Validation of Delphi procedure consensus criteria for defining fetal growth restriction.

OBJECTIVE: Recently, a Delphi procedure was used to establish new criteria for defining fetal growth restriction (FGR). These criteria require clinical validation. We sought to validate the Delphi consensus criteria by comparing their performance with that of our current definition (estimated fetal weight (EFW) < 10th percentile) in predicting adverse neonatal outcome (ANO). METHODS: This was a secondary analysis of data from a prospective cohort study of women referred for fetal growth assessment between 26 and 36 weeks' gestation. The current standard definition of FGR used in our clinical practice is EFW < 10th percentile using Hadlock's fetal growth standard. The Delphi consensus criteria for FGR include either a very small fetus (abdominal circumference (AC) or EFW < 3rd percentile) or a small fetus (AC or EFW < 10th percentile) with additional abnormal Doppler findings or a decrease in AC or EFW by two quartiles or more. The primary outcome was the prediction of a composite of ANO including one or more of: admission to the neonatal intensive care unit, cord pH < 7.1, 5-min Apgar score < 7, respiratory distress syndrome, intraventricular hemorrhage, neonatal seizures or neonatal death. The discriminatory capacities of the two definitions of FGR for composite ANO and delivery of a small-for-gestational-age (SGA) neonate, defined as birth weight < 10th percentile, were compared using area under the receiver-operating-characteristics curve (AUC). The sensitivity, specificity and predictive values of the methods were also compared. RESULTS: Of 1055 pregnancies included in the study, composite ANO occurred in 139 (13.2%). There were only two cases of early FGR (before 32 weeks); therefore, the study focused on late FGR. Our current FGR diagnostic criterion of EFW < 10th percentile was not associated significantly with composite ANO (relative risk (RR), 1.1 (95% CI, 0.6-1.8)), while the Delphi FGR criteria were (RR, 2.0 (95% CI, 1.2-3.3)). Our current definition of FGR showed higher discriminatory ability in the prediction of a SGA neonate (AUC, 0.69 (95% CI, 0.65-0.73)) than did the Delphi definition (AUC, 0.64 (95% CI, 0.60-0.67)) (P = 0.001). The AUCs of both definitions were poor for the prediction of composite ANO, despite slightly improved performance using the Delphi consensus definition of FGR (AUC, 0.53 (95% CI, 0.50-0.55)) compared with that of our current definition (AUC, 0.50 (95% CI, 0.48-0.53)) (P = 0.02). CONCLUSION: The newly postulated criteria for defining FGR based on a Delphi procedure detects fewer cases of neonatal SGA than does our current definition of EFW < 10th percentile, but is associated with a slight improvement in predicting ANO. Copyright © 2020 ISUOG. Published by John Wiley & Sons Ltd.

[Emerging vectorial diseases: West Nile fever, Bluetongue and Schmallenberg]. / Arboviroses émergentes : fièvre West Nile, fièvre catarrhale ovine et virus Schmallenberg.

The increase in international trade over the last thirty years, climate change owing to the industrial revolution, disruption of ecosystems, etc. are some of the factors that may explain the dynamics of disease emergence in regions of the world where they were not present. Thus in 1999, West Nile virus was introduced on the American continent where it spread at high speed. More than 2300 deaths and more than 25,000 neuroinvasive forms were recorded in humans from 1999 to 2019 in the United States of America. In the field of animal diseases, two viruses have made headlines in Europe: bluetongue virus (BTV) and Schmallenberg virus (SBV). The bluetongue virus, previously absent from Europe, was introduced in 1999. Numerous serotypes (1, 2, 4, 6, 8, 9, 25, 27) have since been identified in the European Union. Schmallenberg virus was identified in 2011 in Northern Germany and rapidly spread to other European countries. This virus had never been identified in the world before. These three viruses (WNV, BTV and SBV) are transmitted by arthropod vectors (mosquitoes and Culicoïdes). These emergences are a good illustration of the challenges that our countries will face in the coming years, in public, human and veterinary health.

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.

West Nile virus epizootics in the Camargue (France) in 2015 and reinforcement of surveillance and control networks.

