Molecular Detection of Tick-borne Pathogens in Ticks Collected from Hainan Island, China.

Pathogens like bacteria and protozoa, which affect human and animal health worldwide, can be transmitted by vectors like ticks. To investigate the epidemiology and genetic diversity of bacteria and protozoans carried by ticks in Chengmai county of Hainan province, China, 285 adult hard ticks belonging to two species [ Rhipicephalus sanguineus ( sensu lato): 183, 64.21% and Rhipicephalus microplus: 102, 35.79%] from dogs, cattle, and goats were collected. Microbial families were identified in these ticks by amplifying the 18S rRNA, 16S rRNA ( rrs), citrate synthase ( gltA), and heat shock protein ( groEL) genes. Our data revealed the presence of four recognized species and two Candidatus spp. of Anaplasmataceae and Coxiellaceae. In sum, these data reveal an extensive diversity of Anaplasmataceae bacteria, Coxiellaceae bacteria, Babesiidae, and Hepatozoidae in ticks from Hainan Island, highlighting the need to understand the tick-borne pathogen infection in local animals and humans.

Rickettsia hoogstraalii and a Rickettsiella from the Bat Tick Argas transgariepinus, in Namibia.

Ectoparasites were collected from Eptesicus hottentotus, the long-tailed serotine bat, caught in Namibia as part of an ecological study. Larvae of Argas transgariepinus, a blood-feeding ectoparasite of bats in Africa, were removed from 3 of 18 bats. We present scanning electron microscope images of unengorged larvae. As with other ectoparasites, this bat tick might transmit pathogens such as Borrelia and Rickettsia to their hosts as has been reported for bat ticks in Europe and North America. We screened 3 pools (25 total) of larvae of A. transgariepinus removed from the long-tailed serotine bat Eptesicus hottentotus caught in Namibia. Two microbes of unknown pathogenicity, including Rickettsia hoogstraalii, a spotted fever group pathogen, and a Rickettsiella sp. were detected by molecular techniques.

Isolation of Rickettsia, Rickettsiella, and Spiroplasma from Questing Ticks in Japan Using Arthropod Cells.

Ticks are blood-sucking ectoparasites that transmit zoonotic pathogens to humans and animals. Ticks harbor not only pathogenic microorganisms but also endosymbionts. Although some tick endosymbionts are known to be essential for the survival of ticks, their roles in ticks remain poorly understood. The main aim of this study was to isolate and characterize tick-borne microorganisms from field-collected ticks using two arthropod cell lines derived from Ixodes scapularis embryos (ISE6) and Aedes albopictus larvae (C6/36). A total of 170 tick homogenates originating from 15 different tick species collected in Japan were inoculated into each cell line. Bacterial growth was confirmed by PCR amplification of 16S ribosomal DNA (rDNA) of eubacteria. During the 8-week observation period, bacterial isolation was confirmed in 14 and 4 samples using ISE6 and C6/36 cells, respectively. The sequencing analysis of the 16S rDNA PCR products indicated that they were previously known tick-borne pathogens/endosymbionts in three different genera: Rickettsia, Rickettsiella, and Spiroplasma. These included four previously validated rickettsial species namely Rickettsia asiatica (n = 2), Rickettsia helvetica (n = 3), Rickettsia monacensis (n = 2), and Rickettsia tamurae (n = 3) and one uncharacterized genotype Rickettsia sp. LON (n = 2). Four isolates of Spiroplasma had the highest similarity with previously reported Spiroplasma isolates: Spiroplasma ixodetis obtained from ticks in North America and Spiroplasma sp. Bratislava 1 obtained from Ixodes ricinus in Europe, while two isolates of Rickettsiella showed 100% identity with Rickettsiella sp. detected from Ixodes uriae at Grimsey Island in Iceland. To the best of our knowledge, this is the first report on successful isolation of Rickettsiella from ticks. The isolates obtained in this study can be further analyzed to evaluate their pathogenic potential in animals and their roles as symbionts in ticks.

Genomic Insight into Symbiosis-Induced Insect Color Change by a Facultative Bacterial Endosymbiont, "Candidatus Rickettsiella viridis".

