First Detection and Genetic Identification of Wolbachia Endosymbiont in Field-Caught Aedes aegypti (Diptera: Culicidae) Mosquitoes Collected from Southern Taiwan.

The prevalence and genetic character of Wolbachia endosymbionts in field-collected Aedes aegypti mosquitoes were examined for the first time in Taiwan. A total of 665 Ae. aegypti were screened for Wolbachia infection using a PCR assay targeting the Wolbachia surface protein (wsp) gene. In general, the prevalence of Wolbachia infection was detected in 3.3% Ae. aegypti specimens (2.0% female and 5.2% male). Group-specific Wolbachia infection was detected with an infection rate of 1.8%, 0.8% and 0.8% in groups A, B and A&B, respectively. Genetic analysis demonstrated that all Wolbachia strains from Taiwan were phylogenetically affiliated with Wolbachia belonging to the supergroups A and B, with high sequence similarities of 99.4-100% and 99.2-100%, respectively. Phylogenetic relationships can be easily distinguished by maximum likelihood (ML) analysis and were congruent with the unweighted pair group with the arithmetic mean (UPGMA) method. The intra- and inter-group analysis of genetic distance (GD) values revealed a lower level within the Taiwan strains (GD < 0.006 for group A and GD < 0.008 for group B) and a higher level (GD > 0.498 for group A and GD > 0.286 for group B) as compared with other Wolbachia strains. Our results describe the first detection and molecular identification of Wolbachia endosymbiont in field-caught Ae. aegypti mosquitoes collected from Taiwan, and showed a low Wolbachia infection rate belonging to supergroups A and B in Ae. aegypti mosquitoes.

First report and molecular identification of a reptilian tick, Amblyomma helvolum (Acari: Ixodidae), infesting wild green iguanas, Iguana iguana (Reptilia: Iguanidae), in southern Taiwan.

The genetic identity of the reptilian tick, Amblyomma helvolum, infesting wild green iguanas (Iguana iguana) in Taiwan, was examined. Genetic identity was determined by analyzing the 16S mitochondrial DNA gene sequences obtained from 11 Taiwan A. helvolum compared with other Amblyomma species, with two Dermacentor species and two Rhipicephalus species serving as outgroups. Phylogenetic analysis revealed that all the Taiwan specimens were genetically affiliated with a monophyletic group of A. helvolum and can be discriminated from other Amblyomma species. Our results provide the first genetic identification of adult A. helvolum ticks infesting wild iguanas in Taiwan. Further studies focused on the seasonal prevalence and vectorial capacity of A. helvolum for various tick-borne pathogens will help to clarify the epidemiological significance of this species and its impact on animal and human health in Taiwan.

First detection and molecular identification of Rickettsia massiliae, a human pathogen, in Rhipicephalus sanguineus ticks collected from Southern Taiwan.

The Rickettsia massiliae was firstly detected and identified in Rhipicephalus sanguineus ticks infested on dogs in Taiwan. A total of 1154 Rh. sanguineus ticks collected from 158 dogs of four districts of Tainan city were examined for Rickettsia infection by nested-PCR assay targeting the citrate synthase (gltA) and outer membrane protein B (ompB) genes of Rickettsia. The Rickettsia infection was detected with a general infection rate of 2.77%, and was detected in male, female and nymphal stage with an infection rate of 2.77%, 3.22% and 1.32%, respectively. Phylogenetic relationships were analyzed by comparing the gltA and ompB sequences obtained from 9 Taiwan strains and 16 other strains representing 13 genospecies of Rickettsia. Results revealed that all Taiwan strains were genetically affiliated to the same clades of R. massiliae (spotted fever group) and R. felis (transitional group), and can be discriminated from other genospecies of Rickettsia. This study provides the first evidence of R. massiliae, a pathogenic spotted fever Rickettsia, identified in Rh. sanguineus ticks and highlight the potential threat for the regional transmission of Rickettsia infection among humans in Taiwan.

First zootiological survey of Amblyomma geoemydae ticks (Acari: Ixodidae) infesting a wild turtle (Cuora flavomarginata) in northern Taiwan.

