The importance of passerine birds as tick hosts and in the transmission of Borrelia burgdorferi,the agent of Lyme disease: a case study from Scotland

Borrelia burgdorferi sensu lato (s.l.) is the causative agent of Lyme borreliosis, the most common tick‐borne zoonosis of humans in Europe and North America. Here, we assessed the relative importance of different passerine bird species as tick hosts and their contribution to the B. burgdorferi s.l. transmission cycle in a rural residential area in Scotland. We caught 1229 birds of 22 species during the tick‐questing season. On average, 29% carried larval ticks (0.8 larvae per individual) and 5% carried nymph ticks (0.06 nymphs per individual). All attached ticks tested were Ixodes ricinus. Using a nested‐PCR, we found that 20% of nymphs tested positive to B. burgdorferi s.l. and all these were of the genospecies Borrelia garinii. We identified two new bird species carrying infected nymphs: Eurasian Siskin Carduelis spinus and European Greenfinch Carduelis chloris. Ground‐foraging species were more important than arboreal species in hosting I. ricinus nymphs and B. burgdorferi s.l. Common Blackbirds Turdus merula were the most common hosts, with Song Thrushes Turdus philomelos, Dunnocks Prunella modularis, European Greenfinches and Chaffinches Fringilla coelebs also hosting high rates of infection.     

Differential Transmission of the Genospecies of Borrelia burgdorferi Sensu Lato by Game Birds and Small Rodents in England

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in a focus of Lyme borreliosis in southern Britain dominated by game birds. Ticks, rodents, and pheasants were analyzed for spirochete infections by PCR targeting the 23S-5S rRNA genes, followed by genotyping by the reverse line blot method. In questing Ixodes ricinus ticks, three genospecies of B. burgdorferi sensu lato were detected, with the highest prevalences found for Borrelia garinii and Borrelia valaisiana. B. burgdorferi sensu stricto was rare (<1%) in all tick stages. Borrelia afzelii was not detected in any of the samples. More than 50% of engorged nymphs collected from pheasants were infected with borreliae, mainly B. garinii and/or B. valaisiana. Although 19% of the rodents harbored B. burgdorferi sensu stricto and/or B. garinii in internal organs, only B. burgdorferi sensu stricto was transmitted to xenodiagnostic tick larvae (it was transmitted to 1% of the larvae). The data indicate that different genospecies of B. burgdorferi sensu lato can be maintained in nature by distinct transmission cycles involving the same vector tick species but different vertebrate host species. Wildlife management may have an influence on the relative risk of different clinical forms of Lyme borreliosis.     

Prevalence of Borrelia burgdorferi Sensu Lato in Ticks Collected from Migratory Birds in Switzerland

The prevalence of ticks infected by Borrelia burgdorferi sensu lato on birds during their migrations was studied in Switzerland. A total of 1,270 birds captured at two sites were examined for tick infestation. Ixodes ricinus was the dominant tick species. Prevalences of tick infestation were 6% and 18.2% for birds migrating northward and southward, respectively. Borrelia valaisiana was the species detected most frequently in ticks, followed by Borrelia garinii and Borrelia lusitaniae. Among birds infested by infected ticks, 23% (6/26) were infested by B. lusitaniae-infected larvae. Migratory birds appear to be reservoir hosts for B. lusitaniae.     

Prevalence of Borrelia burgdorferi Sensu Lato Genospecies in Ixodes ricinus Ticks in Europe: a Metaanalysis

In Europe, Borrelia burgdorferi genospecies causing Lyme borreliosis are mainly transmitted by the tick Ixodes ricinus. Since its discovery, B. burgdorferi has been the subject of many epidemiological studies to determine its prevalence and the distribution of the different genospecies in ticks. In the current study we systematically reviewed the literature on epidemiological studies of I. ricinus ticks infected with B. burgdorferi sensu lato. A total of 1,186 abstracts in English published from 1984 to 2003 were identified by a PubMed keyword search and from the compiled article references. A multistep filter process was used to select relevant articles; 110 articles from 24 countries contained data on the rates of infection of I. ricinus with Borrelia in Europe (112,579 ticks), and 44 articles from 21 countries included species-specific analyses (3,273 positive ticks). These data were used to evaluate the overall rate of infection of I. ricinus with Borrelia genospecies, regional distributions within Europe, and changes over time, as well as the influence of different detection methods on the infection rate. While the infection rate was significantly higher in adults (18.6%) than in nymphs (10.1%), no effect of detection method, tick gender, or collection period (1986 to 1993 versus 1994 to 2002) was found. The highest rates of infection of I. ricinus were found in countries in central Europe. B. afzelii and B. garinii are the most common Borrelia species, but the distribution of genospecies seems to vary in different regions in Europe. The most frequent coinfection by Borrelia species was found for B. garinii and B. valaisiana.     

