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Background Borrelia miyamotoi is a newly described relapsing fever spirochete transmitted by ixodid tick species. Little is known about the prevalence of B. miyamotoi infections in humans and ticks in Inner Mongolia, China. Therefore, we investigated the prevalence of B. miyamotoi in Ixodes persulcatus ticks, and we aimed to isolateB. miyamotoi from I. persulcatus from four regions of Greater Khingan, Inner Mongolia, China. Methods From May to June each year during the period 2016–2019, host-seeking adult I. persulcatus ticks were collected from vegetation. Genomic DNA was prepared from half of each tick body for PCR template, and the remaining half was used to cultivate B. miyamotoi in BSK-M medium. We employed quantitative real-time PCR (qPCR) to detect Borrelia DNA in the ticks and to calculate the prevalence of B. miyamotoi and infections with other borreliae. For characterization of the isolated B. miyamotoi, we performed draft genome sequencing and multilocus sequencing analysis (MLSA). Results A total of 2656 adult I. persulcatus ticks were collected. The overall prevalence of relapsing fever (RF) borreliae in ticks was 5.0% (134/2656) and that of Lyme disease (LD) borreliae was 43.8% (1164/2656). Co-infection with RF and LD borreliae was observed in 63 ticks (2.4%). Ticks that were positive for RF borreliae by qPCR were subjected to glycerophosphodiester diester phosphodiesterase gene (glpQ) PCR amplification and sequencing, through which we identified the RF borrelia specimens as B. miyamotoi. Furthermore, the B. miyamotoi strain Hetao-1 was isolated from I. persulcatus, and a draft genome sequence was obtained from the isolate. Sequencing determined the strain Hetao-1 genome to be approximately 906.1 kbp in length (28.9% average GC content), and MLSA identified the strain as ST633, which has previously been reported in Japan and Mongolia. Conclusion We detected B. miyamotoi from I. persulcatus ticks collected in Inner Mongolia, and successfully isolated a B. miyamotoi strain. To our knowledge, this is the first study to culture a B. miyamotoi isolate from China. The data on the prevalence of B. miyamotoi and other borreliae in I. persulcatus ticks will be fundamental for future epidemiological studies of B. miyamotoi disease in Inner Mongolia.

Non-pathogenic Rickettsia species LON strains closely related to an agent of Japanese spotted fever (JSF), R. japonica, were isolated in Japan from Haemaphysalis longicornis ticks in 2001. However, the biological properties of LONs in mammalian host cells are poorly understood. In this study, microscopic analysis showed that LONs in a mouse-derived L929 host cell line were rod shaped with sizes of 0.3-0.5 × 0.5-2.0 μm. Molecular analysis revealed the existence of a LON-specific disrupted open reading frame in R. japonica-related group-specific DNA regions. Growth kinetics of LON-2 and LON-13 strains analyzed by a quantitative real-time PCR showed 100-fold or more increment of LONs cultured in L929 host cells at 30°C and slightly less increment at 33°C, and 25-fold increment in human-derived THP-1 host cells at 35°C on day 7 (168 h) post infection. The generation times of the two LON strains cultured in L929 and THP-1 were estimated to be 9.4-12.9 h and 9.6-10.9 h, respectively. To our knowledge, this is the first report on the biological characteristics of Rickettsia sp. LON strains in mammalian cells, which may provide significant information for the experimental approaches for other rickettsiae.

Ehrlichia species are obligatory intracellular bacteria transmitted by arthropods, and some of these species cause febrile diseases in humans and livestock. Genome sequencing has only been performed with cultured Ehrlichia species, and the taxonomic status of such ehrlichiae has been estimated by core genome-based phylogenetic analysis. However, many uncultured ehrlichiae exist in nature throughout the world, including Japan. This study aimed to conduct a molecular-based taxonomic and ecological characterization of uncultured Ehrlichia species or genotypes from ticks in Japan. We first surveyed 616 Haemaphysalis ticks by p28-PCR screening and analyzed five additional housekeeping genes (16S rRNA, groEL, gltA, ftsZ, and rpoB) from 11 p28-PCR-positive ticks. Phylogenetic analyses of the respective genes showed similar trees but with some differences. Furthermore, we found that V1 in the V1–V9 regions of Ehrlichia 16S rRNA exhibited the greatest variability. From an ecological viewpoint, the amounts of ehrlichiae in a single tick were found to equal approx. 6.3E+3 to 2.0E+6. Subsequently, core-partial-RGGFR-based phylogenetic analysis based on the concatenated sequences of the five housekeeping loci revealed six Ehrlichia genotypes, which included potentially new Ehrlichia species. Thus, our approach contributes to the taxonomic profiling and ecological quantitative analysis of uncultured or unidentified Ehrlichia species or genotypes worldwide.

Staphylococcal enterotoxin A (SEA) functions both as superantigens that stimulate non-specific T cell proliferation as well as potent gastrointestinal toxins. We previously reported that (&minus, )-epigallocatechin gallate (EGCG) binds to SEA. Therefore, the ability of EGCG to inhibit SEA toxin activity was examined. As a result, EGCG significantly decreased SEA-induced expression and production of interferon gamma (IFN-&gamma, ). In addition, EGCG inhibited SEA-induced spleen cell proliferation. To investigate the role of the galloyl group in EGCG on SEA cytotoxicity in more detail, the effect of the binding of a hydroxyl group at position 3 of the galloyl group in EGCG to SEA on SEA cytotoxicity was examined using two methylated EGCG. SEA cytotoxicity was significantly controlled in both (&minus, )-3&prime, &prime, Me-EGCG and (&minus, )-4&prime, Me-EGCG. These results suggest that EGCG inhibits toxic activity via direct interaction with SEA or without any interaction with SEA. The binding affinity between SEA and EGCG under in vivo conditions was examined using a model solution. Although after treatment under acidic and alkaline conditions, the presence of protein and the digestive tract model solution, EGCG still interacted with SEA. Our studies are the first to demonstrate the effect of the binding of EGCG to SEA on toxin activity.

We found Rickettsia raoultii infection in 6/261 brucellosis-negative patients with fever of unknown origin in brucellosis-endemic Inner Mongolia, China. We further identified Hyalomma asiaticum ticks associated with R. raoultii, H. marginatum ticks associated with R. aeschlimannii, and Dermacentor nuttalli ticks associated with both rickettsiae species in the autonomous region.

We herein report a case of suspected Borrelia miyamotoi disease in Hokkaido, Japan. The patient complained of lassitude, arthralgia, and high fever after a tick bite. Furthermore, at the time of consultation, the patient exhibited momentary loss of consciousness and low blood pressure. Laboratory tests revealed elevation of liver enzymes, thrombocytopenia, and increased C-reactive protein. Seroconversion to B. miyamotoi glycerophosphoryl diester phosphodiesterase antigen suggested the patient was infected with a relapsing fever group Borrelia species.

