 countries. The strategy was to interrupt the transmission of O. volvulus for a period longer than the life expectancy of the adult worms, by elimination of the vector. Seven different insecticides were used to spray the breeding rivers of the blackflies, including Bacillus thuringiensis H14 toxin, organophosphates, synthetic pyrethroids or carbamate (Hougard et al. 1997). The success of the OCP was extraordinary. In the original core area, the prevalence dropped from 60-84% in 1974 to 06% in 1996. Great success was also observed in the western and southern extensions (Molyneux et al. 1997). About 2.2 million people are still infected with O. volvulus in the OCP area, but 30 million people are being protected from the risk of infection and about 200,000 individuals from becoming blind. However, there are still a few foci in the OCP area endemic for onchocerciasis due to accidental reinvasion of blackfly vectors, serious logistical problems during control, premature discontinuation of vector control and massive migration of infected persons returning from the southern forest areas. Therefore, it is extremely important to monitor these areas in order to prevent the spreading of infection to adjacent areas. For the long-term maintenance of the success of the OCP, recrudescence of transmission can
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only be prevented if transmittable microfilariae of O. volvulus are eliminated from their human reservoir. For this reason, the OCP will be taken over by the African Programme of Onchocerciasis Control (APOC) in 2002.
CONTROL STRATEGIES INVOLVING IVERMECTIN The introduction of the drug ivermectin in the 1980s was a milestone in the control of onchocerciasis. Following a standard single oral dose of body weight, ivermectin (Mectizan®) shows a strong microfilaricidal effect and a long-term reduction of microfilaria production by female worms. Computer simulations indicate irreversible effects of ivermectin on adult worms, leading to a permanent decline of microfilariae (Plaisier et al. 1995). However, no adulticidal effect could be proven, even following elevated doses (Awadzi et al. 1999). Ivermectin is safe enough to be employed for community-based treatment to control onchocerciasis. In 1987, the manufacturer Merck & Co Inc. made the generous decision to provide ivermectin free of cost for onchocerciasis control for as long as it will be needed. Therefore, the main expenses to control onchocerciasis by mass distribution are the operational costs. Community-based ivermectin treatment should firstly reduce the disease and should finally lead to the interruption of transmission. Since persons with low microfilarial densities are also infectious to blackflies, it is essential to have a sufficient coverage of the population. Independently from the logistic difficulties, this was not achievable in the beginning of the community-based ivermectin treatment because the manufacturer excluded parts of the population, such as pregnant women. Since there is no indication from human or animal use that ivermectin has any embryotoxic or mutagenic effect, pregnant women are no longer excluded from treatment. It was also found that HIV-positive individuals infected with O. volvulus do benefit from ivermectin treatment and do not seem to manifest any additional risk of side effects for community-based treatment as long as they have no acute disease due to AIDS (Fischer et al. 1995). As of today, resistance to ivermectin is unknown in O. volvulus, although it has been observed in some species of animal nematodes. In the ruminant parasite Haemonchus contortus, a four-fold decreased ivermectin sensitivity was found following a selection over 14 generations using the avermectin-related drug moxidectin (Molento et al. 1999). Assuming a period of two years for one generation of O. volvulus and similar mechanisms of ivermectin resistance as in H. contortus, it would take 28 years before decreased sensitivity to the drug appears. In addition, decreased ivermectin sensitivity may be compensated by increasing the dosage. Although a single standard dose of for onchocerciasis is recommended, a dosage of up to is usually well tolerated (Awadzi et al. 1999). Homologs of molecules which play a central role in ivermectin resistance of animal nematodes have been already identified in O. volvulus in order to characterize
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a possible resistance at the molecular level (Huang et al. 1999). The use of ivermectin in combination with other drugs may also reduce the danger of developing ivermectin resistance. Although albendazole probably has no additional effect on O. volvulus when used together with ivermectin (Awadzi et al. 1995), it may help to conserve the efficacy of ivermectin. Following ivermectin therapy, microfilariae disappear from the skin within hours and drain to the local lymph nodes, where they are cleared. Embryogenesis in the female worm is strongly reduced or interrupted for several months. Unfortunately, adult worms slowly resume reproduction thereafter. Three to four months after treatment, a few new skin microfilariae can be detected. Increasing transmission can be observed about three to nine months following community-based treatment. One year after ivermectin, pretreatment values can sometimes be reached. Following multiple rounds of annual treatment, transmission rates may gradually drop, but in many areas annual ivermectin treatment may be not sufficient to interrupt transmission. If interruption of transmission is really required because vectors may spread infection to other areas semiannual treatment may be an option. Whereas increased frequency of treatment can help to interrupt transmission, increasing ivermectin doses may help to prevent the development of ivermectin resistance. The administration of higher ivermectin doses would not increase the logistic costs for drug distribution and would not increase the risk of side effects, if the microfilarial load has already been reduced due to prior treatment. The development of an inexpensive, reliable and self-sustainable distribution system for community-based ivermectin treatment is the most crucial task of the current control efforts. The successful OCP will end in 2002. Computer simulations predict that in onchocerciasis endemic areas of the OCP, 12 years of combined vector control and ivermectin treatment will be sufficient to reduce the risk of recrudescence to below 1%, assuming that 65% of the population participate in the treatment and there is no importation of infection from elsewhere (Plaisier et al. 1997). However, political unrest in Sierra Leone and Liberia impedes effective onchocerciasis control by APOC and infection may be spread from there to the western extension or the Cote d’Ivoire extension area of the OCP, where active foci still exist. Although these new infections may not cause much pathology due to low parasite loads, they put the long-term success of onchocerciasis control at risk. A second program for onchocerciasis intervention using communitybased treatment with ivermectin is the Onchocerciasis Elimination Program of the Americas (OPEA). It was established 1993 and aims at the elimination of all morbidity due to onchocerciasis in Latin America by the year 2007 (Blanks et al. 1998). The isolated nature of onchocerciasis in Latin America is an excellent prerequisite for the permanent elimination of infection. Although the ecology of onchocerciasis in Latin America is different from that in Africa, it may provide a test of whether elimination of infection by community-based ivermectin treatment is feasible.
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The third and largest program for onchocerciasis control is the APOC. It was decided in 1995 to establish, within a period of twelve years, effective and self-sustainable community-based ivermectin treatment throughout the non-OCP areas in Africa to eliminate the disease and to apply vector control only in selected foci (Remme 1995). Although it should be possible to eliminate the disease by annual community-based ivermectin treatment, the question remains whether the disease can be permanently eliminated after cessation of intervention efforts, without eliminating the infection. Apart from ivermectin, a macrofilaricidal or irreversibly sterilizing drug is still desirable to back up the progress of onchocerciasis control. A new approach in filariasis treatment is the use of antibiotics which target the endosymbiontic, intracellular Wolbachia bacteria. First studies using doxycycline showed promise that this approach can be used for the treatment of onchocerciasis (Hoerauf et al. 2001). It is unclear whether and when doxycycline or other anti-Wolbachia drugs can be used for community-based treatment, but doxycycline may be an alternative for the treatment of onchocerciasis patients, especially if they migrate to areas suitable for resumption of transmission.
FILARIAL INFECTIONS VOLVULUS
COENDEMIC
WITH
O.
Ivermectin can be also used for an integrated control of onchocerciasis and lymphatic filariasis in co-endemic areas. Since ivermectin showed better efficacy at a dosage of in Wuchereria bancrofti infection, it should be used at this dosage. Integrated control is essential, because separate programs for the two filarial parasites appear not to be sustainable. For the treatment of W. bancrofti infection, DEC alone or in combination with albendazole or ivermectin is recommended, because in this species these regimens are not only microfilaricidal, but show also limited macrofilaricidal action. However, these multi drug treatments are not recommended for community-based treatment in areas co-endemic for O. volvulus (Ottesen et al. 1997). Severe side effects of single doses of DEC have been only observed in onchocerciasis patients with moderate or high parasite loads. Research is needed to determine whether low doses of DEC can be safely applied for treatment of lymphatic filariasis and co-endemic onchocerciasis in persons with very low parasite densities due to prior ivermectin treatment. In contrast to other filarial infections, severe side effects of ivermectin have been reported in a few persons infected with Loa loa, with high microfilarial loads of more than 50,000 microfilariae per ml (Gardon et al. 1997). Therefore, ivermectin cannot be used for mass treatment in onchocerciasis areas co-endemic for loiasis without prior determination of the intensity of L. loa infection. Ivermectin was shown to be effective for the treatment of M. streptocerca infection (Fischer et al. 1999). The drug has also
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a beneficial effect for persons infected with Ascaris and Strongyloides or scabies (Ottesen et al. 1997). These additional effects of onchocerciasis control by community-based ivermectin treatment can help to insure the compliance in the community over the required period of time.
SUPPORTIVE CONTROL MEASURES Vector control may be still indicated in selected areas where increasing transmission is observed despite the use of community-based ivermectin treatment. Sometimes, health education may strengthen control programs, because it is often difficult to convince individuals to take drugs for altruistic reasons, if they do not suffer from their onchocerciasis. Blackflies are outdoor biting insects and they are a nuisance in many areas. The effect of protective clothes to avoid Simulium bites is often limited. In the Yemen, where people traditionally cover their body with clothing, the local vector of O. volvulus, S. rasyani, rapidly finds the accessible body parts and bites men in the lower leg. The development of a vaccine may be one strategy to back up the success of the current control programs. This vaccine may target the infective larvae to stop the establishment of infection in vaccinated individuals or the microfilariae to reduce pathology and to avoid transmission. The later target may have the advantage that only infected individuals have to be vaccinated. Although vaccination against O. volvulus may be an elegant and efficient strategy to control onchocerciasis, it is also expensive. Once a vaccine candidate has been identified, the development and registration of a new vaccine costs US$500,000,000 or more. If this amount of money were available to support the current control programs, their prospective of success would be marvelous. The Brugia malayi and the O. volvulus genome projects have created a good basis for identification of new vaccine candidates or drug targets. These approaches can take advantage of the success of the current control efforts and the reduced O. volvulus population that remains to be eliminated. The development of new drugs is not an alternative but a much needed back up to the current control strategies.
MONITORING AND LONG CONTROL PROGRAMS
TERM
IMPACT
OF
Monitoring control programs is essential in order to detect recrudescence of human infection and ongoing transmission. The production of microfilariae by female worms is a prerequisite for resuming transmission. The establishment of new infections or re-infections is the consequence of transmission. Most serological tests cannot differentiate between infected individuals with and without microfilariae. They may be useful as monitoring tools in areas cleared of infection or in children and migrants previously known to be infected with O. volvulus. The detection of microfilariae in the resident population by parasitological or PCR-based methods or a positive
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DEC patch test indicates the resumption of microfilarial production in infected individuals or the end of prepatency in fresh infections. Due to the long duration of prepatency, the occurrence of new infections shows that transmission must have occurred at least one year earlier. Therefore, the monitoring of transmission by vector screening is a central issue in order to choose the most promising control strategies in time. An important index for the transmission is the Annual Transmission Potential (ATP), which describes the estimated number of infective larvae transmitted per person per year. The differentiation of infective O. volvulus larvae from larvae of other filarial parasites transmitted by the same vector is often difficult, but O. volvulus larvae can be identified by PCR and DNA probe technology (Fischer et al. 1997b). In hyperendemic onchocerciasis areas without effective control efforts, Simulium infection rates are sometimes extremely high, reaching 40% of parous flies. In these areas vector infection rates and transmission rates can be easily determined by vector dissection using a relatively small sample size. In areas with successful control programs the situation is different. To reliably detect vector infection levels of 0.2%, at least 6,000 parous vectors would have to be examined (Basanez et al. 1998). Examination of large numbers of vectors can be performed cost-effectively by PCR using pools of flies and due to the very low infection rates determination of ATP is unnecessary. In the OCP area, an ATP of 100 infective larvae/ person per year was decided to be tolerable for recommendation of resettlement. The threshold vector infection rate to interrupt transmission may vary depending on the vector biting rate and on local vector species. To interrupt O. volvulus transmission by community-based ivermectin treatment, threshold vector infection rates need to be determined in various geographical settings. It is difficult to predict the long term impact of the current control programs. The elimination of onchocerciasis as a public health problem, as was achieved in the core area of the OCP, may be a realistic goal. It appears difficult to maintain this situation without ongoing control measures, as long as infection is endemic and an increase of prevalence cannot be excluded. On the other hand, if the prevalence of onchocercal infection can be reduced to values which cannot sustain transmission, infection may be eliminated without further control. However, immigration of infected individuals or reinvasion of infected vectors may lead to re-establishment of infection. Therefore, highest priority for control should be given to isolated onchocerciasis foci. The longterm prospective to eliminate infection is probably the best in Latin America, and countries such as Yemen or Tanzania, with few scattered foci. CONCLUDING REMARKS Community-based ivermectin treatment may be effective for reducing onchocerciasis as a public health problem. Essential to this strategy is good community participation. Variations of the treatment schedule may be necessary to interrupt transmission in the early phase of control. Increasing
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doses of ivermectin or a combination of ivermectin and albendazole may help to prevent the development of drug resistance. When microfilaria densities have been reduced, further research is needed if other drugs like DEC or antibiotics can be used instead of albendazole in order to provide a broader panel of drugs in the later phase. Although ivermectin alone may do the job, long-term success can be assured only if additional control strategies are available.
REFERENCES: Awadzi K., E.T. Addy, N.O. Opoku, A. Plenge-Bönig, D.W. Büttner. 1995. The chemotherapy of onchocerciasis XX: ivermectin in combination with albendazole. Trop Med Parasitol 46, 213-220. Awadzi K., S.K. Attah, E.T. Addy, N.O. Opoku, B.T. Quartey. 1999. The effects of high-dose ivermectin regimens on Onchocerca volvulus in onchocerciasis patients. Trans R Soc Trop Med Hyg 93, 189-194. Basanez M.G., M.A. Rodrigues-Perez, F. Reyes-Villanueva, R.C. Collins, M.H. Rodriguez. 1998. Determination of sample sizes for the estimation of Onchocerca volvulus (Filarioidea: Onchocercidae) infection rates in biting populations of Simulium ochraceum s.l. (Diptera: Simuliidae) and its application to ivermectin control programs. J Med Entomol 35, 745-757. Blanks J., F. Richards, F. Beltran, R. Collins, E. Alvarez, G. Zea Flores, B. Bauler, R. Cedillos, M. Heisler, D. Brandling-Bennett, W. Baldwin, M. Bayona, R. Klein, M. Jacox. 1998. The Onchocerciasis Elimination Program for the Americas: a history of partnership. Rev Panam Salud Publica 3, 367-374. Brattig N.W., I. Krawietz, A.Z. Abakar, K.D. Erttmann, T.F. Kruppa, A. Massougbodji. 1994. Strong IgG isotypic antibody response in sowdah type onchocerciasis. J Infect Dis 170, 955-961. Chijioke C.P., R.E. Umeh, A.U. Mbah, P. Nwou, L.L. Fleckenstein, P.O. Okonkwo. 1998. Clinical pharmacokinetics of suramin in patients with onchocerciasis. Eur J Clin Pharmacol 54, 249-251. Cooper P.J., I. Espinel, W. Paredes, R.H. Guderian, T.B. Nutman. 1998. Impaired tetanusspecific cellular and humoral responses following tetanus vaccination in human onchocerciasis: a possible role for interleukin-10. J Infect Dis 178:1133-1138. Davies J.B. 1993. Description of a computer model of forest onchocerciasis transmission and its application to field scenarios of vector control and chemotherapy. Ann Trop Med Parasitol 87, 41-63. Espinel M. 1998. Onchocerciasis: a Latin America perspective. Ann Trop Med Parasitol 92, S157-S160. Fischer P., W. Kipp, J. Bamuhiga, J. Binta-Kahwa, A. Kiefer, D.W. Büttner. 1993. Parasitological and clinical characterization of Simulium neavei-transmitted onchocerciasis in western Uganda. Trop Med Parasitol 44, 311-321. Fischer P., W. Kipp, P. Kabwa, D.W. Büttner. 1995. Onchocerciasis and human immunodeficiency virus in western Uganda: prevalences and treatment with ivermectin. Am J Trop Med Hyg 53, 171-178. Fischer P., J. Bamuhiiga, A.H. Kilian, D.W. Büttner. 1996a. Strain differentiation of Onchocerca volvulus from Uganda using DNA probes. Parasitology 112, 401-408. Fischer P., T. Rubaale, S.E.O. Meredith, D.W. Büttner. 1996b. Sensitivity of a PCR-based assay to detect Onchocerca volvulus DNA in skin biopsies. Parasitol Res 395-401. Fischer P., R. Garms, D.W. Büttner, W. Kipp, J. Bamuhiiga, J. Yocha. 1997a. Reduced prevalence of onchocerciasis in Uganda following either deforestation or vector control with DTT. East Afr Med J 74, 321-326.
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Fischer P., J. Yocha, T. Rubaale, R. Garms. 1997b. PCR and DNA hybridization indicate the absence of animal filariae from vectors of Onchocerca volvulus in Uganda. J Parasitol 83, 1030-1034. Fischer P., E. Tukesiga, D.W. Büttner. 1999. Long-term suppression of Mansonella streptocerca microfilariae following treatment with ivermectin. J Infect Dis 180, 1403-1405. Gardon J., N. Gardon-Wendel, Demanga-Ngangue, J. Kamgno, J.P. Chippaux, M. Boussinesq. 1997. Serious reactions after mass treatment of onchocerciasis with ivermectin in an area endemic for Loa loa infection. Lancet 350, 18-22. Garms R., R.A. Cheke, R. Sachs. 1991. A temporary focus of savanna species of the Simulium damnosum complex in the forest zone of Liberia. Trop Med Parasitol 42, 181-187. Gopinath R., M. Ostrowski, S.J. Justement, A.S. Fauci, T.B. Nutman. 2000. Filarial infections increase susceptibility to human immunodeficiency virus infection in peripheral blood mononuclear cells in vitro. J Infect Dis 182, 1804-1808. Higazi T.B., C.R. Katholi, B.M. Mahmoud, O.Z. Baraka, M.M. Mukhtar, Y. Al Qubat, T.R. Unnasch 2001. Onchocerca volvulus: genetic diversity of parasite isolates from Sudan. Exp Parasitol 97, 24-34. Hoerauf A., S. Mand, O. Adjei, B. Fleischer, D.W. Büttner. 2001. Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilardermia after ivermectin treatment. Lancet 357, 1415-1416. Hourgard J.M., L. Yameogo, A. Seketeli, B. Boatin, H.Y. Dadzie. 1997. Twenty-two years of blackfly control in the Onchocerciasis Control Programme in West Africa. Parasitol Today 13, 425-431. Huang Y.J., R.K. Prichard 1999. Identification and stage-specific expression of two putative Pglycoprotein genes in Onchocerca volvulus. Mol Biochem Parasitol 102, 273-281. Kaiser C., W. Kipp, G. Asaba, C. Mugisa, Kabagambe, D. Rating, M. Leichsenring. 1996. Prevalence of epilepsy follows the distribution of onchocerciasis in a West Ugandan focus. Bull Wld Hlth Org 74, 361-367. Kipp W., G. Burnham, J.B. Bamuhiiga, M. Leichsenring. 1996. The Nakalanga syndrome in Kabarole district, western Uganda. Am J Trop Med Hyg 54, 80-83. Molento M.B., G.T. Wang, R.K. Prichard. 1999. Decreased ivermectin and moxidectin sensitivity in Haemonchus contortus selected with moxidectin over 14 generations. Vet Parasitol 86, 77-81. Molyneux D.H., J.B. Davies. 1997. Onchocerciasis control: moving towards the millenium. Parasitol Today 13, 418-425. Oladepo O., W.R. Brieger, S. Otusanya, O.O. Kale, S. Offiong, M. Titiloye. 1997. Farm land size and onchocerciasis status of peasant farmers in south-western Nigeria. Trop Med Int Hlth 2, 334-340. Ottesen E.A., B.O. Duke, M. Karam, K. Bebehani. 1997. Strategies and tools for the control/elimination of lymphatic filariasis. Bull Wld Hlth Org 75, 491-503. Plaisier A.P., G.J. Van Oortmarssen, J. Remme, E.S. Alley, G.J. Habbema. 1990. Onchosim: a model and computer simulation program for the transmission and control of onchocerciasis. Comput Methods Programs Biomed 31, 43-56. Plaisier A.P., E.S. Alley, B.A. Boatin, G.J. Van Oortmarssen, H. Remme, S.J. De Vlas, L. Bonneux, J.D. Habbema. 1995. Irreversible effects of ivermectin on adult parasites in onchocerciasis patients in the Onchocerciasis Control Programme in West Africa. J Infect Dis 172, 204-210. Plaisier A.P., E.S. Alley, G.J. Van Oortmarssen, B.A. Boatin, J.D.F. Habbema. 1997. Required duration of combined annual ivermectin treatment and vector control in the Onchocerciasis Control Programme in West Africa. Bull Wld Hlth Org 75, 237-244. Remme J.H.F. 1995. The African Programme for Onchocerciasis Control: preparing to launch. Parasitol Today 11, 403-406. Soboslay P.T., C.M. Dreweck, W.H. Hoffmann, C.G.K. Lüder, C. Heuschkel, H. Görgen, M. Banla, H. Schulz-Key. 1992. Ivermectin-facilitated immunity in onchocerciasis.
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Reversal of lymphocytopenia, cellular anergy and and deficient cytokine production after single treatment. Clin Exp Immunol 89, 407-413. Toe L., J. Tang, C. Back, C.R. Katholi, T.R. Unnasch. 1997. Vector-parasite transmission complexes for onchocerciasis in west Africa. Lancet 349, 163-166. Toe L., B.A. Boatin, A. Adjami, C. Back, A. Merriweather, T.R. Unnasch 1998. Detection of Onchocerca volvulus infection by O-150 polymerase chain reaction analysis of skin scratches. J Infect Dis 178, 282-285. Toe L., A.G. Adjami, B.A. Boatin, C. Back, E.S. Alley, N. Dembele, P.G. Brika, E. Pearlman, T.R. Unnasch. 2000. Topical application of diethylcarbamazine to detect onchocerciasis recrudescence. Trans R Soc Trop Med Hyg 94, 519-525. Wahl G., P. Enyong, A. Ngosso, J.M. Schibel, R. Moyou, H. Tubbesing, D. Ekale, A. Renz. 1998. Onchocerca ochengi: epidemiological evidence of cross-protection against Onchocerca volvulus. Parasitology 116, 349-362. Weil G.J., C. Steel, F. Liftis, B.W. Li, G. Mearns, E. Lobos, T.B. Nutman. 2000. A rapidformat antibody card test for diagnosis of onchocerciasis. J Infect Dis 182, 17961799. Whitworth J.A., E. Gemade. 1999. Independent evaluation of onchocerciasis rapid assessment methods in Benue state, Nigeria. Trop Med Int Hlth 4, 26-30. WHO (1987). WHO Expert Committee on Onchocerciasis. Wld Hlth Org Tech Rep Ser 752, 1167. WHO (1995). WHO Expert Committee on Onchocerciasis Control. Wld Hlth Org Tech Rep Ser 852, 1-105. WHO (1998). Guidelines for analayis of REMO data using GIS. WHO/TDR/COMDT/98.3, 136. Wildenburg G., M. Krömer, D.W. Büttner. 1995. Dependence of eosinophil granulocyte infiltration into nodules on the presence of microfilariae producing Onchocerca volvulus. Parasitol Res 82, 117-124. Zimmerman P.A., K.Y. Dadzie, G. de Sole, J. Remme, E.S. Alley, T.R. Unnasch. 1992. Onchocerca volvulus DNA probe classification correlates with epidemiologic patterns of blindness. J Infect Dis 165, 964-968. Zimmerman P.A., C.R. Katholi, M.C. Wooten, N. Lang-Unnasch, T.R. Unnasch. 1994. Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family. Mol Biol Evol 11, 384-392.
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THE EPIDEMIOLOGY CONTROL
OF
FILARIASIS
Edwin Michael Department of Infectious Disease Epidemiology, Imperial College School of Medicine, Norfolk Place, London W2 1PG UK
ABSTRACT The planning and evaluation of parasitic control programmes are complicated by the many interacting epidemiological and programmatic factors that jointly determine infection trends under different control options. By facilitating the integration of these factors, mathematical models of parasite transmission can provide a valuable tool for quantifying the impact of such community-based control measures. Here, one such deterministic modelling tool, which describes Culex-mediated bancroftian filariasis transmission, is used to illustrate the vital role that these frameworks can play in the design and evaluation of effective mass chemotherapy programmes for the control of this parasitic disease. The results show that not only can epidemiological models help resolve the practical questions of the duration and required coverage of various proposed regimens for achieving filariasis control, they can also help assess the impact of uncertainties in key variables and their importance to the predicted results. These applications of parasite transmission models thus highlight not only their usefulness as important decision-making tools for control programming but also their value for suggesting areas requiring further theoretical development and field research. Keywords: Lymphatic filariasis, diethylcarbamazine, ivermectin, albendazole, mass chemotherapy, epidemiological modelling
INTRODUCTION Lymphatic filariasis represents one of the few human helminthiases against which large-scale control programmes at the national level have been successfully attempted. Indeed, some of the earliest and currently longest-running national parasite control programmes have been against this mosquito-borne helminthiasis, which have led to the elimination of the disease in several endemic countries (Michael, 1999). Yet, despite this, the burden of filariasis has hardly changed over decades in most endemic countries (Michael and Bundy, 1997), and may even be on the increase in urban locations (Ottesen and Ramachandran, 1995; Michael et al. 1996). In this context the recent resolution by the World Health Assembly and the resulting Global Programme to Eliminate Filariasis (Ottesen et al. 1999) therefore
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represent a renewed global attempt to achieve control of this parasitic infection. The current renewed interest to initiate national programmes to control filariasis stems primarily from the introduction of new treatment regimens, particularly two-drug single-dose combination drug therapies, which dramatically reduce microfilaraemia (Cao et al. 1997; Ottesen et al. 1999; Plaisier et al. 2000), although impetus has also been provided by recent advances in the development of new diagnostic tools (Ottesen, 2000). Coupled with available estimates regarding the mean adult worm lifespan, this has led to the adoption of an elimination strategy based on once-yearly, two-drug treatment (selecting among albendazole (ALB), diethylcarbamazine (DEC) and ivermectin (IVM)) intervention to be given 4-6 years to entire populations where lymphatic filariasis is endemic (Ottesen et al. 1999). The anticipation (and hope) is that such mass chemotherapy would be effective enough during this period to reduce microfilaraemia (the available drugs being primarily microfilaricidal although albendazole and DEC have some macrofilaricidal effects) to levels below which transmission cannot be sustained any further in the community. The uncertainty regarding operational issues of this strategy, such as the required duration of treatment, arises largely from the difficulty of predicting the long-term impact of repeated chemotherapy on the rate of transmission of parasites, and hence reinfection, within a community (Anderson & May, 1985). This is particularly problematic for parasites with complex life cycles like the lymphatic filarial worms, which have complicated transmission dynamics and are therefore likely to demonstrate complex outcomes in response to chemotherapeutic interventions (Chan et al. 1998; Plaisier et al. 1998; Michael and Bundy, 1998; Norman et al. 2000; Michael, 2000). Empirical field studies alone are unlikely to provide the required information due to constraints on resources and time, and the effects of variations in local transmission conditions make generalizations from individual studies difficult. By contrast, epidemiological modelling can provide a powerful analytical framework in which elements of parasite population biology and epidemiology, including worm lifespan, host acquired immunity and vector competence can be combined with programmatic factors such as drug efficacy and treatment coverage, to examine the epidemiological impact of different control options (Anderson & May, 1985; Plaisier et al. 1990; Habbema et al. 1992; Medley et al. 1993; Chan et al. 1995). Here, I use one available deterministic modelling framework which describes filariasis transmission (Chan et al. 1998; Norman et al. 2000), to demonstrate the critical role that such frameworks can play in resolving these questions and hence aide the planning of the proposed control programmes. The next section begins with a brief description of this deterministic transmission model (Norman et al. 2000), and how it can be used to model the epidemiological impact of control on infection (= microfilaraemia) prevalence. Simulations depicting the likely impact of 5 years of annual mass chemotherapy with the different available treatment regimens on microfilaraemia prevalence are then illustrated, focussing on the utility of models both for permitting comparisons of the long-term
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effectiveness of these measures as well as for addressing existing uncertainties regarding both parasite population biology and drug efficacy. The impact of variations in factors as diverse as treatment coverage, infection endemicity and host acquired immunity on the practical question of the duration of treatment required to achieve threshold infection levels will then be explored, which also underlines the crucial role that modelling can play in rational disease control programming. Finally, comparison of model predictions with field data serves to highlight their applicability not only in reproducing the observed epidemiological effects of control (and hence facilitating realistic evaluations of programme effectiveness) but also in revealing their present limitations and the future work required for the successfully resolution of these gaps in knowledge.
THE MODEL OF FILARIASIS TRANSMISSION AND CONTROL Transmission model The development and validation of the deterministic mathematical model of filariasis transmission used in this study has been described previously (Norman et al. 2000), and will only be briefly outlined here. In essence, the model is a system of partial differential equations which aims to describe patterns of filarial infection over age and time in a host population of a defined demographic structure by considering coupled changes in levels of four population variables, viz. mean adult worm burden (W), mean microfilarial count (M), mean acquired immunity level (I) and mean number of L3 larvae per mosquito (L), as follows:
where for adult worm burden, is the number of bites a host receives per unit time (in which represents the number of bites per mosquito per unit time, V is the number of vectors and H is the number of hosts), is the proportion of L3 larvae which leave the mosquito when it bites; is the proportion of these which enter the host and the proportion of L3 entering the host which survive into mature adult worms. h(a) represents the rate at which individuals of age a is bitten. This
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function is specified to increase linearly up to the age of 9 years, after which it is set to unity to capture the age-dependency in mosquito biting rate typically observed in endemic communities (Michael, Ramaiah et al. 2001). is the equilibrium density of L3 larvae (see below), while and denote the death rate of adult worms (default lifespan set at 8 years) and the strength of acquired immunity respectively (Norman et al. 2000). For microfilariae, is the rate of production of microfilariae per worm (scaled to blood sampling volume) and is the death rate of microfilariae (set at 0.1 per month or a lifespan of 10 months). The level of acquired immunity (I) is assumed to be equivalent to the accumulated experience of worm infection and is specified to operate long-term (Chan et al. 1998; Michael, Simonsen et al. 2001). In eqn 4 for L3 dynamics, additional terms include g, the proportion of bites which are made on infected individuals and which result in the mosquito becoming infected, the age distribution of the population under consideration, and the death rate of L3s. The function f (M) describes the population effect of Wuchereria bancrofti microfilariae uptake and density-dependent development into L3 (specifically the limitation mechanism) in the Culex quinquefasciatus mosquito vector. See Norman et al. (2000) for details of deriving this function from combining the rates of uptake and development of larvae by mosquitoes with an assumed negative binomial distribution of infection in the population. Given that L changes more rapidly than the other variables, we further simplify eqn 4 by deriving the equilibrium number of L3 larvae per mosquito by solving dL/dt = 0 (details in Norman et al. (2000)). The validation of the model against field data from South India for Culex- transmitted bancroftian filariasis is also described in Norman et al. (2000), and parameter values employed and obtained from that study are used in all the simulations carried out here. Modelling the population effects of mass treatment Using parasite transmission models to assess the effect of a community-based chemotherapeutic intervention essentially allows for quantifying how a perturbation on infection in the host population dynamically affects infection levels in the vector population and hence overall parasite transmission in that community. Treatment in such models for helminths is normally modelled as instantaneous reductions in parasite stages in the host population (Anderson & May, 1985). For filariasis, available drugs have impacts on both the adult worm and microfilarial stages, and hence treatment at each time is modelled as instantaneous reductions in both W and M by varying degrees depending on known efficacies of the various proposed treatment regimens. Here, we use the efficacy parameters listed in Table 1 for the various regimens used/proposed in filariasis chemotherapy to carry out the simulations reported in this chapter. Note that while these values represent our best estimates of the efficacies of these drug regimens available from the drug trials literature, an advantage of using mathematical models to explore effectiveness is that the impact of uncertainties in these values can be easily examined and quantified. While the present model simulates the
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effect of several rounds of treatment based on drug effects on parasite levels, we focus here on the outcomes on microfilaraemia prevalence using the following equation to convert from microfilarial intensity to prevalence (Chan et al. 1998; Michael, Simonsen et al. 2001):
Here, is the prevalence in age class a and is the average microfilariae count in age class a, while and are parameters with values 0.0029 and 0.0236 respectively (Norman et al. 2000).
THE POPULATION DYNAMICS OF FILARIASIS CONTROL BY MASS CHEMOTHERAPY Simulation of annual treatment programmes Figure 1 shows the results of the simulation of the different mass treatment regimens, viz. DEC alone, IVM alone, DEC plus IVM given together, DEC plus ALB given together and IVM plus ALB given together, proposed for filariasis control on the community agemicrofilaraemia prevalence during and 10 years following a 5-year repeated annual mass intervention programme. Overall community precontrol microfilaraemia prevalence was set at 10% in all the simulations, and the impact of annual mass chemotherapy on age-patterns of infection was investigated using a microfilaraemia age-prevalence curve which rises with host age to a plateau among the older age-classes. Such agepatterns of infection are thought to imply the lack of operation of acquired immunity in the community (Michael and Bundy, 1998; Michael, 2000; Michael, Simonsen et al. 2001). The simulations for each individual chemotherapy regimen were carried out using the treatment parameters given in Table 1, assuming a random coverage of 80% of the host community in each case. Default assumptions about the life history and transmission dynamics of the parasite, as given in Norman et al (2000) and above, are used throughout. The demographic age pattern for the Indian sub continent (World Bank, 1993) with a maximum human age of 80 years is used in all simulations.
