2008 Annual Report 1a.Objectives (from AD-416) 1. Characterize the interaction of virus replication and macrophage responses. 2. Identify natural genetic variation associated with disease susceptibility. 1b.Approach (from AD-416) Identification of specific pathways that associate with variation in porcine reproductive and respiratory syndrome virus (PRRSV) replication and macrophage function leading to novel gene targets for the control of PRRSV infection. Alveolar macrophages will be obtained from diverse populations of swine and evaluated for their ability to support replication of PRRS viruses. Replication parameters will be estimated and macrophages that support either high or low levels of virus replication will be selected for studies of gene expression. Identifying PRRSV genotypes that confer fitness in macrophages, and host genes that respond to PRRSV fitness, to provide novel targets for intervention and control of PRRSV infections. These studies will use adapted isolates to identify viral genotypes that correlate with fitness of PRRSV in porcine alveolar macrophages and corresponding changes in macrophage transcriptional profiles. Genetic variation in specific ovine genes influences predisposition to ovine lentivirus (OLV) and the associated disease, ovine progressive pneumonia (OPP). We will thoroughly evaluate the most obvious candidate genomic regions for effects on lentiviral disease, like that containing CCR5. Our aim is to evaluate important regions of the genome for allelic association with the OLV disease susceptibility and progression phenotypes. Selection of regions will be based on a variety of scientific observations including, but not limited to, comparative mammalian biology. A selected set of 90 single nucleotide polymorphism (SNP) markers will be identified that are highly-informative in beef and dairy cattle. The development of this marker set represents non-hypothesis-driven research. The markers and genotyping assays for the markers will be readily available for any traceback needs. The same markers are also ideal for animal identification (i.e., sample matching) and routine parentage analysis. After ear tags and other physical identification devices have been removed, an animal’s DNA remains as a stable, accessible, integral, and identifiable component of its products and, thus, provides a gold standard for auditing the fidelity of physical labels and associated records. 3.Progress Report Two porcine monocyte-derived cell lines, designated Cdelta2+ and Cdelta2-, were developed and characterized to determine their usefulness in studies of disease susceptibility. Since peripheral blood monocytes and their tissue counterparts, the macrophages, play an important role as the first immune response responders to infectious disease-causing pathogens, these cell lines will be an additional tool with which to study porcine pathogens in vitro. Analysis of PRRSV-infected swine macrophages. Changes in gene product abundance in PRRSV-infected swine macrophage cell cultures were analyzed as a function of infection time. The experimental serial analysis of gene expression (SAGE) data acquired in FY2007 was used in these analyses. These analyses associated the SAGE data with porcine and PRRSV mRNA sequences, genes, and biological pathways. Changes in gene product abundance were analyzed for statistical significance using multiple tests. National Program 103 - Animal Health; National Program Components - Problem Statements 2C: Genetic and Biological Determinants of Disease Susceptibility - Mucosal Diseases of Livestock and Poultry and 4B: Countermeasures to Prevent and Control Respiratory Diseases - Porcine Respiratory Diseases 4.Accomplishments 1. Association of a bovine prion gene haplotype with atypical BSE. Classical bovine spongiform encephalopathy (BSE), also know as mad cow disease, is a prion disease of cattle that spreads through the consumption of contaminated feed. Recently, atypical BSEs have been discovered in North American, European, and Asian cattle. Atypical BSEs are different cattle prion diseases from classical BSE and are not linked to the consumption of contaminated feed. Genetic variation within the prion gene of cattle was tested for an association with atypical BSE. A particular prion gene sequence (haplotype) was found to associate with atypical BSE cases from the U.S., Canada, and France. This finding indicates that atypical BSE may be managed through the identification of cattle with known genetic risk factors for the disease and their removal from livestock populations. National Program 103 - Animal Health; National Program Components - Problem Statements 2C: Genetic and Biological Determinants of Disease Susceptibility - Mucosal Diseases