Bivalve Pathogen Research Articles

The Vibriolysin-Like Protease VnpA and the Collagenase ColA Are Required for Full Virulence of the Bivalve Mollusks Pathogen Vibrio neptunius.

Vibrio neptunius is an important pathogen of bivalve mollusks worldwide. Several metalloproteases have been described as virulence factors in species of Vibrio that are pathogenic to bivalves, but little is known about the contribution of these potential virulence factors to Vibrio neptunius pathogenesis. In silico analysis of the genome of V. neptunius 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 V. neptunius 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 vnpA resulted in a 3-fold reduction in phospholipase/esterase activity. Inactivation of colA reduced the collagenase activity by 50%. Finally, infection challenges performed in oyster larvae showed that ΔvnpA and ΔcolA—single mutant strains of V. neptunius—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 V. neptunius.

Immunomodulatory and Antiviral Effects of Macroalgae Sulphated Polysaccharides: Case Studies Extend Knowledge on Their Importance in Enhancing Shellfish Health, and the Control of a Global Viral Pathogen Ostreid Herpesvirus-1 microVar

Macroalgae are the primary source of non-animal sulphated polysaccharides (SPs) in the marine environment with fucoidans derived from brown algae (Phaeophyta) and carrageenans from red algae (Rhodophyta). Much research has been carried out on SP effects on Asian shrimp species (genera Penaeus and Metapenaeus) but their effect on commercially important bivalve mollusc species is limited and in Pacific oyster Crassostrea gigas is unknown. Knowledge of their impact on bivalve pathogens and Palaemon shrimp is unknown. The objectives of this study were to assess the effects of Fucus vesiculosus (Phaeophyta), Mastocarpus stellatus (Rhodophyta) and algal derivatives (fucoidan and κ-carrageenan) on C. gigas performance, and on ostreid herpesvirus-1 microvar (OsHV-1 μVar) and bacteria Vibrio spp. development. Both pathogens have been associated with significant oyster mortalities and economic losses globally. The effects of sulphated galactan from Gracilaria fisheri (Rhodophyta) on European common prawn Palaemon serratus, an important fishery species, was also assessed. Findings indicate a rapid and prolonged increase in total blood cell count, lysozyme (enzyme that destroys pathogens), and a difference in the ratio of blood cell types in treated individuals compared to their control counterparts. A significantly lower OsHV-1 μVar prevalence was observed in treated oysters and κ-carrageenan was found to suppress viral replication (loads), while OsHV-1 μVar was not detected in the fucoidan treated oysters from Day 8 of the 26-day trial. No antibacterial effect was observed however, the oysters did not succumb to vibriosis. These findings contribute further knowledge to macroalgae sulphated polysaccharide biotherapeutic properties, their twofold effect on animal health and viral suppression.

Solar UV radiation modulates animal health and pathogen prevalence in coastal habitats—knowledge gaps and implications for bivalve aquaculture

Ultraviolet radiation (UVR) is an important environmental factor that can have an impact directly, or indirectly, on the health of organisms. UVR also has the potential to inactivate pathogens in surface waters. As a result, UVR can alter host-pathogen relationships. Bivalve species are threatened by various pathogens. Here, we assessed the impacts of UVR on (i) bivalves, (ii) bivalve pathogens and (iii) the bivalve host-pathogen relationship. UVR consistently impedes pathogens. However, the effect of UVR on marine animals is variable, with both positive and negative impacts. The limited available data allude to the potential to exploit natural UVR for disease management in aquaculture, but also highlight a striking knowledge gap and uncertainty relating to climate change.

