Ticks In Japan Research Articles

A wide distribution of Beiji nairoviruses and related viruses in Ixodes ticks in Japan

Beiji nairovirus (BJNV), in the family Nairoviridae, the order Bunyavirales, was recently reported as a causative agent of an emerging tick-borne zoonotic infection in China. This study investigated the prevalence of BJNV in ticks in Japan. Screening of over 2,000 ticks from multiple regions revealed a widespread distribution of BJNV and BJNV-related viruses in Japan, particularly in the northern island, and in other high altitude areas with exclusive occurrence of Ixodes ticks. Phylogenetic analysis identified three distinct groups of nairoviruses in ticks in Japan: BJNV, Yichun nairovirus (YCNV) and a newly identified Mikuni nairovirus (MKNV). BJNV and YCNV variants identified in ticks in Japan exhibited high nucleotide sequence identities to those in China and Russia with evidence of non-monophyletic evolution among BJNVs, suggesting multiple cross-border transmission events of BJNV between the Eurasian continent and Japan. Whole genome sequencing of BJNV and MKNV revealed a unique GA-rich region in the S segment, the significance of which remains to be determined. In conclusion, the present study has shown a wide distribution and diversity of BJNV-related nairoviruses in Ixodes ticks in Japan and has identified unique genomic structures. The findings demonstrate the significance of BJNV as well as related viruses in Japan and highlight the necessity of monitoring emerging nairovirus infections and their potential risks to public health.

Development of an entirely cloned cDNA-based reverse genetics system for Tofla virus of orthonairovirus

The genus Orthonairovirus includes highly pathogenic tick-borne viruses such as the Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). A reverse genetics system is an indispensable tool for determining the viral factors related to pathogenicity. Tofla orthonairovirus (TFLV) is a recently identified virus isolated from ticks in Japan and our research has suggested that TFLV is a useful model for studying pathogenic orthonairoviruses. In this study, we successfully established a reverse genetics system for TFLV using T7 RNA polymerase. Recombinant TFLV was generated by transfecting cloned complementary DNAs encoding the TFLV genome into BSR T7/5 cells expressing T7 RNA polymerase. We were able to rescue infectious recombinant TFLV mutant (rTFLVmt) and wild-type TFLV (rTFLVpt) viruses, which exhibited indistinguishable growth kinetics in mammalian cells and pathogenicity in A129 mice compared with the authentic virus. Our approach provides a valuable method for establishing reverse genetics system for orthonairoviruses.

Detection of Japanese Encephalitis Virus RNA in Host-Questing Ticks in Japan, 2019-2020.

Japanese encephalitis virus (JEV), a mosquito-borne virus, causes severe clinical symptoms in humans in the Asian-Pacific region, where it circulates in a primary transmission cycle among Culex tritaeniorhynchus mosquitoes, domestic swine (Sus scrofa domesticus), and wading birds. We report here an anomalous result that mosquito-borne JEV was detected in unfed host-questing ticks collected from the field in Japan. JEV genomic RNA was detected in four pools of Haemaphysalis flava nymphs collected in November and December 2019, and March 2020, when Cx. tritaeniorhynchus adults were not presumed to be active. Moreover, JEV antigenomic RNA was detected in some JEV-positive tick samples, suggesting virus replication in ticks. However, taken together with no infectious virus isolated, the possibility that the antigenomic RNA was derived from the undigested bloodmeal source in ticks cannot be ruled out. Thus, the role of the ticks as a natural reservoir for JEV remains to be confirmed.

Seroepidemiological surveys of tick-borne encephalitis virus and novel tick-borne viruses in wild boar in Nagasaki, Japan

In Japan, tick-borne viruses such as tick-borne encephalitis virus (TBEV) and severe fever with thrombocytopenia syndrome virus have been identified in humans, animals, and ticks. In addition, novel tick-borne viruses have been isolated from ticks in Japan. This study aimed to determine the seroprevalence of TBEV and novel viruses, particularly Tofla virus (TFLV), Kabuto Mountain virus (KAMV), and Muko virus (MUV) in wild boar in Nagasaki, Japan. Enzyme-linked immunosorbent assays and neutralization tests were performed to detect antibodies against each virus. Wild boar serum tested positive for antibodies against KAMV, TFLV, and TBEV, but not MUV. This study revealed the seroprevalence of newly identified tick-borne viruses and TBEV in animals residing in the Nagasaki area. The seroprevalence of these viruses in sentinel animals may inform policies aimed at preventing tick-borne virus disease outbreaks.

