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D J Paton

Publications and source records attributed to D J Paton.

At least 19 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2006

Comparative evaluation of six ELISAs for the detection of antibodies to the non-structural proteins of foot-and-mouth disease virus.

To validate the use of serology in substantiating freedom from infection after foot-and-mouth disease (FMD) outbreaks have been controlled by measures that include vaccination, 3551 sera were tested with six assays that detect antibodies to the non-structural proteins of FMD virus. The sera came from naïve, vaccinated, infected and vaccinated-and-infected animals; two-thirds from cattle, the remainder from sheep and pigs. The assays were covariant for sensitivity, but not necessarily for specificity. A commercial kit from Cedi-diagnostics and an in-house assay from IZS-Brescia were comparable to the NCPanaftosa-screening index method described in the Diagnostic Manual of the World Animal Health Organisation. Using these three tests the specificity and sensitivity for the detection of carriers in vaccinated cattle approaches or exceeds 99% and 90%, respectively.

Animals↗
PubMed2006

Effect of emergency FMD vaccine antigen payload on protection, sub-clinical infection and persistence following direct contact challenge of cattle.

Previous work, in sheep vaccinated with emergency foot-and-mouth disease (FMD) vaccine, indicated the benefit of increasing the antigen payload in inhibiting local virus replication and consequently persistence following an indirect aerosol challenge with a virus homologous to the vaccine strain. The work presented here investigates this possibility further using cattle and a more severe semi-heterologous direct contact challenge. The quantitative dynamics of virus replication and excretion in both vaccinated and non-vaccinated cattle following challenge are examined. Two experiments were carried out each involving 20 vaccinated and 5 non-vaccinated cattle. An O(1) Manisa vaccine (18 PD(50)) was used for the first, previously reported experiment [Cox SJ, Voyce C, Parida S, Reid SM, Hamblin PA, Paton DJ, et al. Protection against direct contact challenge following emergency FMD vaccination of cattle and the effect on virus excretion from the oropharynx. Vaccine 2005;23:1106-13]. The same vaccine was used for the second experiment described in this paper except the antigen payload was increased 10-fold per bovine dose, resulting in significantly higher FMD virus neutralising antibody titres prior to challenge. Twenty-one days post-vaccination the cattle received a 5-day direct contact challenge with FMD virus from five further non-vaccinated cattle infected 24h earlier with O UKG 34/2001. All vaccinated cattle regardless of antigen payload were protected against clinical disease. Sub-clinical oropharyngeal infection was detected in animals from both experiments but the level of virus replication shortly after direct contact challenge was significantly reduced in vaccinated animals. Cattle immunised with the 10-fold antigen payload cleared the virus more readily and consequently at 28 days post-challenge fewer animals were persistently infected compared to the single strength vaccine. Following a severe challenge, the results from both experiments show that use of emergency vaccine can prevent or decrease local virus replication and thereby dramatically reduce the amount of virus released into the environment, particularly during the early post-exposure period. Additionally, increasing the antigen payload of the vaccine may reduce sub-clinical infection, leading to fewer persistently infected virus carrier animals.

Animals↗
PubMed2005

The application of new techniques to the improved detection of persistently infected cattle after vaccination and contact exposure to foot-and-mouth disease.

Detection of antibodies to the non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) was compared with conventional serological and virological methods and with RT-PCR for the identification of FMDV carrier animals obtained after experimental contact challenge of vaccinated cattle. Transmission from carriers to sentinels was also monitored. Twenty FMDV vaccinated and five unvaccinated cattle were challenged by direct contact with five donor cattle excreting FMDV and monitored until 28 days post challenge-exposure . Twelve vaccinated and three unvaccinated animals were retained up to 24 weeks post exposure to FMDV in order to monitor viral persistence, transmission and antibody responses. In nine vaccinated animals, infection persisted beyond 28 days post exposure, virus being detected more frequently and for longer in oesophagopharyngeal samples from these animals when examined by RT-PCR rather than by virus isolation. Although recovery of FMDV RNA became increasingly sporadic over time, the number of RNA copies detected in positive samples declined only slowly. Two naïve sentinel cattle housed with the persistently infected animals between 93 and 168 days after the latter had been challenge-exposed to FMDV did not become infected. There were differences in the ability of commercially available serological tests to detect antibodies to FMDV non-structural proteins (NSP) in vaccinated and subsequently challenged cattle. Although no single test could identify all of the vaccinated cattle that became persistently infected, the most poorly recognised animals were those with the least evidence of virus replication based on other tests. The potential of the detection of antibodies to the 2B NSP of FMDV for diagnosing persistent FMDV infection was demonstrated.

