Diagnosis of Hemorrhagic Disease is Based on:
Compatible gross and microscopic findings supported by:
|The diagnosis of HD can be difficult. Lesions do not differentiate disease caused by BTV from that caused by EHDV. Many of the lesions of HD can be confused with other diseases. To make a diagnosis of HD one must have compatible gross and histopathologic lesions coupled with supportive evidence that BTV or EHDV was present. The gold standard would be a virus isolation of either BTV or EHDV, but RT-PCR and serology can also be helpful. Virus isolation, RT-PCR, and serology must be interpreted with caution in enzootic areas if the classical disease syndrome is not present. Remember, many animals are infected with these viruses but never develop disease. A variety of other tests, including fluorescent antibody testing, immunohistochemistry, or in situ hybridization, can be used to detect virus in tissues. Fluorescent antibody testing is available in a number of labs but does not appear to be too sensitive, especially for EHDV. The same lack of sensitivity is encountered with immunohistochemistry and in situ hybridization.|
Histopathology - Make sure to include buccal papillae, tongue, rumen, pylorus, intestine, lung, and heart, but do not forget other major organs (including eye and brain).
Acutely ill or dead animals may have detectable antibody levels, however, you would need paired sera to demonstrate a rise in titer. Dont forget that many animals are seropositive for BTV and/or EHDV but were previously exposed. Also, remember that not all animals infected with one of these viruses develop disease.
Viral isolation - Whole blood anticoagulated with heparin is the best sample for virus isolation, but spleen and lymph node are also very good samples. When submitting samples for virus isolation one should tell the lab that you are interested in both viruses because the best method of isolation varies between the two viruses. BTV grow better when inoculated into eggs intravascularly, while EHDV seem to do better when isolated in tissue culture systems. A variety of cell cultures will work, but BHK and CPAE cells seem to be the best. Do not freeze samples, but keep refrigerated. These viruses are very sensitive to freeze/thaw.
RT-PCR - There are a number of RT-PCR techniques in the literature, and NVSL will run this test. The sample of choice is EDTA anticoagulated blood. Refrigerate, but do not freeze the blood.
RT-PCR may detect virus RNA long after clinical disease has subsided. This is because these viruses become associated with the red blood cell membrane. Notice the virus particle in a pit in the red blood cell membrane (arrow) in this electron micrograph. In this pit it is sequestered from neutralizing antibodies and will last in circulation for the life of the red cell. Thus, RT-PCR may be positive long after the animal was infected.
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