Rehabilitation and release of orphaned Eurasian lynx (Lynx lynx) in Europe: Implications for management and conservation.

Rehabilitation of injured or immature individuals has become an increasingly used conservation and management tool. However, scientific evaluation of rehabilitations is rare, raising concern about post-release welfare as well as the cost-effectiveness of spending scarce financial resources. Over the past 20 years, events of juvenile Eurasian lynx presumably orphaned have been observed in many European lynx populations. To guide the management of orphaned lynx, we documented survival, rehabilitation and fate after the release and evaluated the potential relevance of lynx orphan rehabilitation for population management and conservation implications. Data on 320 orphaned lynx was collected from 1975 to 2022 from 13 countries and nine populations. The majority of orphaned lynx (55%) were taken to rehabilitation centres or other enclosures. A total of 66 orphans were released back to nature. The portion of rehabilitated lynx who survived at least one year after release was 0.66. Release location was the best predictor for their survival. Of the 66 released lynx, ten have reproduced at least once (8 females and 2 males). Conservation implications of rehabilitation programmes include managing genetic diversity in small, isolated populations and reintroducing species to historical habitats. The lynx is a perfect model species as most reintroduced populations in Central Europe show significantly lower observed heterozygosity than most of the autochthonous populations, indicating that reintroduction bottlenecks, isolation and post-release management have long-term consequences on the genetic composition of populations. The release of translocated orphans could be a valuable contribution to Eurasian lynx conservation in Europe. It is recommended to release orphans at the distribution edge or in the frame of reintroduction projects instead of a release in the core area of a population where it is not necessary from a demographic and genetic point of view. Rehabilitation programmes can have conservation implications that extend far beyond individual welfare benefits.

A new insight into genetic structure of Danube and Italian foci of fascioloidosis.

Human induced translocation and introduction of species have reshaped parasite fauna on a global scale. The introduction of the large American liver fluke Fascioloides magna from North America to Europe is an example of an invasive alien parasite causing significant ecological and economic damage. Recent genetic studies have shown that F. magna was introduced to Europe on multiple occasions forming three permanent foci of infection. This study primarily focuses on the poorly researched genetic structuring of F. magna flukes originating from Croatia and Serbia. Additional samples from USA and Italy are also included, thereby providing novel insights into F. magna's biogeography. Partial cox1 and nad1 genes were amplified from 216 F. magna flukes extracted from red deer, roe deer, fallow deer, white-tailed deer and wild boar. Seven cox1 and nine nad1 haplotypes were identified, of which two cox1 and four nad1 haplotypes have not been not previously found. Our analysis has expanded the knowledge about possible sources of F. magna introduction to Europe, by identifying a cox1 haplotype shared by flukes from the north-eastern parts of the USA and Italy and another cox1 haplotype shared by flukes also from north eastern parts of the USA and the Danube floodplains.

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.

Next-generation phylogeography resolves post-glacial colonization patterns in a widespread carnivore, the red fox (Vulpes vulpes), in Europe.

Carnivores tend to exhibit a lack of (or less pronounced) genetic structure at continental scales in both a geographic and temporal sense and this can confound the identification of post-glacial colonization patterns in this group. In this study we used genome-wide data (using genotyping by sequencing [GBS]) to reconstruct the phylogeographic history of a widespread carnivore, the red fox (Vulpes vulpes), by investigating broad-scale patterns of genomic variation, differentiation and admixture amongst contemporary populations in Europe. Using 15,003 single nucleotide polymorphisms (SNPs) from 524 individuals allowed us to identify the importance of refugial regions for the red fox in terms of endemism (e.g., Iberia). In addition, we tested multiple post-glacial recolonization scenarios of previously glaciated regions during the Last Glacial Maximum using an Approximate Bayesian Computation (ABC) approach that were unresolved from previous studies. This allowed us to identify the role of admixture from multiple source population post-Younger Dryas in the case of Scandinavia and ancient land-bridges in the colonization of the British Isles. A natural colonization of Ireland was deemed more likely than an ancient human-mediated introduction as has previously been proposed and potentially points to a larger mammalian community on the island in the early post-glacial period. Using genome-wide data has allowed us to tease apart broad-scale patterns of structure and diversity in a widespread carnivore in Europe that was not evident from using more limited marker sets and provides a foundation for next-generation phylogeographic studies in other non-model species.

Population genetic structure of European wildcats inhabiting the area between the Dinaric Alps and the Scardo-Pindic mountains.

Habitat fragmentation and loss have contributed significantly to the demographic decline of European wildcat populations and hybridization with domestic cats poses a threat to the loss of genetic purity of the species. In this study we used microsatellite markers to analyse genetic variation and structure of the wildcat populations from the area between the Dinaric Alps and the Scardo-Pindic mountains in Slovenia, Croatia, Serbia and North Macedonia. We also investigated hybridisation between populations of wildcats and domestic cats in the area. One hundred and thirteen samples from free-leaving European wildcats and thirty-two samples from domestic cats were analysed. Allelic richness across populations ranged from 3.61 to 3.98. The observed Ho values ranged between 0.57 and 0.71. The global FST value for the four populations was 0.080 (95% CI 0.056-0.109) and differed significantly from zero (P < 0.001). The highest FST value was observed between the populations North Macedonia and Slovenia and the lowest between Slovenia and Croatia. We also found a signal for the existence of isolation by distance between populations. Our results showed that wildcats are divided in two genetic clusters largely consistent with a geographic division into a genetically diverse northern group (Slovenia, Croatia) and genetically eroded south-eastern group (Serbia, N. Macedonia). Hybridisation rate between wildcats and domestic cats varied between 13% and 52% across the regions.

