Red foxes (Vulpes vulpes) have recently been recognized as potential reservoirs of several vector-borne pathogens and a source of infection for domestic dogs and humans, mostly due to their close vicinity to urban areas and frequent exposure to different arthropod vectors. The aim of this study was to investigate the presence and distribution of Babesia spp., Hepatozoon canis, Anaplasma spp., Bartonella spp., ‘Candidatus Neoehrlichia mikurensis’, Ehrlichia canis, Rickettsia spp. and blood filaroid nematodes in free-ranging red foxes from Bosnia and Herzegovina. Spleen samples from a total of 119 red foxes, shot during the hunting season between October 2013 and April 2014 throughout Bosnia and Herzegovina, were examined for the presence of blood vector-borne pathogens by conventional PCRs and sequencing. In the present study, three species of apicomplexan parasites were molecularly identified in 73 red foxes from the entire sample area, with an overall prevalence of 60.8%. The DNA of B. canis, B. cf. microti and H. canis was found in 1 (0.8%), 38 (31.9%) and 46 (38.6%) spleen samples, respectively. In 11 samples (9.2%) co-infections with B. cf. microti and H. canis were detected and one fox harboured all three parasites (0.8%). There were no statistically significant differences between geographical region, sex or age of the host in the infection prevalence of B. cf. microti, although females (52.9%; 18/34) were significantly more infected with H. canis than males (32.9%; 28/85). The presence of vector-borne bacteria and filaroid nematodes was not detected in our study. This is the first report of B. canis, B. cf. microti and H. canis parasites in foxes from Bosnia and Herzegovina and the data presented here provide a first insight into the distribution of these pathogens among the red fox population. Moreover, the relatively high prevalence of B. cf. microti and H. canis reinforces the assumption that this wild canid species might be a possible reservoir and source of infection for domestic dogs.

Delimitation of species is often complicated by discordance of morphological and genetic data. This may be caused by the existence of cryptic or polymorphic species. The latter case is particularly true for certain snail species showing an exceptionally high intraspecific genetic diversity. The present investigation deals with the Trochulus hispidus complex, which has a complicated taxonomy. Our analyses of the COI sequence revealed that individuals showing a T.hispidus phenotype are distributed in nine highly differentiated mitochondrial clades (showing p-distances up to 19%). The results of a parallel morphometric investigation did not reveal any differentiation between these clades, although the overall variability is quite high. The phylogenetic analyses based on 12S, 16S and COI sequences show that the T.hispidus complex is paraphyletic with respect to several other morphologically well-defined Trochulus species (T.clandestinus, T.villosus, T.villosulus and T.striolatus) which form well-supported monophyletic groups. The nc marker sequence (5.8S-ITS2-28S) shows only a clear separation of T.o.oreinos and T.o.scheerpeltzi, and a weakly supported separation of T.clandestinus, whereas all other species and the clades of the T.hispidus complex appear within one homogeneous group. The paraphyly of the T.hispidus complex reflects its complicated history, which was probably driven by geographic isolation in different glacial refugia and budding speciation. At our present state of knowledge, it cannot be excluded that several cryptic species are embedded within the T.hispidus complex. However, the lack of morphological differentiation of the T.hispidus mitochondrial clades does not provide any hints in this direction. Thus, we currently do not recommend any taxonomic changes. The results of the current investigation exemplify the limitations of barcoding attempts in highly diverse species such as T.hispidus.

Trochulus oreinos oreinos and T. oreinos scheerpeltzi are two land snail taxa endemic to the Northeastern Austrian Alps, which have been regarded as subspecies of the highly variable, widespread land snail T. hispidus. We analysed these three taxa morphologically and genetically to evaluate whether a delimitation between them is possible and, if so, to resolve their phylogenetic relationships. Shell morphological results revealed high similarity between the two T. oreinos taxa, and that they are clearly separated from T. hispidus. Additionally, the T. oreinos subspecies concur with respect to their habitat preferences, as they are both restricted to rocky high alpine areas, whereas the local form of T. hispidus is distributed over a wider altitudinal range in moist areas and scrubby perennial herb vegetation near water bodies. While the morphological and ecological results allow clear differentiation between T. hispidus and T. oreinos only, analyses of the mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes revealed high sequence divergences between all three taxa, which indicates that they represent old lineages. The two T. oreinos taxa appear as distantly related sister groups, well separated from T. hispidus. Whether T. o. oreinos and T. o. scheerpeltzi should be considered as species cannot be decided at the current state of knowledge.

Background: The order Accipitriformes comprises the largest group of birds of prey with 260 species in four families. So far, 21 haemosporidian parasite species have been described from or reported to occur in accipitriform birds. Only five of these parasite species have been characterized molecular genetically. The first part of this study involved molecular genetic screening of accipitriform raptors from Austria and Bosnia-Herzegovina and the first chromogenic in situ hybridization approach targeting parasites in this host group. The aim of the second part of this study was to summarize the CytB sequence data of haemosporidian parasites from accipitriform raptors and to visualize the geographic and host distribution of the lineages. Methods: Blood and tissue samples of 183 accipitriform raptors from Austria and Bosnia-Herzegovina were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR, and tissue samples of 23 PCR-positive birds were subjected to chromogenic in situ hybridization using genus-specific probes targeting the parasites’ 18S rRNAs. All published CytB sequence data from accipitriform raptors were analysed, phylogenetic trees were calculated, and DNA haplotype network analyses were performed with sequences from clades featuring multiple lineages detected in this host group. Results: Of the 183 raptors from Austria and Bosnia-Herzegovina screened by PCR and sequencing, 80 individuals (44%) were infected with haemosporidian parasites. Among the 39 CytB lineages detected, 18 were found for the first time in the present study. The chromogenic in situ hybridization revealed exo-erythrocytic tissue stages of Leucocytozoon parasites belonging to the Leucocytozoon toddi species group in the kidneys of 14 infected birds. The total number of CytB lineages recorded in accipitriform birds worldwide was 57 for Leucocytozoon, 25 for Plasmodium, and 21 for Haemoproteus. Conclusion: The analysis of the DNA haplotype networks allowed identifying numerous distinct groups of lineages, which have not yet been linked to morphospecies, and many of them likely belong to yet undescribed parasite species. Tissue stages of Leucocytozoon parasites developing in accipitriform raptors were discovered and described. The majority of Leucocytozoon and Haemoproteus lineages are specific to this host group, but most Plasmodium lineages were found in birds of other orders. This might indicate local transmission from birds kept at the same facilities (raptor rescue centres and zoos), likely resulting in abortive infections. To clarify the taxonomic and systematic problems, combined morphological and molecular genetic analyses on a wider range of accipitriform host species are needed.