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Gastrointestinal parasites in shelter dogs from Belgrade, Serbia
Veterinary Parasitology: Regional Studies and Reports 7 (2017) 54–57
Contents lists available at ScienceDirect
Veterinary Parasitology: Regional Studies and Reports journal homepage: www.elsevier.com/locate/vprsr
Short Communication
Gastrointestinal parasites in shelter dogs from Belgrade, Serbia Marie Franziska Sommer a,⁎, Nemanja Zdravković b, Ana Vasić b, Felix Grimm c, Cornelia Silaghi a,1 a b c
Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-University, Munich, Germany Faculty of Veterinary Medicine, University of Belgrade, Belgrade, Serbia Institute of Parasitology, University of Zurich, Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 24 May 2016 Accepted 10 January 2017 Available online 12 January 2017 Keywords: MIFC method Coproantigen ELISA Endoparasites Coinfections
a b s t r a c t It is well known that dog shelters are a common source for parasitic infections in different countries worldwide. The present study was conducted in order to determine the prevalence of intestinal parasites and the frequency of polyparasitism in dogs living in two private shelters in Belgrade, Serbia. For this purpose, 134 faecal samples were examined for gastrointestinal parasites with the merthiolate-iodine-formalin concentration (MIFC)-method as well as for Giardia-coproantigen with an enzyme-linked-immunosorbent assay (ELISA). Taeniid eggs were identified by PCR and sequence analysis. Overall, at least one of nine different endoparasites was detected in 75.4% (101/134) of the dogs. Giardia duodenalis coproantigen was found most frequently (45.5%; 61/134), followed by eggs of Ancylostomatidae (41.0%; 55/134), oocysts of Hammondia/Neospora (11.2%; 15/134), eggs of Toxascaris leonina (9.7%; 13/134), oocysts of Isospora canis (8.2%; 11/134), eggs of Trichuris vulpis (6.7%; 9/134), cysts of Sarcocystis spp. (4.5%; 6/134), eggs of Toxocara canis (3.0%; 4/134) and eggs of Taenia spp. (1.5%; 2/ 134). The results of the study confirm a high parasitic burden in the investigated shelter dogs and call for an effective deworming program including an improved hygiene management in the affected facilities. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Shelter dogs living in overcrowded environments under poor zoohygienic conditions are usually exposed to a wide variety of endoparasites (Bugg et al., 1999; Dubná et al., 2007; Tangtrongsup and Scorza, 2010). Dog shelters may serve as parasite reservoirs since the permanent contamination of the facilities by new or untreated dogs leads to a persistent maintenance of endoparasitic infections (Capelli et al., 2003; Martinez-Carrasco et al., 2007). On the contrary, most dogs living in private households suffer less often from infections with endoparasites compared to shelter and street dogs due to regular appropriate antiparasitic treatments and the higher hygienic standard they live in (Martinez-Moreno et al., 2007; Becker et al., 2012). Effective antiparasitic control programmes are required in order to reduce the parasitic burden of shelter dogs (Ortuño et al., 2014; Simonato et al., 2015). Worldwide, shelter dogs have been found infected with numerous endoparasites like Giardia duodenalis, Isospora canis, Hammondia/ Neospora, Sarcocystis spp., Trichuris vulpis, Ascarids, Ancylostomatidae and tapeworms (Bugg et al., 1999; Capelli et al., 2006; Dubná et al., 2007; Martinez-Carrasco et al., 2007; Palmer et al., 2008; Joffe et al., 2011; Ortuño and Castellà, 2011; Simonato et al., 2015). In all these ⁎ Corresponding author at: Leopoldstr. 5, 80802 Munich, Germany. E-mail address: [email protected] (M.F. Sommer). 1 Present address: National Centre for Vector Entomology, Institute of Parasitology, University of Zurich, Zurich, Switzerland.
http://dx.doi.org/10.1016/j.vprsr.2017.01.001 2405-9390/© 2017 Elsevier B.V. All rights reserved.
