Highly sensitive fetal goat tongue cell line for detection and isolation of foot-and-mouth disease virus.

A fetal goat cell line (ZZ-R 127) supplied by the Collection of Cell Lines in Veterinary Medicine of the Friedrich Loeffler Institute was examined for susceptibility to infection by foot-and-mouth disease (FMD) virus (FMDV) and by two other viruses causing clinically indistinguishable vesicular conditions, namely, the viruses of swine vesicular disease and vesicular stomatitis. Primary bovine thyroid (BTY) cells are generally the most sensitive cell culture system for FMDV detection but are problematic to produce, particularly for laboratories that infrequently perform FMD diagnostic tests and for those in countries where FMD is endemic that face problems in sourcing thyroid glands from FMD-negative calves. Strains representing all seven serotypes of FMDV could be isolated in ZZ-R 127 cells with a sensitivity that was considerably higher than that of established cell lines and within 0.5 log of that for BTY cells. The ZZ-R 127 cell line was found to be a sensitive, rapid, and convenient tool for the isolation of FMDV and a useful alternative to BTY cells for FMD diagnosis.

Foot and mouth disease in the Lao People's Democratic Republic: I. A review of recent outbreaks and lessons from control programmes.

Foot and mouth disease (FMD) causes sporadic disease outbreaks in the Lao People's Democratic Republic (Lao PDR). As the Lao PDR is a major thoroughfare for transboundary animal movements, regular FMD outbreaks occur, causing economic hardship for farmers and their families. In this review of the recent history of FMD in the Lao PDR between 1997 and 2006, the authors examine the virological and epidemiological aspects of the disease and appropriate control measures, including the distribution of outbreaks, causative serotypes and the molecular epidemiology of the viruses, as well as large-scale vaccination programmes. The dominant serotype, type O, was reported every year from 1998 to 2005. The majority of outbreaks occurred in Vientiane Capital (n = 42; 28%) and the highest number of outbreaks were reported in cattle (n = 94; 61%); followed by buffalo (n = 41; 27%) and pigs (n = 18; 12%). All type A outbreaks occurred in cattle. Type Asia 1 outbreaks were reported in the central provinces around Vientiane Capital between 1996 and 1998.

Clinical and laboratory investigations of the outbreaks of foot-and-mouth disease in southern England in 2007.

A case of foot-and-mouth disease (fmd) on a cattle farm in Normandy, Surrey, was confirmed on Friday August 3, 2007, the first case in the uk since 2001. The infection was detected nearby on a second farm on August 6. On September 12, fmd was confirmed on a farm approximately 20 km from Normandy in Egham, and this was followed by cases on five more farms in that area in the next three weeks. The majority of the infected farms consisted of multiple beef cattle holdings in semi-urban areas. In total, 1578 animals were culled on the infected farms, and fmd virus infection was confirmed in 278 of them by the detection of viral antigen, genome or antibodies to the virus, or by clinical signs. This paper describes the findings from animal inspections on the infected farms, including the estimated ages of the fmd lesions and the numbers of animals infected. It also summarises the test results from samples taken for investigation, including the detection of preclinically viraemic animals by using real-time reverse transcriptase-pcr.

Evaluation of laboratory tests for SAT serotypes of foot-and-mouth disease virus with specimens collected from convalescent cattle in Zimbabwe.

