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Usefulness of RT-PCR in the diagnostics of foot-and-mouth disease
Abstract
Foot-and-mouth disease virus (FMDV) from the family Picornaviridae, genus Aphthovirus, exists in the form of seven different serotypes: O, A, C, Asia 1 and SAT 1-3. Infection with one serotype does not confer immunity against another. Foot-and-mouth disease is one of the greatest threats to animal health in European countries. The rapid and accurate detection of FMDV is of the utmost importance.The RT-PCR assay was used to detect the presence of FMDV in samples. Positive results of the RT-PCR assay were found in all samples and in the positive control, the negative control reacted negatively. No cyto-athic effects in primary bovine thyroid cells were observed in 2 samples that had been thawed several times.The reference strains of FMDV was used to determine the sensitivity of the test. The sensitivity of RT-PCR for detection of FMDV (serotype O, A) was 1 TCID50and 10 TCID50 (serotype C, Asia 1) by gel electrophoresis.
... It consists of the RNA genome surrounded by a protein shell or capsid (15,23). It causes heavy economic losses to the livestock industry, such as a high morbidity in adult animals, treatment costs, reduced milk production, loss of working ability in draught animals in developing countries, reproductive disorders, and high mortality in young ones (13,19,24). The control of FMD is a national and regional responsibility, and, in many countries, the vaccine may be used only under the control of a veterinary authority (20,35). ...
The aim of the study was to evaluate the cytotoxicity of tulathromycin (macrolide antibiotic) on BHK-21 An30 cells and to determine the effect on FMD (Serotypes; A/TUR/11, O/TUR/07, Asia-1/TUR/15) virus propagation in vitro. In the result of MTT tests and cell culture studies, the non-toxic upper concentration of the tulathromycin limit for BHK-21 An30 cell culture was determined as 30 µg/ml in terms of cell morphologies and numbers. The mean FMD 146S virus particle values produced in BHK-21 An30 cell cultures containing 30 µg/ml of tulathromycin were found to be 0.47, 0.57 and 0.25 µg/ml; the mean infective titer determined as 107.03, 106.23 and 107.40 pfu/ml for A/TUR/11, O/TUR/07 and Asia-1/TUR/15, respectively. In the FMD virus cultures produced with medium containing penicillin-streptomycin, the mean values of 146S virus particles were determined to be A/TUR/11, O/TUR/07 and the Asia-1/TUR/15 serotypes were 0.50, 0.55, and 0.32 µg/ml, respectively, while the mean infective titers were 107.46, 106.62 and 107.73 pfu/ml, respectively. As a result, it was concluded that tulathromycin can be used as an antibiotic up to 30 µg/ml in a BHK-21 An30 cell culture and in the production media of the FMD virus A, O, and Asia-1 serotypes.
Direct detection of foot-and-mouth disease (FMD) virus from infected bovine and porcine tissue was investigated using a modified polymerase chain reaction (PCR) technique. A high degree of conservation was found in the genomic region coding for the viral RNA polymerase among the seven FMD viral (FMDV) serotypes. An oligomeric primer pair and probe were constructed from consensus sequence data within this area. First strand cDNA was synthesized using random hexamers and Moloney MuLV reverse transcriptase. The oligomeric primers used for PCR of the random primed cDNA yielded a 454-base-pair target amplification product. The PCR product was sized by agarose gel electrophoresis and hybridized strongly with the consensus sequence oligomeric probe. The PCR product was further examined by digestion with NcoI, confirming the predicted internal restriction enzyme site. All seven serotypes of FMDV RNA were amplified in a few hours and the PCR product tested positive. The sensitivity of the enzymatic amplification for detection of FMDV was 10 TCID50 by gel electrophoresis and less than 1 TCID50 when combined with hybridization to a labeled probe. The technique was specific, as determined by examination of at least 12 other viruses, including enteroviruses and other agents of vesicular disease. In vitro enzymatic amplification of cDNA from FMDV RNA using the modified PCR technique is highly specific, rapid and at least as sensitive as presently used procedures for FMDV laboratory diagnosis.
The polymerase chain reaction method (PCR) has been applied to the diagnosis of foot-and-mouth disease viral RNA in tissues and, particularly, oesophageal-pharyngeal fluid (probang) samples from cattle. Using primer sets which corresponded to conserved regions of the VP1 sequence of the viral genome, it was possible to amplify sequences regardless of the serotype/strain of the virus. In comparison with infectivity assays, the PCR was generally more sensitive although there were a number of examples where only infectivity was detectable. In experiments with uninfected probang samples deliberately seeded with a dilution series of virus, the PCR proved to be approximately 10(4) times more sensitive than infectivity assays. This greater sensitivity was attributed, in part, to the ability of the PCR to amplify specifically from non-infectious RNA preparations. This enabled the identification, by sequencing, of viral RNA from chemically inactivated virus concentrates typical of those used for commercial vaccine production. Amplification of specific PCR products was also achieved with virus eluted from commercial vaccine, including preparations which had been stored for more than 10 years at 4 degrees C. The PCR technique is of considerable value, therefore, both as a complement to infectivity assays and as a powerful tool in vaccine-associated studies.
A reverse transcription polymerase chain reaction (RT-PCR) method was compared with virus isolation in cell culture and the antigen detection ELISA for the primary diagnosis of foot-and-mouth disease (FMD) on 166 clinical samples from the field. Eighty samples were positive by virus isolation/ELISA and 78 by RT-PCR. The RT-PCR detected FMD viral RNA in 11 of the 86 samples assessed as negative by virus isolation/ELISA but conversely failed to diagnose 13 samples identified as positive by the latter procedures. This RT-PCR is not serotype-specific so a cDNA product is indicative of the presence of FMD viral RNA only. Confirmation of the specificity of the cDNA product and the identification of the serotype requires nucleotide sequence analysis. The value of the RT-PCR is that it can rapidly facilitate the molecular analysis of field isolates and thus provide important epidemiological information regarding the source of outbreaks. However, it is a sophisticated technique requiring specialised equipment, expertise and refined reagents and has to be used in conjunction with current procedures for FMD diagnosis.
Five European reference laboratories participated in an exercise to evaluate the sensitivity and specificity of their routinely employed RT-PCR tests and cell cultures for the detection and isolation of foot-and-mouth disease (FMD) virus. Five identical sets of 20 coded samples were prepared from 10 vesicular epithelia, which were derived from submissions from suspect cases of FMD or swine vesicular disease (SVD). Sixteen samples were derived from six FMD virus positive epithelia representing four different serotypes (two each of types O and A and one each of types Asia 1 and SAT 2), two from samples which had been found to be negative by antigen ELISA and virus isolation (VI) in cell culture and two from SVD virus positive epithelia. Some of the FMD virus positive samples were prepared from 10-fold serial dilutions of three of the initial suspensions. Each laboratory tested the samples by one or more of its available RT-PCR procedures and inoculated cell cultures that it routinely uses for FMD diagnosis in attempts to isolate virus, the specificity of which was confirmed by antigen ELISA. The best of the RT-PCR assays used in each laboratory gave comparable results while the sensitivity of cell cultures was variable from high in one laboratory, moderate in two and low in two others. This prototype panel of samples would appear suitable for external quality assurance of these tests but would benefit from the inclusion of more negative samples and an extension in the serial dilution range of one or more of the FMD positive sample titration series.