The Prevalence of Endoparasites in Slovakian Household Dogs and Cats

Júlia Šmigová Institute of Parasitology of the Slovak Academy of Sciences: Parazitologicky ustav Slovenskej akademie vied Ingrid Papajová (  papaj@saske.sk ) Institute of Parasitology SAS https://orcid.org/0000-0002-2701-6905 Jindřich Šoltys Institute of Parasitology of the Slovak Academy of Sciences: Parazitologicky ustav Slovenskej akademie vied Jana Pipiková Institute of Chemistry Slovak Academy of Sciences: Chemicky ustav Slovenskej akademie vied Ľubomír Šmiga University of Veterinary Medicine and Pharmacy in Kosice: Univerzita veterinarskeho lekarstva a farmacie v Kosiciach Viliam Šnábel Institute of Parasitology of the Slovak Academy of Sciences: Parazitologicky ustav Slovenskej akademie vied Jana Takáčová Univerzita veterinárskeho lekárstva a farmácie v Košiciach: Univerzita veterinarskeho lekarstva a farmacie v Kosiciach Ladislav Takáč Univerzita veterinárskeho lekárstva a farmácie v Košiciach: Univerzita veterinarskeho lekarstva a farmacie v Kosiciach


Introduction
Intestinal parasites are important enteropathogens in dogs and cats. Moreover, several canine/feline intestinal parasites are considered zoonotic and are of great consequence to the public health (Claerebout et al., 2009). Pets play a pivotal role as de nitive or reservoir hosts for many zoonotic parasites, especially in low-income countries and also in socio-economically disadvantaged communities (Traub et al., 2002;Traub et al., 2005;Salb et al., 2008). The period of our time is globalization which increases the likelihood of large-scale and intensive imports and exports of parasites, their vectors and disease vectors, what can lead to local endemics worldwide, or even to pandemics that have a signi cant impact on human and animal health. Thus, there are no longer any tropical diseases that can be avoided by entering these countries. Today we have passenger disease, we have local zoonoses and we have diseases due to imported animals and plants (Melhorn Aspöck ey al., 2008). Zoonotic potential of worldwide spread endoparasites is of major importance due to dwelling proximity and common share of the same living space. Although dogs and cats in the households are usually well cared the frequency of antiparasitic treatments may vary and can be irregular in a great number (Becker el al., 2012). This is the reason why animals without any clinical signs may be a carrier for the infection. Some well-documented and typical canine/feline parasites are responsible for the spread of important zoonotic diseases. Those well-known zoonoses include echinococcosis, larva migrans (toxocariosis, ancylostomatidosis), and emerging and reemerging infections, such as cryptosporidiosis and giardiasis (Martínez-Moreno et al., 2007). All these intestinal parasites have an orofecal transmission cycle and most important factor for dissemination of these parasites is triggered by shedding of eggs or (oo)cysts into the environment (Claerebout et al., 2009. Antiparasitic control measures are based on the prevention (antihelmintic treatments) or reduction of worm eggs and (oo)cysts shedding into the environment. Therefore parasite preventive and control programs should be based and focused on active monitoring of endoparasites distributions, analysis of the environmental contamination and identi cation of potential zoonotic agents. The most commonly protozoan and helminth species occurring in Europe are Giardia spp. and Toxocara canis, respectively. Special veterinary interest is focused on Giardia spp. where this infection is not always easy to cure and their roles as a zoonotic agent is not entirely clear (Pallant et al., 2015). T. canis is zoonotic and poses a health risk especially for the children (Macpherson, 2013). Strongyloides stercoralis represents another potentially zoonotic helminth agent . Others important parasitic diseases are represented by hookworm infections caused by Ancylostoma spp. and Trichuris vulpis (Traversa, 2011).
In contrast with stray dogs and cats, much less data is available for household pets. Despite the increasing numbers of dogs kept as companion animals in Slovakia during the past years, there are no data reporting the situation regarding parasite infections and vector-borne diseases in this group of animals. In Europe, the known works exist regarding parasite fauna in household dogs in Albania Totally 307 feces samples were examined for the endoparasite infection. Animals were sampled by owners during natural emptying for ve consecutive days thus obtaining better chance of Giardia cysts detection. Owners who sent samples provided information on the animal origin, age and gender. Ethical approval was not required since all activities performed on dogs and cats in this study were performed by routine diagnostic procedures performed by veterinarians at each study site. Consent for the examination and sampling from pet owners of were obtained on a case-by-case basis. The research related to animals complied with all the relevant national regulations and institutional policies for the care and use of animals. Therefore, no additional authorization was required. The proposal was reviewed and approved by the Ethical Commission of Institute of Parasitology SAS in Košice.
All samples were stored without any preservation at 4°C and transferred directly to the laboratory at the Institute of Parasitology SAS in Košice, for further parasitological examination. The entire examination was performed within 24 -48 hrs. Fecal samples were examined macroscopically for the detection of proglottids and then screened microscopically for the presence of worm eggs and protozoan oocysts. A otation method with the Shaeter's otation solution with speci c gravity of 1.27 g.ml -1 was used for the coproscopical examinations. Three grams of fecal sample was mixed with water and centrifuged for 5 minutes at 1200 rpm (Eppendorf 5804 centrifuge, Germany). After the pouring out of supernatant, the Shaeter's otation solution was added into 2/3 of the test tube and stirred with the sediment, and then centrifuged once again. The Faust otation solution with speci c gravity of 1.18 g.ml -1 was used for the detection of Giardia cysts. After the centrifugation, 3 drops were taken from the surface of the otation solution and put on a slide. Both reactions were carried on MyCyclerTM Thermal Cycler System (Bio-Rad Laboratories, USA). Known isolate of Giardia spp. was used as a positive control and PCR water was used as a negative control. Both controls were included into each PCR reaction. Obtained amplicons were separated on 1.5% agarose gel stained by GelRed® Nucleic Acid Gel Stain (BIOTIUM, USA) and TAE buffer (40 mM Tris, pH 7.8, 20 mM acetic acid, 2 mM EDTA).
Statistical analysis was done using Windows SPSS® (version 11.5) (StatSoft, Inc. STATISTICA, 2007). Cross tables and Chi-square tests were used to calculate possible correlations between age, sex or purity of breed and potential zoonotic endoparasites agents (Giardia spp., Toxocara spp. Strongyloides stercoralis and family Ancylostomatidae). Differences were considered signi cant at p <0.05 and highly signi cant at p <0.01.

