2002 Annual Report 1.What major problem or issue is being resolved and how are you resolving it? Swine have two prominent foodborne parasitic disease agents: the muscle worm, Trichinella spiralis, and the tissue protozoan parasite, Toxoplasma gondii. Infective eggs from the large roundworm, Ascaris suum, represent an emerging disease where agricultural use of pig manure contaminated with parasite eggs may increase human infection. Outbreaks of human disease from swine products negatively reflect on pork as a safe source of nutritious edible tissue and lowers export potential. Molecular tests to evaluate parasite infections in meat products need to be improved so that they are very sensitive and accurately reflect the true source of the infection. Proper maintenance of herd health precludes infection but requires a large capital investment; breaches in procedure can readily result in new infections. No drugs are available to adequately cure tissue parasites, and the cost of development of new drugs by the pharmaceutical industry is prohibitive and unpredictable. Further, secondary bacterial disease is associated with worm infection in livestock; one such bacteria, Campylobacter, is a major cause of diarrhea in humans. A long-term prevention strategy is to vaccinate against swine parasites. Cooperative efforts between university, industry and government laboratories is directed at molecular regulators of immunity, receptor biology, innate protective mechanisms, and antibody responses targeted at the intestinal immune system of pigs. The basic nature of this work will also provide information for the immunological control of microbial and viral infection in swine. Consumers are now requesting safer food products, and encouraging producers to raise animals without antibiotics. Thus, producers have had to reshape the methods that they employ for raising disease-free pigs. This will require advanced knowledge of neonatal swine immunity and molecular tests for activators of the swine immune system such as recombinant cytokines [interleukin-12 (IL-12) and IL-18]. Description of effects of these cytokines and of synthetic oligonucleotides (CpG) on swine immunity, and their use to enhance neonatal swine immunity and to stimulate appropriate protective mechanisms against foodborne pathogens, will have commercial value. Vaccines incorporating these activators would complement existing control procedures and provide the benefits of long-term protection, cost efficiency and reduced drug residues in the food chain. Studies of toxoplasmosis have shown that strong protective immunity can be induced in pigs by irradiated oocysts and attenuated strains, but that immunity is not complete and may result in low-level infection. Novel cytokine-based procedures that activate porcine immunity by increasing interferon-gamma (IFN-gamma) levels have the potential to enhance resistance to toxoplasmosis. The underdeveloped intestinal immune system of weaned-pigs provides a model to test other prospective immune enhancing agents on development of resistance to infection in neonatal pigs. Swine disease control is most important during the first month of a pig's life due immature immune system development in the newborn pig. Moreover there is rapid exposure of the newborn to infectious agents when it moves from the birthing (farrowing) unit into nursery facilities that house litters from numerous sows. We have asked whether administration of exogenous immune proteins, termed cytokines, enhances neonatal immunity and disease responses, and determine whether this will help protect young pigs from infections and stress-induced immune deficiencies. Our studies are aimed at determining the role of cytokines in the development of the neonatal immune system of swine. Descriptions of the mucosal immune system and the immunobiology of gastrointestinal parasite responses in neonatal and growing pigs will provide useful and largely unique information that will be available to scientists in other federal, academic and industry (producers, vaccine and pharmaceutical companies) laboratories. 