Shellfish hatcheries are often affected by disease outbreaks. Three such episodes were investigated in different Galician hatcheries in order to establish the relationship between present microbiota and mortalities. Isolates were obtained from various parts of the hatcheries. Experimental tests for pathogenicity were carried out in microscale experiments using selected strains on Ostrea edulis larvae. The pathogenicity of 1 strain from each outbreak was demonstrated and shown to cause high mortalities (ranging from 98.5 to 100%) in 72 to 96 h after inoculation of larval cultures. All 3 strains belong to the genus Vibrio. One of the strains was identified as Vibrio neptunius and is the first description of this species as a molluscan pathogen. The other 2 strains showed low similarity with the Vibrio species analysed and may constitute new species within this genus. KEY WORDS: Flat oyster · Vibrio · Vibrio neptunius · Pathogenicity test · Shellfish hatchery Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [209][210][211][212][213][214][215] 2005 Tettelbach 1988, Lodeiros et al. 1992, Riquelme et al. 1995, Nicolas et al. 1996, Sáinz et al. 1998, Sugumar et al. 1998, Araya et al. 1999, Elston et al. 1999, Lacoste et al. 2001, Anguiano-Beltrán et al. 2004, Estes et al. 2004, Gay et al. 2004.In this paper, we present the results of bacteriological studies performed during 3 episodes of severe mortalities in 3 shellfish hatcheries in Galicia. The pathogenicity of isolated bacterial strains was tested in order to identify the aetiological agents. In addition, the first evidence of Vibrio neptunius as mollusc pathogen is presented. MATERIALS AND METHODS Description of facilities and bacterial isolation.The facilities where mortalities occurred are situated at different locations along the Galician coast in NW Spain (Fig. 1). Their management practices vary: one is a commercial hatchery beside a molluscan depuration facility (hatchery A); the second is a plant where molluscs and fishes are cultured (hatchery B); and the third is exclusively dedicated to molluscs (hatchery C). Two of the facilities (B and C) suffered mortalities in oyster larval stages and one (A) in post-larvae.Samples were taken in the different areas of the hatcheries: larvae, spat, broodstock, tank surfaces, phytoplankton and water. Larvae, spat and broodstock gonad (pieces excised aseptically) were washed, ground and homogenized in sterile seawater (SSW). Appropriate dilutions were made with these suspensions as well as with samples of water and phytoplankton used as larval feed. They were spread on Marine Agar (MA, Pronadisa) and Thiosulphate Citrate Bile Sucrose (TCBS, Oxoid). Samples taken directly from inner surfaces of the tanks containing the oysters were spread on the above mentioned media with sterile swabs.Plates were incubated at 22°C and selected colonies were isolated after 24 h (TCBS) or 7 d (MA) on MA, and further restreaked to purity. Pure cultures of strains were frozen at -8...

The aquaculture of bivalve molluscs has attained a considerable level of production but it is not enough to cover the demand of worldwide consumers. In the development of this sector, hatcheries play an important role, as suppliers of competent spat of different bivalves, including species with an aquaculture based on natural extraction present. Besides, these installations may help in the recovery of exhausted natural beds and in the obtaining of populations under genetic selection. Unfortunately, the disease outbreaks caused by bacterial pathogens are frequent, with the loss of complete batches, compromising the regular production and the economic viability of the industry. There are many descriptive studies about these outbreaks, but only a few focused on the control of microbiota. The particularities of bivalve aquaculture in hatchery must be taken into account to design methods of control. A common environment is shared by larvae and bacteria, including both beneficial and potentially pathogenic. The filter-feeding behaviour of larvae increases the strong influence of these populations. The classical treatments are directed toward to the complete elimination of bacteria from culture seawater. That objective is unfeasible, because the cultures are not axenic, and undesirable, since some bacteria enhance larval development. Taking into account these considerations, the most promising alternative is the use of probiotic bacteria. In this review we summarize the scientific literature about this subject, considering the particularities of bivalve larval cultures and the need to adapt the concept of probiotic and the strategies to use in marine bivalve hatcheries.

