Destructive tension: mathematics versus experience--the progress and control of the 2001 foot and mouth disease epidemic in Great Britain.

The 2001 foot and mouth disease epidemic in Great Britain was characterised by control using both traditional and novel methods, some resulting from conclusions of mathematical models. Seven days before the implementation of the novel controversial automatic pre-emptive culling of all susceptible livestock on premises adjacent to infected premises (the 'contiguous cull'), the spread of infection had already been controlled by a combination of the traditional stamping out policy with a national movement ban on livestock. A second controversial novel policy requiring the slaughter of sheep within 3 km of premises on which disease had been confirmed (the 3-km cull) also commenced after the peak of infection spread, was untargeted and took several weeks to complete; serosurveillance of culled sheep detected infection in only one flock, suggesting that cryptic infection of sheep was not propagating the epidemic. Extensive post-epidemic serological surveillance of sheep found only a small number of seropositive animals in a very few flocks, suggesting that foot and mouth disease may self-limit in extensive sheep populations. The epidemic was finally brought to an end following the introduction of enhanced agricultural movement restrictions and biosecurity measures. A welfare culling scheme of unaffected animals was required to support the prolonged national livestock movement ban. The models that supported the contiguous culling policy were severely flawed, being based on data from dissimilar epidemics; used inaccurate background population data, and contained highly improbable biological assumptions about the temporal and quantitative parameters of infection and virus emission in infected herds and flocks.

Certification for regional and international trade in livestock commodities: the need to balance credibility and enterprise.

The current system for the certification of internationally traded animal commodities can act as a barrier to developing countries accessing high-value international markets. In this Viewpoint article, Gavin Thomson and colleagues discuss the situation as it stands and identify inconsistencies with respect to the certification process. They suggest ways to address the lack of capacity for credible certification in some developing countries that will encourage market access for livestock commodities. They emphasise the role of mechanisms other than demonstrating that an area of production is free from a range of animal diseases, arguing that this could be of significant benefit to developing regions and countries, but that a reliable and independent system of certification based on international standards is essential.

Foot-and-mouth disease virus strains isolated from persistently infected cell cultures are attenuated for mice and cattle.

It was previously reported that during serial passage of a BHK-derived cell line persistently infected with foot-and-mouth disease virus, (termed C1-BHK-Rc1) the virus became increasingly more virulent for BHK-21 cells (de la Torre et al 1988). Virus strains isolated from different cell passage levels were tested for virulence in mice and cattle. The results showed that in the course of persistence in BHK cells, FMDV became progressively less virulent for mice and cattle.

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.

Development of a novel real-time RT-PCR assay for quantitation of foot-and-mouth disease virus in diverse porcine tissues.

Pigs are more difficult to immunise and more variable in their response to foot-and-mouth disease (FMD) than other livestock species. This has important consequences for FMD control during both prophylactic vaccination programmes in endemic situations and when emergency vaccination may be used as an adjunct to stamping out during outbreaks in countries normally free from the disease. The rapid and effective control of FMD in pigs is especially important in regions of high pig density since infected pigs have the potential to generate plumes of airborne virus and spread infection beyond the immediate control area. Increased knowledge of the kinetics of FMDV replication in pigs, especially in their respiratory tracts, could create opportunities for strategies to improve FMD vaccines for pigs. A fluorogenic TaqMan RT-PCR assay specific for the IRES (internal ribosome entry site) sequence of the O(1) Kaufbeuren/Lausanne strain of FMDV has been developed for this purpose. The assay had a sensitivity of 0.1 TCID(50)/ml, and a dynamic range of at least eight orders of magnitude. It was found that an established method of quantitation, relative to a housekeeping gene, exhibited two significant shortcomings when applied to a set of 16 anatomically highly diverse solid tissues: (i) tissue differences in housekeeping gene expression caused errors of up to 60-fold in estimated FMDV concentrations; and (ii) variability in total RNA yields caused unpredictable saturation of RT reactions, which in turn caused errors of up to 250-fold in the estimated FMDV concentration. A novel quantitation strategy, designated the C(t)(min) method, was developed to overcome these problems. The C(t)(min) method was based on the the RT-PCR examination of a dilution series of spectrophotometrically quantitated total RNA, spanning the optimum for the RT-PCR system used. The new method was influenced minimally by any tissue-specific RT-PCR inhibitors and was used to determine FMDV concentrations in tissues from four experimentally infected pigs. The results suggest that the lungs play a less important role in the replication of FMDV in pigs than was thought previously.

