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.

Primers were used to amplify a 561-bp region of the 16S rRNA gene of Ehrlichia phagocytophila from Ixodes scapularis ticks and small mammals collected in Rhode Island and Connecticut. DNA sequences for all 50 E. phagocytophila-positive samples collected from 1996 through 1998 in southwestern Connecticut were identical to the sequence reported for E. phagocytophila DNA from confirmed human cases. In contrast, the sequences from 92 of 123 E. phagocytophila-positive Rhode Island samples collected from 1996 through 1999 included several variants differing by 1-2 nucleotides from that in the agent infecting humans. While 11.9% of 67 E. phagocytophila-positive ticks collected during 1997 in Rhode Island harbored ehrlichiae with sequences identical to that of the human agent, 79.1% had a variant sequence not previously described. The low incidence of human ehrlichiosis in Rhode Island may in part result from interference by these variant ehrlichiae with maintenance and transmission of the true agent of human disease.

Piscinckettsia salrnonis, the etiologic agent of piscirickettsiosis, is a systemic disease of salmonid fish. Variations in virulence and mortality have been observed during epizootics at different geographical regions and in laboratory experiments with isolates from these different locations. This raises the possibility that biogeographical patterns of genetic variation might be a significant factor with this disease To assess the genetic variabihty the 16s ribosomal DNA, the internal transcribed spacer (ITS) and the 23s ribosomal DNA of isolates from 3 different hosts and 3 geographic origins were amplified using the polymerase chain reaction (PCR). Results of this analysis confirm that P. salmonis is a member of the gamma subgroup of the Proteobacteria and show that the isolates form a tight monophyletic cluster with 16s rDNA similarities ranging from 99.7 to 98.5%. The ITS regions were 309 base pairs (bp), did not contain tRNA genes, and varied between isolates (95.2 to 99.7 % similarity). Two-thirds of the 23s rRNA gene was sequenced from 5 of the isolates, yielding similarities ranging from 97.9 to 99.8%. Phylogenetic trees were constructed based on the 16s rDNA, ITS and 23s rDNA sequence data and compared. The trees were topologically similar, suggesting that the 3 types of molecules provided similar phylogenetic information. Five of the isolates are closely related (:,99.4 % 16s rDNA s d a r i t y , 99.1 to 99.7 % ITS and 99.3 to 99.8 % 23s rDNA slrmlarities). The sequence of one Chilean isolate, EM-90, was unique, with 16s rDNA similarities to the other isolates ranging from 98.5 to 98.9%, the ITS from 95.2 to 96.9% and the 23s rDNA from 97.6 to 98.5%.

In 2004, cultured Nile tilapia Oreochromis niloticus in several Latin America farms began to succumb to a disease similar to the piscirickettsiosis-like syndrome previously reported in tilapia in Taiwan and the United States. Mortality increased during 2005; reductions in tilapia biomass ranged from 5% to 80% in individual ponds and averaged 50% overall. All ages of fish have been involved. Clinical signs include lethargy, loss of appetite, petechia, exophthalmia, and abnormal swimming behavior. Gross lesions have included splenomegaly, renomegaly, and numerous white nodules observed in the spleen, kidney, testes, heart, ovaries, and occasionally the liver. A previously unreported black granulomatous lesion was reported in up to 30% of the fillets. Histologically, granulomatous infiltrates were observed in the kidney, spleen, liver, testes, ovary, and choroid gland, and rarely in the brain and heart. A small pleomorphic bacterium was observed in Giemsa-stained blood smears and spleen imprints. The bacterium did not grow on standard microbiological media and has not been isolated in cell culture. We obtained a near-complete 16S ribosomal DNA sequence with high similarity to Francisella spp. sequences previously identified in tilapias Oreochromis spp. (Taiwan), Atlantic cod Gadus morhua (Norway), and three-line grunts Parapristipoma trilineatum (Japan).