Edwardsiella tarda is a pathogen with a broad host range infecting animals and humans. We have reported recently that the type III secretion system (TTSS) is essential for intracellular replication of the bacterium in murine macrophages. The present study shows that the TTSS is also needed for intracellular growth of the bacterium in human epithelial cells (HEp-2). However, different from the previous microarray analyses on murine macrophages, upregulation of the mRNA expression level of NF-kappaB target genes was not detected in the infected HEp-2 cells. The wild-type E. tarda, but not its TTSS mutant, actually repressed the tumor necrosis factor alpha-dependent NF-kappaB activation in an NF-kappaB reporter gene assay. These results suggest TTSS-dependent repression of the NF-kappaB activation in HEp-2 cells infected with E. tarda.

Edwardsiella tarda is a broad host-range pathogen infecting both animals and humans. E. tarda isolates from red sea bream Pagrus major are non-motile, whereas isolates from Japanese eel Anguilla japonica and Japanese flounder Paralichthys olivaceus are motile with peritrichous flagella. We compared the fliC gene coding for flagellin (FliC) in motile and non-motile E. tarda strains isolated from diseased fish. Twenty-two amino acid residues differed in the predicted FliC amino acid sequences between non-motile and motile strains. There were no significant differences either in the upstream sequences regulating transcription of the fliC gene or in the fliC transcript levels between motile and non-motile strains. The predicted secondary structure of FliC in non-motile E. tarda differed from that of motile strains, and the modeled data suggested that the secondary structure may be the important factor responsible for non-flagellation in the non-motile strains.KEY WORDS: Edwardsiella tarda · fliC gene sequence · Base changes · Flagellation · Motility · Red sea bream · Japanese flounder Resale or republication not permitted without written consent of the publisherDis Aquat Org 76: [113][114][115][116][117][118][119][120][121] 2007 while the atypical strain exhibits high virulence only in red sea bream.Bacterial flagella are important structures for pathogenic bacteria because they provide motility and increase adhesion to mucosal surfaces (Ramos et al. 2004). The flagellar filament is composed of approximately 20 000 subunits of the protein known as flagellin (FliC). Expression of flagella can be controlled by various genes in response to environmental changes. McIntosh & Austin (1991) showed that another fish pathogen, Aeromonas salmonicida, expresses flagella only at supra-optimal environmental temperatures (from 30 to 37°C). In Edwardsiella tarda, the fliC gene coding for the FliC protein was previously cloned and sequenced from strain PPD130/91, a motile strain (Tan et al. 2002). The other conditions and genes related to the expression of flagella are unknown in E. tarda.In this study we determined the mechanism of motility expression and flagellation in typical and atypical strains of Edwardsiella tarda. We show that non-motile strains do not form flagella in vitro in response to changing environmental temperatures. The data suggests that an alteration of the fliC gene sequence in non-motile strains is likely to be responsible for the deficiency in flagella formation in atypical strains. MATERIALS AND METHODSBacterial strains and media. Thirteen Edwardsiella tarda strains isolated from diseased Japanese flounder or diseased red sea bream were used (Table 1). The E. tarda strains were grown in Triptic-Soy broth (TSB, Eiken) or on Tryptic-Soy agar (TSA, Nissui) at 30°C unless otherwise indicated. Motility tests.The motility of the test strain was examined using a wet mount method with a light microscope (the direct method), and sulfide indole motility (SIM) media (Eiken) (the indirect method) at a vari...

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Short Tandem Repeat (STR) typing plays an important role in forensic science. Currently, many laboratories return the residual unextracted DNA evidence sample to police stations according to regulations in Japan. However, it has not been clarified how well the results of the original DNA analysis can be reproduced in the case of retest of trace or mixed DNA from remaining unextracted DNA evidence samples. In this study, simulated touch DNA samples were prepared, and STR typing was performed to verify the reproducibility of the results. Touch DNA samples were produced by having 25 individuals hold a doorknob, a 50 mL tube, and another personʼs clothing tightly for 1 minute each. These touch DNA samples were divided into two parts and STR typing was performed on each and the results were compared. The results indicated that the number of alleles and peak heights may vary between the original DNA test and retest, and the results are not necessarily identical. The discordance in the results may be due to changes in the amount of DNA extracted from the material, changes in the mixture ratio, or the appearance of alleles of unknown origin. This is due to the heterogeneity of touch DNA samples collected by swabbing, etc., and is not considered to negate the reliability of the respective result of DNA tests. The reproducibility of results when re-extracting from residual touch DNA evidence sample was expected to be difficult due to trace or mixed DNA, but to our knowledge, there are no previous reports verifying this fact. This study reveals for the first time the reality of this situation.