Multiplex PCR for genotyping Flavobacterium columnare

Authors: Lafrentz, Benjamin; Garcia, Julio; Shelley, John

Submitted to: Journal of Fish Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/17/2019
Publication Date: 8/30/2019
Citation: Lafrentz, B.R., Garcia, J.C., Shelley, J.P. 2019. Multiplex PCR for genotyping Flavobacterium columnare. Journal of Fish Diseases. 42:1531-1542. https://doi.org/10.1111/jfd.13068.
DOI: https://doi.org/10.1111/jfd.13068

Interpretive Summary: Columnaris disease is caused by the bacterium Flavobacterium columnare, which has substantial impacts on almost all fin fish aquaculture industries in the United States including catfish, rainbow trout, tilapia, sport fish, baitfish, and ornamental fish. Columnaris disease is one of the top two bacterial pathogens impacting the catfish industry, in which the states of Mississippi and Alabama are the leading producers. Previous research in our laboratory established the existence of four distinct genetic groups within the species F. columnare; however, there are no quick and easy methods to assign an unknown isolate to one of the four genetic groups. In this research, we developed a molecular assay to quickly assign an isolate to genetic group. The results demonstrated that the assay is a rapid, sensitive, and specific molecular tool for genotyping F. columnare. The assay is relatively inexpensive and can be used by any laboratory with polymerase chain reaction (PCR) capabilities. The assay will be useful for facilitating the standard nomenclature for the genetic groups of F. columnare as well as for determining which genetic group(s) are responsible for disease losses in different aquaculture industries impacted by columnaris disease. This knowledge is important because our research has indicated biological relevance to the identified genetic diversity, with some genetic groups isolated preferentially from columnaris disease cases in specific fish species. An increased understanding of this will allow for the development of improved targeted control and treatment measures for columnaris disease.

Technical Abstract: Recent research has identified four distinct genetic groups among isolates of Flavobacterium columnare through multilocus phylogenetic analyses; however, there are no quick methods to determine the genotype of an isolate. The objective of this research was to develop a multiplex PCR to rapidly genotype F. columnare to genetic group. Comparative bacterial genomics was used to identify regions in the genomes unique to each genetic group and primers were designed to specifically amplify different sized amplicons for each genetic group. The optimized assay was demonstrated to be specific for each genetic group and F. columnare, and no specific amplicons were generated using gDNA from a panel of other Flavobacterium spp. and bacterial fish pathogens. The analytical sensitivity of the assay ranged from 209 to 883 genome equivalents depending on the genetic group. The multiplex PCR was evaluated by genotyping a panel of 22 unknown F. columnare isolates and performing DNA sequencing of the dnaK gene in parallel. The results demonstrated 100% accordance between multiplex PCR results and assignment to genetic group via phylogenetic analysis. The multiplex PCR provides a useful tool for assigning an unknown isolate to genetic group and may be used to determine which genetic groups of F. columnare are circulating and most predominant in different aquaculture industries.