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The current study describes the development and application of a TaqMan® real-time PCR assay for the detection of the bacterium Francisella halioticida. Previously, detection of F. halioticida is relied on bacterial culture and conventional PCR; however, the real-time PCR provides many advantages because it is faster, less labour-intensive and reduces the risk of cross-contamination. DNA samples from mussels collected in April 2020 from seven sites in northern Brittany (France) were tested using the newly developed real-time PCR assay. The objective was to screen for the presence of F. halioticida during spring mortality events. The bacterium was detected in 71.4% of the samples tested and was present at all sites except for Saint-Brieuc and Mont-Saint-Michel, two sites which were not concerned by mortality at the time of sampling. Less than a month later, Saint-Brieuc was affected by unusual mortalities and F. halioticida was detected in almost all mussels (81.25%). The findings from this study provide further evidence indicating that F. halioticida may be contributing to mussel mortalities; however, a direct causal relationship has not yet been established. The real-time PCR assay developed in this study allows for rapid, specific and sensitive detection of F. halioticida which should prove useful for future studies concerning the involvement of this bacterium with shellfish mortalities.

Transmissible cancers, in which cancer cells themselves act as an infectious agent, have been identified in Tasmanian devils, dogs, and four bivalves. We investigated a disseminated neoplasia affecting geographically distant populations of two species of mussels (Mytilus chilensis in South America and M. edulis in Europe). Sequencing alleles from four loci (two nuclear and two mitochondrial) provided evidence of transmissible cancer in both species. Phylogenetic analysis of cancer-associated alleles and analysis of diagnostic SNPs showed that cancers in both species likely arose in a third species of mussel (M. trossulus), but these cancer cells are independent from the previously identified transmissible cancer in M. trossulus from Canada. Unexpectedly, cancers from M. chilensis and M. edulis are nearly identical, showing that the same cancer lineage affects both. Thus, a single transmissible cancer lineage has crossed into two new host species and has been transferred across the Atlantic and Pacific Oceans and between the Northern and Southern hemispheres., eLife digest Cancer cells can grow and spread in one individual, but they normally do not spread to others. There are a few exceptions to this rule. For example, there are cancers in Tasmanian devils, dogs and bivalve shellfish that can spread to other members of the same species. In these creatures, cancer from one individual evolved the ability to spread throughout the population. These cancer cells infect animals like a pathogen. A fatal cancer called disseminated neoplasia affects many species of bivalves. In four bivalve species, including the marine mussel Mytilus trossulus, scientists have shown that the cancer can spread from one individual to another. This transmissible cancer has been found in M. trossulus mussels in British Columbia, Canada; but related species of mussels in other parts of the world also develop disseminated neoplasia. It is possible these other cancers are transmissible and have spread from one population of mussels to another. Yonemitsu et al. performed genetic analyses to show that cancers found in two other mussel species – Mytilus chilensis in South America and Mytilus edulis in Europe – are transmissible and arose in M. trossulus. The cancers in the South American and European mussels were nearly identical genetically, which suggests that they came from a single M. trossulus mussel with cancer at some point in the past. Somehow cancer cells spread between the Northern and the Southern Hemispheres and across the Atlantic Ocean, infecting multiple species across the world. The analyses also show that this cancer lineage is different from the one previously identified in British Columbia. These analyses show that bivalve transmissible neoplasia was able to spread worldwide, most likely through accidental transport of infected mussels on international shipping vessels. This suggests that human activities unwittingly introduced the disease to new areas. Learning more about transmissible cancers may help scientists understand how cancers evolve with their hosts in extreme situations.

International audience; 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.

International audience; 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 ¼ 5 oocysts; RR between 5 and 56%); ii) raspberries and basil (LOD ¼ 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

International audience; This study evaluates the viability of Giardia intestinalis cysts, Cryptosporidium spp. and Toxoplasma gondii oocysts, which may be found in water and food matrices subjected to contaminated water. Viability is a key factor to be considered in order to assess parasite infectivity. People living in developing countries are particularly at risk of contamination by these pathogens, which can cause severe diseases particularly in immunocompromised people. In this report, we describe methods combining the mechanical rupture of (oo)cysts and mRNA extraction in order to determine their viability by qRT-PCR. The targeted genes are beta-giardin, hsp70, and SporoSAG for the detection of G. intestinalis, Cryptosporidium spp. and T. gondii respectively. The proposed method is compared to in vivo infectivity tests specific of each parasite. Then, this qRT-PCR method is applied directly on parasite suspensions and subsequently on basil experimentally contaminated with the parasites. For each protozoan, the detection limit of the qRT-PCR method is 2 parasites per reaction (2 μL mRNA analyzed) on parasite suspensions, and 3 parasites per gram of basil matrix. Compared to in vivo methods, qRT-PCR can detect viable but not infectious parasites. This qRT-PCR method could represent an attractive alternative as a specific and sensitive tool for: i) rapid assessment of the risk of human contamination by G. intestinalis, Cryptosporidium spp. and T. gondii parasites; ii) evaluation of the efficiency of industrial process.

Background Massive mortalities have been observed in France since 2008 on spat and juvenile Pacific oysters, Crassostrea gigas. A herpes virus called OsHV-1, easily detectable by PCR, has been implicated in the mortalities as demonstrated by the results of numerous field studies linking mortality with OsHV-1 prevalence. Moreover, experimental infections using viral particles have documented the pathogenicity of OsHV-1 but the physiological responses of host to pathogen are not well known. Results The aim of this study was to understand mechanisms brought into play against the virus during infection in the field. A microarray assay has been developed for a major part of the oyster genome and used for studying the host transcriptome across mortality on field. Spat with and without detectable OsHV-1 infection presenting or not mortality respectively were compared by microarray during mortality episodes. In this study, a number of genes are regulated in the response to pathogen infection on field and seems to argue to an implication of the virus in the observed mortality. The result allowed establishment of a hypothetic scheme of the host cell’s infection by, and response to, the pathogen. Conclusions This response shows a “sensu stricto” innate immunity through genic regulation of the virus OsHV-1 life cycle, but also others biological processes resulting to complex interactions between host and pathogens in general.