A novel nairovirus associated with acute febrile illness in Hokkaido, Japan

The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014–2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan.

Diversity unearthed by the estimated molecular phylogeny and ecologically quantitative characteristics of uncultured Ehrlichia bacteria in Haemaphysalis ticks, Japan

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.

Rickettsia spp. and Ehrlichia spp. in Amblyomma ticks parasitizing wild amphibious sea kraits and yellow-margined box turtles in Okinawa, Japan

Recently, several tick-borne pathogens were detected in reptile-associated ticks. However, studies on the microorganisms in reptile-associated ticks in Japan are limited. This molecular survey thus aimed to identify and characterize tick-borne pathogens (Rickettsiaceae and Anaplasmataceae) in reptile-associated ticks in Japan. In total, 77 Amblyomma nitidum and 104 Amblyomma geoemydae were collected from wild amphibious sea kraits (Laticauda semifasciata, Laticauda colubrina, and Laticauda laticaudata) and from yellow-margined box turtles (Cuora flavomarginata evelynae), respectively. Conventional polymerase chain reaction was performed using the DNA extracted from the ticks to detect the selected pathogens. Sequencing analysis of four Rickettsia genes (gltA, ompA, ompB, and sca4) led to the identification of a putative novel Rickettsia sp. and Rickettsia aeschlimannii-like rickettsia in A. nitidum and A. geoemydae, respectively. Sequencing analysis of gltA and groEL of Anaplasmataceae revealed that the Ehrlichia spp. in these ticks were novel and related to Candidatus Ehrlichia occidentalis. This is the first study on the microorganisms in A. nitidium and the first record of Rickettsia and Ehrlichia in A. geoemydae. Further studies are required to understand their pathogenicity to humans and animals and their life cycle in the wild.

Diversity of spotted fever group rickettsiae and their association with host ticks in Japan

Spotted fever group (SFG) rickettsiae are obligate intracellular Gram-negative bacteria mainly associated with ticks. In Japan, several hundred cases of Japanese spotted fever, caused by Rickettsia japonica, are reported annually. Other Rickettsia species are also known to exist in ixodid ticks; however, their phylogenetic position and pathogenic potential are poorly understood. We conducted a nationwide cross-sectional survey on questing ticks to understand the overall diversity of SFG rickettsiae in Japan. Out of 2,189 individuals (19 tick species in 4 genera), 373 (17.0%) samples were positive for Rickettsia spp. as ascertained by real-time PCR amplification of the citrate synthase gene (gltA). Conventional PCR and sequencing analyses of gltA indicated the presence of 15 different genotypes of SFG rickettsiae. Based on the analysis of five additional genes, we characterised five Rickettsia species; R. asiatica, R. helvetica, R. monacensis (formerly reported as Rickettsia sp. In56 in Japan), R. tamurae, and Candidatus R. tarasevichiae and several unclassified SFG rickettsiae. We also found a strong association between rickettsial genotypes and their host tick species, while there was little association between rickettsial genotypes and their geographical origins. These observations suggested that most of the SFG rickettsiae have a limited host range and are maintained in certain tick species in the natural environment.

Isolation and molecular detection of Ehrlichia species from ticks in western, central, and eastern Japan

