Research Project: Strategies to Control Respiratory Diseases of Cattle

Location: Animal Health Genomics

2022 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
Objective 1A: Whole genome sequence was generated for bovine viral diarrhea virus (BVDV)-susceptible Madin-Darby bovine kidney (MDBK) cells and BVDV-resistant cells resistant to infection with BVDV (CRIB) cells. Comparative genome analyses identified three large deletions in the BVDV-resistant CRIB cell line that were predicted to disrupt the function or expression of the three host genes. CRISPR/Cas9 was used to knockout these genes, individually or in combination, in the parental MDBK cell line to evaluate the role of these genes in virus entry and replication. Infection studies showed that these three genes were not necessary for viral infection in MDBK cells. Through international collaboration, ADAM metallopepidase domain 17 (ADAM17) was identified as the host factor missing in CRIB cells leading to BVDV resistance. Additional studies are ongoing using CRISPR/Cas9 to evaluate the impact of various CD46 variants on BVDV susceptibility. Objective 2A: The design, synthesis, and testing 22-mer CD18 signal peptides in BL3 cell lines for binding affinity with Lkt has begun. In the first round, the effect of an increased positive charge (arginine substitution) at positions 2, 4, 7, 15, and 21 is being tested with in vitro blocking assays. Depending on the results, 2nd round designs will be made accordingly. Objective 2B: A violet laser and associated filter kits have been purchased for the Attune flow cytometer to allow performance of “no wash, no lyse” multiplex assays. Scientists from Thermo Fisher Scientific are helping identify the best labels to use for each multiplex panel to obtain optimal results. Two pilot projects have been performed to show the validity of treating cattle with lipopolysaccharide (LPS) as a model of inflammation/BRDC. In the first project, a group of 4 steers were catheterized for serial collection of blood after intravenous treatment with either 0.25 ug/kg LPS in saline (treated, 2 animals) or saline alone (control, 2 animals). Blood and body temperature was collected at 0, 2, 4, 6, 8, 12, 24, and hours post treatment and serum haptoglobin, an acute stress protein was measured. Body temperature was elevated at 2 hours, peaked by 4 hours, and returned to baseline by 8 hours post treatment. At 2 hours the animals showed signs of malaise and had stopped eating but returned to normal behavior by 4 hours post-treatment. Control animals maintained consistent levels of haptoglobin over the 48 hours trial, however, serum haptoglobin rose in the LPS-treated animals and remained elevated at 48 hours. An additional project was developed using 30 non-catheterized steers that were pen-housed for the trial. Fifteen steers were treated intravenously with 0.25ug/kg LPS in saline and 15 controls were administered saline only. Blood and temperatures were collected at Day 0 before treatment and at Day 1. Animal signs for this trial followed what was observed for the group of four catheterized cattle described above. Blood and temperatures will be obtained weekly for four weeks (Days 7, 14, 21 and 28) and serum haptoglobin concentrations will be obtained. Objective 3A: The identity of 266 Pasteurella multocida strains isolated from North American cattle was 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 degrees C with 5%carbon dioxide. 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 developed and used to demultiplex, or correctly assign pooled sequence reads to each library, and also to assemble the genomes into contigs using the assembly software SKESA. The assembled libraries were analyzed in several phylogenetic software packages to determine strain sub structures. Additional P. multocida strains have been obtained for sequencing and should be processed in 2022.

