- Email: [email protected]
Equine Immunity to Parasites
IMMUNOLOGY
0749-0739/00 $15.00
+ .00
EQUINE IMMUNITY TO PARASITES Thomas R. Klei, PhD
With the exception of Sarcocystis neuron, helminths are the most significant parasites of horses in developed countries, and these are the focus of this review. Immune responses directed against helminth parasites are important in controlling these infections and the disease onditions that they produce. In some instances these immune responses also may significantly exacerbate disease or regulate immune responses to other stimuli. The implied intent of immunologic investigations of infectious agents in any host is to improve the feasibility of the development of vaccines, improve diagnostic procedures, and eradicate disease. Relatively little is know about the immune response to helminth parasites, and it is not surprising that vaccines or diagnostic procedures directed against these agents do not exist at this time. Immunity to helminth parasites is generally judged by a significant reduction in parasite burden in immune individuals. Evidence for a true age resistance independent of acquired immunity is lacking. 12 Thus, the conventional thought is that horses repeatedly exposed to parasites in their environment acquire a specific immunity through this repeated exposure. Helminth parasites can be divided into three categories on the basis of speed and level of immune resistance that they induce. In the first instance, a strong immunity is rapidly acquired to infections of Strongyloides westeri and Parascaris equorum, and these parasites rarely occur as adults in yearling horses. In the second category containing the strongyle nematodes, immunity is slow to develop and incomplete, and some level of infection persists throughout the life of most individuals.
From the Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, and the Department of Veterinary Science, Louisiana Agricultural Experiment Station, Louisiana State Universi~ Baton Rouge, Louisiana
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In the third category, acquired resistance at any level has not been demonstrated and mature horses are believed to harbor as many parasites as immature individuals. Parasites such as stomach bots (Gasterophilus spp), tapeworms (Anoplocephala spp), and pinworms (Oxyuris equi) fall into this group. It is possible with further investigation that acquired immunity will be demonstrated against these parasites and they will thus be grouped with the strongyle nematodes. In instances in which immunity does develop it is most often of a concomitant type. Thus, mares are immune to the establishment of adult S. westeri in the small intestine yet harbor arrested larvae that are activated postpartum and transmitted in the mares' milk to foals. Similarly, horses harbor adult and larval stages of S. vulgaris, which are not affected by the ongoing immune response and yet immune responses prevent the establishment of ingested third stage. Mechanisms associated with these protective immune responses are only partially understood, 'and only the strongyles have been studied in any detail. ll IMMUNITY AGAINST STRONGYLES Strongylus Species
Strongyle nematodes are the most ubiquitous and important internal parasites of horses. With the advent of efficacious anthelmintics the significance of the large strongyles, parasites in the genus Strongylus, has been reduced. s Nonetheless, S. vulgaris remains an excellent model to study mechanisms of equine immunity against helminth parasites and to define certain basic immunologic principles in the horse. There are several technical advantages to using S. vulgaris infections to study the equine immune response to nematodes. Monospecific infections of Strongylus spp can be produced by surgical implantation of adults, and these ponies remain infected for years,2S providing a ready source of feces for third stage infective larvae (L3) culture. This is, as yet, not possible with other equine strongyles. Methods for the in vitro culture of late stage L3 and L4 have been developed1 and provide a source of these stages. S. vulgaris is a highly pathogenic gastrointestinal nematode, and its biology and host interactions have been studied and reviewed. l l In nonimmune equines, L3 are ingested with herbage, exsheath in the intestine, and penetrate the mucosa. These larvae migrate in the submucosa for 4 to 7 days before entering arterioles. At this point, they molt to the fourth stage (L4) and migrate through the mesenteric arteries toward the aorta. Upon reaching the ileo-cecal-colic artery and the cranial mesenteric artery, many of these larvae cease their migration and develop into the fifth stage (L5), which is a neotenic adult form. Upon molting, the L5 migrate distally within these vessels to the cecum and proximal ventral colon where they enter the lumen, develop to adults, mate, and produce eggs that are passed in the feces. This life cycle is prolonged and takes 4 to 6 months to complete. From an experimental
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standpoint, however, the easily demonstrated clinical signs of infection, including pyrexia, anorexia, depression, and abdominal pain, appear within the first week after L3 ingestion and are near a maximal state by 14 days after infection (DPI). These signs are absent in immune ponies, and larvae appear to be killed by the immune response before entry into major arteries or are blocked from entry into the mucosa. If the latter occurs, it is incomplete in that some lesions are found in vaccinated ponies. 29 Although as few as 50 L3 per week for 4 weeks have been shown to be lethal,18 the clinical signs of infection are generally dose related, and infections of 500 to 1000 L3 rarely are fatal and produce only mild colic. This model system has proved to be manipulated easily, and lymphocytes collected from the peripheral blood and surgically from cecal lymph nodes during the 14 to 16 day postchallenge period have been used to recover significant immunologic data. 35 Ponies infected experimentally develop a strong concomitant immunity to reinfection either to a single16 or multiple challenge. 17 Experimental immunization is easily accomplished using two doses of radiation attenuated L3 and avoids the experimental difficulties of massive arterial lesions induced by unattenuated larvae. 16 1trus vaccination regimen produces a strong protective resistance to challenge infection, which reduces challenge larval burdens, eliminates clinical signs of infection, and prevents colic. 15,16 Vaccination of ponies with irradiated L3 induces protection from S. vulgaris infection but not Strongyloides edentatus infection17 and is thus species specific. Vaccination is effective against experimental or field challenges and lasts for at least 9 months. This vaccination protocol has been used to experimentally characterize immune responses to challenge infections. Cytokine mRNA levels before and during challenge infections of vaccinated ponies have been measured in this model using a quantitative Reverse Transcription-Polymerase Chain Reaction procedure. 36 Vaccination induces a strong in situ expression of IL-4 and IL-5 with little interferon gamma (IFN-~), a cytokine profile typical of a dominant type 2 response. 35 Differences between levels of IL-2 and IL-10 using this method were not detected before or after challenge infection. This in situ profile of cytokine expression occurs in both peripheral blood mononuclear cell (PBMC) and cecal lymph node cells (CLN). Although this indicates a lack of completed compartmentalization of this response to the lymphoid tissues of the bowel, expression of IL-4 is first seen in CLN. The most marked increase seen during this challenge infection is the anamnestic IL-5 response seen in vaccinated ponies but not in ponies that received only the challenge infection. This anamnestic increase in IL-5 corresponds to a similar increase in circulating and tissue eosinophil numbers. Similar cytokine responses occur in PBMC and CLN cultured in vitro with adult worm antigens. 37 Subsequent experiments have demonstrated that this expression of IL-4 and IL-5 occurs in CD4 + cells and not CD4- cells separated from these cultures. Immunization of ponies with somatic extracts of adult worms in RIBI adjuvant (Ribi ImmunoChem Research, Inc, Hamilton, MT) produces a strong antibody response to parasite antigens but exacerbates clinical signs and arterial lesions resulting from
