Serological Evidence of Rickettsia, Orientia, and Coxiella in Domestic Animals from Bhutan: Preliminary Findings.

There is no information on rickettsial diseases in domestic animals in Bhutan. This study provides preliminary serological data on exposure of domestic animals to Rickettsia, Orientia, and Coxiella. Animal sera were collected opportunistically from Bhutan and tested in the Australian Rickettsial Reference Laboratory for IgG antibodies against spotted fever group (SFG) and typhus group (TG) Rickettsia, scrub typhus group (STG), and Q fever (QF). Of the 294 animals tested, 136 (46%) showed serological evidence of past exposure to one or more rickettsiae: 106 (36%), 62 (21%), 45 (15%), and 11 (4%) being positive against SFG Rickettsia, Orientia, TG Rickettsia, and Coxiella, respectively. Dogs appeared to exhibit the highest seropositivity against SFG (55%) and TG Rickettsia (45%), horses against STG (91%), while goats were mostly positive for Coxiella (9%). Dogs also appeared to have high risk of being exposed to SFG Rickettsia (odd ratios [OR] 5.71, 95% confidence interval [CI] 3.02-10.80, p < 0.001), TG Rickettsia (OR 48.74, 95% CI 11.29-210.32, p < 0.001), and STG (OR 6.80, 95% CI 3.32-13.95, p < 0.001), but not against QF (OR 1.95, 95% CI 0.42-8.95, p = 0.390). Differences in seropositivity rates between animal species may have been significant for SFG, TG, and STG, but not for QF. The differences in the seropositivity rates of the four infections between districts appeared to be significant for TG and STG, but not for SFG and QF. The seropositivity rates of domestic animals to the four rickettsial infections were consistent with similar studies on the human population in the same areas and appear to demonstrate a high prevalence of exposure to rickettsiae in Bhutan. These preliminary findings constitute baseline data for Bhutan. The findings of this study call for an increased human-livestock sector collaboration in rickettsial diseases research aimed at developing diagnostic and therapeutic guidelines and formulating preventive and control measures through a One Health approach.

Diversity of Rickettsiae in Feeding and Questing Ticks Collected From a Malaysian Forest Reserve Area.

High seropositivity to Rickettsia conorii and Rickettsia felis has been reported in Malaysian indigenous community living in settlements adjacent to forest areas. The current study was conducted to determine the type and distribution of rickettsiae in feeding and questing ticks that were collected from a forest reserve area at Kuala Lompat in Pahang, Malaysia. Using PCR assays targeting citrate synthase (gltA), outer membrane protein A (ompA) and B (ompB) genes, rickettsiae were detected from approximately one-third of 98 ticks (mainly Dermacentor and Haemaphysalis spp.) collected from the forest reserve. BLAST analysis reveals the predominance of Rickettsia sp. RF2125 in both feeding and questing ticks and Rickettsia sp. TCM1 in the questing ticks. Sequences exhibiting close genetic relationship with Rickettsia raoultii, Rickettsia tamurae, Rickettsia heilongjiangensis, and Rickettsia asiatica were also detected from the ticks. This study highlights the diversity of rickettsial species and potential tick vectors which may contribute to the high seropositivity observed among the local communities.

Identification and Characterization of Orientia chuto in Trombiculid Chigger Mites Collected from Wild Rodents in Kenya.

We present data that concurs with the reported geographical expansion of scrub typhus outside the "Tsutsugamushi Triangle" and addition of Orientia chuto as a second species in the Orientia genus. Wild rodents were caught in Marigat, Baringo County, Kenya, and ectoparasites, including chiggers, were recovered. Rodent and chigger species were identified by taxonomic features. DNA was extracted from the chiggers and used to amplify and/or sequence the 47-kDa high temperature transmembrane protein (TSA47), the 56-kDa type-specific antigen (TSA56), and the 16S rRNA (rrs) Orientia genes. The main rodent hosts identified were Acomys wilsoni, Crocidura sp., and Mastomys natalensis, which accounted for 59.2% of the total collection. Of these, A. wilsoni and M. natalensis harbored most of the chiggers that belonged to the Neotrombicula and Microtrombicula genera. A pool of chiggers from one of M. natalensis was positive for Orientia by TSA47 PCR, but Orientia did not amplify with the TSA56 primers. On sequencing the 850 bp of the TSA47 gene, the closest phylogenetic relative was O. chuto, with 97.65% sequence homology compared to 84.63 to 84.76% for O. tsutsugamushi 16S rRNA deep sequencing also revealed O. chuto as the closest phylogenetic relative, with 99.75% sequence homology. These results and the existing immunological and molecular reports are strongly suggestive of the existence of Orientia species in Kenya.

