Josef Harl

A molecular survey of vector-borne pathogens in red foxes (Vulpes vulpes) from Bosnia and Herzegovina.

BackgroundRed 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.MethodsSpleen 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.ResultsIn 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.ConclusionThis 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.

Genetic differentiation and shell morphology of Trochulus oreinos (Wagner, 1915) and T. hispidus (Linnaeus, 1758) (Pulmonata: Hygromiidae) in the northeastern Alps

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.

Screening blood-fed mosquitoes for the diagnosis of filarioid helminths and avian malaria.

BackgroundBoth Dirofilaria repens and recently D. immitis are known to be endemic in Hungary. As one of several recent cases, the fatal case of a dog infested with D. immitis in Szeged, Southern Hungary, received attention from the media. Hence it was decided to catch mosquitoes in the garden where the dog lived to screen for filarioid helminths and Plasmodium spp. using molecular tools.MethodsMosquitoes were caught in Szeged, in the garden where the infected dog was kept, in July 2013 with M-360 electric mosquito traps and were stored in ethanol until further procedure. Female mosquitoes were classified to genus level by morphology. Each mosquito was homogenized and analyzed for filarioid helminths and avian malaria using standardized PCR techniques. Positive mosquito samples were further identified to species level by comparing a section of the mitochondrial COI gene to GenBank® entries.ResultsIn this study, 267 blood-fed mosquitoes were caught in July 2013 in Szeged. Subsequent molecular screening revealed that not only D. immitis was present in the analyzed specimens but also DNA of D. repens, Setaria tundra and Plasmodium spp. was confirmed.ConclusionsThe analysis of blood-fed mosquitoes for the diagnosis of Dirofilaria spp. and other mosquito-borne pathogens seems to be an adequate technique to evaluate if filarioid helminths are present in a certain area. Usually only unfed female mosquitoes are analyzed for epidemiological studies. However, blood-fed mosquitoes can only be used for screening if a pathogen is present because the role of the mosquito as vector cannot be classified (blood of bitten host). Furthermore, Setaria tundra was confirmed for the first time in Hungary.

Phylogeography of the land snail genus Orcula (Orculidae, Stylommatophora) with emphasis on the Eastern Alpine taxa: speciation, hybridization and morphological variation

BackgroundThe Central and Southern European mountain ranges represent important biodiversity hotspots and show high levels of endemism. In the land snail genus Orcula Held, 1837 nine species are distributed in the Alps and a few taxa inhabit the Carpathians, the Dinarids and the Western Black Sea region. In order to elucidate the general patterns of temporal and geographic diversification, mitochondrial and nuclear markers were analyzed in all 13 Orcula species. We particularly aimed to clarify whether the Alpine taxa represent a monophyletic group and if the local species diversity is rather the result of isolation in geographically separated Pleistocene glacial refuges or earlier Tertiary and Quaternary palaeogeographic events. In order to test if patterns of molecular genetic and morphological differentiation were congruent and/or if hybridization had occurred, shell morphometric investigations were performed on the Orcula species endemic to the Alps.ResultsThe phylogenetic trees resulting from the analyses of both the mitochondrial (COI, 12S and 16S) and the nuclear (H4/H3) data sets revealed three main groups, which correspond to the three subgenera Orcula, Illyriobanatica and Hausdorfia. The reconstruction of the historic geographic ranges suggested that the genus originated in the Dinarides during the Middle Miocene and first colonized the Alps during the Late Miocene, giving rise to the most diverse subgenus Orcula. Within the latter subgenus (including all Alpine endemics) almost all species were differentiated by both molecular genetic markers and by shell morphometrics, except O. gularis and O. pseudodolium.ConclusionsThe present study confirms the importance of the Alps as biodiversity hotspot and origin center of land snail diversity. The species diversity in the subgenus Orcula was likely promoted by Miocene to Pliocene palaeogeographic events and the insular distribution of preferred limestone areas. In some cases, speciation events could be linked to the divergence of populations in glacial refuges during the Pleistocene. Sporadic contact between geographically separated and reproductively not yet isolated populations led to intermixture of haplogroups within species and even hybridization and mitochondrial capture between species.