Animals↗
PubMed2005

Interferon-gamma production in vitro from whole blood of foot-and-mouth disease virus (FMDV) vaccinated and infected cattle after incubation with inactivated FMDV.

Studies were performed to determine whether a rapid method to detect cell mediated immune responses to foot-and-mouth disease virus (FMDV) could be used either as a diagnostic test or provide a correlate of protection in animals post-vaccination. Using protocols based on the BOVIGAM assay for tuberculosis, whole blood samples from FMDV vaccinated or control animals, before and after challenge infection, were stimulated overnight with inactivated FMDV antigen. The quantity of interferon gamma (IFN-gamma) produced in the supernatants was measured using an ELISA. Specific induction of IFN-gamma was detected in samples from vaccinated, infected and vaccinated-then-infected cattle. Further development of this assay may provide a useful tool for the diagnosis of FMDV immune animals, including the identification of vaccinated animals that have been subsequently infected with FMDV. In these studies, combining the results of the IFN-gamma assay with virus neutralising antibody titre, in groups of vaccinated animals, provided a correlation with the capacity to control virus replication after subsequent challenge.

Animals↗
PubMed2005

Genetic diversity of BVDV: consequences for classification and molecular epidemiology.

Genetic typing of bovine viral diarrhoea virus (BVDV) is important for the precise classification of viruses as well as for the development of molecular epidemiology. BVDV isolates were usually typed based on comparison of genomic sequences from the 5'-untranslated region (5'-UTR), N(pro) and E2 region. Recently we have identified 11 genetic groups (subgenotypes) of BVDV-1. Our further experiments confirmed a new subgenotype, BVDV-1k, isolated from cattle in Switzerland. BVDV isolates from India were typed as BVDV-1b whereas BVDV-1c is a predominant subgenotype in Australia. The results of genetic typing of BVDV indicate that distribution of subgenotypes has no relationship to the geographic origin of viral isolates.

Animals↗
PubMed2005

Protection against direct-contact challenge following emergency FMD vaccination of cattle and the effect on virus excretion from the oropharynx.

The ability of emergency foot-and-mouth disease (FMD) vaccine to protect cattle from a heterologous direct-contact challenge and the effect on virus excretion from the oropharynx were examined. An oil adjuvant O1 Manisa FMD vaccine protected 20 cattle from clinical disease following 5 days of direct-contact exposure to five infected cattle at 21 days post vaccination. The donor cattle had been infected by tongue inoculation with a different FMD virus of the same serotype (O UKG 2001). Protection from clinical disease did not prevent localised sub-clinical infection at the oropharynx in most animals, although quantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed that the level of virus replication shortly after direct-contact challenge was greatly reduced in vaccinated animals. Nevertheless, 45% of the vaccinated cattle became persistently infected with 10(3)-10(6) RNA copies per millilitre of oropharyngeal fluid at 28 days post challenge. However, since live virus could not be readily isolated, the risk of these animals transmitting disease was probably very low. The findings show that even after an extremely severe challenge, use of an emergency vaccine will prevent or reduce local virus replication and thereby dramatically reduce the amount of virus released into the environment in the all-important early post-exposure period. These data should help to model the dynamics of virus transmission in future outbreaks of disease where vaccination is considered.

Animals↗
PubMed2005

Detection of antibody to the foot-and-mouth disease virus (FMDV) non-structural polyprotein 3ABC in sheep by ELISA.

The specificity and sensitivity of an ELISA for detecting IgG to the 3ABC non-structural protein of foot-and-mouth disease (FMD) virus was evaluated in FMD naive, aerosol-infected, aerosol plus direct contact infected and field-exposed sheep. All 12 sheep that were experimentally infected without prior vaccination seroconverted in the test, although fewer field sera from FMD-exposed sheep were scored seropositive compared to test results for structural protein antibodies. The 3ABC test specificity was 98 or 100% according to whether sera reacting in the doubtful range were scored as positive or negative. The test was then used to investigate the antibody response of sheep vaccinated against FMD and exposed to the virus by an aerosol challenge 4-14 days later. The response of individual animals varied. Whether immunised with high or low doses of vaccine, the development of 3ABC antibody was most likely in sheep from which live virus was recovered at or beyond 9 days post-challenge. Non-structural responses were also more frequent in animals from which multiple incidences of live FMD virus isolation (perhaps more indicative of true virus replication) were demonstrated.

