2003 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 and 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 bacteria disease is associated with worm infection in livestock and bacteria like Campylobacter is a major cause of diarrhea in humans. A long-term preventative control 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, thus 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 food borne 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 to the 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 determines 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. Traditional genetic approaches have been very effectively used by the swine industry to enhance feed efficiency, pig meat production and reproductive traits. Recently, molecular methods have provided new genomic markers to actively select for specific traits such as the ESR gene for improved piglet number per litter. For pig health, inheritance of specific alleles within the swine leukocyte antigen (SLA) complex genes have been known to positively influence disease and vaccine responses. Broader pig health studies to determine whether defined immune markers could be correlated with specific resistance to disease are only now being approached. Results from these studies should provide producers with pigs that would require fewer antibiotic treatments and better disease resistance. 2.How serious is the problem? Why does it matter? Proof of human toxoplasmosis from eating pork products is tenuous, but a high prevalence of 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 serological prevalence 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 food-borne illness that can be transmitted through pork products. Improvements are needed to provide activators of innate immune responses and to make current vaccines more effective, including developing better adjuvants. Moreover, since the World Health Organization and the US Centers for Disease Control have urged that animal producers stop the usage of antibiotics as growth promoters, alternatives to antibiotic treatments are required immediately for pig producers. This has stimulated a very active program, with our colleagues at the Nutrient Requirements and Functions Laboratory, on the use of probiotics to enhance immune system development and limit the spread of swine microbial pathogens. 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. Understanding basic mechanisms of immunological control of infectious agents of swine supports development of vaccines against parasitic, viral and microbial pathogens that compromise animal health and productivity. 4.What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003: Although swine are an inherently important livestock species and serve as a model for human disease, there are limited molecular tools that have been developed to characterize the immune response in swine. We have collaborated with the Nutrient Requirements and Functions Laboratory at the Beltsville Human Nutrition Research Center to develop reagents that assess gene expression of over 230 porcine genes related to immunity and nutrient metabolism. Multiple tissues sites were taken from pigs infected with several important pathogens including Toxoplasma gondii, Salmonella thyphimurium, Campylobacter species, Ascaris suum, Trichuris suis, and Oesophagastomum dentatum in Beltsville or through collaborators at Michigan State University and the Danish Center for Experimental Parasitology, and innate and acquired immune response gene responses were evaluated. The changing gene expression patterns in response to these infections indicate clear differences in immune regulation and suggest strategies to enhance specific arms of the immune response to control or limit the spread of these livestock and zoonotic pathogens. B. Other Significant Accomplishment(s): Inheritance of specific alleles within the swine leukocyte antigen (SLA) complex genes have been known to positively influence disease and vaccine responses in pigs. ARS scientists at Beltsville have collaborated with scientists at Baylor University to rapidly identify different SLA alleles using new molecular tests. Quick PCR tests, requiring only small amounts of blood from each pig, are now available to identify class I and class II alleles of SLA genes using SLA allele specific primers. Results from these studies will provide producers with tools to identify pigs that would respond to vaccines more effectively or be more resistant to certain diseases. There is a need to apply sophisticated molecular and genomic approaches to agriculturally important pathogens. Over 2,000 clones from the forth-stage 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-ß 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. Scientific data over the past 20 years have clearly and consistently demonstrated that recombinant vaccines against animal pathogens have had limited efficacy in reducing disease. As a result, a research void exists in therapeutic approaches to producing pathogen free, healthy animals that are safe to both the environment and the consumer. Immune responses that protect swine from infection are not well understood; however, cytokines are known to be acutely involved. A system of scavenging via soluble receptors exists naturally in most animals but has never been exploited artificially to enhance immune responses against disease. The concept has been demonstrated feasible in mouse models, but a more targeted approach to polarize the immune responses of large animals against disease is a unique, environmentally safe method of enhancing their overall health and well-being. To this end, soluble receptors for IL-4, IL-13alpha1 and IL-13alpha2 have been cloned and are currently being produced as therapeutic agents to help polarize host responses to infection. Commensal microbes in the