Metagenomic bacterial community profiles of chicken embryo gastrointestinal tract by using T-RFLP analysis.

Thirty microbial phylotypes of microorganisms were found in the gastrointestinal tract of chicken belonging to the Hajseks White breed, and 38 phylotypes were found in the gastrointestinal tract of chicken belonging to the Hajseks Brown breed. The microbiome of the gastrointestinal tract of the chicken embryos of the Hajseks White breed was dominated by the typical representatives of avian intestinal microflora--bacteria of the family Enterobacteriaceae (47.3%), orders Actinomycetales (13.6%) and Bifidobacteriales (20.6%), and the family Lachnospiraceae (1.1%). The microbiome of the gastrointestinal tract of the chicken embryos of the Hajseks Brown breed was dominated by the pathogenic bacteria of the order Rickettsiales (94.8%). The metagenome of gastrointestinal tract of both breeds also contained a small number of genes of unidentified bacteria.

Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals.

A novel nested PCR assay was developed to detectRickettsiaspp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) ofRickettsiaspp. The newly designed primers were evaluated using genomic DNA from 11Rickettsiaspecies belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to otherRickettsia-specific PCR targets (ompA,gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11Rickettsiaspp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from "CandidatusRickettsia amblyommii." Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adultDermacentor variabilisticks. The nested 23S-5S IGS assay detectedRickettsiaDNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species ofRickettsia The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species ofRickettsiain the ticks. "CandidatusRickettsia amblyommii,"R. montanensis,R. felis, andR. belliiwere frequently identified species, along with some potentially novelRickettsiastrains that were closely related toR. belliiandR. conorii.

Strategies for detecting rickettsiae and diagnosing rickettsial diseases.

Rickettsial diseases and scrub typhus constitute a group of the oldest known vector-borne diseases. The cosmopolitan distribution of the vectors that transmit rickettsiae and orientiae leads to a worldwide prevalence of these diseases. Despite their significant historical status, detection and diagnosis of these diseases are still evolving today. Serological methods remain among the most prevalent techniques used for the detection/diagnosis of rickettsial diseases and scrub typhus. Molecular techniques have been instrumental in increasing the sensitivity/specificity of diagnosis, identifying new Rickettsia and Orientia species and have enhanced epidemiological capabilities when used in combination with serological methods. In this review, we discuss these techniques and their associated pros and cons.

Distribution of rickettsioses in Oceania: past patterns and implications for the future.

Rickettsioses present a threat to human health worldwide, but relatively little is known on their epidemiology and ecology in Oceania. These bacteria are the cause of potentially fatal febrile illnesses in humans (categorized into scrub typhus, typhus group and spotted fever group rickettsioses). They are transmitted by arthropod vectors such as ticks, mites, fleas and lice, which are associated with vertebrate host animals including rodents and companion animals. We conducted a search in the scientific and grey literature of Rickettsia spp. and Orientia tsutsugamushi within the Oceania region. Human case reports, human serosurveys and PCR-based testing of vectors and host animals reviewed here highlight the widespread distribution of these pathogens in the region, with the majority of human serological and vector surveys reporting positive results. These findings suggest that rickettsioses may have a significantly higher burden of disease in Oceania than is currently appreciated due to diagnostic challenges. Furthermore, consideration of the ecology and risk factors for rickettsioses reported for Oceania suggests that their importance as a cause of undifferentiated acute febrile illness may grow in the future: environmental and social changes driven by predicted climate change and population growth have the potential to lead to the emergence of rickettsioses as a significant public health problem in Oceania.

Vectors of rickettsiae in Africa.