Species Co-Occurrence Patterns among Lyme Borreliosis Pathogens in the Tick Vector Ixodes ricinus

Mixed infections have important consequences for the ecology and evolution of host-parasite interactions. In vector-borne diseases, interactions between pathogens occur in both the vertebrate host and the arthropod vector. Spirochete bacteria belonging to the Borrelia burgdorferi sensu lato genospecies complex are transmitted by Ixodes ticks and cause Lyme borreliosis in humans. In Europe, there is a high diversity of Borrelia pathogens, and the main tick vector, Ixodes ricinus, is often infected with multiple Borrelia genospecies. In the present study, we characterized the pairwise interactions between five B. burgdorferi sensu lato genospecies in a large data set of I. ricinus ticks collected from the same field site in Switzerland. We measured two types of pairwise interactions: (i) co-occurrence, whether double infections occurred more or less often than expected, and (ii) spirochete load additivity, whether the total spirochete load in double infections was greater or less than the sum of the single infections. Mixed infections of Borrelia genospecies specialized on different vertebrate reservoir hosts occurred less frequently than expected (negative co-occurrence) and had joint spirochete loads that were lower than the additive expectation (inhibition). In contrast, mixed infections of genospecies that share the same reservoir hosts were more common than expected (positive co-occurrence) and had joint spirochete loads that were similar to or greater than the additive expectation (facilitation). Our study suggests that the vertebrate host plays an important role in structuring the community of B. burgdorferi sensu lato genospecies inside the tick vector.     

Prevalence and distribution of Borrelia and Babesia species in ticks feeding on dogs in the U.K.

Ticks were collected during March–July 2015 from dogs by veterinarians throughout the U.K. and used to estimate current prevalences and distributions of pathogens. DNA was extracted from 4750 ticks and subjected to polymerase chain reaction and sequence analysis to identify Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) and Babesia (Piroplasmida: Babesiidae) species. Of 4737 ticks [predominantly Ixodes ricinus Linneaus (Ixodida: Ixodidae)], B. burgdorferi s.l. was detected in 94 (2.0%). Four Borrelia genospecies were identified: Borrelia garinii (41.5%); Borrelia afzelli (31.9%); Borrelia burgdorferi sensu stricto (25.5%), and Borrelia spielmanii (1.1%). One Rhipicephalus sanguineus Latreille (Ixodida: Ixodidae), collected from a dog with a history of travel outside the U.K., was positive for B. garinii. Seventy ticks (1.5%) were positive for Babesia spp. Of these, 84.3% were positive for Babesia venatorum, 10.0% for Babesia vulpes sp. nov., 2.9% for Babesia divergens/Babesia capreoli and 1.4% for Babesia microti. One isolate of Babesia canis was detected in a Dermacentor reticulatus (Ixodida: Ixodidae) tick collected from a dog that had recently travelled to France. Prevalences of B. burgdorferi s.l. and Babesia spp. did not differ significantly between different regions of the U.K. The results map the widespread distribution of B. burgdorferi s.l. and Babesia spp. in ticks in the U.K. and highlight the potential for the introduction and establishment of exotic ticks and tick‐borne pathogens.     

First report of Anaplasma phagocytophilum and its co-infections with Borrelia burgdorferi sensu lato in Ixodes ricinus ticks (Acari: Ixodidae) from Republic of Moldova

We examined 198 questing Ixodes ricinus ticks collected in Chisinau City, Republic of Moldova by PCR assays for Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and co-infection of both pathogens, which were detected in 9%, 25.2% and 2.5% of tested ticks, respectively. B. burgdorferi s.l. genotyping revealed the presence of five genospecies with dominance of B. garinii. Our preliminary study provides evidence about occurrence of both pathogens in this populated area, which represent a potential health risk for inhabitants.     

Association of Borrelia garinii and B. valaisiana with Songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.     

