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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Genetics, Breeding, and Animal Health Research » Research » Research Project #432111

Research Project: Genomic Intervention Strategies to Prevent and/or Treat Respiratory Diseases of Ruminants

Location: Genetics, Breeding, and Animal Health Research

2018 Annual Report


1a. Objectives (from AD-416):
Objective 1. Elucidate host response associated with the bovine respiratory disease complex (BRDC) and protective immunity, including discovering genetic and biological determinants associated with bovine respiratory disease susceptibility, tolerance, or resistance, and discovering genetic and biologic determinants associated with good responders to bovine respiratory disease vaccines. Sub-objective 1.A: BVD viral infections play an integral and complicated role in BRDC. Current available technology for preventing BVD virus infection includes vaccination, biosecurity, and the elimination of persistently infected cattle. However, if available, genetic selection for animals less likely to become persistently infected would facilitate control and eradication of BVD. The proposed research will test for genetic risk factors associated with BVD virus infection. Sub-objective 1.B: Ovine progressive pneumonia is one of the most economically important diseases in sheep. A major gene TMEM154 was recently discovered that influences susceptibility to OPP in sheep. However, there are no ovine cell lines with defined TMEM154 diplotypes available to study OPP virus infection in vitro. The proposed research will develop cell lines to enable the study of TMEM154 variants in OPP virus infection.S Objective 2. Develop genomics-based strategies to control respiratory diseases of ruminants, including identifying antibiotic-resistance genes and other virulence determinants of bacteria that associate with increased BRDC severity, and developing intervention strategies to reduce antibiotic use and BRDC severity based on genetic typing of bacteria and cattle. Sub-objective 2.A: M. haemolytica of North America place into two major genotypes (1 and 2). Genotype 2 associates with BRDC and genotype 1 does not. The proposed research will identify genomic determinants specific to genotype 2 that may lead to intervention strategies that reduce the incidence of BRDC caused by genotype 2 M. haemolytica. Sub-objective 2.B: Current interventions for BRDC in beef calves include vaccination and metaphylactic use of antibiotics. However, if we had knowledge of the disease-causing potential of nasopharyngeal bacteria in calves, alternative interventions could be designed to reduce the impact of BRDC outbreaks. The proposed research is designed to identify genetic and biological determinants that may influence the disease-causing potential of nasopharyngeal bacteria. Sub-objective 2.C: BCV is involved in the etiology of three distinct clinical syndromes: calf diarrhea, winter dysentery with hemorrhagic diarrhea in adults, and respiratory infections in cattle of all ages. The biological mechanisms underlying disease presentation and variation in their severity are not well understood. The proposed research will determine the influence of serum antibodies, virus strain, and co-infection with other respiratory pathogens on BCV disease presentation and severity of disease.


1b. Approach (from AD-416):
Infectious respiratory diseases of ruminants are a serious health and economic problem for U.S. agriculture. In cattle alone, the costs of bovine respiratory disease complex (BRDC) exceed one billion dollars annually. Therefore, this research focuses primarily on BRDC with an additional component targeting ovine respiratory disease. Our project vision is to reduce the prevalence and severity of respiratory diseases, thereby promoting livestock welfare, enhancing producer efficiency, and reducing antibiotic use. BRDC is a multi-component disease caused by complex interactions among viral and bacterial pathogens, stress and environmental factors, and host genetics. Consequently, we have developed a multi-component approach focused on the host-pathogen interface to study respiratory disease. On the host side, a genome-wide association study will test for genetic risk factors for bovine viral diarrhea (BVD) virus susceptibility. On the bacterial pathogen side, genomics combined with phenomics will identify the spectrum of genetic determinants of M. haemolytica and other bacteria that associate with BRDC. On the viral pathogen side, genomics combined with serology, and microbial diagnostic testing will determine the contribution of bovine coronavirus (BCV) to BRDC. Lastly, novel ovine cell lines will be developed to test host and virus genetic risk factors for ovine progressive pneumonia (OPP). The knowledge gained from this research will be valuable for developing new intervention strategies for controlling BRDC and producing healthier livestock, and could ultimately benefit animals, producers, veterinarians, diagnostic laboratories, pharmaceutical companies, genetic testing laboratories, and regulatory agencies.


3. Progress Report:
Objective 1.a: Used the newly constructed ARS SciNet/CERES computing resource to re-align whole genome sequences of about 300 cattle. This was needed due to the inclusion of “chromosome unknown” to the UMD3.1 data set. When the new bovine reference assembly (ARS_UCD version 1.2) became available this summer, another massive re-alignment of these 300 animals’ genome was conducted. These alignments produced the needed files to begin analyzing genome-wide variation for association with disease. Objective 1.b: Tissues were harvested from a TMEM154 “1,1” wether. Cells were isolated from the choroid plexus, synovial membranes, alveolar macrophages from the lungs, monocytes from the blood, and kidney, and primary cell cultures were initiated. Cultured cells currently are undergoing expansion in preparation for freezing and characterization. Objective 2.a: The genomes of 34 genotype 2 Mannheimia haemolytica isolates have all been sequenced and assembled into complete, error-corrected, circularized chromosomes. All four known subtypes of genotype 2 are well-represented by the sequenced isolates. All 34 isolate chromosomes have been annotated through the National Center for Biotechnology Information prokaryotic genome annotation pipeline and will become publicly available through the National Center for Biotechnology Information website immediately following the publication of a relevant manuscript. Objective 2.b: 20 round one Pasteurell multocida genomes have been sequenced, assembled and closed. These genomes will be submitted to GenBank. BLAST2GO parser was written for the BioCyc /Pathway Tools inferential systems biology platform. The necessity of using the EDGAR parser diminished as BioCyc /Pathway Tools performed many of the same functions as well as providing additional capabilities in the systems biology domain. Objective for 2.c: Serial blood samples and nasal swabs were collected from 817 beef calves from three herds at pre-determined times from birth though the feed yard. Serum anti-BCV antibody abundance was measured by ELISA and BCV and other viral and bacterial BRD pathogens were detected by real-time PCR methods. Test results were compared among herds, over time, and between calves that did and did not develop BRDC. The associations of various herd and calf factors with test results were also evaluated. In addition, ten full-length BCV genomes were sequenced and annotated in addition to three BCV vaccine strains.