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The plots in Figure 1 highlight the dramatic impact that all the proposed regimens can have on age-infections in the community in the short-term (during the period of the 5-year treatment cycle). A general result, however, is that reinfection of the population will inevitably occur in all cases once mass treatment ceases if transmission is not interrupted. The present model cannot determine this transmission threshold, but theoretical studies on the population dynamics of helminth infections (Anderson and May, 1992) suggest that such breakpoints in transmission are likely to be close to zero worms per host. Here, we therefore used a 0.5% overall community microfilaraemia prevalence as a potential target threshold to assess if any of the proposed treatments could achieve interruption of transmission following the 5-year mass treatment strategy. The results (summarized for overall community infection in Fig. le) indicates that while the studied regimens may induce differential infection reduction and reinfection patterns, none of the drug regimens were able to break parasite transmission based on this criterion for an initial microfilariae endemicity prevalence of 10%. Despite this, one clear finding from these simulations is the superiority of combination therapy compared to using single drugs in reducing community microfilarial prevalence over the studied 10-year period (Fig. 1). Reinfection rates are also lower in general with combination drug therapy. A second important finding from these results is the general superiority of DEC-based regimens in depressing microfilarial prevalence. As noted by Norman et al. (2000), this is mainly due to the higher macrofilaricidal effect of this drug compared to ivermectin (Table 1), which leads to a greater and longer suppression of community infection loads. Thus, of the treatment regimens examined in this study, the best effect on microfilarial prevalence (both depression of infection during treatment and reinfection following the annual treatments) is afforded by DEC (35% worm kill) plus ALB (assumed here to provide a 20% additional worm kill (deduced from Ottesen et al. (1999)) therapy, followed in descending order of effectiveness by DEC (35% worm kill) and IVM (10% worm kill) combination therapy, IVM plus ALB combination therapy (35% worm kill), DEC single therapy (35% worm kill) and lastly IVM single therapy (10% worm kill) (Fig. 1). The key role played by the macrofilaricidal efficacy of an antifilarial drug used is further illustrated by the fact that the IVM/ALB combination therapy (35% worm kill) is only slightly better than DEC single therapy (which also induces 35% killing of worms) despite the greater microfilaricidal effect provided by IVM (Fig. 1). This is further supported by the sensitivity analysis carried out in Figure 2a, which clearly indicates the strong (but non-linear) dependence of effectiveness (% reduction in community microfilariae prevalence averaged over 10
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years) on the worm but not microfilariae killing effect of a drug. These results support the ongoing work seeking more potent macrofilaricidal drug regimens for filariasis mass treatment. However, note that apart from the need to resolve problems with side effects and cost, increasing the macrofilaricidal effect of a regimen once it is already high (say, from 55% to 80%) will lead to only small improvements in effectiveness (Fig. 2a). A second more biological factor which may be expected to influence these results, particularly the long term depression of infection, relates to the mean worm lifespan used in the present analysis. Here, we have assumed the mean expected worm life span to be 8 years (Chan et al. 1998), although values as low as 3.5 years have estimated (Vanamail et al. 1996; Michael, 2000). Figure 2b shows the expected effect of variations in this parameter for programme effectiveness of a 5-year annual treatment intervention in relation to the macrofilaricidal efficacy of the drug regimen used. The result indicates only a small impact of this variable compared to the major effect of the worm killing efficacy of the drug, suggesting that uncertainty regarding worm lifespan is unlikely to greatly influence the prediction presented here. If anything, Figure 2b indicates that using a longer lifespan may represent a best case scenario in assessing the longterm effectiveness of treatment as it slightly increases the reduction in microfilarial prevalence. This is due to the inverse relationship between reinfection rate following the cessation of control and worm lifespan (Anderson and May, 1992). Interestingly, this implies that to achieve similar reductions in infection, the control effort (either population coverage or drug efficacy) required would be somewhat less if the worm were to be long-
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lived. However, note that to achieve eradication especially given low transmission breakpoints, the duration of treatment in general would be longer if the worm were long-lived as opposed to if it were short-lived.
Developing criteria for control The major advantage of using transmission models for the planning of parasite control is that they provide a quantitative framework for exploring the consequences of variations in various programmatic and epidemiological factors on programme effectiveness. With regard to the epidemoiology of helminth control at the community level, these include primarily variations in the impacts of population drug coverage, pre-control endemicity rates and acquired immunity. Here, we illustrate the use of the present model to estimate the impact of these parameters for the two currently proposed treatment regimens for filariasis, viz. DEC plus ALB and IVM plus ALB combination therapies. Figure 3 shows the effect of variations in drug coverage (60 to 90%) on the number of cycles of annual mass treatment required to achieve a defined infection threshold (here set at a microfilariae prevalence of 0.5%) for both these regimens. As expected, a decrease in population coverage can significantly increase the number of years of treatment required to achieve the predefined control threshold. For DEC/ALB, the results show that a decrease in coverage from 90% to 60% can increase the number of treatment years required from 4 to 8 years for a community with a pre-control infection prevalence of 5% (Fig. 3a). For IVM/ALB the same reduction in coverage will extend the number of years of treatment from 8 to more than 10 years (Fig. 3b). Given that typical coverages achieved in large-scale community
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treatment programmes are normally around 65% (Plaisier et al. 2000), the present results clearly highlight the potentially long-term nature of filariasis control programmes. The results also underscore the vital importance of maintaining as high drug coverage as possible in the planned campaigns. The effect of pre-control infection prevalences (2.5 to 20%) on the number of treatment cycles required to meet the same control threshold for both these regimens are depicted in Figure 4. Even when based on a 80% drug coverage, it is clear that variation in endemicity can have a significant impact on the applied control effort. The results for DEC/ALB indicate that a 3-fold variation from 5% to 15% in precontrol microfilaraemia prevalence can increase the number of years required to achieve the same control threshold by up to 3 years, i.e. from 5 to 8 years, in the higher prevalence community (Fig. 4a). For IVM/ALB the effect of precontrol endemicity rate even at high drug coverages (80%) will be even more dramatic, increasing the number of years of treatment required to much greater than 10 years (Fig.4b). Table 2 shows the interaction between drug coverage and local endemicity rates on the number of years required by the more effective DEC/ALB regimen to achieve the pre-defined 0.5% infection threshold. The results illustrate the general point that in practice both these factors will combine to determine the control effort required for a particular community, with some indication that the deleterious effect of falling coverage is likely to be greater for communities with higher pre-control endemicity rates. Recent epidemiological studies have highlighted that one population biological factor that may affect infection control at least in communities with high pre-control prevalences is the likely operation of acquired protective immunity in such communities (Michael and Bundy,
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1998; Michael, Simonsen et al. 2001). The present model can be used to simulate the population dynamic consequences of the impact of this epidemiological factor for the present treatment regimens, Figure 5 illustrating the potential impact of this variable on the age-prevalences of microfilaraemia for the DEC/ALB and IVM/ALB regimens. The results show that compared to the assumption of no immunity (Figs. ld and le), chemotherapy with both regimens in the presence of immunity can result in less marked reductions in prevalence and more rapid reinfections following the cessation of the treatments. Indeed, theoretical studies indicate that if the effect of immunity is strong and if interruption of transmission is not achieved, control can even act perversely to increase parasite loads in the older age classes above the levels pertaining prior to treatment (Anderson and May, 1992). Figure 5c indicates that under certain circumstances, it could lead also to adding the number of years required to achieve control for a given endemic situation compared to when no immunity is involved. Use of epidemiological models for evaluating filariasis control programmes An important function also facilitated by the use of epidemiological models in parasite control programming is that they provide a valuable tool to health planners for undertaking informed evaluations of ongoing programmes (Habbema et al. 1992, Medley et al. 1993; Norman et al. 2000; Plaisier et al. 2000). However, to be used successfully for this purpose, there is a critical requirement for close integration of models with field data, not only in terms of determining the type and quality of data to be collected and analysed but also in terms of the proper calibration of the model to suit local epidemiological conditions. This is indicated in Figure 6, which
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compares the predictions of the present model with observed data from a community trial carried
out recently in India on the effectiveness of DEC and IVM given alone on long-term trends in microfilaremia prevalence (Das et al. 2001). The data represent changes in microfilaraemia prevalence in two groups of villages randomly assigned annual treatments with either DEC or IVM. Published data from 4 cycles of intervention are evaluated in the figure using values of the pre-control mean prevalences and actual treatment coverages obtained in the two village groups. The results show a close correspondence between predicted and observed changes in prevalence for the IVM trial but a poor fit in the case of the DEC intervention. This could imply problems with the drug efficacy rates assumed for this drug regimen (Table 1), unaccounted local variations in epidemiological parameters requiring localized model calibration with field data or problems with the data collected. The possibility of the latter problem afflicting these data is suggested by the unexplained upward blip observed for microfilarial prevalence following the first year of DEC treatment (Fig. 6a). Whichever factor is responsible, it is clear that resolving such questions adequately will ultimately involve close working collaborations between field epidemiologists and theoretical biologists.
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CONCLUDING REMARKS This chapter has highlighted the vital role that epidemiological models can play in the rational planning and evaluation of filariasis control programmes based on repeated annual mass chemotherapy strategies. Indeed, the analyses presented here indicate that for parasitic diseases such as filariasis, which is long-lived and exhibit complex dynamics and for which there is a range of treatment regimens present, accurate predictions of the comparative effectiveness of the available treatment measures at the community level may well be possible only via the development and application of such quantitative frameworks. However, the work described in this chapter also suggests that further progress in integrating filariasis epidemiological modelling tools within control programmes will depend on making advances in two major areas. The first relates to gaining a better knowledge of current uncertainties in at least four aspects of model specification. These include gaining (1) better estimates of the macrofilaricidal and other worm-related effects of the present drug regimens, (2) greater knowledge of the parasite life expectancy, (3) better understanding of the impact of endemicity and acquired immunity on control dynamics, and finally (4) greater appreciation of the patterns of drug coverage to be expected in endemic countries. Ultimately, improving model accuracy and hence model utility for control programming will also require expanding the framework to include the effects of human migration on outcomes in controlled areas. Such more spatially oriented models will also be required to fully deal with variations in endemicity among neighbouring control sites. The present analyses have highlighted the clear superiority of DEC-based and combination regimens over IVM-based and single regimen counterparts. Our analysis has shown that this is primary due the greater assumed macrofilaricidal effects of the former regimens. Given that this result is based on at best educated guestimates regarding the relative macrofilaricidal effects of the presently available regimens, there is clearly a great need to resolve this question (perhaps using antigen assays to determine changes in worm burden (Weil et al. 1997)) if we are to improve predictions regarding the relative effectiveness of the present regimens. Given the present results, however, it would appear that the effective control of filariasis would be somewhat easier in those areas where DEC-based regimens are to be administered compared to those areas where IVM-based treatments are to be the mainstay of control. Regarding the likely impact of gaining a better knowledge of parasite life expectancy on predicting mass treatment outcomes, sensitivity analysis shows that although this is likely to make only a slight difference (owning to the major impact of worm killing action on effectiveness), better estimates may nonetheless allow the specification of less onerous control efforts (for example by lowering the required coverage or using less effective drugs) if it appears that the mean lifespan is indeed long. Note that estimating worm life expectancy is fraught with difficulties given currently available diagnostic tools (Vanamail et al.
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1996; Michael, 2000), and hence in this respect the estimated low impact of this parasite life history parameter is a fortuitous finding of this study. By contrast, the effect of variations in pre-control infection prevalence or endemicity in lengthening the duration of control is much more dramatic and serious. The impact of this variation will be even more problematic if as it has been recently proposed acquired immunity occurs significantly only in areas of higher transmission or endemicity (Michael and Bundy, 1998; Michael, Simonsen et al. 2001). These differential effects of variations in endemicity argues for an endemicity-defined strategy for filariasis control, in which the duration and effort of control (for example maintaining high coverage) will vary with the prevailing precontrol prevalence in a given area. Finally, it is clear that maintaining a high drug coverage is vital if the duration of control required is to be kept within a reasonable time frame. Falling coverages particularly in higher endemicity communities have the potential to extend the duration of control to unsustainable lengths. This will be even more problematic if drug attendance patterns follow semi-systematic or systematic behavioural trends (Plaisier et al. 2000). This implies that effective health education packages to improve coverage requires to be a central component of drug delivery programmes, whether this is through community-directed systems or via the public health system. The second major area of improvement required if quantitative frameworks are to make an important contribution to filariasis control concerns the need to address models more closely to both field data and end-users. The first requirement will not only lead to better parameterization of the model to account for local epidemiological differences, but it will also help steer both field research and the development of sound strategies for the collection of better quality and relevant epidemiological data for programme evaluation and analysis. Closer collaborations between field epidemiologist, health planners and theoretical workers on the other hand will be vital to developing easy to understand and use interfaces to mathematical models, an important requirement if models are to be used effectively by public health managers (Habbema et al. 1992). Mechanisms to achieve such collaborations are now urgently needed if these different strands of work, which represent expertise in population biology, field epidemiology, and health programme planning and implementation, are to be efficiently combined and deployed together for undertaking effective filariasis control.
ACKNOWLEDGEMENTS The simulations carried out in this paper were made possible by the technical development of the EPIFIL deterministic model of filariasis transmission by Man-Suen Chan and Rachel Norman when they were with the author at Oxford University. The author was supported by a UK Medical Research Council Fellowship during the execution of this study.
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REFERENCES Anderson, R.M., and R.M. May. 1985. Helminth infections of humans: mathematical models, population dynamics and control. Advances in Parasitology 24: 1101.Anderson, R.M., and R.M. May. 1992. Infectious diseases of humans. Dynamics and control. Oxford University Press, Oxford, R.K., 757p. Cao, W., C.P.B. van der Ploeg, A.P. Plaisier, I.J.S. van der Sluijs, and J.D.F. Habbema. 1997. Ivermectin for the chemotherapy of bancroftian filariasis: a meta-analysis of the effect of single treatment. Tropical Medicine and International Health 2: 393-403. Chan, M.S., H.L. Guyatt, D.A.P. Bundy, M. Booth, A.J.C. Fulford, and G.F. Medley. 1995. The development of an age structured model of schistosomiasis transmission dynamics and control and its validation for Schistosoma mansoni. Epidemiology and Infection 115: 325-344. Chan, M.S., A. Srividya, R.A. Norman, S.P. Pani, K.D. Ramaiah, P. Vanamail, E. Michael, P.K. Das, and D.A.P. Bundy. 1998. Epifil: a dynamic model of infection and disease in lymphatic filariasis. American Journal of Tropical Medicine and Hygiene 59: 606-614. Das, P.K., K.D. Ramaiah, P. Vanamail, S.P. Pani, J. Yuvaraj, K. Balarajan, and D.A.P. Bundy. 2001. Placebo-controlled community trial of four cycles of single-dose diethylcarbamazine or ivermectin against Wuchereria bancrofti infection and transmission in India. Transactions of the Royal Society of Tropical Hygiene and Medicine 95: 336-341. Habbema, J.D.F., E.S. Alley, A.P. Plaisier, G.J. van Oortmarssen, and J.H.F. Remme. 1992. Epidemiological modelling for onchocerciasis control. Parasitology Today 8; 99-103. Medley, G.F., H.L. Guyatt, and D.A.P. Bundy. 1993. A quantitative framework for evaluating the effect of community treatment on the morbidity due to ascaris. Parasitology 106: 211-221. Michael, E. 1999. The control of the human filariases. Current Opinion in Infectious Disease 12: 565-578. Michael, E. 2000. The population dynamics and epidemiology of lymphatic filariasis. In Lymphatic filariasis, T.B. Nutman (ed.). Imperial College Press, London, U.K., p. 41–81.Michael, E., and D.A.P. Bundy. 1997. Global mapping of lymphatic filariasis. Parasitology Today 13: 472-476. Michael, E., and D.A.P. Bundy. 1998. Herd immunity to filarial infection is a function of vector biting rate. Proceedings of the Royal Society of London B 265: 855-860. Michael, E., D.A.P. Bundy, and B.T. Grenfell. 1996. Re-assessing the global prevalence and distribution of lymphatic filariasis. Parasitology 112: 409-428. Michael, E., P.E. Simonsen, M. Malecela, W.G. Jaoko, E.M. Pedersen, D. Mukoko, R.T. Rwegoshora, and D.W. Meyrowitsch. 2001. Transmission intensity and the immunoepidemiology of bancroftian filariasis in East Africa. Parasite Immunology 23: 373-388. Michael, E., K.D. Ramaiah, S.L. Hoti, G. Barker, M.R. Paul, S.P. Pani, P.K. Das, B.T. Grenfell, and D.A.P. Bundy. 2001. Quantifying mosquito biting patterns on humans by DNA fingerprinting of bloodmeals. American Journal of Tropical Medicine and Hygiene (in press). Norman, R.A., M.S. Chan, A. Srividya, S.P. Pani, K.D. Ramaiah, P. Vanamail, E. Michael, P.K. Das, and D.A.P. Bundy. 2000. EPIFIL: The development of an age-structured model for describing the transmission dynamics and control of lymphatic filariasis. Epidemiology and Infection 124: 529-541. Ottesen, E.A. 2000. Towards eliminating lymphatic filariasis. In Lymphatic filariasis, T.B. Nutman (ed.). Imperial College Press, London, U.K., p. 201-215. Ottesen, E.A., and C.P. Ramachandran. 1995. Lymphatic filariasis infection and disease: control strategies. Parasitology Today 11: 129-131. Ottesen, E.A., M.M. Ismail, and J. Horton. 1999. The role of albendazole in programmes to eliminate lymphatic filariasis. Parasitology Today 15: 382-386. Plaisier, A.P., S. Subramanian, P.K. Das, W. Souza, T. Lapa, A.F. Furtado, C.P. van der Ploeg, J.D. Habbema, and G.J. Oortmarssen. 1998. The LYMFASIM simulation
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program for modelling lymphatic filariasis and its control. Methods of Informatics in Medicine 37: 97-108. Plaisier, A.P., G.J. van Oortmarssen, J.D.F. Habbema, J. Remme, and E.S. Alley. 1990. ONCHOSIM: a model and computer simulation program for the transmission and control of onchocerciasis. Computer Methods and Programs in Biomedicine 31: 43-56. Plaisier, A.P., W.A. Stolk, G.J. van Oortmarssen, and J.D.F. Habbema. 2000. Effectiveness of annual ivermectin treatment for Wuchereria bancrofti infection. Parasitology Today 16: 298-302. Vanamail, P., K.D. Ramaiah, S.P. Pani, P.K. Das, B.T. Grenfell, and D.A.P. Bundy. 1996. Estimation of the fecund lifespan of Wuchereria bancrofti in an endemic area. Transactions of the Royal Society of Tropical Hygiene and Medicine 90: 119121. Weil, G.J., P.J. Lammie, and N. Weiss. 1997. The ICT filariasis test: a rapid format antigen test for diagnosis of bancroftian filariasis. Parasitology Today 13: 401404. World Bank. 1993. World Development Report 1993. Investing in Health. Oxford University Press, New York, 329p.
HOST FACTORS, PARASITE FACTORS, AND EXTERNAL FACTORS INVOLVED IN THE PATHOGENESIS OF FILARIAL INFECTIONS
David O. Freedman Division of Geographic Medicine, University of Alabama at Birmingham
ABSTRACT This chapter describes host, parasite, and extrinsic factors independent of the immune system that contribute to filarial pathogenesis. Those with past infection with lymphatic filariasis but with current inflammation or clinical pathology have been the individuals most studied in the past. A significant proportion of patients in filarial endemic areas with hydroceles and lymphedema have active current filarial infection as defined by circulating antigenemia. These individuals with concurrent inflammation and filarial infection are perhaps the most suitable for studies on the relative roles of the filarial parasite itself, the host immune response, host genetics and extrinsic bacteria in the pathogenesis of inflammatory attacks and disease progression. Keywords: W. bancrofti, B. malayi, O. volvulus, pathogenesis.
INTRODUCTION Filarial tissue inflammation is thought to be substantially more complex than simple blockade of lymphatic vessels or the circumscribed reaction to dying intralymphatic adult worms (Jungmann et al., 1991), pathological events that are now thought to occur only uncommonly (Lichtenberg, 1957; Connor et al., 1986). As will be discussed in this chapter, early damage to lymphatic vessels by Wuchereria bancrofti or Brugia malayi appears to have a number of non-inflammatory components. The evolution of the inflammatory disease that ensues varies from individual to individual. The precise disease-inducing mechanisms in filariasis are not understood but are likely to involve immunemediated damage to the lymphatics, which predisposes to direct lymphatic failure in some, and debilitating secondary infections in others.
PATHOLOGIC LESIONS IN LYMPHATIC FILARIASIS The pathogenesis of the characteristic lymphatic damage is thought to involve three components: 1) mechanical damage by motile parasites (Amaral et al., 1994; Freedman et al., 1994); 2) local immunological responses to
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parasite antigen (Freedman et al., 1995; Freedman, 1998); and 3) bacterial superinfection in previously damaged vessels (Olszewski et al., 1997; Dreyer et al., 1999). Because therapy of known filariasis is nonsurgical, pre-1990’s concepts of pathogenesis had been based almost entirely on observations made from just 3 direct studies of human tissue (O'Connor, 1932; Lichtenberg, 1957; Connor et al., 1986). For example, the most often cited series involved not natives of an endemic area, but some of the 12,000 nonendemic US servicemen who acquired acute filariasis in the South Pacific during WWII. Lymph nodes and vessels that have been examined in these studies have usually been from individuals with acute adenolymphangitis. Only in those cases where dying adult worms are actually seen in inflamed nodes does the case end up classified as filaria-mediated. In areas endemic for bancroftian filariasis, asymptomatic individuals whose tissue specimens were removed for reasons other than filariasis (e.g. suspicion of cancer) had little or no inflammatory reaction around live viable adult worms (Jungmann et al., 1991). In these endemic areas evidence suggests that the insidious onset of inflammatory lymphedema or elephantiasis reflects an immune-mediated response triggered by parasite antigens. Our own study of skin and subcutaneous tissue obtained in 34 Brazilian patients demonstrated abnormal CD3+ cell infiltrates interstitially and around blood capillaries and venules in both clinically symptomatic and asymptomatic individuals (Freedman et al., 1995). A similar subcutaneous infiltrate was demonstrated in tissue from a clinically affected area in 30 Indian patients with filarial lymphedema (Olszewski et al., 1993). Control patients with non-filarial lymphedema do not have these findings.
IMPORTANCE OF PATIENT CLASSIFICATION The relatively recent ability to measure W. bancrofti circulating antigen (CAg) in patient serum, indicative of current infection with live adult worms, has advanced our ability to understand the pathogenesis of lymphatic filariasis by improving the precision of patient classification (Weil et al., 1997; Freedman, 1998). In addition, the knowledge that essentially all patients, including those who are overtly asymptomatic have some underlying pathological damage to lymphatic vessels (Freedman et al., 1994; Freedman et al., 1995), has meant that three distinct patient groups can be defined for detailed study: asymptomatic infected individuals (CAg positive); individuals with overt clinical filariasis and active infection (CAg positive); and those with overt clinical filariasis without active infection (CAg negative). Recent work suggests that the presence (or absence) of antigenemia, rather than overt clinical manifestations of disease, is most closely associated with specific immune responses (de Almeida et al., 1996). Patient classification based on presence of absence of circulating antigenemia should also be useful in dissecting the role of some of the non-immune factors. It is increasingly clear that a significant proportion of patients in filarial endemic areas with
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hydroceles and lymphedema have active current filarial infection (Weller et al., 1982; Kazura et al., 1997; Tisch et al., 2001) as manifest either by microfilaremia or circulating antigenemia. This group with concurrent inflammation and filarial infection is perhaps the most suitable for studies on the relative roles of the filarial parasite itself, the host immune response, host genetics and bacteria in the pathogenesis of inflammatory attacks and disease progression. Those with past filarial infection but with current inflammation or clinical pathology have been extensively studied in the past but form a less defined group for study.
PARASITE FACTORS - MECHANICAL FACTORS, SECRETED MOLECULES, AND STRAINS Mechanical damage to lymph vessels due to the physical action of the constantly motile adult worms has in recent years been implicated early in the clinically asymptomatic noninflammatory stage of infection with the lymphatic filarial parasites (Amaral et al., 1994; Noroes et al., 1996). Ultrasound has emerged as an important tool for the visualization of motile adult W. bancrofti worms in lymphatics of the scrotal area in infected microfilaremic or amicrofilaremic males. The rapid and constant motion of the adult worms in the lymphatics has been dubbed the ‘filaria dance sign’. Abnormal scrotal lymphatic dilatation is universally observed in men, both asymptomatic or clinically symptomatic, who have adult worms visualized by ultrasound (Noroes et al., 1996). In the absence of inflammatory symptoms or immunohistologic evidence of inflammation at this early stage of infection, the likelihood is that mechanical damage to lymphatic endothelium plays an important role. This concept is supported by animal data. There is no animal model of W. bancrofti infection and immunological investigation utilizing Brugia sp. is hampered by the inability of mice to complete the full developmental cycle of this nematode. Mirroring lymphoscintigraphic and ultrasound data from early human disease, both severe combined immunodeficiency and athymic nude mice infected with B. malayi develop a noninflammatory elephantiasislike disease with patent dilated lymphatics in the absence of inflammation (Vincent et al., 1984; Nelson et al., 1991). After immunological reconstitution, circulating mononuclear inflammatory cells migrate through vascular endothelium to initiate a marked inflammatory reaction in infected limbs (Vickery et al., 1991). The B. pahangi-infected cat is the animal model in which the natural course of actual disease development is most closely mimicked. Similar to the case for human disease, a proportion of persistently microfilaremic cats develop lymphedema (Grenfell et al., 1991). A direct role has often been hypothesized for parasite factors, such as excretory-secretory molecules, in mediating endothelial abnormalities or in inducing the inflammatory response to live or dying lymphatic filarial parasites. The bulk of the evidence indicates that inflammatory damage is
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caused by local specific and non-specific immunological responses to these parasite antigens and is not directly mediated. These mechanisms are discussed elsewhere in this volume. Limited data on effects of Brugia excretory secretory molecules show that 1) there is suppression of vascular (not lymphatic) endothelial cell proliferation in in vitro culture (Rao et al., 1996); and 2) depression of endothelium-dependent relaxation occurred in the aortas of infected rats (Kaiser et al., 1991). A body of elegant work in experimental mice has shown the ocular inflammatory response to O. volvulus to be in most part due to the immune response to specific antigens released by dead and dying microfilariae in already sensitized hosts (Pearlman, 1996; Pearlman and Hall, 2000). Recently however, members of a family of recombinant proteins designated Ov-asp have been found to induce an angiogenic response after injection into corneas of naïve mice and new blood vessel formation was associated with only minor inflammatory cell infiltration (Tawe et al., 2000). This suggests a direct effect of a parasite protein in onchocercal keratitis. The population biology and innate genetics of either W. bancrofti or B. malayi are poorly characterized. Live parasite material is difficult to obtain for either species and there are few available tools to distinguish any biological or biochemical differences that may exist amongst different isolates from the same or different regions of the world. Electrophoretic and western blot preparations of parasite material have been extensively published, none of which shows evidence for any population heterogeneity at the protein or antigen levels. Genetic markers offer the best hope to characterize any strain or population differences that might occur. Anecdotally, RFLP and other basic genetic analyses have not demonstrated differences. One preliminary study with B. malayi isolates from Indonesia and Malaysia has described polymorphisms at 2 microsatellite loci (Underwood et al., 2000). No biological or clinico-pathological correlates of these differences were examined in these preliminary studies but further work on a larger battery of microsatellites might allow for the identification of parasite groupings by geographic origin, biological characteristics, or potential for pathology induction. Several lines of evidence, taken together, have led to the hypothesis that two different strains of O. volvulus, a forest and a savannah derived strain, exist which differ in their ability to induce ocular disease. Early clinical and epidemiological studies indicated blinding onchocerciasis to occur predominantly in the savannah bioclime regions of Africa, while equally infected individuals resident in rainforest regions only rarely had blinding disease (Dadzie et al., 1989; Remme et al., 1989). Blinding and nonblinding strains of the parasite have been distinguished by isoenzyme, immunochemical, and genetic differences (Cianchi et al., 1985; Lobos and Weiss, 1985; Flockhart et al., 1986). However, the major and only genetic differences described so far map to a non-coding 150bp repeat sequence family present in the nuclear genome of O. volvulus (Erttmann et al., 1987).
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Recently, work in the murine model of experimental ocular onchocerciasis has identified several discrete antigentic epitopes of O. volvulus capable of inducing T-cell mediated corneal pathology. A study designed to test the hypothesis that the virulence of the blinding and non-blinding strains might be related to qualitative differences in these parasite antigens showed remarkable sequence homogeneity in the genes coding for these discrete antigens between isolates from the forest and savannah bioclimes (Keddie et al., 1999). Even when the study was expanded to include several antigens commonly recognized by infected individuals as well as to the mitochondrial genome of the parasite, this high level of homogeneity was maintained (Rokeach et al., 1994; Keddie et al., 1999).
HOST FACTORS - GENETIC MAKE-UP, GENDER AND AGE Possession of specific HLA genes can influence susceptibility to, and progression of disease. Alleles of individual HLA loci may either act alone or in combination to determine disease outcome. Significant advances in molecular techniques for genetic typing have provided powerful tools for HLA analysis in tandem with epidemiologic studies for a number of parasitic and non-parasitic infectious diseases. The variable outcome of infection with filarial parasites is to a large degree attributable to different patterns of immunological responsiveness to filarial antigens. To this end, several attempts have been made over the past two decades to correlate HLA loci with the clinical outcome of filarial infection. In the only studies of bancroftian filariasis, one from Ottesen et al. reported a familial association with infection but without any linkage to HLA (Ottesen et al., 1981), whereas in another, Sri Lankan investigators found HLA B15 to be associated with elephantiasis (Chan et al., 1984). Neither of these 1980’s studies examined class II loci. In a recent evaluation of an Indonesian population resident in areas endemic for Brugian filariasis in Sumatra, class II antigen frequencies, both DR3 and the 2B3 epitope, were significantly decreased in elephantiasis patients while HLA DQ5 was increased significantly in the control group (Yazdanbakhsh et al., 1995). If corrected for the number of antigens tested, these associations failed to reach statistical significance, leaving open the question of whether there are HLA correlations in this disease population. These results were not reproducible in another small study in Sulawesi by the same group (Yazdanbakhsh et al., 1997). The authors concluded that either HLA -DR and -DQ are not associated with progression to elephantiasis or the associations are too weak to be detected in small studies. Genetic determinants of clinical outcome have been associated with 2 other related human filarial species. Segregation analysis of 74 families in a Loa loa endemic area of Cameroon indicates an undefined genetic predisposition to asymptomatic immunologically hyporesponsive infection
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(Garcia et al., 1999). Two studies on onchocerciasis have strongly suggested immunogenetic associations between HLA class II genes and clinical outcome. In 120 Liberians, higher frequencies of DQA1*0501-DQB*0301 were found in exposed uninfected individuals compared to patients with disease (Meyer et al., 1994). In addition, significant differences were reported for DQA1*0101-DQB1*0501 frequencies in patients with localized versus generalized disease. In a group of 117 Nigerians, individuals with depigmentation had increased frequencies of DQA1*0501 and DQB*0301 compared with highly infected individuals with normal skin (Murdoch et al., 1997). Conversely individuals with depigmentation had a decreased frequency of DQA1*0101 and Cw6 compared to those with normal skin. As the cost and complexity of semi-automated genotyping using PCR techniques are reduced, and study subjects can be better characterized as to CAg status, investigators should be more able to examine large well-defined cohorts in population-based studies. Helminth systems are inherently more complex than are comparable systems in bacteria, viruses or protozoan parasites. So the work will not be straightforward. However, substantive HLA-disease associations could lead to reverse immunogenetic approaches to the elucidation of discrete candidate antigens relevant to pathology induction. A number of studies over the years, carried out in a variety of areas endemic for lymphatic filariasis, have suggested that both infection and disease are more common in men than in women. This differential susceptibility has been thought to be regulated by both immunologic and nonimmunologic mechanisms but the actual mechanism for the differences remain obscure. This conclusion has been borne out in a meta-analysis of the published literature (53 studies), which was also able to discount the suggestion that this was due to less exposure of females to infective vectors (Brabin, 1990). There are also well-documented sex-related differences in susceptibility to infection with B. malayi in the Meriones unguiculatus (Mongolian jird) animal model of infection (Ash, 1971). This is in the face of equivalent innocula of infective larva. These earlier reports of gender differences were based on microfilarial status alone and generally involved smaller numbers of subjects. A recent large scale study of 847 individuals in a W. bancrofti endemic area of Brazil, using CAg status as an indicator of infection, demonstrated the prevalence rate in men to be 25.9% compared to 14.1% in women (Freedman et al., 1997). In contrast, in a study of 1,322 individuals from areas of Papua New Guinea where Anopheles is the vector and with extremely high transmission intensity, there were no gender differences in Og4C3 antigenemia (Tisch et al., 2001). This area has the highest reported transmission rates in the world, so it is possible that any innate differences in gender susceptibility may be overwhelmed in the face of intense vectorial capacity. There are few independent data on the role of age by itself as a factor in filarial pathogenesis. Disease manifestations in those who are predisposed by genetic or other factors to develop them appear to rely more on duration
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and intensity of infection than on age itself and are more likely due to extrinsic factors in the local environment.
