Preliminary study on the prevalence of endoparasite infections and vector-borne diseases in outdoor dogs in Bulgaria

Dogs are still the most common companion animals establishing more frequent and closer contact with humans than any other pets. Furthermore, various canine parasites are involved in the epidemiology of many parasitic diseases affecting a wide range of domestic and wild herbivorous and omnivorous. Also, the canids act as reservoirs and sources of several zoonotic parasites posing a serious threat to the human health (Xhaxhiu et al., 2011). The children are generally at higher risk of acquiring infections with some parasites (e.g. Toxocara canis) than adults due to the habit of placing their fingers in the mouth after playing with dogs or after contact with contaminated soil. In contrast, human hydatid disease is mainly associated with persons, practicing hunting and sheep farming. This dangerous and life-threatening infection is caused by the larval stage of Echinococcus granulosus, a cestode, inhabiting the small intestines of various canids as final hosts (Gillespie & Bradbury, 2017).

The prevalence of parasitic infections in dogs depends on several factors such as the lifestyle, deworming frequency, climate conditions, and contacts with stray dogs or wildlife (Roussel et al., 2019). Additionally, some owners neglect the prophylaxis of their dogs resulting in contamination of environment with infective parasite stages of high tenacity. Also, the shepherd dogs often have no prophylactic therapy, and the occurrence and intensity of parasite infections, especially with tapeworms, usually results from scavenging sheep carcasses (Vasileiou et al., 2015; Rehbein et al., 2016).

Canine parasitic fauna has been a purpose of many studies conducted all around the world over the last few decades. Numerous Bulgarian investigations on the same focus have also been carried out, including different categories of dogs, originating from many regions of the country. In addition, gastrointestinal (GI) parasitism of Trichuris vulpis and Capillaria sp., hookworms (Ancylostoma caninum, Uncinaria stenocephala), ascarids (T. canis, Toxascaris leonina), cestodes (Dipylidium caninum, Mesocestoides lineatus, Taenia hydatigena, Taenia ovis, Taenia multiceps, Taenia pisiformis, E. granulosus), protozoans (Cystoisospora sp., Sarcocystis sp., Giardia sp.) has been recorded in studies based on necropsy (Kamenov et al., 2009); combined necropsy/coproscopy (Georgieva et al., 1999; Lalkovski & Sabev, 2009) or coproscopy (Kirkova et al., 2006; Kirkova et al., 2013; Kanchev et al., 2014; Radev et al., 2016; Iliev et al., 2017).

Canine vector-borne diseases (CVBDs) constitute an important group of illness caused by a diverse range of pathogens, which are transmitted to both animals and humans by different blood-sucking arthropods (Dantas-Torres, 2008). Those diseases are usually endemic in tropics and subtropics, but an increasing prevalence has also been recognized in temperate regions (Dantas-Torres, 2008; Beugnet & Chalvet-Monfray, 2013). The prevalence of CVBDs in Bulgaria has been frequently reported over the past few years, including infections with Dirofilaria immitis, Dirofilaria repens, Ehrlichia canis, Anaplasma sp., and Borrelia burgdorferi (Georgieva et al., 2001; Tsachev et al., 2006; Tsachev et al., 2006a; Tsachev et al., 2008; Panayotova-Pencheva et al., 2016; Radev et al., 2016; Iliev et al., 2017). Considering the data mentioned above as well as the trend towards increase in canine population in the country focused our attention on performing this epidemiological work. We aimed both at corroborating previously published findings and providing updated information on the prevalence of GI parasites as well as some arthropod-transmitted pathogens in outdoor dogs in Bulgaria.

