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
. 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
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 discussed.
Sequencing of the full coding region of both genomic segments of seven betanodavirus strains isolated from different farmed species in Spain and Portugal revealed that six were reassortants, ...exhibiting a red-spotted grouper nervous necrosis virus (RGNNV)-type RNA1 and a striped jack nervous necrosis virus (SJNNV)-type RNA2. Analysis of sequences of reassortant strains at both the genomic and protein levels revealed the existence of differences compared with type strains of both genotypes. These differences were greater in the polymerase sequence, which is remarkable because viral structural proteins generally diverge more rapidly than non-structural proteins. Changes in two amino acids observed in the SJNNV capsid protein might be involved in the colonization of new host species by these reassortant strains. In addition, a more extensive phylogenetic analysis, including partial sequences of both RNA segments of 16 other Iberian nodaviruses, confirmed the existence of reassortment between RGNNV and SJNNV.
The characterization of antibiotic-resistant vibrios isolated from shellfish aquaculture is necessary to elucidate the potential transfer of resistance and to establish effective strategies against ...vibriosis. With this aim, we analyzed a collection of bacterial isolates obtained from 15 failed hatchery larval cultures that, for the most part, had been treated experimentally with chloramphenicol to prevent vibriosis. Isolates were obtained during a 2-year study from experimental cultures of five different clam species. Among a total of 121 Vibrio isolates studied, 28 were found to be chloramphenicol resistant, suggesting that the shellfish hatchery had been using a sublethal concentration of the antibiotic. Interestingly, chloramphenicol-resistant vibrios showed also resistance to tetracycline and amoxicillin (group A; n=19) or to streptomycin (group B; n=9). Chloramphenicol-resistant vibrios were subjected to a PCR amplification and DNA sequencing of the chloramphenicol acetyltransferase genes (cat), and the same approach was followed to study the tetracycline resistance markers (tet). 16S ribosomal RNA (rRNA) gene sequencing revealed that chloramphenicol-resistant vibrios pertained mostly to the Splendidus clade. Conjugation assays demonstrated that various R-plasmids which harbored the cat II/tet(D) genes and cat III gene in groups A and B respectively, were transferred to E. coli and bivalve pathogenic vibrios. Most interestingly, transconjugants exhibited the antibiotic resistance patterns of the donors, despite having been selected only on the basis of chloramphenicol resistance. This is the first report carried out in a bivalve hatchery elucidating the persistence of resistant vibrios, the mechanisms of antibiotic resistance, and the transfer of different R-plasmids.
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
. 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
based on its phenotypic properties, the multilocus sequence analysis (MLSA) of eight housekeeping genes (
Z,
A,
B,
B,
H,
A,
A, and
A) and different
genome-to-genome comparisons. When triplicate cultures of healthy 10 days old scallop larvae were challenged with 1 × 10
colony forming units (CFU) mL
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 (LD
) of VPAP30 strain at 24 and 48 h was 1.3 × 10
and 1.2 × 10
CFU mL
, 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 (
< 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.
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
out of Europe and extends the host range of this species, having demonstrated its pathogenic activity on the Chilean scallop larvae (
). These results supporting the pathogenic potential of
to kill the larvae of a broad range of bivalve species reared in hatcheries located in the Atlantic and the Pacific coasts.
Two
strains (VPAP36 and VPAP40) were isolated from moribund-settled larvae of the Chilean scallop
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
using phenotypic characterization and whole genome sequence analysis. Both strains exhibited the phenotypic properties associated with virulence, gelatin hydrolysis and β-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
VPAP36 and VPAP40 strains exhibited significant (
< 0.05) differences in their larval lethal activity, producing, after 48 h, larval mortalities of 65.51 ± 4.40% and 28.56 ± 5.35%, respectively. Otherwise, the cell-free extracellular products of the VPAP36 and VPAP40 strains produced larval mortalities of 20.86 ± 2.40% and 18.37 ± 2.40%, respectively, after 48 h of exposure. This study reports for the first time the isolation of
from the massive larval mortalities of the farmed scallop (
) in Chile, and demonstrates the pathogenic activity of
towards the Chilean scallop, the second most important species for Chilean mariculture.
Vibrio neptunius
is an inhabitant of mollusc microbiota and an opportunistic pathogen causing disease outbreaks in marine bivalve mollusc species including oysters and clams. Virulence of mollusc ...pathogenic vibrios is mainly associated with the production of extracellular products. However, siderophore production is a common feature in pathogenic marine bacteria but its role in fitness and virulence of mollusc pathogens remains unknown. We previously found that
V. neptunius
produces amphibactin, one of the most abundant siderophores in marine microbes. In this work, synthesis of the siderophore piscibactin was identified as the second siderophore produced by
V. neptunius
. Single and double mutants in biosynthetic genes of each siderophore system, piscibactin and amphibactin, were constructed in
V. neptunius
and their role in growth ability and virulence was characterized. Although the High Pathogenicity Island encoding piscibactin is a major virulence factor in vibrios pathogenic for fish, the
V. neptunius
wild type did not cause mortality in turbot. The results showed that amphibactin contributes more than piscibactin to bacterial fitness
in vitro
. However, infection challenges showed that each siderophore system contributes equally to virulence for molluscs. The
V. neptunius
strain unable to produce any siderophore was severely impaired to cause vibriosis in clams. Although the inactivation of one of the two siderophore systems (either amphibactin or piscibactin) significantly reduced virulence compared to the wild type strain, the ability to produce both siderophores simultaneously maximised the degree of virulence. Evaluation of the gene expression pattern of each siderophore system showed that they are simultaneously expressed when
V. neptunius
is cultivated under low iron availability
in vitro
and
ex vivo
. Finally, the analysis of the distribution of siderophore systems in genomes of
Vibrio
spp. pathogenic for molluscs showed that the gene clusters encoding amphibactin and piscibactin are widespread in the Coralliilyticus clade. Thus, siderophore production would constitute a key virulence factor for bivalve molluscs pathogenic vibrios.
