Harveyi Isolates Research Articles

Development of an in silico serotyping database of Vibrio harveyi by identification and comparison of O-antigen encoding loci

Vibrio harveyi is an important pathogen for aquatic fish and invertebrates, that can lead to significant economic losses in the aquaculture industry. Rapid and accurate serotyping is essential for the diagnosis and epidemiological investigation of bacterial pathogens. In Vibrio spp., serotyping is based on lipopolysaccharide or capsule polysaccharide loci. In this study, we screened and extracted the location and full-length sequences of 12 different lipopolysaccharide biosynthesis loci from all available V. harveyi genome sequences from public database. Herein their gene composition, homology and variation are discussed, and a database is developed to rapidly type V. harveyi isolates based on differences in lipopolysaccharide loci. This study further analyzses the relationship between lipopolysaccharide loci and genome characteristics. The identification and characterization of these lipopolysaccharide loci could help researchers understand the genetic, molecular, and basis serotype structural specificity of this pathogen, and also provide opportunities for further development and improvement of the molecular serotyping method, which would aid the diagnosis and clinical treatment of the disease caused by this pathogen.

Biofilm inhibitor Con A lectin feed additive protects shrimp against pathogenic Vibrio harveyi and V. parahaemolyticus

Extracellular matrix (ECM) is a major component of all bacterial biofilms that protect the bacterial cells living within. It consists mainly of carbohydrates, proteins, lipids, and nucleic acids. We characterized the ECM of an acute hepatopancreatic necrosis disease (AHPND) causing Vibrio parahaemolyticus isolate 3HP and two luminescent shrimp disease-causing V. harveyi isolates (1114GL or VH0 and its Siphoviridae phage-lysogenized isolate, VH1) using 11 FITC-labeled plant lectins with different terminal sugar specificities. Canavalia ensiformis lectin (Con A), wheat germ lectin (Triticum vulgaris, WGA), Griffonia simplicifolia lectin II (GSL-II), and Maclura pomifera Lectin (MPL) were found to react with the biofilms of all three isolates. Based on these results and availability, Con A and WGA and their respective specific sugars (D-mannose or glucose and N-acetyl-D-glucosamine) were tested for effects on V. harveyi and V. parahaemolyticus biofilm formations. Biofilms of these bacteria were evaluated by thickness after growth with and without the two lectins and with and without their respective sugar targets at different concentrations. Only Con A at 0.1 mg/ml and 0.5 mg/ml (with and without D-mannose or d-glucose) were found to inhibit biofilms of all three Vibrio isolates in a dose-dependent manner. Con A was then tested for efficacy in protecting shrimp against V. harveyi (VH1) and V. parahaemolyticus 3-HP by its addition to shrimp feed at 0.05 mg/g and 0.5 mg/g and fed for seven days before they were immersion challenged with either VH1 or 3HP on day 8. After the challenge, the shrimp continued to be fed with their respective feed, and their health and survival were monitored for another 11 and 12 days for the VH1 and 3HP experiments, respectively. From both experiments, the groups fed with Con A-supplemented feed gave significantly higher survival rates [51.9% and 53.6% (VH1) and 52.8% and 62.9% (3HP)] than those fed with un-supplemented feed [38.3% (VH1) and 21.2% (3HP)] (p < 0.05). These results suggested that D-mannose/d-glucose plays an essential role in V. harveyi and V. parahaemolyticus biofilm formations, and Con A lectin might be helpful in monitoring and controlling biofilms to prevent Vibrio diseases.

Antagonistic Potential Marine Bacteria Against Luminescent Vibriosis Pathogen Vibrio Harveyi Obtained from Shrimp Farm, Marakkanam, Tamil Nadu, India

