Detection of Francisella tularensis and analysis of bacterial growth in ticks in Japan.

UNLABELLED: Francisella tularensis is distributed in the Northern hemisphere and it is the bacterial agent responsible for tularaemia, a zoonotic disease. We collected 4 527 samples of DNA from ticks in Japan, which were then analysed by real-time PCR and nested PCR. Francisella DNA was detected by real-time PCR in 2·15% (45/2 093) of Ixodes ovatus, 0·66% (14/2 107) of I. persulcatus, 8·22% (6/73) of I. monospinosus and 0·72% (1/138) of Haemaphysalis flava specimens. Finally, Francisella DNA was detected by nested PCR in 42 and five samples I. ovatus and I. persulcatus, respectively, which were positive according to real-time PCR. Phylogenetic analysis showed that the sequence from I. ovatus and I. persulcatus were clustered with F. tularensis type B strains distributed in Eurasia. Microinjected live F. tularensis persisted in ticks, whereas heat-killed F. tularensis decreased. Microinjected F. tularensis hlyD mutant decreased in ticks significantly compared to parent strain, thereby suggesting that HlyD in F. tularensis contributes to the adaptation or survive of bacterial infection in ticks. SIGNIFICANCE AND IMPACTS OF THE STUDY: Francisella tularensis has been detected in ticks, suggesting that it is a tick-borne pathogen. However, F. tularensis has not been detected in ticks in Japan since 1991. In this study, we performed a large-scale analysis of DNA isolated from ticks in Japan and detected F. tularensis by real-time polymerase chain reaction (PCR) and nested PCR. We found that F. tularensis could survive in ticks based on an experimental tick-infection model. We also identified a bacterial factor that contributes to survival in ticks. Our results suggest that ticks are candidate vectors that mediate F. tularensis infection in Japan.

Infection of different strains of mice with Lawsonia intracellularis derived from rabbit or porcine proliferative enteropathy.

This report describes intestinal lesions in five strains of mice infected orally with Lawsonia intracellularis-infected tissue homogenates from rabbits or pigs (RLI and PLI). BALB/cA, C3H/HeJ, C57BL/6J and ICR mice were susceptible to infection with RLI, whereas only C3H/HeJ, C57BL/6J and ICR strains were susceptible to PLI. In susceptible mice, crypt epithelial hyperplasia occurred in association with an inflammatory reaction, as in proliferative enteropathy (PE) in other species. The intestinal changes in the infected mice varied from mild to severe. Unlike rabbit or porcine PE, in which the changes are confined to the ileum, the lesions in mice were located in the caecum. Immunolabelling of L. intracellularis antigen was abundant in early infection when the epithelial hyperplasia was mild or absent. When the hyperplasia had become severe, however, immunolabelling was weak. For this reason, it is suggested that transitory infection of the epithelium induces epithelial hyperplasia. Genetic differences between mouse strains appeared to play an important role in the response to L. intracellularis infection. Moreover, the susceptibility of BALB/cA mice to RLI but not to PLI suggests that there are significant biological differences between L. intracellularis isolates from rabbit PE and porcine PE.

Disease-specific expression of VEGF and its receptors in AML cells: possible autocrine pathway of VEGF/type1 receptor of VEGF in t(15;17) AML and VEGF/type2 receptor of VEGF in t(8;21) AML.

Various angiogenic factors, such as vascular endothelial growth factor (VEGF) and an associated molecule, placenta growth factor (PlGF), are thought to be important for normal and malignant hematopoiesis. This study examined mRNA expression of VEGF, PlGF and receptors for these molecules in AML cells and identified the disease-specific patterns of expression. AML M3 having t(15;17) abnormality showed highest expression of VEGF and VEGF receptor type 1 (VEGFR1), suggesting the autocrine pathway of VEGF-VEGFR1. Then, t(8;21) AML demonstrated augmented expression of VEGF and VEGF receptor type 2 (VEGFR2), suggesting VEGF-VEGFR2 autocrine pathway. Then, addition of VEGFR2 kinase inhibitor in Kasumi-1, a t(8;21) AML cell line, resulted in marked inhibition of cell growth, although growth inhibitory effect of R2 kinase inhibitor to HL-60 was marginal. In addition, cell cycle analysis study showed S-phase cell population reduction by R2 kinase inhibitor in Kasumi-1, but not in HL-60. This observation is thought to be the rationale for novel molecular target therapy directed to angiogenic molecules.

