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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

2022 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
This is the final report for Project 3040-32000-034-00D which expired in 2021 and was replaced with Project 3040-32000-036-000D, "Strategies to Control Respiratory Diseases of Cattle." Additional information can be found in the Annual Report for Project 3040-32000-036-000D. Substantial progress was made over the life of this project to elucidate host responses associated with bovine respiratory disease complex (BRDC) and protective immunity (Objective 1). We developed 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, to develop intervention strategies to reduce antibiotic use and BRDC severity based on genetic typing of bacteria and cattle (Objective 2). In support of Sub-objective 1.A, we began a systematic program of research to find genetic determinants and risk factors associated with bovine viral diarrhea (BVD) virus infection. The first effort was to assemble a set of "case" samples from feedlot cattle that were persistently infected with BVD virus (BVDPI calves). These calves were confirmed to be persistently infected with the non-cytopathic BVDV genotype 1b. Once the case group was assembled, the whole genome sequence (WGS) for each of 96 BVDPI cases was collected with a minimum of 12x genome coverage. A first analysis of genome-wide association revealed approximately six sites on five chromosomes were significantly associated with persistent BVD virus infection in calves. However, there was a significant imbalance of female animals between the feedlot cases and healthy controls that needed to be corrected before the analysis could proceed. A suitable source of WGS from healthy female feedlot calves was identified from an unrelated project and are being processed for WGS comparison. WGS from two immortalized cell lines were compared in an effort to identify genes that may be associated with BVD virus resistance in vitro. These were the Madin-Darby Bovine Kidney (MDBK) cell line and its mutant daughter cell line named "cells resistant to infection with BVDV-1" (CRIB). Genomic comparison between the two cell lines identified three major deletions spanning three genes on three chromosomes. Gene-editing was used to knock-out all three of these genes in the parent MDBK cell line, however, it remained fully susceptible to the BVD virus infection. In the process of evaluating the role of candidate genes in BVD viral infection, an ARS scientist had a breakthrough discovery with CD46 gene-editing in MDBK cells. This discovery was significant and shifted the focus in the last year of this research project to following this promising line of research. Among the findings was that a slight modification in CD46 amino acid sequence by gene-editing resulted in a dramatic reduction in susceptibility of the MDBK cell line to BVD viruses. At the end of the project, the effects of CD46 editing were being tested in vivo to see if ARS researchers could replicate the phenotypes observed in vitro. In support of Sub-objective 1.B, we developed cell lines to enable the study of transmembrane protein (TMEM) 154 variants in ovine progressive pneumonia (OPP) virus infection. All ewes in the OPP-free flock were genotyped for TMEM, as were rams, to be used each fall for breeding. Rams were tested for OPP by Enzyme-Linked Immunosorbant assay (ELISA) once a month for the three months prior to entry into the flock to ensure that the flock remained OPP-free. Lambs were genotyped for TMEM each spring to select animals for cell line development and to select ewe lambs as replacements for older ewes leaving the flock. A TMEM154 “1,1” ram was selected for the first lamb crop and cell lines were developed from the synovial membranes and choroid plexus of a TMEM154 lamb. Because the pandemic limited time in the laboratory and cell cultures needed to be observed daily once tissues were harvested from the lambs, the development of cell lines with TMEM154 “3,3” and “4,4” is ongoing. In support of Sub-objective 2.A, genomic sequencing and bioinformatic analyses of a diverse set of M. haemolytica genotype 1 and 2 strains led to the identification of 1880 core genes shared between the two genotypes, as well as 112 select genes only found in genotype 1 strains, and 179 found only in genotype 2 strains. Importantly, seven of the 179 genes found only in genotype 2 strains encode predicted outer membrane proteins, including three adhesins. These seven genotype 2-specific outer membrane proteins are ideal candidates for the development of immunological interventions to prevent infection of cattle by genotype 2 M. haemolytica. We further identified outer membrane proteins conserved between Mannheimia haemolytica (both genotypes), and the bovine respiratory disease pathogens Pasteurella multocida and Histophilus somni, as an extension of the approach applied to M. haemolytica. Additionally, two tests were developed to identify M. haemolytica genotypes 1 and 2. The first is run on the matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) platform for use in veterinary diagnostic laboratories, and the other is based on colony morphologies and colors for any lab to perform. The MALDI-TOF MS test is used in veterinary diagnostic labs throughout the U.S. and in Canada, in support of the identification and subsequent treatment of cattle infected with genotype 2 M. haemolytica. In support of Sub-objective 2.B, the complete circularized bacterial genomes of over 30 H. somni, P. multocida and other species were sequenced, analyzed and deposited in GenBank ( A BLAST2GO parser was written for the BioCyc /Pathway Tools inferential systems biology platform. The necessity of using the EDGAR parser diminished as BioCyc /Pathway Tools performed many of the same functions as well as providing additional capabilities in the systems biology domain. The Pathway Tools system went "live" in 2019 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 subspec. enterica, and E. coli 0157:H7. 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 the 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. 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). Work was performed with Iowa State and SRI, International to improve the implementation of Pathway Tools, and the Pathway Tools system itself. Work was done with our liquid chromatography-mass spectrometry (LC-MS) vendor, Waters Corporation, and their software arm, Nonlinear Corporation, and identified a company that will train staff to use the current liquid chromatography-mass spectrometry (LC-MS) system to generate metabolomic and fluxomic data on microbial cultures. These data were generated in pursuit of expanding our 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. The required training, performance metrics and deliverables, and potential vendors were documented in a specification. Liquid Chromatograph-Mass Spectrometry (LC-MS) analyses of BRDC pathogens was used to acquire metabolic phenotypes. The assistance of Iroatech (Sea Girt, New Jersey) was secured to advise about metabolomic laboratory procedures, sample handling, LC-MS performance standards, and data analysis. Work was performed with Clay Center, Nebraska Corelab to tune the microbial culture conditions, sample handling, LC-MS run parameters and the creation of small molecule standard reference spectral libraries. In work supporting Sub-objective 2.C, a longitudinal study demonstrated that bovine coronavirus (BCV) shedding was associated with pre- and post-weaning BRDC and reduced weight gain in nursing calves. Further, anti-BCV antibody titers were inversely related to BRDC incidence in the feedlot. Together, this work suggested that if available, a BCV vaccine could reduce BRDC incidence and improve animal health and production. Also, under Sub-objective 2.C, 78 BCV genomes were sequenced and assembled directly from clinical samples collected between 2013 and 2022 from 27 enteric disease cases and 51 respiratory disease cases from cattle in 12 states, primarily in the Midwestern U.S. This work identified 11 isolates collected between 2020 and 2022 from four states (Nebraska, Colorado, California, and Wisconsin) containing a 12-nucleotide insertion in the receptor-binding domain (RBD) of the hemagglutinin-esterase (HE) gene, identical to one recently reported in China. In addition, a single genome from Nebraska collected in 2020 contained a novel 12-nucleotide deletion in the HE gene RBD. Changes in the HE gene of BCV and related coronaviruses have been associated with changes in cellular tropism and host range. Therefore, further investigation of BCV evolution and in particular, these novel HE gene variants is underway.