Impact of anesthesia on some of physiological parameters in Acipenser nudiventris

Impact of anesthesia with electric shock on some of physiological parameters was studied in in Acipenser nudiventris in this study, we investigated the induction and recovery times and physiological response of Acipenser nudiventris. We anesthetized them by CO2 (mmhg), clove powder (mg/L), and electric shock (V). Shortest and longest induction time were 0.15 min and 4.25 min when electric shock and clove powder were used. On the other hand, shortest and longest time of recovery time were 3.23 min and 2. 20 min respectively when electric shock and CO2 gas were used. Cortisol and glucose levels increased 1 and 6 hours after anesthesia. Changes in plasma osmolality were less among the anesthesia treatments. Results demonstrated that electric shock was a more effective method for quick induction time, although all anesthetic methods were found to be safe

Complete genome sequence of Ostreid herpesvirus type 1 µVar isolated during mortality events in the Pacific oyster Crassostrea gigas in France and Ireland

International audienceInfections with Ostreid herpesvirus 1 (OsHV-1) microvariants in young Pacific oysters are associated with massive mortality events and significant economic losses. Previous studies, focusing on few regions of the genome, have revealed the genomic diversity of these genotypes with respect to the reference type. We used a NGS process to sequence the whole genome of the OsHV-1 µVar in infected individuals, collected during mortality events in France and Ireland. The final genome length of OsHV-1 µVar was approximately 205kbp, shorter than the reference genotype and the overall genome organisation resembled herpes simplex viruses. 94.4% similarity was observed with the OsHV-1 reference genotype. Large indels, including five deletions and three insertions were found to induce the loss and the addition of several ORFs, summed with codon substitutions in 64% of genes shared with the reference type. This diversity raises the question of the exact origin and evolution of OsHV-1 µVar

SARS-CoV-2 RNA isolation method from sewage sludge, application in field samples and comparison with bacteriophage loads

International audienceSevere Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is mainly transmitted through the respiratory tract. It can also be found in faeces leading to its detection in wastewater and potentially in sewage sludge. This one can be used in agriculture as a soil amendment. In France, the spreading of sludge is controlled in order to limit the dissemination of pathogenic microorganisms including SARS-CoV-2 since the pandemic. However, the control only concerns the analysis of bacteriophages. The present study was carried out to assess the presence of the virus in sewage sludge and compare with bacteriophages results. It describes the validation of a method for the isolation of SARS-CoV-2 RNA for detection by RT-PCR, using a surrogate virus. Two virus concentration methods and three nucleic acid extraction methods were compared. After validation, the most efficient method was applied to field samples (n=34) from Normand sewage treatment plants during the pandemic. Then, the results were compared with bacteriophage loads. According to our results, PEG precipitation followed by a nucleic acid extraction based on cleared lysate with phenol:chloroform:isoamyl alcohol, then concentrated and purified on anion-exchange column was selected. This process resulted in a yield of 39.6±37.3%. The field study confirmed the presence of SARS-CoV-2 in both primary and hygienized sludges. The comparative analysis suggested that the study of the effectiveness of sanitation on bacteriophages does not appear representative of that on SARS-CoV-2. In addition to the bacteriophages test, a direct search for the SARS-CoV-2 is recommended to evaluate the sanitation of sludge

SARS-CoV-2 RNA isolation method from sewage sludge, application in field samples and comparison with bacteriophage loads

International audienceSevere Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is mainly transmitted through the respiratory tract. It can also be found in faeces leading to its detection in wastewater and potentially in sewage sludge. This one can be used in agriculture as a soil amendment. In France, the spreading of sludge is controlled in order to limit the dissemination of pathogenic microorganisms including SARS-CoV-2 since the pandemic. However, the control only concerns the analysis of bacteriophages. The present study was carried out to assess the presence of the virus in sewage sludge and compare with bacteriophages results. It describes the validation of a method for the isolation of SARS-CoV-2 RNA for detection by RT-PCR, using a surrogate virus. Two virus concentration methods and three nucleic acid extraction methods were compared. After validation, the most efficient method was applied to field samples (n=34) from Normand sewage treatment plants during the pandemic. Then, the results were compared with bacteriophage loads. According to our results, PEG precipitation followed by a nucleic acid extraction based on cleared lysate with phenol:chloroform:isoamyl alcohol, then concentrated and purified on anion-exchange column was selected. This process resulted in a yield of 39.6±37.3%. The field study confirmed the presence of SARS-CoV-2 in both primary and hygienized sludges. The comparative analysis suggested that the study of the effectiveness of sanitation on bacteriophages does not appear representative of that on SARS-CoV-2. In addition to the bacteriophages test, a direct search for the SARS-CoV-2 is recommended to evaluate the sanitation of sludge

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

Genetic polymorphism of the Ostreid herpesvirus 1 (OsHV-1)

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Haemocytes collected from experimentally infected Pacific oysters, Crassostrea gigas: Detection of ostreid herpesvirus 1 DNA, RNA, and proteins in relation with inhibition of apoptosis.

Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels

Genome exploration of six variants of the Ostreid Herpesvirus 1 and characterization of large deletion in OsHV-1μVar specimens

International audienceThe genetic polymorphism of the Ostreid Herpesvirus 1 (OsHV-1) has generally been investigated in three areas: ORFs 4/5, ORFs 42/43, and ORFs 35 to 38. The present study, however, focuses on 40 ORFs, representing 30% of the OsHV-1 genome, encoding four categories of putative proteins: 4 ORFs encoding putative inhibitor of apoptosis proteins; 17 ORFs encoding membrane proteins; 10 ORFs encoding secreted proteins; and 9 ORFs encoding RING finger proteins. The potential role of these proteins in major steps of the life cycle of the OsHV-1 motivated their selection. Seven specimens have been selected in accordance with their nucleotide variations in the C region (area located between the end of the ORF4 and the beginning of ORF 5): 3 OsHV-1μVar specimens, 2 OsHV-1μVar Δ9, one specimen of OsHV-1μVar Δ15, and one OsHV-1 specimen (reference control) close to the reference genome to validate PCRs. The OsHV-1μVar is mainly characterized by a deletion of 12 consecutive nucleotides followed by a deletion of one adenine in a microsatellite area located in the C region. A representation of nucleotide modifications between the different specimens was performed by building evolutionary trees with respect to the category of ORFs. This phylogenetic analysis revealed two groups: the first one corresponded to the reference control and the reference genome AY509253, and the second one included the 6 OsHV-1 variants. These results suggest that the two main groups come from the same common ancestor, and that the divergence between the reference OsHV-1 and its variants occurred quite far back in time. Moreover, consequences of nucleotide variations in the amino acid sequences, especially the change of the N glycoslyation sites, were investigated. Herein is the first report of four important deletions in these OsHV-1μVar variants: a deletion of 1385bp in ORF 11; a deletion of 599bp in ORF 48; a deletion of 3549bp in ORFs 61 to 64; and a deletion of 712bp in ORF 114. The size of the deletions differed between OsHV-1μVar specimens, OsHV-1μVar Δ9 specimens, and the OsHV-1μVar Δ15 specimen. These zones seem to correspond to special points of gene rearrangements for producing new proteins. Further investigation necessary proves to link such nucleotide modifications with consequences of protein functions in the OsHV-1 life cycle

Development of a semi‐quantitative PCR assay for the detection of Francisella halioticida and its application to field samples

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