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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.

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.

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.

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.

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.

Anaplasma phagocytophilum is an obligate intracellular bacterium that causes febrile illness in humans and livestock. A 49-year-old woman was suffering from feverish symptoms, fatigue, arthralgia, general body pain, and anorexia for 2 weeks. Later, she visited the Bayannur Centers for Disease Control and Prevention Hospital in Inner Mongolia, China. Molecular-based diagnostic analysis of the patient's blood revealed that A. phagocytophilum p44 DNA was positive, but Brucella omp31, spotted fever group Rickettsia gltA, Orientia tsutsugamushi 16S rDNA, and Ehrlichia p28 were negative. The amino acid sequences of 9 A. phagocytophilum p44 clones obtained from the patient shared 44-100% similarity among them and were closely related to those of previously identified p44 clones from Canis familiaris (accession no. KJV64194) and from Ixodes persulcatus tick (no. BAN28309). Serological tests using the patient's serum showed that immunoglobulin M (IgM) and IgG titers to A. phagocytophilum antigens were 160 and 20, respectively, determined using indirect immunofluorescence assay, and the reaction to recombinant P44 proteins (rP44-1, rP44-18ES, and/or rP44-47) was confirmed using Western blot analysis. Thus, the results obtained in this study strongly suggest that the patient was infected with A. phagocytophilum. To our knowledge, this is the first case of human anaplasmosis infection in the Inner Mongolia Autonomous Region.

Anaplasma phagocytophilum, an obligate intracellular bacterium that propagates within host granulocytes, is considered to modify the host intracellular environment for pathogenesis. However, the mechanism(s) underlying such host modifications remain unclear. Here, we aimed to investigate the relation between A. phagocytophilum and endoplasmic reticulum (ER) stress in THP-1 cells. A. phagocytophilum activated the three ER stress sensors: inositol-requiring enzyme-1 (IRE1), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF6). IRE1 activation occurred immediately after host cell invasion by A. phagocytophilum; however, the activated IRE1-induced splicing of X-box-binding protein 1 was not promoted during A. phagocytophilum infection. This suppression was sustained even after the doxycycline-mediated elimination of intracellular A. phagocytophilum. IRE1 knockdown accelerated A. phagocytophilum-induced apoptosis and decreased intracellular A. phagocytophilum. These data suggest that A. phagocytophilum utilizes IRE1 activation to promote its own intracellular proliferation. Moreover, PERK and ATF6 partially mediated A. phagocytophilum-induced apoptosis by promoting the expression of CCAAT/enhancer-binding protein homologous protein, which induces the transcription of several proapoptotic genes. Thus, A. phagocytophilum possibly manipulates the host ER stress signals to facilitate intracellular proliferation and infection of surrounding cells before/after host cell apoptosis.

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

Human granulocytic anaplasmosis (HGA) is caused by Anaplasma phagocytophilum. Indirect immunofluorescence assay (IFA) is generally used for HGA serodiagnosis. A. phagocytophilum immunodominant P44 major outer membrane proteins are encoded by p44/msp2 multigene family, responsible for IFA reactivity. However, because multiple P44-related proteins may involve immunoreactivity in IFA, the available diagnostic antigens remain obscure. In this study, we identified 12 B-cell epitopes on triple P44-related proteins using peptide array that reacted with 4 HGA patients' sera. Then, peptide spot immunoassay using 14 synthetic peptides derived from those 12 epitopes as antigens was applied for the detection of antibody to A. phagocytophilum from patients with fever of unknown origin. The sensitivities and diagnostic efficiencies of this immunoassay were higher than those of Western blot analysis using 3 recombinant proteins previously developed. Thus, the immunoassay using our epitope-derived antigens, which has higher diagnostic performances, may have significant benefit for HGA serodiagnosis.

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.

Tsutsugamushi disease and Japanese spotted fever are representative rickettsioses in Japan, and are caused by infection with Orientia tsutsugamushi and Rickettsia japonica, respectively. For molecular-based diagnosis, conventional PCR assays, which independently amplify respective rickettsial DNA, are usually used; however, this approach is time-consuming. Here, we describe a new duplex real-time PCR assay for the simultaneous detection of O. tsutsugamushi and spotted fever group rickettsiae, and its evaluation using several PCR conditions in 6 public health laboratories. The detection limit of the assay was estimated to be 102 copies and the sensitivity was almost identical to that of 3 conventional PCR methods. A total of 317 febrile patients were selected as clinically suspected or confirmed cases of rickettsioses. The detection efficiency of this assay for O. tsutsugamushi from blood or skin (eschar) specimens appeared to be almost the same as that of the conventional PCR method, even when performed in different laboratories, whereas the efficiency for spotted fever group rickettsiae tended to be higher than that of the 2 traditional double PCR assays. Our duplex real-time PCR is thus a powerful tool for the rapid diagnosis of rickettsioses, especially at the acute stage of infection.

