bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Macrophages in Acute Murine Cytomegalovirus Infection
Authors: Hamano Shinjiro , Yoshida Hiroki , Takimoto Hiroaki , Sonoda Koh‐hei , Osada Kazuhiro , He Xiangdong , Minamishima Yoichi , Kimura Genki , Nomoto Kikuo
Published: November 2013

It has been recognized that macrophages play an important role in controlling virus infection in experimental animal models. To evaluate the role of macrophages in acute murine cytomegalovirus infection, macrophages in the spleen and the liver were eliminated by an intravenous injection of liposomes containing a cytolytic agent, dichloromethylene diphosphonate. The depletion of macrophages led to a significant increase of virus titer in the spleen and lungs in both susceptible BALB/c and resistant C57BL/6 mice during the first three days after intravenous infection. In the spleen, the increase of virus titer in macrophage‐depleted BALB/c mice was much greater than that in NK cell‐depleted mice. These results suggest that macrophages contribute to protection mainly by the mechanisms which are independent of NK cells during the first three days after infection. The increase of virus titer in macrophage‐depleted C57BL/6 mice was as great as that in NK cell‐depleted mice because of the high contribution of NK cells to protection in C57BL/6 mice. In the liver in both strains of mice, the effects of macrophage depletion on virus titer were not as much as those in the spleen and lungs. Furthermore, the local depletion of peritoneal macrophages resulted in a great increase of virus titer in the spleen at three days after intraperitoneal infection. We conclude that macrophages greatly contribute to decreasing the virus load in some organs possibly through either or both intrinsic and extrinsic mechanisms in the early phase of primary infection with murine cytomegalovirus.
Antigenic Differences between Clinical and Environmental Isolates of Burkholderia pseudomallei
Authors: Sirisinha Stitaya , Anuntagool Narisara , Intachote Pakamas , Wuthiekanun Vanaporn , Puthucheary Savithri D. , Vadivelu Jammuna , White Nicholas J.
Published: November 2013

Burkholderia pseudomallei is a free‐living organism that causes the potentially lethal tropical infection melioidosis. The disease is endemic in many parts of eastern Asia and northern Australia. The presence of two distinct biotypes in soil can be reliably distinguished by their ability to assimilate l‐arabinose. Whereas some soil isolates could utilize this substrate (Ara+), the remaining soil isolates and all clinical isolates tested so far could not (Ara−). Only the Ara− isolates were virulent in animal models. We have raised a murine monoclonal antibody (MAb) that can readily distinguish Ara− from Ara+ biotypes. The MAb reacted with a high molecular weight component present only on the Ara− biotype. With this MAb, clinical and soil Ara−isolates gave identical positive reactions in agglutination, immunofluorescence, ELISA and immunoblot assays. Using these same assay systems, the soil Ara+ biotype did not react with the MAb. Similar but distinct immunoblot patterns were also noted when these two Ara biotypes were probed with sera from patients with melioidosis or with polyclonal immune rabbit sera. These data showed that the Ara− biotype from both clinical and environmental isolates is antigenically different from its Ara+ environmental counterpart. The SDS‐PAGE protein and lectin‐binding profiles of both groups of Ara− isolates were also found to be different from those of the Ara+ biotype.
Leishmania donovani Infection of a Susceptible Host Results in Apoptosis of Th1‐Like Cells: Rescue of Anti‐Leishmanial CMI by Providing Th1‐Specific Bystander Costimulation
Authors: Das Gobardhan , Vohra Harpreet , Saha Bhaskar , Agrewala Javed N. , Mishra Gyan C.
Published: November 2013

A protective immune response against Leishmania donovani infection is mediated by T‐helper type 1 (Th1) cells. Th1 induced cell‐mediated immunity (CMI), as assessed by anti‐leishmanial DTH response, is lost in a susceptible host such as BALB/c mice. Although the impaired Th1 function eventuates in unhindered parasite growth and in manifestation of the susceptible phenotype, the mechanism of down‐regulation of the Th1 function is yet to be elucidated. Here, we provide evidence that the parasite downregulates the expression of a Th1‐specific costimulatory molecule, M150, on the surface of infected BALB/c mice‐derived macrophages. Th cells are rendered unresponsive to anti‐CD3 Ab‐mediated stimulation after interaction with infected macrophages. The anergized T cells produce much less IL‐2, IL‐4 and IFN‐γ compared to those T cells which were costimulated using normal macrophages. The defect in proliferation, anti‐CD3 Ab induced unresponsiveness and IFN‐γ but not IL‐4 production can be restored by providing bystander costimulation through M150. These results not only unfold a novel immune evasion strategy used by the parasite but also clarify the mechanism of Th1 cell debilitation during the disease. Recovery of Th1 cytokine production by bystander costimulation through M150 may help in formulating a new strategy for the elimination of intracellular parasites.
Enhancement of the Growth of Helicobacter pylori in Brucella Broth by Hydrogen Peroxide
Authors: Murano Akiyuki , Miyake Masami , Kato Jiro , Tanzawa Hideki , Takeo Kanji , Noda Masatoshi
Published: November 2013

We found that a sub‐lethal concentration of hydrogen peroxide (HPOx) enhanced the growth of Helicobacter pylori in Brucella broth supplemented with 10% fetal bovine serum (BB/FBS). The enhancement was evident at 0.1 mM HPOx and reached a maximun at 3.5 mM. The growth stimulation was dependent on the basal media used; when brain heart infusion broth (BHIB) was used instead of BB, the growth was not altered regardless of the presence or absence of HPOx. Furthermore, the growth in BHIB/FBS was comparable to that in BB/FBS plus 3.5 mM HPOx. This suggested that the enhancement of growth by HPOx resulted from the derepression of the inhibitory factor existing in BB by HPOx. The inhibitory substance seemed to be bisulfite salt since the bacteria grew to a similar extent in bisulfite‐less Brucella broth (BLBB)/FBS compared to the bacterial growth in BHIB/FBS and BB/FBS plus HPOx. These results indicate that the detoxification of bisulfite in BB can be easily achieved by simply adding HPOx to the medium, which causes the oxidation of bisulfite to bisulfate, a less‐toxic compound to the bacterial growth. Since we also found that the morphology and cellular protein profile of BB/FBS‐cultured bacteria were apparently different from those cultured in BLBB/FBS, we propose that the use of BB for primary isolation and cultivation of H. pylori should be limited on certain occasions, or if necessary, BB can be used after detoxification of the bisulfite by the addition of a low concentration of HPOx.
Genetic Variation in the Capsid Region of Human Astrovirus Serotype 4 Isolated in Japan
Authors: Hachiya Masahiko , Matsui Mihoko , Sanogo Mussa , Oseto Mitsuaki , Morooka Keiichi , Ushijima Hiroshi
Published: November 2013

