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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

2019 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
Subobjective 1.a: “Genome wide search for genetic risk factors associated with persistent bovine viral diarrhea (BVD) virus infection in beef calves.” Scientists at ARS used the ARS SciNet/CERES computing resources and GATK software to call 164 M variants in 96 bovine viral diarrhea - persistently infected (BVDPI) cases and 96 controls mapped to the new bovine genome assembly (ARS_UCD version 1.2). These results were filtered with PLINK software to include only 17M single nucleotide polymorphisms (SNPs) that met call rate, frequency, and Hardy-Weinberg cutoffs. A first analysis of genome-wide association revealed approximately six sites on five chromosomes are significantly associated with persistent BVD virus infection in calves. Subobjective 1.b: “Development of an in vitro system for studying ovine progressive pneumonia virus (OPPV).” Four rams from the Easy Care flock with TMEM154 genotype “1,3” were tested once a month for 3 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. Subobjective 2.a: “Identification of genomic determinants specific to virulent “genotype 2” Mannheimia haemolytica for developing interventions strategies to control bovine respiratory disease complex (BRDC).” A collection of 100 M. haemolytica isolates was tested and successfully used to develop a phenotyping test that distinguishes between the two major genotypes of M. haemolytica commonly found in cattle. The collection represented the breadth and depth of diversity between and within the two major genotypes. The test is based on differing, reproducible colony morphologies of the two genotypes under select culture conditions. Previously a matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) test was developed to distinguish the two genotypes for veterinary diagnostic lab use. In conjunction with standard biochemical tests that identify M. haemolytica at the species level, this phenotyping test can be used in any research lab to quickly and effectively identify M. haemolytica genotypes without the use of the commercial MALDI-TOF test. Subobjective 2.b: “Genome sequencing, metabolic and antibiotic resistance phenotyping of diverse nasopharyngeal bacteria isolated from commercial beef calves in an epidemiological study of BRDC.” The Pathway Tools system ( is live and hosts the pathway genome database for a bovine respiratory disease-associated Mannheimia haemolytica isolate as well as for the food-safety pathogen isolates of Salmonella enterica enterica, and E. coli 0157:H7. This milestone was accomplished in two phases, neither of which were a given, as to the best of our knowledge, the work in each phase was never attempted before. In the first phase, new computational approaches were developed to enhance existing GenBank annotation with Gene Ontology terms and Enzyme Commission Numbers to improve the extent and accuracy of the computational inferences that the Pathway Tools system preforms to create a pathway genome database (PGDB) from GenBank file. Additionally, another separate data process outside of the Pathway Tools environment was developed to predict promoters and transcription factor binding sites followed by parsing these predictions into the target PGDB. These predicted regulatory sites provide new contexts to understand the regulation of genes, such as coordinated gene expression, while the Pathway Tools gives the user the ability to compute the statistical significance of the association of the expression of groups of genes to pathways, regulatory elements, or other functional enrichments or depletions. In the second phase under the SCINet research initiative, researchers at SRI International, ARS researchers at Clay Center, Nebraska, BioTeam and Iowa State University configured a cloud instance of Pathway Tools whereby the computational component resides in Amazon AWS Elastic Compute Cloud (EC2) while the data resides in the Amazon AWS Relational Database Service (RDS). This separation of compute and database infrastructure within the cloud provides for more robust database persistence and security but was challenging to accomplish. Planning for PGDB contributions from additional ARS researchers was considered when designing this system. Subobjective 2.c: “Identification of the mechanisms by which bovine coronaviruses (BCVs) induce different diseases, and factors that contribute to disease severity.” Serial blood samples and nasal swabs were collected from 756 beef calves from four herds at pre-determined times from birth though the feed yard. BCV and other viral and bacterial BRD pathogens were detected by real-time PCR. Test results were compared among herds, over time, and between calves that did and did not develop BRDC. Eight full-length BCV genomes from U.S. Meat Animal Research Center (USMARC) and the University of Nebraska Diagnostic Laboratory were sequenced and annotated. A longitudinal study was also initiated to measure the effects of a new modified live BCV vaccine on incidence of naturally occurring respiratory disease and enteritis in calves at USMARC.

