Mutsuo Kobayashi

Epidemiological Potential of Excretion and Regurgitation by Musca domestica (Diptera: Muscidae) in the Dissemination of Escherichia coli O157: H7 to Food

Abstract We previously reported that enterohemorrhagic Escherichia coli O157: H7 (EHEC) proliferates in the mouthparts of the house fly Musca domestica vicina Macquart and are excreted for at least 3 d after ingestion. However, the role of the crop and excretory behavior of the house fly in contamination of human food with EHEC is not known. In the current experiments, EHEC persisted in the crop of house flies for at least 4 d. The number of EHEC in an excreted droplet was ≈104 1 h after bacterial feeding, >1.8 × 105 3 h after feeding, and then drastically decreased after 24 h. Excretion is one of the major mechanisms for decreasing number of EHEC in the crop and gut of the house fly. The frequency of excretion by females with developing eggs in their ovary was clearly higher (6.5 min per drop) than for males or females with mature eggs. Minute eosin-sign around a container filled with eosin-supplemented trypticase soy broth might be derived from frequent contact by house fly contaminated mouthparts. These results show that frequent excretion potentially enhances the dissemination of EHEC to foods, particularly during the first 24 h after ingestion of the bacteria.

Analysis of Northern Distribution of Aedes albopictus (Diptera: Culicidae) in Japan by Geographical Information System

Abstract Aedes albopictus (Skuse), a mosquito vector of the dengue fever virus, is prevalent in Japan, distributed widely in Honshu Island with its northern limits between latitude 38° to 40°degrees north. The factors affecting distribution of the species in the northern part of Japan were studied using the geographical information system (GIS). During 1998–2000, larval surveillance was carried out in 26 urban and rural areas in the Tohoku district, in the northern part of Honshu Island, by collecting larvae from artificial and natural habitats. Climatological analysis, using the GIS, showed that the following conditions accounted for the current distribution of Ae. albopictus: an annual mean temperature higher than 11°C and a mean temperature of the coldest month, January, higher than −2°C. A period with temperature above 11°C in the confirmed area of the mosquito successively continues for more than 186 d per year. The accumulated temperature calculated from a temperature of 11°C, which may be close to the developmental zero of Ae. albopictus, was over 1,350 degree-days. The relationship between the beginning of short-daylength, inducing egg diapause, and the monthly mean temperature during September and October necessary for successful larval development in the Tohoku district is also discussed. We also show the relationship between the current distribution of Ae. albopictus and the annual mean temperature in the United States. From these results it is predicted that Ae. albpictus will be established in some cities in northeast United States.

First Molecular Evidence of Bartonella quintana in Pediculus humanus capitis (Phthiraptera: Pediculidae), Collected from Nepalese Children

Abstract Trench fever is a body louse-borne disease caused by Bartonella quintana Brenner. The recent status of louse infestation in Nepalese children is not well known. We collected head and body lice, Pediculus humanus capitis De Geer and Pediculus humanus humanus L., respectively, from 30 children, including 11 cases of double infestation with both head and body lice. Detection of B. quintana in both louse species identified was carried out by polymerase chain reaction (PCR). PCR products with B. quintana DNA sequences were detected in both head and body lice from two children as well as in body lice derived from two other children. These results demonstrate that head lice may also play a role in the transmission of trench fever.

Further Evidence for the DecayK+→π+νν¯

Additional evidence for the rare kaon decay K+ to pi+ neutrino-antineutrino has been found in a new data set with comparable sensitivity to the previously reported result. One new event was observed in the pion momentum region examined, 211<P<229 MeV/c, bringing the total for the combined data set to two. Including all data taken, the backgrounds were estimated to contribute 0.15 pm 0.05 events. The branching ratio is B=1.57^{+1.75}_{-0.82} 10^{-10}.

Mechanisms of Pyrethroid Resistance in the Dengue Mosquito Vector, Aedes aegypti: Target Site Insensitivity, Penetration, and Metabolism

Aedes aegypti is the major vector of yellow and dengue fevers. After 10 generations of adult selection, an A. aegypti strain (SP) developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel (Vssc, the target site of pyrethroid insecticide) were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can more contribute to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than a susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s) play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2. Transcription levels of these P450s were well correlated with the rate of permethrin excretion and they were certainly capable of detoxifying permethrin to 4′-HO-permethrin. Over expression of CYP9M6 was partially due to gene amplification. There was no significant difference in the rate of permethrin reduction from cuticle between SP and SMK strains.

Symbiotic bacteria associated with stomach discs of human lice

ABSTRACT The symbiotic bacteria associated with the stomach disc, a large aggregate of bacteriocytes on the ventral side of the midgut, of human body and head lice were characterized. Molecular phylogenetic analysis of 16S rRNA gene sequences showed that the symbionts formed a distinct and well-defined clade in the Gammaproteobacteria. The sequences exhibited AT-biased nucleotide composition and accelerated molecular evolution. In situ hybridization revealed that in nymphs and adult males, the symbiont was localized in the stomach disc, while in adult females, the symbiont was not in the stomach disc but in the lateral oviducts and the posterior pole of the oocytes due to female-specific symbiont migration. We propose the designation “Candidatus Riesia pediculicola” for the louse symbionts.

