Exposure assessment of anticoagulant rodenticides in the liver of red foxes (Vulpes vulpes) in Slovenia.

The study deals with the environmental residues of anticoagulant rodenticides (ARs) in Slovenia to evaluate the toxicological risk of secondary poisoning of red foxes (Vulpes vulpes) as representatives of non-target wildlife, and in relation to the investigated use patterns of ARs and specific local parameters in Slovenia. From 2019 to 2022, 148 liver tissue samples of adult red foxes were collected from almost all state geographical regions. The samples were extracted with methanol/water (2:1, v/v), cleaned-up using a solid supported liquid-liquid extraction, and measured by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) with reporting limits of 0.5 to 5.0 ng/g. Residues of at least one rodenticide were detected in 77.7 % of the samples. The second generation ARs of bromadiolone, brodifacoum and difenacoum were the most frequently found, appearing in 75.0, 51.4, and 18.9 % of the samples, respectively. Concentrations of pooled ARs ranged from 1.5 to 2866.5 ng/g with mean and median values of 601.4 and 350.2 ng/g, respectively. We determined bromadiolone and brodifacoum at concentrations of ≥800 ng/g in 10.8 and 10.1 % of the samples, and 1.4 and 0.7 % of the samples contained residues >2000 ng/g, respectively. These concentrations are much higher than those found in comparable studies in Europe and elsewhere in the world. Residues of ARs were detected in all monitored statistical regions of Slovenia, with higher concentrations in the eastern parts of the country. First generation ARs were found in only 9.5 % of samples, and residues were below 10 ng/g with one exception (coumatetralyl with 55 ng/g). The results of the study indicate a serious toxicological risk for red foxes in Slovenia as part of the Western Balkans, and will contribute to the growing body of knowledge about the protection of European ecosystems, as wildlife is not limited by national borders.

Mitochondrial genetic diversity and phylogenetic relationships of Echinococcus multilocularis in Europe.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.

First Molecular Detection of Neospora caninum in Feces of Grey Wolf (Canis lupus) and Golden Jackal (Canis aureus) Populations in Slovenia.

Neospora caninum is an obligate intracellular parasite that causes reproductive disorders and major economic losses in cattle, and induces neuromuscular disorders in canids. Exogenous infections are becoming increasingly important due to disease outbreaks. The sylvatic life cycle of N. caninum interferes with the domestic dog-ruminant life cycle, but understanding of it is scarce. The population of wild canids may play an important role in parasite dispersion. Feces from 42 grey wolves (Canis lupus) and 39 golden jackals (Canis aureus) were analyzed for the N. caninum Nc5 gene using a novel real-time PCR (qPCR) with a detection limit of 5 targets/µL in clinical samples. Three wolves (3/42; 7.1%) and one golden jackal (1/39; 2.6%) tested positive, which is the first detection of N. caninum in the population of grey wolves in Slovenia and the first detection of N. caninum DNA in the feces of a golden jackal. In addition to the grey wolf, we propose the golden jackal as a potential definitive host with hypothetical epidemiological importance for the sylvatic-domestic life cycle of N. caninum, due to its proximity to human habitats and its rapid expansion throughout Europe.

Evaluation of Biosecurity Measures in Pig Holdings in Slovenia as a Risk Assessment for the Introduction of African Swine Fever Virus.

