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Isolation and identification of the foot-and-mouth disease virus
Abstract
Foot-and-mouth disease (FMD) is the most contagious disease of mammals and has great potential for causing severe economic losses in susceptible cloven-hoofed animals. FMD is caused by a virus of the genus Aphthovirus, family Picornaviridae. Serological tests in laboratories have identified seven different serotypes as O, A, C, SAT 1, SAT 2, SAT 3 and Asia 1. FMD is diagnosed by the virus isolation or demonstration of FMD viral antigen or nucleic acid in samples of biological specimens. The purpose of the study was to apply the isolation test in cell culture and a RT-PCR assay for the detection of foot-and-mouth disease virus in biological materials. Out of the total of 14 examined samples, 6 (42.8%) were found positive using these methods. The antigen ELISA was used for the confirmation of specificity of the isolation assay. Primary bovine thyroid cells were found the most sensitive cell culture system for the detection of foot-and-mouth disease virus, followed by secondary lamb kidney and certain IB-RS-2 cells.
... These strains are now presented in most of the Middle Eastern countries and has reached since the beginning of 2009 Bahrain, Kuwait, Lebanon, Libya and Iraq. The new epidemic sub-lineage reported in the above countries and circulating (11) According to the FAO report 1999 the common serotypes isolated in Iraq from 1952 to 1998 were A, O Sat1 and Asia 1 (19) In 1988 Virus was isolation, serotyping and characterization of FMD disease in Iraqi Native Gazella (17). In 1998-2000 outbreak of the disease occur in Iraq, and the virus was isolated from Holstien cattle and characterized as O serotype. ...
... The possibility of using rapid sensitive and specific assays for FMD recognition is a prerequisite it for prevention, control and eradication of the disease. To isolate the FMD virus, the primary bovine thyroid cell culture is the most sensitive system for FMDv detection this view was confirmed by several another's and our previous and recent studies, in agreement with ,but second passage of the cells did not show any Cytopathic effect for three successive passages (4,9,19,23 ) Continuous supply of these cells for diagnostic purposes is not easy because needs both :technical 7 professional preparation .However, when the supply is inadequate, the cells demonstrating comparable similarity in sensitivity may be utilized as we used several types of cell culture for isolation of FMDv and checked the sensitivity of the cells which produced visible CPE. The CPE showed on primary ovine thyroid cells, beside secondary ovine thyroid cells and the other sub passages showed distinctive CPE in less than 20 hours PI. the high sensitivity of the OTY and other cells tested. ...
Seventy four (74) samples from cattle, buffalo, goat and sheep were tested to isolate
the FMD virus, 23 isolates of the virus showed Cytopathic Effect (CPE) on the different types
of cell culture.
The CPE appears after 24 hours on fetal lamb kidney (FLK) cells, 48 hours on the fetal
bovine kidney (FBK), 30 hours on primary Fetal Bovine Thyroid cell (BTY) and 20 hours on
the primary fetal ovine thyroid cells (OTY). Solid phase ELISA test showed three serotypes
of FMD virus O, A and Asia1. One step real time RT-PCR for the diagnosis of FMDV by
using 5UTR FMDV primers and taqman probe (SA-IR-219-246F, SA-IR-315-293R and
SAmulti2-P-IR-292-269R). Samples were tested, showed 23 positive machines by using the
Applied Biosystem Fast 7500 real time PCR.
Virus isolation is the gold standard for foot-and-mouth disease virus (FMDV) detection in diagnostic procedures. This technique is heavily reliant on the use of sensitive cells for rapid and accurate detection of FMDV. To investigate the sensitivity of RM (primary lamb kidney cells), BHK-21 (baby hamster kidney cells) and IR-P1 (a derivative of female pig kidney cells) to infection with FMDV of the Southern African Territories (SAT) serotypes, we examined the virus concentration required to induce cytopathic effect (CPE) on each cell type. The results suggested that sensitivity of RM and IR-P1 cells was high and not significantly different (P < 0.05). BHK-21 however, exhibited low sensitivity to the strains used. Comparisons of three batches of each cell type were also done to establish the consistency of the sensitivity of these cells to FMDV infection. IR-P1 and BHK-21 cell batches gave consistent results for all samples used whereas RM cells showed significant differences (P > 0.05) between batches. TCID 50/ml was used to determine the viral titre required to induce CPE. IR-P1 cell line proved to have consistently higher TCID50/mL for all cell batches while RM cell batches displayed a difference in TCID50/mL values. The IR-P1 cell line was concluded to be a good cell culture system for virus isolation as it showed relatively high and reproducible sensitivity to all the FMDV strains used. The findings of this study indicate that the use of IR-P1 cell line could be considered for FMDV diagnostic work.
Swine vesicular disease virus (SVDV) is a member of the genus Enterovirus in the family Picornaviridae. This virus appears to have evolved from human coxsackievirus B5. Pigs infected with this virus show almost identical clinical signs to foot-and-mouth disease in pigs. Vesicular diseases must be differentiated with laboratory tests. The purpose of the study was to apply the isolation test in cell culture and RT-PCR assay for the detection of swine vesicular disease virus in the epithelial and fecal samples. Out of a total of 11 examined samples, 10 were found positive using these methods. The antigen ELISA was used for the confirmation of specificity of isolation assay. Primary piglet kidney cells and certain IB-RS-2 cells were a sensitive cell culture system for the detection of swine vesicular disease virus, whereas secondary lamb kidney cells not.
Primary bovine thyroid cell cultures and IB-RS-2 continuous cell line were used for foot-and-mouth disease virus (FMDV) isolation. In both cell culture systems, all tested samples gave positive results and the specificity of isolated virus was confirmed by the Ag-ELISA. Results of virus isolation test agreed with those obtained by RT-PCR and rRT-PCR, which enabled detection of the genetic material of FMDV. This indicates a high and comparable sensitivity of the applied diagnostic assays, which permit a reliable detection of FMDV in biological material.
Foot-and-mouth disease virus (FMDV) is an important animal pathogen that belongs to the Aphthovirus genus of the Picornaviridae family and infects cattle and other cloven-hoofed animals. Seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3) have been identified serologically, and multiple subtypes occur within each serotype. FMDV enters cells by receptor-mediated endocytosis. By electron microscopy the FMD virion appears to be a round particle with a smooth surface and a diameter of about 25 nm. The FMD viral particle contains a positive-strand RNA genome of about 8500 nucleotides, enclosed within a protein capsid. The virus capsid is made up from 60 copies each of four virus-encoded proteins VP1 to VP4. The FMDV genome is composed of the 5' non-translated region (5'NTR), the coding region, and the 3' non-translated region (3'NTR). The genome encodes a single polyprotein, from which the different viral polypeptides are derived by viral proteases. FMDV populations are genetically and anti-genetically heterogeneous. FMDV have very high mutation rates.
Sensitive cell cultures, such as primary bovine thyroid cells and primary pig, calf or lamb kidney cells can be used for isolating foot-and-mouth disease virus (FMDV). Established cell lines IB-RS-2 and BHK-21 may also be applied for this purpose. The aim of this study was to assess the efficacy of primary lamb kidney cell culture for detecting FMDV in biological materials. The results of the study demonstrate that this cell culture may be a useful tool in diagnostic studies of FMD.
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.