Evaluation of different yeast cell wall mutants and microalgae strains as feed for gnotobiotically grown brine shrimp Artemia franciscana

The nutritional value of isogenic yeast strains and two microalgal species for gnotobiotically grown Artemia was examined. Yeast cell wall mutants were always better feed for Artemia than their respective wild type. Yeast cells harbouring null mutants for enzymes involved early in the biochemical pathway for cell wall mannoproteins synthesis performed best as feed for Artemia. Yeast cells defective in chitin or β-glucan production were scored in second order. The mnn6 isogenic yeast mutant, harbouring a null mutation for mannoprotein phosphorylation, performed poorly as feed for Artemia, although with good growth. These results suggest that any mutation affecting the yeast cell wall scaffolding by reducing the amount of covalent links between the major components of yeast cell wall, namely mannoproteins, β-glucans and chitin, is sufficient to improve the digestibility for Artemia. The results with microalgae indicated that within one species, strains can have different nutritional value under gnotobiotic conditions. The growth phase was another parameter influencing feed quality, although here it was not possible to reveal the exact cause. It is anticipated that the standard Artemia gnotobiotic growth test is an excellent tool to study the mode of action of bacteria, with a probiotic as well as with a pathogenic character.     

Selected Bacterial Strains Protect Artemia spp. from the Pathogenic Effects of Vibrio proteolyticus CW8T2

In this study Vibrio proteolyticus CW8T2 has been identified as a virulent pathogen for Artemia spp. Its infection route has been visualized with transmission electron microscopy. The pathogen affected microvilli and gut epithelial cells, disrupted epithelial cell junctions, and reached the body cavity, where it devastated cells and tissues. In vivo antagonism tests showed that preemptive colonization of the culture water with nine selected bacterial strains protected Artemia juveniles against the pathogenic effects. Two categories of the selected strains could be distinguished: (i) strains providing total protection, as no mortality occurred 2 days after the experimental infection with V. proteolyticus CW8T2, with strain LVS8 as a representative, and (ii) strains providing partial protection, as significant but not total mortality was observed, with strain LVS2 as a representative. The growth of V. proteolyticus CW8T2 in the culture medium was slowed down in the presence of strains LVS2 and LVS8, but growth suppression was distinctly higher with LVS8 than with LVS2. It was striking that the strains that gave only partial protection against the pathogen in the in vivo antagonism test showed also a restricted capability to colonize the Artemia compared to the strains providing total protection. The in vivo antagonism tests and the filtrate experiments showed that probably no extracellular bacterial compounds were involved in the protective action but that the living cells were required to protect Artemia against V. proteolyticus CW8T2.     

Phenoloxidase and trypsin in germ-free larvae of Artemia fed with cooked unicellular diets: Examining the alimentary and protective effects of putative beneficial bacterium, yeast and microalgae against vibriosis

Three putative beneficial unicellular organisms, the marine bacterium Roseobacter sp, the yeast Saccharomyces cerevisiae mnn9 strain (SC-mnn9) and the microalga Tetraselmis suecica were cooked and offered separately as diets to developing germ-free (GF) Artemia larvae, in order to analyze their alimentary and protective effects. GF Artemia larvae were able to grow with cooked Tetraselmis and SC-mnn9 but failed with cooked Roseobacter. In spite of its high dietary quality, Tetraselmis failed to provide protection against Vibrio proteolyticus infection, while Roseobacter failed as food as well to provide protection. Cooked SC-mnn9 appeared to possess both values, dietary for growth and protective against Vibrio infection. GF Artemia larvae were apparently rapid adapted to dietary swaps; from yeast to algal and from algal to yeast. While the diets swap from algal or yeast, to bacterial diet appeared to be detrimental. Phenoloxidase-L (PO-L) and trypsin-L were used as biochemical indicators of defense and digestive functions, respectively. Developmental trypsin-L patterns were similar when fed on yeast and microalgae diets, suggesting a good digestive adaptation to plant or fungal substrates at early stages. On the contrary, diets swap or Vibrio infection affected PO-L and trypsin-L suggesting a sort of ‘alteration’ of digestive and defense functions.     