Production of specific pathogen free larvae from genetically characterized populations of Anadara tuberculosa (Bivalvia), for stock enhancement and aquaculture in the Peru Northeast Biosphere Reserve

The pustulose ark, Anadara tuberculosa , is considered as an emblematic species of the east Pacific mangrove ecosystem. The decline of its populations is of increasing preoccupation, in particular in Peru. The use of hatchery-produced larvae for stock enhancement or aquaculture is attractive but raises strong criticisms from an ecological point of view, as it could conduce to loss of genetic diversity in wild populations and favor the disease spreading from hatcheries to the environment. In this works, spat production through traditional aquaculture/biotechnology technologies was associated with pathogen molecular diagnostics and with genetic diversity estimation to produce specific pathogen-free (SPF) larvae from genetically characterized populations. All PCR and nested-PCR diagnostics of common bivalve pathogens, such as Bonamia , Martelia , Perkinsus and OsHV1, led to negative results for healthy or moribund adults and spat samples. Microbiological and molecular analyses realized during mortalities led to identifying a pathogenic Pseudomonas strain present on culture tank walls, in moribund spats and adults´ blood. Population genetic variation was assessed by mitochondrial COI gene sequences which showed 36.7% haplotype diversity. This study with hatchery production, pathology and genetic components for an overexploited native bivalve represents an original model for successful mollusk stock enhancement or/and innovative aquaculture programs. Normal 0 21 false false false ES-EC X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:ES-MX; mso-fareast-language:ES-MX;}

Autophagic processes in Mytilus galloprovincialis hemocytes: Effects of Vibrio tapetis

Autophagy is a highly conserved and regulated catabolic process involved in maintaining cell homeostasis in response to different stressors. The autophagic machinery is also used as an innate immune mechanism against microbial infection. In invertebrates, that lack acquired immunity, autophagy may thus play a key role in the protection against potential pathogens. In aquatic molluscs, evidence has been provided for induction of autophagy by starvation and different environmental stressors; however, no information is available on autophagic pathways in the immune cells, the hemocytes.In this work, the autophagic processes were investigated in the hemocytes of the marine bivalve, the mussel Mytilus galloprovincialis. The effects of classical inducers/inhibitors of mammalian autophagy were first tested. Rapamycin induced a decrease in lysosomal membrane stability-LMS that was prevented by the autophagy inhibitor Wortmannin. Increased MDC fluorescence and expression of LC3-II were also observed. Moreover, responses to in vitro challenge with the bivalve pathogen Vibrio tapetis were evaluated. Mussel hemocytes were unable to activate the immune response towards V. tapetis; however, bacterial challenge induced a moderate decrease in LMS, corresponding to lysosomal activation but no cytotoxicity; the effect was prevented by Wortmannin. TEM observations showed that V. tapetis resulted in rapid formation of autophagosomes and autolysosomes. Accordingly, increased LC3-II expression, decreased levels of phosphorylated mTor and of p62 were observed. The results represent the first evidence for autophagic processes in bivalve hemocytes in response to bacterial challenge, and underline the protective role of autophagy towards potential pathogenic vibrios.

Reclassification of the larval pathogen for marine bivalves Vibrio tubiashii subsp. europaeus as Vibrio europaeus sp. nov.

The Orientalis clade has a relevant significance for bivalve aquaculture since it includes the pathogens Vibrio bivalvicida, Vibrio tubiashii subsp. tubiashii and Vibrio tubiashii subsp. europaeus. However, the previous taxonomic description of the subspecies of V. tubiashii shows some incongruities that should be emended. In the genomic age, the comparison between genome assemblies is the key to clarify the taxonomic position of both subspecies. With this purpose, we have tested the ability of multilocus sequence analysis based on eight housekeeping gene sequences (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA and topA), different in silico genome-to-genome comparisons, chemotaxonomic features and phenotypic traits to reclassify the subspecies V. tubiashii subsp. europaeus within the Orientalis clade. This polyphasic approach clearly demonstrated that this subspecies is phylogenetically and phenotypically distinct from V. tubiashii and should be elevated to the rank of species as Vibrio europaeus sp. nov. This reclassification allows us to update the Orientalis clade (V. bivalvicida,V. brasiliensis, V. crosai, V. hepatarius, V. orientalis, V. sinaloensis, V. tubiashii and V. europaeus sp. nov.) and reconstruct a better phylogeny of the genus Vibrio. An emended description of V. tubiashii is provided. Finally, the proposed novel species is represented by emergent bivalve pathogens [type strain PP-638T (=CECT 8136T=DSM 27349T), PP2-843 and 07/118 T2] responsible for high mortalities in Spanish and French hatcheries.

