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.     

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.     

Western-blot detection of RickA within spotted fever group rickettsiae using a specific monoclonal antibody

Spotted fever group rickettsiae are obligate intracellular pathogens able to manipulate the actin cytoskeleton, thus enabling cell-to-cell spreading during infection. While the RickA protein, which has similarity to the WASP family of Arp2/3-complex activators, was described as being responsible for actin-based motility, recent studies demonstrated that another factor, still unidentified, is also involved in this phenomenon. Here, using recombinant protein of Rickettsia conorii as an antigen, we produced a monoclonal antibody (mAb) directed against RickA. Its specificity was checked using two-dimensional polyacrylamide gel electrophoresis coupled with MS analysis. In Western-blot assays, our antibody recognized the RickA protein from all spotted fever group rickettsiae tested. This mAb would be useful to monitor the expression of RickA in spotted fever group rickettsiae grown under various culture conditions, associated or not with the motility phenotype, and thus to gain better knowledge about the molecular mechanisms involved in their cell-to-cell spreading.     

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.     

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 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.     

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.     

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.     

The major outer membrane protein rOmpB of spotted fever group rickettsiae functions in the rickettsial adherence to and invasion of Vero cells

The role of one of the major outer membrane proteins, rOmpB, of spotted fever group rickettsiae was examined. Antibodies generated against native rOmpB inhibited plaque formation by Rickettsia japonica in Vero cells when applied at the time of inoculation of the rickettsiae. However, antibodies to heat-denatured rOmpB did not. Moreover, the soluble recombinant rOmpB also inhibited plaque formation to some extent. Thus it seems that rOmpB functions at least in the adherence of rickettsiae to host cells. To obtain direct evidence of its function in the adherence to and invasion of Vero cells, we generated Escherichia coli transformed by the vector pET-22b(+) inserted with the ompB open reading frame of R. japonica. The recombinant bacteria expressed a 165-kDa protein consistent with the precursor of rOmpB. The protein reacted with monoclonal antibodies to heat-labile epitopes of rOmpB. Immunofluorescence of the recombinant bacteria demonstrated surface expression of the protein. It was shown by light microscopy and transmission and scanning electron microscopy that the bacteria adhered to and invaded Vero cells. Thus, although the recombinant precursor rOmpB was not processed on the outer membrane of E. coli, it functions during these steps. The manner of entry was similar to that of rickettsiae although at a slower rate.     

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.     

Comparative electrophoresis of spotted fever group rickettsial proteins.

Electrophoretic analyses were performed to establish the polypeptide profiles of the following tick-borne typhus rickettsiae of the spotted fever group: $\text{Rickettsia rickettsii}$ (Sheila Smith, Bitter Root, Iowa and R strains), $\text{R. sibirica}$, $\text{R. conorii}$, $\text{R. parkeri}$, $\text{R. australis}$ and $\text{R. akari}$. Organisms were propagated in chick embryo fibroblast cells in roller bottles and purified by density centrifugation. After polyacrylamide gel electrophoresis, comparative electrophoretic profiles were examined by densitometric scanning. Of the more than 20 separate polypeptides detected, six of the most prominent were found in all rickettsiae examined except $\text{R. akari}$. No carbohydrate-containing moieties were detected in electrophoretic gels.     

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.     

Restriction of the growth of a nonpathogenic spotted fever group rickettsia

The growth kinetics of pathogenic and nonpathogenic rickettsiae were compared to elucidate the mechanism responsible for the pathogenicity of rickettsiae. Vero and HeLa cells derived from mammals were inoculated with a nonpathogenic species of spotted fever group rickettsia, Rickettsia montanensis, before being infected with the pathogenic species Rickettsia japonica. The mammalian cells became persistently infected with R. montanensis and produced low levels of rickettsiae. On the other hand, superinfection of the R. montanensis-infected cells with R. japonica resulted in increased yields of R. montanensis accompanied by R. japonica growth. Both rickettsiae also grew well in the R. japonica-infected cells subjected to superinfection with R. montanensis. Western blotting with an antibody to the autophagy-related protein LC3B found that autophagy was induced in the cells infected with R. montanensis alone. On the contrary, autophagy was restricted in the cells that were co-infected with R. japonica. Electron microscopy of the cells infected with R. montanensis alone demonstrated rickettsia particles being digested in intracytoplasmic vacuoles. Conversely, many freely growing rickettsiae were detected in the co-infected cells.     

Serologic survey of spotted fever group rickettsiosis on Hainan Island of China.

A serosurvey for antibodies to Rickettsia japonica was conducted on Hainan Island of China. Serum specimens were collected from 1,030 outpatients at hospitals in different parts of the island regardless of their diagnosis. Only two among 538 serum specimens collected in Baoting and Tongshi counties, located in the southern part of the island, were demonstrated to contain antibodies reactive with R. japonica at a high dilution. The specimens also reacted with R. rickettsii at the same titer as with R. japonica. These two specimens reacted with other pathogenic spotted fever group (SFG) rickettsiae to a lesser extent. On the other hand, the specimens were shown to possess antibodies reactive with R. typhi at a significantly lower dilution or were not reactive at all. The findings suggested the occurrence of an SFG rickettsiosis on Hainan Island. More than half of the serum specimens collected from patients with suspected rickettsial infections in the southern area were found to contain IgM and IgG antibodies to R. typhi, indicating a high incidence of murine typhus.     

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.     

Occurrence of rickettsiosis of spotted fever group in Chiba Prefecture of Japan

Acute- and convalescent-phase sera were obtained from a patient with suspected tsutsugamushi disease in July 1987, in Amatsukominato located in the southeastern area of Chiba Prefecture, and showed negative serologic reactions with Rickettsia tsutsugamushi, while the convalescent-phase serum reacted positively with R. montana and a Japanese isolate of spotted fever group rickettsia at IgM and IgG titers of 1:320 and 1:640, respectively, in the indirect immunofluorescence test. These findings showed that a rickettsiosis of the spotted fever group occurred also in Chiba Prefecture like in the southeastern areas of Shikoku and Kyushu islands of Japan.