2.How serious is the problem? Why does it matter? Proof of human toxoplasmosis from eating pork products is tenuous, but continuing evidence of T. gondii infection in commercial herds is suspicious and generates negative publicity. The reputation of pork as the "other white meat" is undermined by images of pork as "wormy" or requiring greater care in the household. Pro-active biological strategies to control parasite infection compensate for deficiencies in existing control procedures that are inadequate. Vaccination of livestock is a generally acceptable and familiar procedure to the public. Vaccines against gastrointestinal parasites would complement existing control procedures and provide the benefits of long-term protection, cost efficiency and reduced drug residues in the food chain. Outbreaks of trichinellosis are sporadic and limited to peculiar human dietary habits and poor herd management of small farms, while infection of swine with roundworms is ubiquitous. Surveys of the prevalence of toxoplasmosis indicate a high level of herd infection in major swine producing areas and an increase in seroprevalence in humans worldwide. Sensitive tests to prove that pork products are free of parasites will prove that producers are controlling the sources of these infections. Newborn piglets are exposed to a wide array of infectious diseases, particularly as they move from farrowing into nursery facilities. Improvements of the immune capacity of these neonates will decrease disease associated production losses and improve time to market. In addition such improvements may lead to piglets which are less susceptible to secondary infections, such as Campylobacter, another foodborne illness that can be transmitted through pork products. 3.How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? Studies are related to National Program Area 103 in Animal Health (100%). Components: Animal Immunology, Mechanism of Disease, Genetic Resistance to Disease, and Strategies to Control Infectious and Non-Infectious Disease. Studies also apply to National Program Area 108 in Food Safety. This research fits into an overall strategy to reduce the potential for infection of swine with parasitic organisms that could infect humans through ingestion of contaminated food. Studies on the basic mechanism of immunological control of infectious disease of swine supports development of vaccines against parasitic, viral and microbial infections that compromise animal health and productivity. 4.What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment(s) during FY2002 year: A critical aspect of maintaining good animal health is to have an immune system that responds appropriately to infectious agents without inappropriate tissue destruction. We have collaborated with the Nutrient Requirements and Functions Laboratory at the Beltsville Human Nutrition Research Center to develop reagents to measure gene expression of over 100 porcine genes related to immunity and nutrient metabolism. Pigs were infected with either the protozoan parasite, Toxoplasma gondii, a zoonosis that threatens food safety or the economically important round worm, Ascaris suum, and 12 different tissue sites throughout the body of pigs were evaluated for response to infection. The impact of this analysis adds a dynamic and expanding array of genes to determine the response of healthy animals to infectious disease using quantitative measures of gene expression. B. Other Significant Accomplishment(s): There is a need to apply sophisticated molecular and genomic approaches to agriculturally important pathogens. Over 2,000 clones from the forth-stage (L4) larvae of the large roundworm of pigs, Ascaris suum, were cloned and imprinted on glass slides by robotics to create a cDNA microarray. Screening of the microarray with RNA isolated from other stages of A. suum derived from pigs revealed a pattern that indicates successful parasitism correlates with the expression of a TGF-beta homologue present within the parasites, as well as the expression of parasite muscle genes. These results demonstrate that genomic approaches can be applied to a biological question that can lead to a better strategy for control of economically important animal diseases and emerging human infection. Little is known of the mechanisms by which intracellular parasites penetrate and reprogram cells to protect themselves from the host immune system. Understanding one or more of these mechanisms has far reaching implications in both Agricultural and human research, especially when considering the swine muscle parasite Trichinella spiralis as a model. To this end, a protein has been identified specific to the infectious stage that correlates with sequences from protozoan parasites known to associate with the surfaces of infected cells. Results from these studies will assist not only in comprehending mechanisms involved in parasite invasion of the cell, but may serve also