Differential Role of Passerine Birds in Distribution of Borrelia Spirochetes, Based on Data from Ticks Collected from Birds during the Postbreeding Migration Period in Central Europe

Borrelia spirochetes in bird-feeding ticks were studied in the Czech Republic. During the postbreeding period (July to September 2005), 1,080 passerine birds infested by 2,240 Ixodes ricinus subadult ticks were examined. Borrelia garinii was detected in 22.2% of the ticks, Borrelia valaisiana was detected in 12.8% of the ticks, Borrelia afzelii was detected in 1.6% of the ticks, and Borrelia burgdorferi sensu stricto was detected in 0.3% of the ticks. After analysis of infections in which the blood meal volume and the stage of the ticks were considered, we concluded that Eurasian blackbirds (Turdus merula), song thrushes (Turdus philomelos), and great tits (Parus major) are capable of transmitting B. garinii; that juvenile blackbirds and song thrushes are prominent reservoirs for B. garinii spirochetes; that some other passerine birds investigated play minor roles in transmitting B. garinii; and that the presence B. afzelii in ticks results from infection in a former stage. Thus, while B. garinii transmission is associated with only a few passerine bird species, these birds have the potential to distribute millions of Lyme disease spirochetes between urban areas.     

Borrelia burgdorferi sensu lato and co‐infections with Anaplasma phagocytophilum and Rickettsia spp. in Ixodes ricinus in Hamburg,Germany

To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe‐based quantitative real‐time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co‐infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co‐infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia‐positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.     

Longitudinal study of prevalence and spatio-temporal distribution of Borrelia burgdorferi sensu lato in ticks from three defined habitats in Latvia, 1999–2010

Members of the Borrelia burgdorferi sensu lato (s.l.) species complex are known to cause human Lyme borreliosis. Because of longevity of some reservoir hosts and the Ixodes tick vectors' life cycle, long-term studies are required to better understand species and population dynamics of these bacteria in their natural habitats. Ticks were collected between 1999 and 2010 in three ecologically different habitats in Latvia. We used multilocus sequence typing utilizing eight chromosomally located housekeeping genes to obtain information about species and population fluctuations and/or stability of B. burgdorferi s.l. in these habitats. The average prevalence over all years was 18.9%. From initial high-infection prevalences of 25.5%, 33.1% and 31.8%, from 2002 onwards the infection rates steadily decreased to 7.3%. Borrelia afzelii and Borrelia garinii were the most commonly found genospecies but striking local differences were obvious. In one habitat, a significant shift from rodent-associated to bird-associated Borrelia species was noted whilst in the other habitats, Borrelia species composition was relatively stable over time. Sequence types (STs) showed a random spatial and temporal distribution. These results demonstrated that there are temporal regional changes and extrapolations from one habitat to the next are not possible.     

Seasonal dynamics of tick burden and associated Borrelia burgdorferi s.l. and Borrelia miyamotoi infections in rodents in a Dutch forest ecosystem

Ixodes ricinus ticks transmit Borrelia burgdorferi sensu lato (s.l.) as well as Borrelia miyamotoi. Larvae become infected when feeding on infected rodents, with horizontal transmission of B. burgdorferi and horizontal and vertical transmission of B. miyamotoi. We studied seasonal dynamics of infection rates of I. ricinus and their rodent hosts, and hence transmission risk of these two distinctly different Borrelia species. Rodents were live-trapped and inspected for ticks from May to November in 2013 and 2014 in a forest in The Netherlands. Trapped rodents were temporarily housed in the laboratory and detached ticks were collected. Borrelia infections were determined from the trapped rodents and collected ticks. Borrelia burgdorferi s.l. and B. miyamotoi were found in ticks as well as in rodents. Rodent density was higher in 2014, whereas tick burden as well as the Borrelia infection rates in rodents were higher in 2013. The density of B. miyamotoi-infected nymphs did not differ between the years. Tick burdens were higher on Apodemus sylvaticus than on Myodes glareolus, and higher on males than on females. Borrelia-infection rate of rodents varied strongly seasonally, peaking in summer. As the larval tick burden also peaked in summer, the generation of infected nymphs was highest in summer. We conclude that the heterogeneity of environmental and host-specific factors affects the seasonal transmission of Borrelia spp., and that these effects act more strongly on horizontally transmitted B. burgdorferi spp. than on the vertically transmitted B. miyamotoi.

     

Effects of a zoonotic pathogen,Borrelia burgdorferi,on the behavior of a key reservoir host

