Pathogenic bacteria prevalence in cultured Nile tilapia in Southwest Mexico: A real-time PCR analysis.

The present study investigates molecular-based PCR techniques to estimate the prevalence of fish pathogens in southwest Mexico where recurrent mortality in the tilapia cultures has been observed. Sample of internal organs and lesions of Nile tilapia were taken and analysed in 2018, 2019, 2020 and 2022 to detect bacterial pathogens using PCR. No samples were taken in 2021 due to the COVID-19 pandemic. The real-time PCR conditions were optimized to allow a qualitative reliable detection of the bacteria from fixed fish tissue. A total of 599 pond- and cage-cultured tilapia from the southwestern Mexican Pacific (Guerrero, Oaxaca and Chiapas states) were analysed. In this tropical region, during 2018 and 2019 water temperatures of the tilapia cultures were generally with the optimal range to grow Nile tilapia, although extreme values were recorded on some farms. Most of the tilapia sampled were apparently healthy. No Francisella sp. was detected in any sample, and Staphylococcus sp. was the most prevalent (from 0% to 64%) bacteria from the three states over time. Low prevalence of Aeromonas sp. was found, from 0% to 4.3%, although the fish pathogen Aeromonas dhakensis was not detected. Sterptococcus iniae was only detected in Chiapas in 2019 at a low prevalence (1.4%), while the major tilapia pathogen S. agalactiae was detected at a high prevalence (from 0% to 59%) in the three Mexican states. This is the first detection of these pathogenic bacteria in rural farms using real-time PCR and constitutes a great risk for tilapia aquaculture in Mexico, as well as a potential dispersion of these pathogens to other aquaculture areas.

Photobacterium lucens sp. nov., Isolated from a Cultured Shrimp Penaeus vannamei.

Two bacterial strains were isolated from the hepatopancreas of a cultured shrimp (Penaeus vannamei) in Sinaloa, México. Their partial 16S rRNA gene sequences clustered within those of the genus Photobacterium, showing high similarity to the type strains of Photobacterium angustum and Photobacterium leiognathi, were 87.1% and 97.5%, respectively. Multilocus sequence analysis using eight housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and 16S rRNA) and phylogenetic analysis with 139 single-copy genes showed that the new strains form an independent branch whole genome sequencing and genomic analyses (average nucleotide identity, average amino acid identity, and in silico DNA-DNA hybridization) produced values well below the thresholds for species delineation with all methods tested. In addition, a phenotypic characterization was performed to support the description and differentiation of the novel strains from related taxa. The results obtained demonstrate that the two strains represent a novel species for which the name Photobacterium lucens sp. nov. is proposed.

Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp (Litopenaeus vannamei) in Northwestern Mexico.

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.

Virulence of Vibrio harveyi responsible for the "Bright-red" Syndrome in the Pacific white shrimp Litopenaeus vannamei.

Vibrio harveyi (Vh) CAIM 1792 strain was isolated from Litopenaeus vannamei affected with "Bright-red" Syndrome (BRS). The strain grew in 1-10% NaCl, at 15-35°C and was resistant to ampicillin (10 µg), carbenicillin (100 µg) and oxytetracycline (30 µg). The lowest MIC was for enrofloxacine (0.5 µgml(-1)). The in vivo and in vitro toxicity of bacterial cells and the extracellular products (ECPs) of Vh CAIM 1792 grown at 1.0%, 2.0% and 4.0% NaCl were evaluated. Adherence ability, enzymatic activities and siderophore production of bacterial cell was tested. The ECPs exhibited several enzymatic activities, such as gelatinase, amylase, lipase, phospholipase and caseinase. These ECPs displayed a strong cytotoxic effect on HELA cell line at 6 and 24 h. Challenges using 10(3) CFU g(-1) caused opacity at the site of injection and over 80% shrimp mortality before 24 h p.i. (post-injection). Mortality caused by the ECPs was higher than mortalities with bacteria, especially in the first hours p.i. Bacteria were re-isolated from hemolymph samples of moribund shrimp and identified as Vh CAIM 1792 by rep-PCR. Histological analysis of shrimp L. vannamei injected with Vh CAIM 1792 revealed generalized necrosis involving skeletal muscle (MU) at the injection site, the lymphoid organ (LO), heart and connective tissues. Melanization within the MU at the site of injection was also observed as well as hemocytic nodules within the hearth and MU at 168 h p.i. LO was the target organ of BRS. Necrosis of the MU at the injection site was the main difference in comparison to other shrimp vibriosis.