Аpprovals for using animals in the current study were obtained from the Bulgarian Food Safety Agency (registration of the permits: №85/09.01.2014 and №138/28.06.2016). The research has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

The overall prevalence of GI parasites was 64.5 %. Eggs of hookworms were the most commonly observed in feces (54.1 %), followed by T. vulpis (15.1 %), Capillaria sp. (11.0 %), T. canis (6.4 %), Cystoisospora sp. (4.1 %), Sarcocystis sp. (2.3 %), T. leonina (1.7 %) and Taenia sp. (1.2 %) (Table 1). Eggs of the nasal pentastomid Linguatula serrata were also detected (0.6 %). Cases of single infection were found in 38.4 % of dogs sampled (Table 2). Mixed infections with two or more species were observed in 20.3 % and 18.0 % of dogs, respectively (Tables 3). The most frequently detected co-infections were with Capillaria sp./hookworms (4.1 %) and T. vulpis/hookworms (2.9 %).

The serum analysis identified antigens of D. immitis and A. vasorum (10.5 % and 0.6 % of the samples, respectively) and antibodies against Anaplasma sp. (13.4 %), Ehrlichia sp. (13.4 %) and B. burgdorferi (1.7 %). Dirofilaria repens microfilariae were found in 5.8 % of the dogs (Table 1).

The age was identified as a risk factor for D. immitis infection (χ²=4.358). The highest prevalence was observed in dogs above 12 months of age and no case was recognized in younger dogs. Similarly, antibodies against Ehrlichia sp. were more commonly detected in the adult animals (χ²=3.740). Statistical analysis showed no significant association between the gender and prevalence of infections.

The findings of this study showed that more than half of the dogs (64.5 %) were infected by at least one species of GI parasite.

Numerous recent surveys performed on the Balkans have disclosed comparable results indicating the presence of GI parasitism in 67.1 % of dogs in Romania (Ursache et al., 2016), 75.4 % in Serbia (Sommer et al., 2017), between 48.1 % and 64.9 % in Croatia (Brezak et al., 2017), 26 % in Greece (Papazahariadou et al., 2007), 30.4 % in Turkey (Senlik et al., 2006) and 40.7 % in Albania (Shukullari et al., 2015). All the mentioned results were obtained by coprological exaninations. According to the regional reports, between 24.8 % and 65 % of dogs in Bulgaria harbor several species of endoparasites including ascarids, hookworms, whipworms, tapeworms and coccidians (Kirkova et al., 2006; Lalkovski & Sabev, 2009; Kirkova et al., 2013; Radev et al., 2016), which seems to be in general agreement with our finding. The differences between aforementioned results are not unexpected and could be due to various factors. Those investigations include either well- or not well-cared dogs of different ages and categories (e.g. hunting, shepherd, military, pet, shelter and stray dogs) with different deworming frequency (e.g. regular or not), clinically healthy or under veterinary care (with GI disorders). Taking into consideration the above, it can be argued that the combined influence of the age and purpose of dogs, the general hygiene, and the access to regular deworming may exert a marked effect on the prevalence and species variety of GI parasites among the canine population (Shukullari et al., 2015). In addition, the strong influence of the geographic location and climate conditions is confirmed by the results of a recent study, demonstrating lower prevalence of GI parasitic infections (9.4 %) even in stray and not well-cared dogs in Germany (Becker et al., 2012).

In this study, hookworms were recognized as the most common enteric pathogens, which were found in the fecal samples of more than half of dogs (54.1 %) In similar investigation conducted in Stara Zagora (Bulgaria), the prevalence of A. caninum and U. stenocephala in stray dogs was even higher, reaching 90 % and 60 %, respectively (Georgieva et al., 1999). Other local investigations have indicated that between 6.18 % and 37.8 % of the dogs harbor hookworms (Kirkova et al., 2013; Iliev et al., 2017). Those findings were expected, taking into consideration the modes of infection transmission. Also, it should not be underestimated the zoonotic potential of hookworms. Once entered the body, the infective larvae of A. caninum may induce two serious conditions known as human gut disease (eosinophilic enteritis) and cutaneous larva migrans (CLM) or creeping eruption (Katagiri & Oliveira-Sequeira, 2008). The relationship between U. stenocephala and CLM remains unclear and still debated (Villeneuve et al., 2015).

