Stress and reproductive hormones in hair associated with contaminant metal(loid)s of European brown bear (Ursus arctos).

Environmental contaminants like arsenic (As), cadmium (Cd), mercury (Hg) or lead (Pb) may disrupt hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes due to their endocrine toxicity potential. Resulting long-term physiological stress or adverse effects on wildlife reproduction and ontogeny may cause detrimental effects at the individual and population levels. However, data on environmental metal(loid)s' impact on reproductive and stress hormones in wildlife, especially large terrestrial carnivores, are scarce. Hair cortisol, progesterone and testosterone concentrations were quantified and modelled with hair As, Cd, total Hg, Pb, biological, environmental and sampling factors to test for potential effects in free-ranging brown bears (Ursus arctos) from Croatia (N = 46) and Poland (N = 27). Testosterone in males (N = 48) and females (N = 25) showed positive associations with Hg and an interaction between Cd and Pb, but a negative association with interaction between age and Pb. Higher testosterone was found in hair during its growth phase compared to quiescent phase. Body condition index was negatively associated with hair cortisol and positively associated with hair progesterone. Year and conditions of sampling were important for cortisol variation, while maturity stage for progesterone variation (lower concentrations in cubs and yearlings compared to subadult and adult bears). These findings suggest that environmental levels of Cd, Hg and Pb might influence the HPG axis in brown bears. Hair was shown to be a reliable non-invasive sample for investigating hormonal fluctuations in wildlife while addressing individual and sampling specificities.

Metal(loid) exposure assessment and biomarker responses in captive and free-ranging European brown bear (Ursus arctos).

We investigated the level of five non-essential metal(loid)s (As, Cd, Hg, Tl, Pb) and nine essential metals (Mg, Ca, Mn, Fe, Co, Cu, Zn, Se, Mo) in hair and blood components of captive and free-ranging European brown bear populations in Croatia and Poland. Metal(loid) associations with biomarkers of oxidative stress (superoxide dismutase, SOD; glutathione-peroxidase, GSH-Px; malondialdehyde, MDA) and metal exposure (metallothionein, MT) were estimated in this top predatory mammal. Lead was the most abundant non-essential metal(loid) in both blood and hair, with 4 of 35 individuals having blood levels over 100 µg/L. A positive association was found between Pb level and SOD activity in blood. Free-ranging bears had higher blood SOD activity, Mn, Zn and Cd levels, hair Co, Cd, Tl and Pb compared to captive individuals, while the opposite was true for Mg and hair Ca thereby reflecting habitat and diet differences. With increasing age, animals showed lower levels of SOD activity and certain essential metals. Females had higher SOD activity and blood levels of some essential metals than males. Hair showed a higher Fe and Co level when sampled during the growth phase and was not predictive of non-essential metal(loid) blood levels. The established metal(loid) baseline values will enable future risk assessment in both captive and wild European brown bear populations.

A modified method for molecular identification of Baylisascaris transfuga in European brown bears (Ursus arctos).

Baylisascaris transfuga is a roundworm that has been reported worldwide in most bear species. In mammals and possibly humans, the larvae of B. transfuga can migrate in the tissues of aberrant hosts with larva migrans syndrome. The current study was performed to identify B. transfuga in faecal samples from free-ranging brown bears in the Tatra Mountains National Park in southern Poland. A commercial kit was used to extract genomic DNA directly from faecal samples. Additionally, a Chelex resin-based technique was successfully implemented to prepare a PCR template from eggs retrieved by flotation. Based on the flotation results of 32 collected faecal samples, the prevalence of B. transfuga was 15.6%. The parasite was confirmed in samples found to be positive during the initial flotation by a molecular assay using DNA isolated directly from faeces. The retrieved eggs were confirmed as B. transfuga after their DNA was extracted using the Chelex protocol. Based on PCR amplification and sequencing of a 413-bp segment of cytochrome c oxidase 1 (COI), the obtained sequence was 100% identical to the COI segment of B. transfuga after a BLAST comparison to the GenBank™ database. The current study includes the first molecular confirmation of B. transfuga in brown bears in the western part of the Carpathians. We show that direct extraction of parasite DNA from bear faeces is efficient for molecular assays. As an alternative, we present the effectiveness of a Chelex-based technique for fast and convenient DNA isolation from the difficult-to-disrupt eggs of B. transfuga for PCR. Molecular tests of parasite DNA extracted directly from faecal material have limits of detection related to the amount of eggs in the samples. Thus, using classical flotation to obtain eggs for PCR may increase the credibility of the results, particularly in cases with a low number of excreted eggs. The Chelex resin protocol has potential for application in studies of intestinal parasites in wildlife for which conventional flotation is routinely used for microscopy.