The prevalence of Amblyomma geoemydae infesting yellow-margined box turtle (Cuora flavomarginata) was determined for the first time in northern Taiwan. In total, 352 ticks (140 adults, 61 nymphs and 151 larvae) were collected from 56 yellow-margined box turtles. The overall infestation was observed with an average density of 6.3 ticks per turtle and the highest seasonal prevalence was observed on March with an average tick density of 14.3 ticks per turtle. The higher seasonal prevalence was observed on April, May, July and October with an average density of 4.8, 8.3, 5.7 and 8.5 ticks per turtle, respectively. The seasonal activity of these turtles may contribute to the variation in the number of collected ticks. The genetic identity was determined by comparing the sequences of ITS2 genes obtained from seven Taiwan species of A. geoemydae and nine other species representing eight Amblyomma species and Ixodes persulcatus as outgroup. Our results provide the first zootiological survey of A. geoemydae infesting wild turtles in northern Taiwan and highlight the importance of this tick species in the study of parasite dynamics on testudines in Taiwan. Further study focused on the seasonality pattern of turtle ticks will help reveal the ecology of this host-parasite system and its impact on animal/human health.

First detection and molecular identification of a pathogenic spotted fever group Rickettsia, R. massiliae, from Rhipicephalus haemaphysaloides ticks infesting dogs in southern Taiwan.

Tick-borne Rickettsia pathogens become an emerging zoonotic infection worldwide. The prevalence and genetic identity of Rickettsia infection was determined firstly in Rhipicephalus haemaphysaloides ticks collected from dogs in southern Taiwan. A total of 141 Rh. haemaphysaloides ticks were examined for Rickettsia infection by nested-PCR assay targeting the citrate synthase (gltA) and outer membrane protein B (ompB) genes of Rickettsia. The Rickettsia infection was detected with a general infection rate of 2.84%, and was detected in male and female ticks with an infection rate of 3.13% and 2.60%, respectively. Genetic relationships were analyzed by comparing the gltA and ompB sequences obtained from 4 Taiwan strains and 15 other strains representing 13 genospecies of Rickettsia. Phylogenetic analyses reveal that all Taiwan strains were genetically affiliated with the R. massiliae (spotted fever group) and can be distinguished from other genospecies of Rickettsia. These results demonstrate the epidemiological significance of a human pathogenic Rickettsia species (R. massiliae) detected in Rh. haemaphysaloides ticks. Further study focused on the vector competence of this tick species may help to illustrate the potential threat for human infection in southern Taiwan.

Molecular and morphological identification of a reptile-associated tick, Amblyomma geoemydae (Acari: Ixodidae), infesting wild yellow-margined box turtles (Cuora flavomarginata) in northern Taiwan.

The genetic identity and morphological features of a reptile-associated tick, Amblyomma geoemydae, were examined for the first time in Taiwan. The key morphological characters of male and female Am. geoemydae were photographed using a stereo microscope. The genetic identity was analyzed by comparing the 16S mitochondrial DNA gene sequences obtained from 11 Taiwan Am. geoemydae ticks with other Amblyomma species and three Ixodes species as outgroups. All the Taiwan specimens were genetically affiliated to a monophyletic group of Am. geoemydae and could be discriminated from other Amblyomma species. Our results provide the first distinguished colour figures and first genetic identification of adult Am. geoemydae ticks infesting turtles in Taiwan. The host range and vectorial capacity of Am. geoemydae for various tick-borne pathogens need to be further clarified in Taiwan.

First detection and genetic identification of Rickettsia infection in Rhipicephalus sanguineus (Acari: Ixodidae) ticks collected from Southern Taiwan.

