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United States Department of Agriculture

Agricultural Research Service

Research Project: Control of Ovine Respiratory Disease through Genetic and Immunologic Mitigation of Pathogen Transmission and Disease

Location: Animal Disease Research

2012 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.

3. Progress Report:
Multiple investigative studies were completed and reports finalized in FY2012. These included studies on genetic and vaccine-based interventions in pneumonia risk due to specific pathogens, exploration of the role of a critical pathogen following comingling domestic and bighorn sheep, a survey of reasons for bighorn sheep pneumonia epizootics, and multiple studies on mechanisms of ovine immunity to respiratory pathogens. Specifically, overall analysis of TMEM154 genetic risk status for ovine progressive pneumonia virus was updated to improve and finalize estimates of relative risk due to TMEM154 haplotypes. Testing of a multivalent culture supernatant vaccine including virulence factors for M. haemolytica was completed in bighorn sheep. Survival analysis concluded following extended commingling of bighorn sheep and domestic sheep without Mycoplasma ovipneumoniae. Examination of bacterial species prevalence observed in pneumonic bighorn sheep from recent epizootics was completed. Comparative studies of passively transferred maternal antibody titers and lung pathogen clearance in domestic and bighorn sheep ended with evidence for both playing roles in differential levels of immunity to M. haemolytica pneumonia in experimental animals. Targeted sequence capture has begun for loci identified in a large genome-wide association study with seroprevalence and severity of ovine progressive pneumonia virus. Targeted sequence capture has also begun for the major locus identified in association with quantitative shedding of leukotoxin-bearing M. haemolytica. Numerous genetic variants have been identified in many genomic regions. These variants provide input for haplotype-tagging and imputation. Once complete, these steps will allow testing of specific genetic variants for possible functional involvement and/or validation as predictive genetic markers for selective breeding.

4. Accomplishments
1. A genetic marker test to reduce susceptibility to ovine progressive pneumonia virus. It had been known that some sheep breeds were more susceptible to ovine progressive pneumonia virus (OPPV) than others, but the specific genes responsible for these differences were not known. A scan of the genome identified the TMEM154 gene as one component, and further investigation identified three specific variants within the TMEM154 gene. ARS researchers at Pullman, Washington, Clay Center, Nebraska, and Dubois, Idaho, collaborated to show that these gene variants were consistently associated with reduced susceptibility to OPPV in many sheep flocks across the country. Sheep with desirable combinations of TMEM154 variants were almost three times less likely to become infected under field exposure than sheep with other combinations of genetic variants. This was the first genetic test for reduced susceptibility to OPPV validated in multiple flocks and management conditions. This genetic test will enable sheep producers to breed sheep with reduced susceptibility to OPPV.

2. A vaccine to protect bighorn sheep from Mannheimia haemolytica pneumonia. Fatal pneumonia has been an important problem for bighorn sheep, and one research question was whether a vaccine could help bighorn sheep achieve immunity from pneumonia due to Mannheimia haemolytica. An ARS researcher at Pullman, Washington, collaborated with researchers from Washington State University and the University of Guelph to show that repeated vaccination with culture supernatant from Mannheimia haemolytica and Bibersteinia trehalosi enabled bighorn sheep to avoid lethality entirely and prevent all but minor lung lesions on experimental challenge with Mannheimia haemolytica. This was the first experimental demonstration that immunity to Mannheimia haemolytica pneumonia was possible in bighorn sheep. This will stimulate additional research into vaccination and other strategies to enable immune protection of bighorn sheep from fatal pneumonia.

3. Demonstration of dramatically improved survival of bighorn sheep co-housed with domestic sheep without Mycoplasma ovipneumoniae. Much debate has centered on the risk of fatal pneumonia in bighorn sheep following exposure to domestic sheep. An ARS researcher collaborated with researchers from Washington State University, and the Idaho Department of Fish and Game to house domestic sheep without Mycoplasma ovipneumoniae together with bighorn sheep. They noted that the bighorn sheep had very high survival over multiple months following this exposure. This demonstrated the importance of Mycoplasma ovipneumoniae as a potential respiratory pathogen of bighorn sheep. This also demonstrated a set of conditions under which extensive contact between bighorn and domestic sheep can occur with limited impact on bighorn sheep health.

