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Strategies to Control Swine Parasites Affecting Food Safety

Investigators
Dubey, Jitender; Urban, Joseph
Institutions
USDA - Agricultural Research Service
Start date
2001
End date
2006
Objective
Reduce transmission of foodborne pathogens and prevent infections in pigs as a means to improve overall swine health:
  1. Develop reagents and assays to define cytokine-regulated immune mechanisms.
  2. Determine if parasite-induced polarization of swine immunity affects pig health and susceptibility to zoonotic agents.
  3. Assess development of mucosal immune responses & the effect on neonatal pig health and transmission of foodborne pathogens using state-of-the-art methods and cDNA microarray technology.
More information
APPROACH: Test whether immune responses in pigs are polarized: immediate type hypersensitivity at one pole and cellular immunity on the other that regulate pathogen responses. Extremes in response pattern evoke cytokines that are inherently counter-regulatory. Intracellular parasitic infections, e.g. with Toxoplasma gondii, would be expected to drive strong T cell helper 1 (Th1) responses whereas Ascaris suum and Trichuris suis would push toward Th2 responses. The project is fundamental in its description of basic immunity at mucosal surfaces in neonatal and mature pigs. It is technology driven in its description of new reagents for characterization of the pig immune system, and the use of analytical tools that have never been applied to large animal infectious diseases. This knowledge of mucosal immunology, along with the analytical tools generated, will enable the researchers to develop new methods to contol infectious diseases in the swine host and to identify pigs which would likely be more disease resistant. BL-1 exempt 9/5/03.

PROGRESS: 2001/01 TO 2006/01
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Swine have two prominent food borne 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 are 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 such 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 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, e.g., cytokines, enhance 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. 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 pig health or with resistance to specific diseases have now been started. Results from these studies should provide producers with pigs that would require fewer antibiotic treatments and exhibit better disease resistance. Experimental murine models of both protozoan and worm parasitic infections have been utilized to describe novel interactions between the parasite and host in order to develop hypothesis-based testing of immunological control procedures in livestock species. This approach captures the advantage of using well described inbred mouse strains, genetically modified mouse strains and an enormous array of well defined molecular markers to examine detailed changes in immune function during parasitic infection.

A number of important observations have been made in these models over the life of the project that have contributed to the selection of new targets for control of infection and inflammation in swine as well as moving the entire area of immune parasitology and allergic disease forward for the scientific community worldwide. In the last three years the disease target of this CRIS has been expanded to include Porcine Reproductive and Respiratory Syndrome (PRRS) and other respiratory diseases of swine. According to the National Pork Board (NPB), PRRS costs U.S. pork producers at least $560 million annually. Thus, PRRS is the most economically significant disease facing the US industry today, affecting 21.4% of all breeding herd operations and, importantly, 58.3% of operations with >500 sows (NAHMS 2000). From 16.6-50.7% of all grow/finish operations reported having PRRS within the previous 6 months. Losses are due not only to reduced reproduction capacity in gilts or sows, but to other aspects of production, poor growth and increased respiratory problems. Modified live virus vaccines are available for PRRS but protect only against homologous viral challenges. Because PRRSV is an RNA virus, its genome changes and heterologous strains quickly arise. Thus new immune approaches are needed for prevention of PRRS using biotherapeutics, improved vaccines and better adjuvants.

The project addressed these problem areas by working through three broad objectives:
1) Development of reagents and assay to enable definition of cytokine-regulated immune mechanisms that protect pigs from disease.
2) Determination of conditions of parasite-induced polarization of swine immunity that affects pig health and susceptibility to zoonotic agents.
3) Assessment of the mucosal immune response development in pigs and the effect on neonatal pig health and transmission of food borne pathogens using state of the art immunologic methods and cDNA microarray technology.

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.

2. List by year the currently approved milestones (indicators of research progress)

Milestones 1 (12-60 months) represented a major effort initiated in the previous CRIS project to expand the availability of molecular and protein probes for immune, pro-inflammatory and anti-inflammatory cytokines. The expanded repertoire of reagents will find immediate application in the assays described in entire project and be utilized and expanded over the life of the project.

