Foot-and-mouth disease marker vaccine: cattle protection with a partial VP1 G-H loop deleted virus antigen.

Contrary to the dogma that the VP1 G-H loop is essential for FMD vaccine efficacy, it has been previously shown that foot-and-mouth disease 146s antigen containing heterologous VP1 G-H loops confers complete protection in pigs and cattle. Moreover, serological evaluation of cattle vaccinated with an antigen lacking a large proportion of the VP1 G-H loop indicated that these animals should be protected against infection with FMD. Absence of this loop provides opportunity for the development of an FMD negative marker vaccine, allowing infection to be detected by antibodies against this missing region. Cattle vaccinated with this negative marker vaccine were fully protected following virus challenge 28 days post vaccination as determined by the absence of generalised lesions on their feet. Furthermore, use of our improved differentiation ELISA identified animals exposed to infection as early as 7 days post-challenge. We thus demonstrate, for the first time, the ability of this FMD negative marker vaccine to fully protect cattle from experimental challenge and rapidly distinguish animals that are subsequently exposed to infection.

Foot-and-mouth disease viral loads in pigs in the early, acute stage of disease.

The progress and pathogenesis of foot-and-mouth disease virus (FMDV) was studied in infected pigs by observing the development of clinical signs in two separate experiments. Viral loads were determined by real-time quantitative RT-PCR in the liver, spleen, cervical lymph node, mandibular lymph node, retropharyngeal lymph node, soft palate, pharynx, tonsil, tongue and skin (coronary band area). Tissue samples were collected from both inoculated and contact-infected pigs at several time points during infection, and blood samples were collected to assess viraemia and its relationship to tissue viral load. Virus first appeared in the lymph nodes, followed by viraemia and then clinical signs. The results suggested that FMDV accumulated in lymphoid tissue up to six hours after infection, in the tissues drained by the mandibular lymph node and tonsil and then disseminated throughout the body where epithelial cells were the favoured sites of replication.

Toward a global foot and mouth disease vaccine bank network.

A network of foot and mouth (FMD) vaccine banks has been initiated with the support of vaccine bank managers and technical advisors that participated in a workshop held at the Institute for Animal Health, Pirbright, in the United Kingdom in April 2006. Terms of Reference that provide guidance for coordinated activities are under consultation. Practical and economic benefits can be realised from collaboration, which will be achieved through mutually acceptable mechanisms for the exchange of information and materials relevant to vaccine banks and their management. If administrative and technical hurdles can be overcome, the network has the potential to contribute significantly to the improved control of FMD worldwide. A 'global' and interactive vaccine bank association could be created by agreeing a system of resource sharing that could orchestrate additional emergency cover with vaccine or antigen from the reserves of network members.

Making better use of technological advances to meet stakeholder needs.

Controlling transboundary diseases requires an inclusive and collaborative international approach. Decisions should be taken (and seen to be taken) on advice from multidisciplinary teams of scientists and representatives from all groups significantly affected by the disease (the 'stakeholders'). Changes in trade and travel mean that, unless a new model is developed for disease prevention, there is a real possibility that transboundary animal diseases will become increasingly difficult to control. The traditional government approach of dealing almost exclusively with the commercial sector of the livestock industry is no longer sufficient, and new ways must be found to include all sectors, including 'grey' husbandry (fragmented, disparate groups whose involvement with animals may range from the legal and responsible to the unsanctioned and/or illegal). The increasing convergence of human and animal health issues makes it imperative to make the best possible use of new tools. The particular challenges confronting veterinary science are: preventing the introduction of disease, rapidly identifying disease and controlling epidemics. This paper focuses on the United Kingdom to investigate the inadequacies of current approaches, identify needs, offer recommendations and propose a new approach to disease control, which emphasises global considerations. The objectives are: better participation across the entire sector, better communication, better science and better decision-making, all of which should lead to better security from disease.

Cytokine and Toll-like receptor mRNAs in the nasal-associated lymphoid tissues of cattle during foot-and-mouth disease virus infection.

