Validation of a quantitative PCR assay for the detection of 2 Flavobacterium columnare genomovars

Abstract

Flavobacterium columnare is the causative agent of columnaris disease in a variety of fish hosts. Using modifications to previously established protocols, a quantitative PCR (qPCR) assay was validated for the detection of 2 predominant F. columnare genomovars. The oligonucleotide primer and probe combination was designed to amplify a 203-bp region of the chondroitin AC lyase gene (GenBank AY912281) of F. columnare. There were no significant differences in amplification between genomovars. Comparable quantities of genomic DNA from 10 F. columnare strains, 5 representatives of each genomovar, produced similar results. Serial dilutions of purified PCR product demonstrated the limit of sensitivity for the assay was ~ 10 copies per reaction. The presence of gill and spleen tissue did not significantly affect the sensitivity of the assay. Comparably, bacterial DNA detected from the liver and kidney was less sensitive than pure bacterial DNA. However, detection from these tissues was within one order of magnitude of other tissues, indicating this reduction may have minimal analytic significance. This validated assay was used to approximate the minimum infectious dose for F. columnare isolate 94-081 in channel catfish and assess bacterial loads in gill and kidney tissues 48 h post-infection.

Evaluating Stress-Mediated Microbial Pathogenesis in Golden Shiners, Notemigonus crysoleucas

“…An additional concern is most media used in the bacterial plates are not exclusive to F. covae ; often, other bacteria associated with the fish and the environment are also cultured with the target bacteria making counts inaccurate. More recent methods of microbial pathogen identification and enumeration include immunofluorescence antibody technique (IFAT), quantitative polymerase chain reaction (qPCR; Gibbs et al, 2020 ), and immunomagnetic separation ( Fujiwara-Nagata and Eguchi 2009 ). These methods have been more accurate in detecting F. covae.…”