Isolation, Identification, and Pathogenicity of Vibrio gigantis Retrieved from European Seabass (Dicentrarchus labrax) Farmed in Türkiye.

In this study, V. gigantis strain C24 was isolated from cases of winter mortalities of hatchery-reared European seabass (Dicentrarchus labrax) broodstock in Türkiye. The first mortalities were reported in September 2016 and occurred annually in early autumn/late winter until the end of February 2019, when 15% of accumulated mortality was recorded. Diseased moribund fish exhibited general septicemic signs, including dermal ulcerations with hemorrhagic margins, distended abdomens, and hemorrhages below the pectorals, pelvic fins, and at the operculum. Postmortem findings showed congestion in several internal organs, hemorrhagic ascitic fluid, and congested prolapsed anal openings. The representative bacterial isolate V. gigantis strain C24 was characterized as Gram-negative, motile, nitrite-producing, and as vibrio static agent O/129-sensitive. The full-length 16S rRNA sequence (Accession No. ON778781) and gyrB gene sequence (Accession No. ON792326) of the C24 strain showed high similarity to V. gigantis strains. Moreover, the whole-genome average nucleotide identity (ANI) values (ANI > 97.7%) against four V. gigantis strains above the species demarcation limit unambiguously identified the C24 isolate as a member of this species. A preliminary virulence-gene analysis showed that the V. gigantis isolate C24 encoded at least three exotoxins, including two aerolysins and a thermolabile hemolysin. The experimental infection showed that the C24 isolate exhibited low to moderate virulence in experimentally infected European seabass juveniles. Interestingly, antimicrobial susceptibility testing revealed that the C24 isolate was susceptible to nalidixic acid, ciprofloxacin, and several other antibiotics but resistant to tilmicosin, kanamycin, streptomycin, and ampicillin. To our knowledge, this study is the first to report that V. gigantis could be considered an emerging bacterial pathogen in Türkiye, and it may threaten the international European seabass production.

Epidemiological cut-off values for Vibrio anguillarum MIC and disc diffusion data generated by standardised methods.

This work aims to generate the data needed to set epidemiological cut-off values for minimum inhibitory concentration (MIC) and disc-diffusion zone measurements of Vibrio anguillarum. A total of 261 unique isolates were tested, applying standard methods specifying incubation at 28°C for 24-28 h. Aggregated MIC distributions for a total of 247 isolates were determined in 9 laboratories for 11 agents. Data aggregations of the disc zone for the 10 agents analysed contained between 157 and 218 observations made by 4 to 7 laboratories. Acceptable ranges for quality control (QC) reference strains were available for 7 agents and the related multi-laboratory aggregated data were censored, excluding the data of a laboratory that failed to meet QC requirements. Statistical methods were applied to calculate epidemiological cut-off values. Cut-off values for MIC data were calculated for florfenicol (≤1 µg ml-1), gentamicin (≤4 µg ml-1), oxytetracycline (≤0.25 µg ml-1) and trimethoprim/sulfamethoxazole (≤0.125/2.38 µg ml-1). The cut-off values for disc zone data were calculated for enrofloxacin (≥29 mm), florfenicol (≥27 mm), gentamicin (≥19 mm), oxolinic acid (≥24 mm), oxytetracycline (≥24 mm) and trimethoprim/sulfamethoxazole (≥26 mm). MIC and disc-diffusion zone data for the other agents where not supported by QC, thus yielding only provisional cut-off values (meropenem, ceftazidime). Regardless of whether QC is available, some of the aggregated MIC distributions (enrofloxacin, oxolinic acid), disc zone (sulfamethoxazole), and MIC and disc-diffusion distributions (ampicillin, chloramphenicol) did not meet the statistical requirements. The data produced will be submitted to the Clinical Laboratory Standards Institute for their consideration in setting international consensus epidemiological cut-off values.

The influence of diet on the microbiota of live-feed rotifers (Brachionus plicatilis) used in commercial fish larviculture.

