A search for the epidemic typhus agent in Ethiopian ticks

The presence of antibodies to Rickettsia prowazeki in domestic animals from several parts of Africa, and the isolation of this rickettsia from the blood of goats and sheep and from ticks off cattle or camels in Ethiopia, led to the hypothesis that R. prowazeki in nature may occur in an extrahuman cycle involving ticks and domestic animals. This study attempted to recover R. prowazeki from 2 624 ticks (4 genera, 10 species) collected in central and southern Ethiopia. The ticks were examined by the haemolymph test and by the injection of tissues into guineapigs. No strains of typhus rickettsia were received and there was no serologic evidence suggesting the presence of this agent in any of the ticks examined. One Amblyomma cohaerens contained an organism that reacted specifically with fluorescing antibodies against R. prowazeki; attempts to isolate and identify this agent failed. Fifty-seven (2.2%) Amblyomma ticks (26 A. gemma, 17 A. variegatum, 14 A. cohaerens) were infected with rickettsiae of the spotted fever group, and probably represented R. conori or closely related rickettsial agents.     

Borrelia in Ethiopian ticks

Two regions (Jimma and Dire Dawa) in Ethiopia were investigated for the presence of soft ticks. Although no Ornithodoros spp. ticks were collected during this survey, published records of their existence in Ethiopia were found. An overwhelming infestation of Argas persicus was revealed in a village located adjacent to Dire Dawa. These ticks primarily were feeding on poultry, but were also biting humans. Furthermore, hard ticks were collected from livestock and companion animals in these regions.Collected ticks were assessed for Borrelia by real-time PCR followed by conventional PCR and sequencing to identify species present. A. persicus ticks were found to carry B. anserina in 3 of 40 (7.5%) A. persicus tick pools, whilst hard tick pools yielded 2 of 16 (12.5%) positive for B. theileri. Collectively, these borrelial species and their tick vectors are likely to have an important economic impact of particular relevance to subsistence farmers in Ethiopia.     

Rickettsia spp. in Rhipicephalus sanguineus sensu lato ticks collected from stray dogs in Kerman city,Iran

Rickettsial diseases are recognized as one of the most important vector-borne infectious diseases for humans all over the world. Dogs and their ticks are considered the most important reservoirs for Rickettsia spp., especially in spotted fever group rickettsioses. The aim of the study was to investigate Rickettsia infections in ticks collected from stray dogs in southeastern Iran. In this study, 50 stray dogs in Kerman city were randomly selected, of 68% and 52% of which were above 8 months age and male, respectively. Ticks were collected from the dog skins. After identification of collected ticks, genomic DNA of all ticks was extracted. DNA samples were tested using real-time PCR for Rickettsia spp. infections. The species of Rickettsia in positive samples were determined using gltA gene amplification and sequencing. A total of 250 ticks were collected from 50 stray dogs and all of them belonged to Rhipicephalus sanguineus sensu lato. Totally, 10 pooled of 50 pooled ticks were positive for Rickettsia spp. in real-time PCR and the minimal Rickettsial infection rate was 4% in this study. The identified Rickettsia spp. included R. massiliae (n = 5), R. rhipicephali (n = 1), and R. sibirica (n = 1). In this study, molecular evidence of Rickettsia spp. infection was observed in collected ticks from stray dogs in southeast Iran. More sensitivity to human and animal health care systems in southeastern Iran is essential to the diagnosis of suspected clinical cases that are related to rickettsiosis.     

