WILD CANIDS AS HOSTS FOR TICKS AND TICK-BORNE ZOONOTIC PATHOGENS IN SERBIA

Wild canids as hosts for ticks and tick-borne zoonotic pathogens in Abstract Wild canids are recognized as important reservoir hosts for several vector-borne pathogens and are a good sentinel species for epidemiological surveillance. Identifying the reservoir hosts for particular zoonotic pathogens in a specific area enables the implementation of adequate measures in order to prevent the occurrence of zoonotic diseases in humans and domestic animals. The present review aims to summarize the role of wild canids in the epidemiology of tick-borne zoonoses in Serbia based on previously published papers and to discuss the available data from an epidemiological point of view. The results of previously published studies indicate that wild canids are potential reservoir hosts for several zoonotic tick-borne pathogens in Serbia, and hosts for several tick species which are proven competent vectors. Based on molecular studies conducted so far, the presence of Anaplasma phagocytophilum , was confirmed in jackals while the presence of Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto, Borrelia garinii , and Borrelia lusitaniae was proven by PCR in analyzed red foxes. There is a need for continuous epidemiological monitoring of diseases caused by pathogens previously confirmed in wild canids. Also, further research is needed to reveal the role of other wild and domestic animals in the epidemiology of tick-borne zoonoses in Serbia.


INTRODUCTION
In recent decades, vector-borne zoonoses (VBZ) have attracted considerable attention from the scientific community. Due to global climate changes, species that can serve as vectors of infectious agents with zoonotic potential (ticks, sand flies, and mosquitoes) are spreading their range year-to-year. Consequently, some VBZ occur nowadays in regions where they were not registered earlier or they occur more frequently in areas where they occurred sporadically (Dantas-Torres, 2015;Negev et al., 2015). On the other hand, shifts in population dynamics of some vertebrate species that are potential VBZ reservoirs necessitate constant epizootiological surveillance to prevent increasing risks of VBZ for human and animal health.
Wild canids are recognized as important reservoir hosts for several vector-borne pathogens and are good sentinel species for epidemiological surveillance (Tomassone et al., 2018;Aguirre, 2009), while previous faunistic studies also confirm that more tick species now parasitize wild canids in Europe (Gherman and Mihalca, 2017;Tomanović et al., 2013b).
Within the order Carnivora (lat. carne = flesh and vorare = to devour) are more than 280 species of placental mammals, mainly predators, while family Canidae is among the oldest representatives of the order Carnivora. Five wild canid species are autochthonous in Europe: golden jackal (Canis aureus), red fox (Vulpes vulpes), grey wolf (Canis lupus), raccoon dog (Nyctereutes procyonoides) and arctic fox (Alopex lagopus). All but the last are present in Serbia. Due to their abundance and presence over a wide area of the country, jackals and foxes have the greatest epidemiological significance among wild canids in Serbia. Despite the increase in population and the current expansion of the golden jackals' area in southeastern Europe, as well as the animals' tendency to come close to the settlements (Trouwborst et al., 2015), only a few studies so far have addressed the role of jackals in the maintenance and spread of zoonotic agents. Also, although red foxes are the most abundant medium-sized predators and range throughout Serbia (Ćirović, 2000) the role of foxes in enzootic cycles of the vectorborne zoonotic pathogens in our country is still poorly understood.