First description of peritoneal and pleural metacestodosis caused by Mesocestoides vogae in a European wild cat (Felis silvestris silvestris).

Tapeworms of the genus Mesocestoides (Cestoda: Cyclophyllidea: Mesocestoididae) are still enigmatic to scientists, due to their high morphological variability, low host specificity, and unknown details of their life cycle. They are found worldwide, with carnivorous mammals as the main definitive hosts, and the disease is potentially zoonotic. After ingestion by a definitive host, the tetrathyridium can occasionally migrate through the intestinal wall and reach the peritoneal cavity or abdominal organs causing peritoneal metacestodosis. Here, we report on a case of metacestodosis of a European wild cat (Felis silvestris silvestris) found dead in Croatia. At necropsy, a large number of white, rice-like structures were found free in the abdominal and thoracic cavities, as well as along the serous surfaces and in the lungs. DNA isolated from the nodules was genotyped and based on a 320-base pair long 12S fragment classified as Mesocestoides vogae. Although post-mortem changes were advanced, severe emaciation due to the severe parasitic infection and gastrointestinal bleeding was diagnosed as the likely cause of death. Intestinal cestodosis was previously reported in wild cats, but according to our knowledge, this is the first description of peritoneal and pleural metacestodosis caused by M. vogae tetrathyridia (metacestodes) in any wild carnivore species.

Microsatellite based assignment reveals history of extirpated mountain ungulate.

During the early 1900s, Northern chamois (Rupicapra rupicapra) populations in the northern Dinaric Mountains were extirpated. During the 1960s and 1970s there were several reintroductions of individuals from two Northern chamois subspecies (Alpine chamois, R. r. rupicapra and Balkan chamois, R. r. balcanica) from neighbouring areas in the attempt to re-establish the population. Accurate taxonomic classification, at subspecies level, of the autochthonous extirpated population was not known. To clarify which subspecies was present before reintroduction, we genotyped four male chamois skulls originating from Velebit Mountain, collected around 25 years before the population local extinction. DNA was successfully extracted from middle layer and outer sheath of horns. Assignment based on microsatellite loci, using both Bayesian clustering in STRUCTURE (with q values between 0.55 and 0.73) and DAPC (with individual membership probabilities of 0.99 and 1.00) indicated higher assessed likelihood for the Alpine subspecies.

Targeted resequencing of coding DNA sequences for SNP discovery in nonmodel species.

Targeted capture coupled with high-throughput sequencing can be used to gain information about nuclear sequence variation at hundreds to thousands of loci. Divergent reference capture makes use of molecular data of one species to enrich target loci in other (related) species. This is particularly valuable for nonmodel organisms, for which often no a priori knowledge exists regarding these loci. Here, we have used targeted capture to obtain data for 809 nuclear coding DNA sequences (CDS) in a nonmodel organism, the Eurasian lynx Lynx lynx, using baits designed with the help of the published genome of a related model organism (the domestic cat Felis catus). Using this approach, we were able to survey intraspecific variation at hundreds of nuclear loci in L. lynx across the species' European range. A large set of biallelic candidate SNPs was then evaluated using a high-throughput SNP genotyping platform (Fluidigm), which we then reduced to a final 96 SNP-panel based on assay performance and reliability; validation was carried out with 100 additional Eurasian lynx samples not included in the SNP discovery phase. The 96 SNP-panel developed from CDS performed very successfully in the identification of individuals and in population genetic structure inference (including the assignment of individuals to their source population). In keeping with recent studies, our results show that genic SNPs can be valuable for genetic monitoring of wildlife species.

First identification of Echinococcus multilocularis in golden jackals in Croatia.

Alveolar echinococcosis, caused by the tapeworm Echinococcus multilocularis, is one of the world's most dangerous zoonosis and an emerging disease with growing incidence in humans. The disease has been reported in new areas and host species in the last two decades, and the primary hosts of the parasite - red fox, golden jackal and grey wolf - are expanding their distribution in Europe. Here we report the morphological and molecular identification of Echinococcus multilocularis tapeworms in one of 29 carcasses of adult golden jackals in Croatia, where the only previous report of the parasite was in red foxes in 2016. These results suggest that alveolar echinococcosis should be treated as an emerging disease in Croatia.

When things go wrong: Cysticercus longicollis in an adult wild red fox (Vulpes vulpes).

First case of Cysticercus longicollis, larval stage of Taenia crassiceps, was diagnosed in a wild adult male red fox (Vulpes vulpes). The fox was killed by dogs at Nature Park Medvednica and presented to the University of Zagreb Faculty of Veterinary Medicine with history of being unable to run away and having skin lesions on legs that resembled to those of mange. Necropsy revealed whitish fluctuant mass full of cysticercus-like structures, surrounded by fibrous capsule and placed between the leg muscles, and numerous of spherical cysts in the subcutis and in the peritoneal cavity. Cysticerci were identified as C. longicollis based on their size, number and size of the rostellar hooks, mode of proliferation and DNA analysis. To our knowledge, this is the first case of T. crassiceps cysticercosis in a wild carnivore.

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.