The first report of Setaria tundra (Issaitshikoff & Rajewskaya, 1928) in Slovakia by using of molecular methods.

Setaria tundra is a filarioid parasite occurring in the northern hemisphere. Adult forms of helminths are located free in the peritoneal cavity of its definitive host - cervids, while microfilariae are presented in the host's bloodstream. Intermediate hosts are represented by several mosquito species, mainly of the genus Aedes.Nematode S. tundra is well adapted to roe deer (Capreolus capreolus) and therefore is the infection usually asymptomatic. In this study we present the first report of S. tundra in Slovakia. During a period 2022 a total of 6 roe deer coming from eastern Slovakia (Trebisov district) were examined. Nematodes were found during the evisceration process in the abdominal cavity of 3 specimens Intensity of infection was in range from 5 to 38 helminths per host. Mean intensity of infection reached 18.3 parasites per host. The helminths were identified as S. tundra by morphological examination and molecular typing of the COI gene. This study is the first report of S. tundra in Slovakia.

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.

Liver Proteome Alterations in Red Deer (Cervus elaphus) Infected by the Giant Liver Fluke Fascioloides magna.

Liver fluke infections are recognised as diseases with worldwide distribution and considerable veterinary and public health importance. The giant liver fluke, Fascioloides magna, is an important non-native parasite which has been introduced to Europe, posing a threat to the survival of local wildlife populations such as red deer (Cervus elaphus). The aim of the study was to analyse differences in liver proteomes between F. magna-infected and control red deer groups using a label-based high-throughput quantitative proteomics approach. The proteomics analysis identified 234 proteins with differential abundance between the control and infected groups. Our findings showed that F. magna infection in this definitive host is associated with changes in the metabolism of proteins and fatty acids, oxidative stress, fibrosis, and signaling pathways. The identified proteins and associated biological pathways represent a valuable contribution to the understanding of host-parasite interactions and the pathogenesis of liver fluke infection.

Fatty Acid Composition of M. Biceps Femoris of Edible Dormouse (Glis glis L.).

This study aimed to investigate the fatty acid (FA) composition of edible dormouse m. biceps femoris in both sexes. More than 20 FA were identified in the muscle, with the 18:1cis-9 (oleic acid) being the most abundant in both sexes, comprising more than 50% of total FA in muscle. The most dominated FA were monounsaturated (MUFA), followed by saturated FA (SFA) and polyunsaturated FA (PUFA), reaching 54.8%, 25.43% and 19.8% of total FA, respectively. Sums of PUFA and n-3 PUFA tended (p > 0.05) to be higher in males than in females. There were no significant differences between sexes on the FA composition. Nevertheless, the 18:2n-6 tended to differ between sexes (p = 0.063). Several long-chain PUFA (LC-PUFA) were detected in dormouse muscle, with the 20:4 n-6 (arachidonic acid, AA) and the 22:6 n-3 (docosahexaenoic acid, DHA) being the most abundant in both sexes. The relatively high stearoyl-CoA desaturase (SCD) indexes and the large concentration of 18:1cis-9 in dormouse muscle tissues might point to a low mobilization of the SCD products. Furthermore, finding the unusual FA 20:3 ∆5,∆11,∆14, suggests feeding on leaf and wood lipids of Coniferophytes. We demonstrated sexual size monomorphism in edible dormouse. The literature regarding the composition of dormouse meat is scarce and no studies reported the FA composition of muscle, thus, this work can contribute to increasing the knowledge on edible dormouse physiology and nutritional traits.

Wild Boar (Sus scrofa)-Fascioloides magna Interaction from the Perspective of the MHC Genes.