African Swine Fever (ASF) is persistently spreading and hindering pork production in Europe. Slovenia is one of the last countries in Central Europe without a confirmed ASF case in domestic pigs or in wild boar. The aim of this study was to assess the current biosecurity implementation on different types of pig farms. Internal and external biosecurity status was determined in 17 commercial (CF), 15 non-commercial (NC), and 15 outdoor (O) farms. Data were collected using the Biocheck.UGent questionnaire and assessed in combination with the latest information on the wild boar population in Slovenia. Biosecurity was compared between farm types based on the assessment of 12 subcategories. Statistically significant differences (p < 0.05) were found in six subcategories: (i) purchase of pigs and semen, (ii) visitors and farmworkers, (iii) vermin and bird control, (iv) finishing unit, (v) measures between compartments and use of equipment, and (vi) cleaning and disinfection. The highest total biosecurity score (0-100%) was determined on CF with 64.59 ± 16.47%, followed by NC with 55.73 ± 10.67%, and O with 48.47 ± 8.20%. The density of the wild boar population was estimated from the number of wild boars per km2 per year, with 3 or more hunted wild boars per unit representing the highest density. Geolocation of farms on the wild boar population map showed that two O farms are at high risk and seven farms (1 O, 5 NC, and 1 CF) are at medium risk for disease transmission from wild to domestic pigs. Biosecurity measures must be tightened in some subcategories, especially in areas with a high density of wild boar.

Wild Carnivore Survey of Echinococcus Species in Slovenia.

Wild carnivores are definitive hosts and potential reservoirs for the tapeworm Echinococcus sp. which can cause cystic and alveolar echinococcosis. Both are considered neglected and important food-borne pandemics. This study is the first to molecularly test Slovenian wild carnivores for Echinococcus species that can cause disease in humans. Fecal samples from 210 red foxes (Vulpes vulpes), 42 wolves (Canis lupus), 39 golden jackals (Canis aureus), 18 martens (Marten sp.), 2 Eurasian lynx (Lynx lynx), 2 European badger (Meles meles), and 1 Eurasian otter (Lutra lutra) were examined for Echinococcus granulosus sensu lato (EGsl: E. granulosus sensu stricto, E. canadensis) and E. multilocularis (EM) using real-time PCR. Red foxes (29.1%) and golden jackals (18%) were positive for EM. All animals examined were negative for EGsl. Univariate analysis showed no significant differences in EM prevalence with respect to animal species (red fox vs., golden jackal) (p = 0.22), age (p = 0.12), and sex (p = 0.18). Prevalence of EM was associated with the region (p < 0.001), with regions in central and southern Slovenia having higher EM prevalence and risk of infection. Due to the increase in population and expansion of habitat, the golden jackal may soon become as important definitive host for EM as the red fox.

Passive Disease Surveillance of Alpine Chamois (Rupicapra r. rupicapra) in Slovenia between 2000 and 2020.

In this paper, we provide an overview of the causes of death of Alpine chamois (Rupicapra r. rupicapra) diagnosed in the national passive health surveillance of chamois in Slovenia. From 2000 to 2020, 284 free-ranging chamois provided by hunters were necropsied at the Veterinary Faculty, University of Ljubljana, Slovenia. Depending on the results of complete necropsy, histopathological, bacteriological, parasitological, and virological examinations, a descriptive data analysis was performed. The most common causes of death in chamois were infectious diseases (82.2%), followed by non-infectious diseases (11.8%). Of all the causes of death, parasitic infections accounted for 70.3%, trauma for 9.7%, and bacterial infections for 9.3% of all cases. Less common diseases were viral infections, neoplasms, winter starvation, and metabolic disorders.

Serological Survey of Small Ruminant Lentivirus Infections in Free-Ranging Mouflon and Chamois in Slovenia.

Small ruminant lentiviruses (SRLVs) belong to the genus Lentivirus in the Retroviridae family, which are responsible for the diseases maedi-visna and caprine arthritis-encephalitis in sheep and goats worldwide and are also widespread in Slovenian sheep and goats. SRLVs cause lifelong infections with chronic inflammatory lesions in various organ systems. Cross-species transmission of SRLV strains in sheep and goats is well documented, but there are few data on the ability of these viruses to infect wild ruminants. The objective of this study was to investigate whether SRLVs circulate among wild small ruminants in Slovenia. During the 2017-2018 hunting season, a total of 38 blood samples were collected from free-ranging chamois (Rupicapra rupicapra) and European mouflon (Ovis ammon musimon). The serum samples were tested for antibodies against SRLV by enzyme-linked immunosorbent assay (ELISA). The serological tests revealed that of all tested mouflons, 1 animal (11.1%) was seropositive, while all samples from chamois were negative. Based on the results of this study and considering the results of previous studies in which SRLV infections were detected in mouflons with low seroprevalence, it is very likely that the detected seropositive animal was an incidental spillover host for SRLV. Although no seropositive samples were found in chamois, we cannot speculate on whether chamois may not be a host for SRLV infection because of the small sample size and the disadvantages of the ELISA assay used when applied to samples from chamois.

The First Report of Elaphostrongylus cervi Infection in Two Imported Wapitis (Cervus canadensis) in Slovenia.

This study describes two female wapitis (Cervus canadensis) with neurological signs associated with an Elaphostrongylus cervi (E. cervi) infection. The original host of the nematode parasite is the Eurasian red deer (Cervus elaphus), although other cervids and small ruminants may also be affected. The two wapitis imported from Canada were kept in an enclosure with the Slovenian red deer herd. After developing debilitating neurological signs, the wapitis were euthanized and examined for possible causes. A histopathological examination of the brain of the first wapiti revealed severe diffuse perivascular meningoencephalitis with chronic vasculitis, and some cross-sections of nematodes were found in the leptomeninges. A necropsy of the second wapiti revealed severe pachymeningitis and leptomeningitis, where several adult nematode parasites were found. E. cervi was confirmed by molecular methods. The prevalence of E. cervi in the European red deer population is high, but no study has been conducted to assess its prevalence in Slovenia. This was the first confirmation of E. cervi in Slovenia and the first infection with this parasite described in Europe in a wapiti. Elaphostrongylus cervi should also be considered as a differential diagnosis in Europe for all ruminants grazing on pastures frequented by red deer and showing neurological clinical signs.

Exposure of Free-Ranging Wild Animals to Zoonotic Leptospira interrogans Sensu Stricto in Slovenia.

A total of 249 serum samples from 13 wild animal species namely fallow deer (Dama dama, n = 1), roe deer (Capreolus capreolus, n = 80), red deer (Cervus elaphus, n = 22), chamois (Rupicapra rupicapra, n = 21), mouflon (Ovis musimon, n = 4), brown hare (Lepus europaeus, n = 2), nutria (Myocastor coypus, n = 1), red fox (Vulpes vulpes, n = 97), stone marten (Martes foina, n = 12), European badger (Meles meles, n = 2), golden jackal (Canis aureus, n = 2) Eurasian lynx (Lynx lynx, n = 2) and grey wolf (Canis lupus, n = 3) were analysed for the presence of antibodies against Leptospira interrogans sensu stricto. Serum samples were examined via the microscopic agglutination test for the presence of specific antibodies against Leptospira serovars Icterohaemorrhagiae, Bratislava, Pomona, Grippotyphosa, Hardjo, Sejroe, Australis, Autumnalis, Canicola, Saxkoebing and Tarassovi. Antibodies to at least one of the pathogenic serovars were detected in 77 (30.9%; CI = 25-37%) sera. The proportion of positive samples varied intraspecifically and was the biggest in large carnivores (lynx, wolf and jackal; 86%), followed by mezzo predators: stone marten (67%) and red fox (34%), and large herbivores: red deer (32%), roe deer (25%), alpine chamois (10%) and mouflon (0%). Out of the 77 positive samples, 42 samples (53.8%) had positive titres against a single serovar, while 35 (45.4%) samples had positive titres against two or more serovars. The most frequently detected antibodies were those against the serovar Icterohaemorrhagiae. The present study confirmed the presence of multiple pathogenic serovars in wildlife throughout Slovenia. It can be concluded that wild animals are reservoirs for at least some of the leptospiral serovars and are a potential source of leptospirosis for other wild and domestic animals, as well as for humans.

