flaB Sequences Research Articles

Borrelia theileri infections in Rhipicephalus annulatus ticks from the north of Iran.

Ticks serve as vectors and reservoirs of various Borrelia species, potentially causing diseases in humans and animals. Mazandaran, a fertile green land in northern Iran, provides ample grazing grounds for livestock and harbors at least 26 hard tick species. This study investigated Borrelia infection in hard ticks from forest areas in this region and compared their genetic identity with the species data in the GenBank database. A total of 2,049 ticks were collected manually from mammalian hosts or using dragging and flagging methods. These ticks were then grouped into 190 pools and 41 individuals based on host, species, developmental stage, and gender. A real-time PCR (qPCR) detected Borrelia DNA in 26 pools from female, male, and nymph of Rhipicephalus annulatus (n = 17) and Ixodes ricinus (n = 9) ticks and one individual female Haemaphysalis punctata tick. The generated partial flaB and glpQ sequences from qPCR-positive Rh. annulatus ticks exhibited the highest identities of 98.1-100% and 98.2% with Borrelia theileri and closely related undefined isolates. Additionally, in phylogenetic analysis, these sequences clustered within well-supported clades with B. theileri and the closely related undefined isolates from various geographic regions, confirming the presence of B. theileri in the north of Iran. Divergence in B. theileri flaB and glpQ sequences across various geographical areas suggests potential subspeciation driven by adaptations to different tick species. This divergence in our flaB sequences implies the possible introduction of B. theileri-infected ticks from different geographical origins into Iran.

First Detection of Theileria sinensis-like and Anaplasma capra in Ixodes kashmiricus: With Notes on cox1-Based Phylogenetic Position and New Locality Records.

Ixodes ticks transmit Theileria and Anaplasma species to a wide range of animals. The spreading of ticks and tick-borne pathogens has been attributed to transhumant herds, and research on these uninvestigated issues has been neglected in many countries, including Pakistan. Recently, we used internal transcribed spacer (ITS) and 16S ribosomal DNA partial sequences to genetically characterize Ixodes kashmiricus ticks and their associated Rickettsia spp. However, the data on its cox1 sequence and associated Theileria spp. and Anaplasma spp. are missing. This study aimed to genetically characterize I. kashmiricus based on the cox1 sequence and their associated Theileria spp. and Anaplasma spp. The I. kashmiricus ticks were collected from small ruminants: sheep (Ovis aries) and goats (Capra hircus) of transhumant herds in district Shangla, Dir Upper and Chitral, Khyber Pakhtunkhwa (KP), Pakistan. Out of 129 examined hosts, 94 (72.87%) (56 sheep and 38 goats) were infested by 352 ticks, including adult females (175; 49.7%) followed by nymphs (115; 32.7%) and males (62; 17.6%). For molecular analyses, 121 ticks were subjected to DNA isolation and PCR for the amplification of the cox1 sequence for I. kashmiricus, 18S rDNA for Theileria spp. and 16S rDNA sequences for Anaplasma spp. The obtained cox1 sequence showed 89.29%, 88.78%, and 88.71% identity with Ixodes scapularis, Ixodes gibbosus, and Ixodes apronophorus, respectively. Phylogenetically, the present cox1 sequence clustered with the Ixodes ricinus complex. Additionally, the 18S rDNA sequence showed 98.11% maximum identity with Theileria cf. sinensis and 97.99% identity with Theileria sinensis. Phylogenetically, Theileria spp. clustered with the T. cf. sinensis and T. sinensis. In the case of Anaplasma spp., the 16S rDNA sequence showed 100% identity with Anaplasma capra and phylogenetically clustered with the A. capra. PCR-based DNA detection targeting the amplification of groEL and flaB sequences of Coxiella spp. and Borrelia spp., respectively, was unsuccessful. This is the first phylogenetic report based on cox1 and new locality records of I. kashmiricus, and the associated T. sinensis-like and A. capra. Significant tick surveillance studies are needed in order to determine the epidemiology of Ixodes ticks and their associated pathogens.

First molecular detection of Borrelia theileri subclinical infection in a cow from Brazil.