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challenge infections. 29 This RIBI immunization protocol does not induce a type 2 cytokine profile but rather a marked production of IFN-')', which is accompanied by a lack of protection (unpublished results). Although incomplete, these observations support the contention that a type 2 Tcell response is essential for the expression of protective immune response against this parasite. The dominant type 2 response seen in immune animals is consistent with the type 2 cytokine profiles associated with protective immunity in several murine models of gastrointestinal nematode infections and it is interesting to compare the equine responses with those described in the murine mode1. 41 Although a helper 2T (TH 2) cell pattern of cytokine responses is dominant in mice resistant to gastrointestinal helminths, the roles of specific cytokines and effector mechanisms involved are less clear and results vary considerably with the parasite-host system examined. 41 The following examples demonstrate the diversity of results reported from murine systems. Protective resistance of mice against Heligmosomoides polygyrus is abrogated by treatment with anti-IL-4 or anti-IL-4 receptor antibodies40; however, antibodies to IL-3, IL-4, and IL-5, which eliminate mast cell hyperplasia, eosinophilia, and IgE, have no effect on the primary course of infection of Nippostrongylus brasiliensis. 24,44 In other studies, anti-IL-5 treatment that abrogates eosinophilia does not alter acquired protective resistances to Trichinella spiralis 7 or Toxocara canis 31 infections but does eliminate protection against migratory larvae of Strongyloides venezuelensis 19 and Angiostrongylus cantonensis. 33 As indicated, IL-5 expression and eosinophilia are consistent features of the protective immune response to S. vulgaris. In addition, infections of S. vulgaris activate eosinophils and neutrophils in vitro,3 and activated but not normal eosinophils kill L3 in vitro in an antibody-dependent manner,13 whereas other cells do not. Furthermore, antigen stimulation of PBMC cultures from immune but not nonimmune ponies produces a cytokine (or cytokines) in the supernatant that is chemotactic for eosinophils. 5 Interestingl~ this was not the case in similar cultures stimulated with S. edentatus antigen, which correlates with the species-specific nature of the protective immune response previously described. Immunized ponies show an anamnestic eosinophil response that is absent in nonimmune naive or antigen sensitized individuals. 28,48 In recent studies, intestinal eosinophil counts were significantly elevated in vaccinated and protected ponies but not in nonimmune animals 16 days after infection (unpublished results). Interestingl~ increases in mast cell numbers were not seen in the same tissues. These increased eosinophil responses correspond to the increase seen in IL-5 gene expression in vaccinated ponies during the current studies (unpublished results). Although solely correlative in nature, evidence supports the hypothesis that eosinophils are important in the protective immunity against this helminth in the equine. Antibodies generated during the infection are also likely to be important in the protective immune response to S. vulgaris. Vaccinated ponies challenged with L3 have antibodies to L3 surface not seen in nonimmune ponies. 14 These antibodies are specific in that they do not
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react with the L3 surface of the closely related parasite S. edentatus, again supporting the species-specific nature of the response. These antibodies also mediate an in vitro adherence of eosinophils to L3. 13 The speciesspecific antibody response to the surface of the L3 is, however, lost as these larvae molt to the L4 stage and immune serum reacts with L4 of both S. vulgaris and S. edentatus. 29 Other observations also have shown an increase in the antibody response to soluble somatic extracts of adult worms (SAWA) in vaccinated animals as compared with nonvaccinated controls. 29 The isotype showing a significant increase after challenge in vaccinated animals was IgG(T). The isotypic nature of this antibody response to the L3 surface, however, is at this time unknown. Although these observations support the potential role of antibody in protective responses, antibody alone is insufficient to protect against challenge infection. Transfer of 1.5 L of hyperimmune serum per pony while transferring significantly high titers of L3 surface-specific antibody failed to protect against challenge infection. Collectively, these data suggest that the protective immune response to S. vulgaris is species specific, directed at the L3 stage, and mediated by an antibody-dependent eosinophil killing mechanism. It i likely that antigen-specific CD4 + TH 2 cells are required to induce the necessary cellular effector mechanisms involved in this response. The cross regulatory effects of type-1 and type-2 cytokines have not been demonstrated in the equine. Nonetheless, it may be hypothesized that type-2 responses induced by helminth infections may affect the induction or maintenance of type-1 mediated immune responses. Cyathostomes
With the chemotherapeutic control of Strongylus spp, other strongyle nematode parasites, specifically the small strongyles or cyathostomes,ll have been recognized as more relevant to equine health than previously presumed and have gained prominence. These parasites have been associated with poor growth and development, colic, and diarrhea. 21, 23, 30, 38, 39 Some specific differences in the immune responses to Strongylus spp and the cyathostomes likely exist, however, owing to the absence of extensive migrations away from the bowel by the latter group. Nevertheless, horses do naturally acquire an incomplete resistance to cyathostomes. 16,22 Limited data are available on the specific response directed against these parasites but field observation and experimental infections provide some insight about the nature of this immunity. Field studies of cytostome parasite burdens based on fecal egg count (FEC) data indicated that young horses are more heavily parasitized than older horses. Comparisons of FEC of yearlings and mares on the same properties 8 weeks after anthelmintic treatment further show that the distribution of FEC within the older mare population is overdispersed. 12 A pasture challenge experiment comparing the worm burdens of foals raised under parasite-free conditions with those raised
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b.y their dams on cytostome-contaminated pastures also indicated that previous exposure induces a level of resistance to reinfection. Together, these observations are generally similar to those of trichostrongyle nematodes of ruminants and suggest that horses slowly develop a resistance to reinfection and that this resistance is genetically controlled. Other data from necropsy surveys support these field observations and further suggest that some immunity is directed at the production of eggs by female worms. 12 The latter is based on comparisons of FEe with adult worm burdens in young versus old individuals. Studies using controlled experimental infections with cyathostomes for the purpose of examining the acquired immunity are few. These types of studies are limited to the use of infections with mixed cyathostome populations because monospecific infections of these parasites have yet to be established. Nonetheless, some useful data have been gathered. A comparison has been made on the outcome of single challenge infections between foals raised with the benefit of prophylactic anthelmintic treatment. 28 Observations indicated that the challenge infections induce a nonspecific expulsion of existing intestinal parasites. Nonetheless, a reduced take of the challenge infection was noted in the previously exposed individuals. Furthermore, the numbers of mucosal stages of cyathostomes was significantly reduced in animals that were previously exposed and challenged, thus suggesting that some specific immune event was involved in this phenomenon as well. Other experiments have compared the outcome of regular multiple experimental infections (10,000 L3/ day for 44 days) in ponies with different histories of parasite exposure. 27 These exposure levels included young ponies «4 years of age) raised parasite free, young ponies «4 years of age) raised on contaminated pastures; and old ponies (>7 years of age) maintained on contaminated pastures. On the basis of parasite recoveries at necrops~ observations indicated that immunity is acquired during the early stages of life, but this resistance is not as complete as that seen with increased parasite exposure. The data also suggest that protective immune responses are directed at different stages of the life cycle and that these responses are differentially expressed with increased exposure. Thus, although the total worm burden of young ponies previously exposed to parasites is significantly less than that of ponies raised parasite free, the numbers of hypobiotic early L3 (EL3) within the mucosa are not different. The levels of EL3 were significantly reduced in older ponies. ELISA measurements of circulating antibody to somatic antigens of cyathostomes in these ponies did not detect a difference between immune and nonimmune individuals. 12 Interestingl~ serendipitous measurements of cytokine mRNA in a pony undergoing adult worm expulsion suggest that a type-2 response is involved in this phenomenon. 8 Although mechanisms of protective immune responses are not know it can be hypothesized that these are directed at a number of targets. These include the incoming L3, the developing L3 and L4 stages within the mucosa, the lumenal L4 and adult stages, and egg production by adult females.
