Nigel P. Ferris

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 O1 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.

Molecular Epidemiology of the Foot-and-Mouth Disease Virus Outbreak in the United Kingdom in 2001

ABSTRACT The objective of this study was to quantify the extent to which the genetic diversity of foot-and-mouth disease virus (FMDV) arising over the course of infection of an individual animal becomes fixed, is transmitted to other animals, and thereby accumulates over the course of an outbreak. Complete consensus sequences of 23 genomes (each of 8,200 nucleotides) of FMDV were recovered directly from epithelium tissue acquired from 21 farms infected over a nearly 7-month period during the 2001 FMDV outbreak in the United Kingdom. An analysis of these consensus sequences revealed very few apparently ambiguous sites but clear evidence of 197 nucleotide substitutions at 191 different sites. We estimated the rate of nucleotide substitution to be 2.26 × 10−5 per site per day (95% confidence interval [CI], 1.75 × 10−5 to 2.80 × 10−5) and nucleotide substitutions to accrue in the consensus sequence at an average rate of 1.5 substitutions per farm infection. This is a sufficiently high rate showing that detailed histories of the transmission pathways can be reliably reconstructed. Coalescent methods indicated that the date at which FMDV first infected livestock in the United Kingdom was 7 February 2001 (95% CI, 20 January to 19 February 2001), which was identical to estimates obtained on the basis of purely clinical evidence. Nucleotide changes appeared to have occurred evenly across the genome, and within the open reading frame, the ratio of nonsynonymous-to-synonymous change was 0.09. The ability to recover particular transmission pathways of acutely acting RNA pathogens from genetic data will help resolve uncertainties about how virus is spread and could help in the control of future epidemics.

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.

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

Virus diversity indicates a virus reservoir in this country.

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.

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

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.

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.

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

Viruses in 6 genetic groups have caused recent outbreaks in Asia.Emerging outbreaks of zoonotic diseases are affecting humans at an alarming rate. Until the ecological factors associated with zoonoses are better understood, disease emergence will continue. For Lyme disease, disease suppression has been demonstrated by a dilution effect, whereby increasing species diversity decreases disease prevalence in host populations. To test the dilution effect in another disease, we examined 17 ecological variables associated with prevalence of the directly transmitted Sin Nombre virus (genus Hantavirus, etiologic agent of hantavirus pulmonary syndrome) in its wildlife host, the deer mouse (Peromyscus maniculatus). Only species diversity was statistically linked to infection prevalence: as species diversity decreased, infection prevalence increased. The increase was moderate, but prevalence increased exponentially at low levels of diversity, a phenomenon described as zoonotic release. The results suggest that species diversity affects disease emergence.

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.