In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p

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.

SUMMARY: The serotype, Shiga toxin (Stx) type, and antimicrobial resistance patterns of 138 Stxproducing Escherichia coli (STEC) strains isolated from humans between 2003 and 2007 in Shizuoka Prefecture, Japan were characterized. The predominant O serogroups of the STEC isolates were O157, O26, and O111. Antimicrobial susceptibility testing of the STEC isolates showed that 31 of the 138 isolates (22.5z) were resistant to antibiotics. Compared to the results reported in the previous studies, a higher rate of STEC O157 isolates were susceptible to all the antimicrobial agents used in this study. However, antimicrobial susceptibility data from this study showed that antimicrobial resistance patterns have increased by 6 compared to the survey performed by Masuda et al. between 1987 and 2002 (Jpn. J. Food Microbiol., 21, 44–51, 2004). This indicates that STEC isolates have evolved to show a variety of antimicrobial resistance patterns. It is important to consider the population of isolates showing decreased susceptibility to clinically relevant drugs such as ciprofloxacin (CPFX) and fosfomycin (FOM). All the 3 STEC isolates resistant to nalidixic acid showed low susceptibility to CPFX (MIC, 0.25–0.5 mg/ml). In addition, a decreased susceptibility to FOM was clearly observed in the E. coli O26 isolates. Our findings also showed that 1 STEC O26 strain could possibly be a chromosomal AmpC blactamase hyperproducer. These results suggest that antimicrobial therapy may be less effective in patients with non-O157 STEC infections than in those with STEC O157 infections.

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.

A novel concentration method using minute particles of amorphous calcium phosphate (ACP) was developed for the detection of caliciviruses including norovirus and sapovirus, agents of human gastroenteritis, from water. In seeding experiments with feline calicivirus (FCV), ACP particles were able to adsorb efficiently the viruses in water, and the FCV-concentrated solution was obtained by dissolution of the virus-adsorbing ACP particles with citric acid after centrifugation. By quantitative real-time RT-PCR, the recovery efficiencies from 300 ml ultrapure water seeded with 103, 104 and >105 copies of FCV were 48, 68 and >100 %, respectively. A comparative study showed that in the addition of viruses at

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.

Green-mature soybean ( Glycine max) extract (GSE) inhibits the progression of immunoglobulin E-mediated inflammation more than the ordinary yellow-pigmented soybean extract (YSE). The inhibition of nitric oxide (NO) production by GSE in lipopolysaccharide-stimulated RAW264.7 cells has been found to be lower than that by YSE. However, this inhibitory ability became markedly pronounced by visible light irradiation. We investigated the anti-inflammatory effects of GSE and visible light-irradiated GSE (L-GSE) on dextran sodium sulfate (DSS)-induced colitis, immunoglobulin E-independent inflammation, and assessed whether light irradiation affects their anti-inflammatory properties. The severity of DSS-induced colitis was moderated by the ingestion of any of the soybean extracts, and L-GSE was the most effective in moderating the progression in colitis. GSE and L-GSE, except for YSE, inhibited the mRNA expressions of pro-inflammatory cytokines and chemokine in mice colons. All soybean extracts suppressed the mRNA expression of inducible nitric oxide synthase (iNOS), whereas GSE and L-GSE also inhibited the expression of the iNOS protein in the early phase of colitis. These results suggest that GSE is likely to suppress NO production, unlike in vitro results. Unfortunately, a significant difference in the anti-inflammation ability of GSE and L-GSE was not observed in the DSS-induced colitis mice. However, GSE and L-GSE are likely to become new candidate agents for the inhibition of inflammation and excessive NO production.

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.

