Survey of Rickettsia species in hematophagous arthropods from endemic areas for Japanese spotted fever in China.

Japanese spotted fever (JSF) is caused by Rickettsia japonica, mainly vectored by hard ticks. However, whether R. japonica can be transmitted by other arthropods remains unknown. Moreover, it is of interest to investigate whether other Rickettsia species cause spotted fever in endemic areas. In this study, a survey of Rickettsia species was performed in hematophagous arthropods (mosquitoes, tabanids, and ticks) from endemic areas for JSF in Hubei Province, central China. The results showed that the diversity and prevalence of Rickettsia species in mosquitoes are low, suggesting that mosquitoes may not be the vector of zoonotic Rickettsia species. A novel Rickettsia species showed a high prevalence (16.31%, 23/141) in tabanids and was named "Candidatus Rickettsia tabanidii." It is closely related to Rickettsia from fleas and mosquitoes; however, its pathogenicity in humans needs further investigation. Five Rickettsia species were identified in ticks. Rickettsia japonica, the agent of JSF, was detected only in Haemaphysalis longicornis and Haemaphysalis hystricis, suggesting that they may be the major vectors of R. japonica. Notably, two novel species were identified in H. hystricis ticks, one belonging to the spotted fever group and the other potentially belonging to the ancestral group. The latter one named "Candidatus Rickettsia hubeiensis" may provide valuable insight into the evolutionary history of Rickettsia.

Metagenome diversity illuminates the origins of pathogen effectors.

Recent metagenome-assembled genome (MAG) analyses have profoundly impacted Rickettsiology systematics. The discovery of basal lineages (novel families Mitibacteraceae and Athabascaceae) with predicted extracellular lifestyles exposed an evolutionary timepoint for the transition to host dependency, which seemingly occurred independent of mitochondrial evolution. Notably, these basal rickettsiae carry the Rickettsiales vir homolog (rvh) type IV secretion system and purportedly use rvh to kill congener microbes rather than parasitize host cells as described for later-evolving rickettsial pathogens. MAG analysis also substantially increased diversity for the genus Rickettsia and delineated a sister lineage (the novel genus Tisiphia) that stands to inform on the emergence of human pathogens from protist and invertebrate endosymbionts. Herein, we probed Rickettsiales MAG and genomic diversity for the distribution of Rickettsia rvh effectors to ascertain their origins. A sparse distribution of most Rickettsia rvh effectors outside of Rickettsiaceae lineages illuminates unique rvh evolution from basal extracellular species and other rickettsial families. Remarkably, nearly every effector was found in multiple divergent forms with variable architectures, indicating profound roles for gene duplication and recombination in shaping effector repertoires in Rickettsia pathogens. Lateral gene transfer plays a prominent role in shaping the rvh effector landscape, as evinced by the discovery of many effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchange between Rickettsia and Legionella species. Our study exemplifies how MAGs can yield insight into pathogen effector origins, particularly how effector architectures might become tailored to the discrete host cell functions of different eukaryotic hosts.IMPORTANCEWhile rickettsioses are deadly vector-borne human diseases, factors distinguishing Rickettsia pathogens from the innumerable bevy of environmental rickettsial endosymbionts remain lacking. Recent metagenome-assembled genome (MAG) studies revealed evolutionary timepoints for rickettsial transitions to host dependency. The rvh type IV secretion system was likely repurposed from congener killing in basal extracellular species to parasitizing host cells in later-evolving pathogens. Our analysis of MAG diversity for over two dozen rvh effectors unearthed their presence in some non-pathogens. However, most effectors were found in multiple divergent forms with variable architectures, indicating gene duplication and recombination-fashioned effector repertoires of Rickettsia pathogens. Lateral gene transfer substantially shaped pathogen effector arsenals, evinced by the discovery of effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchanges between Rickettsia and Legionella species. Our study exemplifies how MAGs yield insight into pathogen effector origins and evolutionary processes tailoring effectors to eukaryotic host cell biology.

Ticks (Acari: Ixodidae) on resident and migratory wild birds in Orinoquia region, Colombia.

Several species of hard ticks, including those of the genera Ixodes, Haemaphysalis, Amblyomma, and Rhipicephalus, are of medical and veterinary importance and have been reported in association with Neotropical wild birds. Colombia, known for its great bird diversity, has 57 confirmed tick species. However, there are few studies on the association between wild birds and ticks in Colombia. The Orinoquia region, a migratory center in Colombia, provides a unique opportunity to study wild bird-tick associations and their implications for tick-borne disease dynamics. Our study, conducted between October and December 2021, aimed to identify hard ticks infesting resident and migratory wild birds in the department of Arauca and to assess the presence of bacteria from the genera Anaplasma, Borrelia, Ehrlichia, Rickettsia, and piroplasms. A total of 383 birds were examined, of which 21 were infested. We collected 147 ticks, including Amblyomma dissimile (larvae), Amblyomma longirostre (nymphs), Amblyomma mixtum (adults), and Amblyomma nodosum (larvae and nymphs). We did not detect bacterial DNA in the tested ticks; however, piroplasm DNA was detected in ticks from three of the infested birds. Of the 21 bird-tick associations, six are new to the Americas, and interesting documentation of piroplasm DNA in A. longirostre, A. nodosum, and A. dissimile ticks from wild birds in the region. This study provides valuable insights into the ticks associated with wild birds and their role in the dispersal of ticks and pathogens in Colombia, enhancing our understanding of tick life cycles and tick-borne disease dynamics.