Fascioloidosis is a parasitic disease caused by a trematode Fascioloides magna. Since major histocompatibility complex (MHC) genes play an important role in the immune response, the aim of this study was to compare the potential differences in MHC class II SLA-DRB1 exon 2 genes between wild boar populations from infected (cases) and non-infected areas (controls). During the winter of 2021, a total of 136 wild boar tissue samples were collected, 39 cases and 97 controls. DNA was extracted and sequenced using the Illumina platform. Differences in distributions of allele combinations were calculated using the Chi-Square test for homogeneity and between proportions using the large-sample test and Fisher-Irwin test. Analysis revealed 19 previously described swine leucocyte antigen (SLA) alleles. The number of polymorphic sites was 79 (29.6%), with 99 mutations in total. Nucleotide diversity π was estimated at 0.11. Proportions of the alleles SLA-DRB1*12:05 (p = 0.0008379) and SLA-DRB1*0101 (p = 0.0002825) were statistically significantly higher in controls, and proportions of the SLA-DRB1*0602 (p = 0.006059) and SLA-DRB1*0901 (p = 0.0006601) in cases. Alleles SLA-DRB1*04:09, SLA-DRB1*0501, SLA-DRB1*11:09, and SLA-DRB1*1301 were detected only in cases, while SLA-DRB1*0404, SLA-DRB1*0701, SLA-DRB1*02:10, and SLA-DRB1*04:08 were present only in controls. We did not confirm the existence of specific alleles that could be linked to F. magna infection. Detected high variability of the MHC class II SLA-DRB1 exon 2 genes indicate high resistance potential against various pathogens.

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.

Rotavirus A in Domestic Pigs and Wild Boars: High Genetic Diversity and Interspecies Transmission.

Rotavirus A (RVA) is an important pathogen for porcine health. In comparison to humans, RVA in domestic animals and especially in wildlife is under researched. Therefore, the aim of the present study was to investigate the prevalence, genetic diversity, molecular epidemiology and interspecies transmission of RVA in domestic pigs and wild boars. During the three consecutive RVA seasons (2018-2021) we collected 445 and 441 samples from domestic pigs and wild boars, respectively. Samples were tested by real-time RT-PCR, and RVA-positive samples were genotyped in VP7 and VP4 segments. Our results report an RVA prevalence of 49.9% in domestic pigs and 9.3% in wild boars. Outstanding RVA genetic diversity was observed in VP7 and VP4 segments, especially in domestic pigs exhibiting a striking 23 different RVA combinations (G5P[13] and G9P[23] prevailed). Interspecies transmission events were numerous between domestic pigs and wild boars, sharing G3, G5, G6, G9, G11 and P[13] genotypes. Furthermore, our data indicate that such transmission events involved even bovines (G6, P[11]) and, intriguingly, humans (G1P[8]). This study contributes to the basic knowledge that may be considered important for vaccine development and introduction, as a valuable and currently missing tool for efficient pig health management in the EU.

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.

Complementary liver and serum protein profile in wild boars infected by the giant liver fluke Fascioloides magna using tandem mass tags quantitative approach.

Liver fluke, Fascioloides magna, is an important non-native parasite introduced to Europe, posing a threat to survival of local wildlife populations. The aim of this study was to assess the serum and liver protein profile of control and F. magna infected wild boars, by means of shotgun tandem mass tag - based quantitative high resolution proteomics approach. In serum, 4 differentially abundant proteins were found out of total 1073 identified, while in liver from 3520 identified proteins, 116 were differentially abundant between healthy and F. magna infected wild boars. Pathway analysis revealed that most of the proteins differing in abundance are involved in metabolism, biological oxidations, cellular responses to stimuli, fatty acid metabolism, and others. Validation of proteomic results was performed for paraoxonase-1, ceruloplasmin, glutathione S-transferase and liver enzymes by ELISA and automated assays. Complementary analysis of liver and serum in F. magna infection enabled insight into changes of proteome profile of the host at local and sistemic level. Our findings showed that chronic infection with F. magna is associated with immune response in host, oxidative stress and metabolomic changes in liver. SIGNIFICANCE: Liver fluke infections are recognised as worldwide neglected diseases with considerable veterinary and public health importance. Pathological changes, clinical signs and outcome of F. magna infection are strongly related to the type of final hosts and their different tolerance to infection. In order to gain insight into host-parasite interactions in wild boars, dead-end host for F. magna, we assessed proteomics profile of serum and liver of control animals and those infected with F. magna. Proteomics analysis of serum and liver in parallel showed as advantageous and beneficial, demonstrating protein alterations mainly at local level. Bioinformatics analysis enabled elucidation of molecular pathways associated with F. magna infection. Identification and validation of proteins associated with infection may have added value to current tools for efficient liver fluke control.

First record of chronic Fascioloides magna infection in roe deer (Capreolus capreolus).