We recently reported that hypoxia could induce the breakdown of capillary-like tubes formed by human umbilical vein endothelial cells (HUVECs) and that this breakdown was regulated by p38 and not by a caspase cascade, although the exact molecular mechanisms remain unknown. The aim of this study was to identify proteins that regulated hypoxia-induced tube breakdown through p38-regulated and caspase-independent mechanisms. The involvement of adhesion proteins, integrins, VE-cadherin, PECAM-1, and occludin was first investigated. Although some of these proteins decreased after hypoxia, none of them met the conditions of being quantitatively restored by p38 inhibition but not by caspase inhibition. We then conducted 2-D DIGE coupled with MALDI-TOF/TOF-MS to identify altered protein expression. The differential proteomic analysis of tube-forming HUVECs treated with normoxia or hypoxia and treated with hypoxia in the presence or absence of SB203580, a specific p38 inhibitor, revealed the involvement of heat shock proteins in this tube breakdown. We also confirmed that the amount of HSP27 and HSP70 changed in a p38-regulated and caspase-independent manner during hypoxia. Knocking down HSP27 expression using RNAi further augmented hypoxia-induced tube breakdown. Taken together, it was shown that p38-regulated and caspase-independent reduction of HSP27 plays an important role in hypoxia-induced tube breakdown.

Recently, there have been advances in the genome analysis of numerous living organisms, including humans. In one post-genome study, the term “proteomics” has attracted much attention. Although proteomics have been widely investigated in the medical and pharmaceutical fields, only a few proteomic studies in the field of food sciences have been reported. Therefore, in this study, proteomic techniques were applied to an analysis of quality evaluations of fish during storage. Specifically, detailed changes in fish proteins during storage were analyzed using two-dimensional electrophoresis and mass spectrometry. We investigated the early oxidative proteins of bonito muscle during storage, and several proteins were identified by homology search using the NCBI genome database. Thus, the proteomic technique is available to the field of food science.

Human granulocytic anaplasomsis and human monocytic ehrlichiosis are tick-borne emerging infectious diseases which are caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis. These bacteria are obligatory intracellular parasites with tropism for hematopoietic cells. Here, we introduced analysis of protein expression profiles of HL-60 host cells in the response to infection with those bacteria by proteomic approach using fluorescence-prelabeled two dimensional difference gel electrophoresis to clarify the molecular mechanisms of the intracellular parasitism. Three experiments were designed for this purpose: differential analysis of infection at late (i) or early (iii) stages, and time-course analysis postinfection (ii). The detailed technical approaches, advantage of this procedure, and technical improvements desired were described and discussed.

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)

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.

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.

Anaplasma phagocytophila is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis. Immunodominant 44-kDa outer membrane proteins of A. phagocytophila are encoded by a p44 multigene family. In the present study, expression profiles of p44 genes in the blood of acutely infected patients in the year 2000 were characterized. A single p44 gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different p44 genes were transcribed without a single majority in the other two patients. The cDNA sequences of the central hypervariable region of several p44 genes were identical among the isolates from the three patients and a 1995 A. phagocytophila isolate. A. phagocytophila was isolated by cell culture from all of the three 2000 patients. Genomic Southern blot analysis of the three 2000 and two 1995 A. phagocytophila isolates with probes specific to the most dominant p44 transcript in each patient showed that the p44 loci in the A. phagocytophila genome were conserved. Analysis of the predicted amino acid sequences of 43 different p44 genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved. The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains. These results suggest that variations in the sequences of p44 are not random but are restricted. Furthermore, several p44 genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart.

Anaplasma ( Ehrlichia ) phagocytophila and Ehrlichia chaffeensis , the etiologic agents of granulocytic and monocytic ehrlichioses, respectively, are obligatory intracellular bacteria that cause febrile systemic illness in humans. We identified and characterized clusters of genes for a type IV secretion machinery in these two bacteria, and analyzed their gene expression in cell culture and mammalian hosts. Eight virB and virD genes were found in each bacterial genome, and all of the genes were transcribed in cell culture. Although the gene order and orientation were similar to those found in other bacteria, the eight virB and virD genes were clustered at two separate loci in each genome. Five of the genes ( virB8 , virB9 , virB10 , virB11 , and virD4 ) were located downstream from a ribA gene. These five genes in both A. phagocytophila and E. chaffeensis were polycistronically transcribed and controlled through at least two tandem promoters located upstream of the virB8 gene in human leukemia cell lines. The virB9 gene of A. phagocytophila was transcriptionally active in peripheral blood leukocytes from human ehrlichiosis patients and experimentally infected animals. Three of the remaining genes ( virB3 , virB4 , and virB6 ) of both A. phagocytophila and E. chaffeensis were arranged downstream from a sodB gene and cotranscribed with the sodB gene through one or more sodB promoters in human leukocytes. This suggests that transcription of the three virB genes in these two Anaplasma and Ehrlichia spp. is regulated by factors that influence the sodB gene expression. This unique regulation of gene expression for the type IV secretion system may be associated with intracellular survival and replication of Anaplasma and Ehrlichia spp. in granulocytes or monocytes.

