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Martenot C.,French Research Institute for Exploitation of the Sea | Segarra A.,French Research Institute for Exploitation of the Sea | Baillon L.,French Research Institute for Exploitation of the Sea | Faury N.,French Research Institute for Exploitation of the Sea | And 2 more authors.
Journal of Invertebrate Pathology | Year: 2016

Immunohistochemistry (IHC) assays were conducted on paraffin sections from experimentally infected spat and unchallenged spat produced in hatchery to determine the tissue distribution of three viral proteins within the Pacific oyster, Crassostrea gigas. Polyclonal antibodies were produced from recombinant proteins corresponding to two putative membrane proteins and one putative apoptosis inhibitor encoded by ORF 25, 72, and 87, respectively. Results were then compared to those obtained by in situ hybridization performed on the same individuals, and showed a substantial agreement according to Landis and Koch numeric scale. Positive signals were mainly observed in connective tissue of gills, mantle, adductor muscle, heart, digestive gland, labial palps, and gonads of infected spat. Positive signals were also reported in digestive epithelia. However, few positive signals were also observed in healthy appearing oysters (unchallenged spat) and could be due to virus persistence after a primary infection.Cellular localization of staining seemed to be linked to the function of the viral protein targeted. A nucleus staining was preferentially observed with antibodies targeting the putative apoptosis inhibitor protein whereas a cytoplasmic localization was obtained using antibodies recognizing putative membrane proteins. The detection of viral proteins was often associated with histopathological changes previously reported during OsHV-1 infection by histology and transmission electron microscopy. Within the 6 h after viral suspension injection, positive signals were almost at the maximal level with the three antibodies and all studied organs appeared infected at 28 h post viral injection. Connective tissue appeared to be a privileged site for OsHV-1 replication even if positive signals were observed in the epithelium cells of different organs which may be interpreted as a hypothetical portal of entry or release for the virus. IHC constitutes a suited method for analyzing the early infection stages of OsHV-1 infection and a useful tool to investigate interactions between OsHV-1 and its host at a protein level. © 2016. Source

Hohweyer J.,University of Reims Champagne Ardenne | Travaille E.,LABEO Frank Duncombe | Dumetre A.,Aix - Marseille University | Aubert D.,University of Reims Champagne Ardenne | And 6 more authors.
Food Microbiology | Year: 2016

Toxoplasma gondii, Cryptosporidium spp. and Giardia intestinalis are emerging pathogen parasites in the food domain. However, without standardized methods for their detection in food matrices, parasitic foodborne outbreaks remain neglected. In this study, a new immunomagnetic separation assay (IMS Toxo) targeting the oocyst's wall of T. gondii was developed using a specific purified monoclonal antibody. Performance of this IMS Toxo coupled to microscopic and qPCR analyses was evaluated in terms of limit of detection (LOD) and recovery rate (RR) on: i) simple matrix (LOD = 5 oocysts; RR between 5 and 56%); ii) raspberries and basil (LOD = 33 oocysts/g; RR between 0.2 and 35%). Finally, to simultaneously extract the three protozoa from these food matrices, T. gondii oocysts were directly concentrated (without IMS Toxo) from the supernatant of the IMS of Cryptosporidium and Giardia (oo)cysts. This strategy associated to qPCR detection led to LOD <1 to 3 (oo)cysts/g and RR between 2 and 35%. This procedure was coupled to RT-qPCR analyses and showed that the three protozoa persisted on the leaves of basil and remained viable following storage at 4 °C for 8 days. These data strengthen the need to consider these protozoa in food safety. © 2016 Elsevier Ltd. Source

Martenot C.,LABEO Frank Duncombe | Martenot C.,University of Caen Lower Normandy | Martenot C.,French National Center for Scientific Research | Lethuillier O.,LABEO Frank Duncombe | And 11 more authors.
Journal of Invertebrate Pathology | Year: 2015

The 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. © 2015. Source

Martenot C.,LABEO Frank Duncombe | Martenot C.,University of Caen Lower Normandy | Martenot C.,French National Center for Scientific Research | Denechere L.,LABEO Frank Duncombe | And 12 more authors.
Aquaculture | Year: 2015

The Ostreid herpesvirus 1 (OsHV-1) is associated with the mortality of young oysters Crassostrea gigas in France. The virus could infect different marine bivalve species and OsHV-1 DNA was previously detected in crustacean, marine gastropod, and sediment. Nevertheless, the effect of the temperature on the stability of infectious viral particles in sea water remains unpublished. During a mortality event, dead oysters may release virus in the sea water such as free viral particles, viral particles included in cells or attached to a support like plankton, suspended particulate matters, and microalgae. Oysters placed around the infectious source may filtrate the virus and then be infected by OsHV-1. In the present study, we investigated the OsHV-1 μVar virulence in sea water at 16. °C and 25. °C. Filtered infected tissue homogenates were kept at 16. °C or 25. °C prior to injection challenge of uninfected spats. A real-time reverse transcription PCR targeting the OsHV-1 polymerase gene has been developed to confirm the death cause oysters collected during the experiment. The RNA detection of herpesvirus in their hosts is a sign of viral replication and indicates that the virus is certainly infectious. Results showed that the OsHV-1 μVar remained infectious and induced mortality after 33. h at 25. °C and 54. h at 16. °C in controlled conditions. The OsHV-1 μVar is thus able to persist in sea water and high temperatures seem to reduce its infectivity. However, the OsHV-1 μVar virulence may be modulated by biological, physical and chemical factors which are present in sea water. © 2015. Source

Hue E.S.,LABEO Frank Duncombe | Hue E.S.,Normandy University | Fortier C.I.,LABEO Frank Duncombe | Laurent A.M.,LABEO Frank Duncombe | And 7 more authors.
Journal of Visualized Experiments | Year: 2016

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories. © 2016 Journal of Visualized Experiments. Source

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