1. Identification of genes encoding outer membrane proteins specific to genotype 2 Mannheimia haemolytica. There are two major genotypes, or strains of M. haemolytica carried by North American cattle (1 and 2). While both genotypes are commonly found in the upper respiratory tract of cattle, genotype 2 strains are a predominantly more frequent cause of bovine pneumonia then genotype 1 strains. The biological determinants responsible for genotype 2 strains increased propensity to cause pneumonia in cattle are not fully understood. ARS scientists in Clay Center, Nebraska, investigated the genomes of 69 isolates that collectively represent the breadth of diversity between and among the two genotypes were fully sequenced into closed circular genomes. Analysis of the genomes identified a set of genes encoding outer membrane proteins that have known roles in pathogenesis, and that are specific to genotype 2 M. haemolytica isolates. Identification of these genes, and the proteins they encode, may lead to effective intervention strategies that prevent genotype 2 M. haemolytica from causing pneumonia in cattle.

2. A SCINet collaborative research initiative publishes in the Cloud the Pathway Tools symbolic systems biology system for the analysis of agriculturally important bacteria. Advances in DNA sequencing and information technologies have enabled the routine sequencing and assembly of bacterial genomes. Automated algorithms compute the identity and location of genes (annotation) throughout the genome, answering the question “What and where are the genes in the genome?” However, more profound, more impenetrable questions remain such as “How are genes turned on or off in a coordinated fashion to affect phenotype (a physical trait).” The computational modeling of this system of genes, their interactions, and the influence of the environment on the system is the domain of systems biology. Until now, microbiologists and other researchers working on bacteria-related problems in animal disease, food safety, bioengineering and other agricultural domains were not able to efficiently create holistic systems biology models, including regulatory element predictions, for specific field isolates. Through a SCINet collaborative research initiative including ARS researchers at Clay Center, Nebraska, Iowa State University, and industry collaborators, the Pathway Tools systems biology analytical platform was customized to run on Amazon Web Services (AWS) at to host systems biology models of field isolates sequenced and assembled by the USDA. The primary beneficiaries of this resource are microbiologists and others wishing to use, create, and publish systems biology models that relate bacterial gene systems to phenotype. Systems biology models that reproduce past, and predict future, experimental results will yield evidence-based strategies to mitigate the effects of bacterial infection, improve food safety protocols, and promote rational, focused solutions to bacteria-related problems.

3. Leukotoxin susceptibility in cattle. Bovine respiratory disease is the most significant illness in U.S. cattle and a major source of economic loss, exceeding a billion dollars annually. When bacteria invade the lungs, they secrete a toxin that binds to a receptor on bovine white blood cells and kills them, causing rapid inflammation, bleeding, and fluid accumulation in the air sacs of the lungs. This causes a severe and sometimes fatal pneumonia. ARS researchers at Clay Center, Nebraska, used a genome sequencing approach to identify differences in the naturally occurring toxin receptor in more than 1,000 cattle from 46 breeds. One of these receptor variants was 30-times more sensitive to the toxin. These results suggest that cattle with variant receptors may be at increased risk for toxin-related respiratory disease. This also provides new possibilities for disease intervention including: identifying high-risk animals through genetic testing, selectively breeding for less susceptible animals, and developing non-antibiotic treatments that may neutralize the bacterial toxin.

4. Bovine congestive heart failure in feedlot cattle. Congestive heart failure in feedlot cattle has become increasingly common in the Western Great Plains of North America. Reports of cattle losses to this illness (sometimes referred to as “brisket disease”) have exceeded $250,000 annually in individual operations, surpassing losses from bovine respiratory disease. A major gene (EPAS1) had been reported by others to be associated with heart failure in the Rocky Mountains. ARS researchers at Clay Center, Nebraska, tested feedlot cattle raised and finished in the Western Great Plains to determine whether the EPAS1 gene was playing a major role in causing the disease at moderate altitudes. One hundred and two end-stage heart failure cases were genetically evaluated, together with their healthy pen mates originating from more than 30 different ranch operations. No significant genetic association of EPAS1 with heart failure was observed. This result suggests that identifying genetic risk factors underlying bovine congestive heart failure may require a genome-wide search.