4. Accomplishments
1. Development of a test that identifies the two major bacteria species associated with “pinkeye” in cattle. Infectious bovine keratoconjunctivitis (IBK) is a common eye disease in cattle that is also known as “pinkeye”. The test identifies Moraxella bovis, a known pathogen that causes IBK, and Moraxella bovoculi, a recently discovered species that is suspected as having a significant role in the disease. The test was developed in a collaboration between ARS in Clay Center, Nebraska, and the University of Nebraska in Lincoln, Nebraska, and uses matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) to quickly and accurately identify M. bovis and M. bovoculi. Currently offered at the University of Nebraska Diagnostic Center, the test is available for use without restriction, and can assist in the control and reduction of IBK in commercial herds through the identification and management of animals carrying either M. bovis or M. bovoculi.

2. Epidemiological characterization of bovine coronavirus (BCV) in naturally-infected populations of cattle. In recent years, BCV has been found to be associated with respiratory infections in cattle of all ages. However, given the high rates of subclinical infections, the exact role of BCV infection in the development of clinical bovine respiratory disease (BRD) remains unclear. ARS researchers at Clay Center, Nebraska followed multiple herds of calves from birth through the feedlot to determine the relationship between immunity to BCV, virus circulation patterns, and the risk for developing BRD at various production stages. They found that herd prevalence of BCV shedding was the only risk factor associated with both pre- and post-weaning BRD incidence in these herds. Additionally, BCV antibody levels for calves at feed yard entry was inversely related to the subsequent incidence of BRD in that group, meaning that calves with high antibody levels were less likely to develop BRD. Finally, it was found that total serum antibody levels to BCV may not be the best predictor of the probability of BCV shedding and BRD disease development in beef calves. The results from this study suggest that a BCV vaccine may reduce BRD incidence in feedlot cattle. Furthermore, the observations that serum antibodies may not be the best predictor of immune protection has important implications for diagnostic labs, vaccine producers, and researchers investigating the contributions of BCV to BRD, and warrants further study.


Review Publications
Workman, A.M., Zhu, L., Keel, B.N., Smith, T.P.L., Jones, C. 2018. The Wnt signaling pathway is differentially expressed during the bovine herpesvirus 1 latency-reactivation cycle: evidence that two protein kinases associated with neuronal survival (Akt3 and bone morphogenetic protein receptor 2) are expressed at higher levels during latency. Journal of Virology. 92:e01937-17. https//doi.org/10.1128/JVI.01937-17.

Davis, T.Z., Stegelmeier, B.L., Lee, S.T., Green, B.T., Chitko-Mckown, C.G. 2018. Effect of grinding and long-term storage on the toxicity of white snakeroot (Ageratina altissima) in goats. Research in Veterinary Science. 118:419–422. https://doi.org/10.1016/j.rvsc.2018.04.006.

Sanchez, E.G., Riera, E., Nogal, M., Gallardo, C., Fernandez, P., Bello-Morales, R., Lopez-Guerrero, J., Chitko-McKown, C.G., Richt, J.A., Revilla, Y. 2017. Phenotyping and susceptibility of established porcine cells lines to African swine fever virus infection and viral production. Scientific Reports. 7:10369. https://doi.org/10.1038/s41598-017-09948-x.

Workman, A.M., Kuehn, L.A., McDaneld, T.G., Clawson, M.L., Chitko-McKown, C.G., Loy, J.D. 2017. Evaluation of the effect of serum antibody abundance against bovine coronavirus on bovine coronavirus shedding and risk of respiratory tract disease in beef calves from birth through the first five weeks in a feedlot. American Journal of Veterinary Research. 78(9):1065-1076. https://doi.org/10.2460/ajvr.78.9.1065.

Sun, H., Workman, A., Osorio, F.A., Steffen, D., Vu, H.L.X. 2017. Development of a broadly protective modified-live virus vaccine candidate against porcine reproductive and respiratory syndrome virus. Vaccine. 36:66-73. https://doi.org/10.1016/j.vaccine.2017.11.028.

Kalbfleisch, T.S., Murdoch, B.M., Smith, T.P.L., Murdoch, J.D., Heaton, M.P., Mckay, S.D. 2018. A SNP resource for studying North American moose. F1000Research. 7(40):1-17. https://doi.org/10.12688/f1000research.13501.1.

Harhay, G.P., Harhay, D.M., Bono, J.L., Smith, T.P.L., Capik, S.F., DeDonder, K.D., Apley, M.D., Lubbers, B.V., White, B.J., Larson, R.L. 2017. Closed genome sequences of seven histophilus somni isolates from beef calves with bovine respiratory disease complex. Genome Announcements. 5(40):e01099-17. https://doi.org/10.1128/genomeA.01099-17.

Robbins, K., Dickey, A.M., Clawson, M.L., Loy, J.D. 2018. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of Moraxella bovoculi and Moraxella bovis isolates from cattle. Journal of Veterinary Diagnostic Investigation. p. 1-4. Available: jvdi.sagepub.com. https://doi.org/10.1177/1040638718789725.