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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
The most definitive method for diagnosis of EEE or WEE is the isolation of the viruses. EEE virus can usually be isolated from the brains of horses, unless more than 5 days have elapsed between the appearance of clinical signs and the death of the horse. EEE virus can frequently be isolated from brain tissue even in the presence of a high serum antibody titre. WEE virus is rarely isolated from tissues of infected horses. Brain is the tissue of choice for virus isolation, but the virus has been isolated from other tissues, such as the liver and spleen. It is recommended that a complete set of these tissues be collected in duplicate, one set for virus isolation and the other set in formalin for histopathological examination. Specimens for virus isolation should be sent refrigerated if they can be received in the laboratory within 48 hours of collection; otherwise, they should be frozen and sent with dry ice. A complete set of tissues will allow the performance of diagnostic techniques for other diseases. For isolation, a 10% suspension of tissue is prepared in phosphate buffered saline (PBS), pH 7.8, containing bovine serum albumin (BSA) (fraction V; 0.75%), penicillin (100 units/ml), and streptomycin (100 µg/ml). The suspension is clarified by centrifugation at 1500 g for 30 minutes.
The newborn mouse is considered to be a sensitive host system. Inoculate intracranially one or two litters of 1-4-day-old mice with 0.02 ml of inoculum using a 26-gauge 3/8 inch (9.3 mm) needle attached to a 1 ml tuberculin syringe. The inoculation site is just lateral to the midline into the midportion of one lateral hemisphere. Mice are observed for 10 days; dead mice are collected daily and frozen at -70°C. Mouse brains are harvested for virus identification by aspiration using a 20-gauge 1 inch (2.5 cm) needle attached to a 1 ml tuberculin syringe. A second passage is made only if virus cannot be identified from mice that die following inoculation.
The chicken embryo is considered to be less sensitive than newborn mice when used for primary isolation of EEE and WEE viruses. Tissue suspensions can be inoculated by the yolk-sac route into 6-8-day-old embryonating chicken eggs. There are no diagnostic signs or lesions in the embryos infected with these viruses. Inoculated embryos should be incubated for 7 days, but deaths usually occur between 2 and 4 days post-inoculation. Usually only one passage is made unless there are dead embryos from which virus cannot be isolated. Newly hatched chickens are susceptible and have been used for virus isolation. If this method is used, precautions must be taken to prevent aerosol exposure of laboratory personnel, as infected birds can shed highly infectious virus.
EEE and WEE viruses can also be isolated in a number of cell culture systems. The most commonly used cell cultures are primary chicken or duck embryo fibroblasts, continuous cell lines of African green monkey kidney (Vero), rabbit kidney (RK-13), or baby hamster kidney (BHK-21). Isolation is usually attempted in 25 cm2 cell culture flasks. Confluent cells are inoculated with 1.0 ml of tissue suspension.
Following a 1-2-hour absorption period, maintenance medium is added. Cultures are incubated for 7 days, and one blind passage is made. EEE and WEE viruses will produce a cytopathic change in cell culture. Cultures that appear to be infected are frozen. The fluid from the thawed cultures is used for virus identification.
When the complement fixation (CF) test is used, EEE or WEE viruses can be identified in infected mouse or chicken brains, cell culture fluid, or amnionic-allantoic fluid. A 10% brain suspension is prepared in veronal (barbitone) buffer; egg and cell culture fluids are used undiluted or diluted 1/10 in veronal buffer. The fluid or suspension is centrifuged at 9000 g for 30 minutes, and the supernatant fluid is tested against hyperimmune serum or mouse ascitic fluid prepared against EEE and WEE viruses using a standard CF procedure (13). The CF test requires the overnight incubation at 4°C of serum-antigen with 7 units of complement. Virus can be identified in cell culture by direct immunofluorescent staining. The less commonly used method of virus identification is the neutralisation test, as outlined below.
EEE virus nucleic acid in mosquitoes and tissues has been identified by the polymerase chain reaction (PCR) using primers selected from the capsid gene (15). RNA is extracted using guanidium iso-thiocyanate-acid phenol. Forty repetitions of the three-step amplification cycle of nucleic acid denaturisation, primer annealing and primer extension are used. Temperature and duration of each step are optimised for the specific primer pair, reagents and thermal cycler used in the PCR cycles. Reaction products or their fragments are analysed on 2.0-2.6% agarose gels that have been stained with 1 µg/ml of ethidium bromide. An alternate identification procedure is by hybridisation with an oligonucleotide probe. A reverse-transcription PCR method for detection of WEE RNA and alternative methods for EEE RNA detection have also been described (5, 7).
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).
2. Serological tests
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) Complement fixation
The CF test is frequently used for the demonstration of antibodies, although the antibodies detected by the CF test may not persist for as long as those detected by the HI or PRN tests. A sucrose/acetone mouse brain extract is commonly used as antigen. The positive antigen is inactivated by treatment with 0.1% beta-propiolactone.
In the absence of an international standard serum, the antigen should be titrated against a locally prepared positive control serum. The normal antigen, or control antigen, is mouse brain from uninoculated mice similarly extracted and diluted.
