Wolf genetic diversity compared across Europe using the yardstick method.

Integrating data across studies with traditional microsatellite genetic markers requires careful calibration and represents an obstacle for investigation of wide-ranging species where populations require transboundary management. We used the "yardstick" method to compare results published across Europe since 2002 and new wolf (Canis lupus) genetic profiles from the Carpathian Mountains in Central Europe and the Dinaric Mountains in Southeastern Europe, with the latter as our reference population. We compared each population with Dinaric wolves, considering only shared markers (range 4-17). For each population, we calculated standard genetic diversity indices plus calibrated heterozygosity (Hec) and allelic richness (Ac). Hec and Ac in Dinaric (0.704 and 9.394) and Carpathian wolves (0.695 and 7.023) were comparable to those observed in other large and mid-sized European populations, but smaller than those of northeastern Europe. Major discrepancies in marker choices among some studies made comparisons more difficult. However, the yardstick method, including the new measures of Hec and Ac, provided a direct comparison of genetic diversity values among wolf populations and an intuitive interpretation of the results. The yardstick method thus permitted the integration of diverse sources of publicly available microsatellite data for spatiotemporal genetic monitoring of evolutionary potential.

Malassezia pachydermatis from brown bear: A comprehensive analysis reveals novel genotypes and distribution of all detected variants in domestic and wild animals.

Malassezia pachydermatis (phylum Basidiomycota, class Malasseziomycetes) is a zoophilic opportunistic pathogen with recognized potential for invasive infections in humans. Although this pathogenic yeast is widespread in nature, it has been primarily studied in domestic animals, so available data on its genotypes in the wild are limited. In this study, 80 yeast isolates recovered from 42 brown bears (Ursus arctos) were identified as M. pachydermatis by a culture-based approach. MALDI-TOF mass spectrometry (MS) was used to endorse conventional identification. The majority of samples exhibited a high score fluctuation, with 42.5% of isolates generating the best scores in the range confident only for genus identification. However, the use of young biomass significantly improved the identification of M. pachydermatis at the species confidence level (98.8%). Importantly, the same MALDI-TOF MS efficiency would be achieved regardless of colony age if the cut-off value was lowered to ≥1.7. Genotyping of LSU, ITS1, CHS2, and ß-tubulin markers identified four distinct genotypes in M. pachydermatis isolates. The most prevalent among them was the genotype previously found in dogs, indicating its transmission potential and adaptation to distantly related hosts. The other three genotypes are described for the first time in this study. However, only one of the genotypes consisted of all four loci with bear-specific sequences, indicating the formation of a strain specifically adapted to brown bears. Finally, we evaluated the specificity of the spectral profiles of the detected genotypes. MALDI-TOF MS exhibited great potential to detect subtle differences between all M. pachydermatis isolates and revealed distinct spectral profiles of bear-specific genotypes.

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.

Evaluation of physiological stress in free-ranging bears: current knowledge and future directions.

Stress responses, which are mediated by the neurogenic system (NS) and hypothalamic-pituitary-adrenal (HPA) axis help vertebrates maintain physiological homeostasis. Fight-or-flight responses are activated by the NS, which releases norepinephrine/noradrenaline and epinephrine/adrenaline in response to immediate stressors, whilst the HPA axis releases glucocorticoid hormones (e.g. cortisol and corticosterone) to help mitigate allostatic load. There have been many studies on stress responses of captive animals, but they are not truly reflective of typical ranges or the types of stressors encountered by free-ranging wildlife, such as responses and adaptation to environmental change, which are particularly important from a conservation perspective. As stress can influence the composition of age and sex classes of free-ranging populations both directly and indirectly, ecological research must be prioritised towards more vulnerable taxa. Generally, large predators tend to be particularly at risk of anthropogenically driven population declines because they exhibit reduced behavioural plasticity required to adapt to changing landscapes and exist in reduced geographic ranges, have small population sizes, low fecundity rates, large spatial requirements and occupy high trophic positions. As a keystone species with a long history of coexistence with humans in highly anthropogenic landscapes, there has been growing concern about how humans influence bear behaviour and physiology, via numerous short- and long-term stressors. In this review, we synthesise research on the stress response in free-ranging bear populations and evaluate the effectiveness and limitations of current methodology in measuring stress in bears to identify the most effective metrics for future research. Particularly, we integrate research that utilised haematological variables, cardiac monitors and Global Positioning System (GPS) collars, serum/plasma and faecal glucocorticoid concentrations, hair cortisol levels, and morphological metrics (primarily skulls) to investigate the stress response in ursids in both short- and long-term contexts. We found that in free-ranging bears, food availability and consumption have the greatest influence on individual stress, with mixed responses to anthropogenic influences. Effects of sex and age on stress are also mixed, likely attributable to inconsistent methods. We recommend that methodology across all stress indicators used in free-ranging bears should be standardised to improve interpretation of results and that a wider range of species should be incorporated in future studies.

