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

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

Location: Animal Health Genomics

2021 Annual Report

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.

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.

Progress Report
Objective 1A: Gene-editing was used to evaluate the roles of three candidate genes identified in the whole genome sequence comparison between Madin Darby Bovine Kidney (MDBK) and Cells Resistant to Infection with Bovine Viral Diarrhea Virus (BVDV; CRIB) and determined that they were not essential for viral infection. In the process, a breakthrough was discovered with CD46 gene-editing in MDBK cells and shifted our focus in the last year of the current project plan to follow this promising line of research. Specific novel edits were made that appear to vastly reduce permissivity of BVDV in MDBK cell lines. These CD46 edits are being tested in vivo to see if they replicate the phenotype in fetal calf tissues. Objective 1B: Two rams from the Composite IV flock with TMEM 154 genotype "1,3" were tested once a month for three months to determine Ovine Progressive Pneumonia Virus (OPPV) status. All were negative for infection with the virus, and one ram was added to the OPP-free flock for breeding to produce TMEM 154 "3,3" lambs. After lambing, all ewes were tested for OPPV status and all continue to be negative. The lambs were bled in order to submit DNA to a commercial lab for TMEM 154 genotyping. When results are received, TMEM 154 "3,3" lambs will be identified for tissue harvest and cell line development. Objective 2A: A Mannheimia haemolytica genotype 2 specific adhesin was expressed as a recombinant protein and used to develop an enzyme-linked immunosorbent assay (ELISA) test for either cattle serum or plasma reactivity to the adhesin. The ELISA was applied to cattle plasma samples representing animals that were followed serially before and after developing bovine respiratory disease complex (BRDC), with concomitant isolation of genotype 2 M. haemolytica strains from their diseased lungs. The ELISA was additionally applied to cattle serum samples collected before and after vaccination with an attenuated strain of genotype 2 M. haemolytica. For both sample sets, the ELISA results showed the adhesin to be immunogenic in the vaccinated and sick animals. Immunogenicity of the adhesin does not confirm it as an outer cellular membrane protein, but it does support that location and that the adhesin is a viable intervention candidate. An adhesion assay to confirm the outer membrane location of the adhesin was also developed this past year using commercially available bovine turbinate cells and the plasma and sera samples described above. Testing of genotype 2 M. haemolytica with the assay is scheduled for completion by the end of this fiscal year. Objective 2B: Identified metabolic phenotypes of BRDC pathogens using liquid chromatography-mass spectrometry (LC-MS). Initiated work with a scientist in Cambridge, MA to assist in the analysis LC-MS metabolomic and fluxomic data from cultures of BRDC bacteria. Secured the assistance of a cooperator to advise about metabolomic laboratory procedures, sample handling, LC-MS performance standards, and data analysis. Currently working with a cooperator in Clay Center, Nebraska, to tune the microbial culture conditions, sample handling, LC-MS run parameters, and the creation of small molecule standard reference spectral libraries. We have identified reasonable LC-MS run parameters for many non-sugar polar small molecules and some lipids by the judicious selection of solvents and LC columns. Developing methods to tune system for the robust identification of sugars and sugar derivatives. Once accomplished, will run standard compound library through the system to create a spectral library that will be used to identify compound from bacterial culture as well as attempt to follow labeled atoms through metabolic pathways in downstream fluxomics experiments. Objective 2C: Full-length BCV genomes have been sequenced, assembled, and annotated from 52 clinical samples. A novel BCV variant was identified in the U.S. that has not previously been reported. Work is ongoing to characterize this novel BCV variant.

1. Ovine gene variant discovered for Johne’s disease. Johne’s disease is a contagious bacterial disease in ruminants, causing a progressive chronic inflammation of the gut, resulting in significant production loss and economic losses around the world. Infections are life-long with no effective treatments or vaccines. If available, selective breeding strategies for reduced Johne's disease susceptibility would be welcome tools in disease eradication efforts. Collaborating with researchers in Turkey, ARS scientists in Clay Center, Nebraska, identified a variant in the TLR2 gene (Q650) that confers reduced susceptibility to Johne’s disease. Sheep with the Q650 variant were nearly 7-fold less likely to become infected. This raises the possibility for breeding Turkish sheep that are resistant to Johne’s disease. Moreover, this discovery raises the possibility that the same TLR2 gene may positively influence Johne’s disease resistance in other sheep breeds or ruminant species around the world.