The entire capsid regions of 12 serotype‐4 astroviruses from Japan were sequenced and compared with those of other serotypes. Serotype‐4 isolates were divided into two new subgroups. The intrasubgroup nucleic acid and deduced amino acid sequences were quite homologous (more than 93%), but slightly less so between subgroups (almost 85%). However, the serotype‐4 sequences differed from those of serotypes 1,2,3,5,6,7 and 8 (less than 50%). Determining whether these differences significantly alter the epidemiology and antigenicity will require further investigation.
Intracellular Salmonella typhimurium Induce Lysis of Human Polymorphonuclear Leukocytes Which Is Not Associated with the Salmonella Virulence Plasmid
Authors: Chiu Cheng‐hsun , Ou Jonathan T.
Published: November 2013

The interaction between Salmonella typhimurium and human polymorphonuclear leukocytes (PMNs) was analyzed in vitro. Three S. typhimurium strains, the wild‐type strain OU5043, its isogenic virulence plasmid‐cured strain OU5048, and LT2, which represented the types that exhibited three mouse virulence levels, respectively, were used in this study. There was no correlation between the recovery of intracellular S. typhimurium from PMNs and the presence or absence of the virulence plasmid, or the strains' mouse virulence level. When the oxygen‐dependent response of PMNs upon phagocytosis of S. typhimurium was examined by checking the intracellular reduction of nitroblue tetrazolium (NBT), the fraction of PMNs that reduced NBT on phagocytosis of the three strains was around 80%, whereas it was 58% with Escherichia coli, 95% with phorbol 12‐myristate 13‐acetate and 15% with a negative control. Thus there were no significant differences among the three Salmonella strains in terms of their ability to induce the oxidative response in PMNs. Microscopic analysis of Salmonella‐infected PMNs indicated that the intracellular Salmonella induced lysis of PMNs. Both OU5043 and OU5048 exhibited a significant intracellular cytotoxic effect on PMNs after 24 hr of infection and this effect was not associated with the presence or absence of the virulence plasmid. On the other hand, lysis of PMNs was related to the intracellular survival of Salmonella, as ofloxacin, an antibiotic, appeared to be able to protect human PMNs from Salmonella‐induced cytotoxicity when this agent was added into the medium to inactivate the intracellular organism. The ability to induce lysis of PMNs by either wild‐type or plasmid‐cured strains of S. typhimurium may play a crucial role in the pathogenesis of non‐typhoid Salmonella. The contribution of pSTV to humam salmonellosis is likely to be limited. Furthermore, early institution of antibiotics with a high intracellular activity against Salmonella, such as fluoroquinolones, may be useful to prevent the dissemination of Salmonella infection.
Secretion of Hemolysin of Aeromonas sobria as Protoxin and Contribution of the Propeptide Region Removed from the Protoxin to the Proteolytic Stability of the Toxin
Authors: Nomura Tomohiko , Fujii Yoshio , Okamoto Keinosuke
Published: November 2013

The sequence at the amino terminus region of the hemolysin of Aeromonas sobria is homologous with that of aerolysin of A. hydrophila. However, there is no homology between the two toxins in the sequence at the carboxy terminal region. It has been shown that aerolysin is secreted into culture supernatant as a protoxin. This proaerolysin is activated by the proteolytic removal of a carboxy terminal peptide. However, the role of the carboxy terminal region, which is removed in the activation process, has not been elucidated. In this study, we showed that hemolysin is also secreted as a protoxin into culture supernatant and that prohemolysin is cleaved by the protease of A. sobria between Ser‐446 and Ala‐447, resulting in the removal of a 42 amino acid peptide. The removal of the peptide converts the prohemolysin into active hemolysin. Subsequently, we mutated the hemolysin gene to delete the last several amino acid residues and expressed the genes in Escherichia coli, in order to examine the role of the carboxy terminal region of prohemolysin. The amounts of these mutant hemolysins accumulated in the periplasmic space of E. coli were very low compared with that of the wild‐type. This observation indicated that the carboxy terminal region of prohemolysin contributes to the proteolytic stability of the toxin.
Suppression by the DNA Fragment of the motX Promoter Region on Long Flagellar Mutants of Vibrio alginolyticus
Authors: Furuno Masaaki , Nishioka Noriko , Kawagishi Ikuro , Homma Michio
Published: November 2013

The axial length of the polar flagellum (Pof) of Vibrio alginolyticus is about 5 μm. We previously isolated mutants that make abnormally long flagella. The swarm sizes of these mutants in a soft agar plate are smaller than that of a wild‐type strain. We cloned a DNA fragment into the pMF209 plasmid that restored the swarming ability of the long‐Pof strain VIO578. The swimming speed and flagellar length of these transformants were almost equal to the wild‐type values. The amounts of PF47 flagellin and PF60 sheath‐associated protein, which increased in the long‐Pof mutants, were retrieved to almost the wild‐type level in the transformants. The plasmid pMF209 contained only a 143 bp chromosomal fragment whose sequence is about 80% similar to that of the motX promoter region of V. parahaemolyticus. We speculate that this sequence interacts with a regulatory protein that controls Pof expression. The mutation causing the long‐Pof phenotype may be in the gene encoding this protein or in the control region of a structural gene that is regulated by this protein.
Evaluation of an Enzyme‐Linked Immunosorbent Assay Based on Binding Inhibition for Type‐Specific Quantification of Poliovirus Neutralization‐Relevant Antibodies
Authors: Hashido Madoka , Horie Hitoshi , Abe Shinobu , Doi Yutaka , Hashizume So , Agboatwalla Mubina , Isomura Shin , Nishio Osamu , Hagiwara Akio , Inouye Sakae
Published: November 2013

