Identification, characterization and complete genome analysis of a Vibrio anguillarum isolated from Sebastes schlegelii.

Vibrio anguillarum is an important fish pathogen in mariculture, which can infect fish with great economic losses. In this study, a Vibrio anguillarum isolated from Sebastes schlegelii was named VA1 and was identified and characterized from aspects of morphology, physiological and biochemical characteristics, 16SRNA, virulence genes, drug sensitivity, and extracellular enzyme activity. At the same time, The VA1 was investigated at the genomic level. The results showed that a Gram-negative was isolated from the diseased fish. The VA1 was characterized with uneven surface and visible flagella wrapped in a sheath and microbubble structures. The VA1 was identified as Vibrio anguillarum based on the 16S RNA sequence and physiological and biochemical characteristics. The VA1 carried most of the virulence genes (24/29) and was resistant to penicillin, oxacillin, ampicillin, cefradine, neomycin, pipemidic acid, ofloxacin, and norfloxacin. The pathogenicity of the isolated strain was confirmed by an experimental analysis, and its LD50 was 6.43 × 106 CFU/ml. The VA1 had the ability to secrete gelatinase, protease, and amylase, and it had α-hemolysis. The whole genome size of the VA1 was 4232328bp and the G + C content was 44.95 %, consisting of two circular chromosomes, Chromosome1 and Chromosome2, with no plasmid. There were 1006 predicted protein coding sequences (CDSs). A total of 526 genes were predicted as virulence-related genes which could be classified as type IV pili, flagella, hemolysin, siderophore, and type VI secretion system. Virulence genes and correlation data were supported with the histopathological examination of the affected organs and tissues. 194 genes were predicted as antibiotic resistance genes, including fluoroquinolone antibiotic, aminoglycoside antibiotic, and beta-lactam resistant genes, which agreed with the results of the above drug sensitivity, indicating VA1 to be a multidrug-resistant bacterium. This study provided a theoretical basis for a better understanding of pathogenicity and antibiotic resistance, which might contribute to the prevention of V. anguillarum in the future.

Shewanella phaeophyticola sp. nov. and Vibrio algarum sp. nov., isolated from marine brown algae.

Two Gram-stain-negative, rod-shaped bacteria, designated as strains KJ10-1T and KJ40-1T, were isolated from marine brown algae. Both strains were catalase-positive, oxidase-positive, and facultative aerobic. Strain KJ10-1T exhibited optimal growth at 25 °C, pH 7.0, and 3 % NaCl, whereas strain KJ40-1T showed optimal growth at 25 °C, pH 7.0, and 2 % NaCl. The respiratory quinones of strain KJ10-1T were ubiquinone-8, ubiquinone-7, menaquinone-7, and methylated menaquinone-7, while the respiratory quinone of strain KJ40-1T was only ubiquinone-8. As major fatty acids, strain KJ10-1T contained C16 : 0, C17 : 1 ω8c, iso-C15 : 0, and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and strain KJ40-1T contained C16 : 0 and summed features 3 and 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The major polar lipids in strain KJ10-1T were phosphatidylethanolamine, phosphatidylglycerol, and an unidentified aminolipid, whereas those in strain KJ40-1T were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G+C contents of strains KJ10-1T and KJ40-1T were 42.1 and 40.8 mol%, respectively. Based on 16S rRNA gene sequences, strains KJ10-1T and KJ40-1T exhibited the closest relatedness to Shewanella saliphila MMS16-UL250T (98.6 %) and Vibrio rumoiensis S-1T (95.4 %), respectively. Phylogenetic analyses, based on both 16S rRNA and 92 housekeeping genes, showed that the strains formed distinct phylogenic lineages within the genera Shewanella and Vibrio. Digital DNA-DNA hybridization and orthologous average nucleotide identity values between strain KJ10-1T and other Shewanella species, as well as between strain KJ40-1T and other Vibrio species, were below the thresholds commonly accepted for prokaryotic species delineation. Based on the phenotypic, chemotaxonomic, and phylogenetic data, strains KJ10-1T and KJ40-1T represent novel species of the genera Shewanella and Vibrio, respectively, for which the names Shewanella phaeophyticola sp. nov. and Vibrio algarum sp. nov. are proposed, respectively. The type strains of S. phaeophyticola and V. algarum are KJ10-1T (=KACC 22589T=JCM 35409T) and KJ40-1T (=KACC 22588T=JCM 35410T), respectively.

