Managing marine mollusc diseases in the context of regional and international commerce: policy issues and emerging concerns

Marine mollusc production contributes to food and economic security worldwide and provides valuable ecological services, yet diseases threaten these industries and wild populations. Although the infrastructure for mollusc aquaculture health management is well characterized, its foundations are not without flaws. Use of notifiable pathogen lists can leave blind spots with regard to detection of unlisted and emerging pathogens. Increased reliance on molecular tools has come without similar attention to diagnostic validation, raising questions about assay performance, and has been accompanied by a reduced emphasis on microscopic diagnostic expertise that could weaken pathogen detection capabilities. Persistent questions concerning pathogen biology and ecology promote regulatory paralysis that impedes trade and which could weaken biosecurity by driving commerce to surreptitious channels. Solutions that might be pursued to improve shellfish aquaculture health management include the establishment of more broad-based surveillance programmes, wider training and use of general methods like histopathology to ensure alertness to emerging diseases, an increased focus on assay assessment and validation as fundamental to assay development, investment in basic research, and application of risk analyses to improve regulation. A continual sharpening of diagnostic tools and approaches and deepening of scientific knowledge is necessary to manage diseases and promote sustainable molluscan shellfish industries

Characterization of actin genes in Bonamia ostreae and their application to phylogeny of the Haplosporidia

Bonamia ostreae is a protozoan parasite that infects the European flat oyster Ostrea edulis, causing systemic infections and resulting in massive mortalities in populations of this valuable bivalve species. In this work, we have characterized B. ostreae actin genes and used their sequences for a phylogenetic analysis. Design of different primer sets was necessary to amplify the central coding region of actin genes of B. ostreae. Characterization of the sequences and their amplification in different samples demonstrated the presence of 2 intragenomic actin genes in B. ostreae, without any intron. The phylogenetic analysis placed B. ostreae in a clade with Minchinia tapetis, Minchinia teredinis and Haplosporidium costale as its closest relatives, and demonstrated that the paralogous actin genes found in Bonamia resulted from a duplication of the original actin gene after the Bonamia origi

A herpes-like virus infects a non-ostreid bivalve species: virus replication in Ruditapes philippinarum larvae

Sporadic high mortalities were reported in June 1997 among hatchery-reared larval Manila clam Ruditapes philippinarum in a French commercial hatchery. Cellular abnormalities were observed in semi-thin sections in affected animals. Transmission electron microscopy revealed the presence of herpes-like virus particles in larvae. This is the first description of a herpes-like virus infection in larval R. philippinarum, a non-ostreid bivalve species. Virus particles were similar to other herpes-like viruses described from different oyster species with respect to ultrastructure and morphogenesis. Electron microscopic examination also demonstrated cells with condensed chromatin and extensive perinuclear fragmentation of chromatin. Like viruses infecting oysters, the herpes-like virus detected in clams may induce apoptosis in infected animals

Perspectives of diagnostic approaches for mollusc diseases

Surveillance of mollusc diseases is routinely performed by histology and PGR. Efforts made in re-search and development of DNA-based diagnostic methods currently offer a broad panel of probes and tests. TTiese methods have the theoretical advantages of high sensitivity and high specificity and possible rapid screening of molluscs for the presence of a targeted pathogen. However, validation and standardisation of these tests are still needed. In recent years, the European Union Reference Laboratory (EURL) for mollusc diseases has developed and validated a range of duplex Taqman\uae PCR assays aiming at facilitating the detection of bivalve pathogens notifiable to the EU. These assays offer good performances and allow better monitoring and investigation of the epidemiol\uacogy of these different pathogens. Complementary to these PCR assays, new diagnostic approaches based on the use of passive sensors, Magnetic Beads (MBs), electrochemical biosensors, MALDI-TOF MS or Next Generation Sequencing (NGS) have been developed and tested notably in the context of the European project H2020 VIVALDI. The interest of these tools to better detect, characterise and monitor known pathogens is presented and discussed. These new developments will require further assessment, standardisation and validation before being available for routine diagnostics. Additionally, they will certainly not replace but rather complement diagnostic tools currently used to ensure alertness to emerging diseases