Spotted fever group rickettsiae identified in Dermacentor marginatus and Ixodes ricinus ticks in Algeria

Our study was carried out using Ixodes ricinus ticks collected from cattle from Tizi-Ouzou and Dermacentor marginatus ticks collected from the vegetation of the Blida region, a tourist site, both regions situated in northern Algeria. The results of real-time quantitative PCR (qPCR) specific for a partial sequence of the citrate synthase gene (gltA) indicate that Rickettsia spp. were present in 11/23 (48%) and 4/9 (44%) of the examined ticks from Tizi-Ouzou and Blida, respectively. The sequences of Rickettsia helvetica and Ri. monacensis were found in I. ricinus ticks using gltA primers. In addition, Ri. slovaca was detected based on the sequences of the gltA and the outer membrane protein (OmpA) genes in D. marginatus ticks. DNA sequencing to identify the species revealed for the first time the presence of Ri. helvetica in I. ricinus ticks and Ri. slovaca in D. marginatus ticks from Algeria and confirmed the presence of Ri. monacensis.     

Genetic variability of Rickettsia spp. in Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia,Russia: Identification of a new Candidatus Rickettsia species

Rickettsia spp. are the causative agents of a number of diseases in humans. These bacteria are transmitted by arthropods, including ixodid ticks. DNA of several Rickettsia spp. was identified in Ixodes persulcatus ticks, however, the association of Ixodes trianguliceps ticks with Rickettsia spp. is unknown. In our study, blood samples of small mammals (n = 108), unfed adult I. persulcatus ticks (n = 136), and I. persulcatus (n = 12) and I. trianguliceps (n = 34) ticks feeding on voles were collected in two I. persulcatus/I. trianguliceps sympatric areas in Western Siberia. Using nested PCR, ticks and blood samples were studied for the presence of Rickettsia spp. Three distinct Rickettsia species were found in ticks, but no Rickettsia species were found in the blood of examined voles. Candidatus Rickettsia tarasevichiae DNA was detected in 89.7% of unfed I. persulcatus, 91.7% of engorged I. persulcatus and 14.7% of I. trianguliceps ticks. Rickettsia helvetica DNA was detected in 5.9% of I. trianguliceps ticks. In addition, a new Rickettsia genetic variant was found in 32.4% of I. trianguliceps ticks. Sequence analysis of the 16S rRNA, gltA, ompA, оmpB and sca4 genes was performed and, in accordance with genetic criteria, a new Rickettsia genetic variant was classified as a new Candidatus Rickettsia species. We propose to name this species Candidatus Rickettsia uralica, according to the territory where this species was initially identified. Candidatus Rickettsia uralica was found to belong to the spotted fever group. The data obtained in this study leads us to propose that Candidatus Rickettsia uralica is associated with I. trianguliceps ticks.     

Exposure of dogs and cats to Borrelia miyamotoi infected Ixodes ricinus ticks in urban areas of the city of Poznań, west-central Poland

Borrelia miyamotoi is an emerging human pathogen that causes a relapsing fever-like disease named B. miyamotoi disease. The bacterium belongs to the relapsing fever borreliae, and similar to spirochetes of the Borrelia burgdorferi sensu lato group, it is transmitted only by hard ticks of the Ixodes ricinus complex. To date, B. miyamotoi has not been demonstrated to cause illness in dogs or cats, and is poorly documented in veterinary medicine. The aim of this study was to determine the B. miyamotoi presence in (i) host-seeking ticks and (ii) engorged Ixodes sp. ticks collected from dogs and cats during their inspection in veterinary clinics of the city of Poznań, west-central Poland. Host-seeking ticks were sampled in dog walking areas localized in urban forested recreational sites of the city. In this study, 1,059 host-seeking and 837 engorged I. ricinus ticks collected from 680 tick-infested animals (567 dogs and 113 cats) were screened. Additionally, 31 I. hexagonus ticks (one larva, 13 nymphs, and 17 females) were collected from three cats; one larva and one nymph were collected from two dogs; and one dog was infested with a single Dermacentor reticulatus female.Borrelia DNA was identified by the amplification and sequencing of the V4 hypervariable region of the 16S rRNA gene and flaB gene fragments. DNA of B. miyamotoi was detected in 22 (2.1%) of the host-seeking ticks (in all developmental tick stages and in all study areas). In addition, the engorged I. ricinus ticks exhibited a similar B. miyamotoi presence (1.8%). Fifteen I. ricinus ticks collected from animals tested positive for the presence of B. miyamotoi DNA, and the DNA of B. miyamotoi was observed in three (9.1%; one female and two nymphs) I. hexagonus ticks. The single D. reticulatus female collected from a dog tested PCR-negative for the bacterium. The results of this study demonstrated the establishment and broad presence of the bacterium in tick populations from different urban ecosystems of the city of Poznań. The lack of difference in the mean infection presence of animal-derived and host-seeking I. ricinus ticks suggests that the systematic surveillance of pets may be useful for the evaluation of human exposure to B. miyamotoi infected ticks in urban areas. Additional studies are required to further elucidate the role of domestic and wild carnivores in the epidemiology of B. miyamotoi, which remains unknown.     