The Description of Pseudoalteromonas apostichopi sp. nov., Vibrio apostichopi sp. nov., and Marinobacter apostichopi sp. nov. from the Fertilized Eggs and Larvae of Apostichopus japonicus.

Three novel bacterial strains, FE4T, FE10T, and LA51T, which are phylogenetically affiliated to the genera Pseudoalteromonas, Vibrio, or Marinobacter, respectively, isolated from fertilized eggs and juveniles of sea cucumber Apostichopus japonicus were characterized by a genome-based taxonomical approach including multilocus sequence analysis (MLSA) combined with classical phenotypic and chemotaxonomic characterizations. A molecular network reconstructed on the basis of nucleotide sequences of four phylogenetic maker protein genes revealed that the strains FE4T, FE10T, and LA51T were closely related to Pseudoalteromonas shioyasakiensis, Vibrio lentus, and Marinobacter similis, respectively. Average nucleotide identity (ANI) comparisons against phylogenetically related species to FE4T, FE10T, and LA51T demonstrated that each newly described strain could not be identified as any previously described species within each genus showing < 95% ANI: 91.3% of FE4T against P. shioyasakiensis JCM 18891 T, 92.6% of FE10T against "V. bathopelagicus" Sal10, and 92.6% of LA51T against M. similis A3d10T, in maximum, respectively. Here, we show molecular phylogenetic, genomic, phenotypic, and chemotaxonomic features of the newly described species FE4T, FE10T, and LA51T. We also propose Pseudoalteromonas apostichopi sp. nov. with FE4T (JCM 36173 T = LMG 33143 T) as the type strain, Vibrio apostichopi sp. nov. with FE10T (JCM 36174 T = LMG 33144 T) as the type strain, and Marinobacter apostichopi sp. nov. with LA51T (JCM 36175 T = LMG 33145 T) as the type strain.

New Insights into IgZ as a Maternal Transfer Ig Contributing to the Early Defense of Fish against Pathogen Infection.

IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ+ B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ+ B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class.

Environmental parameters associated with incidence and transmission of pathogenic Vibrio spp.

Vibrio spp. thrive in warm water and moderate salinity, and they are associated with aquatic invertebrates, notably crustaceans and zooplankton. At least 12 Vibrio spp. are known to cause infection in humans, and Vibrio cholerae is well documented as the etiological agent of pandemic cholera. Pathogenic non-cholera Vibrio spp., e.g., Vibrio parahaemolyticus and Vibrio vulnificus, cause gastroenteritis, septicemia, and other extra-intestinal infections. Incidence of vibriosis is rising globally, with evidence that anthropogenic factors, primarily emissions of carbon dioxide associated with atmospheric warming and more frequent and intense heatwaves, significantly influence environmental parameters, e.g., temperature, salinity, and nutrients, all of which can enhance growth of Vibrio spp. in aquatic ecosystems. It is not possible to eliminate Vibrio spp., as they are autochthonous to the aquatic environment and many play a critical role in carbon and nitrogen cycling. Risk prediction models provide an early warning that is essential for safeguarding public health. This is especially important for regions of the world vulnerable to infrastructure instability, including lack of 'water, sanitation, and hygiene' (WASH), and a less resilient infrastructure that is vulnerable to natural calamity, e.g., hurricanes, floods, and earthquakes, and/or social disruption and civil unrest, arising from war, coups, political crisis, and economic recession. Incorporating environmental, social, and behavioural parameters into such models allows improved prediction, particularly of cholera epidemics. We have reported that damage to WASH infrastructure, coupled with elevated air temperatures and followed by above average rainfall, promotes exposure of a population to contaminated water and increases the risk of an outbreak of cholera. Interestingly, global predictive risk models successful for cholera have the potential, with modification, to predict diseases caused by other clinically relevant Vibrio spp. In the research reported here, the focus was on environmental parameters associated with incidence and distribution of clinically relevant Vibrio spp. and their role in disease transmission. In addition, molecular methods designed for detection and enumeration proved useful for predictive modelling and are described, namely in the context of prediction of environmental conditions favourable to Vibrio spp., hence human health risk.

