Prevalence of intestinal parasites in street dogs (Canis lupus familiaris) with highlights on zoonosis in Lalitpur, Nepal

Abstract Background The presence of intestinal parasites influences the growth and well‐being of canids. Additionally, infected dogs and their faeces with considerably higher eggs/oocysts released per gram (epg/opg) of zoonotic parasites contribute to parasitic spillover to humans, domestic animals and sympatric wildlife. Objectives The current study aimed to reveal the prevalence of intestinal parasites (protozoa and helminths) and to list the zoonotically significant parasites in free‐roaming street dogs in Lalitpur Metropolitan City, Nepal. Methods Fresh faecal samples (n = 332) were collected from feral dogs of varying ages and sexes and transported to the research laboratory. The copro microscopic examination was carried out via direct wet mount, formalin ethyl acetate sedimentation, saturated salt flotation, acid‐fast staining and sporulation techniques. Results Coproscopy revealed an overall 95.7% (318/332) prevalence rate with 23 diverse species of intestinal parasites (10 protozoa and 13 helminths). Among them, 5 protozoa and 11 helminths possessed zoonotic potential, and their overall prevalence was 92.5%. Helminth's overall prevalence was double that of the protozoa (87.7% vs. 43%). Polyparasitism was dominant over mono parasitism (79.5% vs. 16.3%), and co‐infection of up to seven species of parasites at a time was recorded. Conclusions Urban street dogs harboured a higher prevalence of intestinal parasites that varied with age and sex. Since most of the reported parasites are zoonotic, dog density and parasitic richness indicate a greater spillover risk to humans and domestic animals. Furthermore, this study also provides appropriate ‘baseline’ data for assessing effective control measures against parasitic infestations among street dogs and controlling their transmission to humans.

are important for Hindus who worship them; however, very few people raise them as pets, and most of them are freely living in the human habitat.In rural areas, they are fed by the community, but in urban centres, they aggregate around slaughterhouses, garbage piles, public places, temples and gumbas for their diet.Such free-living canids are predominantly found in the Kathmandu valley and other urban centres in Nepal.Notably, due to the lack of effective deworming, immunization and supplement of adequate feed and shelter, they suffer from malnutrition and diseases (Massei et al., 2017).Such diseases occurring in the intestinal tract involve ancylostomiasis, capillariosis, coccidiosis, giardiasis, spirocercosis, tapeworm infection, toxocariosis, trichuriasis and others.They can induce minor to significant pathogenic effects, including severe morbidity and mortality (Kagira & Kanyari, 2000;Morelli et al., 2021;Schantz, 2007).
Although intestinal infections caused by gastrointestinal (GI) parasites lead to severe effects in dogs, these animals can act as reservoirs, vectors or carriers of bacterial, viral and parasitic agents and pose a threat or negative impact on humans as well as livestock and wildlife (Adhikari, Shrestha, et al., 2020;Fong, 2017;Ghasemzadeh & Namazi, 2015;Traub et al., 2002).Regarding this, several studies have explained the critical role of dogs in the emergence (or re-emergence) of zoonosis (Baneth et al., 2016;Chomel, 2014;Ghasemzadeh & Namazi, 2015;Otranto et al., 2021) signifying, people with chronic illness, immunodeficiency and pregnancy are at the topmost risk.Human acquisition of parasite-borne zoonotic diseases follows direct contact with infected dogs or exposure to the environment, food or water contaminated with dog's faeces (Schantz, 2007).The zoonotic parasites have been predominantly found with the prevalence rates ranging from 44.3% to 100% in feral dog population from global geographies, like India, China, Morocco, Nigeria and Uruguay, especially in the rural setting (Ayinmode et al., 2016;Fang et al., 2015;Idrissi et al., 2022;Malgor et al., 1996;Traub et al., 2002).On the other hand, 15% of clinical cases and 93% sero-prevalence of canine-specific Ancylostoma spp.(Heukelbach et al., 2008) and Toxocara canis (Magnaval et al., 1994) have been reported from Brazil and La Reunion, respectively.Similarly, several cases of canine-specific whipworm (Trichuris vulpis) infection in humans have also been reported from endemic regions like Mexico (Márquez-Navarro et al., 2012), India (Mirdha et al., 1998) and Thailand (Areekul et al., 2010).In these scenarios, external factors like environment and climate alternation, urbanization and habitat fragmentation might be important in parasite survival and transmission.In addition, the predatory pressure on small prey (rodents, birds and reptiles) might be the contributing factor to the acquisition as well as spillover of zoonotic parasites from feral canids (Mendoza Roldan & Otranto, 2023;Wells et al., 2018).In Nepal, a few helminthic faunae, like Ancylostoma sp., Taenia sp., T. canis, Dipylidium caninum, Capillaria sp. and T. vulpis, were recorded in the faecal samples of dogs with higher prevalence ranging from 46.7% to 70% (Sukupayo & Tamang, 2023;Yadav & Shrestha, 2017) and concluded that free-ranging canids had a higher prevalence of zoonotic helminth parasites compared to the pet (Satyal et al., 2013).
However, none of these native reports have discussed the presence of canine-specific protozoan fauna.In addition, these authors have not discussed the presence of intestinal parasites with respect to zoonotic potential.Thus, to fulfil the knowledge gaps, in the current study, we selected free-ranging canid populations from a recently urbanized area in Lalitpur district and aimed to update the information regarding prevalence of different endoparasites, including both protozoa and helminths in the openly defecated faecal samples.In addition, we aimed to explain how these parasites are distributed in different ages and sexes of the dogs and how these hosts can be sources of zoonotic parasitosis to the nearby domestic animals and humans.

