Molecular analysis of scats revealed diet and prey choice of grey wolves and Eurasian lynx in the contact zone between the Dinaric Mountains and the Alps.

A comprehensive understanding of the dietary habits of carnivores is essential to get ecological insights into their role in the ecosystem, potential competition with other carnivorous species, and their effect on prey populations. Genetic analysis of non-invasive samples, such as scats, can supplement behavioural or microscopic diet investigations. The objective of this study was to employ DNA metabarcoding to accurately determine the prey species in grey wolf (Canis lupus) and Eurasian lynx (Lynx lynx) scat samples collected in the Julian Alps and the Dinaric Mountains, Slovenia. The primary prey of wolves were red deer (Cervus elaphus) (detected in 96% scat samples), European roe deer (Capreolus capreolus) (68%), and wild boar (Sus scrofa) (45%). A smaller portion of their diet consisted of mesocarnivores, small mammals, and domestic animals. In contrast, the lynx diet mostly consisted of European roe deer (82%) and red deer (64%). However, small mammals and domestic animals were also present in lynx diet, albeit to a lesser extent. Our findings indicate that the dietary habits of wolves and lynx are influenced by geographical location. Snapshot dietary analyses using metabarcoding are valuable for comprehending the behaviour and ecology of predators, and for devising conservation measures aimed at sustainable management of both their natural habitats and prey populations. However, to gain a more detailed understanding of wolf and lynx dietary habits and ecological impact, it would be essential to conduct long-term genetic monitoring of their diet.

Projection of current and future distribution of adaptive genetic units in an alpine ungulate.

Climate projections predict major changes in alpine environments by the end of the 21st century. To avoid climate-induced maladaptation and extinction, many animal populations will either need to move to more suitable habitats or adapt in situ to novel conditions. Since populations of a species exhibit genetic variation related to local adaptation, it is important to incorporate this variation into predictive models to help assess the ability of the species to survive climate change. Here, we evaluate how the adaptive genetic variation of a mountain ungulate-the Northern chamois (Rupicapra rupicapra)-could be impacted by future global warming. Based on genotype-environment association analyses of 429 chamois using a ddRAD sequencing approach, we identified genetic variation associated with climatic gradients across the European Alps. We then delineated adaptive genetic units and projected the optimal distribution of these adaptive groups in the future. Our results suggest the presence of local adaptation to climate in Northern chamois with similar genetic adaptive responses in geographically distant but climatically similar populations. Furthermore, our results predict that future climatic changes will modify the Northern chamois adaptive landscape considerably, with various degrees of maladaptation risk.

Population genetic structure of the highly endangered butterfly Coenonympha oedippus (Nymphalidae: Satyrinae) at its southern edge of distribution.

The Slovenian False Ringlet Coenonympha oedippus populations are under serious threat, as in the last two decades have witnessed a sharp decline in population distribution and size due to destruction, fragmentation and/or habitat quality degradation through intensive agriculture, abandonment of use and urbanization. We investigated the genetic diversity and structure of C. oedippus from the entire range of the species in Slovenia. Our results showed that the genetic variation in the Slovenian C. oedippus populations agrees with the geographical structure and the ecotypes previously determined by morphological and ecological data. We confirmed the existence of four genetically divergent and spatially non-overlapping geographical groups (Istria, Karst, Gorica, Ljubljansko barje) and two ecotypes ("wet" and "dry"). Despite small sample sizes due to critically threatened species, the results indicated significant nuclear and mitochondrial genetic diversity within the spatial groups. By gaining new insights into the evolutionary history of the C. oedippus population, we have recommended to consider the Slovenian population as one Evolutionary Significant Unit, and four spatial populations as Management Units for conservation purposes.

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.

The impact of illegal waste sites on a transmission of zoonotic viruses.

