Vibrio mediterranei , a potential emerging pathogen of marine fauna: investigation of pathogenicity using a bacterial challenge in Pinna nobilis and development of a species‐specific PCR

Abstract

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 Pinna nobilis collected from local bays were used in a challenge experiment with V. mediterranei (strain IRTA18‐108). The virulence in the host background of P. nobilis was demonstrated at doses of 103 CFUs per animal. An alignment of published Vibrio sp. atpA sequences was used to design V. mediterranei‐specific primers. Furthermore, 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 Vibrio 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 24°C 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.

Multiple Non-Species-Specific Pathogens Possibly Triggered the Mass Mortality in Pinna nobilis

“…Moreover, in accordance with what has already been suggested by Carella et al [ 19 ] (and references therein) for individuals from the northern coast of Spain and by Lattos et al [ 33 ] for Greek populations of the Thermaikos Gulf, our study further supports a hypothesis that is gaining popularity that P. nobilis mass mortality is not linked exclusively to infection by the protozoan H. pinnae [ 16 , 17 , 18 , 19 , 33 ] (and references therein). In this context, we detected no association between the occurrence of the typical signs of disease responsible for the death of individuals of P. nobilis and the presence of H. pinnae in their tissues.…”

NMR Metabolite Profiles of the Bivalve Mollusc Mytilus galloprovincialis Before and After Immune Stimulation With Vibrio splendidus

“…Gram-negative and facultatively anaerobic Vibrio bacteria are widespread in the aquatic environments, and species such as Vibrio anguillarum, Vibrio coralliilyticus, Vibrio splendidus, Vibrio aestuarianus, and Vibrio mediterranei are known to induce severe vibriosis that can occasionally escalate to episodes of bivalve mass mortality (Vezzulli et al, 2010;Sun et al, 2014;Green et al, 2019;Andree et al, 2021;Garcia et al, 2021). Specifically, the strain V. splendidus LGP32 is regarded as a pathogen owing to its association to oysters during summer mortality events and the demonstration of a remarkable virulence in laboratory trials with oysters and clams (Roux et al, 2009).…”

The Implication of Vibrio Bacteria in the Winter Mortalities of the Critically Endangered Pinna nobilis

“…V. splendidus strains, which were abundant in the majority of the examined specimens, have been reported to exhibit their highest virulence at low temperatures, especially considering the presence of virulence genes, while their pathogenicity is reduced at increased temperatures [ 58 , 72 ]. Virulence genes upregulate the transmission and the reproduction rate of the pathogen [ 27 ]. The presence of virulence genes, particularly in V. splendidus and V. mediterranei , support the scenario of pathogenicity of vibrios in the Aegean Sea, as well as their contribution in winter mortalities.…”

“…Using the same software, two phylogenetic dendrograms were constructed implementing the maximum likelihood methodology and applying 1000 bootstrap iterations to evaluate the genetic relationships with closely related haplotypes available in GenBank. Furthermore, to explore the disease pathogenesis of the Vibrio identified bacteria, the presence of virulence factors was examined, i.e., vsm and outer membrane protein ( ompU ) genes in V. splendidus strains and ompU and rtx toxin genes in V. mediterranei strains, using the FastGene Taq 2x Ready Mix with the abovementioned volumes, and primers and conditions as previously described [ 27 , 37 ].…”

“…In a rescue project for P. nobilis conducted in Catalunya, a new pathogen emerged in individuals free from the parasites mentioned above. V. mediterranei was detected in dead individuals when the temperature rose between April and May (17–19 °C) in stable animals, and was later demonstrated to be infectious with the presence of specific virulence genes [ 26 , 27 ]. In the context of exploration in the P. nobilis microbiome, identification of eight different bacterial taxa was performed and the results concluded the presence of V. splendidus clade bacteria [ 28 ].…”