Location: Animal Health Genomics
2024 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: In fiscal year (FY) 2023, Madin-Darby bovine kidney (MDBK) cells were edited using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to identify a minimal CD46 edit that results in reduced bovine viral diarrhea virus (BVDV) susceptibility in vitro. CD46-edits were confirmed with 10x whole genome sequence and evaluated for off-target modifications. The ARS Life Science Patent Committee reviewed the novel edits and determined that a CRISPR-licensed partner is needed to reduce the time from invention to practice and provide experience for seeking U.S. Food and Drug Administration (FDA) approval of animals with intentional genome alterations (IGAs). In FY2024, in vitro susceptibility studies were completed to characterize two novel CD46 edits. In addition, an industry partner was identified and a research agreement was drafted to develop beef cattle with one of the novel CD46 edits.
Sub-objective 2B, progress fully met. We have identified several, unique, single amino acid CD18 signal peptide substitutions that eliminate the toxic effects Mannheimia haemolytica leukotoxin. In addition, we have identified novel residues in peptide analogs that completely block toxin binding. We have submitted an ARS invention disclosure that was selected for ARS patent development. We are collaborating with ARS researchers in Columbia, Missouri, to create BL3-edited cell lines for evaluation of toxin resistance.
Sub-objective 2B progress fully met the third-year goal of the project, which was to complete PacBio sequencing and assembly of P. multocida strains representing the breadth of diversity identified from preliminary Illumina sequencing. Preliminary Illumina sequencing and analyses of 596 P. multocida strains in year two of this project identified three major clades of the bacteria that consist of distinct capsular serotypes. Thirteen strains that represented the diversity of those clades were selected for PacBio sequencing and genome assembly. The strains were cultured in brain heart infusion (BHI) broth at 37 degrees C with 5% CO2, DNAs were extracted, and PacBio genomic DNA libraries were constructed and sequenced on a PacBio instrument to 30X or higher genome coverage. Outer membrane and secreted proteins encoded by P. multocida genomes have been identified, and some are undergoing serological and molecular testing for vaccine development.
Sub-objective 3A progress of the first feedlot trial was postponed due to drought. An experimental outline and animal use protocol has been submitted for approval prior to fall 2024 weaning so that the delayed trial can be initiated.
Accomplishments
1. Detection of infectious bovine keratoconjunctivitis using artificial intelligence-based facial recognition. Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is the most common ocular disease of cattle worldwide, and costs the U.S. cattle industry hundreds of millions of dollars each year. ARS researchers at Clay Center, Nebraska worked with the company MyANiML in the application of MyANiML’s proprietary artificial intelligence (AI) that detects emerging IBK cases using muzzle imagery. The AI detected emerging IBK cases before clinical signs were visible with high sensitivity and specificity. This technology can potentially be used to keep cattle healthy and reduce their need for antibiotic treatments by detecting early cases of IBK and facilitating interventions to prevent large IBK outbreaks.
2. The lung microbiome is altered by feeding probiotics to dairy calves. Probiotics have been investigated for their many health benefits and impact on the microbiota of the gut. However, little research has been performed to determine the effects of oral probiotics on the microbiome of the bovine respiratory tract. Researchers at ARS in Clay Center, Nebraska, and West Lafayette, Indiana, in collaboration with scientists from Purdue University and Chr. Hansen, Inc., compared the lung microbiomes of dairy calves fed either a traditional diet of milk replacer or milk replacer containing probiotics. Lung lavages were performed on five random calves from each treatment and 16S ribosomal RNA gene hypervariable regions 1-3 were amplified by polymerase chain reaction (PCR) and sequenced using next-generation sequencing. The evaluation of these samples revealed that the bacterial genera identified in the lung lavage samples of probiotic-fed calves as compared to control calves were significantly different. Analysis of the respiratory microbiome in lung lavage samples after probiotics provides insight into the distribution of bacterial populations in response to oral probiotics and demonstrates that oral probiotics affect more that the gut microbiome.
