Selection and use of reference panels: a case study highlighting current gaps in the materials available for foot and mouth disease.

The World Organisation for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals describes a diverse array of assays that can be used to detect, characterise and monitor the presence of infectious agents of farmed livestock. These methods have been developed in different laboratories, at different times, and often include tests or kits provided by the commercial sector. Reference panels are essential tools that can be used during assay development and in validation exercises to compare the performance of these varied (and sometimes competing) diagnostic technologies. World Organisation for Animal Health Reference Laboratories already provide approved international standard reagents to help calibrate diagnostic tests for a range of diseases, but there remain important gaps in their availability for comparative purposes and the calibration of test results across different laboratories. Using foot and mouth disease (FMD) as an example, this review highlights four specific areas where new reference reagents are required. These are to: reduce bias in estimates of the diagnostic sensitivity and inter-serotypic specificity of tests used to detect diverse strains of FMD virus (FMDV), provide bio-safe positive controls for new point-of-care test formats that can be deployed outside high containment, harmonise FMDV antigens for post-vaccination serology, and address inter-laboratory differences in serological assays used to measure virus-specific FMD antibody responses. Since there are often limited resources to prepare and distribute these materials, sustainable progress in this arena will only be achievable if there is consensus and coordination of these activities among OIE Reference Laboratories.

Le Manuel des tests de diagnostic et des vaccins pour les animaux terrestres de l'Organisation mondiale de la santé animale (OIE) décrit une vaste panoplie d'essais utilisables pour la détection, la caractérisation et la surveillance des agents pathogènes affectant les animaux d'élevage. Ces méthodes ont été mises au point par des laboratoires différents à diverses périodes et intègrent souvent des tests ou des kits fournis par le secteur privé. Les panels de référence sont des outils essentiels aussi bien lors de la conception d'un essai que lors d'exercices de validation, leur but étant alors de comparer les performances de technologies diagnostiques variées (et parfois concurrentes). Les Laboratoires de référence de l'OIE fournissent des réactifs de référence internationaux validés afin d'aider à calibrer les tests de diagnostic pour un certain nombre de maladies animales ; toutefois, on constate que nombre de ces réactifs ne sont pas disponibles pour la comparaison et le calibrage interlaboratoires des résultats de tests. À partir de l'exemple de la fièvre aphteuse, les auteurs soulignent quatre domaines spécifiques pour lesquels il conviendrait de disposer de nouveaux réactifs de référence. Il s'agit des réactifs nécessaires pour : (1) réduire les biais dans l'estimation de la sensibilité diagnostique et de la spécificité pour différents sérotypes des tests utilisés pour détecter diverses souches du virus de la fièvre aphteuse ; (2) fournir des contrôles positifs sûrs au plan biologique pour les nouveaux formats de tests utilisables sur le lieu d'intervention et non plus dans des laboratoires de confinement à haute sécurité ; (3) harmoniser les antigènes du virus de la fièvre aphteuse pour la sérologie post-vaccinale ; (4) résoudre le problème des différences obtenues entre laboratoires lors d'essais sérologiques visant à mesurer la réponse en anticorps spécifiques du virus de la fièvre aphteuse. Compte tenu des ressources souvent limitées consacrées à la préparation et à la distribution de ces réactifs, des progrès durables ne seront obtenus que s'il existe un consensus en la matière et une coordination de ces activités parmi les Laboratoires de référence de l'OIE.

