In vitro efficacy of antibiotics against different Borrelia isolates.

In the present study, the effectiveness of six antimicrobial agents have been tested against 24 borrelia strains isolated from Ixodes ricinus ticks (11 Borrelia lusitaniae, eight Borrelia afzelii, three Borrelia garinii and two Borrelia valaisiana) and one B. lusitaniae strain isolated from human skin. The minimum inhibitory concentration range of antimicrobial agents was as follows: amoxicillin, 0.125-2 mg/L; doxycycline, 0.125-1 mg/L, ceftriaxone, 0.016-0.063 mg/L; cefuroxime, 0.063-1 mg/L; azithromycin, 0.0017-0.11 mg/L; amikacin 32-512 mg/L. Potentially pathogenic B. lusitaniae and B. valaisiana species were more susceptible to amoxicillin and azithromycin than pathogenic B. afzelii and B. garinii (P < 0.05); B. garinii, B. lusitaniae and B. valaisiana were more susceptible to doxycycline than B. afzelii (P < 0.05) while all species showed same susceptibility to ceftriaxone and cefuroxime (P > 0.05). This study is the first report on in vitro susceptibility of isolates from Serbia to antimicrobial agents and the first report on susceptibility of larger number of isolates of potentially pathogenic species B. lusitaniae. We showed that antimicrobial agents in vitro inhibit growth of borrelia strains very effectively, indicating the potential of their equally beneficial use in the treatment of Lyme borreliosis.

Comparison of growth and morphology of Borrelia burgdorferi sensu lato in BSK-H and BSK-II media stored for prolonged periods.

Barbour-Stoenner-Kelly II (BSK-II) and BSK-H media were used for cultivation and isolation of fastidious Borrelia species - the causative agents of Lyme borreliosis. Culture media have a limited shelf life and require adequate storage. Our goal was to assess how the growth of Borrelia would be affected by prolonged storage of media and inadequate storage conditions (BSK-H stored at +4 °C for 2.5 years and BSK-II stored at -20 °C for 11 years). Growth of different Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae and Borrelia valaisiana strains was assessed during 2 weeks of incubation at 33 °C. Monitored parameters included cell count per mL, morphology and motility. The results of this study have shown weaker growth of borrelia strains in BSK-H at +4 °C (median final cell number of 1.5 × 106 /mL) than in BSK-II at -20 °C (median final cell number of 7.75 × 106 /mL) and in fresh BSK-H media (median final cell number of 8.95 × 106 /mL). Duration of storage of media had no impact on Borrelia morphology and motility. Our results indicate that temperature of -20 °C is optimal for long-term storage of medium, BSK-II stored for 11 years provided effective support to growth of Borrelia and may be employed for cultivation.

Golden jackals (Canis aureus) as hosts for ticks and tick-borne pathogens in Serbia.

The golden jackal (Canis aureus) is a medium-sized canid species native to Europe. This species is characterized by rapid large-scale expansion. A similar trend is also observed in Serbia, where the species is now distributed in more than a half of the territory. Although jackals prefer habitats in human-dominated landscapes, these animals have not been studied well enough from an eco-epidemiological point of view, and little is known about their potential for carrying zoonotic pathogens. In a study conducted during a three-year period (01/2010-02/2013), a total of 216 hunted or road-killed golden jackals were collected from 10 localities in Serbia. Ticks, when present, were removed, and after necropsy, spleen samples were collected from each animal. All tick and spleen samples were tested for the DNA of bacterial and protozoan tick-borne pathogens (Borrelia species, Bartonella species, Rickettsia species, Anaplasma species, Coxiella burnetii, Francisella species and Babesia species) by multiplex real-time PCR, conventional PCR and sequencing analyses. The DNA of Babesia canis was detected in nine out of 216 (4.2%) spleen samples, and two samples (0.9%) tested positive for Anaplasma phagocytophilum. In 118 ticks collected from jackals, the DNA of two Babesia species (Ba. canis and Ba. microti), three Borrelia species (Bo. garinii, Bo. valaisiana, and Bo. lusitaniae) and A. marginale was detected. From the aspect of public health surveillance, the potential role of the golden jackal in the maintenance of vector-borne zoonotic pathogens in Serbia must be considered, and further eco-epidemiological studies should be performed to determine the precise role of this animal species in zoonotic disease transmission cycles.