surveys, 30.0% to over 70.0% of the investigated dogs were infected with at least one of the aforementioned parasites. Information on endoparasitic infections of shelter dogs from South Eastern Europe is limited. For example, data exist for shelter dogs from Romania of which 59.6% were infected with a variety of endoparasites (Sorescu et al., 2014). In comparison to privately owned dogs (50.1%), shelter dogs (73.0%) from rural and urban areas of Hungary were significantly more often infected with gastrointestinal parasites (Fok et al., 2001). Against this background, the aim of the present study was to determine the prevalence of gastrointestinal parasites and the frequency of polyparasitism in shelter dogs from Belgrade, Serbia. Faecal samples were obtained in the framework of a multinational study on multilocus sequence typing of canine Giardia from South Eastern European countries (Sommer et al., 2015). 2. Material and methods In September 2013, 134 faecal samples were collected immediately after defecation from 134 dogs living in two privately owned dog shelters located in the outskirts of Belgrade. All investigated dogs were mixed-breeds older than one year. In the shelters, up to 200 and 400 dogs were accommodated in several kennels separated by fences, respectively. In both shelters, several dogs were kept together in one kennel. The main part of the kennel ground was plain soil and one quarter to one third was covered with concrete. The concrete part of the shelters was cleaned with water at least once a week and excrements on the
M.F. Sommer et al. / Veterinary Parasitology: Regional Studies and Reports 7 (2017) 54–57
other areas of the kennels were removed on a regular basis. All dogs were vaccinated against rabies and were treated orally against helminths with pyrantel and praziquantel once yearly. All 134 faecal samples were investigated with the merthiolate-iodine-formalin concentration (MIFC)-method, which is suitable for all kinds of faecal parasites (Allen and Ridley, 1970; Pfister et al., 2013). In order to screen the samples for the presence of Giardia antigen, the enzyme-linked-immunosorbent assay (ELISA) ProSpecT™ Giardia Microplate assay (Remel, Lenexa, USA) was used according to the manufacturer's instructions. Samples containing taeniid eggs were differentiated by a multiplex PCR (Trachsel et al., 2007). Taenia-positive samples were further analysed by amplifying and sequencing of a fragment of the cox 1 gene (Bowles et al., 1992). The correlation of a Giardia and Ancylostomatidae infection determined by MIFC was evaluated with χ2-test. For parasitic infections occurring less than five times, a Fisher's exact test was performed additionally to determine their correlation (calculation of both tests: http://graphpad.com/quickcalcs/contingency1.cfm). For either test, p values b 0.05 were considered significant. 3. Results At least one gastrointestinal parasitic infection was detected in a total of 101 out of the 134 dogs (75.4%) with MIFC or ELISA. MIFC-positive samples contained developmental stages of different protozoans and eggs of nematodes and tapeworms (Table 1). Cysts of G. duodenalis and eggs of Ancylostomatidae were identified most frequently with 42.5% and 41.0%, respectively. Giardia antigen was detected in four additional samples even though the presence of Giardia cysts could not be confirmed with MIFC. Two samples contained taeniid eggs, which were confirmed to be eggs of Taenia spp. by PCR. The subsequent sequencing of the partial cox 1 gene revealed Taenia hydatigena in one case. No sequencing result was obtained for the second Taenia spp.-positive sample. A total of 41.0% of the investigated dogs were infected with more than one and up to four different parasite species (Table 2). There was no statistically significant correlation of Giardia infection with any of the other parasitic infections (p values from 0.146 to 0.946). The correlation between Ancylostomatidae infection and either Trichuris vulpis, Toxascaris leonina or Sarcocystis spp. was considered statistically significant (p values of 0.0002, 0.002 and 0.042, respectively). The presence of Toxocara canis and Toxascaris leonina eggs correlated significantly (p = 0.047). 4. Discussion The current study confirms the hypothesis of a high parasitic burden in shelter dogs (Martinez-Carrasco et al., 2007; Simonato et al., 2015).
Table 1 Prevalence of intestinal parasites in 134 shelter dogs from Belgrade, Serbia. Parasite
Protozoa
Helminths
a
All dogs (n = 134) Positive (n)
Prevalence (%)
Giardia (ELISA) Giardia (MIFC) Hammondia/Neospora Isospora canis Sarcocystis spp. Ancylostomatidae Toxascaris leonina Trichuris vulpis Toxocara canis Taenia spp.