Animals↗
PubMed2005

Selection of foot and mouth disease vaccine strains--a review.

The choice of the most appropriate strains of foot and mouth disease (FMD) virus vaccines to use in FMD control programmes and to store in vaccine antigen reserves is based on the matching of representative field isolates from outbreaks around the world to available vaccine strains. However, those involved in FMD control at a national level do not always give this work a high priority, while in countries without effective control of FMD there is little incentive to collect samples or to overcome the constraints on submission to international reference laboratories. In the short term, specific initiatives for targeted collection can provide samples on a periodic basis, but a long-term solution requires the development of FMD control measures. This must be underpinned by the strengthening of local Veterinary Services and laboratories, and by demand-driven provision of sufficient amounts of high-quality vaccine. Difficulties may be increased by commercial constraints on disclosure of the strains used for vaccine production and on the supply of reagents needed for matching tests. Vaccine matching tests are mainly based on in vitro methods - such as virus neutralisation, enzyme-linked immunosorbent assay with polyclonal antibodies and complement fixation - and are performed in a relatively small number of laboratories around the world. In addition to the difficulties of gathering representative field and vaccine strains, neither the reagents nor the methods used for vaccine matching are fully harmonised. Consequently, there is no strict equivalence in the results obtained. Alternative approaches using monoclonal antibody panels and/or viral capsid gene sequencing are being developed and could complement the currently employed serological tests. However, there is limited in vivo cross-protection information, more of which is essential for future validation of the vaccine matching methods. In response to the funding and leadership deficit for vaccine strain selection, a network of World Organisation for Animal Health (OIE) and Food and Agriculture Organization FMD reference laboratories has been established; this gives these laboratories the potential to strengthen the coordination of their work and reporting and thereby improve recommendations on vaccine strain selection.

Animals↗
PubMed2004

Mucosal disease-like lesions in sheep infected with Border disease virus.

An enteric disease characterised by diarrhoea and ill thrift affected 12 of a flock of 700 six- to 12-month-old ewe lambs in Cornwall between December 1996 and September 1997. The affected lambs were undersized, became thin and suffered an unremitting diarrhoea until they died. The illness lasted for three to 14 days, although, with hindsight, the owner considered that the lambs had been below average size before the enteric signs developed. The outbreak ceased only as a result of the dispersal sale of the flock as breeding ewes. The flock had been purchased from different sources, but 11 of the cases occurred in a group of 40 purchased from one source. Postmortem, the alimentary changes resembled mucosal disease in cattle, and immunostaining of histological sections of the affected tissues revealed pestiviral antigen. Non-cytopathic pestiviruses were isolated from the lesions of two of the affected lambs and from the blood of several clinically normal ewe lambs from the same group. All the pestivirus isolates were typed as Border disease virus.

Animals↗
PubMed2004

Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs.

Automated real-time RT-PCR was evaluated as a diagnostic tool for swine vesicular disease virus (SVDV) infection on a range of samples (vesicular epithelium, serum, nasal swabs, faeces) from four inoculated and three in-contact pigs over a period of 28 days. Traditional diagnostic procedures (virus isolation, and ELISAs for antigen and antibody) were used in parallel. Each inoculated pig developed a significant viraemia and clinical disease, and excreted virus, which was transmitted to the in-contact animals. The latter, however, developed only a short-lived, low-level viraemia and no clinical disease. The RT-PCR and virus isolation were generally comparable in detecting SVDV in the serum and nasal swabs from inoculated and in-contact pigs up to day 6 after infection; it was possible, however, to isolate virus for a longer period from the faeces of a few pigs. This suggested that further optimization of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs.

Animals↗
PubMed2004

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.

Animals↗
PubMed2003

Classical swine fever--an update.