Coxiella symbiont in the tick Ornithodoros rostratus (Acari: Argasidae)

In the present study, the presence of tick-associated bacteria and protozoa in Ornithodoros rostratus ticks (adults, nymphs, and eggs) from the Pantanal region of Brazil were determined by molecular detection. In these ticks, DNA from protozoa in the genera Babesia and Hepatozoon, and bacteria from the genera Rickettsia, Borrelia, Anaplasma, and Ehrlichia were not detected. Conversely, all tested ticks (100%) yielded PCR products for 3 Coxiella genes (16S rRNA, pyrG, cap). PCR and phylogenetic analysis of 3 amplified genes (16S rRNA, pyrG, cap) demonstrated that the agent infecting O. rostratus ticks was a member of the genus Coxiella. This organism grouped with Coxiella symbionts of other soft tick species (Argasidae), having different isolates of C. burnetii as a sister group, and these 2 groups formed a clade that grouped with another clade containing Coxiella symbionts of hard tick species (Ixodidae). Analysis of tick mitochondrial 16S rRNA gene database composed mostly of tick species previously shown to harbor Coxiella symbionts suggests a phylogenetic congruence of ticks and their Coxiella symbionts. Furthermore, these results suggest a very long period of coevolution between ticks and Coxiella symbionts and indicates that the original infection may have occurred in an ancestor common to the 2 main tick families, Argasidae (soft ticks) and Ixodidae (hard ticks). However, this evolutionary relationship must be confirmed by more extensive testing of additional tick species and expanded populations.     

First report of Candidatus Rickettsia mendelii in Ixodes brunneus from the United States

Candidatus Rickettsia mendelii is a novel rickettsial species recently identified in Ixodes ricinus. In this study, Ixodes brunneus collected from wild birds (n = 77 ticks) or vegetation (n = 4 ticks) in southeastern Virginia were surveyed for rickettsial agents. Candidatus Rickettsia mendelii was confirmed in I. brunneus through sequencing of the gltA and 16S rRNA genes. This is the first report of this rickettsial species in Ixodes ticks in North America.     

Molecular and MALDI-TOF MS characterisation of Hyalomma aegyptium ticks collected from turtles and their associated microorganisms in Algeria

The identification of ticks and their associated pathogens is important for knowledge on tick-borne diseases. The objective of this study was to use morphological, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and/or molecular biology tools to identify ticks collected from turtles in north-eastern Algeria, as well as to investigate the microorganisms associated with these ticks. A total of 471 adult ticks were collected and identified morphologically as Hyalomma aegyptium, of which 248 (52.7%) were female and 223 (47.3%) were male. amongst them, 230 specimens were randomly selected for molecular and MALDI-TOF MS analysis. Molecular biology confirmed that our ticks were Hy. aegyptium. MALDI-TOF MS analysis revealed that 100% of the spectra were of excellent quality. Four spectra were selected to update our own database MALDI-TOF MS arthropod. The blind test of the 226 remaining spectra showed that all ticks were correctly identified, with scores ranging from 1.774 to 2.655 with a mean of 2.271 ± 0.16 of which, 223 (98.6%) had log score value (LSV)>1.8. Molecular biology screening showed that the ticks carried the DNA of Borrelia turcica, Rickettsia africae, Rickettsia aeschlimannii, Rickettsia sibirica mongolitimonae and with the Anaplasmataceae were close to a potentially new, undescribed Ehrlichia sp. This study confirms that MALDI-TOF MS is a reliable tool for the identification of ticks and that ticks collected from turtles in Algeria are carriers of several species of microorganisms which may be responsible for diseases in humans and animals.     

