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SUMMARY
Swine vesicular disease (SVD) is a contagious disease of pigs, caused by an enterovirus and characterised by vesicles on the coronary bands, heels of the feet and occasionally on the lips, tongue, snout and teats. Strains of SVD virus vary in virulence, and the disease may be subclinical, mild or severe, the latter usually only being seen when pigs are housed on abrasive floors in damp conditions. The main importance of SVD is that it is clinically indistinguishable from foot and mouth disease (FMD), and any outbreaks of vesicular disease in pigs must be assumed to be FMD until investigated by laboratory tests and proven otherwise.
Identification of the agent: Where a vesicular condition is seen in pigs, the demonstration by enzyme-linked immunosorbent assay (ELISA) of SVD viral antigen in a sample of lesion material or vesicular fluid is sufficient for a positive diagnosis. If the quantity of lesion material submitted is not sufficient (less than 0.5 g), or if the test results are negative or inconclusive, isolation of virus may be carried out by the inoculation of porcine cell cultures. If any cultures subsequently develop a cytopathic effect, the demonstration of SVD viral antigen by ELISA will suffice to make a positive diagnosis.
Serological tests: Specific antibody to SVD virus can be identified using the microneutralisation test or ELISA. Although the microneutralisation test requires 2-3 days to complete, it remains the definitive test for antibody to SVD virus. A small proportion (up to 0.1%) of normal, uninfected pigs will react positively in serological tests for SVD. These singleton reactors can only be differentiated from infected pigs by resampling of the positive animal and its cohorts.
Requirements for vaccines and diagnostic biologicals: There are currently no commercial vaccines available against SVD. Diagnostic and standard reagents are available from regional reference laboratories.
A. INTRODUCTION
Swine vesicular disease (SVD) can be a subclinical, mild or severe vesicular condition depending on the strain of virus involved, the route and dose of infection, and the husbandry conditions under which the pigs are kept. Clinically, SVD is indistinguishable from foot and mouth disease (FMD) and this is its main importance. It is therefore urgent that cases of SVD be distinguished from FMD by laboratory investigation.
The incubation period for SVD is between 2 and 7 days, after which a transient fever of up to 41°C may occur. Vesicles then develop on the coronary band, typically at the junction with the heel. These may affect the whole coronary band resulting in loss of the hoof. More rarely, vesicles may also appear on the snout, particularly on the dorsal surface, on the lips, tongue and teats, and shallow erosions may be seen on the knees. Affected pigs may be lame and off their feed for a few days. Abortion is not a typical feature of SVD. Recovery is usually complete in 2-3 weeks, with the only evidence of infection being a dark, horizontal line on the hoof where growth has been temporarily interrupted. The clinical signs vary according to the age of pigs affected, the conditions under which they are kept and the strain of SVD virus involved (8). Disease caused by mild strains may remain unobserved, particularly in pigs kept on grass or housed on deep straw. Younger animals are more severely affected, although mortality due to SVD is very rare, in contrast with FMD in young stock. Nervous signs have been reported, but are unusual. Affected pigs may excrete virus from the nose and mouth and in the faeces up to 48 hours before the onset of clinical signs. Most virus is produced in the first 7 days after infection, and virus excretion from the nose and mouth normally stops within 2 weeks. Virus may continue to be shed for up to 3 months in the faeces. The SVD virus is extremely resistant to inactivation in the environment, and is stable in the pH range 2.5-12.0 (9). This is in contrast to the FMD virus, which is very labile outside the pH range 6.0-8.0.
Because SVD may be mild or subclinical, it is essential when submitting samples from suspect clinical cases that serum samples from both the suspect pigs and other apparently unaffected animals in the group be included. It is possible for SVD to circulate unnoticed until it affects a particularly susceptible group, and therefore, in order to ascertain how long infection has been present, it is necessary to look for seroconversion to SVD virus in apparently healthy animals.
SVD is clinically very similar to FMD. Samples for virus isolation or antigen detection must be handled and submitted as though they contained FMD virus and must be transported in phosphate buffered saline (PBS) mixed with glycerol (1/1), pH 7.2-7.6, with antibiotics such as (final concentration per ml) penicillin (1000 International Units [IU]), neomycin sulphate (100 IU), polymyxin B sulphate (50 IU), and mycostatin (100 IU) (6).
