Detection of Vibrio anguillarum and Vibrio alginolyticus by Singleplex and Duplex Loop-Mediated Isothermal Amplification (LAMP) Assays Targeted to groEL and fklB Genes

Singleplex and duplex loop-mediated isothermal amplification (LAMP) assays were developed for detecting Vibrio anguillarum, a major bacterial pathogen of fish, and Vibrio alginolyticus, a pathogen of fish and humans, separately and simultaneously from contaminated seawater by targeting the groEL gene of V. anguillarum, which encodes a molecular chaperone protein, and the fklB gene of V. alginolyticus, which encodes a 22 kilodalton (kDa) peptidyl prolyl isomerase. The optimal reaction conditions to produce consistent results were 65°C for 30 min, 63°C for 30 min, and 63°C for 40 min for the groEL (singleplex for V. anguillarum), fklB (singleplex for V. alginolyticus), and groEL + flkB (duplex) LAMP assays, respectively, analyzed via visual detection methods (use of calcein, and SYBR Green I) and agarose gel electrophoresis. The assays were found to be species-specific, as closely related Vibrio spp. were not detected. The limits of detection (LoDs) of the LAMP assays for DNA template from pure culture and artificially contaminated seawater were 10 and 14 fg (groEL assay; for V. anguillarum), 12.5 and 17 fg (fklB assay; for V. alginolyticus), and 50 and 70 fg (duplex assay) per reaction, respectively, which were much better than the LoDs of conventional polymerase chain reaction (PCR). Singleplex and duplex LAMP assays were found to be rapid, species-specific, and sensitive for the detection of V. anguillarum and V. alginolyticus and are applicable to laboratory and field diagnostics

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Combination ecotoxicity and testing of common chemical discharges to sewer using the Vibrio fischeri luminescence bioassay / Ensayo y ecotoxicidad de productos químicos comúnmente vertidos al alcantarillado, usando el bioensayo de luminiscencia de Vibrio fischeri

In order to investigate possible synergistic or antagonistic (more or less than additive) toxicity effects, mixtures of chemicals were tested in water using a microbial bioassay. Ten toxicants (3,4-dichloroaniline, 3,5-dichlorophenol, cadmium, chromium, copper, Lindane, linear alkylbenzene sulphonate, pentachlorophenol, toluene, zinc) were chosen on the basis of their common occurrence in industrial effluents within local waste water treatment plants. These toxicants also cover a wide range of modes of toxic action, namely, polar and non-polar narcosis, membrane disruption, respiratory disruption, uncouplers of oxidative phosphorylation, biochemical disruption and enzyme inhibition. Efficient screening for possible combination toxicity between toxicants involved testing the chemicals both singly and in triplet combinations. The triplets were based on four replicates of a balanced incomplete block design (BIB). A standardised Vibrio fischeri rapid toxicity bioluminescence assay was used. The combinations tested showed that only one mixture was found to be significantly more toxic than expected from the pure single-toxicant results. Two triplets were significantly less toxic. Further tests on the more toxic triplet showed that the effect was due to only one of the 45 pairs originally screened. It is concluded that synergistic effects in combinations of toxicants are rather rare in bioluminescence systems utilising common effluents discharged to sewer (AU)

Para investigar si mezclas de productos químicos en agua tenían efectos tóxicos sinergísticos o antagónicos (superiores o inferiores a la adición, respectivamente), se utilizó un bioensayo microbiano. Se escogió un total de nueve tóxicos (3,4-dicloroanilina, 3,5-diclorofenol, cadmio, cromo, cobre, sulfonato de alquilbenceno lineal, pentaclorofenol, tolueno, zinc) basándose en la frecuencia con que se encuentran en depuradoras de aguas residuales industriales. Estos productos tóxicos asimismo cubren un amplio espectro de modos de toxicidad, como son narcosis polar y no polar, disrupción de membranas, disrupción de la respiración, desacoplación de la fosforilación oxidativa, disrupción bioquímica e inhibición enzimática. Se ensayaron los productos solos y de tres en tres, con tal de establecer su posible toxicidad combinada. Los tripletes se basaron en cuatro réplicas de un diseño balanceado de bloques incompletos (BIB). Se usó un ensayo estandarizado rápido de toxicidad basado en la bioluminiscencia de Vibrio fischeri. Las combinaciones estudiadas mostraron que solamente una de las mezclas era significativamente más tóxica de lo esperado a partir de los ensayos en solitario. Dos tripletes fueron significativamente menos tóxicos. Sucesivos tests sobre el triplete significativamente más tóxico mostraron que el efecto era debido solamente a uno de los 45 pares inicialmente considerados. El artículo concluye que los efectos sinergísticos al combinar diferentes productos tóxicos son poco frecuentes en sistemas de bioluminiscencia si nos referimos a aguas residuales comunes. (AU)

Hydrophobicity and adhesion to fish cells and mucus of Vibrio strains isolated from infected fish

The hydrophobicity of 44 Vibrio strains isolated from cultured, diseased gilt-head sea bream (Sparus aurata) was determined. Three different methods were used: (1) microbial adhesion to hydrocarbons (MATH), either with phosphate buffer or with phosphate urea magnesium sulfate (PUM) buffer, (2) aggregation in the presence of salt solutions (SAT), and (3) adhesion to nitrocellulose filters (NCF). The results show that experimental conditions exerted a significant influence on hydrophobicity. Thus, Kendall rank coefficients showed the presence of correlation only for SAT and NCF, and for SAT and the MATH assay with PUM buffer. Moreover, no relationships were observed between the bacterial hydrophobicity estimated with the methods mentioned above and the ability of the strains to adhere to fish mucus or cells. These results indicate that adhesion of pathogenic Vibrio strains to host surfaces is mediated mainly by specific receptor interactions, instead of by hydrophobic interactions (AU)

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Gastroenteritis por Vibrio mimicus: reporte de dos casos / Gastroenteritis por Vibrio mimicus: report of two cases

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