Anti-infectious potential of beta-mercapto-ethanol treated baker's yeast in gnotobiotic Artemia challenge test

Aim: To evaluate nutritional and anti‐infectious characteristics of the chemically treated baker’s yeast with 2‐mercapto‐ethanol (2ME) for gnotobiotically grown Artemia. Methods and Results: A selection of isogenic yeast strains was treated with 2ME and fed to gnotobiotically grown Artemia. In the first experiment the effect of the chemical treatment on the yeast nutritional value was studied. In most cases, 2ME‐treated yeast cells were better feed for Artemia than the untreated cells. In the second experiment, a small quantity of 2ME‐treated yeast cells was fed to Vibrio campbellii (VC) challenged Artemia. The 2ME‐treatment on some yeast strains (e.g. gas1, kre6 and chs3) significantly improved Artemia resistance against VC compared with the respective untreated yeast cells. Conclusion: Simple chemical treatment with 2ME could significantly improve the nutritional and anti‐infectious properties of some baker’s yeast strains for gnotobiotically grown Artemia. Significance and Impact of the Study: The gnotobiotic Artemia test system provides a unique opportunity (because of noninterference of other microbial compounds) to investigate how the yeast cell wall composition influences macro parameters (e.g. growth and survival) in an organism. In addition, gene expression studies in these gnotobiotically grown Artemia should provide further documentation on direct effects of yeast cells on the genes involved in immune functions.     

Isolation of lytic bacteriophage against Vibrio harveyi

Aims: The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus. Methods and Results: Water samples from discharge channels and grow‐out ponds of a prawn farm in northeastern Australia were enriched for 24 h in a broth containing four V. harveyi strains. The bacteriophage‐enriched filtrates were spotted onto bacterial lawns demonstrating that the bacteriophage host range for the samples included strains of V. harveyi, Vibrio campbellii, Vibrio rotiferianus, Vibrio parahaemolyticus and Vibrio proteolyticus. Bacteriophage were isolated from eight enriched samples through triple plaque purification. The host range of purified phage included V. harveyi, V. campbellii, V. rotiferianus and V. parahaemolyticus. Transmission electron microscope examination revealed that six purified phage belonged to the family Siphoviridae, whilst two belonged to the family Myoviridae. The Myoviridae appeared to induce bacteriocin production in a limited number of host bacterial strains, suggesting that they were lysogenic rather than lytic. A purified Siphoviridae phage could delay the entry of a broth culture of V. harveyi strain 12 into exponential growth, but could not prevent the overall growth of the bacterial strain. Conclusions: Bacteriophage with lytic activity against V. harveyi were isolated from prawn farm samples. Purified phage of the family Siphoviridae had a clear lytic ability and no apparent transducing properties, indicating they are appropriate for phage therapy. Phage resistance is potentially a major constraint to the use of phage therapy in aquaculture as bacteria are not completely eliminated. Significance and Impact of the Study: Phage therapy is emerging as a potential antibacterial agent that can be used to control pathogenic bacteria in aquaculture systems. The development of phage therapy for aquaculture requires initial isolation and determination of the bacteriophage host range, with subsequent creation of suitable phage cocktails.     

Exposure of gnotobiotic Artemia franciscana larvae to abiotic stress promotes heat shock protein 70 synthesis and enhances resistance to pathogenic Vibrio campbellii

Larvae of the brine shrimp Artemia franciscana serve as important feed in fish and shellfish larviculture; however, they are subject to bacterial diseases that devastate entire populations and consequently hinder their use in aquaculture. Exposure to abiotic stress was shown previously to shield Artemia larvae against infection by pathogenic Vibrio, with the results suggesting a mechanistic role for heat shock protein 70. In the current report, combined hypothermic/hyperthermic shock followed by recovery at ambient temperature induced Hsp70 synthesis in Artemia larvae. Thermotolerance was also increased as was protection against infection by Vibrio campbellii, the latter indicated by reduced mortality and lower bacterial load in challenge tests. Resistance to Vibrio improved in the face of declining body mass as demonstrated by measurement of ash-free dry weight. Hypothermic stress only and acute osmotic insult did not promote Hsp70 expression and thermotolerance in Artemia larvae nor was resistance to Vibrio challenge augmented. The data support a causal link between Hsp70 accumulation induced by abiotic stress and enhanced resistance to infection by V. campbellii, perhaps via stimulation of the Artemia immune system. This possibility is now under investigation, and the work may reveal fundamental properties of crustacean immunity. Additionally, the findings are important in aquaculture where development of procedures to prevent bacterial infection of feed stock such as Artemia larvae is a priority.     