Milestones 2 (12-60 months) tested the use of CpG oligonucleotides with motifs designed for optimal adjuvant effects in swine using whole parasite antigens or attenuated vaccines for T. gondii; later years in the project will extend the analysis to defined recombinant proteins or DNA vaccines. The counter-regulatory effects of T. suis infection on secondary invasion by C. jejuni was documented through cytokine gene expression analysis; later years of the project applied dietary probiotic strategies to provide alternatives to antibiotics for the control of secondary microbial infections of pigs.

Milestones 3 (24-60 months) represents evolving technology that will be applied to tissues collected from experiments described in Objectives 2 and 3. The 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.

4a List the single most significant research accomplishment during FY 2006.
Panels of monoclonal antibodies (mAbs) reactive with human cell subset or cluster of differentiation (CD) markers were evaluated for cross reactivity on swine cell subsets. The immune system is complex and requires new tools to track the numerous cell subsets and activation markers that determine whether an animal makes an effective response to vaccination and to infection with disease organisms. BARC APDL scientists led a major effort to determine the reactivity of 386 mAbs against human immune cells as part of the Animal Homologues section of the 8th Human Leukocyte Differentiation Antigen Workshop (HLDA8). The mAb were exchanged with 12 labs internationally, tested for specific swine cell reactivity and the results statistically analyzed so that cross reactive CD could be affirmed. The swine results have now been compared to reactivity in 17 other species showing that only limited numbers of animal CD markers can be identified with anti-human CD reagents. Scientists worldwide can use these reagents for their species to determine which cells are essential in preventing infections or in stimulating protective vaccine responses. The overall result is that only few anti-CD mAb are cross reactive and, thus, more species specific reagents must be produced for most cell surface CD markers.

4b List other significant research accomplishment(s), if any.
Pig receptors for IL-4 and IL-13 were cloned and functionally evaluated. The cloning of receptors (IL-4R and IL-13Ra1) for two key cytokines was completed and their transcriptional changes were evaluated in parasite infected pigs during the course of infection. Results indicated the importance of evaluating host cellular responses at the site of infection inasmuch as peripheral changes were not predictive of the immune status of the animal. Furthermore, results suggested that evaluating changes in surface receptors may be more critical than circulating immune modulators for the understanding of temporal changes in the host immune response to infection insofar as these molecules are presumed to be leading indicators of the immune status of the host. The receptors for IL-4 and IL-13 are critical for induction of protective responses to worm infection and expression of allergic disease to environmental allergens. International nomenclature for the Major Histocompatibility Complex (MHC) class II complex genes in swine was developed. The genes that are major controls for the intensity of immune and vaccine responses, the MHC class II genes, or for swine, the SLA [Swine Leukocyte Antigen] class II complex genes have been systematically identified. As scientists pursue identification of genes which regulate disease resistance and vaccination responses, it is essential that there be international agreement on the nomenclature for these genes and assignment of their alleles. The SLA Nomenclature Committee of the International Society for Animal Genetics (ISAG) reviewed all of the DNA sequence information for MHC class II genes. SLA class II genes expressed on the cell membrane will be noted as SLA-DRA, SLA-DRB1, SLA-DQA, and SLA-DQB1. The committee will add new SLA class II allele designations, as they are discovered, and will maintain a publicly available list of all recognized genes and alleles using the international database which contains non-human MHC sequences (http://www. ebi.ac.uk/ipd/mhc/sla/). These assignments will facilitate exchange of data as scientists investigate the roles for each SLA class II gene in immune, vaccine and infectious disease responses. An annotated Porcine Immunology and Nutrition (PIN) database is available on the Worldwide Web. Information on pig genes and proteins can help researchers compare expression patterns in pigs with the more extensive databases generated for human and rodent. A porcine immunology and nutrition database was constructed with ARS scientists from BHNRC and ANRI. The database http://www.ars.usda.gov/Services/docs.htm?docid=6065 is largely focused on immune response genes with expression patterns that change as a result of allergy, asthma and atherosclerosis, and further evaluated for associations with disease susceptibility and pathology. The database is also oriented toward genes involved in the transport or metabolism of iron and selenium, with special emphasis on aspects of vitamin D and vitamin A metabolism, and nutrition-related genes with altered expression associated with atherosclerosis, diabetes, and obesity. This process identified 2,599 candidate genes, and designated porcine homologues to human genes of interest by searching several online sources of porcine gene information. The design of this database is being used as a template for similar projects with fish, bovine, rabbit, and chicken. Updating of the database provides an expanding research tool to those interested in veterinary immunology and nutrition. Understanding interrelationships among zoonotic parasites is critical to deciphering variations among host immune responses to these infectious agents.

