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======Serology======
======Serology======
Serological confirmation of EEE or WEE virus infection requires a four-fold or greater increase or decrease in antibody titre in paired serum samples collected 10-14 days apart. Most horses infected with EEE and WEE virus have a high antibody titre when clinical disease is observed. Horses infected with EEE or WEE virus usually have antibody titres in the acute stage of the disease. Consequently, a presumptive diagnosis can be made if an unvaccinated horse with appropriate clinical signs has antibody against only EEE or WEE virus. The detection of IgM antibody by the ELISA can also provide a presumptive diagnosis of acute infection (11). The plaque reduction neutralisation (PRN) test or, preferably, a combination of PRN and haemagglutination inhibition (HI) tests is the procedure most commonly used for the detection of antibody against EEE and WEE viruses. There are cross-reactions between antibody against EEE and WEE virus in the CF and HI tests. CF antibody against both EEE and WEE viruses appears later and does not persist; consequently, it is less useful for the serological diagnosis of disease.
A combination of complement fixation (CF), haemagglutination inhibition (HAI) and cross-serum neutralization assays supports the acquisition of a positive diagnosis. Serological confirmation of EEE or WEE virus infection requires a four-fold or greater increase or decrease in antibody titre in paired serum samples collected 10-14 days apart. Most horses infected with EEE and WEE virus have a high antibody titre when clinical disease is observed. Horses infected with EEE or WEE virus usually have antibody titres in the acute stage of the disease. Consequently, a presumptive diagnosis can be made if an unvaccinated horse with appropriate clinical signs has antibody against only EEE or WEE virus. The detection of IgM antibody by the ELISA can also provide a presumptive diagnosis of acute infection (11). The plaque reduction neutralisation (PRN) test or, preferably, a combination of PRN and haemagglutination inhibition (HI) tests is the procedure most commonly used for the detection of antibody against EEE and WEE viruses. There are cross-reactions between antibody against EEE and WEE virus in the CF and HI tests. CF antibody against both EEE and WEE viruses appears later and does not persist; consequently, it is less useful for the serological diagnosis of disease.
A 4-fold increase in antibody (Ab) titre in convlescent sera is quoted for diagnosis but this test lacks sensitivity. The presence of viral Abs within 24hours of the initial viraemia typically precedes clinical signs. Ab titre increases sharply then deteriorates over 6 months. Samples taken when clinical signs appear are likely to miss the Ab peak and may thus demonstrate a decreasing titre. A single sample demonstrating an increased titre using HAI, CF and neutralizing Ab can provide a presumptive diagnosis. Maternal-derived Ab may interfere with diagnosis in foals. The serum half-life of colostral Ab in foals is around 20days.
a) Complement fixation
a) Complement fixation
To avoid anticomplementary effects, sera should be separated from the blood as soon as possible. Positive and negative control sera should be used in the test.
To avoid anticomplementary effects, sera should be separated from the blood as soon as possible. Positive and negative control sera should be used in the test.
b) Haemagglutination inhibition
b) Haemagglutination inhibition
Titres of 1/10 and 1/20 are suspect, and titres of 1/40 and above are positive.
c) Enzyme-linked immunosorbent assay
c) Enzyme-linked immunosorbent assay
The ELISA is performed by coating flat-bottomed plates with anti-equine IgM capture antibody (11). A test sample is considered to be positive if the absorbance of the test sample in wells containing virus antigen is at least twice the absorbance of negative control serum in wells containing virus antigen and at least twice the absorbance of the sample tested in parallel in wells containing control antigen.
Viral-specific IgM to the surface glycoprotein of Venezuelan EEV may be detected by ELISA, from 3 days post-onset of clinical signs up to 21 days post-infection. The ELISA is useful in acute VEE infections where convalescent serum samples are unobtainable. The ELISA is performed by coating flat-bottomed plates with anti-equine IgM capture antibody (11). A test sample is considered to be positive if the absorbance of the test sample in wells containing virus antigen is at least twice the absorbance of negative control serum in wells containing virus antigen and at least twice the absorbance of the sample tested in parallel in wells containing control antigen.
d) Plaque reduction neutralisation
d) Plaque reduction neutralisation
The PRN test is very specific and can be used to differentiate between EEE and WEE virus infections. The PRN test is performed in duck embryo fibroblast, Vero, or BHK-21 cell cultures. . The test is incubated for 48-72 hours, and endpoints are based on a 90% reduction in the number of plaques compared with the virus control flasks, which should have about 100 plaques.
The PRN test is very specific and can be used to differentiate between EEE and WEE virus infections. The PRN test is performed in duck embryo fibroblast, Vero, or BHK-21 cell cultures. Endpoints are based on a 90% reduction in the number of plaques compared with the virus control flasks.
=====Clinical Pathology=====
=====Clinical Pathology=====