Our results revealed the presence of Anaplasma, Babesia and Theileria species in questing ticks from the northwest of Spain although the prevalence found were lower than 2%.
The percentage of I. ricinus positive to A. phagocytophilum was similar to those (0.4%-0.6%) reported in questing specimens of this tick species from some European countries such as Hungary, Slovenia and The Netherlands [2, 32, 33]. However, our data contrast with previous investigations performed in northern Spain where the percentage of A. phagocytophilum positive questing I. ricinus ranged from 5.6–20.5% [21]. Similarly, prevalence ranging between 1.9% and 23.6% were detected in questing I. ricinus collected in most European countries [1, 5, 13, 34]. These noticeable differences among the prevalence of A. phagocytphilum in I. ricinus throughout Europe may be mainly related to the presence and abundance of reservoirs and susceptible hosts in these areas, although other variables such as the season of study or the number of ticks analysed must be also considered.
A high heterogeneity of A. phagocytophilum groEL-sequences was found (Table 3). Nevertheless, all those single nucleotide polymorphisms do not imply changes in the amino acid sequence except for a single sequence belonging to clade I (GV348) which showed a thymine at the nucleotide position 724 resulting in a change in the codified amino acid (Ala to Ser). This mutation has been observed in A. phagocytophilum variants associated to human or animal clinical cases [10, 35]. In addition, three different variants of A. phagocytophilum were detected at the 16S rRNA gene (Table 3). The most prevalent variants found, “Y” and “X”, are currently considered apathogenic [12, 14]; both variants were the most frequently detected in questing ticks and ticks feeding on both red deer and roe deer from Germany [14] as well as in roe deer from Spain [23]. Variant “W” has been previously found on I. ricinus and some mammalian species, mainly domestic and wild ungulates and it has been identified as pathogenic for cattle and sheep [12, 14, 36, 37]. This variant was detected in sample GV348, so both molecular markers (groEL and 16S rRNA) indicate that this strain may have zoonotic potential [10, 35].
Previous studies performed in Spain have reported the presence of A. phagocytophilum human-pathogenic strains in both questing I. ricinus and blood from roe deer [22–24] and human anaplasmosis cases have been also reported in this country [22]. Although sequence analysis at groEL and 16S rRNA genes does not provide complete information about A. phagocytophilum ecotypes [38] these results provide useful information about A. phagocytophilum pathogenic potential.
Although Ca. N. mikurensis has been found in free-living I. ricinus from several European countries with prevalence ranging from 0.1–24.2% [4, 8, 39], this pathogen was not detected in the present study. In Spain, in fact, Ca. N. mikurensis was only identified in two feeding I. ricinus males collected from a cow in a northern area [24]; however, neither positive questing ticks nor human cases were currently reported [24]. Since a significant number of I. ricinus was analysed in the present study, our results suggest that Ca. N. mikurensis is not present or exists with a very low percentage in free-living ticks from north-western Spain. However, further studies are needed since spreading of tick-borne diseases depends on environmental, socio-economic and demographic factors, among others [40].
The prevalence of Babesia spp. found in questing I. ricinus from the studied area was consistent with those found in previous studies carried out in Spain where a 0.5% of I. ricinus was positive [25]; in contrast, the percentage of I. ricinus positive to Theileria was lower than that found in the previous study (8.3%) [25]. Our data confirm the results reported in other European countries, since the prevalence of both pathogens is usually around 2% in questing I. ricinus [1, 41, 42]. Exceptionally, higher Babesia prevalences have been detected in questing ticks, even above 50%, that could be the consequence of high-density tick populations in sampled areas [43]. The piroplasm species identified in the present study and their diversity were different to those previously reported in I. ricinus from Spain [25] where the most prevalent piroplasms were Theileria ovis followed by Theileria sp. OT3, Theileria annulata and Theileria equi-like; the presence of Babesia spp. was limited since a single isolate of B. caballi, Babesia bigemina, Babesia ovis and Babesia major were identified. These differences could be related to the existing population host in the studied areas [42] since that investigation [25] was performed in a mountainous area from northern Spain where ovine livestock is abundant.
Both Babesia species identified in the present study are considered zoonotic as well as emerging pathogens with special interest in human health. Babesia venatorum, the most prevalent piroplasm in the present study, is frequent in its main vector, I. ricinus [44]. The prevalence found in our study was consistent with those reported (0.3% − 1%) in I. ricinus from Europe [41, 42]. Babesia microti has been detected in European I. ricinus, which could play an important role in the transmission and maintenance of this Babesia species with a prevalence ranging from 0.5–3% [41, 42]. Although B. microti was recently detected in an immunocompetent patient in Spain [45], it is worth noting that not all B. microti can infect humans. Four lineages of B. microti have been described [46] and only some variants of the USA-type are associated with human disease; thus, it has been suggested that most European cases of babesiosis caused by B. microti may be imported [47]. For this reason, the finding of B. microti in questing I. ricinus from north-western Spain may have a limited impact on human health.
