Apart from the concerns about human health, there is an increasing interest in monitoring of canine tick-borne diseases, and how the eco-epidemiological patterns of the spread of ticks and pathogens change through time. This study aimed to investigate the prevalence of the pathogen species in ticks removed from dogs in Latvia and to analyze any possible changes between years 2011 and 2016. The results showed that, in our settings, monitoring at time points six years apart was not sufficient to observe any significant changes in the prevalence rate of the tick-borne pathogens, which have been circulating in the environment for a long time, and/or for already established loci. These results indicate the relative stability of the environmental processes. Similar results were obtained in a French study where no evidence was observed for a climate-associated increase in infection risk over the 7-year period [14]. However, during the last decade, the spread of D. reticulatus has been notable in several European countries, including Latvia and neighboring Lithuania [12, 15]. In this study, almost 7% of ticks removed from dogs in 2016 were D. reticulatus comparing to none in 2011, and the difference was statistically significant. This result is in accordance with previous observations: while no Dermacentor ticks were collected by flagging in years 2005–2007 in Latvian regions, new localities with D. reticulatus occurrence have been found in southern Latvia in the years 2013–2014 [12, 16]. Moreover, our study demonstrated that, until the year 2016, the spreading of D. reticulatus ticks occurred further to the north than previously reported - up to the Gulf of Riga (Fig. 1). Climate is probably the major driver to the presence or absence of a tick species in a given territory [17]. Indeed, the mean annual air temperature in Latvia in years 2011–2016 was 0.2–1.9 °C higher than the usual average + 5.9 °C, while the mean annual precipitation fluctuated (Fig. 2; Data source: Latvian Environment, Geology and Meteorology Centre, https://www.meteo.lv/en/). Importantly, the mean air temperature in the Baltic States including Latvia is increasing fastest in winter and spring, and decrease in the snow cover duration during the 1961–2015 period. In addition, a change of the type of winters after 1989 with a later snow cover formation and earlier snowmelt was observed for this area [18]. Thus, while various biotic and abiotic variables influencing overall D. reticulatus tick abundance have been reported [19], it could be proposed that climate change supported the emergence of D. reticulatus foci in Latvia.
Further, 13 tick-borne human and canine pathogens were detected in ticks removed from dogs this study. In total, 35.8 and 40.0% of ticks were pathogen-positive in 2011 and 2016, respectively; this difference was not statistically significant. The spectrum of detected pathogens was compatible to those reported recently in dog-associated ticks collected in Germany, Italy, Belgium and Poland [20,21,22,23].
The most prevalent pathogen genus was Rickettsia, it was detected in 24.3% of I. ricinus, 8.3% of I. persulcatus and 11.1% of D. reticulatus ticks. Interestingly, in studies of pet-associated ticks, Rickettsia spp. was detected in 18.4 and 14.1% of Ixodes ticks in Italy and Belgium, respectively, while over 50% of I. ricinus were positive for Rickettsia spp. in Poland and Germany; also, Dermacentor ticks were pathogen-negative in Belgium, but 39% of D. reticulatus were found to be Rickettsia–positive in the study in Germany [20,21,22,23]. Such non-uniform prevalence of pathogens could be related to many variables including, among others, existence of natural foci, tick and host abundance, sampling area and sampling season. For example, large differences in pathogen frequencies in questing Dermacentor ticks (31.4–78.3%) were observed between sampling sites in Germany [19]. However, methodology-related factors such as the level of tick engorgement, feeding on an infected animal and sensitivity of the methods used should also be considered.
In our study, three Rickettsia species were identified: R. helvetica, R. monacensis and R. raoultii. R. helvetica has been isolated from Ixodes ticks in many European and Asian countries, however, its pathogenicity is relatively unknown. While there is some evidence that it may cause disease in humans [24], there are no such reports regarding clinical cases in dogs. On the other hand, R. monacensis is recognized as an emerging human pathogen, as cases of infection in humans were reported in Spain, Italy, the Netherlands and South Korea [25]. R. monacensis was also identified in a blood sample of a dog (0.7%) in Maio Island, Cape Verde archipelago, however, its pathogenicity in animals is still unknown [26]. R. raoultii is frequently detected in multiple tick species and, along with R. slovaca, is a causative agent of a syndrome in humans known as DEBONEL/TIBOLA (Dermacentor-borne necrosis erythema and lymphadenopathy/Tick-borne lymphadenopathy) [27]. It is a newly recognized emerging disease, as its incidence has been increasing in Europe during the last decade [28]. Clinical cases in animals induced by R. raoultii have not been described so far, however, in dogs in Germany R. raoultii DNA was detected in 0.68% of samples and a seroprevalence of 2.8% was reported [29, 30].
A. phagocytophilum is the etiologic agent for Human Granulocytic Anaplasmosis (HGA), which occurs in America, Europe and Asia [31]. There are reports on granulocytic anaplasmosis in a variety of domestic and wild animal species including dogs, cats, horses and cattle [32]. The main vector in Europe is I. ricinus, however, A. phagocytophilum has been detected in questing ticks of many species including D. reticulatus and I. persulcatus, and the overall infection rate of I.ricinus ticks in Europe varied from 0.4% to even 33.9% in some localities (reviewed in [32]). Seroprevalence studies in European dogs indicated that 3 to 57% of dogs carried A. phagocytophilum [33], while in Latvia, A. phagocytophilum seroprevalence in dogs was significantly higher in the I. ricinus region than in the I. persulcatus region (12.5% vs 2%) [34]. In the present study, 6% of I. ricinus ticks removed from dogs were A. phagocytophilum-positive, confirming risks present for humans and animals.