The influence of muscle fiber direction (parallel or perpendicular) in relation to the inoculation surface on migration of Salmonella Enteritidis, Staphylococcus aureus, and Escherichia coli into raw chicken breasts was examined. Chicken breast samples with two types of surface fibers (running parallel or perpendicular to the surface) were inoculated with cultures of each bacterium. Inoculated samples were stored for 5 min, 1 h, or 24 h at 4°C. After storage, the samples were divided into segments, and bacterial counts were determined in different regions (inoculation surface, inoculation surface to 1 cm, 1 to 2 cm, 2 to 4 cm, and 4 to 6 cm). The migration of bacteria did not change at 5 min or 1 h regardless of fiber direction. However, after 24 h each bacterium was detected at 4 to 6 cm in the pieces of sample with a perpendicular muscle fiber surface cut. Although these bacteria were detected at 4 to 6 cm in samples with muscle fibers perpendicular to the inoculated surface, these results do not clearly indicate that bacteria migrated into the chicken breast. To monitor actual migration of bacteria into the chicken breast, the tops of the perpendicular muscle fibers of the breast sample were inoculated with bioluminescent E. coli Xen-14. Various regions of the breast sample (inoculation surface and cut surfaces at 1, 2, 4, and 6 cm) were stamped directly on growth medium. Culture revealed that the bacteria migrated directly under the contaminated site and dispersed along the surface of the chicken breast segments. More bacteria distributed laterally than migrated directly below the contamination site. These results suggest that the direction of the muscle fibers is a major factor influencing migration of pathogenic bacteria into chicken breast. HIGHLIGHTS

Anaplasma phagocytophilum is an obligate intracellular bacterium that causes human granulocytic anaplasmosis (HGA), an emerging tick-borne infectious disease. This bacterium expresses various 44-kDa major outer membrane proteins encoded by the p44/msp2 multigene family to avoid the host immune system. We previously detected A. phagocytophilum p44/msp2 from the tick Haemaphysalis longicornis in Mie Prefecture, Japan in 2008. In this study, we further investigated a total of 483 H. longicornis ticks (220 adults and 263 nymphs) collected from the Mie Prefecture by PCR targeting p44/msp2 to characterize the p44/msp2 multigene family of A. phagocytophilum. Six of the 483 ticks tested were PCR-positive for A. phagocytophilum p44/msp2, and these positive individuals were at the nymph stage of the tick life cycle. Cloning, sequencing, and phylogenetic analyses of the amplicons revealed that the 11 p44/msp2 clones obtained from the positive ticks shared a 54.9%-99.3% amino acid sequence similarity with the 27 previously identified clones from HGA patients in Japan. In particular, 6 p44/msp2 clones displayed the highest similarities (97.2%-99.3%) with 3 previously identified clones (FJ417343, FJ417345, FJ417357). Thus, the data from this study provide important public health information regarding A. phagocytophilum infection transmitted by H. longicornis ticks, especially at the nymph stage.

Anaplasma phagocytophilum, an agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium that dominantly produces P44 outer membrane proteins encoded by the p44/msp2 multigene family, which are major antigens for serodiagnosis. However, A. phagocytophilum antigens from cultures with different cell lines seem to have varying reactivities with sera. In this study, we performed RNA-seq to investigate the P44 expression of A. phagocytophilum propagated in 4 cell lines. In infected HL-60 cells, the P44-2b transcript was predominant in the first RNA-seq analysis (HL-60.1). However, the P44-23 transcript was predominant in the second RNA-seq analysis at 1 month after additional passages (HL-60.2). We further analyzed the P44 expression of A. phagocytophilum cultured in THP-1, NB4, and RF/6A cells through consecutive passages in the same cell lines for 1 year after transferring A. phagocytophilum from infected HL-60 cells to the respective cell lines. In the long-term cultures, P44-18, P44-78, and P44-51 were predominantly transcribed in infected THP-1, NB4, and RF/6A cells, respectively. Therefore, the predominant shifts of different P44-expressing transcripts of A. phagocytophilum might occur during cell culture even in the same cell line at different time points of sample harvest (HL-60.1 and HL-60.2), which may be attributed to host cell adaptation/selection/interaction.

Adult Ixodes persulcatus were collected in highly populated districts in Irkutsk city, Russia, and in popular recreational and professional areas in its neighboring territories. Borrelia miyamotoi infection in I. persulcatus was examined using multiplex Taqman-PCR targeting 16S rDNA, and nested PCR and sequencing analyses targeting flaB and 16S rDNA. B. miyamotoi and Lyme disease Borrelia species were detected in 13 (infection rate, 2.9%) and 77 (17.3%) out of 445 I. persulcatus ticks, respectively, collected from 4 sites around the Baikal Lake. The 16S rDNA and flaB sequences of these amplicons were closely related to those of B. miyamotoi detected and/or isolated from I. persulcatus in Japan and Far Eastern Russia, and clustered separately from those of Europe and North America. These results indicate that additional surveillance for B. miyamotoi infection is needed in order to determine how it affects human health in Irkutsk City and its neighboring territories.

The hard tick-borne relapsing fever agent, Borrelia miyamotoi infection in Ixodes ricinus ticks sampled from Istanbul and the countryside of Kirklareli in northwestern Turkey, was examined by TaqMan-PCR targeting 16S rDNA, nested PCR targeting 16S rDNA, the flagellin gene (flaB), and the 16S and 23S rDNA intergenic spacer (IGS), and sequencing analyses of these amplicons. B. miyamotoi was detected in 1 out of 248 I. ricinus ticks (infection rate 0.4%). The tick infected with B. miyamotoi was collected in Longos, Kirklareli province on the European side of Turkey near the Bulgarian border. The 16S rDNA, flaB, and IGS sequences from the infected tick showed high similarities to those of B. miyamotoi detected in I. ricinus in Europe.

In this study, we investigated the interaction between apple polyphenols (AP; mainly consisting of procyanidin (PC) from an apple) and staphylococcal enterotoxin A (SEA), and the inhibitory effects of AP on SEA activity. According to the degree of polymerization, in particularly highly polymerized PC (more than pentamer) strongly interacted with SEA. The binding affinity of AP with SEA molecules was determined using Biacore analysis. AP reacted with SEA immobilized on a Biacore sensor chip. After treatment with pepsin and pancreatin, to examine the changes of binding affinity of AP in intragastric conditions, AP maintained interaction with SEA. We examined whether AP inhibits the proliferation and interferon-γ (IFN-γ) production induced by SEA in mouse spleen cells. AP strongly inactivated the proliferation and IFN-γ production induced by SEA. These results suggest that AP, which has a higher degree of polymerization, inactivates stronger biological activity of SEA through interaction with SEA. Our studies are the first to demonstrate the relationship between the degree of polymerization of AP and the inhibitory effects on SEA activities.

Bioluminescent imaging (BLI) has become a useful tool for monitoring bacterial infections in real time. Citrobacter rodentium and its BLI are widely used as a murine model of enteropathogenic and enterohaemorrhagic Escherichia coli infection. In this study, we evaluated the protective effects of the probiotic Lactobacillus brevis KB290 against C. rodentium infection by the BLI approach. First, we examined several solutions for making the suspension of bioluminescent C. rodentium for an oral inoculation to establish a stable intestinal infection. Three percent NaHCO3 solution was found to be the best. Subsequently, mice were orally administered KB290 once daily for 7 days before inoculation with bioluminescent C. rodentium and for 8 days after infection. The bioluminescence intensity of mice fed with KB290 was significantly lower than that of unfed mice on days 1-3 after infection. The mRNA levels of tumour necrosis factor-α and interferon-γ in the distal colon from KB290-fed mice were shown to be significantly higher than those from unfed mice on day 3 after infection. The results suggested that KB290 intake partially inhibited the proliferation of C. rodentium, especially in the early stages of infection, viathe moderate enhancement of tumour necrosis factor-α and interferon-γ production in the colon.

