2009 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.
The scientists at the Animal Disease Research Unit, in collaboration with Washington State University and University of Wyoming, developed a quick molecular diagnostic assay called multiplex real-time PCR to simultaneously detect and differentiate 5 malignant catarrhal fever viruses in clinical samples in a single reaction. The assay was evaluated with over 100 clinical samples and showed high sensitivity (97.2%) and specificity (100%). The group also identified that lung epithelia are the primary target cells for viral replication during initial infection in sheep and the data have greatly paved the way toward the development of a cell culture system for growing the virus. Experimental infection of bison with low doses of OvHV-2 from sheep nasal secretions was carried out. The data revealed that viral dose is important in determining the incubation period of OvHV-2 infection and that previous infection with an MCF group virus does not provide resistance to OvHV-2 induced MCF in bison after a low dose of challenge. These data are very important to the development of vaccine strategies. Real-time RT-PCR assay, another molecular assay, for measuring the expression of several OvHV-2 genes has been developed, and MCF lesion development in the tissues of experimentally infected bison during preclinical stages has been characterized. Preliminary data indicate that the level of expression of ORF 25 (a gene coding for a viral structural protein), a marker for replication, is associated with lesion severity, suggesting that the development of OvHV-2-induced MCF may be the result of active viral replication, instead of latency-associated cell proliferation as suggested by another research group in a rabbit model with AlHV-1 induced MCF (wildebeest-associated MCF). In addition, an experimental study in bison was initiated to determine virus-host interaction during initial infection and host immune responses to the infection, which is in progress.
Lung cells are associated with malignant catarrhal fever virus during initial infection. Ovine herpesvirus 2 (OvHV-2), the causative virus of sheep-associated malignant catarrhal fever, has never been grown in cell culture and development of such a cell culture system is absolutely required to advance the research. Using the same type of cells that allow the virus to replicate inside of animals would be the best strategy for development of a cell culture. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, identified lung epithelial cells as the primary target cells for viral replication during initial infection in sheep by dual staining fluorescent technology using antibodies specific for the cell types of interest and viral proteins. Identification of the cell type supporting OvHV-2 replication represents a significant step toward development of cell culture systems for manipulating the virus and evaluating specific immune responses in order to understand the disease and develop a vaccine.
A rapid diagnostic test for detection and distinction of malignant catarrhal fever viruses in clinical samples. Current diagnosis of malignant catarrhal fever (MCF) can still pose a significant challenge to veterinary clinicians and pathologists especially for animals in mixed-species operations; there has been no single assay available that is capable of simultaneously differentiating MCF viruses. ARS scientists at the Animal Disease Research Unit, Pullman, Washington, collaborating with faculty at Washington State University, Pullman, Washington, developed a fast diagnostic assay called multiplex real-time Polymerase Chain Reaction (PCR) using a single pair of primers, small fragments of DNA to simultaneously detect and differentiate 5 malignant catarrhal fever viruses in clinical samples in a single reaction. The assay was evaluated using hundreds of known positive and negative samples from clinically affected animals and showed high sensitivity (97.2%) and specificity (100%). The assay represents an important advance in MCF diagnosis, especially for animals in mixed-species operations such as zoos, wildlife parks, and game farms, and this flexible platform can be applied to differential diagnosis of other closely related viruses, especially herpesviruses in humans and animals.
5.Significant Activities that Support Special Target Populations
Research on epidemiology, transmission, and control strategies has a significant impact on MCF control programs for American bison and cattle producers, game farms, wild stocks, zoological collections, and state fair management.
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Mollat, W.H., Gailbreath, K.L., Orbell, G.M. 2009. Metastatic Malignant Melanoma in an alpaca (Vicugna pacos). Journal of Veterinary Diagnostic Investigation. 21(1):141-144.
Alcaraz, A., Warren, A., Jackson, C., Gold, J., Mccoy, M., Cheong, S., Kimball, S., Sells, S., Taus, N.S., Divers, T., Li, H. 2009. Naturally occurring sheep-associated malignant catarrhal fever in North American pigs. Journal of Veterinary Diagnostic Investigation. 21(2):250-253.
Cooley, A.J., Taus, N.S., Li, H. 2008. Development of a Management Program for a Mixed Species Wildlife Park Following an Occurrence of Malignant Catarrhal Fever. Journal of Zoo and Wildlife Medicine. 39(3):380-385.
Gailbreath, K.L., Taus, N.S., Cunha, C.W., Knowles Jr, D.P., Li, H. 2008. Experimental infection of rabbits with ovine herpesvirus 2 from sheep nasal secretions. Veterinary Microbiology. 132:65-73.
Knowles Jr, D.P., Li, H., Pastoret, P.P. 2008. Biotechnology in the diagnosis of infectious diseases and vaccine development. In: Pearson, J.E. Manual of Standards for Diagnostic Tests and Vaccines for Terrestrial Animals. Paris, France. Office des International des Epizooites. 66-89.