61 57 15 11 6 55 13 9 4 2
45.5 42.5 11.2 8.2 4.5 41.0 9.7 6.7 3.0 1.5
Totala
101
75.4
Number of dogs, which were infected with at least one parasite.
55
Three quarters (101/134) of the investigated faecal samples contained developmental stages of at least one endoparasite. All nine different endoparasitic species detected in the investigated canine population have already been reported to occur in dogs from Serbia and other South Eastern European countries. A survey on zoonoses associated with dogs revealed the presence of G. duodenalis, Toxocara canis, Trichuris vulpis, Ancylostomatidae or Taenia-type helminths in 75.5% of a mixed population of household, stray and military working dogs from Belgrade, Serbia (Nikolić et al., 2008). In addition to the mentioned parasitic infections, privately owned dogs from Albania and Romania were infected with Isospora canis, Hammondia/Neospora, Sarcocystis spp. or Toxascaris leonina (Amfim et al., 2011; Xhaxhiu et al., 2011; Shukullari et al., 2015). In the investigated shelter dogs, the intestinal protozoan G. duodenalis was detected most often, not only by the indirect coproantigen ELISA (45.5%) but also by the direct MIFC-method (42.5%). Compared to privately owned dogs, Giardia infections are more prevalent in shelter dogs (Huber et al., 2005; Tangtrongsup and Scorza, 2010). Dubná et al. (2007) detected an 11-fold increase of the prevalence for Giardia infections in dogs during the time of their stay at a shelter in the Czech Republic. This result is not surprising considering that the direct life cycle and the short prepatent period of Giardia facilitate a permanent infection cycle through contaminated food, water or hair coats in shelters with a high concentration of dogs (Papini et al., 2005). Faecal forms of Ancylostomatidae had the second highest frequency of occurrence (41.0%) in the present study. Nikolić et al. (2008) asserted that hookworms belong to the most prevalent endoparasites of dogs in Belgrade. In a worldwide context, the prevalence for infections with Ancylostomatidae in comparable canine populations varies remarkably. For instance, 0.8% of shelter dogs were tested positive for hookworm eggs in the Czech Republic (Dubná et al., 2007), 0.9% in Northern Germany (Becker et al., 2012), 9.3% in Italy (Simonato et al., 2015), 26.8% in the Slovak Republic (Szabová et al., 2007), 62.5% in Mexico (Eguia-Aguilar et al., 2005) and 64.9% in Poland (Borecka, 2005). Compared to Giardia and hookworms, faecal forms of other endoparasites were detected considerably less often in the present study. The prevalence rates for infections with Toxascaris leonina (9.7%), Isospora canis (8.2%) and Taenia spp. (1.5%) are similar to previously reported values (Bugg et al., 1999; Martinez-Carrasco et al., 2007; Miro et al., 2007). The low prevalence for tapeworms might be explained by the fact that a single faecal sample per dog was examined. Proglottids of tapeworms are shed inconsistently and eggs are often erratically distributed in the faeces, which might be the reason for a low sensitivity of single coprological examinations (Simonato et al., 2015). However, the finding of two Taenia-positive dogs indicates that that the shelter dogs have access to meat containing cysticercoids. In comparison to other surveys, a higher prevalence for Hammondia/ Neospora-like oocysts (11.2%) and Trichuris vulpis eggs (6.7%) was found in the present study (Bugg et al., 1999; Dubná et al., 2007; Martinez-Carrasco et al., 2007). Even though oocysts of Hammondia and Neospora share several phenotypical characters, those two members of the Sarcocystidae family are morphologically indistinguishable (Mugridge et al., 1999). Neospora is present in Serbia in both dogs as definite hosts (15.4%) and in cows as intermediate hosts (4.6–17.2%) according to previous seroprevalence studies from Vojvodina and South Banat (Gavrilović et al., 2013; Kuruca et al., 2013). Shelter dogs from other countries harboured Toxocara canis (3.0%) more often than the dogs in the present study (Szabová et al., 2007; Simonato et al., 2015). However, prevalence data in the present study especially for ascarids should be evaluated with care since the habit of coprophagia is not unusual in dogs and may induce the presence of ingested immature ascarid eggs (Houpt, 1991). This behaviour might be even boosted by the hygienic situation in shelters with multiple dogs sharing one kennel.
