1a. Objectives (from AD-416):
1. Discover genetic and biological determinants associated with disease resistance to respiratory diseases of ruminants. 2. Discover effective intervention strategies to prevent and control respiratory diseases of ruminants.
1b. Approach (from AD-416):
Infectious respiratory diseases of ruminants are a serious health and economic problem for U.S. agriculture. In cattle alone, bovine respiratory disease complex (BRDC) costs the feedlot industry approximately 1 billion dollars annually. Accordingly, this research focuses primarily on BRDC with an additional component targeting ovine respiratory disease. These diseases result from host, pathogen and environmental interactions that are incompletely understood. We propose studying these interactions to identify intervention points for producing healthier livestock. For example, failure of passive transfer (FPT) has genetic and environmental components that predispose calves to BRDC. A genome-wide association study will be used to test for FPT genetic risk factors. Also, the predominant pneumonia associated with BRDC is caused by Mannheimia haemolytica. Transcriptome profiling will be used to identify host cellular responses to this pathogen. BRDC research requires development of species-specific tools, thus monocyte cell lines will be developed. Also needed are early indicators of BRDC outbreaks. Changes in the viral metagenomes (VMs) of cattle will be evaluated as potential early indicators of infection. Finally, genetic risk factors for ovine progressive pneumonia (OPP) will be tested as a model for preventing respiratory diseases of ruminants. A reduction of the prevalence, severity and/or treatment of respiratory diseases would enhance producer efficiency and promote the welfare of livestock. This research fills critical gaps in our knowledge of ruminant respiratory disease and could ultimately benefit regulatory agencies, animal health companies, veterinarians, and livestock producers that need information and new approaches for controlling respiratory disease.
3. Progress Report:
To determine the relative susceptibility to ovine progressive pneumonia virus (OPPV) infection of lambs with different transmembrane protein 154 (TMEM154) genotypes, a breeding scheme was developed that produced approximately 250 lambs with TMEM154 genotypes: "1,1"; "1,3"; and "3,3". These lambs received a high level of natural exposure to OPPV from their dams. After weaning, the lambs were isolated from infected adults and tested for OPPV antibodies every 5 weeks over a period of 7 months. As expected, lambs with the TMEM154 "1,1" genotypes were substantially less susceptible to infection than those with "1,3" or "3,3" genotypes. Yearling ewes from this group of lambs were kept in the flock and tested for OPPV as they moved through the production cycle. Vendors, physical resources and livestock operators have been identified to support viral metagenome (VM) activities. We have worked with tangential flow filtration (TFF) system engineers to design and build a TFF system that can filter large volumes of livestock water tank water at pressures that will not damage its viral components. Additionally, protocols, vendors, and veterinary collaborators have been identified to support viral nucleic acid isolation from cattle nasopharyngeal swabs and water tank water. In order to develop stable lines of bovine monocyte-derived macrophage cell lines, peripheral blood mononuclear cells (PBMC) were obtained from animals of varying genetic backgrounds housed at the USMARC feedlot. Monocytes were isolated from PBMC cultures through adherence and culture with growth factors. Several lines have been successfully frozen and thawed, and assay protocols and bovine monocyte/macrophage specific monoclonal antibodies are in place to begin cell line characterization. In the spring of 2012, over 100 neonatal calf serum samples were collected and measured for Immunoglobulin G (IgG) concentration for the failure of passive transfer (FPT) project. Some of these samples, along with others collected over the past ten years, will be used in a genome-wide association study. Additionally, in the spring of 2012, a pilot study was conducted to measure IgG variation in colostrum, passive transfer efficiency, and IgG half-life in calves. These new phenotypes will be further investigated in future studies to complement the FPT project, and fully unravel the genetics responsible for variable passive immunity in neonatal calves. A material transfer agreement was signed enabling us to obtain wild-type and leukotoxin (LKT)-knock out strains of M. haemolytica from Oklahoma State University. These strains will be used to produce LKT for use in in vitro experiments to identify cattle whose blood cells are resistant or susceptible to lysis by this bacterial factor. Equipment and reagents are currently available in our laboratory to culture these bacterial strains.
1. Discovery of a major ovine gene (TMEM154) affecting susceptibility to lentivirus infection. Ovine lentiviruses target the immune system and cause persistent retroviral infections affecting millions of sheep worldwide. Some animals are resistant to infection despite repeated exposure; however, the mechanism of resistance is unknown. ARS scientists at Clay Center, NE designed a case-control genome-wide association study to test whether sheep might have genetic variation that protects against lentivirus infection. The study identified a major susceptibility gene (TMEM154) that is significantly associated with OPP infection. Sheep with some forms of this gene were nearly three times more likely to become infected than those with other forms. Also discovered two mutant forms predicted to abolish the protein’s function and possibly confer a higher level of resistance. Although the biological function of TMEM154 is unknown, the results indicated this gene plays an important role in lentivirus infection in sheep. Producing sheep with the least susceptible form of TMEM154 may help eradicate the ovine disease caused by lentivirus.
2. Risk of lentivirus infection in sheep is affected by genetic variation in both the host and pathogen. Sheep are not uniformly resistant or susceptible to infection by lentiviruses, due in part to genetic variation within the ovine transmembrane gene (TMEM154). However, lentiviruses are genetically diverse, and some viral subtypes may be more adept at infecting sheep with specific TMEM154 variants than others. Accordingly, scientists at Clay Center, NE tested lentivirus subtypes originating from naturally infected sheep for an association with TMEM154 gene variants. Our results indicate that the genetic makeup of both the host (sheep) and lentivirus affect the relative risk of infection. Characterizing viral subtypes in infected sheep, along with TMEM154 genotypes, could assist in the development of management strategies that reduce the prevalence of ovine lentivirus.
Heaton, M.P., Clawson, M.L., Chitko-Mckown, C.G., Leymaster, K.A., Smith, T.P.L., Harhay, G.P., White, S.N., Herrmann-Hoesing, L.M., Mousel, M.R., Lewis, G.S., Kalbfleisch, T.S., Keen, J.E., Laegreid, W.W. 2012. Reduced lentivirus susceptibility in sheep with TMEM154 mutations. PLoS Genetics. 8(1): e1002467. DOI: 10.1371/journal.pgen.1002467.