Screening of Domestic Cats from North-Eastern Hungary for Hepatozoon felis and Cytauxzoon europaeus That Cause Infections in Local Wildcat Populations.

Among vector-borne protozoa Hepatozoon felis and Cytauxzoon europaeus are considered emerging species in felids in Europe. To investigate the presence of these two protozoa 127 domestic cats and 4 wildcats were screened by PCRs targeting the 18S rRNA gene of Hepatozoon spp. and piroplasms, as well as the cytb gene of Cytauxzoon spp. The samples were collected inside and outside a region of Hungary, where both protozoan groups are endemic in wildcats. Among domestic cats, one proved to be infected with H. felis. Furthermore, spleen samples of four wildcats were also examined, among which three tested positive for H. felis, and one had co-infection with C. europaeus. Importantly, H. felis from the co-infected wildcat belonged to genogroup II, similarly to H. felis from the positive domestic cat. Based on phylogenetic evidence, this genogroup probably represents a separate species from genogroup I of H. felis, which was hitherto reported from Mediterranean countries in Europe. The two other wildcats also harbored H. felis from genogroup I. Neither Hepatozoon nor Cytauxzoon infections were detected outside the recently discovered endemic region. In conclusion, this study demonstrates for the first time in Europe that H. felis from genogroup II may emerge in free-roaming domestic cats in regions where this protozoan parasite is endemic in wildcats.

Optimal prey for red fox cubs-An example of dual optimizing foraging strategy in foxes from a dynamic wetland habitat.

The red fox (Vulpes vulpes) is the most abundant mesopredator in the Central European region. Detailed knowledge about their feeding behavior is important both from ecological and wildlife management reasons. Food choices of foxes are poorly predictable in high-biodiversity marshlands. The main aim of our study was to sample parallel the main food-type abundances in the study area and analyze the diet of fox cubs and cohabiting adults across 3 years during the period of maternal dependence of the cubs. According to the optimal foraging theory, we predicted that the cubs' diet would show higher energy content, would be more varied, and the individual prey species fed to the young would be larger. We analyzed the composition of adult fox and cub fecal samples collected separately around dens in a marshland of western Hungary, May 2014, 2017 and 2020, when the abundance values of main food sources differed. Rodents and waterfowl dominated the diet, but their relative occurrence in the samples showed yearly variations. We found that vixens follow a dual optimizing foraging strategy regarding their provisioning of the cubs and their own diet. Adult foxes optimized their diet according to the actual yearly abundances of their main food sources. Additionally, they preferred prey items that can be consumed at the site of capture (large carrion and small individual prey items). Cubs on the other hand were provisioned with optimal high-energy food, even if those in question became less abundant in that year. Vixens mostly fed to their young either larger rodents and waterfowl, or multiple small rodents at a time-these type of prey are both optimal for transportation as a single load. Providing optimal prey at an early age in a changing environment may contribute to the ecological success of the red fox.

European Pond Turtle (Emys orbicularis) Nest Predation: A Study with Artificial Nests.

Nest predation significantly impacts the population decline of the long-living European pond turtle (Emys orbicularis). Kis-Balaton is one of the most important habitats of this species in Hungary, and in May 2017 more than 400 damaged nests were counted. To reveal predation pressure, we conducted a study with artificial nests on three sites in this area. On each site, we used 11 depredated real nests, and near each of them, we created new artificial nests; then in every nest we put one quail egg, one plasticine egg and several turtle egg shells. After that, we sprayed the smoothed surface of the covered holes with water-diluted turtle urine, imitating the turtle's behaviour. Already in the first three nights, 94% of all nests were depredated by the red fox (Vulpes vulpes) and in one case by the European badger (Meles meles), which was confirmed by the bite marks preserved on the plasticine eggs, by the footprints and excrements found near the nests, as well as by camera recordings. Only 6% of the nests survived during the three weeks of our study. Daily survival rates of quail eggs in artificial nests established in both real (damaged) and in new nests were similar, suggesting that estimates obtained with artificial nests reflect the degree of predation pressure on real nests. On the site where the nests were scattered, their daily survival rate (33%) was significantly lower than on the sites where their arrangement was partly scattered, partly linear (83%), or only linear (76%). On two additional sites, by using simulated turtle nests we showed that spraying the nests with diluted turtle urine and marking them with a flag did not affect their survival, although further methodological testing is needed. The information obtained with artificial nests enables the organization of the protection of the nests of the European pond turtle and selective predator control.

Detection and sequence analysis of Canine morbillivirus in multiple species of the Mustelidae family.

