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DETECTION AND ISOLATION OF VIRUSES FROM Courtesy : Festschrift - Dr. S. Ramachandran |
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Introduction The Far East and Middle East countries are the major markets for India, but follow OIE guidelines wherein screening meats for pathogens before exports to any country is mandatory. As a risk-mitigation measure, screening of meats is carried out for several pathogens two of which i.e. Foot and Mouth Disease Virus (FMDV) and Swine Fever Virus (SFV) have been described here. The identification of viruses from meats requires more specialized techniques than those for bacteria and fungi, as viruses are intracellular and are present in minute quantities within the tissues. Secondly, they do not alter the organoleptic qualities and neither they may cause any overt changes in the appearance of the meats. In the literature screened so far, there are no records on the isolation of viruses from frozen buffalo meat and meats of apparently healthy animals. Therefore the present study was conducted to compare the suitability of various serological tests and PCR for the detection of FMDV and ELISA for the detection of SFV from meats of buffalo, cattle, sheep, goat and pig. (b) Sandwich ELISA: The sandwich ELISA was performed at the Disease Investigation Section, Pune which is one of the centres for the All India Co-ordinated Research project on FMD, as per the procedure of Venkataramanan (3) on the PBS extracts as well as the infected cell culture fluids. The optical density was taken in an ELISA reader and the maximum OD obtained at 492 nm after taking into consideration the controls. (c) Suckling Mouse Inoculation Test (SMIT): The test was carried out on the PBS extracts of the samples as described by Rai and Rao (2). Swiss albino suckling mice, 3-5 days old were used for inoculation. One litter (10-12mice) was used for each sample among which one mouse was marked and kept as uninoculated control. (d) Polymerase Chain Reaction (PCR):
The test was carried out at the Indian Veterinary Research Institute, Bangalore as described by Suryanarayana et al. (4). Tissue samples collected from Deonar slaughterhouse were screened by mCFT and those found positive were subjected to PCR. The PCR products were analysed by gel electrophoresis along with marker DNA (lambda phage) using 1% agarose. The PCR products were Southern blotted onto nylon membranes and hybridized with a 35S radiolabelled (0.6kb) probe and autoradiographed. (b) Dot - ELISA:
The samples along with the positive and negative controls were loaded onto nitrocellulose grided membranes (Sartorius) with 1-2 ml quantity of each respectively. The membranes were immersed in 1% BSA for 60 min at 370C. The membranes were dipped in rabbit hyperimmune serum and further incubated in goat antirabbit alkaline phosphatase conjugate. The development of pink dots was due to fast red and naphthol as chromogen substrate mix. (a) Micro Complement Fixation Test: A total of 144 muscle, 69 heart, 58 lymph node and 12 spleen samples were subjected to mCFT for preliminary detection of FMDV from chloroform extracts of samples of buffalo, cattle, sheep, goat and pig. Out of these, 28 muscle, 10 heart, 9 lymph node and one spleen samples were found positive for FMDV. Virus was not detected in 8 muscle samples by direct mCFT but was recovered in cell culture. From cell culture fluids, virus was detected in 24 muscles, 7 hearts and 4 lymph nodes by mCFT. The findings are summarised below species-wise: Buffalo: From frozen meat, serotype “O”; from meat of Thane and Deonar slaughter houses serotypes “O”, “A” and “Asia 1” were detected in chloroform extracts as well as in infected cell culture fluids by mCFT. Sheep: Serotypes “O”, “A” and “Asia1” were detected in chloroform extracts and infected cell culture fluids by mCFT. Goat: Serotypes “A”, “O” and “Asia 1” were detected in chloroform extracts and in cell culture fluids by mCFT. Pigs: Serotypes “O” and “A” were detected in chloroform extracts by mCFT and only serotype “O” was recovered in cell culture and confirmed by mCFT. (b) Sandwich ELISA: FMDV was not detected by sandwich ELISA in the PBS extracts but was detected in cell culture fluids in which serotypes “O” and “Asia 1” could be detected. A few samples showed mixed infection of the two serotypes. The species-wise findings are summarised below: Buffalo: The virus was recovered from one meat sample collected from Deonar slaughter house and 3/5 frozen meat samples and one heart sample from Thane slaughter house. Serotypes “O” and “Asia1” were recovered from these samples. Sheep: The virus was detected in 2/4 muscle samples only from Deonar slaughter house. “Asia1” was recovered from these samples. Goat: FMDV serotype “Asia1” was detected in samples of one lymph node and one spleen. Pig: FMDV was detected in 2 muscle and one heart sample and both “O” and “Asia1” serotypes were identified by ELISA. (c) Suckling mouse inoculation test (SMIT):