EXTRINSIC FACTORS - TRANSMISSION INTENSITY & BACTERIA Recent controlled studies indicate that transmission intensity (the number of incoming L3s) is correlated with the development of clinical disease in bancroftian filariasis. Concomitant entomological and clinical data were collected from 1666 subjects in five similar but distinct W. bancroftiendemic communities in Papua New Guinea within a 20 km radius. For each of the five villages with highly variable annual transmission potentials (ATPs), the prevalence of leg edema was highly positively related to the ATPs (Kazura et al., 1997). Similarly, in a year-long study of 353 episodes of acute filarial adenolymphangitis in a population of 5,246 subjects in Ghana, attacks were very closely correlated with the rainfall pattern. The monthly number of adenolymphangitis attacks in this very stable population decreased by over 50% during the dry season when transmission intensity should be at its lowest (Gyapong et al., 1996). Conversely, it is well documented that when an individual with filariasis moves from an endemic area to an area of non-transmission, the episodes of acute adenolymphangitis subside with no further treatment (Rajan and Gundlapalli, 1997). Thus, when other factors are controlled for, the degree of ongoing exposure of the host immune system to filarial larvae seems to be related to both the acute and chronic sequelae of infection. In a subsequent study in Papua New Guinea, examining antigen levels, there was a direct correlation between transmission potential and antigen levels. However, in the same study there was no significant difference in antigen levels when individuals with clinical disease (lymphedema, hydrocele) were compared to those without clinical disease living in the same villages (Tisch et al., 2001). Taken together, these studies suggest that host reactivity to incoming L3s (transmission intensity) is more important in the pathogenesis of filarial inflammation than the number of adult worms present in an infected host. Some clinicians have noted that at an uncertain point during the clinical evolution of the lymphatic insufficiency in lymphatic filariasis, repeated limb bacterial infections in previously damaged vessels may become superimposed on other processes. The relative contribution to disease evolution of bacterial factors and superinfection is incompletely defined and has been the subject of considerable debate in recent years. One must also consider the likely possibility that the pathogenesis of disease evolution after the onset of clinical manifestation of filariasis may vary from individual to individual. Wolbachia, endosymbiotic bacteria of filarial parasites, are now thought to have an important role in maintaining the life cycle and fertility of mature adult parasites (Taylor et al., 2000). Wolbachia extracts do contain LPS and can induce TNF production in cultured macrophages. However,
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Wolbachia are not known human pathogens and seem unlikely to be causative agents in acute filarial inflammatory episodes even if released during death of the adult worm. No histological evidence was found to support the suggestion of a primary role for bacterial infection in the initiation of filarial inflammation in our studies, conducted in a W. bancrofti endemic area of Brazil (Freedman et al., 1995). Patients included those with recent onset of clinical signs and asymptomatic individuals. In contrast to studies in India, in which all subjects had advanced verrucous and hyperkeratotic skin changes (Olszewski, 1993;Shenoy et al., 1995; Olszewski, 1996; Shenoy et al., 1999) indicative of a role for chronic secondary bacterial superinfection originating in the epidermis, 100% of our subjects had histologically normal epidermis. In tissue specimens that we have examined in Brazil, no dermal reaction was seen consistent with a bacterial process. In addition, in none of the above discussed small animal models of filarial disease is there any pathological evidence for a primary role for bacteria in filarial inflammation. Rhesus monkeys infected with B. malayi and followed closely from the time of infection show evidence of sub-clinical lymphatic pathology by lymphoscintigraphy in the pre-patent period (Dennis et al., 1998). Histopathologically, lymph nodes showed eosinophilic lymphadenitis with no reaction consistent with bacterial infection. Some patients with superinfection may have chronic bacterial colonization or portals of bacterial entry that are not apparent by the time of presentation to the physician. Bacteriologic data are seldom reported from studies that have examined skin, tissue fluid, lymph, and lymph nodes. The largest study to date examined 100 Indian patients with often advanced lymphedema in a unsanitary environment, where 50% or more of individuals do not wear shoes all the time. Bacteria were isolated from 66-75% of specimens but included almost exclusively bacteria that most infectious diseases clinicians would consider to be non-pathogenic for humans and certainly difficult to implicate in acute clinical inflammatory episodes. Species isolated included Bacillus cereus, Staphylococcus epidermidis and other coagulase negative staph species, micrococcus, and aerococcus (Olszewski et al., 1997). An interesting study in 28 patients from a filariasis endemic community in the Dominican Republic showed an acute rise in antistreptococcal antibodies (anti-streptolysin-O; ASOT) after acute attacks of clinical cellulites (Vincent et al., 1998). While 71% of these individuals had irreversible lymphedema between attacks, not every individual in the study population was characterized as to presence of current filarial infection by measurement of CAg levels. Thus, many or most of these cases may have been bacterial superinfection in individuals with residual lymphatic insufficiency due to previous or burnt out filarial infection. In contrast, a study in India of 62 acute filarial attacks showed no rise in ASOT titres but a rise in antifilarial titres (Kar et al., 1993). Perhaps most importantly, this has
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been the only study to examine leukocyte counts, a common marker of bacterial infection, during the acute attacks. No leukocytosis was found in the study group. Two important and well-designed trials comparing antibiotics, diethylcarbamazine, and local foot care in various combinations in individuals with B. malayi infection in India have demonstrated that local foot care and antibiotics play a more important role than DEC in the treatment and prevention of acute episodes of adenolymphangitis. Again most of these individuals had advanced lymphedema and current filarial infection status was unknown. Thus, in superinfection patients, intensive local hygiene (cleaning with soapy water, topical antibiotics) clearly reduces the numbers of attacks of adenolymphangitis (Shenoy et al., 1999). The degree to which attacks of bacterial superinfection promote the progression of lymphedema/elephantiasis in limbs previously damaged during active filarial infection might be considered a separate issue and these individuals studied separately. This is the type of individual that has likely constituted the bulk of the populations who have responded to local and antibiotic therapy in the above cited studies.
REFERENCES Amaral, F., G. Dreyer, J. Figueredo-Silva, J. Noroes, A. Cavalcanti, S. C. Samico, A. Santos, and A. Coutinho. 1994. Live adult worms detected by ultrasonography in human bancroftian filariasis. American Journal of Tropical Medicine and Hygiene 50:753-757. Ash, L. R. 1971. Preferential susceptibility of male jirds (Meriones unguiculatus) to infection with Brugia pahangi. Journal of Parasitology 57:777-780. Brabin, L. 1990. Sex differentials in susceptibility to lymphatic filariasis and implications for maternal child immunity. Epidemiology and Infection 105:335-353. Chan, S. H., S. Dissanayake, J. W. Mak, M. M. Ismail, G. B. Wee, N. Srinivasan, B. H. Soo, and V. Zaman. 1984. HLA and filariasis in Sri Lankans and Indians. Southeast Asian Journal of Tropical Medicine and Public Health 15:281-286. Cianchi, R., M. Karam, M. C. Henry, F. Villani, S. Kumlien, and L. Bullini. 1985. Preliminary data on the genetic differentiation of Onchocerca volvulus in Africa (Nematoda: Filarioidea). Acta Tropica 42:341-351. Connor, D. H., J. R. Palmieri, and D. W. Gibson. 1986. Pathogenesis of lymphatic filariasis in man. Zeitschrift fur Parasitenkunde 72:13-28. Dadzie, K. Y., J. Remme, A. Rolland, and B. Thylefors. 1989. Ocular onchocerciasis and intensity of infection in the community. II. West African rainforest foci of the vector Simulium yahense. Tropical Medicine and Parasitology 40:348-354. de Almeida, A. B., M. C. Maia e Silva, M. A. Maciel, and D. O. Freedman. 1996. The presence or absence of active infection, not clinical status, is most closely associated with cytokine responses in lymphatic filariasis. Journal of Infectious Diseases 173:1453-1459. Dennis, V. A., B. L. Lasater, J. L. Blanchard, R. C. Lowrie, Jr., and R. J. Campeau. 1998. Histopathological, lymphoscintigraphical, and immunological changes in the inguinal lymph nodes of rhesus monkeys during the early course of infection with Brugia malayi. Experimental Parasitology 89:143-152. Dreyer, G., Z. Medeiros, M. J. Netto, N. C. Leal, L. G. de Castro, and W. F. Piessens. 1999. Acute attacks in the extremities of persons living in an area endemic for bancroftian filariasis: differentiation of two syndromes. Transactions of the Royal Society of Tropical Medicine and Hygiene 93:413-417.
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Dreyer, G., J. Noroes, D. Addiss, A. Santos, Z. Medeiros, and J. Figueredo-Silva. 1999. Bancroftian filariasis in a paediatric population: an ultrasonographic study. Transactions of the Royal Society of Tropical Medicine and Hygiene 93:633-636. Erttmann, K. D., T. R. Unnasch, B. M. Greene, E. J. Albiez, J. Boateng, A. M. Denke, J. J. Ferraroni, M. Karam, H. Schulz-Key, and P. N. Williams. 1987. A DNA sequence specific for forest form Onchocerca volvulus. Nature 327:415-417. Flockhart, H. A., R. E. Cibulskis, M. Karam, and E. J. Albiez. 1986. Onchocerca volvulus: enzyme polymorphism in relation to the differentiation of forest and savannah strains of this parasite. Transactions of the Royal Society of Tropical Medicine and Hygiene 80:285292. Freedman, D. O., P. J. de Almeida Filho, S. Besh, M. C. Maia e Silva, C. Braga, and A. Maciel. 1994. Lymphoscintigraphic analysis of lymphatic abnormalities in symptomatic and asymptomatic human filariasis. Journal of Infectious Diseases 170:927-933. Freedman, D. O., P. J. de Almeido Filho, S. Besh, M. C. Maia e Silva, C. Braga, A. Maciel, and A. F. Furtado. 1995. Abnormal lymphatic function in presymptomatic bancroftian filariasis. Journal of Infectious Diseases 171:997-1001. Freedman, D. O., T. D. Horn, C. M. Maia e Silva, C. Braga, and A. Maciel. 1995. Predominant CD8+ infiltrate in limb biopsies of individuals with filarial lymphedema and elephantiasis. American Journal of Tropical Medicine and Hygiene 53:633-638. Freedman, D. O., A. de Almeida, J. Miranda, D. A. Plier, and C. Braga. 1997. Field trial of a rapid card test for Wuchereria bancrofti. Lancet 350:1681. Freedman, D. O. 1998. Immune dynamics in the pathogenesis of human lymphatic filariasis. Parasitology Today 14:229-234. Garcia, A., L. Abel, M. Cot, P. Richard, S. Ranque, J. Feingold, F. Demenais, M. Boussinesq, and J. P. Chippaux. 1999. Genetic epidemiology of host predisposition microfilaraemia in human loiasis. Tropical Medicine and International Health 4:565-574. Grenfell, B. T., E. Michael, and D. A. Denham. 1991. A model for the dynamics of human lymphatic filariasis. Parasitology Today 7:318-323. Gyapong, J. O., M. Gyapong, and S. Adjei. 1996. The epidemiology of acute adenolymphangitis due to lymphatic filariasis in northern Ghana. American Journal of Tropical Medicine and Hygiene 54:591-595. Jungmann, P., J. Figueredo-Silva, and G. Dreyer. 1991. Bancroftian lymphadenopathy: a histopathologic study of fifty-eight cases from northeastern Brazil. American Journal of Tropical Medicine and Hygiene 45:325-331. Kaiser, L., P. K. Tithof, V. L. Lamb, and J. F. Williams. 1991. Depression of endotheliumdependent relaxation in aorta from rats with Brugia pahangi lymphatic filariasis. Circulation Research 68:1703-1712. Kar, S. K., J. Mania, and P. K. Kar. 1993. Humoral immune response during filarial fever in bancroftian filariasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 87:230-233. Kazura, J. W., M. Bockarie, N. Alexander, R. Perry, F. Bockarie, H. Dagoro, Z. Dimber, P. Hyun, and M. P. Alpers. 1997. Transmission intensity and its relationship to infection and disease due to Wuchereria bancrofti in Papua New Guinea. Journal of Infectious Diseases 176:242-246. Keddie, E. M., T. Higazi, D. Boakye, A. Merriweather, M. C. Wooten, and T. R. Unnasch. 1999. Onchocerca volvulus: limited heterogeneity in the nuclear and mitochondrial genomes. Experimental Parasitology 93:198-206. Kurniawan, A., M. Yazdanbakhsh, R. van Ree, R. Aalberse, M. E. Selkirk, F. Partono, and R. M. Maizels. 1993. Differential expression of IgE and IgG4 specific antibody responses in asymptomatic and chronic human filariasis. Journal of Immunology 150:3941-3950. Lichtenberg, F. 1957. The early phase of endemic bancroftian filariasis in the male: Pathological study. Mount Sinai Journal of Medicine 24:983-1000. Lobos, E., and N. Weiss. 1985. Immunochemical comparison between worm extracts of Onchocerca volvulus from savanna and rain forest. Parasite Immunology 7:333-347.
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Maizels, R. M., E. Sartono, A. Kurniawan, F. Partono, M. E. Selkirk, and M. Yazdanbakhsh. 1995. T-cell activation and the balance of antibody isotypes in human lymphatic filariasis. Parasitology Today 11:50-56. Meyer, C. G., M. Gallin, K. D. Erttmann, N. Brattig, L. Schnittger, A. Gelhaus, E. Tannich, A. B. Begovich, H. A. Erlich, and R. D. Horstmann. 1994. HLA-D alleles associated with generalized disease, localized disease, and putative immunity in Onchocerca volvulus infection. Proceedings of the National Academy of Sciences of the United States of America 91:7515-7519. Murdoch, M. E., A. Payton, A. Abiose, W. Thomson, V. K. Panicker, P. A. Dyer, B. R. Jones, R. M. Maizels, and W. E. Oilier. 1997. HLA-DQ alleles associate with cutaneous features of onchocerciasis. The Kaduna-London-Manchester Collaboration for Research on Onchocerciasis. Human Immunology 55:46-52. Nelson, F. K., D. L. Greiner, L. D. Shultz, and T. V. Rajan. 1991. The immunodeficient scid mouse as a model for human lymphatic filariasis. Journal of Experimental Medicine 173:659-663. Noroes, J., D. Addiss, A. Santos, Z. Medeiros, A. Coutinho, and G. Dreyer. 1996. Ultrasonographic evidence of abnormal lymphatic vessels in young men with adult Wuchereria bancrofti infection in the scrotal area. Journal of Urology 156:409-412. O'Connor, F. W. 1932. The aetiology of the disease syndrome in Wuchereria bancrofti infections. Transactions of the Royal Society of Tropical Medicine and Hygiene 26:13-32. Olszewski, W. L., S. Jamal, G. Manokaran, B. Lukomska, and U. Kubicka. 1993. Skin changes in filarial and non-filarial lymphoedema of the lower extremities. Tropical Medicine and Parasitology 44:40-44. Olszewski, W. L. 1996. Recurrent bacterial dermatolymphangioadenitis (DLA) is responsible for progression of lymphedema. Lymphology 29:331-334. Olszewski, W. L., S. Jamal, G. Manokaran, S. Pani, V. Kumaraswami, U. Kubicka, B. Lukomska, A. Dworczynski, E. Swoboda, and F. Meisel Mikolajczyk. 1997. Bacteriologic studies of skin, tissue fluid, lymph, and lymph nodes in patients with filarial lymphedema. American Journal of Tropical Medicine and Hygiene 57:7-15. Ottesen, E. A., N. R. Mendell, J. M. MacQueen, P. F. Weller, D. B. Amos, and F. E. Ward. 1981. Familial predisposition to filarial infection--not linked to HLA-A or-B locus specificities. Acta Tropica 38:205-216. Pearlman, E., and L. R. Hall. 2000. Immune mechanisms in Onchocerca volvulus-mediated corneal disease (river blindness). Parasite Immunology 22:625-631. Rajan, T. V., and A. V. Gundlapalli. 1997. Lymphatic filariasis. Chemical Immunology 66:125-158. Rao, U. R., C. S. Zometa, A. C. Vickery, B. H. Kwa, J. K. Nayar, and E. T. Sutton. 1996. Effect of Brugia malayi on the growth and proliferation of endothelial cells in vitro. Journal of Parasitology 82:550-556. Remme, J., K. Y. Dadzie, A. Rolland, and B. Thylefors. 1989. Ocular onchocerciasis and intensity of infection in the community. I. West African savanna. Tropical Medicine and Parasitology 40:340-347. Rokeach, L. A., P. A. Zimmerman, and T. R. Unnasch. 1994. Epitopes of the Onchocerca volvulus RAL1 antigen, a member of the calreticulin family of proteins, recognized by sera from patients with onchocerciasis. Infection and Immunity 62:3696-3704. Shenoy, R. K., K. Sandhya, T. K. Suma, and V. Kumaraswami. 1995. A preliminary study of filariasis related acute adenolymphangitis with special reference to precipitating factors and treatment modalities. Southeast Asian Journal of Tropical Medicine and Public Health 26:301-305. Shenoy, R. K., V. Kumaraswami, T. K. Suma, K. Rajan, and G. Radhakuttyamma. 1999. A double-blind, placebo-controlled study of the efficacy of oral penicillin, diethylcarbamazine or local treatment of the affected limb in preventing acute adenolymphangitis in lymphoedema caused by Brugian filariasis. Annals of Tropical Medicine and Parasitology 93:367-377.
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Tawe, W., E. Pearlman, T. R. Unnasch, and S. Lustigman. 2000. Angiogenic activity of Onchocerca volvulus recombinant proteins similar to vespid venom antigen 5. Molecular and Biochemical Parasitology 109:91-99. Taylor, M. J., H. F. Cross, and K. Bilo. 2000. Inflammatory responses induced by the filarial nematode Brugia malayi are mediated by lipopolysaccharide-like activity from endosymbiotic Wolbachia bacteria. Journal of Experimental Medicine 191:1429-1436. Tisch, D. J., F. E. Hazlett, W. Kastens, M. P. Alpers, M. J. Bockarie, and J. W. Kazura. 2001. Ecologic and biologic determinants of filarial antigenemia in bancroftian filariasis in Papua New Guinea. Journal of Infectious Diseases in press. Underwood, A. P., T. Supali, Y. Wu, and A. E. Bianco. 2000. Two microsatellite loci from Brugia malayi show polymorphisms among isolates from Indonesia and Malaysia. Molecular and Biochemical Parasitology 106:299-302. Vickery, A. C., K. H. Albertine, J. K. Nayar, and B. H. Kwa. 1991. Histopathology of Brugia malayi-infected nude mice after immune-reconstitution. Acta Tropica 49:45-55. Vincent, A. L., A. C. Vickery, M. J. Lotz, and U. Desai. 1984. The lymphatic pathology of Brugia pahangi in nude (athymic) and thymic mice C3H/HeN. Journal of Parasitology 70:48-56. Vincent, A. L., C. A. Urena Rojas, E. M. Ayoub, E. A. Ottesen, and E. G. Harden. 1998. Filariasis and erisipela in Santo Domingo. Journal of Parasitology 84:557-561. Weil, G. J., P. J. Lammie, and N. Weiss. 1997. The ICT filariasis test: A rapid-format antigen test for diagnosis of bancroftian filariasis. Parasitology Today 13:401-404. Weller, P. F., E. A. Ottesen, L. Heck, T. Tere, and F. A. Neva. 1982. Endemic filariasis on a Pacific island. I. Clinical, epidemiologic, and parasitologic aspects. American Journal of Tropical Medicine and Hygiene 31:942-952. Yazdanbakhsh, M., W. A. Paxton, Y. C. Kruize, E. Sartono, A. Kurniawan, A. van het Wout, M. E. Selkirk, F. Partono, and R. M. Maizels. 1993. T cell responsiveness correlates differentially with antibody isotype levels in clinical and asymptomatic filariasis. Journal of Infectious Diseases 167:925-931. Yazdanbakhsh, M., E. Sartono, Y. C. Kruize, A. Kurniawan, F. Partono, R. M. Maizels, G. M. Schreuder, R. Schipper, and R. R. de Vries. 1995. HLA and elephantiasis in lymphatic filariasis. Human Immunology 44:58-61. Yazdanbakhsh, M., K. Abadi, M. de Roo, L. van Wouwe, D. Denham, F. Medeiros, W. Verduijn, G. M. Schreuder, R. Schipper, M. J. Giphart, and R. R. de Vries. 1997. HLA and elephantiasis revisited. European Journal of Immunogenetics 24:439-442.
NATURAL HISTORY OF HUMAN FILARIASIS – THE ELUSIVE ROAD Balachandran Ravindran Division of Immunology, Regional Medical Research Centre, (Indian Council of Medical Research), Nandankanan Road, Bhubaneswar,751023, India. e-mail :
[email protected]
ABSTRACT There are currently two models to explain the course of lymphatic filarial infection and disease in human populations. I develop a different model that incorporates existing data from animal models as well as epidemiological and longitudinal observations on human populations. The model explains the observations and makes predictions which are testable. Key Words: Natural history, Filariasis, W. bancrofti, Infection and Disease,
INTRODUCTION The natural history of filariasis in human populations continues to be an enigma. Apart from the intellectual challenge that it offers, gaining insights into the natural history of infection and development of chronic disease manifestations has important practical utility for the optimal utilization of available tools and for the development of newer ones for the control and/or management of human filariasis. Unlike the case for several other parasitic diseases, finding the road-map of progression of infection and development of acute/chronic disease manifestations in filariasis has been an arduous task. The persistence of infection for several years, and the equally long, if not greater time span required for the development of disease have been largely responsible for this impasse. Typically, in filariasis endemic areas, one observes groups of a) infected subjects who are often free of overt disease manifestations; b) patients who display one or more of the chronic disease manifestations with or often without current filarial infections and; c) subjects who are free of demonstrable infection or disease. In the absence of comparable clinical features in experimental animal models, the natural history of human filariasis has been deduced by analysis of cross-sectional data on a) prevalence of infection and disease in endemic areas; b) immune response phenotypes, essentially proliferation of filarial specific T-cells and release of IFN- c) clinical presentation; d) ultrasonographic recording; and e) histopathology performed in subjects living in endemic areas. Sequencing the events that take place over a period of several decades by utilizing such “windows” of cross-sectional data has been the mainstay for development of models of natural history of filariasis. The only way of demystifying this puzzle will be to actually follow-up cohorts of individuals presenting with one or the other features of the clinical spectrum in endemic areas over several years – an approach analogous to Jane Goodall’s efforts of living with
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chimpanzees in their natural habitat to understand their behavioral pattern and life style! In the absence of such studies (a moderate beginning has been made in recent years as discussed below) cross-sectional data and follow-up of microfilariae (Mf) carriers have been fundamental to the models proposed on the natural history of the disease (Ottesen,1992; Maizels and Lawrence, 1991, Bundy et al., 1991; Srividya et al., 1991; Chan et al, 1998; Dreyer et al., 2000).
EXISTING MODELS Very broadly, a “static immunological viewpoint” and a “dynamic model” have been put forward. The static immunological view-point proposes that individuals displaying filarial specific T-cell hyporesponsiveness are associated with development and maturation of filarial worms and such individuals harbor microfilaraemia, while those displaying filarial specific Tcell hyperresponsiveness develop pathology and disease and are generally free of patent infection. This implies that differing immune responses predispose individuals either towards harboring infection or developing disease (Ottesen, 1992). The “dynamic model” proposes that there is a sequential progression from infection, microfilaraemia, and amicrofilaraemia to obstructive disease in all individuals who experience microfilaraemia (Bundy et al., 1991; Srividya et al., 1991) and/or that the lymphatic dwelling adult worms essentially mediate pathology and disease Chan et al., 1998. Extending this model, it has been proposed that subclinical lymphangiectasia is caused by lymphatic dwelling adult worms and that loss/death of adult worms would result in an inflammatory reaction leading to development of pathology and consequently chronic disease, often assisted by co-factors such as secondary bacterial infections (Dreyer et al., 2000). A decade ago, it was also proposed that a breakdown of immunological tolerance associated with patent infection would result in recovery of immunological hyperactivity to filarial antigens and lead to development of pathology and chronic disease (Maizels and Lawrence, 1991). While the “immunological view point” was proposed based on immunological read-outs, the dynamic model was proposed on the basis of mathematical derivation using epidemiological data (Bundy et al., 1991; Srividya et al., 1991; Chan et al., 1998) and later by integrating clinical, surgical, ultrasonographic and histopathological data (Dreyer et al., 2000). Both the models continue to be speculative currently, although longitudinal studies reported in recent years have begun to shed light on the validity or otherwise of the two models as elaborated below.
VALIDATION OF CURRENT MODELS The observation that infected nude mice develop pathology on reconstitution with immune spleen cells has been quoted in support of the “immunological viewpoint” and it has also been observed that these infected animals develop lymphatic pathology at a later time point in the absence of
Ravindran 89 immunocompetent lymphocytes (Ottesen, 1992). Proposed nearly two decades ago the 'immunological model' presumes infection and disease to be essentially mutually exclusive. While this may be true in general, there have been notable exceptions. High prevalence of microfilaraemia and more significantly filarial antigenemia (which detects presence of adult filarial worms in the host, a parameter that did not exist at the time when “immunological view point” was proposed) in elephantiasis and hydrocele patients in several geographical areas do not appear to validate this model completely (Ottesen, 1992; Kazura et al., 1997; Addiss et al., 1995; Gyapong, 1998). On the other hand, the “dynamic model”, proposed about a decade ago, suffers from more serious limitations — presence of patent infection or loss of patency leading to development of disease is central to this model. Since vast majority of patients with chronic disease display immunological hyperreactivity to filarial antigens, epidemiological proof for the validity of the “dynamic model” is dependant on demonstration of a switch over from the state of immunological hypo-responsiveness (observed during patency) to that of hyper-reactivity and development of chronic disease over a period of time. Longitudinal studies conducted on the same cohort of subjects, the results of which have been reported in recent years, do not offer credence to such a scenario expected of the “dynamic model”. 1) infected subjects in the Cooke Islands continued to display immunological hyporesponsiveness to filarial antigens after loss of microfilaraemia as well as antigenaemia when examined after 17 years (Ravindran et al., 2000); 2) in two different studies, one after 13 years (Satapathy et al., 2001) and another after 18 years (Simonsen and Meyrowitsch, 1998) of follow-up, it was observed that absence of antibodies to microfilarial sheath (a hallmark of immunological hyporesponsiveness in microfilaraemic subjects) in Mf carriers persist several years after loss of peripheral microfilaremia; 3) chronic filariasis was found to develop in a significant proportion of endemic normals ( asymptomatic subjects without patent infection) after 18 years of follow-up- notably in the same area in Sri Lanka. Mf carriers, on the other hand, continued to be asymptomatic after 18 years even though a significant proportion of them were free of circulating Mf when examined 18 years later (Dissanayake, 2001) very similar observations have been made in two other areas in Orissa, where microfilarial carriers and endemic normals were followed-up for a period of 10-13 years, (Sahoo et al., 2001) and 4) the development of acute filarial episodes in several thousand American troops in South Samoa after an average exposure of 11 months indicated that a patent phase (which generally lasts several years) is not mandatory for development of filarial disease (Wartman, 1947). These long term observations made in different geographical regions do not appear to validate the “dynamic model of disease development” which assumes that the presence of adult filarial worms in the host is a pre-requisite for the development of chronic disease. The above observations on the development of disease in a significant proportion of subjects without demonstrable infection (endemic normals) indicates that with certain
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“modifications and qualifications” the “immunological model” could be refined to draw the road map of natural history of human filariasis as described below.
AN ALTERNATIVE MODEL The different components of an alternative model of progression of filarial infection and disease in naturally exposed human population are shown in Table 1. The model proposed here essentially extrapolates several immunological observations made in experimental animals to development of chronic disease in humans living in endemic areas. In susceptible animal hosts, such as gerbils, dogs, cats, monkeys and chimpanzees, infection with filarial larvae results in inflammatory immune responses followed by downregulation of such responses, after onset of patency. The pre-patent period in infected animals is consistently associated with an immune response phenotype characterized by enhanced filarial specific T-cell proliferation and release of high levels of by the proliferating T-cells. These characteristic features are “switched-off” once patent infections (with microfilariae/adult worms) set in (summarized in Ravindran, 2001) Extending this sequence of events to infected human populations, the proposed model perceives two stages of parasite development. Stage I, during which the filarial larvae are still developing and are yet to reach maturity and thus the infected hosts do not have circulating filarial antigens. This stage is analogous to the pre-patent period observed in experimental animals. Individuals at Stage I display a hyper-responsive immune phenotype characterized by high levels of filarial specific IgG1, IgG2, IgE and presence of antibodies to Mf sheath. During Stage I, filarial specific lymphocytes proliferate vigorously in vitro releasing high levels of and also IL-5. However, production of anti-inflammatory cytokines such as IL-10 and are also released and levels of filarial specific IgG4 are minimal. The model thus places all subjects with the above features described in the literature (Ravindran et al., 2000; Maizels et al., 1995; Freedman, 1998; Dimock et al., 1996; Sartono et al., 1997) at Stage I. Maturation of the developing larvae into adult stage parasites would result in a shift from Stage I to Stage II - a phase in which circulating filarial antigens are detectable; this stage is analogous to the patent phase observed in experimentally infected animals models. The immune response phenotype at Stage II is characterized by lower levels of filarial specific IgG1, IgG2, IgE and absence of antibodies to sheath; proliferation of filarial specific T cells and release of and IL-5 are also significantly down regulated in this stage. This hypo-responsive phase is characterized by production of high levels of filarial specific IgG4 and release of higher levels of antiinflammatory cytokines such as IL-10 and The model thus places at Stage II all infected subjects displaying immunological hyporesponsiveness described by several investigators (Ravindran et al., 2000; Maizels, et al.,
Ravindran 91 1995; Freedman, 1998; Dimock et al., 1996; Sartono et al., 1997; Mahanty et al., 1996; King et al., 1993).
The duration of stay at Stage I could vary between individuals in a given endemic area – a few months in some to a few years in others. Several individuals may never move into the patent phase of Stage II. Host as well as parasite factors would contribute in shifting from Stage I to Stage II. (1) A higher intensity of transmission (greater exposure to infective larvae) would contribute to successful maturation of larvae to adult stage
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parasites in a larger number of individuals in the area and at a shorter duration of time. (2) Adult worms and/or their products could offer the required signal for down-regulation of hyper-responsive inflammatory host responses associated with Stage I. (3) host genetic factors and/or intra-uterine exposure to filarial antigens/parasites would predispose the subjects to readily induce immunological hyporesponsiveness that is required for shifting from Stage I to Stage II; and (4) presence of intestinal worms in the host could augment filarial worms in down-regulating the inflammatory responses associated with Stage I and assist in establishing patent filarial infections. In general, subjects living in low endemic areas would behave more like experimental animals administered with trickle infections of filarial larvae. Susceptible subjects living in areas of high endemicity and satisfying one or more of the above mentioned predisposing factors would shift more readily from Stage I to Stage II, analogous to susceptible animals reaching patency when infected with a large inoculum of infective larvae. Histologically, a lymph-node biopsy taken from individuals at Stage I would reveal dead/degenerating worms associated with a severe inflammatory reaction, while those collected from Stage II would have intact, live mature adult worms in dilated lymphatics without inflammatory reaction. (Jungmann et al., 1992; Jungmann et al., 1991) These are analogous to inflammation and formation of lymph thrombi during pre-patent phase and down regulation of such responses during patent phase in infected animals (Rao et al., 1996). All Mf carriers and those with cryptic infection (as shown by circulating filarial antigens) are those who have moved into Stage II, while endemic normals are those who have remained stationary at Stage I (Sahoo et al., 2000). A majority of patients with chronic filarial disease, particularly lymphedema/ elephantiasis are those who have remained at Stage I. However, infection pressure above a threshold could downregulate the inflammatory responses associated with Stage I and shift some of these patients to Stage II, thus accounting for presence of Mf and/or CFA along with chronic symptoms. The relatively higher prevalence of CFA in patients with hydrocele indicates that shift from Stage I to Stage II takes place more readily in them than in patients with lymphedema/elephantiasis. (Addiss et al., 1995; Ravindran et al., 2000) The model does not exclude pathogenesis of filarial disease mediated per se by lymphatic dwelling adult worms. Parasite-associated factors causing pathology could be operational at Stage II and contribute to the development of disease. Extrapolating from the observations in susceptible animal models of filariasis, the model presumes that a strong inflammatory hyper-responsive state (Stage I) is associated with the growth and development of infective larvae into mature adult worms and that successful persistence of developed worms in the host would depend on rapid down regulation of the
Ravindran 93 inflammatory responses observed in Stage I to an immunologically tolerant Stage II (Ravindran, 2001; Saeftel et al., 2001). The model assumes that the life span of adult filarial worms in infected humans is in the range of 15-20 years, or more. Estimates of the life span of filarial worms are limited to calculations of “fecund life span” only, since they were based on the duration of microfilaraemic phase in Mf carriers (Vanamail et al., 1996). Longitudinal follow-up of Mf carriers for 13-16 years has indicated persistence of adult worms as shown by the presence of CFA several years after loss of circulating microfilariae. (Satapathy et al., 2001; Simonsen and Meyrowitsch, 1998). The long life span of adult filarial worms is further indicated by several immunoepidemiological studies on the prevalence of CFA in age-stratified populations in endemic areas. Unlike intestinal worms, which follow a convex prevalence curve (Anderson, 1986)filarial antigenemia increases in younger age groups (<20 years) and is maintained as a plateau in higher age groups (Ravindran et al., 2000; Simonsen et al., 1996; Day et al., 1991; Weil et al., 1999). Persistence of adult filarial worms for several years (15-20 years or more) would thus maintain the host at the hypo-responsive Stage II. This hypo-responsive state would be irreversible and loss/ death of adult worms would not result in recovery of immunological hyper-reactivity and thus continue to sustain the host at Stage II (Steel and Ottesen, 2001; Satapathy et al., 2001). The loss of microfilariae and /or antigenemia does not result in recovery of immunological hypo-responsiveness. Microfilaraemic subjects continue to display decreased filarial specific T-cell proliferation and IFN-y production and are free of antibodies to microfilarial sheath after loss of circulating Mf/ filarial antigens (Steel and Ottesen, 2001; Satapathy et al., 2001). The current model is partly similar to the “Immunological model” in which adult worm infestation is not considered a pre-requisite (unlike the “dynamic model”) for development of disease. However, it is unique and clearly different from “immunological view-point” which is essentially bidirectional and considers infection and disease to be generally mutually exclusive. The current model is uni-directional and linear. Secondly, apart from a genetic pre-disposition, it considers infection load/transmission intensity as well as intra-uterine exposure to filarial antigens as crucial components for the consequence and progression of infection/disease. The model thus accommodates disease development by inflammatory processes (proposed by immunological viewpoint) as well as by lymphatic dwelling parasites, (proposed by dynamic model) and it explains the presence of filarial infection in patients with chronic disease. It also accommodates the observations made by all the long-term follow-up studies mentioned above (Steel and Ottesen, 2001; Satapathy et al., 2001; Simonsen and Meyrowitsch, 1998; Dissanayake, 2001). Three sets of immuno-epidemiological observations made in endemic areas provide more direct evidence for the major component of the current linear model which proposes that inflammatory Th 1 responses will be observed in the host during the pre-patent
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phase of infection ie., before the onset of antigenemia/microfilaraemia. In endemic areas this phase can be expected to be observed under two circumstances – a) in early childhood or b) in transmigrants who have been moved from non-endemic areas to filarial endemic areas. This implies that Stage I features of the model would be observed in younger age groups and relatively more of Stage II features would be observed in the adult population in endemic areas. Existing epidemiological evidence offer credence to such a possibility (Ravindran et al., 2000; Weil et al., 1999; Lammie et al., 1998). Transmigrants in Indonesia (who were moved from filarial non-endemic areas to endemic zones) displayed more of Stage I immune response phenotype in the early years of exposure (<3 years) and with increasing years of stay in endemic areas many of them moved towards the hypo-responsive Stage II (Yazdanbakhsh, 1999). Finally, in Onchocerciasis, early human infection is associated with an enhanced parasite specific cellular immune responses, which get down-regulated in chronic infections (Cooper et al., 2001)
DEVELOPMENT OF CHRONIC DISEASE IN FILARIASIS Available evidence in the literature indicates a clear role for inflammatory immunological responses as well as the lymphatic dwelling parasites per se in mediating pathology leading to the development of chronic disease manifestations. While development of disease by inflammatory responses is central to the “immunological view-point”, the “dynamic model” lays emphasis on the role of adult worms in development of pathology. One of the critical issues that still needs to be resolved in human filariasis is whether immunological hyporesponsiveness (which is a consistent feature of patent infection) can be reversed to a state of inflammatory immune response to filarial antigens after the loss of Mf/adult worms in an infected human host. Immuno-epidemiological studies reported in recent years however provide evidence to the contrary. In Cook Islands, loss of filarial infection in a cohort of Mf carriers (over a period of 17 years) failed to result in significant recovery of filarial specific T-cell proliferation or enhanced production of (Steel and Ottesen, 2001). In Tanzania (Simonsen and Meyrowitsch, 1998) and India (Satapathy et al., 2001), loss of Mf in parasite carriers after 18 or 13 years respectively failed to result in appearance of antibodies to Mf sheath. Absence of Mf and presence of such Mf sheath reactive antibodies is a consistent feature in vast majority of patients with chronic filarial disease (Ravindran et al., 2000). These observations raise doubts about the proposal that patients with chronic disease would have necessarily gone through a phase of patent infection as proposed by the dynamic model (Maizels and Lawrence, 1991; Bundy et al., 1991; Srividya et al., 1991; Chan et al., 1998; Dreyer et al., 2000). Very recent findings that endemic normals (asymptomatic subjects without demonstrable infection) have a higher predilection for the development of chronic filarial disease than Mf carriers further offers credence to the conclusion that patent infections are not a prerequisite for development of chronic disease (Dissanayake, 2001; Sahoo et al.,
Ravindran 95 2002). Finally three broad areas of filariasis research may have to be considered for possible accommodation in models of chronic disease development – the role of anti-filarial drugs in enhancement/suppression of disease development in Mf carriers, secondary bacterial infections and endosymbionts such as Wolbachia in mediating inflammation and consequently pathology. Convincing evidence for each of the possibilities is expected to emerge in the next few years.
