SummaryThe tick Ixodes ricinus is responsible for the transmission of a number of bacterial, protozoan and viral diseases to humans and animals in Europe and š Č ȇ Northern Africa. Female I. ricinus from England, Switzerland and Italy have been found to harbour an intracellular a-proteobacterium, designated IricES1, within the cells of the ovary. IricES1 is the only prokaryote known to exist within the mitochondria of any animal or multicellular organism. To further examine the distribution, prevalence and mode of transmission of IricES1, we performed polymerase chain reaction screening of I. ricinus adults from 12 countries across its geographic distribution, including tick colonies that have been maintained in the laboratory for varying periods of time. IricES1 was detected in 100% of field-collected female ticks from all countries examined (n = 128), while 44% of males were found to be infected (n = 108). Those males that are infected appear to harbour fewer bacteria than females. Sequencing of fragments of the 16S rRNA and gyrB genes revealed very low nucleotide diversity among various populations of IricES1. Transmission of IricES1 from engorged adult females to eggs was found to be 100% (n = 31). In tick colonies that had been maintained in the laboratory for several years, a relatively low prevalence was found in females (32%; n = 25). To our knowledge, IricES1 is the most widespread and highly prevalent of any tick-associated symbiont.

In order to investigate the presence and prevalence of Lyme borreliosis (Lyme disease) Borrelia species, 312 unfed ticks were collected by flagging at a woodland area in Trakya, in the European side of Turkey, in May 2002. Twelve of 299 Ixodes ricinus ticks were infected with Borrelia spp., as determined by cultivation in BSK medium (prevalence rate 4·0 %). Ten pure cultures were subjected to further characterization by sequencing analysis of the 5S-23S rDNA intergenic spacer, 16S rDNA and flagellin gene. One isolate of Borrelia burgdorferi sensu stricto, two of Borrelia garinii (Eurasian type), two of Borrelia afzelii, four of Borrelia lusitaniae and one of Borrelia valaisiana were identified. However, no Asian-type B. garinii was found. Interestingly, all Borrelia species that are known to be carried by I. ricinus were discovered among the 10 isolates. These results provide the first evidence for the existence of the Lyme borreliosis agent in Turkey. INTRODUCTIONLyme borreliosis is the most prevalent tick-borne zoonosis in Europe, North America and the Far East (Burgdorfer et al., 1982;Gern et al., 1998;Steere, 2001;Yanagihara & Masuzawa, 1997). Borrelia burgdorferi (Johnson et al., 1984) sensu lato is classified into 11 species (Masuzawa et al., 2001). In Europe, Ixodes ricinus is the prominent vector tick and transmits five species, B. burgdorferi sensu stricto, Borrelia garinii (Baranton et al., 1992), Borrelia afzelii (Canica et al., 1993), Borrelia lusitaniae (Le Fleche et al., 1997) and Borrelia valaisiana (Wang et al., 1997). Of these five species, B. burgdorferi, B. afzelii and B. garinii are pathogenic to humans. On the other hand, the pathogenicity of B. lusitaniae and B. valaisiana remains to be elucidated (Escudero et al., 2000;Wang et al., 1999). B. burgdorferi has been isolated and identified in Europe from the vector tick, I. ricinus, wild mammals and patients, in contrast to Turkey, a country that constitutes a bridge between Europe and Asia. Based on RFLP analysis of the 5S-23S rRNA gene spacer, B. garinii were further classified into two subtypes, Eurasian type and Asian type. Eurasian-type B. garinii is found in both Europe and Asia; on the other hand, the Asian type is distributed only in east-Asian countries. Ixodes persulcatus is the vector of the Lyme disease borreliae B. garinii (Eurasian and Asian types) and B. afzelii in Asian countries such as Japan (Masuzawa et al., 1991(Masuzawa et al., , 1996a, far-eastern Russia (Masuzawa et al., 1997;Postic et al., 1997;Sato et al., 1996), north-eastern China (Li et al., 1998;Takada et al., 1998), western China (Uighur; Takada et al., 2001b) and Korea (T. Masuzawa, K. H. Park and M. K. Cho, unpublished). Borrelia strains genetically related to B. valaisiana found in Europe are distributed in the southern part of Asia, on Okinawa island, the southernmost islands of Japan (Takada et al., 2001a), Taiwan (Masuzawa et al., 2000), the southern part of China (Masuzawa et al., 2001) and Korea (Masuzawa et al., 1999).The aims of this study were to is...

Previously, a novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium, which infests tortoises (Testudo graeca), by using Barbour-Stoenner-Kelly (BSK) II medium; the tick samples were taken from the Istanbul area in northwestern Turkey [Gü ner et al. (2003). Microbiology 149, 2539Microbiology 149, -2544. Here is presented a detailed characterization of the spirochaete. Electron microscopy revealed that strain IST7T is morphologically similar to other spirochaetes of the genus Borrelia and possesses 15 to 16 flagellae that emerge from both polar regions. PFGE analysis revealed the genome to comprise a linear chromosome of approximately 1 Mb; two large linear plasmids of approximately 145 and 140 kb, and several small plasmids ranging from 50 to 20 kb in size were also found. The 16S rRNA gene sequence of this Borrelia isolate exhibited 99?4 to 99?8 % identity with other strains isolated from H. aegyptium and less than 99 % similarity with those of other Borrelia species. A phylogenetic tree, generated from 16S rRNA gene sequences, demonstrated that the spirochaete isolates from H. aegyptium clustered together and branched off from both Lyme-disease-related and relapsing-fever-associated Borrelia species. A single copy of the rrs gene was detected in the genome of strain IST7 T by Southern hybridization. DNA-DNA hybridization results showed that strain IST7 T was distinct from Lyme-disease-related Borrelia, Borrelia burgdorferi and the relapsing-fever-associated species Borrelia hermsii. The G+C content of strain IST7 T is 30?0 mol%. From these genetic features, a novel Borrelia species, Borrelia turcica sp. nov., is proposed; the type strain is IST7 T (=JCM 11958 T =DSM 16138 T ).

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 6C, and it could grow at 39 6C. Doubling times at 34 and 37 6C 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.