West Nile virus (WNV) infection is a non-contagious disease mainly transmitted by the bites of infected mosquitoes from the genus Culex. The virus is maintained in a mosquito-bird-mosquito cycle, and can accidentally be transmitted to mammalian hosts. Among mammalian hosts, equines and humans are the most sensitive to WNV infection and can develop severe meningoencephalitis. As WNV infections are zoonotic and can be severe in humans and equines, West Nile fever is considered to be a public and animal health concern. After a silent period of almost ten years, WNV re-emerged in France at the periphery of the Camargue area during the summer of 2015, underlining the fact that the Camargue area creates favourable conditions for WNV emergence and amplification in France. The French Network for Epidemiological Surveillance of Equine Diseases (Réseau d'Épidémio-Surveillance en Pathologie Équine [RESPE]) facilitated the early detection of WNV cases in horses. In total, 49 horses were found to be infected; among them, 44 presented clinical signs, 41 with meningoencephalitis and three with hyperthermia only. Six horses among the 41 with nervous symptoms died from the disease or were euthanised (a case fatality rate of 14.6%). The authors describe the characteristics of the 2015 WNV epizootics, the early detection of the first WNV equine cases via the RESPE network and the coordination of WNV surveillance in France.

L'infection par le virus de West Nile est une maladie non contagieuse essentiellement transmise lors de piqûres de moustiques infectés appartenant au genre Culex ; le virus se maintient dans la nature au moyen d'un cycle moustique­ oiseau­moustique ; la transmission à des hôtes mammifères a lieu de manière accidentelle. Parmi les mammifères hôtes, les plus sensibles à l'infection par le virus de West Nile sont les équidés et l'homme, chez qui l'infection peut se manifester sous forme d'une méningo-encéphalite sévère. Les infections par le virus de West Nile étant des zoonoses potentiellement graves chez l'homme et chez les équidés, la fièvre de West Nile doit être considérée comme une priorité de santé publique et animale. Resté silencieux pendant plus d'une décennie, le virus de West Nile est réapparu en France à l'été 2015 en bordure de la Camargue, confirmant que les conditions de cette région sont favorables à l'émergence et à l'amplification du virus. Le réseau français d'épidémiosurveillance en pathologie équine (RESPE) a contribué à la détection précoce du virus de West Nile chez les chevaux. Au total, 49 chevaux étaient infectés, parmi lesquels 44 présentaient des signes cliniques, correspondant à une méningo-encéphalite pour 41 d'entre eux et à une hyperthermie seule pour les trois autres. Six chevaux parmi les 41 qui présentaient des signes neurologiques ont succombé à la maladie ou ont été euthanasiés (taux de létalité de 14,6 %). Les auteurs de cet article décrivent les principales caractéristiques de l'épizootie de 2015 due au virus de West Nile ainsi que la détection précoce des premiers cas équins grâce au réseau RESPE et la coordination des activités de surveillance du virus en France.

La infección por el virus West Nile es una enfermedad no contagiosa que se transmite básicamente por la picadura de mosquitos infectados del género Culex. El virus, que se instala en un ciclo mosquito­ave­mosquito, también puede transmitirse accidentalmente a mamíferos, de entre los cuales los más sensibles a la infección son los equinos y el ser humano, que pueden contraer graves meningoencefalitis. Puesto que las infecciones por este virus son zoonóticas y pueden revestir gravedad en personas y equinos, se considera que la fiebre West Nile es una enfermedad de importancia sanitaria y zoosanitaria. En Francia, tras un periodo silente de más de diez años, el virus reapareció en verano de 2015 en la periferia de la zona de la Camarga, poniendo así de manifiesto que esta zona genera condiciones propicias al surgimiento y la amplificación del virus en el país. La red francesa de vigilancia epidemiológica de patologías equinas (Réseau d'Épidémio-Surveillance en Pathologie Équine: RESPE]) facilitó la rápida detección de caballos infectados por el virus West Nile. Se detectaron en total 49 animales infectados, entre ellos 44 con signos clínicos, de los que 41 sufrían meningoencefalitis y tres solo presentaban hipertermia. Seis de los 41 caballos que mostraban signos neurológicos murieron a causa de la enfermedad o fueron sacrificados con métodos de eutanasia (lo que supone una tasa de letalidad del 14,6%). Los autores describen las principales características de la epizootia causada por el virus West Nile en 2015, la pronta detección de los primeros casos de caballos infectados gracias a la red RESPE y la coordinación de las labores de vigilancia del virus en Francia.