Italian peninsula as a hybridization zone of Ixodes inopinatus and I. ricinus and the prevalence of tick-borne pathogens in I. inopinatus, I. ricinus, and their hybrids.

BACKGROUND: Ixodes inopinatus was described from Spain on the basis of morphology and partial sequencing of 16S ribosomal DNA. However, several studies suggested that morphological differences between I. inopinatus and Ixodes ricinus are minimal and that 16S rDNA lacks the power to distinguish the two species. Furthermore, nuclear and mitochondrial markers indicated evidence of hybridization between I. inopinatus and I. ricinus. In this study, we tested our hypothesis on tick dispersal from North Africa to Southern Europe and determined the prevalence of selected tick-borne pathogens (TBPs) in I. inopinatus, I. ricinus, and their hybrids. METHODS: Ticks were collected in Italy and Algeria by flagging, identified by sequencing of partial TROSPA and COI genes, and screened for Borrelia burgdorferi s.l., B. miyamotoi, Rickettsia spp., and Anaplasma phagocytophilum by polymerase chain reaction and sequencing of specific markers. RESULTS: Out of the 380 ticks, in Italy, 92 were I. ricinus, 3 were I. inopinatus, and 136 were hybrids of the two species. All 149 ticks from Algeria were I. inopinatus. Overall, 60% of ticks were positive for at least one TBP. Borrelia burgdorferi s.l. was detected in 19.5% of ticks, and it was significantly more prevalent in Ixodes ticks from Algeria than in ticks from Italy. Prevalence of Rickettsia spotted fever group (SFG) was 51.1%, with significantly greater prevalence in ticks from Algeria than in ticks from Italy. Borrelia miyamotoi and A. phagocytophilum were detected in low prevalence (0.9% and 5.2%, respectively) and only in ticks from Italy. CONCLUSIONS: This study indicates that I. inopinatus is a dominant species in Algeria, while I. ricinus and hybrids were common in Italy. The higher prevalence of B. burgdorferi s.l. and Rickettsia SFG in I. inopinatus compared with that in I. ricinus might be due to geographical and ecological differences between these two tick species. The role of I. inopinatus in the epidemiology of TBPs needs further investigation in the Mediterranean Basin.

Dermacentor (Indocentor) auratus Supino 1897: Potential geographic range, and medical and veterinary significance.

Dermacentor (Indocentor) auratus Supino, 1897 occurs in many regions of Southeast Asia and South Asia. In many regions of Southeast Asia and South Asia, targeted tick sampling and subsequent screening of collected D. auratus ticks have detected pathogenic bacteria and viruses in D. auratus. These disease-causing pathogens that have been detected in D. auratus include Anaplasma, Bartonella, Borrelia, Rickettsia (including spotted fever group rickettsiae), African swine fever virus, Lanjan virus, and Kyasanur forest disease virus. Although D. auratus predominantly infests wild pigs, this tick is also an occasional parasite of humans and other animals. Indeed, some 91 % of human otoacariasis cases in Sri Lanka were due to infestation by D. auratus. With the propensity of this tick to feed on multiple species of hosts, including humans, and the detection of pathogenic bacteria and viruses from this tick, D. auratus is a tick of medical, veterinary, and indeed zoonotic concern. The geographic range of this tick, however, is not well known. Therefore, in the present paper, we used the species distribution model, BIOCLIM, to project the potential geographic range of D. auratus, which may aid pathogen and tick-vector surveillance. We showed that the potential geographic range of D. auratus is far wider than the current geographic distribution of this tick, and that regions in Africa, and in North and South America seem to have suitable climates for D. auratus. Interestingly, in Southeast Asia, Borneo and Philippines also have suitable climates for D. auratus, but D. auratus has not been found in these regions yet despite the apparent close proximity of these regions to Mainland Southeast Asia, where D. auratus occurs. We thus hypothesize that the geographic distribution of D. auratus is largely dependent on the movement of wild pigs and whether or not these wild pigs are able to overcome dispersal barriers. We also review the potential pathogens and the diseases that may be associated with D. auratus and provide an updated host index for this tick.

Detection of Rickettsia raoultii in Vermipsylla alakurt-Like Fleas of Sheep in Northwestern China.

