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→Diagnosis
====Diagnosis====
====Diagnosis====
Presumptive based on clinical signs and epidemiological features. Definitive diagnosis requires serological tests and/or post-mortem examination. Virus isolation can be performed from blood or spinal fluid samples
Presumptive based on epidemiology and clinical signs. Definitive diagnosis requires serological tests and/or post-mortem examination. Virus isolation can be performed from blood or spinal fluid samples
=====Laboratory Tests=====
=====Laboratory Tests=====
======Virus Isolation======
The virus is identified by complement fixation (CF), immunofluorescence, or plaque reduction neutralisation (PRN) tests. EEE and WEE viral RNA may also be detected by reverse-transcription polymerase chain reaction methods
The virus is identified by complement fixation (CF), immunofluorescence, or plaque reduction neutralisation (PRN) tests. EEE and WEE viral RNA may also be detected by reverse-transcription polymerase chain reaction methods
1. Identification of the agent
1. Identification of the agent
Antigen-capture enzyme-linked immunosorbent assay (ELISA) has been developed for EEE surveillance in mosquitoes. This can be used in countries that do not have facilities for virus isolation or PCR (1).
Antigen-capture enzyme-linked immunosorbent assay (ELISA) has been developed for EEE surveillance in mosquitoes. This can be used in countries that do not have facilities for virus isolation or PCR (1).
======Serology======
2. Serological tests
2. Serological tests
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 sera can be screened at a 1/10 and 1/100 final dilution. Endpoints can be established using the PRN or HI test. Serum used in the PRN assay is tested against 100 plaque-forming units of virus. The virus/serum mixture is incubated at 37°C for 75 minutes before inoculation on to confluent cell culture monolayers in 25 cm2 flasks. The inoculum is adsorbed for 1 hour, followed by the addition of 6 ml of overlay medium. The overlay medium consists of two solutions that are prepared separately. Solution I contains 2 x Earle's Basic Salts Solution without phenol red, 6.6% yeast extract lactalbumin hydrolysate, 4% fetal bovine serum, 800 units/ml penicillin, 400 µg/ml streptomycin, 200 µg/ml nystatin, 6% of a 7.5% solution of sodium bicarbonate, and 3.3% of a 1/1500 dilution of neutral red (1/8000). Solution II consists of 2% Noble agar that is sterilised and maintained at 47°C. Equal volumes of solutions I and II are adjusted to 47°C and mixed together just before use. 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. The sera can be screened at a 1/10 and 1/100 final dilution. Endpoints can be established using the PRN or HI test. Serum used in the PRN assay is tested against 100 plaque-forming units of virus. The virus/serum mixture is incubated at 37°C for 75 minutes before inoculation on to confluent cell culture monolayers in 25 cm2 flasks. The inoculum is adsorbed for 1 hour, followed by the addition of 6 ml of overlay medium. The overlay medium consists of two solutions that are prepared separately. Solution I contains 2 x Earle's Basic Salts Solution without phenol red, 6.6% yeast extract lactalbumin hydrolysate, 4% fetal bovine serum, 800 units/ml penicillin, 400 µg/ml streptomycin, 200 µg/ml nystatin, 6% of a 7.5% solution of sodium bicarbonate, and 3.3% of a 1/1500 dilution of neutral red (1/8000). Solution II consists of 2% Noble agar that is sterilised and maintained at 47°C. Equal volumes of solutions I and II are adjusted to 47°C and mixed together just before use. 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.
A combination of complement fixation (CF), haemagglutination inhibition (HAI) and cross-serum neutralization assays supports the acquisition of a positive diagnosis. 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.
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. Viral culture may also be useful for acute VEE. Virus may be isolated from the CSF of acutely infected horses. Virus may be found in brain tissue using fluorescent Ab, ELISA and virus isolation.
Maternal-derived Ab may interfere with diagnosis in foals. The serum half-life of colostral Ab in foals is around 20days.
=====Clinical Pathology=====
=====Clinical Pathology=====