First evidence of tick-borne protozoan pathogens, Babesia sp. and Hepatozoon canis, in red foxes (vulpes vulpes) in Serbia.

Tick-borne haematozoans cause severe diseases in domestic animals, and some of them have zoonotic potential. The results of previous studies in Europe point to the important role of foxes in natural endemic cycles of several tick-borne pathogens, including protozoa. The aim of the present research was to acquire information on the prevalence and distribution of tick-borne protozoan parasites among foxes in Serbia. Legally hunted foxes from 14 localities throughout Serbia were analysed. Spleen samples were collected from 129 animals and tested for the presence of Babesia spp. and Hepatozoon spp. by PCR. In total, 79/129 (61.2%) of the tested foxes were positive for H. canis, while the presence of two Babesia species was confirmed: B. vulpes (37/129, 28.7%) and B. canis (1/129, 0.8%). Coinfection with B. vulpes and H. canis was present in 26/129 (20.2%) foxes and one animal (1/129, 0.8%) was co-infected by B. canis and H. canis. The results of this study indicate the important role of foxes in the epizootiology of B. vulpes and H. canis in the Republic of Serbia and stress the need for further research to clarify all elements of the enzootic cycle of the detected pathogens, including other reservoirs, vectors, and transmission routes.

First report of a naturally patent infection with Dirofilaria immitis in an otter (Lutra lutra).

Dirofilaria immitis causes a severe and life-threatening cardio-pulmonary disease in Europe, Asia, Africa, and the Americas; also, zoonotic infections have been reported. This parasitic vector-borne disease is mostly common in domestic and wild canids and felids. Here, we present a rare finding of this roundworm in the heart of a Eurasian otter (Lutra lutra). Four mature specimens of D. immitis were found in the right ventricle of the heart. Necropsy was performed after the animal was found road-killed in Eastern Serbia, close to the Danube River. Two heartworms were females, one was male and sex for one remained unidentified. Microfilariae were present in the uterus of one of the female heartworms, indicating that otters can act as reservoirs of dirofilariasis in the wild. This is the first report of a patent infection in a free-ranging otter.

Concentrations of Selected Elements in Liver Tissue of Grey Wolves (Canis lupus) from Serbia.

The grey wolf (Canis lupus) is a large carnivore species and a top predator in the ecosystems that it inhabits. Considering its role in food webs, wolves may be exposed to high concentrations of potentially harmful elements. Therefore liver samples from 28 legally hunted wolves were analyzed for concentrations of 16 elements using inductively coupled plasma optical emission spectrometry. The Mann-Whitney U test showed a significant difference between the genders only for Li, and there were no differences between individuals caught in different years. The majority of statistically significant correlations between element levels were positive, except for three cases. Compliance with several criteria for suitable bioindicator organisms imply that wolves may serve for monitoring environmental contamination.

Intestinal helminth parasites of the grey wolf (Canis lupus L.) in Serbia.

The grey wolf (Canis lupus L.) is the most widespread large carnivore in Europe with large populations in the Eastern part of Europe and the Balkan Peninsula. In this study, a total of 102 wolves were examined for intestinal helminth parasites. The carcasses were collected within the Serbian part of the wolf's range during the period 2009-2014. Nine helminth species were found: one nematode, Toxocara canis (3.9%), one trematode, Alaria alata (1.0%), and seven cestodes, Taenia pisiformis (1.0%), T. hydatigena (9.8%), T. polyacantha (2.9%), T. taeniaeformis (2.0%), T. (syn. Multiceps) multiceps (3.9%), T. serialis (1.0%) and Mesocestoides litteratus (1.0%). Taenia (syn. Hydatigera) taeniaeformis has been registered for the first time in a wolf from Europe. An overall moderate prevalence (16.7%) of infected wolves was recorded. There was no statistically significant difference in prevalence between sexes. Of the years studied, the highest prevalence was found in 2014 (57.1%). The maximum number of helminth species per host specimen was four.

First findings and prevalence of adult heartworms (Dirofilaria immitis) in wild carnivores from Serbia.