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IMMUNODIAGNOSIS
There is little necessity to diagnose individual infections of most common helminth parasites because this can be accomplished by standard fecal examination techniques. 10 It would be useful, however, for epidemiologic reasons if intensities of cyathostome infections could be estimated or if the presence of some parasites such as S. vulgaris were known. Efforts to diagnosis parasite infections by serologic means have been minimal and ineffective. 10 This is largely due to the complexity of standard helminth antigen preparations and the necessity of developing such tests. Infection of the tapeworm Anoplocephala perfoliata is an exception to the latter point. Anoplocephala perfoliata attaches to the intestinal mucosa at and near the ileocecal junction, producing ulcers and marked inflamation. The extent and severity of these lesions is related to the numbers of parasites present. The presence of these parasites has been implicated in intussusceptions of the ileocecal region. The importance of infections of A. perfoliata in the induction of equine colic, however, has been controversial and universally accepted data supporting this concept have been absent. 6 Recent efforts to diagnose these tapeworm infec 'ons and more importantly the intensity of infection have met with some success. 32 Antibodies to a doublet of protein antigens of 12 and 13 kDa in worm excretory-secretory products were identified in Western blots that were specific for A. perfoliata. These responses were limited to the IgG(T) isotype. Subsequent work used these antigens in an ELISA format to demonstrate the positive relationship of this antibody response to parasite infection intensity and colic. The use of this test in clinical and epidemiologic settings should be explored. VACCINATION
The development of vaccines against parasites has been the goal of the veterinary and medical parasitologist for decades. Early success commercializing the use of radiation attenuated larvae as a vaccine against lung worms of ruminants was encouraging. 42 This development, however, did not lead to additional commercialized products as expected. A notable scientific success and commercial failure of this type is the vaccine developed against hookworms in dogs. 26 As noted previously, vaccination of foals with irradiated L3 clearly has been shown in field studies to be feasible. The diminished occurrence of the parasite in developed areas of the world by the extensive use of avermicitin/ milbymicin anthelmintics, however, made the production of such a product unnecessary. Because of their ubiquitous distribution, importance to equine health, and the widespread prevalence of drug-resistant populations, vaccines against the cyathostomes would be particularly useful. Logical candidates for such a vaccine are molecules that are targets of the equine acquired protective immune response. As noted previously, however,
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little is know of the host or parasite factors involved in this response. In recent years attempts have been made to develop vaccines against gastrointestinal nematodes of ruminants using "hidden" antigens. These are defined as those that do not elicit an immune response during infections yet are important to worm homeostasis and can be disrupted by immune effectors such as antibody. Several of these targets have been found in the worm intestine and are promising. 34 The carbohydrate epitopes of some of these cross-react with equine strongyles including the cyathostomes. 9 It is not clear, however, if cyathostomes ingest sufficient serum to make this approach feasible in the equine. Furthermore, although this approach has been shown to be experimentally feasible for many 'years in sheep it has yet to be demonstrated in controlled field studies. 34 The usefulness of this approach requires further evaluation. It is important to note that many elements must come together for a vaccine to be a useful commercial success, and all of these are not scientific. As an example, excretory-secretory proteins of Taenia cysticerci have been shown to produce protective immunity in sheep. These have been biochemically identified, produced by recombinant DNA technologies, and shown to be an effective vaccine. Nonetheless, this has not led to a commercial product largely because of bureaucratic and/ or political reasons. 20 References 1. Chapman MR, Hutchinson GW, Cenac MJ, et al: In vitro culture of equine strongylidae to the fourth larvae stage in a cell free medium. J Parasitol 80:225-230, 1994 2. Dennis VA, Klei TR, Chapman MR: In vivo activation of equine eosinophils and neutrophils by experimental Strongylus vulgaris infections. Vet Immunol Immunopathol 20:61-74, 1988 3. Dennis VA, Klei TR, Miller MA, et al: Immune responses of pony foals during repeated infections of Strongylus vulgaris and regular ivermectin treatments. Vet Parasitol 42:8399, 1992 4. Dennis VA, Klei TR, Chapman MR: Generation and partial characterization of an eosinophil chemotactic cytokine produced by sensitized equine mononuclear cells. Vet Immunol Immunopathol 37:135-149, 1993 5. DiPietro JA, Klei TR, French DD: Contemporary topics in equine parasitology. Compendium on Continuing Education for the Practicing Veterinarian 12:713-721, 1990 6. French DD, Chapman MR: Tapeworms of the equine gastrointestinal tract. Compendium on Continuing Education for the Practicing Veterinarian 14:655-611, 1992 7. Herndon FJ, Kayes SG: Depletion of eosinophils by anti-IL-5 monoclonal antibody treatment of mice infected with Trichinella spiralis does not alter parasite burden or immunologic resistance to reinfection. J Immunol 149:3642-3647, 1992 8. Horohov DW, Swiderski CE, Robinson JA, et al: Cytokine production in equine disease. In Schijns VECJ, Horzinek MC (eds): Cytokines in Veterinary Medicine. Wallingford, Oxon, UK, CAB International, 1997, pp 167-176 9. Jasmer D~ Perryman LE, Conder GA, et al: Protective immunity to Haemonchus contortus induced by immunoaffinity isolated antigens that share a phylogenetically conserved carbohydrate gut surface epitope. J Immunol151:5450-5460, 1992 10. Klei TR: Laboratory diagnosis. Vet Clin North Am Equine Pract 2:381-394, 1986 11. Klei TR: Recent observations on the epidemiology, pathogenesis and immunology of equine helminth infections. In Equine Infectious Diseases VI, Proceedings of the Sixth International Conference, Newmarket, UK, 1992, pp 129-137
