Moribund shrimp affected by acute hepatopancreatic necrosis disease (AHPND) from farms in northwestern Mexico were sampled for bacteriological and histological analysis. Bacterial isolates were molecularly identified as Vibrio parahaemolyticus by the presence of the tlh gene. The tdh -negative, trh -negative, and tlh -positive V. parahaemolyticus strains were further characterized by repetitive extragenic palindromic element-PCR (rep-PCR), and primers AP1, AP2, AP3, and AP and an ems2 IQ2000 detection kit (GeneReach, Taiwan) were used in the diagnostic tests for AHPND. The V. parahaemolyticus strains were used in immersion challenges with shrimp, and farmed and challenged shrimp presented the same clinical and pathological symptoms: lethargy, empty gut, pale and aqueous hepatopancreas, and expanded chromatophores. Using histological analysis and bacterial density count, three stages of AHNPD (initial, acute, and terminal) were identified in the affected shrimp. The pathognomonic lesions indicating severe desquamation of tubular epithelial cells of the hepatopancreas were observed in both challenged and pond-infected shrimp. The results showed that different V. parahaemolyticus strains have different virulences; some of the less virulent strains do not induce 100% mortality, and mortality rates also rise more slowly than they do for the more virulent strains. The virulence of V. parahaemolyticus strains was dose dependent, where the threshold infective density was 10 ⁴ CFU ml ⁻¹ ; below that density, no mortality was observed. The AP3 primer set had the best sensitivity and specificity. Field and experimental results showed that the V. parahaemolyticus strain that causes AHPND acts as a primary pathogen for shrimp in Mexico compared with the V. parahaemolyticus strains reported to date.

To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera; Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on (1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis); (2) clades amended since the 2007 proposal with recently described new species; (3) orphan clades of genomospecies F6 and F10; (4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3); and (5) description of V. tritonius sp. nov., which is a member of the “Porteresiae” clade.

Aims: To determine the relationship between yellow band disease (YBD)‐associated pathogenic bacteria found in both Caribbean and Indo‐Pacific reefs, and the virulence of these pathogens. YBD is one of the most significant coral diseases of the tropics. Materials and Results: The consortium of four Vibrio species was isolated from YBD tissue on Indo‐Pacific corals: Vibrio rotiferianus, Vibrio harveyi, Vibrio alginolyticus and Vibrio proteolyticus. This consortium affects Symbiodinium (zooxanthellae) in hospite causing symbiotic algal cell dysfunction and disorganization of algal thylakoid membrane‐bound compartment from corals in both field and laboratory. Infected corals have decreased zooxanthella cell division compared with the healthy corals. Vibrios isolated from diseased Diploastrea heliopora, Fungia spp. and Herpolitha spp. of reef‐building corals display pale yellow lesions, which are similar to those found on Caribbean Montastraea spp. with YBD. Conclusions: The Vibrio consortium found in YBD‐infected corals in the Caribbean are close genetic relatives to those in the Indo‐Pacific. The consortium directly attacks Symbiodinium spp. (zooxanthellae) within gastrodermal tissues, causing degenerated and deformed organelles, and depleted photosynthetic pigments in vitro and in situ. Infected Fungia spp. have decreased cell division compared with the healthy zooxanthellae: 4·9%vs 1·9%, (P ≥ 0·0024), and in D. heliopora from 4·7% to 0·7% (P ≥ 0·002). Significance and Impact of the Study: Pathogen virulence has major impacts on the survival of these important reef‐building corals around the tropics.

Fifty strains belonging to Vibrio harveyi, Vibrio campbellii, and the recently described Vibrio rotiferianus, were analysed using phenotypic and genomic techniques with the aim of analysing the usefulness of the different techniques for the identification of V. harveyi-related species. The species V. harveyi and V. campbellii were phenotypically indistinguishable by more than 100 phenotypic features. Thirty-nine experimental strains were phenotypically identified as V. harveyi, but FAFLP, REP-PCR, IGS-PCR and DNA–DNA hybridization proved that they in fact belong to the species V. campbellii. Similar groupings were found among all fingerprinting methodologies (except IGS-PCR). Thirty-two experimental strains clustered with the V. campbellii type and one reference strain; seven strains clustered with the V. harveyi type and three reference strains; and the type and four reference strains of V. rotiferianus grouped together. The correlations between DNA–DNA hybridization and the genomic fingerprinting by FAFLP and (GTG)₅-PCR were found to be above 0·68 and statistically significant, suggesting the value of the latter techniques for the reliable identification of V. harveyi-related species. The results presented indicate that strains phenotypically identified as V. harveyi are in fact V. campbellii; these findings position V. campbellii as an important species involved in diseases of reared aquatic organisms.

Summary: Three notable members of the Harveyi clade, Vibrio harveyi , Vibrio alginolyticus and Vibrio parahaemolyticus , are best known as marine pathogens of commercial and medical import. In spite of this fact, the discrimination of Harveyi clade members remains difficult due to genetic and phenotypic similarities, and this has led to misidentifications and inaccurate estimations of a species' involvement in certain environments. To begin to understand the underlying genetics that complicate species level discrimination, we compared the genomes of Harveyi clade members isolated from different environments (seawater, shrimp, corals, oysters, finfish, humans) using microarray‐based comparative genomic hybridization (CGH) and multilocus sequence analyses (MLSA). Surprisingly, we found that the only two V. harveyi strains that have had their genomes sequenced (strains BAA‐1116 and HY01) have themselves been misidentified. Instead of belonging to the species harveyi , they are actually members of the species campbellii . In total, 28% of the strains tested were found to be misidentified and 42% of these appear to comprise a novel species. Taken together, our findings correct a number of species misidentifications while validating the ability of both CGH and MLSA to distinguish closely related members of the Harveyi clade.