of Livestock and Poultry and 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies - Bovine Spongiform Encephalopathy (BSE) 2. The prevalence of a bovine mutation (E211K) that may cause atypical BSE. A rare type of BSE in cattle, referred to as "atypical BSE," has recently been identified and is of interest because it develops in older animals without apparent exposure to other BSE-contaminated material. Although only 30 atypical BSE cases have been identified worldwide, they are significant because of their possible link to other Creutzfeldt-Jakob Diseases (CJDs) in humans (i.e., other than variant CJD [vCJD]). In 2006, a U.S. case of atypical BSE was discovered in Alabama and later reported to have a mutation (E211K) in the bovine gene required for BSE (i.e., the prion gene). This bovine mutation is strikingly similar to the most commonly inherited defect in humans that causes a type of CJD to develop late in life. This may imply that older cattle with the E211K mutation may also develop atypical BSE late in life. To determine whether this DNA mutation is rare in U.S. cattle, an accurate test was developed and more than 6000 cattle from all parts of the beef and dairy industry were tested for the presence of the mutation. This represents the first prevalence estimate of a mutation that may cause atypical BSE in older animals without prior exposure to BSE-contaminated tissues. None of the cattle tested were found to have the E211K mutation. Thus, the mutation appears to be either exceedingly rare or non-existent among U.S. purebred, crossbred, beef, and dairy cattle. National Program 103 - Animal Health; National Program Component - Problem Statement 8 - Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies (TSE) Diseases - Bovine Spongiform Encephalopathy (BSE) 3. A new class of scrapie-resistant sheep identified. Classical scrapie is a transmissible spongiform encephalopathy (TSE) of sheep. Susceptibility and resistance to this disease is encoded on specific versions of the prion gene. Individuals with the ARR variant of the prion protein are considered resistant, while those with the VRQ and ARQ variants are considered susceptible. However, some sheep with the ARQ variant do not develop clinical scrapie following exposure. Additionally, the ARQ variant has recently been found to have nine genetic subtypes within the prion gene. These observations indicated that a genetic subset of ARQ sheep may actually have resistance to scrapie. Our results identified a scrapie-resistant ARQ subtype with the amino acid threonine at position 112 of the prion protein (T112) rather than a methionine. These results have implications for scrapie eradication programs where ARQ sheep have previously been considered as a homogeneous group, leading to potentially unnecessary losses of economically important germplasm. National Program 103 - Animal Health; National Program Component - Problem Statement 8 - Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies (TSE) Diseases - Scrapie 4. Identified 139 candidate ovine SNPs for parentage-based disease traceback in sheep. Accurate food-animal identification is essential for improving disease control and enhancing food safety. When combined with physical, electronic, and database tools, DNA markers provide a means for verifying animal identification. In the absence of a pre-existing tissue sample for genotype matching, the only available DNA-based method for traceback is parentage testing. In other words, an offspring's point of origin may be inferred if the true parents can be identified by DNA-based methods (i.e., allele sharing). To this end and in collaboration with the International Sheep Genome Consortium (ISGC), we have identified 139 parentage SNP candidates from those 1536 SNP markers used on the 1.5k sheep SNP "chip." All of these markers are scheduled to be included on the ISGC's 60k ovine SNP chip. One hundred percent concordance was observed between genotypes scored by either sequencing genotypes or the 1.5k ovine SNP chip (350 comparisons). Because 8 of the first 11 candidates meet the selection criteria (72%), it is estimated that approximately 100 of the 139 current candidate SNPs could be developed into robust parentage markers for disease traceback in sheep. These markers will also provide high-quality parentage information that many sheep producers desire in their production systems. National Program 103 - Animal Health; National Program Components - Problem Statements 3A&C: Countermeasures to Prevent and Control Zoonotic Diseases - Brucellosis and Tuberculosis, 4A: Countermeasures to Prevent and Control Respiratory Diseases - Ruminant Respiratory Diseases, and 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies (TSE) Diseases - Scrapie 5. Methods for identifying variation throughout the prion gene of BSE-affected cattle. Distinct prion gene variation is known to associate with classical and atypical BSE susceptibility. Accurate detection of genetic variation within the prion gene of BSE-affected cattle is important for determining the full extent of prion gene associations with classical or atypical BSE susceptibility. Methods were developed to: . 