Fascioloidosis is an allochthonous parasitic disease in Europe caused by the digenean trematode Fascioloides magna. The final hosts of F. magna in Europe are defined as definitive, aberrant and dead-end. Roe deer are aberrant hosts in which juvenile flukes permanently migrate through the liver parenchyma. Failure in pseudocysts formation leads to the death of both the host and the parasite. In this paper we present gross and histological findings of F. magna infection in 34 roe deer. The special emphasis is on the pseudocyst formation accompanied with new fluke's migratory channels observed in 7 animals, suggesting reinfection and prolonged survival of roe deer. No F. magna eggs were recovered from the faeces of the infected animals. These findings indicate that pseudocyst formation is essential for roe deer survival, but also point out the potential beginning of adaptation processes in roe deer, altering otherwise acute and fatal disease into a chronic one.

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.

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.

Evaluation of factors affecting the efficacy of treatment against Fascioloides magna in wild red deer population.

Deer fascioloidosis is a serious and potentially fatal disease caused by the non-native trematode Fascioloides magna. Infections of red deer with F. magna in Croatia have been reported for the first time in 2000 in the Baranja region. Subsequently, the disease spread throughout the Eastern parts of the country, involving all 3 deer species (red, roe, and fallow) and mouflons. Within the disease control programme (DCP), livers from all shot deer were thoroughly analysed and all detected trematodes and gross lesions were counted and categorized. Prevalence of positive animals, in this study for Spacva region, in the period ranging from 2007 to 2012 was 36.42% (46.39% when fawns are not considered). Epidemiological analysis was applied to evaluate risk factors and disease patterns at the population level with the aim to understand factors with negative influence on therapeutic effect. Each demographic variable was tested at the seasonal, individual and location level. Model for pathological lesions suggested that the likelihood of lesions was dependent on age (p = 0.003). We did not find any locality or sex related significant differences. Finally, environmental characteristics and migratory patterns were analysed using Geographic Information System (GIS) and showed that Spacva region represents an epidemiological unit for red deer fascioloidosis.

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.

Detection and genetic characterization of tick-borne encephalitis virus (TBEV) derived from ticks removed from red foxes (Vulpes vulpes) and isolated from spleen samples of red deer (Cervus elaphus) in Croatia.

Tick-borne encephalitis (TBE) is a growing public health concern in central and northern European countries. Even though TBE is a notifiable disease in Croatia, there is a significant lack of information in regard to vector tick identification, distribution as well as TBE virus prevalence in ticks or animals. The aim of our study was to identify and to investigate the viral prevalence of TBE virus in ticks removed from red fox (Vulpes vulpes) carcasses hunted in endemic areas in northern Croatia and to gain a better insight in the role of wild ungulates, especially red deer (Cervus elaphus) in the maintenance of the TBE virus in the natural cycle. We identified 5 tick species (Ixodes ricinus, Ixodes hexagonus, Haemaphysalis punctata, Dermacentor reticulatus, Rhipicephalus sanguineus) removed from 40 red foxes. However, TBE virus was isolated only from adult I. ricinus and I. hexagonus ticks showing a viral prevalence (1.6%) similar to or higher than reported in endemic areas of other European countries. Furthermore, 2 positive spleen samples from 182 red deer (1.1%) were found. Croatian TBE virus isolates were genetically analyzed, and they were shown to be closely related, all belonging to the European TBE virus subgroup. However, on the basis of nucleotide and amino acid sequence analysis, 2 clusters were identified. Our results show that further investigation is needed to understand the clustering of isolates and to identify the most common TBE virus reservoir hosts in Croatia. Sentinel surveys based on wild animal species would give a better insight in defining TBE virus-endemic and possible risk areas in Croatia.

Wildlife-vehicle collisions in Croatia--a hazard for humans and animals.

Wildlife-vehicle collisions (WVC) have increased and now there is a world-wide problem related to significant mortality of wildlife, habitat fragmentation, change in behavior and even disappearance of local endangered populations. Along with these deleterious effects on wildlife, WVC can also result in injuries and deaths of humans. During the three-year monitoring, a total of 7,495 wildlife-vehicle collisions were recorded, including mainly roe deer (73%), while other species were less frequently affected (wild boar--9%; brown hare--5%; and red deer and pheasant each with 4%). Incidence of wildlife-vehicle collisions were observed according to territorial distribution, seasonal and daily occurrence and type of road (total and per 1 km).

Cadmium, lead and mercury concentrations in selected red deer (Cervus elaphus L.) tissues from north-eastern Croatia.

