Multiplex PCR for rapid genotyping of Flavobacterium columnare

Authors: Lafrentz, Benjamin; Garcia, Julio

Submitted to: Annual Eastern Fish Health Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 3/20/2017
Publication Date: 3/30/2017
Citation: Lafrentz, B.R., Garcia, J.C. 2017. Multiplex PCR for rapid genotyping of Flavobacterium columnare. Annual Eastern Fish Health Workshop, East Lansing, Michigan, April 3-7, 2017. p. 65.

Interpretive Summary:

Technical Abstract: Columnaris disease, caused by the Gram-negative bacterium Flavobacterium columnare, is one of the leading causes of disease losses to the catfish industry in the Southeast USA. Recent research in our laboratory has deciphered the genetic diversity among F. columnare isolates through whole genome sequencing and four phylogenetically distinct groups were identified. The four genetic groups were highly correlated with genomovar as determined by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene. There is a lack of knowledge of which genetic group(s) is most prevalent in the industry and a better understanding of strain diversity and prevalence will aid in the development of better targeted control measures. The objective of this research was to develop a multiplex PCR to rapidly genotype F. columnare and use the assay to determine the predominant genotype(s) in the catfish industry. Comparative bacterial genomics was used to identify regions in the genomes unique to each genetic group to exploit for developing the assay. PCR primers were designed to amplify different sized amplicons specific for each genetic group. The designed primers were multiplexed and PCR conditions were optimized using purified gDNA as template. Once optimized, the multiplex PCR was tested on gDNA from a panel of F. columnare isolates of unknown genetic group and RFLP analysis was conducted in parallel. The results demonstrated 100% accordance between multiplex PCR results and genomovar assignment. Due to the fastidious nature of F. columnare and difficulty in culturing the bacterium, research was conducted to determine if DNA extracted from necrotic gill tissue and swabs taken from lesions of catfish exhibiting columnaris disease would serve as suitable template for use in the multiplex PCR assay. The multiplex PCR successfully amplified the proper sized amplicon from DNA extracted from both gill tissue and swabs of lesions from laboratory catfish challenged with a F. columnare isolate of known genetic group. The multiplex PCR reported here provides a useful tool for rapidly assigning an unknown isolate to genetic group and for determining the genetic group of F. columnare involved in individual diagnostic cases without the need for bacterial culture. Currently we are collaborating with various diagnostic laboratories in the Southeast USA, in which the labs are collecting swab samples from industry columnaris cases to determine which genetic groups of F. columnare are circulating and most predominant in the catfish industry. An understanding of this will allow for a more focused effort to develop efficacious control and treatment measures for columnaris disease.