Heartworm (Dirofilaria immitis) is a parasitic roundworm that causes a zoonotic disease known as dirofilariosis. Little is known about the role of wild carnivores serving as reservoirs in nature. Therefore, we examined 738 hearts and lungs of free ranging wild carnivores from Serbia to determine the presence of adult heartworms. During the period 2009-2013, the prevalence in golden jackals (Canis aureus) was 7.32%, in red foxes (Vulpes vulpes) 1.55%, in wolves (Canis lupus) 1.43%, and in wild cats (Felis silvestris) 7.69%. No adult heartworm specimens were found in beech martens (Martes foina), stone martens (Martes martes), European polecats (Mustela putorius), badgers (Meles meles) or otter (Lutra lutra). The highest recorded prevalence was in 2013 (7.30%) and the lowest in 2012 (1.6%). In jackals, the prevalence was higher in males (10%) than in females (4.06%), while in foxes the prevalence was 1.75% in males and 1.26% in females. The most infected host was a wolf in which 37 adult specimens were found. Because of the potentially significant role in the life cycle of D. immitis, populations of wild carnivores in Europe should be further examined and tested for heartworm infections.

First records of Dirofilaria repens in wild canids from the region of Central Balkan.

Dirofilaria repens causes an emerging zoonotic disease in Europe, particularly in its southern part, the Mediterranean region. Many reports on human dirofilariosis have been published recently, but little is known about the wildlife hosts and reservoirs of this parasite in nature. This paper presents the first records of adult D. repens specimens from free-ranging carnivores in Central Balkan countries (Serbia and Macedonia). During the period 2009-2013, a total of 145 regularly shot canids were examined for the presence of D. repens adults. In order to investigate their role as hosts and potential wild reservoirs of this zoonosis, 71 wolves (Canis lupus), 48 foxes (Vulpes vulpes) and 26 jackals (Canis aureus) were examined. Under the skin of two wolves (one from Serbia and one from Macedonia) and of a red fox from Serbia D. repens adults were found. In all three cases only one parasite was present. Further research on wild canids is needed, particularly on species widening their range (such as jackals) and those living near human settlements (foxes and jackals), which facilitates the transmission of the parasites to dogs and humans.

The Occurrence and Diversity of Tick-Borne Pathogens in Small Mammals from Serbia.

Background: Despite abundance of small mammals in Serbia, there is no information on their role in the epidemiology of tick-borne diseases (TBDs). This retrospective study aimed to identify different tick-borne pathogens (TBPs) in small mammals in Serbia collected during 2011. Materials and Methods: A total of 179 small mammals were collected from seven different localities in Serbia. The five localities belong to the capital city of Serbia-Belgrade: recreational areas-Ada Ciganlija, Titov gaj, and Kosutnjak as well as mountainous suburban areas used for hiking-Avala and Kosmaj. The locality Veliko Gradiste is a tourist place in northeastern Serbia, whereas the locality Milosev Do is a remote area in western Serbia with minor human impact on the environment. Results: The results of the presented retrospective study are the first findings of Rickettsia helvetica, Rickettsia monacensis, Neoehrlichia mikurensis, Borrelia afzelii, Borrelia miyamotoi, Babesia microti, Hepatozoon canis, and Coxiella burnetii in small mammals in Serbia. The presence of R. helvetica was confirmed in two Apodemus flavicollis, the presence of one of the following pathogens, R. monacensis, B. afzelii, H. canis, Ba. microti, and N. mikurensis was confirmed in one A. flavicollis each, whereas the presence of B. miyamotoi was confirmed in one Apodemus agrarius. Coinfection with B. afzelii and Ba. microti was confirmed in one A. flavicollis. DNA of C. burnetii was detected in 3 of 18 pools. Conclusions: The results confirm that detected pathogens circulate in the sylvatic cycle in Serbia and point to small mammals as potential reservoir hosts for the detected TBPs. Further large-scale studies on contemporary samples are needed to clarify the exact role of particular small mammal species in the epidemiology of TBDs caused by the detected pathogens.

Heartworm Disease in Jackals: Unusual Location of Dirofilaria immitis.

PURPOSE: The knowledge of heartworm disease in free ranging wild canid populations is limited. As it is very difficult to monitor this disease in live animals, sporadic findings are mostly obtained by examining culled individuals of game species. METHODS: As a part of a broader study on jackal (Canis aureus) ecology in Serbia, the necropsy of legally hunted animals was performed in 2020. RESULTS: Two cases of heartworm infection with unusually located adult heartworms were diagnosed. The first case is an adult female jackal harvested in the vicinity of the capital city of Belgrade. Three adult specimens of Dirofilaria immitis were found in the right atrium of the heart, and nine adult specimens were located at an atypical site, in the vena cava caudalis. Very similar, the second case was in an adult female jackal harvested in the vicinity of Backi Monostor. In this case, six adult specimens of D. immitis were found in the right atrium of the heart and six more adult specimens in the vena cava caudalis. CONCLUSION: Although this nematode generally resides in the pulmonary arteries and the right ventricle of the heart, rare findings in other large blood vessels can occur. To the best of our knowledge, these are the first and only two confirmed cases of adult D. immitis found in vena cava caudalis in jackals in Europe.

