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.

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.

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From healthy and diseased penaeid shrimp from Asia and the Americas, 25 luminous and 2 non-luminous bacterial strains were isolated, and 14 were phenotypically identified as Vibrio harveyi; 9 isolates produced significant mortalities (45 to 80%) in Artemia franciscana nauplii at inoculation densities of 10 5 to 10 6 CFU ml -1 compared to the controls (unchallenged nauplii). The maximum number of bacteria ingested (bioencapsulated) by the Artemia nauplii varied from less than 10 to 10 3 CFU nauplius -1 and no significant relationship was observed between the density of bacteria inoculated, the amount of bacteria ingested, and naupliar mortality. Significant correlations were obtained between naupliar mortality and production of proteases, phospholipases or siderophores, but not between mortality and lipase production, gelatinase production, hydrophobicity or hemolytic activity. The results suggest that virulence of the strains tested was more related to the production of particular exoenzymes than to the measured colonization factors.

Aims: To identify bacterial pathogens of diseased NiIe tilapia Oreochromis niloticus and determine their virulence. Methods and Results: Sixteen bacterial isolates were recovered from diseased Nile tilapias (O. niloticus) reared in floating cages in Adolfo Lopez Mateos (ALM), Sanalona and Dique IV dams in Sinaloa, Mexico, from February to May 2009. The bacterial isolates were identified by phenotypic and molecular (rep-PCR and 16S rRNA sequencing) methods and were mostly isolated from the kidneys and the brain of tilapias. Bacterial cells and extracellular products (ECPs) of strains were characterized and used in experimental infections with sole Solea vulgaris and Mozambican tilapia Oreochromis mossambicus. The fish challenged with Aeromonas dhakensis sp. nov. comb nov, Pseudomonas mosselii and Microbacterium paraoxydans (3Á1 9 10 6 CFU g À 1) exhibited mortality between 40 and 100% starting at 6 h postinoculation. The ECPs displayed gelatinase, haemolytic and cytotoxic activity, causing the total destruction of the HeLa cell lines. Conclusions: Aeromonas dhakensis and Ps. mosselii were virulent to O. mossambicus, whereas Mic. paraoxydans displayed virulence to S. vulgaris. Significance and Impact of the Study: This the first time that Aeromonas dhakensis and Ps. mosselii are reported as pathogens to tilapia and Mic. paraoxydans was isolated from fish; then, these fish pathogens could be a threat to farmed Nile tilapia in Mexico. IntroductionSeveral species of tilapia are cultured commercially, but Nile tilapia (Oreochromis niloticus) is the predominant cultured species worldwide (FAO 2012). Strains of Aeromonas hydrophila, Edwarsiella tarda, Pseudomonas fluorescens and Streptococcus sp. have been reported to be virulent to farmed tilapia around the world (Al-Harbi and Uddin 2005; El-Sayed 2006). The heterogeneity of Aeromonas populations has also been observed in wild and farmed freshwater fish (Burr et al. 2012). Aeromonas species, although they have been misidentified many times, are well-known agents of fish disease, and two major groups are recognized. Aeromonas salmonicida sensu stricto causes fish furunculosis, particularly in salmonids, and mesophilic species (Aer. hydrophila and Aer. veronii) cause a similar assortment of diseases in fish, including motile aeromonas septicaemia (MAS), red-sore disease and ulcerative infections in carp, tilapia, perch, catfish, salmon, cod and goby (Joseph and Carnahan 1994).Moreover, the genus Pseudomonas includes metabolically versatile organisms utilizing a wide range of organic compounds. The bacteria belonging to the genus Pseudomonas are present in most natural waters and infect a Journal of Applied Microbiology 115, 654--662