Rapid and effective detection of anthrax spores in soil by PCR.

AIMS: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. METHODS AND RESULTS: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis-specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSIONS: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance.

Expression of endothelial cell-associated molecules in AML cells.

Recently, it has been clarified that interaction between hematopoietic cells and endothelial cells is important in normal hematopoiesis and leukemogenesis. In this study, we examined the relationship between AML cells and endothelial cells by analyzing the expression profile of angiogenic factors, angiopoietin-1 (Ang-1), Ang-2, Tie-2 (a receptor for angiopoietins) and vascular endothelial growth factor (VEGF). Our results demonstrated that CD7(+)AML expressed Ang-2 mRNA frequently and integrin-family adhesion molecules (CD11c and CD18) intensively, suggesting the close correlation with endothelial cells. On the other hand, in t(8;21) AML cells, expression of Ang-2 was infrequent and expression of integrin-family adhesion molecules (CD11b, CD11c and CD18) was weak, suggesting the sparse association with endothelial cells. As for CD7(+)AML cells, despite the frequent and intense expression of endothelial cell-associated molecules (such as Ang-2, CD11c and CD18), intensity of Tie-2 expression was quite low (P < 0.05). Ang-2 expressed in CD7(+)AML cells is not considered to act in an autocrine fashion, but to work on endothelial cells to "feed" leukemic cells. Although Ang-2 is recognized as a natural antagonist for Tie-2, our data presented here suggested the alternative role of Ang-2 in the relationship between endothelial cells and leukemia cells, at least in a subset of leukemia such as CD7(+)AML. These results were supported by the study using AML cell lines, KG-1 (CD7 negative) and its subline KG-1a (CD7 positive); KG-1 had mRNA expression profile of Ang-1(+)Ang-2(-)Tie-2(+), while KG-1a showed Ang-1(+)Ang-2(+)Tie-2(-). These difference in the expression profile of angiogenic factors between CD7(+)AML and t(8;21)AML may explain the characteristic morphological features of these leukemias (CD7(+)AML as blastic type and t(8;21)AML as differentiative type).

Legionella pneumophila is internalized by a macropinocytotic uptake pathway controlled by the Dot/Icm system and the mouse Lgn1 locus.

The products of the Legionella pneumophila dot/icm genes enable the bacterium to replicate within a macrophage vacuole. This study demonstrates that the Dot/Icm machinery promotes macropinocytotic uptake of L. pneumophila into mouse macrophages. In mouse strains harboring a permissive Lgn1 allele, L. pneumophila promoted formation of vacuoles that were morphologically similar to macropinosomes and dependent on the presence of an intact Dot/Icm system. Macropinosome formation appeared to occur during, rather than after, the closure of the plasma membrane about the bacterium, since a fluid-phase marker preloaded into the macrophage endocytic path failed to label the bacterium-laden macropinosome. The resulting macropinosomes were rich in GM1 gangliosides and glycosylphosphatidylinositol-linked proteins. The Lgn1 allele restrictive for L. pneumophila intracellular replication prevented dot/icm-dependent macropinocytosis, with the result that phagosomes bearing the microorganism were targeted into the endocytic network. Analysis of macrophages from recombinant inbred mouse strains support the model that macropinocytotic uptake is controlled by the Lgn1 locus. These results indicate that the products of the dot/icm genes and Lgn1 are involved in controlling an internalization route initiated at the time of bacterial contact with the plasma membrane.

Detection of anthrax spores from the air by real-time PCR.