2. Bison and Simmental genomes released. The first reference-quality genome assemblies for the iconic North American Yellowstone bison and the Simmental cattle breed have been made publicly available. Animal genome assemblies provide genetic “blueprints” for how an animal develops and passes on genetic information to their offspring. Researchers in Clay Center, Nebraska, used in vitro fertilization to cross a bison bull with a Simmental cow. The hybrid animal and its purebred parents were fully sequenced to produce a complete, highly accurate genome assembly for each species. The bison assembly will be used in conservation efforts to maintain genetic diversity and to study bison evolution by comparing DNA obtained from fossil specimens sampled from permafrost in the Arctic Circle. The Simmental cattle assembly will contribute to an international effort to survey existing cattle breeds around the world to preserve their genetic diversity and identify variation useful for improving beef and dairy traits in different environments. These genomic tools will help to speed genetic progress and avoid counterproductive breeding before it happens, providing another means for increasing beef and dairy production important for food security, while maximizing environmental and economic sustainability.

3. Multiplex assay for bovine inflammatory cytokines developed. Commercially available bovine-specific assays are limited in number, and multiplex assays for this species are rare, thus this multiplex assay will be important for scientists studying inflammatory diseases in cattle. Cytokines are proteins produced by cells in the immune system that communicate with other cells to produce additional responses to infection. Assays to measure these proteins are expensive as well as time and labor intensive. ARS scientists in Clay Center, Nebraska, developed a multiplex assay for the bovine inflammatory cytokines IL-1ß, IL-6, and TNF-alpha using the Meso Scale Discovery U-PLEX platform. These proteins when produced in response to infection stimulate fever, stimulate other immune cells, and contribute to inflammation in the area of infection and are elevated during bovine respiratory disease. "Do-It-Yourself" enzyme-linked immunosorbent assay (ELISA) kits available commercially containing polyclonal antibodies. Although there are few commercially available multiplex assays for species others than rodents and humans, the availability of monoclonal and polyclonal antibodies and recombinant proteins specific for other species are becoming more abundant. ARS researchers at Clay Center, Nebraska, have shown that these reagents can be used within the U-PLEX system to allow researchers to more easily customize multiplex assays for their specific analyte- and species-specific needs to more quickly identify proteins associated with immunity and identify infections.

4. Identification of predicted outer membranes in the major bacterial species that cause bovine respiratory disease complex. Bovine respiratory disease complex (BRDC) affects cattle throughout the world and is a serious animal welfare and economic problem. BRDC is the biggest killer of newly weaned cattle in the United States, with costs to the beef industry from treatments, morbidities, and overall reduced productivity exceeding one billion dollars annually. Histophilus somni, Mannheimia haemolytica, and Pasteurella multocida are three major bacterial pathogens that all cause BRDC and are also known to harbor antimicrobial resistance genes. ARS researchers in Clay Center, Nebraska, analyzed available genomes of all three bacteria species using computational and bioinformatic methods and identified a large number of outer membrane proteins (OMPs). OMPs are proteins that are embedded in the outer membrane of the bacteria and protrude outward making them accessible to host immune factors including antibodies. Several specific OMPs were identified in all three of the bacterial species, indicating potential cross-species applications. They, and others are being studied for contributions to pathogenesis, as well as the development of interventions that could reduce BRDC incidence and subsequent needs for antibiotic treatments.

5. Using gene-editing to probe virus entry mechanisms in cattle. BVDV is an important pathogen in cattle that causes reproductive and respiratory diseases costing the industry over a billion dollars annually. Using cattle cell lines, whole genome sequencing, and a gene-editing approach, ARS researchers in Clay Center, Nebraska, were able to identify candidate genes hypothesized to be essential for BVDV entry into cells. In collaboration with a company specializing in genome-editing, three candidate genes were disrupted individually, and in combination, to show they were not necessary for viral infection. This result contributes to our understanding of BVDV pathogenesis and paves the way for identifying genes in cattle that are essential for viral infection. Ultimately, this approach is expected to identify cellular targets for novel intervention strategies to reduce the incidence of BVDV infections in cattle.