as a model for animal and human degenerative muscle diseases, and define targets by which the degenerative process may be abrogated. Determination of the risk of foodborne transmission of Toxoplasma gondii in meat products requires a thorough epidemiological survey to allay consumer fears. Such a survey is underway involving a large statistical sampling of market beef, pork and chicken from numerous cities in the United States. Monitoring the levels of the parasite in actual market samples will involve several approaches, including bioassays, serum and tissue juice antibody assays and a molecular assay developed with the support of National Pork Board funds. Samples are now being tested for T. gondii and the results for each test compared. These results will assure consumers of an accurate risk of exposure to toxoplasmosis through the commercial meat supply. The immune system is complex and requires new tools to track the cells that respond to infectious organisms. Members of this laboratory participated in a major international workshop to determine the reactivity of ~200 monoclonal antibodies (mAb) against swine immune cells. The mAb were exchanged, tested for specific reactivity and the results statistically analyzed so that cluster of differentiation (CD) numbers could be assigned. The results are now internationally accepted as the standard CD designation for pig immune cells, enabling scientists worldwide to exchange data and determine which cells are essential in preventing infections or in stimulating protective vaccine responses. C. Significant Accomplishments/Activities that Suppost Special Target Populations: None D. Progress Report: New approaches are being explored to determine whether pigs that are genetically resistant to disease can be identified as an alternate to using antibiotics to treat early weaned pigs. Agricultural Research Service (ARS) scientists at Beltsville, MD, in collaboration with the Pig Improvement Corporation (PIC) have evaluated piglets blood cell markers associated with immune function. We showed that there were significant increases in specific T cell subsets during the first few weeks of a piglet's life. These results help explain why neonates are more susceptible to disease and may also indicate how producers can plan management changes to avoid immune and disease stressors. Pigs are exposed to infectious agents when they are moved to new facilities and mix with pigs from other areas. The use of antibiotics to control infection is limited and alternate therapeutic approaches are desperately needed. In collaboration with the Biotechnology Research and Development Corporation (BRDC) and their collaborating pharmaceutical and breeding company partners, we have cloned and purified a new pig biotherapeutic, recombinant porcine cytokine interleukin-12 (rPoIL-12). However, rPoIL-12 has limited activity in swine because of low IL-12 receptor expression and therefore, may not, on its own, provide adequate stimulation of the pig immune system. This is essential information for pharmaceutical and vaccine companies as they search for effective immunostimulants for swine. 5.Describe your major accomplishments over the life of the project, including their predicted or actual impact? Molecular markers of distinct Trichinella genotypes/species have been identified and developed into a sensitive diagnostic test. This test will reduce the risk of undetected freeze resistant Trichinella strains that could undermine the frozen meat technology that is the major commercial and consumer-based control strategy for the inactivation of Trichinella in edible tissue. In addition, the epidemiology of the remaining 9 other detectable genotypes can be monitored to prevent their emergence in the U.S. Studies of toxoplasmosis have shown that strong protective immunity can be induced in pigs by irradiated oocysts and attenuated strains, but that immunity is not complete and may result in low level infection. Novel cytokine-based procedures that activate porcine immunity by increasing IFN-gamma levels have the potential to enhance resistance to toxoplasmosis. Details on the underdeveloped nature of the intestinal immune system of weaned-pigs provides a model to test other prospective immune enhancing agents on development of resistance to infection in neonatal pigs. Swine disease control is most important during the first month of a pig's life due to an immature immune system. ARS scientists have established methods to show how the respiratory and intestinal