INTRODUCTION: To date, a total of 2574 validated flea species have been discovered. Vermipsyllidae is a family of fleas that comprises at least eight species. Vermipsylla is a genus of the family Vermipsyllidae within the order Siphonaptera of fleas. Here a novel Vermipsylla species was described, and rickettsial agent was also detected in it. METHODS: A total of 128 fleas were collected directly from 260 pastured sheep in China. Of these, eight representative fleas (four males and four females) were identified by key morphological features. Meanwhile, 120 flea DNAs, including six flea samples for molecular taxonomy, were subjected to Rickettsia spp. DNA detection. The molecular identity of fleas was determined by amplification and sequenmce analysis of four genetic markers (the 28S rDNA genes, the 18S rDNA genes, the mitochondrial cytochrome c oxidase subunit I and subunit II). In addition, five Rickettsia-specific gene fragments were used to identify the species of the rickettsial agents. The amplified products were sequenced and phylogenetically analyzed. RESULTS: The morphological characteristics of the flea species identified in this study were similar to Vermipsylla alakurt, but presented difference in hair number of the metepimeron, the third tergum, the genitals and the tibiae of hind leg. The 18S rDNA, 28S rDNA and COII genetic markers from fleas showed the highest identity to those of V. alakurt, shared 98.45% (954/969), 95.81% (892/931) and 85.86% (571/665) similarities, respectively. However, the COI sequence showed the highest identity to that of Dorcadia ioffi with 88.48% (576/651) similarity. Rickettsia raoutii tested positive in 14.17% (17/120) flea DNA samples. CONCLUSION: Our study reports the detection of R. raoultii in V. alakurt-like fleas infesting sheep in China.

Prevalence of tick infestation and molecular characterization of spotted fever Rickettsia massiliae in Rhipicephalus species parasitizing domestic small ruminants in north-central Nigeria.

Ticks are of great menace to animal and human health. They serve as vectors to both animals and human pathogens including Rickettsia species. Tick-borne rickettsiosis in West Africa remains incompletely understood. We determined the prevalence of tick infestation among small ruminants and molecularly described a clinically significant spotted fever Rickettsia massiliae from Rhipicephalus ticks collected from North-Central, Nigeria. A total of 352 small ruminants comprising of 152 sheep and 200 goats that were brought for slaughter at the major small ruminant slaughterhouse in Ilorin were examined for the presence of ticks. The collected Rhipicephalus species were subjected to molecular studies to detect and characterize Rickettsia massiliae. Of the small ruminants examined, 21 sheep and 46 goats were infested with ticks representing 13.82% and 23.00% respectively. Eight and nine different species of ticks were detected in sheep and goats respectively, with Rhipicephalus (Boophilus) decoloratus being the most prevalent tick species in both sheep and goats. There was a significant difference (p <0.01) in the prevalence of the different tick species collected in sheep and in goats. Based on the PCR amplification of the 23S-5S intergenic spacer (IGS), only 2 of the 142 Rhipicephalus tick samples screened for R. massiliae were positive (1.41%; 95% CI = 0.39-4.99). Rickettsia massiliae was detected from Rhipicephalus turanicus collected from sheep. Sequences obtained from the PCR carried out by amplifying Rickettsia 23S-5S IGS showed 99-100% close identity with members of the R. massiliae group. This study has for the first time confirmed the presence of spotted fever group Rickettsia massiliae from feeding ticks in Nigerian small ruminants. Further investigations to determine the possible pathogenic role of human R. massiliae infection in Nigeria would be beneficial.

Microbiome analyses of 12 psyllid species of the family Psyllidae identified various bacteria including Fukatsuia and Serratia symbiotica, known as secondary symbionts of aphids.

BACKGROUND: Psyllids (Hemiptera: Psylloidea) comprise a group of plant sap-sucking insects that includes important agricultural pests. They have close associations not only with plant pathogens, but also with various microbes, including obligate mutualists and facultative symbionts. Recent studies are revealing that interactions among such bacterial populations are important for psyllid biology and host plant pathology. In the present study, to obtain further insight into the ecological and evolutionary behaviors of bacteria in Psylloidea, we analyzed the microbiomes of 12 psyllid species belonging to the family Psyllidae (11 from Psyllinae and one from Macrocorsinae), using high-throughput amplicon sequencing of the 16S rRNA gene. RESULTS: The analysis showed that all 12 psyllids have the primary symbiont, Candidatus Carsonella ruddii (Gammaproteobacteria: Oceanospirillales), and at least one secondary symbiont. The majority of the secondary symbionts were gammaproteobacteria, especially those of the family Enterobacteriaceae (order: Enterobacteriales). Among them, symbionts belonging to "endosymbionts3", which is a genus-level monophyletic group assigned by the SILVA rRNA database, were the most prevalent and were found in 9 of 11 Psyllinae species. Ca. Fukatsuia symbiotica and Serratia symbiotica, which were recognized only as secondary symbionts of aphids, were also identified. In addition to other Enterobacteriaceae bacteria, including Arsenophonus, Sodalis, and "endosymbionts2", which is another genus-level clade, Pseudomonas (Pseudomonadales: Pseudomonadaceae) and Diplorickettsia (Diplorickettsiales: Diplorickettsiaceae) were identified. Regarding Alphaproteobacteria, the potential plant pathogen Ca. Liberibacter europaeus (Rhizobiales: Rhizobiaceae) was detected for the first time in Anomoneura mori (Psyllinae), a mulberry pest. Wolbachia (Rickettsiales: Anaplasmataceae) and Rickettsia (Rickettsiales: Rickettsiaceae), plausible host reproduction manipulators that are potential tools to control pest insects, were also detected. CONCLUSIONS: The present study identified various bacterial symbionts including previously unexpected lineages in psyllids, suggesting considerable interspecific transfer of arthropod symbionts. The findings provide deeper insights into the evolution of interactions among insects, bacteria, and plants, which may be exploited to facilitate the control of pest psyllids in the future.