Vector-borne diseases are caused by parasites, bacteria, or viruses transmitted by the bites of hematophagous arthropods. In Africa, there has been a recent emergence of new diseases and the re-emergence of existing diseases, usually with changes in disease epidemiology (e.g., geographical distribution, prevalence, and pathogenicity). In Africa, rickettsioses are recognized as important emerging vector-borne infections in humans. Rickettsial diseases are transmitted by different types of arthropods, ticks, fleas, lice, and mites. This review will examine the roles of these different arthropod vectors and their geographical distributions.

Absence of zoonotic Bartonella species in questing ticks: first detection of Bartonella clarridgeiae and Rickettsia felis in cat fleas in the Netherlands.

BACKGROUND: Awareness for flea- and tick-borne infections has grown in recent years and the range of microorganisms associated with these ectoparasites is rising. Bartonella henselae, the causative agent of Cat Scratch Disease, and other Bartonella species have been reported in fleas and ticks. The role of Ixodes ricinus ticks in the natural cycle of Bartonella spp. and the transmission of these bacteria to humans is unclear. Rickettsia spp. have also been reported from as well ticks as also from fleas. However, to date no flea-borne Rickettsia spp. were reported from the Netherlands. Here, the presence of Bartonellaceae and Rickettsiae in ectoparasites was investigated using molecular detection and identification on part of the gltA- and 16S rRNA-genes. RESULTS: The zoonotic Bartonella clarridgeiae and Rickettsia felis were detected for the first time in Dutch cat fleas. B. henselae was found in cat fleas and B. schoenbuchensis in ticks and keds feeding on deer. Two Bartonella species, previously identified in rodents, were found in wild mice and their fleas. However, none of these microorganisms were found in 1719 questing Ixodes ricinus ticks. Notably, the gltA gene amplified from DNA lysates of approximately 10% of the questing nymph and adult ticks was similar to that of an uncultured Bartonella-related species found in other hard tick species. The gltA gene of this Bartonella-related species was also detected in questing larvae for which a 16S rRNA gene PCR also tested positive for "Candidatus Midichloria mitochondrii". The gltA-gene of the Bartonella-related species found in I. ricinus may therefore be from this endosymbiont. CONCLUSIONS: We conclude that the risk of acquiring Cat Scratch Disease or a related bartonellosis from questing ticks in the Netherlands is negligible. On the other hand fleas and deer keds are probable vectors for associated Bartonella species between animals and might also transmit Bartonella spp. to humans.

Isolation of a novel Orientia species (O. chuto sp. nov.) from a patient infected in Dubai.

In July 2006, an Australian tourist returning from Dubai, in the United Arab Emirates (UAE), developed acute scrub typhus. Her signs and symptoms included fever, myalgia, headache, rash, and eschar. Orientia tsutsugamushi serology demonstrated a 4-fold rise in antibody titers in paired serum collections (1:512 to 1:8,192), with the sera reacting strongest against the Gilliam strain antigen. An Orientia species was isolated by the in vitro culture of the patient's acute blood taken prior to antibiotic treatment. The gene sequencing of the 16S rRNA gene (rrs), partial 56-kDa gene, and the full open reading frame 47-kDa gene was performed, and comparisons of this new Orientia sp. isolate to previously characterized strains demonstrated significant sequence diversity. The closest homology to the rrs sequence of the new Orientia sp. isolate was with three strains of O. tsutsugamushi (Ikeda, Kato, and Karp), with a nucleotide sequence similarity of 98.5%. The closest homology to the 47-kDa gene sequence was with O. tsutsugamushi strain Gilliam, with a nucleotide similarity of 82.3%, while the closest homology to the 56-kDa gene sequence was with O. tsutsugamushi strain TA686, with a nucleotide similarity of 53.1%. The molecular divergence and geographically unique origin lead us to believe that this organism should be considered a novel species. Therefore, we have proposed the name "Orientia chuto," and the prototype strain of this species is strain Dubai, named after the location in which the patient was infected.

Rickettsiae in Gulf Coast ticks, Arkansas, USA.