The pharyngeal region is known to play an important role in foot-and-mouth disease virus (FMDV) infection in relation to acute disease and viral persistence. In this study, the local mucosal immune response in nasal-associated lymphoid tissue (NALT) of cattle infected with FMDV (strain O UKG 34/2001) was examined. Quantitative "real-time" RT-PCR assays were used to measure mRNA expression of cytokines (IFN-alpha, beta and gamma, IL-2, IL-1alpha and TNF-alpha) and Toll-like receptors (TLR)-3 and -4. NALTs from dorsal soft palate were collected from cattle at 7 days post-infection (dpi) and from carriers and non-carriers at 64 dpi. Expression of IFN-alpha mRNA was significantly greater in NALT during acute disease than in uninfected animals. Increased expression of IFN-gamma and IL-1alpha mRNA was also observed but was much lower than IFN-alpha expression. There was a slight increase in mRNA expression of TNF-alpha and IL-2. During persistence, TNF-alpha mRNA expression in carrier cattle was much higher than in non-carrier cattle. Expression of TLR-4 in NALT during the acute stage of infection was greater than in uninfected animals. Carrier and non-carrier cattle did not differ in respect of expression of TLR-3 and -4 mRNA in NALT.

Comparison of complement fixation test, immunoblotting, indirect ELISA, and competitive ELISA for detecting antibodies to Mycoplasma mycoides subspecies mycoides small colony (SC) in naturally infected cattle from the 1995 outbreak in Botswana.

Four serological tests were compared in order to evaluate their efficacies in detecting antibodies to M. mycoides subspecies mycoides SC in cattle sera sampled in 1995 from herds affected with contagious bovine pleuropneumonia (CBPP) in the north-western part of Botswana. Tests that were compared included immunoblotting test (IBT), indirect enzyme-linked immunosorbent assay (i-ELISA), competitive enzyme-linked immunosorbent assay (c-ELISA) and complement fixation test (CFT). The percentages of seropositive samples in the iELISA (48%) and in the c-ELISA (48%) were similar but were comparatively lower than those obtained by the IBT (57%) and CFT (61%). The percentages of positive sera in the IBT and CFT were also similar and overall the efficacy of these tests was better than that of the two ELISA tests. There was 95.5% agreement between the IBT and CFT, 85% agreement between the IBT and c-ELISA, 90.9% agreement between the IBT and i-ELISA, 88.6% agreement between the i-ELISA and CFT, 80% agreement between the c-ELISA and CFT and 90% agreement between the two ELISA tests. It became clearly evident from this comparative study that no single serological test was capable of detecting all animals affected by CBPP under natural field conditions of infection.

Confirmation of the presence of Mycoplasma bovis in Hungarian cattle with pneumonia resembling pleuropneumonia.

Cattle from several farms in Hungary were investigated for the presence of mycoplasmal infections after the discovery of pulmonary lesions in some animals at slaughter. The pneumonic lesions, which resembled those of contagious bovine pleuropneumonia (CBPP) macroscopically and histologically were found to be caused by Mycoplasma bovis and not Mycoplasma mycoides subspecies mycoides (MmmSC) which is the causative agent of CBPP. No other bacterial pathogens were isolated. Negative results in complement fixation tests also showed that there was no serological evidence of CBPP. PCR tests for the detection of the M mycoides cluster and specifically for MmmSC were also negative. However, PCR and bacteriological culture detected cases of M bovis and the pneumonias may therefore be attributed to this mycoplasma.

Detection of Mycoplasma mycoides subspecies mycoides in tissues from an outbreak of contagious bovine pleuropneumonia by culture, immunohistochemistry and polymerase chain reaction.

Postmortem observations of 37 cattle from an outbreak of contagious bovine pleuropneumonia (CBPP) in north Italy in 1993 were made at the abattoir, where samples of lung and tracheobronchial lymph node tissues were taken for culture and identification of Mycoplasma mycoides subspecies mycoides (MmmSC), immunohistochemistry with the peroxidase anti-peroxidase (PAP) system, and molecular detection by the polymerase chain reaction (PCR) amplification of specific DNA from MmmSC. Nasal swabs were also taken for testing by PCR Lung pathology typical of CBPP was observed in 38 per cent of the animals, and MmmSC was isolated from 19 per cent DNA of MmmSC was detected by PCR in 64 per cent of lung samples and 35 per cent of the nasal swabs. Staining of lung tissue and lymph node tissue by PAP was positive in 27 per cent and 30 per cent of cases, respectively, and was a useful back-up test. These results suggest that PCR amplification from lung tissue may be used as a rapid and accurate confirmatory test for cases with pathology resembling CBPP.

Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene.