To detect neutralization‐relevant antibodies against 3 types of poliovirus (PV) without using tissue cultures and live viruses, an enzyme‐linked immunosorbent assay (ELISA) based on monoclonal antibody‐binding inhibition was evaluated using sera from 80 vaccinated Japanese children and 60 Pakistani poliomyelitis patients. Compared with the neutralization test, the sensitivity of the inhibition ELISA was 100% (111/111) for detection of anti‐PV1 antibody, 98.3% (118/120) for anti‐PV2, and 96.5% (82/85) for anti‐PV3, and the specificity was 93.1% (27/29), 100% (20/20), and 92.7% (51/55), respectively. Thus, the inhibition ELISA showed excellent potential as a seroepidemiologic tool in both vaccinated and naturally‐infected populations.
Chromosome‐Determined Zinc‐Responsible Operon czr in Staphylococcus aureus Strain 912
Authors: Kuroda Makoto , Hayashi Hideo , Ohta Toshiko
Published: November 2013

A novel operon, czrAB (zinc‐responsible genes), was identified in the chromosome of Staphylococcus aureus. The operon consists of two genes, czrA and czrB. The czrA gene, coding for an 11.5 kDa protein, was homologous to cadC, arsR of S. aureus plasmid pI258 and smtB of Synechococcus PCC7942. The czrB, coding for a 36 kDa membrane spanning protein, was homologous to the czcD gene, cobalt, zinc and the cadmium‐resistant factor of Bacillus subtilis and Alcaligenes eutrophus. In the presence of zinc (0.1‐10 mM), the transcription of czrAB was enhanced in a concentration‐dependent manner. Other heavy metals, such as cobalt, copper, manganese and nickel showed no effect on czrAB expression. The disruptant of the czrB gene became sensitive to zinc ion (MIC, 2 mM; MBC, 10 mM), and the complementation with the plasmid recovered the resistance to zinc at the same concentration as a parental strain (MIC, 5 mM; MBC, 20 mM). The disruptant accumulated intracellular zinc up to 0.4 mg per g dry weight of the organism, while that of the parental strain was 0.25 mg per g dry weight. The findings indicated that the novel operon czrAB should play a role in the transportation of zinc across the cell membrane to maintain the proper intracellular concentration.
Anti‐HIV‐1 Activity of an lonically Modified Porous Polypropylene Membrane Determined by Filtration of a Viral Suspension
Authors: Owada Takashi , Miyashita Yuko , Motomura Tadahiro , Onishi Makoto , Yamashita Shuzo , Yamamoto Naoki
Published: November 2013

We describe here a unique anti‐HIV‐1 membrane, derived from a chemically modified porous polypropylene (PP) membrane, which lowers viral infectivity upon the filtration of HIV‐1 suspension. A cationic polymer, polyethyleneimine (PEI) was graft‐polymerized onto the PP filter membrane (PP‐PEI), and infectious HIV‐1HTLV‐IIIB derived from MOLT‐4/HIV‐1HTLV‐IIIB cells (HIV‐1HTLV‐IIIB(MOLT‐4)) was applied. When a viral suspension of high titer (103.93 TCID50 ml 1) was filtered, efficient reduction (>99%) of gag p24 antigen levels and infectious titer resulted. In a viral suspension of medium titer (102.37 TCID50 ml 1), a significant decrease in the p24 antigen did not occur, although the titer was markedly reduced (>95%). Electron microscopic observation suggested that PEI induced viral aggregations under high titer conditions, and under medium titer conditions, PEI deprived HIV‐1HTLV‐IIIB of its infectivity alone to avoid virus adsorption. In contrast, HIV‐1 propagated in human peripheral blood mononuclear cells (PBMC) such as HIV‐1HTLV‐IIIB(PBMC) was more efficiently trapped by PP‐PEI at lower titers as compared with HIV‐1HTLV‐IIIB(MOLT‐4) from MOLT‐4/HIV‐1HTLV‐IIIB cells. These data suggest host cell modification in the interactions between PP‐PEI and HIV‐1 strains. Since HIV‐1HTLV‐IIIB(MOLT‐4) and HIV‐1HTLV‐IIIB(PBMC) were almost electrically neutral and negative, respectively, we concluded that the divergent effect of PEI on each HIV‐1HTLV‐IIIB resulted from their different electrical characteristics.
Functional Characteristics of Human Peripheral Blood α/βTCR+, CD4‐ and CD8‐Double‐Negative (DN) T Cells
Authors: Niehues Tim , Eichelbauer Dirk , Schneider E. Marion
Published: November 2013

The function of human peripheral blood α/βTCR positive, CD4‐ and CD8‐ double‐negative T lymphocytes (DN cells) in vivo is not completely understood. The response of immunomagnetically isolated DN cells to PHA and anti‐CD3 was compared to the response of single‐positive (SP) CD4 and CD8 subsets. Proliferation of DN cells in response to PHA was largely independent of APC. This suggests activation requirements for DN cells that are different from SP cells. Upon activation, HLA‐DR was found to be upregulated early on DN cells, and IL‐4 and IL‐10 were detected in the supernatants of DN cells. These observations in vitro could correspond with an immunoregulatory role of human DN cells in vivo.
Changes in Prevalence of Herpes Simplex Virus Type 1 and 2 Antibodies from 1973 to 1993 in the Rural Districts of Japan
Authors: Hashido Madoka , Kawana Takashi , Matsunaga Yasuko , Inouye Sakae
Published: November 2013