Study area
The study was conducted in Lalitpur Metropolitan Municipality com/2016/01/26/animal-nepal-launches-lalitpur-dog-populationsurvey-report/), and this population is even more in today's date.
These dogs roam freely in the streets, and their population seems to be highly concentrated towards slaughterhouses, religious sites, butcher shops and dumping sites where they scavenge for food (Figure 2A-D).

Sample collection, preservation and transportation
A total of 332 fresh faecal samples from 332 street dogs of varying age and sex groups were non-invasively collected from 51 different locations within the metropolitan city.Since determining the age of free-ranging canids is a challenging task, we simply categorized them as 'young' and 'adult' based on the differences in their morphology, adopted behaviour and the way of living.Younger ones are more playful and mischievous in nature.Morphologically, they have smaller stature, larger paws, and excess and loose skin and usually live in association with their mother.In contrast to this, adults are physically larger, with greyish and elongated muzzles, and have independent ways of life.On other hand, the sexes of these dogs were confirmed via direct observation of their genitals or via observing their sexually dimorphic urinary behaviour.Male generally lift their one hind leg whereas females squat while urinating (Wirant & McGuire, 2004).Faecal collection was performed on a daily basis early in the morning.For this, the free-ranging canine troops were followed until they defecated and the portion of sample that had not touched the ground was carefully picked up using a metallic spatula and gloved hands.Initially, the faecal samples were macroscopically examined for faecal consistency and the presence of blood, mucus, segments of cestodes and dead nematodes and then kept in screw-capped 20-mL sterile vials.The samples were further added to 2.5% weight/volume (w/v) potassium dichromate solution and transported to the Animal Research Laboratory at Nepal Academy of Science and Technology for microscopic examination and parasitic investigation.Before the investigation, the samples were stored in the refrigerator at 4 • C temperature for 2 weeks.

Direct wet mount technique
A sample drop mixed in 2.5% weight/volume (w/v) potassium dichromate solution was placed on a glass slide and observed under the microscope (40×).

Concentration technique
Concentration techniques were followed by FEA sedimentation and saturated salt floatation methods.For the former process, 2 g of the sample was subsequently centrifuged (1200 rpm × 5 min) initially with normal saline (0.9% w/v NaCl) and then with a mixture of 10 mL of 10% formalin and 4 mL of ethyl acetate.A single drop of the final sediment was then placed on a glass slide and observed under the microscope (10×, 40×).For the saturated salt flotation method, the sediments obtained after the first centrifugation were added to 12 mL of 45% w/v of sodium chloride (NaCl) solution and then followed by centrifugation (1200 rpm × 5 min).Then, the tube was allowed to stand vertically on the test tube stand, and the flotation media was added drop by drop to fill it.The mouth of the tube was covered with a coverslip and left undisturbed for 10 min.At last, the coverslip was recovered and placed on the glass side for microscopic observation (40×).