Members of the genus Rickettsiella are bacterial pathogens of insects and other arthropods. Recently, a novel facultative endosymbiont, "Candidatus Rickettsiella viridis," was described in the pea aphid Acyrthosiphon pisum, whose infection causes a striking host phenotype: red and green genetic color morphs exist in aphid populations, and upon infection with the symbiont, red aphids become green due to increased production of green polycyclic quinone pigments. Here we determined the complete genome sequence of the symbiont. The 1.6-Mb circular genome, harboring some 1,400 protein-coding genes, was similar to the genome of entomopathogenic Rickettsiella grylli (1.6 Mb) but was smaller than the genomes of phylogenetically allied human pathogens Coxiella burnetii (2.0 Mb) and Legionella pneumophila (3.4 Mb). The symbiont's metabolic pathways exhibited little complementarity to those of the coexisting primary symbiont Buchnera aphidicola, reflecting the facultative nature of the symbiont. The symbiont genome harbored neither polyketide synthase genes nor the evolutionarily allied fatty acid synthase genes that are suspected to catalyze the polycyclic quinone synthesis, indicating that the green pigments are produced not by the symbiont but by the host aphid. The symbiont genome retained many type IV secretion system genes and presumable effector protein genes, whose homologues in L. pneumophila were reported to modulate a variety of the host's cellular processes for facilitating infection and virulence. These results suggest the possibility that the symbiont is involved in the green pigment production by affecting the host's metabolism using the secretion machineries for delivering the effector molecules into the host cells.IMPORTANCE Insect body color is relevant to a variety of biological aspects such as species recognition, sexual selection, mimicry, aposematism, and crypsis. Hence, the bacterial endosymbiont "Candidatus Rickettsiella viridis," which alters aphid body color from red to green, is of ecological interest, given that different predators preferentially exploit either red- or green-colored aphids. Here we determined the complete 1.6-Mb genome of the symbiont and uncovered that, although the red-green color transition was ascribed to upregulated production of green polycyclic quinone pigments, the symbiont genome harbored few genes involved in the polycyclic quinone biosynthesis. Meanwhile, the symbiont genome contained type IV secretion system genes and presumable effector protein genes, whose homologues modulate eukaryotic cellular processes for facilitating infection and virulence in the pathogen Legionella pneumophila We propose the hypothesis that the symbiont may upregulate the host's production of polycyclic quinone pigments via cooption of secretion machineries and effector molecules for pathogenicity.

The High Diversity and Global Distribution of the Intracellular Bacterium Rickettsiella in the Polar Seabird Tick Ixodes uriae.

Obligate intracellular bacteria of the Rickettsiella genus are emerging as both widespread and biologically diverse in arthropods. Some Rickettsiella strains are highly virulent entomopathogenic agents, whereas others are maternally inherited endosymbionts exerting very subtle manipulations on host phenotype to promote their own spread. Recently, a variety of Rickettsiella strains have been reported from ticks, but their biology is entirely unknown. In the present study, we examined the incidence and diversity of Rickettsiella in 11 geographically distinct populations of the polar seabird tick Ixodes uriae. We found Rickettsiella in most tick populations with a prevalence ranging from 3 to 24 %. 16S ribosomal RNA (rRNA) and GroEL gene sequences revealed an unexpected diversity of Rickettsiella, with 12 genetically distinct Rickettsiella strains present in populations of I. uriae. Phylogenetic investigations further revealed that these Rickettsiella strains do not cluster within a tick-specific clade but rather exhibit distinct evolutionary origins demonstrating frequent horizontal transfers between distantly related arthropod species. Tick rearing further showed that Rickettsiella are present in eggs laid by infected females with no evidence of abortive development. Using this data set, we discuss the potential biological significance of Rickettsiella in seabird ticks. Most notably, we suggest that these organisms may not be pathogenic forms but rather use more subtle adaptive strategies to persist within tick populations.

The Recent Evolution of a Maternally-Inherited Endosymbiont of Ticks Led to the Emergence of the Q Fever Pathogen, Coxiella burnetii.

Q fever is a highly infectious disease with a worldwide distribution. Its causative agent, the intracellular bacterium Coxiella burnetii, infects a variety of vertebrate species, including humans. Its evolutionary origin remains almost entirely unknown and uncertainty persists regarding the identity and lifestyle of its ancestors. A few tick species were recently found to harbor maternally-inherited Coxiella-like organisms engaged in symbiotic interactions, but their relationships to the Q fever pathogen remain unclear. Here, we extensively sampled ticks, identifying new and atypical Coxiella strains from 40 of 58 examined species, and used this data to infer the evolutionary processes leading to the emergence of C. burnetii. Phylogenetic analyses of multi-locus typing and whole-genome sequencing data revealed that Coxiella-like organisms represent an ancient and monophyletic group allied to ticks. Remarkably, all known C. burnetii strains originate within this group and are the descendants of a Coxiella-like progenitor hosted by ticks. Using both colony-reared and field-collected gravid females, we further establish the presence of highly efficient maternal transmission of these Coxiella-like organisms in four examined tick species, a pattern coherent with an endosymbiotic lifestyle. Our laboratory culture assays also showed that these Coxiella-like organisms were not amenable to culture in the vertebrate cell environment, suggesting different metabolic requirements compared to C. burnetii. Altogether, this corpus of data demonstrates that C. burnetii recently evolved from an inherited symbiont of ticks which succeeded in infecting vertebrate cells, likely by the acquisition of novel virulence factors.

