Occurrence of Dermacentor reticulatus in central-southern Poland, and potential threats to human and animal health.

INTRODUCTION AND OBJECTIVE: Dermacentor reticulatus is one of the tick species of the greatest epidemiological importance in Europe. To date, the Eastern European and Western European populations of this tick species have been separated by an area located in Poland where the species has never been found. In this study, newly discovered D. reticulatus localities in areas transformed by human activities in central-southern Poland are described. MATERIAL AND METHODS: The specimens of the ornate dog tick were identified among ticks collected from companion animals in 2010, 2012, 2013, and 2014. They were examined using PCR methods to detect Borrelia burgdorferi s.l., Rickettsia spp., Anaplasma phagocytophilum, Bartonella spp., Babesia spp., and Toxoplasma gondii. In the case of the positive results, the amplicons were sequenced and examined by a BLAST search. RESULTS: In total, 6 specimens of D. reticulatus were collected (3 females and 3 males). As declared by the owners, animal hosts stayed in the same area throughout the study period and had never travelled outside their place of residence. As many as 3/6 (50%) of D. reticulatus adults removed from dogs were infected with Rickettsia raoultii. CONCLUSIONS: The results expand the available data on the spread of the ornate dog tick and indicate that, since 2010, this tick species and Rickettsia raoultii transmitted by this tick species have probably been present in this area, which has a strongly transformed agricultural structure and and had previously been regarded as a D. reticulatus-free zone. The presence of the ornate dog tick in urban and suburban habitats in central-southern Poland poses new threats to the health of companion animals and humans associated with the transmission of pathogens by this species.

Molecular identification of Trypanosoma theileri complex in Eurasian moose Alces alces (L.).

Although the significance of red deer (Cervus elaphus) and roe deer (Capreolus capreolus) as hosts and their role in the circulation of vector-borne pathogens in Europe is well described, the trypanosomes of moose (Alces alces) are poorly known. As heat sensitive ungulates, moose might be especially vulnerable to the effects of climate change and the associated rise in parasite pressure. Therefore, the aim of our study was to determine the prevalence of trypanosomes in moose in Poland, this being one of the largest populations in Central Europe since the 2001 hunting ban. Molecular analysis revealed the presence of Megatrypanum trypanosomes in almost half of the studied moose. As the population of moose in Central Europe has been recently growing, it is crucial to determine their role in the circulation of vector-borne pathogens in environment. This is the first study of the detection and molecular identification of Trypanosoma theileri complex in moose in central Europe.

The New Haplotypes of Bartonella spp. and Borrelia burgdorferi Sensu Lato Identified in Lipoptena spp. (Diptera: Hippoboscidae) Collected in the Areas of North-Eastern Poland.

Deer keds are hematophagous ectoparasites (Diptera: Hippoboscidae) that mainly parasitize Cervidae. These flies are particularly important for animal health due to the occurrence of numerous pathogenic microorganisms. They may also attack humans and their bites may cause allergenic symptoms. The aim of the study was to identify the molecular characteristics of Borrelia burgdorferi sensu lato and Bartonella spp. pathogens detected in Lipoptena spp. sampled both from the hosts and from the environment. For identification of Bartonella spp and B. burgdorferi s. l., the primers specific to the rpoB and flaB gene fragments were used, respectively. The overall prevalence of B. burgdorferi s.l. DNA in Lipoptena cervi was 14.04%, including 14.8% infection in the tested group of winged specimens. The overall prevalence of Bartonella spp. was 57.02%. The presence of these bacteria was detected in 53.5% of specimens of L. cervi and 75.7% of L. fortisetosa. The phylogenetic analysis showed five new haplotypes of the rpoB gene of Bartonella sp. isolated from L. cervi/Lipoptena fortisetosa. We also identified one new haplotype of B. afzelii and three haplotypes of B. burgdorferi isolated from winged specimens of L. cervi. This is the first study to detect the genetic material of B. burgdorferi s.l. in L. cervi in Poland and the first report on the identification of these bacteria in host-seeking specimens in the environment.

