Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities

Two episodes of mortality of cultured carpet shell clams (Ruditapes decussatus) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain. Vibrio alginolyticus was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events. Vibrio splendidus biovar II, in addition to V. alginolyticus, was isolated as a result of a mixed Vibrio infection from moribund clam larvae obtained from the second mortality event. The larval mortality rates for these events were 62 and 73%, respectively. Mortality was also detected in spat. To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality. The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene. In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion. The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams. V. alginolyticus TA15 and V. splendidus biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms. The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100 degrees C for 10 min). Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15. ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria.     

Incidence of Vibrio alginolyticus and Bacteria of Sanitary Significance in the Bering Sea

The incidence of bacteria of sanitary significance and isolation of Vibrio alginolyticus during a subtidal clam assessment study in the Bering Sea are described. Vibrio parahaemolyticus was not detected in clams, sediment, or seawater.     

Isolation and characterization of a virulent Vibrio sp. bacterium from clams (Meretrix meretrix) with mass mortality

MM5 was a bacterial strain isolated from moribund clam (Meretrix meretrix) collected from a farm with mass mortality outbreak. Primary genotypic and phenotypic identification including 16S rDNA sequence analysis, multilocus sequence analysis (MLSA) of four housekeeping genes (gapA, ftsZ, mreB and topA) and biochemical tests suggested that strain MM5 was a Vibrio species closest to but different from Vibrio furnissii. Our previous study indicated that MM5 could induce a high mortality of M. meretrix (Yue et al., 2010). Quantitative challenge test was performed in this study to further evaluate the pathogenic potential of MM5, which showed that at 84 h post-inoculation, the cumulative mortalities of the MM5-injected group were significantly higher than those of control groups (P < 0.05). Cytopathological and histopathological features of the clam infected by MM5 were carried out by transmission electron microscopy (TEM) and Hematoxylin and Eosin (H&E) staining, respectively. Cytopathologically, foci of MM5 were found in hepatocytes of the clam infected by MM5. In addition, cytopathological lesion was detected in foot of infected clam. Histopathologically, MM5 was detected in different tissues of infected clam, including hepatopancreas, mantle and gill. Challenge test combined with pathological features indicated that MM5 was virulent to M. meretrix.     

Influence of beta-glucans on the immune responses of carpet shell clam (Ruditapes decussatus) and Mediterranean mussel (Mytilus galloprovincialis)

The effects of beta-glucans on several immune functions of carpet shell clam (Ruditapes decussatus) and Mediterranean mussel (Mytilus galloprovincialis) hemocytes were determined. Nitric oxide (NO) production increased significantly in beta-glucan treated mussels and clams. In mussels, beta-glucans increased by themselves the release of free oxygen radicals and also were able to enhance the phorbol 12-myristate 13-acetate (PMA) mediated effect on this hemocyte activity. However, high doses of beta-glucans when combined with zymosan decreased this respiratory burst. In clams, hemolymph treated with several doses of beta-glucans limited the growth of the three bacteria, Vibrio algynolyticus (strain TA15), Vibrio splendidus (strain TA2) and Escherichia coli (strain ATCC 13706). This modulation on the antibacterial activity, however, was not observed when mussel hemolymph was incubated with beta-glucans. These results suggest that the immune responses of these animals can be up and down modulated by external stimuli and, although clams and mussels are both relatively closely related species, their behaviour concerning immune responses can be different.     

Stress and Stress-Induced Neuroendocrine Changes Increase the Susceptibility of Juvenile Oysters (Crassostrea gigas) to Vibrio splendidus

Oysters are permanently exposed to various microbes, and their defense system is continuously solicited to prevent accumulation of invading and pathogenic organisms. Therefore, impairment of the animal's defense system usually results in mass mortalities in cultured oyster stocks or increased bacterial loads in food products intended for human consumption. In the present study, experiments were conducted to examine the effects of stress on the juvenile oyster's resistance to the oyster pathogen Vibrio splendidus. Oysters (Crassostrea gigas) were challenged with a low dose of a pathogenic V. splendidus strain and subjected to a mechanical stress 3 days later. Both mortality and V. splendidus loads increased in stressed oysters, whereas they remained low in unstressed animals. Injection of noradrenaline or adrenocorticotropic hormone, two key components of the oyster neuroendocrine stress response system, also caused higher mortality and increased accumulation of V. splendidus in challenged oysters. These results suggest that the physiological changes imposed by stress, or stress hormones, influenced host-pathogen interactions in oysters and increased juvenile C. gigas vulnerability to Vibrio splendidus.     

Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products of Vibrio splendidus

Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors of Vibrio species; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogen Vibrio splendidus LGP32 and show that toxicity is correlated to the presence of a metalloprotease and its corresponding vsm gene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and a vsm deletion mutant (Δvsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the Δvsm mutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the Δvsm mutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor of Bacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs of V. splendidus.     

Dynamics of Snake-like Swarming Behavior of Vibrio alginolyticus

Swarming represents a special case of bacterial behavior where motile bacteria migrate rapidly and collectively on surfaces. Swarming and swimming motility of bacteria has been studied well for rigid, self-propelled rods. In this study we report a strain of Vibrio alginolyticus, a species that exhibits similar collective motility but a fundamentally different cell morphology with highly flexible snake-like swarming cells. Investigating swarming dynamics requires high-resolution imaging of single cells with coverage over a large area: thousands of square microns. Researchers previously have employed various methods of motion analysis but largely for rod-like bacteria. We employ temporal variance analysis of a short time-lapse microscopic image series to capture the motion dynamics of swarming Vibrio alginolyticus at cellular resolution over hundreds of microns. Temporal variance is a simple and broadly applicable method for analyzing bacterial swarming behavior in two and three dimensions with both high-resolution and wide-spatial coverage. This study provides detailed insights into the swarming architecture and dynamics of Vibrio alginolyticus isolate B522 on carrageenan agar that may lay the foundation for swarming studies of snake-like, nonrod-shaped motile cell types.     

Influence of Alginate on Attachment of Vibrio spp. to Stainless Steel Surfaces in Seawater

The influence of alginate on the attachment of Vibrio alginolyticus and Vibrio pelagius biovar II to stainless steel was investigated. When the bacteria were in stationary phase, alginate decreased the number of attached bacteria in the case of each Vibrio sp. In contrast, when V. pelagius biovar II was grown on alginate and harvested in log phase, attachment was increased. This effect may be due to nutrient availability at the surface or to receptors on the bacterial surface which interact with alginate adsorbed to the metal.     

Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

A Large-Scale Epidemiological Study to Identify Bacteria Pathogenic to Pacific Oyster Crassostrea gigas and Correlation Between Virulence and Metalloprotease-like Activity

A 4-year bacteriological survey (2003-2007) of four molluscs cultivated in France and faced with mortality episodes was performed by the French shellfish pathology network. The more abundant bacteria isolated during 92 mortality episodes, occurring mainly in Pacific oyster Crassostrea gigas, were identified by genotyping methods. It allowed us both to confirm the representativeness of Vibrio splendidus and Vibrio aestuarianus bacterial strains and to identify both a large number of Vibrio harveyi-related strains mainly detected during 2007 oyster mortality outbreaks and to a lesser extent bacterial strains identified as Shewanella colwelliana. Because metalloprotease has been reported to constitute a virulence factor in a few Vibrio strains pathogenic for C. gigas, several bacterial strains isolated in this study were screened to evaluate their pathogenicity in C. gigas spat by experimental infection and their ability to produce metalloprotease-like activity in the culture supernatant fluids. A high level (84%) of concordant results between azocaseinase activities and virulence of strains was obtained in this study. Because bacterial metalloprotease activities appeared as a common feature of pathogenic bacteria strains associated with mortality events of C. gigas reared in France, this phenotypic test could be useful for the evaluation of virulence in bacterial strains associated with such mortality episodes.     

Isolation of <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio splendidus</Emphasis> from captive-bred seahorses with disease symptoms

Vibrio species isolated from diseased seahorses were characterized by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S ribosomal RNA gene sequence analysis. The results demonstrated that Vibrio alginolyticus and Vibrio splendidus were predominant in the lesions of these seahorses. To our knowledge, this is the first time that these bacterial species have been associated with disease symptoms in captive-bred seahorses.     

Identification of Vibrio splendidus as a Member of the Planktonic Luminous Bacteria from the Persian Gulf and Kuwait Region with luxA Probes

Hybridization probes specific for the luxA genes of four groups of luminous bacteria were used to screen luminous isolates obtained from the Persian Gulf, near Al Khiran, Kuwait Nine of these isolates were identified as Vibrio harveyi, a commonly encountered planktonic isolate, while three others showed no hybridization to any of the four probes (V. harveyi, Vibrio fischeri, Photobacterium phosphoreum, or Photobacterium leiognathi) under high-stringency conditions. Polymerase chain reaction amplification was used to prepare a luxA probe against one of these isolates, K-1, and this probe was screened under high-stringency conditions against a collection of DNAs from luminous bacteria; it was found to hybridize specifically to the DNA of the species Vibrio splendidus. A probe prepared against the type strain of V. splendidus (ATCC 33369) was tested against the collection of luminous bacterial DNA preparations and against the Kuwait isolates and was found to hybridize only against the type strain and the three unidentified Kuwait isolates. Extensive taxonomic analysis by standard methods confirmed the identification of the 13 isolates.     

