RESUMO
We developed a simple genotyping method for Flavobacterium psychrophilum for analysing two single nucleotide polymorphisms (SNPs) in the gyrA gene and to distinguish between isolates that are virulent and avirulent to ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel). The genotyping method is an on/off switch assay and is based on the polymerase chain reaction technique with phosphorothioated primers. We classified 232 isolates from four families of fish (i.e. Plecoglossidae, Osmeridae, Cyprinidae and Salmonidae) into four genotypes (G-C, A-T, A-C and G-T). The G-C type isolates exhibited strong pathogenicity to ayu, whereas the A-T and G-T types did not show any pathogenicity to this species. The A-C type exhibited no or weak pathogenicity to ayu. These results indicate that genotyping F. psychrophilum isolates with two SNPs from gyrA can clearly distinguish between isolates potentially harmful to ayu (G-C type) and those that are potentially not harmful or less harmful (A-C, A-T and G-T type). The on/off switch assay provides a quick, simple, and very powerful DNA genotyping technique for F. psychrophilum isolates.
Assuntos
Doenças dos Peixes/microbiologia , Infecções por Flavobacteriaceae/veterinária , Flavobacterium/genética , Flavobacterium/patogenicidade , Técnicas de Genotipagem/veterinária , Osmeriformes , Animais , Sequência de Bases , DNA Girase/genética , Doenças dos Peixes/mortalidade , Infecções por Flavobacteriaceae/microbiologia , Infecções por Flavobacteriaceae/mortalidade , Flavobacterium/classificação , Japão , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Análise de Sobrevida , Virulência/genéticaRESUMO
Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout fry syndrome of salmonids. The pathogen has been reported from all regions in the world involved in salmonid aquaculture, but also from natural fresh-water environments. We established a quantitative loop-mediated isothermal amplification of DNA (LAMP) method to estimate quantities of F. psychrophilum. LAMP primers were designed based on the sequence of the DNA topoisomerase IV subunit B gene, parE, of F. psychrophilum. parE LAMP exhibited a high specificity for the parE gene of F. psychrophilum but not for other related species. parE LAMP detected the gene in a wide range of concentrations from 2.0 x 10(1) to 2.0 x 10(9) copies/reaction within 70 min and revealed a good correlation between threshold times and gene copy number.