SVD virus has been classified as a pig enterovirus, in the family Picornaviridae. Antigenically it is related to the human virus coxsackievirus B5. There are reports of seroconversion to SVD virus in laboratory workers handling the agent. Clinical disease was reported to be mild with the exception of a single case of meningitis associated with SVD virus infection. However, there have been no reported cases of serconversion or disease in farmers or veterinarians working with infected pigs. Under experimental conditions, it has not been possible to show transmission of coxsackievirus B5 between pigs.
B. DIAGNOSTIC TECHNIQUES
| 1. | Identification of the agent
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| | Any vesicular condition in pigs may be FMD. If FMD has been eliminated, the diagnosis of SVD requires the facilities of a specialised laboratory. Countries that lack such a facility should send samples for investigation to the FAO World Reference Laboratory (WRL) for Foot and Mouth Disease (FAO [Food and Agriculture Organization of the United Nations] World Reference Laboratory for FMD, Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, United Kingdom [also an OIE Reference Laboratory for FMD]). In the Americas, parallel testing for vesicular stomatitis viral antigen should also be conducted.
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| | Investigation should start with the examination of a 10% suspension of lesion material in PBS by enzyme-linked immunosorbent assay (ELISA) using antisera specific for SVD and FMD viruses. This suspension should also be inoculated on to monolayers of IB-RS-2 porcine cells (or other susceptible porcine cells), primary calf thyroid cells, and primary (or secondary) calf kidney cells. FMD virus will grow in all three tissue culture systems. Generally SVD virus will grow in cells of porcine origin only, however there is a report that the virus can be isolated in secondary lamb kidney cells. SVD virus can also be isolated from faecal samples.
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| | a) | Culture
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| | | A portion of the clarified epithelial suspension is inoculated on to monolayers of IB-RS-2 cells or other susceptible porcine cells. For differential diagnosis (e.g. FMD) bovine cell culture systems should also be employed. A culture medium found to be satisfactory is 50/50 Eagle's complete medium/yeast lactalbumin hydrolysate (LYH). For cell growth, add 10% bovine serum; for maintenance, add 3% bovine serum; and for virus isolation, add antibiotics only; it is preferable not to add serum.
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| | | Cultures are examined twice daily. If a cytopathic effect (CPE) is observed, the supernatant fluid is harvested and used as antigen in the ELISA for virus identification. Negative cultures are blind-passaged after 48 or 72 hours, and observed for a further 3 days. When isolating virus from faeces in which the amount of virus present may be low, a third tissue culture passage may be required.
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| | b) | Immunological methods
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| | | . | Enzyme-linked immunosorbent assay
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| | | The detection of SVD viral antigen by an indirect sandwich ELISA has replaced the complement fixation test as the method of choice. The test is the same as that used for FMD diagnosis. Duplicate rows in multiwell ELISA plates are coated with rabbit antiserum to SVD virus. This is the capture serum. Test sample suspensions are added to each of the rows. Appropriate controls are also included. Guinea-pig detection serum is added at the next stage followed by rabbit anti-guinea-pig serum conjugated to horseradish 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 (orthophenylenediamine) and substrate (H2O2). With strong positive reactions this will be evident to the naked eye, but results can also be read spectrophotometrically at 492 nm, in which case an absorbance reading greater than or equal to 0.1 above background indicates a positive reaction. As an alternative to guinea-pig and rabbit antisera, suitable monoclonal antibodies (MAbs) can be used, coated to the ELISA plate as the capture antibody, or peroxidase conjugated as tracing antibody.
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| | | An MAb-based ELISA can also be used to study antigenic variation among strains of SVD virus. Tissue-culture grown viral antigens are trapped by a rabbit hyperimmune antiserum to SVD virus adsorbed to the solid phase. Appropriate panels of MAbs are then reacted and the binding of MAbs to field strains is compared with the binding of MAbs to the parental strains. Strong binding indicates the presence of epitopes shared between the parental and the field strains (1).
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| | c) | Faecal samples
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| | | i) | Resuspend faecal material (approximately 20 g) in a minimal amount of phosphate buffer (0.04 M phosphate buffer or PBS).
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| | | ii) | Leave shaking, or on a rotary mixer, overnight at 4°C.
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| | | iii) | Clarify by centrifugation at 10,000 rpm for 30 minutes in a high speed centrifuge.
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| | | iv) | Harvest the supernatant and inoculate five tubes of IB-RS 2 cells with 0.2 ml per tube. Leave in a stationary position to adsorb for 1 hour at 37°C. Wash the tubes three times with PBS. Add serum-free maintenance medium (2 ml/tube) and place in a rolling rack, or suitable alternative that allows agitation of the culture media, at 37°C.