Brown ring disease in clams

Brown Ring Disease (BRD) in the manila clam, Ruditapes philippinarum is a bacterial disease which perturbs the calcification process. This disease occurs in wild and reared clam populations of France, Spain, and occasionally Italy, but has never been reported in Japan from where this species originates. BRD has also been detected in another clam species (Ruditapes decussatus) from France and Spain. Following Koch's classical postulates for pathogen identification, a bacterial strain was identified as the agent responsible for this disease. This bacterium was characterized as belonging to the genus Vibrio and has been termed Vibrio P1 or VP1. The macroscopic sign which characterises this disease is a conchiolin deposit adhering to the inner surface of the shell. To recover from the disease, the clams regenerate their shells by covering the deposit with calcified secretions much like the nacrezation process. Disease and recovery stages have been established for use in epidemiological and experimental studies. Tissue lesions are not systematically observed in diseased clams. Alterations of the digestive gland and the mantle are uniquely detected in the more severe stages of the disease. In all diseased clams, however, the periostracal lamina shows alterations: It is invaded by cell debris and bacteria, and areas of darker melanin-like pigmentation are observed. This disorganized periostracal lamina is not a good substrate for the biomineralization process and, therefore, accumulates forming a deposit on the inner face of the shell. Host-pathogen interactions have been focused at two levels: the internal defense system and the external embedding mechanism. Changes in the hemolymph were observed after challenge with VP1. In the early stages of the disease, an elevation in both circulating hemocytes and peptidase levels appeared; however, a subsequent depression of these parameters was recorded as the disease progressed. An external defense process such as bacterial embedding within periostracal lamina layers is described. Melanization of the periostracal lamina has also been suggested to be an active defense response against bacteria. These new defense processes in Brown Ring Diseased clams could complement the classical hemolymphatic system and, therefore, may act to limit bacterial proliferation within the tissues.     

Labelling strategy and membrane characterization of marine bacteria Vibrio splendidus by in vivo 2H NMR

Vibrio splendidus is a marine bacterium often considered as a threat in aquaculture hatcheries where it is responsible for mass mortality events, notably of bivalves' larvae. This bacterium is highly adapted to dynamic salty ecosystems where it has become an opportunistic and resistant species. To characterize their membranes as a first and necessary step toward studying bacterial interactions with diverse molecules, we established a labelling protocol for in vivo ²H solid-state nuclear magnetic resonance (SS-NMR) analysis of V. splendidus. ²H SS-NMR is a useful tool to study the organization and dynamics of phospholipids at the molecular level, and its application to intact bacteria is further advantageous as it allows probing acyl chains in their natural environment and study membrane interactions. In this study, we showed that V. splendidus can be labelled using deuterated palmitic acid, and demonstrated the importance of surfactant choice in the labelling protocol. Moreover, we assessed the impact of lipid deuteration on the general fitness of the bacteria, as well as the saturated-to-unsaturated fatty acid chains ratio and its impact on the membrane properties. We further characterize the evolution of V. splendidus membrane fluidity during different growth stages and relate it to fatty acid chain composition. Our results show larger membrane fluidity during the stationary growth phase compared to the exponential growth phase under labelling conditions - an information to take into account for future in vivo SS-NMR studies. Our lipid deuteration protocol optimized for V. splendidus is likely applicable other microorganisms for in vivo NMR studies.     

Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector

Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the P_BAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.     

Polymorphism of the multiple hemoglobins in blood clam Tegillarca granosa and its association with disease resistance to Vibrio parahaemolyticus

Hemoglobin (Hb) is the major protein component of erythrocytes in animals with red blood, but it can serve additional functions beyond the transport of oxygen. In this study, we identified polymorphism in the blood clam Tegillarca granosa Hb (Tg-Hb) genes and investigated the association of this polymorphism with resistance/susceptibility to Vibrio parahaemolyticus. Analysis of the 540 sequences revealed 28 SNPs in the coding region of three Tg-Hbs, corresponding to about one SNP per 48 bp. Three SNPS: HbIIA-E2-146, HbIIB-E2-23, HbIIB-E2-121 showed a significant association with resistance/susceptibility to V. parahaemolyticus (P < 0.05). To further demonstrate that three significant SNPs of Tg-Hbs is associated with resistance of clams to V. parahaemolyticus, SNPs were genotyped in V. parahaemolyticus resistant strain clams and the wild base population from which this strain was derived. The results indicated that the nonsynonymous mutation T allele at HbIIA-E2-146 and A allele at HbIIB-E2-23 are associated with V. parahaemolyticus resistance in the blood clam, and its association with disease resistance may be due to its cause changes in amino acid sequences to a functional polymorphism. Together with previous bacterial challenge study, these results provides direct evidence that variation at HbIIA-E2-146 and HbIIB-E2-23 are associated with disease resistance in the blood clam, and these two polymorphic loci could be potential gene markers for the future molecular selection of strains that are resistant to diseases caused by V. parahaemolyticus.     

