Immune response of fish to parasitic protozoa

ParasitologyToday, vol.3, no. 6, 1987

186

Immune Response of Fish to Parasitic Protozoa P,T.K. Woo Epizootic outbreaks of fish diseases are increasingly common as a result of intensive aquaculture, fish farming and sea ranching. Very few drugs are available for treatment or prophylaxis against fish diseases, and development of such compounds is inhibited by different national regulations governing the use of chemicals in.fish for human or animal consumption. Alternative approaches are urgently needed. But although the taxonomy and biology offish parasites have been extensively studied, relatively little is known about protective immunity in fish and the effects of parasites on the piscine immune system I. In this article, Patrick Woo discusses the immune responses of fish "to parasitic protozoa, showing that vaccination is a viable control strategy, and stressing the need for a coordinated global research programme on fish diseases.

Like other vertebrates, fish have both specific and non-specific responses against pathogens. Non-specific (innate) responses occur prior to the activation of specific immune responses; they include agglutinins, lysins, complement, lysozyme and C-reactive protein-like substance. These are effective in the mucous on the body surface and/or in the blood. Interferon in the blood inhibits viral replication - i t s role in parasitic infections is unknown. Nonspecific phagocytic cells occur, including monocytes, granulocytes and thrombocytes; non-specific cytotoxic cells have also been found. Fish produce immunoglobulins against particulate and soluble antigens. In all cases, intraperitoneal injection induces a higher antibody response than spraying or immersing the fish in antibody-containing water. Unlike the mammalian immune system, temperature is important in eliciting primary and anamnestic (immune memory) responses. Fish do not have lymph nodes - their antibodyproducing lymphocytes are in the spleen and anterior kidney but not in the thymus. The distinction between B- and T-lymphocytes is thus not as clear as in mammals. Specific phagocytosis by peritoneal cells and peripheral blood leucocytes is enhanced by antibody and complement as in mammals, and piscine complement can be activated by both the alternate and classical pathways. It is similar to mammalian complement in that free calcium and magnesium ions are needed for lysis, and lytic activity can be reduced by prior incubation in zymosan. Fish complement is also heat labile and some of its components show compatibility with mammalian components.

Acquired Immunity against Protozoa Most of what is known about the immunological response offish to parasites is confined to studies of some of the more common pathogenic protozoa (Box I ), many of which can cause mortality in fish. In some fish species, lack of susceptibility to certain parasites is due to non-specific complement mediated lysis - for example C. catostomi (a parasite of white suckers) and C. salrnositica (a parasite of salmonids) are both lysed by plasma of insusceptible fish such as goldfish7.8. In many cases however, susceptible fish that recover from parasite infection show strong levels of specific acquired immunity. Catfish that recover from Ichthyophthirius infection show protective immunity for up to 8 months 9. This is due to immobilizing antibodies in the mucous, as well as in the blood,,which is thought to immobilize the infective theront and prevent its entry. Fish injected with parasite and adjuvant gave high antibody responses, and all vaccinated fish survived a lethal challenge9. Juvenile carp could also be protected against challenge, but although there was no mortality, some of the immunized fish showed signs of infection on their fins; immune fish could be made again susceptible by injection of corticosteroid ~0. Protective immunity against Ichthyophthirius appears directed against the parasite's cilia. Catfish injected with cilia of a free-living flagellate, Tetrahymena pyriformis, were protected against Ichthyophthirius and showed lower mortality on challenge than those injected with Ichthyophthirius .ciliaI I. Inoculation of 5 I~g