Review Publications
Workman, A.M., Clawson, M.L., Heaton, M.P., Dickey, A.M. 2018. First complete genome sequence of a genotype A2, subgroup 4 small ruminant lentivirus. MICROBIOLOGY RESOURCE ANNOUNCEMENTS. 7(19):e01337-18.
Workman, A.M., Chitko-McKown, C.G., Smith, T.P.L., Bennett, G.L., Kalbfleisch, T.S., Basnayake, V., Heaton, M.P. 2018. A bovine CD18 signal peptide variant with increased binding activity to Mannheimia hemolytica leukotoxin. F1000Research. 7:1985.
Chen, C.T., Perry, T.L., Chitko-Mckown, C.G., Smith, A.D., Cheung, L., Beshah, E., Urban Jr, J.F., Dawson, H.D. 2019. The regulatory actions of retinoic acid on M2 polarization of porcine macrophages. Developmental and Comparative Immunology. 98(9):20-23.
Davis, T.Z., Stegelmeier, B.L., Lee, S.T., Green, B.T., Chitko-McKown, C.G. 2018. Effects of grinding and long-term storage on the toxicity of white snakeroot (Ageratina altissima) in goats. Research in Veterinary Science. 118:419-422.
Lindholm-Perry, A.K., Kuehn, L.A., McDaneld, T.G., Miles, J.R., Workman, A.M., Chitko-McKown, C.G., Keele, J.W. 2018. Complete blood count data and leukocyte expression of cytokine genes and cytokine receptor genes associated with bovine respiratory disease in calves. BMC Research Notes. 11:786.
Loy, J.D., Dickey, A.M., Clawson, M.L. 2018. Complete genome sequence of Moraxella bovis strain Epp63, an etiologic agent of infectious bovine keratoconjunctivitis. Microbiology Resource Announcements. 7(8):1-2.
Workman, A.M., Kuehn, L.A., McDaneld, T.G., Clawson, M.L., Loy, J.D. 2019. Longitudinal study of humoral immunity to bovine coronavirus, virus shedding, and treatment for bovine respiratory disease in pre-weaned beef calves. BioMed Central (BMC) Veterinary Research. 15:161.
Dickey, A.M., Schuller, G., Loy, J.D., Clawson, M.L. 2018. Whole genome sequencing of Moraxella bovoculi reveals high genetic diversity and evidence for interspecies recombination at multiple loci. PLoS One. 13(12):e0209113.
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. 2018. Closed genome sequences and antibiograms of 16 pasteurella multocida isolates from bovine respiratory disease complex cases and apparently healthy controls. Microbiology Resource Announcements. 7(11):e00976-18.
Nguyen, S.V., Harhay, G.P., Bono, J.L., Smith, T.P., Harhay, D.M. 2017. Genome sequence of the thermotolerant foodborne pathogen Salmonella enterica serovar Senftenberg ATCC 43845 and phylogenetic analysis of Loci encoding increased protein quality control mechanisms. mSystems. 2:e00190-16.
Haley, B.J., Smith, T.P., Harhay, G.P., Loneragan, G.H., Webb, H.E., Bugarel, M., Kim, S., Van Kessel, J.S., Harhay, D.M. 2019. Complete genome sequence of a Salmonella enterica subsp. enterica serovar Fresno isolate recovered from beef cattle lymph nodes. Microbiology Resource Announcements. 8(2):e01338-18.
Nguyen, S.V., Harhay, D.M., Bono, J.L., Smith, T.P.L., Fields, P.I., Dinsmore, B.A., Santovina, M., Wang, R., Bosilevac, J.M., Harhay, G.P. 2018. Comparative genomics of Salmonella enterica serovar Montevideo reveals lineage-specific gene differences that may influence ecological niche association. Microbial Genomics. 4:1-17.
Heaton, M.P., Smith, T.P.L., Freking, B.A., Workman, A.M., Bennett, G.L., Carnahan, J.K., Kalbfleisch, T.S. 2017. Using sheep genomes from diverse U.S. breeds to identify missense variants in genes affecting fecundity. F1000Research. 6:1303.
Yaman, Y., Keles, M., Aymaz, R., Sevim, S., Sezenler, T., Onaldi, A.T., Kaptan, C., Baskurt, A., Koncagul, S., Oner, Y., Ozturk, E., Iriadam, M., Un, C., Heaton, M.P. 2019. Association of TMEM154 variants with visna/maedi virus infection in Turkish sheep. Small Ruminant Research. 177:61-67.
Stella, A., Nicolazzi, E.L., Van Tassell, C.P., Rothschild, M., Colli, L., Rosen, B.D., Sonstegard, T.S., Crepaldi, P., Tosser, G., Joost, S., Adaptmap Consortium. 2018. AdaptMap: Exploring goat diversity and adaptation. Genetic Selection Evolution. 50:61.
Neill, J.D., Workman, A.M., Hesse, R., Bai, J., Poulsen-Porter, E., Meadors, B., Anderson, J., Bayles, D.O., Falkenberg, S.M. 2019. Identification of BVDV2b and 2c subgenotypes in the United States: genetic and antigenic characterization. Virology. 528:19-29.