is an important pathogen of bivalve mollusks worldwide. Several metalloproteases have been described as virulence factors in species of
that are pathogenic to bivalves, but little is known about the ...contribution of these potential virulence factors to
pathogenesis. In silico analysis of the genome of
strain PP-145.98 led to the identification of two hitherto uncharacterized chromosomal loci encoding a probable vibriolysin-like metalloprotease and a putative collagenase, which were designated VnpA and ColA, respectively. Single defective mutants of each gene were obtained in
PP-145.98, and the phospholipase, esterase and collagenase activities were studied and compared with those of the wild-type strain. The results showed that the single inactivation of
resulted in a 3-fold reduction in phospholipase/esterase activity. Inactivation of
reduced the collagenase activity by 50%. Finally, infection challenges performed in oyster larvae showed that Δ
and Δ
-single mutant strains of
-are between 2-3-fold less virulent than the wild-type strain. Thus, the present work demonstrates that the production of both VnpA and ColA is required for the full virulence of the bivalve pathogen
.
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
are ...very scarce despite its global significance for aquaculture. Zinc-metalloproteases have been described as a major virulence factor in some
spp., although their contribution and role in the virulence of
is not clear. To address this, we have studied an extracellular zinc-metalloprotease (VemA) encoded by
, 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
gene (Δ
).
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.
Broodstock conditioning in hatcheries is the step previous to spawning and its optimization may be a key to the success of larval cultures. Cleaning and brushing of the broodstock and the utilization ...of antibiotics to reduce their vibrios load are used regularly as routine prophylactic measures prior to spawning induction. The development of protocols to reduce these bacteria using cheap and harmless techniques is of utmost importance for commercial bivalve production in hatcheries. With this aim, we have evaluated initially different conditionings (A–D) during short periods (a total of four weeks): first two weeks under gradient temperature (increasing +0.3°Cday−1 from 14.5°C to 20°C), without (A) or with phytoplankton (B), and constant temperature (20°C) without (C) or with phytoplankton (D). Afterwards, all conditionings were kept at 20°C and fed for two more weeks. Furthermore, broodstock optimal feeding time was re-evaluated during a second trial series. In all conditionings, bacterial loads were determined in terms of marine heterotrophic bacteria (MHB) and presumptive vibrios (PV). Broodstock under the optimal short period of conditioning (conditioning C) obtained the best gonadal development and a significant reduction in PV load at a lower expense. A total of 61 PV were isolated from all conditionings and identified by sequencing the 16S rRNA gene. Splendidus clade was dominant in the samples coming from natural beds. Diversity of vibrios changed throughout the conditionings in the hatchery favoured by exogenous factors whose effect was mainly observed at the end of the trials: Splendidus clade was also dominant in the broodstock conditioned at gradient temperature and Mediterranei and Harveyi clades were prevalent at constant temperature. Moreover, the percentage of transformation to D-larvae was estimated and the vertical transmission of vibrios from broodstock to eggs and D-larvae was suggested. Implementation of conditioning C reduces considerably the Vibrio load of clams without using antibiotics, and thus it represents a novel, cheap, environmental friendly and harmless methodology that can be easily transferred to commercial hatchery.
Routine prophylactic measures for bivalve broodstock arriving hatchery facilities include its cleaning/brushing and the frequent use of antibiotics to reduce its bacterial load, particularly Vibrio, as step previous to spawning induction. The depuration protocol proposed reduces significantly Vibrio load without using antibiotics and represents a novel, cheap, environmental friendly and harmless methodology that can be easily transferred to commercial hatcheries.
•First study in which the Vibrio load of R. decussatus broodstock was evaluated modifying only temperature and food.•Broodstock conditioned for four weeks at 20ºC and fed only during the last two weeks showed the best results.•Depuration protocol used during conditioning represents a novel, cheap, and environmental friendly methodology.•Vibrio load associated to broodstock must be reduced rapidly to guarantee larval culture success in hatcheries.
AIMS: Outbreaks of disease caused by some Vibrio species represent the main production bottleneck in shellfish hatcheries. Although the phytoplankton used as food is one of the main sources of ...bacteria, studies of the associated bacterial populations, specifically vibrios, are scarce. The aim of the study was the microbiological monitoring of the microalgae as the first step in assessing the risk disease for bivalve cultures. METHODS AND RESULTS: Two phytoplankton production systems were sampled weekly throughout 1‐year period in a bivalve hatchery. Quantitative analysis revealed high levels of marine heterotrophic bacteria in both systems throughout the study. Presumptive vibrios were detected occasionally and at low concentrations. In most of the cases, they belonged to the Splendidus and Harveyi clades. CONCLUSIONS: The early detection of vibrios in the microalgae may be the key for a successful bivalve culture. Their abundance and diversity were affected by factors related to the hatchery environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This work represents the first long study where the presence of vibrios was evaluated rigorously in phytoplankton production systems and provides a suitable microbiological protocol to control and guarantee the quality of the algal cultures to avoid the risk of transferring potential pathogens to shellfish larvae and/or broodstock.
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