BACKGROUND: Illegal waste disposal impacts public health and causes aesthetic and environmental pollution. Waste disposed in places without permitted and controlled facilities can provide a ready source of nutrition and shelter for rodents and thus promote the spread of their ecto- and endoparasites. The presence of two distinct zoonotic viruses, lymphocytic choriomeningitis virus (LCMV) and tick-borne encephalitis virus (TBEV), was searched at illegal waste sites. The aim of this study was to determine the prevalence of infection with both viruses in rodents and to discuss the virus-rodent relations in such environments. METHODS: Rodents sampled between October 2011 and April 2013 at 7 locations in the Istrian peninsula, were identified morphologically and genetically to minimize misidentification. Serological and molecular techniques were used to determine seroprevalence of infection in rodents and to detect viral RNAs. Serological testing was performed by immune fluorescence assay for detection of LCMV and TBEV specific antibodies. Real-time RT PCR was used for the detection of LCMV nucleoprotein gene and TBEV 3' non-coding region. Data were statistically analysed using SPSS statistic v2.0. RESULTS: Out of 82 rodent sera tested, the presence of LCMV antibodies was demonstrated in 24.93%. The highest prevalence of LCMV infection was found in commensal Mus musculus (47.37%), followed by 11.53%, 19.04% and 25% prevalence of infection in A. agrarius, A. flavicolis and A. sylvaticus, respectively. The highest prevalence of infection in rodents (53.33%) was found in locations with large waste sites and high anthropogenic influence. LCMV seroprevalence was significantly lower in rodents sampled from natural habitats. Viral nucleic acids were screened in 46 samples but yielded no amplicons of LCMV or TBEV. In addition, TBEV specific antibodies were not detected. CONCLUSIONS: Illegal waste sites have considerable impact on the area where they are located. Results have shown that the transmission of human pathogens can be significantly increased by the presence of waste sites. However, the pathogen must be endemic in the environment where the waste site is located. The introduction of a human pathogen as a consequence of the waste site in the area of interest could not be proven.

First record of Aedes koreicus (Diptera, Culicidae) in Slovenia.

The first record of Aedes koreicus was made in the village of Lovrenc na Dravskem Polju, north-eastern part of the country. The discovery of Ae. koreicus in various continental European countries motivated us to revise samples of the collected Aedes japonicus japonicus. We found Ae. koreicus in samples from 2013, where the larvae were misidentified as Ae. j. japonicus. The species was identified morphologically and molecularly. The first discovery of Ae. koreicus advocates an urgent need for a nationwide mosquito surveillance programme.

Ingenious Architecture and Coloration Generation in Enamel of Rodent Teeth.

Teeth exemplify architectures comprising an interplay of inorganic and organic constituents, resulting in sophisticated natural composites. Rodents (Rodentia) showcase extraordinary adaptations, with their continuously growing incisors surpassing human teeth in functional and structural optimizations. In this study, employing state-of-the-art direct atomic-scale imaging and nanoscale spectroscopies, we present compelling evidence that the release of material from ameloblasts and the subsequent formation of iron-rich enamel and surface layers in the constantly growing incisors of rodents are complex orchestrated processes, intricately regulated and independent of environmental factors. The synergistic fusion of three-dimensional tomography and imaging techniques of etched rodent́s enamel unveils a direct correlation between the presence of pockets infused with ferrihydrite-like material and the acid resistant properties exhibited by the iron-rich enamel, fortifying it as an efficient protective shield. Moreover, observations using optical microscopy shed light on the role of iron-rich enamel as a microstructural element that acts as a path for color transmission, although the native color remains indistinguishable from that of regular enamel, challenging the prevailing paradigms. The redefinition of "pigmented enamel" to encompass ferrihydrite-like infusion in rodent incisors reshapes our perception of incisor microstructure and color generation. The functional significance of acid-resistant iron-rich enamel and the understanding of the underlying coloration mechanism in rodent incisors have far-reaching implications for human health, development of potentially groundbreaking dental materials, and restorative dentistry. These findings enable the creation of an entirely different class of dental biomaterials with enhanced properties, inspired by the ingenious designs found in nature.

Monitoring of species' genetic diversity in Europe varies greatly and overlooks potential climate change impacts.