90Sr and stable element levels in bones of brown bears: long-term trends in bear populations from Croatia and Poland.

The aim of this study was to investigate the temporal trends and geographical differences in 90Sr and stable element (Ba, Ca, Mn, Sr, Pb, Zn) levels in the bones of Croatian and Polish brown bear (Ursus arctos) populations. Experimental data suggest that in the decades after nuclear weapon tests and the Chernobyl accident, 90Sr bone activity concentrations decreased from 352 to 11 Bq kg-1 in the Croatian bear population (period 1982-2015) and from 831 to 27 Bq kg-1 in Polish bears (period 1962-2020). Calculated effective and ecological half-lives were 9 and 13 years for Croatian bears, and 15 and 31 years for Polish bears, respectively. Different temporal trends were noted in levels of Ba, Mn, Pb and Zn between the two countries with majority of bones having lower Pb, Sr and Zn in Croatian than in Polish bears. Estimated values for the soil-to-bear transfer of 90Sr were the same order of magnitude in the studied populations. Contrary to this, the estimated transfer of stable Sr was an order of magnitude lower for the Croatian bear population compared to Polish bears. The observed differences in soil-to-bear transfer between stable Sr and 90Sr found for Croatian bears might suggest the need for careful consideration on the use of stable Sr data as an analogue for 90Sr. To our knowledge, this is the first study that analysed 90Sr activity in tissue of brown bears. As such, it provides insight into the fate and behaviour of one of the most relevant anthropogenic radionuclides at the top of the food chain.

Lead and Other Trace Element Levels in Brains of Croatian Large Terrestrial Carnivores: Influence of Biological and Ecological Factors.

Trace element pollution can adversely affect the brains of individuals and thus impact the entire population of apex predators, such as large European carnivores. We assessed exposure to prominent neurotoxicants As, Cd, Hg and Pb by measuring their brain stem levels in brown bears (n = 114), grey wolves (n = 8), Eurasian lynx (n = 3), and golden jackals (n = 2) sampled in 2015-2022 in Croatia. The highest of the non-essential elements was the Pb level in the bears' brains (median, Q1-Q3; 11.1, 7.13-24.1 µg/kg wet mass), with 4% of animals, all subadults, exceeding the established normal bovine levels (100 µg/kg wet mass). Species-specific differences were noted for Ca, Cd, Cu, Fe, Pb and Se brain levels. Female brown bears had higher As brain levels than males. Cubs and yearlings had lower brain Cd, but higher Zn, while subadults had higher Cu than adult bears. Hepatic As, Cd, Cu and Hg levels were shown to be a moderate proxy for estimating brain levels in bears (rS = 0.30-0.69). Multiple associations of As, Cd, Hg and Pb with essential elements pointed to a possible interaction and disturbance of brain Ca, Cu, Fe, Se and Zn homeostasis. Non-essential element levels in the brains of four studied species were lower than reported earlier for terrestrial meso-carnivores and humans. The age and sex of animals were highlighted as essential factors in interpreting brain element levels in ecotoxicological studies of large carnivores.

Comparison of two non-invasive arterial blood pressure monitoring techniques in brown bears (Ursus arctos).