En el Manual de pruebas de diagnóstico y vacunas para los animales terrestres de la Organización Mundial de Sanidad Animal (OIE) se describe todo un conjunto de ensayos que se pueden emplear para detectar y caracterizar agentes infecciosos del ganado doméstico y hacer así controles sistemáticos de su eventual presencia. Estos métodos, concebidos en distintos laboratorios en distintos momentos, suelen acompañarse de pruebas o estuches analíticos que proporcionan empresas privadas. Los paneles de referencia son una herramienta esencial, que se puede emplear durante la concepción de ensayos y en los procesos de validación para comparar el funcionamiento de estas diferentes técnicas de diagnóstico, que a veces compiten unas con otras. Los laboratorios de referencia de la OIE ya facilitan reactivos de referencia internacional aprobados que ayudan a calibrar las pruebas de diagnóstico de una serie de enfermedades, pero todavía hay importantes carencias por lo que respecta a la posibilidad de procurárselos con fines de comparación y a la calibración de los resultados que obtienen diferentes laboratorios. Sirviéndose del ejemplo de la fiebre aftosa, los autores destacan cuatro aspectos específicos para los que hacen falta nuevos reactivos de referencia. Se trata de los siguientes: reducir el sesgo a la hora de calcular la sensibilidad de diagnóstico y la especificidad interserotípica de las pruebas empleadas para detectar diversas cepas del virus de la fiebre aftosa; proporcionar controles positivos que ofrezcan seguridad biológica para nuevos modalidades de ensayo utilizables en el lugar de consulta, esto es, en condiciones que no sean de alta contención; armonizar los antígenos víricos para la práctica de análisis serológicos tras la vacunación; y solventar las diferencias entre laboratorios por lo que respecta a los ensayos serológicos empleados para medir la respuesta de anticuerpos específicos contra el virus de la fiebre aftosa. Dado que suele haber escasos recursos para preparar y distribuir este tipo de material, solo será posible avanzar duraderamente en la materia si los laboratorios de referencia de la OIE consensúan y coordinan estas actividades.

Estimating the protection afforded by foot-and-mouth disease vaccines in the laboratory.

Foot-and-mouth disease (FMD) vaccines must be carefully selected and their application closely monitored to optimise their effectiveness. This review covers serological techniques for FMD vaccine quality control, including potency testing, vaccine matching and post-vaccination monitoring. It also discusses alternative laboratory procedures, such as antigen quantification and nucleotide sequencing, and briefly compares the approaches for FMD with those for measuring protection against influenza virus, where humoral immunity is also important. Serology is widely used to predict the protection afforded by vaccines and has great practical utility but also limitations. Animals differ in their responses to vaccines and in the protective mechanisms that they develop. Antibodies have a variety of properties and tests differ in what they measure. Antibody-virus interactions may vary between virus serotypes and strains and protection may be affected by the vaccination regime and the nature and timing of field virus challenge. Finally, tests employing biological reagents are difficult to standardise, whilst cross-protection data needed for test calibration and validation are scarce. All of this is difficult to reconcile with the desire for simple and universal criteria and thresholds for evaluating vaccines and vaccination responses and means that oversimplification of test procedures and their interpretation can lead to poor predictions. A holistic approach is therefore recommended, considering multiple sources of field, experimental and laboratory data. New antibody avidity and isotype tests seem promising alternatives to evaluate cross-protective, post-vaccination serological responses, taking account of vaccine potency as well as match. After choosing appropriate serological tests or test combinations and cut-offs, results should be interpreted cautiously and in context. Since opportunities for experimental challenge studies of cross-protection are limited and the approaches incompletely reflect real life, more field studies are needed to quantify cross-protection and its correlation to in vitro measurements.

Acute Global Testicular Infarction Post-EVAR from Cholesterol Embolisation can be Mistaken for Torsion.

INTRODUCTION: Endovascular aneurysm repair (EVAR) is the most commonly used approach for treatment of abdominal aortic aneurysms (AAA). Testicular infarction is a rare complication of EVAR. A novel case of acute global testicular infarction post-EVAR from cholesterol embolisation mimicking torsion is presented. REPORT: A 75 year old man developed acute right testicular ischaemia requiring orchidectomy following EVAR of an infrarenal aortic aneurysm. The patient was initially diagnosed with testicular torsion as the aetiology of the infarction; however, on re-analysis of histopathology it was found to be secondary to cholesterol emboli. DISCUSSION: In patients complaining of groin/scrotal pain following EVAR, it is worth considering testicular ischaemia whether secondary to cholesterol embolisation or gonadal occlusion. Clinicians should be aware that clinical and radiological findings can mimic torsion as this affects management and outcome.

Aerosol transmission of foot-and-mouth disease virus Asia-1 under experimental conditions.

