Detection of selected pathogens in ticks collected from cats and dogs in the Wrocław Agglomeration, South-West Poland

Tick-borne infections are no longer confined to rural areas, they are documented with increasing frequency in urban settlements across the world. They are known to cause diseases in humans as well as in their companion animals. During a period of 2 years, from January 2013 until December 2014, ticks were collected from dogs and cats in 18 veterinary clinics in the Wrocław Agglomeration, Poland. In total, 1455 ticks were found on 931 pets: 760 domestic dogs and 171 cats. For molecular examinations 127 I. ricinus ticks (115 females and 12 males) were randomly selected, all collected I. hexagonus (n = 137, 32 females, 98 nymphs, 7 larvae) and all collected D. reticulatus (n = 46, 31 females, 15 males) were taken. Ixodes ricinus and I. hexagonus ticks were tested for Rickettsia spp., Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis and Babesia spp., while D. reticulatus ticks were investigated for Rickettsia spp. and Babesia spp. only. In total, 65.4 % I. ricinus ticks were infected with at least one pathogen. Over 50 % of I. ricinus were positive for Rickettsia spp. (R. helvetica and R. monacensis). The infection level with A. phagocytophilum was 21.3 %. DNA of Cand. N. mikurensis was detected in 8.1 % I. ricinus ticks. Interestingly only female ticks were infected. The prevalence of Babesia spp. was confirmed in 9.0 % of I. ricinus involving the species B. microti and B. venatorum. A total of nineteen double, one triple and two quadruple infections were found in I. ricinus ticks only. Almost 11 % of I. hexagonus ticks were positive for at least one of the tested pathogens. Rickettsia spp. infection was found in 2.2 %, while A. phagocytophilum was detected in 8.1 % of I. hexagonus ticks. Only one nymph was positive for Cand. N. mikurensis and none of I. hexagonus ticks harbored a Babesia spp. Over 60 % of D. reticulatus ticks were positive for rickettsial DNA, exclusively belonging to the species R. raoultii. The high tick infestation rates and the prevalence of pathogens found in these ticks demonstrate a serious level of encounter to tick-borne diseases in urban dogs in the Wroclaw area, and provide evidence that dogs and cats themselves may substantially contribute to the circulation of the ticks and pathogens in the urban area.


Background
Tick-borne pathogens, such as Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis and Rickettsia spp., belong to the order Rickettsiales, while such as Babesia spp. are parasitic protozoans. All these pathogens, are known to cause diseases in humans as well as in their companion animals, and are considered to be emerging across Europe and other parts of the world [1][2][3][4][5][6].
Rickettsia spp. are divided into four groups: the spotted fever group (SFG), the typhus group, the Rickettsia bellii group, and the Rickettsia canadensis group [7]. Rickettsiae of the SFG are known to be transmitted by ticks and cause DEBONEL (Dermacentor-borne necrosis erythema lymphadenopathy), also known as TIBOLA (tick-borne lymphadenopathy) in humans [8]. In Poland, rickettsioses caused by rickettsiae of the SFG were described in forest workers, dogs and ticks [9][10][11].
Babesiosis is a zoonotic disease occurring worldwide which is caused by intraerythrocytic parasites of the genus Babesia [2]. In Europe, Babesia divergens-like organisms are mainly responsible for the disease in humans. Babesia spp. are reported in Ixodes ricinus and Dermacentor reticulatus from Poland [19]. Nowadays, cases caused by tickborne pathogens are emerging in urban regions in Europe [20,21]. Dogs and cats should be taken into account as important hosts of ticks in urban areas [21,22].
The aim of this follow-up study was to evaluate the prevalence of Babesia spp., A. phagocytophilum, Cand. Neoehrlichia mikurensis and Rickettsia spp. in ticks collected from cats and dogs in the Wrocław Agglomeration, Poland.

