Ticks and associated pathogens in dogs from Greece

With the aim to assess the occurrence of hard ticks and the pathogens they may carry in dogs from Greece, ixodid specimens (n = 757) were collected from 310 animals living in six provinces across the Greek peninsula. All ticks were morphologically identified, and genomic DNA was extracted from 344 (45.5%) representative specimens, according to their species, engorgement status and sampling area. The occurrence of Anaplasma spp., Ehrlichia spp., Hepatozoon spp., Rickettsia spp., Babesia spp., Theileria spp. and Cercopithifilaria spp. was assessed by conventional and quantitative real-time PCR. Overall, 150 dogs (48.4%) were infested by ticks, with Rhipicephalus sanguineus (sensu lato) being the most prevalent (70.1%), followed by Haemaphysalis parva (14.7%), Rhipicephalus turanicus (11.4%), and Haemaphysalis concinna (2.4%). Out of 344 specimens molecularly examined, 41 (11.1%) were positive for at least one microorganism (i.e. 5.5% for Cercopithifilaria bainae, 2.9% for Hepatozoon canis, 1.7% for Rickettsia hoogstraalii, 1.2% for Hepatozoon felis, 0.6% for Rickettsia massiliae, 0.6% for Theileria ovis, 0.3% for Anaplasma platys and 0.3% for Coxiella like-endosymbiont). The results of this study show that different tick species parasitize dogs in Greece, carrying a range of microorganisms potentially pathogenic for dogs and humans. Consequently, control strategies against ticks are of great importance to prevent the risk of tick-borne diseases. The relationship between ticks infesting dogs and associated microorganisms is described according to collection site and dog lifestyle.


Background
Ticks represent a major threat to domestic and wild animals worldwide due to blood depletion, inoculation of toxins and allergens and, importantly, pathogen transmission [1]. A range of viruses, bacteria and protozoa causing tick-borne diseases (TBDs) induces economic losses in livestock production [2] and puts at risk the health of companion animals [3,4]. In addition, several tick-borne pathogens are of zoonotic concern and their transmission to humans is related to a number of driving factors, including the presence of proper vectors and hosts [5][6][7][8]. The distribution of ticks and their vectored pathogens is affected by a plethora of biological and environmental determinants, including climate changes, deforestation, and urbanisation, which may together favour the spreading and establishment of selected vectors into previously free areas [9][10][11]. The scientific knowledge on the ecology of different tick species becomes, therefore, pivotal to assess the risk factors for pathogen transmission.
While most of the studies carried out have focused their attention on the ixodid fauna of livestock, only a few have been performed on dogs, mainly in the northern part of the Greek peninsula [16,17]. Accordingly, selected canine tick-borne pathogens (e.g. Hepatozoon canis, Anaplasma spp., Rickettsia spp. and Cercopithifilaria spp.) have also been detected [21][22][23], although there is no clear association between tick species and their pathogens.
In order to fill this gap in knowledge, this study aimed to investigate the distribution of hard ticks and carried pathogens in dogs living under different conditions across Greece.

Tick collection and identification
From May to August 2015, tick specimens were collected on domestic dogs living in six provinces across Greece (Fig. 1, Table 1), specifically from southern (Corinth, site A; Athens, site B), central (Larisa, site C) and northern regions (i.e. Xanthi, site D; Thessaloniki, site E; Alexandroupoli, site F). Ticks were sampled on animals from rural areas, municipal shelters, temporary kennels, indoor environments, or in hospitalised animals ( Table 1).
All ticks were preserved in 70% ethanol and categorised according to their gender and developing stages. Specimens were morphologically identified at species level using morphological keys [12,24,25].

Pathogen molecular diagnosis
Genomic DNA was extracted from a representative number of tick specimens, according to their species, engorgement status and sampling area. Ticks were cut into small pieces using sterile scalpels, homogenized in 300 μl of DNA extraction buffer (20 mM Tris-HCl pH 8; 100 mM EDTA and 1% SDS, 400 μg proteinase K), and incubated overnight at 37°C. Protein was precipitated using 50 μl of 5 M potassium acetate, before samples were stored on ice for 10 min and centrifuged at 13,000× g for 5 min. The DNA pellet was precipitated using 300 μl of 100% isopropanol followed by centrifugation at 13,000× g for 5 min and a final wash with 300 μl of 70% ethanol and centrifugation at 13,000×g for 5 min. Pellets were air dried and resuspended in 50 μl TE (10 mM Tris-HCl, 1 mM EDTA pH 8).
PCR products were examined on 2% agarose gels stained with GelRed (VWR International PBI, Milano, Italy) and visualised on a GelLogic 100 gel documentation system (Kodak, New York, USA). The amplicons were purified and sequenced in both directions using the same primers used for PCR and qPCR, employing the Big Dye Terminator v.3.1 chemistry in a 3130 genetic analyser (Applied Biosystems, California, USA). Sequences were aligned using the ClustalW program [31] and compared with those available in GenBank by Basic Local Alignment Search Tool (BLAST -http://blast.ncbi .nlm.nih.gov/Blast.cgi).