Population Analysis of Vibrio cholerae in Aquatic Reservoirs Reveals a Novel Sister Species (Vibrio paracholerae sp. nov.) with a History of Association with Humans.

Most efforts to understand the biology of Vibrio cholerae have focused on a single group, the pandemic-generating lineage harboring the strains responsible for all known cholera pandemics. Consequently, little is known about the diversity of this species in its native aquatic environment. To understand the differences in the V. cholerae populations inhabiting regions with a history of cholera cases and those lacking such a history, a comparative analysis of population composition was performed. Little overlap was found in lineage compositions between those in Dhaka, Bangladesh (where cholera is endemic), located in the Ganges Delta, and those in Falmouth, MA (no known history of cholera), a small coastal town on the United States east coast. The most striking difference was the presence of a group of related lineages at high abundance in Dhaka, which was completely absent from Falmouth. Phylogenomic analysis revealed that these lineages form a cluster at the base of the phylogeny for the V. cholerae species and were sufficiently differentiated genetically and phenotypically to form a novel species. A retrospective search revealed that strains from this species have been anecdotally found from around the world and were isolated as early as 1916 from a British soldier in Egypt suffering from choleraic diarrhea. In 1935, Gardner and Venkatraman unofficially referred to a member of this group as Vibrio paracholerae. In recognition of this earlier designation, we propose the name Vibrio paracholerae sp. nov. for this bacterium. Genomic analysis suggests a link with human populations for this novel species and substantial interaction with its better-known sister species. IMPORTANCE Cholera continues to remain a major public health threat around the globe. Understanding the ecology, evolution, and environmental adaptation of the causative agent (Vibrio cholerae) and tracking the emergence of novel lineages with pathogenic potential are essential to combat the problem. In this study, we investigated the population dynamics of Vibrio cholerae in an inland locality, which is known as endemic for cholera, and compared them with those of a cholera-free coastal location. We found the consistent presence of the pandemic-generating lineage of V. cholerae in Dhaka, where cholera is endemic, and an exclusive presence of a lineage phylogenetically distinct from other V. cholerae lineages. Our study suggests that this lineage represents a novel species that has pathogenic potential and a human link to its environmental abundance. The possible association with human populations and coexistence and interaction with toxigenic V. cholerae in the natural environment make this potential human pathogen an important subject for future studies.

Genome sequence of Vibrio fluvialis 362.3 isolated from coral Mussismilia braziliensis reveals genes related to marine environment adaptation.

Vibrio fluvialis is a halophilic bacterium frequently found in estuarine and coastal waters environments. The strain 362.3 was isolated from Mussismilia braziliensis coral of Abrolhos Bank. In this study, to gain insights into the marine adaptation in V. fluvialis, we sequenced the genome of 362.3 strain, which comprised 4,607,294 bp with a G + C content of 50.2%. In silico analysis showed that V. fluvialis 362.2 encodes genes related to chitin catabolic pathway, iron metabolism, osmotic stress and membrane transport.

Vibrio tetraodonis sp. nov.: genomic insights on the secondary metabolites repertoire.

Description of a Gram-negative, motile, circular-shaped bacterial strain, designated A511T obtained from the skin of the pufferfish Sphoeroides spengleri (Family Tetraodontidae), collected in Arraial do Cabo, Brazil. Optimum growth occurs at 20-28 °C in the presence of 3% NaCl. The genome sequence of the novel isolate consisted of 4.36 Mb, 3,976 coding genes and G + C content of 42.5%. Genomic taxonomy analyses based on average amino acid (AAI), genome-to-genome-distance (GGDH) and phylogenetic reconstruction placed A511T (= CBAS 712T = CAIM 1939T) into a new species of the genus Vibrio (Vibrio tetraodonis sp. nov.). The genome of the novel species contains eight genes clusters (~ 183.9 Kbp in total) coding for different types of bioactive compounds that hint to several possible ecological roles in the pufferfish host.

Vibrio aestuarianus subsp. cardii subsp. nov., pathogenic to the edible cockles Cerastoderma edule in France, and establishment of Vibrio aestuarianus subsp. aestuarianus subsp. nov. and Vibrio aestuarianus subsp. francensis subsp. nov.

