Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia

Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (~10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish.