Seroepidemiology of spotted fever group rickettsiae in small field rodents in Japan.

To clarify the geographic distribution of spotted fever group (SFG) rickettsiae in Japan, small field rodents captured in endemic and nonendemic areas were screened for anti-SFG rickettsia antibodies by the immunofluorescence test. Among total 716 specimens tested, 73-75% of rodents were antibody-positive against Rickettsia japonica and/or Rickettsia montana, showing different degree of antibody-positive proportions among each species of the rodents. Interestingly, these profiles were not different in the rodents from each endemic and nonendemic areas, indicating that the SFG rickettsiae are prevailing in the wider areas where patients have not been found yet.     

Seroepidemiology of spotted fever group and typhus group rickettsioses in humans, South Korea

The prevalence of spotted fever group (SFG) and typhus group (TG) rickettsioses was investigated in 3,362 sera by immunofluorescence assay. The serum samples were obtained from patients with acute febrile episodes in South Korea from December 1992 to November 1993. The number of polyvalent positive sera against SFG rickettsial agents at the level of 1: 40 dilution was 269 (8%) in Rickettsia sibirica, 482 (14.34%) in R. conorii, and 546 (16.24%) in R. akari. Many of the positive sera contained immunoglobulin (Ig) M antibodies rather than IgG antibodies. These results strongly suggest that SFG rickettsioses are prevalent in Korea. For TG rickettsial agents, the number of positive sera was 1,096 (32.60%) in R. typhi and 951 (28.29%) in R. prowazekii. Only a few epidemic typhus positive sera contained IgM antibodies. The result suggests that recent and/or primary infections of epidemic typhus were very rare in Korea during the said period. Among seven patients who had high titers (1:5,120) of IgG antibody to R. prowazekii, six were over 50 years old. The result suggests that Brill-Zinsser disease was prevalent in Korea.     

Genetic identification of rickettsiae of the tick-borne spotted fever group, isolated in the foci of tick-borne rickettsiosis

A total of 25 rickettsial cultures of the tick-borne spotted fever (TBSF) group from the collection of the Research Institute of Infections in Omsk, isolated from different sources in the territory of the Russian Federation (from the Urals to the Far East) during the period of 1954-2001) were studied by the methods of genetic analysis. The fragments of the gene coding the outer-membrane protein of 190 kD (ompA) and synthetase citrate (gltA) of the rickettsiae under the study were sequenced. 23 isolates were identified as R. sibirica, among them 3 isolates obtained from patients, 16 isolates obtained from Dermacentor ticks, 2 isolates from Haemaphysalis concinna and 2--from Ixodes persulcatus. The strain Primorye 32/84, isolated from D. silvarum ticks in the Far East and earlier identified as S. sibirica by the results of the PCR-RFLP analysis proved to be a genovariant Rickettsia spBJ-90, i.e. close to this species. Strain Karpunino 19/69, isolated in the Kurgan region, was identified as R. slovaca. The results obtained extended our notions of the spectrum of rickettsiae group TBSF in Russia as well as their vectors.     

Genotypic identification and phylogenetic analysis of the spotted fever group rickettsiae by pulsed-field gel electrophoresis.

Using pulsed-field gel electrophoresis, we studied the chromosomes of spotted fever group rickettsiae. We digested the DNA of 16 species currently known to belong to this group with SmaI, EagI, and BssHII. The genome size of 13 rickettsiae was between 1,200 and 1,300 kb. "Rickettsia massiliae" and "R. helvetica" genome sizes were 1,370 and 1,397 kb, respectively, and that of R. bellii was 1,660 kb. It was possible to obtain distinctive patterns for each species, but in R. conorii, 10 isolates exhibited the same profiles, showing that pulsed-field gel electrophoresis is a good interspecies identification tool. We achieved a phylogenetic analysis of these bacteria by using the Dice coefficient and UPGMA and Package Philip programming. We established a dendrogram of the genetic relationships between the different species showing the existence of a cluster in the spotted fever group rickettsiae including R. conorii, R. rickettsii, R. parkeri, R. sibirica, "R. africae," "R. slovaca," Thai tick typhus rickettsia, and Israeli tick typhus rickettsia. We located three genes previously cloned and sequenced (genes encoding the R. rickettsii surface proteins of 120 and 190 kDa and the R. prowazekii citrate synthase gene), using Southern hybridization. The genes encoding citrate synthase and the surface protein of 190 kDa were usually located on the same band, and it is hypothesized that they are relatively close on the chromosome.     

Identification of the spotted fever group rickettsiae detected from Haemaphysalis longicornis in Korea

Seven Haemaphysalis ticks were found positive in PCR assay of gltA gene to detect the spotted fever group (SFG) rickettsiae DNA from 100 ticks. The nucleotide sequence of 16S rRNA gene was determined from 5 ticks and compared to those of other Rickettsia strains. The nucleotide sequence from 4 ticks showed high homologies (99.7 to 100%) with that of R. japonica YH, and that from 1 tick (tick no. 48) was identical with that of R. rickettsii R, suggesting that SFG rickettsiae exists in Korea. This is the first documentation of SFG rickettsiae in Korea.     

