New Insights into Pathogenic Vibrios Affecting Bivalves in Hatcheries: Present and Future Prospects

Hatcheries constitute nowadays the only viable solution to support the husbandry of bivalve molluscs due to the depletion and/or overexploitation of their natural beds. Hatchery activities include the broodstock conditioning and spawning, rearing larvae and spat, and the production of microalgae to feed all stages of the production cycle. However, outbreaks of disease continue to be the main bottleneck for successful larval and spat production, most of them caused by different representatives of the genus Vibrio. Therefore, attention must be paid on preventive and management measures that allow the control of such undesirable bacterial populations. The present review provides an updated picture of the recently characterized Vibrio species associated with disease of bivalve molluscs during early stages of development, including the controversial taxonomic affiliation of some of them and relevant advances in the knowledge of their virulence determinants. The problematic use of antibiotics, as well as its eco-friendly alternatives are also critically discussedThe studies of the University of Santiago de Compostela reviewed here were supported in part by grants AGL2013-42628R and AGL2014-59655 from the Ministry of Economy and Competitiveness, and GRC-2014/007 from Xunta de Galicia (Spain)S

Isolation and Pathogenic Characterization of Vibrio bivalvicida Associated With a Massive Larval Mortality Event in a Commercial Hatchery of Scallop Argopecten purpuratus in Chile

The VPAP30 strain was isolated as the highly predominant bacteria from an episode of massive larval mortality occurring in a commercial culture of the Chilean scallop Argopecten purpuratus. The main aims of this study were, to characterize and identify the pathogenic strain using biochemical and molecular methods, to demonstrate its pathogenic activity on scallop larvae, to characterize its pathogenic properties and to describe the chronology of the pathology. The pathogenic strain was identified as Vibrio bivalvicida based on its phenotypic properties, the multilocus sequence analysis (MLSA) of eight housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) and different in silico genome-to-genome comparisons. When triplicate cultures of healthy 10 days old scallop larvae were challenged with 1 × 105 colony forming units (CFU) mL-1 of the VPAP30 strain, percentages of larval survival of 78.9 ± 3.3%, 34.3 ± 4.9%, and 0% were observed at 12, 2,4 and 36 h, respectively, whereas uninfected larval cultures showed survival rates of 97.4 ± 1.2% after of 48 h. Clinical symptoms exhibited by the scallop larvae infected with the VPAP30 strain include the accumulation of bacteria around the scallop larvae, velum disruption and necrosis of digestive gland. The 50% lethal dose (LD50) of VPAP30 strain at 24 and 48 h was 1.3 × 104 and 1.2 × 103 CFU mL-1, respectively. The invasive pathogenic activity of the VPAP30 strain was investigated with staining of the bacterial pathogen with 5-DTAF and analyzing bacterial invasion using epifluorescence, and a complete bacterial dissemination inside the larvae at 24 h post-infection was observed. When scallop larvae were inoculated with cell-free extracellular products (ECPs) of VPAP30, the larval survival rate was 59.5 ± 1.7%, significantly (P < 0.001) lower than the control group (97.4 ± 1.2%) whereas larvae treated with heat-treated ECPs exhibited a survival rate of 61.6 ± 1.8% after 48 h of exposure. V. bivalvicida VPAP30 exhibits high pathogenic activity on scallop larvae, mediated both by bacterial invasion and the production of toxigenic heat-stable compounds. This report constitutes the first isolation of V. bivalvicida out of Europe and extends the host range of this species, having demonstrated its pathogenic activity on the Chilean scallop larvae (A. purpuratus). These results supporting the pathogenic potential of V. bivalvicida to kill the larvae of a broad range of bivalve species reared in hatcheries located in the Atlantic and the Pacific coasts.This study was financially supported by the Science and Technology National Council (CONICYT) of Chile by the Postdoctoral Project Grant No. 3150395 and FONDECYT grant No. 1140734 and FONDEF ID16I10291S

Draft Genome Sequence of the Emerging Bivalve Pathogen \u3cem\u3eVibrio tubiashii\u3c/em\u3e subsp. \u3cem\u3eeuropaeus\u3c/em\u3e

Vibrio tubiashii subsp. europaeus is a bivalve pathogen isolated during episodes of mortality affecting larval cultures in different shellfish hatcheries. Here, we announce the draft genome sequence of the type strain PP-638 and describe potential virulence factors, which may provide insight into the mechanism of pathogenicity

