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

Location: Animal Health Genomics

2020 Annual Report

Objectives
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.

Approach
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: The first analysis of genome-wide association of 17M single nucleotide polymorphisms (SNP) variants mapped to the bovine genome assembly (ARS_UCDv1.2) last year revealed approximately six sites on five chromosomes are significantly associated with persistent Bovine Viral Diarrhea (BVD) virus infection in calves. This work was all accomplished with the ARS SciNet/CERES computing resources. For validation ARS researchers at Clay Center, Nebraska, have collected approximately 35 more BVD persistently infected calves, however they have not yet been tested. Additionally, they have also compared whole genome sequence (WGS) from Madin-Darby Bovine kidney and cells resistant to infection with BVDV-1 (CRIB) cell lines and identified three major deletions spanning three genes on three chromosomes and have used gene-editing to knock-out all three of these genes and are evaluating whether they are essential for BVD virus infection. The manuscript describing the gene-editing results is in progress. Objective 1B: Four rams from the Composite IV flock with TMEM154 genotype "1,4" 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 "4,4" 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 "4,1" lambs will be identified for tissue harvest and cell line development. Objective 2A: Phylogenetically distinct Mannheimmia haemolytica strains representing two of five subtypes of genotype 1 and two of four subtypes of genotype 2 were phenotyped on the Biolog platform. Phenotypic differences between the subtypes and genotypes were identified, although additional data is needed for statistical significance. Additionally, a new, fifth subtype of genotype 2 was identified this past year through the sequencing of 714 newly acquired M. haemolytica strains from cattle in the southern U.S. The isolates were sequenced on an Illumina platform and typed using the genotyping and subtyping classification system developed for M. haemolytica by ARS researchers in Clay Center, Nebraska. Representatives of the new subtype have not yet been phenotyped on the Biolog. Objective 2B: ARS researchers are working with Iowa State and SRI, International to improve the implementation of Pathway Tools, and the Pathway Tools system itself. They are also working with the LC-MS vendor Waters Corporation and their software arm, Nonlinear Corporation, and have identified a company that will train ARS staff to use their current liquid chromatography-mass spectrometry (LC-MS) system to generate metabolomic and fluxomic data on microbial cultures. These data will be generated in pursuit of expanding the representation of bacterial biology from a static reference, a series of genes along chromosomes and plasmids, to a dynamic reference that predicts and recapitulates the genome-wide flow of metabolites in bacterial systems. Required training, performance metrics and deliverables, and potential vendors in a specification have been documented and is now awaiting action. Objective 2C: Serial blood samples and nasal swabs were collected from 804 beef calves from four herds at pre-determined times from birth though the feed yard. Bovine coronavirus (BCV) and other viral and bacterial bovine respiratory disease (BRD) pathogens were detected by real-time polymerase chain reaction (PCR). Test results were compared among herds, over time, and between calves that did and did not develop BRD. In addition, a longitudinal study involving 434 calves was completed to measure the effects of a new intranasally administered modified-live bovine coronavirus vaccine on respiratory disease treatment rates, subclinical virus shedding, weight gain, and the frequency or severity of lung lesions observed at slaughter in a population of cattle with endemic BCV. Finally, 18 full-length BCV genomes from an ARS facility in Clay Center, Nebraska, and the University of Nebraska Diagnostic Laboratory were sequenced and annotated.

Accomplishments
1. Culture-based method to distinguish between the two major strain types (genotypes) of M. haemolytica commonly found in cattle. ARS researchers at Clay Center, Nebraska, collaborated with the University of Nebraska-Lincoln to develop a visual culture-based method that distinguishes between the two major strain types (genotypes) of Mannheimmia haemolytica commonly found in cattle. One genotype causes much more bovine respiratory disease than the other. Application of the method can help ensure that both genotypes are detected in samples from diseased cattle. This will help determine the roles, or lack thereof, of these strains (genotypes) in bovine respiratory disease. Understanding the extent of strain involvement in bovine respiratory disease could facilitate better prevention and treatment strategies for outbreaks, and thus reduce the overall prevalence of the disease in U.S. cattle.