Ehrlichiosis is a tick-borne bacterial disease caused by pathogens of the Ehrlichia genus. Although human ehrlichiosis has not been reported in Japan, Ehrlichia spp., which are closely related to Ehrlichia chaffeensis, were detected in several species of ixodid ticks. In this study, the presence of Ehrlichia spp. in ticks in Japan was studied by using isolation and molecular detection methods. In total, 1237 ticks were collected from vegetation in western, central, and eastern parts of Japan. The ticks were tested for detection of ehrlichial DNA with a nested polymerase chain reaction and/or isolation by inoculation of mice with the homogenate. Ehrlichial DNA was detected in 29 of these ticks. The ehrlichial DNAs, groEL and 16S rRNA genes, detected in Ixodes turdus showed a high similarity to those of E. chaffeensis with 94.7% and 99.2% identity, respectively. Ehrlichia sp. HF and Candidatus Neoehrlichia mikurensis were also detected in I. ovatus. Furthermore, Ehrlichia sp. HF was isolated from laboratory mice that were intraperitoneal inoculated with I. ovatus tick homogenate. Some ehrlichial agents detected in Ixodes ticks might be a previously unknown Ehrlichia species. In this study, Candidatus N. mikurensis was detected in I. ovatus ticks. Because I. ovatus is distributed widely and cases of its tick bite in humans are ubiquitously reported in Japan, there is a potential for ehrlichiosis to be endemic to Japan, necessitating intensive surveillance of this infectious disease.

Detection of Francisella tularensis and analysis of bacterial growth in ticks in Japan.

Francisella tularensis is distributed in the Northern hemisphere and it is the bacterial agent responsible for tularaemia, a zoonotic disease. We collected 4527 samples of DNA from ticks in Japan, which were then analysed by real-time PCR and nested PCR. Francisella DNA was detected by real-time PCR in 2·15% (45/2093) of Ixodes ovatus, 0·66% (14/2107) of I.persulcatus, 8·22% (6/73) of I.monospinosus and 0·72% (1/138) of Haemaphysalis flava specimens. Finally, Francisella DNA was detected by nested PCR in 42 and five samples I.ovatus and I.persulcatus, respectively, which were positive according to real-time PCR. Phylogenetic analysis showed that the sequence from I.ovatus and I.persulcatus were clustered with F.tularensis type B strains distributed in Eurasia. Microinjected live F.tularensis persisted in ticks, whereas heat-killed F.tularensis decreased. Microinjected F.tularensis hlyD mutant decreased in ticks significantly compared to parent strain, thereby suggesting that HlyD in F.tularensis contributes to the adaptation or survive of bacterial infection in ticks. Francisella tularensis has been detected in ticks, suggesting that it is a tick-borne pathogen. However, F.tularensis has not been detected in ticks in Japan since 1991. In this study, we performed a large-scale analysis of DNA isolated from ticks in Japan and detected F. tularensis by real-time polymerase chain reaction (PCR) and nested PCR. We found that F.tularensis could survive in ticks based on an experimental tick-infection model. We also identified a bacterial factor that contributes to survival in ticks. Our results suggest that ticks are candidate vectors that mediate F.tularensis infection in Japan.

Tofla virus: A newly identified Nairovirus of the Crimean-Congo hemorrhagic fever group isolated from ticks in Japan.

Ixodid ticks transmit several important viral pathogens. We isolated a new virus (Tofla virus: TFLV) from Heamaphysalis flava and Heamaphysalis formsensis in Japan. The full-genome sequences revealed that TFLV belonged to the genus Nairovirus, family Bunyaviridae. Phylogenetic analyses and neutralization tests suggested that TFLV is closely related to the Hazara virus and that it is classified into the Crimean-Congo hemorrhagic fever group. TFLV caused lethal infection in IFNAR KO mice. The TFLV-infected mice exhibited a gastrointestinal disorder, and positron emission tomography-computed tomography images showed a significant uptake of 18F-fluorodeoxyglucose in the intestinal tract. TFLV was able to infect and propagate in cultured cells of African green monkey-derived Vero E6 cells and human-derived SK-N-SH, T98-G and HEK-293 cells. Although TFLV infections in humans and animals are currently unknown, our findings may provide clues to understand the potential infectivity and to develop of pre-emptive countermeasures against this new tick-borne Nairovirus.

PCR detection of Babesia ovata from questing ticks in Japan

Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n=1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (β-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described β-tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata.

Recent cases of invasive alien mites and ticks in Japan: why is a regulatory framework needed?