immune systems develop in the neonatal piglet. The have shown that the immune system is underdeveloped in the intestine and thus less able to respond effectively against gastrointestinal infections. Future research should determine whether treatment of these young piglets with immune cytokine proteins will stimulate maturation of their immune system and thus enable them to be more resistant to infectious diseases as they move into the nursery, thus preventing piglet losses and saving producers' drug and veterinary costs. Basic information on the swine immune system has been derived from the production of molecular probes for swine cytokines. The current list of reagents developed by ARS scientist in this project include probes for IL-2, IL-4, IL-5, IL-10, IL-12, IL-12 receptor, IL-13, IL-15 and IFN-gamma. In addition, we have cloned and expressed genetically engineered IL-12 and IL-18 permitting us to initiate a study on the influence these cytokines have on attenuating nematode and protozoal infections via an IFN-gamma pathway. The major impact of these reagents is providing valuable information on the genetic changes correlating with immunized and susceptible hosts. Further, the reagents developed have been used to study immunity to a variety of viral and bacterial agents by independent investigators throughout the world. The use of mouse models of immunity to gastrointestinal parasites provides cutting edge information on promising new strategies for the biological control of parasites in livestock. 6.What do you expect to accomplish, year by year, over the next 3 years? Year 1: As part of our National Pork Board grant, the real time PCR (TaqMan) assay for Toxoplasma gondii will be tested with market meat samples, to determine how accurate it is in detecting the parasite in meats and how well it compares to other tests, particularly a standard albeit cumbersome bioassay. Assays will also be performed to determine how soon after infection this assay can detect the parasite in swine tissues. As part of our new BRDC grant, we plan to determine which cytokines are active during different respiratory infections. Swine immune microarrays will be developed so that novel immune regulatory molecules can be identified and results compared to our real time PCR assays of immune molecules in response to infectious diseases. Microarray data should also help to identify new candidates to assess and study changes in host immune responses to parasitic and viral infections. Continue studies on the relationship of TGF-beta homologues within Ascaris suum with the ability of the parasite to escape the expulsion process from the small intestines. Analyze the role played by the highly immunogenic, glutamic acid-rich proteins found within the Trichinella newborn larvae in cell recognition, cell reprogramming and as antigens capable of modifying the host immune response to enhance infectivity. Year 2-3: Begin assessing the role that soluble forms of the cytokine receptors for IL-4 and IL-13 play in modulating local immunity to infection by nematode and protozoan parasites. It is anticipated that an expanded panel of "designer" immune reagents will fit into expanding technologies for veterinary application of cDNA, protein and tissue microarrays, microWestern, microElisa, etc using large format plate readers and robotics. It is anticipated that products from the research will include an expanding base of scientific knowledge on mucosal and neonatal immunology of swine, techniques and reagents for veterinary application, practical outcomes for control of zoonotic pathogens and characterization of products with commercial applications. 7.What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? The results of these studies have been presented to a number of local groups at field days and presentations through the ARS National Visitor's Center and to scientific groups including the American Association of Veterinary Parasitologists, the World Association for the Advancement of Veterinary Parasitology, the Conference of Research Workers in Animal Disease, national and international animal genome meetings, and national and international immunology meetings. Scientists involved in this CRIS serve on the National Pork Board Toxoplasmosis Advisory group and assist in review of the Board's programs and grant submissions. Reagents generated to study immune responses in swine have been made available to the research community worldwide. This has included molecular probes for swine cytokines