We demonstrated the presence of the agent of human granulocytic anaplasmosis (HGA), Anaplasma phagocytophilum, and the agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, in north-western Turkey. A total of 241 questing Ixodes ricinus adult ticks were sampled by flagging from recreational parks of the Asiatic and European sides of the heavily populated Istanbul metropolitan area and rural forests of Kirklareli located in the Thrace region in 2008. Both tick-borne pathogens were detected and identified by PCR and DNA sequencing analysis. A. phagocytophilum infection rates were 2.7% in Istanbul and 17.5% in the Kirklareli area. B. burgdorferi sensu lato infection rates were 38.7% in Istanbul and 11.4% in the Kirklareli area. Only 3 of 241 ticks were coinfected with A. phagocytophilum and B. burgdorferi sensu lato.

application/pdf, We collected a total of 206 Haemaphysalis longicornis ticks by flagging in pastures in Yonaguni Island, Okinawa, Japan, in April 2008. Four of the 206 tick DNA samples tested were positive in a polymerase chain reaction (PCR) screening for the 16SrRNA gene of Anaplasmataceae. Partial sequences of 4 PCR products were identical to each other. Longer sequences of the 16SrRNA gene were successfully determined in 2 of the 4 tick samples, and the obtained 1,392 bp and 1,300 bp sequences revealed high similarity to the 16SrRNA gene sequences of the validated Ehrlichia species, including Ehrlichia ewingii, E. chaffeensis, and E. canis (98.3–98.6%). We also sequenced 1,304 bp of the groEL gene from the 2 tick samples, and found that these had the highest similarity to sequences of E. ewingii (94.0–94.4%) in the validated ehrlichial species. Based on the 16SrRNA and groEL gene sequences, the ehrlichial agents detected in this study were similar to the Ehrlichia species detected in Asia and may compose a new Ehrlichia species with other Ehrlichia species detected in Asia.

L-proline (L-Pro) is a non-essential amino acid, and has become widely used as supplements and health foods, recently. A subchronic oral toxicity study of L-Pro was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.625%, 1.25%, 2.5% and 5.0% of L-Pro for 90 days. No treatment-related clinical signs and mortality were noted. We observed no clear treatment-related effects with regard to body weight, food intake or urinalysis data. The average daily water intakes of the treated female groups were significantly increased compared to the controls. The hematology (red blood cell parameter) and serum biochemistry (glucose, blood urea nitrogen, creatinine or uric acid) of the treated male and/or female groups were lower than those of the control groups. However, these changes were lacked dose-dependence, and no abnormalities were found in corresponding pathological findings. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3mg/kg body weight/day for females) under the present experimental conditions.

Several physical and psychological stresses frequently become triggers for gastrointestinal disorders such as ulcer. In this study, we tried to identify serum proteins as potential biomarkers for the evaluation of stress-induced gastric ulcer. By proteomic analysis using rats with gastric ulcer induced by water immersion and restraint (WIR) stress as an animal model, we found quantitative changes in several serum proteins, including creatine kinase muscle M chain (CK-M) and apolipoprotein A-IV (ApoA4) in the stressed rats. On western blotting and enzyme-linked immunosorbent assay (ELISA), we confirmed that serum CK-M was remarkably increased by WIR stress. However, ApoA4 appeared to be decreased by fasting, but not WIR stress, which is usually applied prior to WIR stress. The findings suggest that these two serum proteins might be useful as biomarkers, CK-M for stress-induced gastric ulcer and ApoA4 for starvation.

Expressed in renal carcinoma (ERC)/mesothelin is a good biomarker for human mesothelioma and has been investigated for its mechanistic rationale during the mesothelioma development. Studies are thus ongoing in our laboratories to assess expression of ERC/mesothelin in sera and normal/proliferative/neoplastic mesothelial tissues of animals untreated or given potentially mesothelioma-inducible xenobiotics, by an enzyme-linked immunosorbent assay (ELISA) for N- and C-(terminal fragments of) ERC/mesothelin and immunohistochemistry for C-ERC/mesothelin. In the present paper, we intend to communicate our preliminary data, because this is the first report to show how and from what stage the ERC/mesothelin expression changes during the chemical induction of mesothelial proliferative/neoplatic lesions. Serum N-ERC/mesothelin levels were 51.4 +/- 5.6 ng/ml in control male Fischer 344 rats, increased to 83.6 +/- 11.2 ng/ml in rats given a single intrascrotal administration of 1 mg/kg body weight of multi-wall carbon nanotube (MWCNT) and bearing mesothelial hyperplasia 52 weeks thereafter, and further elevated to 180 +/- 77 ng/ml in rats similarly treated and becoming moribund 40 weeks thereafter, or killed as scheduled at the end of week 52, bearing mesothelioma. While C-ERC/mesothelin was expressed in normal and hyperplastic mesothelia, the protein was detected only in epithelioid mesothelioma cells at the most superficial layer. It is thus suggested that ERC/mesothelin can be used as a biomarker of mesothelial proliferative lesions also in animals, and that the increase of levels may start from the early stage and be enhanced by the progression of the mesothelioma development.

Anaplasma phagocytophilum, an agent of human granulocytic anaplasmosis, infects neutrophils and causes an emerging tickborne febrile disease. The genome of this bacterium contains a large number of p44/msp2-related genes encoding 44 kDa major outer-membrane proteins, and it is known that a specific p44/msp2 gene is predominantly transcribed from a single expression locus. This study successfully characterized the genomic expression site for p44/msp2 (3.8 kb) in uncultured A. phagocytophilum from Ixodes persulcatus ticks inhabiting a northern part of Japan. Comparative analysis of the sequences revealed that the structures of the expression sites in Japanese A. phagocytophilum were similar to those of US strains from human patients and European strains from a dog and sheep, but omp-1N (upstream from p44/msp2) and a truncated recA (downstream from p44/msp2) in the p44/msp2 expression site seemed to share similarities with those of US and European strains. The central hypervariable region sequences of Japanese p44/msp2 were found to be quite diverse (24.4–100 % amino acid similarities) and distinct from their closest relatives from US human patients or animal host origins (56.3–97.6 % amino acid similarities) with some exceptions. Thus, this study provides significant information about the molecular characteristics of A. phagocytophilum in East Asia, as well as the global diversity of p44/msp2.

Here, we describe for the first time the prevalence and genetic properties of Bartonella organisms in wild rodents in Japan. We captured 685 wild rodents throughout Japan (in 12 prefectures) and successfully isolated Bartonella organisms from 176 of the 685 rodents (isolation rate, 25.7%). Those Bartonella isolates were all obtained from the rodents captured in suburban areas (rate, 51.8%), but no organism was isolated from the animals captured in city areas. Sequence analysis of rpoB and gltA revealed that the Bartonella isolates obtained were classified into eight genetic groups, comprising isolates closely related to B. grahamii (A-I group), B. tribocorum and B. elizabethae (B-J group), B. tribocorum and B. rattimassiliensis (C-K group), B. rattimassiliensis (D-L group), B. phoceensis (F-N group), B. taylorii (G-O group), and probably two additional novel Bartonella species groups (E-M and H-P). B. grahamii , which is one of the potential causative agents of human neuroretinitis, was found to be predominant in Japanese rodents. In terms of the relationships between these Bartonella genetic groups and their rodent species, (i) the A-I, E-M, and H-P groups appear to be associated with Apodemus speciosus and Apodemus argenteus ; (ii) the C-K, D-L, and F-N groups are likely implicated in Rattus rattus ; (iii) the B-J group seems to be involved in Apodemus mice and R. rattus ; and (iv) the G-O group is probably associated with A. speciosus and Clethrionomys voles. Furthermore, dual infections with two different genetic groups of bartonellae were found in A. speciosus and R. rattus . These findings suggest that the rodent in Japan might serve as a reservoir of zoonotic Bartonella infection.