Babesia equi and Babesia caballi are tick-borne haemoparasites that may cause babesiosis of Equidae. In southern Europe B. equi is enzootic and infections may occur asymptomatically and more frequently than those due to B. caballi. Complement fixation test (CFT) is the official serological test for the diagnosis of equine babesiosis, but it has low sensitivity during early and latent stages of the disease. With the aim of developing more sensitive and rapid direct diagnostic alternatives, PCR systems that amplified DNA targets of 664 or 659 bp regions of the 16S rRNA genes were designed and demonstrated to specifically detect the genomes of B. equi and B. caballi, respectively. An approximated parasitaemia of 0.000083% was detected by the PCR system for B. equi compared with reported limits of 0.001% for microscopic examination of stained blood smears, and up to 0.00025% for DNA probes. Although the sensitivity of the PCR system for B. caballi could not be estimated, samples with microscopically undetectable parasitaemia as well as those with 0.017% parasitised red blood cells were detected. DNA extracts of blood collected with EDTA as an anticoagulant from 23 horses from Portugal were tested with both PCR systems. Of these samples, 22 were positive for B. equi and 8 were positive for B. caballi with PCR tests and intraerythrocytic parasites were seen in all samples. Antibodies against both parasites were not detected by CFT in several cases, but in these cases the presence of either or both parasites was apparent by PCR tests. The PCR systems may be useful in the diagnosis of equine babesiosis covering a wider range of clinical disease, as useful adjuncts to serological, microscopic, and cultural methods, especially for the import and export testing of horses.

Detection of Mycoplasma mycoides subspecies mycoides SC in clinical material by a rapid colorimetric PCR.

A colorimetric polymerase chain reaction (PCR) test for the detection of Mycoplasma mycoides subsp. mycoides SC (Mmm SC), the cause of contagious bovine pleuropneumonia (CBPP), was used to test clinical samples from suspect and known cases of the disease. A commercially available detection kit, pleuro TRAP from AMRAD, Australia, which captures amplified double stranded DNA in the wells of a microtitre plate was used. Tissues tested from animals ranging from a variety of anatomical locations included lung, lymph node, kidney, spleen, pleural fluid and nasal swabs. Polymerase chain reaction was conducted on DNA extracted from tissues as well as on culture-derived mycoplasmas. Positive results could be seen by the development of a yellow colour in microtitre wells after the enzymatic detection procedure indicating the presence of specifically amplified products. Spectrophotometric values of OD450for positive samples ranged from 0.211 to 1.409 compared to negative values of around 0.020 which reduced the subjectivity in the interpretation of results. Mmm SC was successfully identified from cultures of reference and field strains, nasal swabs and excised tissue by amplification with primers supplied in the kit. The system is suitable for automation and may be a cost effective alternative to gel electrophoresis for the screening of moderately large sample numbers as is the case in surveillance programmes.

An outbreak of contagious bovine pleuropneumonia in Ngamiland district of north-western Botswana.

An outbreak of contagious bovine pleuropneumonia (CBPP) was detected in Botswana in 1995 after more than half a century of freedom from the disease. Lung tissues, pleural fluids, nasal swabs and serum samples were examined in laboratories in Botswana, South Africa and Namibia and the findings were confirmed in Italy. The disease was confirmed as CBPP from the gross and histopathological changes in the lungs of affected animals and by the culture of the agent of CBPP, Mycoplasma mycoides subspecies mycoides, small colony variant (MmmSC). These findings were supported by the demonstration of specific complement-fixing antibodies and the production of polymerase chain reaction products of MmmSC.

A comparison of serological tests and gross lung pathology for detecting contagious bovine pleuropneumonia in two groups of Italian cattle.

Western and dot blotting techniques were compared with complement fixation tests (CFT), indirect enzyme-linked immunosorbent assays (ELISA), mycoplasma culture and gross lung pathology to detect Mycoplasma mycoides subspecies mycoides SC, the cause of contagious bovine pleuropneumonia (CBPP), in two groups of Italian cattle. None of the animals showed any clinical signs before slaughter. In group A, seven of the 20 cattle had characteristic lung lesions of acute and chronic CBPP but only six were positive by CFT. Western blotting detected antibody in eight of the animals, of which six had lesions and significant CFT titres (> 50 per cent fixation at a serum dilution of 1/10) and two had neither. In group B, seven of the 17 cattle had lesions characteristic of CBPP, and 12 were seropositive by CFT. Western blotting detected antibody in 13 of the animals including one which had a negative CFT titre. The ELISA was less sensitive than either CFT or Western blotting, detecting antibody in five animals in group A and nine animals in group B. The dot blotting test correlated well with Western blotting but gave a small number of ambiguous results. The causative organism was isolated from four of the 20 cattle in group A and six of the 17 cattle in group B.