2. A computational method to quantify the effects of slipped strand mispairing on bacterial tetranucleotide repeats. The genomes of the ruminant respiratory disease pathogens Mannheimia haemolytica, Histophilus somni, and Bibersteinia trehalosi all possess simple sequence repeats (SSRs), genomic features consisting of a single base or multiple bases, repeated in series. In M. haemolytica, and other gram-negative mucosal pathogens, these regions have been associated with hypermutability through slipped-strand mispairing of DNA polymerase that cause SSRs to expand or contract up to one million-fold higher that point mutations. When slipped strand mispairing affects phenotype (e.g. colony morphology, virulence, or pathogenicity), it is referred to as phase variation. Before this method was published, there was no way to directly measure the distribution of SSR length variants (phase variants) in a culture, although indirect measures abound, for example, clones, hybridization, or PCR-based techniques. Directly measuring low-frequency phase variants using DNA sequence has been elusive because they are obscured by sequencer noise. ARS researchers at Clay Center, Nebraska, developed a novel theory to remove sequencer noise, implemented the theory in a computational workflow, and devised the experiments to demonstrate its effectiveness using synthetic DNA duplex controls and bacterial genomic sequence. Many gram-negative mucosal pathogens causing disease in mammals (and birds) contain SSRs, the length and sequence phase variation of which has been associated with host disease and can now be directly assessed for their role in disease. For the first time, researchers using this method can now track slipped-strand mispairing mediated phase variation in bacterial populations and discern their quantifiable subpopulations, each with their own environmental fitness. The fittest members can now be identified as they rapidly expand upon environmental perturbation providing new insights into adaptability biology and new molecular and pathway targets to mitigate disease.