Genetic monitoring of populations currently attracts interest in the context of the Convention on Biological Diversity but needs long-term planning and investments. However, genetic diversity has been largely neglected in biodiversity monitoring, and when addressed, it is treated separately, detached from other conservation issues, such as habitat alteration due to climate change. We report an accounting of efforts to monitor population genetic diversity in Europe (genetic monitoring effort, GME), the evaluation of which can help guide future capacity building and collaboration towards areas most in need of expanded monitoring. Overlaying GME with areas where the ranges of selected species of conservation interest approach current and future climate niche limits helps identify whether GME coincides with anticipated climate change effects on biodiversity. Our analysis suggests that country area, financial resources and conservation policy influence GME, high values of which only partially match species' joint patterns of limits to suitable climatic conditions. Populations at trailing climatic niche margins probably hold genetic diversity that is important for adaptation to changing climate. Our results illuminate the need in Europe for expanded investment in genetic monitoring across climate gradients occupied by focal species, a need arguably greatest in southeastern European countries. This need could be met in part by expanding the European Union's Birds and Habitats Directives to fully address the conservation and monitoring of genetic diversity.

Neutral and Selective Processes Shape MHC Diversity in Roe Deer in Slovenia.

Disease control and containment in free-ranging populations is one of the greatest challenges in wildlife management. Despite the importance of major histocompatibility complex (MHC) genes for immune response, an assessment of the diversity and occurrence of these genes is still rare in European roe deer, the most abundant and widespread large mammal in Europe. Therefore, we examined immunogenetic variation in roe deer in Slovenia to identify species adaptation by comparing the genetic diversity of the MHC genes with the data on neutral microsatellites. We found ten MHC DRB alleles, three of which are novel. Evidence for historical positive selection on the MHC was found using the maximum likelihood codon method. Patterns of MHC allelic distribution were not congruent with neutral population genetic findings. The lack of population genetic differentiation in MHC genes compared to existing structure in neutral markers suggests that MHC polymorphism was influenced primarily by balancing selection and, to a lesser extent, by neutral processes such as genetic drift, with no clear evidence of local adaptation. Selection analyses indicated that approx. 10% of amino acids encoded under episodic positive selection. This study represents one of the first steps towards establishing an immunogenetic map of roe deer populations across Europe, aiming to better support science-based management of this important game species.

Traces of past reintroduction in genetic diversity: The case of the Balkan chamois (Mammalia, Artiodactyla).

The translocation of wild animal species became a common practice worldwide to re-establish local populations threatened with extinction. Archaeological data confirm that chamois once lived in the Biokovo Mountain but, prior to their reintroduction in the 1960s, there was no written evidence of their recent existence in the area. The population was reintroduced in the period 1964-1969, when 48 individuals of Balkan chamois from the neighbouring mountains in Bosnia and Herzegovina were released. The main objective of this study was to determine the accuracy of the existing historical data on the origin of the Balkan chamois population from the Biokovo Mountain and to assess the genetic diversity and population structure of the source and translocated populations 56 years after reintroduction. Sixteen microsatellite loci were used to analyse the genetic structure of three source chamois populations from Prenj, Cvrsnica and Cabulja Mountains and from Biokovo Mountain. Both STRUCTURE and GENELAND analyses showed a clear separation of the reintroduced population on Biokovo from Prenj's chamois and considerable genetic similarity between the Biokovo population and the Cvrsnica-Cabulja population. This suggests that the current genetic composition of the Biokovo population does not derive exclusively from Prenj, as suggested by the available literature and personal interviews, but also from Cvrsnica and Cabulja. GENELAND analysis recognised the Balkan chamois from Prenj as a separate cluster, distinct from the populations of Cvrsnica and Cabulja. Our results thus highlight the need to implement genetic monitoring of both reintroduced and source populations of endangered Balkan chamois to inform sustainable management and conservation strategies in order to maximise the chances of population persistence.

Performance Comparison of Different Approaches in Genotyping MHC-DRB: The Contrast between Single-Locus and Multi-Locus Species.

