Testing of Live
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.
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.