Accomplishments
1. Identification of novel host factor essential for bovine viral diarrhea virus infection. Bovine viral diarrhea virus (BVDV) is an important pathogen in cattle that causes reproductive and respiratory diseases costing the industry over a billion dollars annually. BVDV entry into susceptible host cells is a multi-step process and the complete set of host factors required for virus entry is unknown. ADAM metallopepidase domain 17 (ADAM17) was recently reported to be an essential cellular attachment factor for the closely related classical swine fever virus. To determine whether ADAM17 might be involved in BVDV entry, collaborating scientists from Vienna, Austria measured ADAM17 mRNA and protein expression in two closely related cell lines: Madin-Darby bovine kidney cells (MDBK; fully susceptible to BVDV infection) and cells resistant to infection (CRIB-1) cells (a derivative of MDBK cells resistant to BVDV infection at the level of virus entry). ADAM17 was detectable in MDBK cells but not CRIB-1 cells. They further found that restoring ADAM17 expression in CRIB-1 cells rendered the once resistant cell line susceptible to BVDV infection. ARS scientists at Clay Center, Nebraska, sequenced the complete genomes of MDBK and CRIB-1 cells and analyzed the ADAM17 locus to genetically define the resistant phenotype. This study demonstrated that ADAM17 is a key cellular factor missing in CRIB-1 cells leading to BVDV resistance and establishes an essential role for ADAM17 in the entry of BVDV and related viruses into bovine cells. The identification of cellular factors involved in BVDV entry helps our understanding of BVDV and could facilitate the development of novel intervention strategies against BVDV.

2. Identification of bovine pinkeye intervention targets within Moraxella bovis. Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is the most common ocular disease that affects cattle. IBK is both an animal welfare and production loss concern, as it is painful, can cause partial or complete blindness, and affected cattle can have reduced weight gain. Costs to producers in the U.S. alone are likely in the hundreds of millions of dollars each year. Moraxella bovis is a causative agent of pinkeye. Through whole genome sequencing, ARS researchers at Clay Center, Nebraska, and colleagues at the University of Nebraska, Lincoln, have shown that M. bovis in cattle are comprised of two major strain variants, or genotypes, that have substantial differences in their major virulence factors and outer membrane proteins. Shared determinants between the two genotypes were also identified. New interventions against pinkeye can be developed that comprehensively work to protect cattle from pinkeye infection.

3. Genome-wide association of variants with heart failure in feedlot cattle. Bovine congestive heart failure (BCHF) in feedlot cattle has become increasingly common in the Western Great Plains of North America at moderate elevations (3000 to 4500 ft). This disease is an untreatable, complex condition involving high blood pressure in the lungs, which leads to subsequent heart failure and death. BCHF is fundamentally distinct from the “brisket disease” observed in the high elevations of the Rocky Mountains (>7000 ft), since BCHF occurs at much lower elevations where animals with brisket disease were previously sent to recover. Individual feedlot operations have reported losses from BCHF exceeding $250,000 annually, which are comparable to losses from bovine respiratory disease at similar locations. Cattle herds affected with BCHF are typically bred and managed with the aim of achieving high-quality carcasses. Consequently, reducing the impact of BCHF is a priority for the beef industry. In the present report, ARS researchers at Clay Center, Nebraska, evaluated animals with end-stage heart failure from 30 different ranch sources, together with their healthy penmates. DNA sequence variation in two major genes (ARRDC3 and NFIA) was discovered to be associated with BCHF, and thus, these genes seem to play a role in disease development. Feedlot animals in this study, that were homozygous for the DNA risk markers in both genes, were 28-fold more likely to develop heart failure than those without. A DNA-based test with two markers showed 29% of diseased cattle had homozygous risk alleles in both genes, compared to less than 2.5% in similar unaffected feedlot cattle. This type of testing may be useful for identifying feedlot animals at the highest risk for BCHF in the Western Great Plains of North America. In herds suffering from BCHF, knowledge of which cattle have the highest and lowest genetic risk for disease allows producers to make informed decisions for selective breeding and animal health management.