Spotted Fever Group Rickettsia Trigger Species-Specific Alterations in Macrophage Proteome Signatures with Different Impacts in Host Innate Inflammatory Responses.

The molecular details underlying differences in pathogenicity between Rickettsia species remain to be fully understood. Evidence points to macrophage permissiveness as a key mechanism in rickettsial virulence. Different studies have shown that several rickettsial species responsible for mild forms of rickettsioses can also escape macrophage-mediated killing mechanisms and establish a replicative niche within these cells. However, their manipulative capacity with respect to host cellular processes is far from being understood. A deeper understanding of the interplay between mildly pathogenic rickettsiae and macrophages and the commonalities and specificities of host responses to infection would illuminate differences in immune evasion mechanisms and pathogenicity. We used quantitative proteomics by sequential windowed data independent acquisition of the total high-resolution mass spectra with tandem mass spectrometry (SWATH-MS/MS) to profile alterations resulting from infection of THP-1 macrophages with three mildly pathogenic rickettsiae: Rickettsia parkeri, Rickettsia africae, and Rickettsia massiliae, all successfully proliferating in these cells. We show that all three species trigger different proteome signatures. Our results reveal a significant impact of infection on proteins categorized as type I interferon responses, which here included several components of the retinoic acid-inducible gene I (RIG-1)-like signaling pathway, mRNA splicing, and protein translation. Moreover, significant differences in protein content between infection conditions provide evidence for species-specific induced alterations. Indeed, we confirm distinct impacts on host inflammatory responses between species during infection, demonstrating that these species trigger different levels of beta interferon (IFN-ß), differences in the bioavailability of the proinflammatory cytokine interleukin 1ß (IL-1ß), and differences in triggering of pyroptotic events. This work reveals novel aspects and exciting nuances of macrophage-Rickettsia interactions, adding additional layers of complexity between Rickettsia and host cells' constant arms race for survival. IMPORTANCE The incidence of diseases caused by Rickettsia has been increasing over the years. It has long been known that rickettsioses comprise diseases with a continuous spectrum of severity. There are highly pathogenic species causing diseases that are life threatening if untreated, others causing mild forms of the disease, and a third group for which no pathogenicity to humans has been described. These marked differences likely reflect distinct capacities for manipulation of host cell processes, with macrophage permissiveness emerging as a key virulence trait. However, what defines pathogenicity attributes among rickettsial species is far from being resolved. We demonstrate that the mildly pathogenic Rickettsia parkeri, Rickettsia africae, and Rickettsia massiliae, all successfully proliferating in macrophages, trigger different proteome signatures in these cells and differentially impact critical components of innate immune responses by inducing different levels of beta interferon (IFN-ß) and interleukin 1ß (IL-1ß) and different timing of pyroptotic events during infection. Our work reveals novel nuances in rickettsia-macrophage interactions, offering new clues to understand Rickettsia pathogenicity.

Phylogenetic Differentiation of Rickettsia parkeri Reveals Broad Dispersal and Distinct Clustering within North American Strains.