Aquaculture farming practices in India and other countries are facing a lot of economic loss due to luminescent vibriosis.Ecological and cost-effective practices may have been warranted to combat this tricky disease. A total of 57 morphologicallydifferent marine bacterial isolates were isolated from samples collected at different aquaculture ponds in Marakkanam, Tamil Nadu,India and screened for their antibacterial potential against luminescent vibriosis causing bacteria Vibrio harveyi. All marine bacterialisolates were screened for antibacterial activity by cross streak method against two different isolates V. harveyi GNC01 and V.harveyi GNC03. Secondary screening of antagonistic isolates by dual culture plate method and agar well diffusion method leads tothe identification of antagonistic isolates. The antagonistic isolates were characterized by molecular taxonomy (16S rRNA genesequence) and phylogeny and identified as Neobacillus niacini (GNC04), Bacillus halotolerans (GNC06), Bacillus amyloliquefaciens(GNC16), Lacticaseibacillus paracasei (GNC23) and Lactiplantibacillus plantarum (GNC24). The minimum inhibitory concentrationof all these selected bacterial crude secondary metabolites against both Vibrio harveyi isolates GNC01 and GNC03 ranged between0.062 to 0.50 mg/mL. The GC-MS profiling of the ethyl acetate extract revealed the presence of antibacterial compounds such asOxime-,methoxy-phenyl (26.63%), 2,3-Dihydroxybutane (9.52%), Phosphite, menthyldimethyl- (2.83%), Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- (2.07%). The results of this study suggest that Lactiplantibacillus plantarum (GNC24) is apotential source for antagonistic secondary metabolites against luminescent vibriosis causing bacteria Vibrio harveyi. Further,biological control formulation with these isolates and their metabolites will be a reward for the integrated pest management inthe aquaculture sector.

Vibrio harveyi isolated from marine aquaculture species in eastern China and virulence to the large yellow croaker (Larimichthys crocea).

Diseases of maricultured species caused by Vibrio harveyi are increasing in China and other regions. This study examined the genetic diversity, antimicrobial susceptibility, plasmid profiles and virulence potential of the V. harveyi isolated from marine organisms farmed in two provinces in eastern China between 2014 and 2019. A total of 54 V. harveyi were obtained from seven marine species. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting revealed substantial genetic heterogeneity among the V. harveyi isolates. There was no significant correlation between ERIC-PCR genotypes and host origins or fish farms. All the isolates were resistant to amoxicillin and ampicillin, and 79·6% to kanamycin. We found that 61·1% of the V. harveyi isolates had plasmid(s) and there were 14 different plasmid profiles. Most isolates from fish hosts (76·5%) contained plasmids; however, 75% of isolates from nonfish hosts lacked plasmids. Experimental infection results showed that isolates with plasmid(s) were more virulent to large yellow croaker than isolates lacking plasmids (P<0·05). This study confirmed that V. harveyi isolates obtained from animals farmed in the coastal region of east China were genetically diverse. Our results suggest that the virulence of various V. harveyi strains to fish is associated with the plasmids they carry. More than 50% of the V. harveyi isolates carried one to 11 plasmids. The plasmid-borne traits of V. harveyi strains might be important for host adaptation and virulence, but they were not associated with susceptibility to the tested antibiotics.

Antigenicity of hypothetical protein HP33 of Vibrio harveyi Y6 causing scale drop and muscle necrosis disease in Asian sea bass

A unique strain of Vibrio harveyi is the causative agent of scale drop and muscle necrosis disease (SDMND) in Asian sea bass (Lates calcarifer). This study investigated the protein profiles of SDMND-causing Vibrio harveyi isolates compared to the reference V. harveyi ATCC 14126 strain. A distinct protein band of 33 kDa, namely HP33, found from only V. harveyi SDMND was subjected to analysis by LC-MS/MS and the identified peptide sequences matched to an unknown hypothetical protein. Detection of HP33 coding sequence was investigated at both genomic and transcriptional levels and the results consistently supported the protein analysis. Recombinant HP33 protein was then produced using Escherichia coli system. The rHP33 protein did not cause mortality or visible clinical signs to Asian sea bass. However, the rHP33 protein was able to stimulate antibody response in Asian sea bass as evidenced by Western blotting and agglutination tests. Here, we proposed that rHP33 might be a good protein target for development of subunit vaccine and/or immunostimulant to protect Asian sea bass from SDMND.

Epidemiological situation and phylogenetic relationship of Vibrio harveyi in marine-cultured fishes in China and Southeast Asia

In recent years, marine fish farms in China and Southeast Asian countries, where are the most important maricultural areas around the world, have frequently suffered severe economic losses due to fish diseases. The objective of this study was to clarify the dominant bacterial pathogen in diseased fish collected from China, Vietnam, and Malaysia from 2015 to 2018, and investigate their genetic relationships by multilocus phylogenetic analysis (MLPA). A total of 645 isolates, 371 from diseased fish and 274 from healthy fish, were collected, and Vibrio harveyi was identified as the dominant bacterial pathogen in the samples. V. harveyi was detected in 88.76% of diseased fish and 36.27% of healthy fish, which accounted for 52.02% and 12.77% of the total isolates from diseased and healthy fish, respectively. The intra- and inter-area phylogenetic relationships of 228 V. harveyi isolates were investigated via MLPA based on the concatenated sequences of five house-keeping genes fragments (rpoA-pyrH-topA-ftsZ-mreB, 3, 264 base pairs (bp)). Close genetic relationships were observed among the V. harveyi isolates, including minor difference of nucleotide substitution, and intra- and inter-area phylogenetic depths. The phylogenetic tree also showed that isolates from different areas were randomly distributed on clades, and it was difficult to establish an obvious correlation between the genetic relationships and V. harveyi isolation sites. Therefore, closely related V. harveyi are highly prevalent in marine farms in China and Southeast Asian countries, which is a potentially great challenge to maricultural management and disease prevention.