Classical swine fever (CSF) is a serious and contagious viral disease of pigs and wild boar with a widespread worldwide distribution. The immunopathology of the disease is poorly understood, but the ability of the CSF virus to infect cells without triggering apoptosis and to kill uninfected cells is probably highly significant. The virus may be spread by various direct and indirect methods, but in most cases the exact mechanisms involved in local spread between farms are not known. Excellent diagnostic tools and typing methods are available, but tests that could be performed on-farm, in pre-clinically infected pigs or on meat would also be advantageous. A more complete picture of the viruses circulating in different parts of the world is needed. There is great interest to develop and use marker vaccines for the control of CSF in domestic pigs and in wild boar. Epidemiological modelling is increasingly used to evaluate control options.

Animals↗
PubMed2001

Improved antigen and nucleic acid detection in a bovine virus diarrhoea eradication program.

A bovine viral diarrhoea/mucosal disease (BVD/MD) control and eradication program was introduced in Lower Austria in 1996, according to the Swedish model. An important risk factor for BVD transmission under local conditions is communal grazing where susceptible pregnant cattle from several herds may be mixed with unrecognised persistently infected (PI) animals. A reliable system for identification of PI animals is therefore essential for BVD eradication and steps were taken to improve a commercially available antigen-capture ELISA (Ag-ELISA) by modifying the method for leukocyte preparation and adjusting the negative cut-off value. A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) employing panpestivirus 324/326 primers targeting the 5'-untranslated region of the virus genome was also simplified and used on pooled blood samples to facilitate larger sample throughputs. RT-PCR positive pools were analysed individually to identify infected animals. Seven hundred eighty-six samples were tested by Ag-ELISA according to the instruction manual and 5324 samples with the modified method. All 6110 samples were retested by RT-PCR. The percentage of RT-PCR positive results with doubtful and negative Ag-ELISA samples significantly diminished using the modified method (from 4.71 to 0.82%). Selected BVD viruses were genetically typed by PCR product sequencing; special attention being paid to RT-PCR amplicons from samples which were negative or doubtful by ELISA. However, no correlation was found between the phylogenetic grouping of the viruses and the Ag-ELISA results.

Age Factors↗
PubMed2001

Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups.

Seventy-eight bovine viral diarrhoea viruses (BVDV) recently collected in Austria, France, Hungary, Italy, Slovakia, Spain and UK were genetically typed in the 5'-untranslated (5'UTR) and autoprotease (Npro) regions of the pestivirus genome. Seventy-six of the isolates were BVDV-1 and two French isolates were of the BVDV-2 genotype. Phylogenetic analysis of the 5'UTR (245 nt), including additional BVDV-1 sequences from USA, Canada, Germany, New Zealand, Mozambique and Sweden, taken from GenBank and from our previous works, indicated that these viruses were clustered not only into the two generally accepted groups (BVDV-1a-"NADL like" and BVDV-1b-"Osloss like"), but altogether into 11 phylogenetic groups. Similar clustering was observed with Npro region sequences (385 nt) and the highest bootstrap values (over 95%) were obtained by phylogeny combining 5'UTR and Npro sequences. Some associations between the genetic grouping and the origin of the isolates were apparent, probably reflecting historical trade contacts. Considering the variability of isolates it is recommended that diagnostic PCR primers should be re-examined to ensure coverage of all BVDV-1 groups. The genogroups were less clearly differentiated by monoclonal antibody typing, suggesting significant antigenic similarities within the BVDV-1 genotype.

5' Untranslated Regions↗
PubMed2000

Classical swine fever virus: a second ring test to evaluate RT-PCR detection methods.

Six laboratories participated in a study to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 25 samples of random primed cDNA, synthesised from viral RNA representative of different pestiviruses. The other set comprised samples of blood and serum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols that specified the exact conditions and requirements for inclusion of control samples. Two types of test were evaluated. One amplified a part of the 5'-non coding region of the pestivirus genome by means of a closed, one-tube RT-nested PCR. The other amplified a part of the NS5B gene using non-nested RT-PCR. The results of the laboratories were compared with one another, and with those obtained earlier when similar samples were tested by the same laboratories using non-standardised methods [Paton et al., Classical swine fever virus: a ring test to evaluate RT-PCR detection methods, Vet. Microbiol., in press]. Standardisation of the protocols resulted in a more consistent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test specificity was inadequate from the results obtained with the control samples. Minimum requirements for the inclusion of adequate controls and periodic proficiency testing are proposed.

Animals↗