Improved detection of persistent foot-and-mouth disease infection in cattle by the polymerase chain reaction.

Persistent foot-and-mouth disease (FMD) infection of cattle was established by exposure to infected pigs. Oesophageal-pharyngeal fluid samples collected ante-mortem and tissue samples obtained post-mortem were tested for virus by conventional means and for viral RNA by the polymerase chain reaction (PCR). The results showed that the PCR was superior for detecting the carrier state in tissue samples. A high neutralising activity in tissues probably influenced the yields of virus obtained by infectivity assay.

Detection of foot-and-mouth disease virus RNA in clinical samples and cell culture isolates by amplification of the capsid coding region.

Foot-and-mouth disease is one of the most economically important virus diseases of livestock. Two important requirements for the control of this disease are rapid laboratory diagnosis and epidemiological investigation. The use of the polymerase chain reaction method (PCR) to amplify specific nucleic acid regions offers the unique possibility of combining swift viral detection with the production of genetic material suitable for sequencing and other methods of molecular epidemiological analysis. The sequencing of the region of foot-and-mouth disease virus (FMDV) genome encoding the capsid proteins of the virus (approximately 2260 bps), provides valuable information that adds to the molecular characterisation of an isolate. This paper describes the use of the PCR for the amplification of this region of the FMDV genome from bovine clinical samples and cell culture isolates. Suitable pairs of oligonucleotide primers were selected from the published sequence of FMDV type O1, Kaufbeuren. One primer set amplified 2091 bps of the capsid coding region of all seven serotypes of FMDV. The other primer set amplified 216 bp from this region of FMDV type O1, BFS 1860, in nucleic acid extracts from several clinical samples. Nucleic acid extracts from the picornaviruses, bovine enterovirus and swine vesicular disease virus, which affect the same animals, were not amplified. Direct sequencing was carried out on the amplified fragments and showed that the PCR products were > 98% homologous to published FMDV sequences.

Validation of a foot-and-mouth disease antibody screening solid-phase competition ELISA (SPCE).

This paper describes the validation of a solid-phase competition enzyme-linked immunosorbent assay (SPCE) for the serological detection of antibody to serotype O foot-and-mouth disease (FMD) in sheep, cattle and pigs. The specificity of the SPCE was calculated from the results of testing known negative sera from sheep, cattle and pigs (n=3030, 1418 and 1495, respectively). The mean percentage inhibition (PI) for known negative sheep, cattle and pig sera were 19.3, 24.1 and 20.8%, respectively. The specificity of the SPCE at a cut-off point (COP) of 60 PI was 99.50% for sheep sera, 99.44% for cattle sera and 100% for pig sera. The analytical sensitivity of the SPCE was examined by testing sera from sheep, cattle and pigs. Based on the testing of serial bleeds from experimentally infected animals, seroconversion at the 60 PI COP occurred between 4 and 9 days post-infection or -exposure, similar to that observed using the virus neutralisation test (VNT) with a COP of 1/45. When applied to 267 sheep and 143 pig samples, that were obtained in Great Britain (GB) during the 2001 FMD UK outbreak, the SPCE identified more positive samples than did the VNT. Estimates of the accuracy, repeatability and reproducibility of the SPCE were verified during the large-scale serosurveillance necessitated by the 2001 outbreak. Results from field and experimental sera showed that when compared against the VNT, the sensitivity of the SPCE was less affected by the choice of virus strain used in the test. Using the O(1) UKG 2001 FMD virus in the VNT with samples representative of the uninfected GB sheep population, the test specificity was 100% at a COP of 1/45.

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.

The influence of normal guinea-pig serum and tissue culture assay system on foot-and-mouth disease virus neutralisation.

