SummaryBackgroundThe known bactericidal properties of ozone have not been checked in relation to its action on bacterial biofilms. This is especially true of ozonated fluids. The aim of this study was to investigate the bactericidal activity of ozonated water and that of a mixture of ozone and oxygen against biofilms.Material/MethodsEighteen clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa exhibiting various levels of antibiotic sensitivity were investigated. Bacteria were cultured in biofilm form on polystyrene titration plates for periods of 2 to 72 hours. The biofilms formed in this way were exposed to in statu nascendi ozonated water produced in a prototype device that had been tested in clinical conditions, or to a mixture of oxygen and ozone generated in the same device. Live cells in the biofilm were stained with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide solution. The degree of reduction of viable bacteria following ozone exposure was determined.ResultsOzonated water was found to be an effective bactericidal agent against biofilms after as little as 30 seconds of exposure, while the bactericidal activity of the ozone-oxygen solution was much lower. Prolongation of the duration of biofilm exposure to the gaseous disinfectant to 40 minutes led to a reduction in the viable cell count, which nevertheless remained high.ConclusionsUnlike the ozone-oxygen mixture, ozonated water effectively destroys bacterial biofilms in vitro.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s13364-013-0146-9) contains supplementary material, which is available to authorized users.

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.

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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.

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.

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The abundance and distribution of large carnivores in Europe have been historically reduced. Their recovery requires multilevel coordination, especially regarding transboundary populations. Here, we apply nuclear and mitochondrial genetic markers to test for admixture level and its impact on population genetic structure of contemporary brown bears (Ursus arctos) from the Eastern, Southern, and Western Carpathians. Carpathian Mountains (Europe). Nearly 400 noninvasive brown bear DNA samples from the Western (Poland) and Eastern Carpathians (Bieszczady Mountains in Poland, Slovakia, Ukraine) were collected. Together with DNA isolates from Slovakia and Romania, they were analyzed using the set of eight microsatellite loci and two mtDNA regions (control region and cytochrome b). A set of 113 individuals with complete genotypes was used to investigate genetic differentiation across national boundaries, genetic structuring within and between populations, and movement between populations. Transboundary brown bear subpopulations (Slovakia and Poland) did not show significant internal genetic structure, and thus were treated as cohesive units. All brown bears from the Western Carpathians carried mitochondrial haplotypes from the Eastern lineage, while the Western lineage prevailed in the brown bears from the Bieszczady Mountains. Despite similar levels of microsatellite variability, we documented significant differentiation among the studied populations for nuclear markers and mtDNA. We also detected male‐biased and asymmetrical movement into the Bieszczady Mountains population from the Western Carpathians. Our findings suggest initial colonization of the Western Carpathians by brown bears possessing mtDNA from the Eastern lineage. Genetic structuring among populations at microsatellite loci could be a result of human‐mediated alterations. Detected asymmetric gene flow suggests ongoing expansion from more abundant populations into the Bieszczady Mountains and thus supports a metapopulation model. The knowledge concerning this complex pattern can be implemented in a joint Carpathian brown bear management plan that should allow population mixing by dispersing males.