Transmission pathways of foot-and-mouth disease virus in the United Kingdom in 2007.

Foot-and-mouth disease (FMD) virus causes an acute vesicular disease of domesticated and wild ruminants and pigs. Identifying sources of FMD outbreaks is often confounded by incomplete epidemiological evidence and the numerous routes by which virus can spread (movements of infected animals or their products, contaminated persons, objects, and aerosols). Here, we show that the outbreaks of FMD in the United Kingdom in August 2007 were caused by a derivative of FMDV O(1) BFS 1860, a virus strain handled at two FMD laboratories located on a single site at Pirbright in Surrey. Genetic analysis of complete viral genomes generated in real-time reveals a probable chain of transmission events, predicting undisclosed infected premises, and connecting the second cluster of outbreaks in September to those in August. Complete genome sequence analysis of FMD viruses conducted in real-time have identified the initial and intermediate sources of these outbreaks and demonstrate the value of such techniques in providing information useful to contemporary disease control programmes.

Development and validation of a simplified serotyping ELISA based on monoclonal antibodies for the diagnosis of foot-and-mouth disease virus serotypes O, A, C and Asia 1.

This study describes the development and validation of a simplified enzyme-linked immunosorbent assay (ELISA) for the detection and discrimination of foot-and-mouth disease virus (FMDV) serotypes O, A, C and Asia 1. The multiplex ELISA was designed using selected, type-specific monoclonal antibodies (MAbs) coated onto ELISA plates as catching antibodies and a unique pan-FMDV MAb (1F10) as detector conjugate. Capture MAbs with the broadest intratypic reactivity were selected for each of the four FMDV serotypes by screening large panels of candidate MAbs with a wide spectrum of representative FMDV isolates. An additional pan-FMDV ELISA using 1F10 MAb for both capture and detection was used to complement the specific typing ELISAs to detect virus isolates, which might escape binding to the selected serotype-specific MAbs. This multiplex ELISA was prepared in a stabilized format, with immunoplates pre-coated with six MAbs and positive antigen controls already trapped by the relevant MAb, with the view to make available a diagnostic kit. Diagnostic performance of the MAbs-multiplex ELISA was analysed using 299 FMDV-positive epithelial suspensions representative of the antigenic and genomic variability within each serotype. Overall, the results provided evidence that the diagnostic performance of this assay platform is improved compared to the more complex polyclonal-based antigen detection ELISA; combining data from different serotypes and referring to the gold standard tests (i.e. virus isolation and/or RT-PCR), the MAbs-multiplex ELISA showed a sensitivity of 79% for the serotype-specific ELISA, compared to 72% for the polyclonal ELISA. The absence of reactivity of a minority of FMDV strains using the MAbs-multiplex ELISA can largely be attributed to deteriorated or low antigen concentration in the sample. This multiplex ELISA is simple, rapid and stable. FMDV antigenic diversity was adequately covered by the selected MAbs. Therefore, it can be used to replace existing polyclonal ELISAs for FMDV detection and serotyping.

Genetic characterization of foot-and-mouth disease viruses, Ethiopia, 1981-2007.

Foot-and-mouth disease (FMD) is endemic to sub-Saharan Africa. To further understand its complex epidemiology, which involves multiple virus serotypes and host species, we characterized the viruses recovered from FMD outbreaks in Ethiopia during 1981-2007. We detected 5 of the 7 FMDV serotypes (O, A, C, Southern African Territories [SAT] 1, and SAT 2). Serotype O predominated, followed by serotype A; type C was not recognized after 1983. Phylogenetic analysis of virus protein 1 sequences indicated emergence of a new topotype within serotype O, East Africa 4. In 2007, serotype SAT 1 was detected in Ethiopia and formed a new distinct topotype (IX), and serotype SAT 2 reappeared after an apparent gap of 16 years. The diversity of viruses highlights the role of this region as a reservoir for FMD virus, and their continuing emergence in Ethiopia will greatly affect spread and consequent control strategy of the disease on this continent.

Multiple origins of foot-and-mouth disease virus serotype Asia 1 outbreaks, 2003-2007.