AIMS: To detect and isolate Bacillus anthracis from the air by a simple and rapid procedure. METHODS AND RESULTS: One hundred litres of air were filtered through an air monitor device. After the membrane was suspended in PBS, spores of B. anthracis were added. The suspension was plated on Bacillus cereus selective agar (BCA) plates to detect B. anthracis colonies. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using a Light Cycler system and anthrax-specific primers. CONCLUSION: A single cell of B. anthracis was detected by real-time PCR within 1 h and was also isolated on a BCA plate within two d. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR and a Light Cycler provides a flexible and powerful tool to prevent epidemics.

Phylogenetic diversity and similarity of active sites of Shiga toxin (stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals.

Nucleotide sequences of Shiga toxin (Stx) genes in STEC from various origins were determined and characterized by phylogenetic analysis based on Shiga toxin (Stx) with those deposited in GenBank. The phylogenetic trees placed Stx1 and Stx2 into two and five groups respectively, and indicated that Stx1 in sheep-origin STEC were placed into a different group from those in other STEC, and that Stx2 of deer-origin STEC also belonged to the unique group and appeared to be distantly related to human-origin STEC. On the other hand, Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC. Such a diversity of Stx suggested that STEC might be widely disseminated in many animal species, and be dependent on their host species or their habitat. In addition, the active sites in both toxins were compared; the active sites in both subunits of Stx in all the animal-origin STEC were identical to those in human-origin STEC, suggesting that all the toxin of STEC from animals might be also cytotoxic, and therefore, such animal-origin STEC might have potential pathogenicity for humans.

A simple and sensitive detection system for Bacillus anthracis in meat and tissue.

AIMS: To detect and isolate Bacillus anthracis from meat and tissue by rapid and simple procedures. METHODS AND RESULTS: Bacillus anthracis Pasteur II cells were added to 1 g lymph node and pig meat, which were then cut into small pieces and suspended in PBS. Aliquots were spread on Bacillus cereus selective agar (BCA) plates to isolate B. anthracis cells, and incubated in trypticase soy broth. The enrichment culture was used for nested PCR with B. anthracis specific primers, which were to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSION: One cell of B. anthracis was detected by nested PCR from 1 g of the samples, and was also isolated on BCA plates according to colony morphology within two days. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be useful for detecting animals with latent anthrax, and meat contaminated with B. anthracis, rapidly and simply.

Genetically modified Shiga toxin 2e (Stx2e) producing Escherichia coli is a vaccine candidate for porcine edema disease.

Porcine edema disease (ED) is an enterotoxaemia in pigs after weaning, caused by Shiga toxin 2e (Stx2e) producing Escherichia coli. Recently in Japan, outbreaks of ED are re-emerging in pig production. In this study we constructed a mutant that retained immunogenicity but lost Vero cell cytotoxicity, which produced genetically modified toxin (Stx2e*) by replacing glutamate with glutamine at position 167 and arginine with leucine at position 170 of the A subunit. The stx(2e)* gene was replaced with the stx(2e)gene of the wild type virulent strain by homologous recombination. As the parent wild strain was pathogenic to pigs but the mutant was not, the mutant named as YT106 was given to the pigs to examine its protective immunity against ED. All 20 pigs vaccinated with YT106 survived, but only eight of the 20 non-vaccinated pigs survived after the challenge with a wild strain. Also, the eight pigs that survived had decreased rates of gain relative to those of the controls. Blood IgG and intestinal IgA titres increased 3.3 and 1.6 times more than the control, respectively, showing that YT106 might be a good candidate of a live attenuated vaccine strain to protect against ED.

Formation of a fibrous structure on the surface of Legionella pneumophila associated with exposure of DotH and DotO proteins after intracellular growth.