CONCLUSIONS Building models of natural history of a complex and chronic infection/disease like filariasis and attempting to accommodate every immuno-epidemiological, clinical or histopathological observation into the model has given mixed experience for investigators, frustrating as well as rewarding. Longitudinal studies being undertaken by some groups in recent years have been crucial and have provided valuable insights into the natural history of human filariasis and we currently have what can presumably be labeled as an incomplete road map that still has a few blind ends.
ACKNOWLEDGEMENTS The Regional Medical Research Center is funded by The Indian Council of Medical Research, New Delhi. The work in the author's laboratory is partly funded by the European Commission (IC-18- CT- 970245). Critical reading of the draft manuscript by Tom Nutman, Achim Hoerauf, T.V. Rajan, Swadesah Pani, Pradeep Das and Satyajit Rath are gratefully acknowledged. The author thanks all his laboratory colleagues who have been a constant source of inspiration.
REFERENCES Addiss, A.G. el al. (1995) Clinical, parasitologic, and immunologic observations of patients with hydrocele and elephantiasis in an area with Endemic lymphatic filariasis. J. Infect. Dis. 171, 755-758. Anderson, R.M. (1986) The population dynamics and epidemiology of intestinal nematode infections. Trans R. Soc. Trap. Med. Hyg. 80, 686-696. Bundy, D.A.P., et al. (1991) Immunoepidemiology of lymphatic filariasis: the relationship between infection and disease. Immunoparasitol Today (Gallager, R. and Ash, C., eds Elsevier A71-A75. Chan, M.S. et al. (1998) Epifil: A dynamic model of infection and disease in lymphatic filariasis. Am. J. Trop. Med. Hyg. 59, 606-614. Cooper, P.J. et al (2001) Early human infection with Onchocerca volvulus is associated with an enhanced parasite specific cellular immune response. J. Inf. Dis. 183, 1662-1668. Day, K.P. et al. (1991) Age specific patterns of change in the dynamics of Wuchereria bancrofti infection in Papua New Guinea. Am. J. Trop. Med. Hyg. 44, 518-527. Dimock, K.A. et al. (1996) Th1- Like Antifilarial immune responses predominate in AntigenNegative persons. Infect. Immun. 64, 2962-2967. Dissanayake, S (2001) In Wuchereria bancrofti filariasis, asymptomatic microfilaraemia does not progress to amicrofilaraemic lymphatic disease. Int.J.Epid. 30, 394-399. Dreyer, G. et al . (2000) Pathogenesis of Lymphatic Disease in Bancroftian Filariasis: A Clinical Perspective. Parasitol. Today, 16, 544-548. Freedman, D.O. (1998) Immune Dynamics in the Pathogenesis of human lymphatic filariasis. Parasitol. Today 14, 229-233.
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Gyapong, J.O. (1998) The relation between infection and disease in Wuchereria bancrofii infection in Ghana. Trans. R. Soc. Trop. Med. Hyg. 92, 390-392. Jungmann, P. et al. (1991) Bancroftian lymphadenopathy: A histopathologic study of fiftyeight cases from northeastern Brazil. Am. J. Trop Med. Hyg. 45, 325-331. Jungmann, P. et al. (1992) Bancroftian lymphangitis in northeastern Brazil: A histopathological study of 17 cases. J. Trop. Med. Hyg. 95,114-118. Kazura, J.W. et al. (1997) Transmission intensity and its relationship to infection and disease due to Wuchereria bancrofti in Papua New Guinea. J. Infect. Dis. 176, 242-246. King, C.L. et al. (1993) Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset. J. Clin. Invest. 92, 1667-. Lammie, P.J. et al. (1998) Longitudinal analysis of the development of filarial infection and antifilarial immunity in a cohort of Haitian children. Am. J. Trop. Med. Hyg. 59, 217-221. Mahanty, S. et al. (1996) High levels of spontaneous and parasite antigen-driven interleukin-10 production are associated with antigen-specific hyporesponsiveness in human lymphatic filariasis. J. Infect. Dis. 173, 769-773. Maizels, R.M. et al. (1995) T-cell Activation and the balance of antibody isotypes in human lymphatic filariasis. Parasitol Today 11, 50-56. Maizels, R.M., and R.A. Lawrence. (1991) Immunological tolerance: The key features in human filariasis ? Parasitol Today 7, 271-276. Ottesen, E.A. (1992) Infection and disease in lymphatic filariasis : an immunological perspective. Parasitology 104, S71-S79. Rao, U.R. et al. (1996) Cellular Immune Responses of Jirds to Extracts of life cycle stages and adult excretory products during the Early development of Brugia pahangi. Exp. Parasitol. 82, 255-266. Ravindran, B. (2001) Are inflammation and immunological hyperactivity needed for filarial parasite development? Trends Parasitol. 17, 70-73. Ravindran, B. et al. (2000) Protective immunity in human bancroftian filariasis:inverse relationship between antibodies to microfilarial sheath and circulating filarial antigens. Parasite Immunol. 22, 633-637. Saeftel, M. et al. (2001) Lack of Interferon y confers impaired neutrophil granulocyte function and imparts prolonged survival of adult filarial worms in murine filariasis. Microbes Infect. 3, 203-213. Sahoo, P.K et al. (2000) Bancroftian filariasis : prevalence of antigenaemia and endemic normals in Orissa, India. Trans. R. Soc. Trop Med. Hyg. 94, 515-517. Sahoo, P.K. et al (2002) Bancroftian Filariasis : A 13- year follow-up study of asymptomatic microfilariae carriers and Endemic normals. Parasitology (In Press). Sartono, E. et al. (1997) Depression of Antigen-Specific interleukin-5 and Interferon-y responses in human lymphatic filariasis as a function of clinical status and age. J. Infect. Dis. 175, 1276-1280. Satapathy, A.K. et al. (2001) Human Bancroftian filariasis: Loss of patent microfilaraemia is not associated with production of antibodies to microfilarial sheath Parasite Immunol. 23, 163-167. Simonsen, P.E. et al. (1996) Bancroftian filariasis: The patterns of filarial-specific immunologlobulin G1 (IgGl), IgG4, and circulating antigens in an endemic community of Northeastern Tanzania. Am. J. Trop. Med. Hyg. 55, 69-75. Simonsen, P.E., and D.W. Meyrowitsch. (1998) Bancroftian filariasis in Tanzania: specific antibody responses in relation to long-term observations on microfilaremia. Am. J. Trop. Med. Hyg. 59, 667-672. Srividya, A. et al. (1991) The dynamics of infection and disease in Bancroftian filariasis. Trans. R. Soc. Trop. Med. Hyg. 85, 255-259. Steel, C., and E.A. Ottesen. (2001) Evolution of Immunologic responsiveness of persons living in an area of Endemic Bancroftian Filariasis. J.Inf.Dis. 184, 73-79. Vanamail, P. et al. (1996) Estimation of the fecund life span of Wuchereria bancrofti in an endemic area. Trans. R. Soc. Trop. Med. Hyg. 90, 119-121. Wartman, W.B. (1947) Filariasis in American armed forces in World War II. Medicine. 26, 333-394. Weil, G.J. et al. (1999) A longitudinal study of bancroftian filariasis in the Nile delta of Egypt: Baseline data and one-year follow-up. Am. J. Trop. Med. Hyg. 61, 53-58. Yazdanbakhsh, M. (1999) Common features of T cell reactivity in persistent helminth infections: lymphatic filariasis and schistosomiasis. Immunol. Lett. 65, 109-115.
IN UTERO EXPOSURE TO FILARIAL ANTIGENS AND ITS INFLUENCE ON INFECTION OUTCOMES
Patrick J. Lammie Division of Parasitic Diseases Centers for Disease Control and Prevention
ABSTRACT The earliest potential exposure of the human host to filarial antigens is in the uterine environment. The impact of this exposure on host susceptibility and development of antifilarial immune responses following challenge with infective larvae is unclear. Although a number of studies suggest that children born to infected mothers may have a greater risk of acquiring infection than children of uninfected mothers, it has been difficult to relate these epidemiologic findings to specific alterations in antifilarial immune responsiveness. Immunologic studies of cord blood mononuclear cells (CBMC) have demonstrated that CBMC are capable of responding to crude filarial antigens with the production of a range of cytokines and parasite specific antibodies that are qualitatively similar to those produced by adults. Although these data demonstrate that sensitization to filarial antigens does take place in utero, additional work is needed to critically examine CBMC responses to defined parasite antigens and to conduct quantitative comparisons of B and T cell responses of children born to infected and uninfected mothers. In addition, it is important to consider the possibility that susceptibility to filarial infection may not depend strictly on the expression of filaria-specific immune responses per se, but may be a function of the cytokine environment in which the parasite develops. If so, in utero exposure to filarial antigens may alter this milieu. Keywords: filariasis, antigen sensitization, tolerance.
INTRODUCTION Lymphatic filariasis has always represented a fascinating puzzle to parasitologists and immunologists because of the wide spectrum of clinical outcomes of exposure to infection and their clear association with differential host immune responsiveness (Ottesen, 1992). Exposure to infective stage larvae may lead to the establishment of stable and long term microfilaremia or to the development of distinct disease syndromes, including lymphangitis,
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tropical pulmonary eosinophilia, lymphedema and hydrocele. Our inability to explain why these different outcomes occur in exposed persons reflects our relatively poor understanding of the natural history of filarial infection. More than 30 years ago, Paul Beaver (1970) suggested that the difference between outcomes of filarial infection in endemic and nonendemic persons provided a key to understanding the basis of the host-parasite relationship. Patent infections develop much more commonly among persons born in an endemic area that among visitors or transplants. Beaver noted that, in contrast to endemic residents, long periods of exposure were required to establish patent infections in migrants and proposed “the possibility of tolerance induced by individual and direct contact with the filaria during the fetal and neonatal periods of development” as an explanation of this difference (Beaver, 1970). The concept that tolerance is induced following in utero exposure to filarial antigens has provided the focus for a great deal of interest over the intervening years but, to date, we are still struggling to develop experimental approaches to test this hypothesis. In this review, I will consider evidence from experimental models of filariasis as well as epidemiologic and immunologic studies of filariasis in human populations in my discussion of in utero exposure to filarial antigens. Although most of my comments will focus on lymphatic filariasis, selected studies of onchocerciasis will be considered as well.
ANIMAL STUDIES Studies of animal models of filariasis provide evidence that infection is influenced by in utero events, although differences between model systems complicate direct comparison of outcomes. Microfilariae of Dipetalonema (Acanthocheilonema) viteae can cross the rat placenta and persist for up to 3-4 months in the circulation of young rats. Spleen cells from rats with infections acquired transplacentally fail to proliferate in vitro to parasite extracts while microfilaria persist in the circulation (Haque and Capron, 1982). The relationship between microfilaremia and diminished parasite-specific proliferative responsiveness has been well described in many host-parasite systems; thus, at least in this model, the nonresponsiveness that is observed resembles that noted in microfilaremic infections in both humans and animals. In the jird model, Schrater et al. (1983) demonstrated that female jirds born to infected parents were more susceptible to Brugia malayi infection than female offspring of uninfected animals. In addition, offspring of Brugia pahangi infected jirds demonstrated significantly less lymphatic pathology following infection (Klei et al., 1986). No significant differences were observed in proliferative responsiveness to filarial antigens or in granulomatous response to injections of antigen-coated beads as a function of maternal infection status (Bosshardt et al., 1991). Despite the inability to identify a potential mechanism, taken together, results from these studies suggest that in utero exposure to filarial infection increases host susceptibility to infection while
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diminishing inflammatory responses that could be associated with the development of chronic pathology. Studies by Rajan et al. (1994) of a murine model of filariasis suggest a different possibility. Following crosses of immune-deficient scid/scid mice with immunocompetent mice, susceptibility to Brugia malayi infection was greater in the offspring of scid/scid females than immunocompetent females, independent of maternal infection status. The filarial infection status of the scid/scid mothers did not influence infection outcome. These results suggest that antigen nonspecific factors related to maternal immune status have a greater impact on the developing immune system of offspring and on susceptibility to infection than in utero filarial antigen exposure.
IS THERE EPIDEMIOLOGIC EVIDENCE CLUSTERING OF INFECTION IN HUMANS?
OF
If in utero tolerance does occur as a result of exposure to filarial antigens, then it should be possible to document increased filarial infection levels in offspring of infected mothers or provide evidence of altered filariaspecific immune responsiveness. In studies in Haiti, Lammie and co-workers (1991) showed that maternal filarial infection was a risk factor for infection in children. Children of infected mothers were nearly three-fold more likely to be microfilaremic than children with amicrofilaremic mothers. That paternal infection status was unrelated to the infection status of children was taken as evidence that in utero exposure to filarial antigens altered susceptibility to Wuchereria bancrofti infection (Lammie at al., 1991; Hightower et al., 1993). Although similar results were reported by Simonsen et al. in Tanzania (1995), investigators in India and in Papua New Guinea reported that the familial clustering of infection was related to parental status and not maternal status alone (Das et al.,1997; Alexander et al., 1998). These results imply that differences in exposure at the household level and not in utero exposure per se are the major determinant of infection in children and are consistent with the conclusion that tolerance can be induced peripherally as a consequence of differential larval exposures (Maizels and Lawrence, 1991; Das et al., 1997; King et al., 2001). The reason for the absence of an association between infection in fathers and their children in Haiti and Tanzania is not clear; however, it is possible that differences in the stability of family structures in these settings may obscure an association between paternal infection status and that of their offspring. As pointed out previously (Alexander et al., 1998), the fact that infection in children is associated with the infection status of both parents does not rule out the possibility that in utero exposure to filarial antigens does influence the acquisition of infection and the development of immune responsiveness; on the other hand, it also does not exclude the possibility that any changes in antifilarial immunity which are characterized in children are the consequence of peripheral tolerance rather than in utero events (Maizels and Lawrence, 1991).
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Analyses of familial clustering of Brugia malayi infection reported by Terhell et al. (2000) suggested that host genetics also may influence susceptibility. Lymphedema and elephantiasis may cluster in families, but this may reflect genetic predisposition as well as differential exposure (Ottesen et al, 1981; Cuenco, 2001). Although it has been proposed that risk of immunopathology may be less among children born to infected mothers (Grenfell and Michael, 1992), to date no evidence has been generated to support this hypothesis. Epidemiologic studies of the presumptive impact of in utero exposure to filarial antigens have been problematic for a number of reasons. Most studies have had to rely on the assumption that maternal infection status at the time of the surveys was a reflection of the mother’s status at the time of the birth of the child. Although studies have supported these assumptions by demonstrating the stability of microfilaremia over many years of follow up (Meyrowitsch et al., 1995; Dissanayake, 2001), the absence of contemporaneous information on infection status must be recognized as a study limitation. Initial studies were also limited by the lack of availability of antigen detection assays. These assays have dramatically changed our ability to determine infection status (Weil et al., 1987; More and Copeman, 1990). A follow up study in Haiti suggested that up to one-third of mothers who would have otherwise been classified as microfilaria-negative were, in fact, antigenpositive (Lammie et al., 1998). Given the association between antigen status and altered cytokine responses to parasite antigens (de Almeida et al, 1996; Dimock et al, 1996), antigenemia represents a better marker of maternal infection than microfilaremia. Furthermore, longitudinal studies are expensive and difficult to carry out and are confounded by the ethical requirement to treat infected persons which changes transmission levels at the household level and probably at the community level as well. Finally, without careful assessment of vector indices at the household level, these types of studies do not exclude alternative interpretations of the data, namely that differences in infection outcome are influenced by heterogeneity in exposure level (Das et al., 1997).
DO MICROFILARIA OR FILARIAL ANTIGENS CROSS THE PLACENTA? The most direct route for contact between the parasite and the developing immune system is transfer of the microfilariae across the placenta. Whereas this likely takes place for skin dwelling microfilariae, analysis of cord blood samples collected in Haiti and Brazil suggests that microfilaria rarely cross the placenta at least for Wuchereria bancrofti (Campello, et el., 1993; Eberhard et al., 1993). Definitive demonstration of filarial antigens in cord blood is lacking; Hitch et al. (1997) reported that 10% of cord blood samples were positive using the Og4C3 assay for circulating filarial antigen, but mixtures of cord blood and maternal blood were not specifically excluded
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in this study. In the absence of evidence of direct transfer of antigen across the placenta, the demonstration of antifilarial IgM or IgE in cord blood provides evidence of in utero sensitization to parasite antigens, although whether this takes place via direct transfer of antigen across the placenta or indirectly via stimulation by idiotypes expressed on maternal antibody is not clear. A number of investigators have shown that antifilarial IgM or IgE responses can be detected in neonates born to both infected and uninfected mothers (Dissanayake et al., 1980; Weil et al., 1983; Hitch et al., 1997; Malhotra et al., 1997; King et al., 1998). Typically, responses were more commonly detected in cord bloods of offspring of infected mothers; however, a number of children born to amicrofilaremic mothers also had detectable antifilarial IgM or IgE responses. These latter results are consistent with the hypothesis that maternal antibody, in the absence of filarial antigen may directly stimulate antibody production in utero through idiotypic interactions.
IN UTERO SENSITIZATION OR TOLERANCE? Initial studies of filarial antigen specific responses of uninfected children born to infected mothers indicated that these children were less responsive than children born to uninfected mothers in terms of both proliferative responsiveness and cytokine production (Lammie et al., 1991; Steel et al., 1994). These studies were consistent with the hypothesis that in utero exposure to filarial antigens leads to the development of tolerance and stimulated efforts to monitor responsiveness of cord blood mononuclear cells (CBMC), both in cross-sectional and longitudinal studies. If exposure to filarial antigens in utero uniformly results in tolerance, either through anergy induction or through deletion of immunoreactive cells, these studies might be expected to generate negative data; however, in most studies of lymphatic filariasis and onchocerciasis, measurement of cytokine responses demonstrates that some degree of in utero sensitization occurs (Hitch et al., 1997; Malhotra et al., 1997; Soboslay et al., 1999). In general, proliferative and cytokine responses were more prevalent with CBMC from children born to infected mothers than uninfected mothers. Similarly, CBMC from children of infected mothers also produced more parasite specific IgE and IgG antibody in vitro than CBMC from children of uninfected mothers (King et al, 1998). Thus, sensitized T cells are induced in utero and are able to provide adequate help for antibody responses. Not all infants born to infected mothers have CBMC that produce cytokines or antibody in response to filarial antigen in vitro. Whether the absence of responses reflects tolerance, a low degree of antigen sensitization, or use of crude antigens is not clear. Similarly, the ability to detect CBMC responses to crude extracts of adult or microfilarial antigens does not imply that CBMC are capable of responding to putative protective antigens. Studies of fractionated antigens have demonstrated that different fractions may elicit different cytokine profiles (Dimock et al., 1994). Substantial evidence for direct stimulation of T cells by maternal idiotypes has been generated in studies of schistosomiasis and
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trypanosomiasis, both in experimental animals and in humans (Eloi-Santos et al., 1989; Novato-Silva et al., 1992; Neves et al., 1999; Montesano et al., 1999). In a murine model of schistosomiasis, maternal idiotypes reduce the pathologic response to egg deposition and enhance host survival. Although comparable mechanisms may exist for filariasis, similar studies may require the development of an experimental system that employs well characterized antigenic and idiotypic determinants. Our efforts to address the impact of in utero exposure to filarial antigens on the development of antifilarial immune responses have been complicated by our incomplete understanding of how the developing immune system responds to antigenic exposures, even in relatively simple antigenic systems. Differences in immune responsiveness may be a function of the mode of antigen presentation, where the antigen is presented, antigen concentration and co-stimulatory signals. Filarial antigen specific cellular responses of microfilaremic persons display some characteristics expected of Th2 responses (Maizels et al, 1999). Given this profile of responses among persons with persistent infections, it is possible that in utero exposure to filarial antigens establishes a Th2-biased cytokine environment. Consistent with this hypothesis, peripheral blood mononuclear cells of children of Onchocerca volvulus infected mothers made higher levels of Th-2 cytokines (IL-4 and IL-5) to filarial antigens and lower levels of to recombinant O. volvulus antigens (Elson et al., 1996). In contrast, studies of lymphatic filariasis have not provided any compelling evidence that in vitro cytokine production by CBMC to crude filarial antigens is skewed toward Th2 responses (Malhotra et al, 1997). There is, however, support for the idea that in utero events influence Th-1 immune responses. responses to PPD following BCG immunization were greater among children born to uninfected mothers than children born to infected mothers. Given the association between antigen-negative status and responses and the indirect association through IgG2 (de Almeida et al, 1996; Dimock et al, 1996), it is possible that in utero events lead to subtle alterations in the expression of Thl responses to specific antigens. These responses might not be detected in assays employing crude parasite extracts for antigens.
WHAT DOES INFLUENCE INFECTION OUTCOME? The underlying assumption that protective immunity is a feature of filariasis has provided much of the impetus to study in utero tolerance to filarial antigens. Certainly, our working assumption has been that tolerance increases susceptibility, implying the existence of mechanisms that govern resistance to infection. It is, however, appropriate to ask whether there is evidence that protective immunity does exist and if so, what is it? To date, there is little unequivocal evidence that protective immunity develops in response to filarial infection in humans and it has been difficult to demonstrate that recognition of specific filarial antigens or life cycle stages is
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associated with resistance to infection. On the other hand, epidemiologic evidence is consistent with the existence of protective immunity: 1) The long term stability of amicrofilaremic infections implies that some persons are not susceptible to infection, even in areas of relatively high infection prevalence (Meyrowitsch et al., 1996) ; and 2) The negative association between microfilaremia and lymphedema suggests that persons with lymphedema have a significantly lower risk of re-infection (Addiss et al, 1995; Baird et al., 200l;Dissanayake, 2001). These considerations lead to another question: is immunity directed against parasite antigens or is it related to the host immune environment? Ravindran (2001) has suggested that Thl- and Th-2 biased immune responses provide different developmental cues for larval stages of filarial parasites and hypothesized that persons with Thl-biased responses (i.e., endemic normal individuals and persons with lymphedema) are susceptible to infection, based on an environment which is permissive for larval development. In fact, the persistence of amicrofilaremia and long term stability of Thl responses in persons with lymphedema argue that the Thl environment is not permissive. In addition, L3 induce Th2 responses in vivo very rapidly following infection (Osborne and Devaney, 1998). Perhaps, in utero exposure to filarial antigen leads to subtle down regulation of the expression of Thl-associated responses, thus, promoting the establishment of patent infections. Interestingly, nonendemic persons who are newly exposed to Onchocerca volvulus display elevated and IL-5 responses, similar to putatively immune individuals (Elson et al., 1996; Cooper et al., 2001). It is important to bear in mind that Thl responses, per se, may not be inhibiting parasite development directly, but may be associated with other factors that influence parasite development. Rajan and colleagues have demonstrated that T cells and NK cells promote worm survival to varying degrees in murine models of Brugia infection, but the factors that promote worm survival have not been fully characterized (Babu et al., 1998, 1999). Alternatively, it is possible that our focus on the filarial parasite is misplaced. Kozek (1975) proposed that host response to Wolbachia may play a role in immunopathogenesis and recent results have shown that the presence of Wolbachia is necessary for maintaining the fertility of filarial worms (Hoerauf et al., 1999). Studies in a primate model have shown a correlation between antibody responses to Wolbachia and development of lymphedema in amicrofilaremic animals (Punkosdy et al., 2001). Although it is difficult to understand how the immune response could target Wolbachia within the filarial worm, perhaps such responses contribute to protective immunity, at least among persons with lymphedema. The attractiveness of the concept that in utero exposure to parasite antigens makes it a difficult hypothesis to abandon, particularly when there are strong correlations between immune responses and the presence or absence of infection and when these states tend to be stable over time. It is clear, however, that exposure to infective larvae is a missing variable that has
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not been given enough attention in human studies of antifilarial immunity (Wamae et al., 1998). Maizels and Lawrence (1991) also suggested that larval exposure plays a prominent role in the induction of peripheral tolerance. In a carefully controlled comparison of immune reactivity in high and low transmission villages, King and colleagues (2001) demonstrated that IL-5 responses to parasite antigen were increased and responses to non-parasite antigens were decreased with higher levels of exposure to infective stage larvae. The nonspecific alteration in production is especially intriguing and provides further encouragement for additional study of the influence of the immunologic milieu on parasite development.
REFERENCES Addiss, D.G., K.A. Dimock, M.L. Eberhard and P.J. Lammie. 1995. Clinical, parasitologic and immunologic observations of patients with hydrocele and elephantiasis in an area with endemic lymphatic filariasis. Journal of Infectious Diseases 171:755-8. Alexander, N.D.E., J.W. Kazura, M.J. Bockarie, R.T. Perry, Z.B. Dimber, B.T. Grenfell, and M.P. Alpers. Parental infection confounded with local infection intensity as risk factors for childhood microfilaremia in bancroftian filariasis. 1998. Transactions of the Royal Society of Tropical Medicine and Hygiene 92:23-4. Babu, S., P. Porte, T.R. Klei, L.D. Schulz, and TV Rajan. 1998. Host NK cells are required for the growth of the human filarial parasite Brugia malayi in mice. Journal of Immunology 161:1428-32. Babu, S., L.D. Schulz, and TV Rajan. 1999. T cells facilitate Brugia malayi development in TCR- (null) mice. Experimental Parasitology 93:55-7. Beaver, P.C. 1970. Filariasis without microfilaremia. American Journal of Tropical Medicine and Hygiene 19:181-9. Baird, J.B., P.J. Lammie, J. Louis Charles, T.G. Streit, and D.G. Addiss. 2001. Reactivity to bacterial, fungal, and parasite antigens in patients with lymphedema and elephantiasis. American Journal of Tropical Medicine and Hygiene (In Press). Bosshardt, S.C., C.S. McVay, S.U. Coleman, and T.R. Klei. 1991. Brugia pahangi: circulating antibodies to adult worm antigen in uninfected progeny of homologously infected jirds. Experimental Parasitology 72:440-9, Campello, T.R., R. Santos Ferreira, M.L. Pires, P. Gouveia De Melo, R. Albuquerque, S. Araujo, and G. Dreyer. 1993. A study of placentas from Wuchereria bancrofti microfilaraemic and amicrofilaraemic mothers. Journal of Tropical Medicine and Hygiene. 96:251-5. Cooper, P.J., T. Mancero, M. Espinel, C. Sandoval, R. Lovato, R.H. Guderian, and T.B. Nutman. 2001. Early infection with Onchocerca volvulus is associated with an enhanced parasite-specific cellular response. Journal of Infectious Diseases 183:1662-1668. Cuenco, K.T. 2001. Familial clustering of filarial lymphedema in a Haitian population. Ph.D. Dissertation. Emory University. Das, P.K., A. Sirvidya, P. Vanamail, K.D. Ramaiah, S.P. Pani, E. Michael, and D.A.P. Bundy. 1997. Wuchereria bancrofti microfilaremia in children in relation to parental infection status. Transactions of the Royal Society of Tropical Medicine and Hygiene 91:677-9. DeAlmeida, A.B., M.C. Maia e Silva, M.A. Maciel and D.O. Freedman. The presence or absence of active infection, not clinical status, is most closely associated with cytokine responses in lymphatic filariasis. Journal of Infectious Diseases 173:1453-9. Dimock, K.A., D.G. Addiss, M.L. Eberhard and P.J. Lammie. 1994. Differential proliferative
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and IL-10 responses to fractionated filarial antigens: preferential recognition by patients with chronic lymphatic dysfunction. Journal of Infectious Diseases 170:403-12. --------, M.L. Eberhard, and P.J. Lammie. 1996. Thl-like antifilarial immune responses predominate in antigen-negative persons. Infection and Immunity 64:2962-7. Dissanayake, S., L.V.K. DaSilva, and M.M. Ismail. 1980. IgM antibody to filarial antigens in cord blood: possibility of transplacental infection. Transactions of the Royal Society of Tropical Medicine and Hygiene 74:542-4. Dissanayake, S. 2001. In Wuchereria bancrofti filariasis, asymptomatic microfilaraemia does not progress to amicrofilaraemic disease. International Journal of Epidemiology 30:394-9. Eberhard, M.L., W.L. Hitch, D.F. McNeeley, and P.J. Lammie. 1993. Transplacental transmission of Wuchereria bancrofti in Haitian women. Journal of Parasitology 79:62-6. Eloi-Santos, S., E. Novato-Silva, V.M. Maselli, G. Gazzinelli, D.G. Colley, R. Correa-Oliveira. 1989. Idiotypic sensitization in utero of children born to mother with Schistosomiasis or Chagas’ disease. Journal of Clinical Investigation 84:1028-31. Elson, L.H., A. Days, M. Calvopina, W. Paredes, E. Araujo, R.H. Guderian, J.E. Bradley, and T.B. Nutman. 1996. In utero exposure to Onchocerca volvulus: relationship to subsequent infection intensity and cellular responsiveness. Infection and Immunity 64:5061-5. ---------., M. Calvopina, W. Paredes, E. Araujo,, J.E. Bradley R.H. Guderian, and T.B. Nutman. 1996. Immunity to onchocerciasis: putative immune persons produce a Thl-like immune response to Onchocerca volvulus. Journal of Infectious Diseases 171:652-8. Grenfell, B.T., and E. Michael. 1992. Infection and disease in lymphatic filariasis: an epidemiologic approach. Parasitology 104(Supp1):S81-S90. Haque A., and A. Capron. 1982. Transplacental transfer of rodent microfilariae induces antigen-specific tolerance in rats. Nature 299:361-3. Hightower, A.W., P.J. Lammie, and M.L. Eberhard. 1993. Maternal filarial infection - a persistent risk factor for microfilaremia in offspring. Parasitology Today 9:418-21. Hitch, W.L., M.L. Eberhard, and P.J. Lammie. 1997. Investigation of the influence of maternal infection with Wuchereria bancrofti on the humoral and cellular responses of neonates to filarial antigens. Annals of Tropical Medicine and Parasitology 91:461-9. Hoerauf A, K. Nissen-Pahle, C. Schmetz, K. Henkle-Duhrsen, M.L. Blaxter, D.W. Buttner, M.Y. Gallin, K.M. Al-Qaoud, R. Lucius, and B. Fleischer. Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility. Journal of Clinical Investigation 103:11-8. King, C.L., I. Malhotra, P. Mungai, A. Wamachi, J. Kioko, J.H. Ouma, and J.W. Kazura, 1998. B cell sensitization to helminthic infection develops in utero in humans. Journal of Immunology 160:3578-84. --------, M. Connelly, M.P. Alpers, M. Bockarie, and J.W. Kazura. 2001. Transmission intensity determines lymphocytes responsiveness and cytokine bias in human lymphatic filariasis. Journal of Immunology 166:7427-36. Klei, T.R., D.P. Blanchard, and S.U. Coleman. 1986. Development of Brugia pahangi infections and lymphatic lesions in male offspring of female jirds with homologous infections. Transactions of the Royal Society of Tropical Medicine and Hygiene 80:214-6. Kozek, W.J. 1977. Transovarially-transmitted intracellular microorganisms in adult and larval stages of Brugia malayi. Journal of Parasitology 63:992-1000 Lammie, P.J., W.L. Hitch, E.M. Walker, A.W. Hightower, and M.L. Eberhard. 1991. Maternal filarial infection as risk factor for infection in children. Lancet 337:1005-6. --------, M.D. Reiss, K.A. Dimock, T.G. Streit, J.M. Roberts, and M.L. Eberhard. 1998. Longitudinal analysis of the development of filarial infection and antifilarial immunity in a cohort of Haitian children. American Journal of Tropical Medicine and Hygiene 59:21721. Maizels, R.M., and R.A. Lawrence. 1991. Immunological tolerance: the key feature in lymphatic filariasis. Parasitology Today 7:271-276. --------, M.J. Holland, F.H. Falcone, X.-X. Zang, and M. Yazdanbakhsh. 1999. Vaccination against helminth parasites. Immunological Reviews 171:125-47. Malhotra, I., J.H. Ouma, A. Wamachi, J. Kioko, P. Mungai,, A. Omollo, L. Elson, D. Koech,
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J.W. Kazura, and C.L. King. 1997. In utero responses to helminth and mycobacterial antigens generates cytokine responses similar to that observed in adults. Journal of Clinical Investigation 99:1759-66. --------., P. Mungai, A. Wamachi, J. Kioko, J.H. Ouma, J.W. Kazura, and C.L. King. 1999. Helminth- and Bacillus Calmette-Guerin-induced immunity in children sensitized in utero to filariasis and schistosomiasis. Journal of Immunology 162:6843-8. Meyrowitsch, D.W., P.E. Simonsen, and W.H. Makunde. 1995. Bancroftian filariasis: analysis of infection and disease in five endemic communities of north-eastern Tanzania. Annals of Tropical Medine and Parasitology 89:653-63. Montasano, M.A., D.G. Colley, G.L. Freeman, Jr., and W.E. Secor. 1999. Neonatal exposure to idiotype induces Schistosoma mansoni egg-antigen -specific cellular and humoral immune responses. Journal of Immunology 163:898-905. More, S.J., and D.B. Copeman. 1990. A highly specific and sensitive monoclonal antibodybased ELISA for the detection of circulating antigen in Bancroftian filariasis. Tropical Medicine and Parasitology 41:403-6. Neves S.F., S. Eloi-Santos, R. Ramos, S. Rigueirinho, G. Gazzinelli, R. Correa-Oliveira. 1999. In utero sensitization in Chagas' disease leads to altered lymphocyte phenotypic patterns in the newborn cord blood mononuclear cells. Parasite Immunology 21:631-9. Novato-Silva, E. , G. Gazzinelli, and D.G. Colley. 1992. Immune responses during human schistosomiasis mansoni. XVIII. Immunologic status of pregnant women and their neonates. Scandinavian Journal of Immunology 35:429-37. Osborne, J. and E. Devaney. 1998. The L3 of Brugia induces a Th2-polarized response following activation of an IL-4-producing CD4-CD8T cell population. International Immunology 10:1583-90, Ottesen, E.A. 1992. Infection and disease in lymphatic filariasis: an immunologic perspective. Parasitology 104(Suppl):S71-S79. Punkosdy, G,A., V.A. Dennis., B.L. Lasater., G. Tzertinis, J. Foster, and P.J. Lammie. 2001. Detection of serum IgG antibodies specific for Wolbachia surface protein (WSP) in rhesus monkeys infected with Brugia malayi. Journal of Infectious Diseases (In Press). Rajan, T.V., J.M. Bailis, J.A. Yates, L.D. Schulz, D.L. Greiner, and F.K. Nelson. 1994. Maternal influence on susceptibility of offspring to Brugia malayi infection in a murine model of filariasis. Acta Tropica 58:283-9. Ravindran, B. 2001. Are inflammation and immunological hyperactivity needed for filarial parasite development. TRENDS in Parasitology 17:70-73. Schrater, A.F., A. Spielman and W.F. Piessens. 1983. Predisposition to Brugia malayi microfilaremia in progeny of infected gerbils. American Journal of Tropical Medicine and Hygiene 32:1306-8. Soboslay, P.T., S.M. Geiger, B. Drabner, M. Banla, E. Batchassi, I.A. Kowu, A. Stadler, and H. Schulz-Key. 1999. Prenatal immune priming in onchocerciasis-Onchocerca volvulusspecific cellular responsiveness and cytokine production in newborns from infected mothers. Clinical and Experimental Immunology 117:130-7. Steel, C., A. Guiena, J.S. McCarthy, and E.A. Ottesen. 1994. Long-term effect of prenatal exposure to maternal microfilaremia on immune responsiveness to filarial parasite antigens. Lancet 343:890-3. Terhell, A.J., JJ. Houwing-Duistermaat, Y. Ruiterman, M. Haarbrink, K. Abadi, and M. Yazdanbakhsh. 2000. Clustering of Brugia malayi infection in a community in SouthSulawesi, Indonesia. Parasitology 120:23-9. Wamae, C.N., S.M. Gatika, J.M. Roberts and P.J. Lammie. 1998. Wuchereria_bancrofti in Kwale District, Coastal Kenya. I. Patterns of focal distribution of infection, clinical manifestations and antifilarial IgG responsiveness. Parasitology 116:173-82. Weil, G.J., R. Hussain, V. Kumaraswami, K.S. Phillips, and E.A. Ottesen. 1983. Prenatal allergic sensitization in offspring of parasite-infected mothers. Journal of Clinical Investigation 71:1124-9.