Monitoring arterial blood pressure (BP), represents a more accurate evaluation of hemodynamics than heart rate alone and is essential for preventing and treating intra- and post-operative complications in wildlife chemical immobilization. The objectives of the study were to test the correlation between standard oscillometry and Korotkoff's technique in anesthetized free-ranging brown bears in Croatia and Scandinavia and to assess the blood pressure in both locations. Five bears were snared and darted with xylazine and ketamine in Croatia, and 20 bears were darted from a helicopter with medetomidine and tiletamine-zolazepam in Scandinavia. Blood pressure was simultaneously measured with both techniques every 5 minutes. Correlation between techniques, trends of BP variation, and the factors of the capture which likely influenced BP were assessed. Successful measurements of BP were achieved in 93% of all attempts with the Korotkoff's technique but in only 29% of all attempts with oscillometry. The latter method mostly provided lower values of BP compared to Korotkoff's technique in yearlings. Most bears showed a decreasing trend in systolic and mean BP over time, consistent between the two techniques. All bears were hypertensive: the auscultatory technique detected moderate to severe systolic hypertension in 25% and 84% of bears in Croatia and in Scandinavia, respectively, with significantly higher BP in subadults and adults compared to yearlings. Only Korotkoff's method resulted in a reliable and effective tool for BP assessment in brown bears. The anesthetic protocols used in the present study in association with the capture methods produced hypertension in all animals.

NON-INVASIVE BLOOD PRESSURE AND OTHER PHYSIOLOGICAL DATA IN CHEMICALLY IMMOBILIZED BROWN BEARS (URSUS ARCTOS).

Free-ranging brown bears (Ursus arctos) were snared and subsequently darted with a combination of xylazine-ketamine in Croatia (n = 5) or darted from a helicopter with a combination of medetomidine-tiletamine-zolazepam in Scandinavia (n = 20). Three adults and one yearling (1 year old) bear were captured in Croatia, with one adult being captured twice. The Scandinavian bears were divided into Group A (yearlings, n = 7) and Group B (subadults, n = 2 and adults, n = 11). The exertion time (time from activation of the trap or from the start of the helicopter chase to recumbency) and the induction time (time from darting to recumbency) were recorded. The rectal temperature (Tr) was measured as soon as possible after induction and then monitored at frequent intervals (varied between individuals) in immobilized bears. Blood pressure (BP) was measured with a non-invasive method (Korotkoff's technique) every 5 minutes. The heart rate (HR), respiratory rate (RR), and arterial haemoglobin oxygen saturation (SpO2) were recorded every 5 minutes. Reliability of the BP monitoring technique, trends of variation of the physiological variables, and the factors related to the capture were assessed. Both exertion and induction times were longer in Croatian bears than in Scandinavian bears. In Croatian bears, the Tr was either constant or slightly decreasing, with hyperthermia recorded in two individuals (Tr > 39.0° C). In Scandinavian bears, 17 of 20 individuals developed an initial hyperthermia. Four of five bears in Croatia and 17 of 20 bears in Scandinavia showed a decreasing trend in systolic and mean BP over time. According to the Korotkoff method, all bears were hypertensive (mean BP > 130 mmHg) with varying severity, and the systolic pressure was significantly lower in yearlings when compared to subadults and adults. Yearlings had significantly (p < 0.05) higher HR than subadults and adults, however there was no significant differences in RR, SpO2, and Tr between the age groups. All Croatian bears and 13 of 20 Scandinavian bears were moderately to severely hypoxemic (SpO2 < 90%). Further studies with simultaneous invasive and non-invasive (Korotkoff) BP monitoring techniques are required to confirm the accuracy of methods used in this study. The data presented here provides evidence of the physiological impact of different capture methods and chemical immobilization of brown bears in Croatia and Scandinavia.

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.

Trace and macro elements in the femoral bone as indicators of long-term environmental exposure to toxic metals in European brown bear (Ursus arctos) from Croatia.

We explored the long-term accumulation of aluminium, strontium, cadmium and lead in the compact and trabecular bone of the femoral epiphysis, metaphysis and diaphysis in 41 brown bears (Ursus arctos) from Croatia. Also, we assessed their influence on macro and trace elements (sodium, magnesium, phosphorus, potassium, calcium, manganese, iron, cobalt, copper, zinc and barium) in bears' bone. There were no sex differences in element levels in general, while age was associated with bone length and levels of all elements, except for cadmium. Elements had different levels depending on the part of the bone sampled. More pronounced differences were observed between the compact and trabecular regions, with higher levels of majority of elements found in compact bone. Moderate to high associations (Spearman coefficient, rS = 0.59-0.97) were confirmed between calcium and potassium, magnesium, phosphorus, manganese, cobalt, zinc, strontium and lead. Lead levels in the bone were below those known to cause adverse health effects, but in 4 of 41 animals they exceeded baseline levels for domestic animals. The femoral bone of the brown bear reflected the accumulative nature of lead and strontium well, as it did the impairment of bone-forming essential element levels associated with these two elements. However, the distribution pattern of elements along the bone was not uniform, so additional care should be taken when choosing on the part of the bone sampled.