Using the gG‐capture ELISA, changes in the seroprevalence of HSV‐1 and HSV‐2 from 1973 to 1993 were studied for 614 sera collected from general adults living in rural Japan. The HSV‐1 seroprevalence for men and women decreased from 75.3 and 80.6% in 1973 to 54.4 and 59.6%, respectively, in 1993. The HSV‐2 seroprevalence also decreased from 10.2 and 9.9% in 1973 to 1.8 and 1.2%, respectively, in 1993. Although the decrease in HSV‐2 prevalence seemed to be correlated with the general decrease of sexually transmitted diseases in Japan since the 1950s, these findings should not be interpreted as typical, as HSV‐2 infections are particularly known to distribute unevenly among populations, according to sexual activity and cohorts.
Growth of Starved Escherichia coli O157 Cells in Selective and Non‐Selective Media
Authors: Sata Shin , Osawa Ro , Asai Yoshio , Yamai Shiro
Published: November 2013

Escherichia coli O157 strains starved in sterile deionized water (SDW) and filter‐sterilized natural river water (SRW) were investigated with specific reference to their culturability in selective and non‐selective media. Growth of the strains starved in both SDW and SRW were markedly suppressed with time in selective liquid media such as modified trypticase soy broth supplemented with novobiocin (mTSB+n) and modified E. coli broth supplemented with novobiocin (mEC+n). This suppression was more pronounced when incubated at 42 C than at 37 C, especially with mEC+n. By contrast, such growth suppression was seldom observed when cultured at 37 C in non‐selective liquid media such as trypticase soy broth (TSB) and buffered peptone water. In mEC+n at 42 C, the non‐starved cells from overnight cultures with an initial density of less than 103 colony‐forming units (CFU)/ml grew to the density of over 107 CFU/ml after 24 hr incubation, whereas those starved for 6 weeks in SRW were only to maintain their initial density or died off after 24 hr incubation under the same culturing conditions. These results indicated that the isolation of starved cells of E. coli O157 from water samples would be most difficult with selective enrichment or direct plating on the selective plate media. It is thus highly recommended that a “resuscitation” of the cells with non‐selective enrichment should be performed as a routine practice for maximum recovery of E. coli O157 from water systems.
Genotypic Characterization of Human and Environmental Isolates of Salmonella choleraesuis Subspecies choleraesuis Serovar Infantis by Pulsed‐Field Gel Electrophoresis
Authors: Murakami Koichi , Horikawa Kazumi , Otsuki Koichi
Published: November 2013

To determine the extent of genetic diversity of Salmonella choleraesuis subspecies choleraesuis serovar Infantis and whether environmental isolates were similar or identical to human isolates, a total of 110 isolates from humans, broiler samples, egg production facilities, riverwater, sewage, and chicken meat were analyzed epidemiologically by pulsed‐field gel electrophoresis. While the isolates showed 35 distinct pulsed‐field profiles, none had the genotype of the human isolates. One pulsed‐field profile was shared by 43 (39%) of the 110 isolates. These results indicate that relatively fewer clonal lines of S. serovar Infantis had spread widely while multiple clonal lines, including the strain involved in the outbreak, exist in Western Japan.
Proposal of Sphingomonas suberifaciens (van Bruggen, Jochimsen and Brown 1990) Comb. Nov., Sphingomonas natatoria (Sly 1985) Comb. Nov., Sphingomonas ursincola (Yurkov et al. 1997) Comb. Nov., and Emendation of the Genus Sphingomonas
Authors: Yabuuchi Eiko , Kosako Yoshimasa , Naka Takashi , Suzuki Shinnichiro , Yano Ikuya
Published: November 2013

Based on the results of a phylogenetic analysis of 16S rRNA and the presence of sphingoglycolipid in cellular lipids of the type strains, transfer of “Rhizomonas” suberifaciens, Blastomonas natatoria and Erythromonas ursincola to the genus Sphingomonas as Sphingomonas suberifaciens (van Bruggen et al 1990) comb, nov., Sphingomonas natatoria (Sly 1985) comb, nov., and Sphingomonas ursincola (Yurkov et al 1997) comb. nov. are herein proposed together with the emendation of genus Sphingomonas, The type strain of S. suberifaciens is van Bruggen Ca1=ATCC 49382=NCPPB 3629=IFO 15211=JCM 8521, that of S. natatoria is ATCC 35951=DSM 3183=NCIMB 12085=JCM10396, and that of S. ursincola is DSM 9006=KR‐99.
Detection and Nucleotide Sequence Analysis of Human Caliciviruses (HuCVs) from Samples in Non‐Bacterial Gastroenteritis Outbreaks in Hokkaido, Japan
Authors: Ohyama Tohru , Yoshizumi Shima , Sawada Harumi , Uchiyama Yasuhiro , Katoh Yoshinobu , Hamaoka Naohiro , Utagawa Etsuko
Published: November 2013

Samples of feces and vomit collected from patients during 13 non‐bacterial gastroenteritis outbreaks which occurred in Hokkaido between 1995 and 1998 were examined by electron microscopy (EM) and reverse‐transcription polymerase chain reaction (RT‐PCR) for evidence of infection with human caliciviruses (HuCVs). In 6 food‐borne outbreaks, oysters were the probable source of infection, while the origin of HuCVs was not found out for the other 7 outbreaks. One‐hundred‐eleven of 214 stool, vomit and oyster specimens examined gave positive results by RT‐PCR, while HuCVs were detected by EM in 36 of 121 stool specimens examined. We determined the nucleotide sequences of 470‐bp or 373‐bp PCR products amplified from the RNA polymerase region of the HuCV genomes with primer sets MR3/4 and Yuri22F/R, respectively. The sequences of different strains revealed great heterogenicity, with a range of 60 to 100% homology among strains. In a few cases, a mixed genotype was found in the same patient or same outbreak by means of nested PCR and cloning of PCR products into an appropriate vector. Of the 19 different strains found, 4 strains could be classified as Norwalk virus (genogroup 1) and the other 15 strains as Snow Mountain agent (genogroup 2) based on genotyping with homology analysis. Furthermore, the strains belonging to genogroup 2 could be classified into 4 subgroups with more than 93% homology in amino acids among strains in the subgroup.
Borna Disease Virus Infection in Two Family Clusters of Patients with Chronic Fatigue Syndrome
Authors: Nakaya Takaaki , Takahashi Hirokazu , Nakamura Yurie , Kuratsune Hirohiko , Kitani Teruo , Machii Takashi , Yamanishi Koichi , Ikuta Kazuyoshi
Published: November 2013