Cockle mortality events have been reported in northern France since 2012. In the present study, we describe and investigate the implication of a potential bacterial causative agent in cockle mortality. Bacteria isolated from five different cockle mortality events were characterized and studied. Using phenotypic analysis combined with DNA-DNA hybridization (DDH) and whole genome sequencing, the isolates were shown to belong to Vibrio aestuarianus, a species regularly detected in France during oyster mortality events. Comparison of the strains from cockles with strains from French oysters and the type strain showed that the strains from cockles were genetically different to those from oysters and also different to the V. aestuarianus type strain. Moreover, the cockle and oyster strains were classified into two different, but close, groups both separated from the type strain by: (1) analyses of the ldh gene sequences; (2) DDH assays between 12/122 3T3T (LMG 31436T=DSM 109723T), a representative cockle strain, 02/041T (CIP 109791T=LMG 24517T) representative oyster strain and V. aestuarianus type strain LMG 7909T; (3) average nucleotide identity values calculated on the genomes; and (4) phenotypic traits. Finally, results of MALDI-TOF analyses also revealed specific peaks discriminating the three representative strains. The toxicity of representative strains of these cockle isolates was demonstrated by experimental infection of hatchery-produced cockles. The data therefore allow us to propose two novel subspecies of Vibrio aestuarianus: Vibrio aestuarianus subsp. cardii subsp. nov. for the cockle strains and Vibrio aestuarianus subsp. francensis subsp. nov. for the Pacific oyster strains, in addition to an emended description of the species Vibrio aestuarianus.

Vibrio agarilyticus sp. nov., an agar-digesting marine bacterium isolated from coastal seawater in Daya Bay (Guangdong, China).

A Gram-strain-negative, facultatively anaerobic, motile, rod-shaped and flagellated marine bacterium, designated SM6T, was isolated from surface seawater collected in Daya Bay (Guangdong, China). Phylogenetic analysis based on 16S rRNA gene sequences, multilocus sequence analysis, phylogenomic analysis of single-copy gene families and whole genome data showed that strain SM6T belonged to the genus Vibrio. The closest phylogenetic relatives of SM6T were Vibrio plantisponsor MSSRF60T (97.38 % 16S rRNA gene sequence pairwise similarity), Vibrio variabilis R-40492T (97.27 %), Vibrio aestuarianus ATCC 35048T (97.21 %) and Vibrio sagamiensis LC2-047T (97.3 %). Growth of strain SM6T occurred at 10-45 °C (optimum 30 °C), at pH 6.0-9.0 (optimum 6.0) and in the presence of 0-10 % (w/v) NaCl (optimum 3-8 %). The predominant fatty acids (>10 %) were summed feature 3 (C16 : 1 ω7c or/and C16 : 1 ω6c), C16 : 0 and summed feature 8 (C18 : 1 ω7c or/and C18 : 1 ω6c). The DNA G+C content of the assembled genomic sequences was 47.37 % for strain SM6T. Average nucleotide identity values between SM6T and its reference species were lower than the threshold for species delineation (95-96 %); in silico DNA-DNA hybridization further showed that the strains shared less than 70 % similarity. On the basis of evidence from the present polyphasic study, strain SM6T is considered to represent a novel species of the genus Vibrio, for which the name Vibrio agarilyticus sp. nov. is proposed. The type strain is SM6T (=KCTC 82076T=MCCC 1K04327 T).

Vibrio ulleungensis sp. nov., isolated from Mytilus coruscus.