Phylogeny and evolution of the genus Cervus (Cervidae, Mammalia) as revealed by complete mitochondrial genomes.

Mitochondrial DNA (mtDNA) lineages are recognized as important components of intra- and interspecific biodiversity, and allow to reveal colonization routes and phylogeographic structure of many taxa. Among these is the genus Cervus that is widely distributed across the Holarctic. We obtained sequences of complete mitochondrial genomes from 13 Cervus taxa and included them in global phylogenetic analyses of 71 Cervinae mitogenomes. The well-resolved phylogenetic trees confirmed Cervus to be monophyletic. Molecular dating based on several fossil calibration points revealed that ca. 2.6 Mya two main mitochondrial lineages of Cervus separated in Central Asia, the Western (including C. hanglu and C. elaphus) and the Eastern (comprising C. albirostris, C. canadensis and C. nippon). We also observed convergent changes in the composition of some mitochondrial genes in C. hanglu of the Western lineage and representatives of the Eastern lineage. Several subspecies of C. nippon and C. hanglu have accumulated a large portion of deleterious substitutions in their mitochondrial protein-coding genes, probably due to drift in the wake of decreasing population size. In contrast to previous studies, we found that the relic haplogroup B of C. elaphus was sister to all other red deer lineages and that the Middle-Eastern haplogroup E shared a common ancestor with the Balkan haplogroup C. Comparison of the mtDNA phylogenetic tree with a published nuclear genome tree may imply ancient introgressions of mtDNA between different Cervus species as well as from the common ancestor of South Asian deer, Rusa timorensis and R. unicolor, to the Cervus clade.

New insight into the taxonomic resolution of the genus Bythotrephes Leydig (Crustacea: Cladocera) based on molecular data from Central Europe.

The taxonomic status of the genus Bythotrephes Leydig (Crustacea: Cladocera) has been debated since the second half of the XIX century. The most widespread view of recent decades has been that Bythotrephes is a monotypic genus, which was support by preliminary molecular data. However, the recent detailed morphological revision of this genus clearly distinguishes at least seven species. Therefore, we performed a multi-lake survey in Central Europe to give new insight into the taxonomic status of Bythotrephes by combining genetic analysis with traditional morphology-based taxonomy. Based on the morphology we identified two species in Central Europe, B. brevimanus and B. lilljeborgi, as well as hybrid forms. For the genetic analysis, we used newly obtained 113 sequences of mtDNA COI gene of the 535-bp length Bythotrephes from Central Europe and sequences downloaded from GenBank. There were no significant differences between all analyzed sequences, which supports the hypothesis that Bythotrephes is a monotypic genus, with only one highly polymorphic species. On the other hand, the results of our work could point out that the COI gene is insufficient to evaluate the taxonomic status of Bythotrephes. Nonetheless, we have identified 29 new haplotypes of mtDNA COI, and one which was the same as the haplotype found in North America and Finland. Furthermore, this haplotype was the source variant from which most other haplotypes were derived.

Simultaneous Infection of Elaphostrongylus Nematode Species and Parasite Sharing between Sympatrically Occurring Cervids: Moose, Roe Deer, and Red Deer in Poland.