Assessment of using recombinant Ixodes ricinus AV422 saliva protein for confirmation of tick bites in hunting dogs as naturally infested hosts.

Due to the recorded spreading of ticks in past years, a higher incidence of tick-borne diseases (TBDs) can be expected in the future in endemic areas, but can also pose an emerging public health concern in areas where they have not yet been recognized. Assessment of the exposure of vulnerable hosts to ticks would be a very helpful tool for TBD epidemiological studies, as well as for their proper managing. To confirm previous tick bites, the method of choice is detection of antibodies in host serum as markers developed against injected tick saliva proteins during feeding. We recently showed that the recombinant form of Ixodes ricinus AV422 saliva protein (rIrAV422) can serve for detection of markers in experimentally infested rats. Here we examine whether it can be used in the same manner in naturally exposed hosts. We chose hunting dogs as good sentinel animals. The study group consisted of 15 dogs that varied in breed, age, sex, previous tick infestation history and repellent treatment. Western blot analysis with rIrAV422 as an antigen confirmed the presence of tick bite markers in all analysed dogs. For some of the dogs, their previous tick infestation history was unclear, which emphasizes the usefulness of rIrAV422 for revealing it. Since hunting dogs are naturally infested with different ticks, the potential of rIrAV422 in assessment of general exposure to ticks is highlighted. Use of rIrAV422 can also be helpful in veterinary practice and research as a tool for validation of the efficiency of tick repellent products.

Ixodes ricinus immunogenic saliva protein, homologue to Amblyomma americanum AV422: Determining its potential for use in tick bite confirmation.

Tick bites often go unnoticed, so specific reliable tests are needed to confirm them for prompt diagnosis and treatment of tick-borne diseases. One of the promising candidates for developing such a test is AV422, a tick saliva protein that has been conserved across tick genera. In this study, we demonstrate the potential of the AV422 homologue from Ixodes ricinus to be used for tick bite detection for both Prostriata and Metastriata. We expressed recombinant (r) I. ricinus (Ir) AV422 in E. coli and subjected it to Western blot analysis using rat antibodies to saliva proteins of both I. ricinus (Prostriata) and Dermacentor reticulatus (Metastriata) larvae. Our data demonstrate that rIrAV422 specifically bound to antibodies from sera of rats used for both I. ricinus and D. reticulatus larvae feeding, but not to antibodies from control serum, emphasizing its specificity since tick bites were the sole cause of sera reactivity.

Analysis of pathogen co-occurrence in host-seeking adult hard ticks from Serbia.

Past studies in Serbia have reported concurrent infections of Ixodes ricinus ticks with Borrelia burgdorferi sensu lato genospecies, Anaplasma phagocytophilum and Francisella tularensis. As a step forward, this investigation included a broader range of microorganisms and five most common and abundant tick species in Serbia. Five tick species were identified (Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, H. concinna and I. ricinus) and analyzed for the presence of seven pathogens. Anaplasma ovis, A. phagocytophilum, Babesia canis, B. burgdorferi s.l., Coxiella burnetii, Rickettsia helvetica and R. monacensis were detected. Sequencing of samples positive for F. tularensis revealed the presence of Francisella-like endosymbionts. No Bartonella spp. DNA was amplified. Concurrent infections were present in three tick species (D. reticulatus, H. concinna and I. ricinus). The rate of co-infections was highest in I. ricinus (20/27), while this tick species harbored the broadest range of co-infection combinations, with dual, triple and a quadruple infection(s) being detected.