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12. Klei TR, Chapman MR: Immunity in equine cyathastome infections. Vet Parasitol, in press 13. Klei TR, Chapman MR, Dennis VA: Role of the eosinophil in serum-mediated adherence of equine leukocyte to larvae of Strongylus vulgaris. J Parasitol 68:561-569, 1992 14. Klei TR, Chapman MR, Torbert BJ, et al: Antibody responses of ponies to initial and challenge infections of Strongylus vulgaris. Vet Parasitol 12:187-198, 1983 15. Klei TR, French DO, Chapman MR, et al: Protection of yearling ponies against Strongylus vulgaris by foalhood vaccination. Equine Vet J 7:2-7, 1989 16. Klei TR, Torbert BJ, Chapman MR, et al: Irradiated larvae vaccination of ponies against Strongylus vulgaris. J Parasitol 68:561-569, 1982 17. Klei TR, Turk MAM, McClure JR, et al: Natural and acquired resistance to Strongylus vulgaris: Its associated lesions and colic. In Equine Colic Research. Proceedings of the Second Symposium. 1986, pp 14-18 18. Klei TR, Turk MAM, McClure JR, et al: Effects of repeated experimental Strongylus vulgaris infections and subsequent ivermectin treatments on mesenteric arterial pathology in pony foals. Am J Vet Res 51:654-660, 1990 19. Korenasa M, Hitoshi Y, Yamaguchi N, et al: The role of IL-5 in protective immunity to Strongyloides venezuelensis infection in mice. Immunology 72:502, 1991 20. Lightowers MW: Vaccination against animal parasites. Vet Parasitol 54:177-204, 1994 21. Love S: Parasite associated equine diarrhea. Compendium on Continuing Education for the Practicing Veterinarian 14:642-649, 1992 22. Love S, Duncan JL: The development of naturally acquhed cytostome infection in ponies. Vet Parasitol 44:127-142, 1992 23. Love S, Escola J, Duncan JL, et al: Studies on the pathogenic effects of experirrtental cytostome infections in ponies. In Equine Infectious Diseases VI Proceedings of the 6th International Conference, Newmarket, UK, 1992, pp 149-155 24. Madden KB, Urban JF, Ziltener HJ, et al: Antibodies to IL-3 and IL-4 suppress helminth induced intestinal mastocytosis. J ImmunoI147:1387, 1991 25. McClure JR, Chapman MR, Klei TR: Production and characterization of monospecific adult worm infections of Strongylus vulgaris and Strongylus edentatus in ponies. Vet Parasitol 51:249-254, 1994 26. Miller TA: Vaccination against canine hookworm disease. Adv Parasitol 9:153, 1971 27. Monahan CM, Chapman MR, Taylor HW, et al: Experimental cytostome challenge of ponies maintained with or without benefit of daily pyrantel tartrate feed additive: Comparison of parasite burdens, immunity and colonies pathology. Vet Parasitol 74:229-241, 1998 28. Monahan CM, Chapman MR, Taylor HW, et al: Foals raised on pasture with or without daily pyrantel tartrate feed additive: Comparison of parasite burdens and host responses following experimental challenge with large and small strongyle larvae. Vet Parasitol 73:277-289, 1997 29. Monahan CM, Taylor HW, Chapman MR, et al: Experimental immunization of ponies with Strongylus vulgaris radiation-attenuated larvae or crude soluble somatic extracts from larvae or adult stages. J Parasitol 80:911-923, 1994 30. Ogbourne CP: Pathogenesis of cyathosome (Trichonema) infection in the horse: A review. St. Albans, UK, Miscellaneous Publication No 5, Commonwealth Institute of Parasitolo~ 1978, p 25 31. Parsons JC, Coffman RL, Grieve RR: Antibody to IL-5 prevents blood and tissue eosinophilia but not liver trapping in murine larvae toxocanasis. Parasite Immunol 15:501-508, 1993 32. Proudman CJ, Trees AJ: Tapeworms as a cause of intestinal disease in horses. Parasitology Today 15:156-159, 1999 33. Sasaki 0, Susaya H, Ishida K, et al: Ablation of eosinophils with anti-IL3 antibody enhances the survival of intracranial worms of Angiostrongylus cantonensis in mice. Parasite Immunol 15:349-354, 1993 34. Smith WD: Prospects for vaccines of helminth parasites of grazing ruminants. Int J Parasitol 29:17-24, 1999 35. Swirderski CE, Klei TR, Folsom RW, et al: Vaccination against Strongylus vulgaris in ponies: Comparison of the humoral and cytokine responses of vaccinates and I
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nonvaccinates. In Schultz RD (ed): Advances in Veterinary Medicine, vol 41, Veterinary Vaccines and Diagnostics. New York, Academic Press, 1999, pp. 389-404 Swirderski CE, Klei TR, Horohov DW: Quantitative measurement of equine cytokine mRNA expression by polymerase chain reaction using target-specific standard curves. J Immunol Method 222:155-169, 1999 Swirderski CE, Klei TR, Pourciau SS, et al: T-cell cytokine responses to Strongylus vulgaris in infected and vaccinated ponies. In Proceedings of the 8th Equine Infectious Disease Conference, in press Uhlinger CA: Effects of three anthelmintic schedules on the incidence of colic. Equine Vet J 22:251-254, 1990 Uhlinger CA: Equine small strongyles: Epidemiology, pathology and control. Compend Vet Ed 13:863-869, 1991 Urban JF, Kotona 1M, Paul WE, et al: Interleukin 4 is important in protective immunity to gastrointestinal nematode infection in mice. Proc Nat! Acad Sci USA 88:5513, 1991 Urban JF, Madden KB, Svetic A, et al: Importance of Th2 cytokines in protective immunity to nematodes. Immunol Rev 127:205-220, 1992 Urquhart GM: Field experience with the bovine lungworm vaccine. Dev BioI Stand 64, 109-112, 1985
Address reprint requests to Thomas R. Klei, PhD Department of Veterinary Microbiology and Parasitology School of Veterinary Medicine Louisiana State University S Stadium Road Baton Rouge, LA 70803 e-mail: [email protected]
0749-0739/00 $15.00
+ .00
EQUINE IMMUNITY TO PARASITES Thomas R. Klei, PhD
With the exception of Sarcocystis neuron, helminths are the most significant parasites of horses in developed countries, and these are the focus of this review. Immune responses directed against helminth parasites are important in controlling these infections and the disease onditions that they produce. In some instances these immune responses also may significantly exacerbate disease or regulate immune responses to other stimuli. The implied intent of immunologic investigations of infectious agents in any host is to improve the feasibility of the development of vaccines, improve diagnostic procedures, and eradicate disease. Relatively little is know about the immune response to helminth parasites, and it is not surprising that vaccines or diagnostic procedures directed against these agents do not exist at this time. Immunity to helminth parasites is generally judged by a significant reduction in parasite burden in immune individuals. Evidence for a true age resistance independent of acquired immunity is lacking. 12 Thus, the conventional thought is that horses repeatedly exposed to parasites in their environment acquire a specific immunity through this repeated exposure. Helminth parasites can be divided into three categories on the basis of speed and level of immune resistance that they induce. In the first instance, a strong immunity is rapidly acquired to infections of Strongyloides westeri and Parascaris equorum, and these parasites rarely occur as adults in yearling horses. In the second category containing the strongyle nematodes, immunity is slow to develop and incomplete, and some level of infection persists throughout the life of most individuals.
