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The Epidemiology and Control of Swine Parasites Immunity and Vaccines j. F. Urban,jr., Ph.D.*
More than 15 different species of helminthic parasites infect swine in the United States. Economic loss can be a consequence of infection. 6 , 39 Primary methods for control of infection are proper animal management and proper choice and administration of an array of highly effective anthelminthic drugs. However, the effectiveness of these methods requires knowledge of the parasite and its life cycle. It is apparent that these methods are often inadequate or are poorly implemented because direct swine production and condemnation losses in the United States due to infection with helminthic parasites are currently estimated to exceed $385 million annually.39 An alternative approach or supplemental component of an integrated control strategy is biologic control of parasitism through immunization of the host. However, no economically useful vaccine for swine helminthic parasites currently exists. In fact, except for the rather unique application of radiation-attenuated, live parasite vaccines for cattle and sheep lungworm, no economically useful helminthic vaccine exists for any agriculturally important food-producing animal species. 44 The recent development of effective molecular vaccines against infectious agents of medica1 47 and veterinary 24 importance has stimulated efforts to develop helminthic parasite vaccines. Molecular vaccines currently consist of complete polypeptide antigens or relatively small defined epitopes that are genetically engineered or synthetically constructed. Because they are chemically well defined, they are more advantageous than conventionally prepared vaccines. 25 For example, production is amenable to good quality control, and they can be produced in large quantities, relatively free from the contaminants or byproducts that accompany complex vaccines. Note: Owing to the constraints of size, this article could not be included in the July 1986 issue of Veterinary Clinics of North America: Food Animal Practice on Parasites: Epidemiology and Control (Vol. 2, No.2).
* Helminthic Diseases Laboratory, Animal Parasitology Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland
Veterinary Clinics of North America: Food Animal Practice-Vol. 2, No.3, November 1986
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COMMON NAMES
Large roundworm
Whipworm
Trichinae
Kidney worm
Ascaris suum
Trichuris suis
Trichinella spiralis
Stephanurus dentatus
Free-living, infective larvae in environment (and in earthworms)
Infective larvae in muscle tissue
Eggs in environment
Eggs in environment
SOURCE OF INFECTION
Small intestine (or percutaneous) (or prenatal in utero)
Small intestine
Cecum or colon
Small intestine
infection
I nitial site of
Lymph nodes, liver, perirenal fat, hilus of kidney,
Circulation, liver, muscle
Mucosa of cecum or colon
Circulation, liver, lung, small intestine
Sites during larval migration
LOCATION IN THE HOST
Major Nematode Parasites that Infect Swine in the United States
SCIENTIFIC NAMES
Table 1.
Encysted along ureters
Small intestines
Cecum or colon
Small intestine
Location of adults
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Threadworm
Nodular worm
Lungworm
Red stomach worm
Thick stomach worms
Strongyloides ransomi
Oesophagostomum dentatum O. quadrispinulatum O. brevicaudum O. georgianum
Metastrongylus apri M. pudendotectus M. salmi
Hyostrongylus rubidus
Ascarops strongylina Physocephalus sexalatus Infective larvae within Dung beetle
Free-living infective larvae in environment
Infective larvae within earthworm
Free-living, infective larvae in environment
Free-living infective larvae in environment, parasitic larvae in colostrum derived from mammary fat
Mucosa of stomach
Gastric glands of stomach
Small intestine
Small intestine, colon, cecum
Small intestine (or percutaneous)
Mucosa of stomach
Mucosa of stomach
Lymphatics, circulation, lungs
Mucosa of small intestine, colon, cecum
Circulation, lungs, fat
Stomach
Stomach
Bronchioles and bronchi of lung
Colon, cecum
Small intestines
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The purpose of this report is to discuss the status of two experimental swine helminthic vaccines and how further knowledge of parasite biochemistry and swine immunology and the application of techniques of modern biotechnology may affect their future development. Targeting a Parasite for Development of an Effective Vaccine The most important internal helminthic parasites of swine in the United States are nematodes or roundworms, commonly comprising about 15 species (Table 1). Effective and economically sensible vaccines most probably can be developed within the next few years against Ascaris suum and Trichinella spiralis. This prediction is based on the following facts: 1. Both parasites are of major economic importance to the swine industry. (Infection with A. suum reduces feed efficiency, increases organ condemnation, and decreases herd health both directly and through secondary infection, whereas infection with T. spiralis jeopardizes product safety and threatens public health.) 2. The epidemiology of both parasites is reasonably well understood, and yet they continue to plague selected segments of the swine industry. 3. There is an extensive literature on protective mechanisms to these parasites and on the identification and isolation of their antigens. 4. There is good documentation that protective immunity develops to both parasites in infected swine.
Factors complicating the development of vaccines for the 13 other species of parasites listed in Table 1 are both pragmatic and problematic, but can be loosely catagorized as follows: 1. Recent prevelance studies 39 have indicated a decreasing infection level for lungworms and stomach worms in areas in which confinement rearing of pigs has been adopted. 2. Convenient laboratory animal model systems for passage of many of these parasites and for studies of host resistance are unavailable; and parasite specimens for antigen isolation and characterization are limited. 3. The relative economic importance of infection with these parasites is subordinate to that of A. suum and T. spiralis. However, it should be noted that the prevalence of both Trichuris suis and Strongyloides ransomi infection in pigs is still regarded as important for certain segments of the industry, and economic losses can be significant. 39 In addition, there are defined laboratory animal models available for these parasites, and immunity in swine has been demonstrated.31, 34
It is convenient conceptually that the major site of initial infection of all the common nematode parasites of swine is the mucosa of the alimentary tract (see Table 1) because information gleaned about protective immunity at the mucosal surface to anyone of these parasites would be applicable, in principal, to an understanding of protective imm unity to all. Immunity in S wine to Ascaris suum
Demonstration of Acquired Immunity. Early survey studies of swine for helminthic infestation showed that the prevalence of A.
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suum infection was markedly less in sows than in young pigs, suggesting that resistance to infection developed with age. Clearly, the degree of parasite exposure and not the age of the pig determines the level of acquired immunity to subsequent infection. 41 The first reliable and effective protocol developed for vaccination against A. suum used eggs attenuated by ultraviolet irradiation. 40, 43 The conditions for successful vaccination of pigs and the characteristics of resistance are as follows: 1. Development of attenuated larvae in the host through the fourth stage is sufficient to induce strong protective immunity. 2. Vaccinated pigs have fewer challenge larvae migrating to the lungs and fewer adults developing in the small intestines than challenge-exposed, nonvaccinated pigs. 3. As few as 1500 ultraviolet-irradiated eggs given in two inoculations or 300 ultraviolet-irradiated eggs divided into 3 inoculations can induce marked protection. 4. Oral inoculation is superior to parenteral (intraperitoneal) inoculation, indicating a requirement for local sensitization of the intestine with specific larval stages. 5. Both specific in-vitro cellular lymphoproliferative responses and serum antibody responses to parasite antigens are increased after inoculation. 6. Intestinal immunity is not induced appreciably because larvae from a challenge exposure migrate to the liver and elicit strong milk-spot lesions.
