2013 Annual Report
1a.Objectives (from AD-416):
1. Discover genetic and biological determinants associated with disease resistance to respiratory diseases of ruminants.
2. Discover effective intervention strategies to prevent and control respiratory diseases of ruminants.
1b.Approach (from AD-416):
Infectious respiratory diseases of ruminants are a serious health and economic problem for U.S. agriculture. In cattle alone, bovine respiratory disease complex (BRDC) costs the feedlot industry approximately 1 billion dollars annually. Accordingly, this research focuses primarily on BRDC with an additional component targeting ovine respiratory disease. These diseases result from host, pathogen and environmental interactions that are incompletely understood. We propose studying these interactions to identify intervention points for producing healthier livestock. For example, failure of passive transfer (FPT) has genetic and environmental components that predispose calves to BRDC. A genome-wide association study will be used to test for FPT genetic risk factors. Also, the predominant pneumonia associated with BRDC is caused by Mannheimia haemolytica. Transcriptome profiling will be used to identify host cellular responses to this pathogen. BRDC research requires development of species-specific tools, thus monocyte cell lines will be developed. Also needed are early indicators of BRDC outbreaks. Changes in the viral metagenomes (VMs) of cattle will be evaluated as potential early indicators of infection. Finally, genetic risk factors for ovine progressive pneumonia (OPP) will be tested as a model for preventing respiratory diseases of ruminants. A reduction of the prevalence, severity and/or treatment of respiratory diseases would enhance producer efficiency and promote the welfare of livestock. This research fills critical gaps in our knowledge of ruminant respiratory disease and could ultimately benefit regulatory agencies, animal health companies, veterinarians, and livestock producers that need information and new approaches for controlling respiratory disease.
During the spring and summer of 2013, failure of passive transport of immunoglobulin from cow to calf case and matched control DNAs were scanned to identify areas of the genome associated with failure. However, additional case and control DNAs are required for the test to be conducted with adequate power and statistical significance. To that end, over six hundred calves to be born to pregnant cows in the spring of 2013 were targeted for the project. After excluding calves born at night or in adverse weather conditions, 70 blood samples were collected. Additionally, a pilot study was conducted through the fall of 2012 and spring of 2013 where the immunoglobulin concentrations of 31 calves were serially measured from birth to 170 days of age. The pilot was conducted to assess some of the longer-term effects of high or low passive transfer.
Strains of wild-type and leukotoxin-knock out M. haemolytica, a bacteria associated with respiratory disease in cattle, were obtained from a collaborator along with protocols for establishing and expanding the bacterial cultures, purifying and concentrating the leukotoxin, as well as performing toxicity assays. Additionally, leukotoxin was purified from the wild-type M. heamolytica that was fully sequenced. This leukotoxin is being used to assess differences in sensitivity in cattle with genetic variation within the leukotoxin receptor.
Characterization of a robust, rapidly growing bovine monocyte-derived cell line has been initiated including flow cytometric staining for cell-surface determinants, phagocytosis, testing for contamination with Bovine Viral Diarrhea Virus, and karyotype analysis.
To determine the relative susceptibility to ovine progressive pneumonia virus in sheep with different transmembrane protein 154 genotypes, a breeding scheme was expanded to produce approximately 250 lambs with genotypes: "1,1"; "1,3"; "3,3"; “1,2”; “2,2”, and “2,3”. These lambs are receiving a high level of natural exposure to the virus from their dams and other adult ewes. Yearling ewes from this group of lambs were kept in the flock and tested for the virus as they moved through the production cycle. In addition, approximately 70 lambs were produced with “1,1”; “1,4”; and “4,4” genotypes. These are also receiving a high level of natural exposure.
Sequenced, assembled, annotated and published the first complete circularized chromosomal genomic sequences of Mannheimia haemolytica serotype 1 and serotype 6 clinical isolates as well as a Bibersteinia trehalosi clinical isolate. This was accomplished using in-house DNA sequencers and genome analysis pipelines at the U.S. Meat Animal Research Center, Clay Center, Nebraska. These sequences were deposited in GenBank and are publicly accessible conforming to the Minimum Information about Genomic Sequence sample metadata standard.