Turkey tick news: A molecular investigation into the presence of tick-borne pathogens in host-seeking ticks in Anatolia; Initial evidence of putative vectors and pathogens,and footsteps of a secretly rising vector tick,Haemaphysalis parva

This Turkey-based study investigated the presence of various tick-borne microorganisms in a broad-range of host-seeking ticks (n = 1019) that exhibit both hunter and ambusher characteristics. All collected ticks were analyzed individually via PCR-sequencing, resulting in the identification of 18 different microorganisms: six Babesia spp., including one putative novel species (Ba. occultans, Ba. crassa, Ba. rossi, Babesia sp. tavsan1, Babesia sp. tavsan2, and Babesia sp. nov.); six SFG rickettsiae (Ri. aeschlimannii, Ri. s. mongolitimonae, Ri. slovaca, Ri. raoultii, Ri. monacensis, and Ri. hoogstraalii); two Borrelia burgdorferi sensu lato spp. (Bo. afzelii and Bo. lusitaniae); two unnamed Hepatozoon spp.; Theileria annulata; and Hemolivia mauritanica. This provided evidence for the natural transstadial survival of these tick-borne microorganisms in adult ticks (in addition a nymph) of Turkey. Surprisingly, this study determined the presence of five different microorganisms (Ba. crassa, Ba. rossi, Babesia sp. Ucbas, Hepatozoon sp., and Ri. hoogstraalii) in host-seeking Haemaphysalis parva adults, for which poor data exist on its vectorial competence. Therefore, this study provides important data indicating the potential vectorial capacity of Ha. parva. This study also revealed the presence of the close ecological and evolutionary relationships between two important vector ticks, Hyalomma marginatum and Hy. aegyptium and determined genetic variations (distinct phylogenetic divergences inside the main clades) in some pathogenic SFG rickettsiae that are found in these ticks. Additionally, the presence of two Babesia species described very recently in hares with unknown vectors, namely Babesia sp. tavsan1 and Babesia sp. tavsan2, were detected for the first time in ticks. Finally, two unnamed Hepatozoon spp. were detected in Haemaphysalis ticks and their phylogenetic positions were demonstrated. Consequently, this study provides important data on the diversity of tick-borne microorganisms in host-seeking ticks and on potentially novel microorganisms (Babesia and Hepatozoon species) and their possible vectors (Ha. parva, Ha. sulcata, Hy. aegyptium, Hy. marginatum, and Rh. turanicus).     

New records of Amblyomma gervaisi from Pakistan,with detection of a reptile-associated Borrelia sp.

Ticks are hematophagous ectoparasites of terrestrial and semi-aquatic vertebrates that may transmit microorganisms to their hosts. Spirochetes of the genus Borrelia are common in ticks and an incipient group has been identified in association with reptiles and their tick parasites. To overcome the knowledge deficit, this study aimed to morphologically and molecularly identify ticks infesting wild lizards and to molecularly assess Borrelia spp. associated with these ticks in Pakistan. For this purpose, free-ranging monitor lizards (Varanus bengalensis) from Khyber Pakhtunkhwa province, Pakistan, were examined for tick infestations. A total of 776 ticks were collected from 36/63 lizards, resulting in a prevalence of 57% (95% CI 44.7-69.3%), overall mean intensity of 21.5 (95% CI 18.9-24.1) ticks per infested lizard, and overall mean abundance of 12.3 (95% CI 9.25-15.4) ticks per examined lizard. All ticks were morphologically identified as Amblyomma gervaisi. The morphological identification of the ticks was molecularly confirmed through sequencing fragments of the mitochondrial 16S rRNA gene. In addition, a fragment of nuclear second internal transcribed spacer (ITS2) was generated for the first time for A. gervaisi. Phylogenetic analysis inferred from tick 16S rRNA gene partial sequences predicted a close evolutionary relationship of the collected A. gervaisi ticks with conspecific sequences from India, which shared 94.5% identity. Through two PCR assays targeting fragments of the borrelial genes, 16S rRNA and flaB, 19 (18%) out of 108 ticks yielded borrelial DNA. Phylogenetic analyses inferred from DNA sequences of the two borrelial genes revealed that the Borrelia sp. from A. gervaisi detected in this study belonged to the reptile-associated Borrelia group (REP). This is the first molecular report of ticks infesting monitor lizards and associated Borrelia sp. in Pakistan. The preliminary phylogenetic analyses of A. gervaisi may assist in understanding the molecular epidemiology of Amblyomma spp.     

