The influence of forest habitat type on Ixodes ricinus infections with Rickettsia spp. in south-western Poland.

This study investigates the prevalence of Rickettsia spp. in Ixodes ricinus tick populations in different forest habitat types (broadleaf forest, mixed broadleaf and coniferous forest, and coniferous forest) in south-western Poland. During the survey periods from April to June 2018 and 2019 a total of 494 I. ricinus ticks, including 374 nymphs, 60 females and 60 males, were tested for Rickettsia infections by nested PCR targeting the gltA gene. The overall infection rate was 42.3%; however, we observed statistically significant year-to-year variation. Infection rates varied between tick developmental stages and were significantly influenced by forest habitat type. As assessed by a generalized linear mixed model (GLMM), the highest infection rates were observed in mixed broadleaf and coniferous forests, while coniferous forests had a significant negative effect on infection prevalence. DNA sequencing of selected samples confirmed the predominance of Rickettsia helvetica (91.2%) and less frequent Rickettsia monacensis (8.8%). This study suggests that the forest habitat types can influence Rickettsia spp. infection in tick populations; however, a comprehensive understanding of all factors influencing the level of infection requires future study.

Rickettsiosis in Denmark: A nation-wide survey.

Rickettsia helvetica has been reported at varying prevalences in Danish and other European Ixodes ricinus populations. Though apparently widespread and with reported cases of human infection, the significance of the bacteria as a threat to public health remains unclear. We present a nation-wide survey of rickettsia in ticks, roe deer and humans in Denmark. Ticks were collected by flagging and screened for presence of rickettsial DNA by polymerase chain reaction. Sera from roe deer, hunters, neuroborreliosis patients and blood donors were analyzed for presence of anti-R. helvetica and Rickettsia felis antibodies by immunofluorescence microscopy. The Rickettsia minimum infection rate in ticks was 4.9 % (367/973 pools positive, 7510 ticks in total), with 3.9 % in nymphs and 9.3 % in adults. Rickettsia helvetica accounted for 4.17 % and Rickettsia monacensis for 0.03 %, 0.6 % comprised non-differentiable rickettsial DNA. The prevalence of antibodies against R. helvetica was 2.8 % (9/319) in roe deer, while no hunters (n = 536) or blood donors (n = 181) were positive. The prevalence of anti-R. helvetica antibodies among Lyme neuroborreliosis patients was 6 % (3/47), where it co-occurred with Anaplasma phagocytophilum. Based on our study autochthonous rickettsiosis is of limited concern to the public health in Denmark, but our finding of R. monacensis for the first time in Denmark illustrates the dynamic nature of tick-borne pathogens, emphasizing that continuous surveillance is necessary.

Functional analysis of Rickettsia monacensis strain humboldt folA dihydrofolate reductase gene via complementation assay.

Nutritive symbiosis between bacteria and ticks is observed across a range of ecological contexts; however, little characterization on the molecular components responsible for this symbiosis has been done. Previous studies in our lab demonstrated that Rickettsia monacensis str. Humboldt (strain Humboldt) can synthesize folate de novo via the folate biosynthesis pathway involving folA, folC, folE, folKP, and ptpS genes. In this study, expression of the strain Humboldt folA gene within a folA mutant Escherichia coli construct was used to functionally characterize the strain Humboldt folA folate gene in vivo. The strain Humboldt folA folate gene was subcloned into a TransBac vector and transformed into a folA mutant E. coli construct. The mutant containing strain Humboldt folA subclone and a pFE604 clone of the knocked-out folA gene was cured of pFE604. Curing of the folA mutant E. coli construct was successful using acridine orange and 43.5 °C incubation temperature. The plasmid curing assay showed curing efficiency of the folA mutant at 100%. Functional complementation was assessed by growth phenotype on minimal media with and without IPTG between strain Humboldt folA and E. coli folA. Large and homogenous wild-type colony growth was observed for both strain Humboldt and E. coli folA on minimal media with 0.1 mM IPTG, wild-type growth for strain Humboldt folA and pin-point growth for E. coli folA on 0.01 mM IPTG, and pin-point growth without IPTG for both strain Humboldt and E. coli folA. This study provides evidence substantiating the in vivo functionality of strain Humboldt folA in producing functional gene products for folate biosynthesis.

