Furthermore, the 16S rRNA and groEl gene loci

Furthermore, the
results from the same study showed that foxes can bear unknown blood-associated
organisms (Publication 2). Molecular
and phylogenetic analyses of the 16S rRNA and groEl gene loci discovered a
potentially new bacterial species belonging to the recently recognized cluster
of Candidatus Neoehrlichia, herein
designated as Candidatus Neoehrlichia
sp. (FU98). The nucleotide sequences of both genes analysed in the study are
genetically most closely related to Candidatus
Neoehrlichia lotoris merely found in raccoons (Procyon lotor Linnaeus, 1758) in North America (Yabsley et al.,
2008), but distinct from the sequences of emerging and tick-transmitted Candidatus Neoehrlichia mikurensis
infecting animals and humans in Euroasia (Hornok et al., 2017). After the first
report of Candidatus Neoehrlichia sp.
(FU98) in two foxes from western Austria, the same sequence types have also
been recorded in foxes from the Czech Republic (Hodži? et al., 2017b) and Serbia (data not
published), and a European badger (Meles
meles) from Hungary (Hornok et al., 2017)
displaying a broader geographic and host range of the bacterium, possibly
introduced to Europe via raccoons. However, it is not yet clear whether the
sequences obtained from the wild carnivores belong to Candidatus Neoehrlichia lotoris or represent a new candidate
species, and our attempts to localize and properly characterize the
microorganism by in situ
hybridization and multilocus sequence typing (MLST) approach failed. Therefore,
experimental transmission studies are needed to shed more light on the
classification, biological and pathogenic features and zoonotic potential of
this enigmatic bacterium.

In respect to Babesia cf. microti and H. canis, the red fox fulfils all the criteria required for
reservoir host characterization and fit into the definition of primary
reservoir host, which is defined as “one or more epidemiologically connected
populations or environments in which the pathogen can be permanently maintained
and from which infection is transmitted to the defined target population”
(Haydon et al., 2002). In contrast, foxes are supposedly only accidental hosts
of zoonotic vector-borne bacteria and have limited impact on their circulation
and transmission, as already reported (Henn et al., 2009; Dumitrache et al.,

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The study presented in Publication
3, describes a novel Hepatozoon
species (Adeleorina: Hepatozoidae) principally infecting European wild cats,
named Hepatozoon silvestris sp. nov.,
based on its unique morphological and molecular features. The species
description was done following the rules of International
Code of Zoological Nomenclature (ICZN) and it has been registered in
ZooBank database. Moreover, the study reports the occurrence of Hepatozoon felis for the first time in
this sylvatic felid species. Several parasitic forms at different developmental
stage were recorded in the infected animals by cytology and histopathology.
Heart was the most frequently and severely affected organ indicating a high affinity
of the newly described Hepatozoon
species to cardiac muscle tissue, which harbour the location of merogonic
development. However, there were no significant pathological changes associated
with the presence of the parasite in the investigated animals. Among the Hepatozoon spp. described in carnivores,
H. silvestris sp. nov. is
morphologically and phylogenetically most closely related to H. felis reported from domestic and wild
cats throughout the world. Apart from the genetic differences in the 18S rRNA
gene between these two protozoan species, differences in appearance, size and
shape of the developmental stages can also be used for the species
differentiation. The arthropod vector(s) implicated in the transmission of both
H. silvestris sp. nov. and H. felis is unknown, but I. ricinus and I. hexagonus as the most dominant ticks infesting European wild
cats (Lorusso et al., 2011; Gallusová et al., 2016; D’Amico et al., 2017b)
could be potential candidates. Ixodes
ricinus was also found on a single cat tested in the study however, the
presence of Hepatozoon oocysts was
not confirmed by the tick dissection. Nonetheless, transplacental transmission
of H. felis has been confirmed in
domestic cats (Baneth et al., 2013) and may also represent an optional
transmission pathway of H. silvestris
sp. nov.. Furthermore, the predatory behaviour of the wild cats and the
parasitic forms that occur in different tissues might suggest that infection
with feline Hepatozoon spp. is
transmitted by carnivorism. More recent records of H. silvestris sp. nov. in domestic cats from areas in southern
Italy (Giannelli et al., 2017b) and Switzerland (Walter Basso, personal
communication) where European wild cats are known to be present (IUCN Red List
map, 2018) may indicate the possible cross-infection among the populations of
domestic and wild cats. The cat from Switzerland suffered from severe
myocarditis suggesting the pathogenic impact of this newly characterized
infectious agent causing hepatozoonosis in domestic cats.

