Identification of Edwardsiella ictaluri and E. tarda by Species‐Specific Polymerase Chain Reaction Targeted to the Upstream Region of the Fimbrial Gene

Abstract

Phylogenetic analysis of nine strains of Edwardsiella ictaluri and eight strains of E. tarda (six typical motile strains and two atypical nonmotile strains) isolated from diseased fish was performed using the upstream region of the fimbrial gene cluster. Strains of E. ictaluri and E. tarda were significantly clustered into separate groups. Moreover, atypical E. tarda strains were clustered into a different group from the other strains. Three polymerase chain reaction (PCR) primer sets for differential detection of E. ictaluri as well as typical and atypical E. tarda were developed from the respective characteristic sequences. Strains of E. ictaluri, typical E. tarda, and atypical E. tarda were specifically detected by PCR using each primer set. No amplifications were observed after the use of these three primer sets with 25 other bacterial species, including fish pathogens. In addition, the three primer sets were able to detect the DNA of each target species from fish kidney and liver artificially infected with E. ictaluri or E. tarda.

Edwardsiella piscicida identified in the southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR

“…The DNA Group II gyrB sequences determined in the present study averaged 99.8% (range 99.6 to 100.0%) homology to the gyrB sequence of the Edwardsiella piscicida type strain ET883 (Abayneh et al 2012(Abayneh et al , 2013 (Sakai et al 2009 Table 6. Mean base substitutions per site between gyrB sequences from isolates categorized by Griffin et al (2013) and Edwardsiella piscicida (ET883), E. piscicida-like species (ET080813), E. ictaluri (S97-773), and E. tarda (ATCC #15947).…”

“…The percentage of replicate trees in which the associated taxa clustered together in the boot-strap test (1000 replicates) is shown next to the branches. GenBank accession numbers for sequences used in this analysis are listed in Table 5 agreement with the primers originally designed by Sakai et al (2009), suggesting that what they had classified as typical and atypical E. tarda and what Abayneh et al (2013) classified as E. piscicida and E. piscicida-like species are synonymous. Survey results of the 44 isolates from diseased catfish identified every archived specimen as Edwardsiella piscicida.…”

“…All isolates in the present study were identified as E. tarda upon initial isolation using the BBL TM Crystal TM Enteric/Nonfermenter ID kit (BD) coupled with indole and oxidase tests (Becton Dickinson) (Table 1). In addition, an American Type Culture Collection (ATCC) Escherichia coli isolate (#25942), an ATCC E. tarda isolate (#15947), an Edwardsiella ictaluri isolate (S97-773), and an isolate identified as atypical E. tarda (LADL 05-105) by PCR (Sakai et al 2009 were also included in the analysis. Stoneville, MS S07-348 Channel 2403010153 Stoneville, MS S07-356 Channel 2003010103 Belzoni, MS S07-357 Channel 2003010103 Belzoni, MS S07-358 Channel 2003010113 Belzoni, MS S07-534 Channel 2003110113 Itta Bena, MS S07-907 Channel 2003110113 Tunica, MS S07-1004 Blue 2002010113 Indianola, MS S07-1094 Channel 2003110113 Itta Bena, MS S08-209 Channel 2003010113 Moorhead, MS S10-279 Channel 2003110113 Stoneville, MS S10-430 Hybrid 2402010053 Indianola, MS S10-67 Hybrid 2003010113 Itta Bena, MS S10-512 Hybrid 2402010113 Glen Allen, MS S11-62 Channel 2003110143 Itta Bena, MS S11-159 Channel 2003110113 Itta Bena, MS S11-222 Channel 2003110113 Belzoni, MS S11-233 Channel 2003110013 Itta Bena, MS S11-285 Channel 2403110113 Itta Bena, MS S11-295 Hybrid 2402010113 Inverness, MS S11-310 Channel 2003010113 Moorhead, MS S11-508 Hybrid 2003110113 Indianola, MS S11-509 Channel 2003110013 Indianola, MS S11-534 Hybrid 2503110113 Itta Bena, MS S11-551 Hybrid 2...…”

“…Species-specific PCR primers were developed based on complete and incomplete Edwardsiella genomes (Table 3), targeting the fimbrial subunit, which had been used previously to differentiate between what was termed typical and atypical E. tarda (Sakai et al 2009). Genomic DNA from the 18 isolates used for gyrB sequencing and characterized initially by Griffin et al (2013) was analyzed using the newly developed E. tarda (ET), E. piscicida (EP), and atypical E. piscicida-like (EPL) primers as well as previously established E. ictaluri (ESC) primers (Bilodeau et al 2003, Griffin et al 2011 (Table 3).…”

“…Molecular identification for all 44 survey isolates from farm-raised catfish in Mississippi was carried out using the newly developed ET, EP, and EPL primers, the typical (ETT) and atypical E. tarda (ETA) primers of Sakai et al (2009), and the ESC (Bilodeau et al 2003, Griffin et al 2011. Reaction components and thermal cycling conditions were carried out as described previously, although the ETT and ETA primers used a slightly different cycling profile of 95°C for 5 min, followed by 35 cycles of 95°C for 15 s, 60°C for 15 s, ad 72°C for 15 s, and a final extension of 72°C for 5 min.…”