of cilia protein conferred protection t2, but catfish inoculated with cilia-free antigen .were not protected, nor was protection induced by oral drench or topical application t3. However, trout were immunized by immersion in either T. thermophi/ia or their ciliat4; protection peaked 10 weeks after exposure, and these fish were also more resistant to the ectoparasitic flagellate Ichthyobodo necatrix. Protective immunity against trypanosomes is by humoral antibody. For exampie, mortality is often high in goldfish infected with T. danilewskyi but, depending on the size of the inoculum, 40-90% of fish can recover and resist further challenge3. Trout infected with C. sdmositica typically show two phases of infection. It appears that phagocytosis by peritoneal macrophages is most important during the chronic phase Is, but during the acute phase, parasitaemia can be controlled by complement fixing antibody. Fish that recover from infection resist challenge 16, and passive transfer of lymphocytes from immune fish with antibody can confer partial protection w. Complement-fixing antibody and cellular responses are also shown by summer and winter flounders infected with C. bullocl4, and the humoral antibody may be responsible for the spring decline in prevalence of the parasite 18,t9. Recovered fish are protected from challenge20.

Immunodepression due to Protozoan Infections At present there is insufficient evidence for piscine immunosuppression (i.e. total immunological dysfunction), but their immunological response can be decreased (immunodepression) by several factors including heavy metals, antibiotics, Aeromonas, and low temperatures. Cortisol, corticosteroids and deficient diets (low protein or absence of pantothenic acid) also decrease the immune response. In trout, infection with C. sdlrnositicd depresses the immune response to sheep red blood cells 16 or to Yersinia2k Humoral depression against Y. ruckeri was evident two weeks after parasite infe~on, ~) 1987,ElsevierPublications,Cambridge0169~1758187/$02.00

Parasitology Today, vol. 3, no. 6, 1987 and cellular immunity also appeared depressed. Infected trout exposed to Y. ruckeri suffered higher mortality than those infected with either pathogen alone. Glugea infections also cause depression of the inflammatory response z22. The total immunoglobulin of winter flounder was decreased by injection with G. stephani either as spores or spore homogenate 23. A further decrease fo~lowed a second injection, but there was no decrease in immunoglobulin levels if the fish were also inoculated 2.-3 times per week with indomethacin. These authors suggest that the spores either affect immunoglobulins directly or stimulate macrophages to release immunodepressive factors such as prostaglandins. P r o t e c t i o n against P r o t o z o a n Pathogens

Prote~on of fish against pathogenic parasites would be best achieved by an integrated approach, making use of chemoprophylaxis and chemotherapy, immunization, selective breeding of resistant stocks, environmental manipulation and quarantine if outbreaks occur. In the case of immuniza*don, the objective is to elicit a good response on challenge, so a long-lasting immunological 'memory' is more important and effe~ive than a strong primary response. Immunity can be acquired by allowing infection to run its course; protection on recovery is long-lasting and mortality to subsequent heavy challenge is reduced. Thus, immunization by usi,ng small primary infections and/or less virulent parasite strains, needs further study as a prophylactic measure. Moreover the possibility of cross-protect on between non-pathogenic and pathogenic parasites with similar antigens may also be rewarding. At present, there is no 'live' attenuated nor killed vaccine against protozoal diseases of fish, although they are available for some viral and bacterial diseases 24. Irradiated or killed protozoal vaccines did not confer protection 20,25, but the successful use of crude antigen from Tetrahymena to protect against ichthyophthiriasis shows that this is a viable possibility l 1-14. Many modern approaches to vaccine development against parasites of medical and veterinary importance could be adapted for fish parasites. For example, protective antigens can be characterized, isolated, purified, and synthesized biochemically or using recombinant D N A technology. But in the case of fish parasite vaccines, delivery can be simplified by oral delivery in the food - a practical approach to antigen delivery to

187

Box. I. Some Common Parasitic Protozoa of Fish

I Ichthyophthirius (Order: Hymenosto- I. multifiliis is a common ectoparasitic ciliate with world-wide distribution. It has a free-swimming infective matida) (Fig. I)

stage (theront), produces lesions on the body surface and gills, and causes massive mortality if untreated. Its pathogenicity increases with water temperature.