BACKGROUND: Canine morbillivirus (canine distemper virus, CDV) is a member of the Paramyxoviridae family. Canine distemper is a serious viral disease that affects many mammalian species, including members of the Mustelidae family. These animals have an elusive nature, which makes related virological studies extremely challenging. There is a significant knowledge gap about the evolution of their viruses and about the possible effects of these viruses to the population dynamics of the host animals. Spleen and lung tissue samples of 170 road-killed mustelids belonging to six species were collected between 1997 and 2022 throughout Hungary and tested for CDV with real-time RT-PCR. RESULTS: Three species were positive for viral RNA, 2 out of 64 Steppe polecats (Mustela eversmanii), 1 out of 36 European polecats (Mustela putorius) and 2 out of 36 stone martens (Martes foina); all 18 pine martens (Martes martes), 10 least weasels (Mustela nivalis) and 6 stoats (Mustela erminea) tested negative. The complete CDV genome was sequenced in five samples using pan-genotype CDV-specific, amplicon-based Nanopore sequencing. Based on the phylogenetic analysis, all five viral sequences were grouped to the Europe/South America 1 lineage and the distribution of one sequence among trees indicated recombination of the Hemagglutinin gene. We verified the recombination with SimPlot analysis. CONCLUSIONS: This paper provides the first CDV genome sequences from Steppe polecats and additional complete genomes from European polecats and stone martens. The infected specimens of various species originated from distinct parts of the country over a long time, indicating a wide circulation of CDV among mustelids throughout Hungary. Considering the high virulence of CDV and the presence of the virus in these animals, we highlight the importance of conservation efforts for wild mustelids. In addition, we emphasize the importance of full genomic data acquisition and analysis to better understand the evolution of the virus. Since CDV is prone to recombination, specific genomic segment analyses may provide less representative evolutionary traits than using complete genome sequences.

Retrospective Detection and Complete Genomic Sequencing of Canine morbillivirus in Eurasian Otter (Lutra lutra) Using Nanopore Technology.

The Eurasian otter (Lutra lutra) is a piscivorous apex predator in aquatic habitats, and a flagship species of conservation biology throughout Europe. Despite the wide distribution and ecological relevance of the species, there is a considerable lack of knowledge regarding its virological and veterinary health context, especially in Central Europe. Canine morbillivirus (Canine distemper virus (CDV)) is a highly contagious viral agent of the family Paramyxoviridae with high epizootic potential and veterinary health impact. CDV is present worldwide among a wide range of animals; wild carnivores are at particular risk. As part of a retrospective study, lung-tissue samples (n = 339) from Eurasian otters were collected between 2000 and 2021 throughout Hungary. The samples were screened for CDV using a real-time RT-PCR method. Two specimens proved positive for CDV RNA. In one sample, the complete viral genome was sequenced using a novel, pan-genotype CDV-specific amplicon-based sequencing method with Oxford Nanopore sequencing technology. Both viral sequences were grouped to a European lineage based on the hemagglutinin-gene phylogenetic classification. In this article, we present the feasibility of road-killed animal samples for understanding the long-term dynamics of CDV among wildlife and provide novel virological sequence data to better understand CDV circulation and evolution.

Different increase rate in body mass of two marten species due to climate warming potentially reinforces interspecific competition.

Many species show spatial variation in body size, often associated with climatic patterns. Studying species with contrasting geographical patterns related to climate might help elucidate the role of different drivers. We analysed changes in the body mass of two sympatric medium-sized carnivores-pine marten (Martes martes) and stone marten (Martes foina)-across Europe over 59 years. The body mass of pine marten increased with decreasing latitude, whereas stone marten body mass varied in a more complex pattern across its geographic range. Over time, the average body mass of pine martens increased by 255 g (24%), while stone marten by 86 g (6%). The greatest increase of body mass along both martens' geographic range was observed in central and southern Europe, where both species occur in sympatry. The body mass increase slowed down over time, especially in allopatric regions. The average pine/stone marten body mass ratio increased from 0.87 in 1960 to 0.99 in 2019, potentially strengthening the competition between them. Thus, a differential response in body size to several drivers over time might have led to an adaptive advantage for pine martens. This highlights the importance of considering different responses among interacting species when studying animal adaptation to climate change.

A comparison of microsatellites and genome-wide SNPs for the detection of admixture brings the first molecular evidence for hybridization between Mustela eversmanii and M. putorius (Mustelidae, Carnivora).