Development of a mreB-targeted real-time PCR method for the quantitative detection of Vibrio harveyi in seawater and biofilm from aquaculture systems

Vibrio harveyi is a particularly problematic Gram-negative bacterium because it can form biofilms on aquaculture facility surfaces, leading to resistance of bacteria against antibiotics and water sanitizers. A SYBR Green I quantitative real-time PCR method was developed to detect V. harveyi directly from environmental samples, including seawater and biofilm. Specific primers targeting the mreB gene were designed. The exclusivity and inclusivity of the newly designed primers were evaluated using a panel of 85 bacteria: 58 V. harveyi from multiples origins and 27 non-V. harveyi isolates, and compared with two pairs of primers targeting the topA and toxR genes that were designed previously. All sets of primers were able to distinguish V. harveyi from closely related species belonging to the Harveyi clade. However, the mreB primers showed better inclusivity and were thus used to develop the real-time PCR assay. A quantification curve was obtained from pure culture of V. harveyi and exhibited excellent efficacy with detection levels as low as 5 genome copies in the PCR reaction. After selection of the extraction kit allowing the best DNA quantity and purity, validation was performed on both seawater and biofilm samples collected from a fish farm. The presence of inhibitors in the DNA templates was evaluated and a 10-fold dilution of template DNA was recommended in order to avoid their effects. The assay was able to detect V. harveyi from environmental samples, confirming the validity of the method. This real-time PCR method will help to evaluate the dynamics of V. harveyi in aquaculture facilities. Suitable prophylactic control measures could be designed using this method, instead of the use of curative methods such as antibiotics.

Bacteriophage therapy for the control of Vibrio harveyi in greenlip abalone (Haliotis laevigata)

Bacteriophage therapy has demonstrated promising results towards the control of bacterial infections within the aquaculture industry as an alternative therapy to antibiotics. This current research describes the efficacy of bacteriophage therapy in controlling vibriosis within abalone (Haliotis laevigata). Two bacteriophages were isolated and used in in vitro assays to determine the effect of each specific phage on the growth of specific Vibrio harveyi isolates. To demonstrate efficacy, an in vivo bioassay was performed using abalone (H. laevigata) at a water temperature of 24°C. Characterisation of the two isolated phages revealed they were from the family, Siphoviridae. The phages had different antimicrobial abilities towards different Vibrio harveyi isolates in the in vitro assays and the bioassay demonstrated improved survival of abalone treated with bacteriophage. The results of the bioassay showed that the negative control and phage control were statistically identical, with 100% survival, but they were different from the positive bacterial control, with a 0% survival. The bacteria + phage treated group showed increased accumulative survival of 70% compared to the positive control. This is the first study to demonstrate the efficacy of bacteriophage therapy treatment for abalone. The study demonstrates that if abalones are treated with bacteriophage shortly after Vibrioharveyi infection, the accumulative survival could be significantly increased.

Molecular characterization of antibiotic resistant Vibrio harveyi isolated from shrimp aquaculture environment in the south east coast of India

Vibrio harveyi is a strategic human pathogen that occurs naturally in marine and estuarine environments. The pathogen is believed to cause acute septicemia, gastroenteritis, severe necrotizing soft-tissue infection, and high rate of lethality through ingestion of V. harveyi contaminated seafood. In this study, we isolated and characterized V. harveyi from water suspended sediment samples of black tiger shrimp ponds and from the sea coasts, in the east coast of the Bay of Bengal, India. Initial isolations of putative V. harveyi isolates were grown on thiosulfate-citrate-bill salts-sucrose agar (TCBS) plates for 36 h. Gram-negative and oxidase-positive colonies alone were selected and subsequently identified by 12 different conventional biochemical tests. The species specificity was confirmed by 16S rRNA, hemolysin and toxRvh genes were used through PCR targeted primers. Furthermore, genomic fingerprinting was carried out using randomly amplified polymorphic DNA fingerprinting, which showed that all the five V. harveyi were genetically distinct. From a total of 256 samples, a total of five strains of V. harveyi were isolated, of which three were from various shrimp ponds and two were from the coastal area. These five isolates were subjected to profiling against 15 antibiotics and the perusal results emphasized the V. harveyi resistance to ciprofloxacin, penicillin, rifampicin, and vancomycin compared to other tested antibiotics. The present findings were helpful in understanding the multiple antibiotics resistance of V. harveyi, which indicates the urgent need for targeted alternative biocontrol strategies to enhance the prospects of commercially viable shrimp cultivation.