The inclusion of normal guinea-pig serum in neutralisation reactions involving foot-and-mouth disease virus (FMDV) increased the neutralisation titre and rate of neutralisation by guinea-pig antiserum derived from animals convalescent from FMDV. Such inclusion had little or no effect on neutralisation involving guinea-pig antiserum collected early in infection or early or convalescent bovine antisera. Higher neutralisation titres and more rapid neutralisation were found from assay in bovine thyroid cells than in IB-RS-2 cells. Not all the increase in neutralising activity was removed by heating at 56 degrees C, indicating that other factors in addition to complement were responsible. In some tests a virus fraction resisting neutralisation was found and, when rabbit anti-guinea-pig gamma-globulin was added, an increase in neutralisation resulted, suggesting that most of the persistent virus fraction was due to the presence of infective virus-antibody complexes.

The pathogenesis and diagnosis of foot-and-mouth disease.

The pathogenesis of foot-and-mouth disease (FMD) is reviewed, taking account of knowledge gained from field and experimental studies and embracing investigations at the level of the virus, the cell, the organ, the whole animal and the herd or flock. The review also addresses the immune response and the carrier state in FMD. Progress made in understanding the pathogenesis of the disease is highlighted in relation to developments in diagnosis and methods of control.

Exposure of sheep to natural aerosols of foot-and-mouth disease virus.

Sheep taken individually and allowed to inhale air being drawn along a duct from a cabinet containing pigs acutely infected with foot-and-mouth disease virus for 10 or 15 minute periods were infected by doses as low as 10 TCID50 of virus. The most consistent and reliable indicators of infection were viraemia and seroconversion. The mean times from exposure to onset of viraemia, pyrexia and the appearance of vesicular lesions were 2.5, 3.8 and 4.7 days, respectively. Neither the time from exposure to first detectable viraemia nor vesication correlated with dose. Around 27 per cent of sheep which were known to have been infected did not develop vesicles.

Transmission of foot-and-mouth disease by vaccinated cattle following natural challenge.

Cattle vaccinated with a conventional monovalent type O1 foot-and-mouth disease (FMD) vaccine were challenged between four and 21 days after vaccination by short-term exposure to homologous airborne virus produced by pigs. Transmission was then assessed by housing susceptible cattle with the vaccinated animals and testing and observing all the animals for signs of infection and clinical disease. All 18 cattle vaccinated three weeks before challenge resisted clinical disease and although four contracted subclinical infection, there was no transmission to susceptible cattle in contact. One of the two groups of cattle vaccinated two weeks previously transmitted subclinical infection, but not disease, to susceptible animals housed with them from day 0 after challenge. Subclinical infection was manifested by a transient viraemia which was not followed by a detectable circulating antibody response. Shorter periods (seven or four days) from vaccination to challenge resulted in transmission of disease from clinically normal vaccinated to in-contact animals in one of two experiments. The severe challenge presented by the diseased in-contact animals than overwhelmed the immunity of the vaccinated animals. The results indicate that during emergency vaccination programmes it is advisable to vaccinate all FMD-susceptible animals within the vaccination zone and that at the outer boundary of the zone vaccinated animals should be kept separated from unvaccinated animals for at least three weeks.

Sites of release of airborne foot-and-mouth disease virus from infected pigs.

Pigs infected with foot-and-mouth disease virus by different routes of exposure were air-sampled individually, first as 'intact' (I-) pigs and then as 'intubated' (T-) pigs, using an endotracheal tube. Irrespective of the route of infection it was found that during the early stages of disease more virus was recovered from I-pigs than from T-pigs. Most of the virus from I-pigs during incubation and early disease was associated with large and medium sized particles. T-pigs infected by direct or indirect contact excreted a range of particle sizes at this time but T-pigs infected by inoculation only excreted small particles. During advanced disease all sizes of particle were excreted by I- and T-pigs. Greater amount of airborne virus were recovered at this time from I-pigs than T-pigs infected by indirect contact but I-pigs infected by intravenous or intradermal inoculation excreted less infectivity than T-pigs. The results show that the respiratory tract is involved during the early stages of foot-and-mouth disease in pigs infected by either natural or experimental routes of exposure and suggest that upper respiratory infection precedes lower.

Serological response of pigs infected with Aujeszky's disease virus.

The temporal development of antibody in four groups of pigs infected with different Aujeszky's disease virus isolates was examined. The enzyme-linked immunosorbent assay detected antibody by five to six days after infection and the antibody-dependent cell-mediated cytotoxicity assay detected antibody seven to nine days after infection. Neutralising antibody was first detected nine to 10 days after infection, whereas assays measuring complement mediated antibody lysis did not detect antibody until 10 days after infection. These results are discussed in terms of their importance to the diagnosis of and recovery from Aujeszky's disease.