During a field study in Zimbabwe, clinical specimens were collected from 403 cattle in six herds, in which the history of foot-and-mouth disease (FMD) vaccination and infection appeared to be known with some certainty. Five herds had reported outbreaks of disease one to five months previously but clinical FMD had not been observed in the sixth herd. A trivalent vaccine (South African Territories [SAT] types 1, 2 and 3) had been used in some of the herds at various times either before and/or after the recent outbreaks of FMD. The primary aim of this study was to evaluate the performance of serological tests for the detection of SAT-type FMD virus infection, particularly elisas for antibodies to non-structural proteins (NSPs) of FMD virus and solid phase competition ELISAS (SPCEs) for serotypes SAT1 and SAT2. Secondary aims were to examine NSP seroconversion rates in cattle that had been exposed to infection and to compare virus detection rates by virus isolation and real-time reverse transcriptase-PCR (rtRT-PCR) tests on both oesophagopharyngeal fluids and nasopharyngeal brush swabbings. In addition, the hooves of sampled animals were examined for growth arrest lines as clinical evidence of FMD convalescence. Laboratory tests provided evidence of FMD virus infection in all six herds; SAT2 viruses were isolated from oesophagopharyngeal fluids collected from two herds in northern Zimbabwe, and SAT1 viruses were isolated from three herds in southern Zimbabwe. Optimised rtRT-PCR was more sensitive than virus isolation at detecting FMD virus persistence and when the results of the two methods were combined for oesophagopharyngeal fluids, between 12 and 35 per cent of the cattle sampled in the convalescent herds were deemed to be carriers. In contrast, nasopharyngeal swabs yielded only two virus-positive specimens. The overall seroprevalence in the five affected herds varied with the different NSPS from 56 per cent to 75 per cent, compared with 81 per cent and 91 per cent by homologous SPCE and virus neutralisation tests respectively. However, if serological test results were considered only for the cattle in which persistent infection with FMD virus had been demonstrated, 70 to 90 per cent scored seropositive in the different NSPs.

Foot-and-mouth disease virus: a first inter-laboratory comparison trial to evaluate virus isolation and RT-PCR detection methods.

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.

Comparisons of original laboratory results and retrospective analysis by real-time reverse transcriptase-PCR of virological samples collected from confirmed cases of foot-and-mouth disease in the UK in 2001.

There were 2030 designated cases of foot-and-mouth disease (FMD) during the course of the epidemic in the UK in 2001 (including four from Northern Ireland). Samples from 1720 of the infected premises (IPs) were received in the laboratory and examined for either the presence of FMD virus (virological samples from 1421 IPs) or both FMD virus and antibody (virological and serological samples from 255 IPs) or antibody alone (from 44 IPs). The time taken to issue final diagnostic results ranged from a few hours in cases in which positive results were obtained by ELISA on epithelia containing sufficient virus to be detected, to several days for samples containing small amounts of virus requiring amplification through cell culture, negative samples or samples tested for antibody. Two subsets of samples were analysed retrospectively by real-time reverse transcriptase-PCR (RT-PCR); first, epithelia that were negative by both ELISA and virus isolation (VI) in cell culture, and secondly, samples that were negative by ELISA on epithelial suspension but positive by VI. There was broad agreement between the RT-PCR and VI/ELISA combined, except that the RT-PCR procedure did not detect a group of related virus isolates from Wales. These viruses had evidently evolved during the epidemic and had a nucleotide substitution in the RT-PCR probe site, which prevented them from being detected by the routine diagnostic probe. No evidence of FMD virus, antibody or nucleic acid was found in approximately 23 per cent (390 of 1730) of IPs from which samples were received, suggesting that the incidence of FMD during the outbreak may have been over-reported.

Use of pre-coated immunoplates and freeze-dried reagents for the diagnosis of foot-and-mouth disease and swine vesicular disease by enzyme-linked immunosorbent assay (ELISA).

An indirect sandwich ELISA is used by the World Reference Laboratory for Foot-and-Mouth Disease for the diagnosis of foot-and-mouth disease virus and swine vesicular disease virus. The potential for supplying ELISA 'kits' for diagnosis to other laboratories has been assessed by evaluating the reactivity of (a) immunoplates pre-coated with rabbit antisera to FMDV and SVDV and (b) freeze-dried diluted reference antisera. Immunoplates pre-coated using a sodium carbonate/hydrogen carbonate buffer retained 100% sensitivity at temperatures of 4 degrees C and -20 degrees C over the experimental storage period of 140 days but elevated storage temperatures, 18-24 degrees C and 37 degrees C, produced declining reactivity. There was a marked improvement in retention of reactivity upon storage at 37 degrees C when employing an alternative coating buffer, ammonium hydrogen carbonate. The reactivity of the rabbit antisera diluted in sodium carbonate/hydrogen carbonate solution and freeze-dried was high, as was the freeze-dried guinea pig antisera which had been diluted in each of the test solutions investigated. ELISA 'kits' for diagnosis, therefore, could be supplied using pre-coated immunoplates, with freeze-dried antiserum reagents or a combination of the two.