Borrelia theileri is a relapsing fever group Borrelia that is transmitted to cattle by ticks of the genus Rhipicephalus. In this study, we describe the first molecular detection of B. theileri subclinical infection in a cow in Brazil. During the examination of stained blood smears of 10 cows from a farm with a recent history of fatal Trypanosoma vivax trypanosomiasis, spirochete-like structures were incidentally detected in one of the cows. The animal presented good body score, normal hematocrit and normal-colored ocular mucosa. Temperature, heart rate and respiratory rate were all normal. The animal was infested by ticks, which were morphologically identified as Rhipicephalus microplus. The diagnosis was confirmed by testing DNA extracted from a blood sample using a PCR targeting a ≈ 650bp fragment of the flagellin B (flaB) gene of Borrelia spp. The partial flaB sequence obtained showed 99.83% similarity with B. theileri. Phylogenetically, the flaB partial sequence generated herein clustered with other B. theileri sequences, being separated from B. lonestari. This is the first molecular detection of B. theileri subclinical infection in a cow in Brazil. The possible implications of this finding are discussed.

Carrier Status ofLeptospiraspp. in Healthy Companion Dogs in Sri Lanka

Dogs are asymptomatic chronic carriers of Leptospira spp. and excrete these bacteria in their urine, resulting in environmental contamination and potentially leading to zoonotic transmission. Although a previous study in Sri Lanka detected anti-Leptospira antibodies in companion dogs, the urinary shedding of Leptospira spp. and the Leptospira species and serogroups prevalent in them remain unclear. Thus, the current study identified the prevalent Leptospira serogroups and the carrier status of Leptospira spp. in apparently healthy, client-owned dogs in the Kandy District of Sri Lanka. Serum and urine samples were collected from 96 unvaccinated and 82 vaccinated dogs. Anti-Leptospira antibodies and Leptospira DNA in urine were detected using the microscopic agglutination test (MAT) and nested PCR that targeted the pathogenic leptospiral gene, flaB. The flaB sequences were compared with those of Leptospira spp. using the public databases. MAT detected anti-leptospiral antibodies in 15.6% (15/96) of the unvaccinated dogs, and the reactive serogroups were observed to be Sejroe (11.5%), Canicola (2.1%), Icterohemorrhagiae (1.0%), and Javanica (1.0%). Furthermore, MAT results revealed that 11.0% (9/82) of the vaccinated dogs tested positive for the anti leptospira antibodies and the only reactive serogroup was Sejroe. Leptospira DNA was detected in 15.6% (15/96) and 15.9% (13/82) of urine samples collected from unvaccinated and vaccinated dogs, respectively, and phylogenetic analysis revealed that the animals were infected with L. borgpetersenii, L. interrogans, L. kmetyi, and L. weilii. The L. interrogans sequence detected in the canine sample was identical to the one that was previously reported in a human sample from the Kandy District. This study demonstrated that both unvaccinated and vaccinated dogs excrete various pathogenic Leptospira spp. in their urine, suggesting that they may play an important role in environmental contamination that poses a health risk to the dog owners and the general public.

Rat trade and leptospirosis: Molecular epidemiology of Leptospira species in rats exported from Cambodia to Vietnam.

Rats are an important maintenance host of Leptospira spp., the causative agents of leptospirosis, a zoonotic disease with worldwide distribution. Rats are traded as food in the Mekong Delta, where Cambodia exports tons of rats to Vietnam. Handling wild rats is a potential health risk, but the information on Leptospira spp. carried by rats traded in the region remains limited. In this study, we investigated the carriage of Leptospira spp. in rats exported from Cambodia to Vietnam using bacterial culture, nested PCR and DNA sequencing. Isolates were then assessed using serological analysis and whole-genome sequencing (WGS), and Leptospira DNA detected in rat kidney tissues was also analysed by multilocus sequence typing (MLST). Seventy-two rats (2 Bandicota indica, 57 Rattus argentiventer, 11 R.losea, 1 R.norvegicus and 1 R.rattus) were subjected to bacterial culture, and three L.borgpetersenii serogroup Javanica isolates were obtained from R.argentiventer (5.3%). WGS revealed that although Cambodian isolates were genetically related to L.borgpetersenii serogroup Javanica strains widely distributed in East and South-East Asian countries, they formed a different cluster from other strains. In addition to the three L.borgpetersenii sequences, the flaB sequence of L.interrogans was detected in 18 R.argentiventer and 7 R.losea kidney tissue samples (38.9%) using nested PCR followed by DNA sequencing. The L.interrogans flaB-positive samples were further analysed by MLST, revealing that seven housekeeping genes (glmU, pntA, sucA, tpiA, pfkB, mreA and caiB) contained novel sequences with distinct lineages from other sequence types. This study revealed a high prevalence of Leptospira spp. among rats exported from Cambodia to Vietnam, indicating a potential risk to people engaging in rat trade and demonstrating that a fastidious L.interrogans strain circulates among Cambodian rats.