In Search of Glacial Refuges of the Land Snail Orcula dolium (Pulmonata, Orculidae) - An Integrative Approach Using DNA Sequence and Fossil Data

Harboring a large number of endemic species, the Alps and the Western Carpathians are considered as major centers of biodiversity. Nonetheless, the general opinion until the turn of the millennium was that both Central European mountain regions did not provide suitable habitat during the Last Glacial Maximum, but were colonized later from southern refuges. However, recent molecular genetic studies provide new evidence for peripheral Alpine refuges. We studied the phylogeography of the calciphilous land snail O. dolium across its distribution in the Alps and the Western Carpathians to assess the amount of intraspecific differentiation and to detect potential glacial refuges. A partial sequence of the mitochondrial COI was analyzed in 373 specimens from 135 sampling sites, and for a subset of individuals, partial sequences of the mitochondrial 16S and the nuclear histone H3 and H4 were sequenced. A molecular clock analysis was combined with a reconstruction of the species’ geographic range history to estimate how its lineages spread in the course of time. In order to obtain further information on the species’ past distribution, we also screened its extensive Pleistocene fossil record. The reconstruction of geographic range history suggests that O. dolium is of Western Carpathian origin and diversified already around the Miocene-Pliocene boundary. The fossil record supports the species’ presence at more than 40 sites during the last glacial and earlier cold periods, most of them in the Western Carpathians and the Pannonian Basin. The populations of O. dolium display a high genetic diversity with maximum intraspecific p-distances of 18.4% (COI) and 14.4% (16S). The existence of various diverged clades suggests the survival in several geographically separated refuges. Moreover, the sequence patterns provide evidence of multiple migrations between the Alps and the Western Carpathians. The results indicate that the Southern Calcareous Alps were probably colonized only during the Holocene.

Molecular detection of Theileria sp. ZS TO4 in red deer (Cervus elaphus) and questing Haemaphysalis concinna ticks in Eastern Austria

Theileria spp. are intracellular protozoa transmitted by ixodid ticks. T. parva and T. annulata are highly pathogenic and responsible for serious disease in domestic ruminants in tropical and subtropical countries. However, asymptomatic findings of Theileria sp. in wild ungulates lead to the suggestion that wild ruminants play a role as reservoirs for these piroplasms. In a game enclosure in Eastern Austria (Federal county of Burgenland), piroplasms were detected with molecular analysis in blood samples of all 80 examined asymptomatic red deer (Cervus elaphus). Furthermore, piroplasms were detected in four out of 12 questing nymphs of Haemaphysalis concinna. In 32 Ixodes ticks sampled on-site, no Theileria DNA was detected. Sequence analysis identified these samples from both red deer and ticks as Theileria sp. ZS TO4. Our findings indicate that farmed red deer serve as asymptomatic carriers and adapted intermediate hosts of Theileria sp. in Central Europe and H. concinna was identified as a possible vector species of Theileria sp. ZS TO4.

Candidatus Neoehrlichia sp. in an Austrian fox is distinct from Candidatus Neoehrlichia mikurensis, but closer related to Candidatus Neoehrlichia lotoris

BackgroundCandidatus Neoehrlichia came under the focus of recent research in terms of human and pet relevance. Candidatus Neoehrlichia mikurensis seems to be relatively abundant in animals and humans from Central European countries, whereas Candidatus Neoehrlichia lotoris was found solely in raccoons from the USA.FindingsSpleen samples from a total of 164 red foxes, originating from two western provinces in Austria (Tyrol and Vorarlberg), were collected and examined for the presence of tick-borne bacteria of the family Anaplasmataceae by PCR and sequencing. In a fox sample originating from Vorarlberg Candidatus Neoehrlichia sp. was found, which is genetically (16S rRNA, groEL) closely related to Candidatus Neoehrlichia lotoris but clearly distinct from Candidatus Neoehrlichia mikurensis.ConclusionsThe present study revealed, for the first time, the occurrence of Candidatus Neoehrlichia sp. in a red fox worldwide. A continuing screening of wild carnivores, especially foxes, and ticks for this potential pathogen is required to evaluate the actual occurrence and distribution of these bacteria. Further research is needed to elucidate the relationships of Neoehrlichia, as well as their reservoir and impact on wildlife, pets and humans.