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.

Ehrlichia canis and E. chaffeensis are tick-borne obligatory intramonocytic ehrlichiae that cause febrile systemic illness in humans and dogs, respectively. The current study analyzed the pleomorphic multigene family encoding approximately 30-kDa major outer membrane proteins (OMPs) of E. canis and E. chaffeensis . Upstream from secA and downstream of hypothetical transcriptional regulator, 22 paralogs of the omp gene family were found to be tandemly arranged except for one or two genes with opposite orientations in a 28- and a 27-kb locus in the E. canis and E. chaffeensis genomes, respectively. Each locus consisted of three highly repetitive regions with four nonrepetitive intervening regions. E. canis , in addition, had a 6.9-kb locus which contained a repeat of three tandem paralogs in the 28-kb locus. These total 47 paralogous and orthologous genes encoded OMPs of approximately 30 to 35 kDa consisting of several hypervariable regions alternating with conserved regions. In the 5′-end half of the 27-kb locus or the 28-kb locus of each Ehrlichia species, 14 paralogs were linked by short intergenic spaces ranging from −8 bp (overlapped) to 27 bp, and 8 remaining paralogs in the 3′-end half were connected by longer intergenic spaces ranging from 213 to 632 bp. All 22 paralogs, five unknown genes, and secA in the omp cluster in E. canis were transcriptionally active in the monocyte culture, and the paralogs with short intergenic spaces were cotranscribed with their adjacent genes, including the respective intergenic spaces at both the 5′ and the 3′ sides. Although omp genes are diverse, our results suggest that the gene organization of the clusters and the gene locus are conserved between two species of Ehrlichia to maintain a unique transcriptional mechanism for adaptation to environmental changes common to them.

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.

Ehrlichia chaffeensis and E. sennetsu are genetically divergent obligatory intracellular bacteria of human monocytes and macrophages, and the human granulocytic ehrlichiosis (HGE) agent is an obligatory intracellular bacterium of granulocytes. Infection with both E. chaffeensis and E. sennetsu , but not HGE agent, in the acute monocytic leukemia cell line THP-1 almost completely inhibited by treatment with deferoxamine, a cell-permeable iron chelator. Transferrin receptors (TfRs) accumulated on both E. chaffeensis and E. sennetsu , but not HGE agent, inclusions in THP-1 cells or the cells of the promyelocytic leukemia cell line HL-60. Reverse transcription-PCR showed an increase in the level of TfR mRNA 6 h postinfection which peaked at 24 h postinfection with both E. chaffeensis and E. sennetsu infection in THP-1 or HL-60 cells. In contrast, HGE agent in THP-1 or HL-60 cells induced no increase in TfR mRNA levels. Heat treatment of E. chaffeensis or the addition of monodansylcadaverine, a transglutaminase inhibitor, 3 h prior to infection inhibited the up-regulation of TfR mRNA. The addition of oxytetracycline 6 h after E. chaffeensis infection caused a decrease in TfR mRNA which returned to the basal level by 24 h postinfection. These results indicate that both internalization and continuous proliferation of ehrlichial organisms or the production of ehrlichial proteins are required for the up-regulation of TfR mRNA. Results of electrophoretic mobility shift assays showed that both E. chaffeensis and E. sennetsu infection increased the binding activity of iron-responsive protein 1 (IRP-1) to the iron-responsive element at 6 h postinfection and remained elevated at 24 h postinfection. However, HGE agent infection had no effect on IRP-1 binding activity. This result suggests that activation of IRP-1 and subsequent stabilization of TfR mRNA comprise the mechanism of TfR mRNA up-regulation by E. chaffeensis and E. sennetsu infection.

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.