Legionella pneumophila grows in human alveolar macrophages and resides within a phagosome that initially lacks proteins associated with the endocytic pathway. Required for targeting to this unique location is the Dot/Icm complex, which is highly similar to conjugative DNA transfer apparatuses. Here, we show that exposure to three distinct inducing conditions resulted in the formation of a fibrous structure on the bacterial cell surface that contained the DotH and DotO proteins. These conditions included: (i) incubation for 2 h with mouse bone marrow-derived macrophages; (ii) incubation for 2 h in macrophage-conditioned media; or (iii) replication of bacteria for 22 h within macrophages. Introduction of bacteria harbouring the surface-exposed DotH and DotO onto a fresh monolayer resulted in loss of the surface localization of DotH and DotO shortly after uptake. Treatments that resulted in the production of the fibrous structure enhanced the rate at which the bacteria were internalized, but there was no corresponding increase in the efficiency of intracellular growth compared with bacteria that had been cultured in broth using conditions that resulted in maximal intracellular growth. These data indicate that the surface-exposed DotH and DotO on L. pneumophila may act either just before lysis from the macrophage or at the earliest stages of infection, transiently relocating in a fibrous structure on the bacterial cell surface.

Effects of rearing conditions on the colonization of Salmonella enteritidis in the cecum of chicks.

Salmonella enteritidis is the cause of human salmonellosis associated with contaminated eggs. In this study, we artificially challenged S. enteritidis to chicks just after hatching, and the effects of breeding conditions on the intestinal carriage of S. enteritidis were examined. S. enteritidis was not directly detected from spleen, liver and blood, but were constantly isolated from the cecal contents throughout the experiment. When chicks were reared in the unsanitary conditions and in the high housing density, the numbers of S. enteritidis increased. The subsequent experiment was undertaken to examine whether the antibacterial additive in a feed would have any impact on S. enteritidis colonization in chicks. Some antibiotic effective on the growth promotion had an influence on S. enteritidis colonization.

Detection and characterization of Shiga toxin-producing Escherichia coli from seagulls.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from a seagull in Japan were examined. A total of 50 faecal samples was collected on a harbour bank in Hokkaido, Japan, in July 1998. Two different STEC strains, whose serotypes were O136:H16 and O153:H-, were isolated from the same individual by PCR screening; both of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing active Stx2 and Stx1, respectively. They harboured large plasmids, but did not carry the haemolysin or eaeA genes of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, pulsed-field gel electrophoresis analysis (PFGE), and the stx genes sequences, the isolates were different. Phylogenic analysis of the deduced Stx amino acid sequences demonstrated that the Stx toxins of seagull-origin STEC were closely associated with those of the human-origin, but not those of other animal-origin STEC. In addition, Stx2phi-K7 phage purified from O136 STEC resembled Stx2phi-II from human-origin O157:H7, and was able to convert non-toxigenic E. coli to STEC. These results suggest that birds may be one of the important carriers in terms of the distribution of STEC.

Antihypertensive actions of methylripariochromene A from Orthosiphon aristatus, an Indonesian traditional medicinal plant.

Methylripariochromene A (MRC) was isolated from the leaves of Orthosiphon aristatus (Lamiaceae) and subjected to the examination of several pharmacological actions related to antihypertensive activity. The following four findings were revealed from the present study: 1) MRC caused a continuous decrease in systolic blood pressure and a decrease in heart rate after subcutaneous administration in conscious male SHRSP, 2) MRC exhibited the concentration-dependent suppression of contractions induced by high K+, l-phenylephrine or prostaglandin F2alpha in endothelium-denuded rat thoracic aorta, 3) MRC showed a marked suppression of contractile force without a significant reduction in the beating rate in isolated bilateral guinea pig atria, and 4) MRC increased urinary volume and the excretion of Na+, K+ and Cl- for 3 h after oral administration with a load of saline in fasted rats. These findings indicate that MRC possesses some actions related to a decrease in blood pressure, i.e. vasodilating action, a decrease in cardiac output and diuretic action. Furthermore, it is presumed that the traditional use of this plant in the therapy of hypertension may be partially supported by these actions with MRC.

Amount of metallic ions released from Ti-Ni alloy by abrasion in simulated bioliquids.

