A novel bacterium that infects laboratory rats was isolated from wild Rattus norvegicus rats in Japan. Transmission electron microscopy of the spleen tissue revealed small cocci surrounded by an inner membrane and a thin, rippled outer membrane in a membrane-bound inclusion within the cytoplasm of endothelial cells. Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium found in R. norvegicus rats and Ixodes ovatus ticks in Japan revealed that the organism represents a novel clade in the family Anaplasmataceae, which includes the Schotti variant found in Ixodes ricinus ticks in the Netherlands and the Ehrlichia-like Rattus strain found in R. norvegicus rats from China. The novel clade was confirmed by phylogenetic analysis of groESL sequences found in R. norvegicus rats and Ixodes ovatus ticks in Japan. No serological cross-reactivity was detected between this bacterium and members of the genera Anaplasma, Ehrlichia or Neorickettsia in the family Anaplasmataceae. It is proposed that this new cluster of bacteria should be designated 'Candidatus Neoehrlichia mikurensis'.The family Anaplasmataceae currently includes five genera: Ehrlichia, Anaplasma, Neorickettsia, Aegyptianella and Wolbachia (Dumler et al., 2001;Rikihisa et al., 2003). These are all obligately intracellular bacteria that are capable of infecting invertebrates and/or vertebrates. Species of the genera Ehrlichia and Anaplasma include the emerging tick-borne human pathogens, such as Ehrlichia chaffeensis and Anaplasma phagocytophilum. As it is difficult to isolate and culture this group of bacteria, the ultrastructure and sequences of conserved genes, such as 16S rRNA, groEL and gltA (citrate synthase gene), are primarily used to identify and classify this group of bacteria. The present study describes a novel bacterium in the family Anaplasmataceae that was isolated from wild Rattus norvegicus rats by using laboratory rats. The bacteria were found in R. norvegicus rats and Ixodes ovatus ticks in Japan and represent a novel genetic cluster, based on phylogenetic analysis of 16S rRNA gene sequences that included the bacteria found in R. norvegicus rats in China (Pan et al., 2003) and in Ixodes ricinus ticks in the Netherlands (Schouls et al., 1999). Of note, infection of I. ricinus ticks with similar bacteria in Baltic regions of Russia was reported by Alekseev et al. (2001). A similar 16S

Abstract:The The movement of bacteria is important for their colonization in the animal intestinal tract (5, 6, 9, 10) and the bacterial flagellum is an organelle that supports bacterial locomotion. Bacterial cells can be classified into two groups based on flagella attachments: monotrichous flagellated bacteria with a single flagellum at either one or both ends of the cell; and peritrichous flagellated bacteria with many flagella on the side of the cell. The swimming velocities of these two types of bacterial species are reported to be different (7,8). Bacterial species with polar flagella are reported to be able to move more quickly than peritrichous flagellated bacteria. However, the differences in swimming velocities among species of the monotrichous flagellated bacteria are not well known.Morphological observations of moving bacteria have been carried out by several methods. One method employed recently uses videotape (2,3,7,8). Recent advances in video, microscopy and computer-based image processing have made it possible to observe and analyze the movement of extremely small objects such as bacteria.In this paper we report the results of the measurement of swimming velocity of Vibrio cholerae and Pseudomonas aeruginosa in culture media using a computer-assisted video tracking method. In addition, we aim to show that this method is useful and simple for analysis of the details of such complicated movement. Materials and MethodsBacterial strains and cultures. The bacterial strains used in this experiment were Vibrio cholerae non-O1 strains V018, AP7, AP5 and AI1854, and Pseudomonas aeruginosa strains K, P28, P15, No. 3 and No. 6. These bacterial strains were all obtained from our own stock cultures. Bacterial strains were cultured in nutrient broth consisting of 0.5% Extract Bonito, 1.0% polypepton and 0.5% sodium chloride (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 37 C.Examination of the swimming patterns. The bacteria were cultured in nutrient broth with aeration at 37 C. Concentrations of bacterial cells were estimated from the density of the culture with a photo densitometer by light absorption at 660 nm (Fuji Kogyo Co., Ltd., Tokyo). The bacteria in log phase culture were diluted with nutrient broth up to OD 0.01 which contains approximately 106 CFU/ml of bacterial cells. Then one drop of the diluted culture was placed on a clean microscope slide and covered with a thin cover glass. To prevent evaporation of the medium which usually causes streaming of the media on the slide, the edges of the cover glass were sealed tightly with nail polish. The cover glass was gently pressed with the fingers when it was sealed with nail polish to reduce the thickness of the aqueous layer between the two glass plates.