A Gram-stain-negative, rod-shaped, motile via polar flagellum, facultatively aerobic, light-yellow, bacterium (designated 188UL20-2T) was isolated from a mussel sample of Mytilus coruscus collected on Ulleung Island, Ulleung-gun, Gyeongsangbuk-do, Republic of Korea. On the basis of 16S rRNA gene sequencing results, strain 188UL20-2T clustered with species of the genus Vibrio and appeared closely related to Vibrio marisflavi DSM 23086T (96.59%), Vibrio variabilis DSM 26147T (96.57%), Vibrio penaeicida DSM 14398T (96.37%) and Vibrio litoralis DSM 17657T (95.97%). The average nucleotide identity and digital DNA-DNA hybridization values between strain 188UL20-2T and its closest related strain were 71.3 and 16.4%, indicating that 188UL20-2T represents a novel species of the genus Vibrio. Growth occurred at 18-37 °C on MA medium in the presence of 1-4% NaCl (w/v) and at pH 5.0-10.0. The DNA G+C content of the genomic DNA was 45.4 mol%, and ubiquinone-8 (Q-8) was the major respiratory quinone. The major cellular fatty acids (>5%) were C16:1 ω6c and/or C16:1 ω7c (summed feature 3), C18:1 ω7c and/or C18:1 ω6c (summed feature 8), C16:0, C16:0 iso, C14:0, C14:0 iso and C12:0. The polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids, unidentified aminophospholipid, unidentified glycolipid and seven unidentified lipids. Physiological and biochemical characteristics indicated that strain 188UL20-2T represents a novel species of the genus Vibrio, for which the name Vibrio ulleungensis sp. nov. is proposed. The type strain is 188UL20-2T (=KACC 22258T=LMG 32202T).

Vibrio ziniensis sp. nov., isolated from mangrove sediments.

A novel Gram-staining-negative, catalase- and oxidase-positive, facultatively anaerobic and rod-shaped motile bacterial strain, designated as ZWAL4003T, was isolated from mangrove sediments of the Zini Mangrove Forest, Zhangzhou City, PR China. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that ZWAL4003T was grouped into a separated branch with Vibrio plantisponsor MSSRF60T (97.38% nucleotide sequence identity) and Vibrio diazotrophicus NBRC 103148T (97.27%). The major cellular fatty acids were C14 : 0 (12.6%), C16 : 0 (17.6%), and summed feature 3 (C16 : 1ω6c /C16 : 1 ω7c, 45.6%). Its genome had a length of 4650556 bp with 42.8% DNA G+C content, and contained genes involved in the biosynthesis of bacteriocin, ß-lactone, resorcinol, N-acyl amino acid, and arylpolyene. The in silico DNA-DNA hybridization and average nucleotide identity values for whole-genome sequence comparisons between ZWAL4003T and V. plantisponsor LMG 24470T were clearly below the thresholds used for the delineation of a novel species. The morphological and chemotaxonomic characteristics and the genotypic data of ZWAL4003T indicated that it represented a novel species of the genus Vibrio. Its proposed name is Vibrio ziniensis sp. nov., and the type strain is ZWAL4003T (=KCTC 72971T=MCCC 1A17474T).

Prevalence, antibiogram and virulence characterization of Vibrio isolates from fish and shellfish in Egypt: a possible zoonotic hazard to humans.

AIMS: Infection of seafood with pathogenic species of the genus Vibrio causes human food-borne illnesses. This study was executed to examine the antimicrobial resistance phenotypes, biofilm-forming capability and virulence-associated genes of Vibrio from fish and shellfishes. METHODS AND RESULTS: Three hundred fresh water and marine fish and shellfish samples were collected from wet markets and supermarkets in Mansoura, Egypt. Bacteriological examination and PCR amplification identified 92 Vibrio spp., including 42 Vibrio parahaemolyticus and 50 Vibrio alginolyticus isolates from the examined fish and shellfish (infection rate: 30·67%). However, V. vulnificus was not found in this study. Vibrio spp. exhibited variable frequencies of antimicrobial resistance with higher percentages to ampicillin and penicillin. Multidrug resistance (MDR) was detected in 69·04 and 38% of V. parahaemolyticus and V. alginolyticus respectively. PCR testing of virulence genes, tdh, trh and tlh revealed the presence of tlh and trh in 100 and 11·9% of V. parahaemolyticus isolates respectively and none of V. alginolyticus carried any of these genes. Biofilm-forming capability was displayed by 76% of V. parahaemolyticus and 73·8% of V. alginolyticus isolates. Both V. parahaemolyticus and V. alginolyticus showed nonsignificant weak positive correlations (r < 0·4) between antimicrobial pairs belonging to different classes; however, a significant positive correlation (P <0·05) between trh and resistance to erythromycin (r = 0·45) and imipenem (r = 0·38) was only identified in V. parahaemolyticus. CONCLUSIONS: This study reports the existence of MDR strains of V. parahaemolyticus and V. alginolyticus from the common types of fishes and shellfishes in Egypt. Furthermore, the presence of virulence genes in these isolates and the ability to produce a biofilm in vitro pose potential health hazards to consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: Frequent monitoring of seafood for the presence of Vibrio spp. and their antimicrobial susceptibility, virulence determinants and biofilm-forming capability is important for assessing the risk posed by these organisms to the public and for improving food safety.

