Katja Ebert

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.

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.

Normal variation in thermal radiated temperature in cattle: implications for foot-and-mouth disease detection

BackgroundThermal imagers have been used in a number of disciplines to record animal surface temperatures and as a result detect temperature distributions and abnormalities requiring a particular course of action. Some work, with animals infected with foot-and-mouth disease virus, has suggested that the technique might be used to identify animals in the early stages of disease. In this study, images of 19 healthy cattle have been taken over an extended period to determine hoof and especially coronary band temperatures (a common site for the development of FMD lesions) and eye temperatures (as a surrogate for core body temperature) and to examine how these vary with time and ambient conditions.ResultsThe results showed that under UK conditions an animals hoof temperature varied from 10°C to 36°C and was primarily influenced by the ambient temperature and the animals activity immediately prior to measurement. Eye temperatures were not affected by ambient temperature and are a useful indicator of core body temperature.ConclusionsGiven the variation in temperature of the hooves of normal animals under various environmental conditions the use of a single threshold hoof temperature will be at best a modest predictive indicator of early FMD, even if ambient temperature is factored into the evaluation.

Diagnostic Evaluation of Multiplexed Reverse Transcription-PCR Microsphere Array Assay for Detection of Foot-and-Mouth and Look-Alike Disease Viruses

ABSTRACT 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.

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.

Detection of African swine fever virus by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of African swine fever virus (ASFV). This assay targets the topoisomerase II gene of ASFV and its specificity was confirmed by restriction enzyme digestion of the reaction products. The analytical sensitivity of this ASFV LAMP assay was at least 330 genome copies, and the test was able to detect representative isolates of ASFV (n=38) without cross-reacting with classical swine fever virus. The performance of the LAMP assay was compared with other laboratory tests used for ASF diagnosis. Using blood and tissue samples collected from pigs experimentally infected with ASFV (Malawi isolate), there was good concordance between the LAMP assay and real-time PCR. In addition to detecting the reaction products using either agarose gels or real-time PCR machines, it was possible to visualise dual-labelled biotin and fluorescein ASFV LAMP amplicons using novel lateral flow devices. This assay and detection format represents the first step towards developing a practical, simple-to-use and inexpensive molecular assay format for ASF diagnosis in the field which is especially relevant to Africa where the disease is endemic in many countries.

Development and Initial Results of a Low Cost, Disposable, Point-of-Care Testing Device for Pathogen Detection

Development of small footprint, disposable, fast, and inexpensive devices for pathogen detection in the field and clinic would benefit human and veterinary medicine by allowing evidence-based responses to future out breaks. We designed and tested an integrated nucleic acid extraction and amplification device employing a loop-mediated isothermal amplification (LAMP) or reverse transcriptase-LAMP assay. Our system provides a screening tool with polymerase-chain-reaction-level sensitivity and specificity for outbreak detection, response, and recovery. Time to result is ~90 min. The device utilizes a swab that collects sample and then transfers it to a disc of cellulose-based nucleic acid binding paper. The disc is positioned within a disposable containment tube with a manual loading port. In order to test for the presence of target pathogens, LAMP reagents are loaded through the tubes port into contact with the sample containing cellulose disc. The reagents then are isothermally heated to 63°C for ~1 h to achieve sequence-specific target nucleic acid amplification. Due to the presence of a colorimetric dye, amplification induces visible color change in the reagents from purple to blue. As initial demonstrations, we detected methicillin resistant Staphylococcus aureus genomic DNA, as well as recombinant and live foot-and-mouth disease virus.

Molecular characterisation of foot-and-mouth disease viruses from Pakistan, 2005-2008.

Foot-and-mouth disease (FMD), an economically important disease of cloven-hoofed animals, is endemic in Pakistan where three virus serotypes are present (O, A and Asia 1). Fifty-eight clinical samples collected between 2005 and 2008 from animals with suspected FMD in various locations in Pakistan were subjected to virus isolation on primary cell culture, antigen ELISA and real-time RT-PCR (rRT-PCR). Viruses were isolated from 32 of these samples and identified as FMDV type O (n = 31) or type A (n = 1). Foot-and-mouth disease virus (FMDV) genome was detected in a further 11 samples by real-time RT-PCR. Phylogenetic analyses of the VP1 nucleotide sequences showed that all of the type O viruses belonged to the MIDDLE EAST-SOUTH ASIA topotype with the majority belonging to the PanAsia-2 lineage; a single example of the older PanAsia lineage was identified. The single FMDV type A virus belonged to the ASIA topotype, but did not cluster with known strains that are currently circulating (such as Iran-05) and was not closely related to other type A viruses from the region. These findings demonstrate the widespread distribution of O-PanAsia-2 in Pakistan and the presence of undisclosed novel type A lineages in the region.

Evaluation of automated nucleic acid extraction methods for virus detection in a multicenter comparative trial

Five European veterinary laboratories participated in an exercise to compare the performance of nucleic acid extraction robots. Identical sets of coded samples were prepared using serial dilutions of bovine viral diarrhoea virus (BVDV) from serum and cell culture propagated material. Each laboratory extracted nucleic acid from this panel using available robotic equipment (12 separate instruments, comprising 8 different models), after which the processed samples were frozen and sent to a single laboratory for subsequent testing by real-time RT-PCR. In general, there was good concordance between the results obtained for the different automated extraction platforms. In particular, the limit of detection was identical for 9/12 and 8/12 best performing robots (using dilutions of BVDV infected-serum and cell culture material, respectively), which was similar to a manual extraction method used for comparison. The remaining equipment and protocols used were less sensitive, in an extreme case for serum, by a factor of 1000. There was no evidence for cross-contamination of RNA template in any of the negative samples included in these panels. These results are not intended to replace local optimisation and validation, but provide reassurance to laboratories to indicate that the best performing optimised nucleic acid extraction systems can have similar performance.