Investigations of French mussel mortality events : Is <i>Francisella halioticida</i> the real culprit ?

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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

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

Genomic comparison of Francisella halioticida

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

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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

Evaluation of the relevance of surgery in a retrospective case series of patients who underwent the surgical treatment of a symptomatic spine metastasis from lung cancer

International audienceBackground: The management of spine metastases is an increasing concern for spine surgeons. When considering surgery, it is crucial to ensure that its iatrogenic effects will not exceed its potential benefits, particularly in frail patients with short life expectancy. Among all prognostic factors, the primary site of cancer is the most important, lung cancer being the poorest. Although surgery has shown its effectiveness in the management of spine metastases, there is a lack of studies focusing on lung cancer alone.Purpose: To assess the effectiveness and safety of surgery in the management of symptomatic spine metastases from lung cancer.Methods: We retrospectively reviewed all patients (n = 53) who underwent surgery for spine metastasis from lung cancer at the Lille University Hospital between January 2005 and December 2011. Patients for whom surgery was effective to restore or preserve ambulation, to relieve pain, and to ensure stability without severe complication were considered "surgical success".Results: No patient was lost to follow-up and vital status data were available for all patients. The median survival was 2.1 months and was not influenced by the surgical success (p = 0.1766). We reported seven major complications in seven patients, including three epidural haematoma, two massive pulmonary embolisms and two deaths from cardiopulmonary failure. The surgical success rate was 49 % and on univariate analysis, the factors that have influenced the postoperative outcome were the KPS (p < 0.001), the Frankel grade (p = 0.0217) and the delay between the cancer diagnosis and the occurrence of spine metastases (p = 0.0216).Conclusion: A strict patient selection is required to limit the iatrogenic effect of surgery, which may alter the quality of life of these frail patients with limited life expectancy

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

Clusterin increases post-ischemic damages in organotypic hippocampal slice cultures

Clusterin or apolipoprotein J is a heterodimeric glycoprotein which is known to be increased during tissue involution in response to hormonal changes or injury and under circumstances leading to apoptosis. Previous studies in wild-type (WT) and clusterin-null (Clu-/-) mice indicated a protective role of clusterin over-expression in astrocytes lasting up to 90 days post-ischemia. However, in in vitro and in vivo models of neonatal hypoxia-ischemia, clusterin exacerbates necrotic cell death. We developed recombinant forms of clusterin and examined their effect on propidium iodide uptake, neuronal and synaptic markers as well as electrophysiological recordings in hippocampal slice cultures from Clu-/- and WT mice subjected to oxygen-glucose deprivation (OGD). WT mice displayed a marked up-regulation of clusterin associated with electrophysiological deficits and dramatic increase of propidium iodide uptake 5 days post-OGD. Immunocytochemical and western blot analyses revealed a substantial decrease of neuronal nuclei and synaptophysin immunoreactivity that predominated in WT mice. These findings contrasted with the relative post-OGD resistance of Clu-/- mice. The addition of biologically active recombinant forms of human clusterin for 24 h post-OGD led to the abolishment of the ischemic tolerance in Clu-/- slices. This deleterious effect of clusterin was reverted by the concomitant administration of the NMDA receptor antagonist, d-2-amino-5-phosphonopentanoate. The present data indicate that in an in vitro model of ischemia characterized by the predominance of NMDA-mediated cell death, clusterin exerts a negative effect on the structural integrity and functionality of hippocampal neurons