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Dermacentor marginatus is a known vector of Rickettsia slovaca and Anaplasma ovis distributed across Eurasia. A total of 300 D. marginatus adult male ticks were collected, removed from sheep in East Azerbaijan province, northwestern Iran. The DNA of Rickettsia and Anaplasma were found in 10 (two pools) ticks by PCR amplifying parts of ompA and msp4 genes, respectively. Our study describes the first PCR detection of tick-borne pathogens, R. slovaca and A. ovis, show that they are spread in D. marginatus of Iran highlight their risk for human and animal hosts. Different patterns (ornamentation) were observed. The identity of different patterns in this study was evaluated with cytb, 16S rRNA and ITS2 genes. BLAST analysis of the cytb, 16S rRNA, ITS2 nucleotide sequence was showed 99%, 98%–100% and 98% sequence identity to sequences of D. marginatus, respectively. It was found that all different patterns of this study are in fact a polymorphic species, D. marginatus. These data increase our knowledge of tick and tick-borne microbial agents in Iran.

Spotted fever group rickettsiae, mainly maintained and transmitted by ticks, are important etiological agents of (re)emerging zoonotic diseases worldwide. It is of great significance to investigate spotted fever group rickettsiae in ticks in different areas for the prevention and control of rickettsioses. In this study, a total of 305 ticks were collected from wild and domestic animals in Chongqing, Guizhou, Yunnan, and Guangxi provinces of southwestern China during 2017–2019 and examined for the presence of spotted fever group rickettsiae by PCR with primers targeting the partial gltA, ompA, rrs, and htrA genes. Results showed that two spotted fever group rickettsiae species, including the pathogenic Candidatus Rickettsia jingxinensis (Rickettsiales: Rickettsiaceae) and a potential novel species Rickettsia sp. sw (Rickettsiales: Rickettsiaceae), were identified. The Ca. R. jingxinensis sequences were recovered from Rhipicephalus microplus (Ixodida: Ixodidae) and Haemaphysalis longicornis (Ixodida: Ixodidae) ticks and phylogenetically clustered with previous Ca. R. jingxinensis, Ca. R. longicornii (Rickettsiales: Rickettsiaceae), and Rickettsia sp. XY118 (Rickettsiales: Rickettsiaceae) strains. Rickettsia sp. sw was detected in Amblyomma geoemydae (Ixodida: Ixodidae) and Rh. microplus. Interestingly, as far as we know, this was the first report of Rickettsia (Rickettsiales: Rickettsiaceae) in A. geoemydae. Phylogenetic analyses indicated that this potential novel species was closely related to R. aeschlimannii (Rickettsiales: Rickettsiaceae) with gltA and ompA genes and grouped in a cluster composed of R. montanensis (Rickettsiales: Rickettsiaceae), R. raoultii (Rickettsiales: Rickettsiaceae), R. aeschlimannii, R. massiliae (Rickettsiales: Rickettsiaceae), and R. rhipicephali (Rickettsiales: Rickettsiaceae) with htrA, while formed a separate clade with rrs. The pathogenicity of Rickettsia sp. sw should be further confirmed. These results expand the knowledge of the geographical distribution and vector distribution of spotted fever group rickettsiae in China and are useful for assessing the potential public health risk.

The present study was carried out to identify Rickettsia species with zoonotic potential in ticks collected from dogs in a rural area in Tabasco, Mexico. In total 197 Amblyomma maculatum ticks were collected from 40 domestic dogs. The collected specimens were pooled and subjected to DNA extraction. A fragment (380 bp) of citrate synthase gene (gltA) was amplified by polymerase chain reaction (PCR) using universal primers for Rickettsia. A second PCR was later performed to amplify a fragment (420 bp) of the outer membrane protein B gene (ompB). The PCR products were purified, sequenced and compared using the basic local alignment search tool (BLAST). Twenty out of 40 (50%) tick pools assayed were positive for rickettsial DNA using both primer pairs. The consensus sequence obtained from the ompB gene fragments showed 99.5-100% of identity with strains of Rickettsia parkeri. This study provides the first molecular evidence of the presence of R. parkeri in A. maculatum ticks infesting domestic dogs from southeastern Mexico. Close contact between dogs and humans should lead to consider the infection caused by this species of Rickettsia among the differential diagnoses for people of Tabasco, Mexico, who show acute febrile syndrome associated to inoculation eschar and have a clinical history of tick exposure.

For the last decade, the New Jersey (NJ) Department of Health has reported between 42 and 144 new cases each year of “spotted fever group rickettsiosis” (SFGR), a statistic that reflects uncertainty regarding which rickettsial agents (Proteobacteria: Rickettsiaceae: Rickettsia) are infecting NJ residents. To identify the Rickettsia circulating in NJ ticks, we used a combination of conventional and real time PCR approaches to screen 560 Dermacentor variabilis Say and 245 Amblyomma americanum L. obtained from a 1-day state-wide surveillance in May 2018 and an additional 394 D. variabilis collected across NJ in 2013–2018. We found zero D. variabilis infected with Rickettsia rickettsii, the agent of Rocky Mountain spotted fever and, on average, 1.3% infected with presumed nonpathogenic Rickettsia montanensis. We also found zero A. americanum infected with R. rickettsii, and 20% infected with Rickettsia amblyommatis, a prevalence somewhat lower than in more southern states. Overall, we conclude that it is unlikely that R. rickettsii vectored by D. variabilis is a primary cause of SFGR cases in NJ and discuss our findings in the context of known facts and current limitations. We conclude that understanding the causes of SFGR east of the Mississippi will require collaboration among medical doctors, public health authorities, and medical entomologists to follow up presumptive human cases of SFGR with detailed histories of exposure, species-specific molecular assays, and active surveillance of putative vectors and the pathogens they may carry.

Amblyomma maculatum Koch sensu lato (s.l.) ticks are the vector of Rickettsia parkeri in Arizona, where nine cases of R. parkeri rickettsiosis have been identified since the initial case in 2014. The current study sought to better define the geographic ranges of the vector and pathogen and to assess the potential public health risk posed by R. parkeri in this region of the southwestern United States. A total of 275 A. maculatum s.l. ticks were collected from 34 locations in four counties in Arizona and one county in New Mexico and screened for DNA of Rickettsia species. Rickettsia parkeri was detected in 20.4% of the ticks, including one specimen collected from New Mexico, the first report of R. parkeri in A. maculatum s.l. from this state. This work demonstrates a broader distribution of A. maculatum s.l. ticks and R. parkeri in the southwestern United States than appreciated previously to suggest that R. parkeri rickettsiosis is underrecognized in this region.

Rickettsia parkeri, a tick-borne pathogen distributed throughout several countries of the Americas, causes a mild to moderately severe, eschar-associated spotted fever rickettsiosis. Although most U.S. cases of R. parkeri rickettsiosis are reported from southeastern states, some have been reported recently from remote regions of southern Arizona. These cases are linked to R. parkeri-infected ticks of the Amblyomma maculatum (Acari: Ixodidae) group found in several isolated mountain ranges of southern Arizona and New Mexico, referred to as ‘sky islands’. Archival records also document ticks of the A. maculatum group collected from domestic and wild animals in West Texas. We surveyed sites in two sky island chains of Jeff Davis and Brewster counties to document the off-host occurrence of these ticks and identify the presence of R. parkeri in the Trans-Pecos region of Texas. During August 2019, 43 adult A. maculatum group ticks were flagged from vegetation or removed from a road-killed, female mule deer. Of 39 samples evaluated by PCR, eight contained a partial sca0 sequence with complete identity to R. parkeri and two with complete identity to ‘Candidatus Rickettsia andeanae’, a species of undetermined pathogenicity. Four isolates of R. parkeri were obtained using cell culture. Persons at risk for R. parkeri rickettsiosis include those who work or recreate in these mountains, such as hikers, backpackers, research scientists, foresters, and border enforcement personnel. Additional investigations are needed to define the distribution of these medically important arthropods in other parts of the southwestern United States and northern Mexico.