Pathogenicity of halophilicVibrio harveyiin giant freshwater prawns (Macrobrachium rosenbergiiDe Man)

In this study, the phenotypic and pathogenic properties in freshwater host were characterized in 14 strains of halophilic Vibrio harveyi isolated from infected marine black tiger shrimp, Penaeus monodon. The lysogenic phenotype was assayed via prophage excision and mitomycin C induction. The bacteria were grouped into two types, corresponding to lysogenic and non-lysogenic strains. The pathogenicity was determined via direct injection of bacterial cultures into post-larval juvenile giant freshwater prawns, Macrobrachium rosenbergii De Man. All of the infected prawns showed similar symptoms and inflamed hepatopancreas. The V. harveyi isolates derived from the first-injected infected prawns were re-isolated and re-injected into healthy giant freshwater prawns, in which they retained similar infectivity. Both lysogenic and non-lysogenic Vibrio spp. showed identical virulence associated with 100% mortality within one day post-injection. TEM micrographs showed hepatopancreatic nuclear deformation and lipid breakdown caused by lysogenic γ-hemolytic VL19 and non-lysogenic β-hemolytic V33. However, the V33 strain was associated with severely disrupted mitochondria. None of the V. harveyi strains was able to produce a biofilm. Together, our findings indicate that the lysogenic and non-lysogenic halophilic V. harveyi isolated from marine shrimps may use different virulence factors that are responsible for their pathogenicity in freshwater prawns.

Characterization of four lytic transducing bacteriophages of luminescent Vibrio harveyi isolated from shrimp (Penaeus monodon) hatcheries

Four lytic bacteriophages designated as φVh1, φVh2, φVh3, and φVh4 were isolated from commercial shrimp hatcheries, possessing broad spectrum of infectivity against luminescent Vibrio harveyi isolates, considering their potential as biocontrol agent of luminescent bacterial disease in shrimp hatcheries, and were characterized by electron microscopy, genomic analysis, restriction enzyme analysis (REA), and pulsed-field gel electrophoresis (PFGE). Three phages φVh1, φVh2, and φVh4 had an icosahedral head of 60-115 nm size with a long, noncontractile tail of 130-329 × 1-17 nm, belonged to the family Siphoviridae. φVh3 had an icosahedral head (72 ± 5 nm) with a short tail (27 × 12 nm) and belonged to Podoviridae. REA with DraI and PFGE of genomic DNA digested with ScaI and XbaI and cluster analysis of their banding patterns indicated that φVh3 was distinct from the other three siphophages. PFGE-based genome mean size of the four bacteriophages φVh1, φVh2, φVh3, and φVh4 was estimated to be about 85, 58, 64, and 107 kb, respectively. These phages had the property of generalized transduction as demonstrated by transduction with plasmid pHSG 396 with frequencies ranging from 4.1 × 10(-7) to 2 × 10(-9) per plaque-forming unit, suggesting a potential ecological role in gene transfer among aquatic vibrios.

A pathogenic Vibrio harveyi lineage causes recurrent disease outbreaks in cultured Japanese flounder ( Paralichthys olivaceus) and induces apoptosis in host cells

