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United States Department of Agriculture

Agricultural Research Service

Research Project: CONTROL OF GAMMAHERPESVIRUS-ASSOCIATED MALIGNANT CATARRHAL FEVER IN RUMINANTS
2008 Annual Report


1a.Objectives (from AD-416)
This Project is comprosed of four objectives designed to address key areas of research related to malignant catarrhal fever (MCF) and its causative viruses, particularly ovine herpesvirus 2 (OvHV-2). Discovery of new viruses in the MCF virus group necessitates extension of current nucleic acid based diagnostic tests with an emphasis on developing rapid and reliable assays for use by veterinary diagnosticians. In addition this project seeks to define basic virus-host interactions at the molecular level in order to identify how OvHV-2 causes disease so that control strategies, including immunological based methods such as vaccination, can be developed to protect clinically susceptible ruminants. These four objectives include.
1)extend nucleic acid based diagnostic tests to include newly discovered members in the MCF virus group;.
2)define host-virus interactions in sheep and develop an in vitro propagation system for OvHV-2;.
3)define OvHV-2 gene expression within MCF lesions; and.
4)develop an immunological control strategy for MCF in clinically susceptible ruminants.


1b.Approach (from AD-416)
Extend current nucleic acid-based tests for clinical diagnosis of MCF by validating the recently developed real-time PCR using a large set of defined field samples from animals with clinical MCF and by developing DNA microarray-based PCR for detection and differentiation of MCF group viruses. MCF pathogenesis will be studied in three phases:.
1)characterize lesion development in bison during preclinical and clinical stages;.
2)determine OvHV-2 genes that are highly expressed during preclinical and clinical stages using a gene expression microarray containing all 73 OvHV-2 open reading frames; and.
3)define the role of OvHV-2 gene products in MCF lesion development by developing an infectious OvHV-2 bacterial artificial chromosome (BAC) clone and testing the pathogenicity of infectious OvHV-2 BAC clones in bison, with the deletion of genes associated with lesion development. In developing an immunological control strategy for MCF in clinically susceptible ruminants, we will first characterize bison MHC class I and class II haplotypes and determine any association between MHC specific alleles and MCF resistance/susceptibility. For analysis of immune responses to OvHV-2 and development of vaccines for protection of animals from MCF losses, we will determine if animals that survive initial low dose infection with OvHV-2 are resistant to clinical MCF after a subsequent high dose challenge using our recently established animal model, bison. We will also determine which immune response dominates in the animals that survive the challenge, and finally evaluate vaccine candidates for protection against MCF in clinically susceptible ruminants. Replacing 5348-32000-018-00D October, 2006.


3.Progress Report
The project is a part of NP 103, Animal Health, component 4, Countermeasures to prevent and control respiratory diseases, Problem Statement 4A: Ruminant Respiratory Diseases under output of characterizing mechanisms of infection and protective immunity. The scientists at the Animal Disease Research Unit in collaboration with Washington State University and University of Wyoming, determined OvHV-2 replication sites in sheep and discovered that viral replication in the lung is required for initial infection, and that the replication in the lung was promptly controlled by the host immune mechanism. An immunohistochemical method to detect lytically infected cells in formalin fixed tissue has been developed using a monospecific, polyclonal rabbit antibody against the recombinant OvHV-2 capsid protein. These studies have paved the way toward the development of a cell culture system for growing the virus. Additionally, a set of bison MHC I genes has been cloned and the molecules expressed on the surface of a mouse mastocytoma cell line. Individual genes coding OvHV-2 proteins of interest have also been cloned and the cell lines co-transfected for expressing both MHC class I alleles and the proteins. These cells can be readily used to stimulate peripheral blood mononuclear cells from OvHV-2 infected bison and measure the cell-mediated immune response by IFN-gamma ELISPOT assay and cytotoxicity assays. An experimental animal system using rabbits to mimic a natural route of transmission was evaluated, and indicated that although rabbits are about 1,000 times more resistant to the disease than bison, they may provide an alternative animal model for carefully designed studies on MCF pathogenesis and immune responses that would be difficult or impossible to perform in bison. A pilot experiment for an OvHV-2 gene expression microarray has been carried out to determine the sensitivity of the assay by looking for transcripts from a subset of viral genes involved in different viral replication stages. The preliminary results showed that the conventional gene expression array, even with incorporation of tyramide signal amplification, had inadequate sensitivity for determining OvHV-2 gene expressions during preclinical and clinical stages. A new approach using a real-time PCR array has been initiated and the results are promising. Experimental infection of bison with low doses of OvHV-2 from sheep nasal secretions was evaluated for the ability to induce OvHV-2 infection in bison without clinical disease, a part of an immunological control study. Bison MHC class I haplotypes have also been characterized. Eight potential MHC class I loci, twenty haplotypes, and forty alleles have been identified. This work has positioned us to pursue the evaluation of bison immune responses to OvHV-2 and the development of vaccines for control of the disease. In addition, a five-color, in situ, immunofluorescent method for immunophenotyping immune cells in cattle or bison tissues was developed, and the phenotypes of lymphocytes in the vasculitis lesions of bison with MCF were determined.


4.Accomplishments
1. Determined OvHV-2 in vivo replication sites in sheep during viral shedding and viral entry

Inability to grow ovine herpesvirus 2, the causative agent for sheep-associated malignant catarrhal fever, in cell culture has made it difficult to study virus replication. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, determined the tissue sites of OvHV-2 replication during virus entry and shedding. The data revealed that the virus predominantly replicates in the turbinate during shedding, while it exclusively replicates in the lung in initial infection during entry, suggesting that OvHV-2 might adopt a unique cell tropism-switching mechanism in its replication cycle in sheep, and also suggested that OvHV-2 replication in the lung during initial infection was promptly controlled by a localized host immune mechanism. The discovery of this unique OvHV-2 in vivo replication pattern and the localized host immune control mechanism will lead new strategies for development of an in vitro propagation system and development of vaccines to control viral replication in clinically susceptible hosts. This accomplishment addresses Component 4 of the Animal Health National Program 103, Countermeasures to prevent and control respiratory diseases, Problem Statement 4A: Ruminant Respiratory Diseases under output of characterizing mechanisms of infection and protective immunity.