4,4'-Butylidenebis(6-t-butyl-m-cresol) (BBBC) can be eluted from disposable gloves made of nitrile-butadiene rubber and possibly also detected in food. We have reported that BBBC is an androgen and estrogen antagonist. In this report, BBBC (0.1, 1.0 mg/kg body weight (bw)/day) was subcutaneously administered to pregnant rats from gestation days 11 through 18 and the effects on male offspring (postnatal day (PND) 102) were examined. Body weight at lactation and brain weight at PND 102 were decreased in the 1.0 mg/kg bw BBBC-treated group. Altered levels and turnover of the monoamines dopamine (DA), serotonin (5-HT), and noradrenalin (NA) as well as their metabolites were detected. In the prefrontal cortex DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, 5-hydroxyindole-3-acetic acid (5-HIAA) levels were significantly increased, but homovanillic acid (HVA)/DA was decreased. In the striatum NA level, DOPAC/DA and HVA/DA were significantly increased, but 3-methoxy-4-hydrophenylglycol hemipiperazinium (MHPG) level and MHPG/NA were decreased. In hippocampus MHPG level was significantly decreased. In hypothalamus 5-HIAA level and 5-HIAA/5-HT were significantly increased. These results suggested that prenatal exposure to BBBC affects the central nervous system of male rat offspring, and BBBC may be an endocrine disrupting-chemical during the fetal periods.

We surveyed Rickettsiales bacteria, including Rickettsia, Ehrlichia, Anaplasma, and Neoehrlichia, in wild sika deer (Cervus nippon nippon) from Shizuoka prefecture, Japan. In spleen samples from 187 deer, Anaplasma phagocytophilum (deer type), A. bovis, and A. centrale were successfully detected by PCR assay targeting to 16S rDNA or p44/msp2, and their positive rates were 96.3% (180/187), 53.5% (100/187), and 78.1% (146/187), respectively. Additionally, 2 or 3 Anaplasma species could be detected from a single deer in 165 spleen samples (88.2%), indicating dual or triple infection. In contrast, A. phagocytophilum (human type) 16S rDNA, Rickettsia gltA, Ehrlichia p28/omp-1, and Neoehrlichia 16S rDNA could not be amplified. The serological test of 105 deer serum samples by immunofluorescence assay showed that the detection of antibodies against antigens of A. phagocytophilum HZ (US-human isolate) and Rickettsia japonica YH were 29.5% (31/105) and 75.2% (79/105), respectively. These findings suggest that A. phagocytophilum (deer type), A. centrale, and A. bovis are highly dominant and prevalent in wild sika deer from Shizuoka, a central region of Japan, and that the antibodies against some Rickettsiales bacteria have also been retained in deer blood.

We report Anaplasma phagocytophilum infection of Ixodes persulcatus and I. ovatus ticks in Japan. Unique p44/msp2 paralogs (and/or 16S rRNA genes) were detected in tick tissues, salivary glands, and spleens of experimentally infected mice. These findings indicate the public health threat of anaplasmosis in Japan.

To the Editor: Japanese spotted fever (JSF), caused by Rickettsia japonica, is the most prevalent tickborne infectious disease in Japan (1), occurring most frequently in central and western regions (http://idsc.nih.go.jp/idwr/CDROM/Main.html [in Japanese]). Cases of unknown fever with rickettsiosis-like symptoms not associated with JSF have been reported in JSF-endemic regions of Japan (2). Several spotted fever group (SFG) rickettsiae (R. japonica, R. heilongjiangensis, R. helvetica, R. tamurae, R. asiatica, Candidatus R. tarasevichiae) and other related Rickettsia spp. have been identified in Japan (1,3–6). Human infections with R. heilongjiangensis and R. tamurae have been confirmed (3,5), and Anaplasma phagocytophilum and Ehrlichia chaffeensis, known human pathogens, have been detected in ticks and deer in Japan. We conducted this study to determine the risk in central and western Japan for human exposure to ticks harboring SFG rickettsiae, A. phagocytophilum, or Ehrlichia spp. In 2007–2011, we collected 827 Haemaphysalis, Amblyomma, and Ixodes spp. ticks (392 adults, 435 nymphs) by flagging vegetation in the prefectures of Shizuoka, Mie, Wakayama, Kagoshima, Nagasaki (Goto Island), and Okinawa (the main island and Yonaguni Island) (Technical Appendix Figure 1). We extracted DNA from the salivary glands of each tick and performed PCR to amplify gltA, 16S rDNA, and ompA of SFG rickettsiae. To detect A. phagocytophilum and Ehrlichia spp., we performed nested PCR targeting the p44/msp2 and p28/omp-1 multigenes, respectively. PCR gltA screening revealed SFG rickettsiae in 181 (21.9%) of the 827 ticks (Table). We obtained nearly full-length (1.1-kb) gltA sequences and classified them into 5 groups by phylogenetic analyses (Technical Appendix Figure 2). Sequences for groups 1 (prevalence 1.0%) and 2 (prevalence 3.2%) were identified as R. japonica YH (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AP011533","term_id":"348592266","term_text":"AP011533"}}AP011533) and R. tamurae (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF394896","term_id":"21360604","term_text":"AF394896"}}AF394896), respectively (Table). Group 3 (prevalence 15.1%) sequences were identical to that of Rickettsia sp. LON (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB516964","term_id":"256353425","term_text":"AB516964"}}AB516964). The sequence for group 4 (prevalence 1.6%) was closely related to that for R. raoultii strain Khabarovsk (98.8% similarity), and a part of the sequence (342 bp) was identical to that of Rickettsia sp. Hf 151 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB114815","term_id":"33146352","term_text":"AB114815"}}AB114815). Group 5 consisted of 4 newly identified rickettsiae (Technical Appendix Figure 2). Of these 4 rickettsiae, 3 (Mie311, Goto13, and Mie334) were closely related to R. raoultii strain Khabarovsk (98.0% identity) and 1 (Mie201) was similar to Candidatus R. principis (99.7% identity). Table PCR survey results for Haemaphysalis, Amblyomma, and Ixodes spp. ticks tested for rickettsiae, central and western Japan, 2007–2011* We further analyzed the 16S rDNA and ompA in gltA-positive tick samples. The 16S rDNA and ompA for group 1 samples shared 100% identity with 16S rDNA and ompA of R. japonica YH ({"type":"entrez-nucleotide","attrs":{"text":"AP011533","term_id":"348592266","term_text":"AP011533"}}AP011533). The 16S rDNA of group 2 was identical to that of R. tamurae ({"type":"entrez-nucleotide","attrs":{"text":"AY049981","term_id":"21360334","term_text":"AY049981"}}AY049981). In groups 3–5, some of the specific amplicons in 16S rDNA or ompA could be detected; their sequences were confirmed to be similar (but not identical) to those of several known rickettsial sequences. We amplified the p44/msp2 amplicons of A. phagocytophilum from 25 (3%) of 827 ticks (Table). By cloning (TA Cloning Kit; Life Technologies, Carlsbad, CA, USA) and sequencing these amplicons, we obtained and identified 60 new TA-clone sequences (366–507 bp) for p44/msp2 (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"JQ697880-JQ697950","start_term":"JQ697880","end_term":"JQ697950","start_term_id":"383088493","end_term_id":"383088630"}}JQ697880-JQ697950); these sequences may include a potentially novel Anaplasma species. (7). Ehrlichia p28/omp-1 was detected from 2 (0.2%) of the 827 ticks. Of 5 TA-clone sequences (284–315 bp) obtained from the 2 ticks, 2 from an A. testudinarium tick (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"JQ697886","term_id":"383088503","term_text":"JQ697886"}}JQ697886 and {"type":"entrez-nucleotide","attrs":{"text":"JQ697887","term_id":"383088505","term_text":"JQ697887"}}JQ697887) shared 83.3%–86.7% similarity with E. ruminantium Gardel Map-1 (GenBank accession no. YP196842), and 3 from an H. longicornis tick (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"JQ697888-JQ697890","start_term":"JQ697888","end_term":"JQ697890","start_term_id":"383088507","end_term_id":"383088511"}}JQ697888-JQ697890) showed the closest relationship to E. ewingii omp-1–15 (67%–73% similarity; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF116932","term_id":"133711094","term_text":"EF116932"}}EF116932). We identified the tick species associated with R. japonica as H. formosensis, H. hystricis, and H. cornigera, and another study reported an association with Dermacentor taiwanensis, H. flava, H. longicornis, and I. ovatus (4). In our study and previous studies, the tick species associated with A. phagocytophilum in Japan were identified as H. formosensis, H. longicornis, H. megaspinosa, A. testudinarium, I. ovatus, and I. persulcatus (8). Thus, it appears that 3 tick species (H. formosensis, H. longicornis, and I. ovatus) are associated with R. japonica and A. phagocytophilum. In addition, in an H. formosensis tick, we detected an SFG rickettsia that is closely related to R. raoultii, the etiologic agent of Dermacentor-borne necrosis erythema and lymphadenopathy in Europe and Russia (9). We detected Candidatus R. principis in H. flava in Japan; this species was previously detected in H. japonica douglasi and H. danieli ticks in Russia and China, respectively, (10). And, we found a high prevalence of R. tamurae in A. testidinarium ticks; Imaoka et al. (5) recently reported that R. tamurae causes local skin inflammation without general JSP-like symptoms. We did not detect the human pathogen E. chaffeensis, but we identified 2 potentially new Ehrlichia species. Our findings contribute to the known risks for exposure to Rickettsia-related pathogens in central and western Japan. Further studies may be required for the surveillance of additional pathogens, such as Candidatus Neoehrlichia mikurensis (2), which was recently recognized as a human pathogen. Technical Appendix: Phylogenetic classification of Rickettsia spp. gltA sequences detected in ticks during 2007–2011 in central and western Japan and locations of tick collection sites. Click here to view.(154K, pdf)