Molecular epidemiology of Leptospira interrogans in Rattus norvegicus in Hanoi, Vietnam

Leptospirosis is a zoonotic disease that is caused by pathogenic spirochaetes of Leptospira spp. and it has become a public health concern in urban localities in the tropics. Rats are important reservoir animals for the transmission of leptospirosis in urban areas. Leptospirosis is considered endemic in Vietnam. However, information on the causative Leptospira genotypes and serotypes in the country is limited. We investigated the carrier status of Leptospira spp. in rats captured in Hanoi by culturing and DNA detection. Isolates were characterized using a serological method and multiple-locus variable-number tandem repeat analysis (MLVA).We captured 144 rats (1 Rattus argentiventer, 135 R. norvegicus, and 8 R. rattus) and obtained 17 L. interrogans, determined by rrs sequencing, from R. norvegicus (12.6%). Sixteen of the isolates were serogroup Bataviae. Five of the 16 isolates exhibited an MLVA type identical to that of the serovar Bataviae reference strain Van Tienen, while there were nine repeats for the other 11 isolates at VNTR31 compared with the reference strain. The remaining isolate grew poorly, and we were unable to determine its serogroup. However, it had an MLVA type matching those of serogroup Pomona strains isolated from R. norvegicus in Japan. Three different flaB sequences were detected in 23 out of 81 R. norvegicus kidney tissue samples (28.4%) using nested PCR followed by DNA sequencing. Two of the sequences were identical with those of serogroups Bataviae and Pomona, and no strain with another sequence was detected in the present study.The present study reveals a high prevalence rate of L. interrogans among R. norvegicus in Hanoi, Vietnam, indicating a potential risk of rat-borne leptospirosis in the area. The present study also demonstrates that a fastidious L. interrogans strain circulates among rats and that molecular detection is crucial in facilitating the accurate determination of reservoir animals.

Molecular Epidemiological Survey of Leptospira Infection of Wild Rodents in the Urban Settlement of Cambodia.

Leptospirosis remains a major public health threat in Cambodia. In this study, we aimed at facilitating the development of preventive strategies against leptospirosis in Cambodia by conducting molecular epidemiological surveys of Leptospira infection among wild rodents. One hundred sixty-three wild rodents were captured in the capital Phnom Penh and surrounding areas, and their kidneys and bladders were collected for analysis. Identification of wild rodent species was determined by using the cytochrome c oxidase I gene. TaqMan PCR of the flagellin B gene (flaB) was performed to detect Leptospira, and species of the isolates were identified by flaB sequencing analysis. The species and respective number of rodents collected were as follows: Rattus norvegicus, 80 (49.1%); Rattus argentiventer, 53 (32.5%); Rattus exulans, 6 (3.7%); Rattus indica, 15 (9.2%); Maxomys surifer, 3 (1.8%); and Rattus sp., 6 (3.7%). Leptospira was detected in 20 out of the 163 rodents (12.3%) and was categorized as either Leptospira interrogans or Leptospira noguchii. R. norvegicus had the highest prevalence of Leptospira (17.5%), and R. argentiventer and Rattus sp. showed infection rates of 9.4% and 16.7%, respectively. Based on these results, wild rodents living in urban areas of Cambodia were found to be important reservoirs of highly virulent Leptospira. The flaB sequence results of our study provided information regarding the prevalence of Leptospira species, which was dependent on the rodent species. This study is the first study on leptospirosis in wild rodents in the urban areas of Cambodia, where there is limited information on leptospirosis.

Presence of Borrelia spp. DNA in ticks, but absence of Borrelia spp. and of Leptospira spp. DNA in blood of fever patients in Madagascar

The occurrence of tick-borne relapsing fever and leptospirosis in humans in Madagascar remains unclear despite the presence of their potential vectors and reservoir hosts.We screened 255 Amblyomma variegatum ticks and 148 Rhipicephalus microplus ticks from Zebu cattle in Madagascar for Borrelia-specific DNA. Borrelia spp. DNA was detected in 21 Amblyomma variegatum ticks and 2 Rhipicephalus microplus ticks. One Borrelia found in one Rhipicephalus microplus showed close relationship to Borrelia theileri based on genetic distance and phylogenetic analyses on 16S rRNA and flaB sequences. The borreliae from Amblyomma variegatum could not be identified due to very low quantities of present DNA reflected by high cycle threshold values in real-time-PCR. It is uncertain whether these low numbers of Borrelia spp. are sufficient for transmission of infection from ticks to humans.In order to determine whether spirochaete infections are relevant in humans, blood samples of 1009 patients from the highlands of Madagascar with fever of unknown origin were screened for Borrelia spp. − and in addition for Leptospira spp. − by real-time PCR. No target DNA was detected, indicating a limited relevance of these pathogens for humans in the highlands of Madagascar.

