|Ismaiel, Ed - Ed|
Submitted to: Molecular and Cellular Probes
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/2/2004
Publication Date: 12/1/2004
Citation: Darwish, A.M., Ismaiel, A.A., Newton, J.C., Tang, J. 2004. Identification of Flavobacterium columnare by a species-specific polymerase chain reaction and renaming of atcc43622 strain to Flavobacterium johnsoniae. Molecular and Cellular Probes. 8(6):421-427. Interpretive Summary: Flavobacterium columnare affects many fish species worldwide and is considered one of the most serious pathogens to channel catfish, golden shiners, striped bass, largemouth bass and sunfishes. It is the second most costly pathogen to the channel catfish industry in the United States, second only to enteric septicemia of channel catfish. One of the first steps to control columnaris outbreaks caused by F. columnare is to positively identify the bacterium. Identification of F. columnare is a challenging task because yellow pigmented bacteria as F. columnare are ubiquitous in the aquatic environment and share many characteristics. Therefore, even extensive biochemical testing could prove inconclusive. In this study a polymerase chain reaction (PCR) was developed to definitively identify F. columnare. Phylogenetic analysis was used to design DNA primers that target unique DNA sequences in the 16SrRNA gene of the bacterium. The technique was capable of positively identifying F. columnare isolates and negatively excluding closely related species. Based on this research the American Type Culture Collection (ATCC) isolate 43622, which was deposited in the national collection as F. columnare, has been reclassified as Flavobacterium Johnsoniae. The research will be useful in understanding the bacterial disease mechanisms and epizootics which is essential in devising control methods for columnaris.
Technical Abstract: Species-specific polymerase chain reaction (PCR) primers have been designed to identify the causative agent of columnaris disease, Flavobacterium columnare. The 16S rRNA gene sequences of F. columnare (8 sequences representing the different genotypes of the species) and related species (18 sequences) were aligned and compared to choose specific regions that are unique to F. columnare and do not have significant intraspecies variability. The species-specific regions in the 16S rRNA gene were used to design a pair of species-specific PCR primers, ColF and ColR. The PCR technique produced a specific amplicon of about 675 base pairs (bp) in 27 isolates of F. columnare and there was no amplification in the closely related species. The specificity of the amplified product was confirmed by digesting with HhaI. The PCR primers did not produce a 675 bp product with F. columnare ATCC43622 strain. This ATCC43622 strain was characterized by biochemical and ribotyping methods and renamed Flavobacterium Johnsoniae. The American Type Culture Collection has confirmed these findings and made the change.