Tick-borne rickettsiae in Guinea and Liberia

While the high seroprevalence for the rickettsiae that cause spotted fevers and the multiple pathogenic rickettsiae is known, the data on the distribution of rickettsial diseases in Africa are often incomplete. We collected ticks from domestic or wild animals (generally a source of bushmeat) that were in contact with humans in 2 neighboring countries of tropical West Africa, Guinea and Liberia. In total, 382 ticks representing 6 species were collected in Liberia and 655 ticks representing 7 species were collected in Guinea. We found rickettsiae in 9 different species of ticks from both countries. Rickettsia africae was found in 93–100% of Amblyomma variegatum, in 14–93% of Rhipicephalus (B.) geigyi, Rh. (B.) annulatus, and Rh. (B.) decoloratus, and in several Hyalomma marginatum rufipes and Haemaphysalis paraleachi. A genetic variant of R. africae was found in Amblyomma compressum. R. massiliae was found in 10/61 (16%) of Rh. senegalensis ticks and in 2% of Haemaphysalis paraleachi ticks collected from dogs. We identified a new rickettsia in one of 44 (2%) Ixodes muniensis collected from a dog in Liberia. As this rickettsia is not yet isolated, we propose the provisional name “Candidatus Rickettsia liberiensis” (for the West African country where the host tick was collected).     

Detection of Rickettsia spp. and Anaplasma phagocytophilum in Ixodes ricinus ticks in a region of Middle Germany (Thuringia)

Rickettsia spp. and Anaplasma spp. are regarded as potentially emerging tick-borne pathogens, but so far data on prevalence rates in questing ticks and reports on human diseases in several parts of Europe are rarely available.In this study, 430 nymphs and 570 adult Ixodes (I.) ricinus ticks were collected from a frequently visited forest region of Thuringia (Zeitzgrund, near Stadtroda) in 2006 (n=506) and 2007 (n=494). Individual ticks were investigated for a part of the citrate synthase gene (gltA) of Rickettsia spp. and the 16S rRNA gene of Anaplasma phagocytophilum. Positive amplicons were identified with restriction fragment length polymorphism (RFLP) and/or sequencing. Overall, 14.7% (147/1000) of investigated ticks were infected with Rickettsia spp. After sequencing of 64/147 positive amplicons R. helvetica (29/64) was detected predominantly. Prevalence varied in different developmental stages between 9.3% (40/430) in nymphs and 18.8% (107/570) in adults. A. phagocytophilum-specific DNA was detected in 5.4% (54/1000) of ticks with an infection rate of 4.7% (20/430) in nymphs and 6.0% (34/570) in adults. In 1% (10/1000) of ticks coinfections with Rickettsia spp. and A. phagocytophilum were found. Our study provides interesting insights into the circulation and cocirculation of different rickettsial species and A. phagocytophilum in the same biotope.     