The canine whipworm, T. vulpis, was the second most frequent GI parasite detected in this study (15.1 %). Our results are in general agreement with those of comparable surveys in dogs in Bulgaria (Kirkova et al., 2006; Lalkovski & Sabev, 2009; Kirkova et al., 2013; Radev et al., 2016; Iliev et al., 2017).

The overall number of dogs infected with T. canis and T. leonina in this study was substantially low (6.4 % and 1.7 %, respectively). As observed in similar Bulgarian investigations, the prevalence of those ascarids ranged from 6.8 % to 17.8 % for T. canis and from 0.78 % to 3.1 % for T. leonina (Kirkova et al., 2006; Kirkova et al., 2013; Kanchev et al., 2014; Iliev et al., 2017). More importantly, T. canis is better recognized as the most common causative agent of visceral and ocular larva migrans in people; both syndromes might lead to severe damages of different tissues, especially in children (Villeneuve et al., 2015).

The results of our study showed that 1.2 % of dogs are infected with Taenia sp., which is in general agreement with findings of Kirkova et al. (2006) and Iliev et al. (2017). Those authors also reported a substantially low prevalence of taeniid infections (0.8-1.16 %) in dogs in Bulgaria.

Numerous studies regarding to the prevalence of aforementioned helminth species have been conducted in closer geographical regions, including Croatia (Brezak et al., 2017); Romania (Ursache et al., 2016), Serbia (Sommer et al., 2017); Greece (Papazahariadou et al., 2007); Albania (Shukullari et al., 2015) and Turkey (Senlik et al., 2006). Summarized results indicate the presence of GI parasitism ranging from 1.2 % to 41 % for hookworms, from 2.9 % to 9.6 % for T. vulpis, from 3 % to 34.8 % for T. canis, from 0.7 % to 21.8 % for T. leonina and from 0.3 % to 1.5 % for Taenia sp.

Our results showed Capillaria sp. infection, which is probably caused by the capillariid lungworm Eucoleus aerophilus. We detected eggs of such helminth in 11 % of the fecal samples. That value is very likely to be lower than real percentage because of the eggs may originate not only from adult lungworms, but also after passing through the alimentary tract following ingestion of contaminated food or after coprophagy (Shukullari et al., 2015). The prevalence of E. aerophilus ranges from 0.2 % to 2.8 % in European and Balkan countries (Traversa et al., 2010; Shukullari et al., 2015; Ursache et al., 2016; Brezak et al., 2017) and from 2 % to 6.8 % in Bulgaria (Kirkova et al., 2006; Kirkova et al., 2013). One case of nasal linguatulosis due to the pentastomid L. serrata was recorded here. The adult parasites reside in nasal cavities in dogs but the eggs pass from the respiratory system to intestines and release into the environment through feces. The total prevalence of L. serrata in dogs in Bulgaria reaches to 0.7 % (Kirkova et al., 2013). However, nymphs of this parasite (known as Pentastomum denticulatum) have been recovered from the lungs, liver and mesenteric lymph nodes in Bulgarian goats as intermediate hosts (Ivanov et al., 2012). This pentastomid is considered responsible for important zoonotic disease. Furthermore, visceral linguatulosis (pentastomosis) in Bulgaria was reported in a 9-year-old boy in Pleven (Mateva et al., 2013).

Our investigation presents the first serologically proven case of angiostrongylosis due to the cardiopulmonary nematode A. vasorum. In contrast, several studies have shown such infection in canids in the European countries (Traversa et al., 2010; Elsheikha et al., 2014) and on the Balkans (Papazahariadou et al., 2007; Shukullari et al. 2015; Ilie et al., 2016).

Protozoa infections were less often identified in this study than helminth infections (4.1 % for Cystoisospora sp. and 2.3 % for Sarcocystis sp.). The prevalence of GI protozoa in dogs in Bulgaria varies from 1.97 % to 7.72 % for Cystoisospora sp. and from 0.39 % to 0.5 % for Sarcocystis sp. (Kirkova et al., 2006; Lalkovski & Sabev, 2009; Radev et al., 2016; Iliev et al., 2017). Our findings are in general agreement with those reported in closer geographical region (Croatia, Romania, Albania, Serbia, Greece), where the overall prevalence ranges from 3 % to 16.1 % for Cystoisospora sp. and from 0.3 % to 4.5 % for Sarcocystis sp. (Papazahariadou et al., 2007; Shukullari et al. 2015; Ursache et al., 2016; Brezak et al., 2017; Sommer et al., 2017).