Acid-fast staining technique
Cryptosporidium-and Cyclospora-positive sediments observed in FEA sedimentation were used to prepare a thin smear.The smear was initially fixed in absolute methanol for 2 min, then it was counter-stained with carbol fuchsin for 15 min.Following it, the smear was de-stained with acid alcohol for a while and then subsequently stained with malachite green for a minute.Finally, the slide was allowed for drying, and using immersion oil, the parasite was observed under the microscope (100×).

Sporulation assays
About 5 g of each coccidia (Eimeria, Cystoisospora and Sarcocystis) positive sample was poured into a separate Petri dish.It was then mixed with 2.5% potassium dichromate solution in the ratio 1:5 and then incubated at 28 • C for a week in an incubator.In every 24 h' interval, the sporulation state of the oocysts was checked following the flotation technique.

Measurement of parasitic burden/severity of infection
A 2 Cell McMaster Counting Slide (Hawksley and Sons Ltd) was used to measure the burden of parasite infection.It was measured by quantifying the number of eggs/oocysts of parasites released per gram of faeces (epg/opg).The procedure was followed with the instruction of the manufacturer's company and previously documented literature (Adhikari, Adhikari Dhakal, et al., 2021;Soulsby, 2012).

Data analysis
The data obtained were expressed as the number of positive samples and prevalence rates in the table using Microsoft Word 2010.
Prevalence rates were calculated by dividing the number of GI-positive samples (total or particular species) by the total number of samples observed and multiplied by 100.We used GraphPad Software (Prism 5 for Windows, Version 5.00@1992-2007, GraphPad Software, Inc) to analyse the prevalence rates between different variables like age and sex of dogs by applying Fisher's exact test (two-sided) to assess p-values.Moreover, statistical significance was considered at the 95% confidence interval (p < 0.05).