Foot-and-mouth disease virus (FMDV) control measures rely on understanding of virus transmission mechanisms. Direct contact between naïve and infected animals or spread by contaminated fomites is prevented by quarantines and rigorous decontamination procedures during outbreaks. Transmission of FMDV by aerosol may not be prevented by these control measures and this route of transmission may allow infection of animals at distance from the infection source. Understanding the potential for aerosol spread of specific FMDV strains is important for informing control strategies in an outbreak. Here, the potential for transmission of an FMDV Asia 1 strain between pigs and cattle by indirect aerosol exposure was evaluated in an experimental setting. Four naïve calves were exposed to aerosols emitted from three infected pigs in an adjacent room for a 10h period. Direct contact between pigs and cattle and fomite transfer between rooms was prevented. Viral titres in aerosols emitted by the infected pigs were measured to estimate the dose that calves were exposed to. One of the calves developed clinical signs of FMD, whilst there was serological evidence for spread to cattle by aerosol transmission in the remaining three calves. This highlights the possibility that this FMDV Asia 1 strain could be spread by aerosol transmission given appropriate environmental conditions should an outbreak occur in pigs. Our estimates suggest the exposure dose required for aerosol transmission was higher than has been previously quantified for other serotypes, implying that aerosols are less likely to play a significant role in transmission and spread of this FMDV strain.

Mass vaccination, immunity and coverage: modelling population protection against foot-and-mouth disease in Turkish cattle.

Foot-and-mouth disease (FMD) in Turkey is controlled using biannual mass vaccination of cattle. However, vaccine protection is undermined by population turnover and declining immunity. A dynamic model of the Turkish cattle population was created. Assuming biannual mass vaccination with a single-dose primary course, vaccine history was calculated for the simulated population (number of doses and time since last vaccination). This was used to estimate population immunity. Six months after the last round of vaccination almost half the cattle aged < 24 months remain unvaccinated. Only 50% of all cattle would have received > 1 vaccine dose in their life with the last dose given ≤ 6 months ago. Five months after the last round of vaccination two-thirds of cattle would have low antibody titres (< 70% protection threshold). Giving a two-dose primary vaccination course reduces the proportion of 6-12 month old cattle with low titres by 20-30%. Biannual mass vaccination of cattle leaves significant immunity gaps and over-reliance on vaccine protection should be avoided. Using more effective vaccines and vaccination strategies will increase population immunity, however, the extent to which FMD can be controlled by vaccination alone without effective biosecurity remains uncertain.

Randomised field trial to evaluate serological response after foot-and-mouth disease vaccination in Turkey.

Despite years of biannual mass vaccination of cattle, foot-and-mouth disease (FMD) remains uncontrolled in Anatolian Turkey. To evaluate protection after mass vaccination we measured post-vaccination antibodies in a cohort of cattle (serotypes O, A and Asia-1). To obtain results reflecting typical field protection, participants were randomly sampled from across Central and Western Turkey after routine vaccination. Giving two-doses one month apart is recommended when cattle are first vaccinated against FMD. However, due to cost and logistics, this is not routinely performed in Turkey, and elsewhere. Nested within the cohort, we conducted a randomised trial comparing post-vaccination antibodies after a single-dose versus a two-dose primary vaccination course. Four to five months after vaccination, only a third of single-vaccinated cattle had antibody levels above a threshold associated with protection. A third never reached this threshold, even at peak response one month after vaccination. It was not until animals had received three vaccine doses in their lifetime, vaccinating every six months, that most (64% to 86% depending on serotype) maintained antibody levels above this threshold. By this time cattle would be >20 months old with almost half the population below this age. Consequently, many vaccinated animals will be unprotected for much of the year. Compared to a single-dose, a primary vaccination course of two-doses greatly improved the level and duration of immunity. We concluded that the FMD vaccination programme in Anatolian Turkey did not produce the high levels of immunity required. Higher potency vaccines are now used throughout Turkey, with a two-dose primary course in certain areas. Monitoring post-vaccination serology is an important component of evaluation for FMD vaccination programmes. However, consideration must be given to which antigens are present in the test, the vaccine and the field virus. Differences between these antigens affect the relationship between antibody titre and protection.

Antigenic and genetic comparison of foot-and-mouth disease virus serotype O Indian vaccine strain, O/IND/R2/75 against currently circulating viruses.

