Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 13, 2004
Publication Date: August 1, 2005
Citation: Panangala, V.S., Van Santen, V.L., Shoemaker, C.A., Klesius, P.H. 2005. Analysis of 16s-23s intergenic spacer regions (isrs) of the rRNA operons in Edwardsiella ictaluri and Edwardsiella tarda isolates from fish. Journal of Applied Microbiology 99: 657-669. Interpretive Summary: Edwardsiella ictaluri and E. tarda are important bacterial pathogens of fish and are implicated with a substantial economic loss to the US aquaculture industry amounting to millions of dollars annually. Phenotypically, E. ictaluri has been characterized as a relatively homogeneous species while E. tarda has been widely considered as a heterogeneous bacterium capable of infecting both marine and fresh water fish as well as other terrestrial vertebrates. The present study was initiated to determine a genetic basis for the apparent homogeneity and/or polymorphism among the two species of Edwardsiella with a view to determine the underlying criteria from an epidemiological perspective. Results of this study confirmed that there is a genetic basis for the apparent homogeneity observed among E. ictaluri isolates, concomitantly revealing the degree of diversity among the E. tarda isolates examined. The studies also revealed a close genetic relationship between E. ictaluri and E. tarda which is useful for taxonomic purposes. Several restriction sites that differ between the intergenic spacer region sequences of E. ictaluri and E. tarda were identified that will be useful in distinguishing the two species. The new genetic information generated (for 27 gene sequences) for both E. ictaluri and E. tarda were deposited as 27 accession numbers in the National Center for Biotechnology Information, GenBank data base.
Technical Abstract: The interspecies and intraspecies differences based on the 16S-23S rRNA intergenic spacer region (ISR) sequences of the fish pathogens Edwardsiella ictaluri and E. tarda were analysed. The 16S-23S rRNA spacer regions of 13 E. ictaluri and four E. tarda isolates from four geographic regions were amplified by PCR with primers complementary to conserved sequences within the flanking 16S-23S rRNA coding sequences. Two products were generated from all isolates, without interspecies or intraspecific size polymorphisms. Sequence analysis of the amplified fragments revealed a smaller ISR of 350 bp, which contained a gene for tRNA Glutamine, and a larger ISR of 441 bp, which contained genes for tRNA Isoleucine and tRNA Alanine. The sequences of the smaller ISRs of different E. ictaluri isolates were essentially identical to each other. Partial sequences of larger ISRs from several E. ictaluri isolates also revealed no differences from the one complete E. ictaluri large ISR sequence obtained. The sequences of the smaller ISR of E. tarda were 97% identical to the E. ictaluri smaller ISR and the larger ISRs were 96-98% identical to the E. ictaluri larger ISR sequence. The E. tarda isolates displayed limited ISR sequence heterogeneity, with ' 97% sequence identity among isolates for both small and large ISRs. There is a high degree of size and sequence similarity of 16S-23S ISRs both among isolates within E. ictaluri and E. tarda species and between the two species. Our results confirm a close genetic relationship between E. ictaluri and E. tarda and the relative homogeneity of E. ictaluri isolates compared to E. tarda isolates. Because no differences were found in ISR sequences among E. ictaluri isolates, sequence analysis of the ISR will not be useful for epidemiological studies of E. ictaluri. However, we identified restriction sites that differ between ISR sequences of E. ictaluri and E. tarda, which will be useful in distinguishing the two species.