The tick-borne pathogen Rickettsia parkeri causes a mild rickettsiosis, with cases reported from several countries to its known distribution in the Americas. Molecular analyses have identified a clear distinction between strains of R. parkeri sensu stricto (s. s.) and R. parkeri sensu lato (s. l.) as well as separation between North American and South American R. parkeri s. s. strains. To expand on this previous work, we developed a multilocus sequence typing analysis with two aims: first, to investigate the genetic diversity within strains of North American R. parkeri s. s., and second, to further the understanding of the genetic relationships between R. parkeri s. s. and R. parkeri s. l. Sixty-four R. parkeri isolates and 12 R. parkeri-positive tick lysates were analyzed using a novel typing scheme consisting of four coding regions and two intergenic regions. A concatenated Bayesian phylogeny that identified eight clades was constructed: three represent the R. parkeri s. l. strains, and five represent the R. parkeri s. s. strains. The clades appear to be generally phylogeographically organized and associated with specific tick vectors. However, while one of the four R. parkeri s. s. North American clades appears to be limited to the southwestern United States, the other North American clades exhibit broad dispersal, most notably seen in the largest group, which includes representative samples extending from northern Mexico to Delaware. This work highlights the increasingly recognized geographic range of R. parkeri in the Americas and suggests a potential public health risk for these areas. IMPORTANCE Since 1937, when Rickettsia parkeri was originally identified in Amblyomma maculatum group ticks, the recognized range and associated vectors for this pathogen have expanded significantly. In recent years, R. parkeri has been identified in 12 tick species from seven countries in the Americas. Herein, we provide evidence that the greatest genetic diversity within R. parkeri exists in North America, where one R. parkeri sensu lato and four R. parkeri sensu stricto genotypes are present. While one distinct R. parkeri sensu stricto genotype exists only in the southwestern United States, three genotypes are broadly distributed in the eastern United States, with the largest of these found across the known range of R. parkeri in North America. In contrast, the South American R. parkeri sensu stricto samples represent a single genotype and are completely clonal at the loci analyzed, irrespective of their country of origin.

Characterization of a novel transitional group Rickettsia species (Rickettsia tillamookensis sp. nov.) from the western black-legged tick, Ixodes pacificus.

A previously unrecognized Rickettsia species was isolated in 1976 from a pool of Ixodes pacificus ticks collected in 1967 from Tillamook County, Oregon, USA. The isolate produced low fever and mild scrotal oedema following intraperitoneal injection into male guinea pigs (Cavia porcellus). Subsequent serotyping characterized this isolate as distinct from recognized typhus and spotted fever group Rickettsia species; nonetheless, the isolate remained unevaluated by molecular techniques and was not identified to species level for the subsequent 30 years. Ixodes pacificus is the most frequently identified human-biting tick in the western United States, and as such, formal identification and characterization of this potentially pathogenic Rickettsia species is warranted. Whole-genome sequencing of the Tillamook isolate revealed a genome 1.43 Mbp in size with 32.4 mol% G+C content. Maximum-likelihood phylogeny of core proteins places it in the transitional group of Rickettsia basal to both Rickettsia felis and Rickettsia asembonensis. It is distinct from existing named species, with maximum average nucleotide identity of 95.1% to R. asembonensis and maximum digital DNA-DNA hybridization score similarity to R. felis at 80.1%. The closest similarity at the 16S rRNA gene (97.9%) and sca4 (97.5%/97.6% respectively) is to Candidatus 'Rickettsia senegalensis' and Rickettsia sp. cf9, both isolated from cat fleas (Ctenocephalides felis). We characterized growth at various temperatures and in multiple cell lines. The Tillamook isolate grows aerobically in Vero E6, RF/6A and DH82 cells, and growth is rapid at 28 °C and 32 °C. Using accepted genomic criteria, we propose the name Rickettsia tillamookensis sp. nov., with the type strain Tillamook 23. Strain Tillamook 23 is available from the Centers for Disease Control and Prevention Rickettsial Isolate Reference Collection (WDCM 1093), Atlanta, GA, USA (CRIRC accession number RTI001T) and the Collection de Souches de l'Unité des Rickettsies (WDCM 875), Marseille, France (CSUR accession number R5043). Using accepted genomic criteria, we propose the name Rickettsia tillamookensis sp. nov., with the type strain Tillamook 23 (=CRIRC RTI001=R5043).

Genetic diversity of vector-borne pathogens in spotted and brown hyenas from Namibia and Tanzania relates to ecological conditions rather than host taxonomy.

BACKGROUND: Improved knowledge on vector-borne pathogens in wildlife will help determine their effect on host species at the population and individual level and whether these are affected by anthropogenic factors such as global climate change and landscape changes. Here, samples from brown hyenas (Parahyaena brunnea) from Namibia (BHNA) and spotted hyenas (Crocuta crocuta) from Namibia (SHNA) and Tanzania (SHTZ) were screened for vector-borne pathogens to assess the frequency and genetic diversity of pathogens and the effect of ecological conditions and host taxonomy on this diversity. METHODS: Tissue samples from BHNA (n = 17), SHNA (n = 19) and SHTZ (n = 25) were analysed by PCRs targeting Anaplasmataceae, Rickettsia spp., piroplasms, specifically Babesia lengau-like piroplasms, Hepatozoidae and filarioids. After sequencing, maximum-likelihood phylogenetic analyses were conducted. RESULTS: The relative frequency of Anaplasmataceae was significantly higher in BHNA (82.4%) and SHNA (100.0%) than in SHTZ (32.0%). Only Anaplasma phagocytophilum/platys-like and Anaplasma bovis-like sequences were detected. Rickettsia raoultii was found in one BHNA and three SHTZ. This is the first report of R. raoultii from sub-Saharan Africa. Babesia lengau-like piroplasms were found in 70.6% of BHNA, 88.9% of SHNA and 32.0% of SHTZ, showing higher sequence diversity than B. lengau from South African cheetahs (Acinonyx jubatus). In one SHTZ, a Babesia vogeli-like sequence was identified. Hepatozoon felis-like parasites were identified in 64.7% of BHNA, 36.8% of SHNA and 44.0% of SHTZ. Phylogenetic analysis placed the sequences outside the major H. felis cluster originating from wild and domestic felids. Filarioids were detected in 47.1% of BHNA, 47.4% of SHNA and 36.0% of SHTZ. Phylogenetic analysis revealed high genetic diversity and suggested the presence of several undescribed species. Co-infections were frequently detected in SHNA and BHNA (BHNA median 3 pathogens, range 1-4; SHNA median 3 pathogens, range 2-4) and significantly rarer in SHTZ (median 1, range 0-4, 9 individuals uninfected). CONCLUSIONS: The frequencies of all pathogens groups were high, and except for Rickettsia, multiple species and genotypes were identified for each pathogen group. Ecological conditions explained pathogen identity and diversity better than host taxonomy.

