Microbiology and Immunology

Iwata, Taketoshi; Une, Yumi; Okatani, Alexandre Tomomitsu; Kaneko, Sei‐ichi; Namai, Satoshi; Yoshida, Shin‐ichiro; Horisaka, Tomoko; Horikita, Tetsuya; Nakadai, Aya; Hayashidani, Hideki

doi: 10.1111/j.1348-0421.2005.tb03630.xpmid: 15665447

In the period from December 2002 to January 2003, 5 of 50 squirrel monkeys (Saimiri sciureus) housed at a Zoological Garden in the Kanto region of Japan died following a few days' history of diarrhea. After this outbreak had ended in the squirrel monkeys, 1 of 2 dark‐handed gibbons (Hylobates agilis) died in April of 2003, showing similar clinical signs. We examined the organs of 3 of the dead squirrel monkeys and of the dark‐handed gibbon, and Yersinia enterocolitica serovar O:8, which is the most pathogenic serovar of Y. enterocolitica, was isolated. In order to determine the source and the transmission route of infection, 98 fecal samples (45 from squirrel monkeys, 20 from other monkeys of 18 different species, and 33 from black rats captured around the monkey houses) and 7 water samples were collected in the Zoological Garden, and were examined for the prevalence of Y. enterocolitica serovar O:8. Serovar O:8 was isolated from 21 of 65 monkeys (32.3%) and 5 of 33 (15.2%) black rats (Rattus rattus). Furthermore, we examined the 30 isolates using molecular typing methods, pulsed field gel electrophoresis (PFGE), ribotyping using the RiboPrinter system, and restriction endonuclease analysis of virulence plasmid DNA (REAP), and compared the isolates in this outbreak with Japanese O:8 isolates previously identified. Genotyping showed that almost all the isolates were identical, and the genotype of the isolates was highly similar to that from wild rodents captured in Niigata Prefecture. This is the first report of fatal cases of Y. enterocolitica serovar O:8 infection in monkeys anywhere in the world.

Kook, Joong‐Ki; Sakamoto, Tomonori; Nishi, Kazuya; Kim, Mi‐Kwang; Seong, Jin‐Hyo; Son, Young Nam; Kim, Dong‐Kie

doi: 10.1111/j.1348-0421.2005.tb03634.xpmid: 15665448

A proportion of diseased sites in periodontal disease do not respond to the initial treatment, which might be due in part to the presence of specific microbial pathogens. The aim of this study was to clarify the value of microbial screening for predicting the outcome of periodontal treatment in Koreans using a polymerase chain reaction (PCR). This study enrolled 32 adults with periodontal disease. Microbial and clinical examinations were performed at the baseline and after the initial treatment (professional toothbrushing, scaling, and root planing). Subgingival plaque samples were taken from four sites in each subject (total 128 samples). PCR was used to detect the four putative pathogenic bacteria. There was an improvement in the average of each clinical measurement after the initial treatment. However, approximately half of the sites exhibiting bleeding upon probing (BOP) at the baseline still exhibited bleeding after treatment. There was a close association between the presence of BOP and the presence of Tannerella forsythia (formerly Bacteroides forsythus) and/or Prevotella intermedia. Furthermore, the sites harboring both T. forsythia and P. intermedia at the baseline had a poorer response to treatment than the sites where these two species were not detected. Therefore, microbial screening for T. forsythia and P. intermedia might be useful for predicting the treatment outcome in Koreans.

Jang, Won‐Jong; Choi, Yeon‐Joo; Kim, Jong‐Hyun; Jung, Kwang‐Don; Ryu, Ji‐Sun; Lee, Seung‐Hyun; Yoo, Cheon‐Kwon; Paik, Hyung‐Suk; Choi, Myung‐Sik; Park, Kyung‐Hee; Kim, Ik‐Sang

doi: 10.1111/j.1348-0421.2005.tb03635.x

Kawamura‐Sato, Kumiko; Hirama, Yumi; Agata, Norio; Ito, Hideo; Torii, Kenzo; Takeno, Akira; Hasegawa, Tadao; Shimomura, Yoshiharu; Ohta, Michio