Demonstration of Intestinal Immunity. Ascaris suum larvae penetrate the intestinal mucosa and migrate parenterally, causing severe inflammation, primarily in the liver and lungs, and increasing the risk of secondary infection. Therefore, the ideal target site for induced immunity would be at the level of the intestine. The choice of a strategy that would lead to the development of a practical vaccine for swine ascariasis is dependent on the answer to the following question: Can pigs develop intestinal immunity to a challenge exposure to A. suum eggs that would preclude larval migration to the liver and lungs? Clearly they can because pigs naturally exposed to infection with A. suum on contaminated dirt lots for a prolonged period were nearly free of liver milk spots after a challenge exposure (Fig. lA), whereas pigs receiving a shorter period of exposure developed only a low-level protective immunity and a high degree of liver pathology following challenge exposure (Fig. IB). More direct evidence was the fact that pigs naturally exposed to A. suum and then challenge-exposed orally with eggs were free of surface milk-spot lesions (Fig. 2A), but injection of freshly hatched A. suum larvae directly into the mesenteric veins (Fig. 2B) induced numerous milk-spot lesions on the liver (Fig. 2C).
These results clearly show that the intestine can become an effective barrier against larval migration. This response has recently been reproduced under defined conditions of egg dosage and inoculation frequency in confinement-housed pigs. Therefore, it is induced specifically by exposure to A. suum and is not dependent on
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URBAN,
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Figure 1. Livers obtained after necropsy of pigs 7 days after a challenge exposure to 10,000 A. suum eggs. A, From pigs after 18 weeks (#533 and #538) and 24 weeks (#1571 and #539) of expos ure on contaminated dirt lots, showing few active milk-spot lesions. B, From a confinement-reared pig (#532) and a pig after 6 weeks on dirt followed by 18 weeks in confinement (# 1521), showing numerous milk-spot lesions of varying intensity.
the myriad of other biologic and immunologic stimuli encountered on a dirt lot. Basis of Intestinal Immunity. Stewart's observations on the "self-cure" phenomenon of sheep infected with Haemonchus contortus established the concept that a challenge exposure to nematode larvae could quickly induce a local allergic type reaction, characterized by gastric edema, increased peristalsis, and a rise in whole blood histamine,38 all of which are detrimental to parasite survival in the host. Numerous studies have subsequently established that the cellular and humoral components of allergic responses are characterist-
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Figure 2. Demonstration of gut-level protection to a challenge exposure of 10,000 A . suum eggs in a pig naturally exposed to infection for a prolonged period (16 weeks). A, Liver during laparotomy 7 days after oral challenge ; no visible milk spots. B, Injection of viable second-stage larvae of A. suum into the mesenteric veins during the laparotomy. C, Numerous milk-spot lesions visible at 7 days after the intravenous injection of L2.
ically present following helminth infection, that is, a marked increase in production of reaginic antibody of the IgE class, accumulation of mast cells or basophils, or both, and subsequently eosinophils in parasitized tissues, and the release of preformed and secondary mediators of anaphylaxis. 2 • 19 It is clear that these responses represent the expression of rapid effector mechanisms that potentially can have dramatic direct effects on the parasite or, by markedly altering the ambient environment of the parasite in the host, can indirectly affect parasite survival. Because larvae from infective A. suum eggs begin to migrate from
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the intestinal lumen and establish in the liver within 6 hours after inoculation,12 any intestinal immune response that precludes liver invasion must be of an immediate type. Recent studies have shown that some of the components of immediate-type hypersensitivity responses to helminths are present in pigs with strong intestinal immunity to A. suum. For example, pigs exposed to A. suum eggs on contaminated dirt lots for over 16 weeks have greater numbers of mast cells in the intestinal mucosa that do pigs kept in confinement, free from infection. There is also an increase in eosinophils in the intestinal mucosa, and these pigs show immediate-type skin reactions to a number of A. suum larval and adult antigens, whereas controls that were housed parasitt ':'ree in confinement are unreactive. These observations suggest that the peculiar conditions of chronic exposure to A. suum that appear to be necessary to induce strong intestinal immunity in swine may provide the appropriate stimuli for the local accumulation of most of the components involved in an immediate-type allergic response to infective larvae. Immunization of Swine with Ascaris suum Antigens. The successful vaccination of swine against ascariasis using ultraviolet-attenuated eggs provided implicitly the evidence that products obtained from infective larvae in the egg (first-molt and second-stage larvae) through early fourth-stage larvae contain protective antigens. This was verified by successful immunization of swine with products obtained from eggs of A. suum and from in-vitro cultivation of the larvae. 42 However, two major problems remain for the development of these parasite products into a practical vaccine for ascariasis. First, they are complex and consist of a number of distinct protein components, many of which may be irrelevant to the induction of a protective immune response. Second, the protective immunity that they induce is not expressed strongly at the intestinal level; that is, liver milk-spot lesions are derived from a challenge exposure. The solution to the first problem may be overcome by improvement in antigen isolation procedures similar to those described below for swine trichinosis. A solution to the second may depend upon a better understanding of the regulatory mechanisms necessary to stimulate the local accumulation of the required cellular and humoral components of immunity in the intestinal wall (see section on future research directions). Immunity of Swine to Infection with Trichinella spiralis
Demonstration of Acquired Immunity. Although swine trichinosis is an important zoonotic disease in the United States,35 and vaccination of swine in selected segments of the industry could be a significant component of an effective integrated control program,28 immunity to T. spiralis has been studied primarily in rodent models, and surprisingly little is known of immunity to T. spiralis in swine. Murrell showed recently that adult T. spiralis developing from a primary infection are eventually expelled from the intestines of pigs, and that pigs inoculated with T. spiralis L1 elicit a more rapid intestinal expulsion of adults developing from a secondary challenge expo-