We investigated the molecular epidemiology of foot-and-mouth disease virus (FMDV) serotype Asia 1, which caused outbreaks of disease in Asia during 2003-2007. Since 2004, the region affected by outbreaks of this serotype has increased from disease-endemic countries in southern Asia (Afghanistan, India, Iran, Nepal, Pakistan) northward to encompass Kyrgyzstan, Tajikistan, Uzbekistan, several regions of the People's Republic of China, Mongolia, Eastern Russia, and North Korea. Phylogenetic analysis of complete virus capsid protein 1 (VP1) gene sequences demonstrated that the FMDV isolates responsible for these outbreaks belonged to 6 groups within the Asia 1 serotype. Some contemporary strains were genetically closely related to isolates collected historically from the region as far back as 25 years ago. Our analyses also indicated that some viruses have spread large distances between countries in Asia within a short time.

Development and laboratory validation of a lateral flow device for the detection of foot-and-mouth disease virus in clinical samples.

A lateral flow device (LFD) for the detection of all seven serotypes of foot-and-mouth disease virus (FMDV) was developed using a monoclonal antibody (Mab 1F10) shown to be pan-reactive to FMDV strains of each serotype by ELISA. The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia (304 positive and 1003 negative samples) from suspected cases of vesicular disease collected from 86 countries between 1965 and 2008 and negative samples collected from healthy animals. The diagnostic sensitivity of the LFD for FMDV was similar at 84% compared to 85% obtained by the reference method of antigen ELISA, and the diagnostic specificity of the LFD was approximately 99% compared to 99.9% for the ELISA. The device recognized FMDV strains of wide diversity of all seven serotypes but weaker reactions were often evident with those of type SAT 2, several viruses of which were not detected. Reactions with the viruses of swine vesicular disease and vesicular stomatitis that produce clinically indistinguishable syndromes in pigs and cattle, did not occur. The test procedure was simple and rapid, and typically provided a result within 1-10min of sample addition. Simple homogenizers that could be used in field conditions for preparing epithelial suspensions were demonstrated to be effective for LFD application. These data illustrate the potential for the LFD to be used next to the animal in the pen-side diagnosis of FMD and for providing rapid and objective support to veterinarians in their clinical judgment of the disease.

Molecular epidemiology of foot-and-mouth disease viruses from South East Asia 1998-2006: the Lao perspective.

Foot-and-mouth disease (FMD) causes sporadic disease outbreaks in the Lao People's Democratic Republic (Lao PDR) and appears to be endemic within a livestock population largely susceptible to infection. As Lao PDR is a major thoroughfare for transboundary animal movement, regular FMD outbreaks occur causing economic hardship for farmers and their families. The dominant serotype causing outbreaks between 1998 and 2006 was type O. Using phylogenetic analysis, type O isolated viruses were divided into two topotypes: South East Asia (SEA) and the Middle East-South Asia (ME-SA). Type A virus was reported only in 2003 and 2006 and type Asia 1 only in 1996 and 1998.

Performance of real-time reverse transcription polymerase chain reaction for the detection of foot-and-mouth disease virus during field outbreaks in the United Kingdom in 2007.

Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). The present report describes the practical steps undertaken to deploy a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to process the samples received during the outbreaks of FMD in the United Kingdom in 2007. Two independent real-time RT-PCR assays targeting different regions (5'UTR and 3D) of the FMD virus (FMDV) genome were used to confirm the presence of FMDV in clinical samples collected from the first infected premises. Once the FMDV strain responsible had been sequenced, a single real-time RT-PCR assay (3D) was selected to test a total of 3,216 samples, including material from all 8 infected premises. Using a 96-well automated system to prepare nucleic acid template, up to 84 samples could be processed within 5 hr of submission, and up to 269 samples were tested per working day. A conservative cut-off was used to designate positive samples, giving rise to an assay specificity of 99.9% or 100% for negative control material or samples collected from negative premises, respectively. For the first time, real-time RT-PCR results were used to recognize preclinical FMD in a cattle herd. Furthermore, during the later stages of the outbreaks, the real-time RT-PCR assay supported an active surveillance program within high-risk cattle herds. To the authors' knowledge, this is the first documented use of real-time RT-PCR as a principal laboratory diagnostic tool following introduction of FMD into a country that was FMD-free (without vaccination) and highlights the advantages of this assay to support control decisions during disease outbreaks.

Diagnostic evaluation of multiplexed reverse transcription-PCR microsphere array assay for detection of foot-and-mouth and look-alike disease viruses.