56
M.F. Sommer et al. / Veterinary Parasitology: Regional Studies and Reports 7 (2017) 54–57
Table 2 Distribution of the occurrence of different parasite species in single canine faecal samples. Number of parasite species
Giardia spp.
Four
X
Three
X X X X X
Cystoisospora canis
Hammondia/Neospora Sarcocystis spp. spp.
Ancylostomatidae Toxascaris leonina
X X
X X
X
X X
X
X
X
X X X X
X X X X
X X
X X
X X
X X X X X
One
Taenia spp.
X
X X X X
Trichuris vulpis
X X X
X X X
Two
Toxocara canis
X X X X X X X
X X X X
X
X X X X
None Total
61
11
15
6
Concerning infections with Sarcocystis spp. (4.5%), the prevalence in shelter dog populations varies between 0.6% and 14.0% depending on the study (Bugg et al., 1999; Dubná et al., 2007). The detection of multiple endoparasite species within a single canine faecal sample has previously been described from South Eastern Europe. Similar to the findings in the present study, Nikolić et al. (2008) reported coinfections with up to four different endoparasites in a mixed dog population from Belgrade. In the latter study, the combination of Trichuris vulpis and Ancylostomatidae was most frequent. In the present study, an infection with Ancylostomatidae was correlated not only with the occurrence of Trichuris vulpis but also with Sarcocystis spp. and Toxascaris leonina. An investigation of shelter dogs from Romania revealed a high number of coinfections with Toxocara spp. and Giardia as the most frequent combination (Sorescu et al., 2014). In contrast, the occurrence of Toxocara canis correlated significantly with Toxascaris leonina in the present study. Even though many coinfections with Giardia were detected due to the high prevalence, no significant correlation was found with any other gastrointestinal parasitic infection. The results of the present study call for the implementation of adequate control measures against gastrointestinal parasites in the investigated canine population. The antiparasitic intervention in form of a single treatment per year does not prevent infections with various endoparasites. Palmer et al. (2008) depicted a 50% higher prevalence for helminthic infections in an untreated canine population from Australia compared to dogs treated with anthelmintics. Other surveys have proven that there is a close relation between the occurrence of parasites and shelter management in terms of animal density, hygiene status and deworming strategies (Dubná et al., 2007; Meireles et al., 2008; Simonato et al., 2015). Finally, from the aspect of hygiene and public health, it should be mentioned that potentially zoonotic parasites were detected in the present study, which might also infect humans in contact with infected shelter dogs.
55
13
4
9
2
Number (n) 1 1 1 1 1 3 4 1 2 1 1 1 3 5 14 2 1 3 2 1 5 1 28 1 5 12
2
33
33
16
37
46
134 134
Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements The authors would like to thank Elisabeth Kiess, Kathrin Simon and Claudia Thiel for their excellent assistance in laboratory work. We are also very grateful to Jovan Bojkovski for providing samples. References Allen, A.V., Ridley, D.S., 1970. Further observations on the formol-ether concentration technique for faecal parasites. J. Clin. Pathol. 23, 545–546. Amfim, A., Pârvu, M., Băcescu, B., Simion, V., 2011. Estimation of canine intestinal parasites in Bucharest and their risk to public health. Bulletin UASVM 68. Becker, A.C., Rohen, M., Epe, C., Schnieder, T., 2012. Prevalence of endoparasites in stray and fostered dogs and cats in Northern Germany. Parasitol. Res. 111, 849–857. Borecka, A., 2005. Prevalence of intestinal nematodes of dogs in the Warsaw area, Poland. Helminthologia 41, 35–39. Bowles, J., Blair, D., McManus, D.P., 1992. Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing. Mol. Biochem. Parasitol. 54, 165–173. Bugg, R.J., Robertson, I.D., Elliot, A.D., Thompson, R.C.A., 1999. Gastrointestinal parasites of urban dogs in Perth, Western Australia. Vet. J. 157, 295–301. Capelli, G., Paoletti, B., Iorio, R., Frangipane di Regalbono, A., Pietrobelli, M., Bianciardi, P., Giangaspero, A., 2003. Prevalence of Giardia spp. in dogs and humans in northern and central Italy. Parasitol. Res. 90 (Suppl. 3), 154–155. Capelli, G., Frangipane di Regalbono, A., Iorio, R., Pietrobelli, M., Paoletti, B., Giangaspero, A., 2006. Giardia species and other intestinal parasites in dogs in north-east and central Italy. Vet. Rec. 159, 422–424. Dubná, S., Langrová, I., Nápravník, J., Jankovská, I., Vadlejch, J., Pekár, S., Fechtner, J., 2007. The prevalence of intestinal parasites in dogs from Prague, rural areas, and shelters of the Czech Republic. Vet. Parasitol. 