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.

Detection of Bovine Antibodies against a Conserved Capsid Epitope as the Basis of a Novel Universal Serological Test for Foot-and-Mouth Disease.

Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral nonstructural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus (FMDV) serotype and their sensitivity may be affected by antigenic variability within each serotype and mismatching between test reagents. As a consequence, FMD reference laboratories are required to maintain multiple type-specific SP assays and reagents. A universal SP test would simplify frontline diagnostics and facilitate large-scale serological surveillance and postvaccination monitoring. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterized using a panel of intertype-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide enzyme-linked immunosorbent assay (VP2-ELISA) was optimized using experimental and reference antisera from immunized, convalescent, and naïve animals (n = 172). The VP2-ELISA is universal and simple and provided sensitive (99%) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for serosurveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in countries where the disease is endemic.

The evolution and phylodynamics of serotype A and SAT2 foot-and-mouth disease viruses in endemic regions of Africa.

Foot-and-mouth disease (FMD) is a major livestock disease with direct clinical impacts as well as indirect trade implications. Control through vaccination and stamping-out has successfully reduced or eradicated the disease from Europe and large parts of South America. However, sub-Saharan Africa remains endemically affected with 5/7 serotypes currently known to be circulating across the continent. This has significant implications both locally for livestock production and poverty reduction but also globally as it represents a major reservoir of viruses, which could spark new epidemics in disease free countries or vaccination zones. This paper describes the phylodynamics of serotypes A and SAT2 in Africa including recent isolates from Cameroon in Central Africa. We estimated the most recent common ancestor for serotype A was an East African virus from the 1930s (median 1937; HPD 1922-1950) compared to SAT2 which has a much older common ancestor from the early 1700s (median 1709; HPD 1502-1814). Detailed analysis of the different clades shows clearly that different clades are evolving and diffusing across the landscape at different rates with both serotypes having a particularly recent clade that is evolving and spreading more rapidly than other clades within their serotype. However, the lack of detailed sequence data available for Africa seriously limits our understanding of FMD epidemiology across the continent. A comprehensive view of the evolutionary history and dynamics of FMD viruses is essential to understand many basic epidemiological aspects of FMD in Africa such as the scale of persistence and the role of wildlife and thus the opportunities and scale at which vaccination and other controls could be applied. Finally we ask endemic countries to join the OIE/FAO supported regional networks and take advantage of new cheap technologies being rolled out to collect isolates and submit them to the World Reference Laboratory.

Outbreak investigations and molecular characterization of foot-and-mouth disease viruses circulating in south-west Niger.

In Niger, the epidemiological situation regarding foot-and-mouth disease is unclear as many outbreaks are unreported. This study aimed (i) to identify Foot-and-mouth disease virus (FMDV) strains currently circulating in cattle herds, and (ii) to identify risk factors associated with Foot-and-mouth disease (FMD)-seropositive animals in clinical outbreaks. Epithelial tissues (n = 25) and sera (n = 227) were collected from cattle in eight districts of the south-western part of Niger. Testing of clinical material revealed the presence of FMDV serotype O that was characterized within the O/WEST AFRICA topotype. The antigenic relationship between one of the FMDV isolates from Niger (O/NGR/4/2015) and three reference vaccine strains was determined by the two-dimensional virus neutralization test (2dmVNT), revealing a close antigenic match between the field isolate from Niger and three FMDV serotype O vaccine strains. Serological analyses using a non-structural protein (NSP) test provided evidence for previous FMDV infection in 70% (158/227) of the sera tested. Multivariate logistic regression analysis revealed that only the herd composition (presence of both cattle and small ruminants) was significantly associated with FMDV seropositivity as defined by NSP-positive results (p-value = .006). Of these positive sera, subsequent testing by liquid-phase blocking ELISA (LPBE) showed that 86% (136/158) were positive for one (or more) of four FMDV serotypes (A, O, Southern African Territories (SAT) 1 and SAT 2). This study provides epidemiological information about FMD in the south-western part of Niger and highlights the complex transboundary nature of FMD in Africa. These findings may help to develop effective control and preventive strategies for FMD in Niger as well, as other countries in West Africa.