A PCR-based test for the specific identification of Mycoplasma mycoides subspecies mycoides SC.

The polymerase chain reaction (PCR) was used to develop a test for the detection of Mycoplasma mycoides subspecies mycoides SC in the tissues of animals infected with contagious bovine pleuropneumonia (CBPP). Two sets of primers were designed; one set (MC323/MC358) to amplify a approximately 1.5-kbp DNA fragment from all the members of the M. mycoides 'Cluster' and the other set (MM450/MM451) specifically amplified a 574-bp DNA fragment from M. mycoides subspecies. The PCR products could be differentiated further by digestion with the restriction enzyme AsnI. Enzyme digestion of amplification products from M. m. mycoides SC produced 2 fragments, whereas the other 2 M. mycoides subspecies, M. m. mycoides LC and M. m. capri, produced 3 fragments. This test was shown to be very sensitive, being able to detect between 10 and 100 organisms. Cattle were experimentally infected with the Gladysdale strain of M. m. mycoides SC, and samples of serum and mucus were taken periodically, as were postmortem samples of lung, lymph node, pleural fluid, synovial fluid, and tracheal swabs. Complement fixation test on serum samples, culture of postmortem tissues, and histopathologic examination confirmed disease. DNA was extracted from postmortem samples and amplified by PCR using primers MM450 and MM451. Digestion of products using AsnI allowed the specific identification of M. m. mycoides SC. This test could confirm CBPP in 48 hours and was thus capable of giving a more rapid result than the traditional methods of culture, isolation, and identification using biochemical and serological techniques.

Deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza A viruses of H5 and H7 subtypes.

The amino acid sequences at the haemagglutinin cleavage sites of 9 avian influenza A viruses of H5 subtype (5 high and 4 low pathogenicity for chickens) and 21 of H7 subtype (13 high and 8 low pathogenicity for chickens) were determined by direct RNA sequencing, PCR amplification sequencing or both. None of the viruses of low pathogenicity had multiple basic amino acids at the cleavage site. All highly pathogenic viruses had an insert of basic amino acids at the cleavage site, except A/chicken/Scotland/59 (H5N1) for which the multiple basic amino acids appeared as substitutions and not insertions. All highly pathogenic viruses examined conformed to the amino acid motif of R-X-R/K-R at the cleavage site which is considered to be essential for high pathogenicity in chickens, with the notable exception of highly pathogenic virus A/turkey/England/50-92/91 (H5N1) which had the sequence R-K-R-K-T-R adjacent to the cleavage site.

Deduced amino acid sequences at the fusion protein cleavage site of Newcastle disease viruses showing variation in antigenicity and pathogenicity.

The amino acid sequence at the F2/F1 cleavage site of the F0 fusion protein of 17 strains of Newcastle disease virus (NDV) was deduced from sequencing a 32 nucleotide area of the genome by reverse transcription and polymerase chain reaction (PCR) techniques. With the addition of sequences at the same area previously published for 9 other viruses comparisons were made of a total of 26 NDV strains and isolates (11 of low virulence, 15 of high virulence or mesogenic) covering ten antigenic groups determined by reactions with monoclonal antibodies. All the virulent viruses and the mesogenic strain Komarov showed the amino acid sequence 112R/K-R-Q-K/R-R116 for the C-terminus of the F2 protein and phenylalanine (F) at the N-terminus of the F1 protein, residue 117. The mesogenic isolate of the antigenic variant NDV responsible for the recent panzootic in racing pigeons, often termed "pigeon paramyxovirus type 1", examined in this study had the sequence 112G-R-Q-K-R-F117. The deduced amino acid sequence in the corresponding region of all viruses of low virulence was 112G/E-K/R-Q-G/E-R-L117. The virulent virus, PMV-1/chicken/Ireland/34/90 (34/90), which had a close antigenic relationship to a group of avirulent viruses, three of which were examined in the present study as representatives of the monoclonal antibody group H, showed between 4-6 nucleotide differences from these viruses in the 32 nucleotide region studied. These resulted in differences in the deduced amino acid sequence at residue 112 E-->K, 115 E-->K and 117-->F, giving 34/90 a typical virulent virus motif at the cleavage site. Despite the extremely small portion of the genome studied there were several areas which appeared characteristic for 34/90 and the three group H viruses of low virulence, which suggests that they may have arisen from the same gene pool.