It is important to assess the distribution of parasite species across wildlife populations, to design strategies for game management and effective disease control in nature. In this project we quantified the prevalence of Elaphostrongylus species in eight moose populations. We used molecular methods for identification of parasite species and host individual genotypes from fecal samples. We also demonstrated sharing of parasite species between three cervid hosts sympatrically occurring in the Biebrza River valley, North-Eastern Poland, which is occupied by the largest autochthonous, non-harvested moose population in Central Europe. Nematode species from the genus Elaphostrongylus are ubiquitous in the studied moose populations. The presence of a single parasite species (e.g., E. alces) in moose individuals was more common than simultaneous infection with E. alces and E. cervi. The prevalence of both E. alces and E. cervi was higher in males than females. The distribution of E. alces and E. cervi prevalence in moose, roe deer, and red deer were in accordance with the membership of a host to a subfamily. Simultaneous occurrences of both Elaphostrongylus species were significantly more frequently noted in red deer fecal samples than those collected from moose or roe deer. Thus, we consider red deer to play a dominant role in sharing of those nematodes to other cervids. Our findings promote applications of molecular methods of identifying parasite species and the assessment of the exchange of parasite community between wild ruminant species in management and health monitoring of game animal populations.

Two New Haplotypes of Bartonella sp. Isolated from Lipoptena fortisetosa (Diptera: Hippoboscidae) in SE Poland.

Insects of the genus Lipoptena are parasitic arthropods with a broad host range. Due to the type of parasitism (hematophagy), their potential role as vectors of pathogens, i.e., Bartonella sp., Anaplasma phagocytophilum, Rickettsia spp., and Borrelia burgdorferi is considered. As the range of their occurrence has been changing dynamically in recent years and infestations of humans have increasingly been reported, these organisms are now the subject of numerous studies. Our research aimed to present the molecular characteristics of Bartonella sp. detected in Lipoptena fortisetosa parasitizing wild cervids in south-eastern Poland. Adults of Lipoptena spp. were collected from carcasses of roe deer and red deer between spring and autumn in 2013. The PCR method was used to detect Bartonella sp. in the insects. We report two new haplotypes of the rpoB gene of Bartonella sp. isolated from L. fortisetosa feeding on wild cervids in south-eastern Poland and the presence of this invasive ectoparasitic species in the studied area since 2013. Phylogenetic analyses of newly obtained Bartonella sp. haplotypes confirmed their unique position on the constructed tree and network topology. The rpoB gene sequences found belonging to lineage B support the view that this phylogenetic lineage represents a novel Bartonella species.

Sex-biased polyparasitism in moose (Alces alces) based on molecular analysis of faecal samples.

Simultaneous infection with multiple parasite species in an individual host is often observed in wild populations. The understanding of parasite species distribution across populations of wild animals is of basic and applied importance, because parasites can have pronounced effects on the dynamics of host population. Here, we quantified prevalence and endoparasite species richness in moose and explored sex-biased polyparasitism using diagnostic PCR method coupled with DNA sequencing of moose faecal samples from the Biebrza River valley, North-Eastern Poland. This is the largest moose population in Central Europe that has not been harvested for almost 20 years. We also evaluated the appropriate quantity of faeces for detecting DNA of parasite species. Faecal samples were screened for molecular markers of 10 different species of endoparasites. Endoparasite prevalence was high in the studied population. Almost all of the samples (98%) tested positive for at least one parasite species, and we found polyparasitism in the majority of the tested individuals. The number of different parasite species found in a single individual ranged from 0 to 9. The parasite species richness was significantly higher in male than in female individuals. The most prevalent were liver fluke Parafasciolopsis fasciolaemorpha and gastrointestinal nematodes Ostertargia sp. Of the ten endoparasite species detected, only the prevalence of the tapeworm Moniezia benedeni was significantly higher in males than in females. Additionally, we identified co-occurrence associations of parasite species, which tended to be random, but we noted some evidence of both positive and negative associations. Our findings promote applications of molecular methods for parasite species identification from non-invasively collected faecal samples in management and scientific study of moose population, which should include investigation of parasite status, and in health monitoring programs for other wild cervids.

Patterns of parasite eggs, oocysts and larvae shedding by moose in the Biebrza marshland (NE Poland).