Most emerging infectious diseases of humans are transmitted to humans from other animals. The transmission of these “zoonotic” pathogens is affected by the abundance and behavior of their wildlife hosts. However, the effects of infection with zoonotic pathogens on behavior of wildlife hosts, particularly those that might propagate through ecological communities, are not well understood. Borrelia burgdorferi is a bacterium that causes Lyme disease, the most common vector‐borne disease in the USA and Europe. In its North American range, the pathogen is most frequently transmitted among hosts through the bite of infected blacklegged ticks (Ixodes scapularis). Using sham and true vaccines, we experimentally manipulated infection load with this zoonotic pathogen in its most competent wildlife reservoir host, the white‐footed mouse, Peromyscus leucopus, and quantified the effects of infection on mouse foraging behavior, as well as levels of mouse infestation with ticks. Mice treated with the true vaccine had 20% fewer larval blacklegged ticks infesting them compared to mice treated with the sham vaccine, a significant difference. We observed a nonsignificant trend for mice treated with the true vaccine to be more likely to visit experimental foraging trays (20%–30% effect size) and to prey on gypsy moth pupae (5%–20% effect size) compared to mice treated with the sham vaccine. We observed no difference between mice on true‐ versus sham‐vaccinated grids in risk‐averse foraging. Infection with this zoonotic pathogen appears to elicit behavioral changes that might reduce self‐grooming, but other behaviors were affected subtly or not at all. High titers of B. burgdorferi in mice could elicit a self‐reinforcing feedback loop in which reduced grooming increases tick burdens and hence exposure to tick‐borne pathogens.     

Genetic Variability within Borrelia burgdorferi Sensu Lato Genospecies Established by PCR-Single-Strand Conformation Polymorphism Analysis of the rrfA-rrlB Intergenic Spacer in Ixodes ricinus Ticks from the Czech Republic

In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a ~230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.     

Genomic Analysis of Borrelia japonica Sp. Nov. Isolated from Ixodes ovatus in Japan

Genetic characteristics of 12 Borrelia isolates from the tick, Ixodes ovatus, I. persulcatus, and the rodent, Apodemus speciosus ainu, in Japan were compared to members of the three genospecies of Borrelia burgdorferi sensu lato; B. burgdorferi sensu stricto, B. garinii and group VS461. The methods used in this study were the quantitative microplate DNA hybridization assay and restriction fragment length polymorphism (RFLP) analyses of the flagellin structural genes and the 16S rRNA genes. The six isolates from I. persulcatus and A. speciosus ainu were identified as genospecies B. garinii using RFLP analysis of the flagellin and 16S rRNA genes. In contrast, RFLP analysis of the six isolates from I. ovatus indicated that they were different from the three reported genospecies. DNA homology studies confirmed the RFLP results. The six isolates from I. ovatus had DNA homologies ranging from 85 to 99%, whereas DNA relatedness of the I. ovatus isolate with strains belonging to the three genospecies was 50 to 64%. These results suggest that the strains isolated from I. ovatus in Japan differ from the three genospecies and should be classified as a new genospecies of B. burgdorferi sensu lato. We propose that strains isolated from I. ovatus should be classified as B. japonica sp. nov.     

Genetic Diversity of Borrelia burgdorferi Sensu Lato Isolated in Far Eastern Russia

One-hundred and fifty-seven Borrelia isolated from adult ticks, Ixodes persulcatus, and wild rodents, Clethrionomys rufocanus and Apodemus peninsulae, in the far eastern part of Russia were characterized and identified by restriction fragment length polymorphism (RFLP) of the 5S-23S rRNA intergenic spacer. Some isolates showed unique RFLP patterns and were determined as Borrelia garinii on the basis of a sequence analysis of the intergenic spacer amplicon and reactivity with species-specific monoclonal antibodies (MAbs). 86.5 and 12.7% of the tick isolates, and 74.2 and 12.9% of the rodent isolates were determined as Borrelia garinii and Borrelia afzelii, respectively, but no Borrelia burgdorferi sensu stricto was detected. This finding is similar to the results obtained from Borrelia surveys of I. persulcatus and wild rodents in Hokkaido, Japan.     

An Invasive Mammal (the Gray Squirrel,Sciurus carolinensis) Commonly Hosts Diverse and Atypical Genotypes of the Zoonotic Pathogen Borrelia burgdorferi Sensu Lato

Invasive vertebrate species can act as hosts for endemic pathogens and may alter pathogen community composition and dynamics. For the zoonotic pathogen Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, recent work shows invasive rodent species can be of high epidemiological importance and may support host-specific strains. This study examined the role of gray squirrels (Sciurus carolinensis) (n = 679), an invasive species in the United Kingdom, as B. burgdorferi sensu lato hosts. We found that gray squirrels were frequently infested with Ixodes ricinus, the main vector of B. burgdorferi sensu lato in the United Kingdom, and 11.9% were infected with B. burgdorferi sensu lato. All four genospecies that occur in the United Kingdom were detected in gray squirrels, and unexpectedly, the bird-associated genospecies Borrelia garinii was most common. The second most frequent infection was with Borrelia afzelii. Genotyping of B. garinii and B. afzelii produced no evidence for strains associated with gray squirrels. Generalized linear mixed models (GLMM) identified tick infestation and date of capture as significant factors associated with B. burgdorferi sensu lato infection in gray squirrels, with infection elevated in early summer in squirrels infested with ticks. Invasive gray squirrels appear to become infected with locally circulating strains of B. burgdorferi sensu lato, and further studies are required to determine their role in community disease dynamics. Our findings highlight the fact that the role of introduced host species in B. burgdorferi sensu lato epidemiology can be highly variable and thus difficult to predict.     