Draft Genome Sequence of the New Pathogen for Bivalve Larvae \u3cem\u3eVibrio bivalvicida\u3c/em\u3e

Vibrio bivalvicida is a novel pathogen of bivalve larvae responsible for recent vibriosis outbreaks affecting shellfish hatcheries. Here, we announce the draft genome sequence of V. bivalvicida 605 and describe potential virulence factors

N-Acyl Dehydrotyrosines, Typrosinase Inhibitors from the Marine Bacteriu, Thalasssotalea sp. PP2-459

Thalassotalic acids A–C and thalassotalamides A and B are new N-acyl dehydrotyrosine derivatives produced by Thalassotalea sp. PP2-459, a Gram-negative bacterium isolated from a marine bivalve aquaculture facility. The structures were elucidated via a combination of spectroscopic analyses emphasizing two-dimensional NMR and high-resolution mass spectrometric data. Thalassotalic acid A (1) displays in vitro inhibition of the enzyme tyrosinase with an IC50 value (130 μM) that compares favorably to the commercially used control compounds kojic acid (46 μM) and arbutin (100 μM). These are the first natural products reported from a bacterium belonging to the genus Thalassotalea

Isolation and Pathogenic Characterization of Vibrio bivalvicida Associated With a Massive Larval Mortality Event in a Commercial Hatchery of Scallop Argopecten purpuratus in Chile

The VPAP30 strain was isolated as the highly predominant bacteria from an episode of massive larval mortality occurring in a commercial culture of the Chilean scallop Argopecten purpuratus. The main aims of this study were, to characterize and identify the pathogenic strain using biochemical and molecular methods, to demonstrate its pathogenic activity on scallop larvae, to characterize its pathogenic properties and to describe the chronology of the pathology. The pathogenic strain was identified as Vibrio bivalvicida based on its phenotypic properties, the multilocus sequence analysis (MLSA) of eight housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) and different in silico genome-to-genome comparisons. When triplicate cultures of healthy 10 days old scallop larvae were challenged with 1 × 105 colony forming units (CFU) mL-1 of the VPAP30 strain, percentages of larval survival of 78.9 ± 3.3%, 34.3 ± 4.9%, and 0% were observed at 12, 2,4 and 36 h, respectively, whereas uninfected larval cultures showed survival rates of 97.4 ± 1.2% after of 48 h. Clinical symptoms exhibited by the scallop larvae infected with the VPAP30 strain include the accumulation of bacteria around the scallop larvae, velum disruption and necrosis of digestive gland. The 50% lethal dose (LD50) of VPAP30 strain at 24 and 48 h was 1.3 × 104 and 1.2 × 103 CFU mL-1, respectively. The invasive pathogenic activity of the VPAP30 strain was investigated with staining of the bacterial pathogen with 5-DTAF and analyzing bacterial invasion using epifluorescence, and a complete bacterial dissemination inside the larvae at 24 h post-infection was observed. When scallop larvae were inoculated with cell-free extracellular products (ECPs) of VPAP30, the larval survival rate was 59.5 ± 1.7%, significantly (P &lt; 0.001) lower than the control group (97.4 ± 1.2%) whereas larvae treated with heat-treated ECPs exhibited a survival rate of 61.6 ± 1.8% after 48 h of exposure. V. bivalvicida VPAP30 exhibits high pathogenic activity on scallop larvae, mediated both by bacterial invasion and the production of toxigenic heat-stable compounds. This report constitutes the first isolation of V. bivalvicida out of Europe and extends the host range of this species, having demonstrated its pathogenic activity on the Chilean scallop larvae (A. purpuratus). These results supporting the pathogenic potential of V. bivalvicida to kill the larvae of a broad range of bivalve species reared in hatcheries located in the Atlantic and the Pacific coasts