Draft Genome Sequence of the Emerging Bivalve Pathogen Vibrio tubiashii subsp. europaeus.

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.

Killing of Vibrio cholerae and Escherichia coli Strains Carrying D-mannose-sensitive Ligands by Mytilus Hemocytes is Promoted by a Multifunctional Hemolymph Serum Protein.

In aquatic environments, bivalve mollusks represent an important ecological niche for microorganisms. Persistence of bacteria in bivalve tissues partly depends on their capacity to survive the bactericidal activity of the hemolymph due to both cellular (hemocyes) and soluble serum factors (e.g., enzymes, lectins, opsonins). The extrapallial protein (EP) present in serum of Mytilus galloprovincialis (MgEP) has been recently shown to work as an opsonin promoting D-mannose sensitive (MS) interactions of the bivalve pathogen Vibrio aestuarianus 01/032 strain with the hemocytes. In this study, the role of MgEP in adhesion and killing of other bacteria carrying MS sensitive ligands was investigated. MgEP enhanced adhesion to and killing by hemocytes of Vibrio cholerae ElTor N16961, expressing the MS hemagglutin (MSHA), as well as of Escherichia coli MG1655, carrying type 1 fimbriae. These results further support the recent finding that the multifunctional MgEP also acts as an opsonin involved in mussel defense towards bacteria carrying MS ligands. In addition, these results contribute to elucidate the ecology of bacterial pathogens that can be transmitted to humans via shellfish consumption.

Complete genome sequence of Ostreid herpesvirus-1 associated with mortalities of Scapharca broughtonii broodstocks.

BackgroundOstreid herpesvirus-1 (OsHV-1) is the major bivalve pathogen associated with severe mortality events in a wide host range. In the early summer of 2012 and 2013, mass mortalities of blood clam (Scapharca broughtonii) broodstocks associated with a newly described variant of OsHV-1 (OsHV-1-SB) were reported.MethodsIn this study, the complete genome sequence of the newly described variant was determined through the primer walking approach, and compared with those of the other two OsHV-1 variants.ResultsOsHV-1-SB genome was found to contain 199, 354 bp nucleotides with 38.5 % G/C content, which is highly similar to those of acute viral necrosis virus (AVNV) and OsHV-1 reference type. A total of 123 open reading frames (ORFs) putatively encoding functional proteins were identified; eight of which were duplicated in the major repeat elements of the genome. The genomic organization of OsHV-1-SB could be represented as TRL-UL-IRL-IRS-US-TRS, which is different from that of OsHV-1 reference type and AVNV due to the deletion of a unique region (X, 1.5Kb) between IRL and IRS. The DNA sequence of OsHV-1-SB is 95.2 % and 97.3 % identical to that of OsHV-1 reference type and AVNV respectively. On the basis of nucleotide sequences of 32 ORFs in OsHV-1-SB and the other nine OsHV-1 variants, results from phylogenetic analysis also demonstrated that OsHV-1-SB is most closely related to AVNV.ConclusionsThe determination of the genome of OsHV-1 with distinguished epidemiological features will aid in our better understanding of OsHV-1 diversity, and facilitate further research on the origin, evolution, and epidemiology of the virus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0334-0) contains supplementary material, which is available to authorized users.

Characterization of pathogenic vibrios isolated from bivalve hatcheries in Galicia, NW Atlantic coast of Spain. Description of Vibrio tubiashii subsp. europaensis subsp. nov

The taxonomic position of the bivalve pathogen PP-638 was studied together with five similar isolates. The strains were isolated from flat oyster (Ostrea edulis) and Manila clam (Venerupis philippinarum) cultures during outbreaks of disease in two shellfish hatcheries (Galicia, NW Spain). The pathogenicity, previously established for PP-638, was demonstrated with all isolates and for several bivalve species, including the original hosts. On the basis of phenotypic characterization and 16S rRNA gene sequences, a tight group was defined within the genus Vibrio. Multilocus sequence analysis (MLSA) based on concatenated sequences of the 16S rRNA gene and the five housekeeping genes recA, rpoA, pyrH, gyrB and ftsZ revealed that these strains form a cluster within the Orientalis clade, close to the species Vibrio tubiashii. The results of MLSA, the DDH rate and the phenotypic differences with the type strain of V. tubiashii supported the differentiation of the Galician isolates as a new subspecies within V. tubiashii, for which the name V. tubiashii subsp. europaensis subsp. nov. is proposed (type strain PP-638T=CECT 8136T=DSM 7349T) The emended description of V. tubiashii is included. The pathogenicity assays widen the host range of V. tubiashii to add two unreported species, Venerupis decussata and Donax trunculus, and the described as relatively resistant species V. philippinarum.