Introgressive hybridization can pose a serious threat to endangered species which have an overlapping distribution such as in the case of two polecat species, Mustela eversmanii and M. putorius, in Europe. The population size of steppe polecat is known to continuously shrink, whereas its sister species, the European polecat, is still somehow widespread. In this study, we perform an analysis using microsatellite (SSR) and genomic (SNP) data sets to identify natural hybrids between polecats. Four populations were genotyped for eight polymorphic SSR loci, and thousands of unlinked SNPs were generated using a reduced-representation sequencing approach, RADseq, to characterize the genetic make-up of allopatric populations and to identify hybrids in the sympatric area. We applied standard population genetic analyses to characterize the populations based on their SSR allelic frequency. Only a single sample out of 48 sympatric samples showed exact intermediacy that we identified as an F1 hybrid. Additionally, one specimen was indicated in the genomic data sets as backcrossed. Other backcrosses, indicated by SSRs, were not validated by SNPs, which highlights the higher efficacy of the genomic method to identify backcrossed individuals. The low frequency of hybridization suggests that the difference in habitat preference of the two species may act as a barrier to admixture. Therefore, it is apparently unlikely that polecat populations are threatened by significant introgression. The two species showed a clear genetic differentiation using both techniques. We found higher genetic diversity values in the sympatric steppe polecat population than in the other studies on polecat populations. Although M. putorius is a hunted species in most countries, genetic diversity values indicate worse conditions in Europe than in the protected sibling species M. eversmanii. Suspending hunting and providing protected status of the former seems to be reasonable and timely.

Positive selection on the MHC class II DLA-DQA1 gene in golden jackals (Canis aureus) from their recent expansion range in Europe and its effect on their body mass index.

BACKGROUND: In Europe, golden jackals (Canis aureus) have been expanding their range out of the southern and southeastern Balkans towards central Europe continually since the 1960s. Here, we investigated the level of functional diversity at the MHC class II DLA-DQA1 exon 2 in golden jackal populations from Bulgaria, Serbia, and Hungary. Specifically, we tested for positive selection on and geographic variation at that locus due to adaptation to supposedly regionally varying pathogenic landscapes. To test for potential fitness effects of different protein variants on individual body condition, we used linear modeling of individual body mass indexes (bmi) and accounted for possible age, sex, geographical, and climatic effects. The latter approach was performed, however, only on Serbian individuals with appropriate data. RESULTS: Only three different DLA-DQA1 alleles were detected, all coding for different amino-acid sequences. The neutrality tests revealed no significant but positive values; there was no signal of spatial structuring and no deviation from the Hardy-Weinberg equilibrium across the studied range of expansion. However, we found a signal of trans-species polymorphism and significant test results for positive selection on three codons. Our information-theory based linear modeling results indicated an effect of ambient temperature on the occurrence of individual DLA-DQA1 genotypes in individuals from across the studied expansion range, independent from geographical position. Our linear modeling results of individual bmi values indicated that yearlings homozygous for DLA-DQA1*03001 reached values typical for adults contrary to yearlings carrying other genotypes (protein combinations). This suggested better growth rates and thus a possible fitness advantage of yearlings homozygous for DLA-DQA1*03001. CONCLUSIONS: Our results indicate a demographic (stochastic) signal of reduced DLA-DQA1 exon 2 variation, in line with the documented historical demographic bottleneck. At the same time, however, allelic variation was also affected by positive selection and adaptation to varying ambient temperature, supposedly reflecting geographic variation in the pathogenic landscape. Moreover, an allele effect on body mass index values of yearlings suggested differential fitness associated with growth rates. Overall, a combination of a stochastic effect and positive selection has shaped and is still shaping the variation at the studied MHC locus.

Identification of Hepatitis E Virus in the Feces of Red Foxes (Vulpes vulpes).

Orthohepeviruses (HEV) can infect a wide range of animals, showing a relatively strict host specificity; however, its zoonotic potential, natural transmission in the wildlife are less known. Several new HEV-like viruses have been identified in various animal species, including carnivores; however, the phylogenetic relationship among these viruses is poorly resolved, since some of them were known as rodent-related so far. The red fox, the most widespread carnivore worldwide, is a known reservoir of several viruses that transmit from wildlife to humans or domestic animals; they might have a defined role in the circulation of rodent-borne HEV. In this study, we performed a HEV survey by heminested RT-PCR (Reverse Transcription PCR) on red fox fecal samples to investigate the presence of HEV in red foxes living in natural conditions, and to explore the origin of the virus via phylogenetic analysis. Out of the 26 investigated samples, HEV RNA was identified in one sample. Following Sanger sequencing, the novel sequence displayed 91% identity on the nucleotide level with recently published European common vole-HEV derived from Microtus arvalis. In contrast, it shared 85% nucleotide similarity with HEV strains described previously in red foxes. Our results strongly support "the dietary-origin" of unclassified HEV-like strains described from predators that usually prey on rodents.

