Génotypage BVD par séquençage selon la méthode de Sanger

National audienc

Development of a Taqman® Real-Time PCR for the rapid discrimination of the Vibrio splendidus species among the Splendidus clade

International audienceThe Vibrio splendidus species is ubiquitous in the marine environment but have also been recognized as pathogenic for several aquatic animals. This species belongs to the Splendidus clade which is composed of 16 genetically related species. To our knowledge, there are no available tools able to discriminate the V. splendidus species from the other members of the clade. Thus, we developed a Real Time PCR based on the toxR gene specific to V. splendidus species. Specificity tests were performed on 74 reference strains and 116 field strains and gave 96.05% inclusivity and 100% exclusivity results. The limit of detection of the PCR was determined according to the XP-U47-600-2 AFNOR norm and was estimated at 18 copies genome per reaction. This work describes a reliable, sensible and specific tool to discriminate V. splendidus species from other members of the Splendidus clade and thus could be clearly suitable to perform taxonomic investigations

Comparison of two real-time PCR methods for detection of ostreid herpesvirus 1 in the Pacific oyster Crassostrea gigas

International audienceThe real-time polymerase chain reaction (PCR) is considered to be a suitable tool for nucleic acid quantitation because it is accurate, rapid and reliable. The reference protocol for quantitation of ostreid herpesvirus 1 in Pacific oysters Crassostrea gigas is based on a Sybr(®) Green real-time PCR developed by the IFREMER laboratory. The Frank Duncombe Departmental Laboratory has developed an alternative protocol based on TaqMan(®) chemistry (alternative technique). The quantitation limits were 1000 and 18UG/mg of tissues for the reference method and alternative protocols, respectively, and the latter protocol has a detection limit of 6UG/mg of tissues. The aim of this study was to compare the two protocols using DNA samples obtained from 210 spat. The kappa index (0.41) indicated a moderate concordance between the protocols, according to the measures of Landis and Koch. All samples that were positive by the reference protocol were also positive by the alternative protocol. Of the 76 samples that were negative by the reference protocol, 49 were positives by the alternative protocol. In conclusion, the alternative protocol is an improvement of the reference protocol in terms of sensitivity, specificity and rapidity (<3h)

Detection of different variants of Ostreid Herpesvirus 1 in the Pacific oyster, Crassostrea gigas between 2008 and 2010

International audienceSince summer 2008, high mortality rates of young Pacific oysters Crassostrea gigas have been recorded in association with the detection of the Ostreid Herpesvirus 1 (OsHV-1). A new variant called μVar has been recently described, characterized mainly by 12 consecutive deletions followed by one deletion of an adenine in the C region. The purpose of this study is to characterize the genotype (variants or OsHV-1 reference) of 300 positive samples of C. gigas analyzed between July 2008 and July 2010 collected along the French, Jersey, and Irish coasts. Samples were quantified by TaqMan PCR, amplified with conventional PCR, targeting the area of the deletion, and then sequenced. Eighty-seven percent of the samples were characterized and the OsHV-1 μVar was detected in 257 oyster samples. The genotype OsHV-1 reference was never detected during the 25 months of the present survey. Thirty-eight samples could not be determined and the majority of them had a low viral load. A novel genotype containing only 9 consecutive deletions named OsHV-1 μVar Δ9 was found in 5 samples. These observations indicate the emergence of different OsHV-1 variants

Developement of a novel and efficient equine SNP genotyping method and its utility in studying the prevalence of genetic diseases, coat colorand various traits of interest for equine industry

International audienceNext Generation Sequencing (NGS) have enabled the identification of molecular markers widely used to detect causal mutations in horses for geneticdisorders or traits as coat color. The aim of this study was to develop and evaluate a SNP custom panel as a potential useful tool for equine genotyping. Thispanel was designed with support from Thermo Fisher Scientific, Waltham, Ma, USA and included 108 targets (33 genetic disorder markers, 40 coat colormarkers and 35 gait traits). For all targets, samples of known genotype were analyzed (no mutation, heterozygoty or homozygote mutation) with the AgriSeqtargeted GBS (Genotyping by sequencing) solution. This method utilizes a highly efficient multiplexed PCR chemistry where hundreds of markers can betargeted and uniformly amplified in a single reaction.Briefly, after extraction of DNA from horse hair or blood, sample DNAs were normalized and a multiplexed PCR was performed with the custom primer panel.Librairies were prepared according to the AgriSeqTM HTS library kit and sequenced on the Ion GeneStudioTM S5 system. Genotyping was then carried outautomatically by Torrent Variant Caller (TVC) on the Torrent Suite Software (TS).For the 108 targets, sample results obtained with the custom panel were compared with the expected genotypes for which results were provided fromreference laboratories or obtained after Sanger sequencing analysis. All results were consistent except for 2 markers for which a redesign was needed.Indeed, the custom panel could be if necessary amended or updated if other molecular markers of interest are recently highlighted.This equine SNP genotyping method based on AgriSEQ technology resulted a cost-effective strategy to prevent the disease risk, to study the prevalence oftraits and to improve the knowledge about horse’s performance and abilities. This efficient tool could thus be easily used in veterinary laboratories forlarge-scale routine genetic analyses

Multilocus Sequence Analysis : a powerful tool for the classification of Vibrio splendidus related strains.