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sure. 29 The intensity of both of these events is dependent on the number of LI in the primary infection. Inoculation of pigs with as few as 112 LI evokes a nearly complete resistance to the muscle larva stage developing from a challenge exposure. There is direct evidence that a significant antifecundity response is established in these pigs that accounts for most of the reduction in muscle larvae burdens. However, there also appears to be a direct detrimental effect on the development of newborn larvae in immune pigs because passively transferred immune pig serum reduces muscle larvae burdens without affecting adult worm burdens or fecundity.22 Thus, protective mechanisms induced in swine after a complete infection with T. spiralis appear to be acting at at least three levels: (1) expulsion of adults after a relatively high inoculation dose; (2) an antifecundity effect that markedly reduces the production of newborn larvae; and (3) a stage-specific protective immunity against newborn larvae. Immunization of Swine with Trichinella spiralis Antigens. The demonstrated resistance of hogs to reinfection with T. spiralis LI was the basis for the successful vaccination of swine with radiation-attenuated LIB and, later, the use of antigens derived from in-vitro grown LI for immunization. 45 The association between secreted products of LI and its stichosome l l led to the isolation of stichocyte secretory granules. Antigens from a soluble portion of stichocyte secretory granules (S3) were isolated and used to immunize both mice lO and pigs 30 against a challenge exposure to T. spira lis. However, an immunoaffinity purified fraction (PAW) of S3, although biochemically less complex than S3 and capable of inducing strong immunity in mice, failed to protect pigs when it was administered at the same dose as the S3. There are at least two possible reasons for the failure to observe significant protective immunity in pigs immunized with PAW: (1) the inherent ability of pigs to respond immunologically to the antigens in the PAW preparation could be different from that of mice; or (2) the limited availability of PAW made optimization of an immunizing protocol in pigs impractical. The first explanation describes a mechanistic problem. Individual and strain differences in the ability to invoke immune responses is based on genetic control of immunity. 33 Research on the genetic basis of immunity to helminths is currently very active 27, 46 and includes studies on swine immunogenetics (see below). The marked difference in the kinetics of gut expulsion of adult T. spiralis among mice, rats, and pigs 29 is a notable example of the species variation in host protective responses to helminthic infection. It also emphasizes the danger of generalizing too broadly on the efficacy of particular immune effector mechanisms. The limited availability of purified helminth antigens has seriously hampered progress on vaccine development until recently. A solution to this problem is now possible through techniques derived from the new biotechnologies. Both Silberstein and Despommier37 and, more recently, Gamble 14 used hybridoma technology to generate monoclonal antibodies specific for products derived from the T. spir-
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alis L1 stichosome. They were able to identify one or two very similar antigens from the L1 stichosome that induced high levels of protective immunity in mice against challenge exposure to T. spiralis Ll. Thus, through the use of this very powerful technique, one or a few protective antigens were identified and selected from a complex mixture of antigens contained in the secretory apparatus of T. spiralis. Although this work represents a major advance in the isolation and characterization of protective antigens from nematodes, the relevance of these antigens to the induction of protective immunity in swine, for the reasons stated earlier, must be established experimentally. Immunoaffinity isolation of these antigens in sufficient quantities to test in swine will require a herculean effort. A more practical alternative is the use of recombinant DNA techniques for the production of antigens. Gamble and Zarlenga have used molecular cloning of antigens from T. spiralis L1 to provide a potential source of large quantities of antigens for vaccine development in swine. 15 Future Research Directions The general strategy for development of helminthic vaccines of economic importance requires the following: (1) the identification of vulnerable stages in the parasite's life cycle that are most susceptible to immunologic attack or that are of economic importance; (2) determination of the nature of the host protective mechanism; (3) identification and isolation of the protective antigen(s); and (4) presentation of the antigen(s) to the host to induce the desired form of immunity. Definition of the Vulnerable Stages of the Parasite. Strong mucosal immunity to the parasitic stages of most swine gastrointestinal nematodes would be tantamount to sterilizing immunity (see Table 1); however, for some infections this may be an impossible or at least an unnecessary goal. For example, strong parenteral immunity to newborn larvae of T. spiralis would reduce the frequency of transmission of zoonotic disease and thus would be of practical value, whereas the induction of strong mucosal immunity to S. ransomi third-stage larvae through vaccination of immunologically naive piglets would be most difficult. Investigations of immunity to percutaneous infection with S. ransomi in mature swine or to clearance of an established infection in sows or to the feasibility of passively transferred maternal immunity to piglets would be more practical. 31 Determination of Host Protective Mechanisms. Knowledge of host protective mechanisms against parasite infection is derived from an understanding of the basic cellular and humoral components of the host immune system. Characterization of the immune system of swine is advancing, 7 but most of our understanding of host protective mechanisms against helminthic parasites is derived from laboratory animal models. It is clear from studies in rodents that immune expulsion of intestinal nematodes is a T cell-dependent phenomenon. Production of specific antibody involved in worm damage directly or through association with effector cells or molecules requires the activation of T cells and their collaboration with antibody-producing cells. In ad-
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dition, inflammatory changes in the intestinal mucosa, including villous atrophy, mast cell hyperplasia, and goblet cell proliferation are T cell-dependent. The regulatory role of T cells in the immune system is expressed primarily through the production and release of nonantibody proteins called lymphokines that act as intercellular mediators of lymphocyte function and affect both inflammation and immunity. A lymphokine derived recently from swine T cells 17 can selectively expand parasite antigen-reactive swine T cells in vitro,16 and its in-vivo function during helminthic infection is currently under investigation. Lymphokines have been isolated from the intestinal mucosa of swine. 13 The application of methods developed recently to isolate enriched populations of lymphocytes and effector cells from the intestinal mucosa of man 3 and rodents 4 may define the role of locally produced lymphokines in development of intestinal immunity in swine to parasitic infection. Identification and Isolation of Protective Antigens. Research in this area is stimulated continually by the development of new methodologies and techniques. For example, the discriminating use of monoclonal antibodies in immunoblotting analysis to identify potentially protective antigens and their isolation by affinity columns or high-resolution chromatography in conjunction with molecular cloning techniques for production of defined antigens have been applied to development of several molecular vaccines. 9 The combination of these techniques with analysis of immune responses of genetically inbred mouse strains,26 and eventually genetically defined large animal species,21 could provide a powerful technique for detection and production of protective antigens for swine helminths. Antigen Presentation and Induction of Immunity. The appropriate presentation of antigens to the host to induce an effective immunity has been problematic in the development of immunization protocols for vaccination. The use of defined molecular vaccines will require more powerful adjuvants or delivery systems than conventionally isolated antigens because of the inherently reduced immunogenicity of defined antigens and the genetically based inability of the host to respond immunologically to certain molecules. 33 Sela and Arnon have described the ideal conditions for vaccination in which a synthetic antigen, namely, a 20 amino acid fragment of a protein subunit from the coliphage MS-2, is coupled to a defined adjuvant, muramyl dipeptide, to induce the efficient production of virus neutralizing antibodies after immunization. 36 However, this approach cannot be expected to succeed when cell-mediated immune responses or local induction of immunity are required for effective protection. Many alternate and innovative approaches exist to stimulate components of the immune system selectively or to enhance specific immunity. For example, antigens can be entrapped in the aqueous phase of liposomes or associated with their lipid membrane surface along with adjuvant molecules. 1 In addition, cell-mediated immune responses may be preferentially stimulated by changing the