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant with potential carcinogenicity. It has been shown that BaP, upon UVA irradiation, synergistically induced oxidative DNA damage, but other DNA damage was not confirmed. In this study, we examined whether coexposure to BaP plus UVA induces double strand breaks (DSBs) using xrs-5 cells, deficient in the repair of DSBs (Ku80 mutant), and whether Ku translocates involving the formation of DSBs. BaP plus UVA had a significant cytotoxic effect on CHO-K1 cells and an even more drastic effect on Ku80-deficient, xrs-5 cells, suggesting that the DSBs were generated by coexposure to BaP plus UVA. The DSBs were repaired in CHO-K1 cells within 30 min, but not in xrs-5 cells, indicating the involvement of a non-homologous end joining, which needs Ku proteins. Furthermore, we succeeded in visualizing that Ku80 rapidly assembled to the exposed region, in which DSBs might be generated, and clarified that the presence of both Ku70 and Ku80 was important for their accumulation.

Sixteen of 22 omp-1 paralogs encoding 28-kDa-range immunodominant outer membrane proteins of Ehrlichia chaffeensis were transcribed in blood monocytes of dogs throughout a 56-day infection period. Only one paralog was transcribed by E. chaffeensis in three developmental stages of Amblyomma americanum ticks before or after E. chaffeensis transmission to naïve dogs.

Ehrlichia canis , an obligatory intracellular bacterium of monocytes and macrophages, causes canine monocytic ehrlichiosis. E . canis immunodominant 30-kDa major outer membrane proteins are encoded by a polymorphic multigene family consisting of more than 20 paralogs. In the present study, we analyzed the mRNA expression of 14 paralogs in experimentally infected dogs and Rhipicephalus sanguineus ticks by reverse transcription-PCR using gene-specific primers followed by Southern blotting. Eleven out of 14 paralogs in E . canis were transcribed in increasing numbers and transcription levels, while the mRNA expression of the 3 remaining paralogs was not detected in blood monocytes of infected dogs during the 56-day postinoculation period. Three different groups of R . sanguineus ticks (adult males and females and nymphs) were separately infected with E . canis by feeding on the infected dogs. In these pools of acquisition-fed ticks as well as in the transmission-fed adult ticks, the transcript from only one paralog was detected, suggesting the predominant transcription of that paralog or the suppression of the remaining paralogs in ticks. Expression of the same paralog was higher whereas expression of the remaining paralogs was lower in E . canis cultivated in dog monocyte cell line DH82 at 25°C than in E . canis cultivated at 37°C. Analysis of differential expression of p30 multigenes in dogs, ticks, or monocyte cell cultures would help in understanding the role of these gene products in pathogenesis and E . canis transmission as well as in designing a rational vaccine candidate immunogenic against canine ehrlichiosis.

A 44-kDa major outer membrane protein of the human granulocytic ehrlichiosis (HGE) agent is an immunodominant antigen in human infection. A gene encoding this protein was cloned and sequenced. Southern blot results revealed the existence of multigenes homologous to the P44 gene in the genome of the HGE agent. The recombinant 44-kDa protein (rP44) was expressed by using expression vector pET30a. The reactivity of the affinity-purified rP44 was evaluated by Western immunoblot analysis and dot blot immunoassay. Western immunoblot analysis showed that mouse anti-rP44 serum reacted with 44- to 42-kDa proteins in six different HGE agent strains tested except strain 2, in which three proteins of 42, 40, and 38 kDa were recognized. Eleven HGE patient serum samples, a horse anti-HGE serum, and a horse anti- Ehrlichia equi serum recognized the rP44 protein. This suggests that rP44 is an HGE- E. equi group-specific antigen. Neither human anti- Ehrlichia chaffeensis serum nor rabbit anti- Borrelia burgdorferi serum reacted with rP44. Sera from two patients coinfected with the HGE agent and B. burgdorferi reacted positively with rP44 and the HGE agent. Sera from 20 HGE patients with indirect fluorescent-antibody (IFA) titers ranging from 1:20 to 1:2,560 gave distinct positive reactions in a dot immunoblot assay. There was a positive correlation between the color densities of the dot reactions and the IFA titers when greater than 50 ng of recombinant antigen per dot was used. The use of the affinity-purified rP44 protein as antigen would provide a more specific, consistent, and simpler serodiagnosis for HGE than the use of whole infected cells or purified HGE agents.

Several immunodominant major proteins ranging from 23 to 30 kDa were identified in the outer membrane fractions of Ehrlichia chaffeensis and Ehrlichia canis . The N-terminal amino acid sequence of a 28-kDa protein of E. chaffeensis (one of the major proteins) was determined. The gene ( p28 ), almost full length, encoding the 28-kDa protein was cloned by PCR with primers designed based on the N-terminal sequence of the E. chaffeensis 28-kDa protein and the consensus sequence between the C termini of the Cowdria ruminantium MAP-1 and Anaplasma marginale MSP-4 proteins. The p28 gene was overexpressed, and antibody to the recombinant protein was raised in a rabbit. The antibody and serum from a patient infected with E. chaffeensis reacted with the recombinant protein, three proteins (29, 28, and 25 kDa) of E. chaffeensis , and a 30-kDa protein of E. canis . Immunoelectron microscopy with the rabbit antibody revealed that the antigenic epitope of the 28-kDa protein was exposed on the surface of E. chaffeensis . Southern blot analysis with a 32 P-labeled p28 gene probe revealed multiple copies of genes homologous to p28 in the E. chaffeensis genome. Six copies of the p28 gene were cloned and sequenced from the genomic DNA by using the same probe. The open reading frames of these gene copies were tandemly arranged with intergenic spaces. They were nonidentical genes and contained a semivariable region and three hypervariable regions in the predicted protein molecules. One of the gene copies encoded a protein with an internal amino acid sequence identical to the chemically determined N-terminal amino acid sequence of a 23-kDa protein of E. chaffeensis . Immunization with the recombinant P28 protein protected mice from infection with E. chaffeensis . These findings suggest that the 30-kDa-range proteins of E. chaffeensis represent a family of antigenically related homologous proteins encoded by a single gene family.