Vibrio harveyi is an important aquaculture pathogen that can infect a large number of marine animals. In this study, we examined three V. harveyi strains (VJ1, VJ2, and VJ3) isolated in the years of 2008, 2009, and 2010 respectively from diseased Japanese flounder ( Paralichthys olivaceus) in a fish farm in China that had been inflicted with disease outbreaks. VJ1, VJ2, and VJ3 exhibited comparable virulence in a flounder model and induced symptoms in experimentally infected fish that are indistinguishable from those of naturally diseased fish. 16S rDNA and multilocus sequence analysis indicated that VJ1, VJ2, and VJ3 share 98–100% sequence identities with known V. harveyi isolates in 16S rDNA and four housekeeping genes. VJ1, VJ2, and VJ3 have distinct and identical AFLP banding patterns, which differ profoundly from those of two environmental V. harveyi isolates. VJ1, VJ2, and VJ3 exhibited comparable growth profiles and hemolytic activities and possess the vhh hemolysin gene. Compared to an environmental isolate, VJ1, VJ2, and VJ3 showed comparable and significantly stronger resistances against the bactericidal effect of host serum and induced higher levels of respiratory burst activity in head kidney macrophages. However, VJ1, VJ2, and VJ3 were unable to proliferate in macrophages. When incubated with a cultured cell line of flounder, VJ1, VJ2, and VJ3 induced apoptosis characterized by the occurrence of DNA fragmentation, apoptotic bodies, and elevated caspase 3 activity. Taken together, these results indicate that VJ1, VJ2, and VJ3 are probably clones of the same V. harveyi lineage that has persisted during the examined three-year time frame and likely caused recurrent disease outbreaks via a virulence mechanism involving serum resistance and triggering apoptosis in host cells.

Evaluation of RAPD-PCR and protein profile analysis to differentiate Vibrio harveyi strains prevalent along the southwest coast of India

Sixty five isolates of Vibrio harveyi were subjected to random amplified polymorphic DNA (RAPD)-PCR analysis and protein profiling to investigate the genetic variability among V. harveyi prevalent along the coast and also assess the discriminating ability of these two molecular methods. A total of 10 RAPD primers were assayed for their specificity in detecting V. harveyi, of which only two primers: PM3 and CRA25 were highly reproducible and found suitable for use in RAPD-PCR. The genetic diversity among V. harveyi isolates assessed by RAPD-PCR using PM3 primer yielded 35 different RAPD patterns which clustered the isolates into 15 groups at 72% similarity level. Similarly, RAPD-PCR with CRA25 clustered the 38 patterns into 10 groups at 74% similarity. The discriminatory index (D) value calculated for RAPD fingerprints generated with PM3 and CRA25 were 0.90 and 0.85, respectively. On the other hand, molecular typing of V. harveyi using whole cell proteins generated profiles that showed no major difference indicating the technique to be not useful in typing strains of this bacterium. However, a few of the isolates showed the presence of unique band of 28 kDa that needs to be further investigated to understand the role of the protein in disease process if any.

Shrimp pathogenicity, hemolysis, and the presence of hemolysin and TTSS genes in Vibrio harveyi isolated from Thailand

The virulence factors of Vibrio harveyi, the causative agent of luminous vibriosis, are not completely understood. We investigated the correlations between shrimp mortality, hemolysis, the presence of a hemolysin gene (vhh), and a gene involved in the type III secretion system (the Vibrio calcium response gene vcrD). V harveyi HY01 was isolated from a shrimp that died from vibriosis, and 36 other V. harveyi isolates were obtained from fish and shellfish in Hat Yai city, Thailand. An ocean isolate of V. harveyi BAA-1116 was also included. Thirteen isolates including V harveyi HYO1 caused shrimp death 12 h after injection. Most V harveyi isolates in this group (designated as Group A) caused hemolysis on prawn blood agar. None of the shrimp died after injection with V harveyi BAA-1116. Molecular analysis of all V harveyi isolates revealed the presence of vcrD in both pathogenic and non-pathogenic strains. Although vhh was detected in all V harveyi isolates, some isolates did not cause hemolysis, indicating that vhh gene expression might be regulated. Analysis of the V harveyi HYO1 genome revealed a V cholerae like-hemolysin gene, hlyA (designated as hhl). Specific primers designed for hhl detected this gene in 3 additional V harveyi isolates but the presence of this gene was not correlated with pathogenicity. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity in all V harveyi isolates, and there were no correlations among the hhl-positive isolates or the pathogenic strains.

Identification of vhhP2, a novel genetic marker of Vibrio harveyi, and its application in the quick detection of V. harveyi from animal specimens and environmental samples

To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins. A gene (vhhP2) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24 V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non-V. harveyi species, including V. parahaemolyticus and V. alginolyticus. A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2. This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii, which is most closely related to V. harveyi. One of the V. campbellii strains was falsely identified as V. harveyi. vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non-V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi. However, this method can not distinguish some V. campbellii strains from V. harveyi. the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.