145, 120–128. Eguia-Aguilar, P., Cruz-Reyes, A., Martinez-Maya, J.J., 2005. Ecological analysis and description of the intestinal helminths present in dogs in Mexico City. Vet. Parasitol. 127, 139–146. Fok, E., Szatmari, V., Busak, K., Rozgonyi, F., 2001. Prevalence of intestinal parasites in dogs in some urban and rural areas of Hungary. Vet. Q. 23, 96–98. Gavrilović, P., ŽIivulj, A., Todorović, I., Jovanović, M., Parunović, J., 2013. Investigation of importance of Neospora caninum in aetiology of abortion in dairy cows in Serbia. Revue Méd. Vét. 164, 100–104.
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Contents lists available at ScienceDirect
Veterinary Parasitology: Regional Studies and Reports journal homepage: www.elsevier.com/locate/vprsr
Short Communication
Gastrointestinal parasites in shelter dogs from Belgrade, Serbia Marie Franziska Sommer a,⁎, Nemanja Zdravković b, Ana Vasić b, Felix Grimm c, Cornelia Silaghi a,1 a b c
Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-University, Munich, Germany Faculty of Veterinary Medicine, University of Belgrade, Belgrade, Serbia Institute of Parasitology, University of Zurich, Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 24 May 2016 Accepted 10 January 2017 Available online 12 January 2017 Keywords: MIFC method Coproantigen ELISA Endoparasites Coinfections
a b s t r a c t It is well known that dog shelters are a common source for parasitic infections in different countries worldwide. The present study was conducted in order to determine the prevalence of intestinal parasites and the frequency of polyparasitism in dogs living in two private shelters in Belgrade, Serbia. For this purpose, 134 faecal samples were examined for gastrointestinal parasites with the merthiolate-iodine-formalin concentration (MIFC)-method as well as for Giardia-coproantigen with an enzyme-linked-immunosorbent assay (ELISA). Taeniid eggs were identified by PCR and sequence analysis. Overall, at least one of nine different endoparasites was detected in 75.4% (101/134) of the dogs. Giardia duodenalis coproantigen was found most frequently (45.5%; 61/134), followed by eggs of Ancylostomatidae (41.0%; 55/134), oocysts of Hammondia/Neospora (11.2%; 15/134), eggs of Toxascaris leonina (9.7%; 13/134), oocysts of Isospora canis (8.2%; 11/134), eggs of Trichuris vulpis (6.7%; 9/134), cysts of Sarcocystis spp. (4.5%; 6/134), eggs of Toxocara canis (3.0%; 4/134) and eggs of Taenia spp. (1.5%; 2/ 134). The results of the study confirm a high parasitic burden in the investigated shelter dogs and call for an effective deworming program including an improved hygiene management in the affected facilities. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Shelter dogs living in overcrowded environments under poor zoohygienic conditions are usually exposed to a wide variety of endoparasites (Bugg et al., 1999; Dubná et al., 2007; Tangtrongsup and Scorza, 2010). Dog shelters may serve as parasite reservoirs since the permanent contamination of the facilities by new or untreated dogs leads to a persistent maintenance of endoparasitic infections (Capelli et al., 2003; Martinez-Carrasco et al., 2007). On the contrary, most dogs living in private households suffer less often from infections with endoparasites compared to shelter and street dogs due to regular appropriate antiparasitic treatments and the higher hygienic standard they live in (Martinez-Moreno et al., 2007; Becker et al., 2012). Effective antiparasitic control programmes are required in order to reduce the parasitic burden of shelter dogs (Ortuño et al., 2014; Simonato et al., 2015). Worldwide, shelter dogs have been found infected with numerous endoparasites like Giardia duodenalis, Isospora canis, Hammondia/ Neospora, Sarcocystis spp., Trichuris vulpis, Ascarids, Ancylostomatidae and tapeworms (Bugg et al., 1999; Capelli et al., 2006; Dubná et al., 2007; Martinez-Carrasco et al., 2007; Palmer et al., 2008; Joffe et al., 2011; Ortuño and Castellà, 2011; Simonato et al., 2015). In all these ⁎ Corresponding author at: Leopoldstr. 5, 80802 Munich, Germany. E-mail address: [email protected] (M.F. Sommer). 1 Present address: National Centre for Vector Entomology, Institute of Parasitology, University of Zurich, Zurich, Switzerland.