Molecular characterization of Anaplasma marginale in ticks naturally feeding on buffaloes

Anaplasma marginale is the most prevalent pathogen transmitted by ticks in cattle in tropical and subtropical regions of the world. However, the tick species involved in the transmission of A. marginale in buffaloes in Brazil have not been identified. The objective of the present study was to determine the presence of A. marginale in ticks parasitizing water buffaloes. A total of 200 samples of Rhipicephalus microplus, Dermacentor nitens, Amblyomma cajennense, and Amblyomma maculatum were collected and tested by conventional and quantitative PCR for the presence of the msp1a and msp5 genes. In the present study, 35 ticks (17.5%) were positive for A. marginale DNA by qPCR analysis. The positive ticks belonged to four different species: R. microplus (22.2%), A. cajennense (13.8%), A. maculatum (16.0%), and D. nitens (10.0%). Individuals of the three developmental stages (larvae, nymphs, and adults) of R. microplus and A. cajennense were found to be positive for A. marginale, only nymphs and adults of A. maculatum were found to be positive, and finally, only adults of D. nitens were positive for A. marginale. Our results suggest that R. microplus, A. cajennense, A. maculatum, and D. nitens ticks may be involved in the transmission of A. marginale in buffaloes. However, while A. marginale PCR positive ticks were recorded, this does not indicate vector competence; only that the ticks may contain a blood meal from an infected host. Additionally, the results show that the strains of A. marginale from buffaloes and cattle are phylogenetically related.     

Molecular identification of tick-borne pathogens (Rickettsia spp., Anaplasma phagocytophilum,Borrelia burgdorferi sensu lato,Coxiella burnetii and piroplasms) in questing and feeding hard ticks from North-Western Spain

The occurrence of tick-borne pathogens (TBPs) of human and veterinary interest was studied in questing and feeding ticks collected from wild animals in a region in North-Western Spain. A total of 529 ticks (489 questing, 40 feeding) of seven different species (386 Ixodes ricinus, 53 Haemaphysalis concinna, 27 Haemaphysalis punctata, 25 Dermacentor marginatus, 21 Haemaphysalis inermis, 15 Dermacentor reticulatus, and two Rhipicephalus bursa) were analyzed. Molecular analysis of the 16S rRNA gene in I. ricinus ticks, revealed the presence of two phylogenetic groups in the region. Most of the sequenced ticks (96%) were assigned to I. ricinus haplogroup and 4% of the ticks were phylogenetically related to I. inopinatus haplogroup. Feeding ticks were removed from 17 animals from seven wild species (seven roe deer -Capreolus capreolus-, three wolves -Canis lupus-, two Iberian red deer -Cervus elaphus hispanicus-, two European wild boar -Sus scrofa-, one Cantabrian brown bear -Ursus arctos-, one Eurasian badger -Meles meles-, and one red fox -Vulpes vulpes-). Presence of Rickettsia spp., Anaplasma phagocytophilum, piroplasms, Borrelia burgdorferi sensu lato (s.l.) and Coxiella burnetii were tested in ticks by specific PCR. A total of 92 (17.4%) of the 529 ticks analyzed were positive for at least one of the TBPs tested. Sequencing revealed the presence of the genospecies “Candidatus Rickettsia rioja”, Rickettsia raoultii, and Anaplasma phagocytophilum in both questing and feeding ticks. Rickettsia slovaca, Borrelia lusitaniae, Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi sensu stricto and Babesia bigemina were only detected in questing ticks, while Babesia sp. badger type A, Theileria OT3 and Hepatozoon canis occurred only in engorged ticks. None of the ticks were positive for C. burnetii. The analysis of the 16S rRNA gene sequences of A. phagocytophilum revealed the presence of three variants (I, X and W) circulating in the region. New host-tick-pathogen interactions have been revealed, finding for the first time the human pathogen R. raoultii in D. reticulatus removed from a Cantabrian brown bear. Co-occurrence between different TBPs were detected in 4.3% of the ticks. The association B. burgdorferi s.l./Rickettsia spp. was detected in questing ticks; and Rickettsia spp./piroplasms and A. phagocytophilum/Theileria OT3 in feeding ticks. The presence of pathogenic agents constitutes a threat to human and animal health, and should be considered in the diagnosis and treatment after a tick bite. This study increases the knowledge on TBPs diversity of medical and veterinary interest circulating between ticks and their hosts in North-Western Spain.     

