PCR and in situ hybridization for the detection and localization of a new pathogen Francisella-like bacterium (FLB) in ornamental cichlids

Archived formalin-fixed, paraffin-embedded tissues from 28 diseased ornamental cichlid fish associated with visceral granulomas were examined by polymerase chain reaction (PCR) and in situ hybridization (ISH) for detection of Francisella-like bacteria (FLB). The 16S rDNA FLB-specific primer pair 180f/465r was used on naturally infected ornamental cichlids, resulting in 11 positive cases (39%). Using DNA probes, all 28 cases (100%) showed a positive reaction, and most labeled cells were observed in the visceral granulomas of infected individuals. FLB was detected in cells morphologically resembling epithelioid and endothelioid macrophages. ISH was more sensitive than PCR or routine histopathological examination, based on the examination of archived formalin-fixed, paraffin-embedded tissues in this study. Furthermore, this technique located a new fish pathogen, FLB, in ornamental cichlids. The causative agent was similar to the pathogen inducing systemic granulomas in tilapia.