2. Developed an immunohistochemical method to detect OvHV-2 lytic replication.

Ovine herpesvirus 2 (OvHV-2), the etiological agent of sheep-associated malignant catarrhal fever, has never been propagated in vitro and identification of the cell type(s) in vivo supporting lytic replication is a key step toward development of an in vitro propagation system for the virus. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, developed an immunohistochemical method to detect lytically infected cells in formalin fixed tissue using a monospecific, polyclonal rabbit antibody against ovine herpesvirus 2 capsid protein. The methodology represents a critical step in identifying the cell type(s) in sheep that supports lytic replication in order to develop a culture system for the virus and to understand OvHV-2 pathogenesis. This accomplishment addresses Component 4 of the Animal Health National Program 103, Countermeasures to prevent and control respiratory diseases, Problem Statement 4A: Ruminant Respiratory Diseases under output of characterizing mechanisms of infection and protective immunity.

3. Characterized bison MHC class I haplotypes

Information on bison MHC I haplotypes is essentially needed for evaluation of immune responses in bison, especially the cell-mediated immune response, in order to develop effective immunological control strategies to malignant catarrhal fever in North American bison. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at University of Wyoming and Washington State University, Pullman, Washington, sequenced the bison MHC class I alpha chain precursor and identified 8 potential classical MHC class I loci, 26 class I haplotypes and 40 classical MHC class I alleles. Preliminary data indicated there was a linkage between MHC class I and II haplotypes, suggesting that specific MHC class I alleles might also be associated with disease-resistance. Identification of MHC class I haplotypes of individual bison is a prerequisite for understanding bison cell-mediated immune responses; in addition, it is also first step in developing MCF virus-based vaccines to protect bison from the disease. This accomplishment addresses Component 4 of the Animal Health National Program 103, Countermeasures to prevent and control respiratory diseases, Problem Statement 4A: Ruminant Respiratory Diseases under output of characterizing mechanisms of infection and protective immunity.

4. Determined the phenotypes of lymphocytes in the vasculitis lesions

Little is known about MCF pathogenesis. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with researchers at Washington State University, Pullman, Washington and University of Wyoming, developed a five-color, in situ, immunofluorescent method for immunophenotyping immune cells in cattle or bison tissues, and determined the phenotypes of lymphocytes in the vasculitis lesions of bison with experimentally induced sheep-associated MCF. The results showed that a majority of the cells infiltrating vascular lesions are CD8+ lymphocytes that are NKp40 negative and delta-chain positive; the role of these cells in the lesions is under investigation. Fundamental information on phenotyping immune cells in vascular lesions is necessary to further elucidate pathological mechanisms of vasculitis in MCF and facilitate understanding of immune modulatory mechanisms of gamma-herpesvirus induced vascular diseases. This accomplishment addresses Component 4 of the Animal Health National Program 103, Countermeasures to prevent and control respiratory diseases, Problem Statement 4A: Ruminant Respiratory Diseases under output of characterizing mechanisms of infection and protective immunity.


5.Significant Activities that Support Special Target Populations
Development of immunological control strategies has a significant impact on MCF control programs not only for American bison producers, but also for game farms, wild stocks, and zoological collections.


6.Technology Transfer

Number of Web Sites Managed1
Number of Non-Peer Reviewed Presentations and Proceedings1

Review Publications
Okeson, D.M., Garner, M.M., Taus, N.S., Li, H., Coke, R.L. 2007. IBEX-ASSOCIATED MALIGNANT CATARRHAL FEVER IN A BONGO ANTELOPE (TRAGELAPHUS EURYCEROS). Journal of Zoo and Wildlife Medicine. 38(3): 460-464.

Li, H., Karney, G., O'Toole, D., Crawford, T.B. 2008. Long distance spread of malignant catarrhal fever virus from feedlot lambs to ranch bison. Canadian Veterinary Journal. 49(2):183-185.

O'Toole, D., Taus, N.S., Montgomery, D.L., Oaks, J.L., Crawford, T.B., Li, H. 2007. Intra-nasal Inoculation of American Bison (Bison bison) with Ovine Herpesvirus-2 (OvHV-2) Reliably Reproduces Malignant Catarrhal Fever. Veterinary Pathology. 44(5):655-662

Cunha, C.W., Traul, D., Taus, N.S., Oaks, L., O'Toole, D., Davitt, C.M., Li, H. 2008. Detection of ovine herpesvirus 2 major capsid gene transcripts as an indicator of virus replication in shedding sheep and clinically affected animals. Virus Research. 132(1-2):69-75.

Li, H., Cunha, C.W., Davies, C.J., Gailbreath, K.L., Knowles Jr, D.P., Oaks, J.L., Taus, N.S. 2008. Ovine herpesvirus 2 replicates initially in the lung of experimentally infected sheep. Journal of General Virology. 89(7):1699-1708.

O'Toole, D., Li, H. 2008. Malignant Catarrhal Fever. In: Brown, C., Torres, A., editors. United States Animal Health Association - Foreign Animal Diseases. Seventh Edition. Boca Raton, FL: Boca Publications Group, Inc. p. 325-334.

Last Modified: 10/19/2014
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