Molecular Recalcitrance of Hair Passing the Digestive System of a Canid.

Hair is an important component in scat that is commonly used for prey analyses in carnivores. Chemically, hair predominately consists of keratin. The recalcitrant fiber protein is degraded in nature only by a few insects and soil microorganisms. Common proteases such as pepsin do not decompose keratin. Infrared spectroscopy was used to detect chemical differences caused by pretreatment and fate of hairs. Three sample sets were compared: original untreated hair, original milled hair, and hairs extracted from scats of golden jackals (Canis aureus L.). The results revealed that only milling affected the infrared spectral pattern, whereas digestion had no impact. Moreover, hairs from different species (e.g., boar) could be distinguished due to their spectral characteristics. They did not change through the passage of the digestive system.

Feeding responses of the golden jackal after reduction of anthropogenic food subsidies.

Little is known of the resources that limit or promote the rapidly expanding golden jackal (Canis aureus) population in Europe. We hypothesised that in an area of intensive big game hunting, a reduction of the main food resource (human subsidised big game viscera) would result in dietary switching. We used multivariate analyses to test whether the dietary composition of 200 jackal stomachs varied between two 2-yearly survey occasions, the first without big game viscera removal (availability of 68 kg viscera/year/km2) followed by a period with viscera removal (minimum of 50 kg of viscera/year/km2 removed). The proportion of empty stomachs and the stomach wet content weight did not differ between the two periods. Even after the reduction of food subsidies, the primary food of jackals was viscera and carrion from wild ungulates (frequency of occurrence: 45% vs. 30%; wet weight: 55% vs. 29%, respectively), and scavenging was not affected by season or sex. Log-linear analysis of frequency data revealed no significant differences between survey occasions in consumption of either food type. MANCOVA of wet weight data revealed that in the first period with food subsidies jackals consumed a higher proportion of adult wild boar (11.6% vs. 1.3%; from predation or scavenging), while juvenile wild boar (0 vs. 11.8%; from predation or scavenging), domestic animals (0.8% vs. 6.2%; mostly from scavenging) and invertebrates (2.6% vs. 4.1%) increased in the second period. The stomachs in the second survey occasion contained more varied food items, but the trophic niche was not significantly wider. The feeding responses of this mesopredator to the reduction of food subsidies were less pronounced than expected. Because in high big game density areas, wild ungulate carrion from different mortality causes are available in high quantities throughout the year, predator populations can be maintained despite the high amount of viscera removal.

Diet of otters (Lutra lutra) in various habitat types in the Pannonian biogeographical region compared to other regions of Europe.

Knowledge of the effect of habitat type and region on diet and feeding behaviours of a species facilitates a better understanding of factors impacting populations, which contributes to effective conservation management. Using spraint analysis and relative frequency of occurrence data from the literature, we described the dietary patterns of Eurasian otters (Lutra lutra) in 23 study sites within the Pannonian biogeographical region in Hungary. Our results indicated that diet composition varied by habitat type and is therefore context dependant. The differences among habitat types were however lower than expected. We noticed a decline in the fish consumption with a concomitant increase in trophic niche breadth and amphibian consumption in rivers, ponds (fish farms), backwaters, marshes and small watercourses. The main differences in diet were not attributed to the consumption of primary and secondary food types (fish and amphibians), but rather to differences in other, less important food types (mammals, birds). Using hierarchical cluster analysis, rivers and ponds could clearly be separated from other habitat types. We found the main fish diet of otters in most of these areas consisted of small (<100 g), eurytopic, littoral and non-native, mostly invasive species. Dietary studies from 91 sites in six European biogeographical regions showed that fish are consumed most frequently in the Atlantic and Boreal, less in the Continental and Pannonian, and least in the Alpine and Mediterranean regions. Comparative analysis indicated that the Mediterranean region (with frequent crayfish consumption) and Alpine region (frequent amphibian consumption) cluster separate from the other regions.

A European Concern? Genetic Structure and Expansion of Golden Jackals (Canis aureus) in Europe and the Caucasus.