Molecular investigation of tick-borne pathogens in ticks removed from tick-bitten humans in the southwestern region of the Republic of Korea.

In this study, we investigated the presence of tick-borne pathogens in ticks removed from tick-bitten humans in the southwestern provinces of the Republic of Korea (ROK). We identified 33 ticks from three tick species, namely Amblyomma testudinarium (60.6%), Haemaphysalis longicornis (27.3%), and Ixodes nipponensis (12.1%) in order of occurrence via morphology and 16S rDNA-targeting polymerase chain reaction (PCR). Tick-borne pathogens were detected in 16 ticks using pathogen-specific PCR. From the results, 12 ticks (36.4%) tested positive for spotted fever group (SFG) Rickettsia: Rickettsia monacensis (1/12), R. tamurae (8/12), and Candidatus Rickettsia jingxinensis (3/12). Three ticks (9.1%) were positive for Anaplasma phagocytophilum. In addition, three ticks (9.1%) tested positive for Babesia gibsoni (1/3) and B. microti (2/3). In conclusion, we identified three tick species; the most common species was A. testudinarium, followed by H. longicornis and I. nipponensis. SFG Rickettsia, A. phagocytophilum, and Babesia spp. were the most frequently detected pathogens in ticks removed from tick-bitten humans. To our knowledge, this is the first report of R. tamurae and Ca. R. jingxinensis detection in Korea. The present results will contribute to the understanding of tick-borne infections in animals and humans in the ROK.

Exploring the ecological and evolutionary relationships between Rickettsia and hard ticks in the Neotropical region.

This study addresses a meta-analysis of the distribution of Rickettsia spp. in the Neotropical region, as well as their associations with ticks and vertebrates. A total of 219 published reports on Rickettsia in ticks in the target region were compiled, providing 599 records of 31 species of Rickettsia recorded in 50 species of Ixodidae. The aim is to capture the phylogenetic relationships between rickettsiae and the ticks carrying them in the target region, with a focus on the co-speciation ticks-rickettsiae. We compared the phylogeny of ticks, the records of rickettsiae, the environmental gradients colonized by ticks and the effect of the phylogenetic composition of vertebrates feeding ticks on the detection of Rickettsia in ticks. Results show that differences in rickettsial composition in ticks do not depend on the vertebrate's blood-source. This is the first time this result is demonstrated. This study pinpoints that some Neotropical rickettsial organisms are associated with well-defined phylogenetical clusters of ticks. Secondarily, and probably only in a few cases, rickettsiae share species of phylogenetically distant ticks distributed along a gradient of environmental traits in which the ticks overlap (i.e., the different strains of Rickettsia parkeri sensu lato). We outline the importance of some ticks that share hosts and habitat: these ticks may act as "bridges" for the circulation of rickettsial species. There are also many species of Rickettsia that have been detected so far in only one tick species, pointing to a tight relationship or to the lack of data preventing conclusions about the detection of these bacteria in other ticks. Two species, namely Rickettsia amblyommatis and Rickettsia bellii have been recorded in the majority of ticks in the region (mainly Amblyomma spp.) and seem to be not associated with definite tick species because they may be an essential symbiont of the ticks. We conclude that an adequate analysis of rickettsiae-ticks-habitat is necessary to address the human health issues derived from the infections by rickettsiae.

Lipid A Structural Divergence in Rickettsia Pathogens.

