Location: Animal Disease Research2013 Annual Report
1a. Objectives (from AD-416):
Objective 1: Identification of host genetic markers associated with reduced transmission and replication of ovine progressive pneumonia virus. Subobjective 1.1A: Identification of genetic markers for reduced odds of OPPV infection in domestic sheep. Subobjective 1.1B: Validation of genetic markers for reduced odds of OPPV infection in domestic sheep. Subobjective 1.2A: Identification of genetic markers for reduced OPPV proviral replication in domestic sheep. Subobjective 1.2B: Validation of genetic markers for reduced OPPV proviral replication in domestic sheep. Objective 2: Identification of host genetic factors in domestic and bighorn sheep associated with reduced transmission and disease due to Mannheimia haemolytica. Subobjective 2.1A: Identification of one or more genetic markers in a major gene region for reduced nasal shedding of Mh in domestic sheep. Subobjective 2.1B: Produce a high-throughput diagnostic test for quantifying nasal shedding of Mh in domestic sheep that has high concordance with nasal culture. Subobjective 2.1C: Validation of genetic markers from a major gene locus for reduced nasal shedding of Mh in domestic sheep. Subobjective 2.1D: Genomewide identification of genetic markers for reduced nasal shedding of Mh in domestic sheep. Subobjective 2.2A: Develop comparative transcriptome sequences for domestic versus bighorn sheep neutrophils (PMNs) both with and without exposure to Mh leukotoxin. Subobjective 2.2B: Develop a draft genome assembly for bighorn sheep. Objective 3: Develop intervention strategies to mitigate transmission of Mannheimia haemolytica from domestic sheep to bighorn sheep which will reduce respiratory disease in domestic and bighorn sheep. Subobjective 3.1: Quantify nasal, pharyngeal, pulmonary and serological antibody levels to leukotoxin-bearing Mh serotype A2 [MhA2(+LktA)] in adult domestic sheep with natural nasopharyngeal colonization. Subobjective 3.2: Intranasally immunize domestic and bighorn lambs with genetically modified MhA2 expressing an inactive form of Lkt and compare antibody responses to those measured in subobjective 3.1. Subobjective 3.3: Challenge MhA2(Lkt-mod) immunized domestic and bighorn lambs with wildtype MhA2.
1b. Approach (from AD-416):
Ovine respiratory disease presents many challenges to U.S. agriculture. Respiratory pathogens of domestic sheep like ovine progressive pneumonia virus (OPPV) and Mannheimia haemolytica (Mh) lead to millions of dollars in production losses annually. In addition to production losses in domestic sheep, Mh is an important bacterial pathogen of bighorn sheep. Data indicate that domestic sheep transmit pathogenic subtypes of Mh to bighorn sheep, and a currently debated intervention is separation of domestic from bighorn sheep areas. However, removal of domestic sheep grazing rights threatens a large fraction of the U.S. sheep industry, and additional intervention strategies are urgently needed. Existing data suggest that transmission of both OPPV and Mh has a genetic component in domestic sheep. The development of genetic markers will allow marker-assisted selection for reduced transmission and disease in domestic sheep, as well as reduced transmission to bighorn sheep. Furthermore, there are essentially no genomic and transcriptomic resources for bighorn sheep. However, the development of such resources will allow the formulation of specific biological hypotheses related to differences between bighorn and closely related domestic sheep. Protective immune responses to Mh in domestic and bighorn sheep have not been well-characterized. Documenting the characteristics of protective antibody mediated immunity in domestic sheep is an important first step toward development of a vaccine capable of providing preimmunity in bighorn sheep populations and improving the control of Mh mediated respiratory disease in domestic lambs. A goal of this research project plan is development of an intranasal vaccine utilizing a modified-live Mh expressing an inactive form of leukotoxin. Replacing 5348-32000-029-00D (October 2011).
3. Progress Report:
Multiple investigations were completed and reports finalized in FY2013. These included a sheep co-housing study that provided the first prospective experimental evidence for the importance of Mycoplasma ovipneumoniae in bighorn sheep pneumonia. Another study clarified the role of leukotoxin from Bibersteinia trehalosi in determining lethality of bighorn sheep pneumonia and examined the interaction of viral and bacterial agents in inducing lethal pneumonia. A third study examined the potential of Pasteurella multocida to limit the growth of Mannheimia haemolytica. A comprehensive review of the literature to date on bighorn pneumonia was performed to identify patterns and consider plausibility of competing hypotheses to explain this multifactorial disease. Based on the retrospective and prospective evidence for the role of Mycoplasma ovipneumoniae in bighorn pneumonia, a genome-wide association scan was begun to examine sheep chromosomes for important regions related to transmission of Mycoplasma ovipneumoniae. Furthermore, a genome-wide scan of all sheep chromosomes to identify important genetic regions for ovine lentiviral infection and control post-infection was completed. An evaluation was begun of an existing genetic marker test in current use for predicting probability of ovine lentiviral infection to determine if its use could be extended to prediction of control of ovine lentivirus after infection. Also, an investigation into regulation of genes important for mycobacterial disease was performed.
1. Experimental evidence identifying extended bighorn sheep survival when co-housed with domestic sheep in the absence of Mycoplasma ovipneumoniae. Bighorn sheep have experienced population limiting pneumonia, and attention has focused on exposure to domestic sheep as a potential source. Previous studies showed co-housing bighorn sheep with domestic sheep resulted in rapid, lethal pneumonia in bighorn sheep. ARS researchers in Pullman, Washington, worked in collaboration with researchers from Washington State University and the Idaho Department of Fish and Game to test if using domestic sheep free of Mycoplasma ovipneumoniae in co-housing would influence bighorn sheep survival. Three out of four bighorn sheep lived through the test period of more than 100 days, and the fourth died with pneumonia only after 90 days of co-housing. This unprecedented survival was similar to co-housing bighorn sheep with other ungulate species. These results provide prospective, experimental evidence for the importance of Mycoplasma ovipneumoniae in bighorn sheep pneumonia and support development of intervention strategies based on Mycoplasma ovipneumoniae.
2. Genetic marker test for Johne’s disease in cattle. Johne’s disease is an intestinal disease of ruminants caused by mycobacterial infection and costing hundreds of millions of dollars annually in the U.S. ARS researchers in Pullman, Washington, and Beltsville, Maryland, worked in collaboration with Washington State University and University of Missouri-Columbia to identify specific genetic variants that play roles in regulating the EDN2 gene. These variants can be used in a genetic marker test to predict susceptibility to tissue infection with Mycobacterium paratuberculosis, the pathogen responsible for Johne’s disease. A patent application has been filed for this genetic marker test, and discussions concerning commercialization are underway with multiple commercial enterprises. This genetic marker test may reduce the frequency of Johne’s disease in cattle, potentially reducing need for antibiotic use in livestock. Further, the test may also have additional uses in predicting susceptibility to other mycobacterial diseases of ruminants.
White, S.N., Mousel, M.R., Herrmann-Hoesing, L.M., Reynolds, J.O., Leymaster, K.A., Neibergs, H.L., Lewis, G.S., Knowles Jr, D.P. 2012. Genome-Wide association identifies multiple genomic regions associated with susceptibility to and control of ovine lentivirus. PLoSOne 7(10):e47829.