Motohiko Ogawa

Seroprevalence of Coxiella burnetii Infection in Dairy Cattle and Non-symptomatic People for Routine Health Screening in Korea

We report results on the seroprevalence of antibodies to Coxiella burnetii in cattle and healthy people in Korea. Upon agreement with dairy owners, serum samples from 414 dairy cattle were collected between March and June 2001 and samples from 205 people for health screening were collected between April and December 2002. The sera were analyzed for the presence of anti-C. burnetii phase II antibodies using an indirect microimmunofluorescence test; strong fluorescence at a 1:32 dilution was regarded as positive. The overall seroprevalence of C. burnetii in cattle in Korea was 25.6%, with regional variation from 8.9 to 59.3%. Of the positive serum samples, 75.5% had antibody titers ≥1:256. By contrast, only 1.5% of people in a rural area were seropositive, and most of the positive samples had low antibody titers. In conclusion, this study showed that relatively high seropositivity of C. burnetii in dairy cattle, accordingly, the studies on the high-risk groups are needed to evaluate the seroprevalence for this organism in Korea.

Variations of Plasmid Content in Rickettsia felis

Background Since its first detection, characterization of R. felis has been a matter of debate, mostly due to the contamination of an initial R. felis culture by R. typhi. However, the first stable culture of R. felis allowed its precise phenotypic and genotypic characterization, and demonstrated that this species belonged to the spotted fever group rickettsiae. Later, its genome sequence revealed the presence of two forms of the same plasmid, physically confirmed by biological data. In a recent article, Gillespie et al. (PLoS One. 2007;2(3):e266.) used a bioinformatic approach to refute the presence of the second plasmid form, and proposed the creation of a specific phylogenetic group for R. felis. Methodology/Principal Findings In the present report, we, and five independent international laboratories confirmed unambiguously by PCR the presence of two plasmid forms in R. felis strain URRWXCal2 T, but observed that the plasmid content of this species, from none to 2 plasmid forms, may depend on the culture passage history of the studied strain. We also demonstrated that R. felis does not cultivate in Vero cells at 37°C but generates plaques at 30°C. Finally, using a phylogenetic study based on 667 concatenated core genes, we demonstrated the position of R. felis within the spotted fever group. Significance We demonstrated that R. felis, which unambiguously belongs to the spotted fever group rickettsiae, may contain up to two plasmid forms but this plasmid content is unstable.

Diagnostic Assay for Rickettsia japonica

We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project. The target ORF was present only in R. japonica–related strains.

Murine typhus from Vietnam, imported into Japan.

To the Editor: In Vietnam, many febrile diseases such as malaria, dengue fever, Japanese encephalitis, scrub typhus, and more recently, severe acute respiratory syndrome (SARS) and avian influenza have been reported. Murine typhus cases were also reported during and before the 1960s but not thereafter (1–5). On May 3, 2003, a 54-year-old male resident of Tokushima, Japan, had onset of fever in the suburban town of Cu Chi, ≈60 km northwest of Ho Chi Minh City, Vietnam. Exanthema appeared on his trunk and limbs on May 7. He returned to Japan on May 9 and was admitted to Tokushima University Hospital on May 10. His body temperature was 39.0°C, and serum, C-reactive protein level was high (17.06 mg/dL) on admission (day 8 of illness). Unfortunately, the blood sample taken on that day was discarded. We then collected blood on days 10, 11, 12, 14, 17, and 24 of illness for diagnosis. Minocycline was administered on day 8 and resulted in a gradual decrease in fever and rash. Weil-Felix tests on day 12 showed the serum to be positive for Proteus vulgaris OX19 (titer 160); tests for P. vulgaris OX2 and OXK were negative (titer of 10 for both). We examined blood samples for possible diseases such as malaria, dengue fever, SARS, and rickettsioses. Giemsa-stained peripheral blood samples obtained on day 11 showed no malarial parasites. Results of immunoglobulin M (IgM)-capture ELISA of serum on days 10, 11, and 17 of illness were negative for dengue antibodies. Reverse transcription (RT)–PCR of the serum on day 11 was also negative. RT-PCRs of a pharyngeal swab and urine collected on day 11 were both negative for the SARS coronavirus. These specimens were also injected into Vero cells, and no cytopathic effects were generated. RT-PCR of these cultures was also negative for SARS coronavirus. Moreover, SARS antibodies were not found in serum samples on days 11 and 14 of illness. Serum was also tested for Orientia tsutsugamushi and Coxiella burnttii on day 12 to exclude scrub typhus and Q fever as diagnoses. Indirect immunofluorescence tests for etiologic agents of spotted fever, murine typhus, and epidemic typhus were then performed with serum samples collected on days 10, 14, and 24. We used Rickettsia typhi and R. prowazekii as typhus group (TG) rickettsial antigens and R. japonica and R. conorii as spotted fever group (SFG) rickettsiae. IgM antibody was detected for these antigens, indicating that the disease was a primary infection of rickettsiae (Table). When TG and SFG rickettsioses were compared, TG rickettsiae represented markedly higher elevated titers than SFG rickettsiae, which excluded a diagnosis of SFG rickettsiosis. PCR for the TG rickettsial genome in the convalescent-phase serum on day 10 was negative. Table IFA titers of the patient sera and the cross-absorption test* To demonstrate more detailed antigenic reactivity, Western immunoblotting was performed with serum on day 14 (6). The serum reacted similarly to the ladderlike lipopolysaccharide (LPS) of R. typhi and R. prowazekii. As expected from the group-specific nature of rickettsial LPS, no reaction was demonstrated to LPS of SFG rickettsiae, R. japonica and R. conorii, although weak reactivity, mainly to the major outer member protein of SFG rickettiae, rOmpB, and molecules of smaller sizes was shown (6,7). As described previously, rOmpB has cross-reactive antigenicity between TG and SFG rickettsiae (6). Compared with the trace reaction to rOmpB of SFG rickettsiae, an extremely high level of reaction was demonstrated to rOmpB of TG rickettsiae. These results confirmed the disease to be a TG rickettsiosis. To elucidate whether the disease was murine typhus or epidemic typhus, we conducted cross-absorption tests as described previously (8,9). Serum absorbed by R. typhi showed complete absorption, demonstrating no reaction to R. typhi or R. prowazekii (Table). However, the serum absorbed by R. prowazekii resulted in incomplete absorption, demonstrating no reactivity to R. prowazekii but some reactivity to R. typhi, which was left unabsorbed. Western immunoblotting with the serum absorbed by R. prowazekii showed reactivity only to the rOmpB of R. typhi but not to that of R. prowazekii. These results confirmed the diagnosis of murine typhus. This is the first serodiagnosis of murine typhus in Vietnam since the 1960s (1–5). Since rats inhabit the area where the patient acquired the illness, murine typhus seems to have occurred sporadically or endemically but to have been undiagnosed since the 1960s, maybe because it was thought to have been eradicated and thus widely forgotten. This case was the first imported into Japan since the 1940s, when many Japanese soldiers and residents who returned from abroad had the disease.