Histological, chemical and behavioural evidence of pedal communication in brown bears.

Most mammals rely upon scent for intraspecific communication. As most bear species have large home ranges and are non-territorial, scent deposit while walking could be an effective way to communicate with conspecifics. Here, we investigate the existence of pedal glands in brown bears and their role in chemical communication from a histological, biochemical and behavioural perspective. We found eccrine glands in footpads, and prominent apocrine and sebaceous glands in the interdigital, metacarpal and metatarsal skin sections. Pedal scent contained 26 compounds including carboxylic acids, important constituents of mammalian secretions. Six of these compounds were exclusive for males. Finally, we describe a specific marking gait recorded in the field, mostly performed by males. Our study supports the existence of chemical communication through pedal marking in brown bears and suggests sex-coding potential of pedal scent.

First molecular characterization of Sarcocystis tenella in Tatra chamois (Rupicapra rupicapra tatrica) in Poland.

In this study, sarcocysts from three Polish Tatra chamois were isolated and identified using morphological and molecular methods for the first time. Six cysts were found in the latissimus dorsi muscle and another two in the diaphragm. No sarcocysts were detected in the myocardium, tongue, and esophagus. The isolated cysts were long with rounded ends, 0.35-0.61 mm in length, and 0.02-0.06 mm in width. All the sarcocysts were identified as Sarcocystis tenella on the basis of light microscopy and sequencing of cytochrome C oxidase subunit I (cox1) and small-subunit rRNA (ssu rRNA) genes. Comparative analysis showed a 99.23% identity of the cox1 gene sequences from Tatra chamois and sheep sarcocysts, and an even higher degree of sequence identity (99.88%) was documented in the case of the ssu rRNA gene. When compared at a haplotype level, all the sheep sequences of cox1 differed from those isolated from Tatra chamois. In contrast, one out of the two ssu rRNA haplotypes from the sheep isolates was identical with the haplotype from Tatra chamois. In conclusion, we showed that cox1 and ssu rRNA genes can be used as genetic markers for identification of the S. tenella, with cox1 gene providing better resolution during phylogenetic analyses. However, both genetic population analysis and phylogenetic inference with cox1 and ssu rRNA genes demonstrated that they do not constitute good markers for spatial differentiation of S. tenella.

A survey of intestinal helminths in wild carnivores from the Tatra National Park, southern Poland.

From January 2011 to July 2012, 144 faecal samples of wild carnivores from the Tatra National Park were examined to evaluate the prevalence of intestinal helminths--72 of wolves (Canis lupus), 45 of red foxes (Vulpes vulpes), 15 of pine martens (Martes martes) and 12 of brown bears (Ursus arctos). In wolves, monospecific infection with Trichuris vulpis (13.9%), Toxocara canis (6.9%), Ancylostoma/Uncinaria (5.6%) and taeniids (1.4%) was revealed. In red foxes, the most prevalent infection was 7 vulpis (64.4%), followed by T. canis (11.1%), Ancylostoma/Uncinaria (8.9%) and taeniids (2.2%). Monospecific infection with T. vulpis was more frequent (44.4%), than infection with two species, i.e. T > vulpis with Ancylostoma/Uncinaria, T. vulpis with T. canis or T. vulpis with taeniids (17.8%). In pine martens, Trichuris spp. was the most prevalent (40.0%), while T. cati and Ancylostoma/Uncinaria were found in 13.3% and 6.7% samples, respectively. In faeces from brown bears, no parasite eggs were found. The present survey of wild carnivores revealed a significant prevalence of parasites such as Toxocara spp. and Trichuris spp. (8.3% and 31.0% in all examined samples, respectively), which are hazardous to human and animal health.