Delineating the key virulence factors and intraspecies divergence of Vibrio harveyi via whole-genome sequencing.

Vibrio harveyi is one of the major pathogens in aquaculture. To identify the key virulence factors affecting pathogenesis of V. harveyi towards fish, we conducted a field investigation for three representative fish farms infected with V. harveyi. Multilocus sequence typing (MLST) and whole-genome sequencing were conducted to delineate the phylogenetic relationship and genetic divergence of V. harveyi. A total of 25 V. harveyi strains were isolated from the diseased fish and groundwater and were subtyped into 12 sequence types by MLST. Five virulence genes, mshB, pilA, hutR, ureB, and ureG, were variably presented in the sequenced strains. The virulence gene profiles strongly correlated with the distinct pathogenicity of V. harveyi strains, with a strain harboring all five genes exhibiting the highest virulence towards fish. Phenotype assay confirmed that reduced virulence correlated with decreased motility and biofilm formation ability. Additionally, three types of type VI secretion system, namely T6SS1, T6SS2, and T6SS3, were identified in V. harveyi strains, which can be classified into six, four, and 12 subtypes, respectively. In conclusion, the results indicated that the virulence level of V. harveyi is mainly determined by the above virulence genes, which may play vital roles in environmental adaptation for V. harveyi.

First description outside Europe of the emergent pathogen Vibrio europaeus in shellfish aquaculture.

Vibrio europaeus is an emergent pathogen affecting the most important bivalve species reared in Spanish and French hatcheries. Using a genomic approach, we identified V. europaeus outside Europe for the first time from massive larval mortalities of scallop (Argopecten purpuratus) in Chile and from seawater near a shellfish hatchery in the US West Coast. Results show the worldwide spreading and potential impact of V. europaeus for aquaculture; these four countries are among the 10 major producers of mollusks. Pathogenicity of V. europaeus was demonstrated for the first time towards scallop, the second most important species for Chilean mariculture.

Impact of a reduced water salinity on the composition of Vibrio spp. in recirculating aquaculture systems for Pacific white shrimp (Litopenaeus vannamei) and its possible risks for shrimp health and food safety.

Tropical shrimp, like Litopenaeus vannamei, in land-based recirculating aquaculture systems (RAS) are often kept at low water salinities to reduce costs for artificial sea salt and the amount of salty wastewater. Although these shrimp are tolerant against low salinities, innate immunity suppression and changes in the microbial composition in the water can occur. As especially Vibrio spp. are relevant for shrimp health, alterations in the species composition of the Vibrio community were analysed in water from six RAS, run at 15‰ or 30‰. Additionally, pathogenicity factors including pirA/B, VPI, toxR, toxS, vhh, vfh, tdh, trh, flagellin genes and T6SS1/2 of V. parahaemolyticus were analysed. The Vibrio composition differed significantly depending on water salinity. In RAS at 15‰, higher numbers of the potentially pathogenic species V. parahaemolyticus, V. owensii and V. campbellii were detected, and especially in V. parahaemolyticus, various pathogenicity factors were present. A reduced salinity may therefore pose a higher risk of disease outbreaks in shrimp RAS. Because some of the detected pathogenicity factors are relevant for human health, this might also affect food safety. In order to produce healthy shrimp as a safe food for human consumption, maintaining high water salinities seems to be recommendable.

PCR, in-situ hybridization, and phylogenetic analysis suggest that 'big belly' disease in barramundi, Lates calcarifer (Bloch), is caused by a novel Vibrio species.