Schmallenberg virus: experimental infection in goats and bucks.

BACKGROUND: Schmallenberg virus (SBV) is an emerging Orthobunyavirus of ruminant livestock species currently circulating in Europe. SBV causes a subclinical or mild disease in adult animals but vertical transmission to pregnant dams may lead to severe malformations in the offspring. Data on the onset of clinical signs, viremia and seroconversion in experimentally infected adult animals are available for cattle and sheep but are still lacking for goats. For a better understanding of the pathogenesis of SBV infection in adult ruminants, we carried out experimental infections in adult goats. Our specific objectives were: (i) to record clinical signs, viremia and seroconversion; (ii) to monitor viral excretion in the semen of infected bucks; (iii) to determine in which tissues SBV replication took place and virus-induced lesions developed. RESULTS: Four goats and two bucks were inoculated with SBV. Virus inoculation was followed by a short viremic phase lasting 3 to 4 days and a seroconversion occurring between days 7 and 14 pi in all animals. The inoculated goats did not display any clinical signs, gross lesions or histological lesions. Viral genomic RNA was found in one ovary but could not be detected in other organs. SBV RNA was not found in the semen samples collected from two inoculated bucks. CONCLUSIONS: In the four goats and two bucks, the kinetics of viremia and seroconversion appeared similar to those previously described for sheep and cattle. Our limited set of data provides no evidence of viral excretion in buck semen.

African horse sickness.

African horse sickness (AHS) is a devastating disease of equids caused by an arthropod-borne virus belonging to the Reoviridae family, genus Orbivirus. It is considered a major health threat for horses in endemic areas in sub-Saharan Africa. African horse sickness virus (AHSV) repeatedly caused large epizootics in the Mediterranean region (North Africa and southern Europe in particular) as a result of trade in infected equids. The unexpected emergence of a closely related virus, the bluetongue virus, in northern Europe in 2006 has raised fears about AHSV introduction into Europe, and more specifically into AHSV-free regions that have reported the presence of AHSV vectors, e.g. Culicoides midges. North African and European countries should be prepared to face AHSV incursions in the future, especially since two AHSV serotypes (serotypes 2 and 7) have recently spread northwards to western (e.g. Senegal, Nigeria, Gambia) and eastern Africa (Ethiopia), where historically only serotype 9 had been isolated. The authors review key elements of AHS epidemiology, surveillance and prophylaxis.

Epizootic haemorrhagic disease.

Summary Epizootic haemorrhagic disease (EHD) is an arthropod-transmitted viral disease of certain wild ungulates, notably North American white-tailed deer and, more rarely, cattle. The disease in white-tailed deer results from vascular injury analogous to that caused by bluetongue virus (BTV), to which EHD virus (EHDV) is closely related. There are seven serotypes of EHDV recognised, and Ibaraki virus, which is the cause of sporadic disease outbreaks in cattle in Asia, is included in EHDV serotype 2. The global distribution and epidemiology of BTV and EHDV infections are also similar, as both viruses occur throughout temperate and tropical regions of the world where they are transmitted by biting Culicoides midges and infect a wide variety of domestic and wild ungulates. However, the global distribution and epidemiology of EHDV infection are less well characterised than they are for BTV. Whereas most natural and experimental EHDV infections (other than Ibaraki virus infection) of livestock are subclinical or asymptomatic, outbreaks of EHD have recently been reported among cattle in the Mediterranean Basin, Reunion Island, South Africa, and the United States. Accurate and convenient laboratory tests are increasingly available for the sensitive and specific serological and virological diagnosis of EHDV infection and confirmation of EHD in animals, but commercial vaccines are available only for prevention of Ibaraki disease and not for protection against other strains and serotypes of EHDV.

Bluetongue.