Air sampling of pigs infected with foot-and-mouth disease virus: comparison of Litton and cyclone samplers.

The air in looseboxes containing groups of pigs in the acute stage of foot-and-mouth disease was sampled simultaneously with two air-sampling devices: a large volume sampler (Litton) and a cyclone sampler. Although the cyclone sampler was slightly less efficient at trapping airborne virus it was easier to operate. When pigs were sampled individually within a 610 litre cabinet using the cyclone sampler, the mean recovery of virus over a 24 hour period was log10 8 X 6 TCID50 per animal. This figure is four times higher than the maximum amount estimated in previous studies in which groups of pigs held in looseboxes were sampled with a Litton apparatus.

Functional antibody responses in pigs vaccinated with live and inactivated Aujeszky's disease virus.

Functional antibody tests, including virus neutralising activity of serum, antibody dependent cellular cytotoxicity and complement mediated lysis, were used to measure the response of pigs given either live or inactivated Aujeszky's disease virus vaccines. Pigs were then challenged with virulent Aujeszky's disease virus and antibody responses were analysed and found not to correlate with protection. Reasons for this lack of correlation are discussed and it is suggested that these results indicate that more emphasis should be placed on measuring the local immune response.

Comparative studies of United Kingdom isolates of swine vesicular disease virus.

The characteristics of four United Kingdom isolates of swine vesicular disease (SVD) virus from 1981 to 1982 have been compared with those of an isolate obtained from the first outbreak of swine vesicular disease diagnosed in the United Kingdom in 1972. When the virus structural proteins were examined by polyacrylamide gel electrophoresis the four isolates from 1981-82 all had the same polypeptide pattern, which was different from that of the 1972 isolate. Immunodiffusion tests with the 1972 isolate and one 1982 isolate did not reveal any antigenic difference between the viruses but minor antigenic differences were shown by cross-neutralisation tests between the 1972 isolate and the four isolates from 1981-82. In experimentally infected pigs the 1972 isolate produced typical SVD lesions whereas the four more recent SVD viruses produced only very mild clinical disease. Clinical lesions scored numerically were four- to 10- and five- to 11-fold higher at seven and 14 days after infection for pigs infected with the 1972 isolate than with the four isolates from 1981-82. The serum of pigs infected with the 1972 isolate contained significantly higher levels of neutralising antibody than those of pigs infected with more recent isolates. The antibody titres of pigs with only primary lesions ranged from log10 1.9 to 2.8 and one clinically normal pig had a titre of log10 2.4 at 14 days after infection. Attention is drawn to the implication of these findings for SVD control policies based only on the recognition and reporting of clinical disease.

Haptoglobin response of cattle infected with foot-and-mouth disease virus.

Haptoglobin, a major bovine acute phase protein, was evaluated as a marker of the primary replication of foot-and-mouth disease virus in 12 naturally infected cattle from which blood was collected daily. An acute phase response, as measured by an increase in serum haptoglobin concentration and the presence of fever, was not detected during the previraemic stage of disease, but there was a significant increase in serum haptoglobin after the onset of viraemia. It occurred on the same day as the first appearance of clinical signs in three of the cattle, one day later in four and two days later in the other five cattle.

Infection of cattle by airborne foot-and-mouth disease virus: minimal doses with O1 and SAT 2 strains.

Equipment has been constructed and methods developed for exposing individual cattle to two strains of foot-and-mouth disease (FMD) virus in aerosols to determine the minimal infective dose by the respiratory route. The aerosols used were produced either artificially by a spinning-top aerosol generator, in which case they were of homogeneous small particle size (less than 3 micron in diameter) or else they were derived naturally from infected pigs, in which case the particles were heterogeneous in size. Two strains of FMD virus were used: an O1 strain of UK origin and a SAT 2 strain from South Africa. The lowest doses which initiated infection were 12.5 TCID50 of O1 BFS virus and 25 TCID50 of SAT 2 virus, infectivity having been assayed in primary bovine thyroid cell cultures. Following exposure to low doses of virus (range 12 to 316 TCID50) 33 per cent of the cattle exposed to O1 BFS virus and 27 per cent exposed to SAT 2 virus were infected but did not develop detectable vesicular lesions.