The prevalence and genetic identity of Rickettsia infection in Rhipicephalus sanguineus sensu lato (s.l.) ticks were determined in Taiwan. In total 1153 ticks were examined for Rickettsia infection by PCR assay targeting the citrate synthase gene (gltA) of Rickettsia. The prevalence of Rickettsia infection in R. sanguineus s.l. ticks with a general infection rate of 2.2%, and was detected in nymph, male and female stages of R. sanguineus s.l. ticks with an infection rate of 3.1, 2.0 and 1.1%, respectively. Phylogenetic relationships were analyzed by comparing the gltA sequences obtained from four Taiwan strains and 16 other strains representing 13 genospecies of Rickettsia. Phylogenetic analyses reveal that one Taiwan strain was genetically affiliated with the Japan isolate (GRA-1 strain) which belongs to the spotted fever group (R. parkeri/R. honei) and three Taiwan strains were genetically affiliated with the transitional group of Rickettsia (R. felis). Intra- and inter-species analysis also indicated the genetic distance (GD) of the Taiwan strains with a lower level of GD < 0.011, 0.023 and 0.003 as compared with the type strains of R. parkeri, R. honei and R. felis, respectively. Our findings reveal the first detection of Rickettsia infections (R. parkeri/R. honei and R. felis) in R. sanguineus s.l. ticks of Taiwan. These results also highlight the epidemiological significance of diverse Rickettsia species existed in R. sanguineus s.l. ticks and the potential threat for the geographical transmission of Rickettsia infection in Taiwan.

Molecular Detection and Genetic Identification of Rickettsia Infection in Ixodes granulatus Ticks, an Incriminated Vector for Geographical Transmission in Taiwan.

Tick-borne Rickettsia pathogens have become an emerging source of zoonotic infections and have a major impact on human health worldwide. In this study, the prevalence and genetic identity of Rickettsia infections in Ixodes granulatus ticks was firstly determined in Kinmen Island of Taiwan. A total of 247 I. granulatus ticks were examined for Rickettsia infection by nested-PCR assay targeting the citrate synthase (gltA) gene of Rickettsia. The Rickettsia infection was detected with a general infection rate of 4.86%, and was detected in nymph, male and female stages with an infection rate of 3.81%, 0% and 6.84%, respectively. Phylogenetic relationships were analyzed by comparing the gltA sequences obtained from four Taiwan strains and 19 other strains representing 13 genospecies of Rickettsia. Phylogenetic analyses reveal that all Taiwan strains were genetically affiliated to the genospecies of spotted fever (R. parkeri) and transitional (R. felis) groups of Rickettsia. Our findings reveal the first detection of R. parkeri-like and R. felis in I. granulatus ticks from Kinmen Island. As a tourist island between Taiwan and mainland China, these results demonstrate the epidemiological significance of diverse Rickettsia species existed in I. granulatus ticks and highlight the potential threat of geographical transmission among humans in the Taiwan area.

Molecular Detection and Genetic Identification of Wolbachia Endosymbiont in Wild-Caught Culex quinquefasciatus (Diptera: Culicidae) Mosquitoes from Sumatera Utara, Indonesia.

The genetic identity of Wolbachia endosymbiont in wild-caught Culex quinquefasciatus was determined for the first time in Indonesia. A total of 314 Cx. quinquefasciatus were examined for Wolbachia by PCR assay targeting the Wolbachia surface protein (wsp) gene. The prevalence of Wolbachia infection was detected in 29.94% of Cx. specimens (45.86% female and 8.27% male). The group-specific infection was detected with an infection rate of 0.32%, 28.98%, and 0.64% in groups A, B, and A&B, respectively. Phylogenetic analysis revealed all Wolbachia strains from Indonesia were genetically affiliated to the supergroup A and B with the high sequence similarity of 97.9-100% and 99.7-100%, respectively. Phylogenetic relationships can be easily distinguished by neighbor-joining analysis and were congruent by maximum likelihood method. The genetic distance (GD) values of intra- and inter-group analysis indicated a lower level (GD < 0.007 for group A and GD < 0.003 for group B) within the Indonesia strains and a higher level (GD > 1.125 for group A and GD > 1.129 for group B) as compared with other Wolbachia strains. Our results provide the first genetic identification of Wolbachia endosymbiont in Cx. quinquefasciatus collected from Indonesia, and the phylogenetic analysis revealed a new discovery of group A Wolbachia in wild-caught Cx. quinquefasciatus mosquitoes.

Molecular detection and genetic identification of Wolbachia endosymbiont in Rhipicephalus sanguineus (Acari: Ixodidae) ticks of Taiwan.

The genetic identity of Wolbachia endosymbiont in Rhipicephalus sanguineus ticks was determined for the first time in Taiwan. In total 1004 Rh. sanguineus ticks were examined for Wolbachia by polymerase chain reaction assay targeting the Wolbachia surface protein (wsp) gene. The prevalence of Wolbachia infection was detected in nymphs, females, and males with an infection rate of 55.8, 39.8, and 44%, respectively. The phylogenetic relationships were analyzed by comparing the sequences of wsp gene obtained from 60 strains of Wolbachia representing 11 strains of supergroup A and 10 strains of supergroup B. In general, seven major clades of supergroup A and six major clades of supergroup B can be easily distinguished by neighbour-joining analysis and were congruent by maximum likelihood method. All these Wolbachia strains of Taiwan were genetically affiliated to supergroups A and B with high sequence similarity of 98.3-100% and 98.6-100%, respectively. Intra- and inter-group analysis based on the genetic distance (GD) values indicated a lower level (GD < 0.017) within the group A strains of Taiwan compared with the group B (GD > 0.576) of other Wolbachia strains, as well as a lower level (GD < 0.062) within the group B strains of Taiwan compared with the group A (GD > 0.246) of other Wolbachia strains. Our results provide the first genetic identification of Wolbachia endosymbiont in Rh. sanguineus ticks collected from Taiwan, and detection of Wolbachia in male and nymphal ticks may imply the possible mechanism of transstadial transmission in Rh. sanguineus ticks.

First detection and genetic identification of Hepatozoon canis in Rhipicephalus sanguineus sensu lato ticks collected from dogs of Taiwan.

We determined the prevalence of infection and genetic identity of Hepatozoon spp. harbored by Rhipicephalus sanguineus sensu lato ticks in Taiwan. A total of 1082 ticks were collected from dogs and DNA extraction was performed from individual tick specimens. Hepatozoon infection was detected by performing a nested-PCR assay based on the 18S small subunit ribosomal RNA (ssrRNA) gene. The genetic identity of detected Hepatozoon was identified by gene sequencing and phylogenetic analysis. Hepatozoon infection was detected in nymphs, males and females of R. sanguineus s. l. ticks with an infection rate of 20.8%, 22.8% and 29.4%, respectively. Sequence and phylogenetic analysis revealed that these Hepatozoon spp. of Taiwan were genetically affiliated to the same clade within the genospecies of H. canis and can be discriminated from other genospecies of H. americanum and H. felis. Intraspecies analysis based on the genetic distance (GD) values indicates a lower level (GD < 0.005) genetic divergence within the same genospecies of H. canis detected in Taiwan, Brazil and Spain. Interspecies analysis also reveals a higher heterogeneity of Taiwan strains distinguished from other genospecies of H. felis (GD > 0.040) and H. americanum (GD > 0.056). This study provides the first molecular evidence of H. canis detected and identified in various stages of R. sanguineus s. l. ticks in Taiwan. Detection of H. canis in unfed male ticks may imply the possible mechanism of transstadial survival in R. sanguineus s. l. ticks. Further investigations on Hepatozoon spp. harbored by various vector ticks in Taiwan may illustrate the epidemiological significance of this parasite.

First zootiological survey of hard ticks (Acari: Ixodidae) infesting dogs in northern Taiwan.