Almost half of the dogs (44.8 %) were positive for vector-borne parasites and bacteria such as D. immitis, D. repens, Ehrlichia sp., Anaplasma sp. and B. burgdorferi. The prevalence of those pathogens usually depends on several factors, but the age of dogs is considered as an important parameter as we found for D. immitis and Ehrlichia sp. in current study. Our findings showed that 10.5 % and 5.8 % of dogs were infected with D. immitis and D. repens, respectively. According to other Bulgarian researchers, between 7.4 % and 16.2 % of clinically healthy dogs and 34.33 % of dogs with cardiopulmonary disorders and under veterinary care are infected with D. immitis (Georgieva et al., 1999; Georgieva et al., 2001; Pantchev et al., 2015; Radev et al., 2016; Iliev et al., 2017). The prevalence of canine heartworm disease due to D. immitis is also reported on the Balkans and ranges from 0.7 % to 17.9 % in Greece, from 7.2 % to 22.01 % in Serbia, from 1 % to 27 % in Turkey and from 8 % to 16 % in Croatia (Morchon et al., 2012).

Our results also showed infections with Anaplasma sp. (13.4 %), Ehrlichia sp. (13.4 %) and B. burgdorferi (1.7 %). Data of several researches regarding the overall seroprevalence of those tickborne pathogens among canine population in Bulgaria have been previously reported, indicating occurrence of infections ranging from 21 % to 37.5 % for E. canis, from 3.5 % to 46.1 % for A. phagocytophilum (Tsachev, 2006; Tsachev et al., 2006; Tsachev et al., 2006a; Pantchev et al., 2015) and from 22.74 % to 74.5 % for B. burgdorferi (Angelov et al., 1993; Zarkov & Marinov, 2003). Those findings are much higher and are not in agreement with our results. One reason could be that the authors present data obtained from dogs originated from enzootic regions or under veterinary care. However, our results are similar with the findings published by Pantchev et al. (2015) who also found a low seroprevalence (2.4 %) of B. burgdorferi among dogs in Bulgaria.

The results obtained here indicated that elder dogs (irrespective of sex) were commonly affected by D. immitis and Ehrlichia sp. Similar findings have also been published earlier (Villeneuve et al., 2011; Volgina et al., 2013; Hamel et al., 2016; Pantchev et al., 2015). Other authors have reported that elder dogs, kept outdoor, were more commonly infected by D. immitis (Yildirim et al. 2007; Cardoso et al. 2012; Mircean et al. 2012). According to Hamel et al. (2016), the occurrence of CVBDs is significantly higher in dogs over one year of age. The gender of animals included in our work was not considered as a risk factor for Ehrlichia sp. and Anaplasma sp., which coincides with findings reported from other authors (Solano-Gallego et al., 2006; Tsachev et al., 2006; Tsachev et al., 2006a; Villeneuve et al., 2011; Miro et al., 2013).

This study presents an overview of the prevalence of GI parasites and some vector-borne helminths and bacteria in outdoor dogs in Bulgaria as well as the first report of A. vasorum. Our findings demonstrate a wide variety of endoparasites and high prevalence rates of parasitism, suggesting environmental contamination with infective stages of parasites and presence of arthropods carrying different pathogens. Therefore, both the arthropods and infected dogs could be responsible for occurrence of several zoonotic diseases; particularly T. canis, Taenia sp. (refers to E. granulosus), B. burgdorferi and hookworms. This statement thus should increase the efforts of veterinarians and owners on performing a regular and proper prophylaxis of dogs against ecto and endoparasites resulting in lower levels of parasitism in both animals and humans.