Sera are diluted 1/4 in veronal buffered saline containing 1% gelatin (VBSG), and inactivated at 56°C for 30 minutes. Titrations of positive sera may be performed using additional twofold dilutions. The CF antigens and control antigen (normal mouse brain) are diluted in VBSG to their optimal amount of fixation as determined by titration against the positive sera; guinea-pig complement is diluted in VBSG to contain 5 complement haemolytic units-50% (CH50). Sera, antigen, and complement are reacted in 96-well round-bottom microtitre plates at 4°C for 18 hours. The sheep red blood cells (SRBCs) are standardised to 2.8% concentration. Haemolysin is titrated to determine the optimal dilution for the lot of complement used. Haemolysin is used to sensitise 2.8% SRBCs and the sensitised cells are added to all wells on the microtitre plate. The test is incubated for 30 minutes at 37°C. The plates are then centrifuged (200 g), and the wells are scored for the presence of haemolysis. The following controls are used: (a) serum and control serum each with 5 CH50 and 2.5 CH50 of complement; (b) CF antigen and control antigen each with 5 CH50, and 2.5 CH50 of complement; (c) complement dilutions of 5 CH50, 2.5 CH50, and 1.25 CH50; and (d) cell control wells with only SRBCs and VBSG diluent. These controls test for anticomplementary antigen, anticomplementary serum, activity of complement used in the test, and integrity of the SRBC indicator system in the absence of complement, respectively.
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
The antigen for the HI test is the same as described above for the CF test. The antigen is diluted so that the amount used in each haemagglutinating unit (HAU) is from four to eight times that which agglutinates 50% of the RBCs in the test system. The haemagglutination titre and optimum pH for each antigen are determined with goose RBCs diluted in pH solutions ranging from pH 5.8 to pH 6.6, at 0.2 intervals.
Sera are diluted 1/10 in borate saline, pH 9.0, and then inactivated at 56°C for 30 minutes. Kaolin treatment is used to remove nonspecific serum inhibitors. Sera should be absorbed before use by incubation with a 0.05 ml volume of washed packed goose RBCs for 20 minutes at 4°C.
Following heat inactivation, kaolin treatment and absorption, twofold dilutions of the treated serum are prepared in borate saline, pH 9.0 with 0.4% bovalbumin. Serum dilutions (0.025 ml/well) are prepared in a 96-well round-bottom microtitre plate in twofold dilutions in borate saline, pH 9.0, with 0.4% bovalbumin. Antigen (0.025 ml/well) is added to the serum. Plates are incubated at 4°C overnight. RBCs are derived from normal white male geese (RBCs from adult domestic white male geese are preferred, but RBCs from other male geese can be used. If cells from female geese are used, there may be more test variability. It has been reported that rooster RBCs cause a decrease in the sensitivity of the test) and washed three times in dextrose/gelatin/veronal (DGV), and a 7.0% suspension is prepared in DGV. The 7.0% suspension is then diluted 1/24 in the appropriate pH solution, and 0.05 ml per well is added immediately to the plates. Plates are incubated for 30 minutes at 37°C. Positive and negative control sera are incorporated into each test. A test is considered to be valid only if the control sera give the expected results. Titres of 1/10 and 1/20 are suspect, and titres of 1/40 and above are positive.
c) Enzyme-linked immunosorbent assay
The ELISA is performed by coating flat-bottomed plates with anti-equine IgM capture antibody (11). The antibody is diluted according to the manufacture's recommendations in 0.5 M carbonate buffer, pH 9.6, and 50 µl is added to each well. The plates are incubated at 37°C for 1 hour, and then at 4°C overnight. Prior to use, the coated plates are washed twice with 200 µl/well of 0.01 M PBS containing 0.05% Tween 20. After the second wash, 200 µl/well of PBS/Tween/5% nonfat dried milk is added and the plates are incubated at room temperature for 1 hour. Following incubation, the plates are washed again three times with PBS/Tween. Test and control sera are diluted 1/100 and 1/1000 in 0.01 M PBS, pH 7.2, containing 0.05% Tween 20, and 50 µl is added to each well. The plates are incubated at 37°C for 2 hours and then washed three times. Next, 50 µl of viral antigen is added to all wells. (The dilution of the antigen will depend on the source and should be empirically determined.) The plates are incubated overnight at 4°C, and washed six times. Then, 50 µl of horseradish-peroxidase-conjugated monoclonal antibody (MAb) to encephalitis virus (available from: Centers for Disease Control and Prevention, Biological Reference Reagents, 1600 Clifton Road NE, Mail Stop C21, Atlanta, Georgia 30333, United States of America) is added. The plates are incubated for 60 minutes at 37°C and then washed three times. Finally, 50 µl of freshly prepared ABTS (2,2'-Azino-bis-[3-ethylbenzo-thiazoline-6-sulphonic acid]) substrate + hydrogen peroxidase is added, and the plates are incubated at room temperature for 15-40 minutes The absorbance of the test serum is measured at 405 nm. 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
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.
=====Clinical Pathology=====
=====Clinical Pathology=====