A high rate of Borna disease virus (BDV) infection has been demonstrated in patients with chronic fatigue syndrome (CFS). Herein, we focused on BDV infection in two family clusters of patients with CFS: a father, mother, two sons and one daughter (family #1); and a father, mother, two daughters and one son (family #2). All members, except for the elder son in family #1 and the father and son in family #2, were diagnosed with CFS. The results supported that all the family members with CFS were infected with BDV, as evidenced by the presence of antibodies to viral p40, p24 and/or gp18 and BDV p24 RNA in peripheral blood mononuclear cells. The healthy members, except for the father of family #2 who was positive for antibody to p24, were all negative by both assays. Follow‐up studies in family #1 continued to reveal BDV antibodies and BDV RNA, except in the mother, who lost the RNA upon slight recovery from the disease.
Salt‐Sensitive Growth of Staphylococcus aureus: Stimulation of Salt‐Induced Autolysis by Multiple Environmental Factors
Authors: Ochiai Toshiro
Published: November 2013

The growth of Staphylococcus aureus 209P became extremely sensitive to a high NaCl concentration following lowered temperature, reduced air‐supply, and decreased Ca2+ concentration in the medium. Cells in high‐NaCl and low‐Ca2 concentration media either autolyzed or transformed into protoplast‐like forms during growth when grown standing below 37 C. The abnormal growth, however, was invariably avoided by preliminary supplementation with polyanetholesulfonate (autolysin inhibitor) in the growth media. These results suggested that the autolytic activity of this organism was precisely controlled by multiple environmental factors such as ionic strength, temperature, air supply, and the concentration of Ca2+.
Characterization of the Coxiella burnetii sucB Gene Encoding an Immunogenic Dihydrolipoamide Succinyltransferase
Authors: Van Nguyen Sa , To Ho , Yamaguchi Tsuyoshi , Fukushi Hideto , Hirai Katsuya
Published: November 2013

The Coxiella burnetii sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme was cloned by immunological screening of a λ EMBL3 genomic library prepared from strain Nine Mile DNA and sequenced. The homology of the cloned gene product to the counterpart in Escherichia coli was 54.3%, but the homology of the N‐terminal region was only 42%. The gene was expressed in E. coli as an independent unit from its own promoter, producing an immunoreactive protein of about 50 kDa on SDS‐PAGE which reacted with antisera from laboratory animals and sera from human patients with acute Q fever. The study results suggest that the C. burnetii E2o enzyme may serve as a potential target antigen for diagnostic assays for Q fever.
Survey on Antibody against Parapoxvirus among Cattle in Japan
Authors: Sentsui Hiroshi , Inoshima Yasuo , Minami Akihiro , Yamamoto Yasunori , Murakami Kenji , Shimizu Shinya
Published: November 2013

A seroepidemiological survey was performed on antibody against parapoxvirus among cattle in Japan using the agar gel immunodiffusion test and enzyme‐linked immunosorbent assay. A total 1,819 sera were collected from cattle in various parts of Japan for the survey. The positive rates were in the range of 40 to 98%, and the reactors increased gradually in number with advancing age. These results indicate that parapoxvirus infection is already prevalent among cattle in Japan. It remains to be elucidated, however, whether the antibodies detected have been produced by infection of the same virus or other viruses that belong to the genus parapoxvirus.
Isolation of Orientia tsutsugamushi from Leptotrombidium fuji and Its Characterization
Authors: Tamura Akira , Makisaka Yukie , Kadosaka Teruki , Enatsu Teruyuki , Okubo Koji , Koyama Shoichiro , Yu Qiang , Urakami Hiroshi
Published: November 2013

In our attempts to isolate Orientia tsutsugamushi from trombiculid mites, a strain was successfully isolated from Leptotrombidium fuji collected in Aichi Prefecture, Japan. This is the first case of isolation of O. tsutsugamushi from L. fuji. A phylogenetic analysis based on the base‐sequence homology of the 56‐kDa type‐specific antigen‐gene indicated that the strain is a new type which is not closely related to any strains analyzed previously. Three strains isolated from Leptotrombidium pallidum harvested at the same area were identified as being closely related to the JP‐2 type (subtype‐2 of Karp type in Japan) by phylogenetic analysis.
Inhibition of HIV‐1 Infection by an Intramolecular Antisense Peptide to T20 in gp160
Authors: Imai Masaki , Okada Noriko , Okada Hidechika
Published: November 2013

Antisense amino acids are amino acids which can be translated from the corresponding anti‐codons of a sense amino acid. Antisense peptides encoded by the noncoding DNA strand have a tendency to interact with each other. We have demonstrated that antisense peptide sequences are present intramolecularly, and these may contribute to the folding and maintenance of the tertiary structure of a protein. T20 is a synthetic peptide with an amino acid sequence in the gp41 of HIV‐1 and has been demonstrated to be a potent inhibitor of HIV‐1 infection. We searched for intramolecular peptide sequences which are antisense to portions of T20. A synthetic peptide (TA‐1L) consisting of amino acids 84 to 97 of gp160, which contains an antisense peptide sequence (TA‐1) to T20, was shown to inhibit HIV‐1mB infection of MT‐4 cells. Interaction of these antisense peptides could be involved in sustaining HIV‐1 infectivity. The TA‐1L site, which exists in the C1 domain of gp160, is highly homologous among strains of HIV‐1, especially at TA‐1 and in the amino acids flanking the C terminus. Although the TA‐1 sites of 18 out of 30 HIV‐1 strains were antisense to the T20 region, those of the remaining 12 strains, including HIV‐1MN, were not. However, TA‐1L inhibited infection by HIV‐1MN, which has no antisense peptide in T20 corresponding to TA‐1, although the inhibitory effect was weaker. TA‐1L may thus also interfere with the gp160 interaction with CD4, which has an antisense sequence to TA‐1.
Cloning, Sequencing and Expression of the Gene Encoding the Extracellular Metalloprotease of Aeromonas caviae
Authors: Kawakami Kouji , Toma Claudia , Honma Yasuko
Published: November 2013