Sexual Maturity and Life Stage Influences Toxic Metal Accumulation in Croatian Brown Bears.

The influence of reproductive and (early) life stages on toxic metal levels was investigated in the brown bear (Ursus arctos), the largest mammalian predator species in Croatia. The purpose was to examine critical clusters in a population that might be at a higher risk of adverse health effects caused by metals as environmental contaminants. Levels of cadmium, mercury and lead in muscle, liver and kidney cortex of 325 male and 139 female bears, quantified by inductively coupled plasma mass spectrometry, were analysed according to distinct bear life stages (young: cub, yearling, subadult; mature: adult). Metal levels did not differ among sexes in young animals (< 4 years), except for mercury in muscles (higher in females), and adult females had higher cadmium and mercury. A trend of renal cadmium accumulation with age in immature male animals disappeared once they reached maturity, whereas for females this trend has only slowly declined in mature compared to immature bears. In early life stage (< 1 year), bear cubs had lower cadmium, comparable mercury, and higher lead in the kidneys than the bears of the following age category (yearlings). Due to a higher proportion of renal lead transfer from the mother to the cub compared with cadmium, it may be that the high burden of cadmium found in kidneys of older females has lower toxicological concern for their cubs than the lead content. Sex, reproductive, and life stages of bears were confirmed as important in assessing toxic metal burden.

Apex predatory mammals as bioindicator species in environmental monitoring of elements in Dinaric Alps (Croatia).

Tissue element investigations of apex terrestrial mammals are very scarce in Europe. We quantified 16 essential and nonessential elements in the kidney cortex, liver, and muscle tissue of 467 brown bears (Ursus arctos), 125 gray wolves (Canis lupus), one Eurasian lynx (Lynx lynx), and three golden jackals (Canis aureus) from Croatia by inductively coupled plasma mass spectrometry (ICP-MS). Renal cadmium (0.6% of animals) and lead (1%) and hepatic lead (5%) were found in toxicologically relevant levels for mammals only in bears, while the other elements were within normal range. The association of age, sex, season, and region with measured tissue elements in bear and wolf was estimated by multiple regression analyses. Age-related accumulation of cadmium was observed in bears and wolves. Lead tissue content increased with the age of bears but declined in wolves. Female bears and wolves had higher arsenic, iron, and thallium than males in some tissues. Also, cadmium, mercury, copper, zinc, selenium, molybdenum, and uranium were more abundant only in female bears. Male bears had higher potassium, zinc, and magnesium, while male wolves had higher calcium in some tissues compared to female wolves. Seasonal differences were mainly observed for bears' tissues and region-specific differences only in wolves. The bear kidneys had the highest levels of cobalt, copper, molybdenum, cadmium, and lead among the four studied species. The element levels reported for bears and wolves represent baseline values for the Dinaric population.

The Lipid Composition of Subcutaneous Adipose Tissue of Brown Bears (Ursus arctos) in Croatia.

The composition of adipose tissue in brown bears (Ursus arctos) is highly variable and depends on an individual's feeding habits. Fatty acid composition of subcutaneous adipose tissue (SAT) may provide insight into brown bear feeding habits, for which data are scarce. The aim of this study was to determine the lipid composition of SAT and variations in the composition of fatty substances with regard to gender and to assess SAT relative to season and body mass (BM) of brown bears in Croatia. Seventy-six tissue samples of brown bear SAT were analyzed in this study. We found that gender, season, and BM significantly affected the lipid composition of SAT. Both females and males had higher percentages of saturated fatty acids and monounsaturated fatty acids (MUFAs) in SAT in spring than in autumn, while the percentage of polyunsaturated fatty acids (PUFAs) was higher in autumn. The prevalence of MUFAs in SAT and the greater presence of PUFAs in autumn, together with the presence of odd-chain saturated fatty acids, indicate the importance of these fatty acids in brown bear physiology. We suggest that the lipid content of adipose tissue may provide valuable information on changes in brown bear condition in response to feeding habits and the effects of supplemental feeding.