It was suggested that both Babesia species showed a clear distribution pattern in I. ricinus from Europe [15]. Thus, I. ricinus from Eastern Europe are more frequently infected with B. microti, whereas B. venatorum infection is more common in ticks from western and northern Europe; Germany is considered a transitory area where I. ricinus presents similar rates of infection by both pathogens [15]. This distribution has been related to the distribution of their main vectors and reservoirs in these areas since B. venatorum have been also detected in some wild ungulates such as roe deer (Capreolus capreolus) [48, 49] and mouflons (Ovis aries musimon) [36] and B. microti is closely related to the distribution of its main host, some Microtus species such as Microtus agrestis which is a more specialist species than deer [50].
Only two I. ricinus ticks were positive to Theileria sp. OT3 (0.2%), being the second report of this pathogen in I. ricinus ticks [25]. Since its main vector remains unknown [51], further studies to determine the role of I. ricinus in the transmission of this piroplasm should be performed. This piroplasm has been detected in European wild ungulates such as roe deer, red deer (Cervus elaphus), fallow deer (Dama dama) and chamois (Rupicapra rupicapra) [45, 52] as well as in domestic ruminants such as sheep and goats [48]; however, it has been previously detected only in questing Haemaphysalis punctata and I. ricinus with prevalences of 3.6% and 1.6%, respectively [25].
The present study provides data on the presence of A. phagocytophilum, Ca. N. mikurensis and piroplasms in I. frontalis and I. acuminatus. In this respect, available information on the prevalence of pathogens in those tick species is limited and restricted to a low number of specimens. Our results are consistent with the absence or low prevalence of pathogens previously reported in I. frontalis specimens collected from birds and nest boxes since it shows endophilic behaviour and all its life stages feed mainly on birds [53]; thus, only the 2.3% and 3.6% of the specimens analysed were positive to Anaplasma bovis and A. phagocytophilum, respectively [54] and Ca. N. mikurensis has been also detected in one I. frontalis specimen feeding on a common blackbird (Turdus merula) from Russia [55]. In addition, only one investigation analysed the presence of piroplasms in I. frontalis but no positive specimens were detected [56]. It is worth noting that the close relationship between this tick species and birds hampers the transmission of those pathogens to both humans and other animals [53] suggesting a low impact on human health. Regarding I. acuminatus, it has been reported that may be involved in the endophilic cycle of some pathogens such as Borrelia afzelii, Borrelia valaisiana, Coxiella burnetii, Francisella tularensis and Rickettsia helvetica [35], although there is a lack of information concerning the vectorial capacity of this tick species [53] and no association with Anaplasma spp. or Ca. N. mikurensis has been reported up-to-now. Only one study on the presence of Babesia DNA in questing I. acuminatus was performed, although only three specimens were tested and all were negative [57].
Anaplasma phagocytophilum and Ca. N. mikurensis infection has been also reported in Dermacentor ticks such as Dermacentor reticulatus [8]; in contrast, only the former pathogen was currently identified in D. marginatus [1]. However, the vector capacity of both Dermacentor species for the transmission of A. phagocytophilum and Ca. N. mikurensis remains unknown [8]. Current available data suggest a lower prevalence of both pathogens in Dermacentor spp. than in I. ricinus; since our data show a low percentage of positive I. ricinus it is reasonable that no positive Dermacentor spp. were found. Nevertheless, further studies are needed to determine the real situation of both pathogens in Dermacentor ticks from NW Spain. Although both Dermacentor species are competent vectors of some Babesia and Theileria species such as B. caballi and T. equi [58], no specimens resulted positive. Some authors have detected Dermacentor positive to Babesia spp. and Theileria spp. in some European countries such as Slovakia, France and Poland with prevalences up to 5% [1]. In Spain, Babesia and Theileria positive questing Dermacentor were previously reported [25] and seven piroplasm species were identified, namely Theileria equi, Theileria sp. OT1, Theileria annae, Babesia canis, Babesia bigemina, Babesia divergens and Babesia caballi-like; however, the number of processed Dermacentor spp. was higher (n = 97) than in our study. In addition, although B. microti has been previously detected in questing D. reticulatus ticks [15], the role of this tick species as a vector of this piroplasm is currently unknown.