From the Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, and the Department of Veterinary Science, Louisiana Agricultural Experiment Station, Louisiana State Universi~ Baton Rouge, Louisiana
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In the third category, acquired resistance at any level has not been demonstrated and mature horses are believed to harbor as many parasites as immature individuals. Parasites such as stomach bots (Gasterophilus spp), tapeworms (Anoplocephala spp), and pinworms (Oxyuris equi) fall into this group. It is possible with further investigation that acquired immunity will be demonstrated against these parasites and they will thus be grouped with the strongyle nematodes. In instances in which immunity does develop it is most often of a concomitant type. Thus, mares are immune to the establishment of adult S. westeri in the small intestine yet harbor arrested larvae that are activated postpartum and transmitted in the mares' milk to foals. Similarly, horses harbor adult and larval stages of S. vulgaris, which are not affected by the ongoing immune response and yet immune responses prevent the establishment of ingested third stage. Mechanisms associated with these protective immune responses are only partially understood, 'and only the strongyles have been studied in any detail. ll IMMUNITY AGAINST STRONGYLES Strongylus Species
Strongyle nematodes are the most ubiquitous and important internal parasites of horses. With the advent of efficacious anthelmintics the significance of the large strongyles, parasites in the genus Strongylus, has been reduced. s Nonetheless, S. vulgaris remains an excellent model to study mechanisms of equine immunity against helminth parasites and to define certain basic immunologic principles in the horse. There are several technical advantages to using S. vulgaris infections to study the equine immune response to nematodes. Monospecific infections of Strongylus spp can be produced by surgical implantation of adults, and these ponies remain infected for years,2S providing a ready source of feces for third stage infective larvae (L3) culture. This is, as yet, not possible with other equine strongyles. Methods for the in vitro culture of late stage L3 and L4 have been developed1 and provide a source of these stages. S. vulgaris is a highly pathogenic gastrointestinal nematode, and its biology and host interactions have been studied and reviewed. l l In nonimmune equines, L3 are ingested with herbage, exsheath in the intestine, and penetrate the mucosa. These larvae migrate in the submucosa for 4 to 7 days before entering arterioles. At this point, they molt to the fourth stage (L4) and migrate through the mesenteric arteries toward the aorta. Upon reaching the ileo-cecal-colic artery and the cranial mesenteric artery, many of these larvae cease their migration and develop into the fifth stage (L5), which is a neotenic adult form. Upon molting, the L5 migrate distally within these vessels to the cecum and proximal ventral colon where they enter the lumen, develop to adults, mate, and produce eggs that are passed in the feces. This life cycle is prolonged and takes 4 to 6 months to complete. From an experimental
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standpoint, however, the easily demonstrated clinical signs of infection, including pyrexia, anorexia, depression, and abdominal pain, appear within the first week after L3 ingestion and are near a maximal state by 14 days after infection (DPI). These signs are absent in immune ponies, and larvae appear to be killed by the immune response before entry into major arteries or are blocked from entry into the mucosa. If the latter occurs, it is incomplete in that some lesions are found in vaccinated ponies. 29 Although as few as 50 L3 per week for 4 weeks have been shown to be lethal,18 the clinical signs of infection are generally dose related, and infections of 500 to 1000 L3 rarely are fatal and produce only mild colic. This model system has proved to be manipulated easily, and lymphocytes collected from the peripheral blood and surgically from cecal lymph nodes during the 14 to 16 day postchallenge period have been used to recover significant immunologic data. 35 Ponies infected experimentally develop a strong concomitant immunity to reinfection either to a single16 or multiple challenge. 17 Experimental immunization is easily accomplished using two doses of radiation attenuated L3 and avoids the experimental difficulties of massive arterial lesions induced by unattenuated larvae. 16 1trus vaccination regimen produces a strong protective resistance to challenge infection, which reduces challenge larval burdens, eliminates clinical signs of infection, and prevents colic. 15,16 Vaccination of ponies with irradiated L3 induces protection from S. vulgaris infection but not Strongyloides edentatus infection17 and is thus species specific. Vaccination is effective against experimental or field challenges and lasts for at least 9 months. This vaccination protocol has been used to experimentally characterize immune responses to challenge infections. Cytokine mRNA levels before and during challenge infections of vaccinated ponies have been measured in this model using a quantitative Reverse Transcription-Polymerase Chain Reaction procedure. 36 Vaccination induces a strong in situ expression of IL-4 and IL-5 with little interferon gamma (IFN-~), a cytokine profile typical of a dominant type 2 response. 35 Differences between levels of IL-2 and IL-10 using this method were not detected before or after challenge infection. This in situ profile of cytokine expression occurs in both peripheral blood mononuclear cell (PBMC) and cecal lymph node cells (CLN). Although this indicates a lack of completed compartmentalization of this response to the lymphoid tissues of the bowel, expression of IL-4 is first seen in CLN. The most marked increase seen during this challenge infection is the anamnestic IL-5 response seen in vaccinated ponies but not in ponies that received only the challenge infection. This anamnestic increase in IL-5 corresponds to a similar increase in circulating and tissue eosinophil numbers. Similar cytokine responses occur in PBMC and CLN cultured in vitro with adult worm antigens. 37 Subsequent experiments have demonstrated that this expression of IL-4 and IL-5 occurs in CD4 + cells and not CD4- cells separated from these cultures. Immunization of ponies with somatic extracts of adult worms in RIBI adjuvant (Ribi ImmunoChem Research, Inc, Hamilton, MT) produces a strong antibody response to parasite antigens but exacerbates clinical signs and arterial lesions resulting from
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challenge infections. 29 This RIBI immunization protocol does not induce a type 2 cytokine profile but rather a marked production of IFN-')', which is accompanied by a lack of protection (unpublished results). Although incomplete, these observations support the contention that a type 2 Tcell response is essential for the expression of protective immune response against this parasite. The dominant type 2 response seen in immune animals is consistent with the type 2 cytokine profiles associated with protective immunity in several murine models of gastrointestinal nematode infections and it is interesting to compare the equine responses with those described in the murine mode1. 41 Although a helper 2T (TH 2) cell pattern of cytokine responses is dominant in mice resistant to gastrointestinal helminths, the roles of specific cytokines and effector mechanisms involved are less clear and results vary considerably with the parasite-host system examined. 41 The following examples demonstrate the diversity of results reported from murine systems. Protective resistance of mice against Heligmosomoides polygyrus is abrogated by treatment with anti-IL-4 or anti-IL-4 receptor antibodies40; however, antibodies to IL-3, IL-4, and IL-5, which eliminate mast cell hyperplasia, eosinophilia, and IgE, have no effect on the primary course of infection of Nippostrongylus brasiliensis. 