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.

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.

Extended phylogeny of equine arteritis virus: division into new subgroups.

To determine a conclusive phylogeny, equine arteritis viruses from Italy, Austria, Hungary, Sweden, South Africa and other parts of the world were analysed by reverse-transcription polymerase chain reaction amplification and direct sequencing. The nucleotide sequences corresponding to the variable part of the large glycoprotein GP5, specified by open reading frame 5, were compared and added to a previously published phylogenetic tree in which a clear division between 'European' and 'American' type viruses had been established. Adding the sequences determined in this study and new sequences retrieved from GenBank revealed additional diversity and new subgroups.

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.

Utility of automated real-time RT-PCR for the detection of foot-and-mouth disease virus excreted in milk.

Foot-and-mouth disease virus (FMDV) can be excreted in milk and thereby spread infection to susceptible animals in other holdings. The feasibility of using real-time reverse transcription polymerase chain reaction (rRT-PCR) as a diagnostic tool for detection of FMDV in milk was assessed by studying the excretion of virus from experimentally-infected cattle. Fore- and machine milk samples were collected over a 4-week period from two dairy cows infected with FMDV and from two in-contact cows held in the same pen.

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.

Secretory IgA as an indicator of oro-pharyngeal foot-and-mouth disease virus replication and as a tool for post vaccination surveillance.

A serotype-specific ELISA was developed to detect foot-and-mouth disease virus (FMDV) specific IgA antibody in the saliva of cattle, and the method was evaluated for its feasibility in detecting serotype O FMDV carrier animals, particularly amongst vaccinated cattle that had subsequently become sub-clinically infected. For this purpose, saliva samples were collected from naïve cattle (n = 173), FMDV challenged cattle (n = 10), FMDV vaccinated cattle (n = 40) and FMDV vaccinated-and-challenged cattle (n = 40). A subset of 29 cattle was sampled for 105-168 days after challenge.

Sequence analysis of the 5' untranslated region of swine vesicular disease virus reveals block deletions between the end of the internal ribosomal entry site and the initiation codon.

Swine vesicular disease virus (SVDV) is a picornavirus closely related to the human pathogen coxsackievirus B5. In common with other picornaviruses, the 5' untranslated region (5' UTR) of SVDV contains an internal ribosomal entry site (IRES) that plays an important role in cap-independent translation. The aim of this study was to use RT-PCR and sequencing to characterize a fragment of the 5' UTR encompassing the entire IRES.

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.

Noninfectious virus-like particle antigen for detection of swine vesicular disease virus antibodies in pigs by enzyme-linked immunosorbent assay.

An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits.

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 .

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).

Multiplexed microsatellite markers for the genetic analysis of Eucalyptus leucoxylon (Myrtaceae) and their utility for ecological and breeding studies in other eucalyptus species.

Eucalyptus leucoxylon is a widespread woodland tree species found in southeastern Australia that has suffered from, and continues to be, threatened by the impacts of habitat clearance and degradation. Populations now consist predominantly of scattered individuals, and their conservation status is of increasing concern. We report the development and characterization of a set of eight highly polymorphic microsatellite loci for E.

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.

Accumulation of selenium and lack of severe effects on productivity of American dippers (Cinclus mexicanus) and spotted sandpipers (Actitis macularia).

Selenium has been found at elevated concentrations in water, sediments, and aquatic biota in the Elk River (British Columbia, Canada) and some of its tributaries downstream of several coal mines. Selenium water concentrations in those areas exceed Canadian and British Columbia guidelines and are above levels at which adverse effects to fish and waterfowl could occur. We compared selenium concentrations in the eggs of two riverine waterbirds, American dippers and spotted sandpipers, with measures of productivity: the number of eggs laid, egg hatchability, and nestling survival.

Making a vaccinate-to-live policy a reality in foot-and-mouth disease.

Public opinion and the availability of new technologies are making the use of 'stamping- out' an increasingly unattractive option as the method of first choice for foot-and-mouth disease (FMD) control in FMD-free countries or zones seeking to control incursion of disease. There is therefore increasing pressure to adopt a 'vaccinate-to-live' policy in these circumstances. For a successful vaccinate-to-live policy, veterinary services need access to appropriate, licensed vaccines; to have adequate contingency plans to ensure that they can deliver the required vaccine, where and when it is needed; and to have developed an 'exit strategy' that enables recognition of freedom from disease as quickly as possible.

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.

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).

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.

Genetic diversity of international bovine viral diarrhoea virus (BVDV) isolates: identification of a new BVDV-1 genetic group.

In the last decade, several studies were performed to characterise bovine viral diarrhoea virus (BVDV) isolates and define genetic groups by genotyping. Much data is now available from GenBank, predominantly sequences from the 5' untranslated region (5'-UTR). In order to find out whether genetic grouping of isolates from different countries could be harmonised, 22 new isolates from five countries were analysed in combination with published sequences.

Genetic typing of bovine viral diarrhoea virus: most Slovenian isolates are of genotypes 1d and 1f.

A selection of 43 bovine viral diarrhoea viruses isolated from mainly persistently infected cattle on 23 Slovenian farms between 1997 and 2001 were characterised genetically. Viral RNA was extracted from infected cell cultures, reverse transcribed and amplified by PCR with primers targeting the 5'-UTR and the N(pro) gene, followed by direct sequencing of purified PCR products obtained for both genomic regions. The N(pro) sequences provided the best genetic resolution, and gave also higher statistical support for phylogenetic classification of the viruses.

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.

Remarkable cross-reaction of pan-pestivirus PCR primers with poliovirus genome.

When several human vaccines were tested for pestivirus contamination using a one-tube closed nested RT-PCR method employing pan-pestivirus primers selected from the 5'-untranslated region (5'-UTR) of the pestivirus genome, a 224 bp DNA product was produced from a poliovirus vaccine. Although this amplicon was of the size expected for pestiviruses, its sequence showed a 100% similarity with the corresponding reverse complement of a nucleotide sequence from the VP2 gene of the poliovirus type 1 Sabin strain. It is recommended that all positive PCR products, especially those prepared using pan-pestivirus primers, obtained from screening biological substances for pestivirus contamination should be checked by use of a specific hybridization probe and preferably by sequencing.