A gene (apk) encoding the extracellular protease of Aeromonas caviae Ae6 has been cloned and sequenced. For cloning the gene, the DNA genomic library was screened using skim milk LB agar. One clone harboring plasmid pKK3 was selected for sequencing. Nucleotide sequencing of the 3.5 kb region of pKK3 revealed a single open reading frame (ORF) of 1,785 bp encoding 595 amino acids. The deduced polypeptide contained a putative 16‐amino acid signal peptide followed by a large propeptide. The N‐terminal amino acid sequence of purified recombinant protein (APK) was consistent with the DNA sequence. This result suggested a mature protein of 412 amino acids with a molecular mass of 44 kDa. However, the molecular mass of purified recombinant APK revealed 34 kDa by SDS‐PAGE, suggesting that further processing at the C‐terminal region took place. The 2 motifs of zinc binding sites deduced are highly conserved in the APK as well as in other zinc metalloproteases including Vibrio proteolyticus neutral protease, Emp V from Vibrio vulnificus, HA/P from Vibrio cholerae, and Pseudomonas aeruginosa elastase. Proteolytic activity was inhibited by EDTA, Zincov, 1,10‐phenanthroline and tetraethylenepentamine while unaffected by the other inhibitors tested. The protease showed maximum activity at pH 7.0 and was inactivated by heating at 80 C for 15 min. These results together suggest that APK belongs to the thermolysin family of metalloendopeptidases.
Constitutively Expressed phoP Inhibits Mouse‐Virulence of Salmonella typhimurium in an Spv‐Dependent Manner
Authors: Matsui Hidenori , Kawakami Takatoshi , Ishikawa Satoshi , Danbara Hirofumi , Gulig Paul A.
Published: November 2013

In Salmonella typhimurium, the transcription of several virulence genes including spvB is regulated by the PhoP/PhoQ regulatory system. To further examine the relationship between the PhoP/PhoQ and Spv systems for virulence in mice, we examined a non‐polar phoP mutation combined with different virulence plasmid genotypes for effects on virulence of S. typhimurium in the mouse model. PhoP–/Spv+ PhoP–/ Spv– mutants were not detectably recovered from the spleens of subcutaneously or orally inoculated mice. The phoP gene constitutively expressed from the lacZ promoter of a low copy number vector (phoPC) only partially complemented the non‐polar phoP mutation for mouse‐virulence in both the Spv+ Spv– backgrounds; both PhoPC strains exhibited virulence equal only to a PhoP+/Spv– strain. Interestingly, in a PhoP+ background, the phoPC gene reduced splenic infection of the Spv+ but not Spv– salmonellae after subcutaneous or oral inoculation compared with the PhoP+ parents. Additionally, the phoPC gene in an Spv+ background reduced the net growth of salmonellae in macrophages in vitro; phoPC in an Spv– background was without effect. These data suggest that the constitutive expression of the phoP gene attenuates the virulence of S. typhimurium in mice in an Spv‐dependent manner.
Clinical and Bacteriological Profiles of Patients with Typhoid Fever Treated during 1975–1998 in the Tokyo Metropolitan Komagome Hospital
Authors: Hoshino Yoshihiko , Masuda Gohta , Negishi Masayoshi , Ajisawa Atsushi , Imamura Akifumi , Hachimori Kei , Takayama Naohide , Yamaguchi Tsuyoshi , Kimura Mikio
Published: November 2013

Patients with typhoid fever presenting to the Tokyo Metropolitan Komagome Hospital during the period 1975–1998 were retrospectively investigated. All cases were diagnosed by a positive culture for Salmonella typhi in either of their clinical specimens. Of the total number of 130 patients, 57% contracted the disease abroad; this population increased in later years as the total numbers of cases decreased. The period from disease onset to diagnosis averaged 14 days with 20% of the cases requiring over three weeks to establish a diagnosis. As for symptomatology relative bradycardia was seen in less than half of the cases, and rose spots or splenomegaly in less than one third. A positive blood culture was the most frequent test establishing the diagnosis followed by a positive stool culture. Intestinal bleeding was recognized in as many as 35 cases (27%) and even intestinal perforation occurred in two cases (1.5%). Chloramphenicol was most commonly employed during the early study period, however, during the late period it was replaced by fluoroquinolones. The clinical cure rate was 98% with regimens that include fluoroquinolones/quinolone; however it was 87% with the other antimicrobial regimens. Bacteriological relapse occurred in 25% of the non‐fluoroquinolone group while only in 2.0% in the fluoroquinolone/quinolone group. Four strains of Salmonella typhi that were multi‐resistant to chloramphenicol, ampicillin and cotrimoxazole were isolated in travelers from Asia. Early diagnosis by appropriate bacteriological examination regardless of classical symptomatology should be stressed and the use of fluoroquinolones is warranted in the treatment of typhoid fever.
Antibacterial Properties of Pseudomonas aeruginosa Immunotype 1 Lipopolysaccharide‐Specific Monoclonal Antibody (MAb) in a Murine Thigh Infection Model: Combined Effects of MAb and Ceftazidime
Authors: Akiyama Moritoshi , Oishi Kazunori , Tao Misao , Matsumoto Keizo , Pollack Matthew
Published: November 2013