To determine the cause of spotted fever cases in the southern United States, we screened Gulf Coast ticks (Amblyomma maculatum) collected in Arkansas for rickettsiae. Of the screened ticks, 30% had PCR amplicons consistent with Rickettsia parkeri or Candidatus Rickettsia amblyommii.

Detection and identification of rickettsial agents in ticks from domestic mammals in eastern Panama.

Several outbreaks of Rocky Mountain spotted fever have occurred in recent years in Colombian communities close to the border with Panama. However, little is known about rickettsiae and rickettsial diseases in eastern Panamanian provinces, the Darien Province and the Kuna Yala, located north of the endemic area in Colombia. In 2007, 289 ticks were collected in several towns from dogs, horses, mules, cows, and pigs. DNA was extracted from 124 Dermacentor nitens, 64 Rhipicephalus sanguineus, 43 Amblyomma ovale, 35 A. cajennense, 10 Boophilus microplus, 4 A. oblongoguttatum, and 9 A. cajennense nymphs. SYBR-Green polymerase chain reaction assays targeting a fragment of the OmpA and 16S rRNA genes were used for detection of DNA of the spotted fever group rickettsiae (SFGR) and Anaplasmataceae (Anaplasma and Ehrlichia), respectively. In total, 37.4% ticks were positive for SFGR, including 20.3% R. sanguineus, 27.9% A. ovale, 25.8% D. nitens, 50% B. microplus, 50% A. oblongoguttatum, and 100% A. cajennense. The presence of Rickettsia amblyommii DNA was confirmed by sequencing in A. cajennense, A. oblongoguttatum, A. ovale, B. microplus, and R. sanguineus. DNA of R. rickettsii was only detected in one D. nitens collected from a horse in Santa Fe, Darien Province. Prevalence of Anaplasmataceae varied from 6.3% in R. sanguineus to 26.5% in A. cajennense. DNA of Ehrlichia chaffensis was found in three D. nitens and three A. cajennense from horses. This is the first study providing molecular characterization and prevalence information on SFGR in ticks from these areas and thus will be helpful for future evaluations of the risk of rickettsial diseases for individuals living in this region.

Rickettsial infections and their clinical presentations in the Western Province of Sri Lanka: a hospital-based study.

BACKGROUND: Rickettsial infections are re-emerging. A study of the geographical distribution of rickettsial infections, their clinical manifestations, and their complications would facilitate early diagnosis. METHODS: Thirty-one selected patients from the Western Province of Sri Lanka were studied for rickettsial species, clinical manifestations, and complications. RESULTS: Of 31 patients with possible rickettsioses, 29 (94%) fell into the categories of confirmed, presumptive, or exposed cases of acute rickettsial infections (scrub typhus was diagnosed in 19 (66%), spotted fever group in eight (28%)). Early acute infection or past exposure was suggested in two (7%) cases; cross-reactivity of antigens or past exposure to one or more species was suggested in nine (31%). Seventeen out of 19 (89%) patients with scrub typhus had eschars. Nine out of 29 (32%) patients had a discrete erythematous papular rash: seven caused by spotted fever group, two by scrub typhus. Severe complications were pneumonitis in eight (28%), myocarditis in five (17%), deafness in four (14%), and tinnitus in two (7%). The mean duration of illness before onset of complications was 12.0 (SD 1.4) days. All patients except one made a good clinical recovery with doxycycline or a combination of doxycycline and chloramphenicol. CONCLUSIONS: In a region representing the low country wet zone of Sri Lanka, the main rickettsial agent seems to be Orientia tsutsugamushi. Delay in diagnosis may result in complications. All species responded well to current treatment.

Molecular detection of various rickettsiae in mites (acari: trombiculidae) in southern Jeolla Province, Korea.

This study revealed the presence of various rickettsial agents in mites from wild rodents collected in Southern Jeolla Province, Korea, by nested polymerase chain reaction (PCR) and sequence analysis of a partial citrate synthase and rickettsia outer membrane protein B genes. Rickettsial agents closely related to the Rickettsia species TwKM02, R. australis, and the Rickettsia species Cf15 were successfully identified in this study, for the first time in Korea, and R. japonica, R. akari, R. conorii, R. felis, and R. typhi were also detected, as previously described. The data presented in this paper extend knowledge on the geographic distribution of SFG rickettsiae in eastern Asia and it may necessary to consider the role of mites in rickettsial transmission.