To date, work has focused upon parasite identification, speciation and dissemination in the genus Trichinella because this genus presents a unique opportunity for studying and dissecting swine protective immune responses to nematodes. Prior work has demonstrated that extant species of Trichinella are genetically very similar and diverged within the past 20 million years. In addition, research has shown that the most recently evolved species, i.e., Trichinella nativa and Trichinella T6, as well as the most common Nearctic species, T. murrelli, elicit characteristic B-cell immune responses when experimentally-presented to pigs, yet these species do not infect pigs. The disparity in these rates of infection between crown species and T. spiralis, the common swine pathogen, form the basis of current studies to evaluate mechanisms involved in host protection. Differentially perturbed host immune responses to infectious versus non-infectious Trichinella whose immunodominant antigens are cross-reactive should provide important information on the discrepancy between infection rates as it pertains to host immune responses and tissue location.

Currently, work is aimed at determining if the protective immune response(s) in animals subjected to infection by crown species can be used as a mechanism to vaccinate against T. spiralis. Understanding disease and vaccine responses requires improved antibody reagents for pigs. Current pig antibody reagents can distinguish the broad classes of immunoglobulins (Igs), the IgGs from IgM and IgA; however, there are no IgG subclass reagents, nor reagents for IgE, the Ig associated with allergies and asthma. This new National Pork Board grant (ARS project 1265-32000-064-10R) to BARC APDL and University of Iowa scientists will enable them to develop, for all swine researchers, monoclonal antibodies (mAb) reactive with the different IgG subclass proteins and with IgE. Such tools will enable researchers to better define, and ultimately target specific antibody (Ig) responses for infectious disease and vaccine studies. Ultimately, these anti-Ig mAb tools will help determine which swine Ig subclass genes and proteins are most important for specific immune functions, e.g., clearing bacterial infections or neutralizing viral infections. The conjunctiva can serve as a mucosal site to evaluate responses to parasite and non-parasite allergens. A mouse model was developed to measure local responses to allergens in the eye. This provides both a non-invasive read out for allergen responsiveness due to the redness and swelling that accompanies a local allergic reaction, and also can be used for quantitative measurements of cytokine gene expression and histological evaluation of infiltrating inflammatory cells. The study also revealed that a concurrent infection with A. suum eggs would enhance the response to non-parasite allergens in the eye. This study was documented by a recent publication in one of the major journals of eye research, and has been duplicated in pigs for description as a proof of principal study for measuring allergic responses to parasite and non- parasite allergens. The gene expression pattern in the conjunctiva of allergen-exposed eyes shows a prominent enhancement in allergy/asthma- associated genes that are signatures for allergic disease expression in humans. This represents both a novel mucosal tissue readout for assessing allergic disease in swine, and a model for studies of mechanisms of allergic sensitization and induction for humans.

4d Progress report. Detailed descriptions are in sections #4 A and B. This project is in its last year and the reporting of results and planning for the new project will complement the completion of studies outlined for the milestones shown in 3b.