Identification of a Novel Papillomavirus Type (MfoiPV1) Associated with Acrochordon in a Stone Marten (Martes foina).

Papillomaviruses (PVs) are an extremely large group of viruses that cause skin and mucosal infections in humans and various domestic and wild animals. Nevertheless, there is limited knowledge about PVs in wildlife hosts, including mustelid species. This study describes a case in stone marten (Martes foina) with a clinical manifestation of skin tumor, which is rather atypical for infections with PVs. The result of the papillomavirus PCR performed on the skin tumor sample was positive, and the complete PV genome was determined in the studied sample using next-generation sequencing technology. The analysis of the PV genome revealed infection of the stone marten with a putative new PV type belonging to the Dyonupapillomavirus genus. The proposed new stone marten PV type was named MfoiPV1.

Twenty Years of Passive Disease Surveillance of Roe Deer (Capreolus capreolus) in Slovenia.

In this paper, we provide an overview of the causes of death of roe deer (Capreolus capreolus) diagnosed within the national passive health surveillance of roe deer in Slovenia. From 2000 to 2019, postmortem examinations of 510 free-ranging roe deer provided by hunters were conducted at the Veterinary Faculty, Slovenia. A comprehensive necropsy was performed. According to the results of the necropsy, the samples were subjected to microscopic, histopathological, bacteriological, parasitological, or virological examination. The most frequent causes of death in roe deer were infectious diseases (67%), followed by noninfectious diseases (28%). Of all deaths, parasitic infections represented 48%, bacterial infections 14.8%, trauma 12.5%, and metabolic disorders 9.8%. Less frequent causes were diseases like neoplasia and mycotic infections, winter starvation, hernias, and lightning strike. This study covered an estimated 1% of the total disease-related mortality of roe deer in Slovenia. Comparisons of sex/age structure indicated that hunters did not provide random samples (e.g., young males were disproportionately represented). Therefore, such monitoring does not ensure an unbiased assessment of the significance of the individual disease for the mortality of the population; however, it can provide credible evidence of whether or not a particular disease is present in a population. We show that no identified disease in roe deer in Slovenia can be considered a significant health threat to roe deer, other wildlife species, or humans.

A Comprehensive Study of Cutaneous Fibropapillomatosis in Free-Ranging Roe Deer (Capreolus capreolus) and Red Deer (Cervus elaphus): from Clinical Manifestations to Whole-Genome Sequencing of Papillomaviruses.

Papillomaviruses (PVs) are an extremely large group of viruses that cause skin and mucosa infections in humans and various animals. In roe deer and red deer, most PVs belong to the Deltapapillomavirus genus and cause neoplastic changes that are generally described as fibropapillomas. Despite the wide distribution of roe and red deer throughout Europe and beyond, the data in the scientific literature regarding the widespread distribution of PVs and the genetic variability of PV genomes in these species are rather scarce. This study describes cutaneous fibropapillomatosis cases in roe and red deer with clinical manifestations that are typical of infections with PVs. In all cases, the presence of PV DNA was confirmed using PCR, followed by Sanger sequencing of the partial L1 gene. The complete PV genomes were determined in all the investigated samples using next-generation sequencing technology, revealing infections of roe deer with the CcaPV1-type and red deer with the CePV1v-type variant. A comparison of the complete CcaPV1-type and CePV1v-type variant genome sequences reported here with already available complete genome sequences in GenBank revealed their great genetic stability across time and space.

Post-epidemic investigation of Schmallenberg virus in wild ruminants in Slovenia.