Novel Borrelia species detected in echidna ticks, Bothriocroton concolor, in Australia.

BackgroundTo date, little has been documented about microorganisms harboured within Australian native ticks or their pathogenic potential. Recently, a Borrelia sp. related to the Relapsing Fever (RF) group was identified in a single tick removed from a wild echidna (Tachyglossus aculeatus). The present study investigated the presence of Borrelia in 97 Bothriocroton concolor ticks parasitizing echidnas in Queensland, New South Wales, and Victoria, Australia, using nested PCR with Borrelia-specific primers targeting the 16S rRNA (16S) and flaB genes.ResultsBorrelia-specific PCR assays confirmed the presence of a novel Borrelia sp. related to the RF and reptile-associated (REP) spirochaetes in 38 (39 %) B. concolor ticks. This novel Borrelia sp. was identified in 41 % of the B. concolor ticks in Queensland and New South Wales, but not in any ticks from Victoria. The resulting flaB sequences (407 bp) were 88 and 86 % similar to the flaB sequences from Borrelia turcica and Borrelia hermsii, respectively. Of the ticks confirmed as Borrelia-positive following the flaB assay, 28 were positive with the 16S assay. Phylogenetic analysis of the 16S sequences (1097 bp) suggests that these sequences belong to a novel Borrelia sp., which forms a unique monophyletic clade that is similar to, but distinct from, RF Borrelia spp. and REP-associated Borrelia spp.ConclusionsWe conclude that the novel Borrelia sp. identified in this study does not belong to the Borrelia burgdorferi (sensu lato) complex, and that the phylogenetic analysis of the partial 16S gene sequences suggests it forms a unique monophyletic cluster in the genus Borrelia, potentially forming a fourth major group in this genus associated with monotremes in Australia. However, a thorough molecular characterisation will be required to confirm the phylogenetic position of this unique Borrelia sp. The zoonotic potential and pathogenic consequences of this novel Borrelia sp. are unknown at the current time.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1627-x) contains supplementary material, which is available to authorized users.

Molecular epidemiology of pathogenic Leptospira spp. among large ruminants in the Philippines.

The extent of Leptospira infection in large ruminants resulting to economic problems in livestock industry in a leptospirosis-endemic country like the Philippines has not been extensively explored. Therefore, we determined the prevalence and carrier status of leptospirosis in large ruminants using molecular techniques and assessed the risk factors of acquiring leptospirosis in these animals. Water buffalo and cattle urine samples (n=831) collected from 21 farms during 2013–2015 were subjected to flaB-nested PCR to detect pathogenic Leptospira spp. Leptospiral flaB was detected in both species with a detection rate of 16.1%. Leptospiral DNA was detected only in samples from animals managed in communal farms. Sequence analysis of Leptospira flaB in large ruminants revealed the formation of three major clusters with L. borgpetersenii or L. kirschneri. One farm contained Leptospira flaB sequences from all clusters identified in this study, suggesting this farm was the main source of leptospires for other farms. This study suggested that these large ruminants are infected with various pathogenic Leptospira species causing possible major economic loss in the livestock industry as well as potential Leptospira reservoirs that can transmit infection to humans and other animals in the Philippines.

Prevalence of Borrelia miyamotoi in Ixodes persulcatus in Irkutsk City and its neighboring territories, Russia

Adult Ixodes persulcatus were collected in highly populated districts in Irkutsk city, Russia, and in popular recreational and professional areas in its neighboring territories. Borrelia miyamotoi infection in I. persulcatus was examined using multiplex Taqman-PCR targeting 16S rDNA, and nested PCR and sequencing analyses targeting flaB and 16S rDNA. B. miyamotoi and Lyme disease Borrelia species were detected in 13 (infection rate, 2.9%) and 77 (17.3%) out of 445 I. persulcatus ticks, respectively, collected from 4 sites around the Baikal Lake. The 16S rDNA and flaB sequences of these amplicons were closely related to those of B. miyamotoi detected and/or isolated from I. persulcatus in Japan and Far Eastern Russia, and clustered separately from those of Europe and North America. These results indicate that additional surveillance for B. miyamotoi infection is needed in order to determine how it affects human health in Irkutsk City and its neighboring territories.