'Big belly' disease is a chronic, granulomatous bacterial enteritis and peritonitis, first reported in 3- to 4-week-old Asian seabass or barramundi, Lates calcarifer Bloch fry. Affected fry are emaciated and have a swollen abdomen, and the condition is referred to as 'skinny pot-belly' or 'big belly' disease. In this study, histopathological examinations of diseased fish from a batch of 2-month-old, 6- to 8-cm L. calcarifer fingerlings, kept in seawater recirculating aquaculture systems, showed pathology resembling 'big belly' disease. Ethanol-fixed tissues were tested positive using specific PCR primers based on 16S rRNA genes. In situ hybridization using dioxygenin-labelled positive PCR products on formalin-fixed paraffin-embedded tissues showed positive reactions with intralesional, clusters of the large, 'big belly' coccobacilli. A phylogenetic tree constructed based on analyses of these 16S rRNA gene PCR products from five positive fish suggests that the 'big belly' bacterium is most likely a novel Vibrio species.

Characterization of a novel shrimp pathogen, Vibrio brasiliensis, isolated from Pacific white shrimp, Penaeus vannamei.

A novel pathogenic strain Vibrio 20190611023 was isolated from the hepatopancreas of moribund cultured Penaeus vannamei suffering from black gill disease. This strain was identified as V. brasiliensis based on the phylogenetic analyses of 16S rDNA gene and five other housekeeping genes (i.e., gapA, ftsZ, mreB, topA and gyrB). Some biochemical features of this strain were determined with an API 20NE system, and its haemolytic activity was determined using a sheep blood agar plate. The pathogenicity of this isolate 20190611023 was confirmed by the experimental challenge tests and histopathological examinations. P. vannamei were challenged via reverse gavage with different doses of bacterial suspensions. The calculated median lethal dose (LD50 ) was (3.16 ± 1.78) × 105  CFU/g (body weight). Moreover, antibiotic susceptibility tests were performed, the results of which showed that the strain 20190611023 was sensitive to chloramphenicol, compound sulphamethoxazole, ciprofloxacin, doxycycline and oxacillin, but resistant to erythromycin, kanamycin, gentamicin, cefoperazone, ceftriaxone, cefamezin and piperacillin. To our knowledge, this is the first report for demonstrating V. brasiliensis as a shrimp pathogen, which expands the host range of V. brasiliensis infection. The present study highlights that more attention should be paid to this novel pathogen in intensive shrimp aquaculture.

Non-O1/non-O139 vibrios - occurrence not only in Europe in recent years.

Non-O1/non-O139 vibrios refer to all vibrios except toxin producing Vibrio cholerae serogroups O1 and O139. The prevalence of illness caused by non-O1/non-O139 vibrios steadily increases all over the world in the last 20 years, which is very probably related to global warming. These infections are reported year-round from tropical and subtropical climate zones, but they were also detected in the mild climate zone of the United States of America and Europe. In mild climate, they have markedly seasonal occurrence, typically peaking in May to October. A human can be infected after ingestion of contaminated food, especially seafood and fish, or water or while bathing. In Europe, non-O1/non-O139 vibrios were detected in the Baltic Sea, North Sea and Mediterranean Sea but also in ponds and rivers. Depending on the pathogen entry route, the clinical manifestation may appear as gastroenteritis, otitis, wound infection or severe up to fatal illness, predominantly in immunocompromised patients. There is no specific prevention. Non-specific prevention includes good personal and food handling hygiene practices and avoiding contact of unhealed wounds with sea or surface swimming water. Given the severity and increasing frequency of infections caused by non-O1/non-O139 vibrios, they should be considered in differential diagnosis of gastrointestinal and wound infections, especially in patients with a history of consumption of fish and seafood or with a history of contact of unhealed wounds with sea or other open swimming water.

Zebrafish Models for Pathogenic Vibrios.

Vibrio is a large and diverse genus of bacteria, of which most are nonpathogenic species found in the aquatic environment. However, a subset of the Vibrio genus includes several species that are highly pathogenic, either to humans or to aquatic animals. In recent years, Danio rerio, commonly known as the zebrafish, has emerged as a major animal model used for studying nearly every aspect of biology, including infectious diseases. Zebrafish are especially useful because the embryos are transparent, larvae are small and facilitate imaging studies, and numerous transgenic fish strains have been constructed. Zebrafish models for several pathogenic Vibrio species have been described, and indeed a fish model is highly relevant for the study of aquatic bacterial pathogens. Here, we summarize the zebrafish models that have been used to study pathogenic Vibrio species to date.