Review Publications
Carlson, J.M., Vander Ley, B.L., Lee, S.I., Grotelueschen, D.M., Walz, P.H., Workman, A.M., Heaton, M.P., Boxler, D.J. 2020. Detection of bovine viral diarrhea virus in stable flies following consumption of blood from persistently infected cattle. Journal of Veterinary Diagnostic Investigation. 32(1):108-111. https://doi.org/10.1177/1040638719898688.
Harhay, G.P., Harhay, D.M., Bono, J.L., Capik, S.F., DeDonder, K.D., Apley, M.D., Lubbers, B.V., White, B.J., Larson, R.L., Smith, T.P.L. 2019. A computational method to quantify the effects of slipped strand mispairing on bacterial tetranucleotide repeats. Nature Scientific Reports. 9:18087. https://doi.org/10.1038/s41598-019-53866-z.
Nilson, S.M., Workman, A.M., Sjeklocha, D., Brodersen, B., Grotelueschen, D.M., Petersen, J.L. 2020. Upregulation of the type I interferon pathway in feedlot cattle persistently infected with bovine viral diarrhea virus. Virus Research. 278:197862. https://doi.org/10.1016/j.virusres.2020.197862.
Wynn, E.L., Schuller, G., Loy, J.D., Workman, A.M., McDaneld, T.G., Clawson, M.L. 2020. Differentiation of Mannheimia haemolytica genotype 1 and 2 strains by visible phenotypic characteristics on solid media. Journal of Microbiological Methods. 171. Article 105877. https://doi.org/10.1016/j.mimet.2020.105877.
Eicher, S.D., Chitko-McKown, C.G., Bryan, K. 2020. Variation in the response of bovine alveolar lavage cells to diverse species of probiotic bacteria. BMC Research Notes. https://doi.org/10.1186/s13104-020-4921-9.
Harhay, D.M., Smith, T.P.L., Harhay, G.P., Loneragan, G.H., Webb, H.E., Bugarel, M., Haley, B.J., Kim, S.W., Van Kessel, J.S. 2018. Complete closed genome sequences of three Salmonella enterica subsp. enterica Serovar Dublin strains isolated from cattle at harvest. Microbiology Resource Announcements. 7:e01334-18. https://doi.org/10.1128/MRA.01334-18.
Whitman, K.J., Bono, J.L., Clawson, M.L., Loy, J.D., Bosilevac, J.M., Arthur, T.M., Ondrak, J.D. 2020. Genomic-based identification of environmental and clinical Listeria monocytogenes strains associated with an abortion outbreak in beef heifers. BMC Veterinary Research. 16:70. https://doi.org/10.1186/s12917-020-2276-z.
Hille, M., Dickey, A.M., Robbins, K., Clawson, M.L., Loy, J.D. 2020. Rapid differentiation of Moraxella bovoculi genotypes 1 and 2 using MALDI-TOF mass spectrometry profiles. Journal of Microbiological Methods. 173:105942. https://doi.org/10.1016/j.mimet.2020.105942.
Heaton, M.P., Bassett, A.S., Whitman, K.J., Krafsur, G.M., Lee, S., Carlson, J.M., Clark, H.J., Smith, H.R., Pelster, M.C., Basnayake, V., Grotelueschen, D.M., Vander Ley, B.L. 2019. Evaluation of EPAS1 variants for association with bovine congestive heart failure. F1000Research. 8:1189. https://doi.org/10.12688/f1000research.19951.1.
Rice, E.S., Koren, S., Rhie, A., Heaton, M.P., Kalbfleisch, T., Hardy, T., Hackett, P., Bickhart, D.M., Rosen, B.D., Vander Ley, B., Maurer, N.W., Green, R.E., Phillippy, A.M., Petersen, J.L., Smith, T.P. 2020. Continuous chromosome-scale haplotypes assembled from a single interspecies F1 hybrid of yak and cattle. GigaScience. 9(4):1-9. https://doi.org/10.1093/gigascience/giaa029.
Krafsur, G.M., Neary, J.M., Garry, F., Holt, T., Gould, D.H., Mason, G.L., Thomas, M.G., Enns, R.M., Tuder, R.M., Heaton, M.P., Brown, R.D., Stenmark, K.R. 2019. Cardiopulmonary remodeling in fattened beef cattle: A naturally occurring large animal model of obesity-associated pulmonary hypertension with left heart disease. Pulmonary Circulation. 9(1):1-13. https://doi.org/10.1177/2045894018796804.
Abdullah, N., Kelly, J.T., Graham, S.C., Birch, J., Goncalves-Carneiro, D., Mitchell, T., Thompson, R.N., Lythgoe, K.A., Logan, N., Hosie, M.J., Bavro, V.N., Willett, B.J., Heaton, M.P., Bailey, D. 2018. Structure-guided identification of a nonhuman morbillivirus with zoonotic potential. Journal of Virology. 92(23):e01248-18. https://doi.org/10.1128/jvi.01248-18.
Low, W., Tearle, R., Liu, C., Koren, S., Rhie, A., Bickhart, D.M., Rosen, B.D., Kronenberg, Z.N., Kingan, S.B., Tseng, E., Thibaud-Nissen, F., Martin, F., Billis, K., Ghurye, J., Hastie, A.R., Lee, J., Pang, A., Heaton, M.P., Phillippy, A.M., Hiendleder, S., Smith, T.P., Williams, J.L. 2020. Haplotype-resolved genomes provide insights into structural variation and gene content in Angus and Brahman cattle. Nature Communications. 11:2071. https://doi.org/10.1038/s41467-020-15848-y.
Wynn, E.L., Purfeerst, E., Christensen, A.C. 2020. Mitochondrial DNA repair in an Arabidopsis thaliana uracil N-glycosylase mutant. Plants. 9(2):261. https://doi.org/10.3390/plants9020261.
Clawson, M.L., Schuller, G., Dickey, A.M., Bono, J.L., Murray, R.W., Sweeney, M.T., Apley, M.D., DeDonder, K.D., Capik, S.F., Larson, R.L., Lubbers, B.V., White, B.J., Blom, J., Chitko-McKown, C.G., Brichta-Harhay, D.M., Smith, T.P.L. 2020. Differences between predicted outer membrane proteins of genotype 1 and 2 Mannheimia haemolytica. BMC Microbiology. 20:250. https://doi.org/10.1186/s12866-020-01932-2.
Chaudhari, J., Liew, C., Workman, A.M., Riethoven, J.M., Steffen, D., Sillman, S., Vu, H.L.X. 2020. Host transcriptional response to persistent infection with a live-attenuated porcine reproductive and respiratory syndrome virus strain. Viruses. 12(8):817. https://doi.org/10.3390/v12080817.