Role of jackals and foxes in the epidemiology of tick-borne zoonoses in Serbia
The epidemiology of tick-borne diseases is complex since several factors are involved in their maintenance in the environment. The most important factors are: the biology of the microorganism itself, vectors, hosts, and environmental factors.
In addition to the epizootiological role that jackals and foxes have as potential reservoir hosts for tick-borne pathogens, these wild canids could also be responsible for the mechanical transmission and geographic expansion of specific tick species. Since jackals and foxes live near human settlements, it is likely that some of the life stages of the important tick species in Serbia parasitize domestic animals or humans, including I. ricinus, the most common anthropophilic species. These factors delineate the importance of jackals and foxes in enzootic cycles of tick-borne pathogens.
To reveal the exact roles of some wild canid species in enzootic cycles of vectorborne zoonotic pathogens, molecular methods are of great importance. Even though the confirmed presence of a particular pathogen by PCR assay in wild canid tissue is not valid evidence of that same species being a reservoir, this approach gives valuable information that can help elucidate the complete enzootic cycle of the tickborne pathogen in the investigated location. Research based on molecular methods conducted in Serbia identified jackals as potential reservoirs for A. phagocytophilum, while the presence of Candidatus Neoehrlichia micurensis (FU98), Borrelia burgdorferi sensu stricto (s.s.), B. garinii, and B. lusitaniae were confirmed in analyzed red foxes. Still, the epidemiology of VBZ caused by these agents (anaplasmosis, neoehrlichiosis, Lyme borreliosis) is not completely elucidated in Serbia.
A. phagocytophilum is an emerging tick-borne pathogen with a global distribution that infects humans and a wide range of domestic and wild animals. The main vector of A. phagocytophilum in Europe is I. ricinus, while wild animals are considered as important reservoirs of different strains of A. phagocytophilum. The role of certain wildlife species as potential reservoirs is still unknown and justifies further epidemiological studies (Stuen et al., 2013).
Data on the role of wild canids in the maintenance of A. phagocytophilum are scarce. The presence of DNA of this zoonotic pathogen was confirmed in foxes from Poland (2.7%), Czech Republic (4.0%), and Italy (16.6%), indicating the importance of foxes in the sylvatic cycle of A. phagocytophilum (Ebani et al., 2011;Karbowiak et al., 2009;Hulinska et al., 2004). One case of acute granulocyte anaplasmosis in a captive wolf in Austria has also been described (Leschnik et al., 2012).
The eco-epidemiology of A. phagocytophilum in Serbia is largely unknown and insufficiently researched. The presence of A. phagocytophilum was confirmed by molecular methods in I. ricinus ticks collected from vegetation at several localities in central Serbia, Vojvodina, and the Belgrade area, with average prevalence of 13.9% . No cases of human granulocytic anaplasmosis (HGA), a disease caused in humans by A. phagocytophilum, have been officially confirmed in Serbia (Dumler, 2005). So far, only two cases of human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis belonging to the Anaplasmataceae family have been described (Arsić et al., 2014;Đokić et al., 2006). There is a possibility that HGA occurs as a febrile illness of unclear etiology, but remains undiagnosed due to its nonspecific symptoms.
Serological studies conducted in dogs in Serbia confirmed they are exposed to A. phagocytophilum. Using the immunofluorescence antibody test (IFAT), a seroprevalence of 15.5% was recorded in analyzed dogs from Vojvodina (Potkonjak et al., 2015), while dogs from Belgrade had a seroprevalence of 26.1% (Kovačević Filipović et al., 2018). Although previous studies indicate that dogs are exposed to the causative agent of canine anaplasmosis, there have been no confirmed cases of this disease in Serbia to date. A recent study conducted in dairy cows confirmed the presence of antibodies to A. phagocytophilum in 2.45% of the tested animals from the areas of Gruža and Požarevac (Vasić et al., 2018).
For the first time in Europe, a recently conducted study in Serbia has indicated to jackals as potential reservoirs of A. phagocytophilum. Two animals (0.9%) originating from the locality Smederevo and Velika Plana were positive for A. phagocytophilum DNA (Sukara et al., 2018). This finding and the fact that jackals inhabit around 70% of the country (Ćirović et al., 2018) together with the confirmed presence of I. ricinus as the main vector of A. phagocytophilum on jackals (Sukara et al., 2018), give us a basis to conclude that jackals have a potential role in the maintenance of the causative agent of human and animal anaplasmosis in Serbia. Further research is needed on jackals and other wild canids (foxes and wolves) to elucidate the role of these animals in the epidemiology of A. phagocytophilum.
A recent study on the role of foxes in the epidemiology of VBZ in Serbia, has confirmed the presence of a newly discovered member of the family Anaplasmataceae, Candidatus Neoehrlichia sp., sequence type FU98, and indicated foxes could be potential reservoir hosts for the causative agents of Lyme borreliosis, the spirochetes Borrelia burgdorferi s.l. .

