Molecular diagnosis of francisellosis, a systemic granulomatous inflammatory disease in Atlantic cod, Gadus morhua L

Abstract

A PCR-based assay for the detection of Francisella noatunensis causing francisellosis in Atlantic cod, Gadus morhua has been developed. Seven sets of primers targeting the flanking regions of the genes (rpoA, sdhA, atpA, rpoB, pgm, groEL and 16S rRNA) of the pathogen were designed. Among the primers, groEL was found to be the most suitable gene candidate for detecting the pathogen, due to its high sensitivity at various annealing temperatures and specificity in detection. The detection limit of the assay was 100 pg of bacterial DNA per milliliter or 100 fg bacterial DNA (approximately 50 genome equivalents) per PCR reaction, however, the sensitivity of the reaction decreased by 1 log dilution in the presence of 1 mg mL(-1) of serum and mucus samples as inhibitors. Nevertheless, the assay can potentially be used as a direct and non-lethal method to detect the pathogen in fish. Thus this PCR assay is a specific and sensitive molecular method to diagnose francisellosis in Atlantic cod, and will be helpful for controlling the infection through prompt detection of the disease in farms.

Real-time PCR assays targeting unique DNA sequences of fish-pathogenic Francisella noatunensis subspecies noatunensis and orientalis

“…noatunensis, but its specificity against F. noatunensis subsp. orientalis and other close relatives was not tested (Kulkarni et al 2011). Nevertheless, the presence of large numbers of closely related Francisella species in the environment (Barns et al 2005, Berrada & Telford 2010, Duodu et al 2012 should be borne in mind, particularly when identifying infection in species of fish that have been previously unexamined.…”