Schmallenberg virus (SBV) is a vector-borne virus belonging to the genus Orthobunyavirus within the Bunyaviridae family. SBV emerged in Europe in 2011 and was characterized by epidemics of abortions, stillbirths and congenital malformations in domestic ruminants. The first evidence of SBV infection in Slovenia was from an ELISA-positive sample from a cow collected in August 2012; clinical manifestations of SBV disease in sheep and cattle were observed in 2013, with SBV RNA detected in samples collected from a total of 28 herds. A potential re-emergence of SBV in Europe is predicted to occur when population-level immunity declines. SBV is also capable of infecting several wild ruminant species, although clinical disease has not yet been described in these species. Data on SBV-positive wild ruminants suggest that these species might be possible sources for the re-emergence of SBV. The aim of this study was to investigate whether SBV was circulating among wild ruminants in Slovenia and whether these species can act as a virus reservoir. A total of 281 blood and spleen samples from wild ruminants, including roe deer, red deer, chamois and European mouflon, were collected during the 2017-2018 hunting season. Serum samples were tested for antibodies against SBV by ELISA; the overall seroprevalence was 18.1%. Seropositive samples were reported from all over the country in examined animal species from 1 to 15 years of age. Spleen samples from the seropositive animals and serum samples from the seronegative animals were tested for the presence of SBV RNA using real-time RT-PCR; all the samples tested negative. Based on the results of the seropositive animals, it was demonstrated that SBV was circulating in wild ruminant populations in Slovenia even after the epidemic, as almost half (23/51) of the seropositive animals were 1 or 2 years old.

The prevalence of Coxiella burnetii in ticks and animals in Slovenia.

BACKGROUND: The obligate intracellular bacterium Coxiella burnetii causes globally distributed zoonotic Q fever. Ruminant livestock are common reservoirs of C. burnetii. Coxiella burnetii are shed in large numbers in the waste of infected animals and are transmitted by inhalation of contaminated aerosols. This study was conducted to evaluate the prevalence of C. burnetii infection in domestic animals and ticks in areas of Slovenia associated with a history of Q fever outbreaks. RESULTS: A total of 701 ticks were collected and identified from vegetation, domestic animals and wild animals. C. burnetii DNA was detected in 17 out of 701 (2.4%) ticks. No C. burnetii DNA was found in male ticks. Ticks that tested positive in the PCR-based assay were most commonly sampled from wild deer (5.09%), followed by ticks collected from domestic animals (1.16%) and ticks collected by flagging vegetation (0.79%). Additionally, 150 animal blood samples were investigated for the presence of C. burnetii-specific antibodies and pathogen DNA. The presence of pathogen DNA was confirmed in 14 out of 150 (9.3%) blood samples, while specific antibodies were detected in sera from 60 out of 150 (40.4%) animals. CONCLUSIONS: Our results indicate that ticks, although not the primary source of the bacteria, are infected with C. burnetii and may represent a potential source of infection for humans and animals. Ticks collected from animals were most likely found to harbor C. burnetii DNA, and the infection was not lost during molting. The persistence and distribution of pathogens in cattle and sheep indicates that C. burnetii is constantly present in Slovenia.

Hypertrophic Osteopathy Associated with Mycotic Pneumonia in a Roe Deer ( Capreolus capreolus).

Aspergillus fumigatus is one of the most common Aspergillus species causing disease in individual animals but it rarely affects deer species. We report a case of A. fumigatus infection causing mycotic pneumonia and hypertrophic osteopathy in a roe deer ( Capreolus capreolus).

Complete Genome Sequence of Roe Deer Picobirnavirus Strain PBV/roe_deer/SLO/D38-14/2014.

Picobirnaviruses (PBVs) have been detected in feces from various animal species and humans. Here, we report the complete genome sequence of the PBV/roe_deer/SLO/D38-14/2014 strain, which is the first PBV detected in roe deer, providing additional knowledge about the high diversity and host range of PBVs.

Prevalence of Anti-Hepatitis E Virus Antibodies and First Detection of Hepatitis E Virus in Wild Boar in Slovenia.