A murine monoclonal antibody (MAb) specific for the Pseudomonas aeruginosa immunotype 1 (It‐1) lipopolysaccharide (LPS) O‐side chain was evaluated in terms of its in vitro bactericidal opsonophagocytic activity and in vivo bacterial killing in a mouse thigh infection model. An immunoglobulin (Ig) G2a MAb Ld3–2F2, specific for It‐1 LPS, mediated in vitro complement‐dependent opsonophagocytic killing at a concentration of 10 μg/ml. MAb‐mediated, complement‐dependent killing also occurred in the absence of neutrophils at serum concentrations in excess of 20%. A remarkable synergy was observed in opsonophagocytic assays between MAb Ld3–2F2 (0.5 μg/ml) and ceftazidime (1/4 MIC). The administration of MAb Ld3–2F2 at a level of 1 μg resulted in a significant decrease in the number of bacteria in the thigh muscles of normal mice, while 100 μg of the same MAb was required for one log of reduction in the number of bacteria at the same site in neutropenic mice. The combined therapy with MAb Ld3–2F2 and ceftazidime provided a significant reduction in the density of bacteria in the thigh muscle at 9 hr post‐infection in normal and neutropenic mice as compared with those after treatment alone or with no treatment (P < 0.01). These favorable in vitro and in vivo interactions of an LPS‐specific IgG MAb and ceftazidime strongly support their potential for use in therapy, combined with an LPS‐reactive MAb and parenteral antipseudomonas β‐lactam antibiotics in the therapy of systemic Pseudomonas infections in normal and neutropenic hosts.
Effect of Ions on Antibacterial Activity of Human Beta Defensin 2
Authors: Tomita Tetsuji , Hitomi Shigemi , Nagase Takahide , Matsui Hirotoshi , Matsuse Takeshi , Kimura Satoshi , Ouchi Yasuyoshi
Published: November 2013

Human beta defensin 2 (HBD‐2), the most recently discovered human defensin, has been considered to work as a host defense substance against microbial infection. Using Escherichia coli ATCC 25922, we investigated how some cations and anions influenced the antimicrobial activity of HBD‐2. This activity, measured in 10 mm phosphate buffer at a concentration of 20 μg/ml, reduced significantly in the presence of 100 and 150 mm sodium or potassium chloride. The reduction was not significantly different when the total amounts of sodium and potassium ions were equal. The kind and the valence of anions (chlorine and sulfate ions) did not affect the bactericidal activity as long as the concentrations of sodium ions were equal. Divalent ions (calcium and magnesium ions) added to 10 mm of Tris buffer significantly inactivated HBD‐2 at much lower concentrations (more than or equal to 0.01 mm and 0.05 mm, respectively) than the monovalent ions did. These findings suggest that HBD‐2 kills the bacteria through at least two phases, which are affected independently by either monovalent or divalent ions and unaffected by anions.
Mechanism of High Susceptibility of Iron‐Overloaded Mouse to Vibrio vulnificus Infection
Authors: Hor Lien‐I , Chang Yir‐kun , Chang Chien‐chung , Lei Huan‐yao , Ou Jonathan T.
Published: November 2013

Vibrio vulnificus produces fulminant septicemia in humans with underlying conditions, particularly those with diseases that elevate the iron level. The effect of a high iron level on the virulence of V. vulnificus was therefore investigated in mice treated with iron dextran. The mice loaded with iron became highly susceptible to V. vulnificus infection, the LD50 (50% lethal dose) decreased five logs when infected per peritoneum. However, when infected via the oral route, the LD50 was affected little unless the mouse was treated with an additional drug such as cyclophosphamide or d‐galactosamine. Mice with or without iron‐overloading died when the bacterial concentration in the blood reached 105 cfu/ml or above. Iron increased the growth rate of the bacteria, both inside and outside of the animal, quickly reaching a lethal concentration in the iron‐overloaded mouse. V. vulnificus, grown with or without the addition of iron, showed strong cytotoxicity on the isolated cells or within the animal at high bacterial concentration. Iron overload stimulated the production of tumor necrosis factor a (TNF‐α), a major factor of septic shock, in mice upon infection with the bacteria, probably caused by the endotoxin; however, the neutrophils, whose migration is effected by TNF‐α, appeared to be less active. Taken together, the major virulence factor of V. vulnificus appeared to be the accelerated growth of bacteria to quickly reach the lethal level and the lower activity of immune cells including neutrophil as a result of iron‐overloading. These two effects manifest other virulence factors, the host's as well as bacterial. Such factors, other than TNF‐α stimulated by the endotoxin, enhanced cytotoxicity, which kills the host cells including the host's immune cells.
Serological Properties of Abiotrophia and Granulicatella Species (Nutritionally Variant Streptococci)
Authors: Kitada Katsuhiro , Okada Yasuko , Kanamoto Taisei , Inoue Masakazu
Published: November 2013

Serological variations were examined among 12 type or reference strains and 91 oral isolates of vitamin B6‐dependent Abiotrophia and Granulicatella spp. Rabbits were immunized with whole cells of 12 selected strains and 10 typing antisera were obtained, which were unreactive with the Lancefield group A to G antigen preparations. The reactivity of the antisera and autoclaved cell surface antigen extracts was tested by double diffusion in agar gel and a capillary precipitin test. These typing antisera categorized all Abiotrophia defectiva strains, all except one Granulicatella elegans strain, three‐quarters of the Granulicatella adiacens, and half of the Granulicatella paraadiacens into 8 serotypes and 2 subserotypes. The Granulicatella balaenopterae type strain was unserotypable. All A. defectiva strains were serotype I, some of which were divided into subserotype I‐1 and/or I‐5. The G. adiacens strains generally belonged to serotype II or III, and the G. paraadiacens strains to serotype IV, V or VI. All G. adiacens or G. paraadiacens serotype II strains were also subserotype I‐5. The G. elegans strains were serotype VII or VIII. These Abiotrophia and Granulicatella serotypes were undetectable among 33 strains of the other 11 species including the bacteriolytic enzyme‐producing but vitamin B6‐independent strains of Streptococcus, Enterococcus, Dolosigranulum and Aerococcus. The proposed serotyping system for Abiotrophia and Granulicatella spp. would be helpful in the identification and classification of these unique coccal isolates in ecological and epidemiological studies.
Acute Primary Infection with Cytomegalovirus (CMV) in Kidney Transplant Recipients Results in the Appearance of a Phenotypically Aberrant CD8+ T Cell Population
Authors: Van Dam Joanne G. , Damoiseaux Jan G.M.C. , Christiaans Maarten H.L. , Bruggeman Cathrien A.
Published: November 2013