Hard ticks infesting canine hosts were determined for the first time in northern Taiwan. Between May 2010 and April 2011, a total of 9467 ticks were collected from 2025 dogs. They were identified based on pictorial keys of their morphological characteristics. These ticks belong to three genus and six species: Rhipicephalus sanguineus, R. haemaphysaloides, Haemaphysalis hystricis, H. lagrangei, H. formosensis and Ixodes ovatus. Rhipicephalus sanguineus was the most dominant species (92.5%), followed by H. hystricis (4.6%), R. haemaphysaloides (2.3%), I. ovatus (0.54%), H. lagrangei (0.04%) and H. formosensis (0.01%). The overall density was 4.7 ticks per dog (ranging from 1.8 to 20.8) and the highest seasonal prevalence was observed in September with an average density of 8.2 ticks per dog. Our results not only provide the first survey of hard ticks infesting dogs in northern Taiwan, but also highlight the possible impact of these tick species on human health.

Molecular and morphological identification of a human biting tick, Amblyomma testudinarium (Acari: Ixodidae), in Taiwan.

Genetic identity and morphological features of a human biting tick, Amblyomma testudinarium, were determined for the first time in Taiwan. Morphological features of adult male and female ticks of Am. testudinarium were observed and photographed by a stereo- microscope. The genetic identity was analyzed by comparing the sequences of mitochondrial 16S ribosomal DNA gene obtained from 18 strains of ticks representing 10 species of Amblyomma, and four outgroup species of Dermacentor and Rhipicephalus ticks. Nine major clades could be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these Am. testudinarium ticks collected from Taiwan and Japan were genetically affiliated to a monophyletic group with highly homogeneous sequence (99.8-100% similarity), and can be discriminated from other species of Amblyomma and other genera of ticks (Dermacentor and Rhipicephalus) with a sequence divergence ranging from 6.9 to 23.9%. Moreover, intra- and inter-species analysis based on the genetic distance (GD) values indicated a lower level (GD < 0.003) within the same lineage of Am. testudinarium ticks collected from Taiwan and Japan, as compared with other lineage groups (GD > 0.108) of Amblyomma ticks, as well as outgroup (GD > 0.172) species. Our results provide the first distinguished features of adult Am. testudinarium ticks and the first genetic identification of Am. testudinarium ticks collected from humans in Taiwan. Seasonal prevalence, host range, and vectorial capacity of this tick species in Taiwan need to be further clarified.

First detection and molecular identification of Babesia microti in Rattus losea captured from the offshore Kinmen Island of Taiwan.

Babesia microti was firstly detected and identified in brown country rats (Rattus losea, Swinhoe) captured from the offshore Kinmen Island of Taiwan. The prevalence of Babesia infection in 283 rodents was screened by polymerase chain reaction (PCR) assay using a piroplasma-conserved primer set (Piro A/B) and the thirty-seven PCR-positive rodents were further examined by PCR using a species-specific primer set (Bab 1/4) targeting the gene encoding the nuclear small-subunit ribosomal RNA (18S rRNA) of Babesia species. B. microti was detected only in Rattus losea with a total infection rate of 9.9% (28/283). Positivity examined by species-specific PCR (9.9%) is higher than examined by blood smear (4.6%). Sequence and phylogenetic analyses revealed that Babesia species detected in Taiwan were genetically affiliated to the genotypes of B. microti, and can be easily distinguished from other genotypes of Babesia parasites by neighbour-joining and maximum-parsimony methods. Intra- and inter-species analysis also indicate that all these Taiwan species have a lower level of genetic divergence (genetic distance values <0.084) within the genotypes of B. microti, and were genetically more distant to other genotypes (>0.218) of Babesia parasites. This study provides the first evidence of B. microti identified in R. losea in Taiwan, and the high prevalence of Babesia infection in R. losea may imply its possible role served as reservoir host for maintaining an enzoonotic cycle of Babesia transmission in Kinmen Island. The possible vector tick responsible for the transmission of Babesia infection need to be further identified.

First detection and molecular identification of Babesia gibsoni from Rhipicephalus sanguineus ticks.