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--------, D.C. Jain, S. Santhanam, A. Malhotra, H. Kumar, K.V.P. Sethumadhavan, F. Liftis, and T.K. Ghosh. 1987. A monoclonal antibody-based enzyme immunoassay for detecting parasite antigenemia in Bancroftian filariasis. Journal of Infectious Diseases 156:350-5.
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IMMUNE EFFECTORS IMPORTANT IN PROTECTIVE RESISTANCE
Achim Hoerauf Department of Helminthology, Bernhard Nocht Institute of Tropical Medicine, Hamburg, Germany
ABSTRACT This chapter describes immune effector mechanisms against the different developmental stages of filarial nematodes in humans and animal models of filarial infection. Over the last few years, significant insight has been gained from work using laboratory mice with engineered genetic defects leading to lack of immune cell subsets or cytokine responses. These data have provided evidence that both T helper 1 and T helper 2 responses as well as pathways of the innate system are essential for parasite containment. As data from animal and human studies reveal, these pathways are counterregulated by suppressor responses involving recently described regulatory T cells. Keywords: B. malayi, W. bancrofti, O. volvulus, L. sigmodontis, T helper cells, T regulatory cells.
DIFFERENT FORMS OF IMMUNITY ARE DIRECTED AT DIFFERENT DEVELOPMENTAL STAGES OF THE FILARIAE Complex, multicellular parasites like filarial nematodes undergo several stages of development within their hosts. Each developmental stage presents the host with not only overlapping, but also many stage specific antigens (Hunter et al., 1999). As a result, immunity to filarial nematodes is a network of multiple interacting, in part counteracting, immune responses. One can classify the immune responses simply according to the developmental stage of the worm to which they are directed: i) against infective third stage 3 (L3) and fourth stage 4 (L4) larvae; ii) against adult worms; and iii) against microfilariae (mf). In animal models, immune reactions against any stage can lead to some level of protection or reduction of worm numbers; in humans, important phenomena of immunity with the potential of application for vaccines comprise mainly the reactions detailed under i) and iii).
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IMMUNITY AGAINST L3 Immunity against L3/4 can be further subgrouped in humans: a) there is the phenomenon of putative immunity, impeding development of patent infection in individuals in endemic areas, despite exposure to L3 which is equivalent to that in individuals who aquire patency (Ottesen, 1995); b) the phenomenon of concomitant immunity individuals with patent infection. There is probably a high degree of immunity since despite annual transmissions of several 100 to 1000 L3 in areas endemic for a given filarial infection, once a certain number of adult worms are in the body, the rate of new infections levels off. For example, if there are 20 adult worms in the body, all living 10 years, an average replacement of 2 worms will be necessary to keep adult worm numbers stable. Assuming an annual transmission potential of 100 L3, concomitant immunity may account for up to 98% protection (Schulz-Key, 1990); and c) vaccine induced immunity. Enhancement of immunity to L3 and L4 is a prime goal of vaccine development against filarial nematodes. The mechanisms underlying vaccineinduced immunity and its potential for vaccination have been assessed using various animal models (see below). While neither concomitant immunity nor vaccine induced immunity has been examined by intervention studies in human populations, a key to understanding immunity against filariae (and in particular, against L3) has been to analyze the different clinical presentations in filariasis patients after exposure to the parasite. In both onchocerciasis and lymphatic filariasis, a focus of interest have been individuals who remain free from infection, although they have an equivalent degree of exposure compared to patently infected individuals and even get infected by L3 (Maizels et al., 1995; Ottesen, 1995). These individuals have been termed either "endemic normals" (EN) or "putatively immune individuals" (PI). Putative immunity against onchocerciasis does not result from low vector attractiveness of the respective individuals (Kruppa and Burchard, 1999). Rather, a different activity of the immune system compared to patently infected individuals accounts for an inhibition of L3 from developing into adult worms. The analysis of parasite molecules to which the immune response of PI/EN is directed has been critical in defining potential vaccine candidates for testing in animal models (Cook et al., 2001). In the 1980s it was demonstrated, at first in mice, that T helper cell responses could be divided into two major mutually cross-regulating subgroups, termed Th1 and Th2 (Mosmann et al., 1986). This paradigm was also applied to the human immune response (King and Nutman, 1991), and it was further studied if a Th1/2 dichotomy was associated with the different clinical presentations, such as PI/EN vs. patent infections. Few reports indeed observed a tendency towards a Th1 response in PI/EN (Elson et al., 1995), or downregulation of a Th1 response in
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microfilaremic patients infected with Brugia malayi (Maizels et al., 1995), a finding which would be consistent with higher specific lymphocyte proliferation rates found in this group. However, in the majority of other studies, including more recent ones, PBMC from PI/EN were shown to display a mixed Th1/2 response (Doetze et al., 1997; Hoerauf and Brattig, 2002; Sartono et al., 1997; Soboslay et al., 1997; Turaga et al., 2000), being elevated in immune individuals in comparison to patients with patent infections. Often, and IL-5 were major cytokines found to be jointly produced. Can a Th1, a Th2 type of response, or both, be responsible for a protective immune response? While the mechanisms of immune-dependent destruction of L3 have been elusive to analysis of human material ex vivo (e.g. by histology), animal models of filarial infection have been of tremendous help. Clearly defined primary infections with L3 can be induced in animal models, in contrast to humans. The immunity which is active in containing larval development during a primary infection has, however, to be distinguished from the immunity induced before infection, e.g. by vaccination. B. malayi infection of mice has been the only model where a Th1 type immune response has been found responsible for the impairment of L3 into adult worm development during primary infection: KO mice show significantly higher adult worm loads after the end of larval development (Babu et al., 2000), as do mice where nitric oxide has been depleted by aminoguanidine blockade of the NO producing enzyme iNOS (Rajan et al., 1996). It is not known if mechanisms similar to those observed in schistosomiasis for schistosomulae exist where activated macrophages release ROI and NO, thereby destroying the parasites (those in vitro analyses were done only with mf (Taylor et al., 1996), see below). Interestingly, IL-4 KO mice which have higher NO production during filarial infection (Volkmann et al., 2001), also show increased larval development into adult worms (Babu et al., 2000), suggesting that more than a single effector pathway limits the development of L3 to adults in a primary infection with B. malayi in mice. Despite the evidence for a role of endogenous in the containment of larval development cited above, the capacity of L3 to induce production in mice, if observed at all (Taylor et al., 1994), seems to be low in a majority of reports. Consistently, infection with Brugia L3 did not result in any Th1 priming in vivo, even when IL-4 was depleted either by antibodies or by genetic KO (in IL-4 KO mice), underscoring that the lack of was not the result of a suppression by IL-4 (Lawrence et al., 1995; Osborne et al., 1996). In contrast, a strong, T cell driven Th2 response is induced by L3, whether injected into non-permissive (Bancroft et al., 1993) or permissive (Al-Qaoud et al., 1998; Al-Qaoud et al., 1997) hosts.
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Infection of mice with the rodent filaria Litomosoides sigmodontis is the only fully permissive model using laboratory mice. There, dependence upon CD4+ cells for protection even in primary infection was observed (AlQaoud et al., 1997), in contrast to other, not fully permissive models where such depletion had no additional effect to the already rather fast worm elimination (possibly due to host-parasite incompatibility) (Rajan et al., 1994; Rajan et al., 1992). L3 or adult worm antigens stimulate an early Th2 response (Le Goff et al., 2000b). IL-5 but not IL-4 KO mice allow a higher percentage of adult worms to develop from L3 (Volkmann and Hoerauf, submitted). In this model (Al-Qaoud et al., 1998), as well as in B. malayi (Paciorkowski et al., 2000), a unique role for a subset of B lymphocytes, socalled Bl cells, has been observed. Transfer of Bl cells into T and B celldevoid mutant mice (RAG KO) completely restored resistance against B. malayi infection (Paciorkowski et al., 2000). It is not known at present if the major role of this B cell subset lies in the production of particular antibodies, or in driving the immune reaction into a polarized direction. In contrast to primary infection, available evidence suggests that the immune reaction following vaccination is Th2-mediated. In a surrogate model for onchocerciasis, mice can be s.c. implanted a diffusion chamber filled with O. volvulus L3. The chamber allows the entry of fluid and all types of granulocytes and lymphocytes but confines the L3. Although L3 develop only into L4 in this model, it was shown that prior vaccination of these mice with irradiated L3 led to significant reductions in the survival of the larvae (Lange et al., 1993). This model has been used to test the protective potential (as assessed by larval survival time and integrity) of many recombinant O. volvulus antigens as vaccines (Abraham et al., 2001), and also to define protective immune mechanisms. Both IL-4 and IL-5 were cytokines essential for protection, while was not (Johnson et al., 1998; Lange et al., 1994). In addition, B cells (or antibodies) are also an essential component of the protective response (David Abraham, pers. comm.). It can be concluded that antibody-dependent cytotoxicity mediated primarily by eosinophil granulocytes is the most important mechanism which has to be induced for successful protective immunity in this system. Using the L. sigmodontis model described above, essential new insights on the (Th2-dependent) mechanisms of immunity to L3 after successful vaccination have been established. This model has the advantage that the parasitological readout is not limited to larval survival but can be measured through the whole developmental cycle of the worm. In this system there is an essential requirement for IL-5 and eosinophils for protective immunity following vaccination with irradiated L3 (Le Goff et al., 2000a; Martin et al., 2000). Eosinophils must be present in the s.c. tissue, "waiting" for incoming challenge L3 in order to quickly attack them (Martin et al., 2000). If a critical time point of probably only a few hours has been passed,
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then no further reduction is observed in the recovery rate of worms (Le Goff et al., 1998). IL-5 neutralization shortly before challenge abolishes protective immunity in vaccinated WT BALB/c mice (Martin et al., 2000). In this model eosinophils must have immunoglobulins at their surface. Further, vaccination of B cell-devoid BALB/c mice does not lead to protective immunity against challenge L3. Although eosinophils are present in the s.c. tissue, in the absence of antibodies there is no crosslinking signal necessary for eosinophil degranulation and release of toxic proteins (e.g. major basic protein) which are probably the molecules responsible for larval destruction (Martin et al., 2001). In summary, the results from vaccination studies in rodent models point to an essential role for antibody-dependent, eosinophil-mediated destruction of L3 shortly after host entry and possibly during the molt to the L4 (Eisenbeiss et al., 1994), but not of later developmental stages of the parasite. Both antibodies and cells seem to be essential but neither alone is sufficient for L3 destruction in immune hosts: evidently, the above mentioned mechanisms and the dependence on IL-4 and IL-5 qualify this type of reaction as Th2. A shortcoming of the rodent models is that they have no correlate in the field, i.e. exposure of vaccinated animals to natural infection over long time. In this regard, bovine onchocerciasis may bridge this gap for the studies in Onchocerca research. Cattle can be protectively vaccinated with irradiated L3 (studies with a large panel of recombinant antigens are underway), and interestingly, putative immunity has been reported under conditions of natural exposure (Trees et al., 2000). The immune pathways seem to closely mimic those in human infection (Graham et al., 2001). This model may lead to further insights into the nature of a protective response against L3.
IMMUNITY AGAINST ADULT WORMS Human immune reactions against adult worms can be readily studied in onchocerciasis, using onchocercomas resected under local anesthesia as a therapeutic measure to limit the infectious burden (Albiez et al., 1988). Histology then reveals the nature of the inflammatory cells, and their activity. There is an inner ring of granulocytes and macrophages adjacent to the worms (Wildenburg et al., 1997), with an outer ring consisting mainly of macrophages, some scattered granulocytes and T cells (Parkhouse et al., 1985). T cells probably keep the balance between inflammatory and downregulatory reactions (see below). The presence of eosinophils requires the release of mf from female worms (Wildenburg et al., 1996). Of interest is the presence of neutrophil granulocytes around virtually every female (Brattig et al., 2001; Rubio de Krömer et al., 1998), predominantly at the cranial end around the vulva where live, motile mf are being released. At first sight, the attraction of neutrophils is unusual for a helminth infection, but
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neutrophils seem to be triggered for chemotaxis by bacterial products as usual. Most filarial species including O. volvulus contain, as a long and stable mutualistic association, endobacteria of the genus Wolbachia, order Rickettsiales (Bandi et al., 1998; Taylor and Hoerauf, 1999). There is a stringent association between the presence of Wolbachia and the presence of neutrophils: i) the one Onchocerca species being naturally devoid of endobacteria, O. flexuosa, induces nodules without neutrophils; ii) the presence of neutrophils around the worms in Wolbachia-containing O. volvulus is lost when the worms have been depleted from Wolbachia by doxycycline therapy (Brattig et al., 2001). The investigation of immune reactions induced by adult worms causing lymphatic filariasis, e.g. Wuchereria bancrofti, also relies on the histological evaluation of biopsy material, as is the case with nodulectomies of onchocercomas. In contrast to the latter, resection is not by itself a therapeutic measure but is only used for differential- diagnostic reasons, e.g. in form of a lymph node biopsy when ruling out other causes for lymphadenitis or lymphadenopathy (Jungmann et al., 1991). Although not many studies exist on this issue, significant knowledge has been gained by the work of Dreyer and her co-workers. According to their studies, lymphatic fluid around worms collected from intrascrotal lymphatics contains almost no inflammatory cells, as long as the worm is alive. This strongly points to antigen-specific suppression by adult worms (Dreyer et al., 2000a; Dreyer et al., 2000b). In lymph nodes the process is different since there are many circulating lymphocytes in the lymphatic vessels inside the lymph nodes. Again, these lymphocytes do not seem to be reactive (Jungmann et al., 1991). As with onchocerciasis, neutrophils were observed in some lymph nodes which lie adjacent to worms, forming microabscesses (Jungmann et al., 1991). It remains to be seen whether there is the same stringent association with the Wolbachia endobacteria which are present in all species causing human filariasis. At some distance to the worms, reactive tissue apparently exists, with follicular hyperplasia, and many eosinophils (Jungmann et al., 1991). In conclusion, there is rather little inflammatory reaction around adult Wuchereria bancrofti in lymphatic filariasis, suggesting active suppression of an immune response (Dreyer et al., 2000a). Despite inflammatory cells taking part in the onchocercoma formation in onchocerciasis, suppressive mechanisms similar to those in lymphatic filariasis may operate because suppressor T cells can be generated from onchocercomas in generalized onchocerciasis (see below). Further, in the non-suppressive, hyperreactive form of onchocerciasis (sowda), there is a different nodule architecture with approximately 10 -20 times as much host cell biomass per worm on average compared to generalized onchocerciasis (Hoerauf and Brattig, 2002). This suggests antigen-specific suppression is also present to some degree in the generalized form of onchocerciasis. Conversely, the hyperreactive form in lymphatic filariasis, elephantiasis, is
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also associated with a strong immune response and attack on mf begins at their birthplace, the adult female. Both Th1 and Th2 type of immune reactions are associated with this form (Maizels et al., 1995). In both human lymphatic filariasis and onchocerciasis it cannot be proven that the proinflammatory immune reactions of either the Th1 or Th2 type indeed limit the survival of adult worms. However, information on this topic has been gathered from work in animal models. In B. malayi, both and IL-4 KO mice show a delayed clearance of adult worms, indicating that both cytokines contribute to limiting the life of adult worms (Babu et al., 2000). In L sigmodontis, lack of was found to lead to a delayed formation of inflammatory nodules around adult worms (Saeftel et al., 2001). These nodules resemble histologically the onchocercomas and can thus serve as a model for immune reactions observed there. In contrast to onchocercomas, however, L. sigmodontis inflammatory nodules are shorterlived and part of a process associated with the degeneration of adult worms. Similar to KO, delayed inflammatory nodule formation was also observed when IL-5 was depleted either by monoclonal antibodies or by genetic KO. Both cytokines thus seem to synergize in the production of neutrophil chemotactic cytokines such as and KC, the murine homologue of IL-8, leading to less neutrophil influx to the site of infection (Al-Qaoud et al., 2000). Indeed, using a neutrophil depleting antibody, it could be shown that neutrophil were essential for the adult worm killing in this model (Al-Qaoud et al., 2000). These observations established for the first time a link between neutrophils and IL-5 (Al-Qaoud et al., 2000). In contrast to B. malayi, IL-4 seems merely to limit adult worm size and fertility in L. sigmodontis infections. Worms in IL-4 KO mice were larger in size and displayed a higher degree of fertility. So, the main activity of IL-4 in L. sigmodontis infection seems to be against production of the mf stage (Volkmann et al., 2001). While one would assume that IL-4, IL-5 and are primarily T cell derived, a recent study of ours shows that production is also derived from NK cells which seem to limit adult worm survival on the one hand (Korten et al., submitted) but also promote larval development on the other (Babu et al., 1998, and Korten et al., unpublished). In summary, immune responses against adult worms are apparently of both the Th1 and Th2 type and involve cells of the innate immune system. This has been demonstrated in animal models using KO mice and histologically in human tissue when studying the inflammatory cell types involved. In animal models the immune responses are proven to limit adult worm survival. It may well be that the dominant trigger for Th1 are Wolbachia. A possible way to test this would be to use the naturally Wolbachia-devoid species Acanthocheilonema viteae; however, mice are not permissive for this filarial species (Lucius et al., 1991).
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IMMUNITY AGAINST MF The mf is the developmental stage studied most extensively using animal models (reviewed in Hoerauf and Fleischer, 1997). In many studies, due to non-permissivity of the host for other worms stages, mf have been injected i.p. or i.v. In summary, results obtained point to an involvement of antibodies, including natural IgM antibodies to phosphorylcholine, and a role for both (Taylor et al., 1996) and IL-5 (Al-Qaoud et al., 2000; Folkard et al., 1996) dependent pathways. Results on the requirement for T cells seem to be equivocal, either depending on the filarial species (dispensable for B. malayi and A. viteae (Haque et al., 1980; Kazura and Davis, 1982; Thompson et al., 1981) but required for O. lienalis (Folkard and Bianco, 1995)) or on the experimental read-out. Unaltered mf clearance in the absence of IL-4 (Lawrence et al., 1995) or after addition of IL-12 (Pearlman et al., 1995) may lead to the speculation that there is a limited contribution of the adaptive immune system to mf containment. However, this conclusion may be hasty: it is known that injection of mf in the absence of females does not reflect the situation in a natural infection since female worms are well known to suppress immune responses directed against mf (Lawrence et al., 1994). Recently, in an elegant study it has been suggested that this suppression is IL10 dependent (see below) (Hoffmann et al., 2001). It is therefore useful to study the relevance of certain immune pathways for mf containment again in a fully permissive infection, namely L sigmodontis infection of BALB/c mice. Here, the two dominant cytokines for mf permissivity are IL-4 and IL-5. IL-4 KO mice (on a BALB/c background) display a more than 100 fold higher microfilarial load. Accordingly, the mf are present in the peripheral blood 6 times as long as in WT mice (120 days instead of 20 days) (Volkmann et al., 2001). The effect of a genetic KO of IL5 is more pronounced than that of IL-4 with regard to mf loads. This is a finding not surprising given that IL-5 in addition to its direct effect on mf survival also limits the survival of adult worms (see above, (Al-Qaoud et al., 2000); Volkmann et al., in prep.). In human filarial infections, attack against mf can again be observed ex vivo in nodulectomized onchocercomas (Darge et al., 1991). Similar to what is known from animal models, the cells directed against mf consist of eosinophils, macrophages, and also neutrophils as long as Wolbachia endobacteria are present in the mf (Gutierrez-Pena et al., 1996). Antibody dependency can be assumed from the fact that the response is greater in the presence of increased numbers of plasma cells (mainly IgE bearing) and IgEcoated mast cells can be found in the nodular tissue (Korten et al., 1998). However, the striking feature is that these reactions against mf do not take place frequently in individuals with generalized onchocerciasis, but can only be observed in histology after damage to mf induced by microfilaricidal drugs DEC or ivermectin (Darge et al., 1991). In these individuals, the immune
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system which would normally rapidly attack the many mf in the skin, has been instructed antigen-specifically to tolerate these targets. This mechanism is beneficial to the host, and prevents the severe skin inflammation which otherwise would occur, given a daily turnover of >50,000 mf (Duke, 1993). When this tolerance is broken in onchocerciasis, the clinical picture of the hyperreactive "sowda" form of the disease results, with strong Th2 responses (Brattig et al., 1997; Brattig et al, 1994) and frequent histologically detectable attack against mf (Büttner and Racz, 1983). Clinically one can often observe unilateral papular dermatitis, hyperpigmented lichenified lesions, pruritus and lymphadenitis (McMahon and Simonsen, 1996). With regard to mf control, however, sowda is the successful form of the infection since mf loads are very low compared to the generalized infection. This is, however, at the expense of host tissue inflammation (recent overview in (Hoerauf and Brattig, 2002)). In conclusion, immune mechanisms directed against mf and limiting their presence are mostly of the Th2 type, both in permissive mouse models and in human disease such as onchocerciasis. In order to prevent overreaction with damage to the host, and in order to facilitate survival of the mf, downregulatory mechanisms exist which are detailed in the next section (for a review regarding this topic with a focus on onchocerciasis see (Hoerauf and Brattig, 2002).)
DOWNREGULATION OF IMMUNE EFFECTOR PATHWAYS IN HUMAN FILARIASIS - THE TH3/TR1 CONCEPT When mf are accidentally transferred during transfusion of blood from a person infected with lymphatic filariasis, the mf are eliminated in the new host much more quickly than in the donor, provided that the recipient has not had prior exposure to filariae (Hira and Husein, 1979). This scenario has been reproduced recently in L. sigmodontis infection. Mf injected i.v. live longer if gravid female worms are co-transplanted into the peritoneal cavity. The mechanism of female-induced suppression is apparently dependent on IL-10 induction in the murine host since in IL-10 KO mice, the mf do not live longer in the presence of female worms (Hoffmann et al., 2001). Over the last years, there has been increasing evidence for an IL-10 (plus -dependent mechanism of antigen-specific suppression of T cell activation and inflammation in human onchocerciasis as well as in lymphatic filariasis. This type of reaction seems not to be part of a Th2 response, and results in lower worm-specific proliferation of PBMC, compared to PI/EN individuals (Doetze et al., 2000; Elson et al., 1995; Soboslay et al., 1992; Soboslay et al., 1997; Steel and Nutman, 1993), or hyperreactive forms such as sowda (Hoerauf and Brattig, 2002), and is
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mediated by IL-10 and another downregulatory cytokine (Brattig et al., 1997; Cooper et al., 1998; Doetze et al., 2000; Elson et al., 1995; Henry et al., 2001; Mahanty and Nutman, 1995; Soboslay et al., 1997). Reversal of the low proliferation to levels which are equivalent to PI requires neutralization of IL-10 and (Doetze et al., 2000). Interestingly, T cell clones could be generated from PBMC of patients with generalized onchocerciasis (Doetze et al., 2000) with a cytokine profile characteristic of a new type of suppressor cell (IL-2–, and which so far has been found in tolerization against autoimmunity and against alloreactivity (Groux et al., 1997; Levings and Roncarolo, 2000; Weiner, 1997)) and recently also against Th2-dependent asthma (Cottrez et al., 2000), but not in infectious diseases. T cells of the type discussed above have been termed either Th3 or Tr1 (T regulatory 1). We have followed this nomenclature and have suggested calling the downregulatory response in filariasis Th3/Tr1 (Doetze et al., 2000), in order to make it clear that in a chronic helminth infection downregulation requires a novel, non-Th1 - nonTh2, type of response (Fig. 1). T cell effector pathways have also been found in lymphatic filariasis and schistosomiasis to be of a mixed Thl/Th2 type (Grogan et al., 1998a; Sartono et al., 1997), while cellular hypoproliferation to specific antigen is mediated by IL-10 and (Grogan et al., 1998b; King et al., 1993). It is very likely that T cell clones of the Th3/Trl type will also be found in these diseases. The occurrence of macrophages which produce IL-10 and are suppressive has been recently reported in lymphatic filariasis (Mahanty et al., 1996; Osborne and Devaney, 1999; Ravichandran et al., 1997) as well as onchocerciasis (Brattig et al., 2000). These results indicate the expression of a type 2 macrophage (Kalinski et al., 1999) which can influence the Th cell dichotomy by IL-10. Thus, not only regulatory suppressor Th3/Trl cells but also macrophages may contribute to the downregulation of cellular responses. What are the final effector pathways in humans that are actually suppressed along with cellular hyporeactivity in peripheral blood? The attack against mf, at least in onchocerciasis, relies on granulocytes and macrophages and seems dependent on antibodies. T cell suppression of Th 1/2 responses could thus diminish the presence of both, effector cells and antibodies. In summary, available results strongly support a concept where hyporeactivity of immune cells which is a hallmark of individuals with high mf loads, is not the result of induction of a Th2 but of a truly antagonistic Th3/Trl response (Fig. 1).
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CONCLUSION The immune mechanisms which regulate protection against filariae are very complex. Yet over recent years, "strategies" have been defined by which the hosts's immune system apparently controls parasite infection. A major task of the world community is to control human filarial infection and to prevent new infections, adding to the approximately 150 million already present. The opinion has been brought forward that to achieve this goal, the public health community can live virtually without taking into account antifilarial immune mechanisms. This argument has been proven wrong, because in areas where onchocerciasis had been eliminated, a reimportation of the infection occurred much more quickly than anticipated by mathematical models, which did not take into account concomitant immunity and because it has become increasingly clear that current programs relying on vector control and mass chemotherapy are not sufficient for achieving eradication (Abiose et al., 2000; Richards et al., 2000). There is good reason to assume that the development of recombinant vaccines (Abraham et al., 2001) which are now in the stage of field testing (Trees et al., 2000) will provide the "second punch" (Cook et al., 2001) needed to eventually control these diseases.
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ACKNOWLEDGEMENTS I would like to express my thanks to Dr. N. Brattig for critical reading of the manuscript, to him and to Prof. Dr. D. Büttner for many stimulating discussions during our collaborations, and to the members of my department for their support. Support from the German Research Foundation (grant Ho 2009/1-3), from the European Community (grant ICA4-Ct-1999-10002) and the VW-Foundation (grant I/73952) is gratefully acknowledged.
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Groux, H., A. O'Garra, M. Bigler, M. Rouleau, S. Antonenko, J.E. de Vries, and M.G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389, 737-42. Gutierrez-Pena, E.J., J. Knab, and D.W. Büttner. 1996. Neutrophil granule proteins: evidence for the participation in the host reaction to skin microfilariae of Onchocerca volvulus after diethylcarbamazine administration. Parasitology 113, 403-14. Haque, A., M.J. Worms, B.M. Ogilvie, and A. Capron. 1980. Dipetalonema viteae : microfilariae production in various mouse strains and in nude mice. Exp. Parasitol. 49, 398-404. Henry, N.L., M. Law, T.B. Nutman, and A.D. Klion. 2001. Onchocerciasis in a nonendemic population: clinical and immunologic assessment before treatment and at the time of presumed cure. J. Infect. Dis. 183, 512-6. Hira, P.R., and S.F. Husein. 1979. Some transfusion-induced parasitic infections in Zambia. J. Hyg. Epidemiol. Microbiol. Immunol. 23, 436-44. Hoerauf, A., and N. Brattig. 2002. Resistance and susceptibility in human onchocerciasis – beyond Th1 vs. Th2. Trends Parasitol. 18, 25-31. Hoerauf, A., and B. Fleischer. 1997. Immune responses to filarial infection in laboratory mice. Med. Microbiol. Immunol. 185, 207-15. Hoerauf, A., S. Mand, O. Adjei, B. Fleischer, and D.W. Büttner. 2001. Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet 357, 1415-6. Hoerauf, A., K. Nissen-Pähle, C. Schmetz, K. Henkle-Duhrsen, M.L. Blaxter, D.W. Büttner, M.Y. Gallin, K.M. Al-Qaoud, R. Lucius, and B. Fleischer. 1999. Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility. J. Clin. Invest. 103, 11-8. Hoerauf, A., L. Volkmann, C. Hamelmann, O. Adjei, I.B. Autenrieth, B. Fleischer, and D.W. Büttner. 2000. Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis. Lancet 355, 1242-3. Hoffmann, W.H., A.W. Pfaff, H. Schulz-Key, and P.T. Soboslay. 2001. Determinants for resistance and susceptibility to microfilaraemia in Litomosoides sigmodontis filariasis. Parasitology 122, 641-9. Hunter, S.J., S.A. Martin, F.J. Thompson, L. Tetley, and E. Devaney. 1999. The isolation of differentially expressed cDNA clones from the filarial nematode Brugia pahangi. Parasitology 119, 189-98. Johnson, E.H., S. Schynder-Candrian, T.V. Rajan, F.K. Nelson, S. Lustigman, and D. Abraham. 1998. Immune responses to third stage larvae of Onchocerca volvulus in interferon-gamma and interleukin-4 knockout mice. Parasite Immunol. 20, 319-24. Jungmann, P., J. Figueredo-Silva, and G. Dreyer. 1991. Bancroftian lymphadenopathy: a histopathologic study of fifty-eight cases from northeastern Brazil. Am. J. Trop. Med. Hyg. 45, 325-31. Kalinski, P., C.M. Hilkens, E.A. Wierenga, and M.L. Kapsenberg. 1999. T-cell priming by type-1 and type-2 polarized dendritic cells: the concept of a third signal. Immunol. Today 20, 561-7. Kazura, J.W., and R.S. Davis. 1982. Soluble Brugia malayi microfilarial antigens protect mice against challenge by an antibody-dependent mechanism. J. Immunol. 128, 1792-6. King, C.L., S. Mahanty, V. Kumaraswami, J.S. Abrams, J. Regunathan, K. Jayaraman, E.A. Ottesen, and T.B. Nutman. 1993. Cytokine control of parasite-specific anergy in human lymphatic filariasis. Preferential induction of a regulatory T helper type 2 lymphocyte subset. J. Clin. Invest. 92, 1667-73. King, C.L., and T.B. Nutman. 1991. Regulation of the immune response in lymphatic filariasis and onchocerciasis. Immunol. Today 12, A54-8. Korten, S., M. Saeftel, L. Volkmann, K. Fischer, B. Fleischer, and A. Hoerauf. 2001. Expansion of NK cells with reduction of their inhibitory Ly-49C and Ly-49G2 expressing
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Mosmann, T.R., H. Cherwinski, M.W. Bond, M.A. Giedlin, and R.L. Coffman. 1986. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136, 2348-57. Osborne, J., and E. Devaney. 1999. Interleukin-10 and antigen-presenting cells actively suppress Th1 cells in BALB/c mice infected with the filarial parasite Brugia pahangi. Infect. Immun. 67, 1599-605. Osborne, J., S.J. Hunter, and E. Devaney. 1996. Anti-interleukin-4 modulation of the Th2 polarized response to the parasitic nematode Brugia pahangi. Infect. Immun. 64, 3461-6. Ottesen, E.A. 1995. Immune responsiveness and the pathogenesis of human onchocerciasis. J. Infect. Dis. 171, 659-71. Paciorkowski, N., P. Porte, L.D. Shultz, and T.V. Rajan. 2000. B1 B lymphocytes play a critical role in host protection against lymphatic filarial parasites. J. Exp. Med. 191, 731-6. Parkhouse, R.M., M. Bofill, A. Gomez-Priego, and G. Janossy. 1985. Human macrophages and T-lymphocyte subsets infiltrating nodules of Onchocerca volvulus. Clin. Exp. Immunol. 62, 13-8. Pearlman, E., F.P. Heinzel, F.E. Hazlett, and J.W. Kazura. 1995. IL-12 modulation of T helper responses to the filarial helminth, Brugia malayi. J. Immunol. 154, 4658-64. Rajan, T.V., N. Kenneth, N. Killeen, L.D. Shultz, J.A. Yates, J.M. Bailis, D.R. Littman, and D.L. Greiner. 1994. CD4+ T-lymphocytes are not required for murine resistance to the human filarial parasite, Brugia malayi. Exp. Parasitol. 78, 352-60. Rajan, T.V., F.K. Nelson, L.D. Shultz, B.H. Koller, and D.L. Greiner. 1992. CD8+ T lymphocytes are not required for murine resistance to human filarial parasites. J. Parasitol. 78, 744-6. Rajan, T.V., P. Porte, J.A. Yates, L. Keefer, and L.D. Shultz. 1996. Role of nitric oxide in host defense against an extracellular, metazoan parasite, Brugia malayi. Infect. Immun. 64, 3351-3. Rao, U.R., A.C. Vickery, B.H. Kwa, J.K. Nayar, and D. Subrahmanyam. 1992. Effect of carrageenan on the resistance of congenitally athymic nude and normal BALB/c mice to infective larvae of Brugia malayi. Parasitol. Res. 78, 235-40. Ravichandran, M., S. Mahanty, V. Kumaraswami, T.B. Nutman, and K. Jayaraman. 1997. Elevated IL-10 mRNA expression and downregulation of Th1-type cytokines in microfilaraemic individuals with Wuchereria bancrofti infection. Parasite Immunol. 19, 69-77. Richards, F., D. Hopkins, and E. Cupp. 2000. Programmatic goals and approaches to onchocerciasis. Lancet 355, 1663-4. Rubio de Krömer, M.T., M. Krömer, K. Luersen, and N.W. Brattig. 1998. Detection of a chemotactic factor for neutrophils in extracts of female Onchocerca volvulus. Acta Trop. 71, 45-56. Saeftel, M., L. Volkmann, S. Korten, N. Brattig, K.M. Al-Qaoud, B. Fleischer, and A. Hoerauf. 2001. Lack of IFN-γ confers impaired neutrophil granulocyte function and imparts prolonged survival of adult filarial worms in murine filariasis. Microbes Infect. 3, 203-13. Sartono, E., Y.C. Kruize, A. Kurniawan, R.M. Maizels, and M. Yazdanbakhsh. 1997. Depression of antigen-specific interleukin-5 and interferon-gamma responses in human lymphatic filariasis as a function of clinical status and age. J. Infect. Dis. 175, 1276-80. Schulz-Key, H. 1990. Observations on the reproductive biology of Onchocerca volvulus. Acta Leiden. 59, 27-44. Soboslay, P.T., C.M. Dreweck, W.H. Hoffmann, C.G. Lüder, C. Heuschkel, H. Gorgen, M. Banla, and H. Schulz-Key. 1992. Ivermectin-facilitated immunity in onchocerciasis. Reversal of lymphocytopenia, cellular anergy and deficient cytokine production after single treatment. Clin. Exp. Immunol. 89, 407-13. Soboslay, P.T., S.M. Geiger, N. Weiss, M. Banla, C.G. Lüder, C.M. Dreweck, E. Batchassi, B.A. Boatin, A. Stadler, and H. Schulz-Key. 1997. The diverse expression of immunity in humans at distinct states of Onchocerca volvulus infection. Immunology 90, 592-9.