Human cytomegalovirus (CMV) is a β‐herpesvirus that causes a chronic subclinical infection in healthy man. The immune system is unable to eliminate the virus completely, allowing virus to persist in a latent state. In the immunocompromised host, this equilibrium is disturbed, resulting in a clinical infection. In immunocompromised rats, clinical CMV infection is associated with an increase in NK cells and CD8+ T cells, including a phenotypically aberrant CD8+ T cell population. Using flow cytometry, we examined the effect of acute CMV infection on the composition of leukocyte subsets in immunocompromised patients. Therefore, we used peripheral blood of CMV seronegative patients receiving a kidney from a seronegative (control group) or a seropositive donor. Of the patients receiving a seropositive kidney, only the patients undergoing acute CMV infection were included (experimental group). Special attention was paid to the phenotype of the cytotoxic T cells. The development of acute CMV infection resulted in an increased NK cell number and an activation of both CD4+ and CD8+ T cells, as determined by HLA‐DR expression. An aberrant CD8+ T cell subset with decreased expression of CD8 and TCRαβ appeared in the infected patients. Furthermore, the size of this subpopulation of CD8+ T cells is positively correlated with the viral load.
Seroprevalence of Antibodies against Spotted Fever Group Rickettsia among Dogs and Humans in Okinawa, Japan
Authors: Satoh Hiroshi , Tsuneki Akemi , Inokuma Hisashi , Kumazawa Norichika , Jahana Yoshihide , Kiyuuna Tsuyoshi , Okabayashi Tamaki , Muramatsu Yasukazu , Ueno Hiroshi , Morita Chiharu
Published: November 2013

We evaluated serum antibodies against Rickettsia japonica in 517 dogs (430 stray dogs and 87 pet dogs) and 164 humans in Okinawa, Japan, by indirect immunofluorescence assay. The seropositive rate in stray dogs was significantly higher than that in pet dogs (30.7 versus 4.6%, P<0.01). This high prevalence rate is attributed to the understandably frequent environmental exposure of stray dogs to tick infestation. Human samples obtained from Okinawa and Sapporo also showed a significant difference in seropositive antibody percentages (45.1 and 12.0%, respectively, P<0.01). This result suggests that there has been preexposure to spotted fever group rickettsia in humans in Okinawa.
Serological Analysis of Human Leptospirosis in the Philippines
Authors: Masuzawa Toshiyuki , Dancel Louella A. , Miyake Masaki , Yanagihara Yasutake
Published: November 2013

Leptospirosis in the Philippines is an underrepresented disease. To achieve an accurate means of serodiagnosis, we demonstrated antibodies to the prevalent Leptospira serovars in sera of 71 patients from three major hospitals in Manila by the microscopic agglutination test and Western blot analysis. Sera of 53 patients contained antibody against 8 serovars poi, tarassovi, manilae, pyrogenes, australis, grippotyphosa, javanica, and autumnalis.
Molecular Characterization in the VP7, VP4 and NSP4 Genes of Human Rotavirus Serotype 4 (G4) Isolated in Japan and Kenya
Authors: Kudo Satoshi , Zhou Yumei , Cao Xin‐Ru , Yamanishi Shigeki , Nakata Shuji , Ushijima Hiroshi
Published: November 2013

The VP7, VP4 and NSP4 genes of human rotavirus serotype 4 (G4) were analyzed to investigate intraserotypic variations. The techniques used included reverse transcription polymerase chain reaction with subtype specific primers, restriction fragment length polymorphism analysis and sequence analysis. Twelve isolates (nine from Japan and three from Kenya) and two standard strains (Hochi, Odelia) were G4A P[8] Wa group NSP4. A standard strain (ST3) was G4A P[6] Wa group NSP4 and a strain (VA70) was G4B P[8] Wa group NSP4. These results show G4 rotaviruses can be divided into three combinations at the moment.
Iron‐Mediated Regulation of Alkaline Proteinase Production in Pseudomonas aeruginosa
Authors: Shigematsu Takashi , Fukushima Jun , Oyama Masatsune , Tsuda Masataka , Kawamoto Susumu , Okuda Kenji
Published: November 2013

We analyzed the regulation by iron of alkaline proteinase (AP) production in Pseudomonas aeruginosa. Extracellular AP production was detected from the mid‐logarithmic to the stationary phase by an antibody‐based assay system, and was strongly repressed by iron in the medium. This repression was shown by Northern hybridization and primer extension to occur at the level of transcription. The primer extension analysis revealed that the start point of transcription of AP gene was the nucleotide position −84 from the start point of translation. Furthermore, we investigated whether this transcriptional repression involved PvdS protein. Using the mutant strain of pvdS, the alternative σ factor gene revealed that the PvdS protein is required for the full expression of AP, and a previous study showed that expression of pvdS is also repressed by iron. Therefore, we thought that one mechanism of repression of AP production operated through reduction of the PvdS protein level. Purified AP decomposed the transferrin, and released iron from it Purified AP added to the medium containing transferrin as the only iron source enhanced the growth of P. aeruginosa. Moreover, mutation in the AP gene decreased the growth rate in the medium containing the transferrin as the only iron source. These results clearly indicated that AP expression should occur in a free‐iron‐deficient environment and emphasized the importance of AP to iron acquisition in the infection site.