Computational modeling of differences in the quorum sensing induced luminescence phenotypes of Vibrio harveyi and Vibrio cholerae

Vibrio harveyi and Vibrio cholerae have quorum sensing pathways with similar design and highly homologous components including multiple small RNAs (sRNAs). However, the associated luminescence phenotypes of strains with sRNA deletions differ dramatically: in V. harveyi, the sRNAs act additively; however, in V. cholerae, the sRNAs act redundantly. Furthermore, there are striking differences in the luminescence phenotypes for different pathway mutants in V. harveyi and V. cholerae. However, these differences have not been connected with the observed differences for the sRNA deletion strains in these bacteria. In this work, we present a model for quorum sensing induced luminescence phenotypes focusing on the interactions of multiple sRNAs with target mRNA. Within our model, we find that one key parameter - the fold-change in protein concentration necessary for luminescence activation - can control whether the sRNAs appear to act additively or redundantly. For specific parameter choices, we find that differences in this key parameter can also explain hitherto unconnected luminescence phenotypes differences for various pathway mutants in V. harveyi and V. cholerae. The model can thus provide a unifying explanation for observed differences in luminescence phenotypes and can also be used to make testable predictions for future experiments.     

Ingestion of bacteria overproducing DnaK attenuates Vibrio infection of Artemia franciscana larvae

Feeding of bacterially encapsulated heat shock proteins (Hsps) to invertebrates is a novel way to limit Vibrio infection. As an example, ingestion of Escherichia coli overproducing prokaryotic Hsps significantly improves survival of gnotobiotically cultured Artemia larvae upon challenge with pathogenic Vibrio campbellii. The relationship between Hsp accumulation and enhanced resistance to infection may involve DnaK, the prokaryotic equivalent to Hsp70, a major molecular chaperone in eukaryotic cells. In support of this proposal, heat-stressed bacterial strains LVS 2 (Bacillus sp.), LVS 3 (Aeromonas hydrophila), LVS 8 (Vibrio sp.), GR 8 (Cytophaga sp.), and GR 10 (Roseobacter sp.) were shown in this work to be more effective than nonheated bacteria in protecting gnotobiotic Artemia larvae against V. campbellii challenge. Immunoprobing of Western blots and quantification by enzyme-linked immunosorbent assay revealed that the amount of DnaK in bacteria and their ability to enhance larval resistance to infection by V. campbellii are correlated. Although the function of DnaK is uncertain, it may improve tolerance to V. campbellii via immune stimulation, a possibility of significance from a fundamental perspective and also because it could be applied in aquaculture, a major method of food production.     

Influence of different yeast cell-wall mutants on performance and protection against pathogenic bacteria (Vibrio campbellii) in gnotobiotically-grown Artemia

A selection of isogenic yeast strains (with deletion for genes involved in cell-wall synthesis) was used to evaluate their nutritional and immunostimulatory characteristics for gnotobiotically-grown Artemia. In the first set of experiments the nutritional value of isogenic yeast strains (effected in mannoproteins, glucan, chitin and cell-wall bound protein synthesis) for gnotobiotically-grown Artemia was studied. Yeast cell-wall mutants were always better feed for Artemia than the isogenic wild type mainly because they supported a higher survival but not a stronger individual growth. The difference in Artemia performance between WT and mutants feeding was reduced when stationary-phase grown cells were used. These results suggest that any mutation affecting the yeast cell-wall make-up is sufficient to improve the digestibility in Artemia. The second set of experiments, investigates the use of a small amount of yeast cells in gnotobiotic Artemia to overcome pathogenicity of Vibrio campbellii (VC). Among all yeast cell strains used in this study, only mnn9 yeast (less cell-wall bound mannoproteins and more glucan and chitin) seems to completely protect Artemia against the pathogen. Incomplete protection against the pathogen was obtained by the gas1 and chs3 mutants, which are lacking the gene for a particular cell-wall protein and chitin synthesis, respectively, resulting in more glucan. The result with the chs3 mutant is of particular interest, as its nutritional value for Artemia is comparable to the wild type. Hence, only with the chs3 strain, in contrast to the gas1 or mnn9 strains, the temporary protection to VC is not concomitant with a better growth performance under non-challenged conditions, suggesting non-interference of general nutritional effects.     