Concentrations of cadmium, lead and mercury were determined in muscle, liver and kidney tissue from three different age groups of red deer (young animals aged 6 or 7 months, middle-aged animals aged 3 to 5 years, old animals aged over 8 years) from the Baranja region of Croatia. Median cadmium concentrations were low in the muscles of all investigated age groups (0.0002; 0.0009 and 0.0020 µg/g), higher in the liver (0.0279; 0.0656 and 0.1463 µg/g) and highest in the kidneys (0.4792; 2.8531 and 6.1657 µg/g). A positive correlation was established between cadmium concentration and age. In all analyzed tissues the median lead concentration was higher in young (muscle 0.0024; liver 0.0364 and kidney 0.0618 µg/g), compared to middle-aged animals (muscle 0.0001; liver 0.0184 and kidney 0.0160 µg/g). In contrast to cadmium, mercury had a negative correlation to age but median concentrations were very low (in muscle: 0.0011; 0.0001 and 0.0006 µg/g; in liver: 0.0051; 0.0037 and 0.0022 µg/g and in kidney: 0.0145; 0.0183 and 0.0106 µg/g) especially compared to cadmium. From the hygienic point of view all the examined tissues are edible since concentrations of the analyzed metals do not exceed values proposed by the official regulations, with the exception of a few kidney samples, which contained cadmium above the recommended value. Furthermore, mercury concentrations are no longer (since 2008) a matter of legislation.

Organochlorine compounds in red deer (Cervus elaphus L.) and fallow deer (Dama dama L.) from inland and coastal Croatia.

Polychlorinated biphenyl (PCB) and organochlorine pesticide (OCP) levels in fat tissue of red and fallow deer (Cervus elaphus L. and Dama dama L.) from two inland and an Adriatic area were established. Of 17 analysed PCBs, PCB-28, PCB-138, PCB-153, PCB-180 and PCB-118 were found in all samples, whilst PCB-101 and PCB-170 were found in more than 50% of samples. They ranged between 0.03 and 5.98 ng g(-1) fat weight. Of seven analysed OCPs, HCB, γ-HCH and 1,1-dichloro-2,2-di(4-chlorophenyl)ethylene (DDE) were found in all samples, whilst ß-HCH was found in more than 50% of samples. They ranged between 0.17 and 22.14 ng g(-1) fat weight. The dominating compounds were DDE, PCB-138, PCB153, PCB-118 and PCB-180. According to the Duncan multistage test, the levels of PCB-138, PCB-153, PCB 170 and PCB-180 were significantly higher in perirenal fat samples of specimens taken from the Adriatic area. DDE was significantly higher in the inland deer samples. Some species differences were determined and were mostly related to higher PCB and ß-HCH levels in fallow deer samples and higher DDE levels in red deer samples. No sex difference was established. As for age, significantly higher levels of PCB-118 and PCB-52 were found in fawns.

Prevalence of mandibular osteomyelitis in roe deer (Capreolus capreolus) in Slovenia.

Mandibular osteomyelitis in free-ranging cervids is a rare, but eventually fatal, disease. We examined 41,895 defleshed mandibles of roe deer collected throughout Slovenia in 2007. Mandibles from 14,679 fawns had no signs of osteomyelitis, and were excluded from further analysis. Of the remaining 27,216 specimens, chronic osteomyelitis ("lumpy jaw") was found in 113 mandibles (4.2%; 7.0% of adults). The majority of cases were observed from the Mediterranean and subalpine regions, near larger cities and thermal power plants. There was no statistically significant correlation between severity of the mandibular osteomyelitis and body weight. Females were more frequently affected than males. Coarse and abrasive food, and to some extent dental fluorosis, are the most probable triggers for development of lesions.

The centenary progress of molecular genetics. A 100th anniversary of T. H. Morgan's discoveries.

A century ago, Thomas Hunt Morgan, the American scientist, studied the cytogenetic changes of drosophila and came to cytogenetic explanation of Mendel's basic laws of genetic heredity. These studies resulted in today's Mendel-Morgan chromosomal theory of heredity. On the occasion of the hundredth anniversary of this important discovery the authors have decided to give a review of the most significant achievements in the field of molecular genetics until the completion of the Human Genome Project. The most important points concerning the technology of DNA recombination and genetic engineering are also presented. The final section discusses the significance of previous achievements of molecular genetics in biomedicine and other related fields. There is also a tabular presentation of the sequence of the most important findings in the field of molecular genetics through time.