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United States Department of Agriculture

Agricultural Research Service

2010 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
1) Genetic predisposition to ovine progressive pneumonia virus (OPPV) - A genome-wide association study of OPPV in sheep has revealed specific gene sequences that may predispose sheep to infection under some production environments. OPP research from four perspectives has been conducted: i) Host genetics - We have typed 54,241 SNPs on a set of case-control sheep and identified a gene and haplotypes associated with OPPV resistance/susceptibility. The expressed mRNA haplotypes of this gene have also been sequenced. ii) Pathogen genetics - PCR-based assays have been developed for detecting infected sheep by measuring OPPV proviral DNA integrated in the host genome and for characterizing OPPV strain variation. iii) Host serology - More than 4,500 U.S. Meat Animal Research Center (USMARC) sheep have been phenotyped for OPP using a competitive ELISA for OPPV serological status. iv) Bioinformatics - We have sequenced, assembled, and annotated the complete ovine gene that appears to influence OPP infection in our sheep (78 kb total, from four BACs, 170-fold coverage). The sequence and information have been deposited in GenBank for concurrent release with manuscript publication. 2) Informatics for studying the transcriptome of swine infected with PRRSV - Collaborated with National Animal Disease Center (NADC) to investigate genes involved in the progression of PRSSV, PRV, SIV, and PCV2 in infected swine. Next-generation sequencing was used to create libraries of 10 million “SAGE-like” 20-bp tags at days 1, 3, 7 and 14 post-infection in both an infected and control group of swine. Data has been processed though our pipeline that has associated the tags with transcripts, genes, and pathways. 3) Effect of shade on heat-stress induced immune function changes in cattle - Bovine Respiratory Disease Complex (BRDC) is one of the most costly diseases affecting cattle. Although BRDC is composed of an assortment of bacterial and viral pathogens, conditions which place cattle under physiological stress can exacerbate the disease. We have initiated a study to determine if the presence or absence of shade alters immune parameters in cattle, in particular, during times of extreme heat. Cattle are currently being exposed to four different types/densities of shade, and blood values are being collected weekly during this eight-week study. 4) Evaluation of antibody titers to BRDC vaccination and association with respiratory disease - It may be possible to identify animals that are more susceptible to BRDC soon after their arrival at a feedlot by measuring their immune response to prophylactic vaccination. Whole blood and serum have been collected from the USMARC's “disease resistance” herd of cattle to determine complete blood counts and antibody titers to IBR, PI3, and BRSV using a single three-way assay. This ImmunoComb® assay will allow us to calculate the association between high and/or low antibody titers and illness recorded during the animals’ feeding-out period in the feedlot.

1. Improvement of a nucleotide polymorphism-based typing system for Escherichia coli O157:H7. Cattle are a reservoir of the recently emerged human pathogen E. coli O157:H7 and harbor distinct subtypes that do not all associate with human disease. A set of 178 nucleotide polymorphisms was developed in 2009 by ARS scientists at Clay Center, NE, that showed great utility in classifying E. coli O157:H7 genetic subtypes of cattle and/or human origin. To achieve higher resolution with this system, the scientists developed and applied a set of 720 nucleotide polymorphisms to 420 E. coli O157:H7 strains. This set effectively tags E. coli O157:H7 genetic diversity associated with humans and cattle, and an evolutionary analysis of the diversity indicates that cattle harbor subtypes that have evolved away from an association with human disease. Consequently, the nucleotide polymorphism set depicts E. coli O157:H7 genetic diversity and shows how strains are evolutionarily related with high resolution. These qualities define it as a preferential method for detecting and distinguishing E. coli O157:H7 genetic subtypes involved in epidemiological investigations.

Review Publications
Decker, J.E., Pires, J.C., Conant, G.C., McKay, S.D., Heaton, M.P., Chen, K., Cooper, A., Vilkki, J., Seabury, C.M., Caetano, A.R., Johnson, G.S., Brenneman, R.A., Hanotte, O., Eggert, L.S., Wiener, P., Kim, J.J., Kim, K.S., Sonstegard, T.S., Van Tassell, C.P., Neibergs, H.L., McEwan, J.C., Brauning, R., Coutinho, L.L., Babar, M.E., Wilson, G.A., McClure, M.C., Rolf, M.M., Kim, J., Schnabel, R.D., Taylor, J.F. 2009. Resolving the Evolution of Extant and Extinct Ruminants With High-Throughput Phylogenomics. Proceedings of the National Academy of Sciences. 106(44):18644-18649.

Daude, N., Wohlgemuth, S., Rogaeva, E., Farid, A.H., Heaton, M.P., Westaway, D. 2009. Frequent Missense and Insertion/Deletion Polymorphisms in the Ovine Shadoo Gene Parallel Species-Specific Variation in PrP. PLoS One [serial online]. 4(8):e6538.

Bono, J.L. 2009. Genotyping Escherichia coli O157:H7 for Its Ability to Cause Disease in Humans. Current Protocols in Microbiology. 14:5A.3.1-5A.3.10.

Heaton, M.P., Leymaster, K.A., Kalbfleisch, T.S., Freking, B.A., Smith, T.P., Clawson, M.L., Laegreid, W.W. 2010. Ovine Reference Materials and Assays for Prion Genetic Testing. BioMed Central (BMC) Veterinary Research [serial online]. 6:23. Available:

Durso, L.M., Harhay, G.P., Smith, T.P.L., Bono, J.L., Desantis, T.Z., Harhay, D.M., Andersen, G.L., Keen, J.E., Laegreid, W.W., Clawson, M.L. 2010. Animal-to-Animal Variation in Fecal Microbial Diversity among Beef Cattle. Applied and Environmental Microbiology. 76(14):4858-4862.

Donthu, R., Larkin, D.M., Heaton, M.P., Lewin, H.A. 2010. Discovery, Validation and Characterization of 1039 Cattle Single Nucleotide Polymorphisms. Animal Genetics. 41(4):421-423.

Keen, J.E., Laegreid, W.W., Chitko Mckown, C.G., Durso, L.M., Bono, J.L. 2010. Distribution of Shiga-Toxigenic Escherichia coli O157 in the Gastrointestinal Tract of Naturally O157-Shedding Cattle at Necropsy. Applied and Environmental Microbiology. 76(15):5278-5281.

Chitko Mckown, C.G., Macneil, M.D. 2010. Development of Fibroblast Cell Lines From the Cow Used to Sequence the Bovine Genome. Animal Genetics. 41(4):445.

Murdoch, B.M., Clawson, M.L., Laegreid, W.W., Stothard, P., Settles, M., McKay, S., Prasad, A., Wang, Z., Moore, S.S., Williams, J.L. 2010. A 2cM Genome-Wide Scan of European Holstein Cattle Affected by Classical BSE. BioMed Central (BMC) Genetics [serial online]. 11:20. Available:

Last Modified: 4/21/2014
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