FMDV was detected in meat from various sources using SMIT. Cattle: A total of 7 muscle, one heart and 2 lymph node samples had viable virus by SMIT out of 18 positive by mCFT. The serotypes identified include 4 “O”, 3 “Asia 1” and 3 “A” from the various tissues. Buffalo: Out of a total of 11 tissues positive for the presence of FMDV by mCFT, 9 samples contained live virus as detected by SMIT, which included 5 muscles, 3 heart and 2 lymph node samples. Altogether, 7 “O”, 1 “A” and 1 “Asia 1” were identified. Sheep: Out of 6 tissues positive by mCFT, only one tissue had viable virus detected by SMIT and that belonged to “A” serotype. Goat: Out of 9 samples positive by mCFT, 4 samples had viable virus as detected by SMIT, which consisted of 1 muscle, 1 heart and 2 lymph nodes. In these tissues, 2 “O” and 2 “Asia 1” serotypes were identified. Pig: Out of 4 tissues positive by mCFT, only one tissue had live virus as detected by SMIT i.e. one muscle sample having “O” serotype of FMDV. (d) Polymerase Chain Reaction: PCR was performed on samples of cattle, buffalo, sheep and pig collected from Deonar slaughter house. Cattle: Viral RNA was detected in 7 muscle and one heart samples out of 13 samples subjected to PCR. Out of these, virus was recovered in tissue culture in 3/7 muscles and 1/1 heart sample as detected by ELISA, indicating the specificity of the test in detecting FMDV from meat samples. Buffalo: Viral RNA was detected from one sample each of muscle and heart tissues, out of which viable virus was recovered from muscle only and it was serotype “Asia 1”. Sheep: Out of the 6 samples tested by PCR, only 3 were found to contain viral RNA and viable virus was detected in one muscle sample only. Viral RNA was detected in one muscle in which virus was detected only by mCFT and not by ELISA. Only in one muscle sample viable virus was detected both by ELISA and mCFT but no viral RNA could be detected. This may be due to total degradation of viral RNA while processing. In the rest of the samples no viral RNA or viral antigens could be detected by PCR and ELISA respectively. In one of the sheep muscle sample neither viral RNA nor viable virus was detected by any of the tests, indicating a good correlation between the 3 tests. Pig:
In 2/4 samples viral RNA was detected by PCR and virus was isolated only from one heart sample. From the other sample (muscle) no virus was isolated, although viral RNA was detected by PCR and virus was detected in the chloroform extract by mCFT. In 2 samples viable virus could be detected by mCFT and ELISA but no viral RNA was detected by PCR. When tested by dot-ELISA, the PBS extracts of all the 22 samples were found to contain virus indicated by pink dots on the membrane. FMDV serotypes were detected from PBS extracts by mCFT but not by ELISA. The reason for the lack of sensitivity of ELISA may be due to the predominance of 12 ‘S’ subunits rather than 146 S units which may constitute only a minor fraction of FMDV antigens in the samples, (13,14). This phenomenon may also be due to exclusive use of 146 S guinea pig sera in the test (15). In meat samples, the presence of serous exudates may have also resulted in a greater opportunity for antigen-antibody complex formation thereby blocking antigen detection both by mCFT and ELISA (16). Hence a combined use of mCFT and ELISA has been advocated, since the percentage of typed samples increased considerably when both tests were used in combination instead of singly (14). It was also observed that maximum sensitivity and type specificity cannot be achieved within the same diagnostic test (13). Therefore, virus isolation was used as a definitive test of FMDV infectivity and was the most sensitive procedure for detection of live FMDV. Another important observation was the presence of two serotypes of FMDV in some of the samples of cattle and pigs. Such multiple infected samples are common when samples are screened by indirect sandwich ELISA (17). Apart from the World Reference Laboratory, Pirbright, most