This study provides the first evidence for the occurrence of any Cytauxzoon species and of three Hepatozoon species (H. sciuri, H. felis, H. martis) in Hungary, as well as for the occurrence of latter species in central Europe. Two further mustelid-associated Hepatozoon genotypes were also detected, one of which was new and closely related to isolates reported so far only in Eastern Asia but not yet in Europe. In addition, the two H. felis genotypes found in wild cats in Hungary are new in the phylogenetic context. To the best of our knowledge, this is also the first report of H. felis and C. europaeus coinfection in any felid in Europe (where this was evaluated, no coinfection was found ).
Cytauxzoon europaeus, which was identified in a wild cat in the present study, appears to be the most widespread species of its genus in Europe, reported so far in Germany, the Czech Republic, Luxembourg, Bosnia and Herzegovina, Italy, Switzerland and France [14, 31]. While the clinical signs of infection in wild cats, if any, remain to be explored (taking into account that all studies conducted so far used postmortem sampling), domestic cats most often do not show relevant symptoms (e.g. anemia) and remain clinically unaffected . This is in contrast to what is known of C. felis in North America . Another major difference in this intercontinental comparison is that while the tick vectors of C. felis are known , this is not true for any of the European Cytauxzoon species. In this study, one of the two I. ricinus females that were collected from the Cytauxzoon-infected wild cat showed PCR positivity, and sequencing verified the presence of Cytauxzoon DNA in this tick. It cannot be ascertained if this is a consequence of taking up piroplasms with the blood meal from the infected cat, or whether this tick may have been the source of infection for its feline host. Nevertheless, as already suggested , I. ricinus is a likely candidate for a biological vector in the case of Cytauxzoon species in Europe.
Among the wild cats included in the present study, the rate of infection was higher with H. felis (3 of 4 cats found to be infected) than with C. europaeus. Autochthonous infections of domestic cats with H. felis have mostly been reported from Mediterranean region of Europe, including Portugal, Spain, France, Italy, Greece and Cyprus . More recently, an isolated case of clinical infection has also been reported in a domestic cat from eastern Austria, the country neighboring western Hungary , and imported cases have also been reported in Germany . Nevertheless, to the best of our knowledge, we report here the first case of autochthonous H. felis infection in wild cats north of the Mediterranean Basin (previously reported in Bosnia and Herzegovina ).
Importantly, the molecular and phylogenetic analyses indicated that two remarkably different genotypes of H. felis are present in wild cats in Hungary, clustering within both genogroups “I” and “II”. The first of these (in “genogroup I”) is also remarkably different from other previously reported genotypes of its clade, i.e. from H. felis reported from domestic cats (Spain) or Pantherinae (India) and ticks (Rhipicephalus sanguineus: Thailand, Turkey). Therefore, the findings of the present study add to the genetic diversity of H. felis, as reported previously , and warrant further investigations on its taxonomic status and heterogeneity.
The detection of H. martis in Hungary in two mustelid species, namely the European pine marten and the beech marten from which it has recently been described (in Bosnia and Herzegovina, as well as in Croatia ), is not surprising. However, both of these Balkan countries lie south of Hungary, and thus their mustelid populations are probably well-separated from those in northeastern Hungary sampled in the present study. Since the occurrence of H. martis has been reported from south European countries (including Spain ) and a sequence is also available in GenBank from Western Europe (MH656728: the Netherlands), the present finding in central Europe adds to the known geographical distribution of this recently described species.
Regarding the two other Hepatozoon genotypes detected in three mustelids in Hungary, the Hepatozoon sp. from the least weasel showed close molecular and phylogenetic properties to an isolate from host-seeking H. parva tick collected in Turkey . In Hungary, H. parva has also been reported, but only in the southern part of the country . To the best of our knowledge, this is the first report of this or a similar genotype in Europe. In addition, a new Hepatozoon genotype was detected in the known hosts of H. martis (i.e. the European pine marten and the beech marten) which showed a significant 18S rRNA sequence divergence from H. martis and clustered separately from this species. The two genotypes most closely related to this variant were reported in Asia, most importantly in Japanese martens where the infection was associated with granulomatous myocarditis . To the best of our knowledge, this is the first report of this genotype or any other from its phylogenetic group (Fig. 1) in Europe.
In agreement with the overall genetic similarity of H. sciuri and the monophyletic clade formed by its sequences from different European countries , the 18S rRNA gene sequences of this species from Hungary were also identical with each other and with those of this parasite from the Czech Republic, the Netherlands and Spain. It was also reported that PCR-positivity for H. sciuri occurred significantly more often for spleen DNA extracts than for liver samples . Consistent with this finding, in both PCR-positive squirrels in our study, only the spleen (but not the liver) was PCR-positive for H. sciuri. Although H. sciuri is known to infect both of these organs belonging to the haemolymphatic system , there still might be differences in the tissue tropism and frequency of Hepatozoon developmental stages, and the spleen may harbor more infected white blood cells. In line with this, the rate of positivity was found to be higher in spleen samples than in liver samples among Cytauxzoon-infected wild cats . However, in the present study, in a wild cat and a beech marten, the liver DNA extract was PCR-positive for Hepatozoon DNA, but not the spleen, highlighting the importance to evaluate both organs simultaneously if possible (and also the myocardium if the expected Hepatozoon species has muscle affinity).