Major histocompatibility complex (MHC) genes are widely recognised as valuable markers for wildlife genetic studies given their extreme polymorphism and functional importance in fitness-related traits. Newly developed genotyping methods, which rely on the use of next-generation sequencing (NGS), are gradually replacing traditional cloning and Sanger sequencing methods in MHC genotyping studies. Allele calling in NGS methods remains challenging due to extreme polymorphism and locus multiplication in the MHC coupled with allele amplification bias and the generation of artificial sequences. In this study, we compared the performance of molecular cloning with Illumina and Ion Torrent NGS sequencing in MHC-DRB genotyping of single-locus species (roe deer) and species with multiple DRB loci (red deer) in an attempt to adopt a reliable and straightforward method that does not require complex bioinformatic analyses. Our results show that all methods work similarly well in roe deer, but we demonstrate non-consistency in results across methods in red deer. With Illumina sequencing, we detected a maximum number of alleles in 10 red deer individuals (42), while other methods were somewhat less accurate as they scored 69-81% of alleles detected with Illumina sequencing.

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.

A Mother's Story, Mitogenome Relationships in the Genus Rupicapra.

Although the two species of chamois (Rupicapra rupicapra and R. pyrenaica) are currently classified as least-concern by the IUCN (International Union for Conservation of Nature), inconsistencies on the subspecies classification reported in literature make it challenging to assess the conservation status of the single subspecies. Previous studies relying on mitochondrial genes, sometimes in combination with nuclear or Y-chromosome markers, reported the presence of clusters corresponding to the geographic distribution but highlighting ambiguities in the genus phylogeny. Here we report novel de novo assembled sequences of the mitochondrial genome from nine individuals, including previously unpublished R. r. balcanica and R. r. tatrica subspecies, and use them to untangle the genus phylogeny. Our results based on the full mitogenome inferred phylogeny confirm the previously reported genus subdivision in three clades and its monophyletic positioning within the Caprinae. Phylogeny and taxonomy of Rupicapra species thus remain controversial prompting for the inclusion of archeological remains to solve the controversy.

Geographical Distribution and Genetic Diversity of Bank Vole Hepaciviruses in Europe.

The development of new diagnostic methods resulted in the discovery of novel hepaciviruses in wild populations of the bank vole (Myodes glareolus, syn. Clethrionomys glareolus). The naturally infected voles demonstrate signs of hepatitis similar to those induced by hepatitis C virus (HCV) in humans. The aim of the present research was to investigate the geographical distribution of bank vole-associated hepaciviruses (BvHVs) and their genetic diversity in Europe. Real-time reverse transcription polymerase chain reaction (RT-qPCR) screening revealed BvHV RNA in 442 out of 1838 (24.0%) bank voles from nine European countries and in one of seven northern red-backed voles (Myodes rutilus, syn. Clethrionomys rutilus). BvHV RNA was not found in any other small mammal species (n = 23) tested here. Phylogenetic and isolation-by-distance analyses confirmed the occurrence of both BvHV species (Hepacivirus F and Hepacivirus J) and their sympatric occurrence at several trapping sites in two countries. The broad geographical distribution of BvHVs across Europe was associated with their presence in bank voles of different evolutionary lineages. The extensive geographical distribution and high levels of genetic diversity of BvHVs, as well as the high population fluctuations of bank voles and occasional commensalism in some parts of Europe warrant future studies on the zoonotic potential of BvHVs.

Pilot Study on Genetic Associations With Age-Related Sarcopenia.

Despite strong evidence of an inheritable component of muscle phenotypes, little progress has been made in identifying the specific genetic factors involved in the development of sarcopenia. Even rarer are studies that focus on predicting the risk of sarcopenia based on a genetic risk score. In the present study, we tested the single and combined effect of seven candidate gene variants on the risk of sarcopenia. Single nucleotide polymorphisms in candidate genes were genotyped using the KASP assay. We examined 190 older adults that were classified as non-sarcopenic or sarcopenic according to the diagnostic criteria of the European Working Group on Sarcopenia in Older People. Sarcopenia was associated with Methylenetetrahydrofolate reductase, Alpha-actinin-3, and Nuclear respiratory factor 2 genotypes. The combined effect of all three polymorphisms explained 39% of the interindividual variation in sarcopenia risk. Our results suggest that the single and combined effect of Methylenetetrahydrofolate reductase, Alpha-actinin-3, and Nuclear respiratory factor 2 polymorphism is associated with sarcopenia risk in older adults. Nowadays, as the population is getting older and older, great efforts are being made to research the etiology, diagnosis and treatment of sarcopenia. At the same time, small progress has been made in understanding the genetic etiology of sarcopenia. Given the importance of research on this disease, further genetic studies are needed to better understand the genetic risk underlying sarcopenia. We believe that this small-scale study will help to demonstrate that there is still much to be discovered in this field.