Between 1983 and 1999, 27 human cases of scrub typhus (two fatal) occurred in the Nodagawa River basin of northern Kyoto, Japan, an area where no cases had been previously reported. Antibody screening of infected patients' sera showed that nine of 15 patients had high titers against the Gilliam type of Orientia tsutsugamushi (Hayashi). To determine the vector mite transmitting the disease, we studied rodent and chigger populations in and near a rice field alongside the Nodagawa River between 1996 and 1999. The most common rodent species was Microtus montebelli (Milne-Edwards), representing 73.3% (33/45) of the population. The mite index (average number of mites per infested host) was highest (190.8) in Leptotrombidium pallidum Nagayo, Mitamura & Tamiya parasitizing on M. montebelli, followed by Leptotrombidium intermedium (Nagayo, Mitamura & Tamiya) (174.9) on the same host species. Orientia tsutsugamushi was isolated from 60.5% (23/38) of rodents and from 71.2% (37/52) of pools of engorged L. pallidum. The Gilliam type of O. tsutsugamushi was most prevalent in rodents, and in engorged L. pallidum and it was the only type recovered from 10 isolates inoculated into L 929 cells for indirect immunofluorescence examination. Orientia tsutsugamushi infected 14.3% (181/1263) and 14.8% (306/2066) of engorged and unfed L. pallidum larvae, respectively, and was also detected in 0.055% (2/3634) of unfed L. intermedium, although previous studies suggest that this mite rarely bites humans. These results show that L. pallidum is the primary vector species of scrub typhus in this new endemic area in Japan.

Engorged larvae of Leptotrombidium pallidum Nagayo, Miyagawa, Mitamura & Tamiya, the vector mite of scrub typhus in Japan, were reared by feeding them with fresh eggs of the collembolan Sinella curviseta Brook while confined in small plastic containers under natural conditions in a copse. The larvae were collected from wild rodents (Apodemus speciosus) in autumn 1985 and spring 1986. Adults were kept alive for 2 yr or longer. The larvae obtained in autumn became dormant in the cold winter season, and growth recommenced in the spring. Thus, the development of mites collected in April became synchronized with that of larvae obtained in the autumn. Most larvae developed into protonymphs in May, deutonymphs in June, tritonymphs in July, and adults in August. The females laid eggs in two consecutive summers. Some larvae collected in autumn were kept in a refrigerator until the following summer. They developed into deutonymphs, tritonymphs, or adults and then became dormant in the winter. Development restarted the next spring and all became adult by summer, when the females laid eggs. Under experimental conditions, all larvae are hatched in the autumn, unlike the natural situation in which two peaks of larval occurrence on wild rodents are observed in autumn and spring.

The stylostome of larvae of the trombiculids Leptotrombidium scutellare (Nagayo et al.), Leptotrombidium fletcheri (Womersley et Heaslip) and Leptotrombidium deliense (Walch) was studied experimentally at different time intervals after larval attachment using the histological method. The stylostome of these species has the same organization and belongs to the epidermal combined with the mixed type, developing more in width than in length. Neither transverse nor conspicuous longitudinal layers are present within the stylostome walls, which stain predominantly in red with Azan, also showing longitudinal portions with blue staining. Larvae tend to attach closely to each other and scabs, consisting of the hyperkeratotic epidermal layers fusing with migrating inflammatory cells, develop around the attachment sites. The dermis shows inflammatory foci with dilated capillaries and inflammatory cells inserting in the connective tissue layer underneath the stylostome. The feeding cavity, which is moderately expressed, may be found either in the epidermis or in the dermis. It contains inflammatory cells and their debris in the liquefied host tissues. The stylostome length depends on the character of the attachment site (the thicker epidermis or scab the longer the stylostome), and does not directly correspond to the stages of larval feeding. Nevertheless, at the 48-h time interval, nearly all attached larvae are found to be fully fed and their midgut cells are filled with nutritional globules.

Larvae of Leptotrombidium pallidum (Nagayo, Miyagawa, Mitamura & Tamiya) from uninfected laboratory colonies were fed on mice infected with Rickettsia tsutsugamushi (Hayashi) Ogata. Infection of the chiggers with R. tsutsugamushi was determined by passage of chigger exudates into ddY mice. The passage method was modified so that an inoculum was considered to be positive when R. tsutsugamushi or anti-R. tsutsugamushi antibody, or both, were detected in mice up to the third blind passage. R. tsutsugamushi was detected in six of 18 larvae (33.3%) and in all developmental stages. In adults, five of 18 males and 10 of 46 females were infected with R. tsutsugamushi. In L. fuji (Kuwata, Berge & Philip), R. tsutsugamushi was not found in 57 engorged larvae fed on rickettsemic mice but was found in a very low percentage of deutonymphs and adults. Female L. pallidum from larvae fed on infected mice were paired individually, and F1 larvae were collected. Although eight females were found to be positive for R. tsutsugamushi, the rickettsia was not detected in 12 pools (249 larvae) of F1 larvae from these infected females. We concluded that uninfected mites became infected by feeding on rickettsemic mice at comparatively high rates depending on the species and transmitted this infection transstadially to succeeding life stages, but not vertically to larvae in the following F1 generation.

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