A clone expressing an immunoreactive 55-kilodalton (kDa) protein of Ehrlichia sennetsu, the causative agent of human Sennetsu ehrlichiosis, was isolated from a gene library of this organism. Sequence analysis of the DNA insert revealed two open reading frames, encoding proteins of 10,620 and 58,225 kDa, respectively. These deduced amino acid sequences were homologous to those of the GroES and GroEL heat shock proteins (HSP) of other bacteria, respectively. Phylogenetic trees based on GroES and GroEL homologs of several bacteria including E. sennetsu showed a relationship similar to that based on 16S rRNA gene sequences. The recombinant and native 55-kDa proteins of E. sennetsu, GroEL homolog, reacted with a monoclonal antibody (SPA807) which recognizes a homologous sequence between human and mycobacterial HSP60 and a polyclonal antibody (SPA804) to cyanobacteria HSP60, but not with antibodies to HSP60 of several other organisms used. Furthermore, anti-recombinant E. sennetsu 55-kDa protein antibody prepared in a rabbit was reactive to HSP60 antigens of other Ehrlichia and Rickettsia species, but not GroEL of E. coli. The recombinant 55-kDa protein would be a useful tool for studying the role of this antigen in the immune response to E. sennetsu infection.

We conducted a preliminary investigation of the effects of visible light irradiation on plant extracts and we observed a strong suppressive effect on interleukin (IL) 2 expression with the inhibition of c-Jun amino-terminal kinase (JNK) phosphorylation in Jurkat cells by visible light irradiation to ethanol extract from green soybeans (LIEGS). This effect was produced only by extracts from green soybeans (Glycine max) and not other-color soybeans. LIEGS suppressed the lipopolysaccharide-induced IL-6, IL-12 and TNF-α expression levels in human monocyte THP-1 cells in a concentration-dependent manner. LIEGS was applied for 8 weeks to NC/Nga mice. LIEGS suppressed the development of atopic dermatitis (AD)-like skin lesions and reduced the dermatitis scores of the mice. The light irradiation changed the various types of small-molecule compounds in extracts. Visible light irradiation to daidzein with chlorophyll b induced a novel oxidative product of daidzein. This product suppressed IL-2 expression in Jurkat cells.

To clarify the factors for occurrence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in broilers, two flocks (1 day of age) fed a diet with or without antibiotics were kept in a broiler house sanitized with disinfectants. ESBL-producing E. coli, however, was detected at a concentration of over 10(6) CFU/g of feces at 9 days of age to 49 days of age in both broiler flocks. Therefore, this indicated that the antibiotics other than cephalosporins used in this study had no effect due to co-selection on the numbers of ESBL-producing E. coli in broiler feces during this period. When a flock was kept with diet containing antibiotics for 49 days in a laboratory animal room, no ESBL-producing E. coli was detected in the flock. These results suggest that the occurrence of ESBL-producing E. coli may not be related to feeding with antibiotics and that the contamination of broiler houses with ESBL-producing E. coli might be an important factor.

To evaluate the diversity of extended-spectrum β-lactamases (ESBL) genes among food-producing animals, 48 isolates of ESBL-producing Escherichia coli isolates were obtained from rectal samples of broilers, layers, beef cattle and pigs, at the slaughterhouse level. ESBL-carrying E. coli were isolated from 60.0% of individual broiler rectal samples, 5.9% of layers, 12.5% of beef cattle and 3% of pigs. One ESBL-producing Klebsiella pneumoniae was isolated from a broiler. The ESBL-positive E. coli isolates from broilers harbored various ESBL genes: bla (SHV-12), bla(CTX-M-2), bla(CTX-M-14), bla(CTX-M-15) and bla(CTX-M-44). The plasmid DNAs were analyzed by restriction patterns. Homogeneous band patterns were yielded in those of K. pneumoniae and E. coli isolates harboring the bla(CTX-M-2) gene from different farms. No genetic relation between the 2 CTX-M-14 ESBL-producing strains was found by pulsed-field gel electrophoresis, although 2 plasmids in these strains, obtained from different broiler farms, were similar to each other. This study provides evidence that the proliferation of CTX-M-producing E. coli is due to the growth of indigenous CTX-M-producing strains and the possible emergence of strains that acquired CTX-M genes by horizontal transfer in different broiler farms. CTX-M-producing coliforms in broilers should be controlled due to the critical importance of cephalosporins and the zoonotic potential of ESBL-producing bacteria.

We surveyed β-lactamase-producing Escherichia coli from farm animals (chickens, pigs, and cattle) and raw retail meat in Shizuoka Prefecture, Japan. In total 305 E. coli isolates, 15 isolates collected from broilers, beef cattle, chicken meat, and pork meat, were found to have β-lactamase genes encoding CTX-M-2, CTX-M-14, CMY-2, SHV-2, and/or TEM-1, whereas 7 possessed mutations in the ampC promoter region. The findings suggest that broilers are more important than other farm animals with regards to the surveillance of β-lactamase-producing E. coli in this region.

Angiogenesis is crucial for tumor growth and hematogenous metastasis. Specifically expressed and functional protein molecules in angiogenic endothelial cells, especially on the plasma membrane, may be molecular targets for antiangiogenic drugs and drug delivery systems (DDS) in cancer therapy. To discover such target molecules, we performed subcellular proteome analysis of human umbilical vein endothelial cells (HUVECs) treated with or without vascular endothelial growth factor (VEGF) using 2-dimensional difference in-gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Among the identified proteins, BiP/GRP78, a molecular chaperone, was highly expressed in the membrane/organelle fraction of HUVECs after VEGF treatment. The involvement of BiP in VEGF-induced angiogenesis was examined by RNA interference. BiP knockdown significantly suppressed VEGF-induced endothelial cell proliferation and VEGF-induced phosphorylation of extracellular-regulated kinase 1/2, phospholipase C-γ, and VEGF receptor-2 in HUVECs. Cell surface biotinylation analysis revealed that the cell surface expression of BiP was elevated in VEGF-activated HUVECs. Aiming to apply BiP to a target molecule in liposomal DDS, we developed liposomes modified with the WIFPWIQL peptide, which has been shown to bind to BiP, and investigated its potential for cancer therapy. The WIFPWIQL-modified liposomes (WIFPWIQL liposomes) were significantly taken up by VEGF-activated HUVECs as compared to peptide-unmodified liposomes. WIFPWIQL liposomes appeared to accumulate in tumor endothelial cells in vivo. WIFPWIQL liposomes containing doxorubicin significantly suppressed tumor growth and prolonged the survival of colon26 NL-17 carcinoma cell-bearing mice. In summary, BiP may regulate VEGF-induced endothelial cell proliferation through VEGFR-2-mediated signaling and be an effective target molecule for cancer antineovascular therapy.