24,44 In other studies, anti-IL-5 treatment that abrogates eosinophilia does not alter acquired protective resistances to Trichinella spiralis 7 or Toxocara canis 31 infections but does eliminate protection against migratory larvae of Strongyloides venezuelensis 19 and Angiostrongylus cantonensis. 33 As indicated, IL-5 expression and eosinophilia are consistent features of the protective immune response to S. vulgaris. In addition, infections of S. vulgaris activate eosinophils and neutrophils in vitro,3 and activated but not normal eosinophils kill L3 in vitro in an antibody-dependent manner,13 whereas other cells do not. Furthermore, antigen stimulation of PBMC cultures from immune but not nonimmune ponies produces a cytokine (or cytokines) in the supernatant that is chemotactic for eosinophils. 5 Interestingl~ this was not the case in similar cultures stimulated with S. edentatus antigen, which correlates with the species-specific nature of the protective immune response previously described. Immunized ponies show an anamnestic eosinophil response that is absent in nonimmune naive or antigen sensitized individuals. 28,48 In recent studies, intestinal eosinophil counts were significantly elevated in vaccinated and protected ponies but not in nonimmune animals 16 days after infection (unpublished results). Interestingl~ increases in mast cell numbers were not seen in the same tissues. These increased eosinophil responses correspond to the increase seen in IL-5 gene expression in vaccinated ponies during the current studies (unpublished results). Although solely correlative in nature, evidence supports the hypothesis that eosinophils are important in the protective immunity against this helminth in the equine. Antibodies generated during the infection are also likely to be important in the protective immune response to S. vulgaris. Vaccinated ponies challenged with L3 have antibodies to L3 surface not seen in nonimmune ponies. 14 These antibodies are specific in that they do not
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react with the L3 surface of the closely related parasite S. edentatus, again supporting the species-specific nature of the response. These antibodies also mediate an in vitro adherence of eosinophils to L3. 13 The speciesspecific antibody response to the surface of the L3 is, however, lost as these larvae molt to the L4 stage and immune serum reacts with L4 of both S. vulgaris and S. edentatus. 29 Other observations also have shown an increase in the antibody response to soluble somatic extracts of adult worms (SAWA) in vaccinated animals as compared with nonvaccinated controls. 29 The isotype showing a significant increase after challenge in vaccinated animals was IgG(T). The isotypic nature of this antibody response to the L3 surface, however, is at this time unknown. Although these observations support the potential role of antibody in protective responses, antibody alone is insufficient to protect against challenge infection. Transfer of 1.5 L of hyperimmune serum per pony while transferring significantly high titers of L3 surface-specific antibody failed to protect against challenge infection. Collectively, these data suggest that the protective immune response to S. vulgaris is species specific, directed at the L3 stage, and mediated by an antibody-dependent eosinophil killing mechanism. It i likely that antigen-specific CD4 + TH 2 cells are required to induce the necessary cellular effector mechanisms involved in this response. The cross regulatory effects of type-1 and type-2 cytokines have not been demonstrated in the equine. Nonetheless, it may be hypothesized that type-2 responses induced by helminth infections may affect the induction or maintenance of type-1 mediated immune responses. Cyathostomes
With the chemotherapeutic control of Strongylus spp, other strongyle nematode parasites, specifically the small strongyles or cyathostomes,ll have been recognized as more relevant to equine health than previously presumed and have gained prominence. These parasites have been associated with poor growth and development, colic, and diarrhea. 21, 23, 30, 38, 39 Some specific differences in the immune responses to Strongylus spp and the cyathostomes likely exist, however, owing to the absence of extensive migrations away from the bowel by the latter group. Nevertheless, horses do naturally acquire an incomplete resistance to cyathostomes. 16,22 Limited data are available on the specific response directed against these parasites but field observation and experimental infections provide some insight about the nature of this immunity. Field studies of cytostome parasite burdens based on fecal egg count (FEC) data indicated that young horses are more heavily parasitized than older horses. Comparisons of FEC of yearlings and mares on the same properties 8 weeks after anthelmintic treatment further show that the distribution of FEC within the older mare population is overdispersed. 12 A pasture challenge experiment comparing the worm burdens of foals raised under parasite-free conditions with those raised
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b.y their dams on cytostome-contaminated pastures also indicated that previous exposure induces a level of resistance to reinfection. Together, these observations are generally similar to those of trichostrongyle nematodes of ruminants and suggest that horses slowly develop a resistance to reinfection and that this resistance is genetically controlled. Other data from necropsy surveys support these field observations and further suggest that some immunity is directed at the production of eggs by female worms. 12 The latter is based on comparisons of FEe with adult worm burdens in young versus old individuals. Studies using controlled experimental infections with cyathostomes for the purpose of examining the acquired immunity are few. These types of studies are limited to the use of infections with mixed cyathostome populations because monospecific infections of these parasites have yet to be established. Nonetheless, some useful data have been gathered. A comparison has been made on the outcome of single challenge infections between foals raised with the benefit of prophylactic anthelmintic treatment. 28 Observations indicated that the challenge infections induce a nonspecific expulsion of existing intestinal parasites. Nonetheless, a reduced take of the challenge infection was noted in the previously exposed individuals. Furthermore, the numbers of mucosal stages of cyathostomes was significantly reduced in animals that were previously exposed and challenged, thus suggesting that some specific immune event was involved in this phenomenon as well. Other experiments have compared the outcome of regular multiple experimental infections (10,000 L3/ day for 44 days) in ponies with different histories of parasite exposure. 27 These exposure levels included young ponies «4 years of age) raised parasite free, young ponies «4 years of age) raised on contaminated pastures; and old ponies (>7 years of age) maintained on contaminated pastures. On the basis of parasite recoveries at necrops~ observations indicated that immunity is acquired during the early stages of life, but this resistance is not as complete as that seen with increased parasite exposure. The data also suggest that protective immune responses are directed at different stages of the life cycle and that these responses are differentially expressed with increased exposure. Thus, although the total worm burden of young ponies previously exposed to parasites is significantly less than that of ponies raised parasite free, the numbers of hypobiotic early L3 (EL3) within the mucosa are not different. The levels of EL3 were significantly reduced in older ponies. ELISA measurements of circulating antibody to somatic antigens of cyathostomes in these ponies did not detect a difference between immune and nonimmune individuals. 12 Interestingl~ serendipitous measurements of cytokine mRNA in a pony undergoing adult worm expulsion suggest that a type-2 response is involved in this phenomenon. 8 Although mechanisms of protective immune responses are not know it can be hypothesized that these are directed at a number of targets. These include the incoming L3, the developing L3 and L4 stages within the mucosa, the lumenal L4 and adult stages, and egg production by adult females.