In I. ricinus, a member of the relapsing fever group spirochete B. miyamotoi, as well as Lyme-disease borrelia B. afzelii, B. garinii, B. valaisiana, B. spielmanii were present, but no Borrelia-positive D. reticulatus ticks were detected in our study. While these pathogens are of a high importance to human health, studies in Europe have shown that many dogs are exposed to Borrelia, but only a small number of seropositive animals ever have a clinical disease (reviewed in [35]). According to the American College of Veterinary Internal Medicine consensus update on Lyme borreliosis in dogs and cats it is stated that in dogs residing in North America Lyme borreliosis is associated only with B. burgdorferi sensu stricto, and it has not yet been proven that borrelia found in Europe can cause clinical signs in dogs [36]. Nevertheless, for the future reference and from a standpoint of the OneHealth perspective it is important to find out, which borrelia species are prevalent in ticks removed from animals.
Among Babesia, B. microti, B. venatorum, B. capreoli and B. canis were detected in this study. Both B. microti and B. venatorum are considered to pose a zoonotic risk to humans, but no reports exist that they either infect or cause disease in dogs. On the other hand, 1.6% of ticks carried B. canis, the agent of canine babesiosis. Importantly, none B. canis-positive field-collected ticks were detected in Latvia in the 2005–2007 time frame, while the first autochthonous canine babesiosis cases in the country were reported between years 2009 and 2011, and B. canis was detected in D. reticulatus ticks in Latvia in the 2013–2015 time frame [11, 16, 37]. Unsurprisingly, in this study, the prevalence of B. canis in D. reticulatus was significantly higher than in I. ricinus ticks (14.8% vs 1%; P ≤ 0.05). The presence of B. canis in I. ricinus observed here is in accordance with the study of Cieniuch and colleagues [38] in Poland, which found out that around 1% of field-collected I. ricinus ticks were infected, and may explain the cases of autochthonous canine babesiosis in Latvia in 2009–2011, when Dermacentor ticks were apparently absent in the country [11]. However, future studies are needed to confirm the role of this tick species. On the other hand, a possibility of a cross-infection exists, when multiple ticks, including infected D. reticulatus, are feeding on one animal, or if the dog itself is a carrier of the disease.
While most people in Latvia are aware of human tick-borne diseases, information to the general public on the same topic in house-hold pets is insufficient in Latvia, leading to common misconceptions such “my dog/cat is exposed to ticks so frequently that it is immune to them” (Seleznova M., personal communication). This lack of awareness also means that a lot of pet owners disregard regular anti-tick treatments to their animals during the active season as unnecessary. This situation was exacerbated during the last decade because of the speedy spreading of D. reticulatus, which were previously absent in the country, and the risk of infection arising from this tick species populations is not sufficiently investigated. The results of this study confirmed that the spread of novel vectors could bring additional risk of exposure to novel emerging pathogens to pets and their owners, as both B. canis and R. raoultii were shown to be highly associated with D. reticulatus ticks. In addition, the clear possibility of dog’s infestation with several ticks in the sympatric areas for Ixodes and Dermacentor tick species increases the probability of the co-infection with several pathogens, which, in turn, could increase the risk of severe pathology, and complicates both, diagnosis and therapy. Infection with tick-borne pathogens can also be complicated by other arthropod-borne diseases that share the tick biohabitat, and co-infection could partially explain variations in clinical presentation, pathogenicity and response to therapy in dogs [10].
In this study, a co-infection with two and three tick-borne pathogens was detected in 7.9 and 7.4% of I. ricinus and D. reticulatus samples, respectively, and a high variability of co-infections was observed. In total, 19 different pathogen combinations were detected among the samples, none of which appeared to be a dominant pattern. This result indicates that a substantial risk of the co-infection with multiple tick-borne pathogens exists, and the combination of pathogens appears to be a dynamic process which varies depending on the changes in the prevalence of separate pathogens in nature. Thus the awareness regarding possible co-infections in ticks should be increased and further studies are needed, especially against the background of the climate change, the emergence and the spread of the sympatric areas for Ixodes and Dermacentor tick species, and increasing importance of pet travel.
Several limitations of this study should be outlined. First, only adult ticks were included in this study. While the removal of the adult ticks from animals is relatively easy, the molecular analysis of nymphs could provide additional important data. Secondly, the ticks were collected from dogs and were partially or fully engorged. Thus, a possibility exists that some tick samples were pathogen-positive because of feeding on a positive dog. Also, the number of I. persulcatus ticks available for this study was very low and did not mirror the actual spread of this tick species in Latvia (according to data presented in [16, 34, 39]. Only R. helvetica was identified in these samples, however, the presence of different pathogens in the field-collected I. persulcatus ticks such as Babesia, Borrelia and Ehrlichia have been shown previously in Latvia.
