2008 Annual Report 1a.Objectives (from AD-416) (1) Determine the genetic organization and expression of Ovine MHC Class I, IIa, and IIb loci, (2) Determine and validate that specific MHC Class I and/or MCH Class II alleles associate with OPPV clinical disease phenotypes and, (3) Determine the functional significance and subsequent immune responses of MHC alleles that associate with OPPV clinical disease phenotypes. 1b.Approach (from AD-416) OPPV is a sheep lentivirus that infects 24% of U.S. sheep flocks and causes economic losses to the sheep producer due to mastitis, dypsnea, and lameness. Serological diagnostic tests which test for the presence of anti-OPPV antibodies have provided a highly sensitive and specific means of testing but have not shown statistical correlations with pathology or other clinical markers of disease. Therefore, an OPPV test that predicts or determines which infected sheep will proceed to clinical disease progression is highly sought. Our laboratory is currently evaluating two different tests for the prediction or determination of OPPV clinical disease. One test is a quantitative PCR test utilizing real time technology, which targets a conserved region of the transmembrane protein of OPP provirus, and the second test is an immunogenetics test for MHC Class II DRB1. With one or both of these tests, we hope to provide a diagnostic OPPV test that determines or predicts whether the sheep will progress to OPPV clinical signs. In addition, these types of tests will significantly reduce the number of other tests necessary for determining infection and possibly lower the transmission potential in a flock. Therefore, these new tests offer significant long-term economic advantages for the producer over conventional serological diagnostic tests. Replacing 5348-32000-025-00D, April 2007. 3.Progress Report OPPV persistent infection in sheep is defined by the presence of anti-OPPV antibodies in serum above a specific detection threshold level. Fifty percent of 3-year old sheep in Dubois, Idaho are persistently infected with OPPV. However, a recent research project conducted in Pullman, Washington revealed that although OPPV transmission occurs naturally from ewe (n=10) to lambs (n=22) via colostrum and milk, persistent infection does not result in 95% of lambs (21/22) after 6.0 years. Differences accounting for the discrepant results between Dubois and Pullman include husbandry issues such as restricting lambs from exposure to the persistently infected flock after weaning, not re-using needles during vaccination, and selenium/vitamin E supplementation. In 2001, the Sheep National Animal Health Monitoring System (NAHMS) and showed that needle re-use had a positive correlation with flock size, and OPPV seroprevalence had a positive correlation with flock size. With these correlations in mind, a pneumatic needle-less system was tested for its ability to administer vaccines as compared to a traditional needle system. It was found that the pneumatic needle-less system administered vaccines as well as a traditional needle system. If implemented, this needle-less system has the potential to reduce iatrogenic transmission of OPPV and other blood-born infectious diseases. The main focus of this research project is to evaluate whether OPP provirus levels or specific sheep immune response genes can be utilized as predictive tools of the severity of OPPV induced disease. As a first step, development and validation of new OPPV quantitative PCR assay, which measures OPP provirus levels in peripheral blood leukocytes, ensued. This OPPV qPCR is the only validated qPCR assay for detection of OPPV provirus in peripheral blood leukocytes. OPP provirus levels may allow sheep producers to cull these sheep and lower overall levels in their flocks, and this would reduce ewe production losses worldwide. Current results indicate that OPP provirus levels correlate with the severity of disease. Since the Ovis aries (Ovar) major histocompatibility complex (MHC) class II proteins are putative receptors for OPPV, these sheep immune response genes were the first examined as candidate immune response genes that could influence OPP provirus levels and disease progression. As a first step, a bacterial artificial chromosome genomic DNA library was constructed and a clone was identified containing the Ovar-DQB2, DQA2, DQB1, DQA1 and DRB1 loci. This research yielded the first complete ruminant allele containing the DQB2, DQA2, DQB1, DQA1, and DRB1 loci, and this information is critical for understanding cell-mediated and humoral immune responses to OPPV and other pathogens. We are currently evaluating a high-throughput sequencing assay for Ovar-DRB1 and have recently submitted a manuscript describing significant Ovar-DRB1 associations with OPP provirus levels. These