Isolation of spotted fever group rickettsia from Apodemus speciosus in an endemic area in Japan.

A spotted fever group rickettsia was isolated from Apodemus mice captured near the site where a person had been infected in Miyazaki Prefecture. Antigenic characteristics of the isolate designated as strain TO-1 were compared with those of Rickettsia japonica (strain YH) and Rickettsia montana (ATCC VR611) by the indirect fluorescent antibody test with 14 serum specimens obtained from Apodemus speciosus, immune rat antisera against the strains TO-1 and YH, and three patients' sera. The titers of these sera measured with strain TO-1 were identical to those with strain YH, suggesting the antigenic similarity between these two organisms. It was also suggested that Apodemus speciosus is likely to be an important vertebrate host for a spotted fever group rickettsia in Japan.     

Characterization of spotted fever group rickettsiae detected in dogs and ticks in Okinawa,Japan

Spotted fever group (SFG) rickettsial DNAs were detected in 2.4% of 340 canine blood samples and a pool of 84 tick pool samples (229 ticks) collected in Okinawa, Japan by PCR using a citrate synthase and an SFG rickettsial 190-kDa surface antigen gene primer pair. The sequences of both genes from canine blood and tick samples showed high levels of similarity with those of Rickettsiajaponica and several SFG rickettsiae (R. aeschlimannii, R. massiliae, R. rhipicephali and Bar-29 strain). Phylogenesis of canine blood and tick samples was closely related to that of reference SFG rickettsiae. Serological evidence of SFG rickettsial infection in dogs and humans in Okinawa, where no clinical human cases have been reported, has been obtained. In this study, genetical characterization of SFG rickettsia in Okinawa was investigated phylogenetically.     

Spotted fever group rickettsiae in immature and adult ticks (Acari: Ixodidae) from a focus of Rocky Mountain spotted fever in Connecticut

Immature and adult ixodid ticks were collected during 1983 and 1984 in Newtown, Connecticut, an area endemic for Rocky Mountain spotted fever (RMSF), to determine prevalence of infection by spotted fever group (SFG) rickettsiae. Direct fluorescent-antibody (FA) staining revealed SFG organisms in 6 (1.8%) of 332 Dermacentor variabilis larvae, 5 (7.8%) of 64 D. variabilis nymphs, and in 2 (40%) of 5 Ixodes cookei nymphs removed from small- and medium-sized mammals. Hemolymph tests detected rickettsia-like organisms in 15 (8.8%) of 170 D. variabilis adults; 8 specimens retested by direct FA were negative. In contrast, hemocytes from 5 (8.6%) of 58 Ixodes texanus females contained organisms that stained positively in both hemolymph and direct FA tests. An indirect microimmunofluorescence test identified specific antibodies to Rickettsia rickettsii, the etiologic agent of RMSF, in serum samples from a chipmunk, raccoons, and white-footed mice. Results indicate that immature or adult ticks of at least three species may be involved in the maintenance and transmission of SFG rickettsiae at Newtown.     

Epigenetic polymorphism in populations of Apodemus sylvaticus and Apodemus flavicollis (Rodentia, Muridae)

The main object of this study was to use discontinuous variation as a means of measuring divergence between populations of A. sylvaticus (L.) and A. flavicollis (Melchior) from Britain and Eurasia. Samples from 17 populations (671 skulls) were each classified for 20 discontinuous morphological variants. The investigation has shown that certain epigenetic variants have a characteristic incidence in populations of the two species and that the relative incidence of the variants could be used as an aid to specific separation. The calculation of degree of divergence between the populations has made it possible to suggest the order in which populations are related to each other.     

Evidence for infection caused by spotted fever group Rickettsia in Kyushu, Japan

Of 50 cases that gave negative immunofluorescence reaction with Rickettsia tsutsugamushi among patients with suspected tsutsugamushi disease encountered in Miyazaki Prefecture during the last four years, three showed a significant rise in the antibody titer to Rickettsia montana, a species of spotted fever group. This paper reports evidence for the occurrence of the rickettsial infection of the spotted fever group in Kyushu, Japan.     

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.     

