Tick transmitted diseases are a serious and permanent public health problem. In Europe, the most frequent and most epidemiologically important vector is the hard tick Ixodes ricinus. It transmits viral, bacterial and protozoan agents to humans and animals. The most common and important tick-transmitted disease in the northern hemisphere, Lyme borreliosis, is caused by spirochetes from the Borrelia burgdorferi sensu lato (s.l.) complex. It currently includes 19 different genospecies . The considerable genotypic and phenotypic heterogeneity of the B. burgdorferi s.l. complex has been linked to differences in pathogenicity, clinical symptoms and ecology [2–4]. The Borrelia genus also includes a second group of spirochetes, called the relapsing fever group. The spirochetes of this group are transmitted mainly by soft ticks, but can also utilize some hard ticks as vectors .
Anaplasmoses are also common tick-borne, zoonotic bacterial diseases. The causative agents are intracellular gram-negative bacteria that belong to the family Anaplasmataceae . The genus Anaplasma consists of Anaplasma marginale, Anaplasma ovis, Anaplasma bovis and Anaplasma platys. While they are primary of veterinary significance, A. phagocytophilum can cause granulocytic anaplasmosis in humans as well as horses and dogs and tick-borne fever in ruminants . A relatively new member of the family Anaplasmataceae is Candidatus Neoehrlichia mikurensis . It infects endothelial cells and most infection symptoms depend on the physical status of the patient. The illness predominantly develops in immunocompromised patients [9–12].
Less common bacteria that may be transmitted by Ixodes ricinus amongst other tick species include Francisella spp. and Coxiella spp. Coxiella burnetii is the causative agent of Q-fever, which can be either an acute or chronic disease. Francisella tularensis causes tularemia, a febrile disease with myalgia and headache and when left untreated, it can cause a high mortality rate . Most cases of disease caused by both C. burnetii and F. tularensis result from non-vector transmission.
Another European tick-borne obligate intracellular parasite, which is also globally distributed, is Rickettsia spp. The genus Rickettsia contains many species which form several biogroups, including the typhus fever group, the spotted fever group and the group causing tick-borne lymphadenopathy or Dermacentor spp. - borne necrosis - erythema - lymphadenopathy (TIBOLA or DEBONEL) . Many other Rickettsia species have been recently identified, but are not yet well described, including the human pathogens R. helvetica and R. aeschlimannii[15, 16].
Considering all the serious diseases that humans can potentially be exposed to after a tick-bite, an unambiguous diagnostic tool is essential for identifying them. The most reliable modern diagnostic tools employ serological tests, including ELISA (enzyme linked immunoabsorbent assay), Western blot, indirect immunofluorescence assay (IFA), a microagglutination test, and in the case of rickettsial infection, the Weil-Felix test . Unfortunately, these methods are only indirect and do not allow illnesses to be diagnosed in the early stages of infection. Another major limitation of serology is cross-reactivity , application of the non-standardized antigen preparations and discrepancies in test procedures among laboratories can lead to different test results. Furthermore, identification of Candidatus N. mikurensis using serology is presently not possible and A. phagocytophilum and E. chaffeensis antigens do not interact with Candidatus N. mikurensis antibodies . The primary approach for detecting Candidatus N. mikurensis therefore relies on PCR-based methods.
Molecular biology approaches offer the advantages of directly detecting these pathogens during early infection along with better taxonomic classification. The most common techniques employ conventional, nested, or quantitative PCR (qPCR) targeted to a genus or species specific gene, such as 16S rDNA gene (rrs), gltA, omp, ospA or ospC[20–23]. Another method, commonly used for identifying B. burgdorferi s.l., targets the 5S-23S rDNA (rrfA-rrlB) intergenic spacer followed by genotyping using RFLP or SSCP [24, 25]. These tests target the rDNA genes because they are minimally affected by horizontal gene transfer. Typically, these genes have hypervariable regions, specific for each bacterial genus, which are flanked by conserved regions .
The more recent, microarray-based techniques are high-throughput large-scale screening systems for the simultaneous identification of several target amplicons. DNA microarrays are used in many fields of research, including transcription profile analysis and DNA-DNA or protein–protein interactions. Microarrays have been developed for the identification of microorganisms in soil extracts , for the detection of multiple pathogens [28–30] and for differentiating between different Borrelia genospecies . These techniques employ DNA or RNA as a template for the preparation of a target product which is suitable for passive hybridization with complementary DNA fragments or oligonucleotides bound to the surface of a slide. The stringency and hybridization efficiency is regulated by solution composition and temperature.
An alternative to the DNA microarray is an electronic microarray - biosensor, which can be prepared using standard complementary metal oxide semiconductor (CMOS) technology. This “smart” biosensor uses an electric field to regulate the stringency, transport and active hybridization of nucleic acids [32, 33]. An electronic microarray based on the genus-specific variability of the rrs gene has already been developed for the detection of marine bacterial species .
In this study, we report the development of a detection system combining a second generation DNA microarray with qPCR for the detection of pathogens in vectors or in clinical samples. A second generation DNA microarray is basically an epoxy glass slide with bound capture oligonucleotides, which code for the hypervariable regions of the rrs gene, specific for each bacterial genus. The target DNA is amplified, Cy5-labeled using nested PCR and passively hybridized with capture probes on the microarray. We also developed qPCRs employing the genus-specific, hypervariable regions of rrs for Coxiella spp., Francisella spp. and Rickettsia spp. to confirm the DNA microarray results.