The current density of Ti-56mass%Ni (Ti-50at.%) alloy after abrasion in simulated bioliquids was measured using a potentiostat to estimate the amount of metallic ions released from the alloy during repassivation and maturation. The current density in saline, saline with and without N2 bubbling, and Hanks' solutions with and without proteins after abrasion was measured and the amount of released ion was calculated from the integrated current density with time, assuming that Ti4+ and Ni2+ are equivalently released. No difference in the amount of released ion was observed between saline with and without N2 bubbling. Also, no difference was observed between saline and pH 7.4 Hanks' solution. More Ti4+ and Ni2+ were released in bioliquids with proteins than in saline with and without N2 bubbling (p < 0.05). That is, dissolved oxygen and inorganic ions in Hanks' solution did not influence the amount of released ion, but proteins influenced it. The release of metallic ions from metals and alloys in biological systems can be estimated by the methodology employed in this study.

Identification and characterization of a newly isolated shiga toxin 2-converting phage from shiga toxin-producing Escherichia coli.

Shiga toxins 1 (Stx1) and 2 (Stx2) are encoded by toxin-converting bacteriophages of Stx-producing Escherichia coli (STEC), and so far two Stx1- and one Stx2-converting phages have been isolated from two STEC strains (A. D. O'Brien, J. W. Newlands, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal, Science 226:694-696, 1984). In this study, we isolated two Stx2-converting phages, designated Stx2Phi-I and Stx2Phi-II, from two clinical strains of STEC associated with the outbreaks in Japan in 1996 and found that Stx2Phi-I resembled 933W, the previously reported Stx2-converting phage, in its infective properties for E. coli K-12 strain C600 while Stx2Phi-II was distinct from them. The sizes of the plaques of Stx2Phi-I and Stx2Phi-II in C600 were different; the former was larger than the latter. The restriction maps of Stx2Phi-I and Stx2Phi-II were not identical; rather, Stx2Phi-II DNA was approximately 3 kb larger than Stx2Phi-I DNA. Furthermore, Stx2Phi-I and Stx2Phi-II showed different phage immunity, with Stx2Phi-I and 933W belonging to the same group. Infection of C600 by Stx2Phi-I or 933W was affected by environmental osmolarity differently from that by Stx2Phi-II. When C600 was grown under conditions of high osmolarity, the infectivity of Stx2Phi-I and 933W was greatly decreased compared with that of Stx2Phi-II. Examination of the plating efficiency of the three phages for the defined mutations in C600 revealed that the efficiency of Stx2Phi-I and 933W for the fadL mutant decreased to less than 10(-7) compared with that for C600 whereas the efficiency of Stx2Phi-II decreased to 0.1% of that for C600. In contrast, while the plating efficiency of Stx2Phi-II for the lamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2Phi-I and 933W were not changed. This was confirmed by the phage neutralization experiments with isolated outer membrane fractions from C600, fadL mutant, or lamB mutant or the purified His6-tagged FadL and LamB proteins. Based on the data, we concluded that FadL acts as the receptor for Stx2Phi-I and Stx2Phi-II whereas LamB acts as the receptor only for Stx2Phi-II.

A matrix form of fibronectin mediates enhanced binding of Streptococcus pyogenes to host tissue.

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) binds to fibronectin via protein F. In this study, we have investigated the binding properties of protein F to various multimeric tissue forms of fibronectin that appear on cell surfaces and in the extracellular matrix. We show that binding of S. pyogenes through protein F is more efficient to an in vitro-derived polymerized form of fibronectin (superfibronectin) than to soluble fibronectin immobilized in a solid phase. In addition, Chinese hamster ovary cells overexpressing the alpha5beta1 integrin produced an increased amount of a fibronectin matrix and consequently bound a higher number of S. pyogenes cells. Inhibition and direct binding assays using purified proteins demonstrated that binding to a fibronectin matrix involved both domains of protein F (UR and RD2) that have previously been implicated in interactions with fibronectin. Using intact S. pyogenes bacteria in which various domains of protein F were expressed as hybrids with the surface-exposed region of an unrelated protein, we revealed that, in contrast to the predominantly UR-mediated binding to soluble fibronectin, the maximal binding to the fibronectin matrix required RD2 in addition to UR. Since in some infections S. pyogenes may initially encounter a matrix form of fibronectin, these results suggest that UR and RD2 may be important for the initiation of streptococcal infectious processes.

rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells.

Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamister ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.