Summary Bluetongue (BT) is an arthropod-transmitted viral disease of non-African ungulates, principally sheep. The disease results from vascular injury analogous to that of human haemorrhagic viral fevers, with characteristic tissue infarction, haemorrhage, vascular leakage, oedema, and hypovolaemic shock. Importantly, BT is not zoonotic. Bluetongue virus (BTV) infection of ruminants and vector Culicoides midges is endemic throughout many tropical and temperate regions of the world; however, within this global range the virus exists within relatively discrete ecosystems (syn. episystems) where specific constellations of BTV serotypes are spread by different species of biting Culicoides midges. Recently discovered goat-associated BTVs, notably BTV serotype 25 (BTV-25) in central Europe, appear to have distinctive biological properties and an epidemiology that is not reliant on Culicoides midges as vectors for virus transmission. Bluetongue virus infection of ruminants is often subclinical, but outbreaks of severe disease occur regularly at the upper and lower limits of the virus's global range, where infection is distinctly seasonal. There have been recent regional alterations in the global distribution of BTV infection, particularly in Europe. It is proposed that climate change is responsible for these events through its impact on vector midges. However, the role of anthropogenic factors in mediating emergence of BTV into new areas remains poorly defined; for example, it is not clear to what extent anthropogenic factors were responsible for the recent translocation to northern and eastern Europe of live attenuated vaccine viruses and an especially virulent strain of BTV-8 with distinctive properties. Without thorough characterisation of all environmental and anthropogenic drivers of the recent emergence of BT in northern Europe and elsewhere, it is difficult to predict what the future holds in terms of global emergence of BTV infection. Accurate and convenient laboratory tests are available for the sensitive and specific serological and virological diagnosis of BTV infection and confirmation of BT in animals. Prevention and control strategies for BT are largely reactive in nature, and typically are reliant on vaccination of susceptible livestock and restrictions on animal trade and movement.

Prevalence of equine viral arteritis in Algeria.

In order to determine the prevalence of equine viral arteritis in Algeria, 268 sera from non-vaccinated horses were collected from the western and eastern regions. Serological analysis of the sera, which were collected from 2009 to 2011, was performed using the virus neutralisation test, as described by the World Organisation for Animal Health. Overall, 20 sera (7.46%) were seropositive, 152 (56.71%) were negative and 96 sera (35.82%) were cytotoxic. Equine arteritis virus (EAV) seroprevalence was significantly higher in the western region (Tiaret) than in the eastern region (Barika and El-Eulma). Interestingly, more than 20% of the tested horses over 16 years old were seropositive for EAV. However, EAV prevalence did not depend on either horse breed or horse gender. This study is the first to describe the circulation of EAV in the Algerian horse population.

Encephalomyocarditis virus 2A protein is required for viral pathogenesis and inhibition of apoptosis.

The encephalomyocarditis virus (EMCV), a Picornaviridae virus, has a wide host spectrum and can cause various diseases. EMCV virulence factors, however, are as yet ill defined. Here, we demonstrate that the EMCV 2A protein is essential for the pathogenesis of EMCV. Infection of mice with the B279/95 strain of EMCV resulted in acute fatal disease, while the clone C9, derived by serial in vitro passage of the B279/95 strain, was avirulent. C9 harbored a large deletion in the gene encoding the 2A protein. This deletion was incorporated into the cDNA of a pathogenic EMCV1.26 strain. The new virus, EMCV1.26Δ2A, was capable of replicating in vitro, albeit more slowly than EMCV1.26. Only mice inoculated with EMCV1.26 triggered death within a few days. Mice infected with EMCV1.26Δ2A did not exhibit clinical signs, and histopathological analyses showed no damage in the central nervous system, unlike EMCV1.26-infected mice. In vitro, EMCV1.26Δ2A presented a defect in viral particle release correlating with prolonged cell viability. Unlike EMCV1.26, which induced cytopathic cell death, EMCV1.26Δ2A induced apoptosis via caspase 3 activation. This strongly suggests that the 2A protein is required for inhibition of apoptosis during EMCV infection. All together, our data indicate that the EMCV 2A protein is important for the virus in counteracting host defenses, since Δ2A viruses were no longer pathogenic and were unable to inhibit apoptosis in vitro.

Effect of pooling and multiplexing on the detection of bluetongue virus RNA by real-time RT-PCR.

