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

International audienc

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

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

International audienceThe 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

Influence of strains in development of francisellosis in the blue mussel Mytilus edulis during experimental challenges

International audienceThe bacterium Francisella halioticida, known to induce francisellosis in abalone and Yesso scallop, is suspected of being involved in the blue mussel mortalities observed in France. Recently, several isolates of F. halioticida were obtained from moribund mussels and categorized into two type strains, FR21 and FR22. Two other strains, AG1 and AG3, determined to belong to the genus Francisella were isolated. To determine the virulence of these isolates, juvenile and adult blue mussels were injected with bacterial solutions at high dose and monitored for 11 days. FR22c and FR22d were found to induce 80% mortality in less than seven days. The isolates AG1 and AG3 led to over 50% mortality in adult mussels but only AG1 led to significant mortality in juveniles (41%). FR22c and FR22d, the most virulent isolates, were selected to determine their respective lethal dose at 50% (LD50) in juveniles and adults. This analysis was performed with bacterial solutions ranging from 10$^2$ to 10$^6$ CFU/mussel and monitored for 30 days. The isolate FR22c was found to be the most virulent. Observed LD50 for the isolate FR22c was 4.14 ×10$^3$ CFU/juvenile and 3.45 ×10$^3$ CFU/adult and for the isolate FR22d, 1.89 ×10$^4$ CFU/juvenile and 1.52 ×10$^4$ CFU/adult. To confirm Koch’s postulate, a selection of moribund, surviving and control animals were plated on specific media. The isolates were reisolated from moribund animals but not from surviving or controls. To confirm the proliferation, a specific real-time PCR was performed. All moribund individuals were positive by PCR. The main Ct values were lower for moribund compared to surviving animals and a dose effect was observed in DNA bacterial load. This study shows that some F. halioticida isolates are able to induce francisellosis in mussels and lead to high mortality, highlighting differences in virulence among the strains

Genomic comparison of Francisella halioticida

International audienc

<i>Francisella halioticida</i>'s towards the mussel <i>Mytilus edulis</i> is strain-dependent

International audienc

First isolation of Francisella halioticida strains from blue mussel (Mytilus edulis) in Normandy, France

International audienceMass mortality events affecting the blue mussels Mytilus edulis have been observed in France since 2014. The DNA of the bacterium Francisella halioticida, reported as pathogen of giant abalone (Haliotis gigantea) and Yesso scallop (Mizuhopecten yessoensis) has been detected recently in mussels from areas suffering mortalities. Isolation of this bacterium was attempted from individuals collected during mortality events. Identification was performed by16S rRNA gene sequencing, real-time specific PCR and MALDI-ToF using spectra produced from the strain 8472-13A isolated from diseased Yesso scallop in Canada. Five isolates were identified as F. halioticida by real-time specific PCR and 16S rRNA sequencing. MALDI-ToF allowed the direct identification of four isolates(FR22a,b,c,d) which had 100% identity on the 16S rRNA gene with the known strains. On the other hand, one isolate (FR21) was not recognized by MALDI-ToF and had 99.9% identity on the 16S rRNA gene. The FR22 isolates showed difficult growth and required media optimization, which was not the case with the FR21 isolate. For these reasons, it was hypothesized that two type strains are present on French coasts, named FR21 and FR22. The FR21 isolate was selected for phenotypic analysis (growth curve, biochemical characteristics, electron microscopy), phylogenetic analysis and an experimental challenge.This isolate showed distinct differences compared to published F. halioticida strains, both at phenotypic and genotypic levels. Experimental infections of adult mussels led to 36% mortalities in 23 days following intramuscular injection with 3 x 107 CFU while a lower dose (3 x 103 CFU) did not lead to significant mortalities. In the context of this study, the strain FR21 was not virulent towards adult mussels