International audienc

Multilocus sequence analysis of Vibrio splendidus related-strains isolated from blue mussel Mytilus sp. during mortality events

International audienceOne of the most widely European farmed mollusc, the mussel Mytilus sp., has been subjected to massive mortalities located in Charente-Maritime (France) in spring 2014. The national surveillance network for mollusc health has reported a systematic detection of V. splendidus in all dying batches. V. splendidus is the type species of a clade composed of almost 20 known strains with variable pathogenicity on bivalves. In our study, we first developed a Multi Locus Sequence Analysis (MLSA), specific to V. splendidus group, based on fragments of 5 housekeeping genes: atpA, ftsZ, mreB, rpoD and topA. This tool was validated on reference strains and compared with individual gene analyses. It allowed a useful and reliable classification of V. splendidus closely-related strains. Thanks to MLSA, we then tried to classify genetically 23 strains isolated from healthy or dying mussels. 21 were classified within the Splendidus clade: in splendidus cluster (38%), in tasmaniensis cluster (24%), in artabrorum cluster (24%) and on distinct branches (14%). All of them were tested by injection to healthy adult mussels to identify possible pathogenic strains. Experimental trials revealed the presence of a strain called M3H, allied with the splendidus cluster and able to induce mortality in mussels with rates up to 80%. The M3H virulence was demonstrated by the recovery of the injected strain in dying animals, resulting from repeated experimental infections. Further work should be now conducted to explore the pathogenicity of the M3H strain towards different mussel batches and under various conditions

Intense hemocyte infiltration in gonadal tubules of ripe triploid Pacific oyster Crassostrea gigas (Thunberg, 1793): Correlation with mortality

International audienceMortality outbreaks in adult oysters (Crassostrea gigas) have been reported since 1994 in Normandy. Recent work has shown that triploid oysters tend to be subject to higher mortality than diploids. Many parameters could be involved in this phenomenon including pathogens, environmental conditions, the physiological and genetic states of the oysters. With respect to reproductive physiology, studies showed that triploid oysters have gamete production. In order to understand the abnormal mortalities, we focused on gametogenesis and more specifically on hemocyte infiltration in gonadal tubules. Fourteen batches of oysters with different origins and ploidy were followed during two years. The results of the study showed intense hemocyte infiltration in gonadal tubules during the ripe stage of gametogenesis in triploid oysters. Finally, this article suggests a link between the frequency of individuals with hemocyte infiltration in gonadal tubules and the proportion of mortality within the triploid groups

Detection of undescribed ostreid herpesvirus 1 (OsHV-1) specimens from Pacific oyster, Crassostrea gigas

International audienceThe ostreid herpesvirus 1 (OsHV-1) and variants were implicated in mass mortality affecting the young Pacific cupped oysters, Crassostrea gigas, in European countries and those around the world. From 2008 onwards, oyster mortality had greatly increased on the French coast and was associated with the detection of a new OsHV-1 variant, entitled OsHV-1 μVar. The OsHV-1 μVar is predominant in oysters; however, other OsHV-1 variants have been detected in samples collected during mortality periods or collected out of mortality periods in France, Ireland, Spain, Portugal, Italy, Mexico, United States, South Korea, Australia, and New Zealand. A retrospective study conducted on 1047 OsHV-1 specimens sampled mainly in France between 2009 and 2012, revealed 17 undescribed OsHV-1 variants found in 65 oyster samples. These specimens presented point mutations situated downstream and upstream from the microsatellite area in the C region (ORF 4/5) which were different from the OsHV-1 reference and the OsHV-1 μVar. In the present work, investigation was performed to further characterize these OsHV-1 specimens by sequencing two habitually targeted regions to study genetic polymorphism of the virus: ORF 41/42 and ORF 35-38. An OsHV-1 variant detected in six oyster samples, contained a nucleotide substitution in the C region which impacted the amino acid sequence and might modify the function of the unknown protein encoding by ORF 4. For the ORF 41/42 region, only two specimens presented a synonymous mutation in comparison with the OsHV-1 μVar. All specimens contained the same deletion with the OsHV-1 μVar in ORF 35-38. Then, a phylogenetic analysis based on the C region was performed to investigate the distribution of undescribed specimens among 21 OsHV-1 DNA sequences notified in GenBank and collected from different countries (France, Japan, New Zealand, China, Ireland, and United States) between 1995 and 2012. All analyzed samples and the OsHV-1 μVar were placed in the same group, excepted for a Japan specimen. Our results contribute to improve the description of the genetic diversity of the OsHV-1 and the C region (ORF 4/5) appears to be a better target than ORF 42/42 and 35-38 to distinguish variants between themselves

Abnormal mortality of triploid adult Pacific oysters: Is there a correlation with high gametogenesis in Normandy, France?

International audienceSummer mortalities of the adult Pacific oysters Crassostrea gigas are an important economic concern. In 2015 and 2016, the mortality of hatchery origin spat comprising 4 diploid batches and 9 triploid batches as well as 12 batches of wild caught diploids was followed at 3 sites in Normandy. Abnormal mortalities (> 20%) were observed at one site only and were significantly higher in triploid animals (P < .05). Triploid oysters are believed to be partially sterile but a high level of gametogenesis was observed in all monitored triploid batches. In 2015, preliminary results revealed that triploid oysters underwent strong gametogenesis with mature gametes (stage 3) and 50% of them were non-perturbed (i.e. named alpha). In 2016, 42% of triploid oysters were alpha. This study revealed that triploid oyster maturation occurred in September, which corresponded to the time of high mortalities in Normandy