Vibrios en criaderos de bivalvos. Una visión integrada

Los criaderos de bivalvos constituyen en la actualidad la única solución viable para satisfacer la elevada demanda de semilla para una actividad intensiva como es la acuicultura, debido al agotamiento de los bancos naturales y/o sobreexplotación. En este sector, los aspectos microbiológicos juegan un papel fundamental en el éxito de los cultivos, tanto en el ambiente natural como en el ecosistema artificial de un criadero. En estas instalaciones, las mortalidades causadas por especies patógenas del género Vibrio constituyen el principal factor limitante en la producción de bivalvos durante las primeras fases de desarrollo. A pesar de la relevancia de los vibrios en los cultivos de bivalvos, existen pocos estudios en los que se analice la presencia y el papel de estos microorganismos entre las diferentes secciones del criadero y ofrezcan una visión integrada durante un período prolongado de tiempo. A lo largo de esta Memoria hemos podido identificar tres nuevos taxones patógenos dentro del género Vibrio, responsables de diferentes episodios de mortalidad larvaria. Además, hemos contribuido al avance en el esclarecimiento de la patogénesis de la vibriosis al determinar por primera vez la ruta de colonización de las larvas de bivalvos por diferentes vibrios patógenos y su relación con los signos clínicos que manifiestan dichas larvas. Se ha demostrado también la utilización inadecuada de cloranfenicol para prevenir la vibriosis ya que no garantiza el éxito de los cultivos y favorece el rápido desarrollo de vibrios resistentes a diferentes antibióticos. Además, hemos analizado las poblaciones de vibrios presentes en los distintos compartimentos del criadero integrando la información para relacionar estos resultados con el éxito/fracaso de los cultivos larvarios y poder así minimizar los episodios de vibriosis. A partir de los resultados obtenidos, hemos determinado la existencia de una estrecha relación entre los vibrios y el criadero. Por tanto, los criaderos actúan como reservorio bacteriano y el flujo entre los diferentes compartimentos es inevitable. La mejor estrategia para garantizar el éxito de los cultivos es reducir la concentración de vibrios estableciendo un control microbiológico sobre los diferentes compartimentos del criadero para evitar su proliferación. En este sentido, nuestro estudio proporciona un protocolo microbiológico para controlar y garantizar la calidad de los cultivos de microalgas, evitando el riesgo de transferencia de patógenos potenciales para las larvas y/o a los reproductores. Además, la implementación del tratamiento de acondicionamiento evaluado para R. decussatus mejoraría el manejo de los reproductores y reduciría la entrada de vibrios con potencial patogénico en los cultivos larvarios a través de una transmisión vertical bacteriana

First study of Vibrios in larval cultures of pullet carpet shell clam (<i>Venerupis corrugata</i>) in hatchery

Protocol for hatchery culture of the pullet carpet shell clam Venerupis corrugata spat is currently under development, as the only reliable means of providing spat to replenish natural beds or to support aquaculture activities. Among other variables, the microbiota has been demonstrated to be critical for successful bivalve culture. Shellfish hatcheries are hindered by fatal outbreaks of disease, regardless the bivalve species. These mass mortalities are mainly caused by opportunistic bacteria belonging to genus Vibrio and constitute one bottleneck for this economic activity. Different species, as V. tubiashii, V. pectenicida, V. splendidus, V. neptunius, V. ostreicida and V. bivalvicida, have been identified as responsible of mortalities in hatchery-reared larvae, affecting a wide range of bivalves. This is the first report of the microbiota associated with larval cultures of the pullet carpet shell clam. We present the results of the microbiological analyses of two larval cultures of pullet carpet shell reared in the Centro de Investigacións Mariñas (CIMA, Xunta de Galicia) de Ribadeo (Galicia, NW Spain) following the procedures developed in the institution. Each batch, A and B, was obtained from broodstocks collected in natural environment but in different geographical locations, the stock A (SW Galicia) and the stock B (NW Galicia). Previous records of mortalities led us to divide each batch in two. One sub-batch (A1 and B1) was cultured following the routine procedures. Antibiotic was experimentally added to the other sub-batch (A2 and B2) with the aim of evaluating the effects on the culturable bacterial population (total marine bacteria and presumptive vibrios) and on larval survival. Chloramphenicol, formerly the most commonly used antibiotic in bivalve hatcheries, was supplied with each change of seawater during larval development. Microbiological samples of broodstock, larvae and seawater in culture tanks were taken and processed immediately following Prado et al. (2005, 2014). The bacteriological media used were Marine Agar, for heterotrophic marine bacteria, and Thiosulphate-Citrate-Bile-Sucrose, selective for vibrios. Bacterial counts were determined in all samples and expressed in colony forming unit (cfu) per millilitre or gram. The different types of colony in the samples of larval cultures were isolated, purified and subjected to a basic phenotypic characterization. Further studies were carried out with the isolates that shared the main features of the genus Vibrio, including phenotypic tests and the genetic characterization by sequencing of 16S rRNA gene, as described by Prado et al. (2014). The counts of marine heterotrophic bacteria associated to broodstock gonad were similar in both groups (≈105 cfu/g). However, substantial differences were observed in the counts of presumptive vibrios, with higher values in the stock B (≈104 cfu/g) than in the stock A (≈102 cfu/g). Interestingly, the larval batch A showed high bacterial counts in the initial samples, including presumptive vibrios, while the batch B began with lower numbers of total marine bacteria and vibrios. The survival rates at the end of the larval development were higher in batch B (40% B1, 32% B2) than in batch A (24% A1, 27% A2). The use of antibiotic did not imply a significant enhancement of the survival. The total counts in the seawater from the culture tanks were similar. The presumptive vibrios were found mainly associated to larvae, regardless the batch or the use of antibiotic. The differences on the composition of bacterial populations will be analysed, focused on the identification of the presumptive vibrios. Preliminary results showed the dominance of species within the Splendidus clade of the genus Vibrio, as expected taking into account that its members have been isolated from marine environments and many of them from bivalves. Due to the high phenotypic diversity within this clade and the difficult to differentiate species, the correlation between phenotypic and genotypic identification will be evaluated

