A Two‐Step PCR Workflow Reveals Predominance of <i>Flavobacterium covae</i> in Ornamental Fish With Clinical Signs of Columnaris Disease

Columnaris is an important bacterial disease of freshwater fish and is increasingly recognised as being caused by a species complex historically referred to as Flavobacterium columnare. A two-step molecular workflow was applied to ornamental fish with clinical signs of columnaris disease from a multispecies commercial distributor operating a shared recirculating system in São Paulo, Brazil. Skin and fin lesion scrapings were Gram stained, and lesion-derived tissue DNA was screened by ISR-PCR for the columnaris disease complex. ISR-PCR yielded the expected amplicon in 61/123 samples (49.6%) from 28 ornamental fish species. All ISR-PCR-positive samples were then analysed by multiplex PCR for species assignment. Group-specific amplicons were obtained from 29/61 samples (47.5%): Flavobacterium covae in 27/61 (44.3%) and F. davisii in 2/61 (3.3%); no GG1 or GG4 amplicons were detected. The remaining 32/61 samples (52.5%) were not typeable by multiplex PCR. These findings provide baseline species-level data and support combined molecular surveillance in multispecies ornamental fish systems.