Multilocus sequence typing and DNA similarity analysis implicates that a Borrelia valaisiana-related sp. isolated in Japan is distinguishable from European B. valaisiana.

Lyme disease Borrelia spp. are transmitted by Ixodes ticks, and more than 10 species of borreliae have been identified around the world. Recently, another Borrelia sp. has been reported in Asia (Japan, Korea, China, Taiwan and Thailand) as Borrelia valaisiana-related sp. In the present study, we obtained and genetically characterized 19 B. valaisiana-related sp. strains from mammals and ticks. Genetic analyses showed that the Borrelia strains were distinct from B. valaisiana found in Europe. Multilocus sequence typing revealed that these Borrelia isolates formed a monophyletic group with B. yangtze strains in China. Some of the strains were isolated from the bladders of small mammals, and also two strains were experimentally confirmed to be infectious in C3H/HeN mice. We observed that the Borrelia sp. was maintained in the Ixodes granulatus tick after molting. These results suggested that small mammals and I. granulatus were possible reservoir hosts and the vector tick for the Borrelia sp., respectively. B. valaisiana, originally found in Europe, was transmitted mainly by I. ricinus, and birds were mainly thought to be reservoir hosts. Our results suggested that Japanese isolates of B. yangtze (formerly B. valaisiana-related sp.) were distinguishable from B. valaisiana according to the reservoir host and its vector tick. In this study, we also deposited borrelia strain Okinawa-CW62 into bioresource centers as a reference strain of B. yangtze(=DSM 24625, JCM 17189).

Observation of live ticks (Haemaphysalis flava) by scanning electron microscopy under high vacuum pressure.

Scanning electron microscopes (SEM), which image sample surfaces by scanning with an electron beam, are widely used for steric observations of resting samples in basic and applied biology. Various conventional methods exist for SEM sample preparation. However, conventional SEM is not a good tool to observe living organisms because of the associated exposure to high vacuum pressure and electron beam radiation. Here we attempted SEM observations of live ticks. During 1.5×10(-3) Pa vacuum pressure and electron beam irradiation with accelerated voltages (2-5 kV), many ticks remained alive and moved their legs. After 30-min observation, we removed the ticks from the SEM stage; they could walk actively under atmospheric pressure. When we tested 20 ticks (8 female adults and 12 nymphs), they survived for two days after SEM observation. These results indicate the resistance of ticks against SEM observation. Our second survival test showed that the electron beam, not vacuum conditions, results in tick death. Moreover, we describe the reaction of their legs to electron beam exposure. These findings open the new possibility of SEM observation of living organisms and showed the resistance of living ticks to vacuum condition in SEM. These data also indicate, for the first time, the usefulness of tick as a model system for biology under extreme condition.

Dermoscopy for tick bite: reconfirmation of the usefulness for the initial diagnosis.

Ticks transmit a variety of infectious diseases. Diagnosis requires verification of a tick's presence. Here, we describe a 61-year-old woman bitten by an eight-legged nymphal Amblyomma testudinarium. We re-emphasize the usefulness of dermoscopy for identifying signs of the bite and determining the species of the biting tick.

Multilocus sequence typing implicates rodents as the main reservoir host of human-pathogenic Borrelia garinii in Japan.

Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.

Human Rickettsia heilongjiangensis infection, Japan.

A case of Rickettsia heilongjiangensis infection in Japan was identified in a 35-year-old man who had rash, fever, and eschars. Serum contained R. heilongjiangensis antibodies, and eschars contained R. heilongjiangensis DNA. R. heilongjiangensis was also isolated from ticks in the suspected geographic area of infection.

Survey of the vectorial competence of ticks in an endemic area of spotted fever group rickettsioses in Fukui Prefecture, Japan.

The prevalence of SFGR in ixodid ticks in the Mt. Arashima-dake area in the northern part of Fukui Prefecture was surveyed, because of strong suspicions that the first case identified in this Prefecture had become infected with R. helvetica in this region. The ticks identified consisted of three genera and six species; I.ovatus, I. persulcatus, I. monospinosus, H. flava, H. japonica and D. taiwanensis. Of all 222 ticks collected, only I. monospinosus ticks (8 of 32 examined) were positive for SFGR isolates, which were genetically identified as R. helvetica. Ticks (157 of all 222) positive for SFGR-DNA fragments consisted of I. monospinosus (14 of 32), I. persulcatus (11 of 55), I. ovatus (3 of 38), H. flava (5 of 21) and H. japonica (2 of 9). Of these, thirteen I. monospinosus, eight I. persulcatus, three I. ovatus, two H. flava and one H. japonica were identified by nucleotide sequences as positive for R. helvetica. DNA fragments from three H. flava and one H. japonica showed greater homology to R. japonica than to R. helvetica or R. asiatica. The present results indicate that it is most likely that the vector tick of R. helvetica infection in Fukui Prefecture is I. monospinosus.