Prevalence of Spotted Fever Group Rickettsia and Candidatus Lariskella in Multiple Tick Species from Guizhou Province, China.

Rickettsiales (Rickettsia spp., Ehrlichia spp., and Anaplasma spp., etc.) are generally recognized as potentially emerging tick-borne pathogens. However, some bacteria and areas in China remain uninvestigated. In this study, we collected 113 ticks from mammals in Guizhou Province, Southwest China, and screened for the Rickettsiales bacteria. Subsequently, two spotted fever group Rickettsia species and one Candidatus Lariskella sp. were detected and characterized. "Candidatus Rickettsia jingxinensis" was detected in Rhipicephalus microplus (1/1), Haemaphysalis flava (1/3, 33.33%), Haemaphysalis kitaokai (1/3), and Ixodes sinensis (4/101, 3.96%), whereas Rickettsia monacensis was positive in H. flava (1/3), H. kitaokai (2/3), and I. sinensis ticks (74/101, 73.27%). At least two variants/sub-genotypes were identified in the R. monacensis isolates, and the strikingly high prevalence of R. monacensis may suggest a risk of human infection. Unexpectedly, a Candidatus Lariskella sp. belonging to the family Candidatus Midichloriaceae was detected from Ixodes ovatus (1/4) and I. sinensis (10/101, 9.90%). The gltA and groEL gene sequences were successfully obtained, and they show the highest (74.63-74.89% and 73.31%) similarities to "Candidatus Midichloria mitochondrii", respectively. Herein, we name the species "Candidatus Lariskella guizhouensis". These may be the first recovered gltA and groEL sequences of the genus Candidatus Lariskella.

Rare Case of Rickettsiosis Caused by Rickettsia monacensis, Portugal, 2021.

We report a case of rickettsiosis caused by Rickettsia monacensis in an immunocompetent 67-year-old man in Portugal who had eschar, erythematous rash, and an attached Ixodes ricinus tick. Seroconversion and eschar biopsy led to confirmed diagnosis by PCR. Physicians should be aware of this rare rickettsiosis, especially in geographic regions with the vector.

Examination of Rickettsial Host Range for Shuttle Vectors Based on dnaA and parA Genes from the pRM Plasmid of Rickettsia monacensis.

The genus Rickettsia encompasses a diverse group of obligate intracellular bacteria that are highly virulent disease agents of mankind as well as symbionts of arthropods. Native plasmids of Rickettsia amblyommatis (AaR/SC) have been used as models to construct shuttle vectors for genetic manipulation of several Rickettsia species. Here, we report on the isolation of the complete plasmid (pRM658B) from Rickettsia monacensis IrR/Munich mutant Rmona658B and the construction of shuttle vectors based on pRM. To identify regions essential for replication, we made vectors containing the dnaA and parA genes of pRM with various portions of the region surrounding these genes and a selection reporter cassette conferring resistance to spectinomycin and expression of green fluorescent protein. Rickettsia amblyommatis (AaR/SC), R. monacensis (IrR/Munich), Rickettsia bellii (RML 369-C), Rickettsia parkeri (Tate's Hell), and Rickettsia montanensis (M5/6) were successfully transformed with shuttle vectors containing pRM parA and dnaA. PCR assays targeting pRM regions not included in the vectors revealed that native pRM was retained in R. monacensis transformants. Determination of native pRM copy number using a plasmid-carried gene (RM_p5) in comparison to chromosomally carried gltA indicated reduced copy numbers in R. monacensis transformants. In transformed R. monacensis strains, native pRM and shuttle vectors with homologous parA and dnaA formed native plasmid-shuttle vector complexes. These studies provide insight on the maintenance of plasmids and shuttle vectors in rickettsiae. IMPORTANCERickettsia spp. are found in a diverse array of organisms, from ticks, mites, and fleas to leeches and insects. Many are not pathogenic, but others, such as Rickettsia rickettsii and Rickettsia prowazeckii, can cause severe illness or death. Plasmids are found in a large percentage of nonpathogenic rickettsiae, but not in species that cause severe disease. Studying these plasmids can reveal their role in the biology of these bacteria, as well as the molecular mechanism whereby they are maintained and replicate in rickettsiae. Here, we describe a new series of shuttle plasmids for the transformation of rickettsiae based on parA and dnaA sequences of plasmid pRM from Rickettsia monacensis. These shuttle vectors support transformation of diverse rickettsiae, including the native host of pRM, and are useful for investigating genetic determinants that govern rickettsial virulence or their ability to function as symbionts.