Aims Insulin resistance caused by a high-fat diet induces type 2 diabetes and its complications. In this study, we investigated gene expression changes in peripheral leukocytes with insulin resistance by conducting microarray analyses in rats with high-fat diet-induced insulin resistance. Main methods After assessing insulin resistance in rats by an oral glucose tolerance test, we performed microarray analyses using peripheral leukocytes from normal rats and insulin-resistant rats after fasting. Real-time RT-PCR analyses were performed for several upregulated genes in the microarray data after fasting and at 3 h after a single oral glucose load. Key findings Feeding rats a high-fat diet for 77 days induced moderate insulin resistance. Microarray analysis showed that the high-fat diet enhances many genes related to leukocyte activation. These upregulated genes included genes related to host defense, and many genes related to G-protein-coupled receptor/tyrosine receptor signaling. Moreover, many genes, such as Anxa1 , S100a8 , Il22ra2 , Gng10 , Csf3r and Cd302 , showed further upregulation of their expression after a single oral glucose load. Exposure to high glucose and/or tumor necrosis factor-α which is known to be a factor that induces insulin resistance, enhanced the mRNA levels of DUSP1 , ANXA1 , IL1B , S100A8 , IL22RA2, S100A9 and IRF1 in human monocyte-like U937 cells. Significance These results suggest that the expression of genes related to leukocyte activation in peripheral leukocytes is associated with the development of moderate insulin resistance.

Rats and mice (8 animals per species per group) were injected subcutaneously or intraperitoneally with paradichlorobenzene (PDCB) at doses of 0, 100, 200 and 400 mg/kg/day, 4-5 days a week, for 8 weeks (for rats) and either 2 or 6 weeks (for mice). Prostate and seminal vesicle weights were significantly increased in PDCB-treated rats but not in mice. Major histopathologic injuries were not found in testis and epididymis of both species. Daily sperm production was depressed in both species in a dose-response manner. Serum testosterone levels were not significantly changed in both species. Sperm morphology was evaluated in rats intraperitoneally administered PDCB at a dose of 800 mg/kg. Abnormal sperms with reduced hook, bent neck, coiled flagellum, bent flagellum and bent flagellum tip were significantly increased in treated rats. In Hershberger assay, PDCB administration increased weights of ventral prostate gland, seminal vesicle, levator ani/bulbocavernosus muscle and glans penis in castrated rats, and also weights of ventral prostate gland and glans penis in castrated mice. PDCB and 2,5-dichlorophenol (the major metabolite) did not bind androgen receptor (AR) up to 10 mM. In conclusion, PDCB affects sperm production and morphology but is somewhat androgenic independently from AR binding in rats and mice.

The present study assessed a carcinogenic hazard of multi-wall carbon nanotube (MWCNT) in intact (not genetically modified) rodents. MWCNT (1 mg/kg body weight, 7 animals), crocidolite (2 mg/kg body weight, 10 animals) or vehicle (2% carboxymethyl cellulose, 5 animals) was administered to male Fischer 344 rats (12 weeks old) by a single intrascrotal injection. Rats were autopsied immediately after death, when becoming moribund or at the end of the maximal observation period scheduled to be 52 weeks. After 37-40 weeks, however, 6 MWCNT-treated animals died or became moribund due to intraperitoneally disseminated mesothelioma (6/7, 85.7%) with bloody ascites. Peritoneal mesothelium was generally hypertrophic, and numerous nodular or papillary lesions of mesothelioma and mesothelial hyperplasia were developed. While mesothelioid cells were predominant in relatively early stage tumors, advanced stage mesotheliomas were constituted by 2 portions occupied by mesothelioid cells on the surface and spindle-shaped sarcomatous cells in the depth. In the latter, the histological transition was apparently observed between these 2 portions. Mesotheliomas were invasive to adjacent organs and tissues, and frequently metastasized into the pleura. Only 1 rat survived for 52 weeks in the MWCNT-treated group, and similar findings except mesothelioma were observed. All 10 crocidolite-treated and 5 vehicle-treated rats survived for 52 weeks without any particular changes except deposition of asbestos in the former case. It is thus indicated that MWCNT possesses carcinogenicity causing mesothelioma at a high rate in intact male rats under the present experimental conditions. The present data identifies a carcinogenic hazard of MWCNT and will serve as one of the indispensable evidences to be used for the risk assessment crucial for not only protection and improvement of human health and welfare, but also safe and acceptable development and prevalence of this and similar upcoming materials.

Nanomaterials of carbon origin tend to form various shapes of particles in micrometer dimensions. Among them, multi-wall carbon nanotubes (MWCNT) form fibrous or rod-shaped particles of length around 10 to 20 micrometers with an aspect ratio of more than three. Fibrous particles of this dimension including asbestos and some man-made fibers are reported to be carcinogenic, typically inducing mesothelioma. Here we report that MWCNT induces mesothelioma along with a positive control, crocidolite (blue asbestos), when administered intraperitoneally to p53 heterozygous mice that have been reported to be sensitive to asbestos. Our results point out the possibility that carbon-made fibrous or rod-shaped micrometer particles may share the carcinogenic mechanisms postulated for asbestos. To maintain sound activity of industrialization of nanomaterials, it would be prudent to implement strategies to keep good control of exposure to fibrous or rod-shaped carbon materials both in the workplace and in the future market until the biological/ carcinogenic properties, especially of their long-term biodurability, are fully assessed.

Anaplasma phagocytophilum, an obligate intracellular bacterium, is the aetiologic agent of human granulocytic anaplasmosis (HGA). A. phagocytophilum virB/D operons encoding type IV secretion system are expressed in cell culture and in the blood of HGA patients. In the present study, their expression across the A. phagocytophilum intracellular developmental cycle was investigated. We found that mRNA levels of both virB9 and virB6 were upregulated during infection of human neutrophils in vitro. The antibody against the recombinant VirB9 protein was prepared and immunogold and immunofluorescence labelling were used to determine the VirB9 protein expression by individual organisms. Majority of A. phagocytophilum spontaneously released from the infected host cells poorly expressed VirB9. At 1 h post infection, VirB9 was not detectable on most bacteria associated with neutrophils. However, VirB9 was strongly expressed by A. phagocytophilum during proliferation in neutrophils. In contrast, with HL-60 cells, approximately 80% of A. phagocytophilum organisms associated at 1 h post infection expressed VirB9 protein and were colocalized with lysosome-associated membrane protein-1 (LAMP-1), whereas, VirB9-undetectable bacteria were not colocalized with LAMP-1. These results indicate developmental regulation of expression of components of type IV secretion system during A. phagocytophilum intracellular life cycle and suggest that bacterial developmental stages influence the nature of binding to the hosts and early avoidance of late endosome-lysosome pathway.

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens., Synopsis Ehrlichiosis is an acute disease that triggers flu-like symptoms in both humans and animals. It is caused by a range of bacteria transmitted by ticks or flukes. Because these bacteria are difficult to culture, however, the organisms are poorly understood. The genomes of three emerging human pathogens causing ehrlichiosis were sequenced. A database was designed to allow the comparison of these three genomes to sixteen other bacteria with similar lifestyles. Analysis from this database reveals new species-specific and disease-specific genes indicating niche adaptations, pathogenic traits, and other features. In particular, one of the organisms contains more than 100 copies of a single gene involved in interactions with the host(s). These comparisons also enabled a reconstruction of the metabolic potential of five representative genomes from these bacteria and their close relatives. With this work, scientists can study these emerging pathogens in earnest.