Importation of Hyalomma marginatum,vector of Crimean-Congo haemorrhagic fever virus,into the United Kingdom by migratory birds

Hyalomma marginatum ticks are an important vector of Crimean-Congo haemorrhagic fever virus which can result in a severe and potentially fatal disease in humans. Given the continued emergence of clinical cases in Eurasia and focalised upsurges of H. marginatum populations in Europe, it seemed prudent to assess the potential of this vector species to be introduced into the United Kingdom. Immature forms of H. marginatum are frequent ectoparasites of passerine birds many of which migrate from Africa to the UK each spring. Incoming birds were inspected for ticks during the spring migration in 2010 and 2011. A total of 68 ticks was collected from 971 birds (29 bird species), 21% (14) of the ticks were identified as H. marginatum. Oenanthe oenanthe (Northern wheatear) and Sylvia communis (Whitethroat) were found to be infested by this tick in both years and with multiple ticks. Single specimens were also removed from Acrocephalus schoenobaenus (Sedge warbler) and Phoenicurus phoenicurus (Common redstart) in 2010. This study provides the first contemporary evidence for substantial importation of this tick species into the UK.     

First detection of “Candidatus Rickettsia wissemanii” in Ornithodoros hasei (Schulze, 1935) (Acari: Argasidae) from Argentina

The aim of this study was to assess the presence of Rickettsia in soft ticks (Acari: Argasidae) collected from insectivorous bats (Chiroptera) in Santa Fe province, Argentina. First, a subset of ticks were mounted in Hoyer’s medium to be determined by morphological characters and then confirmed by sequencing the mitochondrial 16S rRNA gene. Also ticks were processed by PCR assays using primers CS-78 and CS-323, which amplify a fragment of the Rickettsia spp. gltA gene. Positive ticks were subjected to a second PCR round with primers Rr190.70p and Rr190.701n of the spotted fever group rickettsiae ompA gene. A phylogenetic analysis was performed with Maximum-likelihood method, and the best fitting substitution models were determined with the Akaike Information Criterion. Five bats of the species Eptesicus diminutus Osgood, 1915, Eptesicus furinalis (d’Orbigny and Gervais, 1847), Eptesicus spp. (Vespertilionidae), and Molossops temminckii Burmeister, 1854 (Molossidae) were parasitized with Ornithodoros hasei (Schulze, 1935) larvae. One E. diminutus ticks’ tested positive to “Candidatus Rickettsia wissemanii”, a spotted fever group rickettsiae. The association O. hasei –“Ca. R. wissemanii” detected in this study represents the first evidence of a Rickettsia in Ornithodoros ticks in Argentina and the third report of this association in America. Also, this finding constitutes the first record of “Ca. R. wissemanii” in Argentina. Finally, we found for the first time the insectivorous bats E. diminutus and E. furinalis parasitized with O. hasei larvae. These findings add two new hosts and a new location, the southernmost recorded to date, for O. hasei.     

Growth dynamics and tissue localization of a Coxiella-like endosymbiont in the tick Haemaphysalis longicornis