Foot-and-mouth disease (FMD) virus serotype O is the most common cause of FMD outbreaks in India and three of the six lineages that have been described are most frequently detected, namely Ind2001, PanAsia and PanAsia 2. We report the full capsid sequence of 21 serotype O viruses isolated from India between 2002 and 2012. All these viruses belong to the Middle East-South Asia (ME-SA) topotype. The serological cross-reactivity of a bovine post-vaccination serum pool raised against the current Indian vaccine strain, O/IND/R2/75,was tested by virus neutralisation test with the 23 Indian field isolates, revealing a good match between the vaccine and the field isolates. The cross reactivity of the O/IND/R2/75 vaccine with 19 field isolates from other countries (mainly from Asia and Africa) revealed a good match to 79% of the viruses indicating that the vaccine strain is broadly cross-reactive and could be used to control FMD in other countries. Comparison of the capsid sequences of the serologically non-matching isolates with the vaccine strain sequence identified substitutions in neutralising antigenic sites 1 and 2, which could explain the observed serological differences.

The use of serosurveys following emergency vaccination, to recover the status of "foot-and-mouth disease free where vaccination is not practised".

To eliminate incursions of foot-and-mouth disease (FMD) quickly, a combination of measures, including emergency vaccination, can help block the spread of infection. For the earliest recovery of the FMD-free status for trade, without the slaughter of uninfected vaccinated animals, a serosurvey for antibodies to FMD virus non-structural proteins (NSP) must be used to substantiate absence of occult virus infections. Areas of doubt over requirements for post-vaccination serosurveillance and its feasibility include the required and achievable confidence, the amount of sampling necessary, and the appropriate responses to and consequences of different seropositive findings. This derives largely from uncertainty over the extent of localised pockets of virus infection that may remain within vaccinated populations and the circumstances that permit this. The question therefore remains whether tests are sufficiently sensitive and specific to detect and eliminate infected animals, without excessive culling of uninfected animals, before vaccinated animals mix with non-vaccinated livestock when movement restrictions are lifted. It is recommended to change the rationale for serosurveillance after emergency vaccination. Only when emergency vaccination is used in limited outbreaks is it possible to test and cull comprehensively, an approach compatible with a three-month minimum period to recover the FMD-free status. In other situations, where emergency vaccination is used, such as dealing with large outbreaks in animal-dense regions and where the onset of vaccination has been delayed, post-vaccination serosurveys should be targeted and focus on providing an assurance to detect higher levels of infection, in case of inadequate control measures. As this provides less assurance of absence of infection, the approach would be compatible with a six-month waiting period for free-status recovery and should be complemented by other methods to provide evidence that vaccination and control measures have been effectively implemented, as these are the best guarantee against continuing virus transmission.

Within herd transmission and evaluation of the performance of clinical and serological diagnosis of foot-and-mouth disease in partially immune cattle herds.

The control of foot-and-mouth disease (FMD) in vaccinated populations relies upon surveillance activities such as clinical inspections (CI) and serological monitoring. New evidence to refine current surveillance guidelines has been provided by evaluating (1) the diagnostic performance of CI and serological tests for detection of FMD virus (FMDV) non-structural proteins (NSP), and (2) the within-herd transmission of the virus in partially immune cattle. Data came from 23 affected herds during an epidemic of FMDV type O in Bolivia, in 2007. All cattle (n=957) in these herds were clinically inspected and serum samples were collected one month after the last animal with clinical signs was detected. Samples were tested for the presence of antibodies against NSP using the PANAFTOSA 3ABC-ELISA test and a subset of samples were tested using the enzyme-linked immunoelectrotransfer blot assay (EITB). Data from clinical and serological diagnoses were analysed using a Bayesian model. The sensitivity Se and specificity Sp of the tests, as well as the prevalence and the within-herd reproduction ratio R of FMDV were estimated. In addition, risk factors for infection were identified. The Se of CI, the 3ABC-ELISA and the EITB tests were estimated to be 0.30, 0.88 and 0.96 respectively. The estimated Sp, in the same order, were 0.88, 0.93 and 0.97. The within-herd prevalence of infected animals ranged from 0.04 to 0.91 and R ranged from 1.02 to 2.68. It was observed that cattle coming from areas with high vaccination coverage had a lower risk of becoming infected than home-bred cattle from the affected herds, where vaccination coverage was thought to be low. Although these estimates come from herds kept under specific conditions, they provide a reference for future surveillance design and can inform simulation models for surveillance and control of FMD in similar cattle populations.