A total of 293 ticks and 111 wild rodents that were collected in Shizuoka and Nagano Prefectures, Japan, were examined for infection of Ehrlichia species and 'Candidatus Neoehrlichia mikurensis.' The 16S rDNA or the omp-1 gene of these bacterial DNAs were detected from the spleens of tick-inoculated mice (5 positive/total 29 mice) or from the spleens of wild rodents (25 positive/total 111 rodents) by PCR amplifi-cation. Sequencing of the 16S rDNA revealed Ehrlichia spp. from the 5 tick-inoculated mice and 8 wild rodents, and 'Candidatus N. mikurensis' from 17 wild rodents. The data suggest the presence of additional genetic variants, and potential vectors and/or reservoirs for these bacteria in central Japan.

Ultraviolet (UV) irradiation is well known to induce apoptosis, a hallmark event of which is the occurrence of sunburn cells in the epidermis. Keratinocytes in which DNA damaged by UV irradiation is not repaired undergo apoptosis as sunburn cells. However, we have previously reported that low-dose UV-B irradiation (approximately 0.1 J/cm2) suppressed the apoptosis induced by cell detachment and serum depletion. Dysregulation of apoptosis is important in tumor progression and malignancy and in promoting resistance to cancer therapy. To develop a better understanding of the antiapoptotic effect of UV irradiation, and to design the effective induction of apoptosis, we tried the proteome analysis of the molecules regulating apoptosis in low-dose UV-B-irradiated NIH3T3 cells, using two-dimensional difference gel electrophoresis (DIGE). Of a total of 3811 protein spots detected, 42 were found to be different between the cells undergoing apoptosis and cells after the irradiation. Of the spots selected, 25 were identified using MALDI-TOF/TOF-MS, some as structural proteins. Although typical apoptosis-related molecules were not detected, possibly because proteins with low molecular weights were difficult to identify in the gel conditions used in this study, some of the proteins were considered to be involved in apoptosis. The DIGE system used in this experiment has advantages (including a high level of statistical confidence) for discovering new functional proteins related to the regulation of apoptosis.

A total of 390 adult ticks (288 Ixodes ovatus and 102 I. persulcatus ) collected at the foot of Mt. Fuji and two near cities in Shizuoka prefecture, Japan, were examined for Ehrlichia infection by isolation with laboratory mice from whole tick tissues. Ehrlichial DNAs were detected from the spleens of mice inoculated with tissues from I. ovatus, but not I. persulcatus. The prevalence of ehrlichiae in the ticks was estimated to be ca. 3%. The 16S rDNA analysis revealed that the sequences of 8 ehrlichial isolates (termed "Shizuoka" isolates) obtained were identical, and they were very similar, but not identical, to those of two Ehrlichia species strain variants recently isolated in Japan, followed by Ehrlichia chaffeensis in the US. Analysis of parts of the omp-1 multigene family specific for monocytic ehrlichiosis agents showed that the Shizuoka isolates were distinct from other ehrlichial organisms. The Shizuoka isolates caused death in immunocompetent laboratory mice, suggesting that they are highly pathogenic in mice. The data show that the Shizuoka isolates are likely to be a new strain variant of Ehrlichia species in Japan. Further characterization and surveillance will be required in Japan due to the presence of these human ehrlichiosis agent-like organisms.

The p51 gene that encodes the major antigenic 51-kDa protein in Neorickettsia risticii was identified in strains of Neorickettsia sennetsu and the Stellantchasmus falcatus agent but not in Neorickettsia helminthoeca , suggesting that p51 -based diagnosis would be useful to distinguish among them. groESL sequencing results delineated the phylogenic relationships among Neorickettsia spp.

Operculate snails (the family Pleuroceridae:Elimia livescens) were collected between June and October 1998 from a river in central Ohio where repeated cases of Potomac horse fever (PHF) have occurred. Of collected snails, consistently 50 to 80% carried a combination of cercariae and sporocysts of digenetic virgulate trematodes. The trematodes obtained from each snail were pooled and tested for Ehrlichia risticii, the agent of PHF, by nested PCR using primers specific to the 16S rRNA gene. Out of a total of 209 trematode pools, 50 pools were found to be positive by PCR. The DNA sequence of the 16S rRNA gene identified in one trematode pool was identical to that of the type strain of E. risticii, and the sequence of the gene identified in another pool differed from that of the type strain by 1 nucleotide. Comparison of the deduced amino acid sequence of the partial 51-kDa antigen gene from various sources revealed that Maryland, Ohio (except Ohio 081), and Kentucky strains are in a cluster distinct from the sequences obtained from sources in California and Oregon. Ohio 081 was shown previously by antigenic composition analysis to be distinct from other groups. However, all sequences examined were not segregated according to their sources: horse blood or infected trematodes. E. risticiiwas found to be transmittable from trematodes to mice and was subsequently passaged from infected mice to additional mice, as determined by PCR analysis. Our findings suggest the evolution ofE. risticii in the natural reservoir in separate geographic regions and persistent infection of trematode populations with E. risticii during summer and early fall. The study also suggests that the mouse can be used to isolate E. risticii from the infected trematode.

Human monocytic ehrlichiosis is an emerging infectious disease caused by Ehrlichia chaffeensis , a gram-negative obligatory intracellular bacterium closely related to E. canis . The immunoreactive recombinant fusion proteins rP28 and rP30 have become available after cloning and expressing of the 28- and 30-kDa major outer membrane protein genes of E. chaffeensis and E. canis , respectively. Western immunoblotting was performed to analyze the antibody responses of the 37 E. chaffeensis indirect fluorescent-antibody assay (IFA)-positive and 20 IFA-negative serum specimens with purified whole organisms, rP28, and rP30. All IFA-negative sera were negative with purified whole organisms, rP28, or rP30 by Western immunoblot analysis (100% relative diagnostic specificity). Of 37 IFA-positive sera, 34 sera reacted with any native proteins of E. chaffeensis ranging from 44 to 110 kDa, and 30 sera reacted with 44- to 110-kDa native E. canis antigens. The 28-kDa E. chaffeensis and 30-kDa E. canis native proteins were recognized by 25 IFA-positive sera. Fifteen IFA-positive sera reacted with rP28 by Western blot analysis, whereas 34 IFA-positive sera reacted with rP30 (92% relative diagnostic specificity), indicating that rP30 is more sensitive than rP28 for detecting the antibodies in IFA-positive sera. These 34 IFA-positive sera were positive by the dot blot assay with rP30, distinguishing them from IFA-negative sera. Except for three rP30-negative but IFA-positive specimens that instead showed an E. ewingii infection-like profile by Western immunoblotting, the results of Western and dot blot assays with rP30 matched 100% with the IFA test results. Densitometric analysis of dot blot reactions showed a positive correlation between the dot density and the IFA titer. These results suggest that rP30 antigen would provide a simple, consistent, and rapid serodiagnosis for human monocytic ehrlichiosis.

A 30-kDa major outer membrane protein of Ehrlichia canis , the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene ( omp-1 ) family of Ehrlichia chaffeensis . Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis . Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene ( p30 , p30-1 , and p30a ) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies ( p30 and p30-1 ) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp., p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale , and map-1 from Cowdria ruminantium . Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis . Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis . To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of E. canis will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.

A total of 10 strains of Orientia tsutsugamushi were isolated from field rodents and chiggers in Taiwan, and characterized by immunofluorescence analysis with monoclonal antibodies and by restriction fragment length polymorphism analysis of the 56-kilodalton (kDa) protein gene. The isolates were divided into two groups consisting of 1 and 9 strains which showed some relation to Gilliam and Karp type strains, respectively. However, all these isolates possessed characteristics distinct not only from those of known prototype strains including Gilliam and Karp but also from all isolates from Japan. These findings suggest the existence of a large number of immunotypic and genotypic variants among the strains of O. tsutsugamushi, and the distribution of distinguishable strains in each area to which this species is endemic.

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type-specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)-amplified 56-kilodalton type-specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.