Species of Rickettsia (Alphaproteobacteria: Rickettsiales) are obligate intracellular parasites of a wide range of eukaryotes, with recognized arthropod-borne human pathogens belonging to the transitional group (TRG), typhus group (TG), and spotted fever group (SFG) rickettsiae. Growing in the host cytosol, rickettsiae pilfer numerous metabolites to make a typical Gram-negative bacterial cell envelope. The O-antigen of rickettsial lipopolysaccharide (LPS) is immunogenic and has been shown to tether the S-layer to the rickettsial surface; however, little is known about the structure and immunogenicity of the Rickettsia lipid A moiety. The structure of lipid A, the membrane anchor of LPS, affects the ability of this molecule to interact with components of the host innate immune system, specifically the MD-2/TLR4 receptor complex. To dissect the host responses that can occur during Rickettsia in vitro and in vivo infection, structural analysis of Rickettsia lipid A is needed. Lipid A was extracted from four Rickettsia species and structurally analyzed. R. akari (TRG), R. typhi (TG), and R. montanensis (SFG) produced a similar structure, whereas R. rickettsii (SFG) altered the length of a secondary acyl group. While all structures have longer acyl chains than known highly inflammatory hexa-acylated lipid A structures, the R. rickettsii modification should differentially alter interactions with the hydrophobic internal pocket in MD2. The significance of these characteristics toward inflammatory potential as well as membrane dynamics between arthropod and vertebrate cellular environments warrants further investigation. Our work adds lipid A to the secretome and O-antigen as variable factors possibly correlating with phenotypically diverse rickettsioses.IMPORTANCE Spikes in rickettsioses occur as deforestation, urbanization, and homelessness increase human exposure to blood-feeding arthropods. Still, effective Rickettsia vaccines remain elusive. Recent studies have determined that Rickettsia lipopolysaccharide anchors the protective S-layer to the bacterial surface and elicits bactericidal antibodies. Furthermore, growing immunological evidence suggests vertebrate sensors (MD-2/TLR4 and noncanonical inflammasome) typically triggered by the lipid A portion of lipopolysaccharide are activated during Rickettsia infection. However, the immunopotency of Rickettsia lipid A is unknown due to poor appreciation for its structure. We determined lipid A structures for four distinct rickettsiae, revealing longer acyl chains relative to highly inflammatory bacterial lipid A. Surprisingly, lipid A of the Rocky Mountain spotted fever agent deviates in structure from other rickettsiae. Thus, lipid A divergence may contribute to variable disease phenotypes, sounding an alarm for determining its immunopotency and possible utility (i.e., as an adjuvant or anti-inflammatory) for development of more prudent rickettsiacidal therapies.

Rickettsiae in red fox (Vulpes vulpes), marbled polecat (Vormela peregusna) and their ticks in northwestern China.

BACKGROUND: Previously, twelve Rickettsia species were identified in ticks, fleas, sheep keds (Melophagus ovinus), bats (Pipistrellus pipistrellus) and a tick-bitten patient in the Xinjiang Uygur Autonomous Region (XUAR) in northwestern China. Here we aimed to molecularly detect rickettsial agents in red fox (Vulpes vulpes), marbled polecat (Vormela peregusna) and their ticks. METHODS: During 2018-2019, 12 red foxes, one marbled polecat and their ticks were sampled in two counties and a city of the XUAR. The heart, liver, spleen, lung and kidney of these 13 carnivores were dissected, followed by DNA extraction. Hard ticks were identified both morphologically and molecularly. All samples were examined for the presence of rickettsiae by amplifying four genetic markers (17-kDa, gltA, ompA, sca1). RESULTS: A total of 26 adult ticks and 28 nymphs (38 Ixodes canisuga, nine Ixodes kaiseri, six Haemaphysalis erinacei and one Dermacentor marginatus) were collected from red foxes, and four Ha. erinacei ticks were removed from the marbled polecat. Analysis of cytochrome c oxidase subunit I (COI) gene sequences indicated that 2-32 nucleotides differed between I. canisuga, I. kaiseri and Ha. erinacei from northwestern China and Europe. Rickettsia raoultii was detected in three red foxes, Candidatus Rickettsia barbariae in a red fox, Rickettsia sibirica in a red fox and a marbled polecat, and R. raoultii in two tick species (I. canisuga and D. marginatus). CONCLUSIONS: To the best of our knowledge, I. canisuga and I. kaiseri have not been previously reported from red foxes in China. The DNA of R. sibirica and R. raoultii was detected for the first time in the organs of red foxes, and R. sibirica in the organs of a marbled polecat. This is also the first molecular evidence for the presence of R. raoultii in I. canisuga. Our findings expand the range of tick-borne pathogens in wildlife species and associated ticks in China.

Diversity of Rickettsia in ticks collected from wild animals in Panama.