A Coxiella-like endosymbiont (Coxiella-LE hereinafter) stably infects and influences Haemaphysalis longicornis development, indicating a mutualistic relationship of Coxiella-LE and ticks. To further elucidate the patterns of growth dynamics and tissue localization of Coxiella-LE in H. longicornis, 16S rRNA high-throughput sequencing, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH) were used in this study. The density of Coxiella-LE varied among different tick life stages, and fed female ticks had the highest density, followed by unfed female and unfed larval ticks. In the four organs that were dissected from fed female ticks, the ovary carried the highest density of Coxiella-LE, which was significantly different from salivary glands, midgut and Malpighian tubules. The high abundance of Coxiella-LE in fed female ticks and in the ovaries of fed female ticks in the bacterial microbiota analyses further confirmed that Coxiella-LE rapidly proliferates in the ovary after blood feeding. The ovaries continued to develop after engorgement and oviposition began on day 5, with a significant decrease in the density of Coxiella-LE in the ovaries occurring on day 7. FISH results indicated that Coxiella-LE is mainly colonized in the cytoplasm of the oocyte and proliferates with oogenesis. Coxiella-LE was expelled from the body with the mature oocyte, ensuring its vertical transmission. In the Malpighian tubules at different days after engorgement, the white flocculent materials were increasing, and the density of Coxiella-LE raised significantly on day 7. Unlike the localization pattern in the ovary, Coxiella-LE was initially distributed in a mass and continually increased during the development of Malpighian tubules until it filled the Malpighian tubules. These findings provide new insights on the growth dynamics and tissue localization of Coxiella-LE in ticks and are useful for further investigation on the interactions of symbiont and ticks .     

Spotted fever group rickettsiae in ticks and fleas from the Democratic Republic of the Congo

Little is known about the prevalence of spotted fever group (SFG) rickettsiae in ticks and fleas in the Democratic Republic of the Congo. In 2008, 12 Amblyomma compressum ticks were collected from 3 pangolins (Manis gigantea). Two Haemaphysalis punctaleachi ticks were collected from 2 African civets (Civettictis civetta congica), and one was collected from an antelope (Onotragus leche). A total of 111 Rhipicephalus sanguineus ticks, 23 Ctenocephalides canis fleas, 39 C. felis fleas, and 5 Trichodectes canis lice were sampled from 19 dogs. One C. canis flea was collected from a human. Six of the 12 A. compressum ticks were positive for rickettsial DNA, as determined by genus-specific qPCR. The ompA gene sequences amplified from positive samples showed 100% homology with Rickettsia africae (GenBank accession number CP001612). The detection of Ri. africae in A. compressum ticks, which are highly specialized parasites of pangolins, is consistent with our previous data showing the presence of Ri. africae in A. compressum ticks from Liberia. No other ticks contained rickettsial DNA. A total of 9 C. canis fleas (39%, 9/23) and 37 C. felis fleas (95%, 37/39) that was collected from dogs and one C. canis flea collected from a human harbored Ri. felis.     

Molecular detection of Rickettsia,Anaplasma, and Bartonella in ticks from free-ranging sheep in Gansu Province,China

Ticks pose a serious threat to public health as carriers and often vectors of zoonotic pathogens. There are few systematic studies on the prevalence and genetic diversity of tick-borne bacterial pathogens in Western China. In this study, 465 ticks were collected from free-ranging sheep in Gansu Province in China. Ticks were divided into 113 pools and tick DNA was extracted from these ticks. PCR assays were performed using specific primers to screen for tick-borne pathogens as well as sequence analysis based on the 16S rRNA (rrs), ompB, gltA, ompA genes for Rickettsia, rrs, groEL genes for Anaplasma, and ssrA and rpoB genes for Bartonella. The PCR results showed that the minimum infection rates with Rickettsia, Anaplasma, and Bartonella were 16.8% (78/465), 18.9% (88/465), and 0.9% (4/465), respectively. Sequence analysis based on the concatenated sequences of rrs-ompB-gltA-ompA indicated that the Rickettsia species identified in the ticks belonged to Rickettsia raoultii, Rickettsia slovaca, and Rickettsia sibirica, respectively; phylogenetic analysis based on the groEL gene showed that all Anaplasma strains identified were Anaplasma ovis; and phylogenetic analysis based on the ssrA and rpoB genes indicated that all Bartonella strains in the ticks belonged to Bartonella melophagi. The results of this study showed that ticks in Gansu Province harbored multiple pathogens that may cause rickettsial diseases and bartonellosis. These diseases were neglected in the area and physicians and public health workers need to pay attention to their diagnoses to prevent human infection.