Survey of Babesia microti infection in field rodents in Japan: records of the Kobe-type in new foci and findings of a new type related to the Otsu-type.

Of 247 rodents comprising 5 genera and 7 species collected at 17 sites throughout Japan from 2003 to 2005, Babesia microti was detected microscopically and by polymerase chain reaction (PCR) in 36 rodents comprising 2 genera and 3 species from 12 sites. Based on the analysis of small subunit ribosomal RNA gene (SSUrDNA) sequences, the Kobe-type, the etiological type of the first Japanese case of human infection was found in Apodemus speciosus and Apodemus argenteus in Aomori, the northernmost prefecture of the Japanese mainland, while the U.S.-type was found on Hokkaido Island and the Otsu-type was widely distributed. In addition, a new Otsu-related type was detected exclusively in Eothenomys andersoni in Nagano, a prefecture in central Japan. The sequences of internal transcribed spacer 1 to 2 (ITS1/2) of the present Kobe- and Otsu-types were almost identical to those of the same types previously identified. The ITS1/2 sequence of the U.S.-type identified in Hokkaido in this survey was somewhat different from that of the U.S.-type strain originating from the U.S.A., with approximately 95% identity. This value was similar to the 94% identity found between the ITS1/2 sequences of the Otsu-type and the new Otsu-related type. The new Otsu-related type of B. microti was isolated as the Nagano strain, which was serologically differentiated from the other type strains of B. microti. The divergence and distribution of genotypes are important factors in investigating the epidemiology of human B. microti infection in Japan.

Japanese Babesia microti cytologically detected in salivary glands of naturally infected tick Ixodes ovatus.

Babesia microti protozoa were detected by light and electron microscopy in the salivary glands of field-collected Ixodes ovatus ticks; 6 of 85 adult ticks were demonstrated to be positive for B. microti DNA by polymerase chain reaction assays. In the salivary glands of unfed ticks, B. microti existed in the sporoblast stage in the granular acinus cells, and developed into the sporozoite stage during feeding on the host for 2 days. The present results indicated for the first time that I. ovatus can indeed carry B. microti and is not infected mechanically with the parasites by blood-sucking. This frequent infection of I. ovatus with B. microti demonstrates the significance of such a vector-pathogen relationship in Japan, and strongly suggests that I. ovatus is involved in the maintenance of B. microti in the fauna of Japanese rodents.

The first reported case of spotted fever in Fukui Prefecture, the northern part of central Japan.

A 53-year-old man visited Mt. Arashima-dake in Fukui Prefecture, and was infested by a tick-like organism. He visited a local clinic on July 12, 2004, complaining of high fever, general fatigue and rash. After several days without definite diagnosis, he was admitted to the Fukui Prefectural Hospital, where he was treated with minocycline hydrochloride for 10 days until recovery. His clinical symptoms on admission were high fever (39.6 degrees C), erythematous eruption, eschar on the right upper arm, and regional lymphoadenopathy. The epidemiological status and some clinical findings strongly suggested spotted fever (SF), and SF was confirmed based on the finding that his sera were reactive only to antigens of the SF group rickettsiae in the indirect immunoperoxidase analysis. This case is the first official report of SF rickettsiosis in Fukui Prefecture, the northern part of central Japan.

Ultrastructure of a Japanese Rickettsial strain genetically identified as Rickettsia helvetica which was originally found in Europe.

A rickettsial strain IO-1 has been isolated from a tick, Ixodes ovatus, in Japan and genetically identified as Rickettsia helvetica, a member of the spotted fever group rickettsiae. Ultrastructural observations were made on the microorganism. The ultrastructure of R. helvetica IO-1 appeared to be generally the same as that previously shown for other rickettsiae of the spotted fever and typhus groups. The rickettsiae were primarily found free in the cytoplasm of L929 cultured cells. Occasionally, the rickettsiae may also invade the host cell nucleus; however, the frequency of the nuclear localization was very low.

Survey of rodents and ticks in human babesiosis emergence area in Japan: first detection of Babesia microti-like parasites in Ixodes ovatus.

Babesia microti-like parasites were detected for the first time in Ixodes ovatus in Hyogo Prefecture, Japan, where two reported types of B. microti-like parasites were recognized in many rodents. Of 80 adult I. ovatus ticks collected, 5 possessed the reported type and 1 possessed a new type of B. microti-like parasite.