Amblyomma maculatum Koch is the primary vector of Rickettsia parkeri, the etiologic agent of tidewater spotted fever, and can also carry and transmit a variety of other pathogens. This tick historically has been a costly nuisance to livestock owners in the southeastern United States. Over the past 6 yr, A. maculatum has been collected in numbers sufficient to demonstrate their establishment in Kent County, Delaware, and the presence of R. parkeri has been documented. Our goals were to determine the geographic distribution of A. maculatum and R. parkeri in Delaware, and to equate this to relative risk to the public of encountering R. parkeri-infected ticks. We surveyed for ticks in four locations throughout the state from May to August 2019, and found established A. maculatum populations in all three counties. Laboratory analysis of collected specimens by quantitative polymerase chain reaction detected R. parkeri in A. maculatum populations across the state. These results indicate that A. maculatum could present a health risk to inhabitants of the state, and they also historically have posed a risk to the livestock industry, making them an important consideration in the development and implementation of continued tick surveillance efforts and future policies regarding tick management.

The western black-legged tick (Ixodes pacificus) is the most frequently identified human-biting tick species in the western United States and the principal vector of at least three recognized bacterial pathogens of humans. A potentially pathogenic Rickettsia species, first described in 1978 and recently characterized as a novel transitional group agent designated as Rickettsia tillamookensis, also exists among populations of I. pacificus, although the distribution and frequency of this agent are poorly known. We evaluated DNA extracts from 348 host-seeking I. pacificus nymphs collected from 9 locations in five California counties, and from 916 I. pacificus adults collected from 24 locations in 13 counties, by using a real-time PCR designed specifically to detect DNA of R. tillamookensis. DNA of R. tillamookensis was detected in 10 (2.9%) nymphs (95% CI: 1.6–5.2%) and 17 (1.9%) adults (95% CI: 1.2–3.0%) from 11 counties of northern California. Although site-specific infection rates varied greatly, frequencies of infection remained consistently low when aggregated by stage, sex, habitat type, or geographical region. Four novel isolates of R. tillamookensis were cultivated in Vero E6 cells from individual adult ticks collected from Alameda, Nevada, and Yolo counties. Four historical isolates, serotyped previously as ‘Tillamook-like’ strains over 40 yr ago, were revived from long-term storage in liquid nitrogen and confirmed subsequently by molecular methods as isolates of R. tillamookensis. The potential public health impact of R. tillamookensis requires further investigation.

A total of 482 bats representing 32 species and two families were captured in the Amazon forests of the Amapá state in northern Brazil. Nineteen Artibeus planirostris bats (3.9 %) were infested with 160 ticks, all identified as Ornithodoros hasei. Three pools of larvae were screened for rickettsial DNA via polymerase chain reaction (PCR) targeting three rickettsial genes: gltA, ompA and htrA. Only one of them yielded an amplicons of the expected size for all three molecular assays. Comparisons of the obtained sequences including a phylogenetic analysis confirmed the occurrence of "Candidatus Rickettsia wissemanii" in Brazil.

Rickettsia monacensis and Orientia tsutsugamushi are bacteria of the family Rickettsiaceae, which causes fever, rash, and eschar formation; outdoor activities are a risk factor for Rickettsiaceae infection. A 75-year-old woman presented with fever, rash, and eschar and was confirmed as being scrub typhus based on a nested-polymerase chain reaction (N-PCR) test for a 56-kDa gene of O. tsutsugamushi; the genome was identified as the Boryong genotype. In addition, a pan-Rickettsia real-time PCR test was positive and a N-PCR test using a Rickettsia-specific partial outer membrane protein A (rOmpA) confirmed R. monacensis. This is the first case wherein a patient suspected of having scrub typhus owing to the presence of rash and eschar was also found to be coinfected with O. tsutsugamushi and R. monacensis based on molecular testing.

Close reciprocal interactions in symbiotic systems have suggested the holobiont concept, in which the host and its microbiota are considered as a single entity. Ciliates are known for their ability to form symbiotic associations with prokaryotes. Relationships between the partners in such systems vary from mutualism to parasitism and differ significantly in their robustness. We assessed the viability of the ciliate Paramecium multimicronucleatum and its ability to maintain its intranuclear endosymbiont Ca. Trichorickettsia mobilis (Rickettsiaceae) after treatment with antibiotics characterized by different mode of action, such as ampicillin, streptomycin, chloramphenicol, tetracycline. The presence of endosymbionts in the host cell was determined by means of living cell observations made using differential interference contrast or fluorescence in situ hybridization with the species-specific oligonucleotide probe (FISH). Administration of antibiotics traditionally used in treatments of rickettsioses, tetracycline and chloramphenicol, depending on the concentration used and the ciliate strain treated, either caused death of both, infected and control cells, or did not affect the ability of the host to maintain the intranuclear endosymbiont. The surviving cells always manifested motile bacteria in the macronucleus. Streptomycin treatment never led to the loss of endosymbionts in any of the four infected strains, and nearly all ciliates remained viable. Ampicillin treatment never caused host cell death, but resulted in formation of filamentous and immobile oval bacterial forms. Under repeated ampicillin treatments, a part of endosymbionts was registered in the host cytoplasm, as evidenced both by FISH and transmission electron microscopy. Endosymbionts located in the host cytoplasm were enclosed in vacuoles, apparently, corresponding to autophagosomes. Nevertheless, the bacteria seemed to persist in this compartment and might cause relapse of the infection. Although the antibiotic sensitivity profile of Trichorickettsia seems to resemble that of other representatives of Rickettsiaceae, causative agents of severe diseases in humans, neither of the antibiotic treatments used in this study resulted in an aposymbiotic cell line, apparently, due to the protists’ sensitivity to tetracyclines, the drugs of preference in rickettsiosis treatment. The observed robustness of this symbiotic system makes it a good model for further elaboration of the holobiont concept.

We identified an established population of the Gulf Coast tick (Amblyomma maculatum Koch) infected with Rickettsia parkeri in Connecticut, representing the northernmost range limit of this medically relevant tick species. Our finding highlights the importance of tick surveillance and public health challenges posed by geographic expansion of tick vectors and their pathogens.

Over the last decades, rickettsioses are emerging worldwide. These diseases are caused by intracellular bacteria. Although rickettsioses can be treated with antibiotics, a vaccine against rickettsiae is highly desired for several reasons. Rickettsioses are highly prevalent, especially in poor countries, and there are indications of the development of antibiotic resistance. In addition, some rickettsiae can persist and cause recurrent disease. The development of a vaccine requires the understanding of the immune mechanisms that are involved in protection as well as in immunopathology. Knowledge about these immune responses is accumulating, and efforts have been undertaken to identify antigenic components of rickettsiae that may be useful as a vaccine. This review provides an overview on current knowledge of adaptive immunity against rickettsiae, which is essential for defense, rickettsial antigens that have been identified so far, and on vaccination strategies that have been used in animal models of rickettsial infections.