Real-time RT-PCR (RT-qPCR) was used routinely for laboratory diagnosis during the 2006/2007 bluetongue virus (BTV) serotype 8 epidemic. In the present study the impact of pooling and multiplexing strategies on RT-qPCR are assessed. To avoid any bias in the pooling experiments, 121 BTV-8 positive blood samples with a low to high viral load were selected and pooled individually with nine negative blood samples. Analyses of the individually and pooled samples indicated an overall mean difference of 4.32 Ct-values. The most pronounced differences were observed in samples with the lowest viral load of which 70% could no longer be detected after pooling. The pooling strategy is therefore not suitable for BTV detection at the individual level since animals infected recently may be missed. An alternative approach to reduce costs and workload is to apply a multiplexing strategy in which the viral RNA and internal beta-actin control RNA are detected in a single reaction. Parallel analysis (singleplex versus multiplex) of a 10-fold dilution series and 546 field samples proved that the sensitivity of the BTV RT-qPCR was not affected whereas the beta-actin reaction was reduced only slightly. Without the use of an internal control, 0.6% of 1985 field samples is at risk of being diagnosed incorrectly as negative.

Description of the outbreak of bluetongue in Corsica in 2003, and lessons for surveillance.

Since 1999, several serotypes of bluetongue virus (btv) have been isolated in the western part of the Mediterranean basin, and since 2000, Corsica has been exposed to three different serotypes: BTV serotype 2 in 2000, BTV serotype 4 (BTV-4) in 2003 and BTV serotype 16 in 2004. In 2000 there were no surveillance systems for bluetongue, but in 2003, active surveillance of the circulation of BTV and its vector Culicoides species, aided by a raised level of awareness in farmers and veterinarians, made it possible to study the introduction of BTV-4. The monitoring and analysis of the seroconversions of sentinel herds of goats, clinical signs and meteorological variables showed that the serotype had been present in the island since May that year, but clinical signs were first observed only in October. Moreover, the weather conditions and wind patterns were suitable for the transport of Culicoides species from Sardinia in May. These observations suggest that btv had been transported on air currents from a southern infected area, and that it could have spread without causing clinical signs of disease for a few months.

Molecular characterization of equine infectious anaemia virus from a major outbreak in southeastern France.

In 2009, a major outbreak of equine infectious anaemia (EIA) was reported in the south-east of France. This outbreak affected three premises located in the Var region where the index case, a 10-year-old mare that exhibited clinical signs consistent with EIA, occurred at a riding school. Overall, more than 250 horses were tested for EIAV (equine infectious anaemia virus) antibodies, using agar gel immunodiffusion test, and 16 horses were positive in three different holdings. Epidemiological survey confirmed that the three premises were related through the purchase/sale of horses and the use of shared or nearby pastures. Molecular characterization of viruses was performed by sequencing the full gag gene sequence (1,400 bp) of the proviral DNAs retrieved from the spleen of infected animals collected post-mortem. Phylogenetic analysis confirmed epidemiological data from the field, as viruses isolated from the three premises were clustering together suggesting a common origin whereas some premises were 50 km apart. Moreover, viruses characterized during this outbreak are different from European strains described so far, underlying the high genetic diversity of EIAV in Europe.

Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador.

Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV; however, no data are available about the presence of EHDV. In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT-qPCRs revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV1) and BTV serotypes 9 (BTV9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador.

Safe and cost-effective protocol for shipment of samples from Foot-and-Mouth Disease suspected cases for laboratory diagnostic.

An essential step towards the global control and eradication of foot-and-mouth disease (FMD) is the identification of circulating virus strains in endemic regions to implement adequate outbreak control measures. However, due to the high biological risk and the requirement for biological samples to be shipped frozen, the cost of shipping samples becomes one of major obstacles hindering submission of suspected samples to reference laboratories for virus identification. In this study, we report the development of a cost-effective and safe method for shipment of FMD samples. The protocol is based on the inactivation of FMD virus (FMDV) on lateral flow device (LFD, penside test routinely used in the field for rapid immunodetection of FMDV), allowing its subsequent detection and typing by RT-PCR and recovery of live virus upon RNA transfection into permissive cells. After live FMDV collection onto LFD strip and soaking in 0.2% citric acid solution, the virus is totally inactivated. Viral RNA is still detectable by real-time RT-PCR following inactivation, and the virus strain can be characterized by sequencing of the VP1 coding region. In addition, live virus can be rescued by transfecting RNA extract from treated LFD into cells. This protocol should help promoting submission of FMD suspected samples to reference laboratories (by reducing the cost of sample shipping) and thus characterization of FMDV strains circulating in endemic regions.

