Đuro Huber

A Case of Visceral Leishmaniosis in a Gray Wolf (Canis lupus) from Croatia

The southern habitats of Croatias gray wolf (Canis lupus) population are found in central and southern parts of Dalmatia. This region is recognized as an endemic region for canine visceral leishmaniosis, caused by Leishmania infantum. In November 2003, a 4-yr-old male gray wolf was found dead in the northwestern border of this endemic region. Pathologic and parasitologic analysis, confirmed by polymerase chain reaction, indicated that lesions associated with infection by Leishmania infantum are, in this case, typical for visceral leshmaniosis commonly described in dogs. Review of the literature suggests that this is the first reported case of gray wolf death due to lesions caused by L. infantum.

Extensive polymorphism and evidence of selection pressure on major histocompatibility complex DLA-DRB1, DQA1 and DQB1 class II genes in Croatian grey wolves

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for measuring fitness-related genetic variation in wildlife populations. Because of human persecution and habitat fragmentation, the grey wolf has become extinct from a large part of Western and Central Europe, and remaining populations have become isolated. In Croatia, the grey wolf population, part of the Dinaric-Balkan population, shrank nearly to extinction during the 20th century, and is now legally protected. Using the cloning-sequencing method, we investigated the genetic diversity and evolutionary history of exon 2 of MHC class II DLA-DRB1, DQA1 and DQB1 genes in 77 individuals. We identified 13 DRB1, 7 DQA1 and 11 DQB1 highly divergent alleles, and 13 DLA-DRB1/DQA1/DQB1 haplotypes. Selection analysis comparing the relative rates of non-synonymous to synonymous mutations (d(N)/d(S)) showed evidence of positive selection pressure acting on all three loci. Trans-species polymorphism was found, suggesting the existence of balancing selection. Evolutionary codon models detected considerable difference between alpha and beta chain gene selection patterns: DRB1 and DQB1 appeared to be under stronger selection pressure, while DQA1 showed signs of moderate selection. Our results suggest that, despite the recent contraction of the Croatian wolf population, genetic variability in selectively maintained immune genes has been preserved.

Presence of the Mediterranean monk meal (Monachus monachus) in the Croatian part of the Adriatic sea

All findings and observations included in this note suggest that monk seals can easily reach all parts on the Croatian side of the Adriatic Sea.

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, β-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.

Abstract 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. Sažetak KADMIJ I OLOVO U UZORCIMA VUČJE JETRE: OPTIMIZACIJA METODE RAZGRADNJE UZORAKA UZ POMOĆ MIKROVALOVA Certificirani referenti materijal (CRM) govedskih jetara (BCR-185R) i uzorci vučjih jetara koristili su se za optimizaciju metode razgradnje uzoraka prije mjerenja kadmija (Cd) i olova (Pb) masenom spektrometrijom induktivno spregnute plazme (ICP-MS). Ispitivani su različiti uvjeti koji utječu na učinkovitost razgradnje (temperatura, vrijeme, sastav otopine za razgradnju, masa uzorka). Validacijom je obuhvaćeno ispitivanje linearnosti (do 200 μg L-1 Cd/Pb), granice detekcije (0,003 μg L-1 za Cd, 0.035 μg L-1 za Pb) i kvantifikacije (0,008 μg L-1 za Cd, 0,081 μg L-1 za Pb). Postignuto je dobro slaganje izmjerenih i certificiranih vrijednosti u svim ispitivanim uvjetima uz izračunati raspon iskorištenja 94 % do 111 % za Cd i 95 % do 105 % za Pb. Najveća relativna standardna devijacija, kao mjera preciznosti, iznosila je 3 % za Cd i 8 % za Pb. Najbolji uvjeti za razgradnju (260 °C, 30 min, 1 mL HNO3+4 mL H2O, 0,2 g CRM), izabrani prema točnosti i preciznosti, primijenjeni su na uzorke vučjih jetara pri procjeni odgovarajuće mase uzorka i potrebe za uvođenjem dodatnog koraka prije razgradnje (liofilizacije). Liofilizacija poboljšava preciznost pa je preporučujemo pri pripremi uzoraka tkiva. Također, najmanja masa uzorka izabrana je ne samo zbog bolje preciznosti nego i zbog najmanjeg utjecaja matrice te potrebne količine tkiva. Medijani Cd (0,055 μg g-1) i Pb (0,107 μg g-1 mokre mase) dobiveni analizom 40 uzoraka vučjih jetara skupljenih u Hrvatskoj bili su u rasponu vrijednosti objavljenih u literaturi za sivog vuka.

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 Ab-positive 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 (Icterohaemorrhagiae, 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.

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.

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.

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.