All species of hard ticks associated with reptiles as hosts throughout their life cycle, are currently assigned to genera including Amblyomma and Africaniella. Among these species, based on literature data, Africaniella transversale has never been investigated for the presence of tick-borne pathogens. In this study, seven DNA extracts (two from A. transversale and five from Amblyomma exornatum) were screened for the presence of important tick-borne protozoa (piroplasms) and bacteria (Anaplasmataceae and Rickettsiaceae) with conventional PCRs and sequencing. A new heat shock protein chaperonin (groEL) gene-specific PCR was also developed to identify Occidentia spp. in these samples. In A. transversale, Occidentia massiliensis (previously detected in rodent-associated soft ticks) and Rickettsia hoogstraalii were present. While the latter was molecularly identical with formerly reported sequences of this rickettsia, the genotype of O. massiliensis was new based on sequence and phylogenetic analyses of its groEL gene. In A. exornatum, a Rickettsia genotype closely related to R. tamurae and R. monacensis, was detected. The ompA sequence of this genotype was identical to that of Rickettsia sp. Ae-8 reported from A. exornatum in a reptile breeding facility in the USA. These results show that A. transversale might carry O. massiliensis which (unless having a symbiotic nature in ticks) may originate either from the reptile host of this hard tick species or the rodent prey of reptiles. This is also the first detection of the reptile tick-associated Rickettsia sp. Ae-8 (phylogenetically aligning with R. tamurae, R. monacensis) in Africa, i.e. within the original geographical range of A. exornatum.

The bacterium Rickettsia rickettsii is the agent of Brazilian spotted fever (BSF), a highly fatal disease that is transmitted in Brazil mainly by the tick Amblyomma sculptum Berlese, which uses capybaras (Hydrochoerus hydrochaeris Linnaeus) as major hosts. In 2015, we captured nine capybaras in a BSF-endemic area of southeastern Brazil. From each capybara, we collected blood sera that were tested through the immunofluorescence assay using Rickettsia spp. antigens, and A. sculptum ticks, processed for isolation of R. rickettsii through guinea pig inoculation. All capybaras (100%) were seroreactive to Rickettsia spp., with highest titers to R. rickettsii. A total of 166 A. sculptum ticks were macerated and inoculated into nine guinea pigs, from which only one presented high fever and seroconverted to R. rickettsii. Blood from this febrile animal was inoculated into additional guinea pigs (guinea pig passages), which also became febrile and seroconverted, or when euthanized during the fever period, their internal organs (spleen, lung) were shown to contain R. rickettsii DNA. The present rickettsial isolate has been maintained cryopreserved as infected guinea pig organs. There was at least one R. rickettsii-infected tick among the 166 macerated ticks, giving a minimal infection rate of 0.6% (1/166). This infection rate is within the range of previous studies, which reported that only 0.05% to at most 1.28% A. sculptum ticks were infected by R. rickettsii in other BSF-endemic areas. These low infection rates support the low incidence of BSF, despite of A. sculptum being the most frequent human-biting tick in southeastern Brazil.

Haemaphysalis longicornis, the cattle tick or bush tick, has an extended distribution throughout Asia and the Pacific region, including China, Russia, the Republic of Korea (ROK), Japan, Australia, New Zealand, and the South Pacific islands. It is an obligate ectoparasite found commonly on medium to large sized wild and domestic animals, with humans as an accidental host. Haemaphysalis longicornis transmits a number of pathogens, including severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses, bacteria, helminths, and protozoans, that impact on veterinary (wild and domestic animals) and human health. Surveys of rickettsial pathogens associated with H. longicornis from China, the ROK, and Japan have resulted in the discovery of more than 35 incompletely characterized molecular isolates of Rickettsia. In response to the increased global threat of tick-borne rickettsial diseases, H. longicornis collected in the ROK and China were assessed in our laboratory and two additional Rickettsia spp. isolates (ROK-HL727 and XinXian HL9) were identified. These agents were fully characterized by multilocus sequence typing using partial gene fragment sequences of rrs, gltA, ompA, ompB, and sca4. Phylogenetic comparisons of these Rickettsia isolates with known Rickettsia species and other molecular isolates identified from H. longicornis were performed to better understand their interrelationships. Phylogenetic analysis of the sequences from these 5 gene fragments showed that ROK-HL727 was closely related to rickettsial isolates of H. longicornis previously reported from China, the ROK and Japan, but distinct from any currently recognized Rickettsia species. It therefore qualifies genetically as a new species, introduced herein as Candidatus Rickettsia longicornii. The XinXian-HL9 isolate detected from China was determined to be genetically similar to the human pathogen Rickettsia heilongjiangensis. People living and working in areas where H. longicornis is endemic should be aware of the potential for rickettsial diseases.

Rickettsia parkeri is an emerging human pathogen transmitted by Amblyomma ticks in predominately tropical and subtropical regions of the western hemisphere. In 2014 and 2015, one confirmed case and one probable case of R. parkeri rickettsiosis were reported from the Pajarita Wilderness Area, a semi-arid mountainous region in southern Arizona. To examine more closely the potential public health risk of R. parkeri in this region, a study was initiated to investigate the pervasiveness of Amblyomma maculatum Koch group ticks in mountainous areas of southern Arizona and to ascertain the infection frequencies of R. parkeri in these ticks. During July 2016, a total of 182 adult ticks were collected and evaluated from the Pajarita Wilderness Area in Santa Cruz County and two additional sites in Cochise and Santa Cruz counties in southern Arizona. DNA of R. parkeri was detected in a total of 44 (24%) of these ticks. DNA of "Candidatus Rickettsia andeanae" and Rickettsia rhipicephali was detected in three (2%) and one (0.5%) of the samples, respectively. These observations corroborate previous collection records and indicate that established populations of A. maculatum group ticks exist in multiple foci in southern Arizona. The high frequency of R. parkeri in these tick populations suggests a public health risk as well as the need to increase education of R. parkeri rickettsiosis for those residing, working in, or visiting this area.

It has been reported that starving ticks do not transmit spotted fever group Rickettsia immediately upon attachment because pathogenic bacteria exist in a dormant, uninfectious state and require time for ‘reactivation’ before transmission to a susceptible host. To clarify the length of reactivation period, we exposed guinea pigs to bites of Rickettsia rickettsii-infected Dermacentor variabilis (Say) and allowed ticks to remain attached for predetermined time periods from 0 to 48 h. Following removal of attached ticks, salivary glands were immediately tested by PCR, while guinea pigs were observed for 10–12 d post-exposure. Guinea pigs in a control group were subcutaneously inoculated with salivary glands from unfed D. variabilis from the same cohort. In a parallel experiment, skin at the location of tick bite was also excised at the time of tick removal to ascertain dissemination of pathogen from the inoculation site. Animals in every exposure group developed clinical and pathological signs of infection. The severity of rickettsial infection in animals increased with the length of tick attachment, but even attachments for less than 8 h resulted in clinically identifiable infection in some guinea pigs. Guinea pigs inoculated with salivary glands from unfed ticks also became severely ill. Results of our study indicate that R. rickettsii residing in salivary glands of unfed questing ticks does not necessarily require a period of reactivation to precede the salivary transmission and ticks can transmit infectious Rickettsia virtually as soon as they attach to the host.

Tick-borne rickettsiae are undergoing epidemiological changes in the eastern United States while human encounters with lone star ticks (Amblyomma americanum L.) have increased substantially. We used real-time polymerase chain reaction assays to test for three species of spotted fever group rickettsiae (SFGR) (Rickettsiales: Rickettsiaceae) in 1,858 nymphal A. americanum collected from Monmouth County, New Jersey, a coastal county with endemic Lyme disease and established tick surveillance. Out of the 1,858 tested, 465 (25.0%) were infected with Rickettsia amblyommatis Karpathy, a species of undetermined pathogenicity found frequently in A. americanum, while 1/1,858 (0.05%) contained Rickettsia rickettsii Brumpt, the agent of Rocky Mountain spotted fever. No ticks tested positive for mildly pathogenic Rickettsia parkeri Lackman, and no ticks were co-infected with multiple Rickettsia spp. Our results indicate that A. americanum could be involved in transmission of R. rickettsii to humans in New Jersey, albeit rarely. The much higher rates of R. amblyommatis infection are consistent with hypotheses that human sera reacting to this species could contribute to reports of mild SFGR cases.

