Investigation of The Occurrence and Genetic Diversity of Tick-associated Pathogens in Foxes

Wild animals are obviously important for tick maintenance and long-term persistence of pathogens often serving as a blood meal resource and reservoirs or amplification hosts, respectively (Lorusso et al., 2011; Rizzoli et al., 2014). Among others, red foxes are the most scrutinized wild carnivore species in Europe mostly because of their high population densities and widespread distribution (Mitková et al., 2017).

As a result, they have been suggested as reservoirs for several TBPs including those affecting companion animals and humans (Cardoso et al., 2015; Hodžić et al., 2015; Liesner et al., 2016; Ebani et al., 2017), but their reservoir status has yet to be proven (Lorusso et al., 2011). The main aim of the present project was to investigate the occurrence and genetic diversity of tick-associated pathogens in foxes and to estimate their reservoir competence based on the data herein obtained and those available in the published scientific literature.

To test our hypothesis (H1), we analyzed blood and spleen samples from 506 foxes originating from two westernmost Austrian provinces i.e., Tyrol and Vorarlberg for common pathogens derived by arthropod vectors (Publication 1). As an outcome of the comprehensive molecular study, it was found that foxes in Austria harbour a substantial number of pathogenic agents of veterinary and public health significance, and these include: Babesia Canis, Babesia cf. microti (syn. Theileria annae, Babesia microti-like, Babesia vulpes), Hepatozoon canis, Anaplasma phagocytophilum, Candidatus Neoehrlichia sp. (FU98) and Bartonella rochalimae. Correspondingly to the results of other studies from Europe (Duscher et al., 2014; Hodžić et al., 2015; Tolnai et al., 2015; Ebani et al., 2017), Babesia cf. microti and H. cranes were the most prevalent pathogens in the foxes investigated.

The high prevalence and wide geographic range of these two haematozoan parasites infecting foxes nearly all over Europe are the main reasons why they have been proposed as the main reservoir candidate. However, the high rate of infections itself does not necessarily qualify the host as a reservoir (Rizzoli et al., 2014; Alvarado-Rybak et al., 2016; Hodo and Hamer, 2017) and only indicates the exposure to the pathogen or its carrier status (Estrada-Peña and de la Fuente, 2014; Hodo and Hamer, 2017). Therefore, the reservoir role of a certain animal species can unequivocally be demonstrated only through xenodiagnostic and transmission experiments (Rizzoli et al., 2014). Unfortunately, such studies are rare and have not been performed for most TBPs, so the reservoir status of wild animals, particularly carnivores, involved in their natural transmission cycles still remains unknown (Rizzoli et al., 2014). This mainly reflects the difficulties in the long-term keeping of wild animal colonies in captivity for experimental transmission studies (Roque and Jansen, 2014).

However, the prevalence in combination with other data provides a constructive framework for estimation of the reservoir potential in absence of such experimental studies (Gürtler and Cardinal, 2015; Hodo and Hamer, 2017). The conceptual approach has already been employed for assessing reservoir host competence and the role of domestic and wildlife species in the transmission of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae), and it can be applicable to any multihost pathogen transmission system (Gürtler and Cardinal, 2015; Hodo and Hamer, 2017), including Babesia cf. microti and H. canis. Therefore, the results of the thesis are discussed in the light of the following criteria: (1) host susceptibility, (2) host infectiousness to tick vector, (3) tick-host contact, and (4) host-parasite haplotype associations.

The relative host susceptibility is defined as a proportion or probability of exposed animal host to become infected, and it can be computed from the epidemiological studies reporting the prevalence of infections (Hodo and Hamer, 2017). Babesia cf. microti and H. canis are evidently the most common parasites hosted by red foxes in Europe, with an overall rates of detection ranging from 1% (Zanet et al., 2014) to 69.2% (Cardoso et al., 2013), and 7.8% (Farkas et al., 2014) to 100% (Criado-Fornelio et al., 2003), respectively (Supplementary material).

The large discrepancy in the prevalence between the studies may be attributed to the geographic locality, abundance and density of tick vectors, red fox population size, and sensitivity of PCR assays (Cardoso et al., 2013). However, the results of our study revealed the infection rate highly depends on the tissue utilized for the molecular detection. We observed that blood is statistically more frequently infected with Babesia cf. microti compared to the spleen, while spleen exhibits the higher level of H. canis infection than blood. Therefore, an aggregate overall prevalence of infection i.e., the total number of positive animals/ a total number of tested animals in all published reports should be used for the comparative purposes (Hodo and Hamer, 2017) instead of the direct prevalence comparison since it is often deceptive (Gürtler and Cardinal, 2015). Overall infection rate higher than 20% has been proposed as one of the criteria used for reservoir host identification (Gürtler and Cardinal, 2015). The aggregate overall prevalence of Babesia cf. microti and H. canis in foxes in Europe, calculated from the available molecular genetic studies are estimated to be 23.9% and 28.4%, respectively (Supplementary material). Nonetheless, golden jackals (Canis aureus) and raccoon dogs (Nyctereutes procyonoides) are two other wild carnivore species recently recognized as suitable hosts and potential reservoirs for Babesia cf. microti (Mitková et al., 2017; Duscher et al., 2017) and H. canis (Duscher et al., 2013; Farkas et al., 2014; Mitková et al., 2017). However, their role in the eco-epidemiology of the blood parasites is uncertain and requires more in-depth studies involving a larger number of samples from different geographical regions.

Furthermore, the capacity of a suspected reservoir host to infect a tick vector (host infectiousness) is not equally distributed in the host population, and the transmission pattern is mainly determined by host (e.g. genetic constitution, body mass, sex, behaviour) and environmental factors (Hersh et al., 2012; Roque and Jansen, 2014).

Accordingly, the reservoir capacity of a given animal host may be different at different localities and time points (Estrada-Peña and de la Fuente, 2014). In absence of the xenodiagnostic surveys, the presence of a parasite in the blood observed by cytology or PCR can be used as an indicator to calculate the infectiousness index (Hodo and Hamer, 2017). The number of studies in which blood has been used for the parasite detection in foxes is considerably smaller compared to those using spleen, and showed an aggregate infectiousness index for Babesia cf. microti and H. canis of 39.1% and 19.5%, respectively (Supplementary material).

Molecular detection of these two blood parasites by PCR is the gold standard method for diagnosis of the acute and chronic phase of the infections in dogs (Otranto et al., 2011; Miró et al, 2015). The high level of parasitemia in a juvenile red fox naturally infected by H. canis, with gamonts circulating in 60% of peripheral blood neutrophils has recently been reported (Giannelli et al., 2017a). Moreover, the blood of the particular fox was proven to be infective for Rhipicephalus turanicus, but not for Ixodes hexagonus and Haemaphysalis erinacei ticks collected from the animal, which clearly denotes the host infectiousness to the competent tick vector and further emphasized the fox reservoir competence for H. canis.