2 Trypanosoma (Order: Kinetoplastida) (Fig. 2) - blood flagellates. Like their better known relatives that parasitize terrestrial vertebrates, aquatic trypanosomes are often transmitted by blood-sucking invertebrates, such as leeches. Most piscine trypanosomes are considered non-pathogenic, but T.. variabile causes inflammation of the brain, fatty degeneration, anaemia and eosinophilia; T.

rF

also causes pathological lesions and anaemia, often resulting in high mortal-

danilewskyi

ityZ.3. 3 Cryptobia (Order. Kinetoplastida) (Fig. 3) - species of Cryptobia have been described from blood, intestine, and body surface both of freshwater and marine fish. Some are nonpathogenic (e.g.C. catostom O, while others cause diseaseand high mortality in susceptible fish. Clinical signs of crytobiosis are anaemia, exophthalmia, and abdominal distension with ascites4.

4 Glugea (Order: Pleistophorida) (Fig. 4) intracellular microslx~ridia with infective spores. G. stephani infects many species of flatfish in North America and EuropeS; it progressively replaces the submucosa of the intestine and rectum, resulting in massive mortality6.

Fig. I. (Top). Theront 0flchthyophthirius multifiliis. Fig. 2. (Middle left). Trypanosoma danilewskyi.

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Fig. 3. (Middle right). Cryptobia salmositica.

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k-

Fig. 4. (Bottom). Spore of Glugea arnericanus (courtesy ofJ. Protozool.). fish of any size that requires little effort and imposes no stress on the fish 26. Vaccines could also I~e delivered by hyperosmotic immersion 27, although this is more stressful, or by spraying the antigen under pressure as the fish swim through a shallow channel. Intraperitoneal injection of antigen is most effective, but is labour intensive, time consuming, stressful and unsuitable for small fish. Alternatives to immunization could include selective breeding of refractory fish stocks - for example mortality of different salmon stocks after experimental infection with C. salmositica varies from 0 to 100%, which may be due to genetic factors 4. Manipulation of the environment can also help control infections, particularly

by changing the water temperature (which has a direct effect on the parasites). Trout infected with C. salmositica lost their infections or had greatly reduced parasitaemia when the water temperature was raised from 11-20°C28; similarly there was no mortality in infected sockeye salmon after the temperature was raised 29. In contrast, the proliferation of G. stephani was arrested when the infected fish were returned to colder waters 30. Although various drugs and chemicals are effective against Ichthyophthirius, there are no therapeutic or prophylactic drugs against the majority of fish endoparasites. Development of such drugs by screening and testing would be an expensive and time-consuming pro-

188 cedure with no guarantee of success. Fish for human or animal consumption can only be treated with chemicals in accordance with national regulations - which vary widely between countries. For these reasons, alternative approaches to the control of fish parasites are urgently needed, There is now increasing evidence that the fish immune system plays an important role in the control of fish diseases, and with further research this could be usefully exploited in the protection and further commercialization of fish stocks. Already, prospects for vaccination against Ichthyophthirius appear promising, and there is reason to hope that other vaccines could soon be within reach. Moreover, better understanding of the fish immune system can suggest ways in which the immune response can be boosted through suitable environmental modification. But in spite of recent research, there remain many gaps in our understanding of the fish immune system, It is similar in many respects to that of mammals (e.g. production of specific immunoglobulins, phagocytic and cytotoxic cells) but has its own distinctive features (e.g. reduced number and structural differences in immunoglobulin classes, absence of lymph nodes). O f key importance is the depression in immune response caused by even sublethal infections of some parasitic p r o t o z o a this probably exposes the fish to concomitant infections, yet we know little about the effects of multiple infections, nor about the interaction between environmental and dietary factors with infection. We know nothing about immunomodulation due to other common fish parasites such as platyhelminths, copepods, acanthocephala and nematodes. But given the importance of marine and freshwater fish as a source of protein in many countries, a better understanding of fish parasites and their control becomes essential, This could best be achieved by a coordinated international research programme - similar perhaps to the W H O Special Programme for Research and Training in Tropical Diseases. I hope this communication will help to stimulate discussion on support for such an approach for fish diseases.