The importance of ticks in veterinary and medical science has received much attention. The dominant tick species in northeastern China, Ixodes persulcatus tick can transmit various pathogens to humans and animals and there are some studies on the microbiome composition of this tick. Our study characterized the bacterial communities in I. persulcatus by 16S amplicon pyrosequencing and described the differences of microorganisms in male and female tick and assessed the variation of microorganisms in the development stages in northeastern China. We mainly found the following bacteria genera: Pseudomonas (Pseudomonadales: Pseudomonadaceae), Citrobacter (Enterobacteriales: Enterobacteriaceae), and Rickettsia (Rickettsiales: Rickettsiaceae). Rickettsia is common and harmful pathogen transmitted by ticks. Meanwhile, we found there were differences between male and female tick of microbiomes, and the diversity of microbiome increased from engorged female ticks to eggs, but decreased when the eggs were molting into larvae. Our data showed that male ticks exhibited greater microbial diversity than female I. persulcatus tick and larvae presented with a different bacterial community compared to engorged female tick and hatched eggs. These findings may be useful for further understanding the interaction between I. persulcatus and microbiome biology.

Rickettsia typhi and Rickettsia felis (Rickettsiales: Rickettsiaceae) are flea-transmitted pathogens. They are important causes of acute febrile illness throughout the world. We, therefore, sought to identify the rickettsial species present in the fleas of dogs and cats in the department of Cauca, Colombia. In this study, we collected 1,242 fleas from 132 dogs and 43 fleas from 11 cats. All fleas were morphologically identified as Ctenocephalides felis (Bouché) adults and organized in pools for DNA extraction (234 pools from dogs and 11 from cats). The gltA gene from rickettsiae was targeted for screening amplification using conventional PCR. In total, 144 of the 245 pools (58.7%) were positive. The positive samples were then processed for the amplification of the 17kDa antigen gene (144/144; 100% positive) and sca5 gene (140/144; 97.2% positive). In addition, restriction enzyme length polymorphism analysis using NlaIV on the amplified product of the sca5 gene demonstrated several organisms: 21/140 (15%) were R. felis, 118/140 (84.3%) were Rickettsia asemboensis, and 1/140 (0.7%) were Candidatus Rickettsia senegalensis. Subsequent sequencing confirmed Candidatus Rickettsia senegalensis in C. felis collected from dogs the first reported from Colombia.

Ixodes lividus Koch, 1844 (Ixodida: Ixodidae) ticks are parasites specific to the sand martin (Riparia riparia). The distribution range of I. lividus covers Europe and Asia and they are found wherever sand martins nest. Borrelia burgdorferi sensu stricto, Borrelia garinii and Rickettsia vini have recently been detected in I. lividus ticks in Central Europe. The aim of this study was to investigate the presence of Rickettsia spp. (Rickettsiales: Rickettsiaceae) and Borrelia spp. (Spirochaetales: Spirochaetaceae) in I. lividus ticks in Lithuania. A total of 2622 non-engorged I. lividus from two different colonies comprising 40 sand martin nests were collected in Lithuania in 2013 and 2015. Rickettsial DNA was detected in 106 of the 117 pooled larvae samples, in all examined seven nymphs, four females and one male specimen. Sequence analysis of partial gltA, ompA and htrA genes showed 99-100 % identity with the corresponding R. vini sequences deposited in GenBank. None of I. lividus tested was infected with Borrelia burgdorferi sensu lato. This is the first time that the presence of R. vini has been reported in Lithuania.

Tick-borne rickettsiosis is becoming a common emergent disease in many tropical countries, and the rapid detection of this disease could reduce the impact on wild life and public health. Pools of larvae and nymphs, and individual adult ticks are used for detection of Rickettsia species (Rickettsiaceae), but the number of larvae and nymphs in the pools likely affect the probability of detection of Rickettsia spp. We tested whether the number of larvae or nymphs of Amblyomma (Ixodidae) included in the pools affected the probability of detection. The number of larvae in each pool did not affect the probability of detecting Rickettsia spp., but the probability of detection increased rapidly with the number of nymphs. In this study, 20 nymphs are enough to approach a probability of 1 of detecting Rickettsia La rickettsiosis transmitida por garrapatas es una enfermedad emergente cada vez más común en muchos países tropicales, por lo que su rápida detección podría reducir su impacto negativo en la salud humana. Grupos (pozos) de larvas y ninfas de garrapatas, así como individuos adultos son usados para detectar la presencia de diferentes especies de Rickettsia. Sin embargo, el número de larvas y ninfas incluidos en estos grupos probablemente afecta la detección de Rickettsia spp. Nosotros probamos si el número de larvas o ninfas incluidas en estos grupos afecta la probabilidad de detección. Encontramos que el número de larvas incluidas en cada grupo no afectó la probabilidad de detección de Rickettsia spp., pero la probabilidad de detección aumenta rápidamente con el número de ninfas incluidas en los grupos. En este estudio, 20 ninfas por grupo fueron suficientes para tener una probabilidad cercana a 1 de detectar Rickettsia

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.

Rickettsioses are caused by intracellular bacteria of the family of Rickettsiaceae. Rickettsia (R.) typhi is the causative agent of endemic typhus. The disease occurs worldwide and is one of the most prevalent rickettsioses. Rickettsial diseases, however, are generally underdiagnosed which is mainly due to the lack of sensitive and specific methods. In addition, methods for quantitative detection of the bacteria for research purposes are rare. We established two qPCRs for the detection of R. typhi by amplification of the outer membrane protein B (ompB) and parvulin-type PPIase (prsA) genes. Both qPCRs are specific and exclusively recognize R. typhi but no other rickettsiae including the closest relative, R. prowazekii. The prsA-based qPCR revealed to be much more sensitive than the amplification of ompB and provided highly reproducible results in the detection of R. typhi in organs of infected mice. Furthermore, as a nested PCR the prsA qPCR was applicable for the detection of R. typhi in human blood samples. Collectively, the prsA-based qPCR represents a reliable method for the quantitative detection of R. typhi for research purposes and is a promising candidate for differential diagnosis.

Rickettsioses are re-emerging vector-borne zoonoses with a global distribution. Recently, Rickettsia sp. strain Atlantic rainforest has been associated with new human spotted-fever (SF) cases in Brazil, featuring particular clinical signs: eschar formation and lymphadenopathy. These cases have been associated with the tick species, Amblyomma ovale From 2010 until 2015, the Brazilian Health Department confirmed 11 human SF cases in the Maciço de Baturité region, Ceará, Brazil. The present study reports the circulation of Rickettsia spp. in vectors from this entirely new endemic area for SF. A total of 1,727 ectoparasites were collected in this area from the environment, humans, and wild and domestic animals. Samples (n = 887) were screened by polymerase chain reaction (PCR), targeting the gltA and ompA rickettsial genes. Sequencing and phylogenetic analyses of gltA gene amplicons were carried out for 13 samples positive for both screening PCRs. Fragments of gltA and ompA from three samples were cloned, sequenced, and analyzed further. A. ovale and Rhipicephalus sanguineus specimens, collected from dogs, were found to be infected with Rickettsia sp. str. Atlantic rainforest, suggesting the importance of dogs in the epidemic cycle. Candidatus Rickettsia andeanae, Rickettsia felis, and Rickettsia bellii were also found infecting ticks and fleas in five municipalities, demonstrating the broad diversity of rickettsiae in circulation in the studied area. This study reports, for the first time, evidence of infection with Rickettsia sp. strain Atlantic rainforest in A. ovale and R. sanguineus in Ceará, and Ca. R. andeanae in an Atlantic rainforest environment of Brazil.