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lipid composition of the liposome,l and intestinal absorption 18 of stable liposomes offers the advantage of antigen presentation to mucosal surfaces. Novel approaches to the presentation of parasite antigens to agriculturally important animal species may include the complexing of protective antigens to biologically active molecules capable of binding to mucosal cell surfaces, for example, cholera toxin or the fimbrial adhesins of E. coli, which have receptors on swine enterocytes. 32 Further, the preparation of vaccinia virus recombinants as expression vectors for DNA fragments coding for protective parasite antigens may soon become widely applied to veterinary use: Vaccinia virus has the capacity to accept large DNA inserts; it can provide glycosylated forms of the translated polypeptide antigens derived from the DNA insert; it can express recombinant antigens on the cell surface of infected host cells and induce cytolytic T cells 5 that are genetically restricted in their cell killing; it has the ability to infect a variety of domesticated animals; and it has been used recently to induce protection in cattle to a recombinant polypeptide from vesicular stomatitus virus. 23 Genetic regulation of immune responses to synthetic antigens and to low concentrations of certain protein antigens has been observed in swine inbred for genes of the major histocompatibility complex. 21 One of the implications of this observation is that genes that control immune responses necessary for disease resistance may soon be identified. Cloned DNA fragments of immune response genes could be incorporated into the genome of embryos of agriculturally important species by microinjection. The resulting transgenic animals might then be made responsive to specific protective antigens without a significant alteration of its existing genetic background. 20
REFERENCES 1. Alving, C. R., BaneIji, B., Shiba, T., et al.: Liposomes as vehicles for vaccines. In Mizrahi, A., Hertman, I., Klingberg, M. A., et al. (eds.): New Developments With Human and Veterinary Vaccines. New York, Alan R. Liss, Inc., 1980. 2. Befus, D., and Bienenstock, J.: Factors involved in symbiosis and host resistance at the mucosa-parasite interface. Prog. Allergy, 31 :76-177, 1982. 3. Befus, D., Goodacre, R., Dyck, N., et al.: Mast cell heterogeneity in man: I. Histologic studies of the intestine. Int. Arch. Allergy Appl. Immunol., 76:232-236,
1985. 4. Befus, A. D., Pearce, F. L., Gauldi, J., et al.: Mucosal mast cells: I. Isolation and functional characteristics of rat intestinal mast cells. J. Immunol., 128:2475-2480, 1982. 5. Bennink, J. R., Yewdell, J. W., and Smith, G. L.: Recombinant vaccinia virus primes and stimulates influenza haemagglutinin-specific cytotoxic T cells. Nature, 311:578-579, 1984. 6. Biehl, L. G.: Common internal parasites of swine. Vet. Clin. North Am., 4:355-375, 1982. 7. Binns, R. M.: Organization of the lymphoreticular system and lymphocyte markers in the pig. Vet. Immunol. Immunopathol., 3:95-146, 1982. 8. Cabrera, P. B., and Gould, S. E.: Resistance to trichinosis in swine induced by administration of irradiated larvae. J. Parasitol., 50:681-686, 1964.
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9. Cobon, G., and Willetts, N. S.: The application of recombinant DNA techniques to the production of antihelminth vaccines. In Dineen, J. K, and Outteridge, P. M. (eds.): Immunogenetic approaches to the control of endoparasites-with particular referance to parasites of sheep. Sydney, Australia, Sydney University, Division of Animal Health, 1984. 10. Despommier, D. D., and Laccetti, A.: Trichinella spiralis: Proteins and antigens isolated from a large-particle fraction derived from the muscle larva. Exp. Parasitol., 51 :279-295, 1981. 11. Despommier, D. D., and Muller, M.: The stichosome and its secretion granules in the mature muscle larva of Trichinella spiralis. J. Parasitol., 62:775-785, 1976. 12. Douvres, F. W., Tromba, F. G., and Malakatis, G. M.: Morphogenesis and migration of Ascaris suum larvae developing to fourth stage in swine. J. Parasitol., 55:689712,1969. 13. Frederich, G. T., and Bohl, E. H.: Local and systemic cell-mediated immunity against transmissible gastroenteritis and intestinal viral infection of swine. J. Immunol., 116:1000-1005, 1976. 14. Gamble, H. R: Trichinella spiralis: Immunization of mice using monoclonal antibody affinity-isolated antigens. Exp. Parasitol., 59:398-404, 1985. 15. Gamble, H. R, and Zarlenga, D. S.: Biotechnology in the development of vaccines for animal parasites. Vet. Parasitol., 1986 (in press). 16. Gasbarre, L. C., and Urban, J. F., Jr.: Assessment of T lymphocyte responses induced by parasite antigens. Vet. Parasitol., 10:119-129, 1982. 17. Gasbarre, L. C., Urban, J. F., Jr., and Romanowski, R D.: Porcine interleukin 2: Parameters of production and biochemical characterization. Vet. Immunol. Immunopathol., 5:221-236, 1984. 18. Genco, R J., Linzer, R, and Evans, R T.: Effect of adjuvants on orally administered antigens. In McGhee, J. R, and Mestecky, J. (eds.): The Secretory Immune System. Annals N.Y. Acad. ScL, 409:637-649, 1983. 19. Jarrett, E. E. E., and Miller, H. R P.: Production and activities of IgE in helminth infection. Prog. Allergy, 31 :178-233, 1982. 20. Lunney, J. K: Genetic control of host resistance to disease. In Augustine, P., Danforth, H. D., and Bakst, M. R (eds.): Proceedings of the Beltsville Symposium X: Biotechnology for Solving Agriculture Problems. Netherlands, Martinus Nijhoff, 1986 (in press). 21. Lunney, J. K, Pescovitz, M. D., and Sachs, D. H.: The swine major histocompatibility complex: Its structure and function. In Tumbleson, M. E. (ed.): Swine in Biomedical Research, New York, Plenum Press, 1986 (in press). 22. Marti, H. P., and Murrell, K D.: Antifecundity effect on Trichinella spiralis in immune pigs. [Abstract.] Proceeding of the 60th Annual Meeting of the American Society of Parasitologists, 1985. 23. Mackett, M., Yilma, T., Rose, J. K, et al.: Vaccinia virus recombinants: Expression ofVSV genes and protective immunization of mice and cattle. Science, 227:433435, 1985. 24. McKercher, P. D., Moore, D. M., Morgan, D.O., et al.: Dose-response evaluation of a genetically engineered foot-and-mouth disease virus polypeptide immunogen in cattle. Am. J. Vet. Res., 45:587-590, 1985. 25. Mitchell, G. F.: Towards molecular vaccines against parasites. Parasite Immunol., 6:493-498, 1984. 26. Mitchell, G. F., and Anders, R F.: Parasite antigens and their immunogenicity in infected hosts. In Sela, M. (ed.): The Antigens, Volume 6. New York, Academic Press, 1982. 27. Mitchell, G. F., Anders, R F., Brown, G. V., et al.: Analysis of infection characteristics and antiparasite immune responses in resistant compared with susceptible hosts. Immunol. Rev., 61 :138-188, 1982. 28. Murrell, K D.: Strategies for the control of human trichinosis transmitted by pork. Food Technology, 39:65-68, 1985. 29. Murrell, K D.: Trichinella spiralis: Acquired immunity in swine. Exp. Parasitol., 59:347-354, 1985. 30. Murrell, K D., and Despommier, D. D.: Immunization of swine against Trichinella spiralis. Vet. Parasitol., 15:263-270, 1984.
