Edwardsiella tarda, a Gram-negative member of the family Enterobacteriaceae, has been implicated in significant losses in aquaculture facilities worldwide. Here, we assessed the intra-specific variability of E. tarda isolates from 4 different fish species in the eastern United States. Repetitive sequence mediated PCR (rep-PCR) using 4 different primer sets (ERIC I & II, ERIC II, BOX, and GTG5) and multi-locus sequence analysis of 16S SSU rDNA, groEl, gyrA, gyrB, pho, pgi, pgm, and rpoA gene fragments identified two distinct genotypes of E. tarda (DNA group I; DNA group II). Isolates that fell into DNA group II demonstrated more similarity to E. ictaluri than DNA group I, which contained the reference E. tarda strain (ATCC #15947). Conventional PCR analysis using published E. tarda-specific primer sets yielded variable results, with several primer sets producing no observable amplification of target DNA from some isolates. Fluorometric determination of G+C content demonstrated 56.4% G+C content for DNA group I, 60.2% for DNA group II, and 58.4% for E. ictaluri. Surprisingly, these isolates were indistinguishable using conventional biochemical techniques, with all isolates demonstrating phenotypic characteristics consistent with E. tarda. Analysis using two commercial test kits identified multiple phenotypes, although no single metabolic characteristic could reliably discriminate between genetic groups. Additionally, anti-microbial susceptibility and fatty acid profiles did not demonstrate remarkable differences between groups. The significant genetic variation (<90% similarity at gyrA, gyrB, pho, phi and pgm; <40% similarity by rep-PCR) between these groups suggests organisms from DNA group II may represent an unrecognized, genetically distinct taxa of Edwardsiella that is phenotypically indistinguishable from E. tarda.

spp. are responsible for significant losses in important wild and cultured fish species worldwide. Recent phylogenomic investigations have determined that bacteria historically classified as actually represent three genetically distinct yet phenotypically ambiguous taxa with various degrees of pathogenicity in different hosts. Previous recognition of these taxa was hampered by the lack of a distinguishing phenotypic character. Commercial test panel configurations are relatively constant over time, and as new species are defined, appropriate discriminatory tests may not be present in current test panel arrangements. While phenobiochemical tests fail to discriminate between these taxa, data presented here revealed discriminatory peaks for each species using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) methodology, suggesting that MALDI-TOF can offer rapid, reliable identification in line with current systematic classifications. Furthermore, a multiplex PCR assay was validated for rapid molecular differentiation of the spp. affecting fish. Moreover, the limitations of relying on partial 16S rRNA for discrimination of spp. and advantages of employing alternative single-copy genes and for molecular identification and classification of were demonstrated. Last, sequencing confirmed that isolates previously defined as typical motile fish-pathogenic are synonymous with, while atypical nonmotile fish-pathogenic isolates are equivalent to Fish-nonpathogenic isolates are consistent with as it is currently defined. These analyses help deconvolute the scientific literature regarding these organisms and provide baseline information to better facilitate proper taxonomic assignment and minimize erroneous identifications of isolates in clinical and research settings.

This study indicated that the bacterium Flavobacterium psychrophilum induced an infection within eggs of Atlantic salmon Salmo salar that were held at federal New England restoration facilities. The pathogen, which originated from the Connecticut, Penobscot, Machias, East Machias, Dennys, Narraguagus, and Sheepscot rivers, was obtained from these eggs at concentrations that ranged from 5.0 ϫ 10 2 to 2.5 ϫ 10 8 colony-forming units per gram of egg, despite successive treatments with povidone iodine (I 2 ). Treatments consisted of 50 mg/L of water for 30 min, then 100 mg/L for 10 min, followed at the eyed egg stage by 100 mg/L for 60 min. Collectively, 63% of the egg lots (77 of 122) obtained from paired matings of these captive broodfish were infected; 39% of these lots contained 10 or fewer infected eggs (60 eggs sampled per lot), and less than 10% of the lots contained more than 20 positive eggs. Consequently, standard iodophor disinfection procedures were ineffective. Eggs were positive from each of the river-specific captive brood populations during both of the spawning cycles that were studied. I concluded that F. psychrophilum established an intraovum infection that was prevalent among captive brood lots from different New England watersheds.

Coomassie Brilliant Blue Agar was used to quantify the frequency of the A-layer phenotype in different isolates of Aeromonas salmonicida. Hydrophilic, non-clumping isolates of A. salmonicida consisted predominantly of the A-layer minus phenotype. These bacteria were avirulent by intraperitoneal injection into susceptible brook trout (Salvelinus fontinalis) and could not be reisolated from infected fish. By contrast, hydrophobic, clumping isolates were predominantly of the A-layer positive phenotype, highly virulent in brook trout, and easily recovered from dead or moribund fish. A-layer positive and negative clones of A. salmonicida were derived by plating bacteria on Coomassie Blue Agar. The plating showed clearly that Coomassie Blue Agar could be used as a highly selective in vitro screening method to reclaim the virulence of certain isolates of A. salmonicida having a relatively low percentage of A-layer positive phenotypes.

Mucus of salmonids was evaluated as a source for nonlethal detection of the pathogen Aeromonas salmonicida in fish. The bacterium was readily isolated from mucus on dilution plates when Coomassie Brilliant Blue agar was the primary plating medium. Kidney samples from the fish that served as sources of mucus were similarly processed. Infection was detected in 56% of mucus samples from lake trout Salvelinus namaycush that were undergoing an epizootic of furunculosis, but only 6% of the kidneys from these fish were positive for the pathogen. Only 1% of asymptomatic brown trout Salmo trutta sampled at another fish hatchery had A. salmonicida in their mucus, and none had a kidney infection. Combined results from the examination of two pools of Atlantic salmon Salmo salar reared at a third hatchery indicated that 37% of these fish had mucus infections caused by A. salmonicida, but only 4% of the kidneys were infected.