Emergence of an exotic strain of serotype O foot-and-mouth disease virus O/ME-SA/Ind-2001d in South-East Asia in 2015.

The O/Middle East-South Asia (ME-SA)/Ind-2001 lineage of foot-and-mouth disease virus (FMDV) is endemic in the Indian subcontinent and has been reported in the Middle East and North Africa, but it had not been detected in South-East Asia (SEA) before 2015. This study reports the recent incursions of this viral lineage into SEA, which caused outbreaks in Vientiane Capital of Lao People's Democratic Republic (PDR) in April 2015, in Dak Nong, Dak Lak and Ninh Thuan Provinces of Vietnam from May to October 2015, and in Rakhine State of Myanmar in October 2015. Disease investigations were conducted during the outbreaks and followed up after laboratory results confirmed the involvement of FMDV O/ME-SA/Ind-2001 sublineage d (O/ME-SA/Ind-2001d). Affected host species included cattle, buffalo and pig, and all the outbreaks resolved within 2 months. Animals with clinical signs were separated, and affected premises were disinfected. However, strict movement restrictions were not enforced, and emergency vaccinations were only implemented in Vientiane Capital of Lao PDR and Dak Nong and Ninh Thuan Provinces of Vietnam. Clinical samples were collected from each outbreak and examined by nucleotide sequencing of the FMDV viral protein 1 coding region. Sequence analysis revealed that the O/ME-SA/Ind-2001d isolates from Lao PDR and Vietnam were closely related to each other and similar to viruses previously circulating in India in 2013. Viruses collected from Myanmar were divergent from viruses of the same sublineage recovered from Lao PDR and Vietnam but were closely related to viruses present in Bangladesh in 2015. These findings imply that at least two independent introductions of O/ME-SA/Ind-2001d into SEA have occurred. Our study highlights the transboundary nature of foot-and-mouth disease (FMD) and reinforces the importance of improved FMD surveillance and promotion of safer cross-border trade in SEA to control the risk of introduction and spread of exotic FMDV strains.

Direct detection and characterization of foot-and-mouth disease virus in East Africa using a field-ready real-time PCR platform.

Effective control and monitoring of foot-and-mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype-specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD-endemic countries have motivated the development of simple diagnostic platforms to support local decision-making. Using a portable thermocycler, the T-COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan-serotype-specific real-time RT-PCR (rRT-PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa-specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan-serotype-specific lyophilized assay were comparable to that of an OIE-recommended laboratory-based rRT-PCR (determined using a panel of 57 FMDV-positive samples and six non-FMDV vesicular disease samples for differential diagnosis). The FMDV-typing assay was able to correctly identify the serotype of 33/36 FMDV-positive samples (no cross-reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal-pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre-clinical, clinical and clinically recovered cattle. These data support the use of field-ready rRT-PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine.

Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/µl for ASFV, PCV2 and PRRSV, 100 copies/µl for SVDV, CSFV, VESV and 1,000 copies/µl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases.

Rapid and simple detection of foot-and-mouth disease virus: Evaluation of a cartridge-based molecular detection system for use in basic laboratories.

Highly contagious transboundary animal diseases such as foot-and-mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen-side. Here, we report on a study to evaluate a fully automated cartridge-based real-time RT-PCR diagnostic system (Enigma MiniLab® ) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real-time RT-PCR (rRT-PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter-laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge-based automated real-time RT-PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.

Technological advances in veterinary diagnostics: opportunities to deploy rapid decentralised tests to detect pathogens affecting livestock.