DNA isolation and biological material
All collected ticks were kept in 70 % ethanol until isolation of DNA was performed. Before DNA extraction, ticks were washed in sterile water. All ticks were individually homogenized using sterile polystyrene pistils and then genomic DNA was extracted by using a Tissue Genomic Extraction GPB Mini Kit with proteinase K (Genoplast Biochemicals, Poland) according to the manufacturer's instructions. All of the obtained lysates were stored at -20°C until examined.
For detection of Rickettsia spp., a real-time PCR targeting the gltA genome region (70 bp) was used [31]. A real-time PCR targeting msp2 gene fragment (77 bp) was performed to detect A. phagocytophilum [32]. In order to detect Candidatus Neoehrlichia mikurensis, a realtime PCR targeting the partial groEL gene (99 bp) was used [33,34]. All PCR methods were carried out using the Mx3000P real-time cycler (Stratagene). For detection of Babesia spp., a conventional PCR amplification of the small 18S subunit of the rRNA gene (411-452 bp) with primers BJ1 and BN2 was performed [35]. Samples positive for Rickettsia spp. DNA by realtime PCR were further investigated using a conventional PCR in which a 811-bp fragment of the ompB gene was amplified [36]. The PCR products were visualized by electrophoresis on 1.5 % agarose gels stained with Midori Green (NIPPON Genetics, Düren, Germany). Randomly selected positive PCR products (n = 22) were purified using the NucleoSpin® and PCR Clean-up Kit (MACHEREY-NAGEL, Düren, Germany) according to the manufacturer's instructions. Purified PCR products were sequenced (Interdisziplinäres Zentrum für Klinische Forschung, Leipzig, Germany) with forward and reverse primers, and analyzed with Chromas Lite (Technelysium Pty Ltd, Australia). Nucleotide sequences were compared with GenBank entries using NCBI BLAST.

Statistical analysis
The chi-square test was used to compare infected and not infected ticks (STATISTICA ver. 9.0). Yates' correction was used for 1-df tests when expected frequencies were less than 5. The significance level was set at 0.05.

Discussion
From three ticks species identified as parasites of dogs and cats in the Wrocław Agglomeration, Ixodes ricinus was predominant, followed by I. hexagonus and D. reticulatus. Similar findings were obtained in Belgium [37], Switzerland [38], Germany [39] and Great Britain [40], as well as in Bosnia and Herzegovina [41].
The prevalence of pathogens differed between the tick species. Ixodes ricinus individuals were the most often infected species. The lowest infection levels were observed in I. hexagonus ticks. From all tested pathogens (Rickettsia spp., Babesia spp., Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum), rickettsial infections were the most common. The infection levels of A. phagocytophilum, Cand. N. mikurensis and Babesia spp. (B. microti, B. venatorum) were the highest in I. ricinus ticks and co-infections were detected only in this species. Interestingly, Babesia spp. DNA was only found in I. ricinus ticks, none of the D. reticulatus, the main vector for B. canis [42], was infected. The most often detected pathogen in I. hexagonus specimens was A. phagocytophilum. There were no statistically significant differences in infection levels between different tick life stages and between ticks collected from cats and dogs in any case. In Switzerland [43], researchers observed a difference in Rickettsia spp. infection levels between ticks from cats (40 %) and dogs (18 %), which was not found in this study.
Anaplasma phagocytophilum infection levels of I. ricinus in this research were comparable to those reported in Belgium but much lower for I. hexagonus [37]. However, in The Netherlands and Germany, the prevalence was lower in I. ricinus ticks but similar to I. hexagonus [44,50]. In Poland, also in Lower Silesia, the infection of I. ricinus ticks (questing as well as collected from dogs) was lower than the data presented here [51][52][53].
Candidatus N. mikurensis was previously found in ticks in Poland [18], however, the detection in feeding I. hexagonus is the first one in the country. In Germany, the prevalence of this pathogen in I. ricinus and I. hexagonus from pets was 4 % and 6 %, respectively [45], while in Denmark only 1 % of I. ricinus was infected [46]. The prevalence in I. hexagonus ticks in the present study (0.7 %) is lower than in other countries; however, it is comparable to results obtained in a former study from Poland conducted on other tick species, I. ricinus (0.5 %) [18].
The prevalence of Babesia spp. (B. microti and B. venatorum) in I. ricinus was higher than in Belgium [54] and Germany [45], where the infection was also detected in I. hexagonus ticks. In Poland, also in Lower Silesia, only 1-3 % of questing I. ricinus ticks were infected [52, Table 4 Co-infections in I. ricinus ticks collected from pets in the Wrocław Agglomeration (Poland), 2013-2014