Live-feed is indispensable to commercial fish larviculture. However, high bacterial loads in rotifers could pose a biosecurity risk. While this may be true, live-feed associated bacteria could also be beneficial to fish larvae through improved feed utilization or pathogen inhibition following host microbiota modification. The study objective was to elucidate the largely unexplored microbiota of rotifers propagated on five different diets through bacterial community profiling by 16S rRNA gene amplicon sequencing. Investigated rotifer samples had a median observed alpha-diversity of 338 ± 87 bacterial species. Alpha- and Gamma-Proteobacteria dominated the rotifer microbiota followed by members of classes Flavobacteriia, Cytophagia, Mollicutes, Phycisphaerae and Bacteroidia. Different diets significantly altered the bacterial communities associated with rotifers according to PERMANOVA test results and beta dispersion calculations. A common core rotifer microbiome included 31 bacterial species present in relative abundances over 0.01%. We discuss the functional role of some microbiome members. Our data suggested the presence of several known fish pathogens in stock rotifers. However, we found no evidence for increased loads of these presumptive taxa in propagated live-feed rotifers during this field trial.

Multilocus Sequence Analysis of Close Relatives Vibrio anguillarum and Vibrio ordalii.

UNLABELLED: The genetic heterogeneity of the close relatives Vibrio anguillarum and Vibrio ordalii, both serious pathogens of fish causing extensive losses in aquaculture, was studied. Eight housekeeping genes, i.e., atpA, ftsZ, gapA, gyrB, mreB, rpoA, topA, and pyrH, were partially sequenced in 116 isolates from diverse fish species and geographical areas. The eight genes appear to be under purifying selection, and the genetic diversity in the total data set was estimated to be 0.767 ± 0.026. Our multilocus sequence analysis (MLSA) scheme identified several widespread clonal complexes and resolved the isolates, for the most part, according to serotype. Serotype O2b isolates from diseased cod in Norway, Ireland, and Scotland were found to be extremely homogeneous. Horizontal gene transfer appears to be fairly common within and between clonal complexes. Taken together, MLSA and in silico DNA-DNA hybridization (DDH) calculations suggest that some isolates previously characterized as V ordalii, i.e., 12B09, FF93, FS144, and FS238, are in fact V. anguillarum isolates. The precise taxonomic situation for two isolates from Atlantic cod that display several traits consistent with V. ordalii, i.e., NVI 5286 and NVI 5918, and a single environmental strain that was previously considered to represent V. ordalii, i.e., FF167, is less clear. IMPORTANCE: It is still being debated whether V. anguillarum and V ordalii represent separate bacterial species. Our study addresses this issue and elucidates the degree of genetic variability within this group of closely related bacteria, based on a substantial number of isolates. Our results clearly illustrate the existence of different populations among putative V ordalii isolates. On the basis of additional full-length genomic analysis, we conclude that most environmental isolates previously identified as V ordalii lie firmly within the species V. anguillarum While bona fide fish-pathogenic V ordalii isolates display a very close genetic relationship with V. anguillarum, they combine a clearly divergent evolutionary pattern with clear phenotypic differences. The study also highlights the need for further characterization of fish-pathogenic isolates from the northern Atlantic region that share phenotypic characteristics with V. ordalii but are genetically closer to V. anguillarum The retention of taxonomic distinctions between the phenotypically different groups of bacteria is of practical advantage to microbial ecologists and veterinarians.

Effect of linear alkylbenzene sulphonate (LAS) on non-specific defence mechanisms in rainbow trout (Oncorhynchus mykiss).

Linear alkylbenzene sulphonate (LAS) is among the most widely disseminated xenobiotics to enter waste streams and the aquatic environment. In the present investigation, we present a novel approach to evaluate in toxicity of LAS. The effects of sublethal levels (0.2 and 0.4 mg/l) of LAS on non-specific immune system, phagocytosis, respiratory burst and lyzosyme activity, and specific growth rate in the rainbow trout, Oncorhynchus mykiss, during a 54-day exposure were examined by a static bioassay test procedure. The phagocytic activity of leukocytes from fish exposed to 0.4 mg/l LAS statistically decreased compared with the control fish values. No significant reductions were observed in the extra-intracellular respiratory burst and lysozyme activities after exposure to LAS at any of the concentrations tested. The final body weight in fish groups exposed to the LAS were found to be significantly lower than in the control. The specific growth rate results also supported the result above. The results of this study showed sublethal doses (0.2-0.4 mg/l) of LAS caused to statistically insignificant suppression of non-specific immune system mechanisms excluding phagocytosis in fish at laboratory conditions. These doses of LAS may produce potential synergism on immune system when presented with other environmental pollutants.