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

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

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Resistance to OsHV-1 Infection in Crassostrea gigas Larvae

The ostreid herpesvirus(OsHV-1) is one of the major diseases that affect the Pacific oyster Crassostrea gigas. Selective breeding programs were recently shown to improve resistance easily to OsHV-1 infections in spat, juvenile, and adult oysters. Nevertheless, this resistance has never been investigated in larvae, whereas this developmental stage has crucial importance for the production of commercial hatcheries, as well as explaining the abundance of spatfall. A first trial tested several viral suspensions at several concentrations using contaminated water with OsHV-1 in 4- and 10-day-old larvae that we reproduced from an unselected broodstock. In follow up on the results, one viral suspension at a final concentration of 10+6 OsHV-1 DNA copies per L was used to assess resistance to OsHV-1 infection in C.gigas larvae that we reproduced from selected and unselected broodstock. A second trial evaluated OsHV-1 resistance in larvae from both broodstocks intrials 2a, 2b, and 2c with 4,10, and 16-day-old larvae for 7 days, which corresponded to post D larvae, umbo larvae, and eyed larvae, respectively. The mortality of unchallenged larvae for both stocks were low (<15%) at day 7 intrials 2a and 2b, whereas it ranged from 48 to 56% in trial 2c. More interestingly, selected larvae had significantly lower mortality than unselected larvae when exposed to OsHV-1 in all of the trials. Thus, themortality was 11 and 49% for the selected larvae at day 7 post-exposure in trials 2a and 2c, respectively, in comparison with 84 and 97% for the unselected larvae. Although this difference in mortality was observed at day 5 in trial 2b, it was reduced at day 7, to 86 and 98% for the selected and unselected larvae, respectively. For the first time in the literature, the difference in mortality or the delayed on set of mortality between selected and unselected larvae have indicated a genetic resistance to OsHV-1 infection at the larval stage. Such finding should facilitate the selective breeding programs focusing on resistance to OsHV-1 infection by reducing the span of the genetic evaluation, and thus decreasing its cost

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

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

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Detection of the protistan parasite, Haplosporidium costale in Crassostrea gigas oysters from the French coast: A retrospective study

International audienceThe parasite Haplosporidium costale is known to infect and cause mortality in the oyster Crassostrea virginica in the USA. Decades after its first description in the 1960s, this parasite was detected in Crassostrea gigas in the USA and China. However, it presented a low prevalence and no mortality was associated with it. More recently, in 2019, H. costale was detected in France in a batch of moribund oysters. In order to observe how long this parasite has been present on French coasts, from Normandy to Thau lagoon, a retrospective investigation was conducted on 871 adult and spat oyster batches from 2004 to 2020. To allow rapid detection on a large panel of samples, a real-time PCR for the H. costale actin gene was developed. This method allowed the detection of H. costale DNA in adults from 2005 and in spat from 2008. The H. costale prevalence in spat appeared higher than in adults over the years studied, 14.59 % compared to 6.50 %, respectively. All samples presenting positive results were then sequenced on two targets, H. costale rRNA and actin genes. The actin gene sequencing highlighted the presence of two H. costale strains. Adult C. gigas as well as spat batches coming from hatcheries and DNA controls from C. virginica all presented with the Profile 1 H. costale strain. The Profile 2 H. costale strain was detected only in C. gigas spat coming from natural sources. These observations suggest a correlation between the origin of oysters and H. costale strains which may have been caused by commercial imports between Japan, USA and France back to the 1970s. Over the positive samples studied, only few batches (n = 3) suffered mortalities which could be hypothesized to be caused by H. costale, all presenting the Profile 1 H. costale strain

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

First detection of Francisella halioticida in mussels Mytilus spp. experiencing mortalities in France

International audiencehis note describes the first detection of the bacteria Francisella halioticida in mussels Mytilus spp. from locations in Normandy and northern Brittany (France) experiencing high mussel mortalities, while it was not detected in the Bay of St Brieuc (northern Brittany), an area which was not affected by abnormal mussel mortality. The distribution of the bacteria in mussels seems to be restricted to inflammatory granulomas as observed in Yesso scallops Mizuhopecten yessoensis from Canada and Japan. F. halioticida has been identified as being involved in mass (>80%) mortality of abalones Haliotis gigantea in Japan and high (up to 40%) mortality of Yesso scallops Mizuhopecten yessoensis in Canada as well as in lesions reducing marketability of Yesso scallops in Japan. The impact of this bacterium on the health of mussels needs to be investigated in future research, especially since the cause of high mussel mortalities that have been occurring in France for the past few years is still undetermined