Detection of Tick-Borne Pathogens in Ticks from Dogs and Cats in the Yamagata Prefecture of Japan in 2018.

Companion animals can become infected with tick-borne diseases (TBDs) becoming a reservoir for human transfer, thereby damaging human health. To evaluate whether companion animals are infested with ticks harboring human TBD pathogens, we detected TBD pathogens in ticks collected from dogs and cats brought to animal hospitals in the Yamagata prefecture of Japan. An investigation of 164 adult ticks collected from 88 dogs and 41 cats between March and July 2018 revealed that this region was dominated by three tick species, Ixodes ovatus (n = 95, 57.9%), Ixodes nipponensis (n = 37, 22.6%) and Haemaphysalis flava (n = 10, 6.1%). To evaluate their pathogenic potential, we went on to test each tick for spotted fever group rickettsiae, Lyme disease borreliae, relapsing fever borreliae, tick-borne encephalitis virus, and Huaiyangshan banyangvirus (formerly SFTS virus). Our results identified two I. ovatus ticks infected with Borrelia miyamotoi, which causes emerging relapsing fever; several I. nipponensis ticks infected with Rickettsia monacensis, which cause rickettsiosis; and several Ixodes persulcatus ticks infected with Rickettsia helvetica, which can also cause rickettsiosis. These results suggest that dogs and cats, and veterinary professionals and pet owners, in the Yamagata prefecture have some risk of exposure to several TBDs. This means that there should be continuous monitoring and reporting of TBDs, even those known to be uncommon in Japan, in both companion animals and humans to ensure the health and safety of both humans and animals in Japan.

Tick-borne rickettsiae in Midwestern region of Republic of Korea.

To identify spotted fever group (SFG) rickettsiae among ticks collected by dragging at eight sites in three provinces of the midwestern region of the Republic of Korea (ROK), genus- and species-specific quantitative real-time PCR (qPCR) assays and sequencing were performed. DNA was extracted from a total of 2,312 ticks that were assayed individually (n=140) or in pools (n=444), resulting in a total of 584 individual and pooled tick samples. The 584 tick samples were screened with the genus-specific qPCR assay (Rick17b) and produced 265 (45.38%) positive reactions [individual (n=64) and pooled (n=101) samples]. Of these genus-specific positive samples, 57 (21.51%) were identified as Candidatus Rickettsia longicornii and 48 (18.11%) were identified as R. monacensis by species-specific qPCR assays. Subsequently, nested PCR (nPCR) was performed with 120 samples, which tested positive samples for genus-specific, but not species-specific, qPCR assays. The sequences of ompA and ompB genes showed how many close relatedness to Ca. R. longicornii and Ca. R. jingxinensis isolate Xian Hl-79, uncultured Rickettsia sp. Y27-1, Ca. R. tasmanensis strain T152, R. endosymbiont of H. longicornis tick 47, and R. koreansis strain CNH17-7. In conclusion, we successfully detected specific rickettsial agents using qPCR and a sequence-based analysis approach that demonstrated the prevalence of various tick-borne Rickettsia spp. in midwestern ROK.

GTP cyclohydrolase I activity from Rickettsia monacensis strain Humboldt, a rickettsial endosymbiont of Ixodes pacificus.