workers in India (18), Bangladesh (19), Saudi Arabia (20) and Thailand (21) found sandwich ELISA more sensitive than mCFT in detection of virus from clinical samples and use of cell culture as the most efficient means of virus isolation, with further confirmation by sandwich ELISA. SMIT is one of the reliable tests in detection and confirmation of FMDV (2,22). FMDV has also been isolated from tissues of experimentally infected pigs after storage for 55 days at -20°C in suckling mice (23). From most of the samples found positive for FMDV by mCFT, the virus could be recovered in BHK 21 cells by 24-48 hours post infection (PI). The FMDV isolates so obtained were also confirmed by ELISA. Among the FMDV types encountered, ‘O’ was the most common followed by ‘Asia 1’ and ‘A’ in that order. The CPE such as rounding of cells and syncytia were seen by 24 hours PI and there was detachment of the cell sheet by 48 hours PI. The stages of CPE such as margination of chromatin, bar shaped eosinophilic inclusions and pyknotic nuclei characteristic of FMDV were seen in the infected BHK 21 cells (24) by May- Gruenwald Giemsa staining. CPE on fibroblastic cells of hamster kidney could be detected within 16-24 hours PI and completed by 72 hours PI. Therefore, BHK 21 was found suitable for isolation of FMDV from meat and other organs of infected cattle and of other species obtained from different sources. BHK 21 was also used in earlier studies for isolation of FMDV from beef for studying the survival time of FMDV in uncooked and cooked beef products (25). FMDV was detected in tissues of cattle, buffalo, sheep, goat and pig indicating the wide spectrum of hosts susceptible to the virus and its implications in the role of the virus in evolving a carrier status in the above mentioned species. India being endemic for FMDV, spread of the virus occurs irrespective of vaccination. Several factors such as the epidemiology and persistence of FMDV in the animals are responsible for the development of carriers, which play an important role in the spread of FMDV through meat (26). Serotype ‘Asia 1’ was isolated from meat and other organs of cattle, buffalo, sheep and goat but not from pigs whereas type ‘O’ was isolated from all species. These animals were from the abattoir and were apparently healthy at the time of slaughter at antemortem inspection and hence isolation of FMDV from these animals indicated that they were preclinically infected (27) or they were carriers. Virus was also detected in the muscle of incontact pigs 20 hours before rise in temperature or the appearance of aphthae, suggesting a carrier status (28). All the types grew well in BHK 21 cells and the results correlated well with those of SMIT and mCFT. Highest percentage of tissues positive for ‘O’ was from buffalo and that of ‘Asia 1’ from cattle. The overall percentage of tissues from various species with ‘O’ serotype was highest followed by ‘Asia 1’. ‘O’ serotype was most commonly detected from field specimens by mCFT followed by ‘Asia 1’ mainly from cattle and buffalo and other species ‘A’ followed by ‘O’ and ‘Asia 1’. With the advent of newer specific techniques such as PCR minute quantities of the virus genome can be amplified to an amount detectable by radiolabelled probe or by restriction enzyme digests or by direct sequencing and therefore can be used instead of ELISA for the diagnosis of FMDV (26). Since muscle is not the site of prediliction of FMDV, titers of virus may have been too low for detection by ELISA (29) and hence virus was not detected in the tissue extracts by ELISA. Secondly, detection of FMDV by serological tests and virus isolation are time consuming and use of either test may not be reliable enough to confirm the diagnosis. Under such circumstances, PCR has shown to be of great value in detection of viral RNA from meat and other tissues (30). The results of PCR correlated well with isolation of FMDV on BHK 21 cells. Reverse transcriptase (rt) PCR has simplified the detection of FMDV-RNA from tissue samples making it more sensitive than ELISA (31). Since pre and post PCR contamination of the product with other extraneous DNA is possible, PCR coupled with hybridisation with a specific radiolabelled probe, (0.6 kb) increased the specificity of the test as also confirmed in beef of experimentally infected cattle (30). In the present study, 15 out of 24 samples of meat and other organs from Deonar slaughterhouse contained FMDV-RNA which correlated well with other tests. Dot-blot