Sustainable food production capable of feeding a growing human population is a significant global challenge, and is a priority encompassed within the United Nations Millennium Development Goal to 'eradicate extreme poverty and hunger'. Infectious diseases reduce the productivity of farm animals, and the globalised trade of animals and their products increases the threat of disease incursion. Accurate and rapid diagnostic tests are an essential component of contingency plans to detect, control and eradicate such diseases. Diagnosis involves a 'pipeline' that normally starts with clinical suspicion, followed by collecting samples, transporting specimens to a centralised laboratory setting (e.g. national/international Reference Laboratories), analysing these samples using a range of diagnostic tests and reporting the results. However, the transport of specimens from the field to the laboratory can be a lengthy process that can delay critical decision-making and severely affect the quality of the samples. This important limitation of centralised diagnostic testing has motivated the development of tools for the rapid, simple detection of livestock pathogens. Recent advances in the development of technologies for personalised human medicine have motivated the development of prototype diagnostic tests for a wide selection of diseases of livestock. However, many of these tests are not yet routinely used or commercially available. This paper critically reviews the most promising examples of such assays, and highlights the challenges that remain to transition these tests from applied research and development into routine use.

La production durable de denrées alimentaires pour nourrir une population humaine en constante augmentation constitue un vaste enjeu planétaire ainsi que l'une des priorités définies par les Nations Unies dans le cadre des Objectifs du Millénaire pour le développement visant à « éradiquer l'extrême pauvreté et la faim dans le monde ¼. D'une part, les maladies animales réduisent la productivité des animaux d'élevage ; d'autre part, la mondialisation des échanges d'animaux et de produits d'origine animale intensifie les risques d'incursion de maladies. L'utilisation de tests de diagnostic rapides et fiables est une composante essentielle des plans d'urgence visant à détecter, contrôler et éradiquer ces maladies. Une procédure de diagnostic est généralement constituée de plusieurs opérations, depuis la suspicion clinique, la collecte d'échantillons, leur transport vers un laboratoire central (par exemple un laboratoire de référence national/international), jusqu'à l'analyse de ces échantillons au moyen d'une série de tests diagnostiques et la notification des résultats. Néanmoins, le transport des échantillons depuis le terrain jusqu'au laboratoire est parfois un processus très long qui peut retarder la prise de décisions cruciales, voire compromettre gravement la qualité des échantillons. Cette limitation importante des procédures diagnostiques centralisées a incité à mettre au point des outils permettant une détection rapide et aisée des agents pathogènes affectant le bétail. Les progrès récents accomplis dans les technologies relevant de la médecine humaine personnalisée ont encouragé le développement de prototypes d'épreuves de diagnostic pour nombre de maladies du bétail. Toutefois, plusieurs de ces tests ne sont pas encore utilisés en routine ni disponibles commercialement. Les auteurs font le point sur les exemples les plus prometteurs de ces tests et soulignent les difficultés restant à résoudre pour que ces tests puissent évoluer d'une application en recherche et développement à une utilisation en routine.

El logro de una producción sostenible de alimentos en cantidad suficiente para abastecer a una población humana cada vez más numerosa es una difícil empresa que el mundo tiene ante sí, que además entronca con una de las prioridades plasmadas en los Objetivos de Desarrollo del Milenio de las Naciones Unidas: «erradicar la pobreza extrema y el hambre¼. Las enfermedades infecciosas merman la productividad de los animales de granja, al tiempo que el comercio mundializado de animales y sus derivados amplifica la amenaza de incursiones infecciosas. La existencia de pruebas de diagnóstico rápidas y exactas es un elemento básico de todo plan de emergencia encaminado a detectar, controlar y erradicar esas enfermedades. Las labores de diagnóstico entrañan un «circuito¼ que normalmente empieza con la sospecha clínica, sigue con la obtención de muestras, su transporte a un laboratorio central (como un laboratorio de referencia nacional o internacional) y su análisis mediante diversas pruebas de diagnóstico y culmina con la notificación de los resultados. Sin embargo, el transporte hasta un laboratorio de las muestras obtenidas sobre el terreno es a veces un proceso lento, que puede retrasar la adopción de decisiones cruciales y mermar sensiblemente la calidad de las muestras. Este importante inconveniente derivado de la realización centralizada de pruebas ha llevado a concebir herramientas que permitan detectar de forma rápida y sencilla patógenos presentes en el ganado. Los avances registrados últimamente en la obtención de tecnologías destinadas a la medicina humana personalizada han propiciado también la elaboración de prototipos de pruebas para diagnosticar numerosas enfermedades del ganado, aunque muchas de ellas todavía no se utilizan sistemáticamente ni están comercializadas. Los autores, tras examinar en clave crítica los más prometedores ejemplos de estos nuevos ensayos, señalan las dificultades que aún subsisten para que estas pruebas puedan pasar del ámbito de la investigación aplicada y el desarrollo al de su utilización sistemática.