Japan's economy depends on the importation of natural resources, and as a result, Japan is subjected to a high risk of biological invasion. Although Japan has quarantine systems to protect ecosystems, agriculture, forestry, fisheries, and human health against alien species, economic globalization has resulted in an ever-increasing risk of invasion. Mite invasion is no exception. Alien species that impact natural ecosystems are regulated in Japan by the Invasive Alien Species Act. However, the law focuses only on visibly recognizable species, so that species too small to see, such as viruses, bacteria, fungi, and mites, are beyond the scope of this law. The Plant Protection Law has limited the introduction of alien pests, including mites, that are harmful to agricultural crops. Recently, the liberalization of global trade policies have increased pressure to loosen regulations on various pests, including spider mites. Infectious diseases and their causative species are quarantined under the Rabies Prevention Law, the Domestic Animal Infectious Diseases Control Law, and the Human Infectious Diseases Control Law, but these laws do not cover wildlife diseases. The most serious problem is that wild reptiles, which can be carriers of ticks and tick-borne diseases, can be freely introduced to Japan. These loopholes in Japan's regulatory system have resulted in mite and tick invasions, which affect not only wildlife communities and human society but also endemism and biological diversity of natural mite populations.

Isolation and preliminary characterisation of ‘Rickettsia monacensis’ in south-eastern Germany

G. Dobler, S. Essbauer and R. Wo¨lfelDepartment of Virology & Rickettsiology, Bundeswehr Institute of Microbiology, Munich, Germany‘Rickettsia (R.) monacensis’ is a member of thespotted fever group of rickettsiae. It was firstisolated in ticks from a city park in Munich,Germany [1]. Since then, it has been detected bymolecular biological tests (polymerase chain reac-tion, PCR) in several European countries, mainlyin southern and south-eastern Europe (Spain,Italy, Slovakia, Albania and Bulgaria) and in ticksin Japan [2]. Only recently, ‘R. monacensis’ wasdetected in patients with spotted fever, andtherefore its pathogenic potential for humanswas proven for the first time [3]. Besides theoriginal isolate, no isolates from ticks are avail-able. Two recent isolates from patients in Spainare actually available beside the original isolateform Ixodes (I.) ricinus. We report the isolation andpreliminary characterisation of ‘R. monacensis’from ticks (I. ricinus) from an area some 200 kmnorth of the location of original isolation.Ticks of the species I. ricinus were collected in amixed pine-leaf wood forest and transported aliveto the laboratory. They were homogenised indi-vidually (adults) or in pools of five individuals(nymphs, larvae) and the nucleic acid wasextracted automatically (MagnaPure, Roche,Mannheim). Rickettsial DNA was detected usingan in-house pan-rickettsial real-time PCR directedagainst part of the gltA gene of rickettsiae [4]. Ticksuspensions were kept at 4 C until PCR resultswere available. PCR-positive tick suspensionswere used in inoculation of Vero cells. Suspen-sions were centrifuged on cell monolayers for60 min at 2000 g at room temperature and furtherincubated for 60 min at 37 C. The inoculates wereremoved and replaced by cell culture mediumplus penicillin⁄streptomycin⁄amphothericin.After incubation overnight at 28 C, the cell cul-ture medium was replaced by antibiotic-freemedium and incubated for up to 14 days at28 C. Supernatants were tested for presence ofrickettsiae by real-time PCR after 7 days and14 days. In case of rickettsial growth sub-passageswere made using the described procedures. Iden-tification of rickettsial species was done usingsequencing of PCR amplicons of the ompA, theompB and the gltA genes [5].In a total of 260 collected ticks two positive ticksfor ‘R. monacensis’ could be identified, corre-sponding to a minimal infection rate (MIR) of0.77%. In contrast, 14 tick samples were foundpositive for ‘R. helvetica’, resulting in a MIR of5.4% for this species. We succeeded in isolatingtwo isolates (Hoegling-1, Hoegling-2). The furthercharacterisation resulted in the identification of‘R. monacensis’ and ‘R. IRS4⁄3’, respectively [1,5].For further molecular characterisation of strainHoegling-1 amplicons for partial ompA, ompB andgltA genes were used. A comparison of sequencehomologies with nucleotide sequences of otherrickettsiae identified the isolate as ‘R. monacensis’with 100% homology of the amplicons to thecorresponding genes of the original isolateIrRMunich, and 99% homology to ‘R. IrS4’ and‘R. IrS3’ sequences (Table 1).The rickettsial isolates identified as ‘R. monac-ensis’ enlarge the known geographical distribu-tion of this Rickettsia species in Germany up to200 km to the north. In fact, it is the most northerndetection of this rickettsial species so far. Besidethe original isolate IrRMunich [1] and two recentpatient isolates in Spain [3], the isolates increasethe available collection of geographically differentstrains of ‘R. monacensis’. All other detections ofthis rickettsial species in Europe were molecularbiological detections of small amplicons. Theavailability of the strains will enable moredetailed research in the evolution of this rickett-sial species. Both isolates Hoegling-1 and Hoe-gling-2 grew at 28 C in cell culture, but no growthat 37 C could be observed. This observation isinteresting as only recently ‘R. monacensis’ wasfound to cause spotted fever in two patients.