doi: 10.1111/j.1348-0421.2005.tb03636.xpmid: 15665450

An emetic toxin cereulide, produced by Bacillus cereus, causes emetic food poisonings, but a method for quantitative measurement of cereulide has not been well established. A current detection method is a bioassay method using the HEp‐2 cell vacuolation test, but it was unable to measure an accurate concentration. We established a quantitative assay for cereulide based on its mitochondrial respiratory uncoupling activity. The oxygen consumption in a reaction medium containing rat liver mitochondria was rapid in the presence of cereulide. Thus uncoupling effect of cereulide on mitochondrial respiration was similar to those of uncouplers 2,4‐dinitrophenol (DNP), carbonylcyanide m‐chlorophenylhydrazone (CCCP), and valinomycin. This method gave constant results over a wide range of cereulide concentrations, ranging from 0.05 to 100 μg/ml. The minimum cereulide concentration to detect uncoupled oxygen consumption was 50 ng/ml and increased dose‐dependently to the maximum level. Semi‐log relationship between the oxygen consumption rate and the cereulide concentraiton enables this method to quantify cereulide. The results of this method were highly reproducible as compared with the HEp‐2 cell vacuolation test and were in good agreement with those of the HEp‐2 cell vacuolation test. The enterotoxin of B. cereus or Staphylococcus aureus did not show any effect on the oxygen consumption, indicating this method is specific for the identification of cereulide as a causative agent of emetic food poisonings.

Woo, Patrick C.Y.; Teng, Jade L.L.; Leung, Kit‐wah; Lau, Susanna K.P.; Woo, Gibson K.S.; Wong, Amy C.Y.; Wong, Michelle K.M.; Yuen, Kwok‐yung

doi: 10.1111/j.1348-0421.2005.tb03637.xpmid: 15665451

A bacterium was isolated from the blood culture of an intravenous drug abuser with pseudobacteremia. The cells were strictly anaerobic, straight or slightly curved, sporulating, Gram‐negative rods. It grew on sheep blood agar as non‐hemolytic, pinpoint colonies after 48 hr of incubation at 37 C in an anaerobic environment. It was motile but did not produce catalase or cytochrome oxidase. 16S ribosomal DNA (rDNA) sequencing revealed three different copies of 16S rDNA sequences. More than 90% of the differences among them were due to differences in the lengths of the sequences. Phylogenetically, the bacterium is clustered with Dendrosporobacter, Sporomusa, and Propionispora, the other three genera of anaerobic, sporulating, Gram‐negative rods. There were 8.6–11.1% differences between the 16S rDNA sequences of the bacterium and that of D. quercicolus, 4.7–15.1% differences between the 16S rDNA sequences of it and those of S. acidovorans, S. aerivorans, S. malonica, S. ovata, S. paucivorans, S. silvacetica, S. spaeroides, and S. termitida, and 7.6–13.1% differences between the 16S rDNA sequences of it and those of P. hippei and P. vibrioides. The G+C content of the bacterium (mean±SD) was 46.8±3.2%. For these reasons, a new genus and species, Anaerospora hongkongensis gen. nov. sp. nov., is proposed, for which HKU15T is the type strain.

Imanishi, Nobuko; Andoh, Tsugunobu; Sakai, Shinya; Satoh, Miyuki; Katada, Yuko; Ueda, Kyouka; Terasawa, Katsutoshi; Ochiai, Hiroshi

doi: 10.1111/j.1348-0421.2005.tb03638.xpmid: 15665452

We investigated the inductive activity of infective influenza A/PR/8/34 (PR8) virus and its ether‐split product (ESP) on the expression of inducible nitric oxide (NO) synthase (iNOS) and NO production in RAW264.7 (RAW) cells, a murine macrophage (MΦ) cell line, and thioglycolate‐elicited peritoneal MΦ (TPM). In both cells, PR8 virus infection induced iNOS mRNA between 4 hr and 24 hr, attaining a peak value at 12 hr. In correlation with induction of iNOS mRNA, NO amounts increased significantly from 12 to 24 hr. Moreover, this study demonstrated that ESP with the same hemagglutination titer as PR8 virus could induce iNOS mRNA and NO production, although the inductive activity of ESP was weaker than that of PR8 virus. Considering the dual role (beneficial and detrimental roles) of NO on certain inflammatory disorders and virus infections, the inductive activity of influenza virus on the iNOS‐mediated NO production independent of its infectivity might contribute to a modification of influenza virus infection.

