Disease is an increasing threat for marine bivalves worldwide. Recently, a mass mortality event (MME) impacting the bivalve Pinna nobilis was detected across a wide geographical area of the Spanish Mediterranean Sea and linked to a haplosporidian parasite. In 2017–2018, mass mortality events affecting the pen shell Pinna nobilis were recorded in two different regions of Italy, Campania and Sicily, in the Tyrrhenian Sea (Mediterranean Sea). Histopathological and molecular examinations of specimens showed the presence of Haplosporidium sp. in only one specimen in one area. Conversely, in all of the surveyed moribund animals, strong inflammatory lesions at the level of connective tissue surrounding the digestive system and gonads and linked to the presence of intracellular Zhiel-Neelsen-positive bacteria were observed. Molecular analysis of all of the diseased specimens (13) confirmed the presence of a Mycobacterium. Blast analysis of the sequences from all of the areas revealed that they were grouped together with the human mycobacterium M. sherrisii close to the group including M. shigaense, M. lentiflavum and M. simiae. Based on pathological and molecular findings, it is proposed that a mycobacterial disease is associated with the mortality episodes of Pinna nobilis, indicating that, at this time, Haplosporidium sp. is not responsible for these events in Campanian and Sicilian waters.

Summary: Infectious agents such as the bacteria Vibrio aestuarianus or Ostreid herpesvirus 1 have been repeatedly associated with dramatic disease outbreaks of Crassostrea gigas beds in Europe. Beside roles played by these pathogens, microbial infections in C . gigas may derive from the contribution of a larger number of microorganisms than previously thought, according to an emerging view supporting the polymicrobial nature of bivalve diseases. In this study, the microbial communities associated with a large number of C . gigas samples collected during recurrent mortality episodes at different European sites were investigated by real‐time PCR and 16SrRNA gene‐based microbial profiling. A new target enrichment next‐generation sequencing protocol for selective capturing of 884 phylogenetic and virulence markers of the potential microbial pathogenic community in oyster tissue was developed allowing high taxonomic resolution analysis of the bivalve pathobiota. Comparative analysis of contrasting C . gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter . The role of these species and their consortia should be targeted by future studies aiming to shed light on mechanisms underlying polymicrobial infections in C . gigas .

Aims Extreme mortality events affecting Pinna nobilis, some associated to Vibrio mediterranei, have depleted many populations of this bivalve. The objective of this study was to demonstrate pathogenicity of V. mediterranei in the host P. nobilis by performing a bacterial challenge in P. nobilis to understand if V. mediterranei has specific virulence in this host. To assist this objective, a secondary objective was to develop a species‐specific DNA diagnostic test. Methods and Results P. nobilis collected from local bays were used in a challenge experiment with V. mediterranei (strain IRTA18‐108). Virulence in the host background of P. nobilis was demonstrated at doses of 10³ CFUs / animal. An alignment of published Vibrio spp. atpA sequences was used to design V. mediterranei ‐specific primers. Further, data mining of published literature and V. mediterranei genomes identified multiple virulence‐related genes (vir genes) from which specific primers were designed for PCR detection of selected genes. Conclusion V. mediterranei strain IRTA18‐108 is pathogenic in the host P. nobilis . The virulence genes sod, rtx , and mshA were identified in this strain. Temperatures of 24ºC or higher appear to trigger onset of virulence. Sensitivity and specificity of the Vm atpA PCR is useful for diagnosis of Vibriosis in shellfish. Significance and Impact of the Study The presence of previously described virulence genes have been confirmed in this strain. The specific Vm atpA PCR assay will aid management of future epizootics of this emerging pathogen of aquatic fauna, and improve surveillance capabilities for mortality events where Vibrios are suspect.