Rickettsia parkeri Luckman (Rickettsiales: Rickettsiaceae) is a pathogenic spotted fever group Rickettsia transmitted by Amblyomma maculatum Koch (Acari: Ixodidae) in the United States. The acute innate immune response to this pathogen and the effect of tick feeding or salivary components on this response is largely unknown. We hypothesized that A. maculatum saliva enhances R. parkeri infection via downregulation of the acute cellular and cytokine immune response. C3H/HeN mice were intradermally inoculated with R. parkeri both with and without A. maculatum saliva. Flow cytometry and microscopic evaluation of inoculation site skin suspensions revealed that neutrophils and macrophages predominated at 6 and 24 h post R. parkeri inoculation, respectively. This cellular influx was significantly downregulated when A. maculatum saliva was inoculated along with R. parkeri . Inflammatory cytokines (interferon γ and interleukins 6 and 10) were significantly elevated after R. parkeri inoculation. However, cytokine concentration and rickettsial load were not significantly modified by A. maculatum saliva during the acute phase of infection. These results revealed that tick saliva inhibits the cutaneous cellular influx during the acute phase of rickettsial infection. Further study is needed to determine the overall impact of this effect on the establishment of rickettsiosis in the host and development of disease.

Bacterial endosymbionts, particularly Wolbachia (Rickettsiales: Rickettsiaceae), Rickettsia (Rickettsiales: Rickettsiaceae), and Cardinium (Bacteroidales: Bacteroidaceae), are commonly found in several arthropod groups, including insects. Most estimates of the global infection rate of Wolbachia (52% [95% credible intervals: 44-60]) show that these bacteria infect more than half of all insect species. Other endosymbionts, such as Rickettsia (24% [confidence intervals [CIs] 20-42]) and Cardinium (13% [CIs 13-55]), infect a smaller but still substantial proportion of insect species. In spite of these observations, it is unclear what proportion of individuals within those species are infected. Here, we used published databases to estimate the proportion of individuals that are infected with either Wolbachia, Rickettsia, or Cardinium. We found that the majority (69%) of Wolbachia-infected species have less than half of their individuals infected with Wolbachia, indicating that although the bacterium may be common among species, it is not common within species. The same was true for Rickettsia (81%) and Cardinium (87%). This discrepancy was consistent across orders, in which less than 10% of individuals were typically infected, even though more than 50% of species within orders were infected. For example, according to our model, nearly 50% of beetle (Coleoptera) species are infected with Wolbachia (i.e., contain at least one individual that has tested positive for Wolbachia), but less than 5% of all individuals are infected. These results add to the growing knowledge base about endosymbionts in insects and should guide future sampling efforts and investigations on the role that these bacteria play in populations.

Background Pathogen colonization inside tick tissues is a significant aspect of the overall competence of a vector. Amblyomma maculatum is a competent vector of the spotted fever group rickettsiae, Rickettsia parkeri. When R. parkeri colonizes its tick host, it has the opportunity to dynamically interact with not just its host but with the endosymbionts living within it, and this enables it to modulate the tick’s defenses by regulating tick gene expression. The microbiome in A. maculatum is dominated by two endosymbiont microbes: a Francisella-like endosymbiont (FLE) and Candidatus Midichloria mitochondrii (CMM). A range of selenium-containing proteins (selenoproteins) in A. maculatum ticks protects them from oxidative stress during blood feeding and pathogen infections. Here, we investigated rickettsial multiplication in the presence of tick endosymbionts and characterized the functional significance of selenoproteins during R. parkeri replication in the tick. Results FLE and CMM were quantified throughout the tick life stages by quantitative PCR in R. parkeri-infected and uninfected ticks. R. parkeri infection was found to decrease the FLE numbers but CMM thrived across the tick life cycle. Our qRT-PCR analysis indicated that the transcripts of genes with functions related to redox (selenogenes) were upregulated in ticks infected with R. parkeri. Three differentially expressed proteins, selenoprotein M, selenoprotein O, and selenoprotein S were silenced to examine their functional significance during rickettsial replication within the tick tissues. Gene silencing of the target genes was found to impair R. parkeri colonization in the tick vector. Knockdown of the selenogenes triggered a compensatory response from other selenogenes, as observed by changes in gene expression, but oxidative stress levels and endoplasmic reticulum stress inside the ticks were also found to have heightened. Conclusions This study illustrates the potential of this new research model for augmenting our understanding of the pathogen interactions occurring within tick hosts and the important roles that symbionts and various tick factors play in regulating pathogen growth. Electronic supplementary material The online version of this article (10.1186/s40168-018-0524-2) contains supplementary material, which is available to authorized users.

Rickettsia felis (Rickettsiales: Rickettsiaceae) is an emergent human pathogen that causes febrile illnesses in various parts of the world. This study describes the identification and growth characteristics of a R. felis-like organism (designated as Rickettsia sp. TH2014) cultured from Ctenocephalides orientis fleas in rural Malaysia. In this study, culturing of rickettsiae from filtered triturated flea lysates was performed in Aedes albopictus C6/36 cells. Cytopathic effects were observed from one of the samples 4 d post-inoculation. Electron microscopy revealed actively replicating intracytosolic coccobacillary organisms in the rickettsia-infected cells. Sequence analysis of amplified citrate synthase (gltA) gene fragment shows complete match of the rickettsia with Rickettsia sp. Rf31 in Southeast Asia, and 'Candidatus Rickettsia senegalensis' strain PU01-02 in Africa. The whole-genome sequence of Rickettsia sp. TH2014 was determined and assembled. The estimated genome size and guanine + cytosine content of the rickettsia are 1.37 Mb and 32.9%, respectively. The high values of average nucleotide identity and tetra-nucleotide signature correlation index obtained from pairwise genome comparison study suggest the identification of the rickettsia as R. felis. The whole-genome single-nucleotide polymorphism analysis demonstrates close genetic relatedness of the rickettsia with R. felis and Rickettsia asemboensis. However, based on sequence analyses of rickettsial genes (16S rDNA, gltA, ompB, and sca4), Rickettsia sp. TH2014 is found to be distinct from R. felis and R. asemboensis. The sequence analyses reveal that Rickettsia sp. TH2014 is highly similar to 'Ca. Rickettsia senegalensis' detected in fleas from Africa, Asia, and North America. Further investigation to provide insights on pathogenic potential and transmission dynamics of the rickettsia is warranted.

Espírito Santo state (southeastern Brazil) is considered an endemic area for spotted fever group rickettsioses. In February 2017, we received in our laboratory seven unfed Amblyomma ovale adult ticks collected by a farmer from his clothes and body (not attached) during a working day in the rural area of Ibiraçu municipality, Espírito Santo state. By polymerase chain reaction (PCR) analyses, targeting gltA and ompA rickettsial genes, the DNA of Rickettsia was detected in 6/7 (85.7%) A. ovale. In all cases, DNA sequencing of PCR products revealed that consensus sequences of both genes were 100% identical to gltA and ompA corresponding sequences of Rickettsia sp. strain Atlantic rainforest retrieved from GenBank. This study reports the first molecular detection of Rickettsia sp. strain Atlantic rainforest in A. ovale ticks from Espírito Santo state. Our findings indicate a new Brazilian state in the southeast region at risk of human infection with this tick-borne emerging rickettsial agent.