Prevalence and Heterogeneity of Hemolysin Gene vhh Among Hatchery Isolates of Vibrio harveyi in India

Vibrio harveyi, pathogenic to fish, harbor a hemolysin gene vhh, the homologues of which are found in many species of the Genus Vibrio. In this study, we investigated the prevalence of vhh gene among V. harveyi isolated from Penaeus monodon hatcheries in India by polymerase chain reaction (PCR). The vhh was detected in 67 of the 70 V. harveyi isolates tested in this study using different combinations of PCR primers. A variant vhh gene detected in a minority of strains was cloned, sequenced, and the recombinant protein was expressed in Escherichia coli. The deduced amino acid sequence of the cloned gene was 86% similar to the previously reported amino acid sequences of VHH. The results of this study suggest that though V. harveyi strains invariably harbor vhh, the sequence variants of the hemolysin gene exist that may impede their detection by PCR.

Distribution of five vibrio virulence-related genes among Vibrio harveyi isolates

Distribution of five vibrio virulence-related genes among Vibrio harveyi isolates

Luminescence, virulence and quorum sensing signal production by pathogenic Vibrio campbellii and Vibrio harveyi isolates

To study the relationship between luminescence, autoinducer production and virulence of pathogenic vibrios. Luminescence, quorum sensing signal production and virulence towards brine shrimp nauplii of 13 Vibrio campbellii and Vibrio harveyi strains were studied. Although only two of the tested strains were brightly luminescent, all of them were shown to produce the three different types of quorum sensing signals known to be produced by Vibrio harveyi. Cell-free culture fluids of all strains significantly induced bioluminescence in the cholerae autoinducer 1, autoinducer 2 and harveyi autoinducer 1 reporter strains JAF375, JMH597 and JMH612, respectively. There was no relation between luminescence and signal production and virulence towards brine shrimp. There is a large difference between different strains of Vibrio campbellii and Vibrio harveyi with respect to bioluminescence. However, this is not reflected in signal production and virulence towards gnotobiotic brine shrimp. Moreover, there seems to be no relation between quorum sensing signal production and virulence towards brine shrimp. The results presented here indicate that strains that are most brightly luminescent are not necessarily the most virulent ones and that the lower virulence of some of the strains is not due to a lack of autoinducer production.

Biocontrol of pathogens in shrimp hatcheries using bacteriophages

Microflora associated with larval stages of shrimp could affect the health and development of the larvae. Some bacteria such as luminous Vibrio harveyi cause serious mortalities. Consequent to the ban on use of most antibiotics in aquaculture, there is a need for alternate technologies for control of bacterial pathogens. Bacteriophages have a potential to control bacterial pathogens. This paper reports isolation of lytic bacteriophages against V. harveyi. Four bacteriophages were isolated, three from oyster tissue and one from shrimp hatchery water. The bacteriophages lysed 55–70% of the 100 V. harveyi isolates tested. Two bacteriophages subjected to further study were found to belong to family Siphoviridae and were effective in reducing V. harveyi population in biofilm formed on high density polyethylene (HDPE) surface. In hatchery trials, bacteriophage treatment at 2 × 10 6 pfu ml − 1 level resulted in over 85% survival of Penaeus monodon larvae suggesting that bacteriophage therapy would be an effective alternative to antibiotics in shrimp hatcheries.

Phenotypic and molecular typing ofVibrio harveyiisolates and their pathogenicity to tiger shrimp larvae

The objective of the present study was to identify the biotype(s) and molecular type(s) of Vibrio harveyi associated with pathogenicity in tiger shrimp (Penaeus monodon) larvae. Five luminescent and four nonluminescent V. harveyi isolates were subjected to phenotyping and random amplified polymorphic DNA (RAPD) fingerprinting, and pathogenicity testing to P. monodon mysis. Four isolates induced 34-41% mortality of P. monodon mysis when challenged at the rate of 10(6) CFU ml(-1) within 60 h. Sucrose-fermenting biotypes of V. harveyi appeared to be associated with pathogenicity to larval shrimp. Higher temperature and salinity appeared to play a role on the onset of vibriosis and mortality in the challenged larval shrimp. Pathogenic isolates of V. harveyi could be demarcated as revealed by their clustering in the dendrogram constructed based on the RAPD fingerprints. Nonluminescent V. harveyi also appear to be important aetiological agents of vibriosis of shrimp larvae. Sucrose-fermenting biotypes are likely to be pathogenic. High temperature may trigger onset of vibriosis. Biotyping of V. harveyi isolates and looking for traits, such as ability to ferment sucrose may be helpful in identifying the pathogenic forms, and such approach requires to be investigated further with larger number of isolates.