http://dx.doi.org/10.1016/j.vprsr.2017.01.001 2405-9390/© 2017 Elsevier B.V. All rights reserved.
surveys, 30.0% to over 70.0% of the investigated dogs were infected with at least one of the aforementioned parasites. Information on endoparasitic infections of shelter dogs from South Eastern Europe is limited. For example, data exist for shelter dogs from Romania of which 59.6% were infected with a variety of endoparasites (Sorescu et al., 2014). In comparison to privately owned dogs (50.1%), shelter dogs (73.0%) from rural and urban areas of Hungary were significantly more often infected with gastrointestinal parasites (Fok et al., 2001). Against this background, the aim of the present study was to determine the prevalence of gastrointestinal parasites and the frequency of polyparasitism in shelter dogs from Belgrade, Serbia. Faecal samples were obtained in the framework of a multinational study on multilocus sequence typing of canine Giardia from South Eastern European countries (Sommer et al., 2015). 2. Material and methods In September 2013, 134 faecal samples were collected immediately after defecation from 134 dogs living in two privately owned dog shelters located in the outskirts of Belgrade. All investigated dogs were mixed-breeds older than one year. In the shelters, up to 200 and 400 dogs were accommodated in several kennels separated by fences, respectively. In both shelters, several dogs were kept together in one kennel. The main part of the kennel ground was plain soil and one quarter to one third was covered with concrete. The concrete part of the shelters was cleaned with water at least once a week and excrements on the
M.F. Sommer et al. / Veterinary Parasitology: Regional Studies and Reports 7 (2017) 54–57
other areas of the kennels were removed on a regular basis. All dogs were vaccinated against rabies and were treated orally against helminths with pyrantel and praziquantel once yearly. All 134 faecal samples were investigated with the merthiolate-iodine-formalin concentration (MIFC)-method, which is suitable for all kinds of faecal parasites (Allen and Ridley, 1970; Pfister et al., 2013). In order to screen the samples for the presence of Giardia antigen, the enzyme-linked-immunosorbent assay (ELISA) ProSpecT™ Giardia Microplate assay (Remel, Lenexa, USA) was used according to the manufacturer's instructions. Samples containing taeniid eggs were differentiated by a multiplex PCR (Trachsel et al., 2007). Taenia-positive samples were further analysed by amplifying and sequencing of a fragment of the cox 1 gene (Bowles et al., 1992). The correlation of a Giardia and Ancylostomatidae infection determined by MIFC was evaluated with χ2-test. For parasitic infections occurring less than five times, a Fisher's exact test was performed additionally to determine their correlation (calculation of both tests: http://graphpad.com/quickcalcs/contingency1.cfm). For either test, p values b 0.05 were considered significant. 3. Results At least one gastrointestinal parasitic infection was detected in a total of 101 out of the 134 dogs (75.4%) with MIFC or ELISA. MIFC-positive samples contained developmental stages of different protozoans and eggs of nematodes and tapeworms (Table 1). Cysts of G. duodenalis and eggs of Ancylostomatidae were identified most frequently with 42.5% and 41.0%, respectively. Giardia antigen was detected in four additional samples even though the presence of Giardia cysts could not be confirmed with MIFC. Two samples contained taeniid eggs, which were confirmed to be eggs of Taenia spp. by PCR. The subsequent sequencing of the partial cox 1 gene revealed Taenia hydatigena in one case. No sequencing result was obtained for the second Taenia spp.-positive sample. A total of 41.0% of the investigated dogs were infected with more than one and up to four different parasite species (Table 2). There was no statistically significant correlation of Giardia infection with any of the other parasitic infections (p values from 0.146 to 0.946). The correlation between Ancylostomatidae infection and either Trichuris vulpis, Toxascaris leonina or Sarcocystis spp. was considered statistically significant (p values of 0.0002, 0.002 and 0.042, respectively). The presence of Toxocara canis and Toxascaris leonina eggs correlated significantly (p = 0.047). 4. Discussion The current study confirms the hypothesis of a high parasitic burden in shelter dogs (Martinez-Carrasco et al., 2007; Simonato et al., 2015).