Acanthamoeba polyphaga is a possible host for Vibrio cholerae in aquatic environments

Acanthamoeba is a genus of free-living amoebae found to be able to host many bacterial species living in the environment. Acanthamoebae and Vibrio cholerae are found in the aquatic environments of cholera endemic areas. Previously it has been shown that V. cholerae O1 and O139 can survive and grow in Acanthamoeba castellanii. The aim of this study was to examine the ability of Acanthamoeba polyphaga to host V. cholerae O1 and O139. The interaction between A. polyphaga and V. cholerae strains was studied by means of viable amoeba cell counts and viable count of the bacteria in the absence and presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Electron microscopy was used to determine the localization of V. cholerae inside A. polyphaga. The results showed that A. polyphaga enhanced growth and survival of V. cholerae, which grew and survived inside the amoeba cells for 2 weeks. The electron microscopy showed that A. polyphaga hosted intracellular V. cholerae localized in the vacuoles of amoeba cell. Neither the presence of V. cholerae together with A. polyphaga nor the intracellular localization of the bacteria inhibited growth and survival of A. polyphaga. The outcome of the interaction between these microorganisms may support strongly the role of A. polyphaga as host for V. cholerae O1 and O139.     

Tracing Vibrio parahaemolyticus in oysters (Tiostrea chilensis) using a Green Fluorescent Protein tag

Oysters feed by removing particles from the water. This food is composed of complex mixtures of living microorganisms, detritus, and inorganic particles that widely range in size. It has been speculated that some marine heterotrophic microorganisms, such as Vibrio parahaemolyticus, could enter in this digestive process and persist in the oyster tissue. Since some strains of V. parahaemolyticus are pathogenic for humans, these bacteria are considered to be a constant menace for health and aquaculture. In order to improve the safety of marine products it is imperative to obtain more knowledge about Tiostrea chilensis and its interactions with V. parahaemolyticus. In this study V. parahaemolyticus ATCC 17802 was tagged using plasmid pKV111, which carries the gfp gene that codifies a Green Fluorescent Protein (GFP), thereby allowing these strains (VpGFP) to be detected under epifluorescence microscopy. Results obtained showed that T. chilensis can filter VpGFP directly from sea water and suggested that most of them were digested by oysters. However, in the postharvest stage, a small fraction can remain in oyster tissues after depuration and VpGFP can rapidly grow if the bivalves are stored at room temperature.     

Effect of the addition of four potential probiotic strains on the survival of pacific white shrimp (Litopenaeus vannamei) following immersion challenge with Vibrio parahaemolyticus

Four bacterial strains isolated from the gastrointestinal tract of adult shrimp Litopenaeus vannamei, Vibrio alginolyticus UTM 102, Bacillus subtilis UTM 126, Roseobacter gallaeciensis SLV03, and Pseudomonas aestumarina SLV22, were evaluated for potential use as probiotics for shrimp. In vitro studies demonstrated antagonism against the shrimp-pathogenic bacterium, Vibrio parahaemolyticus PS-017. Feeding shrimp with diets containing the potential probiotics showed the best feed conversion ratio in comparison with the control groups. After feeding with the potential probiotics for 28 days, challenge by immersion indicated effectiveness at reducing disease caused by V. parahaemolyticus in shrimp.     

Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi

Two hundred and two strains of lactic acid bacteria (LAB) isolated from digestive tracts of cultivated and wild adult shrimp, including Litopenaeus vannamei, Metapenaeus brevicornis and Penaeus merguiensis were selected based on their antibacterial activity against Vibrio harveyi. LAB strain of MRO3.12 exhibiting highest reduction of V. harveyi was identified as Lactobacillus plantarum MRO3.12 based on the nucleotide sequence of its 16S rDNA, which showed 99% (780/786 bp) homology to L. plantarum strain L5 (GenBank accession number DQ 239698.1). Co-cultivation of V. harveyi and L. plantarum MRO3.12 showed complete reduction of V. harveyi at 24 h under aerobic and anaerobic conditions, whereas L. plantarum increased from 5.29 to 9.47 log CFU ml⁻¹. After 6-week feeding trial with L. plantarum supplemented diet, white shrimp (L. vannamei) exhibited significant differences (p < 0.05) in relative growth rate (% RGR), feed conversion ratio (FCR) and survival compared to the control group fed with non-supplemented diet. LAB-fed group showed 98.89% survival, whereas only 68.89% survival was observed in the control group. LAB from the digestive tract of probiotic-fed shrimp showed higher level of 5.0 ± 0.14 log CFU/g than the non-supplemented ones (3.34 ± 0.21 log CFU/g). However, total bacterial and non-fermenting vibrios counts decreased in shrimps fed on L. plantarum. Ten days after infection with V. harveyi (5.3-5.5 log CFU ml⁻¹), significant survival (p < 0.05) of 77% was observed in LAB supplemented shrimp, while only 67% survival was observed in the control.     