First complete genomes assembled for pathogens that cause shipping fever in cattle. Mannheimia haemolytica and Bibersteinia trehalosi are respiratory pathogens affecting cattle and related ruminants worldwide. M. haemolytica is commonly associated with bovine respiratory disease complex, a polymicrobial multifactorial disease. B. trehalosi is less commonly associated with outbreaks of this disease, but anecdotal evidence suggests that this pathogen may be associated with increased cattle morbidity and mortality. The impact of respiratory disease on the U.S. beef cattle industry is estimated to be at least $1 billion/year. Before this study, the genomic circular "blueprint" for M. haemolytica was broken up into 151 separate sequences. ARS researchers at Clay Center, Nebraska finished the first complete circular single chromosomal sequence for M. haemolytica and B. trehalosi. These reference sequences provide the pathogens’ complete genomic blueprints that serves as their playbooks for causing disease. With the playbook in hand, researchers hope to develop strategies for thwarting these pathogens.
A DNA test for susceptibility to ovine progressive pneumonia. A genetic test was developed to help sheep producers identify animals at high risk for ovine progressive pneumonia. This disease is an incurable wasting disease, that affects millions of sheep worldwide. Infected sheep are less productive and have fewer lambs. In addition to pneumonia, animals show signs of lameness and “hard bag” syndrome, which causes udders to become hard and produce little milk. ARS researchers at Clay Center, Nebraska, working with researchers from the International Sheep Genomics Consortium and a commercial company, developed a publicly available genetic test. The test detects 12 different versions (haplotypes) of the sheep gene TMEM154. Different haplotypes of the gene are known to affect susceptibility to progressive pneumonia virus infection. Producers use the test to detect sheep that are genetically less susceptible to the virus, decrease the risk of animals becoming infected, and select breeding stock with low-risk genetic factors, thereby reducing the prevalence of the virus in flocks.
Miller, J.M., Kijas, J.W., Heaton, M.P., McEwan, J.C., Coltman, D.W. 2012. Consistent divergence times and allele sharing measured from cross-species application of SNP chips developed for three domestic species. Molecular Ecology Resources. 12(6):1145-1150. doi:10.1111/1755-0998.12017.
Heaton, M.P., Kalbfleisch, T.S., Petrik, D.T., Simpson, B., Kijas, J.W., Clawson, M.L., Chitko MckKown, C.G., Harhay, G.P., Leymaster, K.A. 2013. Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep. PLoS One. 8(2):e55490. doi:10.1371/journal.pone.0055490.
Birch, J., Juleff, N., Heaton, M.P., Kalbfleisch, T., Kijas, J., Bailey, D. 2013. Characterization of ovine Nectin-4, a novel peste des petits ruminants virus (PPRV) receptor. Journal of Virology. 87(8):4756-4761.
Chitko McKown, C.G., Chapes, S.K., Miller, L.C., Riggs, P.K., Ortega, M.T., Green, B.T., McKown, R.D. 2013. Development and characterization of two porcine monocyte-derived macrophage cell lines. Results in Immunology. 3:26-31.
Chitko-McKown, C.G., Leymaster, K.A., Heaton, M.P., Griffin, D.D., Veatch, J.K., Jones, S., Clawson, M.L. 2013. Rhodotorula minuta fungemia in a ewe lamb. Transboundary and Emerging Diseases. doi:10.1111/tbed.12062. [Epub ahead of print].
Harhay, G.P., Koren, S., Phillippy, A.M., McVey, D.S., Kuszak, J., Clawson, M.L., Harhay, D.M., Heaton, M.P., Chitko-McKown, C.G., Smith, T.P.L. 2013. Complete closed genome sequences of Mannheimia haemolytica serotypes A1 and A6 isolated from cattle. Genome Announcements. 1(3):e00188-13. DOI: 10.1128/GENOMEA.00188-13.
Durso, L.M., Wells, J., Harhay, G.P., Rice, W.C., Kuehn, L.A., Bono, J.L., Shackelford, S.D., Wheeler, T.L., Smith, T.P. 2012. Comparison of bacterial communities in faeces of beef cattle fed diets containing corn and wet distillers grain with solubles. Letters in Applied Microbiology. 55(2):109-14. DOI: 10.1111/J.1472-765X.2012.03265.X.
Jiang, Z., Zhou, X., Michal, J.J., Wu, X.-L., Zhang, L., Zhang, M., Ding, B., Liu, B., Manoranjan, V.S., Neill, J.D., Harhay, G.P., Kehrli, Jr., M.E., Miller, L.C. 2013. Reactomes of porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus. PLoS One. 8(3):e59229.
Sider, L.H., Heaton, M.P., Chitko-Mckown, C.G., Harhay, G.P., Smith, T.P.L., Leymaster, K.A., Laegreid, W.W., and Clawson, M.L. 2013. Small ruminant lentivirus genetic subgroups associate with sheep TMEM154 genotypes. Veterinary Research. 44:64. DOI: 10.1186/1297-9716-44-64.