Research Project: Strategies to Control Respiratory Diseases of Cattle

Location: Animal Health Genomics

2023 Annual Report

Objectives
Objective 1. Elucidate genotypic and phenotypic factors affecting host susceptibility to viral and bacterial pathogens associated with bovine respiratory disease complex (BRDC) in order to rationally design tools and approaches for increasing host resilience. Sub-objective 1A: Genomic comparisons of BVDV-susceptible and -resistant bovine cell lines to identify host factors required for virus entry. Objective 2. Elucidate virulence mechanisms of BRDC pathogens to rationally design effective strategies that reduce antibiotic use in the prevention and treatment of BRDC. Sub-objective 2A: Reducing bovine CD18 binding to bacterial leukotoxin. Sub-objective 2B: Identify outer membrane proteins of BRD bacterial pathogens suitable for vaccine testing and development. Objective 3. Develop alternatives to antibiotics for the prevention and treatment of BRDC. Sub-objective 3A: Identify changes in immune cell populations and the respiratory microbiome associated with administering probiotics to feeder cattle.

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. 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 whole genome sequencing approach, combined with in vitro cell line gene-editing, will be used to identify bovine genes affecting susceptibility to bovine viral diarrhea virus infections. In addition, novel bovine CD18 sequences will be tested in vitro for reduced binding to bacterial leukotoxin, a major pathological cause of BRDC pneumonia. The impact of toxin-resistant CD18 sequences on cellular health will be tested with gene-editing approaches focused on cell lines. On the bacterial pathogen side, genomics will be used to identify and compare outer membrane proteins of Histophilus somni, Mannheimia haemolytica and Pasteurella multocida that could be developed into vaccines. Lastly, our approach will measure changes in the immune cell population and the respiratory microbiome associated with administering probiotics to feeder cattle. The knowledge gained from this research will be useful in 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
Sub-objective 1A: Clustered regularly interspaced short palindromic repeats (CRISPR) gene-editing technology was used to make two novel CD46 gene edits in Madin-Darby bovine kidney (MDBK) cells. Targeted sequencing was used to identify clones homozygous for the intended CD46 edits. These clones were then screened to identify those that retained normal cellular morphology, growth rates, and CD46 protein expression levels compared to the parental MDBK cells. Preliminary virus susceptibility testing was then completed to determine the smallest possible edit that still confers resistance to bovine viral diarrhea virus (BVDV). Sub-objective 2A: In the second year of the Project Plan cycle our aim was to design, synthesize, and begin testing CD18 SP peptides in bovine lymphocyte cell lines (BL3) for binding affinity with LktA. We have accomplished that milestone and have identified a promising novel CD18 signal peptide sequence that does not bind Lkt. This milestone relates directly to Objective 2: Elucidating virulence mechanisms of BRDC pathogens to rationally design effective strategies that reduce antibiotic use in the prevention and treatment of bovine respiratory disease complex (BRDC). Sub-objective 2B: The identities of 321 H. somni strains isolated from North America cattle were determined in a veterinary diagnostic lab using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The strains were cultured in brain heart infusion (BHI) broth at 37 deg C with 5% CO2. DNAs were extracted and genomic DNA libraries were constructed using Twist RipTide high throughput rapid library prep kits. The libraries were pooled and sequenced on an Illumina platform. A custom in-house pipeline was used to demultiplex, or correctly assign pooled sequence reads to each library, and to assemble the genomes into contigs using the assembly software strategic k-mer extension for scrupulous assemblies (SKESA). The assembled libraries were analyzed in several phylogenetic software packages to determine strain sub structures. A subset of diverse strains was identified from the Illumina dataset for more comprehensive PacBio sequencing that will be used with the Illumina dataset generated this year to identify outer membrane proteins of H. somni, a BRDC pathogen, that are suitable for vaccine testing and development. Sub-objective 3A: In year one we performed a pilot project on 30 non-catheterized steers that were pen-housed. Fifteen steers received lipopolysaccharides (LPS) in saline intravenously, and fifteen control steers received saline only. We obtained body temperatures, weight, and peripheral blood at day 0 before treatment and at days 1, 7, 14, and 28 after treatment. All animals included in the study had normal body temperatures when measured. Animals in the LPS treatment group showed decreased weight gain the first week after treatment, but weight gain was equal to or better than the control group by day 28. Inflammation as measured by haptoglobin in plasma returned to normal levels by day seven indicating that the LPS-treated animals had physiologically recovered from LPS-treatment. This data showed that LPS-treatment as a physiologic stressor and model of bacterial infection can be used in pen-housed cattle without the need for veterinary intervention. Based on this information, the feed trial with or without probiotics in diets of control- of LPS-treated feedlot cattle will proceed this fall depending on the environment (drought) and available cattle numbers.

Accomplishments
1. First gene-edited calf with resistance to common cattle virus. Bovine viral diarrhea virus (BVDV) is one of the most widespread and economically important viral infections in cattle, with annual losses approaching $1 billion in the U.S. alone. The main host receptor mediating BVDV infection is CD46. ARS researchers at Clay Center, Nebraska, published a proof-of-concept study showing that substituting six amino acids in CD46 caused a dramatic reduction in BVDV susceptibility in a gene-edited calf without causing any obvious adverse effects in the first two years of life. This provides the first example of gene editing in cattle to reduce the impact of a major viral disease. This approach could significantly improve animal welfare, increase the long-term sustainability of cattle production, and provide an opportunity to reduce antibiotic use in agriculture, given that BVDV infection puts calves at risk for secondary bacterial diseases.