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31. Murrell, K. D., and Urban, J. F., Jr.: Induction of immunity by transcolostrally passed Strongyloides ransomi larvae in neonatal pigs. J. Parasitol., 69:74-77, 1983. 32. Newby, T. J.: Protective immune responses in the intestinal tract. In Newby, T. J., and Stokes, e. R (eds.): Local Immune Responses of the Gut. Boca Raton, Florida, eRe Press, Inc., 1984. 33. Paul, W. E.: Immune response genes. In Fundamental Immunology. New York, Raven Press, 1984. 34. Powers, K. G., Todd, A. C., and Goldsby, M. S.: Swine whipworm in Wisconsin. Vet. Med., 54:396-397, 1959. . 35. Schantz, P. M.: Trichinosis in the United States. Food Technology, 37:83-86, 1983. 36. Sela, M., and Arnon, R: Antiviral antibodies obtained with aqueous solution of a synthetic antigen. In Mizrahi, A., Hertman, I., Klingberg, M. A., et al. (eds.): New Developments With Human and Veterinary Vaccines, New York, Alan R Liss, Inc., 1980. 37. Silberstein, D. S., and Despommier, D. D.: Antigens from Trichinella spiralis that induce a protective response in the mouse. J. Immunol., 132:898-904, 1984. 38. Stewart, D. F.: Studies on resistance of sheep to infestation with Haemonchus contortus and Trichostrongylus spp. and on the immunological reactions of sheep exposed to infestation: V. The nature of the "self-cure" phenomenon. Aust. J. Agric. Res., 4:100-117, 1953. 39. Stewart, T. B., Batte, G., Connell, H. E., et al.: Research needs and priorities for swine internal parasites in the United States. Am. J. Vet. Res., 46:1029-1033, 1985. 40. Tromba, F. G.: Immunization of pigs against experimental Ascaris suum infection by feeding ultraviolet-attenuated eggs. J. Parasitol., 64:651-656, 1978. 41. Urban, J. F., Jr., Alizadeh, H., and Romanowski, R: Acquired gut immunity to the large roundworm (Ascaris suum) of swine. In Proceedings of the Pig International Veterinary Society Congress, 1984. 42. Urban, J. F., Jr., and Romanowski, R D.: Ascaris suum: Protective immunity in pigs immunized with products from eggs and larvae. Exp. Parasitol., 60:245-254, 1985. 43. Urban, J. F., Jr., and Tromba, F. G.: An ultraviolet-attentuated egg vaccine for swine ascariasis: Parameters affecting the development of protective immunity. Am. J. Vet. Res., 45:2104-2108, 1984. 44. Urquhart, G. A.: Irradiated vaccines. In Capron, A., Lambert, P. H., Ogilvie, B., et al. (eds.): Immunity in Parasitic Diseases, Colloque Inserm-INRA, ThivervalGrignon, les Editions de L'Institut National de la Sante et de la Recherche Medicale, 1977. 45. Vernes, A.: Immunization of the mouse and minipig against Trichinella spiralis. In Van der Bossche, H. (ed.): Biochemistry of Parasites and Host-Parasite Relationships, Amsterdam, Elsevier/North Holland Press, 1976. 46. Wakelin, D.: Genetic control of immunity to helminth infections. Parasitology Today, 1:17-23,1985. 47. Zavala, F., Tam, J. P., Cochrane, A. H., et al.: Rationale for development of a synthetic vaccine against Plasmodium Jalciparum malaria. Science, 228: 1436-1440, 1985. Helminthic Diseases Laboratory Animal Parasitology Institute U.S. Department of Agriculture Beltsville, Maryland 20705
0749-0720/86
$00.00 + $.20
The Epidemiology and Control of Swine Parasites Immunity and Vaccines j. F. Urban,jr., Ph.D.*
More than 15 different species of helminthic parasites infect swine in the United States. Economic loss can be a consequence of infection. 6 , 39 Primary methods for control of infection are proper animal management and proper choice and administration of an array of highly effective anthelminthic drugs. However, the effectiveness of these methods requires knowledge of the parasite and its life cycle. It is apparent that these methods are often inadequate or are poorly implemented because direct swine production and condemnation losses in the United States due to infection with helminthic parasites are currently estimated to exceed $385 million annually.39 An alternative approach or supplemental component of an integrated control strategy is biologic control of parasitism through immunization of the host. However, no economically useful vaccine for swine helminthic parasites currently exists. In fact, except for the rather unique application of radiation-attenuated, live parasite vaccines for cattle and sheep lungworm, no economically useful helminthic vaccine exists for any agriculturally important food-producing animal species. 44 The recent development of effective molecular vaccines against infectious agents of medica1 47 and veterinary 24 importance has stimulated efforts to develop helminthic parasite vaccines. Molecular vaccines currently consist of complete polypeptide antigens or relatively small defined epitopes that are genetically engineered or synthetically constructed. Because they are chemically well defined, they are more advantageous than conventionally prepared vaccines. 25 For example, production is amenable to good quality control, and they can be produced in large quantities, relatively free from the contaminants or byproducts that accompany complex vaccines. Note: Owing to the constraints of size, this article could not be included in the July 1986 issue of Veterinary Clinics of North America: Food Animal Practice on Parasites: Epidemiology and Control (Vol. 2, No.2).
* Helminthic Diseases Laboratory, Animal Parasitology Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland
Veterinary Clinics of North America: Food Animal Practice-Vol. 2, No.3, November 1986
765
COMMON NAMES
Large roundworm
Whipworm
Trichinae
Kidney worm
Ascaris suum
Trichuris suis
Trichinella spiralis
Stephanurus dentatus
Free-living, infective larvae in environment (and in earthworms)
Infective larvae in muscle tissue
Eggs in environment
Eggs in environment
SOURCE OF INFECTION
Small intestine (or percutaneous) (or prenatal in utero)
Small intestine
Cecum or colon
Small intestine
infection
I nitial site of
Lymph nodes, liver, perirenal fat, hilus of kidney,
Circulation, liver, muscle
Mucosa of cecum or colon
Circulation, liver, lung, small intestine
Sites during larval migration
LOCATION IN THE HOST
Major Nematode Parasites that Infect Swine in the United States
SCIENTIFIC NAMES
Table 1.