4. Causes of population limiting pneumonia among bighorn sheep, western United States, 2008-2010. During the recent decades pneumonia outbreaks in bighorn sheep have thought to be due primarily to transmission of Mannheimia haemolytic from domestic to bighorn sheep. This accomplishment performed at Washington State University and co-authored and co-investigated by an ARS researcher with the Animal Disease Research Unit, Pullman, Washington, revealed a statistically significant association between pneumonia in bighorn sheep and infection with Mycoplasma ovipneumoniae. Pneumonia in bighorn sheep populations remains a significant problem and recently has had immense negative impact on the domestic sheep industry in the western United States. This accomplishment demonstrates that early assumptions about a single cause and source of transmission of pneumonia causing agents to bighorn sheep were inaccurate. Therefore the view that contact with domestic sheep is the highest risk factor for pneumonia in bighorn sheep should be reviewed.

5. Demonstration of lower passively transferred antibodies to Mannheimia haemolytica in bighorn versus domestic sheep lambs. Bighorn sheep lambs have had higher susceptibility to Mannheimia haemolytica pneumonia than domestic sheep lambs. An important question being if this was related to lower antibody levels from the mothers of these lambs. A study performed at Washington State University through collaboration with an ARS researcher at Pullman, Washington, demonstrated for the first time that the domestic sheep mothers had higher antibody levels to Mannheimia haemolytica than bighorn mothers. Further, the domestic sheep lambs received higher antibody levels from their mothers than the bighorn lambs. This suggested antibody-mediated immune responses could play an important role in Mannheimia haemolytica pneumonia.

6. Demonstration of reduced bacterial clearance from lungs in bighorn versus domestic sheep with pneumonia. Bighorn sheep have had more severe lung damage than domestic sheep with Mannheimia haemolytica pneumonia, but the reasons had not been clear. Researchers from Washington State University and ARS scientist at Pullman, Washington, provided bighorn and domestic sheep the same dose of Mannheimia haemolytica at the same time, but the bighorn sheep failed to remove the bacteria from their lungs as quickly or as thoroughly as the domestic sheep. This did not rule out overactive immune responses as part of the explanation for differing lung damage between these closely related species. However, it demonstrated differences in bacterial clearance between them and provided baseline data for future studies to determine mechanisms underlying these differences in ability to remove Mannheimia haemolytica bacteria from the lungs.

Review Publications
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.

Subramaniam, R., Herndon, C.N., Shanthalingam, S., Dassanayake, R.P., Bavananthasivam, J., Potter, K.A., Knowles Jr, D.P., Foreyt, W.J., Srikumaran, S. 2011. Defective bacterial clearance is responsible for the enhanced lung pathology characteristic of Mannheimia haemolytica pneumonia in bighorn sheep. Veterinary Microbiology. 153(3-4):332-8.

Aoki, T., Scandiani, M.M., O Donnell, K. 2011. Phenotypic, molecular phylogenetic, and pathogenetic characterization of Fusarium crassistipitatum sp. nov., a novel soybean sudden death syndrome pathogen from Argentina and Brazil. Mycoscience. 53(3):167-186.

Herndon, C.N., Shanthalingam, S., Knowles Jr, D.P., Call, D.R., Srikumaran, S. 2011. Comparison of Passively Transferred Antibodies in Bighorn and Domestic Lambs Reveals One Factor in Differential Susceptibility of These Species to Mannheimia haemolytica-Induced Pneumonia. Clinical and Vaccine Immunology. (7):1133-8.

Subramaniam, R., White, S.N., Herrmann-Hoesing, L.M., Subramaniam, S. 2012. MHC class II DR allelic diversity in bighorn sheep. Gene. GENE2012506(1):217-222.

Besser, T.E., Highland, M.A., Baker, K., Cassirer, F.E., Anderson, N.J., Ramsey, J.M., Mansfield, K., Bruning, D.L., Wolff, P., Smith, J.B., Jenks, J.A. 2012. Causes of pneumonia epizootics among bighorn sheep, western United States, 2008-2010. Emerging Infectious Diseases. 18(3):406-14.

Meurs, K.M., Lahmers, S., Keene, B.W., White, S.N., Oyama, M.A., Mauceli, E., Lindblad-Toh, K. 2012. A splice site mutation in a gene encoding for PDK4, a mitochondrial protein, is associated with the development of dilated cardiomyopathy in the Doberman pinscher. Human Genetics. 131(8):1319-25.

Last Modified: 06/25/2017
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