Characterization of Foot-and-Mouth Disease Viruses Collected in Nigeria Between 2007 and 2014: Evidence for Epidemiological Links Between West and East Africa.

This study describes the molecular characterization of 47 foot-and-mouth disease (FMD) viruses recovered from field outbreaks in Nigeria between 2007 and 2014. Antigen ELISA of viral isolates was used to identify FMD virus serotypes O, A and SAT 2. Phylogenetic analyses of VP1 nucleotide sequences provide evidence for the presence of multiple sublineages of serotype SAT 2, and O/EAST AFRICA 3 (EA-3) and O/WEST AFRICA topotypes in the country. In contrast, for serotype A, a single monophyletic cluster of viruses has persisted within Nigeria (2009-2013). These results demonstrate the close genetic relatedness of viruses in Nigeria to those from other African countries, including the first formal characterization of serotype O/EA-3 viruses in Nigeria. The introductions and persistence of certain viral lineages in Nigeria may reflect transmission patterns via nomadic pastoralism and animal trade. Continuous monitoring of field outbreaks is necessary to dissect the complexity of FMD epidemiology in sub-Saharan Africa.

Multiplex RT-PCR and Automated Microarray for Detection of Eight Bovine Viruses.

Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)-PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non-specific reactions were observed with a panel of 23 non-target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post-inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID50 /ml for RPV. The novel microarray platform automates the entire post-PCR steps of the assay and integrates electrophoretic-driven capture probe printing in a single user-friendly instrument that allows array layout and assay configuration to be user-customized on-site.

Detection of Capripoxvirus DNA Using a Field-Ready Nucleic Acid Extraction and Real-Time PCR Platform.

Capripoxviruses, comprising sheep pox virus, goat pox virus and lumpy skin disease virus cause serious diseases of domesticated ruminants, notifiable to The World Organization for Animal Health. This report describes the evaluation of a mobile diagnostic system (Enigma Field Laboratory) that performs automated sequential steps for nucleic acid extraction and real-time PCR to detect capripoxvirus DNA within laboratory and endemic field settings. To prepare stable reagents that could be deployed into field settings, lyophilized reagents were used that employed an established diagnostic PCR assay. These stabilized reagents demonstrated an analytical sensitivity that was equivalent, or greater than the established laboratory-based PCR test which utilizes wet reagents, and the limit of detection for the complete assay pipeline was approximately one log10 more sensitive than the laboratory-based PCR assay. Concordant results were generated when the mobile PCR system was compared to the laboratory-based PCR using samples collected from Africa, Asia and Europe (n = 10) and experimental studies (n = 9) representing clinical cases of sheep pox, goat pox and lumpy skin disease. Furthermore, this mobile assay reported positive results in situ using specimens that were collected from a dairy cow in Morogoro, Tanzania, which was exhibiting clinical signs of lumpy skin disease. These data support the use of mobile PCR systems for the rapid and sensitive detection of capripoxvirus DNA in endemic field settings.

Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings.

Accurate, timely diagnosis is essential for the control, monitoring and eradication of foot-and-mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple-to-use technologies, including molecular-based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time RT-PCR (rRT-PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory-based rRT-PCR. However, the lack of robust 'ready-to-use kits' that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT-PCR and RT-LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real-time, and for the RT-LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV.

Genomics and outbreaks: foot and mouth disease.

Foot and mouth disease virus (FMDV) is an animal pathogen of global economic significance. Identifying the sources of outbreaks plays an important role in disease control; however, this can be confounded by the ease with which FMDV can spread via movement of infected livestock and animal products, aerosols or fomites, e.g. contaminated persons and objects. As sequencing technologies have advanced, this review highlights the uses of viral genomic data in helping to understand the global distribution and transboundary movements of FMDV, and the role that these approaches have played in control and surveillance programmes. The recent application of next-generation sequencing platforms to address important epidemiological and evolutionary challenges is discussed with particular reference to the advent of 'omics' technologies.