Recombinant pestivirus E2 glycoproteins prevent viral attachment to permissive and non permissive cells with different efficiency.

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen, which like other pestiviruses has similar molecular biological features to hepaciviruses, including human Hepatitis C virus. The pestivirus E2 glycoproteins are the major target for virus-neutralising antibodies, as well as playing a role in receptor binding and host range restriction. In this study, recombinant E2 glycoproteins (rE2) derived from three different pestivirus species were examined for their inhibitory effects on pestivirus infectivity in cell culture. Histidine-tagged rE2 glycoproteins of BVDV type 2 strain 178003, BVDV type 1 strain Oregon C24V and CSFV strain Alfort 187 were produced in Spodoptera frugiperda insect cells and purified under native conditions. The ability of rE2 glycoprotein to inhibit the infection of permissive cells by both homologous and heterologous virus was compared, revealing that the inhibitory effects of rE2 glycoproteins correlated with the predicted similarity of the E2 structures in the recombinant protein and the test virus. This result suggests that the sequence and structure of E2 are likely to be involved in the host specificity of pestiviruses at their point of uptake into cells.

Veterinary and human vaccine evaluation methods.

Despite the universal importance of vaccines, approaches to human and veterinary vaccine evaluation differ markedly. For human vaccines, vaccine efficacy is the proportion of vaccinated individuals protected by the vaccine against a defined outcome under ideal conditions, whereas for veterinary vaccines the term is used for a range of measures of vaccine protection. The evaluation of vaccine effectiveness, vaccine protection assessed under routine programme conditions, is largely limited to human vaccines. Challenge studies under controlled conditions and sero-conversion studies are widely used when evaluating veterinary vaccines, whereas human vaccines are generally evaluated in terms of protection against natural challenge assessed in trials or post-marketing observational studies. Although challenge studies provide a standardized platform on which to compare different vaccines, they do not capture the variation that occurs under field conditions. Field studies of vaccine effectiveness are needed to assess the performance of a vaccination programme. However, if vaccination is performed without central co-ordination, as is often the case for veterinary vaccines, evaluation will be limited. This paper reviews approaches to veterinary vaccine evaluation in comparison to evaluation methods used for human vaccines. Foot-and-mouth disease has been used to illustrate the veterinary approach. Recommendations are made for standardization of terminology and for rigorous evaluation of veterinary vaccines.

Retrospective evaluation of foot-and-mouth disease vaccine effectiveness in Turkey.

Foot-and-mouth disease (FMD) is present in much of Turkey and its control is largely based on vaccination. The arrival of the FMD Asia-1 serotype in Turkey in 2011 caused particular concern, spreading rapidly westwards across the country towards the FMD free European Union. With no prior natural immunity, control of spread would rely heavily on vaccination. Unlike human vaccines, field protection is rarely evaluated directly for FMD vaccines. Between September 2011 and July 2012 we performed four retrospective outbreak investigations to assess the vaccine effectiveness (VE) of FMD Asia-1 vaccines in Turkey. Vaccine effectiveness is defined as the reduction in risk in vaccinated compared to unvaccinated individuals with similar virus exposure in the field. The four investigations included 12 villages and 1230 cattle >4 months of age. One investigation assessed the FMD Asia-1 Shamir vaccine, the other three evaluated the recently introduced FMD Asia-1 TUR 11 vaccine made using a field isolate of the FMD Asia-1 Sindh-08 lineage that had recently entered Turkey. After adjustment for confounding, the TUR 11 vaccine provided moderate protection against both clinical disease VE=69% [95% CI: 50%-81%] and infection VE=63% [95% CI: 29%-81%]. However, protection was variable with some herds with high vaccine coverage still experiencing high disease incidence. Some of this variability will be the result of the variation in virus challenge and immunity that occurs under field conditions. In the outbreak investigated there was no evidence that the Asia-1 Shamir vaccine provided adequate protection against clinical FMD with an incidence of 89% in single vaccinated cattle and 69% in those vaccinated two to five times. Based on these effectiveness estimates, vaccination alone is unlikely to produce the high levels of herd immunity needed to control FMD without additional control measures.

Antigenic variation of foot-and-mouth disease virus serotype A.