findings address Component 2, Genetic and Biological Determinants of Disease Susceptibility in NP 103 Action Plan. 4.Accomplishments 1. Development of a pneumatic needle-system for vaccination of sheep. Management practices concerning disease control in sheep often include re-use of needles for vaccination and this practice enhances transmission of infectious diseases. The goal of reducing disease transmission by this management procedure led to the collaboration between scientists at the U.S. Sheep Experiment Station (USSES) in Dubois, ID and scientists at the Animal Disease Research Unit (ADRU) in Pullman, WA to evaluate a pneumatic needle-less system against standard needle administration for vaccinations in sheep. Utilization of a pneumatic needle-less system will reduce management induced transmission of ovine progressive pneumonia virus (OPPV), an economically important disease of sheep and other blood born infectious pathogens. Adoption of this practice by sheep producers will contribute to economic gain by decreasing disease transmission. This accomplishment addresses NP 103 Component 2, Animal Genomics and Immunology, Problem Statement 2C, Mucosal Diseases of Livestock and Poultry. 2. Development and validation of an Ovine Progressive Pneumonia (OPP) quantitative (q) polymerase chain reaction (PCR) assay for detection of OPP provirus in peripheral blood leukocytes. An important need of the United States Sheep Industry is testing methods which are direct measures of pathogen level and directly reflect an animal's ability to control infection and limit transmission. Scientists at the United States Sheep Experiment Station (USSES) in Dubois, ID and the Animal Disease Research Unit (ADRU), Pullman, WA developed and validated an ovine progressive pneumonia virus (OPPV) quantitative polymerase chain reaction (qPCR) assay for detection of OPPV in sheep blood. Quantifying OPPV levels allows sheep producers to identify and cull sheep likely to experience loss of condition from OPPV and lower the overall level of the virus in their flocks. This accomplishment addresses NP 103 Component 2, Animal Genomics and Immunology, Problem Statement 2C, Mucosal Diseases of Livestock and Poultry. 3. Determined the genetic sequence and transcription of the sheep major histocompatibility complex (MHC) class IIa region. An important need of the United States Sheep Industry is to determine which sheep will lose condition due to infectious diseases such as ovine progressive pneumonia virus (OPPV). Genetic markers in immune response genes provide potential tools to provide such predictions. A bacterial artificial chromosome (BAC) library of genomic DNA from peripheral blood leukocytes of a Rambouillet ram was constructed, and scientists at the Animal Disease Research Unit (ADRU) in Pullman, WA determined the genetic sequence and transcription of the sheep major histocompatibility complex (MHC) class IIa loci: Ovis aries (Ovar)-DQB2, Ovar-DQA2, Ovar-DQB1, Ovar-DQA1 and Ovar-DRB1. This is the first completed sequence for the ovine (sheep) MHC class II region spanning DQB2 through DRB1, and these genetic sequences and expressed products of these sheep MHC class II DR and DQ molecules are now available for determination of their usefulness as markers for disease progression in individual sheep. This accomplishment addresses NP 103, Component 2, Animal Genomics and Immunology, Problem Statement 2C, Mucosal Diseases of Livestock and Poultry. 5.Significant Activities that Support Special Target Populations We are currently testing the efficacy of a new OPPV quantitative PCR assay against a competitive ELISA within differing production and regional sheep populations. We continue work with small and medium size sheep operations in the U.S. states of New York, Colorado, Minnesota, and Oregon. We also have been teaching and mentoring Sumner, Washington High School students that are part of Future Farmers of America to utilize the OPPV qPCR assay and ELISAs in testing sheep for OPPV. 6.Technology Transfer Number of Active CRADAs 1 Number of New Commercial Licenses Executed 5 Number of Web Sites Managed 1 Number of Non-Peer Reviewed Presentations and Proceedings 1 Review Publications Hoesing, L.M., White, S.N., Lewis, G.S., Mousel, M.R., Knowles Jr, D.P. 2007. Development and Validation of an Ovine Progressive Pneumonia Virus Quantitative PCR. Clinical and Vaccine Immunology. 14(10):1274-1278 Hoesing, L.M., White, S.N., Kappmeyer, L.S., Herndon, D.R., Knowles Jr, D.P. 2008. Genomic analysis of Ovis aries (Ovar)MHC Class IIa loci. Immunogenetics. 60(3):167-176. Mousel, M.R., Leeds, T.D., White, S.N., Hoesing, L.M. 2008. Technical Note: Comparison of traditional needle vaccination to pneumatic, needle-free vaccination in sheep. Journal of Animal Science. 86:1468-1471.