The study analyses patterns of endoparasite eggs, oocysts and larvae shedding by moose from the relict population in the Biebrza marshland, NE Poland, which has grown to be one of the largest in Central Europe since the ban on hunting imposed in 2001. The analysis identified 10 species or groups of parasites among 230 faecal moose samples collected over 16 consequent months. The most prevalent were the eggs of Trichostrongylidae, Trichuris spp., Nematodirella alcidis, Parafasciolopsis fasciolaemorpha and the larvae of Elaphostrongylus sp. Four parasite species were more prevalent in males, indicating male-biased parasitism, and the studied moose population exhibited a female-skewed sex ratio. Nematodirella alcidis eggs and Protostrongylid larvae were more prevalent during winter, which indicated their resistance to harsh weather conditions. The prevalence of Eimeria alces and Aonchotheca sp. increased during the growing season, as did the number of eggs per gram of faeces (EPG) of P. fasciolaemorpha, possibly due to the availability of water sources. Higher mean monthly temperature was also found to have a positive effect on the excretion of Trichostrongylidae and Moniezia spp. eggs. In addition, the time of infection and the specificity of the parasite life cycle, being sensitive to certain climatic conditions, also appeared to have a strong influence on eggs, oocysts and larvae shedding in this non-harvested moose population.

Purifying Selection, Density Blocking and Unnoticed Mitochondrial DNA Diversity in the Red Deer, Cervus elaphus.

The trajectories of postglacial range expansions, the occurrence of lineage patches and the formation and maintenance of secondary contact between lineages may mostly reflect neutral demographic processes, including density blocking, that may leave long-lasting genetic signatures. However, a few studies have recently shown that climate may also play a role. We used red deer, a large, mobile herbivore that is assumed to be sensitive to climate change, to test hypotheses of possible selection on the mitochondrial DNA cytochrome b gene (mtDNA cytb) and competitive and/or density-blocking (using mtDNA control region). We searched for a possible link between the phylogeographic structure and abiotic climatic variables. Finally, we tested for isolation by distance and isolation by environment and assessed the impact of human-mediated translocations on the genetic structure of red deer. Our analysis of 30 red deer populations in Poland using the mtDNA control region (N = 357) and cytochrome b (N = 50) markers not only confirmed the presence of the Western and South-Eastern lineages of the species but also indicated the presence of a previously unnoticed, rare relic haplotype that grouped together C. e. italicus from Italy (the Mesola deer). No significant signs of positive selection were detected for the mtDNA cytb gene in the studied red deer. However, a significant signal for purifying selection was found in our study that may explain the narrowness of the contact zone because gene flow between the Western and South-Eastern lineages should drive relatively strong mito-nuclear incompatibilities. MtDNA control region differentiation among red deer populations in Poland correlated with different abiotic climatic variables. Strikingly, the southernmost ice sheet limits during the Elsterian was the most important factor, and it explained the largest amount of variation. However, neither isolation by distance (IBD) nor isolation by environment (IBE) were recorded, and a very limited impact of human translocations was evident. The above-mentioned results suggest that in contemporary red deer populations in Poland, the phylogeographic pattern is well preserved, and long-term processes (density and/or competitive blocking) still play a major role.

Unexpected population genetic structure of European roe deer in Poland: an invasion of the mtDNA genome from Siberian roe deer.

Introgressive hybridization is a widespread evolutionary phenomenon which may lead to increased allelic variation at selective neutral loci and to transfer of fitness-related traits to introgressed lineages. We inferred the population genetic structure of the European roe deer (Capreolus capreolus) in Poland from mitochondrial (CR and cyt b) and sex-linked markers (ZFX, SRY, DBY4 and DBY8). Analyses of CR mtDNA sequences from 452 individuals indicated widespread introgression of Siberian roe deer (C. pygargus) mtDNA in the European roe deer genome, 2000 km from the current distribution range of C. pygargus. Introgressed individuals constituted 16.6% of the deer studied. Nearly 75% of them possessed haplotypes belonging to the group which arose 23 kyr ago and have not been detected within the natural range of Siberian roe deer, indicating that majority of present introgression has ancient origin. Unlike the mtDNA results, sex-specific markers did not show signs of introgression. Species distribution modelling analyses suggested that C. pygargus could have extended its range as far west as Central Europe after last glacial maximum. The main hybridization event was probably associated with range expansion of the most abundant European roe deer lineage from western refugia and took place in Central Europe after the Younger Dryas (10.8-10.0 ka BP). Initially, introgressed mtDNA variants could have spread out on the wave of expansion through the mechanism of gene surfing, reaching high frequencies in European roe deer populations and leading to observed asymmetrical gene flow. Human-mediated introductions of C. pygargus had minimal effect on the extent of mtDNA introgression.