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Steel, C., and T.B. Nutman. 1993. Regulation of IL-5 in onchocerciasis. A critical role for IL-2. J. Immunol. 150, 5511-8. Suswillo, R.R., D.G. Owen, and D.A. Denham. 1980. Infections of Brugia pahangi in conventional and nude (athymic) mice. Acta Trop. 37, 327-35. Taylor, M, and A. Hoerauf. 1999. Wolbachia bacteria of filarial nematodes. Parasitol. Today 15, 437-42. Taylor, M.J., H.F. Cross, A.A. Mohammed, A.J. Trees, and A.E. Bianco. 1996. Susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to nitric oxide and hydrogen peroxide in cell free culture and from IFN-γ-activated macrophages. Parasitology 112, 315-22. Taylor, M.J., and A. Hoerauf. 2001. A new approach to the treatment of filariasis. Curr. Opin. Infect. Dis., in press. Taylor, M.J., R.P. Van Es, K. Shay, S.G. Folkard, S. Townson, and A.E. Bianco. 1994. Protective immunity to Onchocerca volvulus and O. lienalis infective larvae in mice. Trop. Med. Parasitol. 45, 17-23. Thompson, J.P., R.B. Crandall, C.A. Crandall, and J.T. Neilson. 1981. Microfilaremia and antibody responses in CBA/H and CBA/N mice following injection of microfilariae of Brugia malayi. J. Parasitol. 67, 728-30. Trees, A.J., S.P. Graham, A. Renz, A.E. Bianco, and V. Tanya. 2000. Onchocerca ochengi infections in cattle as a model for human onchocerciasis: recent developments. Parasitology 120 Suppl, S133-42. Turaga, P.S., T.J. Tierney, K.E. Bennett, M.C. McCarthy, S.C. Simonek, P.A. Enyong, D.W. Moukatte, and S. Lustigman. 2000. Immunity to onchocerciasis: cells from putatively immune individuals produce enhanced levels of interleukin-5, gamma interferon, and granulocyte-macrophage colony-stimulating factor in response to Onchocerca volvulus larval and male worm antigens. Infect. Immun. 68, 1905-11. Vincent, A.L., W.A. Soderman, and A. Winters. 1980. Development of Brugia pahangi in normal and nude mice. J. Parasitol. 66, 648. Volkmann, L., M. Saeftel, B. Fleischer, and A. Hoerauf. 2001. IL-4 is essential for the control of microfilariae in murine infection with the filaria Litomosoides sigmodontis. Infect. Immun. 69, 2950-6. Weiner, H.L. 1997. Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol. Today 18, 335-43. Wildenburg, G., M. Krömer, and D.W. Büttner. 1996. Dependence of eosinophil granulocyte infiltration into nodules on the presence of microfilariae producing Onchocerca volvulus. Parasitol. Res. 82, 117-24. Wildenburg, G., A. Plenge-Bönig, A. Renz, P. Fischer, and D.W. Büttner. 1997. Distribution of mast cells and their correlation with inflammatory cells around Onchocerca gutturosa, O. tarsicola, O. ochengi, and O. flexuosa. Parasitol. Res. 83, 109-20.
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IMMUNE REGULATION AND THE SPECTRUM OF FILARIAL DISEASE
Christopher L. King Division of Geographic Medicine Case Western Reserve University
ABSTRACT Only a minority of individuals infected with lymphatic filariasis develop clinically overt disease during their lifelong residence in endemic areas. Why this occurs is poorly understood and underscores our continuing ignorance about the pathogenesis of this disease. Here the role of the host immune response in the development and modulation of inflammatory response associated acute disease such as filarial fevers, lymphangitis and lymphadenitis that may lead to chronic lymphedema in some infected subjects and not others is examined. The role of the host immune response in the development of hydrocele, the most common form of clinical filariasis, is less clear although mechanisms of inflammation and disease are likely to be similar to that observed with acute and chronic lymphedema. Postulated mechanisms of filarial antigen-specific immune hyporesponsiveness are examined. Keywords: Filariasis, immunoregulation, disease
INTRODUCTION There is a broad range of clinical manifestations associated with filarial infection discussed at length in other sections of this book. Many risks factors have been attributed to why some individuals develop disease and others not. The diversity of clinical responses to filarial infection partially reflects the intensity and type of immune response to the parasite or its products. Other factors such as duration and intensity infection, prior treatment, coinfections, genetic susceptibility and differences in parasite stains shape the nature of host immune response. But ultimately pathology, as in many chronic diseases, reflects the collateral damage generated by host inflammatory response in its efforts to rid itself of foreign material. A central theme with lymphatic filariasis, as in other chronic tissue dwelling parasitic infections, is the capacity of the host to modulate the host immune response and therefore reduce disease. Most infected individuals in endemic populations are clinically asymptomatic and immunologically hyporesponsive
128 King to filarial antigens compared to uninfected subjects. This review relates the spectrum of host immune response with disease and its regulation.
SPECTRUM FILARIASIS
OF
HOST
IMMUNE
RESPONSE
TO
Hyper-responsiveness: Hyper-responsiveness to filarial infection has been characterized by markedly increased lymphocyte proliferation responses to filarial antigens, a high frequency of filarial antigen-specific lymphocytes (King, et al. 1993), and intense production of a mixture of cytokines including Interleukin (IL)-2, IL-4 and IL-5 (Mahanty, et al. 1993). Infected subjects develop a peripheral eosinophilia and elevated levels of filarial specific antibodies, particularly IgE, often in the absence of IgG4. Such a pattern of immune responses generally occur in three circumstances; a) tropical pulmonary eosinophilia (TPE) which is a very exaggerated form of immune hyper-responsiveness, b) acute infections and c) in some individuals with chronic lymphatic disease. Tropical pulmonary eosinophilia is rare form of lymphatic filariasis. The primary organ affected is the lung rather than the lymphatics. A syndrome of eosinophilic alveolitis and marked elevations of serum IgE and eosinophils develops. These individuals have been found to be uniformly amicrofilaremic, but infected as determined by the presence of circulating filarial antigen and adult worms detected by ultrasound (Dreyer, et al. 1996). This immune hyper-responsiveness appears to be directed at the microfilarial stage of disease as they become trapped in the lung (Ottesen, et al. 1979). The reason why some individuals develop this very uncommon condition remains a mystery. A strong genetic predisposition to this illness has never been reported, although such individuals may preferentially recognize a subset of filarial antigens (Nutman, et al. 1989). Alternatively some individuals who develop TPE may initially acquire light transient infections beginning in adulthood. Upon re-infection, often many years later, they develop symptoms associated with TPE. This implies that transient infection acquired in adulthood may fail to induce immunoregulatory mechanisms. Acute infections in previously unexposed adults typically develop an immune hyper-responsive state. Expatriates, transmigrants, infected volunteers or military personnel are typical examples. Such individuals often present with acute adenolymphangitis (ADL), the absence of microfilaremia and accelerated worm death as described above. Studies of permissive animal models infected with Brugia malayi infections also show acute disease (jirds, dogs, monkeys & cats) and immune hyper-responsiveness (Klei, et al. 1990) that becomes down-modulated with chronic infection. Symptoms of infection can be more severe in nonresidents of filarial endemic regions than in long term residents of these regions. This was seen in military personnel transiently exposed in the Pacific Islands during World War II
King 129 (Warton 1947) and more recently in Indonesia transmigrantes (Portono et al. 1978). Susceptibility to acute disease and the capacity to down-modulate infection may also be age-dependent. Although children become infected early in life (Lammie, et al. 1998) and develop similar disease manifestations to those observed in adults (Gyapong, et al. 1998; Alexander, et al. 1999; Dreyer, et al. 2001) their frequency is much lower than that observed in adults. This protection from disease cannot be attributed to perinatal exposure. Prospective studies of children from birth indicate no evidence of acute disease with early infection (personal observations). This reduced frequency of acute disease also occurs in children whose mothers were uninfected with filariasis at birth thereby eliminating the likelihood of prenatal tolerance. In children < 10 years of age that have been exposed/infected with filariasis, cytokine responses to filarial antigens are robust, stimulating strong lymphocyte proliferation responses and IL-2, IL-4, IL-5 and IL-13 production of similar magnitude to that observed in exposed, but uninfected adults (personal observations). These observations suggest that children generate brisk immune responses of a mixed phenotype in response to filarial infection, but may not develop overt clinical disease as readily as adults. Therefore immune hyper-responsiveness associated with acute infection may be attenuated or does not necessary result in disease in children, but appears to do so with greater frequency in acutely infected previously unexposed adults. This development of less severe illness with acute infection in children compared to adults has been recognized with others infectious agents, particularly acute viral infections such as measles and chickenpox (Gremillion and Crawford 1981). The reasons for these differences are unclear, but may relate to the increased presence of immunoregulatory cells in children, e.g. CD5+ B cells, or CD4+, CD25+, CD62L+ suppressor T cells (Chatenoud, et al. 2001). Alternatively adults have more immunologic experience and therefore greater numbers of crossreactive clones of lymphocytes than children and that these lymphocytes expand rapidly with infection and may contribute to disease. Immune hyporesponsiveness: At the other end of the spectrum of host immune response to lymphatic filariasis is the development of impaired lymphocyte proliferation and reduced and IL-2 production to filarial antigens in vitro. This is seen in actively infected individuals (reviewed in Nutman and Kumaraswami 2001). Generally, responses to non-parasite antigens and mitogens are retained, although in some instances they are suppressed, particularly among heavily infected individuals or those living in communities subject to intense transmission of filariasis (King, et al. 2001). These responses are in marked contrast to stronger filarial antigen-specific proliferation and responses among presumably uninfected subjects (mf and circulating antigen negative individuals) with chronic lymphatic obstruction (e.g. elephantiasis), individuals with TPE, acute infections (see
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above) or parasite-exposed, but uninfected individuals (often referred to as endemic normal or putatively immune subject). Most immunological research in human lymphatic filariasis has focused on the mechanisms of this hyporesponsiveness and its association with infection and disease. The nature of immune hyporesponsiveness has been studied in some detail, particularly among patients in South India (King 2001; Nutman and Kumaraswami 2001). Although these results generally mirror immunologic studies in other filarial endemic areas (Ottesen, et al. 1977; Piessens, McGreevy, et al. 1980), generalizations are limited by two factors. First, the presence of active infection was determined by the presence of microfilaria in the blood alone. Some subjects who were classified as uninfected were actually infected, as subsequently determined by the circulating antigen assay (Nutman and Kumaraswami 2001). This obscured some differences, but does not negate their general conclusions as confirmed in subsequent studies using the circulating antigen assay (Maizels, et al. 1991; Lammie, et al. 1993; Sartono, Kruize et al. 1995b; de Almedia, et al. 1996; Dimock, et al. 1996; Freedman 1998; King, et al. 2001). Second, treatment for lymphatic filariasis using DEC is more widely available in India compared to many filarial endemic areas. Many subjects studied in India had been previously treated, which may modify the immune responses compared to previously untreated subjects, although cellular hyporesponsiveness re-established itself several years after DEC treatment in this population (Gopinath, et al. 1999). This immune hyporesponsiveness in filarial infected subjects from India corresponded to an absolute reduction in the frequency of filarialspecific T and B lymphocyte precursors (King, et al. 1993; Mahanty, et al. 1993). These studies showed that the frequency of proliferating CD3+ T cell responding to extracts of adult B. malayi was significantly lower among microfilaremic (1/3,757) compared to amicrofilaremic subjects (1/1,513). Similarly, the proportion of B cells producing parasite-specific IgG and IgE was lower in the microfilaremic group. The mechanism of hyporesponsiveness or partial anergy among infected individuals is in part related to the dominance of Th2 subset of CD4+ T cells. Using IL-4 as a marker of a Th2 type response and as an indicator of a Th 1 type response, microfilaremic subjects had significantly fewer secreting cells compared to amicrofilaremic subjects. The absolute number of IL-4 secreting cells however, was equivalent between the 2 groups. Overall, microfilaremic individuals had 8-fold more filarial-specific IL-4 relative to secreting ceils. By contrast, amicrofilaremic individuals had a predominance of relative to IL-4 secreting cells. It should be pointed out that the absolute number of filarial-specific lymphocytes, both Th 1 and Th2 type cells, were diminished in microfilaremic compared to amicrofilaremic individuals. The relative reduction in Th 1-type cells is in part due to IL-10 and/or TGF-beta, since in vitro neutralization of these cytokines partially reversed lymphocyte hyporesponsiveness among
King 131 microfilaremic subjects as measured by proliferation and cytokine production in some (King, et al. 1993; Mahanty, et al. 1997), but not all studies (Sartono, Kruize et al. 1995b). It is important to note that this increased proliferative responses in the presence of neutralizing anti-IL-10 and anti-TGF-beta among infected subjects was never restored the level of responsiveness observed in uninfected subjects. This impaired proliferation responses and and IL-2 proliferation responses appears to be primarily directed by microfilarial antigens (e.g. microfilaria and adult female worms) (Steel, et al. 1994; Mahanty, Luke et al. 1996). Microfilarial antigens likely represent the bulk of filarial antigen in chronically infected subjects, but this may not be the case in a more recently infected or lightly infected individual. To attribute this antigen-specific hyporesponsiveness among infected patients to immune deviation towards Th2 is probably an oversimplification for a number of reasons. First, impaired lymphocyte proliferation should affect both Th 1 and Th 2 type cells, although it appears that Th 1 type lymphocytes may be more affected. Second, Th1 and Th2 type cells as well as monocytes produce IL-10 and its suppressive effect on lymphocyte proliferation cannot be explained by a simple Th2/Thl dichotomy. Third, the depressed production of parasite-specific IgE is discordant with increased IL4 and IgG4 production characteristic of infected individuals. It is important to remember that other cell types such as basophils mast cells and macrophages may also contribute IL-4, IL-13 and IL-10 production (see below).
MECHANISMS OF IMMUNE HYPORESPONSIVENESS Perinatal Influence: The mechanisms associated with partial anergy observed in filarial infected patients can be considered in the context of “central tolerance” verses “peripheral tolerance”. Central tolerance relates to the selective depletion or anergy that may develop as a consequence of in utero exposure to filarial antigens. Such a mechanism may be important in communities with high prevalence of lymphatic filariasis among pregnant mothers. Considerable indirect evidence supports a critical role for T cell hyporesponsive in children of mothers known to be microfilaremic (Steel, et al. 1994) or likely to be microfilaremic during pregnancy (Lammie, et al. 1991). Some of these studies also showed children born to mothers microfilaremic during pregnancy were more likely to be infected later in childhood compared to children born of amicrofilaremic mothers in the same villages or hamlets. These observations were interpreted to mean that “central tolerance” impairs the host immune response to potentially protective antigens, thereby increasing their risk for infection. Other studies, however, suggest that the increased risk for infection in children born to infected mothers can be better explained by differences in exposure rather than a maternal effect on the fetus (Das, et al. 1997; Alexander, et al. 1998).
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Whether the development of immune hyporesponsiveness observed in children of filarial infected mothers protect them from subsequent disease has never been directly demonstrated. Acquired or peripheral tolerance: Peripheral “tolerance” may be considered as the effect of persistent or intermittent release of high amounts of antigen by the parasite on the host immune response, the direct of effect of parasite itself or by parasite-derived molecules that may mimic or block host cytokines or receptors. Peripheral tolerance is likely to be only partial and can be reversed with elimination of the parasite infection after chemotherapy. This peripheral tolerance may take many years to develop beginning with initial infections with bancroftian filariasis during childhood. As infection load increases with cumulative exposure, we and others have observed that antigen-specific immune responses to microfilarial antigens diminish (Sartono, et al. 1997). In addition, high levels of antigenemia and microfilaremia have been shown to lead to down-regulation of levels of all parasite-specific immunoglobulin isotypes (Marley, et al. 1995) and lymphocyte proliferative responses. Similar observations have been made from jirds infected with B. malayi that showed a negative correlation between antigen-specific proliferation and microfilarial burden (Bosshardt, et al. 1995) while in humans, changes in antigen–driven IFN-• (and not IL-4) production were associated with seasonal fluctuations in microfilarial density (Sartono, et al. 1999). Reduction in parasite burden, by DEC treatment which effectively clears the microfilaria and kills some adult worms, produces a partial reversal of the impaired host lymphocyte responsiveness to filarial antigens (Lammie, et al. 1988; Sartono, Kruize et al. 1995a). Long term follow-up after treatment has shown that some individuals revert to their prior levels of immune hyporesponsiveness even though microfilaremia fails to reoccur. However, some of these individuals became positive for circulating antigen (Gopinath et al. 1999). It should be pointed out, however, that this inverse relationship between intensity of infection and magnitude of host immune hyporesponsiveness is not absolute. There are infected adults with undetectable peripheral blood microfilaremia who have immunological profiles similar to individuals with microfilaremia. This suggests that the relationship between the duration and intensity of infection and down-regulating host immune responsiveness is complex and other mechanisms are involved such as exposure to infective stage larval antigens, or the presence of immunoregulatory cells (Pieces, Ratiwaytano, et al. 1980; Piessenset, et al. 1982; Osborne and Devaney 1998; King, Connelly et al. 2001). The relationship between host immune responsiveness and duration and intensity of infection with clinically overt disease is less clear. In many populations (Estambale et al. 1994; Albuquerque et al. 1995; Kazura et al. 1997) the frequency of clinical disease increases with age, even though host immune responsiveness declines. At an individual level, subjects with
King 133 chronic lymphedema have the most robust host immune responsiveness compared to infected asymptomatic individuals (King and Nutman 1991). However these studies were generally conducted in populations referred to clinics and that have been frequently treated with DEC. Generally reduced host immune responsiveness is more closely associated with infection rather than the presence or absence of disease (de Almedia et al. 1996; de Almeidaet al. 1998; Freedman 1998), although this relationship has not been examined in populations without prior treatment. One interpretation of these data is that active infection suppresses the host immune responsiveness, which slows progression to clinical disease. To test this hypothesis would require longterm prospective studies of infected subjects without administering treatment, which would raise serious ethical questions. Effect of chronic infection on antigen presenting cell (APC) function. Filarial antigens, particularly microfilarial antigens, can directly suppress the ability of dendritic cells (DC) to generate IL-12 and present antigen in an allogenic mixed lymphocyte reaction (Semnani et al. 2001). Monocytes isolated from microfilaremic individuals also produce large amounts IL-10 spontaneously ex vivo (Mahanty, Mollis et al. 1996) and PBMC and monocytes from these individuals express reduced amounts of CD80 (Ravichandran et al. 1997; Nutman and Kumaraswami 2001), an important costimulatory molecule for T cell activation. Filarial infected individuals exposed to intense transmission in Papua New Guinea have markedly impaired lymphocyte proliferation and production in responses to APC-dependent compared to APC-independent T cell mitogens (King et al. 2001). These observations indicate defective APC function in chronically infected individuals, and suggest that larval as well and microfilarial antigens suppress host immune capacity. It has been shown that addition of autologous DC matured with IL-4 and GMCSF from PBMC taken from uninfected individuals can partially reverse the immune hyporesponsive observed with chronic human schistosomiasis (van Den Biggelaaret al. 2000). Although parallel experiments have not been done in human filariasis, shared mechanisms of immune hyporesponsiveness likely exist for these chronic tissue dwelling helminth infections (Yazdanbakhsh 1999). Filarial infection may also preferentially activate a subset immunoregulatory APC (Allen and Loke 2001). Specific filarial antigens such as homologues to human cystatins, which are protein inhibitors of cysteine proteases, have been shown to directly block antigen-presentation by the MHC class II pathway (Manoury et al. 2001). Filarial-derived products can directly suppress lymphocyte function: Filarial-derived products that contain the hapten phosphorylcholine may directly act as an inhibitor to T and B cell reactivity (Lal et al. 1990; Harnett et al. 1999). The most well studied molecule of this type is the excretory-secretory molecule ES-62 produced of the filarial nematode
134 King Acanthocheilonema viteae. A homologue exists for the human parasite Brugia malayi (Harnettet al. 1999). This molecule has been shown to have remarkable immunoregulatory properties. It can selectively desensitize T and B cells by uncoupling of the antigen receptors from key intracellular proliferative signaling pathways (Deehan, et al. 2001). It can modify APC function by suppressing the pro-inflammatory cytokines IL-12, and IL-6 release and favor maturation of dendritic cells that promote Th2 type T cell differentiation (Whelan et al. 2000; Goodridge, et al. 2001). Filarial parasites, like some viruses (Tortorellaet, et al. 2000), may also subvert the host immune response by producing molecules that are homologous to host immunoregulatory cytokines. Two TGF-beta homologues has been identified in B. malayi and one has been shown to be secreted by adult parasites in vitro and able to bind to host TGF-beta receptors (Maizels, et al. 2001). TGF-beta is potent inhibitor of lymphocyte proliferation. Likewise, B. malayi expresses a homologue of mammalian macrophage inhibitory factor (MIF), that has potent immunoregulatory functions in humans (Petrovsky and Bucala 2000) and is similar in both structure and function to the host protein. The parasite protein has a direct effect on human macrophages (Falcone, et al. 2001). As more of the filarial genome becomes sequenced, additional genes with homologies to human immunoregulatory molecules are likely to be identified. Regulatory cells: Antigen-specific suppressor cells were first postulated to account for the specific immune hyporesponsiveness (Ottesen, et al. 1977) based on observations that partial depletion of potentially inhibitory cells populations enhanced lymphocyte proliferative responses to filarial antigens in patients with brugian filariasis (Piessens, Ratiwaytano, et al. 1980; Piessens, Partono et al. 1982). Similar studies in bancroftian filariasis failed to reverse the suppressed lymphocyte proliferative responses (Nutman, et al. 1987a; Nutman et al. 1987b). This suggests that regulatory cells may differ between the two species of human lymphatic filariasis. Subsequent to these early studies, our knowledge about the regulatory cells has greatly expanded to suggest they represent relatively rare cells strategically located in host lymphocyte organs (Groux, 2001). The cytokines IL-10 and TGF-beta are major regulators of T cells responses in humans and as mentioned earlier in this review participate in the down-modulation filarial antigen-driven lymphocyte proliferation and production (King, et al. 1993; Mahanty, Mollis, et al. 1996; Ravichandran, et al. 1997). Recently a population of regulatory antigen-specific T (Trl/Th3) cells has been identified. These cells are defined by their ability to produce high levels of IL-10 and TGF-beta and little IL-2 (Roncarolo, et al. 2001), may play a key role in the maintenance of peripheral tolerance. These cells are relatively rare and because of their poor ability to proliferate, are difficult to isolate. Such cells have been found to participate in modulating T cell responses in human onchocerciasis and
King 135 intestinal helminth infections (Doetze, et al. 2000), Whether such cells exist or other regulatory T cells participate in modulating human lymphatic filariasis remains to be determined. Effect of infective stage larvae on Th2 bias and immune suppression: Most studies have appropriately focused on microfilarial antigens, the most abundant antigens in chronically infected individuals, in regulating host immune responses. Recent studies however indicate that infective stage larvae may participate in immune modulation. Subcutaneous infection of mice with infective stage L3 larvae of Brugia pahangi suppresses of lymphocyte proliferation and production by splenocytes to mitogens and parasite antigens within 12 days, yet a strong IL-4 response is initiated within 24 h of infection and retained over the following 1-2 weeks (Osborne et al. 1996). This corresponds to a time when infective larvae have migrated to the lymphatics and begin to undergo their molt to L4. This active suppression has been shown to involve IL-10 and resident APC (Osborne and Devaney 1999). In a separate set of experiments, APC obtained from mouse peritoneum after inoculation of L3 markedly suppress various T cell lines in vitro (MacDonald et al. 1998). This suppression is dependent on IL-4, although IL-4 itself is not directly suppressive, and is not dependent on IL-10. Although these studies differ somewhat in proposed mechanisms of L3mediated immunosuppression they suggest that frequent exposure of mammalian host to developing larvae may play a critical role in Th2 bias and the development of immune hyporesponsiveness. This is not surprising since L3 stage parasites must also evade immune defenses on penetration of the host. The murine studies appear to mirror a similar induction of partial immune hyporesponsiveness and Th2 bias in human filariasis. Residents of a high transmission village had 4- to 11-fold lower proliferation and responses to filarial Ags compared to residents of a village more than 60-fold lower exposure to L3 even when subjects were matched for the intensity of infection (King et al. 2001). This immune hyporesponsive extended to immune responses to nonfilarial antigens and APC-dependent, but not T cell mitogens (King et al. 2001). These results suggest that the incoming and developing larvae non-specifically depress of lymphocyte proliferation and production, analogous to that observed in murine models. Intense exposure to L3 was also associated with increased IL-4 (particularly in the plasma) and IL-5 production, but not IL-10 or TGF-beta (King et al. 2001). This suggests that developing larvae may suppress host immune responses differently from that observed with chronic infection. This immune suppression may be directly mediated by a cysteine protease inhibitor that has been to shown in other filarial parasites to suppression lymphocyte proliferation and enhance IL-10 release (Hartmann, et al. 1997). Recently, a homologue to this molecule has been identified in the human filarial parasite, Brugia malayi. It inhibits MHC class II-restricted antigen processing and is one of the most abundant transcripts produced by L3 larvae (Manoury, et
136 King al. 2001). Its production may participate in maintaining host immune hyporesponsiveness in these heavily exposed individuals and avoid its immune destruction within the host.
Basophils and mast cells and larval antigens: The high level of IL-4 in plasma among individuals filarial infected individuals, especially those with intense exposure to L3 larvae (King et al. 2001), the consistent production of this cytokine in most infected individuals irrespective of disease or age, and discordant production with respect to other Th2-type cytokines like IL-5 (Sartono, et al. 1997) suggests that a major source of this cytokine may be cells other than filarial specific T cells. Mast cells and basophils that are plentiful in dermal tissues where L3 are inoculated may be a major source of IL-4 release. Persons exposed repeatedly to large numbers of L3 and preadult W. bancrofti may thus experience sustained increases in IL-4 production by activation and degranulation of cells located in the dermis, particularly mast cells bearing cytophilic filarial-specific IgE bound to (Fig. 1).
We have observed that L3 preferentially stimulate IL-4 and IL-13 release from basophils as well as histamine release (King 2001). In addition, basophils comprise approximately 1% of cells in PBMC and their contribution to the observed cytokine production can be substantial. Therefore mast cells and basophils may play an important role in regulating the host response to filarial infection by affecting T cell differentiation, local blood flow, lymphocyte proliferation or by release of histamine or other
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prostanoids (Hofstetter, et al. 1983; Gauchat, et al. 1993; Devouassoux, et al. 1999; Schroeder, et al. 1999; Matsuoka, et al. 2000).
THE RELATIONSHIP OF INFECTION, HOST IMMUNE RESPONSE AND DISEASE An overview of the interaction of host immune response, infection and disease is shown in Figure 2. Initial infection with L3 and subsequent molt to L4 contribute to a Th2 bias and help to shape of the overall immune response to infection as well as to contribute to filarial-specific immune suppression. As larvae mature into adult worms and produce microfilariae that enter peripheral circulation, down modulation of the host innate and acquired immune response extends throughout the remaining lymphoid organs that experience exposure to circulating microfilariae. Production of IL-4 is retained even as IL-5 and IL-10 release decline in some chronically infected patients may be at increased risk for clinical overt disease. Where host and parasite are equivalent and a constant rate of worm death is present three major factors determine the risk for clinically overt disease. These are the worm burden, the duration of infection and the degree of T cell reactivity to filarial antigens as defined by lymphocyte proliferation and production and likely other unidentified factors. Ideally a strong Th2-type cytokine response would result in increased levels of protective filarial-specific IgE, particularly directed at the infective stage larvae, that reduces worm burdens and strongly modulates host inflammatory responses to living and most importantly dying worms. Even under natural conditions, in areas of intense transmission and in individuals who never receive treatment (as in Papua New Guinea), this immunoregulatory system is likely
138 King to be very leaky and people may progress to clinical disease. Treatment, coinfections or changes in exposure may alter this balance and lead to accelerated disease progression. A comparison of immune responses before and after treatment over the course of several years may provide further insights into the mechanisms of immune regulation.
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WOLBACHIA BACTERIAL ENDOSYMBIONTS Mark J. Taylor Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK e-mail:
[email protected], Phone: 44 151 708 9393 Fax: 44 151 708 9007
ABSTRACT Wolbachia intracellular bacteria appear to have evolved as essential symbionts of their filarial nematode hosts. Antibiotic depletion of bacteria shows that they are required for normal fertility and development of the worm. The bacteria are transmitted maternally via the egg and are necessary for normal embryogenesis. In addition to their role in the biology of filarial nematodes, Wolbachia also contributes to the inflammatory pathogenesis of filarial disease. This together with the possible role of endosymbionts in the long-term survival of nematodes has been exploited by using antibiotic therapy as a new approach to the treatment of filariasis. Keywords: Filariasis, Wolbachia, bacteria, symbiosis, pathogenesis, therapy.
INTRODUCTION The fact that filarial parasites consist of two organisms, a nematode and symbiotic bacteria, has until recently been a well-kept secret in filarial research. The discovery that intracellular bacteria inhabit the hypodermis and female reproductive organs of filarial nematodes came from the use of electron microscopy to study the ultrastructure of these parasites (McLaren et al. 1975, Vincent et al. 1975, Kosek 1977, Kosek and Figueroa 1977). It was another 20 years before these organisms were identified as belonging to the Wolbachia complex, a group of intracellular bacterial symbionts of arthropods (Sironi et al. 1995). Over the past five years research on these bacteria has increased dramatically and uncovered important roles for these symbionts in the biology of filarial nematodes (Taylor and Hoerauf 1999, Taylor 2000). Here I will summarize our current knowledge of this symbiosis and consider Wolbachia as a novel target for control and in the pathogenesis of filarial disease.
DISTRIBUTION, TRANSMISSION AND PHYLOGENY We now know that almost all filarial nematodes are infected with Wolbachia, including the major pathogenic species of humans and animals (Bandi et al. 1998, Taylor and Hoerauf 1999, unpublished observations). Each species of nematode is infected with its own ‘strain’ of symbiont throughout their geographical distribution. Studies on large numbers of individual adults of Brugia malayi and Onchocerca ochengi have failed to detect any uninfected worms (Bandi et al 1998, Taylor et al. 1999, Taylor et al. 2000a,
144 Taylor unpublished observation). All developmental stages of the nematode are infected with bacteria, although the intensity of infection appears to vary throughout the parasite’s life cycle and is lower in male worms than females (Taylor et al. 2000a). The bacteria are confined to the hypodermal tissues of male and female worms and the reproductive organs of female worms (Figure 1). Organisms are located within host derived vacuoles and can occupy large areas of the hypodermal cords. Bacteria are also present in large numbers throughout the female reproductive tissues and intrauterine developmental stages. This, together with crossing experiments between Brugia species, shows that transmission occurs vertically via the eggs of female worms A) Wolbachia bacteria in the lateral cord of Brugia malayi, stained red using the APAAP method with antisera raised to Wolbachia surface protein (WSP) (Photograph, D. Buttner, A. Hoerauf, BNI Hamburg) B) Wolbachia distributed throughout the median and lateral cords of Onchocerca volvulus. Bacteria can be seen as numerous black particles scattered throughout the cord cells. Stained using the Warthin-Starry method (Photograph, B.O. Duke and the American Registry of Pathology)
(Kosek 1977, Taylor et al. 1999).
Wolbachia are related to other rickettsial bacteria including Anaplasma, Cowdria and Ehrlichia, which are responsible for a number of arthropod transmitted diseases in humans and animals (Raoult & Roux 1997). Molecular phylogeny of filarial Wolbachia has been studied extensively by Bandi and colleagues using comparisons of DNA sequence from 16S rDNA, ftsZ, and Wolbachia surface protein (wsp) derived from symbionts of several species of filarial nematode (Bandi et al. 1998, Bazzocchi et al. 2000, Casiraghi et al 2001). These studies have identified two clades of filarial Wolbachia (C including symbionts from Onchocerca and
Taylor 145 Dirofilaria species; and D, symbionts of lymphatic filariae and Litomosoides), which are most closely related to the two clades of Wolbachia (A and B) previously described in arthropods. In contrast to arthropod Wolbachia, which can exhibit extensive horizontal transmission between hosts, filarial Wolbachia phylogeny corresponds to that of their nematode host suggesting a stable and long-term association (Casiraghi et al 2001). This evolutionary pattern is also consistent with an obligate requirement for symbionts in filarial nematodes, as opposed to the facultative associations of arthropod Wolbachia, a feature which is also reflected in the relative size of Wolbachia genomes (arthropod 1.36-1.66 Mb, filarial 0.95-1.1 Mb) (Sun et al. 2001). Phylogenetic studies have so far failed to resolve the question concerning the absence of Wolbachia from the rodent filarial nematode Acanthocheilonema viteae (Casiraghi et al 2001). Analyses are ambiguous in determining whether A. viteae have ‘lost’ their symbionts, or diverged from other filarial species prior to the acquisition of Wolbachia (or a variety of other possibilities, see Casiraghi et al 2001). The absence of Wolbachia from this species has so far only been demonstrated in a laboratory-maintained isolate (McLaren et al 1975, Bandi et al 1998, Taylor & Hoerauf 1999). Studies on field isolates of A. viteae will be needed to determine if the absence of Wolbachia occurs in the wild. The absence of bacteria from Onchocerca flexuosa, a parasite of deer, has been shown in wild isolates using microscopy and nested PCR (Plenge-Bonig et al 1995, Brattig et al. 2001, unpublished observation). The presence of Wolbachia in the 10 other species of Onchocerca so far studied would suggest that O. flexuosa has lost its symbiont. While these observations show that life as a filarial nematode is possible without Wolbachia symbiosis, these species may hold important clues in understanding how Wolbachia contributes to filarial biology. One striking feature of O. flexuosa, which sets it apart from all other symbiontcontaining filariae, is its short life span (Klager 1989). Adult worms live for only ~18 months compared to the 10 years or more estimated for symbiont containing Onchocerca species (Karam, et al. 1987). As a parasite of rodents A. viteae need only survive for the 1 or 2 year life span of its host, and so has no requirement to live as long as the Wolbachia infected filariae of ungulates and humans. Although these observations are limited to only two species, it does support the idea that Wolbachia may be important for the long-term survival of filarial nematodes in mammalian hosts. Further evidence for this comes from studies using antibiotics to eliminate symbionts from their hosts.