In this study, the amorphous calcium phosphate (ACP) method developed previously for calicivirus concentration from water was applied for norovirus detection from food. The viral recovery from cabbage, lettuce, or ham (10g of each) was firstly examined in seeding experiments with feline caliciviruses (FCVs). The viruses were concentrated by viral adsorption to ACP particles (0.3g) in the eluent solution (40ml) from foods, collection of the particles by centrifugation, followed by dissolution of the particles with 3.3M citric acid (3ml). In ham, FCV recovery was improved by addition of ascorbic acids into the eluent solution before ACP-particle adsorption. Quantitative real-time reverse transcription-PCR (qRT-PCR) revealed that FCV recoveries were 32-33%, 50-55%, and 37-46% from cabbage, lettuce, and ham, respectively, when seeded with 10(3)-10(4) viruses, and detection limits were estimated ∼10(3) genomic copies in all 3 foods. Subsequently, the ACP-concentration method was evaluated for norovirus (NoV) detection from these 3 foods. The recoveries and detection limit of NoVs determined by qRT-PCR were 12-41% and 10(3) (genomic copies) from cabbage, 30-57% and 10(3) from lettuce, and 20-26% and 10(4) from ham, when seeded with 10(3)-10(5) viruses. This simple method may be suitable for NoV detection from these foods.

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.

To the Editor: Human granulocytic anaplasmosis (HGA) is an emerging tick-borne infectious disease caused by Anaplasma phagocytophilum, an obligatory intracellular bacterium (1). Recently, 2 cases of HGA were identified by a retrospective study in Japan (2). For serodiagnosis of HGA, A. phagocytophilum propagated in HL60 cells is usually used as an antigen, especially by indirect immunofluorescent assay (IFA) (3). However, the serum from these 2 patients in Japan reacted with antigens of A. phagocytophilum cultured in THP-1 cells rather than in HL60 cells in IFA (2). In A. phagocytophilum, a p44/msp2 multigene family encoding multiple 44-kDa immunodominant major outer membrane protein species (so-called P44) exists on the genome, and these multigenes are similar, but not identical, to each other, and the bacterium generates antigenic variations because of gene conversion (4). The previous studies showed that A. phagocytophilum expresses predominantly 2 species of p44/msp2 transcripts in THP-1 cells, but it produces the variation of P44 protein species in HL60 cells (2,5). This finding strongly suggested that A. phagocytophilum grown in THP-1 cells differs serologically from that in HL60 cells. Our serologic analysis found 4 recent cases of HGA in Japan by using infected THP-1 and HL60 cells as antigens, and some P44 immunoreactive protein species of A. phagocytophilum that were associated with the respective cell line cultures, binding to antibodies from the 4 patients’ serum, also were identified.

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.

Monoclonal antibodies (MAbs) against Rickettsia tsutsugamushi Kawasaki were prepared. The crossreactivity tests of the MAbs performed by using antigenically distinct strains of R. tsutsugamushi in immunofluorescence and immunoblotting analyses indicated that the Kawasaki strain contains a strain-specific epitope and also contains a common epitope on the 56-kDa polypeptide cross-reactive with the Gilliam strain, group- and subgroup-specific epitopes on the 46-kDa polypeptide, and a subgroup-specific epitope on the 25-kDa polypeptide. By using the strain-specific MAb for serodiagnosis of tsutsugamushi disease (or scrub typhus fever), we have established a method which was designated the inhibition enzyme-linked immunosorbent assay. The principle of the method is to measure the percentage of inhibition of antigen absorption on a MAb-coated plate by antibody-positive sample sera which were mixed with the antigen suspension. The advantages of this test for practical use are that (i) crude antigen can be used, i.e., purification of the antigen is not required; (ii) the test is more sensitive than immunofluorescence; (iii) the final judgment of plus or minus is clear-cut; and (iv) rickettsial antigenic types in the patients can be distinguished by this test.

Diabetic patients are prone to severe bacterial infections. The functional alterations of neutrophils by hyperglycemia are thought to be partially responsible for such infections. In this study, we investigated the functional changes of neutrophil-like differentiated cell lines (dHL-60, dTHP-1, and dNB-4) by treatment with 5.5 mM, 11 mM, or 35 mM of glucose. In dHL-60 cells, the incubation with high glucose (35 mM) resulted in the enhancement of cell aggregation, the suppression of cellular fragility, the induction of reactive-oxygen species (ROS) production by phorbol myristate acetate (PMA) stimulation, and the impairment of phagocytosis. In dTHP-1 cells, the treatment with higher glucose generated the suppression of cellular fragility and extremely impaired phagocytosis (by 35 mM), and induced ROS production due to PMA stimulation (by 11 mM). Furthermore, the higher glucose exposure to dNB-4 cells enlarged intracellular vacuoles (by 35 mM) and induced ROS production due to PMA stimulation (by 11 mM). Since the ROS generation of those cells was enhanced only after PMA stimulation under the higher glucose conditions, glucose may have a priming effect rather than a triggering effect. These extraordinary sensitivities caused by the higher glucose treatments may reflect the dysfunction or overactivation of neutrophils.