Primary diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction.

Universal and serotype-specific primer sets were used in simple reverse transcription polymerase chain reaction (RT-PCR) assays on field samples of epithelium and vesicular fluid to determine their suitability for primary diagnosis of all seven serotypes of foot-and-mouth disease (FMD). The specificity of reactions was confirmed by using other vesicular disease viruses, namely: swine vesicular disease virus, vesicular stomatitis virus and three vesiviruses. This resulted in the identification of a universal O/A/C/Asia 1 primer set (1F/1R) located in the 5' untranslated region (UTR) of the FMD virus genome for the successful detection of virus of these serotypes in clinical samples although this primer set detected FMD virus of the SAT1/2/3 serotypes less efficiently. The 5' UTR universal primer set could be used for the primary diagnosis of FMD in conjunction with the routine diagnostic methods of virus isolation in cell culture and ELISA, although a more favourable reaction would be expected with FMD viruses of the O/A/C/Asia 1 group than with those of the SAT serotypes. The other examined universal and serotype-specific primer sets, located principally in the P1 capsid-coding region, were generally inferior to the 5' UTR universal primer set. It is envisaged that this evaluation of primers will lead to the development of alternative PCR strategies, for example nested PCR formats, with concomitant improvement in the speed of primary diagnosis of FMD which under present procedures can be lengthy.

A hemi-nested PCR assay for the detection and identification of vesicular stomatitis virus nucleic acid.

This paper is the first to describe the development of a hemi-nested PCR assay for the detection of vesicular stomatitis virus (VSV) nucleic acid. This assay was developed as it combines high sensitivity for virus genome detection with the identification of the external amplification product in the reamplification step, thus confirming the specificity of the reaction. The assay did not depend on the presence of infectious virus in samples, as demonstrated by its detection of VSV in blood samples which were non-infectious in tissue culture. One further advantage was that the VSV-New Jersey and VSV-Indiana serotypes could be differentiated through the selective use of the appropriate hemi-nested primer. This assay is ideal for the study of VSV pathogenesis and persistence.

Comparison of reverse transcription polymerase chain reaction, enzyme linked immunosorbent assay and virus isolation for the routine diagnosis of foot-and-mouth disease.

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.

Diagnosis of foot-and-mouth disease by RT-PCR: evaluation of primers for serotypic characterisation of viral RNA in clinical samples.

Multiple primers designed from the 1D and 2AB regions of the foot-and-mouth disease (FMD) viral genome were evaluated extensively for the detection of all seven serotypes of the virus by reverse transcription polymerase chain reaction (RT-PCR) at the OIE/FAO World Reference Laboratory for FMD (WRL), Pirbright. The primers had been characterised previously elsewhere on a relatively small number of cell culture grown isolates and epithelial suspensions and had been shown to identify and differentiate all seven serotypes of FMD virus. The extended study evaluated several RT-PCR protocols on epithelial suspensions and supernatant fluids, resulting from their passage in cell culture, derived from clinical samples of diverse molecular characteristics. Each of the serotype-specific primers in selected RT-PCR protocols demonstrated suitable specificity and detected cell culture passaged isolates with some success but were not adequate for the serotyping of suspensions prepared from clinical samples of epithelium. The results showed that the primers can be used in RT-PCR procedures in conjunction with the routine detection methods of virus isolation and ELISA for the diagnosis and serotyping of FMD virus.

A solid-phase competition ELISA for measuring antibody to foot-and-mouth disease virus.

A solid-phase competition ELISA has been developed to measure antibodies to foot-and-mouth disease (FMD) virus and has been validated using an extensive range of sera from cattle. The assay uses polyclonal antisera and inactivated purified 146S antigens of FMD virus and was compared with the liquid-phase blocking ELISA and the virus neutralisation test on a range of serum sets. When examining test sera at a 1:5 dilution with a cut-off point of 30% inhibition of reaction, the solid-phase competition ELISA was as sensitive as the liquid-phase blocking ELISA for sera from infected or vaccinated animals. The limit of detection of the solid-phase ELISA was similar to that of the liquid-phase assay and both tests had lower limit of detection (i.e. were able to detect lower amounts of antibody) than the virus neutralisation test. The specificity of the solid-phase ELISA was considerably higher than that of the liquid-phase blocking ELISA and almost equivalent to that of the virus neutralisation test. The assay thus retains the sensitivity of the liquid-phase blocking ELISA whilst being easier to use, more robust and specific, and therefore offers an improvement for FMD virus antibody detection.