Encysted along ureters
Small intestines
Cecum or colon
Small intestine
Location of adults
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Threadworm
Nodular worm
Lungworm
Red stomach worm
Thick stomach worms
Strongyloides ransomi
Oesophagostomum dentatum O. quadrispinulatum O. brevicaudum O. georgianum
Metastrongylus apri M. pudendotectus M. salmi
Hyostrongylus rubidus
Ascarops strongylina Physocephalus sexalatus Infective larvae within Dung beetle
Free-living infective larvae in environment
Infective larvae within earthworm
Free-living, infective larvae in environment
Free-living infective larvae in environment, parasitic larvae in colostrum derived from mammary fat
Mucosa of stomach
Gastric glands of stomach
Small intestine
Small intestine, colon, cecum
Small intestine (or percutaneous)
Mucosa of stomach
Mucosa of stomach
Lymphatics, circulation, lungs
Mucosa of small intestine, colon, cecum
Circulation, lungs, fat
Stomach
Stomach
Bronchioles and bronchi of lung
Colon, cecum
Small intestines
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The purpose of this report is to discuss the status of two experimental swine helminthic vaccines and how further knowledge of parasite biochemistry and swine immunology and the application of techniques of modern biotechnology may affect their future development. Targeting a Parasite for Development of an Effective Vaccine The most important internal helminthic parasites of swine in the United States are nematodes or roundworms, commonly comprising about 15 species (Table 1). Effective and economically sensible vaccines most probably can be developed within the next few years against Ascaris suum and Trichinella spiralis. This prediction is based on the following facts: 1. Both parasites are of major economic importance to the swine industry. (Infection with A. suum reduces feed efficiency, increases organ condemnation, and decreases herd health both directly and through secondary infection, whereas infection with T. spiralis jeopardizes product safety and threatens public health.) 2. The epidemiology of both parasites is reasonably well understood, and yet they continue to plague selected segments of the swine industry. 3. There is an extensive literature on protective mechanisms to these parasites and on the identification and isolation of their antigens. 4. There is good documentation that protective immunity develops to both parasites in infected swine.
Factors complicating the development of vaccines for the 13 other species of parasites listed in Table 1 are both pragmatic and problematic, but can be loosely catagorized as follows: 1. Recent prevelance studies 39 have indicated a decreasing infection level for lungworms and stomach worms in areas in which confinement rearing of pigs has been adopted. 2. Convenient laboratory animal model systems for passage of many of these parasites and for studies of host resistance are unavailable; and parasite specimens for antigen isolation and characterization are limited. 3. The relative economic importance of infection with these parasites is subordinate to that of A. suum and T. spiralis. However, it should be noted that the prevalence of both Trichuris suis and Strongyloides ransomi infection in pigs is still regarded as important for certain segments of the industry, and economic losses can be significant. 39 In addition, there are defined laboratory animal models available for these parasites, and immunity in swine has been demonstrated.31, 34
It is convenient conceptually that the major site of initial infection of all the common nematode parasites of swine is the mucosa of the alimentary tract (see Table 1) because information gleaned about protective immunity at the mucosal surface to anyone of these parasites would be applicable, in principal, to an understanding of protective imm unity to all. Immunity in S wine to Ascaris suum
Demonstration of Acquired Immunity. Early survey studies of swine for helminthic infestation showed that the prevalence of A.
SWINE PARASITES: IMMUNITY AND VACCINES
769
suum infection was markedly less in sows than in young pigs, suggesting that resistance to infection developed with age. Clearly, the degree of parasite exposure and not the age of the pig determines the level of acquired immunity to subsequent infection. 41 The first reliable and effective protocol developed for vaccination against A. suum used eggs attenuated by ultraviolet irradiation. 40, 43 The conditions for successful vaccination of pigs and the characteristics of resistance are as follows: 1. Development of attenuated larvae in the host through the fourth stage is sufficient to induce strong protective immunity. 2. Vaccinated pigs have fewer challenge larvae migrating to the lungs and fewer adults developing in the small intestines than challenge-exposed, nonvaccinated pigs. 3. As few as 1500 ultraviolet-irradiated eggs given in two inoculations or 300 ultraviolet-irradiated eggs divided into 3 inoculations can induce marked protection. 4. Oral inoculation is superior to parenteral (intraperitoneal) inoculation, indicating a requirement for local sensitization of the intestine with specific larval stages. 5. Both specific in-vitro cellular lymphoproliferative responses and serum antibody responses to parasite antigens are increased after inoculation. 6. Intestinal immunity is not induced appreciably because larvae from a challenge exposure migrate to the liver and elicit strong milk-spot lesions.
Demonstration of Intestinal Immunity. Ascaris suum larvae penetrate the intestinal mucosa and migrate parenterally, causing severe inflammation, primarily in the liver and lungs, and increasing the risk of secondary infection. Therefore, the ideal target site for induced immunity would be at the level of the intestine. The choice of a strategy that would lead to the development of a practical vaccine for swine ascariasis is dependent on the answer to the following question: Can pigs develop intestinal immunity to a challenge exposure to A. suum eggs that would preclude larval migration to the liver and lungs? Clearly they can because pigs naturally exposed to infection with A. suum on contaminated dirt lots for a prolonged period were nearly free of liver milk spots after a challenge exposure (Fig. lA), whereas pigs receiving a shorter period of exposure developed only a low-level protective immunity and a high degree of liver pathology following challenge exposure (Fig. IB). More direct evidence was the fact that pigs naturally exposed to A. suum and then challenge-exposed orally with eggs were free of surface milk-spot lesions (Fig. 2A), but injection of freshly hatched A. suum larvae directly into the mesenteric veins (Fig. 2B) induced numerous milk-spot lesions on the liver (Fig. 2C).
These results clearly show that the intestine can become an effective barrier against larval migration. This response has recently been reproduced under defined conditions of egg dosage and inoculation frequency in confinement-housed pigs. Therefore, it is induced specifically by exposure to A. suum and is not dependent on
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URBAN,
In.
Figure 1. Livers obtained after necropsy of pigs 7 days after a challenge exposure to 10,000 A. suum eggs. A, From pigs after 18 weeks (#533 and #538) and 24 weeks (#1571 and #539) of expos ure on contaminated dirt lots, showing few active milk-spot lesions. B, From a confinement-reared pig (#532) and a pig after 6 weeks on dirt followed by 18 weeks in confinement (# 1521), showing numerous milk-spot lesions of varying intensity.
the myriad of other biologic and immunologic stimuli encountered on a dirt lot. Basis of Intestinal Immunity. Stewart's observations on the "self-cure" phenomenon of sheep infected with Haemonchus contortus established the concept that a challenge exposure to nematode larvae could quickly induce a local allergic type reaction, characterized by gastric edema, increased peristalsis, and a rise in whole blood histamine,38 all of which are detrimental to parasite survival in the host. Numerous studies have subsequently established that the cellular and humoral components of allergic responses are characterist-
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Figure 2. Demonstration of gut-level protection to a challenge exposure of 10,000 A . suum eggs in a pig naturally exposed to infection for a prolonged period (16 weeks). A, Liver during laparotomy 7 days after oral challenge ; no visible milk spots. B, Injection of viable second-stage larvae of A. suum into the mesenteric veins during the laparotomy. C, Numerous milk-spot lesions visible at 7 days after the intravenous injection of L2.