Le virus de la fièvre aphteuse est un agent pathogène affectant les animaux d'élevage, avec des conséquences économiques considérables à l'échelle mondiale. La détection des sources des foyers est un aspect important de la lutte contre cette maladie ; l'efficacité de cette stratégie est toutefois compromise par la facilité avec laquelle le virus de la fièvre aphteuse se propage à la faveur des mouvements d'animaux ou de produits d'origine animale infectés, d'aérosols ou de personnes ou matières contaminées. Les auteurs décrivent, au fur et à mesure des avancées des technologies du séquençage, les données de la génomique virale qui ont permis de mieux comprendre la distribution mondiale et la propagation transfrontalière du virus de la fièvre aphteuse et le rôle que ces approches ont commencé à jouer dans les programmes de contrôle et de surveillance. Les auteurs examinent également les applications récentes des plates-formes de séquençage de nouvelle génération pour résoudre des problèmes épidémiologiques et évolutifs importants, en se référant particulièrement à l'avènement des technologies dites «­omiques ¼.

El virus de la fiebre aftosa es un patógeno animal que reviste importancia planetaria. A la hora de combatir la enfermedad es útil poder determinar el origen de los brotes, tarea que sin embargo puede verse frustrada por la facilidad con que el virus es capaz de diseminarse siguiendo los desplazamientos de animales o derivados animales infectados o por aerosoles o fómites (por ejemplo personas u objetos contaminados). Los autores hacen hincapié en la utilización de datos de genómica vírica para ayudar a aprehender la distribución mundial y los movimientos transfronterizos del virus de la fiebre aftosa, lo cual es posible gracias a los avances que han conocido las técnicas de secuenciación, así como en la función que pueden cumplir estos métodos dentro de los programas de control y vigilancia. También examinan la reciente aplicación de dispositivos de secuenciación de próxima generación para abordar importantes problemas epidemiológicos y evolutivos, refiriéndose especialmente al advenimiento de las técnicas «ómicas¼.

Genome Sequence of Foot-and-Mouth Disease Virus Serotype O Isolated from Morocco in 2015.

The genome of a virus isolated from an outbreak of foot-and-mouth disease (FMD) in Morocco in 2015 is described here. This virus is classified as lineage Ind-2001d within serotype O, topotype ME-SA (Middle East-South Asia). This lineage is endemic on the Indian subcontinent but has caused outbreaks in the Middle East and North Africa since 2013.

Complete Genome Sequence of a Serotype A Foot-and-Mouth Disease Virus from an Outbreak in Saudi Arabia during 2015.

The complete genome of a foot-and-mouth disease (FMD) type A virus isolated from cattle in Saudi Arabia in 2015 is described here. This virus belongs to an FMD virus lineage named genotype VII, which is normally endemic on the Indian subcontinent.

Outbreaks of Foot-and-Mouth Disease in Libya and Saudi Arabia During 2013 Due to an Exotic O/ME-SA/Ind-2001 Lineage Virus.

Foot-and-mouth disease viruses are often restricted to specific geographical regions and spread to new areas may lead to significant epidemics. Phylogenetic analysis of sequences of the VP1 genome region of recent outbreak viruses from Libya and Saudi Arabia has revealed a lineage, O-Ind-2001, normally found in the Indian subcontinent. This paper describes the characterization of field viruses collected from these cases and provides information about a new real-time RT-PCR assay that can be used to detect viruses from this lineage and discriminate them from other endemic FMD viruses that are co-circulating in North Africa and western Eurasia.

VP1 sequencing protocol for foot and mouth disease virus molecular epidemiology.

Nucleotide sequences of field strains of foot and mouth disease virus (FMDV) contribute to our understanding of the distribution and evolution of viral lineages that circulate in different regions of the world. This paper outlines a practical reversetranscription polymerase chain reaction (RT-PCR) and sequencing strategy that can be used to generate RNA sequences encoding the VP1 (1D) region of FMDV. The protocol contains a panel of PCR and sequencing primers that can be selected to characterise genetically diverse isolates representing all seven FMDV serotypes. A list of sequences is also described, comprising prototype sequences for all proposed FMDV topotypes, in order to provide a framework for phylogenetic analysis. The technical details and prototype sequences provided in this paper can be employed by FMD Reference Laboratories and others in an approach to harmonise the molecular epidemiology of FMDV.