The current measures to control foot-and-mouth disease (FMD) include vaccination, movement control and slaughter of infected or susceptible animals. One of the difficulties in controlling FMD by vaccination arises due to the substantial diversity found among the seven serotypes of FMD virus (FMDV) and the strains within these serotypes. Therefore, vaccination using a single vaccine strain may not fully cross-protect against all strains within that serotype, and therefore selection of appropriate vaccines requires serological comparison of the field virus and potential vaccine viruses using relationship coefficients (r1 values). Limitations of this approach are that antigenic relationships among field viruses are not addressed, as comparisons are only with potential vaccine virus. Furthermore, inherent variation among vaccine sera may impair reproducibility of one-way relationship scores. Here, we used antigenic cartography to quantify and visualize the antigenic relationships among FMD serotype A viruses, aiming to improve the understanding of FMDV antigenic evolution and the scope and reliability of vaccine matching. Our results suggest that predicting antigenic difference using genetic sequence alone or by geographical location is not currently reliable. We found co-circulating lineages in one region that were genetically similar but antigenically distinct. Nevertheless, by comparing antigenic distances measured from the antigenic maps with the full capsid (P1) sequence, we identified a specific amino acid substitution associated with an antigenic mismatch among field viruses and a commonly used prototype vaccine strain, A22/IRQ/24/64.

Diagnosis of foot-and-mouth disease.

Foot-and-mouth disease virus (FMDV) exists as multiple serotypes and strains that infect a range of cloven-hoofed animals with variable severity. Clinical diagnosis reinforced by diagnostic tests support timely intervention, whilst virus characterisation helps trace routes of spread and select appropriate vaccine strains. To speed up and simplify diagnosis, penside tests have recently been developed. Serology is used to identify undisclosed infection and substantiate freedom from infection and specific tests are needed to detect infected animals in vaccinated populations. Serology is also used to estimate post-vaccinal population immunity. Contingency plans are required to enable countries to scale up diagnosis at short notice. Improvements are needed in preclinical and penside diagnosis and in our ability to model vaccine effectiveness.

Foot-and-mouth disease virus epitope dominance in the antibody response of vaccinated animals.

Five neutralizing antigenic sites have been identified on the surface of serotype O foot-and-mouth disease virus (FMDV). A set of mAb neutralization-escape mutant viruses was used for the first time to evaluate the relative use of known binding sites by polyclonal antibodies from three target species: cattle, sheep and pigs. Antibodies to all five neutralizing antigenic sites were detected in all three species, with most antibodies directed against antigenic site 2, followed by antigenic site 1. In 76 % of cattle, 65 % of sheep and 58 % of pigs, most antibodies were directed against site 2. Antibodies specific to antigenic sites 3, 4 and 5 were found to be minor constituents in the sera of each of the target species. This implies that antigenic site 2 is a dominant neutralization immunogenic site in serotype O FMDV and may therefore be a good candidate for designing novel vaccines.

Combining livestock trade patterns with phylogenetics to help understand the spread of foot and mouth disease in sub-Saharan Africa, the Middle East and Southeast Asia.

International trade in animals and their products is recognised as a primary determinant of the global epidemiology of transboundary diseases such as foot and mouth disease (FMD). As well as causing serious production losses, FMD is highly contagious, being transmitted through multiple routes and hosts, which makes it one of the most important diseases affecting trade in livestock. Its occurrence has dramatic consequences for the agricultural economy of a normally disease-free country, as well as for the livelihoods and income generation of developing countries where the disease continues to be endemic. In the dynamic of FMD virus (FMDV) dispersal across the globe, phylogenetic inference from molecular sequences of isolated viruses makes a significant contribution to investigating the evolutionary and spatial pathways underlying the source of FMD epidemics. Matching data on livestock movement with molecular epidemiology can enhance our fundamental understanding when reconstructing the spread of the virus between geographical regions, which is essential for the development of FMD control strategies worldwide. This paper reviews the global situation of FMD in the last ten years, combining phylogenetic insights with information on livestock production systems and international trade to analyse the epidemiological dynamics of FMD and the sources of FMDV introductions at a regional level in sub-Saharan Africa, the Middle East and Southeast Asia.

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.