A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and-mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the 17 primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true-negative samples collected from healthy animals. The mRT-PCR assay results were compared to those of two singleplex rRT-PCR assays, using virus isolation with antigen enzyme-linked immunosorbent assays as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 99.3%) for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses, such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n = 2) and bovine viral diarrhea virus (n = 2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized by using focused single-target rRT-PCR assays.

Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus.

A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody was developed and evaluated for the detection of foot-and-mouth disease virus (FMDV) in clinical samples collected from field cases of disease. The FMDV-specific PLA was found to be 100 times more sensitive for virus detection than the commonly used antigen capture-ELISA (AgELISA). As few as five TCID50 were detected in individual assays, which was comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was capable of detecting diverse isolates from all seven FMDV serotypes, the diagnostic sensitivity of the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid and simple tool for use in FMD diagnosis.

Detection of foot-and-mouth disease virus by nucleic acid sequence-based amplification (NASBA).

A study was conducted to evaluate the performance of a nucleic acid sequence-based amplification (NASBA) assay for the detection of foot-and-mouth disease virus (FMDV). Two detection methods: NASBA-electrochemiluminescence (NASBA-ECL) and a newly developed NASBA-enzyme-linked oligonucleotide capture (NASBA-EOC) were evaluated. The diagnostic sensitivity of these assays was compared with other laboratory-based methods using 200 clinical samples collected from different regions of the world. Assay specificity was also assessed using samples (n=43) of other viruses that cause vesicular disease in livestock and genetic relatives of FMDV. Concordant results were generated for 174/200 (87.0%) of suspect FMD samples between NASBA-ECL and real-time RT-PCR. In comparison with the virus isolation (VI) data, the sensitivity of the NASBA-ECL assay was 92.9%, which was almost identical to that of the real-time RT-PCR (92.4%) for the same set of samples. There was broad agreement between the results of the NASBA-ECL and the simpler NASBA-EOC detection method for 97.1% of samples. In conclusion, this study provides further data to support the use of NASBA as a rapid and sensitive diagnostic method for the detection and surveillance of FMDV.

Foot-and-mouth disease virus serotype A in Egypt.

We describe the characterization of a foot-and-mouth disease (FMD) serotype A virus responsible for recent outbreaks of disease in Egypt. Phylogenetic analysis of VP1 nucleotide sequences demonstrated a close relationship to recent FMD virus isolates from East Africa, rather than to viruses currently circulating in the Middle East.

Implementation of a one-step real-time RT-PCR protocol for diagnosis of foot-and-mouth disease.

An automated one-step real-time reverse transcription polymerase chain reaction (rRT-PCR) protocol was optimised and evaluated for the routine diagnosis of foot-and-mouth disease (FMD). Parallel testing of RNA samples (n=257) indicated that this assay has a diagnostic sensitivity at least equivalent to the automated two-step rRT-PCR protocol previously used for the laboratory detection of FMD virus (FMDV). This more rapid and economical one-step protocol will play a key role in contingency planning for any future outbreaks of FMD in the United Kingdom (UK).

Development of a real-time reverse transcription polymerase chain reaction assay for detection of marine caliciviruses (genus Vesivirus).

Marine caliciviruses form a distinct lineage within the genus Vesivirus (family Caliciviridae). This group includes vesicular exanthema of swine virus (VESV) and San Miguel sea lion virus (SMSV) and other related viruses which have been proposed to be marine in origin isolated from a variety of terrestrial and marine animals. Rapid and reliable detection of marine caliciviruses is important as these viruses appear to be widespread and can cause vesicular disease in a wide variety of susceptible hosts including pigs and experimentally infected cattle where clinical signs cannot be easily distinguished from foot-and-mouth disease (FMD), swine vesicular disease (SVD) and vesicular stomatitis (VS). A real-time RT-PCR assay targeting conserved nucleotide sequences in the RNA-dependent RNA polymerase (3D) region of the genome successfully detected cell culture-grown virus preparations of more than thirty marine calicivirus serotypes. Only the atypical SMSV serotypes 8 and 12 failed to be detected, which provided further indication of genetic divergence between these and the other calicivirus serotypes said to be marine in origin. The real-time RT-PCR assay also specifically amplified RNA from samples collected following experimental inoculation of pigs with VESV. No cross-reactivity was demonstrated when the assay was tested with RNA prepared from representative viruses of FMD, SVD and VS. The real-time RT-PCR assay described is a sensitive and specific tool for detection and differential diagnosis of these viruses from other vesicular-disease causing viruses.