Organochlorine pesticides and polychlorinated biphenyl congeners in wild terrestrial mammals from Croatia: Interspecies comparison of residue levels and compositions.

In this pilot study, we investigated levels of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in the adipose tissues of two free-ranging terrestrial carnivores from Croatia sampled in 2010 and 2011: the brown bear (Ursus arctos; N=32) and the grey wolf (Canis lupus; N=29). Concentrations of ∑OCPs and ΣPCBs ranged from 0.45 to 4.09 ng g(-1) lipid mass (lm) and from 0.93 to 8.52 ng g(-1) lm in brown bear, and from 1.18 to 5.67 ng g(-1) lm and 2.68 to 48.9 ng g(-1) lm in grey wolf adipose tissues, respectively. PCBs were dominant accounting for over 72% of total analyzed persistent organic pollutants (POPs). The sum of six indicator non-dioxin-like PCBs (Σ6IndNDL PCBs) made up 60-93% and 58-85% of the total congener concentrations in brown bears and wolves, respectively. Although the levels of the measured parameters were significantly higher in grey wolves than in bears, the contaminant profiles of the two species were similar, with γ-HCH, HCB, ß-HCH and DDE as major OCP contaminants, and PCB-153>PCB-180≈PCB-170>PCB-138 as the dominant congeners. The sum of hexachlorocyclohexanes (ΣHCHs) and 8 toxicologically relevant dioxin-like PCBs (Σ8ToxDL PCBs) was higher in the males than in the females of the brown bear. Concentrations of ΣDDTs, HCB, ΣOCP, ΣPCBs, Σ6IndNDL PCBs, and toxicologically relevant non-dioxin-like PCBs (ΣToxNDL PCBs) were significantly positively correlated with lipid content in the grey wolf. Concentrations of OCPs and PCBs in brown bears and wolves from Croatia were low and normal for large terrestrial mammals.

Selenium in brown bears (Ursus arctos) from Croatia: Relation to cadmium and mercury.

Muscle (n = 111), liver (n = 111), and kidney cortex (n = 101) samples from brown bears (Ursus arctos) were collected in the 2009 and 2010 hunting seasons in Croatia and analysed for selenium (Se), cadmium (Cd), and total mercury (Hg). The aim was to assess the levels of these elements according to age, sex, and season of collection, and to investigate possible Se/Cd and Se/Hg interactions. Median Se concentrations were 0.139 µg/g in muscle, 0.409 µg/g in liver and 1.75 µg/g wet mass in kidney cortex. Median Cd and Hg were 0.0078 and 0.0018 µg/g in muscle, 1.09 and 0.031 µg/g in liver, and 16.5 and 0.206 µg/g wet mass in the renal cortex, respectively. Se/Cd molar ratios were less than 1 in the kidney cortex, and close to or above 1 in liver and muscle, respectively. Toxic Cd and Hg correlated with Se in all of the studied tissues. Sex differences were found for all three elements (except Se in liver), with females having higher tissue concentration than males. Only Cd showed age-dependence. Bear samples collected in fall had higher Se in muscles, and Hg in muscles and liver compared to samples collected in spring. Element concentrations in brown bear tissues were within the range of previously reported studies. Bear meat is considered a rich source of Se, safe for consumption with regard to its Cd and Hg content. According to the molar ratio and correlation results, we assume that Se binding is not the primary detoxification pathway for Cd and Hg in brown bears.

Cadmium and lead in grey wolf liver samples: optimisation of a microwave-assisted digestion method.

A microwave-assisted digestion method for the determination of cadmium (Cd) and lead (Pb) by inductively coupled plasma mass spectrometry (ICP-MS) was optimised on certified reference material (CRM) (bovine liver, BCR-185R) and wolf liver samples. Different factors influencing digestion efficiency (temperature, time, composition of the digestion mixture, sample mass) were tested. Validation included linearity (up to 200 µg L(-1) for Cd and Pb), detection (0.003 µg L(-1) for Cd and 0.035 µg L(-1) for Pb), and quantification (0.008 µg L(-1) for Cd and 0.081 µg L(-1) for Pb) limits. Good agreement between measured and certified values was achieved in all conditions, with recoveries ranging from 94 % to 111 % for Cd and from 95 % to 105 % for Pb. The precision of the method, expressed as relative standard deviation, was up to 3 % for Cd and 8 % for Pb. The best digestion parameters (260 °C, 30 min, 1 mL HNO3+4 mL H2O, 0.1 g of CRM) based on accuracy and precision were applied on two wolf liver samples to evaluate the need for the predigestion step (freeze-drying) and appropriate mass of the sample. Freeze-drying improved precision and minimising the tissue mass to 0.1 g reduced the matrix effect. Using these optimised digestion conditions, we determined Cd and Pb in 40 wolf livers collected in Croatia, and their medians (0.055 µg g(-1) and 0.107 µg g(-1), respectively) were in the range of previously reported data for the grey wolf.