In the first continent-wide study of the golden jackal (Canis aureus), we characterised its population genetic structure and attempted to identify the origin of European populations. This provided a unique insight into genetic characteristics of a native carnivore population with rapid large-scale expansion. We analysed 15 microsatellite markers and a 406 base-pair fragment of the mitochondrial control region. Bayesian-based and principal components methods were applied to evaluate whether the geographical grouping of samples corresponded with genetic groups. Our analysis revealed low levels of genetic diversity, reflecting the unique history of the golden jackal among Europe's native carnivores. The results suggest ongoing gene flow between south-eastern Europe and the Caucasus, with both contributing to the Baltic population, which appeared only recently. The population from the Peloponnese Peninsula in southern Greece forms a common genetic cluster with samples from south-eastern Europe (ΔK approach in STRUCTURE, Principal Components Analysis [PCA]), although the results based on BAPS and the estimated likelihood in STRUCTURE indicate that Peloponnesian jackals may represent a distinct population. Moreover, analyses of population structure also suggest either genetic distinctiveness of the island population from Samos near the coast of Asia Minor (BAPS, most STRUCTURE, PCA), or possibly its connection with the Caucasus population (one analysis in STRUCTURE). We speculate from our results that ancient Mediterranean jackal populations have persisted to the present day, and have merged with jackals colonising from Asia. These data also suggest that new populations of the golden jackal may be founded by long-distance dispersal, and thus should not be treated as an invasive alien species, i.e. an organism that is "non-native to an ecosystem, and which may cause economic or environmental harm or adversely affect human health". These insights into the genetic structure and ancestry of Baltic jackals have important implications for management and conservation of jackals in Europe. The golden jackal is listed as an Annex V species in the EU Habitats Directive and as such, considering also the results presented here, should be legally protected in all EU member states.

Data on the parasitological status of golden jackal (Canis aureus L., 1758) in Hungary.

In Hungary, twenty Canis aureus individuals were submitted to parasitological examinations in 2010-2012. Two Coccidia: Cystoisospora canis (15%) and Toxoplasma-type oocysts (5%), one Trematoda: Alaria alata (10%), six Cestoda: Mesocestoides lineatus (20%), Echinococcus granulosus (10%), Dipylidium caninums (5%), Taenia hydatigena (15%), Taenia pisiformis (20%), Taenia crassiceps (40%), and nine Nematoda: Angiostrongylus vasorum (10%), Crenosoma vulpis (30%), Capillaria aerophila (5%), Toxocara canis (20%), Toxascaris leonina (15%), Trichuris vulpis (10%), Ancylostoma caninum (45%), Uncinaria stenocephala (40%), Capillaria plica (45%) have been identified. Angiostronglyus vasorum has been reported from carnivores in Europe, Africa, South America and North America. The helminth A. vasorum or French heartworm is a metastrongylid nematode, widely distributed in Western Europe, that infects the pulmonary arterial tree of dogs, various species of foxes, wolves, Eurasian badgers, coyotes and stoats. To our knowledge, this is the first report of natural A. vasorum infection in golden jackal.

Diet of wolves Canis lupus returning to Hungary.

At the end of the nineteenth century, the wolf Canis lupus was extinct in Hungary and in recent decades has returned to the northern highland area of the country. The diet of wolves living in groups in Aggteleki National Park was investigated using scat analysis (n = 81 scats) and prey remains (n = 31 carcasses). Throughout the year wolves (average, minimum two wolves per year) consumed mostly wild-living ungulates (mean percent of biomass consumed, B% 97.2%; relative frequency of occurrence, %O 74.0%). The wild boar Sus scrofa was the most common prey item found in wolf scat (%B 35.6%) and is also the most commonly occurring ungulate in the study areas. The second most commonly occurring prey item in wolf scat was red deer Cervus elaphus (B% 32.8%). Conversely, prey remain analyses revealed wild boar as the second most commonly utilised prey species (%O 16.1%) after red deer (%O 67.7%). The roe deer Capreolus capreolus that occurs at lower population densities was the third most commonly utilised prey species. The importance of low population density mouflon Ovis aries, livestock and other food types was low. The results are similar to those found in the northern part of the Carpathian Mountains.

Metal levels in tissues of Eurasian otters (Lutra lutra) from Hungary: variation with sex, age, condition and location.

Liver samples of Eurasian otters from various parts of Hungary were analysed for mercury, copper, zinc, lead and cadmium. Only zinc concentration was significantly higher in females. Higher mercury and cadmium concentrations in adults and higher zinc values in immature otters were measured. Accumulation of mercury, copper and zinc in tissues increased with the declining condition of animals. Mercury and copper were detected with higher values in samples from large rivers.