intestine are considered as essential to the initial priming signals that can lead to a balanced development of the immune system and enhanced mucosal barrier function. Studies done in collaboration with scientists at the Nutrient Requirements and Functions Laboratory were conducted with commercially available probiotic stains from lyophilized Lactic Acid Bacteria (LAB) commonly used by humans that were given to neonatal pigs and colonization of the LAB was tracked by real-time PCR using highly sensitive and specific probes. The immune response of probiotic treated neonates indicated that there is an activation of their innate and adaptive immune system. These results indicate that dietary administration of probiotics can stimulate the neonatal immune system and improve its responsiveness to challenge with infectious agents or allergens. Wild type or Stat6 deficient mice were infected with the enteric nematode parasites, Heligmosomoides polygyrus or Nippostrongylus brasiliensis; H. polygyrus-infected mice were cured of the infection and then re-infected. Separate groups of mice were treated with IL-4 or IL-13 for 7 days and intestinal epithelial cell function was assessed using Ussing chambers to measure tissue permeability, glucose absorption, as well as secretion in response to a number of endogenous mediators, and organ baths were used to determine smooth muscle responses to acetylcholine and to nerves stimulation. Smooth muscle responses were elevated in wild type mice after infection or treatment with Th2 cytokines and these changes were attenuated or inhibited in Stat6 deficient mice. Nematode infection may negatively impact normal gut function leading to nutritional deficiencies in severe infection. Wild type or Stat6 deficient mice were infected with, H.polygyrus or N. brasiliensis; H. polygyrus infected mice were cured of the infection and then re-infected. Separate groups of mice were treated with IL-4 or IL-13 for 7 days, and intestinal epithelial cell function was assessed using Ussing chambers to measure secretion in response to a number of protease-activated receptor (PAR) agonists, while organ baths were used to determine smooth muscle responses to acetylcholine and to nerve stimulation in the presence and absence of PAR agonists. PAR agonists induce a contraction of smooth muscle that is dependent on substance P, and PAR expression is enhanced after treatment with IL-13 or enteric nematode infection that is associated with an increase in smooth muscle contraction, but epithelial cell secretion is reduced after infection. PAR are activated by digestive enzymes such as trypsin and are elevated in inflammatory diseases, thus, they are a potential target for the nutritional regulation of inflammation. C. Significant Accomplishments/Activities that Support Special Target Populations: None. D. Progress Report: The health of pigs in typical production facilities is challenged by porcine respiratory disease complex (PRDC), characterized by infections with porcine reproductive and respiratory syndrome virus (PRRSV), along with several other organisms, e.g., swine influenza virus (SIV), Mycoplasma hyopneumonia. Such infections require veterinary attention and the inclusion of antibiotics for effective pig production. Scientists at ARS Beltsville, MD, and the Univ. Illinois-Urbana, through the BRDC and their collaborating pharmaceutical and breeding company partners, have assessed porcine immune cytokine expression in lines of pigs which genetically differ in the amounts of the virus fighting cytokine, interferon-gamma, produced following PRRSV vaccination. Results from this work should help identify PRRS resistant pigs and mechanisms by which such pigs more effectively produce cytokines and resist viral infections. Current PRRSV vaccines are only partially protective against this major pig infection. Scientists at the Univ. Illinois-Urbana and ARS Beltsville, MD, and, through the BRDC and their collaborating pharmaceutical and breeding company partners, have used immune gene expression assays to compare pigs vaccinated with normal modified live vaccine to those vaccinated against PRRSV using the cytokine, interferon-alpha, as an adjuvant. Interferon-alpha clearly causes differences in PRRSV vaccine responses; investigations correlating these responses with immune gene expression differences are underway. This research should provide information for pharmaceutical and vaccine companies as they search for the best immune stimulants for swine viral vaccines. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. 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 clusters 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. 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 under developed 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 and have shown that the immune system is under-developed 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. ARS scientists at Beltsville, MD, with PIC/Sygen collaborators, have phenotyped hundreds of young piglets from PIC stock for early indicators of immune functions. This project was established to aid producers looking for alternatives to using antibiotics for early weaned pigs and researched methods to identify pigs which are genetically more disease resistant. The statistical analyses have indicated that some immune correlates of healthier and more productive pigs do exist; further studies will determine whether healthier pigs can be identified using simpler genetic markers. With such markers, pigs which are more disease resistant could be made available so that consumers would be provided with healthier pork products that contained less drug residues. 