ANTIBIOTIC ELIMINATION OF WOLBACHIA Antibiotics have been used against a variety of filarial species to determine if Wolbachia elimination can affect filarial nematodes (Taylor et al. 2000b, Taylor 2000). As antibiotic therapy appears to have no direct effect on nematodes, as suggested by the absence of any influence on A. viteae infection (Hoerauf et al. 1999, McCall, et al. 1999), these studies are useful in
Taylor 146 identifying which features of filarial biology are influenced by Wolbachia. Antibiotics shown to be active against Wolbachia are restricted to the antirickettsial antibiotics tetracycline and rifampicin, with other antibiotics having no or marginal effects (Taylor 2000, Hoerauf et al. 2000, Townson et al. 2000). Long periods of treatment are required to deplete bacteria, but once bacterial numbers are reduced a number of profound effects on worm development, fertility and viability occur (Hoerauf et al 1999, Taylor 2000, Langworthy et al. 2000, Townson et al 2000). The initial effect of tetracycline therapy on adult female worms appears to be an inhibition of the transmission of bacteria to eggs, and their depletion from other developmental stages in the uterus (Bandi et al. 1999). This leads to profound embryotoxicity and reduced fertility (Bandi et al 1999, Hoerauf et al. 1999, Townson et al. 2000). If treatment is given during the growth of infective larvae to adults, reduced numbers of adult worms, which are infertile and stunted develop (Hoerauf et al. 1999). These studies suggest that Wolbachia are important for the normal development of embryos and larvae. The ability of tetracycline to inhibit the molting of infective third-stage larvae in vitro may partly explain the effect on prepatent development (Smith & Rajan 2000, Rao et al. personal com). Whether this reflects an inhibition in development preventing molting or a direct effect on the molting process requires further studies and these observations should be extended to determine the effect of antibiotics on the molting of other developmental stages and on species without Wolbachia, such as A. viteae. Although the effects on embryogenesis and fertility occur soon after bacterial depletion, the effects on parasite viability take much longer to appear. A study using intermittent oxytetracycline dosing of cattle infected with O. ochengi for 6 months resulted in a dramatic killing of adult worms 9 months post treatment (Langworthy et al. 2000). This study is strong evidence that Wolbachia are essential for the survival of the nematode in its natural host and show that antibiotic therapy of Wolbachia can have adulticidal activity. Depletion of bacteria and abnormal embryogenesis occurred 2-3 months after the start of treatment showing that the loss of bacteria from the hypodermis and reproductive organs has little immediate effect on worm viability (other than embryonic stages). This would imply that Wolbachia are not necessary for any critical physiological processes of adult worms (unless redundant compensatory pathways or use of stored reserves occurs). Instead the bacteria may make an important contribution to the long-term survival of filarial nematodes, such as evasion or modulation of host immunity. Filarial Wolbachia have been shown to contain a catalase enzyme, which may serve to protect both bacteria and nematode from oxidative damage (Henkle et al 1998). The loss of this protective mechanism following antibiotic depletion of Wolbachia may then leave the nematode susceptible to host immunity resulting in its eventual death. Worms recovered from antibiotic treated animals are often encapsulated in granulomatous inflammatory cells (Hoerauf
Taylor 147 et al. 1999, Townson et al. 2000). Overall, the use of antibiotics to deplete Wolbachia has a number of significant consequences on filarial nematode biology supporting the view that these bacteria are an essential symbiont of filarial nematodes. They have also led to the use of antibiotic therapy in a novel approach to the control of human filariasis.
WOLBACHIA AS A TARGET FOR CONTROL The development of long-term sterility and effects on parasite development and viability following antibiotic therapy make Wolbachia an attractive target for a novel approach to the control of filariasis (Taylor et al. 2000, Taylor 2000). The availability of antibiotics in endemic areas, widely used to treat other bacterial diseases, has led to the opportunity to test the efficacy of these drugs in clinical trials on human filariasis. The first of these trials on onchocerciasis used a 6 week course of doxycycline (Hoerauf et al 2000). This was shown to be effective in depleting bacteria from adult worms resulting in profound and extensive embryotoxicity at four months after the end of the treatment. Follow up studies in these patients following ivermectin treatment at 2.5 or 6 months after treatment with or without doxycycline, showed that antibiotic depletion of bacteria and sterility was sustained 18 months after the start of antibiotic treatment. This resulted in extended reductions in skin microfilarial load in patients treated with doxycycline, in contrast to those receiving ivermectin alone (Hoerauf et al. 2001). These studies confirm that Wolbachia is a target for antibiotic therapy in human filariasis and that depletion of the bacteria appears to be long-term and with dramatic consequences on worm fertility. A question remains as to whether or not the bacteria are completely eliminated, as worms derived from antibiotic treated individuals are still positive (although reduced more than 10 fold) using PCR detection of Wolbachia 16S rDNA (Hoerauf et al 2000). If these results are derived from residual viable bacteria, then they appear to be unable to repopulate worm tissues more than a year and a half after antibiotic depletion (Hoerauf et al. 2001). Although this treatment regime is clearly inappropriate as a community mass treatment strategy, the development of long-term sterility would have major advantages in the interruption of transmission, potentially reducing the period for sustained community mass treatment strategies currently used (for debate see Richards et al. 2000 and Ambiose et al. 2000). Trials which aim to identify the period of antibiotic therapy that is sufficient to deplete Wolbachia and to determine the possibility of long-term effects on adult worm viability are currently underway on onchocerciasis and lymphatic filariasis, using different regimes and antibiotics.
PATHOGENESIS AND IMMUNE RESPONSE Another important area in which filarial Wolbachia play a major role is in the inflammatory pathogenesis of disease. Filarial disease is associated
148 Taylor with episodes of acute and chronic inflammation leading to pathologies including hydrocele, lymphedema and elephantiasis in lymphatic filariasis, and skin disease and blindness in onchocerciasis. Studies aimed at characterizing the nature of inflammatory stimuli derived from the human filarial parasite B. malayi showed that lipopolysaccharide (LPS)-like molecules from Wolbachia are responsible for the potent inflammatory activity of parasite extracts (Taylor et al 2000a). Soluble extracts of parasites induced tumor necrosis factor and nitric oxide (NO) responses from macrophages. The active component of the extract reacted positively in the Limulus Amebocyte Lysate (LAL) endotoxin test, was resistant to heat, and could be inhibited by incubation with Polymyxin B. Further studies showed that inflammatory responses of macrophages required CD 14 and Toll-like receptor 4 (TLR4), two innate pattern recognition receptors of bacterial endotoxin. Evidence to show that these LPS-like molecules were derived from Wolbachia included experiments with A. viteae (which is free of Wolbachia endosymbionts), and Wolbachia derived from a mosquito cell-line. Soluble extracts of A. viteae failed to stimulate any inflammatory responses from macrophages and were negative in the LAL endotoxin test whereas extracts derived from the Wolbachia infected cell-line did induce LPS-like responses, which were lost following antibiotic treatment (Taylor et al. 2000a). Studies by Brattig and co-workers on O. volvulus have also shown that LPS-like molecules derived from Wolbachia are responsible for inducing and IL-10 from human monocytes (Brattig et al. 2000). Analysis of nematode extracts using the LAL endotoxin assay showed positive reactions with extracts of Onchocerca parasites, but were negative using extracts of the Wolbachia-free A. viteae. LPS-like responses were inhibited with antibodies to CD 14 and with a lipid A antagonist. These studies also showed the LPSlike stimulation of induced IL-10 responses leading to the down regulation of HLA and co-stimulatory molecule expression. This suggests Wolbachia LPS-like molecules induce both inflammatory and antiinflammatory responses from human monocytes, which could affect antigen presentation and modulate lymphocyte responses. Together, these studies suggest that following death of parasites, the liberation of LPS-like molecules can induce potent activation of innate inflammatory responses. One situation in which the death of large numbers of parasites can lead to inflammation is in the adverse reaction to anti-filarial chemotherapy. In animal models, it has been shown that injection of filarial extracts into dogs can result in severe shock-like systemic responses (Kitoh et al. 1998). Furthermore, if rodents are sensitized to the hepatotoxicity of TNF a using D-galactosamine, lethal shock-like responses occur following drug treatment of filarial infections (Zahner 1995). To investigate the contribution of Wolbachia LPS-like molecules to inflammatory responses post-treatment, we infected LPS responsive (C3H/HeN) and non-responsive (C3H/HeJ) mice
Taylor 149 with B. malayi microfilariae and treated animals with the anti-filarial drug ivermectin. responses were only observed in the LPS-responsive mice post treatment (Taylor et al. 2000a). To determine if similar mechanisms account for the adverse reaction to filarial chemotherapy in humans, we analyzed plasma samples from individuals infected with B. malayi taken before and after treatment with the anti-filarial drug diethylcarbamazine (DEC). Adverse reactions to treatment included fever, headache, dizziness, myalgia, arthralgia and lymph-node enlargement, which were scored to categorize reactions into none/mild, moderate and severe. Severe systemic inflammatory reactions were shown to be associated with high levels of microfilariaemia pre-treatment and an increase in IL-6, IL-10, lipopolysaccharide-binding protein (LBP) and soluble tumor necrosis factor receptors post-treatment, with IL-6 and LBP most significantly associated with severe reactions (Haarbrink et al. 2000). Wolbachia specific PCR and immunogold electron microscopy analysis of the plasma samples showed that in all individuals with severe reactions, bacteria could be detected in the blood from 4h post-treatment and persisted for 8-20h, coinciding with the onset of systemic inflammatory responses (Cross et al., 2001). As well as showing that Wolbachia are strongly implicated in the systemic inflammatory responses of adverse drug reactions, they show that on death of the nematode, bacterial cells are released into the hosts tissues, exposing Wolbachia to the innate and acquired immune system. A recent study by Brattig et al (2001) has shown that Wolbachia are also responsible for the recruitment of neutrophils into nodules and adjacent to living adult O. volvulus. Neutrophils disappear from the nodules of O. volvulus depleted of bacteria by doxycycline treatment. Neutrophils are also absent from the nodules of O. flexuosa, a species without Wolbachia, but occur in the nodules of endosymbiont infected O. jakutensis in the same red deer host. Extracts of O. volvulus induced neutrophil chemotaxis and activated monocytes/macrophages to produce and IL-8, cytokines which can activate and recruit neutrophils. These responses were dramatically reduced or absent when extracts from O. volvulus depleted of bacteria by doxycycline, or A. viteae were used. This study suggests that Wolbachia and/or their products are released by living worms, which directly or indirectly activate innate inflammatory responses. Neutrophil-mediated inflammation also occurs in response to parasite extracts in corneal inflammatory pathology in a mouse model of onchocerciasis. The development of neutrophil-mediated inflammation is only induced by parasite extracts derived from Wolbachia infected species (O. volvulus and B. malayi) and not from those which are free of endosymbionts (A. viteae) or from extracts derived from O. volvulus depleted of Wolbachia using antibiotics. Inflammatory pathology is also dependent on TLR4, suggesting that Wolbachia LPS-like molecules induce neutrophil-mediated corneal inflammation (Saint André et al, submitted manuscript).
150
Taylor Overall these studies show that endotoxin-like activity of Wolbachia is responsible for the activation of innate inflammatory responses and bacteria and/or their products are associated with acute and chronic inflammatory responses in human filariasis. They also show that endosymbionts are exposed to the host immune system and so may contribute to acquired immune responses in filariasis. A number of Wolbachia antigens have been shown to induce antibody responses in animal models and humans (Taylor et al. 2001). In particular, studies have investigated antibody responses to a major surface protein of Wolbachia (WSP). Antibodies to WSP are produced in cats experimentally infected with Dirofilaria immitis and in jirds infected with B. malayi (Bazzocchi et al 2000, Tzertzinis et al. submitted manuscript). However, in rhesus monkeys infected with B. malayi, antibody responses were detected in only two out of twelve infected animals (Punkosdy et al. 2001). Interestingly these monkeys were among three animals that developed lymphedema. Strikingly, WSP antibody responses only occurred just prior to and throughout episodes of lymphedema. Although further studies are needed, these observations suggest exposure to Wolbachia may be associated with the onset of chronic lymphatic pathology. Taken together these studies highlight the contribution of Wolbachia to the inflammatory reactions caused by filarial nematodes. This provides a new insight into the pathogenesis of filarial disease that should be integrated into our current understanding of the pathological processes that underlie disease progression (Dreyer and Piessens 2000). In lymphatic filariasis, the death of worms and the subsequent release of Wolbachia will result in acute inflammation associated with acute filarial lymphangitis (AFL). The cumulative effect of multiple episodes of AFL may lead to lymphatic damage and desensitization of innate immune responses, both of which would promote the establishment of opportunistic infections associated with acute dermatolymphangioadenitis (ADLA) in elephantiasis (for further discussion see Taylor et al. 2001). In onchocerciasis, inflammatory responses induced by Wolbachia from dead microfilaria may contribute to skin pathology and inflammation in the eye leading eventually to blindness. If Wolbachia are the major cause of filarial pathology then antibiotic therapy, in addition to any anti-nematode activity, may well be useful in preventing the onset or development of filarial pathology.
CONCLUSION In the space of a few years the Wolbachia endosymbionts of filarial nematodes have risen from obscurity to providing new insights into filarial biology, disease pathogenesis and treatment. Yet we know next to nothing of the nature of this symbiosis and the interaction between bacteria and nematode. At the time of writing, DNA from Wolbachia of B. malayi and O. volvulus is being prepared for genome sequencing, which will undoubtedly
Taylor 151 provide new and fascinating insights into this association. This together with the filarial genome project (Williams et al. 2000) and genome sequencing projects for arthropod Wolbachia (Bandi et al. 1999) will provide important tools to dissect the molecular cellular microbiology of Wolbachia. Understanding the role of this symbiosis in the biology of filarial nematodes will, however, still rely on the type of fundamental parasitological research which led to their discovery and current status as a potential target for the control of filariasis.
ACKNOWLEDGEMENTS I would like to thank all of the people that have generously provided access to unpublished information and images for use in this chapter: N. Brattig, D. Büttner, J. Foster, A. Hoerauf, S. Klager, P. Lammie, E. Pearlman, G. Punkosdy, U.R. Rao, A. v. Saint André, G. Tzertzinis and colleagues at the Liverpool School of Tropical Medicine. I thank the Wellcome Trust for Fellowship support. Brian Duke and the American Registry of Pathology slide series on "Filarial Diseases" for the image of O. volvulus Wolbachia.
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APPROACHES TO THE CONTROL AND ELIMINATION OF THE CLINICALLY IMPORTANT FILARIAL DISEASES C. D. Mackenzie, M. Malecela, Mueller I. and M.A. Homeida. Filarial Diseases Unit, Michigan State University, East Lansing and Biological Imaging Center, Western Michigan University, Kalamazoo USA; National Institute for Medical Research, Ocean Road, Dar Es Salaam, Tanzania; HealthNet International, Nairobi, Kenya; The Academy for Medical Sciences and Technology, Khartoum, Sudan.
ABSTRACT Mass drug administration programs are the mainstay of current attempts to control and rid the globe of the two major filarial diseases of humans, onchocerciasis and lymphatic filariasis. There are a number of important components to developing and maintaining a treatment program; these include advocacy, communication, financial support, and the presence of local "champions" to lead the program. Two active country programs - lymphatic filariasis elimination in Tanzania and onchocerciasis control in Sudan - provide practical examples of these program needs. There are a number of positive consequences of a successful program that extend outside the primary goal of disease control or elimination; these include national infrastructure and personnel development. The challenges and consequent actions that could be taken to ensure the success of control programs are discussed. Keywords: Filariasis, onchocerciasis, mass drug administration, Mectizan®, albendazole, diethylcarbamazine, control, elimination.
BACKGROUND Mass drug administration (MDA) of anthelminthics is the current key to combating two major human filarial diseases, lymphatic filariasis and onchocerciasis (Bebehani, 1998, Cox 2000). In the lymphatic filariasis program, more than 1.2 billion people are targeted to be treated with chemotherapy within the next ten years, and more than 30 million people are expected to be treated for onchocerciasis within the next fifteen or so years. There are approximately 80 million people at risk of onchocerciasis in the world. These efforts represent a massive logistic and practical challenge that requires the development of new approaches to health care distribution and is catalyzing an unprecedented interest in these important, debilitating diseases. The immunology of filarial diseases is thought to be extremely complicated, and these parasites flout the host by living in the very immune
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system we would wish to utilize: lymph nodes, blood and lymphatic vessels. Although inroads have been made into the understanding of the immunological basis of the clinical spectra of both onchocerciasis and lymphatic filariasis, the development of agents such as vaccines for use in treatment, control or elimination programs has not yet been achieved. Thus chemotherapeutic approaches are used. The onchocercal disease control program (ODCP) was first based on the goal of eliminating blindness in affected communities; in contrast, the lymphatic filariasis elimination program (LFEP) is from the onset aimed at the complete eradication of the infection from the endemic areas (Maher and Ottesen 2000). These two different approaches carry with them differences in implementation, and in many associated managerial matters such as the agencies providing fiscal support and the forms of scientific evaluation used. It is important to constantly reevaluate the progress and goals of these programs as they proceed. This is especially important in Africa where in many areas there is an overlap of the two programs due to co-edemicity for these two filariae. The search for the optimal treatment regime for these two diseases has been long and often frustrating. There is still no obviously effective and safe adulticidal anthelmintic for Onchocerca volvulus, and the data remains unclear as far as adulticidal efficacy of anthelminthics currently used against Wuchereria bancrofti. Four drugs predominate in the filarial literature diethylcarbamazine (still used for LFEPs), suramin (an adultacide now not used), ivermectin (an important veterinary drug, central to today’s ODCPs and LFEPs), and albendazole (a well known anthelminthic used for many years against intestinal helminthoses). After dismissal of suramin as an unsafe drug, and after many attempts to redefine the mode of application of diethylcarbamazine to reduce the unacceptable side effects, ivermectin (subsequently called Mectizan® for use in humans) was introduced in 1987 for use in onchocerciasis (Aziz, 1986). When the LFEP was initiated in 1998, the combination of either Mectizan® (Merck & Co. Inc.) or diethylcarbamazine (DEC) both given with the potential adulticidal benzimidazole, albendazole (GlaxoSmithKline), was defined for the start of the Program. The current recommended chemotherapeutic regimes are shown in Table 1.
Mackenzie 157 The donation of ivermectin for onchocerciasis in 1987 by Merck & Co. Inc. has been one of the most important events in filarial disease control for many decades. Indeed, MDA initiated programmes through this donation have radically changed the nature of applied tropical medicine itself. Merck & Co. Inc.’s and then GSK's donations have renewed interest first in onchocerciasis and now lymphatic filariasis, diseases that were often ignored compared to the more lethal and acute tropical infections such as malaria. The targets for these MDA Programs are a very diverse group of countries: varied in many aspects including politics, geography, health systems and disease characteristics. The ODC Program is divided into the Americas (known as OEPA) (Blanks et al, 1998) and the multifaceted African Program (which includes the Arabian Peninsula). In recent times, spearheaded by the Global 2000 of The Carter Center, the OEPA America’s program is approaching elimination of onchocerciasis in the Region. Although Africa has the majority of disease, success in the Americas would be a major demonstration of the feasibility of eliminating a parasitic disease. The lymphatic filariasis program aims to eliminate an infection which affects many more people and countries than does the onchocerciasis program. The endemic regions encompass virtually all the tropical regions of the world, and this Program has been regionalized to improve management. As shown in Table 1 different combinations of drugs are used. Merck & Co. Inc. has provided Mectizan® for use against LF in countries where there is already onchocerciasis present - thus avoiding any dangers that might arise from onchocerciasis patients using DEC in these countries. Thus the LFEP is divided into those countries with onchocerciasis - where Mectizan® is combined with albendazole and the remainder of the world where DEC is used with albendazole. Most of the anthelminthics used in humans have originated in veterinary science, where parasitic diseases have always been a major economic issue in production animals. This is the case with ivermectin and albendazole. However diethylcarbamazine (DEC), which although widely used in veterinary medicine against canine heartworm, was developed by medical laboratories and first applied by Mexican physicians in filariasis (Mackenzie and Kron 1985). Our knowledge of how these agents actually work is still surprisingly limited. An agent best suited for MDA can be characterized as 1) safe, even in the young, the aged and the pregnant, 2) effective in a single, and no more than annual dose, 2) macrofilariacidal, 4) easily administered, and finally 5) with a long shelf life and resistant to harsh environmental conditions. The drugs in use today do approach these ideals in many aspects. One of the important issues that remains with the use of filariacidal agents is controlling the reactions to the dead and dying parasites. These reactions plagued the use of DEC in killing the microfilariae of O. volvulus and may also cause potentially serious reactions with the death of adult LF parasites. It is ironic that Luis Mazzotti, working in Chiapas, Mexico, who described the first clinical use of a useful chemotherapeutic agent for filariasis, is actually best
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known for the clinical problems that are associated with the treatment - the anaphylactoid reactions termed "mazzotti reactions". Ivermectin fortunately induces fewer of these unwanted responses. Importantly, it does not cause the damage to the ocular tissues that DEC induces in patients carrying O.volvulus microfilariae in the eye (visual loss and dangerous retinal vessel leakage). DEC cannot be used at present for LF treatment in onchocerciasis endemic areas, at least not without developing methods of ensuring that no ocular pathology can occur. A serious and sometimes lethal situation does however occur with ivermectin in patients with large numbers of another filarial parasite, Loa loa. An encephalopathy with coma characterizes the fatal cases (Boussinesq et al, 1998). Management of this relatively rare condition poses a problem for the ODCP and potentially also for the LFEP.
EARLY PROGRAMS Once onchocerciasis was clearly defined in the first third of the last century, some countries developed their own approach to controlling their disease. Mexico and Guatemala developed nodulectomy campaigns to surgically remove palpable nodules containing adult O.volvulus worms. These efforts were usually not well accepted by the population but may have served to at least limit the infection in these countries. Sudan, where the disease was well described and its severity understood early last century, developed a country wide treatment program based first on the use of suramin – in a protocol known as the “Sudanese regimen. This program, although well integrated into the existing health system, did not achieve much in terms of real
Mackenzie 159 control of the disease. However the medical system set up for this program has provided a strong logistical basis for the new treatments as they came along. The first major multi-country program was developed in late 1970s in West Africa, based on aerial spraying to destroy the vector and its breeding sites in the rivers of the program area. The success of this program was limited, in part due to the unrecognized reinvasion capability of the vector Simulium blackflies. However, when combined with the newly introduced Mectizan®, great successes began to appear and in some areas in the OCP area, no child under 8 years is infected with this parasite (Hougard et al 2001). Although successful programs based solely on entomological approaches are uncommon, they have occurred in limited situations, such as river dam areas in Uganda. The World Health Organization, in conjunction with NGDO partners (the ODCP has always preferred the involvement of NGOs in country based programs), developed a MDA system to involve the countries in Africa not originally part of the West African OCP project. This was called the African Program for Onchocerciasis Control (APOC), a centrally managed program which has managed to bring many countries in Africa into the effort to reduce blindness due to onchocerciasis. Major programs have been in place for LF using DEC in countries such as India and Sri Lanka for decades; these countries have a vast amount of experience and have developed very efficient managerial systems for parasitological assessment and drug distribution. At the present time, India is treating over 20 million people with the new DEC/albendazole regime and will increase their numbers greatly in the near future. An important successful program was set up in China approximately thirty years ago using DEC. The first doses of tablets were backed up with the use of this anthelminthic administered in the salt used for cooking. This was remarkably effective in reducing the prevalence of filariasis in the region. Saltbased treatments are currently being initiated in Haiti and elsewhere.
CURRENT CONTROL PROGRAMS The mainstay of control in onchocerciasis today is ivermectin (Brown and Neu 1990). African countries that have shown interest in being part of the APOC effort must apply for matching funding. APOC (based in Ougadougou) will grant financial support, but the country must still request the drugs by itself from the Mectizan® Donation Program based in Atlanta - the agency set up by Merck & Co., Inc. to distribute the drug to the countries. The APOC program is a community based distribution system with the village expected to take responsibility for the actual administration of the drug to the people. Many countries remain to be included in the APOC system. Some of them, such as the Democratic Republic of the Congo, are countries with large numbers of people needing treatment. APOC does not include those people residing in areas that are hypoendemic for O. volvulus based on their programmatic focus of preventing ocular disease - which they associate with hyper and meso-endemic regions. A
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criticism of this approach is that in many cases, the worst forms of dermal disease are actually seen in hypo-endemic areas are not included in APOC activities. Thus, it is arguably time to reassess the target populations in the MDA programs for onchocerciasis, as well as the goals of the program. Those involved with the OEPA activities in the Americas are already reassessing the criteria and methods used to achieve their goal of elimination of the disease in this region. Lymphatic filariasis with its two different regimes and its enormous target population (made greater by virtue of being an "elimination" program) has been divided up into five major areas (Africa, Asia, the Middle East, the Americas and Pacific Regions). A Global Alliance made up of all interested partners in the elimination effort is managed by World Health Organization and serves as a forum for communication. In recent times The Bill and Melinda Gates Foundation, the UK's DFID and other major agencies have provided funding for selected model country programs. Drug requests for those countries using Mectizan® are, as with onchocerciasis, handled by the MDP in Atlanta. It has been predicted that this program should take as little as 5-6 years to have the desired effect of elimination in an area (based on the adult worms life span). Whether this is an overly ambitious program target remains to be seen.
ESSENTIAL COMPONENTS OF CONTROL PROGRAMS Experience has shown that there are a number of important components and characteristics that are needed for successful filarial disease control programs. Communication and education are major components and successful programs devote much effort in these areas. They are the basis of good advocacy and when used with all the appropriate groups of participants politicians, medical community and the target populations themselves - reap great rewards in terms of final MDA coverage. Leadership is always essential and the presence of an effective “champion” for the cause at the country level is almost always necessary for success. The vigorous endorsement of the programs by Ministries of Health of the participating country is also an essential ingredient. It is important for these, the country’s official agencies for health, to take responsibility for these diseases and their control or elimination. Financial support is also a basic necessity and although many believe that the country, even the population themselves, should cover the costs of administration of the drugs, in reality this is not readily feasible and external support must nearly always be found. This does not exclude the need for the filarial programs to be quickly integrated into a country's health system administration wherever possible. The issue of integrating sector wide reform activities and these vertical programs must be addressed on a country-bycountry basis. The participation of western medical scientists in these programs should be one of providing technical support and information, and never one of decision-
Mackenzie 161 making or outright leadership. These MDA programs have an important catalytic role in infrastructure and country personnel building according to a specific country’s needs and situation. Many drug donations are occurring and countries are being asked to become involved in more and more of these. Indeed, there is an argument that governments should set up special sections to bring together these programs and maximize the limited resources available. These new efforts in tropical medicine bring with them a number of social and ethical issues as well as responsibilities. The countries themselves and their MOH have a central responsibility for these programs and their implementation; western medical experts should support these national efforts in every way they can.
PROGRAMS IN PRACTICE It is useful to look at two examples of filarial control programs in practice, Tanzania and Sudan, and see how these principles have been put to good use. In 2001, the Tanzanian LF Elimination Program began its challenge to eliminate LF from the exposed population of probably more than 12 million people. LF is an important and devastating clinical disease in Tanzania, especially along the Coast. To date more than 500,000 people have been treated with Mectizan and albendazole through drug distribution carried out by village health workers. There is a great deal of enthusiasm for the program in the country. One senior politician stated, when asked why there was such a high level of support and compliance, that “it is because we know why we are taking the pills". Thus he underlines the importance of communication and acceptance by both the political arm and the public for success of such programs. It is expected that coverage of the target population will well exceed 70% in 2001. Good communication of both the need for treatment to the public and the procedures for treatment to the medical staff have been central to the success of distribution in Tanzania. Involvement of those already affected with the disease in the advocacy activities with a clear demonstration of actions to help those affected e.g. hygiene procedures for elephantiasis, surgery for hydrocoele, all serve well to promote the program. Treatment regimes, both drug administration and hygiene/surgery activities, are extremely well accepted by those already with disease. They perceive a marked reduction in the number and intensity of their acute filarial events; these latter events are debilitating and can be very severe. This unexpectedly positive effect of treatment on those already with disease is serving as an "auto-advocate" for the program. Sudan has some of the worst onchocercal disease known. Extensive clinical, epidemiological and pathogenesis studies were carried out into Sudanese onchocerciasis during the 1980’s. After a period of inability to reach the worst affected areas of endemic disease during to civil disturbance in 1975, a cease fire set up by President Jimmy Carter allowed the initiation of the first Mectizan® distribution in Southern Sudan. Shortly after, spearheaded by leadership from the Academy of Medical Sciences and Technology in
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Khartoum, a detailed ODCP was established. As the country was in a state of civil unrest, this required a diplomatic approach - a country specific approach so that all endemic areas could be covered. The establishment of a Government component and Southern Sudan component (through the NGOs coordinating group in the rebel held areas of the country), has allowed the program to function politically. These areas often vary with the changing political situation, but at any one time the people of the area covered one or the other of the program’s components. In the rebel held regions in the South, over 40 NGO groups have been involved in the distribution of the drugs for onchocerciasis. The characteristics of the Sudanese program that have allowed this unique and difficult situation to be functional include: local understanding and commitment, separation from political bias, a local champion with the ability to perform, and team dedication (in fact three members of the Program have died carrying out their duties). The importance of giving the country the right responsibility and credit, as well as external support (from The Carter Center and Michigan State University in this case), is apparent in this Program.
CHALLENGES TO SUCCESS Geographic difficulties, such as the size and isolation of the areas to be treated together with the lack of infrastructure (roads and other forms of communication) are major hurdles in many countries. In contrast, it is thought that MDA in the large urban areas of many African countries will pose its own set of problems. However, the very successful and well organized system of distribution in place in urban areas in India and Sri Lanka give hope. Migratory populations, especially those in ODC Programs in Africa are a challenge, especially in countries with civil unrest or who are in conflict with their neighbors. The need to integrate across country borders is important in certain areas and will be a problem where there is not a good relationship between the countries involved - this has been a problem in Southern Sudan. Although country based development of these programs is the main approach needed, there is a need at some point for the individual countries to combine to address across border issues. Financial support is obviously a central and fundamental issue. There are always many needs seeking the world's available donated finance. What makes the filarial diseases inviting to donors is that the programs have a high likelihood of success in a relatively short period of time - unlike arguably "more deserving" causes such as the acute killing diseases and disasters. The economic importance of these two filarial diseases has been examined in considerable detail in recent years and there is strong association between poverty and this disease. Continual advocacy for the programs is needed especially in the later years, when there will be less overt reason for villagers to continue treatment, less clinical disease to improve and “auto-advocate” for the programs. Issues such
Mackenzie 163 as that of the Loa loa adverse reactions need to be addressed and managed, as they could be very devastating to these programs in the short and long term. Finally there is the issue that should not be a challenge in this day and age the investment of fully responsibility and decision making to the nationals of the countries in question. The diseases are the responsibility of the governments of the countries affected and the appointed physicians in these countries. The MOH are asked to sign off on all programs. It is the responsibility of the international experts and individuals to provide the medical authorities of the affected countries with all the best information available and allow them to make the decisions for their countries.
THE FUTURE Control programs often begin with much hope and enthusiasm, but with a number of questions - such as the period required to achieve the goals, the best means for evaluation, and other important issues - unanswered. Sometimes the scientific data needed to answer these important questions is lacking. Both these filarial control programs began this way, but as time passes the questions are being answered. It is essential to continue to gather practical data that addresses the parameters that will define the progress, conclusion, and the success of the program (Mackenzie 2000). Will drug resistance occur? This is remains a threat. Certainly resistance to ivermectin has occurred in veterinary medicine in intestinal helminths. It is not clear yet if resistance to ivermectin is inevitable with filariae. There are as yet unsubstantiated suggestions of unresponsiveness to ivermectin in onchocerciasis patients in Africa. Whether this is drug resistance or other phenomena, such as variation in host responses, remains to be seen. Fortunately there are no credible descriptions yet of albendazole resistance in these helminths. New drugs would be good insurance against any loss of effectiveness of ivermectin and many believe there is a need for a macrofilariacide to enhance elimination. To achieve these goals, model systems must be supported and there must be more basic research into candidate targets for such chemotherapeutics. There are few substitutes for these human conditions: the cattle parasite Onchocerca ochengi is arguably the best surrogate for O.volvulus and Brugia sp in cats best for lymphatic filariasis. The current interesting approaches involving Wolbachia are important and hopeful. However, many basic scientific approaches to the search for adulticides should be still pursued using modern biotechnology (Mackenzie 2000). It is important to involve national and research based institutions in the continuing investigation and research, as well as participants in the international bodies that decide on the aid and advice provided to endemic countries. Distributing chemotherapeutic agents in mass distribution systems at the village level, with the physician at least three if not more stations removed from the patients, an essential characteristic of mass drug administration
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systems, comes with heightened medical responsibilities. MDA is an important approach that will enhance the health of rural populations in a way not possible before. For example, medical systems must be developed and implemented for responding to severe adverse reactions in a timely and responsible manner. Although these three drugs are regarded as being very safe, it is important to continue to monitor their combined use, and to understand better any situations in which the drugs might be contraindicated - as one would do with any drug in medical use. Nevertheless it must also be expected that drug reactions will occur at some time, especially with the huge numbers of people to be eventually treated. It goes without saying that methods for monitoring and evaluation are key to the all important measurements of success of such program. Such evaluations should be published in peer-reviewed forums as often as possible. There is a tendency to confine such data to internal documents. There is a great need to publish success wherever possible, to share experiences and to obtain a wide acceptance of the Programs. This will aid the continuing need for fiscal and political support. To this end, the achievement of success in specific locations such as the onchocerciasis in the Americas would be a tremendous boost to the program and demonstrate feasibility. Among the most important data needed are the definition of success, the most appropriate disease-alleviation procedures for both onchocerciasis (the visually impaired and the dermatologically afflicted) and those with the various forms of lymphatic filariasis (hydrocoele, AFE and ADL and elephantiasis), as well as the importance of measuring entomological criteria. Disease alleviation mechanisms must be given significant emphasis. Although much has been stated about this aspect of the programs, the financial support is still unclear and needs attention. These efforts, however, need to be country specific, with each locality developing its own programs to fit the available facilities and personnel. The OCDP currently are very poor in this area, the visually impaired being largely ignored and forgotten in most countries. Care must be taken to maintain and support the rights of the countries that the programs aim to assist. Nationals from the endemic countries must participate fully in decision-making at the regional and global level, as well as obviously for their own country. If carried out in the right manner and with sensitivity to these issues, these disease control programs will not only be medically successful but will also advance the development of health systems in those countries. To achieve control, a high level of enthusiasm must be maintained and must be further embraced by the wider international community, both scientific and donor alike. Thus communication of success must be at the highest and most proactive of levels. Are these programs going to achieve their original goals? Goals will be reached and much will be attained: but perhaps not quite as originally intended. What will be achieved, aside from the elimination or control of these two dreadful diseases, is the building of a country's medical infrastructure. The training of national medical personnel and improvement in health systems will
Mackenzie 165 occur. Importantly a positive sense of achievement against an infectious disease - at a time when new infectious diseases seem to be confronting us will be a welcome boost to tropical medicine. These programs will improve the well being of villagers, give those affected a better quality of life, and should promote economic activity, and hopefully social stability.