Characterisation and epitope mapping of neutralising monoclonal antibodies to A24 Cruzeiro strain of FMDV.

Characterisation of seven neutralising monoclonal antibodies (mAbs) produced against foot-and-mouth disease virus A(24) Cruzeiro revealed three reactivity groups. Gr-I recognised linear epitopes where as Gr-II was conformation-dependent and trypsin-insensitive. The Gr-III was also conformation-dependent, but trypsin-sensitive. Mar (mAb neutralisation resistant)-mutants could only be produced against Gr-I and Gr-III mAbs. Capsid sequence comparison of Gr-I mar-mutants with parent virus revealed changes in the G-H loop of VP1 at positions 141, 143 and 147. Similarly, a Gr-III mar-mutant showed a change from a highly conserved glycine to a tryptophan at position 148 of VP1 along with three additional changes at the N-terminus of VP1, VP2 and VP4. This residue at 148 of VP1 is located at +2 position after "RGD" and is equivalent to the position identified by the mAb recognising site 5 in serotype O viruses. This site is probably formed because of the interaction of the G-H loop with other residues in different structural proteins.

Molecular characterization of foot-and-mouth disease viruses collected from Sudan.

The aim of this study was to characterize foot-and-mouth disease (FMD) viruses collected between 2004 and 2008 from Sudan, a country where FMD is endemic. Using virus isolation and antigen ELISA, three FMD virus serotypes (O, A and SAT2) were detected in 24 samples that were submitted to the FAO World Reference Laboratory for FMD. Pan-serotypic real-time RT-PCR assays targeting the 5' untranslated region (5'UTR) and 3D genes of FMD virus were also used to contribute to the laboratory diagnosis of these cases. The lack of concordant results between the real-time RT-PCR assays for three serotype O viruses was attributed to four nucleotide mismatches in the 5'UTR PCR primer and probe sites (three substitutions for the sense-primer and one in the TaqMan(®) probe region). Taken together, the laboratory results showed that recent FMD outbreaks that occurred during 2008 in northern and central Sudan were caused by serotypes O and SAT2, while serotype A was last detected in 2006. Phylogenetic analyses of VP1 sequences from these viruses were used to determine the relationships with 23 older viruses from Sudan and other viruses from West and East Africa. For serotype O, closest genetic identities were between concurrent and historical Sudanese isolates, indicating that within-country circulation is an important mechanism by which FMD is maintained year-on-year in Sudan. A similar pattern was also evident for serotype A and SAT2 viruses; however, these lineages also contained recent representative FMD viral isolates from other countries in the region suggesting that long-distance animal movement can also contribute to FMD dispersal across sub-Saharan Africa. These findings provide the first molecular description of FMD viruses that are circulating in Sudan, and highlight that further sampling of representative viruses from the region is required before the complex epidemiology of FMD in sub-Saharan Africa can be fully understood.

Qualitative assessment of the commodity risk for spread of foot-and-mouth disease associated with international trade in deboned beef.

The risk of importing foot-and-mouth disease virus (FMDV) restricts trade in livestock and their products from parts of the world where the virus is present. This reduces trade opportunities and investment in the livestock sector of many developing countries and constrains global food supply. This review focuses on the risks associated with trade in deboned beef (DB) from foot-and-mouth disease (FMD)-infected cattle, countries or zones. A definition of DB is provided along with a description of the procedures for its preparation within beef slaughtering operations. Evidence is reviewed for circumstances under which DB can be contaminated with FMDV, and a commodity risk factor approach is used to consider the mitigating efficacy of slaughterhouse procedures. A combination of pre-slaughter and slaughterhouse measures has enabled DB to be safely imported into FMD-free countries from countries that were not nationally or zonally FMD-free. Nevertheless, current evidence does not provide absolute assurance that abattoir procedures for producing DB can result, by themselves, in a commodity with a negligible risk of transmitting FMDV without complementary measures to reduce the likelihood of slaughtering infected cattle. The main areas of uncertainty are the amounts of residual FMDV-harbouring tissues within DB, and our understanding of what constitutes a safe level of contamination. More detailed guidance should be developed to specify the mitigating measures needed in support of the export of DB from regions that are not officially FMD-free. This will help to avoid differences in interpretation of what is needed that give rise to obstacles to trade.