Vibrio ostreicida sp. nov., a new pathogen of bivalve larvae

The taxonomic position of the bivalve pathogen PP-203T was studied together with those of two similar isolates (PP-200 and PP-204). The bacterial strains were isolated from samples of young oyster spat in a bivalve hatchery in Galicia (NW Spain), which was continually affected by outbreaks of disease and severe mortalities. On the basis of 16S rRNA gene sequencing, the three strains formed a cluster within the genus Vibrio and were most closely related to Vibrio pectenicida DSM 19585T (97.9% similarity). Additional multilocus sequence analysis, including sequences of the housekeeping genes rpoA, recA, pyrH, gyrB and ftsZ, and DNA-DNA hybridization experiments indicated that the strains were distinct from currently known species of the genus Vibrio and confirmed the clustering of the three isolates. Several phenotypic features, such as growth in TCBS medium and nitrate reduction, proved useful for distinguishing the proposed novel species from its closest relatives. The findings support the description of a novel species to include the three isolates, for which the name Vibrio ostreicida sp. nov. (type strain PP-203T=CECT 7398T=DSM 21433T) is proposed.

Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32

Marine bivalves can accumulate large numbers of bacteria, in particular Vibrio species, whose persistence in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating hemocytes and hemolymph soluble factors. The interactions between vibrios and hemolymph have been investigated, in particular in bivalve species susceptible to infection by certain Vibrio spp. and strains.In this work, the effects of two bivalve pathogens, Vibrio splendidus LGP32 (V.s.) and Vibrio aestuarianus 01/032 (V.a.), isolated from oyster mortality outbreaks, on the hemocytes of Mytilus galloprovincialis were investigated. In vitro, V.s., but not V.a., induced a dramatic decrease in lysosomal membrane stability-LMS in the hemocytes; both vibrios induced a moderate lysozyme release, with V.s. > V.a.. The V.s.-induced decrease in LMS was mediated by activation of PI-3Kinase, as shown by use of different kinase inhibitors. TEM analysis showed rapid internalization of both vibrios; however, V.s. lead to cellular and lysosomal damage and was able to survive within the hemocytes, whereas significant killing of V.a. was observed. In vivo, in mussels challenged with either vibrio and sampled at 6, 24 and 96 h post-injection, transient decreases in hemocyte LMS and progressive increases in serum lysozyme activity were observed, with V.s. > V.a.. Moreover, whereas V.a. was efficiently cleared from hemolymph, V.s. showed significant growth, that was maximal at 24 h p.i. when lowest LMS values were recorded in the hemocytes. Both vibrios also induced significant decreases in LMS in the digestive gland, again with V.s. > V.a..The results indicate distinct interactions between mussel hemocytes and the two vibrio strains tested. The effects of V.s. may be due to the capacity of this strain to interfere with the signaling pathways involved in hemocyte function, thus escaping the bactericidal activity of the host cell, as observed for certain mammalian pathogens. Although V.s. is considered not pathogenic to Mytilus, this vibrio strain can affect the lysosomal function at the cellular and tissue level, thus leading to stressful conditions.