5. Describe the major accomplishments to date and their predicted or actual impact.
Determined critical innate immune markers required for effective immune responses against porcine reproductive and respiratory syndrome (PRRS). The most economically significant disease facing the swine industry today is PRRS; it costs U.S. pork producers at least $600 million annually. Biosecurity is only partially effective in preventing spread of the virus and new, heterologous viral infections are rapidly developing nationwide. Efforts at BARC APDL, under the national PRRS grant (ARS project 1265- 32000-064-06R); have been aimed at understanding protective anti-PRRS immunity and designing more effective vaccines. Vaccine trials with the University Illinois-Urbana scientists showed that the use of interferon- alpha (IFNA) as a novel cytokine vaccine adjuvant did not improve protection against viral challenge. Ongoing genetic studies with University Nebraska-Lincoln scientists indicate that there may be correlations between levels of certain innate cytokines and resistance to PRRS associated pathologies. These studies potentially will help pig producers to genetically select pigs with improved resistance to PRRSV infections. First usage of NRSP8-Qiagen 13,000 swine gene oligo microarray to assess tissue specific gene expression and responses to Salmonella infections. This USDA CSREES NRI grant to Iowa State University, Oklahoma State and USDA ARS BARC APDL scientists (ARS project 1265-32000-064-05R) is aimed at analyzing genes involved in causing the major reproduction losses to pig producers; 40 to 50% of fertilized ova do not survive through gestation. Prior to use with valuable embryonic samples, the 13,000 swine gene NRSP8-Qiagen oligo microarray was first analyzed for differentially expressed genes in normal porcine tissues and, in collaboration with NADC and BARC NRFL scientists, in lung tissue during Salmonella enterica serovar choleraesuis infection. Transcriptional profiling results, confirmed with real-time gene expression studies, proved that numerous genes and underlying molecular mechanisms are correlated with tissue specific responses and a subset of these are critical for effective lung responses to control Salmonella infections. These studies have expanded our ongoing immune analyses of pig disease responses and identified a broad range of mechanisms controlling respiratory immune responses to bacterial and viral infections. Functional Th1/Th2 paradigm validated in swine response to parasitic infection. Understanding immune regulation to infectious agents has been driven largely by work in experimental rodent models of disease. Yet there are unique differences between mammalian species that require that fundamental response patterns are validated to explore hypothesis based research of specific infections important to swine. Published work from ARS scientists at BHNRC and ANRI have shown that pigs respond to protozoan and helminth parasite infections with immune and physiological patterns that are generally comparable to that observed in mice and humans. There are, however, unique gene expression patterns which show that pigs are more like humans than mice providing a basis for model testing of human diseases in pigs. The studies also help provide a basis for understanding the immunity to important viral and bacterial disease of swine including PRRS virus and bacterial infections that are food borne. Major expansion of toolkit for assessing swine immunity. A comprehensive panel of "designer" immune reagents has been developed to assess swine immune responses, for use in analyses of disease and vaccine studies and neonatal pig development. Real-time gene expression tests for >300 swine immune and metabolic pathway genes, were developed in a collaborative venture between the Animal Parasitic Diseases Lab, ANRI, and the Nutrient Requirements and Functions Laboratory at the Beltsville Human Nutrition Research Center. Samples were taken from multiple tissues sites from pigs infected with several important pathogens including T. gondii, PRRSV, Salmonella typhimurium, Campylobacter species, A. suum, T. suis, and Oesophagostomum dentatum in Beltsville or through collaborators at ARS NADC, University of Illinois, Iowa State University, Michigan State University and the Danish Center for Experimental Parasitology. Innate and acquired immune responses were evaluated in infected, vaccinated and control pigs using this expanded toolkit. The changing gene expression patterns in response to these infections or vaccinations 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. Studies of whipworm infection in pigs provide therapeutic approach to inflammatory bowel disease in humans. Immune response patterns in the large intestine of pigs infected with whipworm revealed the expression of genes that are anti-inflammatory and likely to regulate the pathology associated with inflammatory bowel disease. ARS scientists helped research physicians at the School of Medicine, University of Iowa to design a treatment protocol that fed Trichuris suis eggs to patients with intractable ulcerative colitis and Crohns disease. Results of double- blind and placebo controlled studies have been reported showing that this procedure is efficacious in the improvement of clinical scores of disease. This is an important example of the modeling of human diseases in pigs and the need for basic research in pig immune function to provide important information on immune regulation. Food-borne transmission of Toxoplasma gondii in meat products was evaluated. Determination of the