Hepatitis E is an emerging zoonotic disease caused by hepatitis E virus (HEV). In this study, we investigated HEV presence in a wild boar (Sus scrofa) population of Slovenia. A total of 288 wild boar serum samples were collected throughout the country, and HEV infection was investigated by serology, using enzyme-linked immunosorbent assay (ELISA) and by HEV RNA detection using a real-time PCR assay. Antibodies against HEV were detected in 30.2% (87/288) of animals tested, whereas HEV RNA was detected in only one sample. This is the first evidence of HEV presence in the wild boar population in Slovenia, and these results suggest that these animals are part of the HEV epidemiological cycle in the country.

Mycobacterium spp. in wild game in Slovenia.

Wildlife species are an important reservoir of mycobacterial infections that may jeopardise efforts to control and eradicate bovine tuberculosis (bTB), caused by Mycobacterium bovis. Slovenia is officially free of bTB, but no data on the presence of mycobacteria in wild animals has been reported. In this study, samples of liver and lymph nodes were examined from 306 apparently healthy free-range wild animals of 13 species in Slovenia belonging to the families Cervidae, Suidae, Canidae, Mustelidae and Bovidae. Mycobacteria were isolated from 36/306 (11.8%) animals (red deer, roe deer, fallow deer, wild boar and jackal) and identified by PCR, commercial diagnostic kits and sequencing. Non-tuberculous mycobacteria identified in five species were Mycobacterium peregrinum, M. avium subsp. hominissuis, M. intracellulare, M. confluentis, M. fortuitum, M. terrae, M. avium subsp. avium, M. celatum, M. engbaekii, M. neoaurum, M. nonchromogenicum and M. vaccae.

Intestinal parasites of the red fox (Vulpes vulpes) in Slovenia.

In the present study, 428 foxes were collected and examined for intestinal helminths using the washing-out method. Parasites were found in 93.2% of the examined animals. The most frequently identified nematodes were Uncinaria stenocephala (58.9%), Toxocara canis (38.3%) and Molineus patens (30.6%). Other nematodes found were Pterygodermatites affinis (4.2%), Capillaria sp. (2.8%), Crenosoma vulpis (2.8%), Toxascaris leonina (2.5%), Trichuris vulpis (0.7%) and Physaloptera sp. (0.2%). Mesocestoides sp. (27.6%) and Taenia crassiceps (22.2%) were the most prevalent cestodes, followed by T. polyacantha (6.5%), Hymenolepis nana (2.1%), T. pisiformis (2.1%) and Dipylidium caninum (1.4%). The study also revealed four trematode species: Rossicotrema donicum (1.6%), Heterophyes heterophyes (1.1%), Metagonimus yokogawai (1.1%), Prohemistomum appendiculatum (0.4%) and two protozoan species: oocysts of Sarcocystis (2.8%) and Isospora (0.4%). This is the first extensive study on the intestinal parasites of the red fox (Vulpes vulpes) in Slovenia. The 2.6% prevalence of Echinococcus multilocularis in the same sample population as investigated herein has been reported previously (Vergles Rataj et al., 2010).

Evidence of Anaplasma phagocytophilum in game animals from Slovenia.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen responsible for granulocytic anaplasmosis in mammalian hosts including humans. Wild animals may play an important role in the epidemiology of this disease. The aim of this study was to estimate the prevalence of infection with A. phagocytophilum among wildlife in Slovenia. Serum samples (n = 376) from the most important game species [red deer (Cervus elaphus), roe deer (Capreolus capreolus), wild boar (Sus scrofa), chamois (Rupicapra rupicapra) and brown bear (Ursus arctos)] were examined by A. phagocytophilum-specific indirect fluorescent-antibody assay (IFA) and wild boar spleen samples (n = 160) were tested by polymerase chain reaction (PCR). A. phagocytophilum-specific antibodies were found in 72% of sera and A. phagocytophilum DNA was present in 6.2% of spleens. The data indicate that A. phagocytophilum is present and widespread in Slovenian game animals and that game species are involved in the natural life cycle of A. phagocytophilum.