Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment

Authors: Lafrentz, Benjamin; Waldbieser, Geoffrey - Geoff; Welch, Timothy - Tim; Shoemaker, Craig

Submitted to: Journal of Fish Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/12/2013
Publication Date: 10/22/2013
Publication URL: http://handle.nal.usda.gov/10113/60392
Citation: Lafrentz, B.R., Waldbieser, G.C., Welch, T.J., Shoemaker, C.A. 2014. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and standard protocol for genomovar assignment. Journal of Fish Diseases. 37(7):657-669.

Interpretive Summary: Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and may be emerging as an important pathogen in the rainbow trout industry. The 16S rRNA gene is a housekeeping gene commonly used for bacterial taxonomy and genotyping. Genetic variability in the sequence of this gene has been demonstrated among isolates of F. columnare. A genetic typing system has been developed for F. columnare in which a portion of the 16S rRNA gene is amplified by PCR and then digested with an enzyme that will cut the DNA at specific sites. Based on the number and size of DNA fragments generated, isolates of the bacterium are assigned to a genomovar (i.e., genetic type). However, interpretation of results can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated. In this study, partial 16S rRNA gene sequences (ca. 1250 bp fragment) from isolates representing each described genomovar and isolates generating unique DNA fragment patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different patterns due to DNA nucleotide variability at the sites where the enzyme cuts the DNA. This research provides a standard protocol for the genetic typing of F. columnare and a formal description of the expected number and sizes of DNA fragments generated for each genomovar. The new knowledge obtained from this research will allow for the proper assignment of an unknown isolate to a genetic type. Use of the standard protocol will allow for monitoring of the different genetic types in aquaculture reared fish species, which is important because research has suggested an association between genetic type of F. columnare and virulence.

Technical Abstract: Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250 bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B, and III. Additionally, we describe a new genomovar, I/II.