Results
Total prevalence of endoparasites The population of examined animals consisted of household owned dogs and cats. Collected fecal samples of 257 dogs and 50 cats were examined coproscopically for the presence of endoparasites. Out of 307 investigated fecal samples 107 were positive for the propagative parasitic stages of what represented the overall prevalence of 34.9%. Examination con rmed that 86 dogs (33.5%) and 21 cats (42.0%) were positive with one or more different parasitic species and genera. However, the majority of animals (25.4%) were infected with only one parasite species. Mixed infections were found in 9.8% of examined samples.
The examined dogs were divided by age into two groups. 89 (34.6%) of them were within the age of 1 year. Dogs up to 1 year were infected more often than older dogs. Distribution of endoparasites according the age, sex and breed is shown on the Table 2. years. Cats younger than 1-year old were infected more frequently what represented 61.9% of all cases in this study. Parasites detected were Giardia spp., Cystoisopsora spp., and T. cati. The rst stage larvae of pulmonary lungworm Aelurostrongylus abstrusus was detected during the coproscopical examination as well. Though, it was found only in 2% of all examined samples. The most prevalent parasite was Giardia spp. (36.0%). This was followed by Cystoisospora spp. (4.0%). Helminth T. cati was found in 6.0% of samples. Cats up to 1 year of age were likewise dogs infected more signi cantly (53.3 %) than the older animals (20.0%; p < 0.01).
Out of the 18 cat feces samples, which were positive for Giardia cysts during coproscopically examination and submitted to the nested and semi -nested PCR, 16 were con rmed to be positive at the ß giardin loci. Only two isolates were successfully ampli ed at both the gdh and ß giardin markers.

Correlation between potential zoonotic parasite infection and gender/breed
Gender in dogs was distributed unevenly and were composed of 105 males (40.9 %) and 152 females (59.1 %). Gender distribution in cats was more even and consisted of 30 males (56.0 %) and 20 females (44.0 %). There was no signi cant correlation found between breed (pure breed vs. mixed-breed) and gender. It appears those two examined factors do not belong to the risk factors for potential zoonotic endoparasitism spread involving household dogs and cats population.