The complete folate biosynthesis pathway exists in the genome of a rickettsial endosymbiont of Ixodes pacificus, Rickettsia monacensis strain Humboldt (formerly known as Rickettsia species phylotype G021). Recently, our lab demonstrated that the folA gene of strain Humboldt, the final gene in the folate biosynthesis pathway, encodes a functional dihydrofolate reductase enzyme. In this study, we report R. monacensis strain Humboldt has a functional GTP cyclohydrolase I (GCH1), an enzyme required for the hydrolysis of GTP to form 7,8-dihydroneopterin triphosphate in the folate biosynthesis pathway. The GCH1 gene of R. monacensis, folE, share homology with the folE gene of R. monacensis strain IrR/Munich, with a nucleotide sequence identity of 99%. Amino acid alignment and comparative protein structure modeling have shown that the FolE protein of R. monacensis has a conserved core subunit of GCH1 from the T-fold structural superfamily. All amino acid residues, including conserved GTP binding sites and zinc binding sites, are preserved in the FolE protein of R. monacensis. A recombinant GST-FolE protein from R. monacensis was overexpressed in Escherichia coli, purified by affinity chromatography, and assayed for enzyme activity in vitro. The in vitro enzymatic assay described in this study accorded the recombinant GCH1 enzyme of R. monacensis with a specific activity of 0.81 U/mg. Our data suggest folate genes of R. monacensis strain Humboldt have the potential to produce biochemically active enzymes for de novo folate synthesis, addressing the physioecological underpinnings behind tick-Rickettsia symbioses.

The Prevalence of Tick-Borne Pathogens in Ticks Collected from the Northernmost Province (Sinop) of Turkey.

Ixodes ricinus is a potential vector for some of the tick-borne microorganisms that can cause significant diseases in animals and humans. This study aims to determine the prevalence of Anaplasma, Rickettsia, Bartonella, and Francisella species in host-seeking ticks collected from the forest areas in the Sinop region located in the northernmost part of Turkey. Between May and July 2017, a total of 135 tick pools formed from 2571 of the 2734 ticks collected out of the vegetation. Samples of each pool were homogenized and analyzed by PCR. Infection prevalence was statistically analyzed in view of the maximum likelihood estimation (MLE) with a 95% confidence interval (CI). DNA of the infectious agents was determined only in the adult and nymph pools of I. ricinus. MLE values of Anaplasma spp. and Bartonella spp. in 58 pools formed from 517 of I. ricinus adults were 1.20% (95% CI: 0.50-2.49) and 0.80% (95% CI: 0.26-1.91), respectively. In 42 pools generated from 1222 of I. ricinus nymph, MLE values of infection prevalence for Anaplasma spp. and Bartonella spp. were calculated to be 0.17% (95% CI: 0.03-0.54) and 0.34% (95% CI: 0.11-0.82) in respective order. MLE values for Rickettsia spp. were 7.55% (95% CI: 5.21-10.69) and 0.52% (95% CI: 0.22-1.083) for the adult and nymph I. ricinus, respectively. The DNA of Francisella tularensis was not detected in any tick pool. The outcomes of this research are the first molecular evidence of Bartonella spp. and Bartonella henselae in questing I. ricinus in Turkey. The results also suggested that I. ricinus plays considerable roles in enzootic transmission cycles of Anaplasma phagocytophilum, B. henselae, and Rickettsia monacensis in the northernmost region of Turkey.

Isolation and characterization of a Rickettsia from the ovary of a Western black-legged tick, Ixodes pacificus.

A rickettsial isolate was obtained from a partially engorged Ixodes pacificus female, which was collected from Humboldt County, California. The isolate was provisionally named Rickettsia endosymbiont Ixodes pacificus (REIP). The REIP isolate displayed the highest nucleotide sequence identity to Rickettsia species phylotype G021 in I. pacificus (99%, 99%, and 100% for ompA, 16S rRNA, and gltA, respectively), a bacterium that was previously identified in I. pacifiucs by PCR. Analysis of sequences from complete opening frames of five genes, 16S rRNA, gltA, ompA, ompB, and sca4, provided inference to the bacteria's classification among other Rickettsia species. The REIP isolate displayed 99.8%, 99.4%, 99.2%, 99.5%, and 99.6% nucleotide sequence identity for 16S rRNA, gltA, ompA, ompB, and sca4 gene, respectively, with genes of 'R. monacensis' str. IrR/Munich, indicating the REIP isolate is closely related to 'R. monacensis'. Our suggestion was further supported by phylogenetic analysis using concatenated sequences of 16S rRNA, gltA, ompA, ompB, and sca4 genes, concatenated sequences of dksA-xerC, mppA-purC, and rpmE-tRNAfMet intergenic spacer regions. Both phylogenetic trees implied that the REIP isolate is most closely related to 'R. monacensis' str. IrR/Munich. We propose the bacterium be considered as 'Rickettsia monacensis' str. Humboldt for its closest phylogenetic relative (=DSM 103975 T = ATCC TSD-94 T).