that have been shared internationally. Numerous monoclonal antibodies (mAb) reactive with swine immune cell subsets (CD antigen) and cytokines are now available through commercial sources worldwide. The cloning and expression of a functional interleukin-12 (IL-12) cytokine was conducted under a BRDC funded grant. Sponsors from the BRDC legally reviewed the patent potential of porcine IL-12. No patent application was submitted. Availability of the cloned, expressed IL-12 is decided by BRDC. The patent application for the real-time, PCR-based, diagnostic test to identify Toxoplasma DNA in tissues and other biological samples, including meat products, has been submitted. Further tests to determine usefulness on commercial pork products are underway through support of a new National Pork Board grant under the new CRIS project. Transfer of this technology to scientists at the Centers for Disease Control has been underway this year. 8.List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) Core,J. "Researchers Investigate Immunity in Pigs" Healthy Animals, Issue 11, online at: http://www.ars.usda.gov/is/np/ha/han11.htm Review Publications Dawson,H.,Beshah,E.,Nishi,S.,Lunney,J.K.,Urban,J.F. Proceedings Experimental Biology. 2002. Abstract p.182. Thacker,E.,Summerfield,A.,McCullough,K.,Ezquerra,A.,Dominquez,J.,Alonso,F., Lunney,J.,Sinkora,J.,Haverson,K. Summary of workshop findings for porcine myelomonocytic markers. Veterinary Immunology and Immunopathology. 2001. v.80. p.93-109. Davis,W.C.,Haverson,K.,Saalmuller,A.,Yang,H.,Lunney,J.K.,Hamilton,M.J., Pescovitz,M.D. Analysis of monoclonal antibodies reacting with molecules expressed on T cells. Veterinary Immunology and Immunopathology. 2001. v.80. p.53-62. HAVERSON, K., SAALMULLER, A., ALVAREZ, B., DAVIS, W.C., LEDBETTER, J.A., LUNNEY, J.K., NIELSEN, J.K., VALPOTIC, I., ZUCKERMANN, F.A., ZWART, R. OVERVIEW OF THE THIRD INTERNATIONAL WORKSHOP OF SWINE LEUKOCYTE DIFFERENTIATION ANTIGENS. VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY. 80:5-23. 2001. Lunney,J.K.,Fossum,C.,Alm,G.V.,Steinbach,F.,Wattrang,E. Veterinary immunology: opportunities and challenges. Trends in Immunology. 2002. v.23. p.4-6. Jauregui, L.H.,Nishi,S.,Higgins,J.,Zarlenga,D.,Dubey,J.P.,Lunney,J.K. Real-time PCR assay for the detection of toxoplasma gondii in pig tissues. Proceedings 82nd Annual CRWAD Meeting. 2001. p.86. Zarlenga,D.S.,Geary,T.G. Special Issue: "Advances in Molecular Parasitology" Veterinary Parasitology. 2001. v.101. p.171-414. Zarlenga, D.S., Morimoto, M., Urban Jr, J.F., Mccarter, J.P. 2002. A tgf-beta homologue within populations of ascaris suum 4th stage larvae (l4): evidence for multiple spliting and regulated transcription between l4 in the jejunum and ileum following spontaneously cure [abstract]. American Association of Veterinary Parasitologists Proceedings. Shea-Donohue,T.,Sullivan,C.,Finkelman,F.D.,Madden,K.B.,Morris,S.C.,Goldhill,J., Piñeiro-Carrero,V.,Urban,Jr.,J.F. The Role of Interleukin-4 in Heligmosomoides polygyrus induced alterations in murine intestinal epithelial cell function. Journal of Immunology. 2001. v.167. p.2234-2239. Urban,Jr.,J.F.,Noben-Trauth,N.,Schopf,L.,Madden,K.,Finkelman,F.D. Cutting edge: IL-4 receptor expression by non-bone marrow-derived cells is required to expel gastrointestinal nematode parasites. Journal of Immunology. 2001. v.167. p.6078-6081. Schopf,L.R.,Hoffmann,K.F.,Cheever,A.W.,Urban,Jr.,J.F.,Wynn,T.A. IL-10 is critical for resistance and survival during gastrointestinal helminth infection. Journal of Immunology. 2002. v.168. p.2383-2392. Ekkens,M.,Liu,Z.,Liu,Q.,Foster,A.,Whitmire,J.,Pesce,J.,Urban,Jr.,J.F.,Gause, W.C. Memory Th2 effector cells can develop in the absence of B7-1/B7-2, CD28 interactions, and effector Th cells after priming with an intestinal nematode parasite. Journal of Immunology. 2002. v.168. p.6344-6351. Nishi,S.,Lunney,J.,Beshah,E.,Solano-Aguilar,G.,Dubey,J.P.,Urban,Jr.J.F.,Dawson, H. Use of real-time RT-PCR analysis to detect local responses to Toxoplasma gondii in swine. Proceedings of the American Association of Veterinary Parasitology. 2002. Abstract p.25. Dawson,H.,Beshah,E.,Nishi,S.,Solano-Aguilar,G.,Morimoto,M.,Zhao,A.,Madden,K., Kringel,H.,Ledbetter,T.,Dubey,J.P.,Shea-Donoghue,T.,Lunney,J.L.,Urban,Jr.J.F. Verification of a Th1/Th2 paradigm of cytokine gene expression in swine infected with Toxoplasma gondii and Ascaris suum using real-time RT-PCR analysis. Proceedings of the American Association of Veterinary Parasitology. 2002. Abstract p.25.