This paper presents new data about Rickettsia species detected in ticks collected from wild animals, using 16S rRNA, gltA and ompA. Rickettsia DNA was found in 66 of 101 ticks. Using EZ BioCloud libraries were produced reads that identified Rickettsia aeschlimannii, and Illumina BaseSpace produced reads of Rickettsia rickettsii group, Rickettsia bellii group, and unclassified Rickettsia. Using gltA and ompA gene-specific primers, R. aeschlimannii could not be confirmed, but detection of Rickettsia amblyommatis was achieved in Amblyomma auricularium, Amblyomma geayi, Amblyomma mixtum, and Amblyomma pacae; R. bellii from Amblyomma dissimile, "Candidatus Rickettsia colombianensi" from A. dissimile, Rickettsia spp. closely related to R. raoultii from A. geayi, Rickettsia tamurae from A. dissimile, and Rickettsia endosymbionts of Ixodes from Ixodes affinis. There were no databases available specifically for 16S rRNA of Neotropical Rickettsia, highlighting the need to use species primers over only 16S rRNA primers to achieve more accurate interpretations and identifications. These findings increase the number of Rickettsia species detected in Panama and highlight the need to establish isolates to further characterize the nature of Rickettsia in the area.

First report of the molecular detection of human pathogen Rickettsia raoultii in ticks from the Republic of Korea.

BACKGROUND: Rickettsial diseases associated with the spotted fever group constitute a growing number of newly identified Rickettsia pathogens and their tick vectors in various parts of the world. At least 15 distinct tick species belonging to six genera have shown the presence of Rickettsia raoultii. Herein, we report the detection of R. raoultii in ticks from the Republic of Korea (ROK). METHODS: Thirty-five ticks were collected from 29 patients with tick bites in Gwangju Metropolitan City, Jeollanam Province, ROK. The ticks were identified using molecular, morphological, and taxonomic characteristics. All samples were screened for presence of Rickettsia species using nested polymerase chain reactions of their outer membrane protein (ompA) and citrate synthase (gltA) genes. The amplified products were sequenced for subsequent phylogenetic analyses. RESULTS: Sequencing data showed the DNA sequences of R. raoultii in three Haemaphysalis longicornis ticks. All three tick samples were 99.4-100% similar to previously reported partial sequences of ompA of R. raoultii strains CP019435 and MF002523, which formed a single clade with the reference strains. CONCLUSIONS: We provide the first description and molecular identification of R. raoultii detected in H. longicornis ticks in the ROK. This observation extends the geographical distribution of R. raoultii. Screening of human samples for this pathogen will provide information about the prevalence of rickettsial infections in this region.

Molecular detection and genetic diversity of Rickettsia spp. in pet dogs and their infesting ticks in Harbin, northeastern China.

BACKGROUND: Pet dogs are important companion animals that share the environment within households, and play an important role in local community life. In addition, pet dogs also are reservoirs of zoonotic agents, including Rickettsia spp., thus increasing the risk of rickettsial infections in humans. It's meaningful to investigate the epidemiology of rickettsial agents in pet dogs, and make contribute to the surveillance of rickettsioses in human in China. RESULTS: In this study, a total of 496 pet dogs' blood samples and 343 ticks infested in pet dogs were collected, and the presence and prevalence of Rickettsia were determined by amplifying the partial gltA and 17-kDa genes, with an overall positive rate of 8.1 % in blood samples and 14.0 % in tick samples. In addition, the rrs, gltA, groEL, and ompA genes of rickettsial were also recovered to determine the species of Rickettsia detected furtherly. Sequencing blast and phylogenetic analyses revealed the presence of three human pathogenic Rickettsia species (Rickettsia raoultii, Candidatus Rickettsia tarasevichiae and Rickettsia felis) in samples associated with pet dogs. Moreover, all the sequences of Rickettsia that we obtained presented close relationship with others available in GenBank, and Rickettsia raoultii was the most predominant Rickettsia species infected in pet dogs' blood samples or in tick samples. CONCLUSIONS: This study provides the molecular epidemiology data about the Rickettsia spp. infection associated with pet dogs in urban areas of Harbin city. Three rickettisae species pathogenic to humans were identified from pet dogs' blood and the infested ticks in urban areas of Harbin city. Considering the intimate relationship between human and pets, these results indicate the potential transmission risk of human rickettisal infections from pet dogs through ectoparasites, and also highlighting that more attention should be paid to rickettsial infection in pet dogs and the infested ticks from the "One health" perspective.

Rickettsia-host interaction: strategies of intracytosolic host colonization.

Bacterial infection is a highly complex biological process involving a dynamic interaction between the invading microorganism and the host. Specifically, intracellular pathogens seize control over the host cellular processes including membrane dynamics, actin cytoskeleton, phosphoinositide metabolism, intracellular trafficking and immune defense mechanisms to promote their host colonization. To accomplish such challenging tasks, virulent bacteria deploy unique species-specific secreted effectors to evade and/or subvert cellular defense surveillance mechanisms to establish a replication niche. However, despite superficially similar infection strategies, diverse Rickettsia species utilize different effector repertoires to promote host colonization. This review will discuss our current understandings on how different Rickettsia species deploy their effector arsenal to manipulate host cellular processes to promote their intracytosolic life within the mammalian host.