risk of food-borne 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. International nomenclature for the Major Histocompatibility Complex (MHC) class I complex genes in swine was developed. The MHC class I genes, or for swine, the SLA [Swine Leukocyte Antigen] class I complex genes are known to be major determinants of disease resistance regulating immune and vaccine responses, particularly for viruses. As scientists pursue identification of genes which regulate disease resistance and vaccination responses, it is essential that there be international agreement on the nomenclature for these genes and assignment of their alleles. The SLA Nomenclature Committee of the International Society for Animal Genetics (ISAG) reviewed all of the DNA sequence information for MHC class I genes and assigned the classical class I SLA genes as SLA-1, SLA-2 and SLA-3, and the non-classical as SLA-6, SLA-7, and SLA-8. All sequences will be available from the international database which contains non-human MHC sequences (http://www.ebi.ac.uk/ipd/mhc/sla/). These assignments will facilitate exchange of data as scientists investigate the roles for each SLA class I gene in immune, vaccine and infectious disease responses. New tools to identify Major Histocompatibility Complex (MHC) alleles in swine were developed. Many genes control how well a pig will grow and reproduce, and the quality of its carcass and meat; independently, there are numerous genes which determine animal health. It has been known, and proven in many species, that there is one set of genes that are major determinants of disease resistance and some vaccine responses; these are the MHC genes, or for swine, the SLA [Swine Leukocyte Antigen] complex. Thus, as we pursue genes which regulate disease resistance and vaccination responses, it is essential that we can identify alleles at the many SLA loci as was accomplished by scientists at Baylor University in collaboration with scientists at USDA ARS BARC using molecular, PCR [Polymerase chain reaction] procedures with site specific primers [PCR- ssp]. These assays enable scientists to investigate and assign roles for each SLA gene in immune, vaccine and infectious disease responses. Standardization of mAb reactive with swine cell surface (CD) markers was continued. 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. Cytokines that enhance protective immunity against toxoplasmosis in swine were evaluated. 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. Respiratory and intestinal immune system development in the neonatal piglets was examined as sites of infectious agent entry. 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. Procedure for detection of early indicators of immune functions to identify healthier piglets was developed. ARS scientists at Beltsville, MD, with PIC/Sygen collaborators, phenotype 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 indicated that some immune correlates of healthier and more productive pigs do exist in blood; further studies will determine whether healthier pigs can be identified using simpler genetic markers. 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. 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. Collaborative production and evaluation of pig IL-12 as a potential pig therapeutic was completed. 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, unlike other animal species, there is low up- regulation of IL-12 receptor expression and therefore, rPoIL-12 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. Key swine immune markers were cloned to measure gene expression. Researchers have limited reagents to use to predict swine protective disease and vaccine responses. Thus, efforts were made to clone and sequence pig TBX21 (T-bet), MYD88, ICSBP1, CD8A (CD8alpha), CD8B (CD8beta) , and CD28 cDNA as new tools to measure T helper (Th) cell immune responses. Real-time PCR assays were developed for quantitative measurement of these and other immune genes. When used for examining temporal immune gene expression in the liver of Toxoplasma gondii infected pigs, the positive regulators of Th1 responses, IRF1, MYD88, and STAT1, were found to be expressed prior to the simultaneous up regulation of interferon gamma (IFNG), HLX1 and TBX21 gene expression. Based on their demonstrated utility in establishing an immune response pathway, these PCR assays should be valuable additions to our swine immune toolkit. This data represented the first report of induced TBX21 expression during a Th1-associated response in an animal not of human or rodent origin. A robust molecular technology to determine phylogeny for the genus Trichinella was developed. Molecular methods have been developed to predict parasites within this important genus. The tremendous disparity in infectivity for swine among species of Trichinella has raised important questions regarding host parasite relationships and the evolution of differential host immune response, primarily as it relates to the domestication of swine and genetic selection. The most recently evolved species of Trichinella have showed the lowest infectivity for swine suggesting that episodic biotic expansion, subsequent isolation, and host switching played important roles in the evolution of Trichinella and its ability to adapt or circumvent host immune responses. Equally important is the role that the human/swine relationship played in this expansion. Understanding the phylogeny will shed light on the disparity in infectivity and host immunity. Molecular tools to track freezer resistant Trichinella species. There are no data available on the population