Genetic characterisation of wild ungulates: successful isolation and analysis of DNA from widely available bones can be cheap, fast and easy.

Genetic characterisation of wild ungulates can be a useful tool in wildlife management and in obtaining a greater understanding of their biological and ecological roles in a wider spatiotemporal context. Different ways of optimising methodologies and reducing the costs of genetic analyses using widely available bone tissues collected within regular hunting allocations were examined. Successful isolation and analysis of DNA from widely available bones can be cheap, fast and easy. In particular, this study explored the possibility of using bones for extracting high quality nuclear DNA for microsatellite analysis. The utility of applying a modified demineralisation process using two commercially available DNA isolation kits, which differ significantly in price, was evaluated. The sample sets included bones and, for comparison, muscle tissues from four wild ungulate species: chamois (Rupicapra rupicapra), roe deer (Capreolus capreolus), wild boar (Sus scrofa), and Alpine ibex (Capra ibex). For the recent bones, these results confirmed that the DNA concentrations and microsatellite amplification were sufficiently high, even when using low-cost kits, after prior demineralisation. For old bones, prior demineralisation and use of a specially designed isolation kit led to a more successful extraction of DNA. Besides reducing kit-related costs, low-cost kits are much faster and therefore make genetic analysis more efficient.

MHC Genotyping by SSCP and Amplicon-Based NGS Approach in Chamois.

Genes of the major histocompatibility complex (MHC) code for cell surface proteins essential for adaptive immunity. They show the most outstanding genetic diversity in vertebrates, which has been connected with various fitness traits and thus with the long-term persistence of populations. In this study, polymorphism of the MHC class II DRB locus was investigated in chamois with Single-Strand Conformation Polymorphism (SSCP)/Sanger genotyping and Ion Torrent S5 next-generation sequencing (NGS). From eight identified DRB variants in 28 individuals, five had already been described, and three were new, undescribed alleles. With conventional SSCP/Sanger sequencing, we were able to detect seven alleles, all of which were also detected with NGS. We found inconsistencies in the individual genotypes between the two methods, which were mainly caused by allelic dropout in the SSCP/Sanger method. Six out of 28 individuals were falsely classified as homozygous with SSCP/Sanger analysis. Overall, 25% of the individuals were identified as genotyping discrepancies between the two methods. Our results show that NGS technologies are better performing in sequencing highly variable regions such as the MHC, and they also have a higher detection capacity, thus allowing a more accurate description of the genetic composition, which is crucial for evolutionary and population genetic studies.

First Insights into the Population Genetic Structure and Heterozygosity-Fitness Relationship in Roe Deer Inhabiting the Area between the Alps and Dinaric Mountains.