Structural analysis of a p44/msp2 expression site of Anaplasma phagocytophilum in naturally infected ticks in Japan

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.

Rickettsioses in Japan and the Far East

Three rickettsial diseases are known to exist in Japan currently: Japanese spotted fever (JSF), Tsutsugamushi disease (TD; scrub typhus), and Q fever. Since April 1999, the system for infection control and prevention in Japan has changed drastically. JSF, Q fever, and TD, as emerging infectious diseases, are designated as national notifiable diseases.The geographic distribution of JSF patients is along the coast of central and southwestern Japan, whereas TD and Q fever occur almost all over the country. The number of JSF patients reported was 216 cases during 1984-1998 and 268 cases, under the revised law, in 1999-2004. About 300-1000 cases of TD occur every year, and 7-46 cases of Q fever in 1999-2004. The number of cases of JSF and its endemic area are gradually increasing. There was only one fatality due to JSF until 2003, whereas two patients died of JSF in 2004, so JSF is still a life-threatening disease in Japan. Treatment of fulminant JSF consists of prompt administration of a combination of tetracycline and quinolone. Recent tick surveys revealed that the most probable vectors of JSF are Haemophysalis flava and Haemophysalis hystericis. In addition to R. japonica, two serotypes or species of spotted fever group rickettsiae have been isolated from ticks in Japan; one is closely related to R. helvetica and the other is a new genotype of unknown genotype AT, which is closely related to a Slovakian genotype. These serotypes are of uncertain clinical significance. Epidemiology of rickettsioses in the Far East is mentioned briefly.

Phylogenetic analysis of spotted fever group rickettsiae based on gltA, 17-kDa, and rOmpA genes amplified by nested PCR from ticks in Japan.

In order to understand the natural situation of rickettsiae in the ticks in Japan, the rickettsial genes, gltA gene, rOmpA gene, and 17-kDa gene, were amplified from the ticks by nested PCR. The prevalences of rickettsial gltA genes among Haemaphysalis formosensis, H. longicornis, H. megaspinosa, Ixodes ovatus, H. flava, H. kitaokai, and I. persulcatus were 62, 57, 24, 24, 19, 13, and 10%, respectively; 26% (186/722) being the average. The gltA genes amplified from the ticks were classified into 9 genotypes (I to IX) by the difference in nucleotide sequences. Genotype I was detected from 7 species of ticks. Genotype II mainly was detected from H. longicornis and H. formosensis. Genotypes III and VII mainly were detected from H. flava and I. ovatus. The polarization in the distribution of genotypes among regions where the ticks were collected was not clear. Based on the phylogenetic analysis of the three genes presented here, genotypes I, III, and IV (detected from H. formosensis, H. hystricia, and I. ovatus ) are genetically close with each other, but rickettsiae of the same property still have not been isolated from ticks anywhere in the world. These genotypes should be considered as new species among SFG rickettsiae. Genotype II was identical with strain FUJ-98, genetically close to R. japonica which has been isolated from ticks in China. Genotype V was identical with R. felis and strain California 2 isolated from the cat flea. This is the first report on the detection of R. felis from ticks. Genotype VI detected from Ixodes sp. did not seem to belong to genus Rickettsia. Based on the previous antigenic data and the phylogenetic analysis presented here, Genotype VII should be considered a variant of R. helvetica and genotype VIII detected from I. ovatus and I. persulcatus were identical with R. helvetica. Genotype IX detected from I. nipponensis was genetically close to the strains IRS3, IRS4, and IrR/Munich isolated from I. ricinus in Slovakia and German.