Babesia gibsoni was firstly identified in Rhipicephalus sanguineus ticks infested on dogs in Asia. A total of 1195 ticks collected from veterinary practitioners and pet clinics were examined for Babesia by nested-polymerase chain reaction assay based on the 18S small subunit ribosomal RNA (ssrRNA) gene. Babesia infection was detected in nymph, male and female ticks with an infection rate of 2.42%, 0.98% and 1.97%, respectively. Sequence and phylogenetic analysis revealed that these Babesia spp. were genetically affiliated to the same clades within the genospecies of B. gibsoni and B. vogeli, and can be discriminated from other genospecies of Babesia. Intra- and inter-species analysis based on the genetic distance (GD) values indicated a lower level in B. gibsoni (GD<0.011) compared with other genospecies of Babesia (GD>0.106) and out-group protozoa (GD>0.244). This study provides the first molecular evidence of B. gibsoni identified in nymphs of R. sanguineus ticks in Asia, and further confirms the detection of B. vogeli in nymphs and adults of R. sanguineus in northern Taiwan. The epizootiological significance of canine Babesia spp. transmitted by R. sanguineus tick needs to be further identified.

Molecular analysis of Rhipicephalus sanguineus (Acari: Ixodidae), an incriminated vector tick for Babesia vogeli in Taiwan.

The genetic identity of Rhipicephalus sanguineus tick was determined for the first time in Taiwan. The phylogenetic relationships were analyzed by comparing the sequences of mitochondrial 16S ribosomal DNA gene obtained from 32 strains of ticks representing six species of Rhipicephalus, two species of Dermacentor and two outgroup species (Haemaphysalis inermis and Ixodes ricinus). Seven major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All R. sanguineus ticks of Taiwan were genetically affiliated to the tropical lineage group of R. sanguineus sensu lato with highly homogeneous sequence (99.7-100% similarity), and can be discriminated from the temperate lineage group of Rhipicephalus sp. II and R. turanicus with a sequence divergence ranging from 1.7 to 5.2%. In contrast, the nucleotide variations among other Rhipicephalus spp. and other species/genus of ticks compared with the R. sanguineus ticks of Taiwan were measured from 10.6 to 25.5%. Moreover, intra- and inter-species analysis based on the genetic distance (GD) values indicated a lower level (GD < 0.003) within tropical lineage group compared with temperate lineage group (GD > 0.055) of Rhipicephalus, as well as other (GD > 0.129) and outgroup (GD > 0.236) species. Our results provide the first genetic identification of R. sanguineus ticks collected from Taiwan and demonstrate that all these R. sanguineus of Taiwan affiliated to the tropical lineage group of R. sanguineus sensu lato.

Time-Lagging Interplay Effect and Excess Risk of Meteorological/Mosquito Parameters and Petrochemical Gas Explosion on Dengue Incidence.

In Kaohsiung, a metropolitan city in Taiwan at high risk of dengue epidemic, weather factors combined with an accidental petrochemical gas explosion (PGE) may affect mosquito‒human dynamics in 2014. Generalized estimating equations with lagged-time Poisson regression analyses were used to evaluate the effect of meteorological/mosquito parameters and PGE on dengue incidences (2000-2014) in Kaohsiung. Increased minimum temperatures rendered a 2- and 3-month lagging interactive effect on higher dengue risks, and higher rainfall exhibited a 1- and 2-month lagging interplay effect on lower risks (interaction, P ≤ 0.001). The dengue risk was significantly higher than that in a large-scale outbreak year (2002) from week 5 after PGE accident in 2014 (2.9‒8.3-fold for weeks 5‒22). The greatest cross-correlation of dengue incidences in the PGE-affected and PGE-neighboring districts was identified at weeks 1 after the PGE (rs = 0.956, P < 0.001). Compared with the reference years, the combined effect of minimum temperature, rainfall, and PGE accounted for 75.1% of excess dengue risk in 2014. In conclusion, time-lagging interplay effects from minimum temperature and rainfall may be respectively associated with early and near environments facilitating dengue transmission. Events that interact with weather and influence mosquito‒human dynamics, such as PGEs, should not be ignored in dengue prevention and control.

First detection and molecular identification of Babesia vogeli from Rhipicephalus sanguineus (Acari: Ixodidae) in Taiwan.