Prevalence of Perkinsus marinus (Dermo), Haplosporidium nelsoni (MSX), and QPX in Bivalves of Delaware's Inland Bays and Quantitative, High‐Throughput Diagnosis of Dermo by QPCR

Restoration of oyster reef habitat in the Inland Bays of Delaware was accompanied by an effort to detect and determine relative abundance of the bivalve pathogens Perkinsus marinus, Haplosporidium nelsoni, and QPX. Both the oyster Crassostrea virginica and the clam Mercenaria mercenaria were sampled from the bays. In addition, oysters were deployed at eight sites around the bays as sentinels for the three parasites. Perkinsus marinus prevalence was measured with a real-time, quantitative polymerase chain reaction (PCR) methodology that enabled high-throughput detection of as few as 31 copies of the ribosomal non-transcribed spacer region in 500 ng oyster DNA. The other pathogens were assayed using PCR with species-specific primers. Perkinsus marinus was identified in Indian River Bay at moderate prevalence ( approximately 40%) in both an artificial reef and a wild oyster population whereas sentinel oysters were PCR-negative after 3-months exposure during summer and early fall. Haplosporidium nelsoni was restricted to one oyster deployed in Little Assawoman Bay. QPX and P. marinus were not detected among wild clams. While oysters in these bays have historically been under the greatest threat by MSX, it is apparent that P. marinus currently poses a greater threat to recovery of oyster aquaculture in Delaware's Inland Bays.

Bacterial disease in marine bivalves, a review of recent studies: Trends and evolution

The main microbial diseases affecting marine cultured bivalves have been revised on the basis of the eti- ologic agents, pathogenesis and pathogenicity. Several recent bivalve-interaction models have been studied, including Pecten larvae-Vibrio pectinicida, brown ring disease, juvenile oyster disease, Pacific oyster nocardiosis and summer mortalities of oysters. In addition, the taxonomy and phylogeny of new potential bivalve pathogens and their virulence factors have been established. Facing the difficulty of identifying bacterial strains associated with molluscan diseases (mainly vibriosis), polyphasic approaches have been developed to correlate the phenotype and genotype of potential pathogens. By evaluating likely virulence mechanisms, developing biotests to screen virulent strains and characterising the genes implicated in pathogenesis, a new generation of diagnostic tools, based on potential virulence, will be devel- oped. Acquisition of pertinent diagnostic tools will be of major benefit in disease management, health surveillance and monitoring will contribute to maintaining sustainable aquaculture industries.

In VitroActivity of the Limulus Antimicrobial Peptide Tachyplesin I on Marine Bivalve Pathogens

Tachyplesin 1 is an antimicrobial peptide extracted from hemocytes of the Japanese horseshoe crabTachypleus tridentatus. We studied thein vitroactivity of tachyplesin I against bivalve pathogens: the oyster parasitesBonamia ostreae,the intrahemocytic parasite of the flat oysterOstrea edulisandPerkinsus marinus,the histozoic parasite of the Eastern oysterCrassostrea virginica,and the bacteriumVibrioP1, pathogenic for the clamTapes philippinarum.Viability of the protozoans was assessed microscopically by the uptake of the vital dyes acridine orange and ethidium bromide. Following exposure to tachyplesin I,B. ostreaeandP. marinusviabilities were reduced in a dose-dependent manner, up to, respectively, 94 and 62% within a 500 μg/ml peptide concentration. The fine structure ofP. marinuswas highly altered by the peptide. Tachyplesin I also displayed a potent activity against marine vibrios, with a MIC of 0.4–0.8 μg/ml againstVibrioP1. We examined the morphology of oyster hemocytes treated by tachyplesin I, together with the cell functional capabilities to produce chemiluminescence. No effect of the peptide was found on bivalve host cells. As transgenic technology is currently being applied to marine invertebrates, these results indicate that tachyplesin I may provide effective gene sequences to be manipulated in order to produce disease-resistant bivalves.

Purification of a proteinase produced by the bivalve pathogen Vibrio alginolyticus NCMB 1339

Abstract. A proteinase produced by the bivalve pathogen Vibrio alginolyticus NCMB 1339 was purified by preparative isoelectric focusing and gel filtration. Proteinase activity was associated with two components of molecular weights 43000 and 41000 and was toxic to larval Ostrea edulis. Production of this enzyme was maximal during the late exponential phase of growth and it remained stable throughout the stationary phase of growth. At 37°C, activity against azocasein was optimal at pH 7–5. The enzyme was inhibited by mercuric chloride, dithiothreitol and ethylene diamine tetra‐acetic acid, but not by pepstatin‐A, phenylmethylsulphonyl‐fluoride or tosyl‐L‐lysine chloromethylketone.