Indirect sandwich ELISA for antigen detection of African swine fever virus: comparison of polyclonal and monoclonal antibodies.

Two indirect, sandwich ELISAs are described for use in African swine fever (ASF) diagnosis. One assay uses polyclonal serum raised in rabbits and guinea pigs against the cytoplasmic soluble ASF virus protein and the second, a combination of monoclonal antibody raised against the VP73 protein and rabbit polyclonal serum. Both assays have been shown to detect antigen of representative field strains of phylogenetically distinct groupings of ASF virus but the ELISA, which utilises polyclonal antisera was slightly more sensitive than that using the monoclonal antibody.

Detection of foot-and-mouth disease virus: comparative diagnostic sensitivity of two independent real-time reverse transcription-polymerase chain reaction assays.

Rapid and accurate diagnosis is central to the effective control of foot-and-mouth disease (FMD). It is now recognized that reverse-transcription polymerase chain reaction (RT-PCR) assays can play an important role in the routine detection of FMD virus (FMDV) in clinical samples. The aim of this study was to compare the ability of 2 independent real-time RT-PCR (rRT-PCR) assays targeting the 5' untranslated region (5'UTR) and RNA polymerase (3D) to detect FMDV in clinical samples. There was concordance between the results generated by the 2 assays for 88.1% (347 of 394) of RNA samples extracted from suspensions of epithelial tissue obtained from suspect FMD cases. The comparison between the 2 tests highlighted 19 FMDV isolates (13 for the 5'UTR and 6 for the 3D assay), which failed to produce a signal in 1 assay but gave a positive signal in the other. The sequence of the genomic targets of selected isolates highlighted nucleotide substitutions in the primer or probe regions, thereby providing an explanation for negative results generated in the rRT-PCR assays. These data illustrate the importance of the continuous monitoring of circulating FMDV field strains to ensure the design of the rRT-PCR assay remains fit for purpose and suggest that the use of multiple diagnostic targets could further enhance the sensitivity of molecular methods for the detection of FMDV.

Utility of recombinant integrin alpha v beta6 as a capture reagent in immunoassays for the diagnosis of foot-and-mouth disease.

Recombinant integrin alpha v beta6 was evaluated as a capture ligand in a sandwich ELISA for the detection and serotyping of foot-and-mouth disease (FMD) virus. Our routinely applied method employs seven serotype-specific rabbit polyclonal antibodies as capture ligands and seven serotype-specific guinea pig polyclonal antibodies as detecting reagents. The recombinant integrin bound FMD virus of all seven serotypes but not that of another vesicular disease, swine vesicular disease (SVD). Considerable heterotypic cross-reactions were evident when using the integrin capture ligand in combination with guinea pig detecting antibodies but totally type-specific reactions resulted when serotype-specific monoclonal antibodies (mabs) were used instead of the guinea pig reagents. The specificity of reaction of the integrin capture/mab detector combination was superior to that of our routinely employed rabbit/guinea pig ELISA and offers an improvement for test interpretation. As a universal trapping reagent for all FMD virus serotypes the alpha v beta6 recombinant protein also has the potential for application in other test procedures for viral identification (e.g. pen-side chromatographic strip-tests, biosensors, immunocapture RT-PCR, antigenic characterization procedures and monoclonal antibody profiling of emerging field virus strains) and in antibody detection assays employed for the diagnosis of FMD.

Evaluation of real-time reverse transcription polymerase chain reaction assays for the detection of swine vesicular disease virus.