Experimental infection of sheep, goats and cattle with a bluetongue virus serotype 4 field strain from Bulgaria, 2014.

In 2014, a new bluetongue virus serotype 4 (BTV-4) strain was detected in southern Greece and spread rapidly throughout the Balkan Peninsula and adjacent countries. Within half a year, more than 7,068 outbreaks were reported in ruminants, particularly in sheep. However, the reported morbidity and case fatality rates in ruminants varied. The pathogenesis of a Bulgarian BTV-4 strain isolated from sheep during the BTV-4 epizootic was studied in different species. Therefore, four sheep, three goats and three cattle were experimentally infected with the isolate BTV-4/BUL2014/15 and monitored for clinical signs up to several weeks. Serum and whole-blood samples were collected at regular intervals and subjected to serological and virological analyses. In this context, BTV-4-specific real-time RT-PCR assays were developed. The infection kinetics were similar to those known for other traditional BTV serotypes, and only mild BT-like clinical signs were observed in goats and sheep. In cattle, no obvious clinical signs were observed, except a transient increase in body temperature. The study results contrast with the severe clinical signs reported in sheep experimentally infected with an African BTV-4 strain and with the reports of BT-like clinical signs in a considerable proportion of different ruminant species infected with BTV-4 in the Balkan region and Italy. The discrepancies between the results of these animal trials and observations of BTV-4 infection in the field may be explained by the influence of various factors on the manifestation of BT disease, such as animal breed, fitness and virus strain, as described previously.

Complete genome sequence of bluetongue virus serotype 4 that emerged on the French island of Corsica in December 2016.

In November 2016, sheep located in the south of Corsica island exhibited clinical signs suggestive of bluetongue virus (BTV) infection. Laboratory analyses allowed to isolate and identify a BTV strain of serotype 4. The analysis of the full viral genome showed that all the 10 genomic segments were closely related to those of the BTV-4 present in Hungary in 2014 and involved in a large BT outbreak in the Balkan Peninsula. These results together with epidemiological data suggest that BTV-4 has been introduced to Corsica from Italy (Sardinia) where BTV-4 outbreaks have been reported in autumn 2016. This is the first report of the introduction in Corsica of a BTV strain previously spreading in eastern Europe.

First molecular characterisation of a Brazilian Burkholderia mallei strain isolated from a mule in 2016.

We present the first molecular characterisation based on MLVA and SNP analysis of a strain of Burkholderia mallei isolated from a mule found dead in Brazil in 2016.

Bluetongue virus serotype 27: Experimental infection of goats, sheep and cattle with three BTV-27 variants reveal atypical characteristics and likely direct contact transmission BTV-27 between goats.

Bluetongue virus (BTV) hitherto consisted of 26 recognized serotypes, of which all except BTV-26 are primarily transmitted by certain species of Culicoides biting midges. Three variants of an additional 27th bluetongue virus serotype (BTV-27v01-v03) were recently detected in asymptomatic goats in Corsica, France, 2014-2015. Molecular characterization revealed genetic differences between the three variants. Therefore, in vivo characteristics were investigated by experimental infection of a total of 15 goats, 11 sheep and 4 cattle with any one of the three variants in separated animal trials. In goat trials, BTV-naïve animals of the same species were kept in a facility where direct contact was unhindered. Of the 15 inoculated goats, 13 and 14 animals were found positive for BTV-RNA and antibodies (Ab), respectively, until the end of the experiments. Surprisingly, BTV-Ab levels as measured with ELISA and neutralization test (SNT) were remarkably low in all seropositive goats. Virus isolation from whole-blood was possible at the peak of viremia until 49 dpi. Moreover, detection of BTV-27v02-RNA and Ab in one contact goat indicated that-similar to BTV-26-at least one of three BTV-27 variants may be transmitted by contact between goats. In the field, BTV-27 RNA can be detected up to 6 months in the whole-blood of BTV-27-infected Corsican goats. In contrast, BTV RNA was not detected in the blood of cattle or sheep. In addition, BTV-27 Abs were not detected in cattle and only a transient increase in Ab levels was observed in some sheep. None of the 30 animals showed obvious BT-like clinical signs. In summary, the phenotypes observed for BTV-27v01-v03 phenotypes correspond to a mixture of characteristics known for BTV-25 and 26.