2. Identification of protein targets for a Mycoplasma bovis vaccine. M. bovis causes respiratory disease in cattle and bison. In cattle, M. bovis interacts with other pathogens as part of a polymicrobial complex involved with disease. In bison, M. bovis is a primary pathogen that causes pneumonia with high fatality rates. Infections in bison are so problematic to treat successfully that treatments are not recommended. Thus, there is a need for vaccines to control M. bovis in both cattle and bison. Researchers at ARS in Clay Center, Nebraska, analyzed the genomes of 240 M. bovis strains isolated from either cattle or bison and analyzed the vaccine potential of their proteins. They focused on proteins that either reside on the outer membrane of M. bovis or are secreted, as both types can work effectively in a vaccine. Regions of genetic diversity and immunological potentials were mapped for each protein of interest. This work provides a novel reference for the research community to use in the design of vaccines that could protect both cattle and bison from M. bovis and reduce devasting losses of bison to this pathogen.

Review Publications
Loy, D., Clawson, M.L. 2023. From genomics to MALDI-TOF MS: Diagnostic identification and typing of bacteria in veterinary clinical laboratories. In: Shah, H.N., Gharbia, S.E., Shah, A.J., Tranfield, E.Y., Thompson, K.C., editors. Microbiological Identification using MALDI-TOF and Tandem Mass Spectrometry - Industrial and Environmental Applications. April 2023 edition. New York, NY: Wiley Press. p. 283-302.
Wynn, E.L., Hille, M.M., Loy, J.D., Schuller, G., Kuhn, K.L., Dickey, A.M., Bono, J.L., Clawson, M.L. 2022. Whole genome sequencing of Moraxella bovis strains from North America reveals two genotypes with different genetic determinants. BMC Microbiology. 22(1). Article 258. https://doi.org/10.1186/s12866-022-02670-3.
Toomer, G., Workman, A.M., Harrison, K.S., Stayton, E., Hoyt, P.R., Jones, C. 2022. Stress triggers expression of bovine herpesvirus 1 infected cell protein 4 (bICP4) RNA during early stages of reactivation from latency in pharyngeal tonsil. Journal of Virology. 96(23). Article e0101022. https://doi.org/10.1128/jvi.01010-22.
Quinn, L., Garcia-Erill, G., Santander, C., Bruniche-Olsen, A., Liu, X., Sinding, M.S., Heaton, M.P., Smith, T.P.L., Pecnerova, P., Bertola, L.D., Hanghoj, K., Rasmussen, M.S., De Jager, D., Siegismund, H.R., Albrechtsen, A., Heller, R., Moltke, I. 2023. Colonialism in Southern Africa leaves a lasting legacy of reduced genetic diversity in Cape buffalo. Molecular Ecology. Article 16851. https://doi.org/10.1111/mec.16851.
Chaudhari, J., Leme, R.A., Durazo-Martinez, K., Sillman, S., Workman, A.M., Vu, H.L.X. 2022. A single amino acid substitution in porcine reproductive and respiratory syndrome virus glycoprotein 2 significantly impairs its infectivity in macrophages. Viruses. 14(12). Article 2822. https://doi.org/10.3390/v14122822.
Loy, J.D., Clawson, M.L., Adkins, P.R.F., Middleton, J.R. 2023. Current and emerging diagnostic approaches to bacterial diseases of ruminants. Veterinary Clinics of North America, Food Animal Practice. 39:93-114. https://doi.org/10.1016/j.cvfa.2022.10.006.
Olson, H.G., Loy, J.D., Clawson, M.L., Wynn, E.L., Hille, M.M. 2022. Genotype classification of Moraxella bovis using MALDI-TOF MS profiles. Frontiers in Microbiology. 13. Article 1057621. https://doi.org/10.3389/fmicb.2022.1057621.
Poonsuk, K., Kordik, C., Hille, M., Cheng, T., Crosby, W.B., Woolums, A., Clawson, M.L., Chitko-McKown, C., Brodersen, B., Loy, J.D. 2023. Detection of Mannheimia haemolytica-specific IgG, IgM and IgA in sera and their relationship to respiratory disease in cattle. Animals. 13(9). Article 1531. https://doi.org/10.3390/ani13091531.
Workman, A.M., Heaton, M.P., Vander Ley, B., Webster, D., Sherry, L., Bostrom, J.R., Larson, S., Kalbfleisch, T., Harhay, G.P., Jobman, E., Carlson, D., Sonstegard, T.S. 2023. First gene-edited calf with reduced susceptibility to a major viral pathogen. Proceedings of the National Academy of Sciences-Nexus. 2(5). Article pgad125. https://doi.org/10.1093/pnasnexus/pgad125.
Stegemiller, M.R., Redden, R.R., Notter, D.R., Taylor, T., Taylor, J.B., Cockett, N.E., Heaton, M.P., Kalbfleisch, T.S., Murdoch, B.M. 2023. Using whole genome sequence to compare variant callers and breed differences of US sheep. Frontiers in Genetics. 13. Article 1060882. https://doi.org/10.3389/fgene.2022.1060882.
Workman, A.M., McDaneld, T.G., Harhay, G.P., Das, S., Loy, J.D., Hause, B.M. 2022. Recent emergence of bovine coronavirus variants with mutations in the hemagglutinin-esterase receptor binding domain in U.S. cattle. Viruses. 14(10). Article 2125. https://doi.org/10.3390/v14102125.
Freking, B.A., Murphy, T.W., Chitko-McKown, C.G., Workman, A.M., Heaton, M.P. 2022. Impact of four ovine TMEM154 haplotypes on ewes during multiyear lentivirus exposure. International Journal of Molecular Sciences. 23(23). Article 14966. https://doi.org/10.3390/ijms232314966.