Ultrastructural characterization and multilocus sequence analysis (MLSA) of 'Candidatus Rickettsiella isopodorum', a new lineage of intracellular bacteria infecting woodlice (Crustacea: Isopoda).

The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation 'Candidatus Rickettsiella isopodorum' is introduced for this new lineage of isopod-associated Rickettsiella bacteria.

Discovery of novel Rickettsiella spp. in ixodid ticks from Western Canada.

The genomic DNA from four species of ixodid ticks in western Canada was tested for the presence of Rickettsiella by PCR analyses targeting the 16S rRNA gene. Eighty-eight percent of the Ixodes angustus (n = 270), 43% of the I. sculptus (n = 61), and 4% of the I. kingi (n = 93) individuals examined were PCR positive for Rickettsiella, whereas there was no evidence for the presence of Rickettsiella in Dermacentor andersoni (n = 45). Three different single-strand conformation polymorphism profiles of the 16S rRNA gene were detected among amplicons derived from Rickettsiella-positive ticks, each corresponding to a different sequence type. Furthermore, each sequence type was associated with a different tick species. Phylogenetic analyses of sequence data of the 16S rRNA gene and three other genes (rpsA, gidA, and sucB) revealed that all three sequence types were placed in a clade that contained species and pathotypes of the genus Rickettsiella. The bacterium in I. kingi represented the sister taxon to the Rickettsiella in I. sculptus, and both formed a clade with Rickettsiella grylli from crickets (Gryllus bimaculatus) and "R. ixodidis" from I. woodi. In contrast, the Rickettsiella in I. angustus was not a member of this clade but was placed external to the clade comprising the pathotypes of R. popilliae. The results indicate the existence of at least two new species of Rickettsiella: one in I. angustus and another in I. kingi and I. sculptus. However, the Rickettsiella strains in I. kingi and I. sculptus may also represent different species because each had unique sequences for all four genes.

Novel Rickettsiella bacterium in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae).

Bacteria in the genus Rickettsiella (Coxiellaceae), which are mainly known as arthropod pathogens, are emerging as excellent models to study transitions between mutualism and pathogenicity. The current report characterizes a novel Rickettsiella found in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae), a major vector of phytoplasma diseases in Europe and Asia. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to survey the main symbionts of O. albicinctus, revealing the obligate symbionts Sulcia and Nasuia, and the facultative symbionts Arsenophonus and Wolbachia, in addition to Rickettsiella. The leafhopper Rickettsiella is allied with bacteria found in ticks. Screening O. albicinctus from the field showed that Rickettsiella is highly prevalent, with over 60% of individuals infected. A stable Rickettsiella infection was maintained in a leafhopper laboratory colony for at least 10 generations, and fluorescence microscopy localized bacteria to accessory glands of the female reproductive tract, suggesting that the bacterium is vertically transmitted. Future studies will be needed to examine how Rickettsiella affects host fitess and its ability to vector phytopathogens.

Multilocus sequence analysis (MLSA) of 'Rickettsiella agriotidis', an intracellular bacterial pathogen of Agriotes wireworms.

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae) are severe and widespread agricultural pests that affect numerous crops globally. A new bacterial specimen identified in diseased wireworms had previously been shown by microscopy and 16S ribosomal RNA (rRNA) gene-based phylogenetic reconstruction to belong to the taxonomic genus Rickettsiella (Gammaproteobacteria) that comprises intracellular bacteria associated with and typically pathogenic for a wide range of arthropods. Going beyond these earlier results obtained from rRNA phylogenies, multilocus sequence analysis (MLSA) using a four marker scheme has been employed in the molecular taxonomic characterization of the new Rickettsiella pathotype, referred to as 'Rickettsiella agriotidis'. In combination with likelihood-based significance testing, the MLSA approach demonstrated the close phylogenetic relationship of 'R. agriotidis' to the pathotypes 'Rickettsiella melolonthae' and 'Rickettsiella tipulae', i.e., subjective synonyms of the nomenclatural type species, Rickettsiella popilliae. 'R. agriotidis' forms, therefore, part of a Rickettsiella pathotype complex that most likely represents the species R. popilliae. As there are currently no genetic data available from the R. popilliae type strain, the respective assignment cannot be corroborated directly. However, an alternative taxonomic assignment to the species Rickettsiella grylli has been positively ruled out by significance testing. MLSA has been shown to provide a more powerful tool for taxonomic delineation within the genus Rickettsiella as compared to 16S rRNA phylogenetics. However, the limitations of the present MLSA scheme for the sub-species level classification of 'R. agriotidis' and further R. popilliae synonyms has been critically evaluated.