Neoehrlichia mikurensis, an emerging tick-borne zoonotic pathogen, and Candidatus
Neoehrlichia lotoris (non-zoonotic) are currently proposed to be members of the family Anaplasmataceae. Candidatus Neoehrlichia sp. FU98, which is distinct from N. mikurensis, but more closely related to Candidatus Neoehrlichia lotoris, was confirmed for the first time in foxes from Austria (Hodžić et al., 2015), then in Czech Republic (Hodžić et al., 2017), and recently in Serbia, where an overall prevalence of 4.7% was recorded . Positive foxes originated from three localities: Svilajnac, Veliko Gradište, and Surčin.
The potential reservoir role of other wild animals in Europe has been proposed after the confirmed presence of Candidatus Neoehrlichia sp. FU98 in raccoon dogs and badgers (Hildebrand et al., 2018;Hornok et al., 2017b). More recently, a strain similar to Candidatus Neoehrlichia sp. FU98 was reported in domestic dogs from Hungary (Hornok et al., 2018). Further research is needed to investigate the pathogenic and zoonotic potential of this zoonotic pathogen and the exact role of foxes and other animals in the epizootiology of Candidatus Neoehrlichia sp. FU98.
Maintenance of the causative agents of Lyme borreliosis in the environment occurs in enzoonotic cycles that include vertebrates and ticks. In Europe, I. ricinus is the main vector, but the roles of many vertebrates as potential reservoir hosts are still unknown (Franke et al., 2013). Previous seroepidemiological studies in Serbia confirmed that domestic dogs are exposed to B. burgdorferi s.l (Obrenović et al., 2015;Potkonjak et al., 2013), while a high diversity of Borrelia species (B. burgdorferi s.s., Borrelia afzelii, B. lusitaniae, B. garinii, Borrelia bavariensis, and Borrelia valaisiana) were confirmed in I. ricinus ticks collected from vegetation (Ćakić et al., 2019;Tomanović et al., 2008).
Although it has been previously confirmed that foxes are competent reservoirs for B. burgdorferi s.l. (Liebisch et al., 1995), only a few studies to date have addressed the role of red foxes in the epizootiology of Lyme borreliosis in Europe (Dumitrache et al., 2015;Heidrich et al., 1999). Recently, for the first time, the potential role of red foxes as reservoir hosts for B. burgdorferi s.s., Borrelia g (proven causative agents of Lyme borreliosis), and B. lusitaniae was proposed in Serbia. The total prevalence of these zoonotic pathogens in red foxes was 5.4%, recorded by molecular methods . So far there is no available data on the role of other wild animals in the epidemiology of Lyme disease in our country. Future research should be directed towards identifying the exact role of foxes and other vertebrate hosts in the maintenance of Lyme borreliosis agents in Serbia.

CONCLUSION
Due to increased contact between wildlife, domestic animals, and humans worldwide, and the fact that these interactions in the future will be more frequent, there is a need for constant epidemiological surveillance of VBZ in wildlife populations. Identification of the reservoir hosts in a specific area is a prerequisite for the implementation of adequate measures to prevent the occurrence of zoonotic diseases in domestic animals and humans. The results of previous studies conducted in Serbia confirmed that jackals and red foxes are good hosts for several tick species and are potential reservoir hosts for several tick-borne zoonotic pathogens. Further research is needed to discover the exact role of wild canids and other wild and domestic animals in the epidemiology of VBZ in Serbia.