Repetitive sequences in Eurasian lynx (Lynx lynx L.) mitochondrial DNA control region.

Mitochondrial DNA (mtDNA) control region (CR) of numerous species is known to include up to five different repetitive sequences (RS1-RS5) that are found at various locations, involving motifs of different length and extensive length heteroplasmy. Two repetitive sequences (RS2 and RS3) on opposite sides of mtDNA central conserved region have been described in domestic cat (Felis catus) and some other felid species. However, the presence of repetitive sequence RS3 has not been detected in Eurasian lynx (Lynx lynx) yet. We analyzed mtDNA CR of 35 Eurasian lynx (L. lynx L.) samples to characterize repetitive sequences and to compare them with those found in other felid species. We confirmed the presence of 80 base pairs (bp) repetitive sequence (RS2) at the 5' end of the Eurasian lynx mtDNA CR L strand and for the first time we described RS3 repetitive sequence at its 3' end, consisting of an array of tandem repeats five to ten bp long. We found that felid species share similar RS3 repetitive pattern and fundamental repeat motif TACAC.

Genotyping success of historical Eurasian lynx (Lynx lynx L.) samples.

Historical samples, like tanned hides and trophy skulls, can be extremely important for genetic studies of endangered or elusive species. Selection of a sampling protocol that is likely to provide sufficient amount and quality of DNA with a minimum damage to the original specimen is often critical for a success of the study. We investigated microsatellite genotyping success of DNA isolated from three different types of Eurasian lynx historical samples. We analysed a total of 20 microsatellite loci in 106 historical samples from the endangered Dinaric lynx population, established from re-introduction of three pairs of lynx in 1973 from Slovakian Carpathians. Of the three tested sample types, turbinal bone and septum from the nasal cavity of the trophy skulls had the lowest percentage of samples successfully genotyped for all 20 microsatellite loci. Footpad samples, collected using a cork drill, exhibited better results in polymerase chain reaction amplification and genotyping than samples of footpad epidermis cut with a scalpel. We report simple and efficient sampling protocols, which could be widely applied for future studies utilizing historical samples.

Serologic evidence of Leptospira spp. serovars in brown bears (Ursus arctos) from Croatia.

Serum samples from 52 free-ranging brown bears (Ursus arctos) collected in Croatia over a period of 10 yr (1998-2007) were tested by microscopic agglutination test for specific antibodies (Ab) to 12 Leptospira spp. pathogenic serovars. At titers ranging from 1:100 to 1:2,000, 19 samples (36.5%) were Abpositive to at least one serovar. Antibodies for 10 Leptospira spp. serovars were detected: Icterohaemorrhagiae, Australis, Sejroe, Canicola, Poi, Hardjo, Ballum, Saxkoebing, Pomona, and Grippotyphosa. In comparison to previous reports, the prevalence of Ab to serovar Icterohaemorrhagiae (52.6%) was significantly higher. Other common serovars were Australis (47.4%) and Sejroe (42.1%). High Ab titers for serovars Canicola (1:500) and Grippotyphosa (1:1,000) were detected for the first time in free-ranging bears from Croatia. A significant correlation between the age of the bears and detection of Ab to Leptospira spp. serovars suggested the presence of pathogenic agents in the natural habitats, whereas increasing trends of Ab prevalence for specific serovars (Icter-ohaemorrhagiae, Australis, and Sejroe) confirmed cohabitation of bears with rats and other small terrestrial mammals on garbage dumps and at bear feeding stations. To prevent cohabitation of bears and rodents, improvements in Croatian waste treatment, big game management, and rodent control programs are strongly recommended, especially in Lika and Gorski Kotar, regions that have high-quality natural habitats for brown bears in Croatia.