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| | | v) | Centrifuge the remaining supernatant at 28,000 rpm for 3 hours. This will concentrate any virus present in the supernatant in low amounts.
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| | | vi) | Discard the supernatant and resuspend the pellet in 2 ml of PBS by brief sonication. Add an equal volume of Freon and vortex 2-3 ml. Spin at 4000 rpm for 10 minutes on a bench centrifuge. Remove the supernatant fluid.
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| | | vii) | Inoculate five tissue culture tubes as in step iv above.
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| | | viii) | Incubate the tubes at 37°C for 3 days inspecting daily for evidence of CPE.
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| | | ix) | After 3 days, if there is no evidence of CPE, freeze-thaw the cell cultures and perform a blind passage into fresh tubes of IB-RS-2 cells. Incubate for a further 3 days, inspecting the tubes daily as before.
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| | | x) | Harvest the supernatant of any tubes showing CPE and confirm the presence of SVD virus by ELISA (or other appropriate test).
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| | | xi) | If no CPE is evident after the second passage the sample will be recorded as NVD (no virus detected).
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| | d) | Nucleic acid recognition methods
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| | | Nucleic acid recognition methods can be used to detect SVD viral genome in clinical material using reverse transcription followed by the polymerase chain reaction (PCR) and to establish relationships between isolates of SVD virus by determining the nucleotide sequence of part of the genome. By sequencing approximately 200 nucleotides within the 1D gene, which codes for the major structural protein VP1, it has been possible to group strains of SVD virus according to their sequence homology, and to relate epidemiologically strains causing disease in different regions or at different times (2). Techniques using the PCR have been developed to improve the sensitivity of diagnosis. A PCR has been described that combines extraction of RNA using commercially available silica gel columns with a reverse-transcription PCR (RT-PCR) using primers corresponding to highly conserved regions in the 1C and 1D genes (7). The technique is rapid, detects all genotypes of SVD virus, and is sufficiently sensitive for use on samples collected from cases of suspect clinical disease. Where subclinical infection is suspected, or when samples are collected after the resolution of clinical disease, a more sensitive nested RT-PCR can be combined with a more elaborate RNA extraction method to produce a detection system at least as sensitive, and considerably more rapid, than multiple passage on tissue culture. Several laboratories have developed alternative PCRs using a variety of protocols (3, 10, 11).
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| 2. | Serological tests
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| | SVD is often diagnosed solely on the evidence of serological tests. Because of the subclinical or mild nature of the disease, it is often first suspected following routine serology for disease surveillance or export certification. The virus neutralisation (VN) test, the double immunodiffusion test, the radial immunodiffusion test, the counter immunoelectrophoresis test and the ELISA have all been used for the detection of antibodies to SVD virus (1, 4, 5). The VN test and the ELISA are used most frequently. The VN test is the accepted standard test, but has the disadvantage that it takes 2-3 days to complete and requires tissue culture facilities. The ELISA is more rapid and can be more easily standardised. A small proportion of sera from animals with no previous exposure to SVD virus will react positively in serological tests for antibody to SVD virus. The 5B7 MAb competitive ELISA (MAC-ELISA) has been a reliable technique for detecting SVD antibody (1). Results from up to approximately 1% of sera from normal pigs are borderline or positive by the MAC-ELISA and should be retested by the VN test. Up to approximately 10% of these sera will also be positive by the VN test (i.e. 0.1% of the original population). Animals positive by ELISA, but negative by VN test can be regarded as uninfected. Repeat samples should be collected from animals positive in both tests and from cohorts. A constant or declining titre in the positive animal and the absence of antibody to SVD virus in cohorts confirms the status of the positive animal as a 'singleton reactor'. The factors responsible for 'singleton reactors' are unknown. Serological cross-reactivity with SVD virus might arise due to infection with another, as yet unidentified, picornavirus or may be due to other nonspecific factors present in the serum. Identification of the isotype of antibody present in positive sera (1) can be helpful as sera from infected pigs usually contain specific IgG alone or both IgG and IgM, whereas sera from 'singleton' reactors contain exclusively IgM.
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a) |
Virus neutralisation (the prescribed test for international trade)
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The quantitative VN microtest for antibody to SVD virus is performed using IB-RS-2 cells (or suitable susceptible porcine cells) in flat-bottomed tissue-culture grade microtitre plates.