Extremely acidophilic protists from acid mine drainage host Rickettsiales-lineage endosymbionts that have intervening sequences in their 16S rRNA genes.

During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name "Candidatus Captivus acidiprotistae." To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat.

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.

Rickettsiella-like bacteria in Ixodes woodi (Acari: Ixodidae).

We examined a parthenogenetic strain of the hard tick Ixodes woodi Bishopp for the presence of endosymbiotic bacteria. Electron microscopic examination revealed the ovarian tissues and Malpighian tubules were infected with pleomorphic bacteria. Two basic types were observed: a larger granular cell and a smaller condensed cell. Cloning and sequence analysis of polymerase chain reaction (PCR) amplified 16S rRNA gene yielded a single sequence from bacteria present in I. woodi tissues. Phylogenetic analysis of the nearly complete 16S rDNA indicated that the ticks were infected with an endosymbiont belonging to the gamma subdivision of the Proteobacteria. It clustered with the insect pathogenic species Rickettsiellagrylli (Vago and Martoja 1963) and the animal pathogen Coxiella burnetii (Derrick 1939) Philip 1948. Our results suggest that the I. woodi females harbored a single endosymbiotic bacterium related to selected Rickettsiella species and to C burnetii.

[Detection of north-Asia tick-borne spotted fever in ticks and rodents along the Heilongjiang river-side by restriction fragment length polymorphism of PCR products].

In order to elucidate the natural foci of North-Asia tick-borne spotted fever along the bank of Heilongjiang river, we used PCR/RFLP to detect spotted fever group rickettsiae in ticks and rodents. The results showed that the wild samples of Dermacentor silvarum, Haemaphysalis concinna and Apodemus agrarius, Microtus fortis, Clethrionomys rufocanus and Ondatra zibethica were all positive with amplification, but typhus rickettsiae, tsutsugamushi fever rickettsiae and Q fever rickettsiae were all negative. Futher RFLP analysis of amplified products with PstI and Rsal demonstrated that their restriction endonuclease profiles were identical to Rickettsia sibirica, but were different from the other prototype strains of SFG rickettsiae, suggesting the possible existance of natural foci of North-Asia tick borne spotted fever in these areas.

Unknown species of rickettsiae isolated from Ixodes ricinus tick in Walcz.

Ticks Ixodes ricinus have been harvested in park down-town Walcz in June 1994. Presence of Borrelia burgdorferi and rickettsiae has been tested with PCR. B. burgdorferi and Coxiella burnetii have not been detected in any tick whereas characteristic for other rickettsiae DNA fragment of gene encoding citrate-synthase has been found. Bacterial strain has been cultured from the tick. Biochemical properties of isolated strain has strongly suggested that these bacteria belong to genus Bartonella (Rochalimaea). Further identification of bacterial DNA with RFLP-PCR (restriction fragment length polymorphism-PCR) has shown characteristics of Bartonella bacilliformis species not recognised in Poland until recently.

Bacteremia due to Rochalimaea henselae in a child: practical identification of isolates in the clinical laboratory.

Two closely related species of Rochalimaea, Rochalimaea quintana and Rochalimaea henselae, are nutritionally fastidious but can be cultivated on bacteriologic media from the blood of patients with diverse clinical presentations. We report a case of culture-proven R. henselae bacteremia in a child with persistent fever. Serologic evidence of infection by R. henselae was ascertained by testing sera at two intervals for immunoglobulin G or immunoglobulin M antibodies by enzyme immunoassay and immunoblot. The case isolate and a collection of other strains (R. henselae, R. quintana, and related organisms) were used to test commercial identification systems for their comparative utility in the identification of Rochalimaea spp. on a practical basis. Of six systems designed for testing of either fastidious or anaerobic isolates of bacteria, the MicroScan Rapid Anaerobe Panel was the only system that distinguished R. henselae from R. quintana. Four of five others gave reactions that were unique within their data bases but did not distinguish Rochalimaea isolates at the species level.