Bacteria of Rickettsiaceae and Anaplasmataceae families include disease agents spread by Ixodes ricinus ticks, the most common tick vector in Europe. The aim of the study was to compare the prevalence and co-infection prevalence of particular tick-transmitted Rickettsiales members: Rickettsia spp. (further referred as Rs), Anaplasma phagocytophilum (Ap), and “Candidatus Neoehrlichia mikurensis” (CNM) in I. ricinus ticks in two types of areas, different in terms of human impact: natural and urban. Using additional data, we aimed at investigating co-occurrence of these Rickettsiales with Borreliella spp. A total of 4189 tick specimens, 2363 from the urban area (Warsaw park and forests) and 1826 from the natural area (forests and park in the vicinity of National Parks), were tested for the presence of Rickettsiales DNA by PCRs. The prevalence of selected Rickettsiales was twice higher in urban than natural areas (13.2% vs. 6.9%, respectively). In total ticks, the prevalence of Rs, Ap, and CNM was 6.5%, 5.3%, and 3.6% in urban areas vs. 4.4%, 1.1%, and 2.1% in natural areas, respectively. Co-infections of Rickettsiales were also more prevalent in urban areas (2.6% vs. 0.3%, respectively). The most common Rs was R. helvetica; also R. monacensis and novel “Candidatus Rickettsia mendelii” were detected. Positive association between Ap and CNM infections was discovered. Rickettsiales bacteria occurrence was not associated with Borreliella occurrence, but co-infections with these two groups were more common in ticks in urban areas. In conclusion, three groups of Rickettsiales constituted the important part of the tick pathogen community in Poland, especially in the urbanized central Poland (Mazovia). In the Warsaw agglomeration, there is a greater risk of encountering the I. ricinus tick infected with Rickettsiales and co-infected with Lyme spirochaetes, in comparison to natural areas. This finding raises the question whether cities might in fact be the hot spots for TBDs. Electronic supplementary material The online version of this article (10.1007/s00248-018-1269-y) contains supplementary material, which is available to authorized users.

The American dog tick, Dermacentor variabilis (Say), is a vector of spotted fever group (SFG) rickettsiae, including Rickettsia rickettsii the causative organism of Rocky Mountain spotted fever (RMSF). In North Carolina, SFG rickettsioses (including RMSF) are a leading cause of tick-borne illness. Knowledge of the infection rate and geographic distribution of D. variabilis ticks infected with Rickettsia spp. provides information on the spatial distribution of public health risk. Accordingly, we extracted genomic DNA from adult D. variabilis collected from field habitats in 32 North Carolina counties from 2009 to 2013. A nested PCR assay of the 23S-5S intergenic spacer (IGS) region of Rickettsia coupled with reverse line blot hybridization (RLBH) with species-specific probes was used to detect and identify rickettsiae to species. Approximately half of the 532 tick DNA samples exhibited a band of the expected size on agarose gels, indicating infection with Rickettsia spp. RLBH analyses showed R. amblyommatis (formerly ‘Candidatus R. amblyommii’), R. parkeri, and R. montanensis were predominant, while other Rickettsia species detected included R. conorii-like, R. massiliae, R. rhipicephali, R. canadensis, R. bellii, and some unknown Rickettsia spp. Some ticks were infected with more than one Rickettsia species. Notably, several Rickettsia-positive ticks harbored R. rickettsii. DNA sequencing was performed on a portion of the 23S-5S IGS amplicons and the results were concordant with RLB assay results. We conclude that Rickettsia spp. are common in D. variabilis in North Carolina. Geographic patterns in the occurrence of Rickettsia-infected D. variabilis ticks across the counties sampled are discussed.

The Cerrado Biome is the second largest in Brazil covering roughly 2 million km2, with varying features throughout its area. The Biome is endangered but it is also source of animal species for rural, green urban and degraded rainforest areas. Ticks are among Cerrado species that establish at anthropogenic sites and although information about them is steadily increasing, several features are unknown. We herein report tick species, abundance and some ecological relationships within natural areas of the Cerrado at higher altitudes (800–1500 m) within and around Serra da Canastra National Park, in Minas Gerais State Brazil. In total of 1196 ticks were collected in the environment along 10 campaigns held in 3 years (2007–2009). Amblyomma sculptum was the most numerous species followed by Amblyomma dubitatum and Amblyomma brasiliense. Distribution of these species was very uneven and an established population of A. brasiliense in the Cerrado is reported for the first time. Other tick species (Amblyomma ovale, Amblyomma nodosum, Amblyomma parvum, Ixodes schulzei and Haemaphysalis leporispalustris) were found in lesser numbers. Domestic animals displayed tick infestations of both rural and urban origin as well as from natural areas (Rhipicephalus sanguineus sensu lato, Rhipicephalus microplus, Dermacentor nitens, A. sculptum, A. ovale, Amblyomma tigrinum, Argas miniatus). Amblyomma sculptum had the widest domestic host spectrum among all tick species. DNA of only one Rickettsia species, R. bellii, was found in an A. dubitatum tick. Several biological and ecological features of ticks of the studied areas are discussed.

Made available in DSpace on 2019-10-06T16:03:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 The aim of this study was to perform a molecular survey and determine the genetic diversity of Bartonella spp. (Rhizobiales: Bartonellaceae) and Rickettsia spp. (Rickettsiales: Rickettsiaceae) in cat feas (Siphonaptera: Pulicidae) from Southern Chile. Fleas (n = 251) were collected from 150 cats in Valdivia city and identified using morphological keys. Fleas belonging to the same cat were pooled (two to seven feas per pool). DNA was purified from individual (n = 92) and pooled (n = 58) feas and submitted to conventional polymerase chain reaction assays targeting Bartonella spp. (gltA) and Rickettsia spp. (ompA). Selected positive samples were sequenced for Bartonella gltA (n = 19), Rickettsia ompA (n = 14), and Rickettsia gltA (n = 11) for speciation, phylogenetic, and diversity analyses. All feas (n = 251) were identified as Ctenocephalides felis felis (Bouché) (Siphonaptera: Pulicidae). Bartonella and Rickettsia occurrences in C. felis felis were 39.3% (59/150) and 76.6% (115/150), respectively. From sequenced Bartonella spp., 47.3% (9/19) were identified as Bartonella clarridgeiae, 42.1% (8/19) as Bartonella henselae, 5.3% (1/19) as Bartonella koehlerae, and 5.3% (1/19) as Bartonella sp. Rickettsia felis was the only Rickettsiaceae species identified in both ompA (14/14) and gltA (11/11) products. B. henselae and B. clarridgeiae presented five genotypes. R. felis ompA sequences presented seven genotypes. On the other hand, R. felis gltA sequences showed only one genotype. Bartonella spp. and R. felis are described for the first time in C. felis felis feas from Southern Chile, highlighting the importance of these vectors as a source of zoonotic agents. Instituto de Ciencias Clínicas Veterinarias Facultad de Ciencias Veterinarias Universidad Austral de Chile Escuela de Medicina Veterinaria Facultad de Ciencias Universidad Mayor Departamento de Patologia Veterinária Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista (FCAV/UNESP) Departamento de Patologia Veterinária Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista (FCAV/UNESP)

Rickettsial-like cells are reported from the body cavity of the Myanmar amber larval tick, Cornupalpatum burmanicum (Ixodida: Ixodidae). These cells are characterized and described in a new collective fossil genus erected for putative rickettsia in fossil ticks. The size and shape of the fossil cells resemble those of present day members of the Rickettsiaceae, many of which occur in the body cavity of present day ticks.

Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia ( Alphaproteobacteria ; Rickettsiaceae ). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell. IMPORTANCE A hallmark of obligate intracellular bacteria is the tradeoff of metabolic genes for the ability to acquire host metabolites. For species of Rickettsia , arthropod-borne parasites with the potential to cause serious human disease, the range of pilfered host metabolites is unknown. This information is critical for dissociating rickettsiae from eukaryotic cells to facilitate rickettsial genetic manipulation. In this study, we reconstructed the Rickettsia metabolic network and identified 51 host metabolites required to compensate patchwork Rickettsia biosynthesis pathways. Remarkably, some metabolites are not known to be transported by any bacteria, and overall, few cognate transporters were identified. Several pathways contain missing enzymes, yet similar pathways in unrelated bacteria indicate convergence and possible novel enzymes awaiting characterization. Our work illuminates the parasitic nature by which rickettsiae hijack host metabolism to counterbalance numerous disintegrated biosynthesis pathways that have arisen through evolution within the eukaryotic cell. This metabolic blueprint reveals what a Rickettsia axenic medium might entail.

The genus Rickettsia (Rickettsiales: Rickettsiaceae) includes Gram-negative, small, obligate intracellular, nonmotile, pleomorphic coccobacilli bacteria transmitted by arthropods. Some of them cause human and probably also animal disease (life threatening in some patients). In these guidelines, we give clinical practice advices (microscopy, serology, molecular tools, and culture) for the microbiological study of these microorganisms in clinical samples. Since in our environment rickettsioses are mainly transmitted by ticks, practical information for the identification of these arthropods and for the study of Rickettsia infections in ticks has also been added. info:eu-repo/semantics/publishedVersion

Rickettsioses are emerging zoonotic diseases reported worldwide. In spite of the serological evidence of spotted fever group rickettsioses in febrile patients in Malaysia, limited studies have been conducted to identify the animal reservoirs and vectors of rickettsioses. This study investigated the presence of rickettsiae in the tissue homogenates of 95 wild rats and 589 animal ectoparasites. Using PCR assays targeting the citrate synthase gene (gltA), rickettsial DNA was detected in the tissue homogenates of 13 (13.7%) wild rats. Sequence analysis of the gltA amplicons showed 98.6-100% similarity with those of Rickettsia honei/R. conorii/R. raoultii (Rickettsiales: Rickettsiaceae). Sequence analysis of outer membrane protein A gene (ompA) identified Rickettsia sp. TCM1 strain from two rats. No rickettsia was detected from Laelaps mites, Rhipicephalus sanguineus and Haemaphysalis bispinosa ticks, and Felicola subrostratus lice in this study. R. felis was identified from 32.2% of 177 Ctenocephalides felis fleas. Sequence analysis of the gltA amplicons revealed two genotypes of R. felis (Rf31 and RF2125) in the fleas. As wild rats and cat fleas play an important role in the enzoonotic maintenance of rickettsiae, control of rodent and flea populations may be able to reduce transmission of rickettsioses in the local setting.

Background & objectives: Mosquito surveillance is one of the critical functions of local health departments, particularly in the context of outbreaks of severe mosquito-borne viral infections. Unfortunately, some viral and parasitic infections transmitted by mosquitoes, manifests non-specific clinical symptoms which may actually be of rickettsial etiology, including Rickettsia felis infections. This study tested the hypothesis that mosquitoes from southeastern Georgia, USA may be infected with Rickettsia felis and Wolbachia, an endosymbiotic bacterium of the order Rickettsiales. Methods: Specimens of the five most common mosquito species occurring in the region were collected using gravid and light-traps and identified using morphological keys. Mosquitoes were then pooled by species, sex, trap and collection site and their DNA was extracted. Molecular methods were used to confirm mosquito identification, and presence of Wolbachia and R. felis. Results: Wolbachia DNA was detected in 90.8% of the mosquito pools tested, which included 98% pools of Cx. quinquefasciatus Say (Diptera: Culicidae), 95% pools of Ae. albopictus Skuse (Diptera: Culicidae), and 66.7% of pools of Cx. pipiens complex. Samples of An. punctipennis Say (Diptera: Culicidae) and An. crucians Wiedemann (Diptera: Culicidae) were tested negative for Wolbachia DNA. Three genotypes of Wolbachia sp. belonging to Group A (1 type) and Group B (2 types) were identified. DNA of R. felis was not found in any pool of mosquitoes tested. Interpretation & conclusions: This study provides a pilot data on the high presence of Wolbachia in Cx. quinque-fasciatus and Ae. albopictus mosquitoes prevalent in the study region. Whether the high prevalence of Wolbachia and its genetic diversity in mosquitoes affects the mosquitoes’ susceptibility to R. felis infection in Georgia will need further evaluation.

The Gulf Coast tick, Amblyomma maculatum Koch, has become increasingly important in public health for its role as a vector of the recently recognized human pathogen, Rickettsia parkeri. More recently, these ticks were also found to harbor a novel spotted fever group rickettsia, "Candidatus Rickettsia andeanae." First identified in Peru, and subsequently reported in ticks collected in the United States, Chile, and Argentina, "Ca. R. andeanae" remains largely uncharacterized, in part because of the lack of a stable isolate. Although the isolation of "Ca. R. andeanae" was recently described in DH82, Vero, and Drosophila S2 cells, its stability in these cell lines was not shown. To evaluate "Ca. R. andeanae" transmission and pathogenicity in vertebrates, as well as further describe biological characteristics of this candidate species to fulfill criteria for its establishment as a new species, availability of a stable isolate is essential. Here we describe the propagation of "Ca. R. andeanae" by using a primary culture derived from naturally infected A. maculatum embryos. Subsequent passage of the "Ca. R. andeanae" isolate to ISE6 (Ixodes scapularis embryonic) and Vero (African green monkey kidney epithelial) cell lines demonstrated limited propagation of the rickettsiae. Treatment of the infected primary cells with tetracycline resulted in cultures negative for "Ca. R. andeanae" by polymerase chain reaction and microscopy. Establishment of an isolate of "Ca. R. andeanae" will promote further investigation into the significance of this tick-associated rickettsia, including its role in spotted fever and interactions with the sympatric species, R. parkeri in A.

An engorged female Amblyomma helvolum Koch tick was removed from an adult Varanus salvator Laurenti lizard during field collection in Thailand. After using polymerase chain reaction to amplify three genes (16S rDNA, gltA, and OmpA), we discovered the presence of a Rickettsia sp. of the Spotted Fever Group. Phylogenetic analysis revealed that this Rickettsia sp. is closely related to Rickettsia raoultii Mediannikov. Therefore, we report herein for the first time the detection of a novel Spotted Fever Group Rickettsia in an Amblyomma helvolum from a Varanus salvator in Thailand.