Table 1 Prevalence of intestinal parasites in 134 shelter dogs from Belgrade, Serbia. Parasite
Protozoa
Helminths
a
All dogs (n = 134) Positive (n)
Prevalence (%)
Giardia (ELISA) Giardia (MIFC) Hammondia/Neospora Isospora canis Sarcocystis spp. Ancylostomatidae Toxascaris leonina Trichuris vulpis Toxocara canis Taenia spp.
61 57 15 11 6 55 13 9 4 2
45.5 42.5 11.2 8.2 4.5 41.0 9.7 6.7 3.0 1.5
Totala
101
75.4
Number of dogs, which were infected with at least one parasite.
55
Three quarters (101/134) of the investigated faecal samples contained developmental stages of at least one endoparasite. All nine different endoparasitic species detected in the investigated canine population have already been reported to occur in dogs from Serbia and other South Eastern European countries. A survey on zoonoses associated with dogs revealed the presence of G. duodenalis, Toxocara canis, Trichuris vulpis, Ancylostomatidae or Taenia-type helminths in 75.5% of a mixed population of household, stray and military working dogs from Belgrade, Serbia (Nikolić et al., 2008). In addition to the mentioned parasitic infections, privately owned dogs from Albania and Romania were infected with Isospora canis, Hammondia/Neospora, Sarcocystis spp. or Toxascaris leonina (Amfim et al., 2011; Xhaxhiu et al., 2011; Shukullari et al., 2015). In the investigated shelter dogs, the intestinal protozoan G. duodenalis was detected most often, not only by the indirect coproantigen ELISA (45.5%) but also by the direct MIFC-method (42.5%). Compared to privately owned dogs, Giardia infections are more prevalent in shelter dogs (Huber et al., 2005; Tangtrongsup and Scorza, 2010). Dubná et al. (2007) detected an 11-fold increase of the prevalence for Giardia infections in dogs during the time of their stay at a shelter in the Czech Republic. This result is not surprising considering that the direct life cycle and the short prepatent period of Giardia facilitate a permanent infection cycle through contaminated food, water or hair coats in shelters with a high concentration of dogs (Papini et al., 2005). Faecal forms of Ancylostomatidae had the second highest frequency of occurrence (41.0%) in the present study. Nikolić et al. (2008) asserted that hookworms belong to the most prevalent endoparasites of dogs in Belgrade. In a worldwide context, the prevalence for infections with Ancylostomatidae in comparable canine populations varies remarkably. For instance, 0.8% of shelter dogs were tested positive for hookworm eggs in the Czech Republic (Dubná et al., 2007), 0.9% in Northern Germany (Becker et al., 2012), 9.3% in Italy (Simonato et al., 2015), 26.8% in the Slovak Republic (Szabová et al., 2007), 62.5% in Mexico (Eguia-Aguilar et al., 2005) and 64.9% in Poland (Borecka, 2005). Compared to Giardia and hookworms, faecal forms of other endoparasites were detected considerably less often in the present study. The prevalence rates for infections with Toxascaris leonina (9.7%), Isospora canis (8.2%) and Taenia spp. (1.5%) are similar to previously reported values (Bugg et al., 1999; Martinez-Carrasco et al., 2007; Miro et al., 2007). The low prevalence for tapeworms might be explained by the fact that a single faecal sample per dog was examined. Proglottids of tapeworms are shed inconsistently and eggs are often erratically distributed in the faeces, which might be the reason for a low sensitivity of single coprological examinations (Simonato et al., 2015). However, the finding of two Taenia-positive dogs indicates that that the shelter dogs have access to meat containing cysticercoids. In comparison to other surveys, a higher prevalence for Hammondia/ Neospora-like oocysts (11.2%) and Trichuris vulpis eggs (6.7%) was found in the present study (Bugg et al., 1999; Dubná et al., 