Involvement of the JNK-like protein of the Aedes albopictus mosquito cell line, C6/36, in phagocytosis, endocytosis and infection of West Nile virus

We recently cloned a c‐Jun amino‐terminal kinase (JNK) sequence from the C6/36 cell line, derived from the mosquito Aedes albopictus. We showed that SP600125, an inhibitor of JNK proteins, inhibits phagocytosis by C6/36 cells, suggesting that the JNK‐like protein regulates phagocytosis. Here, we show that C6/36 cells constitutively express low levels of mRNA encoding the antibacterial peptides, cecropin and defensin, but that these mRNAs were up‐regulated upon stimulation by lipopolysaccharide (LPS). Thus, the C6/36 cells have properties similar to those of mammalian macrophages. To characterize further the functional properties of C6/36 cells, we have assayed the role of the JNK‐like protein in phagocytosis, endocytosis, and viral infection. C6/36 cells phagocytosed bacteria and artificial beads, and this was only slightly up‐regulated following LPS stimulation, suggesting that newly stimulated JNK‐like protein was not necessary for phagocytosis. SP600125 inhibited the acidification of intracellular compartments, including those involved in the endocytic pathway. Pretreatment of C6/36 cells with SP600125 or bafilomycin A1, but not cytochalasin D, inhibited the entry of West Nile virus (WNV), suggesting that WNV is internalized mainly by endocytosis, and that the JNK signalling pathway is important for endocytic entry. These findings indicate that the JNK‐like protein regulates basic physiological functions, including phagocytosis and endocytosis and infection of WNV.

Host-Feeding Habits of Culex pipiens and Aedes albopictus (Diptera: Culicidae) Collected at the Urban and Suburban Residential Areas of Japan

ABSTRACT To evaluate the vectorial capacity of mosquitoes for viruses in Japan, the host-feeding habits of the mosquitoes were analyzed by sequencing polymerase chain reaction-amplified fragments of the cytochrome b and 16S ribosomal RNA regions of the mitochondrial DNA of 516 mosquitoes of 15 species from seven genera that were collected from residential areas during 2003–2006. Culex pipiens L. and Aedes albopictus Skuse were the most commonly collected species in urban and suburban residential areas. Anautogenous Culex pipiens pallens Coquillett was distinguished from the autogenous Cx. pipiens form molestus Forskal using a polymerase chain reaction-based identification method. Both Cx. p. pollens and Cx. p. form molestus exhibited similar host-feeding habits, broadly preferring avian (50.0 and 42.5% of avian, respectively) and mammalian (38.6 and 45.0% of avian, respectively) hosts, such as tree sparrows, ducks, and humans. Conversely, Ae. albopictus exhibited a highly mammalophilic and anthropophilic feeding pattern, with 84.2% feeding on mammalian hosts and 68.5% of these on humans. We concluded that in Japan, Cx. pipiens might play a significant role in the avian-to-mammal transmission of viruses, such as West Nile virus, whereas Ae. albopictus might play a role in the human-human transmission of dengue and Chikungunya viruses.

Identification and molecular characterization of a new nonsegmented double-stranded RNA virus isolated from Culex mosquitoes in Japan.

Two infectious agents were isolated from Culex species mosquitoes in Japan and were identified as distinct strains of a new RNA virus by a method for sequence-independent amplification of viral nucleic acids. The virus designated Omono River virus (OMRV) replicated in mosquito cells in which it produced a severe cytopathic effect. Icosahedral virus particles of approximately 40 nm in diameter were detected in the cytoplasm of infected cells. The OMRV genome was observed to consist of a nonsegmented, 7.6-kb double-stranded RNA (dsRNA) and contain two overlapping open reading frames (ORFs), namely ORF1 and ORF2. ORF1 was found to encode a putative dsRNA-binding protein, a major capsid protein, and other putative proteins, which might be generated by co- and/or post-translational processing of the ORF1 polyprotein precursor, and ORF2 was found to encode a putative RNA-dependent RNA polymerase (RdRp), which could be translated as a fusion with the ORF1 product by a -1 ribosomal frameshift. Phylogenetic analysis based on RdRp revealed that OMRV is closely related to penaeid shrimp infectious myonecrosis virus and Drosophila totivirus, which are tentatively assigned to the family Totiviridae. These results indicated that OMRV is a new member of the family of nonsegmented dsRNA viruses infecting arthropod hosts, but not fungal or protozoan hosts.

Detection of Bartonella quintana from Body Lice (Anoplura: Pediculidae) Infesting Homeless People in Tokyo by Molecular Technique

Abstract We report detection of Bartonella quintana Brenner, the pathogenic agent of trench fever, from body lice, Pediculus humanus L., infesting homeless people in Tokyo by polymerase chain reaction. Two of 12 (16.7%) homeless were infested with Bartonella-positive body lice. From the current status of the recent increase of homeless people in many large cities of the developed countries, a medical examination of homeless people should be carefully performed in the consideration of trench fever. Sampling of body lice from clothing of homeless people is recommended for quick and accurate diagnosis of trench fever through the detection of B. quintana DNA.