Ticks and bacterial tick-borne pathogens in Piemonte region, Northwest Italy.

A molecular screening for tick-borne pathogens was carried out in engorged and in questing ticks collected in Verbano Cusio Ossola county, Piemonte region, Italy. Engorged ticks were removed from wild and domestic animal hosts. The most abundant and common tick species in the area was Ixodes ricinus (192 adults, 907 nymphs). Few individuals of Ixodes hexagonus (15) and Rhipicephalus sanguineus (7) were found among the ticks removed from domestic animals (46 examined ticks). The presence of Rickettsia spp., Borrelia burgdorferi sensu latu, Francisella tularensis and Coxiella burnetii was evaluated by PCR and sequencing in 392 individuals of I. ricinus (adult and nymphal stages) and 22 individuals of the two other tick species. Five Borrelia species (i.e. B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana and B. lusitaniae), proved or suspected to cause clinical manifestations of Lyme disease in humans, showed 10.5 and 2.2% combined prevalence in questing and engorged I. ricinus, respectively. In addition, two species of rickettsiae (R. helvetica and R. monacensis) were identified and reported with 14.5 and 24.8% overall prevalence in questing and in engorged ticks. The prevalence of F. tularensis in the ticks collected on two wild ungulate species (Capreolus capreolus and Cervus elaphus) was 5.7%. This work provided further data and broadened our knowledge on bacterial pathogens present in ticks in Northwest Italy.

First isolation of Rickettsia monacensis from a patient in South Korea.

A Rickettsia sp. was isolated from the blood of a patient with an acute febrile illness using the shell vial technique; the isolate was named CN45Kr and was identified by molecular assay as Rickettsia monacensis, which was first recognized as a pathogen in Spain. Sequencing analysis showed that the gltA sequence of the isolate was identical to that of Rickettsia sp. IRS3. The ompA-5mp fragment sequence showed 100% identity to those of R. monacensis and Rickettsia sp. In56 and ompA-3pA In56 and 100% identity to that of Rickettsia sp. IRS3. The ompB sequence was found to have 99.9% similarity to that of R. monacensis IrR/Munich. This study confirms the pathogenicity of this agent and provides additional information about its geographic distribution.

Rickettsia helvetica and R. monacensis infections in immature Ixodes ricinus ticks derived from sylvatic passerine birds in west-central Poland.

The aim of this study was to assess the importance of forest passerine birds in spreading ixodid ticks infected with rickettsiae of spotted fever group (SFG) in sylvatic habitats in western Poland. In total, 834 immature Ixodes ricinus ticks were found on 64 birds of 11 species which were captured during the tick-questing season between May and September of 2006. Ground-foraging passerines hosted most of the ticks compared with arboreal species, and therefore, only the former group was included into a detailed analysis. Significant predominance of larvae over nymphs was observed (581 vs. 253, respectively). Blackbirds and song thrushes hosted 82 % (n = 681) of the ticks collected from all infested passerines. The overall prevalence range of SF rickettsiae (including Rickettsia helvetica and Rickettsia monacensis) in bird-derived ticks was 10.5-26.9 %, exceeding that in questing ticks, and in ticks feeding on rodents and deer reported earlier from the same study area. This high prevalence of infection in immature I. ricinus ticks feeding on passerine birds strongly implies that they are involved in the enzootic maintenance of spotted fever group rickettsiae in the tick vector populations occurring in sylvatic habitats.

Serological and molecular evidence for spotted fever group Rickettsia and Borrelia burgdorferi sensu lato co-infections in The Netherlands.