A novel duplex real-time PCR permits simultaneous detection and differentiation of Borrelia miyamotoi and Borrelia burgdorferi sensu lato

For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection. Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 10(4) to 10(-1)) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment. The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 10(2) genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2% for B. miyamotoi and 11.8% for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples. The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.

Novel Relapsing Fever Spirochete in Bat Tick

To the Editor: Tick-borne relapsing fever in western North America is a zoonosis caused by spirochetes in the genus Borrelia that are transmitted by argasid ticks of the genus Ornithodoros (1). Human disease occurs in many focal areas and is associated with infections of Borrelia hermsii, B. turicatae, and possibly B. parkeri (2,3). Although the ecologic parameters that maintain B. hermsii and B. turicatae differ, human infections usually occur in rustic cabins (B. hermsii) and caves (B. turicata) inhabited by ticks and their terrestrial vertebrate hosts (1). Recently, Gill et al. (4) provided evidence that the argasid bat tick, Carios kelleyi, feeds upon humans. Subsequently, Loftis et al. (5) used PCR analysis and DNA sequencing to detect in C. kelleyi an unidentified Borrelia species that was closely related to B. turicatae and B. parkeri. We report the partial molecular characterization of another novel tick-borne relapsing fever spirochete in C. kelleyi, which expands our knowledge for this group of pathogenic spirochetes and their potential vertebrate hosts and tick vectors. C. kelleyi were collected August 18, 2005, from a house in Jones County, Iowa, built in 1857. Bats had been excluded from the attic since 1992. Nine months before ticks were collected, bats were prevented from roosting under the eaves. DNA was extracted from 31 nymphal C. kelleyi, as described previously (6). For each tick, regions of the glpQ, flaB, and 16S rRNA genes were amplified and sequenced as described (3,7,8). Sequences were assembled by using the SeqMan program in the Lasergene software package (DNASTAR, Madison, WI, USA). Fourteen (45.1%) of 31 ticks were positive by PCR for >1 of the genes tested. Partial DNA sequences were determined from tick no. 16, for which amplicons for all 3 genes were obtained. The partial flaB sequence had 4 bases different from the 300-base sequence (98.66% identity) reported previously (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY763104,term_id:54610284}}AY763104) for another Borrelia sp. found in C. kelleyi (5). We constructed a 1,992-bp concatenated sequence that contained 1,273 bp of the 16S rRNA, 351 bp of flaB, and 368 bp of glpQ. This concatenated sequence was aligned with homologous, trimmed DNA sequences of the same length obtained from representative full-length sequences determined previously for B. hermsii, B. turicatae, and B. parkeri (3,9) (Figure). This C. kelleyi spirochete was more closely related to B. turicatae and B. parkeri than to B. hermsii but was clearly distinct from all 3 species (DNA sequence identities of 98.89%, 98.75%, and 95.98% to B. turicatae, B. parkeri, and B. hermsii, respectively). Figure Phylogram comparing the novel spirochete in the bat tick Carios kelleyi with Borrelia parkeri, B. turicatae, and B. hermsii based on the concatenated partial 16S rRNA-flaB-glpQ DNA sequences in the Carios spirochete (1,992 bp total) (produced with ClustalV ... A glpQ amplicon from another nymphal tick (no. 3) was equenced (GenBank accession no. {type:entrez-nucleotide,attrs:{text:EF688578,term_id:157418962}}EF688578) and was unique in the database; it was also considerably different from the glpQ sequence determined from tick 16, with 325 of 368 bases matching (88.3% identity). The Borrelia glpQ sequence from tick 3 had 85.1%–89.1% identity compared with glpQ sequences from B. hermsii, B. turicatae, and B. parkeri. This finding suggests the presence of at least 2 relapsing fever group spirochetes in C. kelleyi that await further characterization. We found a novel Borrelia in bat ticks that is closely related to, but distinct from, the other known species of tick-borne relapsing fever spirochetes in North America. The human health implications of the new relapsing fever group spirochete are not yet known. The willingness of C. kelleyi to feed on humans and the fact that infection with bacteria closely related to true relapsing fever spirochetes occurs in these ticks suggest that human habitation near bats and their associated tick colonies could pose a public health risk. Growth in laboratory animals or culture could help isolate these novel organisms for further studies to establish the distribution and public health implications of this newly identified Borrelia sp.