The intracellular pathogen Orientia tsutsugamushi responsible for scrub typhus induces lipid droplet formation in mouse fibroblasts.

Mammalian cells store excess fatty acids in the form of triglycerides within lipid droplets. The intracellular bacterium Orientia tsutsugamush is the causative agent of severe human rickettiosis. We found that O. tsutsugamushi infection induces the formation of lipid droplets in mouse L-929 fibroblasts. In infected cells, a parallel increase in the number of lipid droplets and pathogens was observed. Interestingly, the pathogen-infection induced the accumulation of triglycerides even without external supply of fatty acids. These results suggest that O. tsutsugamushi alters lipid metabolism of host cells to induce lipid droplets.

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.

Multilocus VNTR analysis-ompA typing of venereal isolates of Chlamydia trachomatis in Japan.

In this study, we investigated the prevalence of genital Chlamydia trachomatis isolated in Japan using a high-resolution genotyping method, the multilocus VNTR analysis (MLVA)-ompA typing method. Seventeen serotypes of C. trachomatis standard strain (A-L3) and 44 clinical isolates were obtained from clinical settings. Genotyping of the ompA gene allowed clinical isolates to be divided into nine serotypes: B (6.8%), D (15.9%), E (25%), F (20.5%), G (18.1%), H (6.8%), Ia (2.3%), J (2.3%), and K (2.3%). These isolates were further divided into 28 types after combining ompA genotyping data with MLVA data (Hunter-Gaston discriminatory index D, 0.949). Thus, our results demonstrated that MLVA could identify clinical isolates that could not be distinguished by ompA typing.

Complete Genomic DNA Sequence of the East Asian Spotted Fever Disease Agent Rickettsia japonica

Rickettsia japonica is an obligate intracellular alphaproteobacteria that causes tick-borne Japanese spotted fever, which has spread throughout East Asia. We determined the complete genomic DNA sequence of R. japonica type strain YH (VR-1363), which consists of 1,283,087 base pairs (bp) and 971 protein-coding genes. Comparison of the genomic DNA sequence of R. japonica with other rickettsiae in the public databases showed that 2 regions (4,323 and 216 bp) were conserved in a very narrow range of Rickettsia species, and the shorter one was inserted in, and disrupted, a preexisting open reading frame (ORF). While it is unknown how the DNA sequences were acquired in R. japonica genomes, it may be a useful signature for the diagnosis of Rickettsia species. Instead of the species-specific inserted DNA sequences, rickettsial genomes contain Rickettsia-specific palindromic elements (RPEs), which are also capable of locating in preexisting ORFs. Precise alignments of protein and DNA sequences involving RPEs showed that when a gene contains an inserted DNA sequence, each rickettsial ortholog carried an inserted DNA sequence at the same locus. The sequence, ATGAC, was shown to be highly frequent and thus characteristic in certain RPEs (RPE-4, RPE-6, and RPE-7). This finding implies that RPE-4, RPE-6, and RPE-7 were derived from a common inserted DNA sequence.