Only a few reported cases indicate that Rickettsia helvetica and Rickettsia monacensis can cause disease in humans. Exposure to these two spotted fever group (SFG) rickettsiae occurs through bites of Ixodes ricinus, also the primary vector of Lyme borreliosis in Europe. To date, it is unclear how often exposure to these two microorganisms results in infection or disease. We show that of all the Borrelia burgdorferi s.l.-positive ticks, 25% were co-infected with rickettsiae. Predominantly R. helvetica was detected while R. monacensis was only found in approximately 2% of the ticks. In addition, exposure to tick-borne pathogens was compared by serology in healthy blood donors, erythema migrans (EM)-patients, and patients suspected of Lyme neuroborreliosis (LNB). As could be expected, seroreactivity against B. burgdorferi sensu lato was lower in blood donors (6%) compared to EM patients (34%) and suspected LNB cases (64%). Interestingly, seroreactivity against SFG Rickettsia antigens was not detected in serum samples from blood donors (0%), but 6% of the EM patients and 21% of the LNB suspects showed anti-rickettsial antibodies. Finally, the presence of B. burgdorferi s.l. and Rickettsia spp. in cerebrospinal fluid samples of a large cohort of patients suspected of LNB (n=208) was investigated by PCR. DNA of B. burgdorferi s.l., R. helvetica and R. monacensis was detected in seventeen, four and one patient, respectively. In conclusion, our data show that B. burgdorferi s.l. and SFG rickettsiae co-infection occurs in Dutch I. ricinus and that Lyme borreliosis patients, or patients suspected of Lyme borreliosis, are indeed exposed to both tick-borne pathogens. Whether SFG rickettsiae actually cause disease, and whether co-infections alter the clinical course of Lyme borreliosis, is not clear from our data, and warrants further investigation.

Prevalence and diversity of human pathogenic rickettsiae in urban versus rural habitats, Hungary.

Tick-borne rickettsioses belong to the important emerging infectious diseases worldwide. We investigated the potential human exposure to rickettsiae by determining their presence in questing ticks collected in an urban park of Budapest and a popular hunting and recreational forest area in southern Hungary. Differences were found in the infectious risk between the two habitats. Rickettsia monacensis and Rickettsia helvetica were identified with sequencing in questing Ixodes ricinus, the only ticks species collected in the city park. Female I. ricinus had a particularly high prevalence of R. helvetica (45%). Tick community was more diverse in the rural habitat with Dermacentor reticulatus ticks having especially high percentage (58%) of Rickettsia raoultii infection. We conclude that despite the distinct eco-epidemiological traits, the risk (hazard and exposure) of acquiring human pathogenic rickettsial infections in both the urban and the rural study sites exists.

Seasonal analysis of Rickettsia species in ticks in an agricultural site of Slovakia.

Many rickettsiae of the spotted fever group are emerging pathogens causing serious diseases associated with vertebrate hosts. Ixodidae ticks are known as their vectors. Investigation of the relative abundance of questing Ixodes ricinus and their infection with Rickettsia spp. in an agricultural site comprising a game reserve in Slovakia was the aim of this study. In total, 2198 I. ricinus (492 larvae, 1503 nymphs and 203 adults) were collected by flagging the vegetation along 100 m(2) transects in Rozhanovce (eastern Slovakia): 334, 595 and 1269 in 2011, 2012 and 2013, respectively. Considering questing nymphs and adults, the highest relative density of 81 individuals/100 m(2) was observed in May 2013, the lowest of 0.3 individuals/100 m(2) in March 2012. A total of 1056 ticks (853 nymphs, 100 females and 103 males; 2011: n = 329, 2012: n = 509 and 2013: n = 218) were individually screened by PCR-based methods for the presence of Rickettsia spp. The overall prevalences were 7.3% for nymphs, 15% for females, 7.8% for males; 7.0% in 2011, 8.4% in 2012, and 8.7% in 2013. The maximum prevalences were observed in July in nymphs and in May in adults. Sequencing showed infection with R. helvetica in 73 ticks (72.6% nymphs, 16.4% females, 11% males) and with R. monacensis in 11 ticks (8 nymphs, 3 females). The results showed the circulation of pathogenic Rickettsia species in the agricultural site and a potential risk for humans to encounter infected ticks.

Spotted fever group rickettsiae detected in immature stages of ticks parasitizing on Iberian endemic lizard Lacerta schreiberi Bedriaga, 1878.