Review Publications
Zaruba, M., Chen, H., Pietsch, O.F., Szakmary-Braendle, K., Auer, A., Motz, M., Seitz, K., Dusterhoft, S., Workman, A.M., Rumenapf, T., Riedel, C. 2022. ADAM17 is an essential factor for the infection of bovine cells with pestiviruses. Viruses. 14(2). Article 381. https://doi.org/10.3390/v14020381.
Heaton, M.P., Harhay, G.P., Bassett, A.S., Clark, H.J., Carlson, J.M., Jobman, E.E., Sadd, H.R., Pelster, M.C., Workman, A.M., Kuehn, L.A., Kalbfleisch, T.S., Piscatelli, H., Carrie, M., Krafsur, G.M., Grotelueschen, D.M., Vander Ley, B.L. 2022. Association of ARRDC3 and NFIA variants with bovine congestive heart failure in feedlot cattle. F1000Research. 11. Article 385. https://doi.org/10.12688/f1000research.109488.1 .
Murphy, T.W., Chitko-McKown, C.G., Heaton, M.P., Freking, B.A. 2021. Effect of TMEM154 E35K variant (haplotypes 1 and 3) on the incidence of ovine lentivirus infection and ewe productivity during lifetime exposure. Journal of Animal Science. 99(11). Article skab304. https://doi.org/10.1093/jas/skab304.
Leonard, A.S., Crysnanto, D., Fang, Z., Heaton, M.P., Vander Ley, B.L., Herrera, C., Bollwein, H., Bickhart, D.M., Kuhn, K.L., Smith, T.P.L., Rosen, B.D., Pausch, H. 2022. Structural variant-based pangenome construction has low sensitivity to variability of haplotype-resolved bovine assemblies. Nature Communications. 13. Article 3012. https://doi.org/10.1038/s41467-022-30680-2.
Hille, M.M., Spangler, M.L., Clawson, M.L., Heath, K.D., Vu, H.L.X., Rogers, R.E.S., Loy, J.D. 2022. A five year randomized controlled trial to assess the efficacy and antibody responses of a commercial and autogenous vaccine for the prevention of infectious bovine keratoconjunctivitis. Vaccines. 10(6). Article 916. https://doi.org/10.3390/vaccines10060916.
Workman, A.M., Heaton, M.P., Webster, D.A., Harhay, G.P., Kalbfleisch, T.S., Smith, T.P.L., Falkenberg, S.M., Carlson, D.F., Sonstegard, T.S. 2021. Evaluating large spontaneous deletions in a bovine cell line selected for bovine viral diarrhea virus resistance. Viruses. 13(11). Article 2147. https://doi.org/10.3390/v13112147.
Adetunji, S.A., Smolensky, D., Mitzel, D.N., Chitko-Mckown, C.G., Cernicchiaro, N., Noronha, L.E., Owens, J.L. 2021. In vitro infection dynamics of Japanese encephalitis virus in established porcine cell lines. Pathogens. 10(11). Article 1468. https://doi.org/10.3390/pathogens10111468.
Lindholm-Perry, A.K., Kuehn, L.A., Wells, J., Rempel, L.A., Chitko-McKown, C.G., Keel, B.N., Oliver, W.T. 2021. Hematology parameters as potential indicators of feed efficiency in pigs. Translational Animal Science. 5(4). Article txab219. https://doi.org/10.1093/tas/txab219.
Davenport, K.M., Bickhart, D.M., Worley, K.C., Murali, S.C., Salavati, M., Clark, E.L., Cockett, N., Heaton, M.P., Smith, T.P., Murdoch, B.M., Rosen, B.D. 2022. An improved ovine reference genome assembly to facilitate in depth functional annotation of the sheep genome. Gigascience. 11. Article giab096. https://doi.org/10.1093/gigascience/giab096.
Low, W.Y., Rosen, B.D., Ren, Y., Bickhart, D.M., To, T., Martin, F.J., Billis, K., Sonstegard, T.S., Sullivan, S.T., Hiendleder, S., Williams, J.L., Heaton, M.P., Smith, T.P. 2022. Gaur genome reveals expansion of sperm odorant receptors in domesticated cattle. BMC Genomics. 23. Article 344. https://doi.org/10.1186/s12864-022-08561-1.