Decontamination of mycoplasma-contaminated Orientia tsutsugamushi strains by repeating passages through cell cultures with antibiotics

BackgroundMycoplasmas-contamination of Orientia tsutsugamushi, one of the obligated intracellular bacteria, is a very serious problem in in vitro studies using cell cultures because mycoplasmas have significant influence on the results of scientific studies. Only a recommended decontamination method is to passage the contaminated O. tsutsugamushi strains through mice to eliminate only mycoplasmas under influence of their immunity. However, this method sometimes does not work especially for low virulent strains of O. tsutsugamushi which are difficult to propagate in mice. In this study, we tried to eliminate mycoplasmas contaminants from both high virulent and low virulent strains of the contaminated O. tsutsugamushi by repeating passage through cell cultures with antibiotics in vitro.ResultsWe cultured a contaminated, high virulent strain of O. tsutsugamushi using a mouse lung fibroblasts cell line, L-929 cell in the culture medium containing lincomycin at various concentrations and repeated passages about every seven days. At the passage 5 only with 10 μg/ml of lincomycin, we did not detect mycoplasmas by two PCR based methods whereas O. tsutsugamushi continued good growth. During following four passages without lincomycin, mycoplasmas did not recover. These results suggested that mycoplasmas were completely eliminated from the high virulent strain of O. tsutsugamushi. Furthermore, by the same procedures with 10 μg/ml of lincomycin, we also eliminated mycoplasmas from a contaminated, low virulent strain of O. tsutsugamushi. Our additional assay showed that 50 μg/ml of lyncomycin did not inhibit the growth of O. tsutsugamushi, although MICs of many mycoplasmas contaminants were less than 6 μg/ml as shown previously.ConclusionOur results showed an alternative method to eliminate mycoplasmas from the contaminated O. tsutsugamushi strains in place of in vivo passage through mice. Especially this notable method works for the decontamination not only from the high virulent strain also from the low virulent strain of O. tsutsugamushi. For further elimination, lincomycin at the limit concentration, which does not inhibit the growth of O. tsutsugamushi, can possibly eliminate most mycoplasmas from contaminated O. tsutsugamushi strains.

Shotgun proteomics of Orientia tsutsugamushi

Orientia tsutsugamushi is one of the obligate intracellular bacteria, and causes Tsutsugamushi disease (scrub typhus). The whole genome sequence of Boryong strain revealed some characteristics of this bacterium, such as a type IV secretion system, many histidine kinases, SpoT, Tra, and ankyrin repeatand tetratricopeptide repeat (TPR)-containing proteins [1]. Additionally, the structure of the genome of O. tsutsugamushi was completely different from those of other rickettsia that have been previously reported. We performed a combination assay of SDS-PAGE and LC-MSMS (GeLC-MSMS) to identify expressed proteins and study the protein profile of this bacterium. The Kuroki strain of O. tsutsugamsuhi, which is very close to the Boryong strain, was propagated in L929 cells and purified by the Renografin density method. About 50 lg of the purified rickettsia was subjected to SDS-PAGE, and the gel was cut into about 40 pieces. Then, the pieces of gel were subjected to in-gel digestion with trypsin or endoproteinase Lys-C. Trypsin digests proteins at the arginine and lysine sites, whereas Lys-C digests only at the lysine site. Finally, the digested peptides were subjected to LC-MSMS analysis (Magic2002 Michrom Biosource Inc., Auburn, CA, USA and LCQ-DECAXP Thermo Fisher Scientific, San Jose, CA, USA). Two different procedures were used, involving different sizes of column (InertsilODS3; GL Science Inc., Shinjuku, Tokyo, Japan) and acetonitrile concentration gradient times: a 0.1 mm · 5 cm column and a 60-min gradient time; and a 0.1 mm · 15 cm column and a 120-min gradient time. For data analysis, proteins were identified using the TurboSequest algorithm in the Bioworks 3.1 package software (Thermo fisher Scientific) and the O. tsutsugamushi database (NCBI, National Center for Biotechnology Information, Bethesda, MD, USA). The criteria for positive identification of peptides were cross-correlation number (Xcorr) >1.0 for singly charged ions, Xcorr >1.5 for doubly charged ions, and Xcorr >2.0 for triply charged ions. Only the best-matched peptides were considered. The strategy using both trypsin and Lys-C for in-gel digestion and two sets of columns and gradient times for LC-MSMS had the great advantage of identifying proteins, especially proteins of lower molecular mass (<50 kDa). One hundred and sixty-five proteins were identified by both trypsin and Lys-C digestion, 68 proteins only by Lys-C, and 351 only by trypsin digestion. Finally, the strategy allowed us to identify 584 of 1152 proteins that are annotated on the genome of O. tsutsugamsuhi (49.4%). The identified proteins included 67.9% (127 ⁄ 187) of essential proteins of the potential minimum bacterium as previously proposed by Gil et al. [2] (Table 1). The identified proteins were classified into COG functional categories [3,4] and compared with those of other rickettsia and bacteria [5]. Comparatively, in O. tsutsugamushi, the category ‘translation’ was higher than the other categories, whereas categories of the entire metabolism were not as high as in other rickettsia and obligate intracellular