RESUMO
The enzyme nitric oxide synthase 2 or inducible NOS (NOS2), reactive oxygen species (ROS) and nitric oxide (NO) are important participants in various inflammatory and immune responses. However, the functional significances of the correlations among piscine NOS2, ROS and NO during pathogen infection remain unclear. In teleost, there are two nos2 genes (nos2a and nos2b). It has been previously reported that zebrafish nos2a behaves as a classical inducible NOS, and nos2b exerts some functions similar to mammalian NOS3. In the present study, we reported the functional characterization of zebrafish nos2a during bacterial infection. We found that zebrafish nos2a promoted bacterial proliferation, accompanied by an increased susceptibility to Edwardsiella piscicida infection. The nagative regulation of zebrafish nos2a during E. piscicida infection was characterized by the impaired ROS levels, the induced NO production and the decreased expressions of proinflammatory cytokines, antibacterial genes and oxidant factors. Furthermore, although both inducing ROS and inhibiting NO production significantly inhibited bacterial proliferation, only inhibiting NO production but not inducing ROS significantly increased resistance to E. piscicida infection. More importantly, ROS supplementation and inhibition of NO completely abolished this detrimental consequence mediated by zebrafish nos2a during E. piscicida infection. All together, these results firstly demonstrate that the innate response mediated by zebrafish nos2a in promoting bacterial proliferation is dependent on the lower ROS level and higher NO production. The present study also reveals that inhibition of NO can be effective in the protection against E. piscicida infection.
Assuntos
Edwardsiella , Infecções por Enterobacteriaceae , Animais , Citocinas , Peixe-Zebra , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Proliferação de Células , Edwardsiella/fisiologia , Mamíferos/metabolismoRESUMO
Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1 pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5 pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.
Assuntos
Aeromonas hydrophila/isolamento & purificação , Densovirinae/isolamento & purificação , Edwardsiella tarda/isolamento & purificação , Iridoviridae/isolamento & purificação , Microfluídica/métodos , Técnicas de Diagnóstico Molecular/veterinária , Técnicas de Amplificação de Ácido Nucleico/veterinária , Vibrio/isolamento & purificação , Vírus da Síndrome da Mancha Branca 1/isolamento & purificação , Animais , Crustáceos/microbiologia , Crustáceos/virologia , Infecções por Vírus de DNA/diagnóstico , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/microbiologia , Doenças dos Peixes/virologia , Peixes/microbiologia , Peixes/virologia , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Limite de Detecção , Técnicas de Diagnóstico Molecular/métodos , Moluscos/microbiologia , Moluscos/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Waterborne pathogens are becoming a serious worldwide health hazard; thus, the regular monitoring of epidemic pathogens is urgently required for public safety. In the present study, we developed a microfluidic chip integrated loop-mediated isothermal amplification technique (on-chip LAMP) to simultaneously detect 10 waterborne pathogenic bacteria, Campylobacter jejuni, Listeria monocytogenes, Salmonella enterica, Shigella flexneri, Staphylococcus aureus, Vibrio alginolyticus, V. cholerae, V. parahemolyticus, V. vulnificus, and Yersinia enterocolitica. This method was capable of simultaneously completing 22 genetic analyses of two specimens and achieved limits of detection ranging from 7.92 × 10-3 to 9.54 × 10-1 pg of genomic DNA of pure bacteria per reaction. The processes from sample loading to microfluidic operation were in a highly automated format, and the LAMP reaction ran to completion within 35 minutes, with a minimal volume of 22 µl per each half of a single chip. The coefficient of variation for the time-to-positive value was less than 0.1, indicating an excellent reproducibility of the dual-sample on-chip LAMP assay. The clinical sensitivity and specificity in analyses of coastal water samples were 93.1% and 98.0%, respectively, in comparison with traditional microbiological methods. Our established dual-sample on-chip LAMP assay provides an effective multiple-pathogen analysis of waterborne bacterial pathogens. This indicates that the method is applicable for on-site detection and routine monitoring of waterborne bacteria in aquatic environments.
Assuntos
Listeria monocytogenes , Microfluídica , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: We isolated bacterial strain named M-1 from ill Sciaenops ocellatus with ulceration, and further analyzed the phylogenetics of the bacterial strain. METHODS: We characterized the strain on the basis of its morphology, physiology, and sequence comparison of 16SrRNA and HSP60 genes. RESULTS: Strain M-1 was identified as Vibrio splendidus. 16SrRNA gene of strain M-1 and Vibrio splendidus (AJ874361) had nearest kinship, and the analysis of HSP60 gene sequence indicated that strain M-1 and Vibrio (EU653883, AY837440) located in the same phylogenetic tree. CONCLUSION: The results suggested that strain M-1 was a member of Vibrio splendidus.