Differential detection of swine vesicular disease virus (SVDV) from the other vesicular disease viruses of foot-and-mouth disease (FMD), vesicular stomatitis (VS) and vesivirus is important as the vesicular lesions produced by these viruses are indistinguishable in pigs. Two independent sets of primers and probe, designed from nucleotide sequences within the 5' untranslated region (UTR) of the SVDV genome, were evaluated in a real-time (5' nuclease probe-based or fluorogenic) PCR format. Although both primers/probe sets failed to detect one isolate, the assays successfully amplified RNA extracted from epithelial suspensions (ES) and cell culture grown virus preparations from clinical samples representing all currently designated phylogenetic groups of SVDV. Furthermore, no cross-reactivity was demonstrated when these primer/probe sets were tested with RNA prepared from all seven serotypes of FMD virus (FMDV) and from selected isolates of VS virus (VSV), vesivirus and teschoviruses. These assays provide sensitive and rapid alternatives to supplement the routine procedures of ELISA and virus isolation for SVDV diagnosis. The two independent sets of primers/probe can be used routinely while only one of the primers/probe sets would typically be used in SVDV diagnosis during an outbreak.

Evaluation of automated RT-PCR to accelerate the laboratory diagnosis of foot-and-mouth disease virus.

Automated fluorogenic (5' nuclease probe-based) reverse transcription polymerase chain reaction (RT-PCR) procedures were evaluated for the diagnosis of foot-and-mouth disease (FMD) using suspensions of vesicular epithelium, heparinised or clotted blood, milk and oesophageal-pharyngeal fluid ('probang') samples from the United Kingdom (UK) 2001 epidemic and on sera from animals experimentally infected with the outbreak serotype O FMD virus strain. A MagNA Pure LC was initially programmed to automate the nucleic acid extraction and RT procedures with the PCR amplification carried out manually by fluorogenic assay in a GeneAmp 5700 Sequence Detection System. This allowed 32 samples to be tested by one person in a typical working day or 64 samples by two people within 10-12 h. The PCR amplification was later automated and a protocol developed for one person to complete a single test incorporating 96 RT-PCR results within 2 working days or for two people to do the same thing in around 12 h. The RT-PCR results were directly compared with those obtained by the routine diagnostic tests of ELISA and virus isolation in cell culture. The results on blood, probang and milk samples were in broad agreement between the three procedures but specific RT-PCR protocols for such material have to be fully optimised as perhaps have the positive-negative acceptance criteria. However, the automated RT-PCR achieved definitive diagnostic results (positive or negative) on supernatant fluids from first passage inoculated cell cultures and its sensitivity was greater than ELISA on suspensions of vesicular epithelium (ES) and at least equivalent to that of virus isolation in cell culture. The combined tests of ELISA, virus isolation in cell culture and RT-PCR might, therefore, only be required for confirmation of a first outbreak of FMD in a previously FMD-free country. Should a prolonged outbreak subsequently occur, then either ELISA plus RT-PCR or else RT-PCR alone could be used as the laboratory diagnostic tool(s). Either approach would eliminate the requirement for sample passage in cell culture and considerably advance the issue of laboratory diagnostic test results.

Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay.

A fluorogenic RT-PCR (5'-nuclease probe-based) assay using a primer/probe set designed from the internal ribosomal entry site region of the virus genome was developed for the specific detection of all seven serotypes of foot-and-mouth disease (FMD) virus in epithelial suspensions and cell culture virus preparations. The reverse transcription polymerase chain reaction (RT-PCR) specifically detected FMD virus in sample submissions from the UK 2001 FMD outbreak with greater sensitivity than our conventional RT-PCR procedure and our routine diagnostic procedures of ELISA and virus isolation in cell culture. The fluorogenic RT-PCR provides relatively fast results, enables a quantitative assessment to be made of virus amounts and can handle more samples and/or replicates of samples in a single assay than the conventional RT-PCR procedure. Therefore it is seen as a valuable tool to complement the routine diagnostic procedures for FMD virus diagnosis.

Sensitivity of primary cells immortalised by oncogene transfection for the detection and isolation of foot-and-mouth disease and swine vesicular disease viruses.

Primary cells derived from calf thyroid (CTY), calf kidney (CK) and piglet kidney (PK) were immortalised by oncogene transfection and their susceptibility to infection by foot-and-mouth disease (FMD) virus and swine vesicular disease (SVD) virus examined. Eighty-five immortalised cell lines (47 CTY, 20 CK and 18 PK) proved stable upon repeated cell culture passage and many supported the growth of FMD virus and several of the PK cell lines supported SVD virus. However, none of the immortalised lines exhibited either the degree of sensitivity or the specificity for all virus serotypes and strains as shown by primary CTY and IB-RS-2 cell cultures which are routinely employed for vesicular virus diagnosis.