An Ixodes minor and Borrelia carolinensis enzootic cycle involving a critically endangered Mojave Desert rodent

Microtus californicus scirpensis is an endangered, isolated subspecies of California vole. It requires water pools and riparian bulrush (Schoenoplectus americanus) and occupies some of the rarest habitat of any North American mammal. The minimally vegetated, extremely arid desert surrounding the pools is essentially uninhabitable for Ixodes species ticks. We describe an enzootic cycle of Borrelia carolinensis in Ixodes minor ticks at a site 3500 km distant from the region in which I. minor is known to occur in Tecopa Host Springs, Inyo County, eastern Mojave Desert, California. Voles were live‐trapped, and ticks and blood samples queried by PCR and DNA sequencing for identification and determination of the presence of Borrelia spp. Between 2011–2013, we found 21 Ixodes minor ticks (prevalence 4–8%) on Amargosa voles and Reithrodontomys megalotis. DNA sequencing of 16S rRNA from ticks yielded 99% identity to I. minor. There was 92% identity with I. minor in the calreticulin gene fragment. Three ticks (23.1%), 15 (24%) voles, three (27%) house mice, and one (7%) harvest mice were PCR positive for Borrelia spp. Sequencing of the 5S‐23S intergenic spacer region and flagellin gene assigned Amargosa vole Borrelia strains to B. carolinensis. Ixodes minor, first described in 1902 from a single Guatemalan record, reportedly occurs only in the southeast American on small mammals and birds. The source of this tick in the Mojave Desert and time scale for introduction is not known but likely via migratory birds. Borrelia strains in the Amargosa ecosystem most closely resemble B. carolinensis. B. carolinensis occurs in a rodent‐I. minor enzootic cycle in the southeast U.S. although its epidemiological significance for people or rodents is unknown. The presence of a tick and Borrelia spp. only known from southeast U.S. in this extremely isolated habitat on the other side of the continent is of serious concern because it suggests that the animals in the ecosystem could be vulnerable to further incursions of pathogens and parasites.     

Detection of Lyme disease spirochete,Borrelia burgdorferi sensu lato,including three novel genotypes in ticks (Acari: Ixodidae) collected from songbirds (Passeriformes) across Canada

Lyme disease is reported across Canada, but pinpointing the source of infection has been problematic. In this three‐year, bird‐tick‐pathogen study (2004–2006), 366 ticks representing 12 species were collected from 151 songbirds (31 passerine species/subspecies) at 16 locations Canada‐wide. Of the 167 ticks/pools tested, 19 (11.4%) were infected with Borrelia burgdorferi sensu lato (s.l.). Sequencing of the rrf‐rrl intergenic spacer gene revealed four Borrelia genotypes: B. burgdorferi sensu stricto (s.s.) and three novel genotypes (BC genotype 1, BC genotype 2, BC genotype 3). All four genotypes were detected in spirochete‐infected Ixodes auritulus (females, nymphs, larvae) suggesting this tick species is a vector for B. burgdorferi s.l. We provide first‐time records for: ticks in the Yukon (north of 60° latitude), northernmost collection of Amblyomma americanum in North America, and Amblyomma imitator in Canada. First reports of bird‐derived ticks infected with B. burgdorferi s.l. include: live culture of spirochetes from Ixodes pacificus (nymph) plus detection in I. auritulus nymphs, Ixodes scapularis in New Brunswick, and an I. scapularis larva in Canada. We provide the first account of B. burgdorferi s. l. in an Ixodes muris tick collected from a songbird anywhere. Congruent with previous data for the American Robin, we suggest that the Common Yellowthroat, Golden‐crowned Sparrow, Song Sparrow, and Swainson's Thrush are reservoir‐competent hosts. Song Sparrows, the predominant hosts, were parasitized by I. auritulus harboring all four Borrelia genotypes. Our results show that songbirds import B. burgdorferi s.l.‐infected ticks into Canada. Bird‐feeding I. scapularis subadults were infected with Lyme spirochetes during both spring and fall migration in eastern Canada. Because songbirds disperse millions of infected ticks across Canada, people and domestic animals contract Lyme disease outside of the known and expected range.     

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.