Spotted fever rickettsioses are tick-borne diseases of growing public health concern. The prevalence of rickettsia-infected ticks and their ability to parasitize humans significantly influence the risk of human infection. Altogether 466 Ixodes ricinus ticks (428 nymphs and 38 larvae) collected from 73 Lacerta schreiberi lizards were examined by PCR targeting the citrate synthetase gene gltA for the presence of Rickettsia spp. Rickettsial DNA was detected in 47% of nymphs and 31.6% of larvae. They were subsequently subjected to a second PCR reaction using primers derived from the outer membrane protein rOmpA encoding gene (ompA) to detect spotted fever group rickettsiae (SFG). This analysis shows that 41.4% of nymphs and 7.9% of larvae collected from the lizards contain DNA of SFG rickettsiae. Sequencing of 43 randomly selected samples revealed two different haplotypes, both closely related to R. monacensis (39 and 4 samples, respectively). The remaining ompA negative Rickettsia spp. samples were determined to be R. helvetica based on sequencing of ompB and gltA fragments. Our results indicate that the role of Iberian endemic lizard L. schreiberi and its ectoparasites in the ecology and epidemiology of zoonotic SFG rickettsioses may be appreciable.

Detection and identification of Rickettsia species in Ixodes tick populations from Estonia.

A total of 1640 ticks collected in different geographical parts of Estonia were screened for the presence of Rickettsia species DNA by real-time PCR. DNA of Rickettsia was detected in 83 out of 1640 questing ticks with an overall prevalence of 5.1%. The majority of the ticks infected by rickettsiae were Ixodes ricinus (74 of 83), while 9 of the 83 positive ticks were Ixodes persulcatus. For rickettsial species identification, a part of the citrate synthase gltA gene was sequenced. The majority of the positive samples were identified as Rickettsia helvetica (81 out of 83) and two of the samples were identified as Rickettsia monacensis and Candidatus R. tarasevichiae, respectively. Genetic characterization based on the partial gltA gene showed that the Estonian sequences within the R. helvetica, R. monacensis and Candidatus R. tarasevichiae species demonstrated 100% similarity with sequences deposited in GenBank, originating from Rickettsia species distributed over large territories from Europe to Asia.

Are Apodemus spp. mice and Myodes glareolus reservoirs for Borrelia miyamotoi, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, R. monacensis and Anaplasma phagocytophilum?

In Europe, in addition to Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus, other zoonotic pathogens, like B. miyamotoi, a species related to the relapsing fever spirochaetes, Candidatus Neoehrlichia mikurensis (N. mikurensis), Rickettsia helvetica, Rickettsia monacensis, and Anaplasma phagocytophilum have been reported in the ixodid tick Ixodes ricinus. No study was conducted to identify reservoir hosts for these pathogens. Here, we investigated the role played by wild rodents in the natural transmission cycle of B. miyamotoi, N. mikurensis, R. helvetica, R. monacensis, and A. phagocytophilum in Switzerland. In 2011 and 2012, small mammals were captured in an area where these pathogens occur in questing ticks. Ixodes ricinus ticks infesting captured small mammals were analysed after their moult by PCR followed by reverse line blot to detect the different pathogens. Xenodiagnostic larvae were used to evaluate the role of rodents as reservoirs and analysed after their moult. Most of the 108 captured rodents (95.4%) were infested by I. ricinus ticks; 4.9%, 3.9%, 24.0%, and 0% of the rodents were infested by Borrelia, N. mikurensis, Rickettsia spp., and A. phagocytophilum-infected larvae, respectively. Borrelia afzelii, B. miyamotoi, N. mikurensis, Rickettsia spp., and A. phagocytophilum were detected in 2.8%, 0.17%, 2.6%, 6.8%, and 0% of the ticks attached to rodents, respectively. Borrelia afzelii was transmitted by 4 rodents to 41.2% of the xenodiagnostic ticks, B. miyamotoi by 3 rodents to 23.8%, and N. mikurensis was transmitted by 6 rodents to 41.0% of the xenodiagnostic ticks. None of the tested rodent transmitted Rickettsia spp. or A. phagocytophilum to I. ricinus xenodiagnostic larvae. This study showed that rodents are reservoir hosts for B. miyamotoi and N. mikurensis in Europe.