ically present following helminth infection, that is, a marked increase in production of reaginic antibody of the IgE class, accumulation of mast cells or basophils, or both, and subsequently eosinophils in parasitized tissues, and the release of preformed and secondary mediators of anaphylaxis. 2 • 19 It is clear that these responses represent the expression of rapid effector mechanisms that potentially can have dramatic direct effects on the parasite or, by markedly altering the ambient environment of the parasite in the host, can indirectly affect parasite survival. Because larvae from infective A. suum eggs begin to migrate from
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the intestinal lumen and establish in the liver within 6 hours after inoculation,12 any intestinal immune response that precludes liver invasion must be of an immediate type. Recent studies have shown that some of the components of immediate-type hypersensitivity responses to helminths are present in pigs with strong intestinal immunity to A. suum. For example, pigs exposed to A. suum eggs on contaminated dirt lots for over 16 weeks have greater numbers of mast cells in the intestinal mucosa that do pigs kept in confinement, free from infection. There is also an increase in eosinophils in the intestinal mucosa, and these pigs show immediate-type skin reactions to a number of A. suum larval and adult antigens, whereas controls that were housed parasitt ':'ree in confinement are unreactive. These observations suggest that the peculiar conditions of chronic exposure to A. suum that appear to be necessary to induce strong intestinal immunity in swine may provide the appropriate stimuli for the local accumulation of most of the components involved in an immediate-type allergic response to infective larvae. Immunization of Swine with Ascaris suum Antigens. The successful vaccination of swine against ascariasis using ultraviolet-attenuated eggs provided implicitly the evidence that products obtained from infective larvae in the egg (first-molt and second-stage larvae) through early fourth-stage larvae contain protective antigens. This was verified by successful immunization of swine with products obtained from eggs of A. suum and from in-vitro cultivation of the larvae. 42 However, two major problems remain for the development of these parasite products into a practical vaccine for ascariasis. First, they are complex and consist of a number of distinct protein components, many of which may be irrelevant to the induction of a protective immune response. Second, the protective immunity that they induce is not expressed strongly at the intestinal level; that is, liver milk-spot lesions are derived from a challenge exposure. The solution to the first problem may be overcome by improvement in antigen isolation procedures similar to those described below for swine trichinosis. A solution to the second may depend upon a better understanding of the regulatory mechanisms necessary to stimulate the local accumulation of the required cellular and humoral components of immunity in the intestinal wall (see section on future research directions). Immunity of Swine to Infection with Trichinella spiralis
Demonstration of Acquired Immunity. Although swine trichinosis is an important zoonotic disease in the United States,35 and vaccination of swine in selected segments of the industry could be a significant component of an effective integrated control program,28 immunity to T. spiralis has been studied primarily in rodent models, and surprisingly little is known of immunity to T. spiralis in swine. Murrell showed recently that adult T. spiralis developing from a primary infection are eventually expelled from the intestines of pigs, and that pigs inoculated with T. spiralis L1 elicit a more rapid intestinal expulsion of adults developing from a secondary challenge expo-
SWINE PARASITES: IMMUNITY AND VACCINES
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sure. 29 The intensity of both of these events is dependent on the number of LI in the primary infection. Inoculation of pigs with as few as 112 LI evokes a nearly complete resistance to the muscle larva stage developing from a challenge exposure. There is direct evidence that a significant antifecundity response is established in these pigs that accounts for most of the reduction in muscle larvae burdens. However, there also appears to be a direct detrimental effect on the development of newborn larvae in immune pigs because passively transferred immune pig serum reduces muscle larvae burdens without affecting adult worm burdens or fecundity.22 Thus, protective mechanisms induced in swine after a complete infection with T. spiralis appear to be acting at at least three levels: (1) expulsion of adults after a relatively high inoculation dose; (2) an antifecundity effect that markedly reduces the production of newborn larvae; and (3) a stage-specific protective immunity against newborn larvae. Immunization of Swine with Trichinella spiralis Antigens. The demonstrated resistance of hogs to reinfection with T. spiralis LI was the basis for the successful vaccination of swine with radiation-attenuated LIB and, later, the use of antigens derived from in-vitro grown LI for immunization. 45 The association between secreted products of LI and its stichosome l l led to the isolation of stichocyte secretory granules. Antigens from a soluble portion of stichocyte secretory granules (S3) were isolated and used to immunize both mice lO and pigs 30 against a challenge exposure to T. spira lis. However, an immunoaffinity purified fraction (PAW) of S3, although biochemically less complex than S3 and capable of inducing strong immunity in mice, failed to protect pigs when it was administered at the same dose as the S3. There are at least two possible reasons for the failure to observe significant protective immunity in pigs immunized with PAW: (1) the inherent ability of pigs to respond immunologically to the antigens in the PAW preparation could be different from that of mice; or (2) the limited availability of PAW made optimization of an immunizing protocol in pigs impractical. The first explanation describes a mechanistic problem. Individual and strain differences in the ability to invoke immune responses is based on genetic control of immunity. 33 Research on the genetic basis of immunity to helminths is currently very active 27, 46 and includes studies on swine immunogenetics (see below). The marked difference in the kinetics of gut expulsion of adult T. spiralis among mice, rats, and pigs 29 is a notable example of the species variation in host protective responses to helminthic infection. It also emphasizes the danger of generalizing too broadly on the efficacy of particular immune effector mechanisms. The limited availability of purified helminth antigens has seriously hampered progress on vaccine development until recently. A solution to this problem is now possible through techniques derived from the new biotechnologies. Both Silberstein and Despommier37 and, more recently, Gamble 14 used hybridoma technology to generate monoclonal antibodies specific for products derived from the T. spir-
