Testing of Live Equine Suspects

Diagnosis of WNV infection in live horses with neurologic signs is made by testing blood or cerebrospinal fluid for antibodies against the virus. The most widely used test is the IgM capture ELISA (enzyme-linked immunosorbent assay). The USDA and many state veterinary diagnostic laboratories now conduct this test. Positive results by this assay, in conjunction with compatible clinical signs, is strong support for WNV as the causative agent. Laboratories which are equipped to handle live virus may also do a plaque reduction virus neutralization assay, in which paired serum samples (taken 2-3 weeks apart) are measured for WNV antibodies; a 4-fold or greater rise in titer is indicative of active infection.

At the DCPAH, the IgM capture ELISA test will be performed on serum and cerebrospinal fluid (CSF) from horses showing acute neurological signs. The Equine Arbovirus Encephalitis Reporting Form should be filled out completely and should accompany the sample. The test requires approximately 2 ml of serum, 5 ml of clotted whole blood or 1 ml of CSF, and takes 2 days to complete. The Virology Section of DCPAH will perform this test on Tuesdays and Thursdays and the results will be faxed. Samples have to be in the laboratory by noon at the latest on the days the test is performed. The fee for this testing is $25.00.

The IgG capture ELISA is a screening test for previous exposure to WNV. The test requires approximately 2 ml of serum or 5 ml of clotted whole blood and takes 2 days to complete. Sera will be tested at a dilution of 1:100 and the results will be reported as positive or negative. Positive results indicate that the horse was previously vaccinated or exposed to field virus, but cannot be used to determine whether the horse has protective immunity to WNV. The Virology Section of DCPAH will perform this test on Thursdays and the results will be faxed. Samples have to be in the laboratory by noon at the latest on the days the test is performed. The fee for this testing is $25.00.

The Virus Neutralization (VN) test will be performed in the Immunodiagnostics Section of DCPAH. It can be used in conjunction with, or instead of, the IgG capture ELISA. Two ml of serum or 5 ml of whole blood are required. This test provides information on the titer of viral neutralizing antibodies in serum. However, at this time, no information or standard exists on correlation of neutralizing titers and protective immunity in the horse. The fee for this test is $25.00.

Testing of Dead Equine Suspects

Unlike the situation in birds, WNV infection in horses affects only the nervous system. In horses which die or are euthanized with WNV infection, necropsy findings indicate inflammation in the brain (encephalitis) and/or spinal cord (myelitis). Cranial and peripheral nerves also may be involved. Immunohistochemical (IHC) staining of the nervous tissues for WNV antigen, however, generally yields minimal reaction. For this reason, IHC has not been a useful confirmatory test for pathologic diagnosis of WNV infection in horses. Rather, fresh brain or spinal cord tissue is tested by RT-PCR or virus isolation to confirm a clinical or necropsy diagnosis.

Horses and other equidae with acute neurologic signs that die or are euthanized are tested not only for WNV infection, but also for rabies virus, eastern equine encephalitis (EEE) virus, and western equine encephalitis (WEE) virus infections, and possibly other diseases. The head of the animal should be submitted to the MSU DCPAH. Brain tissue is first collected and submitted to the MDCH for rabies testing. At the same time, brain and possibly spinal cord tissues are examined microscopically for evidence of disease. If rabies results are negative, and arbovirus encephalitis (caused by WNV, EEE, or WEE) is suspected following microscopic examination, then the nervous tissue is tested by reverse transcription-polymerase chain reaction (RT-PCR) for these viruses.

The RT-PCR tests for the presence of WNV-specific RNA. This test requires two days to complete and will be set up on Monday afternoon and Wednesday afternoon. The fee for this test is $35.00.

The Michigan Department of Agriculture facilitates testing of suspect cases by providing transportation of specimens to the MSU DCPAH, and also covers test expenses.