Discussion
A key objective for the control of dogs and cats parasites is reduction the risk of transmission for zoonotic diseases. Parasitic fauna and endoparasites prevalence in household pets depend on many abiotic and biotic factors such as geographic location, climate, demographic factors, sampling protocols, and diagnostic techniques, status of animal ownership, veterinary facilities, antihelmintic usage and public awareness (Katagiri and Oliveira-Sequeira, 2008). Our study detected high parasitic prevalences in dogs and cats of which most of them are of zoonotic importance. The diversity in contamination of canine and feline feces with different groups of parasites points out on the substantial potential of public spaces contamination and presents a constant and non-diminishing hazard to the public health. The occurrence of relatively high infection intensities can be result of deworming programs absence or bad selection of the antiparasitic drug administered without adequate copro-parasitological examination.
Recent study is the rst who provides data on gastrointestinal endoparasites in household dogs and cats in Slovakia. Almost 30.7% of dogs and 36% of cats were positive for the protozoan infections (mainly Giardia cysts) and nematode endoparasites. Sofar the known endoparasites occurrence in Slovakia is known from coproscopical feces examinations from parks and playgrounds in urban settlements The nematodes infections identi ed in the dogs and cats feces may cause zoonotic diseases and pose a risk for human health. T. canis and T. cati may lead to the visceral and ocular larva migrans, which may trigger blindness. Similarly to hookworm A. caninum which also may set off cutaneous larva migrans (Heukelbach and Hengge, 2009). The study of Fahrion et al. (2011) showed that a high percentage of Toxocara eggs found in the dogs feces identi ed by PCR analysis were actually T. cati where coprophagy of cat feces is quite common in dogs. However, Toxocara eggs in the present study have not been distinguished by PCR. We do not know how many of the Toxocara eggs found coproscopically were actually only contaminants from ingested cat feces. Since there is no difference in the zoonotic potential between T. canis and T. cati (Cardillo et al., 2009) this nding deserves special attention.
The overall endoparasites prevalence in household pets in this study was 34.9%. Detected prevalence was comparable to that obtained in studies conducted in Albania and Brazil (Shukullari et al., 2015;Curia et al., 2017) where the majority of animals were infected with only one parasite species (25.4%). In our study the majority of infection was represented by protozoa (31.6%) and remaining (17.9%) were helminthic infections. Combined infections were found in 9.8% of all examined cases.
The rst stage larvae of Angiostrongylus vasorum were diagnosed in two dog fecal samples. This cardiopulmonary nematode, which can be found in dogs and several wild canines has been reported by identi cation of rst-stage larvae from fecal examination what was similar to Greece ndings (Diakou, 1995;Founta et al., 2000).
Our study showed that endoparasites prevalence in domestic cats (42.0%) is higher than the prevalence reported in a study from Canada, where only 6. . In contrast to dogs less data is available for the cats. In our study, the prevalence of T. cacti was found to be about 6.0%, Cystoisospora spp. 4.0%, and A. abstrusus 2.0%. The most common parasite was Giardia spp. with prevalence of 36.0%.
Helminth T. cati was found in 6.0% of examined samples and when compared with results stray cats results in Iran (44%) and Germany (27.1%) (Becker el al., 2012; Sharif et al., 2010), the prevalence in our study was signi cantly lower. This observation points out on the importance of adequate treatment and animals deworming. Work of Barutzki and Shaper (2013) found that the prevalence of T. cati in Germany was 4.7%. This is comparable to our results. In contrast to our data Capari et al. (2013)  Parasite Aelurostrongylus abstrusus is the most well-known nematode infecting cat respiratory tract as its natural host (Traversa et al., 2010). In our study, the larvae of A. abstrusus feline lungworm, were found only in one of fecal sample, but the Baermann technique was not used. Nematode larvae were detected by the oatation technique; and therefore, the actual infection with A. abstrusus is presumably underestimated.
In general canine and feline feces samples were coinfected largely with protozoan cysts of Giardia spp. Since Giardia is a common cause of diarrheal disease in humans the companion animals are able to transmit it on the owners. Depending on the investigated animal population the prevalence data of Giardia infections in dogs and cats may vary extremely. As a result, the utilized diagnostic method is the most important factor affecting the prevalence rate (Bouzid et al., 2015). Our study demonstrated that the overall prevalence of Giardia spp. in domestic dogs and cats was 22.1% what is similar to study from Albania, where Giardia spp. prevalence was 26.4% (Shukullari et al., 2015). The overall Giardia spp. prevalence in cats was 36.0% and in dogs 20.2% this indicates that every third cat and every fth dog were infected with this potentially zoonotic protozoan parasite -Giardia spp. Because of various Giardia hosts-speci c genotypes/species in dogs and cats more studies are required on genotypisation of Giardia isolates in order to better understand the giardiasis epidemiology and transmission ecology. The infection prevalence in this study in household kept animals was much more higher when compared with the study of Bouzid et al. (2015), where the Giardia spp. occurrence was 15.2% in dogs and 12% in cats. We assume that the difference between those studies was due to use of different quantitative and qualitative diagnostic methodology.
Potential zoonotic agents represented by T. canis and eggs from family Ancylostomatidae were found in 7.4% and 4.3% of samples, respectively. Our study revealed that T. canis is the most common nematode in examined domestic dogs (7.39%). animals. This survey is the rst which evaluated the endoparasite status of client-owned, veterinary cared dogs and cats in Slovakia. The data presented in this study indicate the presence of a wide variety of endoparasites and demonstrate relatively high prevalence of potentially zoonotic agents. Therefore, the veterinary doctors and pets owners need to step up their interest on periodical endoparasites examination and antihelmintic treatment. Only these approaches will reduce the parasitism in pets and consequently lower the potential for transmission of zoonotic agents to humans.

Conclusions
Close and frequent contact between pets and people increases the risks for the transmission of zoonotic diseases. The contamination of the environment by dogs and cats feces represents an everyday serious health hazards. It is due to the spread of parasite developmental (cysts, eggs and larval) stages which can survive in the ecosystems for a very long time. We recommend the use of appropriate fecal and molecular diagnostic techniques to detect the spread of gastrointestinal parasites. The proper and timely diagnosis of zoonotic agents is very important. The utilization of the proper treatments and control strategies will lessen the risk of infection spread to the other household animals, and also reduce the subsequent exposure of humans to zoonotic parasites.

Declarations Ethics declarations
Ethics approval and consent to participate The research related to animals complied with all the relevant national regulations and institutional policies for the care and use of animals. The proposal was reviewed and approved by the Ethical Commission of Institute of Parasitology SAS in Košice. Consent for the examination and sampling from pet owners of were obtained on a case-by-case basis.