Identification of potential hosts and vectors of scrub typhus and tick-borne spotted fever group rickettsiae in eastern Taiwan

Scrub typhus and tick-borne spotted fever group (SFG) rickettsioses are transmitted by chiggers (larval trombiculid mites) and hard ticks, respectively. We assessed exposure to these disease vectors by extensively sampling both chiggers and ticks and their small mammal hosts in eastern Taiwan during 2007 and 2008. The striped field mouse Apodemus agrarius Pallas (Rodentia: Muridae) was the most common of the small mammals (36.1% of 1393 captures) and presented the highest rate of infestation with both chiggers (47.8% of 110 760) and ticks (78.1% of 1431). Leptotrombidium imphalum Vercammen-Grandjean & Langston (Trombidiformes: Trombiculidae) and immature Rhipicephalus haemaphysaloides Supino (Ixodida: Ixodidae) were the most abundant chiggers (84.5%) and ticks (>99%) identified, respectively. Immunofluorescent antibody assay revealed high seropositive rates of rodents against Orientia tsutsugamushi Hyashi (Rickettsiales: Rickettsiaceae), the aetiological agent of scrub typhus (70.0% of 437 rodents), and tick-borne SFG rickettsiae (91.9% of 418 rodents). The current study represents a first step towards elucidating the potential hosts and vectors in the enzootic transmission of O. tsutsugamushi and tick-borne SFG rickettsiae in Taiwan. Further studies should focus on characterizing pathogens in L. imphalum and R. haemaphysaloides, as well as the proclivity of both vectors to humans. Uncovering the main hosts of adult ticks is also critical for the prevention of SFG rickettsial infections.     

Ultrastructure of Rickettsia rhipicephali, a new member of the spotted fever group rickettsiae in tissues of the host vector Rhipicephalus sanguineus.

Rickettsia rhipicephali is similar in ultrastructure to R. rickettsii while differing from other rickettsiae of the typhus group and of Q fever and others by its lack of a prominently reticulated cytoplasmic matrix and in the thickness of the inner osmophilic layer of the cell wall. In tissues of the tick vector Rhipicephalus sanguineus, R. rhipicephali had a mean length and width of 1.2 and 0.46 micrometer, respectively. It possessed a trilaminar cell wall with an adhering capsule-like layer. The trilaminar cell wall was approximately 12 to 18 nm thick; its inner osmophilic layer was thicker than that previously reported for other rickettsiae. The capsule-like layer varied from 7 to 18 nm thick. The plasma membrane was similar in structure, measurement, and appearance to that of other reported rickettsiae. The cytoplasm appeared to be composed of a finely granular, amorphous, ground substance and randomly dispersed ribosomes and lacked a reticular matrix or nuclear fibrils. In massively infected salivary glands and ovarial tissues of its tick vector, R. rhipicephali produced a low degree of histopathology which does not appear to affect the engorgement and egg-laying process of the ticks.     

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.     

Electron microscopic studies on the in vitro proliferation of spotted fever group rickettsia isolated in Japan.

Rickettsia was isolated from a patient with Japanese spotted fever, and its proliferation in cultured green monkey kidney cells was observed by electron microscopy. In the course of this study, we observed fusion of infected cells to uninfected cells which may be a way of spreading the rickettsiae from a cell to another. On the other hand, whirlpool-like, multilayer membranous structures, similar to the mesosomes of gram-negative bacteria, were sometimes seen in the rickettsial cells. The other profiles common to the other rickettsiae in spotted fever group were observed, such as the electron-lucent halo zone around the rickettsiae, and external fibrous materials on their surface, but intranuclear multiplication was rarely observed.     

Studies on the distributions of Apodemus sylvaticus (L.) and A. flavicollis (Melchior) in Britain

The known distributions of A. sylvaticus and A. flavicollis were examined with respect to four woodland classifications and two soil types. The distribution of A. flavicollis in Britain is associated with drier areas and with the distribution of mature, deciduous woodland. A. sylvaticus is found in both wet and dry areas and is distributed independently of deciduous woodland.     

MHC diversity and the association to nematode parasitism in the yellow-necked mouse (Apodemus flavicollis)

In vertebrates, the genes of the major histocompatibility complex (MHC) are among the most debated candidates accounting for co-evolutionary processes of host-parasite interaction at the molecular level. The exceptionally high allelic polymorphism found in MHC loci is believed to be maintained by pathogen-driven selection, mediated either through heterozygous advantage or rare allele advantage (= frequency dependent selection). While investigations under natural conditions are still very rare, studies on humans or mice under laboratory conditions revealed support for both hypotheses. We investigated nematode burden and allelic diversity of a functional important MHC class II gene (DRB exon2) in free-ranging yellow-necked mice (Apodemus flavicollis). Twenty-seven distinct Apfl-DRB alleles were detected in 146 individuals with high levels of amino acid sequence divergence, especially at the antigen binding sites (ABS), indicating selection processes acting on this locus. Heterozygosity had no influence on the infection status (being infected or not), the number of different nematode infections (NNI) or the intensity of infection, measured as the individual faecal egg count (FEC). However, significant associations of specific Apfl-DRB alleles to both nematode susceptibility and resistance were found, for all nematodes as well as in separate analyses of the two most common nematodes. Apodemus flavicollis individuals carrying the alleles Apfl-DRB*5 or Apfl-DRB*15 revealed significantly higher FEC than individuals with other alleles. In contrast, the allele Apfl-DRB*23 showed a significant association to low FEC of the most common nematode. Thus, our results provide evidence for pathogen-driven selection acting through rare allele advantage under natural conditions.