Identification and Phylogenetic Analysis of Flavobacterium spp. Associated with Aquaculture Fish Diseased from Brazil

Due to the recent taxonomic reclassification of the species <i>Flavobacterium columnare</i> into four new species-<i>Flavobacterium columnare</i>, <i>Flavobacterium davisii</i>, <i>Flavobacterium covae</i>, and <i>Flavobacterium oreochromis</i>-it is necessary to re-evaluate isolates of previous outbreaks to better understand the epidemiology related to this bacterial group. Therefore, the aim of this study was to determine the taxonomic profile of Brazilian isolates of <i>Flavobacterium</i> spp. associated with columnaris disease using available diagnostic methods. Fifty isolates from different outbreaks (17 clinical cases) occurring in five different Brazilian states previously identified as <i>F. columnare</i> were selected and identified by multiplex PCR and MALDI-ToF methods. In addition, at least one isolate from each clinical case was analyzed by <i>16S rRNA</i> gene sequencing. After inclusion of the MSPs (main spectra profiles), the isolates were identifiable, and when compared with the multiplex PCR results, they showed almost perfect agreement (94.2% Kappa = 0.85). Only <i>F. davisii</i>, <i>F. covae</i>, and <i>F. oreochromis</i> were found among the Brazilian isolates, with these species causing disease in neotropical fish hosts not previously reported (e.g., Siluriformes, Serrasalmidae, and Bryconidae), while <i>F. columnare</i> was not detected. This study provides evidence of <i>Flavobacterium</i> species associated with columnaris disease circulating in various aquaculture facilities across different regions of Brazil. This information is crucial for developing control programs and advancing epidemiologic studies on columnaris disease in Brazilian aquaculture.