REFERENCES Aziz, M.A. 1986. Chemotherapeutic approach to control of onchocerciasis. Reviews in Infectious Diseases 8:500-4. Behbehani, K. 1998. Candidate parasitic diseases. Bulletin of the World Health Organization. 76:(Suppl 2) 64-7. Blanks, J., F. Richards, F. Beltran, R. Collins, E. Alvarez, G. Zea Flores, B. Bauler, R. Cedillos, M. Heisler, D. Brandling-Bennett, W. Baldwin, M. Bayona, R. Klein, and M. Jacox. 1998. The Onchocerciasis Elimination Program for the Americas: a history of partnership. Revista de Panama. Salud Publica 3:367-74. Brown, K.R., and D.C. Neu. 1990. Ivermectin - clinical trials and treatment schedules in onchocerciasis. Acta Leiden 59:69-75. Bussinesq, M., J. Garden, N. Gardon-Wendel, J. Kamgno, P. Ngoumou, and J.P. Chippaux. 1998. Three probable cases of Loa loa encephalopathy following ivermectin treatment for onchocerciasis. American Journal of Tropical Medicine and Hygiene. 58:461-9. Cox, F.E. 2000. Elimination of lymphatic filariasis as a public health problem. Parasitol Today 16:135. Hougard, J.M., E.S. Alley, L. Yameogo, Y. Dadzie, and B.A. Boatin. 2001. Eliminating onchocerciasis after 14 years of vector control strategy. Journal of Infectious Diseases. 184:497-503. Mackenzie, C.D. 2000. Human onchocerciasis: essential research needs for control. Current Opinions in Infectious Diseases 13:1513-21. Mackenzie, C.D., and M.A. Kron. 1985.Diethylcarbamazine: a review of its action in onchocerciasis, lymphatic filariasis and inflammation. Tropical Diseases Bulletin. 82: R1–R37. Maher, D., and E.A. Ottesen. 2000. The Global Lymphatic Filariasis Initiative. Tropical Doctor. 30:178-9.
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VACCINES FOR FILARIAL INFECTIONS
Paul B. Keiser and Thomas B. Nutman Helminth Immunology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 USA
ABSTRACT The development of an effective vaccine against lymphatic filariasis and onchocerciasis would improve efforts to eradicate these infections. Despite the limited availability of parasites and animal models, a number of strategies have emerged to identify targets for protective and transmission-blocking immune responses. This chapter reviews these strategies and discusses some of the vaccine candidates that are currently being evaluated. Keywords: Antibodies, Helminth/blood, Brugia malayi, Chitinase, Disease Models, animal, human, immunization, filariasis/immunology, Onchocerca volvulus, Onchocerciasis/immunology, Paramyosin, Tropomyosin, Vaccines, Wuchereria bancrofti.
INTRODUCTION Lymphatic filariasis and onchocerciasis remain persistent and significant causes of morbidity in tropical areas worldwide. Despite the use of vector control measures and the institution of widespread distribution of microfilaricidal agents for transmission reduction and symptom amelioration, neither of these strategies, as currently employed, can provide a longstanding and self-sustaining solution to the global eradication of filarial parasites. While the absence of an animal reservoir for most filarial pathogens suggests that a vaccination strategy could lead to global eradication (Ada et al., 2001), an effective vaccine has yet to be developed. Efforts have been made to understand the immunological underpinnings of protective immunity in filarial infections and to develop vaccines to aid in controlling the spread of these infections. Thus far, most successful vaccines have been directed against viral and bacterial pathogens associated with acute clinical disease exhibiting stable expression of surface antigens (Ada et al., 2001). The chronic extracellular nature of filarial parasitism, however, is indicative of a more complex relationship with the host immune system. The progression through multiple life cycle stages, with corresponding differences in tissue tropism and antigen expression within the host suggests that immune targeting and elimination of these pathogens may not be as straightforward as it has been with other controllable diseases.
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CHALLENGES IN FILARIAL VACCINE DEVELOPMENT The host parasite relationship for each of the filarial infections is both complex and incompletely understood. While most filarial-infected individuals are clinically asymptomatic, tolerating enormous quantities of foreign antigen, the development of an inflammatory response to these parasites can be associated with the development of debilitating and potentially irreversible pathology (Dreyer et al., 2000). The possibility exists, therefore, that a vaccine that stimulates host immune responses may actually predispose to or precipitate immune-mediated pathology. Immunopathology has not, thus far, been reported in animal studies of filarial vaccines, though it has not been looked for with the same care as protective efficacy. For most of the filarial parasites that infect humans, availability of parasite material is limited. Wuchereria bancrofti and Onchocerca volvulus cannot be maintained indefinitely in the laboratory (Lok 1992). Therefore, parasite material is often drawn from the more readily available Brugia species or from related filarial species obtained from their natural animal hosts (e.g Onchocerca spp in ungulates, Brugia spp in cats, Dirofilaria in dogs). Although this paucity of material has limited to a great degree biochemical and protein chemical approaches to antigen identification, as expected, molecular biology, genomics and proteomics have provided alternative approaches to both identification and production of potential vaccine targets. Animal models for vaccine experiments have significant limitations. Only the silvered leaf monkey (Presbytis cristatus) is permissive to Wuchereria bancrofti for life cycle progression. Jirds (Meriones unguiculatus) and ferrets (Mustela putorius) are fully permissive for Brugia species and develop lymphatic pathology to an extent, but reagents for working with these animals are limited (Lok 1992). Only chimpanzees and mangabey monkeys are fully susceptible to Onchocerca volvulus. Though mice are not permissive hosts for the filarial pathogens of humans, the ready availability of reagents and various mouse strains with targeted deletions of known genes has encouraged their use in filarial research. Experiments with immunocompromised (thymectomized, SCID or athymic) mice have shown increased susceptibility to both larval (Babu et al., 1999) and microfilarial (Townson et al., 1984) infection, implying that the non-permissiveness of mice has an immunological basis and justifying their use for studies on protective immunity. A number of natural host-parasite systems with filariae not normally pathogenic in humans have also been used to study protective immunity in filarial infections. Besides host-parasite incompatibilities, animal studies are constrained by practical considerations. Recovery of parasites from animal hosts is very low, even within hours of inoculating infective stage larvae into a permissive host. To demonstrate significant differences in viable parasites between control and vaccinated animals, the number of infective larvae in a typical challenge far exceeds what would normally be encountered at a single site in natural
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exposure. Such bolus challenges might overwhelm a level of immunity that could be protective in a natural infection. Other, non-immunological factors may also be introduced by these bolus inoculations. For example, in the A. viteae-jird model, a negative correlation has been shown between inoculum dose and recovery rate as well as worm length (Barthold and Wenk 1992), perhaps resulting from local parasite crowding. Despite these limitations, a number of strategies have been used to identify vaccine candidates.
STRATEGIES FOR IDENTIFICATION OF VACCINE CANDIDATES PUTATIVE IMMUNITY Individuals living in endemic regions who have no past or present evidence of infection have been referred to as endemic normals and are considered by some investigators to be “putatively immune” (PI). There appear to be prevalence differences in different areas, due at least in part to the intensity of transmission, but perhaps also due in part to genetic differences in either the host or the parasite. For lymphatic filariasis, the prevalence of putative immunity varies from zero in the East Sepik province of Papua New Guinea (Day 1991) to over 40% in Mauke, Cook Islands (Steel et al., 1996). For onchocerciasis, the prevalence of putative immunity is reported to be around 5% (Boyer et al., 1991). Criteria for categorizing an individual as putatively immune have evolved in parallel with the development of more sensitive and specific diagnostic modalities. Generally, the state of putative immunity is defined as: 1) the absence of patent infection, 2) longstanding historical evidence of being infection free, and 3) the absence of clinical findings suggestive of past or current infection. The lack of a clinical reaction to a challenge dose of diethylcarbamazine has been used as further evidence of being free of infection (Ward et al., 1988; Freedman et al., 1989). Some investigators have used the term PI to refer to individuals with detectable but very low microfilarial levels (Kazura et al., 1992) while historical information suggestive of filarial infection has been disregarded by others as too nonspecific (Zhang et al., 1999). Application of modern diagnostic techniques, such as scrotal ultrasound (Dreyer et al., 1999), antigen detection or PCR (Day 1991; Elson et al., 1994) has caused some PI individuals to be reclassified as having occult infection. Differential antibody recognition of specific filarial proteins, particularly larval stage proteins, by PI versus infected patients has become one method of identifying vaccine candidates (Freedman et al., 1989). Not all investigators have actually found differences in the pattern of antigen recognition (Zhang et al., 1999), however, and it has been shown that antigens differentially recognized by PI sera in one endemic area differ significantly from those identified in another (Irvine et al., 1997). The antigens identified also differ
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significantly depending on the filarial species used to screen the sera (Nutman et al., 1991). While earlier studies used sera to screen parasite extracts, the ability to induce protein expression directly or indirectly from cDNA libraries has greatly enhanced the ability to identify and test candidate antigens (Lizotte-Waniewski et al., 2000). One potential problem with this strategy is that studies of differential recognition usually compare putatively immune individuals with those with asymptomatic patent infections, as opposed to those with chronic or hyperreactive pathology (Freedman et al., 1989; Peralta et al., 1999). As both of these latter clinical scenarios are associated with an increased T cell response to parasite antigens, it is not clear how precisely to create a protective immune profile without creating a predisposition to pathology. Secondly, it is difficult to know with certainty whether the differential recognition of parasite antigens is the cause or effect of increased parasite killing. The third problem with this strategy has been the experience that antigens identified in this fashion have failed to remain differentially recognized by the same serum samples when expressed in recombinant form (McCarthy 2001). In onchocerciasis, differential recognition by putatively immune sera was the rationale for further experiments with Ov-aldolase (McCarthy 2001), Ov7 (oncocystatin), Ov64, OvB8, and Ov73k (Abraham et al., 2001). Of these, Ov-aldolase, Ov7, Ov64 and OvB8 showed modest (34 – 50%) reduction in survival of O. volvulus L3’s planted in diffusion chambers in BALB/c mice. Interestingly, a cocktail of multiple antigens failed to improve on the protection achieved when they were used individually (Abraham et al., 2001). For lymphatic filariasis, recombinant OvG15 (filarial HSP-70) was identified in this fashion but failed to induce protection in jirds challenged with Brugia malayi (Peralta et al., 1999).
ANIMAL STUDIES WITH RADIATION ATTENUATED PARASITES It has been known since 1969 that prior exposure to radiation-attenuated filarial larvae confers a degree of protection to subsequent challenge (Wong et al., 1969). The degree of protection, as determined by percent decrease in survival of challenge larvae, is greater in animals exposed to radiationattenuated larvae than in those previously exposed to intact larvae or larval extracts (Lucius et al., 1986; Lange et al., 1993). What precisely the radiation does to the larvae to result in this improved protection is not certain. It has been observed that immunizing doses of radiation prevent or delay larval molting, so the operative mechanism may be prolonging the duration the host is exposed to infective stage antigens, allowing an immune response to mature before the immune hyporesponsiveness associated with patent infection occurs (Nasarre et al., 1997). Another possibility is that radiation induces increased expression of protective antigens, or decreased expression of
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“decoy” antigens on the larval surface, although no differences could be seen in the protein expression of irradiated and non-irradiated Brugia pahangi, even up to 12 days post-inoculation (Devaney et al., 1993). It is possible that different factors or combinations of factors are responsible for the protective effect in different host-parasite systems, or perhaps even at different doses of radiation. The possibility that radiation increases expression and exposure to potentially protective antigens has inspired a number of studies to determine what these protective antigens might be. The procedure followed in a number of host-parasite systems has been as follows: 1) determine the optimal dose of radiation required for the larvae to induce protection, which has been shown to vary considerably among host-parasite systems (Chusattayanond and Denham 1986); (2) inoculate subcutaneously to mimic natural exposure, or intraperitoneally to improve exposure to antigen presenting cells, the numbers of larvae inoculated having been determined empirically, although protection has resulted from one dose of 5 irradiated larvae (Lucius et al., 1991) to multiple inoculations of thousands of larvae (Oothuman et al., 1979); (3) challenge with a sufficient number of unattenuated larvae to detect a difference in survival compared with control animals. In general, the challenge dose is given subcutaneously, to mimic natural exposure, intraperitoneally to facilitate recovery (Yates and Higashi 1985; Chusattayanond and Denham 1986), or surgically implanted in diffusion chambers to facilitate recovery and allow a more detailed examination of the local immune response (Lange et al., 1993). Results obtained from different routes of challenge do not always concur (Weil et al., 1992). Some investigators have used different larval species for the immunization and challenge, showing some degree of cross-protection (Oothuman et al., 1979; Storey and Al-Mukhtar 1982). After demonstrating a protective effect, serum from the immune animals is then used to identify vaccine candidates from parasite extracts or recombinant proteins from cDNA libraries. Although antibody responses against the vaccinating strain can be demonstrated in protected animals, antibody levels do not always correlate with the degree of protection (Rao et al., 1977). The pattern of antigens recognized is complex (Yutanawiboonchai et al., 1996), and antibodies to larval surface antigens are frequently (Weil et al., 1992) but not always (Lucius et al., 1991) detected. Protection induced by radiation attenuated infective larvae is, in general through a Type-2 helper T cell response, with increased parasite-specific IL-5 production (Le Goff et al., 2000) and local increases in IL-4, IL-5 (Taylor et al., 1994; Johnson et al., 1998) and eosinophil infiltration (Yates and Higashi 1985; Yates and Higashi 1986; Lange et al., 1994; Taylor et al., 1994). Paramyosin Paramyosin was identified as preferentially recognized by jirds immunized by radiation attenuated Brugia malayi larvae (Li et al., 1991).
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Although filarial paramyosin is not expressed on the surface, Dirofilaria paramyosin had previously been shown to be preferentially recognized among putatively immune persons from two different onchocerciasis-endemic regions (Steel et al., 1990), supporting a role in protective immunity and raising hopes of cross-protection from other filarial species. Vaccination of jirds with recombinant Brugia paramyosin showed a slight decrease in the number of adults that could be recovered from a subcutaneous challenge (40% of the 13% of the challenge inoculum that could be recovered in control jirds), though no protection was seen from the 98% identical recombinant Dirofilaria paramyosin in the same experiment (Li et al., 1993). DNA immunization was attempted with the coding sequence, stimulating both antibody and cellular proliferative responses to the antigen, but failing to decrease the number of adult parasites recovered compared to controls (Li et al., 2000). OvB20 The antigen OvB20 was identified from cattle experimentally inoculated with irradiated Onchocerca lienalis larvae (Abdel-Wahab et al., 1996). Subsequent characterization of homologous proteins showed localization to discrete patches of the cuticle and hypodermis of O. volvulus L3’s as well as culture supernatants of A. viteae. Jirds vaccinated with recombinant OvB20 showed a significant reduction in adult A. viteae recovered from an L3 challenge, and a 97% reduction in the level of microfilaremia. Vaccinated BALB/c mice inoculated intradermally with microfilariae of O. lienalis did not show protection relative to unvaccinated controls (Taylor et al., 1995). Further evaluation is ongoing in a bovine model (Graham et al., 1999).
STUDIES IN NON-PERMISSIVE HOSTS One advantage of the non-permissiveness of readily available laboratory animals to filarial infection is the ability to isolate the immune response to a single parasite stage. Tropomyosin Antisera were produced to Onchocerca infective larvae were produced by multiple subcutaneous injections of live or freeze-killed O. lienalis L3’s into rabbits. Subsequent screening of a cDNA library from adult female O. volvulus led to the identification of tropomyosin as one of the antigens recognized (Jenkins et al., 1998). Tropomyosin is expressed by all stages of O. volvulus in both muscular and cuticular layers, and a cross-reactive epitope was found in the excretory-secretory product of A. viteae L3’s (Taylor et al., 1996). Reactivity of sera from infected humans seemed to correlate inversely with infection intensity. Vaccination with recombinant Ov-tropomyosin decreased skin microfilarial density in BALB/c mice intradermally challenged with O. lienalis, and decreased the adults and microfilariae that could be recovered from jirds inoculated with A. viteae L3’s (Taylor et al., 1996).
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However, subsequent studies in mice immunized with the DNA sequence of Ov-tropomyosin failed to decrease the numbers of O. volvulus larvae that could be recovered from subcutaneously implanted diffusion chambers (Harrison and Bianco 2000). Enthusiasm for this vaccine is also constrained by the observation that there is significant homology between O. volvulus and human tropomyosins, as well as a highly allergenic IgE-binding crustacean tropomyosin (Jenkins et al., 1998), raising the possibility of vaccine-induced autoimmune or allergic disease. Further evaluation is ongoing in a bovine model (Graham et al., 1999). Paramyosin In a conscious effort to identify antigens present at all stages of the filarial life cycle, antisera from rabbits immunized with Brugia malayi L3s were used to screen Mf and adult antigens. Based on reactivity on Western blots, an antigen designated Bm97, and subsequently identified as paramyosin, was purified for use in transmission-blocking experiments. Mice immunized with purified B. malayi and C. elegans paramyosin did show a modest decrease in the degree of microfilaremia, but also developed significant footpad swelling, indicative of an Arthus or delayed type hypersensitivity reaction, when a second dose antigen was injected (Nanduri and Kazura 1989). Subsequent work with paramyosin, which was also preferentially recognized following immunization with radiation-attenuated larvae, was described above. Chitinase A potential transmission-blocking vaccine for lymphatic filariasis was sought by inoculating BALB/c mice intravenously in the Brugia microfilariae or microfilarial antigens. A resulting monoclonal antibody (MF1), when transferred intraperitoneally into patently infected jirds, transiently (up to two weeks) decreased the level of microfilaremia (Canlas et al., 1984). This is consistent with the notion that immunity to microfilariae can be predominantly humoral (Carlow and Bianco 1987). The antigen recognized by this antibody was subsequently identified as filarial chitinase (Fuhrman et al., 1992), which was produced in recombinant form and used in seroreactivity and immunization experiments. The specific epitope corresponding to MF1 was found by competitive ELISA to be recognized preferentially by amicrofilaremic individuals with either brugian or bancroftian filariasis when compared with microfilaremics from the same population (Dissanayake et al., 1995). Vaccinating jirds with a recombinant Brugia chitinase had little impact on microfilaremia, whether administered before infection, or during pre-patent or patent infection (Wang et al., 1997). Unlike the human sera that were initially screened, however, sera from microfilaremic jirds do recognize chitinase, emphasizing the limitations of even a permissive animal model. However this has led to some promising work with a DNA vaccine for O. volvulus chitinase in the mouse model (Harrison et al., 1999).
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GENOMIC APPROACHES TO IDENTIFYING VACCINE CANDIDATES Another approach to vaccine identification is to select antigens that are up-regulated or unique to a particular parasite stage of interest. Proteins expressed at particular times might be critical to the establishment of infection. Furthermore, if pathological reactions are most often directed against adult and microfilarial stages, a vaccine directed uniquely to larval stage antigens may avoid precipitating such an inflammatory response. Targeting filarial gene products with critical roles in parasite development (e.g. specific enzymes, endosymbiont gene products, and key metabolites) based on sequence data is now possible. Indeed, many more vaccine candidates have been identified and await further testing (Lizotte-Waniewski et al., 2000). Bm-ALT-1 Analysis of the Brugia malayi L3-expressed cDNA’s in the EST database led to the identification of the highly expressed abundant larval transcripts, ALT-1 and –2 (Gregory et al., 2000). Immunization of jirds with ALT-1 resulted in a 76% reduction in the number of inoculated Brugia malayi larvae that could be recovered. Interestingly, sera from both putatively immune and infected individuals recognize these antigens, demonstrating that vaccine candidates identified by one strategy might be dismissed by another. Studies with the Ov-ALT-1 are also ongoing (Graham et al., 1999).
CONCLUSIONS Over two hundred years since Jenner developed a vaccine for smallpox and twenty years after natural smallpox has been declared eradicated, vaccines remain a desirable strategy to control, if not eradicate, many human pathogens. The filarial parasites of humans, however, are among the most complex Animaliae for which vaccines have been considered. Though the potential for pathology exists, it appears that stage-specific antigen expression and immune clearance mechanisms might allow the immune responses to be targeted accordingly. (Canlas et al., 1984; Taylor et al., 1995). The lack of a permissive animal model for which reagents and targeted gene deletions are readily available is a limitation, but not one that is unique to the field of filarial vaccine development. As a number of antigens identified in animal models are recognized by human sera from endemic areas, it would appear that these host-parasite systems are sufficiently similar to what humans encounter to draw some conclusions about mechanisms of protective immunity. The existence of multiple host-parasite systems will enable vaccine candidates to be studied more thoroughly before proceeding to human trials. The use of non-permissive models that limit progression to a single stage has actually improved our understanding of stage-specific immune responses.
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Each of the strategies that has been used to identify vaccine candidates has shown some legitimacy in animal models. A number of vaccines have been shown to confer a degree of protection and are being evaluated further. Emerging technologies in protein identification and DNA vaccination have accelerated the pace at which vaccine candidates are identified, characterized, and tested. Many promising strategies for vaccine delivery are emerging from research with other pathogens that await evaluation with filarial parasites. Vaccination against filarial parasites may be the “ultimate challenge” for vaccinologists (Maizels et al., 1999), but it is a challenge that is being met head on.
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Lizotte-Waniewski, M., W. Tawe, D. B. Guiliano, W. Lu, J. Liu, S. A. Williams and S. Lustigman. 2000. Identification of potential vaccine and drug target candidates by expressed sequence tag analysis and immunoscreening of Onchocerca volvulus larval cDNA libraries. Infect Immun 68: 3491-501. Lok, J. B., Abraham D. 1992. Animal models for the study of immunity in human filariasis. Parasitology Today 8: 168-71. Lucius, R., A. Ruppel and H. J. Diesfeld. 1986. Dipetalonema viteae: resistance in Meriones unguiculatus with multiple infections of stage-3 larvae. Exp Parasitol 62: 237-46. Lucius, R., G. Textor, A. Kern and C. Kirsten. 1991. Acanthocheilonema viteae: vaccination of jirds with irradiation-attenuated stage-3 larvae and with exported larval antigens. Exp Parasitol 73: 184-96. Maizels, R. M., M. J. Holland, F. H. Falcone, X. X. Zang and M. Yazdanbakhsh. 1999. Vaccination against helminth parasites--the ultimate challenge for vaccinologists? Immunol Rev 171: 125-47. McCarthy, J. S., Wieseman M., Tropea J., Kaslow D., Abraham D., Lustigman S., Tuan R., Guderian R.H., Nutman T.B. 2001. Onchocerca volvulus fructose 1,6 bisphosphate aldolase: a parasite glycolytic enzyme as target for a protective human immune response. in press. Nanduri, J. and J. W. Kazura. 1989. Paramyosin-enhanced clearance of Brugia malayi microfilaremia in mice. J Immunol 143: 3359-63. Nasarre, C., U. R. Rao, S. U. Coleman and T. R. Klei. 1997. Effect of gamma radiation on Brugia L3 development in vivo and the kinetics of granulomatous inflammation induced by these parasites. J Parasitol 83: 1119-23. Nutman, T. B., C. Steel, D. J. Ward, G. Zea-Flores and E. A. Ottesen. 1991. Immunity to onchocerciasis: recognition of larval antigens by humans putatively immune to Onchocerca volvulus infection. J Infect Dis 163: 1128-33. Oothuman, P., D. A. Denham, P. B. Mcgreevy, G. S. Nelson and R. Rogers. 1979. Successful vaccination of cats against Brugia pahangi with larvae attenuated by irradiation with 10 krad cobalt 60. Parasite Immunol 1: 209-16. Peralta, M. E., K. A. Schmitz and T. V. Rajan. 1999. Failure of highly immunogenic filarial proteins to provide host-protective immunity. Exp Parasitol 91: 334-40. Rao, Y. V. B. G., K. Mehta and D. Subrahmanyam. 1977. Litomosoides carinii: effect of irradiation on the development and immunogenicity of the larval forms. Exp Parasitol 43: 39-44. Steel, C., A. Guinea and E. A. Ottesen. 1996. Evidence for protective immunity to bancroftian filariasis in the Cook Islands. J Infect Dis 174: 598-605. Steel, C., R. J. Limberger, L. A. Mcreynolds, E. A. Ottesen and T. B. Nutman. 1990. B cell responses to paramyosin. Isotypic analysis and epitope mapping of filarial paramyosin in patients with onchocerciasis. J Immunol 145: 3917-23. Storey, D. M. and A. S. Al-Mukhtar. 1982. Vaccination of Jirds, Meriones unguiculatus, against Litomosoides carinii and Brugia pahangi using irradiate larvae of L. carinii. Tropenmed Parasitol 33: 23-4. Taylor, M. J., N. Abdel-Wahab, Y. Wu, R. E. Jenkins and A. E. Bianco. 1995. Onchocerca volvulus larval antigen, OvB20, induces partial protection in a rodent model of onchocerciasis. Infect Immun 63: 4417-22. Taylor, M. J., R. E. Jenkins and A. E. Bianco. 1996. Protective immunity induced by vaccination with Onchocerca volvulus tropomyosin in rodents. Parasite Immunol 18: 21925. Taylor, M. J., R. P. Van Es, K. Shay, S. G. Folkard, S. Townson and A. E. Bianco. 1994. Protective immunity against Onchocerca volvulus and O. lienalis infective larvae in mice. Trop Med Parasitol 45: 17-23. Townson, S., A. E. Bianco, M. J. Doenhoff and R. Muller. 1984. Immunity to onchocerca lienalis microfilariae in mice. I. Resistance induced by the homologous parasite. Tropenmed Parasitol 35: 202-8.
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Wang, S. H., H. J. Zheng, S. Dissanayake, W. F. Cheng, Z. H. Tao, S. Z. Lin and W. F. Piessens. 1997. Evaluation of recombinant chitinase and SXP1 antigens as antimicrofilarial vaccines. Am J Trop Med Hyg 56: 474-81. Ward, D. J., T. B. Nutman, G. Zea-Flores, C. Portocarrero, A. Lujan and E. A. Ottesen. 1988. Onchocerciasis and immunity in humans: enhanced T cell responsiveness to parasite antigen in putatively immune individuals. J Infect Dis 157: 536-43. Weil, G. J., B. W. Li, F. Liftis and R. Chandrashekar. 1992. Brugia malayi: antibody responses to larval antigens in infected and immunized jirds. Exp Parasitol 74: 315-23. Wong, M. M., H. J. Fredericks and C. P. Ramachandran. 1969. Studies on immunization against Brugia malayi infection in the rhesus monkey. Bull Wld Hlth Org 40: 493-501. Yates, J. A. and G. I. Higashi. 1985. Brugia malayi: vaccination of jirds with 60cobaltattenuated infective stage larvae protects against homologous challenge. Am J Trop Med Hyg 34: 1132-7. Yates, J. A. and G. I. Higashi. 1986. Ultrastructural observations on the fate of Brugia malayi in jirds previously vaccinated with irradiated infective stage larvae. Am J Trop Med Hyg 35: 982-7. Yutanawiboonchai, W., R. A. Brigandi, H. L. Rotman and D. Abraham. 1996. Structural and molecular specificity of antibody responses in mice immune to third stage larvae of Onchocerca volvulus. Parasite Immunol 18: 95-102. Zhang, S., B. Li and G. J. Weil. 1999. Human antibody responses to Brugia malayi antigens in brugian filariasis. Int J Parasitol 29: 429-36.
INDEX adenolymphangitis 4, 76, 81, 128, Aedes aegypti 9, 11, 12, 13, 14, 15, 16 Aedes 2, 9, 11 AIDS 50 albendazole 5, 51, 52, 59, 60, 63, 64, 67, 68, 69, 70, 155, 156, 157, 159, 161, 163 An. stephensi 16 Anopheles 2, 11, 80 Antibiotics 145 APOC 50, 51, 52, 159 Armigeres subalbatus 11, 13, 14 Ascaris 1, 53 asymptomatic state 3 Brugia (B.) malayi 1, 9, 31, 32, 53, 75, 98, 100, 111, 128, 133, 135, 143, 167, 170, 171, 173, 174, 175, 176, 177, 178 Brugia (B.) pahangi 1, 98, 135, 171, 175, 177 Brugia (B.) timori 1, 4, 25 Brugia (B.) tupaiae 25 Bacillus thuringiensis 49 Bancroftian filariasis 175 Blackfly (Simulium) 44, 46, 53, 54, 159 Brugia 1, 2, 3, 9, 11, 21, 24, 25, 31, 32, 33, 34, 37, 38, 53, 75, 77, 98, 100, 103, 111, 128, 133, 135, 143, 163, 167, 168, 170, 171, 173, 174, 175, 176, 177, 178 brugian filariasis 1, 134, 178 Central America 49 Cercopithifilaria 24 chyluria 3 class II loci, 79 cryopreserved 11
Culex 2, 10, 11, 15, 59, 62 Cw6, 80 Cx. pipiens pipiens 12, 13 Cx. pipiens quinquefasciatus 16 Diethlycarbamazine (DEC), 5, 7, 48, 49, 52, 54, 60, 63, 64, 67, 68, 69, 70, 71, 116, 130, 132, 133, 149, 156,157,159 Dirofilaria 12, 144, 150, 168, 172 DQA1*0101-DQB1*0501, 80 DR3, 79 dynamic model 88, 89, 92, 93 elephantiasis 3, 4, 76, 77, 79, 89, 91, 100, 114, 129, 147, 150, 161, 164 embryonic eggshell 2 epididymitis 4 Esslingeria 26 Expatriates 128 exsheathment 10 filaria dance sign 77 funiculitis 4 Genome projects 32 Green fluorescent protein (GFP) 16 hemocoel 10, 11, 13 hemocytes 13 hemolymph 9, 10, 13, 14, 15, 16 Hermes 16 HIV-1 47 HLA B15 79 HLA DQ5 79 HLA genes 79 Homo sapiens 1 hydrocele 3, 4, 5, 81, 89, 92, 98, 127, 147 hyperresponsiveness, 88
180
IL-4 102, 111, 112, 113, 115, 116, 128, 129, 130, 131, 132, 133, 135, 136, 137, 171, 176 IL-5 90, 91, 102, 103, 111, 112, 113, 115, 116, 128, 129, 135, 136, 137, 171, 176 immunological model, 89 in utero exposure 97, 98, 99, 100, 101, 102, 103, 131 in utero tolerance 99, 102 Ivermectin (IVM) 5, 7, 8, 43, 47, 50, 51, 52, 53, 54, 59, 60, 63, 64, 67, 68, 69, 70, 71, 116, 147, 148, 156, 157, 158, 159, 163 L3 2, 4, 33, 36, 38, 61, 103, 109, 110, 111, 112, 113, 135, 136, 137, 143, 170, 172, 174, 176, 177 L4 2, 4, 33, 35, 36, 38, 109, 110, 112,113,135,137 Litomosoides 24, 112, 144, 176, 177 Loa loa 2, 52, 79, 158, 163 Loa 1, 2, 21, 52, 79, 158, 163 lymphatic filariasis elimination program (LFEP) 156 Lymphatic filariasis 59, 97, 160, 167 M. (E.) perstans 26 M. (E.) streptocerca 26 M. (M.) ozzardi 26 Mansonella rodhaini 27 Mansonella 1 Mansonella 21, 26, 27 Mansonia 2, 11 Mass drug administration (MDA), 155 Mazzotti test 48 Meningonema perruzzi 27
Meriones unguiculatus (jird) 9 mf sheath 13 Microfilaria semiclarum 27 Microfilaria vauceli 27 microfilariae 2, 5, 10, 11, 13, 14, 21, 24, 25, 27, 36, 38, 43, 47, 48, 49, 50, 51, 52, 53, 62, 63, 64, 67, 70, 88, 90, 92, 100, 109, 111, 113, 115, 116, 117, 118, 129, 137, 148, 157, 172, 173, 175, 176, 177 Minos 16 mitochondrial genome 38, 39, 79 Mosl 16 murine model 79, 135, 99, 102, 103 Nakalanga 46 nocturnal periodicity 2 O. volvulus 2, 8, 32, 38, 43, 102, 103, 156, 167, 168, 175, 176, 177, 178 O. gibsoni 27 O. ochengi 27, 146 O-150 47, 48 OCP 44, 47, 49, 51,52, 54, 159 Og4C3 8, 80, 100 Onchocerca ochengi 46, 143 Onchocerciasis Elimination Program of the Americas (OPEA) 51 Onchocerciasis 27, 43, 44, 50, 51, 93, 156, 159, 167, 178 Onchocercidae 21, 22, 23 ONCHOSIM 48 Oswaldofilaria 22 Papua New Guinea 7, 80, 81, 99, 133, 169
patent infection 88, 89, 90, 93, 98, 103, 110, 111, 169, 170, 173 PCR 33, 35, 37, 38, 45, 48, 54, 80, 145, 147, 149, 169 PCR-based tests 45 Piggybac 16 Plasmodium gallinaceum 16 Procyon 26 River blindness 43 S. rasyani 53 Sandnema 26 sheath 2, 10, 89, 90, 92, 94 SIMON 49 Strongyloides 53 suramin 49, 156, 158 T-cell hyporesponsiveness 88 Tetrapetalonema 26 The Bill and Melinda Gates Foundation 160 The Carter Center 157, 162 transmigrants 93, 128 transmission intensity 4, 5, 80, 81, 93 tropical (pulmonary) eosinophilia (TPE) 3 Tupainema 26 ultrasound examination 3 ultrasound 3, 5, 77, 128, 169, 176 Ursus 26 Vector control 49, 53 W. bancrofti 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 25, 32, 33, 52, 62, 75, 76, 77, 80, 81, 87, 99, 100, 109, 114, 136, 156, 167, 168 W. kalimantani 25 Wolbachia 6, 8, 25, 38, 39, 52, 81, 94, 103, 114, 115, 116, 143,
181 144, 145, 146, 147, 148, 149, 150, 163 Wuchereria 1, 2, 7, 8, 9, 10, 21, 25, 32, 52, 62, 75, 99, 100, 114, 156, 167, 168