The new tick-host associations observed in this study include Dermacentor species on European pine marten, H. concinna on European pine marten and least weasel and I. hexagonus on the wolverine; however, the infestation of most of the mustelid species examined here (and indigenous in Hungary) with I. ricinus and of the least weasel with I. acuminatus have already been reported . Ixodes kaiseri is known to infest wild cats, as reported in the Caucasus region  and in the present study, and I. canisuga was reported from Eurasian otter in Germany , similar to our report in Hungary in the present study. Nevertheless, these are the first records of these tick-host associations in Europe and Hungary, respectively . To the best of our knowledge, the presence of D. marginatus is also new on red squirrels, both in Hungary and in a broader geographical context. Since mustelids and squirrels have a comparable preference to use enclosures (burrows or tree holes), they frequently harbor tick species or stages that are endophilic [38, 41], in particular, species of the subgenus Pholeoixodes (as exemplified by I. kaiseri, I. hexagonus and I. canisuga), I. acuminatus and Dermacentor larvae, as supported by findings of the present study.
The results of this study on the presence of protozoan DNA in ticks should be interpreted carefully. It is evident that in the great majority of cases the reason for finding the same Hepatozoon species/genotype in a host and its tick(s) is that these were probably ingested with the blood meal, and this scenario does not indicate a vector role. However, the situation is different in the case of the wild cat that was infected with a specific H. felis genotype, but from which two engorged female I. ricinus ticks were collected with a significantly different H. felis genotype. This finding raises the possibility that the latter genotype of H. felis may be transmitted transstadially from the nymphal to the adult stage in I. ricinus. However, an experimental transmission study is required to ultimately confirm this possibility. The complete life-cycle and the tick vector of H. felis are still unknown , but this protozoan parasite has already been reported from I. ricinus . In line with this finding, during our study, both I. ricinus females collected from the infected wild cat contained the DNA of H. felis. On the other hand, the I. kaiseri larva removed from another infected wild cat was PCR-negative for H. felis DNA.
Among the four H. martis-infected mustelids only one was infected with ticks. Interestingly, all I. ricinus specimens (including 1 male, 5 females and 1 larva) collected from the PCR-positive host contained the DNA of H. martis, unlike in the case of any other Hepatozoon-infected host in the present study. The Hepatozoon genotype closely related to H. martis was detected in two tick species collected from its host, the least weasel, i.e. I. acuminatus and H. concinna, with a low rate of PCR-positivity. On the other hand, the DNA of the Hepatozoon genotype more distantly related to H. martis was detectable in all four tick species collected from their host, a European pine marten (Table 1).
These data do not specify potential vectors of the detected Hepatozoon spp., but they do indicate which tick species infest relevant hosts and have access to Hepatozoon gamonts in the study region, and the biological vectors are probably among them. On the contrary, ticks that are not vectors of Hepatozoon species may soon digest protozoan DNA together with the blood meal; therefore, negative PCR test results for all engorged larvae or nymphs of a tick species from a PCR-positive host may indicate that the tick species is not a suitable vector. This is well exemplified by our finding that all 21 ticks collected from the H. sciuri-infected squirrels had negative PCR test results. In line with this, as already suggested, the biological vectors of H. sciuri are most likely not ticks but other arthropods, probably fleas [43, 44].
Last but not least, the DNA of H. martis was demonstrated here retrospectively from a pool of questing H. concinna nymphs collected in 2007 . The corresponding sequence was identical with that of H. martis from two host species examined in this study in Hungary, suggesting that this tick species is a carrier (possibly a vector) of H. martis. This is in line with the proposed role of Haemaphysalis ticks in the transmission of certain Hepatozoon species [23, 24]. In particular, the possibility that H. concinna may play a vector role in the transmission of H. martis has already been mentioned in the context of unpublished previous data , and this is confirmed by the present results. It is noteworthy that only nymphs were found to be PCR-positive from a large set of various tick stages, indicating that in the case of transstadial transmission characteristic of Hepatozoon spp. , these may have acquired H. martis in the larval stage. This possibility is further supported by observations that typically larvae and nymphs, but not adults, of H. concinna use mustelids as hosts (Table 1; ). However, ultimately only transmission studies can confirm the biological vector role of H. concinna in the epidemiology of H. martis.
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