1)extract DNA from small amounts of brain tissue (obex) from BSE-affected cattle and. 2)detect variation throughout both protein coding and non-coding regions of the prion gene for association testing. The methods were successfully applied to the U.S. case of classical BSE (2003) and are currently being applied to classical and atypical BSE cases of European and/or North American origin. This may result in the identification of prion gene variation that is highly predictive of BSE susceptibility. National Program 103 – Animal Health; National Program Components - Problem Statements 2C: Genetic and Biological Determinants of Disease Susceptibility - Mucosal Diseases of Livestock and Poultry and 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies - Bovine Spongiform Encephalopathy (BSE) 6. Development and characterization of two porcine monocyte-derived cell lines. The study of macrophage-tropic pathogens is made more difficult in pigs by the paucity of monocyte/macrophage cell lines available from this species. Therefore, it was our objective to develop macrophage-like cell lines from porcine peripheral blood mononuclear cells. Two cell lines, designated Cdelta2+ and Cdelta2-, were developed from a cross-bred grower pig. These cell lines morphologically resemble macrophages, and positively stain for the production of alpha-naphthyl esterase and negatively stain for peroxidase, consistent with macrophages. The lines produce inducible nitric oxide synthase, are phagocytic against latex beads, opsonize and non-opsonized bacteria, and are bactericidal against both Gram-negative and Gram-positive organisms. The pattern of cytokine expression as measured by nested real-time PCR is consistent with monocyte/macrophage lineage. Additionally, both cell lines are positive for the monocyte/macrophage markers CD11b, CD14, CD16, and CD172. The cells have been grown continuously for more than two years without any apparent changes in morphology or activity. These cells may be a suitable substitute for primary porcine monocytes in in vitro experiments. National Program 103 – Animal Health; National Program Components - Problem Statements 2C: Genetic and Biological Determinants of Disease Susceptibility - Mucosal Diseases of Livestock and Poultry and 4B: Countermeasures to Prevent and Control Respiratory Diseases - Porcine Respiratory Diseases 7. Analysis of PRRSV-infected swine macrophages. Porcine reproductive and respiratory syndrome virus (PRRSV) infection has been estimated to cost the U.S. pork industry over $500 million annually. Determining novel porcine and PRRSV gene or gene product targets for intervention and control of PRRSV infection will likely have a large impact on the U.S. and global pork industries. This work is the first comprehensive evaluation of changes in gene product abundance in PRRSV-infected swine macrophage cell cultures. Pathways have been identified that may contain potential molecular targets for controlling PRRSV infection. The initial analysis of the SAGE data was published in Developments in Biologicals. This report documents a resource that can be used by PRRSV researchers as a source for potential molecular targets for controlling PRRSV infection. National Program 103 - Animal Health; National Program Components - Problem Statements 2C: Genetic and Biological Determinants of Disease Susceptibility - Mucosal Diseases of Livestock and Poultry and 4B: Countermeasures to Prevent and Control Respiratory Diseases - Porcine Respiratory Diseases 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of New Commercial Licenses Executed 3 Review Publications Chitko Mckown, C.G., Leonard, J.C., Moscatello, D., Miller, L.C., Freking, B.A. 2008. Development of cell lines from the sheep used to construct the CHORI-243 ovine BAC library. Animal Biotechnology. 19(2):84-88. Bono, J.L., Keen, J.E., Clawson, M.L., Durso, L.M., Heaton, M.P., Laegreid, W.W. 2007. Association of Escherichia coli O157:H7 tir polymorphisms with human infection. BMC Infectious Diseases. 7:98. Miller, L.C., Harhay, G.P., Lager, K.M., Smith, T.P., Neill, J.D. 2008. Effect of porcine reproductive and respiratory syndrome virus on porcine alveolar macrophage function as determined using serial analysis of gene expression (SAGE). Developments in Biologicals. 132:169-174.