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IMMUNODIAGNOSIS
There is little necessity to diagnose individual infections of most common helminth parasites because this can be accomplished by standard fecal examination techniques. 10 It would be useful, however, for epidemiologic reasons if intensities of cyathostome infections could be estimated or if the presence of some parasites such as S. vulgaris were known. Efforts to diagnosis parasite infections by serologic means have been minimal and ineffective. 10 This is largely due to the complexity of standard helminth antigen preparations and the necessity of developing such tests. Infection of the tapeworm Anoplocephala perfoliata is an exception to the latter point. Anoplocephala perfoliata attaches to the intestinal mucosa at and near the ileocecal junction, producing ulcers and marked inflamation. The extent and severity of these lesions is related to the numbers of parasites present. The presence of these parasites has been implicated in intussusceptions of the ileocecal region. The importance of infections of A. perfoliata in the induction of equine colic, however, has been controversial and universally accepted data supporting this concept have been absent. 6 Recent efforts to diagnose these tapeworm infec 'ons and more importantly the intensity of infection have met with some success. 32 Antibodies to a doublet of protein antigens of 12 and 13 kDa in worm excretory-secretory products were identified in Western blots that were specific for A. perfoliata. These responses were limited to the IgG(T) isotype. Subsequent work used these antigens in an ELISA format to demonstrate the positive relationship of this antibody response to parasite infection intensity and colic. The use of this test in clinical and epidemiologic settings should be explored. VACCINATION
The development of vaccines against parasites has been the goal of the veterinary and medical parasitologist for decades. Early success commercializing the use of radiation attenuated larvae as a vaccine against lung worms of ruminants was encouraging. 42 This development, however, did not lead to additional commercialized products as expected. A notable scientific success and commercial failure of this type is the vaccine developed against hookworms in dogs. 26 As noted previously, vaccination of foals with irradiated L3 clearly has been shown in field studies to be feasible. The diminished occurrence of the parasite in developed areas of the world by the extensive use of avermicitin/ milbymicin anthelmintics, however, made the production of such a product unnecessary. Because of their ubiquitous distribution, importance to equine health, and the widespread prevalence of drug-resistant populations, vaccines against the cyathostomes would be particularly useful. Logical candidates for such a vaccine are molecules that are targets of the equine acquired protective immune response. As noted previously, however,
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little is know of the host or parasite factors involved in this response. In recent years attempts have been made to develop vaccines against gastrointestinal nematodes of ruminants using "hidden" antigens. These are defined as those that do not elicit an immune response during infections yet are important to worm homeostasis and can be disrupted by immune effectors such as antibody. Several of these targets have been found in the worm intestine and are promising. 34 The carbohydrate epitopes of some of these cross-react with equine strongyles including the cyathostomes. 9 It is not clear, however, if cyathostomes ingest sufficient serum to make this approach feasible in the equine. Furthermore, although this approach has been shown to be experimentally feasible for many 'years in sheep it has yet to be demonstrated in controlled field studies. 34 The usefulness of this approach requires further evaluation. It is important to note that many elements must come together for a vaccine to be a useful commercial success, and all of these are not scientific. As an example, excretory-secretory proteins of Taenia cysticerci have been shown to produce protective immunity in sheep. These have been biochemically identified, produced by recombinant DNA technologies, and shown to be an effective vaccine. Nonetheless, this has not led to a commercial product largely because of bureaucratic and/ or political reasons. 20 References 1. Chapman MR, Hutchinson GW, Cenac MJ, et al: In vitro culture of equine strongylidae to the fourth larvae stage in a cell free medium. J Parasitol 80:225-230, 1994 2. Dennis VA, Klei TR, Chapman MR: In vivo activation of equine eosinophils and neutrophils by experimental Strongylus vulgaris infections. Vet Immunol Immunopathol 20:61-74, 1988 3. Dennis VA, Klei TR, Miller MA, et al: Immune responses of pony foals during repeated infections of Strongylus vulgaris and regular ivermectin treatments. Vet Parasitol 42:8399, 1992 4. Dennis VA, Klei TR, Chapman MR: Generation and partial characterization of an eosinophil chemotactic cytokine produced by sensitized equine mononuclear cells. Vet Immunol Immunopathol 37:135-149, 1993 5. DiPietro JA, Klei TR, French DD: Contemporary topics in equine parasitology. Compendium on Continuing Education for the Practicing Veterinarian 12:713-721, 1990 6. French DD, Chapman MR: Tapeworms of the equine gastrointestinal tract. Compendium on Continuing Education for the Practicing Veterinarian 14:655-611, 1992 7. Herndon FJ, Kayes SG: Depletion of eosinophils by anti-IL-5 monoclonal antibody treatment of mice infected with Trichinella spiralis does not alter parasite burden or immunologic resistance to reinfection. J Immunol 149:3642-3647, 1992 8. Horohov DW, Swiderski CE, Robinson JA, et al: Cytokine production in equine disease. In Schijns VECJ, Horzinek MC (eds): Cytokines in Veterinary Medicine. Wallingford, Oxon, UK, CAB International, 1997, pp 167-176 9. Jasmer D~ Perryman LE, Conder GA, et al: Protective immunity to Haemonchus contortus induced by immunoaffinity isolated antigens that share a phylogenetically conserved carbohydrate gut surface epitope. J Immunol151:5450-5460, 1992 10. Klei TR: Laboratory diagnosis. Vet Clin North Am Equine Pract 2:381-394, 1986 11. Klei TR: Recent observations on the epidemiology, pathogenesis and immunology of equine helminth infections. In Equine Infectious Diseases VI, Proceedings of the Sixth International Conference, Newmarket, UK, 1992, pp 129-137