Fast and efficient DNA-based method for winter diet analysis from stools of three cervids: moose, red deer, and roe deer.

Effects of cervid browsing on timber production, especially during winter, lead to economic losses in forest management. The aim of this study was to present an efficient DNA-based method which allows qualitative assessment of the winter diet from stools of moose (Alces alces), roe deer (Capreolus capreolus), and red deer (Cervus elaphus). The preliminary results of the diet composition of the three cervids from Poland were also presented with a special emphasis on moose. The electropherograms of the chloroplast intron trnL (UAA) P6 loop amplification products using g (fluorescence-labeled) and h primers revealed differences in the length of PCR products among various plant species eaten by these herbivores. In addition, the usage of species-specific primers allowed unambiguous identification of different gymnosperms and angiosperms. The preliminary moose diet analysis, based on winter fecal samples from the entire range of moose occurrence in Poland, revealed the presence of 15 to 24 tree, shrub, and herbaceous species. This fast, cost-efficient, and simple method proved also to be reliable for the diet analysis of red deer and roe deer. It may be a valuable tool in forest and conservation management, as well as a way of enhancing ecological studies focusing on the impact of herbivores on the ecosystems and their possible food niche overlap.

Complex patterns of population genetic structure of moose, Alces alces, after recent spatial expansion in Poland revealed by sex-linked markers.

In recent years, human activity directly and indirectly influenced the demography of moose in Poland. The species was close to extinction, and only a few isolated populations survived after the Second World War; then, unprecedented demographic and spatial expansions had occurred, possibly generating a very complex pattern of population genetic structure at the present-day margins of the species range in Poland. Over 370 moose from seven populations were collected from Poland, and partial sequences of the mitochondrial control region (mtDNA-cr; 607 bp) were obtained. In addition, the entire mtDNA cytochrome b gene (1,140 bp) and Y-chromosome markers (1,982 bp in total) were studied in a chosen set of individuals. Twelve mtDNA haplotypes that all belonged to the European moose phylogroup were recorded. They could be divided into two distinct clades: Central Europe and the Ural Mountains. The first clade consists of three distinct groups/branches: Biebrza, Polesie, and Fennoscandia. The Biebrza group has experienced spatial and demographic expansion in the recent past. Average genetic differentiation among moose populations in Poland at mtDNA-cr was great and significant (ΦST = 0.407, p < 0.001). Using mtDNA-cr data, four separate groups of population were recognized using spatial analysis of molecular variance and principal coordinate analysis, including a relict population in Biebrza National Park, a reintroduced Kampinos National Park population, as well as populations that were descendants of moose that colonized Poland from the east (Lithuania, Belarus, and Ukraine) and the north (former East Prussia). Among all the sequenced Y-chromosome markers, polymorphisms were found in the DBY14 marker in three populations only; four haplotypes were recorded in total. No significant differentiation was detected for this Y-linked marker among moose populations in Poland. Our mtDNA study revealed that a variety of different factors-bottleneck, the presence of relict, autochthonous populations, translocations, limited female dispersal, and the colonization from the east and north-are responsible for the observed complex pattern of population genetic structure after demographic and spatial expansion of moose in Poland.