A total of 578 Rhipicephalus sanguineus ticks collected from dogs in Taiwan were examined for Babesia by species-specific polymerase chain reaction assay based on the 18S small subunit ribosomal RNA (ssrRNA) gene. Babesia DNA was detected in 1.04 % (6/578) of Rh. sanguineus ticks. Phylogenetic analysis revealed that these Babesia spp. were genetically linked to the same clade within the genospecies of Babesia vogeli and could be discriminated from other genospecies of Babesia. Intra-species analysis based on the genetic distance values indicated a lower level (0.079) compared with other genospecies of Babesia (GD > 0.094) and out-group protozoa (GD > 0.236). This study provides the first molecular evidence of B. vogeli detected and identified in various stages of Rh. sanguineus ticks removed from dogs in Taiwan. Detection of Rh. sanguineus in flat male ticks may imply the possible mechanism of transstadial transmission in Rh. sanguineus ticks. The vector competence and the diversity of Babesia species harbored by Rh. sanguineus ticks need to be further investigated.

First detection and molecular identification of Borrelia garinii spirochete from Ixodes ovatus tick ectoparasitized on stray cat in Taiwan.

Borrelia garinii spirochete was detected for the first time in Ixodes ovatus tick ectoparasitized on stray cat in Taiwan. The genetic identity of this detected spirochete was determined by analyzing the gene sequence amplified by genospecies-specific polymerase chain reaction assays based on the 5S-23S intergenic spacer amplicon (rrf-rrl) and outer surface protein A (ospA) genes of B. burgdorferi sensu lato. Phylogenetic relationships were analyzed by comparing the sequences of rrf-rrl and ospA genes obtained from 27 strains of Borrelia spirochetes representing six genospecies of Borrelia. Seven major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. Phylogenetic analysis based on rrf-rrl gene revealed that this detected spirochete (strain IO-TP-TW) was genetically affiliated to the same clade with a high homogeneous sequences (96.7 to 98.1% similarity) within the genospecies of B. garinii and can be discriminated from other genospecies of Borrelia spirochetes. Interspecies analysis based on the genetic distance values indicates a lower level (<0.022) of genetic divergence (GD) within the genospecies of B. garinii, and strain IO-TP-TW was genetically more distant ( >0.113) to the strains identified in I. ovatus collected from Japan and China. Intraspecies analysis also reveals a higher homogeneity (GD<0.005) between tick (strain IO-TP-TW) and human (strain Bg-PP-TW1) isolates of B. garinii in Taiwan. This study provides the first evidence of B. garinii isolated and identified in an I. ovatus tick in Asia, and the higher homogeneity of B. garinii between tick and human strain may imply the risk of human infection by I. ovatus bite.

Molecular detection and genetic identification of Borrelia garinii and Borrelia afzelii from patients presenting with a rare skin manifestation of prurigo pigmentosa in Taiwan.

OBJECTIVES: To determine the genetic identity of Borrelia spirochetes isolated from patients with an unusual skin lesion of prurigo pigmentosa (PP) in Taiwan. The causative agents responsible for human borreliosis were clarified. METHODS: Serum samples and skin specimens were collected from 14 patients with suspected PP and five controls. Serological testing by Western immunoblot analysis and isolation of Borrelia spirochetes from skin specimens were used to verify the Borrelia infection. Genetic identities of isolated spirochetes were determined by analyzing the gene sequences amplified by PCR assay based on the 5S (rrf)-23S (rrl) intergenic spacer amplicon gene of Borrelia burgdorferi sensu lato. RESULTS: Borrelia spirochetes were isolated from skin biopsies of three patients. Serological evidence of Borrelia infection in these patients was also confirmed by elevated IgG and IgM antibodies against the major protein antigens of B. burgdorferi. Phylogenetic analysis revealed that these detected spirochetes are genetically affiliated to the genospecies of Borrelia garinii and Borrelia afzelii with high sequence homology within the genospecies of B. garinii (91.0-98.7%) and B. afzelii (97%). CONCLUSIONS: This study provides the first evidence of B. garinii and B. afzelii isolated and identified in patients with PP. Whether this unusual skin lesion is a new manifestation of Lyme disease needs to be studied further.