First Report, Characterization and Pathogenicity of Vibrio chagasii Isolated from Diseased Reared Larvae of Chilean Scallop, Argopecten purpuratus (Lamarck, 1819)

Two Vibrio strains (VPAP36 and VPAP40) were isolated from moribund-settled larvae of the Chilean scallop Argopecten purpuratus during vibriosis outbreaks that occurred in two commercial scallop larvae hatcheries located in the Inglesa and Tongoy bays in Northern Chile. The strains were identified as Vibrio chagasii using phenotypic characterization and whole genome sequence analysis. Both strains exhibited the phenotypic properties associated with virulence, gelatin hydrolysis and &beta;-hemolysis, whereas only VPAP36 produced phospholipase and only VPAP40 produced caseinase. The whole genome analysis showed that the strains harbored genes encoding for the virulence factors, the EPS type II secretion system, and Quorum Sensing (auto-inductor 1 and auto-inductor 2), whereas genes encoding a metalloproteinase and a capsular polysaccharide were detected only in the VPAP40 genome. When challenge bioassays using healthy 11-day-old scallop larvae were performed, the V. chagasii VPAP36 and VPAP40 strains exhibited significant (p &lt; 0.05) differences in their larval lethal activity, producing, after 48 h, larval mortalities of 65.51 &plusmn; 4.40% and 28.56 &plusmn; 5.35%, respectively. Otherwise, the cell-free extracellular products of the VPAP36 and VPAP40 strains produced larval mortalities of 20.86 &plusmn; 2.40% and 18.37 &plusmn; 2.40%, respectively, after 48 h of exposure. This study reports for the first time the isolation of V. chagasii from the massive larval mortalities of the farmed scallop (Argopecten purpuratus) in Chile, and demonstrates the pathogenic activity of V. chagasii towards the Chilean scallop, the second most important species for Chilean mariculture

Role of the Vibriolysin VemA Secreted by the Emergent Pathogen Vibrio europaeus in the Colonization of Manila Clam Mucus

Vibrio europaeus is an emergent pathogen affecting clams, oysters and scallops produced in the most important countries for bivalve aquaculture. Studies concerning virulence factors involved in the virulence of V. europaeus are very scarce despite its global significance for aquaculture. Zinc-metalloproteases have been described as a major virulence factor in some Vibrio spp., although their contribution and role in the virulence of V. europaeus is not clear. To address this, we have studied an extracellular zinc-metalloprotease (VemA) encoded by V. europaeus, which was identified as a vibriolysin, highly conserved in this species and homologous in other pathogenic and non-pathogenic species. Virulence challenge experiments demonstrated that infection processes were faster when Manila clam larvae and juveniles were infected with the wildtype rather than with a mutant defective in the vemA gene (&Delta;vemA). V. europaeus was able to resist the bactericidal action of mucus and displayed a chemotaxis ability favoured by VemA to colonize the body mucus of clams and form a biofilm. The overall results suggest that VemA, although it is not a major virulence factor, plays a role in the colonization of the Manila clam mucus, and thus boosts the infection process as we observed in virulence challenge experiments