Prevalence and antibiotic resistance patterns of Vibrio parahaemolyticus isolated from different types of seafood in Selangor,Malaysia

Vibrio parahaemolyticus is a foodborne bacterial pathogen that may cause gastroenteritis in humans through the consumption of seafood contaminated with this microorganism. The emergence of antimicrobial and multidrug-resistant bacteria is another serious public health threat worldwide. In this study, the prevalence and antibiotic susceptibility test of V. parahaemolyticus in blood clams, shrimps, surf clams, and squids were determined. The overall prevalence of V. parahaemolyticus in seafood was 85.71% (120/140), consisting of 91.43% (32/35) in blood clam, 88.57% (31/35) in shrimps, 82.86% (29/35) in surf clams, and 80% (28/35) in squids. The majority of V. parahaemolyticus isolates from the seafood samples were found to be susceptible to most antibiotics except ampicillin, cefazolin, and penicillin. The MAR indices of V. parahaemolyticus isolates ranged from 0.04 to 0.71 and about 90.83% of isolates were found resistant to more than one antibiotic. The high prevalence of V. parahaemolyticus in seafood and multidrug-resistant isolates detected in this study could pose a potential risk to human health and hence appropriate control methods should be in place to minimize the potential contamination and prevent the emergence of antibiotic resistance.     

Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: Changes in cell structure, numbers and adherence

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24 h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p < 0.01) than with 7SHRW. The cell adherence was markedly diminished (p < 0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p < 0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.     

Impact of Brown Ring Disease on the energy budget of the Manila clam Ruditapes philippinarum

Brown Ring Disease (BRD) is a bacterial disease caused by the pathogen, Vibrio tapetis. The disease induces formation of a brown deposit on inner shell of the Manila clam, Ruditapes philippinarum. Development of this disease is correlated with a decrease in the condition index of infected clams. Experiments were conduced in order to assess the effect of the development of BRD on two parameters affecting the energy balance of the clams: the clearance and the respiration rates. Experiments were performed in a physiological measurement system that allowed simultaneous measures of clearance and respiration rates. During both acclimation and measurements clams were fed with cultured T-iso and temperature was close to seasonal field temperature (10°C). Our results showed that severely diseased clams (conchiolin deposit stage, CDS ≥ 4) are subject to weight loss in comparison to uninfected ones, indicating that BRD induces a disequilibrium in the energy balance. We demonstrated a reduction of the clearance rate of severely diseased clams which led to a decrease in energy acquisition. Respiration rate showed a significant decrease with BRD symptoms, but evidence in the literature allowed us to hypothesize that energy mobilised for an immune response and lesion repair increases overall organism maintenance costs. Both factors should thus contribute to the degradation of the energy balance of diseased clams. Because effects of BRD on naturally infected clams only appears significant for CDS ≥ 4, when brown ring assumes a significant place on the inner shell, we consider that the Manila clam is tolerant of low disease levels.     

Investigating the action of the microalgal pigment marennine on Vibrio splendidus by in vivo 2H and 31P solid-state NMR

This work investigates the potential probiotic effect of marennine - a natural pigment produced by the diatom Haslea ostrearia - on Vibrio splendidus. These marine bacteria are often considered a threat for aquaculture; therefore, chemical antibiotics can be required to reduce bacterial outbreaks. In vivo ²H solid-state NMR was used to probe the effects of marennine on the bacterial membrane in the exponential and stationary phases. Comparisons were made with polymyxin B (PxB) - an antibiotic used in aquaculture and known to interact with Gram(?) bacteria membranes. We also investigated the effect of marennine using ³¹P solid-state NMR on model membranes. Our results show that marennine has little effect on phospholipid headgroups dynamics, but reduces the acyl chain fluidity. Our data suggest that the two antimicrobial agents perturb V. splendidus membranes through different mechanisms. While PxB would alter the bacterial outer and inner membranes, marennine would act through a membrane stiffening mechanism, without affecting the bilayer integrity. Our study proposes this microalgal pigment, which is harmless for humans, as a potential treatment against vibriosis.