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alis L1 stichosome. They were able to identify one or two very similar antigens from the L1 stichosome that induced high levels of protective immunity in mice against challenge exposure to T. spiralis Ll. Thus, through the use of this very powerful technique, one or a few protective antigens were identified and selected from a complex mixture of antigens contained in the secretory apparatus of T. spiralis. Although this work represents a major advance in the isolation and characterization of protective antigens from nematodes, the relevance of these antigens to the induction of protective immunity in swine, for the reasons stated earlier, must be established experimentally. Immunoaffinity isolation of these antigens in sufficient quantities to test in swine will require a herculean effort. A more practical alternative is the use of recombinant DNA techniques for the production of antigens. Gamble and Zarlenga have used molecular cloning of antigens from T. spiralis L1 to provide a potential source of large quantities of antigens for vaccine development in swine. 15 Future Research Directions The general strategy for development of helminthic vaccines of economic importance requires the following: (1) the identification of vulnerable stages in the parasite's life cycle that are most susceptible to immunologic attack or that are of economic importance; (2) determination of the nature of the host protective mechanism; (3) identification and isolation of the protective antigen(s); and (4) presentation of the antigen(s) to the host to induce the desired form of immunity. Definition of the Vulnerable Stages of the Parasite. Strong mucosal immunity to the parasitic stages of most swine gastrointestinal nematodes would be tantamount to sterilizing immunity (see Table 1); however, for some infections this may be an impossible or at least an unnecessary goal. For example, strong parenteral immunity to newborn larvae of T. spiralis would reduce the frequency of transmission of zoonotic disease and thus would be of practical value, whereas the induction of strong mucosal immunity to S. ransomi third-stage larvae through vaccination of immunologically naive piglets would be most difficult. Investigations of immunity to percutaneous infection with S. ransomi in mature swine or to clearance of an established infection in sows or to the feasibility of passively transferred maternal immunity to piglets would be more practical. 31 Determination of Host Protective Mechanisms. Knowledge of host protective mechanisms against parasite infection is derived from an understanding of the basic cellular and humoral components of the host immune system. Characterization of the immune system of swine is advancing, 7 but most of our understanding of host protective mechanisms against helminthic parasites is derived from laboratory animal models. It is clear from studies in rodents that immune expulsion of intestinal nematodes is a T cell-dependent phenomenon. Production of specific antibody involved in worm damage directly or through association with effector cells or molecules requires the activation of T cells and their collaboration with antibody-producing cells. In ad-
SWINE PARASITES: IMMUNITY AND VACCINES
775
dition, inflammatory changes in the intestinal mucosa, including villous atrophy, mast cell hyperplasia, and goblet cell proliferation are T cell-dependent. The regulatory role of T cells in the immune system is expressed primarily through the production and release of nonantibody proteins called lymphokines that act as intercellular mediators of lymphocyte function and affect both inflammation and immunity. A lymphokine derived recently from swine T cells 17 can selectively expand parasite antigen-reactive swine T cells in vitro,16 and its in-vivo function during helminthic infection is currently under investigation. Lymphokines have been isolated from the intestinal mucosa of swine. 13 The application of methods developed recently to isolate enriched populations of lymphocytes and effector cells from the intestinal mucosa of man 3 and rodents 4 may define the role of locally produced lymphokines in development of intestinal immunity in swine to parasitic infection. Identification and Isolation of Protective Antigens. Research in this area is stimulated continually by the development of new methodologies and techniques. For example, the discriminating use of monoclonal antibodies in immunoblotting analysis to identify potentially protective antigens and their isolation by affinity columns or high-resolution chromatography in conjunction with molecular cloning techniques for production of defined antigens have been applied to development of several molecular vaccines. 9 The combination of these techniques with analysis of immune responses of genetically inbred mouse strains,26 and eventually genetically defined large animal species,21 could provide a powerful technique for detection and production of protective antigens for swine helminths. Antigen Presentation and Induction of Immunity. The appropriate presentation of antigens to the host to induce an effective immunity has been problematic in the development of immunization protocols for vaccination. The use of defined molecular vaccines will require more powerful adjuvants or delivery systems than conventionally isolated antigens because of the inherently reduced immunogenicity of defined antigens and the genetically based inability of the host to respond immunologically to certain molecules. 33 Sela and Arnon have described the ideal conditions for vaccination in which a synthetic antigen, namely, a 20 amino acid fragment of a protein subunit from the coliphage MS-2, is coupled to a defined adjuvant, muramyl dipeptide, to induce the efficient production of virus neutralizing antibodies after immunization. 36 However, this approach cannot be expected to succeed when cell-mediated immune responses or local induction of immunity are required for effective protection. Many alternate and innovative approaches exist to stimulate components of the immune system selectively or to enhance specific immunity. For example, antigens can be entrapped in the aqueous phase of liposomes or associated with their lipid membrane surface along with adjuvant molecules. 1 In addition, cell-mediated immune responses may be preferentially stimulated by changing the
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lipid composition of the liposome,l and intestinal absorption 18 of stable liposomes offers the advantage of antigen presentation to mucosal surfaces. Novel approaches to the presentation of parasite antigens to agriculturally important animal species may include the complexing of protective antigens to biologically active molecules capable of binding to mucosal cell surfaces, for example, cholera toxin or the fimbrial adhesins of E. coli, which have receptors on swine enterocytes. 32 Further, the preparation of vaccinia virus recombinants as expression vectors for DNA fragments coding for protective parasite antigens may soon become widely applied to veterinary use: Vaccinia virus has the capacity to accept large DNA inserts; it can provide glycosylated forms of the translated polypeptide antigens derived from the DNA insert; it can express recombinant antigens on the cell surface of infected host cells and induce cytolytic T cells 5 that are genetically restricted in their cell killing; it has the ability to infect a variety of domesticated animals; and it has been used recently to induce protection in cattle to a recombinant polypeptide from vesicular stomatitus virus. 23 Genetic regulation of immune responses to synthetic antigens and to low concentrations of certain protein antigens has been observed in swine inbred for genes of the major histocompatibility complex. 21 One of the implications of this observation is that genes that control immune responses necessary for disease resistance may soon be identified. Cloned DNA fragments of immune response genes could be incorporated into the genome of embryos of agriculturally important species by microinjection. The resulting transgenic animals might then be made responsive to specific protective antigens without a significant alteration of its existing genetic background. 20
REFERENCES 1. Alving, C. R., BaneIji, B., Shiba, T., et al.: Liposomes as vehicles for vaccines. In Mizrahi, A., Hertman, I., Klingberg, M. A., et al. (eds.): New Developments With Human and Veterinary Vaccines. New York, Alan R. Liss, Inc., 1980. 2. Befus, D., and Bienenstock, J.: Factors involved in symbiosis and host resistance at the mucosa-parasite interface. Prog. Allergy, 31 :76-177, 1982. 3. Befus, D., Goodacre, R., Dyck, N., et al.: Mast cell heterogeneity in man: I. Histologic studies of the intestine. Int. Arch. Allergy Appl. Immunol., 76:232-236,
1985. 4. Befus, A. D., Pearce, F. L., Gauldi, J., et al.: Mucosal mast cells: I. Isolation and functional characteristics of rat intestinal mast cells. J. Immunol., 128:2475-2480, 1982. 5. Bennink, J. R., Yewdell, J. W., and Smith, G. L.: Recombinant vaccinia virus primes and stimulates influenza haemagglutinin-specific cytotoxic T cells. Nature, 311:578-579, 1984. 6. Biehl, L. G.: Common internal parasites of swine. Vet. Clin. North Am., 4:355-375, 1982. 7. Binns, R. M.: Organization of the lymphoreticular system and lymphocyte markers in the pig. Vet. Immunol. Immunopathol., 3:95-146, 1982. 8. Cabrera, P. B., and Gould, S. E.: Resistance to trichinosis in swine induced by administration of irradiated larvae. J. Parasitol., 50:681-686, 1964.
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