Members of the genus Bartonella are small, fastidious Gram-negative rod-shaped bacteria.
They are emerging arthropod-borne pathogens that can cause severe disease in humans and animals [1,2]. B. henselae is the causative agent of cat scratch disease, characterized by clinical symptoms with varying severity . B. henselae is found in cats, and cat fleas are well established vectors for B. henselae transmission among cats . Infection of humans seems to occur via cat scratches or bites, however, transmission by other arthropods, in particular ticks, is suggested [5,6].
Ticks are known to transmit a large spectrum of emerging bacterial zoonotic pathogens  including Borrelia burgdorferi sensu lato (sl) , Anaplasma phagocytophilum , and Babesia spp. . B. henselae DNA was detected in Ixodes (I.) pacificus and I. persulcatus ticks in North America, Eastern Europe, and Russia [11–15]. Furthermore, in I. scapularis, co-infection with Bartonella spp. and Borrelia burgdorferi sl has been described . I. ricinus is the most widespread and abundant ixodid tick in Western Europe feeding on humans or domestic animals [17–19]. More recently it was shown that B. henselae persists during the developmental stages of I. ricinus ticks and that inoculation of cats with salivary glands of these ticks resulted in B. henselae bacteremia. These results strongly indicate a putative transmission of B. henselae to mammals by ticks .
The impact of this vector bound transmission of B. henselae on humans is still unknown. Evidence for tick transmission of Bartonella is supported by the study of Angelakis et al. (2008)  who identified B. henselae in tissues samples from patients with a syndrome characterized by scalp eschar and lymphadenopathy in close temporal sequence to a tick bite. Moreover, B. henselae has been detected in I. ricinus ticks collected from humans in Italy .
Interestingly, Eskow et al. (2001) related cases with ongoing symptoms of chronic Lyme disease, in which B. henselae and Borrelia burgdorferi sl were simultaneously detected, to tick contact of the patients. They concluded that a concomitant B. henselae infection might be the reason for unresponsiveness to standard therapy .
More recently it was shown that the prevalence of B. henselae and Borrelia burgdorferi sl DNA in the environmental I. ricinus populations sampled in different European sites was up to 40%. However, ticks co-infected with B. henselae and Borrelia burgdorferi sl were not found ., To date, co-infection of ticks with B. henselae and Borrelia burgdorferi sl has not been shown to occur at all in Germany.
In the present study we analysed 230 I. ricinus ticks from humans for the presence B. henselae and Borrelia burgdorferi sl DNA (Table 1). DNA was extracted from ticks using the QIAamp® DNA Blood Mini kit (Qiagen). Molecular detection of Bartonella spp. was performed using a quantitative real time PCR analysis targeting the citrate synthase (gltA). A 249 bp fragment was amplified with the oligonucleotide primers strat1 (5′-GGGGACCAGCTCATGGTGG-3′) and strat2 (5′-GCGTGATAGCAATATCAAGAAGTGG-3′). Signals were related to serial dilution of B. henselae genomic DNA, which was included as standard control. The species differentiation was then performed with PCR-restriction enzyme analysis (PCR-REA). PCR-REA was performed according to the method of Mietze et al. (2010) . The amplified product of the gltA gene was digested with the restriction endonucleases TaqI and MseI or BspHI (New England Biolabs). The digested samples were run on a 10% polyacrylamid gel and visualized by ethidiumbromide staining. Molecular diagnosis of Borrelia burgdorferi sl was performed in the ISO/IEC 17025 accredited diagnostic laboratory of the Institute for Parasitology, University of Veterinary Medicine Hannover, using the standard diagnostic procedure (TaqManTM minor groove binder probe-based quantitative real time PCR).
Table 1. PCR results of ticks for Bartonella spp. and Borrelia burgdorferi sl
|B. henselae||B. clarridgeiae||B. burgdorferi sl||B. henselae + B. burgdorferi sl||Total|
Among a total of 230 ticks, seven were classified as larvae, 161 as nymphs and 62 as adults (Table 1). Bartonella spp. DNA was detected in 16 ticks, resulting in a prevalence of 6.9%. In the PCR-REA, 15 of the PCR positive samples showed the characteristic restriction pattern of B. henselae, while one showed the pattern typical for B. clarridgeiae. Thirteen (8%) of the positive ticks were nymphs, 2 (3.2%) were adults and one was a larva. The prevalence of Borrelia burgdorferi sl in this tick collective was 23.4%. Fifty-five out of 230 ticks showed positive amplification, 35 of them were nymphs and 20 adults. Four nymphs of the 16 B. henselea positive ticks were found positive for Borrelia burgdorferi sl. The statistical analysis with fourfold test revealed no significance between nymphs and adults of I. ricinus and the detection of Bartonella spp. or Borrelia burgdorferi sl, respectively (p 0.2455). Furthermore, the simple kappa coefficient (κ = 0.0119) revealed no significant correlation between the diagnostic findings related to Bartonella spp. and Borrelia burgdorferi sl.
There is an ongoing controversial discussion for B. henselae transmission to humans and animals by ticks, since experimental evidence confirming ticks as true vectors is still missing . On the other hand, the increasing number of clinical and epidemiological cases support potential tick transmission of Bartonella [5,6].
In summary, our analyses show that ticks are infected with Bartonella spp., mostly B. henselae. Furthermore, we demonstrate the occurrence of co-infections of ticks with B. henselae and Borrelia burgdorferi sl., suggesting humans may be at risk of getting infected with both pathogens concurrently by ticks.