Review Publications
Yaman, Y., Aymaz, R., Keles, M., Bay, V., Hatipoglu, E., Kaptan, C., Baskurt, A., Yilmaz, O., Heaton, M.P. 2021. Evaluation of CD109, PCP4, and SEMA3D genes for their association with Ovine Johne’s disease in Turkish sheep. Animal Biotechnology. Article 1887880.
Chitko-McKown, C.G., Bennett, G.L., Kuehn, L.A., DeDonder, K.D., Apley, M.D., Harhay, G.P., Clawson, M.L., Workman, A.M., White, B.J., Larson, R.L., Capik, S.F., Lubbers, B.V. 2021. Cytokine and haptoglobin profiles from shipping through sickness and recovery in metaphylaxis- or un-treated cattle. Frontiers in Veterinary Science. 8. Article 611927.
Dickey, A.M., Smith, T.P.L., Clawson, M.L., Heaton, M.P., Workman, A.M. 2020. Classification of small ruminant lentivirus subtype A2, subgroups 1 and 2 based on whole genome comparisons and complex recombination patterns. F1000Research. 9:1449.
Hille, M.M., Clawson, M.L., Dickey, A.M., Lowery, J.H., Loy, J.D. 2021. MALDI-TOF MS biomarker detection models to distinguish RTX toxin phenotypes of Moraxella bovoculi strains are enhanced using calcium chloride supplemented agar. Frontiers in Cellular and Infection Microbiology. 11. Article 632647.
Heaton, M.P., Smith, T.P.L., Bickhart, D.M., Vander Ley, B.L., Kuehn, L.A., Oppenheimer, J., Shafer, W.R., Schuetze, F.T., Stroud, B., McClure, J.C., Barfield, J.P., Blackburn, H.D., Kalbfleisch, T.S., Davenport, K.M., Kuhn, K.L., Green, R.E., Shapiro, B., Rosen, B.D. 2021. A reference genome assembly of Simmental cattle, Bos taurus taurus. Journal of Heredity. 112(2):184-191.
Oppenheimer, J., Rosen, B.D., Heaton, M.P., Vander Ley, B.L., Shafer, W.R., Schuetze, F.T., Stroud, B., Kuehn, L.A., McClure, J.C., Barfield, J.P., Blackburn, H.D., Kalbfleisch, T.S., Bickhart, D.M., Davenport, K.M., Kuhn, K.L., Green, R.E., Shapiro, B., Smith, T.P.L. 2021. A reference genome assembly of American bison, Bison bison bison. Journal of Heredity. 112(2):174-183.
Dutta, E., Loy, J.D., Deal, C.A., Wynn, E.L., Clawson, M.L., Clarke, J., Wang, B. 2021. Development of a multiplex real-time PCR assay for predicting macrolide and tetracycline resistance associated with bacterial pathogens of bovine respiratory disease. Pathogens. 10:64.
Yaman, Y., Aymaz, R., Keles, M., Bay, V., Ün, C., Heaton, M.P. 2021. Association of TLR2 haplotypes encoding Q650 with reduced susceptibility to ovine Johne’s disease in Turkish sheep. Scientific Reports. 11. Article 7088.
Loy, J.D., Hille, M., Maier, G., Clawson, M.L. 2021. Component causes of infectious bovine keratoconjunctivitis - the role of Moraxella species in the epidemiology of infectious bovine keratoconjunctivitis. Veterinary Clinics of North America:Food Animal Practice. 37(2):279-293.
Chitko-McKown, C.G., Bierman, S.L., Kuehn, L.A., Bennett, G.L., DeDonder, K.D., Apley, M.D., Harhay, G.P., Clawson, M.L., White, B.J., Larson, R.L., Capik, S.F., Lubbers, B.V. 2021. Detection of bovine inflammatory cytokines IL-1ß, IL-6, and TNF-a with a multiplex electrochemiluminescent assay platform. Veterinary Immunology and Immunopathology. 237. Article 110274.
Wynn, E.L., Clawson, M.L. 2021. Differences between predicted outer membrane proteins of Pasteurella multocida, Histophilus somni, and genotype 1 and 2 Mannheimia haemolytica strains isolated from cattle. Genome.