genetics of Trichinella species due to the lack of genetic markers and the difficulty of working with such small parasites. In the Arctic region of North America and along the Rocky Mountains, there exist two genotypes of Trichinella, Trichinella nativa and Trichinella T6, respectively, which are well differentiated by biochemical and molecular characters. However, both are resistant to freezing, show other common biological characters (e.g. low or no infectivity to rodents and swine) and produce fertile F1 offspring upon interbreeding; a single GTT trinucleotide present in the ITS-2 sequence of T. nativa but not in Trichinella T6 was used as a genetic marker to study gene flow. Both genotypes were detected in wolves of the same wolf packs in Alaska, suggesting sympatry. Only F1 larvae originating from a cross between T. nativa male and Trichinella T6 female were able to produce F2 offspring; all F1 larvae showed a heterozygote pattern for the GTT character upon heteroduplex analysis. The data suggest that T. nativa and Trichinella T6 live in sympatry at least in Alaskan wolves, where T. nativa occurs more frequently (69%) than Trichinella T6 (31%) due to geographical relocation, colonization and independent evolution of T. nativa within the Rocky Mountains, resulting in a bifurcation of the freeze-resistant genotype. Interferon-alpha was evaluated as a PRRSV vaccine adjuvant. Porcine Reproductive and Respiratory Syndrome (PRRS) is the most economically significant disease facing the swine industry today, costing U.S. pork producers at least $600 million annually. Current modified live vaccines (MLV) are only partially effective against virus infection because they elicit a weak immune response that is not fully protective, particularly for a heterologous PRRSV challenge, as is frequently found in field situations. To improve PRRSV vaccines scientists at USDA ARS BARC worked with the Univ. Illinois-Urbana scientists to use a novel cytokine adjuvant, interferon-alpha (IFNA), and sophisticated molecular tests to evaluate immune factors that influence vaccine efficacy. Results showed that PRRSV vaccination, with or without IFNA, stimulated low levels of protective IFN-gamma (IFNG) and only limited amounts of innate immune markers, interleukin-1 beta (IL1B), IL6 and IL8 that should enhance immunity. Further exploration will be required to decipher the immunobiology of PRRSV and to increase the immunogenicity of conventional MLV vaccines using IFNA and other adjuvants to insure protection even from heterologous PRRSV isolates. CpG oligodeoxynucleotides tested as an adjuvant for swine parasite infections. Protection against a challenge infection with T. gondii VEG strain oocysts was examined in pigs after vaccination with T. gondii RH strain tachyzoites with or without a porcine specific synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. Protection from challenge was significantly improved in pigs vaccinated using CpG ODN as an adjuvant with tachyzoites compared to all other groups. The CpG ODN tachyzoite-immunized pigs also had higher serum parasite specific IgG antibody, no clinical signs of disease, and 52% had no demonstrable tissue cysts after the challenge infection. These data indicate that CpG ODN is a potential safe and effective adjuvant for the T. gondii RH strain vaccine and should be considered for other vaccinations in pigs. Are ubiquitous ectoparasites really commensals? Mice lacking 2 important T helper 2 (Th2) markers, CD28, a T-cell co stimulatory molecule, and STAT6, a transcription factor that mediates interleukin-4 (IL-4) signaling, were developed; at birth they had a normal phenotype with positive IL-4 production following infection with nematode parasites. Between 4 and 8 months old, however, all double mutant mice examined spontaneously developed severe chronic dermatitis associated with large numbers of Demodex ectoparasites and pronounced CD4+ and CD8+ T-cell infiltrates in the dermis and subcutaneous fat, along with increased serum immunoglobulin G2a levels, and lymphadenopathy associated with increased gamma interferon and IL-12 expression were observed. To distinguish whether the ectoparasite Demodex or Th1 immunity was the proximal cause of the inflammatory skin disease, STAT6/CD28(-/-) mice was treated with a miticide; that treatment eliminated the ectoparasites and markedly reduced the severity of the dermatitis and the associated lymphoid infiltrates. These findings suggest that ubiquitous ectoparasites, which are generally considered to be commensal, may contribute to disease when specific molecules required for an effective Th2 immune response are blocked. Localized immune memory detected at the host-parasite interface. Memory peripheral T helper 2 (Th2) immune responses to infectious pathogens are not well studied due to the lack of suitable models and the difficulty of assessing Th2 cytokine expression at sites of inflammation. A localized immune response to a nematode parasite (Heligmosomoides polygyrus) that encysts in the small intestine was examined. An unexpected architecture was observed on day 4 of the memory response, with granulocytes and macrophages infiltrating the cyst and CD4(+), TCR-alpha/beta(+) T cells surrounding the cyst. Laser capture micro dissection analysis showed a pronounced CD4-dependent Th2 cytokine pattern at the cyst region but only during the memory response, demonstrating that the Th2 memory response is readily distinguished from the primary response by the rapid accumulation of Th2 effector cells at the host-parasite interface. Mouse responses to worm infections provide