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 and showed that there were significant increases in specific T cell subsets during the first few weeks of a piglet's life. These results may 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, ARS scientists cloned and purified a new pig therapeutic, recombinant porcine cytokine interleukin-12 (rPoIL-12). However, rPoIL-12 has limited activity in swine because of low IL-12 receptor expression [unlike other animal species] 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 immune stimulants for swine. 6.What do you expect to accomplish, year by year, over the next 3 years? Year 1: It is anticipated that a comprehensive 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 this CRIS's research will enhance 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. 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 BRDC grant with UIL scientists, 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. The effect of human-derived probiotic species on intestinal function and responses to parasitic diseases of swine will be evaluated with scientists at the Nutrient Requirements and Functions Laboratory. Molecular probes for host immune and metabolic genes will be analyzed with the expanding panel of reagents developed during this collaborative interaction. New probes for detecting the colonization with probiotics in swine have also been developed to associate the presence of bacteria at mucosal sites with host responses. Year 2-3: Begin assessing the role of soluble forms of the cytokine receptors for IL-4 and IL-13 in modulating local immunity to infection by nematode and protozoan parasites. 7.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? 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 antigens) 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 in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Lunney, J.K. 2003. In Search of Disease-Resistant Pigs. National Hog Farmer, Apr 15, 2003. p.30-34. Tabitha Powledge. BioMedNet bmn.com. Improving disease resistance in the pig. Investigator: Joan Lunney. Available from: http://news.bmn.com/conferences/list/view?fileyear=2003&fileacronyn=PAGXI&fileday=day1&pagefile=story_4.html [2003] Review Publications Solano Aguilar, G., Zarlenga, D.S., Beshah, E., Gasbarre, L.C., Junker, D., Cochran, M., Dawson, H.D., Urban Jr, J.F., Lunney, J.K. 2002. Limited effect of recombinant porcine interleukin 12 on porcine lymphocytes due to a low level of il-12 beta 2 receptor. Veterinary Immunology and Immunopathology. 89:133-148 (2002) Finkelman, F., Urban Jr, J.F. The other side of the coin: the protective role of the type 2(th2) cytokines. Journal of Allergy Clinical Immunology. 107:772-780 (2001) Abner, S.R., Hill, D.E., Turner, J.R., Black, E.D., Bartlett, P., Urban Jr, J.F., Mansfield, L.S. Response of intestinal epithelial cells to trichuris suis excretory- secretory products and the influence on campylobacter jejuni invasion under in vitro conditions.. Journal of Parasitology. Zarlenga, D.S., Boyd, P., Lichtenfels, J.R., Hill, D.E., Gamble, H.R. 2002. Identification and partial characterization of a cdna sequence encoding a glutamic acid-rich protein specifically transcribed in trichinella spiralis newborn larvae and recognized by swine infection serum. International Journal for Parasitology. LAROSA, G., MARUCCI, G., ZARLENGA, D.S., CASULLI, A., ZARNKE, R.L., POZIO, E. A STUDY OF THE ITS-2 LOCUS IN LABORATORY AND NATURAL ISOLATES OF TRICHINELLA NATIVE AND TRICHINELLA T6 SUGGESTS AN INCIPIENT GENETIC DIVERGENCE BETWEEN THE TWO GENOTYPES. INTERNATIONAL JOURNAL FOR PARASITOLOGY. 2003. Madden, K., Whitman, L., Sullivan, C., Gause, W., Donaldson, D., Urban Jr, J.F., Katona, I., Finkelman, F., Shea Donohue, P.T. 2002. Role of stat6 and mast cells in il4-and il-13-induced alterations in murine intestinal epithelial cell function. Journal of Immunology. 169:4417-4432 (2002) Gause, W., Urban Jr, J.F., Stadecker, M. 2003. The immune response to parasitic helminths: insight from murine models. Trends in Immunology. 24:269-277. Liu, Q., Liu, Z., Ekkens, M., Fang, H., Whitmire, J., Chen, A., Sharpe, A., Urban Jr, J.F., Gause, W. 2002. Host protection and cytokine production are b7-independent in the th2 immune response to the gastrointestinal nematode parasite, nippostrongylus brasiliensis. Journal of Immunology. 169:6959-6968 (2002) Ekkens, M., Liu, Z., Liu, Q., Foster, A., Ehitmire, J., Pesce, J., Vannoy, J., Sharpe, A., Urban Jr, J.F., Gause, W. 2003. The role of ox40l interaction in the development of the primary and memory th2 response to the gastrointestinal nematode parasite heligmosomoides polygyrus. Journal of Immunology. 170:384-393 (2003) Strait, R., Morris, S., Smiley, K., Urban Jr, J.F., Finkelman, F. 2003. Il-4 exacerbates anaphylaxis. Journal of Immunology. 170:3835-3842 (2003) 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. Solano Aguilar, G., Dawson, H.D., Ledbetter, T., Shea Donohue, P.T., Schoene, N.W., Call, J., Beshah, E., Hare Jr, W.R., Urban Jr, J.F. 2003. The effects of dietary probiotics on immunity to ascaris suum in pigs [abstract]. Proceedings of American Association of Veterinary Parasitologists. Paper No. 56-54. Nishi, S., Dawson, H., Dubey, J.P., Urban, J.F., Lunney,J. Immune reponses controlling Toxoplasma gondii infection in pigs. Proceedings of the American Association of Veterinary Parasitologists. 2003. v.48. Abstract p.52. Solano Aguilar, G., Ledbetter, T., Dawson, H.D., Schoene, N.W., Urban Jr, J.F. 2003. The effect of dietary probiotic in the immune response of pigs [abstract]. 9th International Symposium on Digestive Physiology in Pigs. 2:69. Zarlenga, D.S., Morimoto, M., Urban Jr, J.F., Mccarter, J.P. 2003. Cloning and characterization of a tgf-beta homologue within populations of ascaris suum 4th stage larvae (l4): regulated transcription and multiple splicing differentiative l4 in the jejunum and ileum during spontaneous cure [abstract]. World Association for the Advancement of Veterinary Parasitology.