Differentiation in the Trochulus hispidus complex and related taxa (Pulmonata: Hygromiidae): morphology, ecology and their relation to phylogeography

In this study we investigated the morphology and ecology of representatives of the taxonomically ambiguous genus Trochulus. The main focus was on the T. hispidus complex, which comprises several genetically highly divergent mitochondrial clades, as determined in a parallel molecular genetic study. We analysed shell morphology and anatomical traits and asked whether the clades are differentiated in these characters. In addition, the related species T. oreinos and T. striolatus were investigated and compared with the T. hispidus complex. Finally, we compared the ecological requirements of the taxa. Among the genetic clades of the T. hispidus complex there was no clear morphological differentiation and geographic populations could not be distinguished based on their morphology. The investigated characters of the genital anatomy did not allow discrimination of any of the T. hispidus clades and were not even diagnostic for the group as a whole. The morphotype of T. sericeus is present in all clades and thus cannot be assigned to a genetic group or any specific population. Thus, our morphological data do not provide evidence that any of the mitochondrial T. hispidus clades represent separate species. Concerning interspecific delimitation, the T. hispidus complex was clearly differentiated from T. striolatus and T. oreinos by shell morphological and anatomical characters, e.g. sculpture of shell surface and details of the penis. Finally, the habitat of T. oreinos is different from those of the other two species. In contrast to the lack of correspondence between genetic and morphological differentiation within the T. hispidus complex, related species display intraspecific morphological differentiation corresponding with mitochondrial clades: within T. striolatus there was a slight morphological differentiation between the subspecies T. s. striolatus, T. s. juvavensis and T. s. danubialis. The two subspecies of T. oreinos could be discriminated by a small but consistent difference in the cross-section of the penis. The unequal levels of intraspecific differentiation are caused by different evolutionary histories as a consequence of disparities in ecological demands, dispersal ability and use of glacial refugia: both the T. hispidus complex and T. striolatus are fast-spreading, euryoecious organisms which are able to (re-)colonize habitats and survive under different climate conditions. While the T. hispidus complex probably survived the Pleistocene in several glacial refugia, for T. striolatus one glacial refugium is suggested. Trochulus oreinos differs from the other taxa, as it is a slow disperser with a narrow ecological niche. We suggest that its subspecies spent at least the last glaciation in or close to the presently inhabited areas.

Evidence of Troglotrema acutum and Skrjabingylus sp. coinfection in a polecat from Lower Austria

Summary The trematode Troglotrema acutum and nematodes of the genus Skrjabingylus are parasitic helminths infecting nasal sinuses of mustelids. Despite different infection routes of these parasites, their occurrence becomes evident due to their destructive lesions of the bone structure of the head, which appears almost similar in both cases. This is a report of coinfection of both the trematode and the nematode, in a polecat from Lower Austria, as well as the first attempt to barcode T. acutum. The nematode could only be found fragmen-tally, therefore accurate morphological determination was not possible. DNA barcoding was successful, however, a clear species assignment was not possible as the similarity with published COI sequences of other nematodes was only 87 % or less. The influence of both parasitic helminths on the health condition of the hosts remains elusive and has to be evaluated in separate studies.

Subgeneric division of the genus Orcula Held 1837 with remarks on Romanian orculid data (Gastropoda, Pulmonata, Orculidae)

Abstract The genital anatomy of Orcula jetschini (Romania), Orcula zilchi (Bulgaria), and Orcula wagneri (Albania) is described. Based on anatomical features (morphology of the penial caecum) shell characters (sculpture and shape) and unpublished molecular data the genus Orcula is subdivided into three subgenera. Orcula zilchi was classified within the monotypic subgenus Orcula (Hausdorfia) subgen. n.; Orcula jetschini, Orcula wagneri, and Orcula schmidtii were classified to Orcula (Illyriobanatica) subgen. n. (type species: Pupa schmidtii) whereas the other Orcula species remain in the nominotypical subgenus. Orcula (Hausdorfia) is known from South-Eastern Bulgaria and North-Western Turkey, Orcula (Illyriobanatica) inhabits Western Romania, North-Western Greece, Albania, Macedonia, Kosovo, and Montenegro. The nine species of Orcula (Orcula) are known mainly from the Alps and the Western Carpathians (from Eastern France to Eastern Hungary and Slovakia). The occurrence of only one Orcula species namely Orcula jetschini is verified from Romania. Available information suggests that data on the Romanian occurrence of Orcula dolium and Orcula gularis were based on wrongly identified specimens. Sphyradium dobrogicum (=Orcula dobrogica) is considered as a synonym of Sphyradium doliolum.