Ixodid tick species recovered from domestic dogs in Japan.

The species of ixodid ticks (Acari: Ixodidae) recovered from domestic dogs in Japan between September to November 2000 and April to June 2001 were identified. A total of 4122 ticks, including 1624 larvae, 1200 nymphs, 1016 females and 282 males were removed from 1221 dogs during these periods. Haemaphysalis longicornis (Neumann) was the most frequently found (40.3% of dogs), followed by H. flava (Neumann) (16.1% of dogs), Rhipicephalus sanguineus (Latreille) (4.8% of dogs) and Ixodes ovatus (Neumann) (4.1% of dogs). Small numbers of H. hystricis (Supino), H. campanulata (Warburton), H. japonica (Warburton), H. ias (Nakamura and Yajima), I. persulcatus (Schulze), I. nipponensis (Kitaoka and Saito) and Amblyomma testudinarium (Koch) were also recovered. In the spring sample, a total of 1408 ticks (78 larvae, 411 nymphs, 792 adult females and 127 adult males) were recovered from 570 dogs. The autumn sample included a larger proportion of larval stage and fewer adult ticks (1546 larvae, 789 nymphs, 224 adult females and 155 adult males). Haemaphysalis longicornis, H. flava and I. ovatus showed a wide geographical distribution from northern to southern Japan, whereas R. sanguineus were mainly distributed in the subtropical Okinawa prefecture with a few exceptions. Dogs in rural areas more frequently carried H. longicornis, H. flava and I. ovatus than dogs in urban or suburban areas, whereas R. sanguineus was more associated with the dogs in urban/suburban areas. Exposure to a garden was significantly associated with R. sanguineus and exposure to woodland was significantly associated with H. flava and I. ovatus. This is the first systematic survey of canine ticks in Japan.

Genetic identification of rickettsiae isolated from ticks in Japan.

Following the description in Japan of Japanese spotted fever, caused by Rickettsia japonica, a search for the vector of this disease led to the isolation of several rickettsiae from various tick species. Sixty-three rickettsial isolates were obtained from six different tick species, and six type strains were described by PCR and monoclonal antibody testing. We identified these six strains by amplification and sequencing of the genes encoding 16S rRNA and citrate synthase. We confirmed that the isolates from Dermacentor taiwanensis and Haemaphysalis flava ticks were R. japonica isolates. In Ixodes ovatus, Ixodes persulcatus, and Ixodes monospinosus, we identified a Rickettsia identical or closely related to Rickettsia helvetica, a species that is pathogenic for humans and that to date has only been found in Europe. Finally, we identified a new genotype of unknown pathogenicity, genotype AT, that was isolated from Amblyomma testudinarium ticks and that is closely related to a Slovakian genotype obtained from Ixodes ricinus ticks.

Phylogenetic analysis of spotted fever group Rickettsiae isolated from ticks in Japan.

Eight spotted fever group (SFG) rickettsiae isolated from ticks in Japan were classified by phylogenetic analysis based on the nucleotide sequences of both the citrate synthase-encoding gene (gltA) and 190-kDa antigen-encoding gene (rOmpA). In the phylogenetic tree of gltA, strains DT-1 and FLA-1 isolated from the Dermacentor taiwanensis and Haemaphysalis frava ticks, respectively, were placed as Rickettsia japonica, and strains IO-1, IO-2, IO-25, IM-1 and IP-2 from genus Ixodes ticks were placed as Rickettsia helvetica. Strain AT-1 isolated from the Amblyomma testudinarium belonged to the cluster including Rickettsia akari, Rickettsia australis and Rickettsia felis. In the phylogenetic tree of the rOmpA, strains DT-1 and FLA-1 were placed as R. japonica, and strain AT-1 belonged to the cluster including Rickettsia cooleyi and the symbiont of Ixodes scapularis. The rOmpA fragments of 5 Ixodes isolates could not be amplified by PCR. The present study showed that strains DT-1 and FLA-1 were genotypically identical to R. japonica, and 5 Ixodes isolates were associated with the R. helvetica. Based on previous genotypic and antigenic data, and the phylogenetic analysis presented here, strain AT-1 should be considered as a new species among SFG rickettsiae.