Statistical analysis
Prevalence (proportion of hosts infested by ticks and of tick species positive for a given pathogen) and tick infestation burden (arithmetic mean count of ticks on each infested hosts) were assessed. For prevalence rates > 5%, Fisher's exact test was used to compare the prevalence of infection among sampling areas, and among dog keeping conditions. Differences were considered significant when P <0.05. Statistical analyses were performed using BioEstat 5.0.
Though not statistically significant (P > 0.05), the tick burden varied according to dog type (i.e. dogs sheltered in temporary kennels or living in rural areas were more often infested than those referred to private veterinary clinics or housed indoor) and the sampling sites. Dogs from site D (northern Greece) harboured more ticks than those from sites B and C (southern and central Greece) (Table 1). While R. sanguineus (s.l.) was found in all the sampling areas, R. turanicus and H. parva were not detected in site C and sites A, B or D, respectively ( Table 2). Specimens of I. ricinus, H. scupense and R. bursa were found only in northern regions (Fig. 1, Table  2). Rhipicephalus sanguineus (s.l.), R. turanicus, R. bursa and H. scupense were thoroughly collected from dogs living in rural areas, municipal shelter, and referred to private clinics, whereas Haemaphysalis spp. were mainly found on animals from temporary kennels ( Table 2).
Out of 344 specimens analysed, 41 (11.1%) were positive for at least one microorganism based on PCR (Table  2), with the largest number of positives being detected in the northern regions (Fig. 1). Nineteen ticks (5.5%; nine females, two larvae, and eight nymphs) were positive for Cercopithifilaria bainae, 10 (2.9%; two males, four females, one larva, and three nymphs) for H. canis and six (1.7%; one male and five females) for Rickettsia hoogstraalii. Other microorganisms were detected less frequently, such as Hepatozoon felis (1.2%; one male, one female, and two nymphs), Rickettsia massiliae (0.6%; one larva and one nymph), Theileria ovis (0.6%; two males), A. platys (0.3%; one nymph) and for a Coxiella-likeendosymbiont (0.3%; one female) ( Table 2, Fig. 1). Co-infections with multiple microorganisms were detected in six specimens. In particular, C. bainae was detected in combination with H. felis or H. canis or R. massiliae in R. sanguineus (s.l.), and with H. canis in R. turanicus. Rickettsia hoogstraalii was simultaneously diagnosed with H. felis or H. canis and C. bainae in H. parva. Associations between tick developmental stages, dog lifestyles, collection site and microorganisms are reported in Table 2, and none of the parameters evaluated was statistically significant (P > 0.05).

Discussion
Data from this study indicate that dogs from Greece are exposed to different tick species and, potentially, to   several tick-borne pathogens, whose occurrence is not strictly influenced by the conditions in which dogs live. Nonetheless, the finding of a higher tick burden in animals from kennels, rural areas or municipal shelters compared to those kept indoor is presumably related to the frequency of treatment against ectoparasites, which is performed more frequently in pet than in shepherd or kennelled dogs [32]. Overall, the collection of different tick species in each of the geographical areas confirms the existence of a marked ixodid diversity in the Greek peninsula [16,20,22] with a higher number of ticks sampled in northern areas bordering continental Europe (i.e. Macedonia, Turkey and Albania) [16,32,33] than southern regions [16,20].
Rhipicephalus sanguineus (s.l.) was the most prevalent species throughout Greece, most likely due to its strict affiliation to canids [25], and/or its ability to survive in a large array of environmental conditions [13,16,[34][35][36]. Conversely, the finding of R. turanicus on dogs is probably related to its adaptability to several vertebrate animals, including goats and sheep [25], which have a major role in the economy of this country. Along with R. bursa and H. scupense, R. turanicus can often be detected on livestock in the northern regions of Greece [20], where they often parasitize dogs [16]. Haemaphysalis parva and H. concinna usually parasitize birds as larvae and nymphs, and herbivores as adults [24]. Finding these species on dogs was likely due to the location of animal shelters, close to forested, meadow and rural habitats [37].
Amongst the microorganisms detected, the filarioid C. bainae was the most common, being found in R. sanguineus (s.l.), as previously reported [22]. The detection of C. bainae in H. parva probably occurred during the ingestion of skin-dwelling microfilariae during the tick blood meal. Nonetheless, considering that the same tick species was found positive for H. canis and H. felis, its implication as a vector for these pathogens cannot be ruled out. Though H. felis has been detected in R. sanguineus (s.l.) [38,39], the vector of this protozoon remains unknown, whereas H. canis has been so far been detected in a number of other tick species, including Haemphysalis spp. and R. turanicus [40,41]. In the current study, H. parva specimens were positive for R. hoogstraalii, adding new scientific information to knowledge on this Rickettsia species. Rickettsia hoogstraalii was originally isolated from H. sulcata from sheep and goats in Croatia [42], H. punctata and H. sulcata from Spain [43] and, in the same tick species from foxes in Cyprus [44]. Rickettsia massiliae was here found in R. sanguineus (s.l.) and R. turanicus collected from dogs living in municipal shelters in northern Greece, close to the Turkish boundaries. This finding is not surprising when considering that the transstadial and transovarial transmission of R. massiliae has been described in both Rhipicephalus species [45]. Besides the current report, R. massiliae has been detected in R. sanguineus (s.l.) and R. turanicus from Greece and other countries (e.g. Spain, Portugal, Switzerland, France, Algeria, Morocco, Israel and Italy) [6,46], as well as in Rhipicephalus mushamae, Rhipicephalus lunulatus, Rhipicephalus sulcatus and Rhipicephalus guilhoni [6,25].
Remarkably, R. massiliae has not yet been isolated from humans in Greece, but its detection in Rhipicephalus spp. ticks suggest that the risk for human infections is probably underestimated [46,47]. The absence of E. canis and Babesia vogeli positive ticks in this study was surprising as both pathogens are transmitted by R. sanguineus (s.l.) ticks [48,49], and could be explained by their transitory parasitaemia and by the number of ticks examined.

Conclusions
The results of this study show that different tick species parasitize dogs in Greece, carrying a range of microorganisms potentially pathogenic for dogs and humans. As such, control strategies against ticks are of great importance to prevent the risk of TBDs.