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Virus is grown on IB-RS-2 cell monolayers and stored at -20°C after the addition of an equal volume of glycerol. SVD virus has been found to be stable under these conditions for at least 1 year. The sera are inactivated at 56°C for 30 minutes before testing. A suitable medium is Eagle's complete medium/LYH with antibiotics.
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The test is an equal volume test in 50 µl volumes:
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i) |
Starting from a 1/4 dilution, sera are diluted in a twofold dilution series across the plate, two rows of wells per serum.
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ii) |
Previously titrated virus is added; each 50 µl unit volume of virus suspension contains about 100 TCID50 (50% tissue culture infective dose).
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iii) |
Controls include a strong positive serum, a weak positive serum and a negative serum, a cell control, a medium control and a virus titration used to calculate the actual virus titre used in the test.
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Incubate at 37°C for 1 hour with the plates covered.
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v) |
A cell suspension at 106 cells/ml is prepared in medium containing 10% bovine serum for cell growth. 50 µl of cell suspension is added to each well.
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vi) |
Plates are sealed with pressure-sensitive tape and incubated at 37°C for 2-3 days. Alternatively, the plates may be covered with loosely fitting lids and incubated in an atmosphere of 5% carbon dioxide at 37°C for 2-3 days.
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vii) |
Microscopic readings may be feasible after 48 hours; the plates are finally fixed and stained routinely on the third day. Fixation is effected with 10% formalin/saline for 30 minutes; staining is done by immersion in 0.05% methylene blue in 10% formalin for 30 minutes. The plates are rinsed in tapwater.
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viii) |
Positive results are blue-stained cell sheets; the negative wells are empty. Titres are expressed as the final dilution of serum present in the serum/virus mixture at the 50% end-point. The test is considered to be valid when the amount of virus actually used per well is between 101.5 and 102.5 TCID50, and the positive standard sera are within twofold of their expected titre.
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ix) |
Interpretation of the results: At the OIE/FAO WRL for FMD, VN titres less than or equal to 1/11 are considered to be negative. Titres of 1/16 to 1/32 are doubtful and VN titres of 1/45 or more are regarded as positive. However, as titres depend on the cell system used, laboratories should establish their own criteria by reference to standard reagents available from the OIE/FAO WRL for FMD.
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| | b) | Enzyme-linked immunosorbent assay
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| | | In the ELISA developed by Brocchi et al. (1), the SVD viral antigen is trapped to the solid phase using the MAb 5B7. The ability of test sera to inhibit the binding of peroxidase-conjugated MAb 5B7 to the trapped antigen is then evaluated. Finally, the amount of conjugated MAb bound is detected by the addition of substrate and chromogen.
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| | | i) | ELISA plates are coated with 50 µl/well of MAb 5B7 at a dilution of 10 µg/ml in carbonate/bicarbonate buffer, pH 9.6, by overnight incubation at 4°C.
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| | | ii) | The plates are washed three times with PBS containing 0.05% Tween 20, and 50 µl of SVD antigen (SVD virus grown in IB-RS-2 cells, clarified, filtered and inactivated) at a predetermined optimal dilution, is added to each well. The optimal dilution of antigen is determined by checkerboard titrations of antigen and conjugated MAb that define the working dilution giving an absorbance on the upper part of the linear region of the antigen titration curve (between 1.5 and 2.0 optical density units). Plates are then incubated for 1 hour at 37°C.
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| | | iii) | After three additional washes, 50 µl of diluted test sera (not inactivated) and control sera are incubated with the trapped antigen for 1 hour at 37°C. Three-fold dilution series of sera are obtained directly in ELISA wells by adding 10 µl of serum to 65 µl of buffer (1/7.5 dilution) then transferring 25 µl to sequential wells containing 50 µl of buffer, mixing, and finally discarding 25 µl.
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| | | iv) | After incubation for 1 hour, 25 µl of an optimal dilution of peroxidase-conjugated MAb 5B7 (see step ii above) is added to each well and the plates are incubated at 37°C for a further 1 hour.
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| | | v) | After a final series of washes, the colorimetric reaction is developed by distributing 50 µl per well of the substrate solution (0.5 mg/ml orthophenylene-diamine in phosphate/citrate buffer, pH 5, containing 0.02% H2O2).
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| | | vi) | The reaction is stopped after 10 minutes by adding 50 µl of 2 N H2SO4. The absorbance is read at 492 nm using a microplate reader.