Review Publications
Liu, X., Lin, L., Sinding, M.S., Bertola, L.D., Hanghoj, K., Quinn, L., Garcia-Erill, G., Rasmussen, M.S., Schubert, M., Pecnerova, P., Balboa, R.F., Li, Z., Heaton, M.P., Smith, T.P.L., Pinto, R., Wang, X., Kuja, J., Bruniche-Olsen, A., Meisner, J., Santander, C.G., Ogutu, J.O., Masembe, C., da Fonseca, R.R., Muwanika, V., Siegismund, H.R., Albrechtsen, A., Moltke, I., Heller, R. 2024. Introgression and disruption of migration routes have shaped the genetic integrity of wildebeest populations. Nature Communications. 15. Article 2921. https://doi.org/10.1038/s41467-024-47015-y.
Wynn, E.L., Browne, A.S., Clawson, M.L. 2024. Diversity and antigenic potentials of Mycoplasmopsis bovis secreted and outer membrane proteins within a core genome of strains isolated from North American bison and cattle. Genome. https://doi.org/10.1139/gen-2023-0084.
Eicher, S.D., Kritshevsky, J.E., Bryan, K.A., Chitko-Mckown, C.G. 2023. The effect of probiotics in a milk replacer on leukocyte differential counts, phenotype, and function in neonatal dairy calves. Microorganisms. 11(11). Article 2620. https://doi.org/10.3390/microorganisms11112620.
Balboa, R.F., Bertola, L., Bruniche-Olsen, A., Rasmussen, M., Liu, X., Besnard, G., Salmona, J., Santander, C.G., He, S., Zinner, D., Heaton, M.P., Smith, T.P., Moltke, I., Albrechtsen, A., Heller, R. et al. 2024. African bush pigs exhibit porous species boundaries and appeared in Madagascar concurrently with human arrival. Nature Communications. 15. Article 172. https://doi.org/10.1038/s41467-023-44105-1.
Gupta, S., Kuehn, L.A., Clawson, M.L. 2023. Early detection of infectious bovine keratoconjunctivitis with artificial intelligence. Veterinary Research. https://doi.org/10.1186/s13567-023-01255-w.
Workman, A.M., Harhay, G.P., Groves, J.T., Vander Ley, B.L. 2024. Two bovine hepacivirus genome sequences from U.S. cattle. Journal of Veterinary Diagnostic Investigation. 36(2):274-277. https://doi.org/10.1177/10406387231225656.
Weinroth, M.D., Clawson, M.L., Harhay, G.P., Eppinger, M., Harhay, D.M., Smith, T.P.L., Bono, J.L. 2023. Escherichia coli O157:H7 tir 255 T > A allele strains differ in chromosomal and plasmid composition. Frontiers in Microbiology. 14. Article 1303387. https://doi.org/10.3389/fmicb.2023.1303387.
Ma, H., Alt, D.P., Falkenberg, S.M., Briggs, R.E., Tatum, F.M., Clawson, M.L., Casas, E., Dassanayake, R.P. 2024. Transcriptomic profiles of Mannheimia haemolytica planktonic and biofilm associated cells. PLOS ONE. 19(2). Article e0297692. https://doi.org/10.1371/journal.pone.0297692.
McDaneld, T.G., Eicher, S.D., Dickey, A.M., Kritchevsky, J.E., Bryan, K.A., Chitko-McKown, C.G. 2024. Probiotics in milk replacer affect the microbiome of the lung in neonatal dairy calves. Frontiers in Microbiology. 14. Article 1298570. https://doi.org/10.3389/fmicb.2023.1298570.
Rosenblatt, E., Gieder, K., Donovan, T., Murdoch, J., Smith, T.P.L., Heaton, M.P., Kalbfleisch, T.S., Murdoch, B.M., Bhattarai, S., Pacht, E., Verbist, E., Basnayake, V., McKay, S. 2023. Genetic diversity and connectivity of moose (Alces americanus americanus) in eastern North America. Conservation Genetics. 24:235-248. https://doi.org/10.1007/s10592-022-01496-w.