2007; Martinez-Carrasco et al., 2007). Even though oocysts of Hammondia and Neospora share several phenotypical characters, those two members of the Sarcocystidae family are morphologically indistinguishable (Mugridge et al., 1999). Neospora is present in Serbia in both dogs as definite hosts (15.4%) and in cows as intermediate hosts (4.6–17.2%) according to previous seroprevalence studies from Vojvodina and South Banat (Gavrilović et al., 2013; Kuruca et al., 2013). Shelter dogs from other countries harboured Toxocara canis (3.0%) more often than the dogs in the present study (Szabová et al., 2007; Simonato et al., 2015). However, prevalence data in the present study especially for ascarids should be evaluated with care since the habit of coprophagia is not unusual in dogs and may induce the presence of ingested immature ascarid eggs (Houpt, 1991). This behaviour might be even boosted by the hygienic situation in shelters with multiple dogs sharing one kennel.
56
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Table 2 Distribution of the occurrence of different parasite species in single canine faecal samples. Number of parasite species
Giardia spp.
Four
X
Three
X X X X X
Cystoisospora canis
Hammondia/Neospora Sarcocystis spp. spp.
Ancylostomatidae Toxascaris leonina
X X
X X
X
X X
X
X
X
X X X X
X X X X
X X
X X
X X
X X X X X
One
Taenia spp.
X
X X X X
Trichuris vulpis
X X X
X X X
Two
Toxocara canis
X X X X X X X
X X X X
X
X X X X
None Total
61
11
15
6
Concerning infections with Sarcocystis spp. (4.5%), the prevalence in shelter dog populations varies between 0.6% and 14.0% depending on the study (Bugg et al., 1999; Dubná et al., 2007). The detection of multiple endoparasite species within a single canine faecal sample has previously been described from South Eastern Europe. Similar to the findings in the present study, Nikolić et al. (2008) reported coinfections with up to four different endoparasites in a mixed dog population from Belgrade. In the latter study, the combination of Trichuris vulpis and Ancylostomatidae was most frequent. In the present study, an infection with Ancylostomatidae was correlated not only with the occurrence of Trichuris vulpis but also with Sarcocystis spp. and Toxascaris leonina. An investigation of shelter dogs from Romania revealed a high number of coinfections with Toxocara spp. and Giardia as the most frequent combination (Sorescu et al., 2014). In contrast, the occurrence of Toxocara canis correlated significantly with Toxascaris leonina in the present study. Even though many coinfections with Giardia were detected due to the high prevalence, no significant correlation was found with any other gastrointestinal parasitic infection. The results of the present study call for the implementation of adequate control measures against gastrointestinal parasites in the investigated canine population. The antiparasitic intervention in form of a single treatment per year does not prevent infections with various endoparasites. Palmer et al. (2008) depicted a 50% higher prevalence for helminthic infections in an untreated canine population from Australia compared to dogs treated with anthelmintics. Other surveys have proven that there is a close relation between the occurrence of parasites and shelter management in terms of animal density, hygiene status and deworming strategies (Dubná et al., 2007; Meireles et al., 2008; Simonato et al., 2015). Finally, from the aspect of hygiene and public health, it should be mentioned that potentially zoonotic parasites were detected in the present study, which might also infect humans in contact with infected shelter dogs.
55
13
4
9
2
Number (n) 1 1 1 1 1 3 4 1 2 1 1 1 3 5 14 2 1 3 2 1 5 1 28 1 5 12
2
33
33
16
37
46
134 134
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