Freeze-drying foot-and-mouth disease virus antigens. I. Infectivity studies.

The ability of foot-and-mouth disease virus strains type O1 BFS 1860 and type A22 IRQ 24/64 to retain infectivity after freeze-drying with or without additives being made to virus suspensions was studied. The infectivity titres of freeze-dried antigens was assessed at intervals over a six month storage period at various temperatures and also after reconstitution to the liquid phase and storage with or without glycerination. Certain additive solutions were necessary to prevent degradation of virus during the freeze-drying procedure which reduced any loss of infectivity caused by storage of products at 4 degrees C and 20 degrees C. Additive solutions composed of 10% sucrose and 5% lactalbumin hydrolysate; 10% skimmed milk; 4% peptone and 1% gelatin; and 5% dextran, 1% sodium glutamate and 5% sucrose all prolonged the keeping qualities of virus at the elevated temperature of 37 degrees C. The results indicate that short-term storage and shipment of freeze-dried foot-and-mouth disease virus antigens is possible without the need for refrigeration, thereby reducing transportation and storage costs. Reconstituted antigens survived better after glycerination and storage at -20 degrees C than did non-glycerinated samples stored at 4 degrees C.

Freeze-drying foot-and-mouth disease virus antigens. II. For use in the ELISA.

Live and inactivated preparations of foot-and-mouth disease virus strains 01 BFS 1860 and A22 IRQ 24/64 were freeze-dried in the presence or absence of additive solutions and assessed for their reactivity by ELISA at intervals over a six month storage period at various temperatures and also after reconstitution and subsequent storage with or without glycerination. The type specificity of all antigen preparations was maintained throughout the study period and the potency of antigens, judged by titration in ELISA, remained constant during the freeze-drying procedure and throughout subsequent storage at -20 degrees C and 4 degrees C with or without additives having been made to virus suspensions prior to freeze-drying. This was also the case with antigens reconstituted and stored at either -20 degrees C with glycerol or at 4 degrees C without glycerol. Certain additive solutions were necessary, however, to preserve the activity of antigens stored at the elevated temperature of 37 degrees C. The reactivity of all freeze-dried antigens was not unduly affected in the liquid-phase blocking ELISA using bovine convalescent antisera of each of the seven serotypes of foot-and-mouth disease virus and known negative, non-immune bovine sera. The results suggest that shipment and long-term storage of freeze-dried foot-and-mouth disease virus antigens is possible for use in the ELISA in the absence of refrigeration. This has attractive advantages for reducing both shipment and storage costs of antigens and for the development of ELISA kits for the diagnosis of foot-and-mouth disease virus.

Use of inactivated foot-and-mouth disease virus antigen in liquid-phase blocking ELISA.

A liquid-phase blocking ELISA is used by the World Reference Laboratory for Foot-and-Mouth Disease for the quantification of antibodies to foot-and-mouth disease virus. The potential for using inactivated FMDV antigens in the assay has been assessed by titrating bovine convalescent sera to all seven serotypes and comparing the titres obtained with live or inactivated antigens. The titres were similar indicating that either live or inactivated antigens can be used in the liquid-phase blocking ELISA. Removing the need to use live antigens in tests for FMD antibody would reduce disease security risk and widen the acceptability of kits for FMD antibody detection and assay.

Development of a rapid chromatographic strip test for the pen-side detection of foot-and-mouth disease virus antigen.