Kondo, Nobuhiko; Ichimiya, Shingo; Tamura, Yasuaki; Tonooka, Akiko; Koshiba, Shigeru; Torigoe, Toshihiko; Kamiguchi, Kenjiro; Takenaga, Keizo; Sato9, Noriyuki

doi: 10.1111/j.1348-0421.2005.tb03639.xpmid: 15665453

The nature of the target molecule of TCRγδ T cell‐mediated lysis remains to be determined. As we previously reported, #067 monoclonal antibody (mAb) recognizes one of the transformation‐associated antigens, designated as #067 antigen. This antigen is expressed on the cell surface of rat fibrosarcoma W31 cells, which are established by transformation of fetal fibroblastic WFB cells with H‐ras oncogene. It has been suggested that the #067 antigen is a target molecule for TCRγδ T cells since #067 mAb inhibited TCRγδ T cell‐mediated lysis against #067 positive cells. In this study we attempted to identify the protein sequence of the #067 antigen. By using molecular cloning techniques, we demonstrated that a calcium binding protein, S100A4, was possibly one and the same molecule as the #067 antigen. It was shown that the expression of S100A4 was higher in W31 cells than in WFB cells at transcription and protein level. Flow cytometry and immunocytochemical studies showed that #067 antigen partially co‐localized with S100A4 on the cell surface as well as the cytoplasm of W31 cells. Moreover, rabbit anti‐S100A4 polyclonal antibodies (pAb) inhibited TCRγδ T cell‐mediated lysis against #067 positive cells. Our results indicated that S100A4 may play a role as a possible target molecule for TCRγδ T cell‐mediated lysis although how S100A4 is involved in TCRγδ T cell‐mediated lysis remains to be determined.

Karasawa, Rie; Ozaki, Shoichi; Nishioka, Kusuki; Kato, Tomohiro

doi: 10.1111/j.1348-0421.2005.tb03640.xpmid: 15665454

Anti‐oxidative enzymes protect living bodies from various oxidative stresses. In the systemic autoimmune diseases, autoantibodies to oxidized molecules and to anti‐oxidative enzymes have been reported. To promote understanding of the relationships between autoimmunity and oxidative stress, we here investigate whether autoimmunity to the anti‐oxidative peroxiredoxin (Prxs) enzymes exists in patients with systemic autoimmune diseases. Specifically, we detected autoantibodies to recombinant Prx I and Prx IV respectively by ELISA and western blotting. Next, clinical parameters were compared between the anti‐Prx I or IV‐positive and ‐negative patients. We found that 33% of the 92 patients with autoimmune diseases tested possessed autoantibodies to Prx I (57% in systemic lupus erythematosus (SLE), 19% in rheumatoid arthritis (RA), 5% in Behçet disease, and 46% in primary vasculitis syndrome). In contrast, autoantibodies to Prx IV were detected in only 17% of the same patients. No significant correlation was found between occurrence of the two autoantibodies. Clinically, possession of anti‐Prx I autoantibodies correlated with lower serum levels of CH50, C3, and C4. Taken together, our data demonstrate the existence of autoantibodies to Prxs for the first time. The autoantibodies to Prx I may be involved in the pathophysiology of systemic autoimmune diseases such as SLE and vasculitis.

Tay, Sun Tee; Rohani, Yasin Mohd; Ho, Tze Ming; Shamala, Devi

doi: 10.1111/j.1348-0421.2005.tb03641.xpmid: 15665455

The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95–99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA678 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.

Li, Yunshan; Okamoto, Keinosuke; Takahashi, Eizo; Miyoshi, Shin‐ichi; Shinoda, Sumio; Tsuji, Takao; Fujii, Yoshio

doi: 10.1111/j.1348-0421.2005.tb03631.xpmid: 15665456

The hemolysin of Vibrio mimicus (VMH) is a pore‐forming toxin with both enterotoxic and hemolytic activity. The hemolysis by VMH is induced by creation of pores in the membrane of erythrocyte; however, the mechanism for the enterotoxic action of VMH has remained unclear. In order to clarify the mechanism, we incubated T84 cells (a human colon carcinoma cell line) with VMH and found that the levels of ATP and cyclic AMP of culture medium increased after exposure of the cells to VMH. Subsequently, we found that the fluid accumulating activity of VMH in a mouse internal loop assay was reduced by administration of glibenclamide, an inhibitor of cyclic AMP‐dependent chloride channels, into the intestinal loop. These results suggest that the stimulation of cells to produce nucleotides by VMH is linked to the enterotoxic activity of the toxin.