Morphological similarities of ticks from the subgenus Pholeoixodes (Ixodidae: Ixodes) feeding on carnivores and
uninformative description of some tick species (i.e. Ixodes crenulatus) often lead to misidentification and incorrect
definition of their geographical distribution. Therefore, the aims of the last
study (Publication 4) were to
identify ticks collected from dogs, foxes and badgers based on the
morphological features, and to revise their taxonomic status employing the
molecular and phylogenetic analyses of two mitochondrial gene markers, cox1 and 16S rRNA. The ticks identified
as Ixodes canisuga, Ixodes kaiseri and I. hexagonus were collected from the animals in nine European
countries. The analyses of the cox1
nucleotide sequences revealed a higher level of intraspecific divergence
compared to 16S rRNA gene, represented by 11 (referred as A to K), nine (L to
T) and five (U to Y) haplotypes of I.
canisuga, I kaiseri and I. hexagonus, respectively. These
findings advocate the continuous gene flow among different tick populations in
Europe. However, dispersal and distribution of ticks over long distances depend
on the vertebrate host mobility (Aguirre, 2009), and red fox as an important
and widespread host for all three tick species most likely contribute to the
gene exchange and low level of contemporary genetic structuring (Araya-Anchetta
et al., 2015). The information on population dynamics and distribution of ticks
may help to estimate the differences in pathogen transmission pattern between
population sub-groups (Araya-Anchetta et al., 2015). Furthermore, the
phylogenetic analyses demonstrated a closer genetic relationship between I. kaiseri and I. hexagonus than to I.
canisuga, which is congruent to the morphological observations. The
findings of the study also recommend the shape of anterior surface of basis capituli should always be taken
into account for the traditional morphology-based identifications of females of
Pholeoixodes ticks from carnivores.

In conclusion,
the results obtained by using molecular tools revealed that foxes are hosts for
a diversity of pathogens and further advocate their high reservoir competence
for Babesia cf. microti and H. canis.
Moreover, population genetic study performed within the thesis project
demonstrate the circulation of the protozoan parasites between foxes and dogs.
In areas where the tick vectors of H.
canis are not present, foxes can play an extremely important role in the
pathogen maintenance through the transplacental transmission herein observed.
Therefore, our results provide highly relevant information for better
understanding the epidemiology of TBDs and may represent a solid base for the
future research studies on host-tick-pathogen interactions. However, multi-component
and complex life cycles of the microorganisms may change over the time
following variations in the animal population size, geographical range and
community composition (Tomassone et al., 2018). Consequently, continuous
screening of wild carnivores for ticks and the associated pathogens is of a
great importance for determining the risks of infections and identification of
previously unrecognized agents with disease-causing potential in domestic and
wild animals. Characterization of disease reservoirs and pathogens circulating
in environments are also critical for conservation and success of endangered
animal species (Lesniak et al., 2017), including recently expanding populations
of large carnivores, i.e. brown bears (Ursus
arctos), Eurasian lynx (Lynx lynx),
gray wolves (Canis lupus), and
wolverines (Gulo gulo) (Chapron et
al., 2014).

            Future research should be focused on the transmission experiments in
order to identify the competent arthropod vectors implicated in the life cycles
of many pathogens and to elucidate the potential impact of the pathogens on the
wild carnivore conservation. This would also contribute to the control of existing
and identification of new foci of TBDs.