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12. Klei TR, Chapman MR: Immunity in equine cyathastome infections. Vet Parasitol, in press 13. Klei TR, Chapman MR, Dennis VA: Role of the eosinophil in serum-mediated adherence of equine leukocyte to larvae of Strongylus vulgaris. J Parasitol 68:561-569, 1992 14. Klei TR, Chapman MR, Torbert BJ, et al: Antibody responses of ponies to initial and challenge infections of Strongylus vulgaris. Vet Parasitol 12:187-198, 1983 15. Klei TR, French DO, Chapman MR, et al: Protection of yearling ponies against Strongylus vulgaris by foalhood vaccination. Equine Vet J 7:2-7, 1989 16. Klei TR, Torbert BJ, Chapman MR, et al: Irradiated larvae vaccination of ponies against Strongylus vulgaris. J Parasitol 68:561-569, 1982 17. Klei TR, Turk MAM, McClure JR, et al: Natural and acquired resistance to Strongylus vulgaris: Its associated lesions and colic. In Equine Colic Research. Proceedings of the Second Symposium. 1986, pp 14-18 18. Klei TR, Turk MAM, McClure JR, et al: Effects of repeated experimental Strongylus vulgaris infections and subsequent ivermectin treatments on mesenteric arterial pathology in pony foals. Am J Vet Res 51:654-660, 1990 19. Korenasa M, Hitoshi Y, Yamaguchi N, et al: The role of IL-5 in protective immunity to Strongyloides venezuelensis infection in mice. Immunology 72:502, 1991 20. Lightowers MW: Vaccination against animal parasites. Vet Parasitol 54:177-204, 1994 21. Love S: Parasite associated equine diarrhea. Compendium on Continuing Education for the Practicing Veterinarian 14:642-649, 1992 22. Love S, Duncan JL: The development of naturally acquhed cytostome infection in ponies. Vet Parasitol 44:127-142, 1992 23. Love S, Escola J, Duncan JL, et al: Studies on the pathogenic effects of experirrtental cytostome infections in ponies. In Equine Infectious Diseases VI Proceedings of the 6th International Conference, Newmarket, UK, 1992, pp 149-155 24. Madden KB, Urban JF, Ziltener HJ, et al: Antibodies to IL-3 and IL-4 suppress helminth induced intestinal mastocytosis. J ImmunoI147:1387, 1991 25. McClure JR, Chapman MR, Klei TR: Production and characterization of monospecific adult worm infections of Strongylus vulgaris and Strongylus edentatus in ponies. Vet Parasitol 51:249-254, 1994 26. Miller TA: Vaccination against canine hookworm disease. Adv Parasitol 9:153, 1971 27. Monahan CM, Chapman MR, Taylor HW, et al: Experimental cytostome challenge of ponies maintained with or without benefit of daily pyrantel tartrate feed additive: Comparison of parasite burdens, immunity and colonies pathology. Vet Parasitol 74:229-241, 1998 28. Monahan CM, Chapman MR, Taylor HW, et al: Foals raised on pasture with or without daily pyrantel tartrate feed additive: Comparison of parasite burdens and host responses following experimental challenge with large and small strongyle larvae. Vet Parasitol 73:277-289, 1997 29. Monahan CM, Taylor HW, Chapman MR, et al: Experimental immunization of ponies with Strongylus vulgaris radiation-attenuated larvae or crude soluble somatic extracts from larvae or adult stages. J Parasitol 80:911-923, 1994 30. Ogbourne CP: Pathogenesis of cyathosome (Trichonema) infection in the horse: A review. St. Albans, UK, Miscellaneous Publication No 5, Commonwealth Institute of Parasitolo~ 1978, p 25 31. Parsons JC, Coffman RL, Grieve RR: Antibody to IL-5 prevents blood and tissue eosinophilia but not liver trapping in murine larvae toxocanasis. Parasite Immunol 15:501-508, 1993 32. Proudman CJ, Trees AJ: Tapeworms as a cause of intestinal disease in horses. Parasitology Today 15:156-159, 1999 33. Sasaki 0, Susaya H, Ishida K, et al: Ablation of eosinophils with anti-IL3 antibody enhances the survival of intracranial worms of Angiostrongylus cantonensis in mice. Parasite Immunol 15:349-354, 1993 34. Smith WD: Prospects for vaccines of helminth parasites of grazing ruminants. Int J Parasitol 29:17-24, 1999 35. Swirderski CE, Klei TR, Folsom RW, et al: Vaccination against Strongylus vulgaris in ponies: Comparison of the humoral and cytokine responses of vaccinates and I
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36. 37. 38. 39. 40. 41. 42.
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nonvaccinates. In Schultz RD (ed): Advances in Veterinary Medicine, vol 41, Veterinary Vaccines and Diagnostics. New York, Academic Press, 1999, pp. 389-404 Swirderski CE, Klei TR, Horohov DW: Quantitative measurement of equine cytokine mRNA expression by polymerase chain reaction using target-specific standard curves. J Immunol Method 222:155-169, 1999 Swirderski CE, Klei TR, Pourciau SS, et al: T-cell cytokine responses to Strongylus vulgaris in infected and vaccinated ponies. In Proceedings of the 8th Equine Infectious Disease Conference, in press Uhlinger CA: Effects of three anthelmintic schedules on the incidence of colic. Equine Vet J 22:251-254, 1990 Uhlinger CA: Equine small strongyles: Epidemiology, pathology and control. Compend Vet Ed 13:863-869, 1991 Urban JF, Kotona 1M, Paul WE, et al: Interleukin 4 is important in protective immunity to gastrointestinal nematode infection in mice. Proc Nat! Acad Sci USA 88:5513, 1991 Urban JF, Madden KB, Svetic A, et al: Importance of Th2 cytokines in protective immunity to nematodes. Immunol Rev 127:205-220, 1992 Urquhart GM: Field experience with the bovine lungworm vaccine. Dev BioI Stand 64, 109-112, 1985
Address reprint requests to Thomas R. Klei, PhD Department of Veterinary Microbiology and Parasitology School of Veterinary Medicine Louisiana State University S Stadium Road Baton Rouge, LA 70803 e-mail: [email protected]