basic information on immune function. Several key observations of mouse responses to worm infection have provided basic information to the research community and helped in the interpretation of immunity of pigs to infectious disease. Studies demonstrated that knocking out the GATA-3 gene in mice leaves them unable to respond appropriately to worm infection. This was supported in pigs infected with whipworm showing that GATA-3 is strongly up regulated during infection. The mouse basophil was shown to be a early producer of IL-4 which drives the immune response to worms in mice and pigs. This will require confirmation in pigs which also have a strong basophil response during worm infection. The infection of mice with Ascaris suum and products from this roundworm that is most prevalent in pigs showed that it enhances the allergenicity of ragweed protein. This observation demonstrates how worm infection can influence the response to environmental and food proteins that are common to humans and pigs. A series of reports and review articles on these subjects have been completed or are anticipated for the final year of the project. Differential role for interleukin-4 (IL-4) and IL-13 in intestinal smooth muscle contractility was observed. The T helper 2 (Th2) cytokines, interleukin-4 (IL-4) and IL-13 promote worm expulsion in part through effects on non lymphoid cells, such as intestinal smooth muscle cells. The roles of Stat6 in IL-4-, IL-13-, and parasitic nematode-induced effects on small intestinal smooth muscle contractility were investigated in mice treated with a long-lasting formulation of recombinant mouse IL-4 (IL-4C) or IL-13. Separate groups of BALB/c mice were infected with Nippostrongylus brasiliensis or were drug-cured of an initial Heligmosomoides polygyrus infection and later re-infected. Both IL-4 and IL-13 increased smooth muscle responses to nerve stimulation in wild-type mice, but the effects were greater in IL-13-treated mice and were absent in IL-13-treated Stat6-deficient mice. Similarly, hyper contractile responses to nerve stimulation in H. polygyrus- and N. brasiliensis- infected mice were dependent in part on Stat6. IL-13, H. polygyrus, and N. brasiliensis, but not IL-4, also increased contractility to acetylcholine by mechanisms that involved Stat6 and enteric nerves. These studies demonstrate that both IL-4 and IL-13 promote intestinal smooth muscle contractility, but by different mechanisms. 6. 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 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 American Association of Swine Veterinarians, the International PRRS Symposium, and the Conference of Research Workers in Animal Disease, national and international animal genome meetings, and national and international immunology meetings. Scientists involved in this project serve on the National Pork Boards Toxoplasmosis Advisory and Genomics of PRRS groups and assist in review of the Board's programs and grant submissions for food borne diseases and for PRRS. ARS scientists are also involved in multi state projects, NC-1004 and NRSP8 for animal genome studies (serving in 2004-2005 as NRSP8 Chair), and NC-229 for PRRS research (serving in 2006 as NC229 Secretary). Most recently this has been evidenced by active participation in the multi state USDA CSREES Animal and Plant Bio security grant #2003-05164: Integrated Control and Elimination of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in the U.S. Reagents generated to study immune responses in swine have been made available to the research community worldwide. This has included molecular probes and real-time PCR primers and probes for swine cytokines and immune markers that have been shared internationally. Numerous mAb reactive with swine immune cell subsets (CD antigens) and cytokines are now available through commercial sources. The results of international CD workshops are now widely accepted, enabling scientists worldwide to exchange data and determine which cells are essential in preventing infections or in stimulating protective vaccine responses. In 2003-2006, a major international effort has been completed to test for cross- reactivity of anti-human CD mAb on swine cells as part of the Human Leukocyte Differentiation Antigens (HLDA8) workshop Animal Homologues Section. This will further increase availability of crucial anti-CD mAb for immune research in pigs. 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, was issued by the U.S. patent office. Further tests to determine usefulness on commercial pork products are underway through support of a new National Pork Board grant under this project. This technology has been transferred to scientists at the Centers for Disease Control and other institutions.

7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Johnson, R., Petry, D., Lunney, J.K. Genetic resistance to PRRS virus studied. National Hog Farmer. December, 15, 2005, p.19-22. Agricultural Research Service (ARS) scientists have updated to version 3. 6 that is scheduled for public release in August 2006 a searchable database on the World Wide Web where users can find comparative information on 2,600 annotated swine genes and proteins related to nutrition and immunity. The resource is called the "Porcine Immunology and Nutrition (PIN) Database" http://www.ars.usda.gov/Services/docs.htm? docid=6065

Funding Source
Agricultural Research Service
Project number
1265-32000-064-00D
Accession number
403992
Categories
Parasites
Sanitation and Quality Standards
Natural Toxins
Bacterial Pathogens
Prevention and Control
Commodities
Meat, Poultry, Game