Detection and genotyping of Toxoplasma gondii in wild canids in Serbia.

To gain insight into the population structure of the protozoan parasite Toxoplasma gondii in wildlife in Serbia, multiplex nested PCR- RFLP was performed on T. gondii DNA positive samples of heart tissue of three wild canid species, including red foxes (Vulpes vulpes), golden jackals (Canis aureus) and grey wolves (Canis lupus). Out of 110 samples (28 red foxes, 72 golden jackals, 10 wolves), 30 (27.3%) were positive for T. gondii DNA, of which 24 were genotyped (nine red foxes, 13 golden jackals and two wolves). The results showed a large dominance of lineage II (n = 20, 83.4%) over lineage III (n = 4, 16.7%) strains. Lineage II comprised four archetypes, 14 variants and one which remained undetermined, while within lineage III there were three archetypes and one variant strain. However, a notable difference in diversity was observed among the examined canids, with eight (88.9%) of the nine strains detected in red foxes distinct, vs. six (46.1%) distinct strains of the 13 detected in golden jackals. Since anthropogenic food sources are a major reservoir of archetypal lineage II and III T. gondii strains, the results suggest that the golden jackal may be outcompeting the red fox, forcing it to hunt wild mammals and birds for sustenance, thereby increasing its chances of infection with variant strains. In addition to presenting the first data on the T. gondii population structure in wild canids in Serbia, this is the first report ever on T. gondii genotypes in golden jackals, a species with an increasing presence in Europe and another important reservoir of domestically circulating T. gondii strains.

Golden jackals (Canis aureus) as hosts for ticks and tick-borne pathogens in Serbia.

The golden jackal (Canis aureus) is a medium-sized canid species native to Europe. This species is characterized by rapid large-scale expansion. A similar trend is also observed in Serbia, where the species is now distributed in more than a half of the territory. Although jackals prefer habitats in human-dominated landscapes, these animals have not been studied well enough from an eco-epidemiological point of view, and little is known about their potential for carrying zoonotic pathogens. In a study conducted during a three-year period (01/2010-02/2013), a total of 216 hunted or road-killed golden jackals were collected from 10 localities in Serbia. Ticks, when present, were removed, and after necropsy, spleen samples were collected from each animal. All tick and spleen samples were tested for the DNA of bacterial and protozoan tick-borne pathogens (Borrelia species, Bartonella species, Rickettsia species, Anaplasma species, Coxiella burnetii, Francisella species and Babesia species) by multiplex real-time PCR, conventional PCR and sequencing analyses. The DNA of Babesia canis was detected in nine out of 216 (4.2%) spleen samples, and two samples (0.9%) tested positive for Anaplasma phagocytophilum. In 118 ticks collected from jackals, the DNA of two Babesia species (Ba. canis and Ba. microti), three Borrelia species (Bo. garinii, Bo. valaisiana, and Bo. lusitaniae) and A. marginale was detected. From the aspect of public health surveillance, the potential role of the golden jackal in the maintenance of vector-borne zoonotic pathogens in Serbia must be considered, and further eco-epidemiological studies should be performed to determine the precise role of this animal species in zoonotic disease transmission cycles.

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.

Presence of Leishmania and Brucella species in the golden jackal Canis aureus in Serbia.

The golden jackal Canis aureus occurs in south-eastern Europe, Asia, the Middle East, the Caucasus, and Africa. In Serbia, jackals neared extinction; however, during the last 30 years, the species started to spread quickly and to increase in number. Few studies in the past have revealed their potential role as carriers of zoonotic diseases. Animal samples were collected over a three-year period (01/2010-02/2013) from 12 sites all over Serbia. Of the tissue samples collected, spleen was chosen as the tissue to proceed; all samples were tested for Leishmania species and Brucella species by real-time PCR. Of the 216 samples collected, 15 (6.9%) were positive for Leishmania species, while four (1.9%) were positive for B. canis. The potential epidemiologic role of the golden jackal in carrying and dispersing zoonotic diseases in Serbia should be taken under consideration when applying surveillance monitoring schemes.