References

I Woo, P.T.K.andJones,S.R.M.(1987)in Current Concepts in Parasitology (R.C. Ko, ed.), University of Hong KongPress(in press) 2 Dykova, I. and Lom, J.(I 979)J. Fish Dis. 2, 38 I 390 3 Woo, P.T.K.(198 I) Parasitology 83,343-346 4 Woo, P.T.K.(I 987)Adv.ParasitoL 26, 199-237 5 Sprague,V. (1977)in Systematics of the Microsporida (L. Bellaand T.C. Cheng,eds), pp. 31334, PlenumPress,New York

Parasitology Today, vol. 3, no. 6, 1987

6 Takvorian, P.M.and Call, A. (1981)J. Fish Biol. 18,491-501 7 Bower, S.M. and Woo, P.T.K (1977) Exp Parasitol. 43, 63-68 8 Wehne~, S.D. and Woo, P.T.K.(1980)J Wildf Dis. 16, 183 187 9 Hines,R.S.and Spira, D.T. ( 1974)J.Fish Biol. 6, 373-378 10 Houghton,G. and Matthews, R.A. (1986) Vet tmmunol. Immunopathot. t 2, 413-419 II Goven, B.A, Dawe, D.L. and Gratzek, J.B. ( 1980)J.Fish Biol. 17, 31 I-316 12 Goven, B.A, Dawe, D.L. and Gratzek, J.B. ( 1981)Aquaculture 23,269 273 13 Pyle,S.W. and Dawe, D.W (1985)Aquaculture 46, 1-10 14 Wolf, K. and Martin,M.E.(1982)Can.j. Fish. and Aquat. Sci. 39, 1722 1725 15 Woo, P.T.K.(1979)Exp. Parasitol. 47, 36-48 16 Wehnert, S.D. and Woo, P.T.K.(1981) Cand. Sac. Zool. Bulletin I I, 100 (abstract) 17 Jones,S.R.M.and Woo, P.T.K.(I 986) Parasitology - Quo Vadit? Handbook 6th Int. Cong. Parasitol. Brisbane,Australia,p. 333 (abstract) 18 Sypek, J.P. and Burreson, E.M. (1983) Dev. Comp. Immunof. 7, 277-286 19 Sypek, J.P. and Howe, A.B. (1985) Int. Meet. Fish lmmunol, p. P3 (abstract)

20 Burreson, E.M.and Frizzell,L.J.(I 986) Vet. Immunol. Immunopathol. 12, 395-402 21 Jones, S.R.M., Woo, P.T.K. and Stevenson, R.M.W. (I 986)J. Fish Dis. 9, 431-438 22 Dykova, I., Lom, J. and Egusa,S. (1980) Fotia Parasit. 27, 213-216 23 Laudan, R., Stolen,J.S. and A. Call, A. (1986) Vet. Immunol. Immunopathol. 12,403-412 24 Anderson,D.P.,Dorson, M and Dubourget, P. (eds) (1982) Antigens of Fish Pathogens - Development and Production for Vaccines and Serodiagnostics (1982) Collection Fondation

Marcel Merieux, Lyon,273 pp. 25 Lom~ J. (1979) in Biology of the Kinetoplastida vol. 2 (W.H.R. Lumsdenand D.A. Evans,eds), pp. 269 337, Academic Press,London 26 Fryer, L. et al. (1976)Fish Pathot. I0, 155-164 27 Antipa, R. and Amend, D.F. (1977)J. Fish. Res. Bd. Canada 34,203-208 28 Woo, PT.K, Wehnert, S.D. and Rodgers, D. (1983) Parasitology 87, 385 392 29 Bower, S.M. and Margolis,L. (I 985)j. Fish Dis. 8, 25-33 30 Olson, R.E ( 1981)j. Wltdl. Dis. 17, 559-562 Patrick Woo is at the Department of Zoology, University of Guelph, Ontario N I G 2 W l , Canada.

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