Genome sequence of Diplorickettsia massiliensis, an emerging Ixodes ricinus-associated human pathogen.

Diplorickettsia massiliensis is a gammaproteobacterium in the order Legionellales and an agent of tick-borne infection. We sequenced the genome from strain 20B, isolated from an Ixodes ricinus tick. The genome consists of a 1,727,973-bp chromosome but no plasmid and includes 2,269 protein-coding genes and 42 RNA genes, including 3 rRNA genes.

Multilocus sequence typing (MLST) for the infra-generic taxonomic classification of entomopathogenic Rickettsiella bacteria.

The genus Rickettsiella comprises intracellular bacterial pathogens of a wide range of arthropods that are currently classified in four recognized species and numerous further pathotypes. However, both the delineation of and the synonymization of pathotypes with species are highly problematic. In the sequel of a previous phylogenomic study at the supra-generic level, nine selected genes - the 16S and 23S rRNA genes and the protein-encoding genes dnaG, ftsY, gidA, ksgA, rpoB, rpsA, and sucB - were evaluated for their potential as markers for the generic and infra-generic taxonomic classification of Rickettsiella-like bacteria. A methodological approach combining phylogenetic reconstruction with likelihood-based significance testing was employed on the basis of sequence data from the species Rickettsiella grylli and Rickettsiella popilliae, pathotypes 'Rickettsiella melolonthae' and 'Rickettsiella tipulae'. This study provides the first multilocus sequence typing (MLST) data for the genus Rickettsiella and identifies two new genetic markers, gidA and sucB, for the infra-generic classification within this taxon. In particular, aforesaid genes were found more reliable and informative markers than the corresponding 16S rRNA-encoding sequences that failed to produce strictly significant infra-generic taxonomic assignments. However, gidA- and sucB-based phylogenies were consistent with the currently accepted view of species delineation and species-pathotype synonymization within the genus Rickettsiella.

Genetic and electron-microscopic characterization of 'Rickettsiella agriotidis', a new Rickettsiella pathotype associated with wireworm, Agriotes sp. (Coleoptera: Elateridae).

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae), are severe and widespread agricultural pests affecting numerous crops. A previously unknown intracellular bacterium has been identified in a diseased Agriotes larva. Microscopic studies revealed the subcellular structures characteristic of Rickettsiella infections. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the wireworm pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the new pathotype designation 'R. agriotidis' is proposed to refer to this organism. Moreover, genetic analysis makes it likely that--on the basis of the currently accepted organization of the genus Rickettsiella--this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.

Molecular detection of Rickettsia, Coxiella and Rickettsiella DNA in three native Australian tick species.

Three Australian native animal species yielded 60 samples composed of three indigenous ticks. Hosts included twelve koalas, two echidnas and one wombat from Victoria, and ticks were of the species Ixodes tasmani (n = 42), Bothriocroton concolor (n = 8) and B. auruginans (n = 10), respectively. PCR screening and sequencing detected a species of Coxiella, sharing closest sequence identity to C. burnetii (>98%), in all B. auruginans, as well as a species of Rickettsia, matching closest to R. massiliae, in 70% of the same samples. A genotype sharing closest similarity to Rickettsia bellii (>99%) was identified in three female B. concolor collected from one of the echidnas. Three samples of I. tasmani, taken from three koalas, yielded different genotypes of Rickettsiella. These results represent the first detection of the three genera in each tick species and identify a high level of previously undetected bacterial diversity in Australian ticks.

Endosymbiotic bacteria living inside the poultry red mite (Dermanyssus gallinae).

This study investigated the endosymbiotic bacteria living inside the poultry red mite collected from five samples of one commercial farm from the UK and 16 farms from France using genus-specific PCR, PCR-TTGE and DNA sequencing. Endosymbiotic bacteria are intracellular obligate organisms that can cause several phenotypic and reproductive anomalies to their host and they are found widespread living inside arthropods. The farm sampled from the UK was positive for bacteria of the genera Cardinium sp. and Spiroplasma sp. From France, 7 farms were positive for Cardinium sp., 1 farm was positive for Spiroplasma sp., 1 farm was positive for Rickettsiella sp. and 2 farms were positive for Schineria sp. However, it was not possible to detect the presence of the genus Wolbachia sp. which has been observed in other ectoparasites. This study is the first report of the presence of endosymbionts living inside the poultry red mite. The results obtained suggest that it may be possible that these bacterial endosymbionts cause biological modifications to the poultry red mite.