Vibriosis represents the main bottleneck for the larval production process in shellfish aquaculture. While the signs of this disease in bivalve larvae are well known, the infection process by pathogenic Vibrio spp. during episodes of vibriosis has not been elucidated. To investigate the infection process in bivalves, the pathogens of larvae as V. tubiashii subsp. europaensis, V. neptunius and V. bivalvicida were tagged with green fluorescent protein (GFP). Larvae of Manila clam (Ruditapes philippinarum) were inoculated with the GFP-labeled pathogens in different infection assays and monitored by microscopy. Manila clam larvae infected by distinct GFP-tagged Vibrio spp. in different challenges showed the same progression in the infection process, defining three infection stages. GFP-tagged Vibrio spp. were filtered by the larvae through the vellum and entered in the digestive system through the esophagus and stomach and colonized the digestive gland and particularly the intestine, where they proliferated during the first 2h of contact (Stage I), suggesting a chemotactic response. Then, GFP-tagged Vibrio spp. expanded rapidly to the surrounding organs in the body cavity from the dorsal to ventral region (Stage II; 6-8h), colonizing the larvae completely at the peak of infection (Stage III) (14-24h). Results demonstrated for the first time that the vibriosis is asymptomatic in Manila clam larvae during the early infection stages. Thus, the early colonization and the rapid proliferation of Vibrio pathogens within the body cavity supported the sudden and fatal effect of the vibriosis, since the larvae exhibited the first signs of disease when the infection process is advanced. As a first step in the elucidation of the potential mechanisms of bacterial pathogenesis in bivalve larvae the enzymatic activities of the extracellular products released from the wild type V. neptunius, V. tubiashii subsp. europaensis and V. bivalvicida were determined and their cytotoxicity was demonstrated in fish and homeothermic cell lines for the first time. That activity was lost after heat treatment.

The health of marine bivalve larvae is greatly affected by bacteria in the environment particularly when reared in marine hatcheries. This is generally because high stocking densities resulting in high organic loads of both food and faeces, can support increased bacterial growth and biomass levels. Increased bacterial load can lead to larval disease referred to as bacillary necrosis (BN) leading in turn to rapid larval mortality and loss of production. Despite more than 50 years since the first detailed description of BN, we still do not fully understand its causes and mechanisms. Through the manipulation of a model larval culture of the Australian blue mussels (Mytilus galloprovincialis), we determined that BN is linked with rapid and systematic changes in the bacterial community. Early investigation of larval mortality in bivalve larval cultures in the 1950s reported mortality associated with infection by gram negative bacilli that necrotised larval tissues, leading to the descriptive term bacillary necrosis (BN) 1. The disease is capable of causing total collapse of larval cultures (larval crash) in a period of 24-48 hours and is today, the most prevalent hatchery disease worldwide, affecting more than 20 bivalve species. Whilst it is difficult to quantify the impact of BN in shellfish hatcheries, frequent recurrence can severely impact hatchery production with repercussions often felt throughout the supply chains. The prevailing view that BN is an opportunistic disease leads to the emphasis on sound husbandry practices primarily to reduce excess build-up of organic matter. However, whether and how enriched organic conditions are linked with development of BN is unclear. Efforts to study BN have also been complicated by the unpredictable nature of the outbreaks. To address this problem, we deliberately overfed a series of identical small-scale larval cultures with microalgae to create an environment that would increase the incidence of the disease. In cultures that developed mass mortal-ities, automated ribosomal intergenic spaces analysis (ARISA) demonstrated that BN involves rapid and systematic changes in the bacterial community, firstly in the seawater, then rapidly proceeding to the larvae as the disease progresses and necrosis occurs (Figure 1). This study shows that, at least within the system analysed here, BN is a condition of abnormal changes in seawater-associated communities that are capable of affecting the larvae, suggestive of seawater-to-larvae infectivity. The similarity of bacterial communities in seawater and larvae at the onset of mortality suggest swamping by outgrowth of particular bacteria. Bacterial diversity examination using Illumina MiSeq sequencing of 16S rRNA ampli-cons showed that mortality in the model systems was linked with a bacterial community increasingly dominated by Psychroserpens, Polaribacter, Marinomonas, and members of the Candidatus phylum Gracilibacteria. The observation that BN did not occur in all overfed cultures suggests variability in causation made it difficul...

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