Les séquences de nucléotides des souches de terrain du virus de la fièvre aphteuse nous aident à comprendre la distribution et l'évolution des lignées virales présentes dans les différentes régions du monde. Les auteurs décrivent les grandes lignes d'un protocole pratique, basé sur l'amplification en chaîne par polymérase couplée à une transcription inverse (RT-PCR) et sur le séquençage, qui peut être utilisé pour générer des séquences d'ARN codant pour la région VP1 (1D) du virus de la fièvre aphteuse. Le protocole permet de procéder à une sélection parmi un panel de PCR et de marqueurs de séquençage dans le but de caractériser les gènes de divers isolats représentant les sept sérotypes du virus de la fièvre aphteuse. Les auteurs décrivent également une liste de séquences pouvant servir de cadre à l'analyse phylogénétique, dont des séquences prototypes pour tous les topotypes proposés du virus de la fièvre aphteuse. Les données techniques détaillées et les séquences prototypes fournies par les auteurs peuvent être utilisées par les Laboratoires de référence pour la fièvre aphteuse et d'autres institutions, en vue d'harmoniser l'épidémiologie moléculaire du virus de la fièvre aphteuse.

Las secuencias nucleotídicas de las cepas salvajes del virus de la fiebre aftosa nos ayudan a entender la distribución y evolución de los linajes víricos circulantes en distintas regiones del mundo. Los autores exponen sucintamente un práctico procedimiento de reacción en cadena de la polimerasa acoplada a transcripción inversa (RT-PCR) y de secuenciación que se puede utilizar para generar secuencias de ARN que codifican la región VP1 (1D) del virus de la fiebre aftosa. El protocolo ofrece la posibilidad de elegir entre todo un repertorio de cebadores de PCR y de secuenciación en el que están representados los siete serotipos víricos existentes con objeto de caracterizar genéticamente diversas cepas aisladas sobre el terreno. Los autores también presentan una lista de secuencias que comprende secuencias prototípicas de todos los topotipos propuestos del virus, a fin de proporcionar un marco de referencia para el análisis filogenético. Los laboratorios de referencia para la fiebre aftosa, así como otros establecimientos, pueden servirse de las detalladas técnicas y las secuencias prototípicas aquí presentadas para armonizar el estudio de la epidemiología molecular del virus de la fiebre aftosa.

Molecular Characterization of Foot-and-Mouth Disease Viruses Collected in Tanzania Between 1967 and 2009.

This paper describes the molecular characterization of foot-and-mouth disease viruses (FMDV) recovered from outbreaks in Tanzania that occurred between 1967 and 2009. A total of 44 FMDV isolates, containing representatives of serotypes O, A, SAT 1 and SAT 2 from 13 regions of Tanzania, were selected from the FAO World Reference Laboratory for FMD (WRLFMD) virus collection. VP1 nucleotide sequences were determined for RT-PCR amplicons, and phylogenetic reconstructions were determined by maximum likelihood and neighbour-joining methods. These analyses showed that Tanzanian type O viruses fell into the EAST AFRICA 2 (EA-2) topotype, type A viruses fell into the AFRICA topotype (genotype I), type SAT 1 viruses into topotype I and type SAT 2 viruses into topotype IV. Taken together, these findings reveal that serotypes O, A, SAT 1 and SAT 2 that caused FMD outbreaks in Tanzania were genetically related to lineages and topotypes occurring in the East African region. The close genetic relationship of viruses in Tanzania to those from other countries suggests that animal movements can contribute to virus dispersal in sub-Saharan Africa. This is the first molecular description of viruses circulating in Tanzania and highlights the need for further sampling of representative viruses from the region so as to elucidate the complex epidemiology of FMD in Tanzania and sub-Saharan Africa.

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.