New Insights into the Mechanism of Action of PirAB from Vibrio Parahaemolyticus.

PirAB toxins secreted by Vibrio parahaemolyticus (Vp) harbor the pVA1 virulence plasmid, which causes acute hepatopancreatic necrosis disease (AHPND), an emerging disease in Penaeid shrimp that can cause 70-100% mortality and that has resulted in great economic losses since its first appearance. The cytotoxic effect of PirABVp on the epithelial cells of the shrimp hepatopancreas (Hp) has been extensively documented. New insights into the biological role of the PirBVp subunit show that it has lectin-like activity and recognizes mucin-like O-glycosidic structures in the shrimp Hp. The search for toxin receptors can lead to a better understanding of the infection mechanisms of the pathogen and the prevention of the host disease by blocking toxin-receptor interactions using a mimetic antagonist. There is also evidence that Vp AHPND changes the community structure of the microbiota in the surrounding water, resulting in a significant reduction of several bacterial taxa, especially Neptuniibacter spp. Considering these findings, the PirABvp toxin could exhibit a dual role of damaging the shrimp Hp while killing the surrounding bacteria.

The Vibrio parahaemolyticus subunit toxin PirBvp recognizes glycoproteins on the epithelium of the Penaeus vannamei hepatopancreas.

Vibrio parahaemolyticus toxin PirABvp is the major virulence factor exotoxin that contributes to the disruption of the hepatopancreatic epithelium in acute hepatopancreatic necrosis disease in shrimp. The PirBvp subunit is a lectin that recognizes amino sugars; however, its potential role in recognition of the hepatopancreas has not been identified. In the present work, we identified the cellular receptor for PirBvp in the shrimp hepatopancreas. A ligand blot assay of hepatopancreas lysate showed that the PirBvp subunit recognizes two glycoprotein bands of 60 and 70 kDa (Gc60 and Gc70). The hepatopancreas lysate was fractionated by anion-exchange chromatography, and the three main fractions obtained contained the recognized Gc60 and Gc70 protein bands. LC-MS/MS indicated that beta-hexosaminidases subunit beta and mucin-like 5 AC corresponded to the 60 and 70 kDa bands, respectively, which seem to be expressed in the epithelial cells of the hepatopancreas. Endoglycosidase treatment of hepatopancreas lysate with the O-glycosidase from Enterococcus faecalis, inhibits the binding of PirBvp. Altogether, these results suggest the relevance of the interaction of PirBvp with the hepatopancreas in the pathogenesis of acute hepatopancreatic necrosis disease in shrimp.

'Bright-red' syndrome in Pacific white shrimp Litopenaeus vannamei is caused by Vibrio harveyi.

Since July 2005, recurrent outbreaks of vibriosis have occurred in shrimp farms in northwestern Mexico. Moribund Litopenaeus vannamei associated with mass mortalities were lethargic and displayed red discoloration spots on their abdomen, and hence were called 'bright-reds' by farmers. Shrimp submitted for diagnosis were examined using wet tissue mounts, bacteriological assays and their respective minimum inhibitory concentration (MIC), and histology. A dominant yellow bacterial colony was isolated in thiosulphate citrate bile salts-sucrose (TCBS) agar and identified by molecular methods as Vibrio harveyi strain CAIM 1792. Pathogenicity of the V. harveyi strain was demonstrated in L. vannamei. The lowest MIC against Vibrio isolates from bright-red shrimp was obtained with enrofloxacine (3.01, SD = 5.96 pg ml(-1)). Histology detected severe necrosis in lymphoid organ tubules, muscle fibers, and connective tissue, as well as melanization and hemocytic nodules associate with microcolonies of Gram-negative bacilli. Bacteria from severely affected shrimp were dispersed from the haemocoel to other tissues causing a systemic vibriosis. The data indicate that V. harveyi strain CAIM 1792 is the cause of bright-red syndrome (BRS) and represents a threat to the Mexican shrimp farming industry.