Production and characterization of a novel monoclonal antibody against Vibrio parahaemolyticus F0F1 ATP synthase's delta subunit and its application for rapid identification of the pathogen

We raised monoclonal antibodies (MAbs) against Vibrio parahaemolyticus cell extracts. One of the MAbs, designated MAb-VP34, reacted in enzyme-linked immunosorbent assays (ELISAs) with 140 V. parahaemolyticus strains, regardless of serotype or origin. MAb-VP34 did not detectably react with 96 strains belonging to 27 other Vibrio species (except for Vibrio natriegens) or with 29 non-Vibrio species. These results show that MAb-VP34 is highly specific for V. parahaemolyticus. Western blotting and mass spectrometry analyses revealed that MAb-VP34 recognized V. parahaemolyticus F₀F₁ ATP synthase's delta subunit.Using MAb-VP34, a rapid and simple immunodot blotting assay (VP-Dot) was developed to determine whether bacterial colonies growing on selective agar, represented V. parahaemolyticus. To evaluate VP-Dot, 20 V. parahaemolyticus strains and 19 non-related strains were tested. The results indicated that VP-Dot is a reliable tool for identification of V. parahaemolyticus colonies. The simple VP-Dot procedure took 40 min, indicating that the MAb-VP34 based immunological method will greatly reduce labor, time, and costs required to verify V. parahaemolyticus colonies as compared with the conventional biochemical test.     

Genomic analysis of a novel integrative conjugative element in Vibrio cholerae

Integrative conjugative elements (ICEs) are a class of self-transmissible mobile elements that mediate horizontal gene transfer in bacteria, and play an important role in bacterial evolution. Since 1992, ICEs of the SXT/R391 family have been found to be widely distributed among Vibrio cholerae strains isolated in Asian countries. Here we describe ICEVchB33, an ICE found in the genomes of two V. cholerae O1 Eltor strains, one isolated in India, 1994, and the other from Mozambique, 2004. ICEVchB33 revealed a new genetic organization, different from other ICEs of the SXT/R391 family, demonstrating the genomic plasticity of these elements.     

Development of a haemolysin gene‐based multiplex PCR for simultaneous detection of Vibrio campbellii,Vibrio harveyi and Vibrio parahaemolyticus

Aim: To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species‐specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost‐effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.     

Vibrio gallaecicus sp. nov. isolated from cultured clams in north-western Spain

A group of three motile facultative anaerobic marine bacteria were isolated from cultured Manila clams (Ruditapes philippinarum) in Galicia, north-western Spain. The strains were characterized phenotypically and genotypically. Phylogenetic analysis of the 16S rRNA gene and four housekeeping genes, RNA polymerase α-chain (rpoA), RecA protein (recA), the α-subunit of bacterial ATP synthase (atpA) and the uridine monophosphate (UMP) kinase (pyrH), indicated that these strains were closely related to the Vibrio splendidus clade. The amplified fragment length polymorphism (AFLP) fingerprints, DNA–DNA hybridizations and phylogenies of the housekeeping and 16S rRNA gene sequences showed that the three strains represented a different species from all currently described vibrios. The new species could be differentiated from its nearest neighbours on the basis of several phenotypic features. The three strains are therefore a novel species within the genus Vibrio, for which the name Vibrio gallaecicus is proposed, with the type strain being VB 8.9^T(=CECT 7244^T=LMG 24045^T).     

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.     

Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida

Taxonomic characterization was performed on the putative N₂-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of São Sebastião (São Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n=76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n=7), V. alginolyticus (n=8), V. campbellii (n=3), and V. parahaemolyticus (n=1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N₂ might explain why they are so abundant in the mucus of different coral species.