Foot-and-mouth disease (FMD) is the most contagious animal virus disease of cloven-hoofed livestock and requires reliable and accurate diagnosis for the implementation of measures to control effectively its spread. Routine diagnosis of FMD is carried out at the OIE/FAO World Reference Laboratory for Foot-and-Mouth Disease (WRL for FMD), Pirbright by the combined use of ELISA and virus isolation in cell culture supplemented by reverse transcription polymerase chain reaction (RT-PCR) methods. These techniques require skilled personnel and dedicated laboratory facilities which are expensive. The development of a rapid and simple test for the detection of FMD virus antigen using Clearview chromatographic strip test technology for field application is described. This device detected FMD viral antigen in nasal swabs, epithelial suspensions and probangs from clinical samples submitted from the field, from animals infected experimentally and in supernatant fluids resulting from their passage in cell culture. The test system was more sensitive than ELISA for the diagnosis of all seven serotypes of FMD virus in the epithelial suspensions and nasal swabs and had equivalent sensitivity to the ELISA for the detection of contemporary virus strains in cell culture supernatant fluids. The study demonstrated the potential for this device to confirm a clinical diagnosis at the site of a suspected FMD outbreak, thereby offering the possibility of implementing control procedures more rapidly. Such pen-side diagnosis would have particular benefits in FMD emergencies, relevance to FMD control programmes which operate in endemic regions of the world such as South East Asia and for increasing disease awareness in other areas where efforts to control disease may be difficult. In each circumstance the availability of a pen-side device for diagnosis would reduce the necessity for sending routine diagnostic samples to an FMD laboratory and thereby reduce the delay in diagnosis, which can in some areas be considerable.

Development of a reverse transcription polymerase chain reaction procedure for the detection of marine caliciviruses with potential application for nucleotide sequencing.

A reverse transcription polymerase chain reaction (RT-PCR) procedure is described for the detection of marine caliciviruses including vesicular exanthema of swine virus (VESV), San Miguel sea lion virus (SMSV), bovine Tillamook virus (BCV Bos-1) and caliciviruses (CV) isolated from dolphin (Cetacean CV), gorilla (Primate CV) and rattlesnake (Reptile CV) using primers (1F and 1R) designed from the capsid-coding region of the viral genome. These primers were compared with those described by Neill, J.D. and Seal, B.S., 1995: Development of PCR primers for specific amplification of two distinct regions of the genomes of San Miguel sea lion and vesicular exanthema of swine viruses, Mol. Cell. Probes 9, 33-38 (Hel1/Hel2), which had been designed from the 2C-like region of the calicivirus genome. Both sets proved to be extremely useful diagnostic tools for all of the known marine calicivirus serotypes with the exception of three: SMSV-8 and -12 and mink CV suggesting that these three caliciviruses may belong to a different group. Neither of the two primer sets reacted with strains of the vesicular disease viruses of foot-and-mouth disease (FMD), swine vesicular disease (SVD) or vesicular stomatitis (VS) nor with two feline caliciviruses (FCV). The 1F/1R primer set has the advantage over the Hel1/Hel2 set in that it generates a larger PCR product for nucleotide sequence investigations and so provides greater opportunity for identifying molecular differences between the viruses.

Routine application of enzyme-linked immunosorbent assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular diseases.

An indirect, sandwich enzyme-linked immunosorbent assay (ELISA) has been compared in parallel with the standard complement-fixation typing test (CFT) for the diagnosis of foot-and-mouth and swine vesicular diseases. The superior sensitivity, reproducibility, economical use of reagents and ease of performance of the ELISA confirm the hypothesis that it should replace the CFT as the routine test of choice.

An enzyme-linked immunosorbent assay for the detection of vesicular stomatitis virus antigen.

An indirect, sandwich enzyme-linked immunosorbent assay (ELISA) has been developed for the laboratory diagnosis of vesicular stomatitis (VS). The assay which uses rabbit and guinea-pig antisera to the purified glycoprotein antigens of Serotypes New Jersey (VSV-NJ) and Indiana (VSV-IND-1), has both high sensitivity and specificity, but has lower reactivity for the two other Indiana subtypes VSV-IND-2 and VSV-IND-3.

An enzyme-linked immunosorbent assay for the detection of marine caliciviruses.

An indirect, sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of antigens of vesicular exanthema, San Miguel sea lion viruses and other marine caliciviruses is described. The assay which uses rabbit and guinea-pig antisera to purified antigens of each calicivirus serotype has high sensitivity and is almost totally type-specific.