hybridization signals also confirmed the presence of FMDV in the meat and other tissues. The probe used for hybridization was from the highly conserved region of the FMDV genome i.e. the 3D site for all 4 serotypes consisting of 620 bp and inserted in phagemed bluescript SK+. The probe was radiolabelled with 35S and used for hybridization. On dot-blot hybridization, the probe had specifically hybridised with the DNA in 9 samples, probably in the rest of the samples, the quantity of cDNA was very less and therefore could not be observed. Using the primer pair of P1 and SV1 the presence of FMDV nucleic acid in meat and other organs could be detected. The use of PCR for confirmation of FMD-RNA from meat of freshly slaughtered animals may be the first attempt since, from the literature reviewed so far, none of the workers have used PCR for the above purpose. The ability of PCR to reliably and rapidly obtain a positive or negative result in one or two days is a significant advantage particularly as the free exchange of biological and animal products is of increasing importance. ELISA was used to detect SFV from muscle, heart and lymph nodes of apparently healthy pigs. ELISA was also used to detect virus antigen from lymph nodes and other tissues of experimentally infected pigs (35). The recovery of virus from the muscle was in very low titers due to the fact that muscle is not the site of primary multiplication of the virus. Therefore, the probability of such pigs harbouring virus in the preclinical stages is very high, as may also have been the case at the Deonar abattoir. Virus was also detected in tissues from pigs of infected herds sent for slaughter using FAT and ELISA (36). During this study, virus was detected by dot-ELISA and ELISA and the results of virus detection by dot-ELISA correlated well with ELISA. On putting the samples to cell culture, it was observed that SFV showed better titers in PK-15 cells rather than SPKT and apart from rounding of the cells, there were no overt degenerative changes. PK-15 has been used by various workers for isolation of SFV, the reason being that the latent period is shorter in PK-15 than SPKT. Most of the viral antigen remained intracellular and hence there was no CPE (37). Hence detection of virus from the sample extracts by ELISA and subsequent isolation in PK-15 cells is required to confirm the survival of virus in the tissues (38). As observed in the present study, the mild cytopathogenicity of the isolates could be due to the presence of Defective Interfering (DI) particles since most of the isolates of SFV were non-cytopathogenic on tissue culture cells (39). When screening the samples for SFV, cross-reactions are common with bovine viral diarrhoea (BVD) virus (40). In the present study this probability was eliminated by using either new born calf serum/FCS (Sigma) tested for absence of BVDV, in the growth medium used for PK-15 cells and SPKT. Similarly, the use of anti-rabbit hyperimmune serum in ELISA also ruled out the possibility of cross-reactions with BVDV, lending more specificity to the test (41). The presence of live viruses in frozen meat indicated that viruses survived freezing at -20°C since they were recovered in cell cultures. The use of susceptible cell cultures proved to be easy and cost effective for isolation of the above viruses. Since the viruses isolated in the present study are virulent or infectious, their detection in meat focuses on the endemicity and the carrier status that these diseases produce in a population, which may or may not have undergone vaccination prior to slaughter. Hence ELISA can be adopted as a screening test for FMDV and SFV, at abattoirs to screen the carcasses and those testing positive may subsequently be further tested for viable virus in cell cultures. Only steps taken towards eradication of these diseases may help to alleviate this problem to some extent and reduce the restrictions imposed on the trade of meat to countries free of these diseases. The elimination of these viruses by suitable techniques such as irradiation treatment and use of chemical sanitizers in combination with irradiation opens up a new area of future research. |
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Authors Corresponding address: Dr. (Mrs.) Sharmila Majee |
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The views expressed in this article are of the author(s), and any clarifications can be obtained from the author(s). |
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