Outbreaks of spotted fevers have been reported in Costa Rica since the 1950s, although vectors responsible for transmission to humans have not been directly identified. In this study, species of Rickettsia were detected in ectoparasites from Costa Rica, mostly from five study sites where cases of spotted fevers have been reported. Ticks and fleas were collected using drag cloths or directly from domestic and wild animals and pooled according to species, host, and location. Pools were analyzed initially by PCR to detect a fragment of Rickettsia spp. specific gltA gene, and those positive were confirmed by detection of htrA and/or ompA gene fragments. Partial sequences of the gltA gene were obtained, as well as at least one ompA and/or ompB partial sequence of each species. Rickettsia spp. were confirmed in 119 of 497 (23.9%) pools of ticks and fleas analyzed. Rickettsia rickettsii was identified in one nymph of Amblyomma mixtum and one nymph of Amblyomma varium. Other rickettsiae present were ‘Candidatus Rickettsia amblyommii’ in A. mixtum, Amblyomma ovale, Dermacentor nitens, and Rhipicephalus sanguineus s. l.; Rickettsia bellii in Amblyomma sabanerae; Rickettsia felis in Ctenocephalides felis; and Rickettsia sp. similar to ‘Candidatus R. asemboensis’ in C. felis, Pulex simulans, A. ovale, and Rhipicephalus microplus. Results show the presence of rickettsiae in vectors that may be responsible for transmission to humans in Costa Rica, and evidence suggests exposure to rickettsial organisms in the human environment may be common. This is the first study to report R. rickettsii in A. varium and in A. mixtum in Costa Rica. Universidad de Costa Rica/[803-A8-127]/UCR/Costa Rica Universidad de Costa Rica/[803-B1-041]/UCR/Costa Rica Universidad de Costa Rica/[ED-548]/UCR/Costa Rica Red para la Investigación y el entrenamiento de enfermedades tropicales/[9-N-2008]/NETROPICA/ UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET)

Bei den Bakterien der Familien Rickettsiaceae, Anaplasmataceae und Coxiellaceae handelt es sich um kleine, gramnegative und obligat intrazellulare Stabchen. Mit Ausnahme der Coxiellen erfolgt die Ubertragung dieser Bakterien im Allgemeinen durch Vektoren wie z. B. Zecken. Die ausgelosten akuten Erkrankungen sind durch unspezifische Allgemeinsymptome, unterschiedliche Organmanifestationen und oft durch Blutbildveranderungen und Exantheme gekennzeichnet.

Brazilian spotted fever (BSF) is a fatal zoonosis because of the difficulties in its early diagnosis and treatment. Occurrences of BSF in the northeast of the state of Paraná prompted investigation of areas at risk of this rickettsiosis in the municipalities of Japira, Jaboti, Pinhalão and Tomazina. To determine the areas at risk, 592 serum samples from dogs and 230 from equids were analyzed by means of the indirect immunofluorescence assay (IFA) for Rickettsia rickettsii and R. parkeri . In addition, risk probability maps were drawn up using the kriging indicator technique. Among the samples tested, 5.3% (43/822) indicated presence of antibodies reactive to at least one of the two Rickettsia species tested: 7.8% of the equids (18/230) and 4.2% of the dogs (25/592) were positive. Geostatistical analysis showed that the average seropositivity rate was 5 to 6%. Although the average seropositivity rates observed among these dogs and equids were lower than those reported from endemic areas of Brazil, the biotic components (etiological agent, vector and reservoirs) and environmental aspects of BSF epidemiology were present in these municipalities. Resumo A febre maculosa brasileira (FMB) é uma zoonose fatal devido às dificuldades para diagnosticá-la e tratá-la precocemente. A ocorrência de casos de FMB no Estado do Paraná suscitou a investigação de áreas de risco desta rickettsiose nos municípios de Japira, Jaboti, Pinhalão e Tomazina, na mesorregião norte pioneiro do Paraná. Para determinar as áreas de risco foram analisadas amostras de soro de 592 cães e 230 equídeos submetidos à reação de imunofluorescência indireta para Rickettsia rickettsii e R. parkeri. Além disto, foram construídos mapas de probabilidade de risco pela técnica de krigagem indicatriz. Das amostras testadas 5,3% (43/822) continham anticorpos para pelo menos uma das duas rickettsias testadas. Os equídeos apresentaram uma positividade de 7,8% (18/230) e os cães de 4,2% (25/592). A análise geoestatística mostrou que a soropositividade média é de 5 a 6%. Embora as soropositividade médias de cães e equídeos constatadas tenham sido menores do que as relatadas em áreas endêmicas do território brasileiro, os componentes bióticos (agente etiológico, vetor e reservatórios) e ambientais da epidemiologia da FMB se fazem presentes nos municípios referidos.

Rocky Mountain spotted fever, Lyme disease, and ehrlichiosis are tick-borne diseases that are reported annually in Kentucky. We conducted a survey to describe infection prevalence of tick-borne pathogens in Amblyomma americanum and Dermacentor variabilis ticks collected in Kentucky. During 2007-2008, we collected 287 ticks (179 D. variabilis and 108 A. americanum ) from canine, feral hog, horse, raccoon, white-tailed deer, and human hosts in six counties in Kentucky. Ticks were screened for Rickettsia spp., Borrelia spp., and Ehrlichia spp. by using polymerase chain reaction. Forty-one (14.3%) ticks (31 A. americanum and 10 D. variabilis ) were polymerase chain reaction-positive for a Rickettsia spp. Fourteen (4.9%) ticks (6 A. americanum and 8 D. variabilis ) were positive for E. chaffeensis , and 4 A. americanum (1.4%) were positive for E. ewingii . One (0.4%) A. americanum was positive for Borrelia lonestari . Although Rocky Mountain spotted fever is diagnosed in Kentucky, no R. rickettsii was found in ticks in this study.

TOC Summary: Rickettsiae closely related to the scrub typhus agent are present in the Western Hemisphere., We report a case of scrub typhus in a 54-year-old man who was bitten by several terrestrial leeches during a trip to Chiloé Island in southern Chile in 2006. A molecular sample, identified as related to Orientia tsutsugamushi based on the sequence of the16S rRNA gene, was obtained from a biopsy specimen of the eschar on the patient’s leg. Serologic analysis showed immunoglobulin G conversion against O. tsutsugamushi whole cell antigen. This case and its associated molecular analyses suggest that an Orientia-like agent is present in the Western Hemisphere that can produce scrub typhus–like illness. The molecular analysis suggests that the infectious agent is closely related, although not identical, to members of the Orientia sp. from Asia.

Salivary glands, midgut, Malpighian tubules, and ovaries were dissected from infected, colony-derived Amblyomma maculatum (Gulf Coast ticks) injected as nymphs with either Rickettsia parkeri (a spotted fever group rickettsia [SFGR]; treatment) or phosphate-buffered saline (negative control). For comparison, similar tissues were dissected from hemolymph-positive, field-collected ticks. Tissues were analyzed by indirect fluorescent antibody (IFA) tests. All phosphate-buffered saline-injected ticks were IFA negative, whereas SFGR were detected by IFA in 100% of the salivary glands and ovaries and 78 and 75% of midgut and Malpighian tubule samples, respectively, of R. parkeri-injected ticks. Nearly 22% (10/46) of the field-collected ticks were hemolymph positive. Of those, SFGR were detected by IFA in 80% of the salivary glands, 67% of the ovaries, and 60% in the midgut and Malpighian tubules. This is the first study to assess the distribution of SFGR in select tissues of A. maculatum ticks.

Adult ticks of the species Amblyomma parvum were collected from the vegetation in the Pantanal biome (state of Mato Grosso do Sul) and from horses in the Cerrado biome (state of Piauí) in Brazil. The ticks were individually tested for rickettsial infection via polymerase chain reaction (PCR) targeting three rickettsial genes, gltA, ompA and ompB. Overall, 63.5% (40/63) and 66.7% (2/3) of A. parvum ticks from Pantanal and Cerrado, respectively, contained rickettsial DNA, which were all confirmed by DNA sequencing to be 100% identical to the corresponding fragments of the gltA, ompA and ompB genes of Candidatus Rickettsia andeanae. This report is the first to describe Ca. R. andeanae in Brazil.