2000/428/EC: Commission DecisionShow full title

CHAPTER VII U.K. Principles and applications of virological tests and evaluation of their results

A. Detection of virus antigen U.K.

1. An indirect sandwich ELISA has replaced the complement fixation test as the method of choice for the detection of swine vesicular disease viral antigen. The test is the same as that used for foot-and-mouth disease diagnosis. The tests for the two diseases must be performed at the same time, unless foot-and-mouth disease has already been ruled out. It is recommended in particular on samples of epithelium or fluid from vesicular lesions, in which both swine vesicular disease and foot-and-mouth disease viruses can be present at high titers in acutely infected pigs and detected in a few hours(1). U.K.

Duplicate rows in multiwell ELISA plates are coated with rabbit antiserum to swine vesicular disease virus and to each of the seven serotypes of foot and mouth disease virus. These are the trapping sera. Test sample suspensions are added to each of the rows. Appropriate controls are also included. Homologous guinea-pig detection serum is added in the respective rows at the next stage followed by rabbit anti-guinea pig serum conjugated to an enzyme such as horse-radish peroxidase. Extensive washing is carried out between each stage to remove unbound reagents. A positive reaction is indicated if there is a colour reaction on the addition of chromogen and substrate. With strong positive reactions this will be evident to the naked eye, but results can also be read spectrophotometrically, in which case an absorbance reading 0,1 above background indicates a positive reaction.

2. Alternative monoclonal antibody-based ELISA systems, using selected monoclonal antibodies as trapping antibody and peroxidase-conjugated monoclonal antibodies as detecting antibody, may be used for swine vesicular disease antigen detection and for differential diagnosis with foot-and-mouth disease in epithelium samples, vesicular fluid or infected tissue culture. U.K.

3. A monoclonal antibody-based ELISA can be used to study antigenic variation among strains of swine vesicular disease virus. Tissue-culture grown viral antigens are trapped by a rabbit hyperimmune anti-serum to swine vesicular disease adsorbed to the solid phase. Appropriate panels of monoclonal antibodies are then reacted and the binding of monoclonal antibodies to field strains is compared to the binding of monoclonal antibodies to the parental strains. Similar binding indicates the presence of epitopes shared between the parental and the field strains. U.K.

B. Isolation and growth of virus U.K.

1. As a routine, clarified suspensions of samples of epithelium, vesicular fluid or faeces suspected to contain swine vesicular disease virus must be inoculated onto sensitive cell cultures. If the quantity and quality of samples from vesicular lesions submitted for examination is insufficient for immediate examination by ELISA, the growth of virus in tissue culture will be necessary to amplify viral antigen. U.K.

2. To isolate and grow virus, clarified epithelial suspension is inoculated onto monolayer cultures of IB-RS-2 cells. Two dilutions of epithelial suspension, one high (1/500) and one low (1/10) should be used to avoid interference with virus growth by interferon, the release of which will interfere with the growth of swine vesicular disease virus. For virus isolation only antibiotics are added to maintenance medium. For differential diagnosis from foot-and-mouth disease virus, primary bovine thyroid cells, or baby hamster kidney cells (BHK-21) must also be inoculated. U.K.

3. If a cytopathic effect develops, the supernatant fluid must be harvested from positive cultures when the effect is complete and used in the ELISA for virus identification. Negative cultures must be inoculated on fresh tissue cultures at 48 or 72 hours and this blind passage examined up to 72 hours later. In the absence of cytopathic effect after a further blind passage the sample can be declared negative for the presence of live virus. U.K.

4. Suspensions of faecal samples can be processed as described in 1. As there is generally less virus in faeces than in epithelium, it is essential that in the absence of a cytopathic effect in the first two passages, a third blind passage is included. U.K.

5. The simultaneous inoculation of a porcine cell line and one of the above-mentioned tissue culture systems (preferably primary bovine thyroid cells) is a useful guide as to whether vesicular samples contain swine vesicular disease virus or foot and mouth disease virus, as the former will only grow in cells of porcine origin. However, foot-and-mouth disease virus isolates with a prolonged history of transmission between pigs may also preferentially grow in porcine cell culture systems. U.K.

C. The polymerase chain reaction (PCR) for genome detection U.K.

1. Nucleic acid recognition methods can be used to detect swine vesicular disease viral genome in clinical material using the PCR and to establish relationships between isolates of swine vesicular disease virus by determining the nucleotide sequence of part of the genome. Techniques using the PCR have been developed to improve the sensitivity of diagnosis. Slightly different reverse transcriptase-PCR procedures have been described using primers corresponding to highly conserved regions in the 1C and 1D genes. U.K.

2. The PCR technique is rapid (the results are usually available within 24 hours), detects all genotypes of swine vesicular disease virus, and is sufficiently sensitive for use on samples collected from cases of suspect clinical disease. U.K.

3. Where sub-clinical infection is suspected, or when samples are collected after the resolution of clinical disease or when processing faecal samples, enhanced RT-PCR techniques, like nested RT-PCR, immune-PCR, ELISA-PCR and more elaborate RNA extraction methods produce a detection system at least as sensitive, but considerably more rapid, than multiple passage on tissue culture. U.K.

4. By sequencing approximately 200 nucleotides within the 1D gene which codes for the major structural protein VP1, it is possible to group strains of swine vesicular disease virus according to their sequence homology, and epidemiologically relate strains causing disease in different regions or at different times. U.K.

D. Evaluation of the results of virological tests U.K.

The detection of antigens or genome of swine vesicular disease virus by means of ELISA and PCR has the same diagnostic value as virus isolation.

However, virus isolation must be considered as the reference test and must be used as confirmatory test when necessary, in particular if a positive ELISA or PCR result is not associated with:

(a)

the detection of clinical signs of disease,

(b)

the detection of seropositive pigs, or

(c)

a direct epidemiological connection with a confirmed outbreak.

(1)

Positive ELISA results are associated with the presence of at least 105 TCID50 (tissue culture infectious doses) of virus in the sample.