A Hard Tick Relapsing Fever Group Spirochete in a BrazilianRhipicephalus(Boophilus)microplus

Tick-borne diseases usually comprise a complex epidemiological and ecological network connecting the vector, pathogen, and a group of host species. Symptoms associated with Lyme disease have been reported in Brazil, but no Borrelia sp. has been definitively related to these events. Here we have identified a B. lonestari/B. theileri-related spirochete DNA in the cattle tick Rhipicephalus (Boophilus) microplus from Brazil. Four hundred R. microplus and 80 Amblyomma cajennense ticks were screened, and only 1 horse-fed R. microplus was infected. A Borrelia sp. 16S rDNA sequence was amplified by polymerase chain reaction (PCR) from the total tick DNA with 99% similarity to B. theileri and B. lonestari. Partial flaB sequence was also obtained, demonstrating 96% similarity to the B. lonestari flagellin gene, and the resultant putative amino acid sequence demonstrated 97% identity to B. lonestari flagellin. Moreover, partial glpQ sequence demonstrated 92% similarity to the B. lonestari gene, with a putative amino acid sequence 90% identical to the B. lonestari glycerophosphodiester phosphodiesterase. Phylogenetic analyses clearly include this Brazilian Borrelia sp., denoted "Borrelia," sp-BR in a group of spirochetes aligned with B. theileri and B. lonestari. Thus, hard tick relapsing fever group spirochetes represent a clade of widespread bacteria and herein we describe the first molecular identification of a Borrelia sp. in South America.

Isolation and molecular characterization of a Brazilian strain of Borrelia anserina, the agent of fowl spirochaetosis

Borrelia anserina the agent of fowl spirochaetosis, has a worldwide distribution, where it is transmitted by Argas spp. ticks. The present study reports the first molecular characterization and in vitro isolation of an avian spirochaete strain from Brazil, presumably identified as B. anserina originated from naturally infected Argas miniatus ticks. DNA fragments of the rrs and flab genes were amplified by PCR and sequenced to determine phylogenetic similarities. The resulting sequences were 99.8% (483 of 484) and 98.7% (754 of 764) similar to GenBank corresponding sequences of B. anserina rrs and flaB genes, respectively. By neighbor-joining phylogenetic analysis, the flaB sequence of the Brazilian strain clustered in a monophyletic group with the sequence of B. anserina under 100% bootstrap support. The isolate was successfully isolated in BSK medium, with seven passages performed. The spirochaete strain isolated in the present study was genetically identified as B. anserina labeled as strain PL.

A novel, fast-growing Borrelia sp. isolated from the hard tick Hyalomma aegyptium in Turkey.

A novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium (family Ixodidae, subfamily Metastriata) using Barbour-Stoenner-Kelly (BSK) II medium. Tick samples were taken during the summer of 2000 from the Istanbul area in northwestern Turkey. Sixty-seven of 153 adults (44%) and 72 of 185 nymphs (39%) were infected with the novel spirochaete, whereas none of the 20 larvae examined were infected. The optimal growth temperature of the spirochaete in BSK II medium was 34-37 degrees C, and it could grow at 39 degrees C. Doubling times at 34 and 37 degrees C were 5.3 and 5.1 h, respectively. Six pure cultures of the spirochaete were obtained and characterized by microscopic observation, sequence analysis of the flagellin gene (flaB), SDS-PAGE and Western blotting. The spirochaete was morphologically similar to those of the genus Borrelia and contained a 41 kDa protein reactive with mAb H9724 specific to the flagellin of a Borrelia species. Polyclonal antibody raised to this spirochaete reacted with several antigen bands, whereas no bands were detected with Borrelia burgdorferi, Borrelia hermsii, Borrelia turicatae and Borrelia parkeri. The flaB sequences of the six isolates showed high similarity, with sequence similarity values ranging from 99.2 to 100%; however, the similarity of the isolates' flaB sequences to those of the Lyme-disease-related Borrelia and relapsing-fever-associated Borrelia species was less than 90%. These findings suggest that the unique spirochaete is a member of the genus Borrelia, and differs from previously described Borrelia species.