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| | | Antigen, sera and conjugate are diluted in PBS, pH 7.4, containing 0.05% Tween 20 and 1% yeast extract; the dilution buffer for sera contains, in addition, 1.0% mouse serum to prevent nonspecific binding of pig serum to MAb 5B7 either coated to the plate or conjugated to peroxidase.
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| | | vii) | Controls: Four wells on each plate containing all reactants except test serum confirm the maximum absorbance reading for the antigen; convalescent pig serum at four selected dilutions; negative pig serum; a low positive standard pig serum.
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| | | viii) | Interpretation of the results: Reactions are expressed as the percentage inhibition by each test serum of the MAb reaction with the SVD antigen. Sera are considered to be strongly positive if the mean per cent inhibition at both 1/7.5 and 1/22.5 dilutions is more than 70%. Sera registering a mean of more than 70% inhibition at the 1/7.5 dilution but less than 70% inhibition at the 1/22.5 dilution are considered to be low positive or borderline. Sera showing less than 70% inhibition at both dilutions are considered negative. All positive and borderline sera should be confirmed using the VN test.
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STANDARD REFERENCE SERA FOR SVD SEROLOGY
The FAO/OIE WRL for FMD maintains a panel of reference sera that have been extensively validated by the National SVD Reference Laboratories of the Member States of the European Union.
C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
No commercial SVD vaccines are currently available. Standard sera can be obtained from the OIE/FAO WRL for FMD. MAb 5B7 is available from the OIE Reference Laboratory for swine vesicular disease in Italy (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
REFERENCES
| 1. | Brocchi E., Berlinzani A., Gamba D. & De Simone F. (1995). Development of two novel monoclonal antibody-based ELISAs for the detection of antibodies and the identification of swine isotypes against swine vesicular disease virus. J. Virol. Methods, 52, 155-167.
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| 2. | Brocchi E., Zhang G., Knowles N.J., Wilsden G., McCawley J.W., Marquardt O., Ohlinger V.F. & De Simone F. (1997). Molecular epidemiology of recent outbreaks of swine vesicular disease: two genetically and antigenically distinct variants in Europe, 1987-1994. Epidemiol. Infect., 118, 51-61.
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| 3. | Callens M. & De Clercq K. (1999). Highly sensitive detection of swine vesicular disease virus based on a single tube RT-PCR system and DIG-ELISA detection. J. Virol. Methods, 77, 87-99.
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| 4. | Donaldson A.I., Ferris N.P., Knowles N.J. & Barnett I.T.R. (1983). Comparative studies of United Kingdom isolates of swine vesicular disease virus. Res. Vet. Sci., 35, 295-300.
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| 5. | Golding S.M., Hedger R.S., Talbot P. & Watson J. (1976). Radial immunodiffusion and serum neutralisation techniques for the assay of antibodies to swine vesicular disease. Res. Vet. Sci., 20, 142-147.
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| 6. | Kitching R.P. & Donaldson A.I. (1987). Collection and transportation of specimens for vesicular virus investigation. Rev. sci. tech. Off. int. Epiz., 6, 263-272.
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| 7. | Lin F., Mackay D.K.J. & Knowles N.J. (1997). Detection of swine vesicular disease virus RNA by reverse transcription-polymerase chain reaction. J. Virol. Methods, 65, 111-121.
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| 8. | Loxam J.R. & Hedger R.S. (1983). Swine vesicular disease: clinical signs, diagnosis, epidemiology and control. Rec. sci. tech. Off. int. Epiz., 2, 11-24.
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| 9. | Mann J.A. (1981). Swine vesicular disease. In: Virus Diseases of Farm Animals, Vol. 2, Gibbs E.P.J., ed. Academic Press, London, UK, 365-381.
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| 10. | Nunez J.I., Blanco E., Hernandez T., Gomex-Tejedor C., Martin M.I., Dopazo J. & Sobrino F. (1998). A RT-PCR assay for the differential diagnosis of vesicular viral diseases of swine. J. Virol. Methods, 72, 227-235.
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| 11. | Vangrysperre W. & De Clercq K. (1996). Rapid and sensitive polymerase chain reaction based on detection and typing of foot-and-mouth disease virus in clinical samples and cell culture isolates, combined with a simultaneous differentiation with other genomically and/or symptomatically related viruses. Arch. Virol., 141, 331-344.
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NB: There are OIE Reference Laboratories for Swine vesicular disease (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
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