Evidence for interspecies transmission of oyster herpesvirus in marine bivalves

Since 1991, numerous herpesvirus infections associated with high mortality have been reported around the world in various marine bivalve species. In order to determine whether these infections are due to ostreid herpesvirus-1 (OsHV1), a previously characterized pathogen of the Japanese oyster (Crassostrea gigas), PCR analysis was carried out on 30 samples of larvae collected from four bivalve species (C. gigas, Ostrea edulis, Ruditapes decussatus and Ruditapes philippinarum), most exhibiting mortality prior to collection. All samples were shown to be infected by OsHV1. Viral genomes in three samples of C. gigas and three of R. philippinarum that originated from the same hatchery were unusual in bearing a deletion of at least 2.8 kbp in an inverted repeat region. The results demonstrate that OsHV1 is capable of infecting several bivalve species, and this raises the possibility that interspecies transmission may be promoted by intensive rearing in modern hatcheries

Bonamia parasites: a rapidly changing perspective on a genus of important mollusc pathogens

Organisms of the genus Bonamia are intracellular protistan parasites of oysters. To date, 4 species have been described (B. ostreae, B. exitiosa, B. perspora and B. roughleyi), although the status of B. roughleyi is controversial. Introduction especially of B. ostreae and B. exitiosa to naive host populations has been shown to cause mass mortalities in the past and has had a dramatic impact on oyster production. Both B. ostreae and B. exitiosa are pathogens notifiable to the World Organisation for Animal Health (OIE) and the European Union. Effective management of the disease caused by these pathogens is complicated by the extensive nature of the oyster production process and limited options for disease control of the cultured stocks in open water. This review focuses on the recent advances in research on genetic relationships between Bonamia isolates, geographical distribution, susceptible host species, diagnostics, epizootiology, host-parasite interactions, and disease resistance and control of this globally important genus of oyster pathogens

A new multiplex real-time PCR assay to improve the diagnosis of shellfish regulated parasites of the genus Marteilia and Bonamia

Aquaculture including shellfish production is an important food resource worldwide which is particularly vulnerable to infectious diseases. Marteilia refringens, Bonamia ostreae and Bonamia exitiosa are regulated protozoan parasites infecting flat oysters Ostrea edulis that are endemic in Europe. Although some PCR assays have been already developed for their detection, a formal validation to assess the performances of those tools is often lacking. In order to facilitate the diagnosis of flat oyster regulated diseases, we have developed and evaluated a new multiplex Taqman (R) PCR allowing the detection of both M. refringens and Bonamia sp. parasites in one step. First part of this work consisted in assessing analytical sensitivity and specificity of the new PCR assay. Then, diagnostic performances were assessed by testing a panel of field samples with the new real-time PCR and currently recommended conventional PCR methods for the detection of M. refringens and Bonamia sp. Samples were collected from the main flat oyster production sites in France (N = 386 for M. refringens and N = 349 for B. ostreae). In the absence of gold standard, diagnostic sensitivity and specificity of the new PCR were estimated through Bayesian latent class analysis (DSe 87,2% and DSp 98,4% for the detection M. refringens, DSe 77,5% and DSp 98,4% for the detection of Bonamia sp.). Those results suggest equivalent performances for the detection of Bonamia sp. and an improved sensitivity for the detection of M. refringens compared to commonly used conventional protocols. Finally, the new PCR was evaluated in the context of an inter-laboratory comparison study including 17 European laboratories. Results revealed a very good reproducibility with a global accordance (intralaboratory precision) >96% and a global concordance (inter-laboratory precision) >93% for both targets, demonstrating that this new tool is easily transferable to different laboratory settings. This is the first assay designed to detect both Marteilia refringens and Bonamia sp. in a single step and it should allow reducing the number of analysis to monitor both diseases, and where relevant to demonstrate freedom from infection