Across its pan-European distribution, the European roe deer (Capreolus capreolus) faces a wide diversity of environmental and climatic conditions; therefore, several factors, including intrinsic ones, shape life-history traits and cause significant variability in parameters of fitness. By utilizing microsatellite variations in 214 roe deer females collected throughout Slovenia, Central Europe, we determined the genetic variability and population structure of this species in the contact zone between the Alps and the Dinaric Mountains, i.e., over a wider area where data on the genetic outlook for this-the most common and widespread European wild ungulate-have been completely lacking so far. Throughout the country, we found moderate microsatellite diversity (Ho = 0.57-0.65) in relation to the observed heterozygosity reported for other roe deer populations in Europe. Spatial differences in genetic diversity of the species in Slovenia can be explained by population history linked to varying approaches to population management and/or different connectivity among subpopulations in topographically differentiated habitats. A country-wide pattern of genetic structure is clearly defined by separation of the populations into three groups present in the following regions: (i) Southern sub-Mediterranean and Karst regions, (ii) Central Slovenia, and (iii) the Sub-Pannonian Region in the north-east. This is also confirmed by evidencing a moderate isolation by distance, especially by separating southern samples (coastal Slovenia) from others. Levels of genetic differentiation vary among populations, which can be explained by the effect of natural geographical barriers or the presence of anthropogenic barriers such as urban areas and highways. In the subset of 172 yearling females, we analyzed the influence of genetic advantage (individual heterozygosity) and other genetic data (reflected in the structuring of the population) on body mass and reproductive ability. We found evidence that genetic factors influence the body mass of roe deer yearling females (explaining altogether 18.8% of body mass variance), and the level of individual heterozygosity alone also positively affected body mass, which is in accordance with the theory that heterozygosity is commonly positively correlated with fitness in wild populations. However, we did not uncover any effect of heterozygosity on two parameters of reproductive ability (fertility and potential reproductive outcome), indicating that several other factors, especially environmental ones, have a predominant effect on the parameters of fitness in roe deer.

Geographical Distribution of Ljungan Virus in Small Mammals in Europe.

Ljungan virus (LV), which belongs to the Parechovirus genus in the Picornaviridae family, was first isolated from bank voles (Myodes glareolus) in Sweden in 1998 and proposed as a zoonotic agent. To improve knowledge of the host association and geographical distribution of LV, tissues from 1685 animals belonging to multiple rodent and insectivore species from 12 European countries were screened for LV-RNA using reverse transcriptase (RT)-PCR. In addition, we investigated how the prevalence of LV-RNA in bank voles is associated with various intrinsic and extrinsic factors. We show that LV is widespread geographically, having been detected in at least one host species in nine European countries. Twelve out of 21 species screened were LV-RNA PCR positive, including, for the first time, the red vole (Myodes rutilus) and the root or tundra vole (Alexandromys formerly Microtus oeconomus), as well as in insectivores, including the bicolored white-toothed shrew (Crocidura leucodon) and the Valais shrew (Sorex antinorii). Results indicated that bank voles are the main rodent host for this virus (overall RT-PCR prevalence: 15.2%). Linear modeling of intrinsic and extrinsic factors that could impact LV prevalence showed a concave-down relationship between body mass and LV occurrence, so that subadults had the highest LV positivity, but LV in older animals was less prevalent. Also, LV prevalence was higher in autumn and lower in spring, and the amount of precipitation recorded during the 6 months preceding the trapping date was negatively correlated with the presence of the virus. Phylogenetic analysis on the 185 base pair species-specific sequence of the 5' untranslated region identified high genetic diversity (46.5%) between 80 haplotypes, although no geographical or host-specific patterns of diversity were detected.

Prevalence and genotype identification of Toxoplasma gondii in suburban rodents collected at waste disposal sites.

To assess the prevalence of Toxoplasma gondii infection in native and commensal rodents as indicators of environmental pollution, we analyzed brain tissue from small mammals collected on legal and illegal waste sites in the Slovenian and Croatian parts of Istria. A total of 136 animals and five species of the family Muridae were analyzed: black rat (Rattus rattus), domestic mouse (Mus musculus), wood mouse (Apodemus sylvaticus), striped field mouse (Apodemus agrarius), and yellow-necked mouse (Apodemus flavicollis). Using quantitative Polymerase Chain Reaction (qPCR), T. gondii DNA was detected in four homogenized brain tissue samples (2.94%), from all of the analyzed species, except black rat. Out of these, two samples, domestic mouse (Mus musculus) and wood mouse (Apodemus sylvaticus) had sufficient DNA for genotyping of T. gondii isolates in which we demonstrated the presence of clonal type II using RFLP PCR with four markers (SAG1, SAG2, GRA6 and GRA7). Three of four infected animals (75%) were collected on dumpsites.