RESULTS
In

DISCUSSION
The current study first identified a considerable diversity of intestinal parasites from free-ranging canids in the study area and Nepal.
Furthermore, stray dogs are reservoirs of many intestinal parasites, including zoonotic ones (Trasviña-Muñoz et al., 2020;Traub et al., 2014).Current research shows that the overall prevalence of enteric parasites and zoonotic species depends on the age of the canids.Similar results with greater prevalence of enteric parasites in young were obtained from previous studies from Nepal (Satyal et al., 2013;Yadav & Shrestha, 2017), and Ecuador (Sangache et al., 2020), whereas studies from Zambia (Mugala et al., 2018) and Mexico (Cantó et al., 2011;Trasviña-Muñoz et al., 2020) reported contrasting results.Currently, zoonotic species like Entamoeba sp., Giardia sp., Cryptosporidium sp. and T. canis were predominant in the young, but Ancylostoma spp., Taeniid, Strongyloides sp., D. caninum, Strongyle, T. salmincola, T. vulpis and Capillaria spp.were dominant in the adults.It is not easy to explain this predilection.However, it has been discussed that due to naïve immune status, the newborn offspring can be infected by protozoa with high pressure (Barutzki & Schaper, 2011).A similar result has been obtained in the context of T. canis nematodes in young compared to adults (Nijsse et al., 2016).Prior infection by parasites in the young generates a protective immunological memory in adults and protects them from new infection (Jensen et al., 2015) resulting in reduced parasitic prevalence.Therefore, further studies are necessary to assess why the parasite-specific predominance differs in different ages.
Regarding the sex, prevalence of overall enteric parasites in females had higher than males, although similar ranges of zoonotic parasites were detected in both sexes.These findings are in agreement with the previous reports from Nepal (Satyal et al., 2013), Mexico (Trasviña-Muñoz et al., 2020) and Zambia (Mugala et al., 2018).However, other studies from Nepal (Yadav & Shrestha, 2017), Mexico (Cantó et al., 2011) and Ecuador (Sangache et al., 2020) completely disagreed with it.Physiologically, female canids have higher cortisol concentrations than males (Sundman et al., 2019).Thus, it is speculated that they may have higher stress, which might cause weak immunity and greater susceptibility to parasitic infections (Fleming, 1997;Wakelin, 1984).In addition, owing to the partially suppressed immune system at whelping, females may acquire a wide variety of parasites (Fleming, 1997;Pereira et al., 2019;Wakelin, 1984).Furthermore, unlike the female, males' fitness increases with promiscuous mating behaviour (insemination to multiple females) (Scandurra et al., 2018;Shuster & Wade, 2019) which might decrease parasitic acceptance and establishment.However, further studies should confirm these hypotheses.
Noticeably, helminth parasites had a higher prevalence and a wider variety than protozoa.A higher prevalence of helminth parasites in stray dogs has already been reported in India (Sudan et al., 2015) and South Africa (Mukaratirwa & Singh, 2010).These regions exhibit subtropical to tropical climatic conditions.In this study, Ancylostoma spp. was the most prevalent parasite.This indicates that egg hatching and larval development of ancylostomatid worms are favoured by the subtropical climatic condition of the study areas.Hookworm induces threatening anaemia in canids and causes eosinophilic enteritis and larval migrans syndromes in humans (Croese et al., 1994;McCarthy & Moore, 2000).Next to the hookworm was the Taeniid.The Taeniid is transmitted via scavenging on the carcasses of infected domestic herbivores.Although Echinococcus granulosus, Echinococcus multilocularis, Taenia ovis, Taenia multiceps and Taenia hydatigena are reported from canids, only, E. granulosus and E. multilocularis possess significant medical and public health risks (Cardona & Carmena, 2013;Eckert & Deplazes, 2004) suggesting a need of further molecular diagnosis.
D. caninum is another zoonotic helminth that rarely infects humans; however, the zoonotic risk increases with the availability of a high proportion of canids excreting eggs to the environment (Gutema et al., 2021).Interestingly, T. canis can easily cross the host barrier and infect a wide range of hosts like cats and wild canids, including humans (Macpherson, 2013).With increasing cases of visceral toxocariasis, ocular toxocariasis, common toxocariasis and covert toxocariasis in humans from different geography (Magnaval et al., 1994;Magnaval et al., 2001;Singh et al., 2007;Chen et al., 2018), T. canis has been a concern for public health.However, the zoonotic potential of the currently detected canid Toxascaris leonina is still questionable.However, a case of ocular involvement in a child from East Africa had been reported (Beaver & Bowman, 1984).In addition, its larvae have been shown to invade the tissues of some laboratory animals (Despommier, 2003).Uniquely, this study first reported T. salmincola (Nanophyetus salmincola) in Nepalese dogs.The canids acquire their infection upon the ingestion of raw or undercooked flesh of fish encysted with infective metacercaria larva (Lin et al., 2017).Although non-pathogenic, it acts as a vector of Neorickettsia helminthoeca, or salmon poisoning disease, and can result in higher mortality in dogs (Fritsche et al., 1989;Lin et al., 2017).It is critical for public health because it may infect humans as well as cats, raccoons, foxes and a few species of birds (Eastburn et al., 1987;Fritsche et al., 1989).Furthermore, C. plica and C. aerophila detected in this study have a history of human infection (Cross 1992).In the same way, a few clinical cases have also been attributed to T. vulpis (Areekul et al., 2010;Dunn et al., 2002), indicating zoonotic transmission in endemic regions.
Furthermore, regarding the protozoa, coccidia were the most dominant parasitic species.Except for the common Cystoisospora spp., the presence of Cryptosporidium sp. and Sarcocystis spp.might be critical for both canids and sympatric hosts.Canine cryptosporidiosis is caused by C. canis, Cryptosporidium parvum, Cryptosporidium muris and Cryptosporidium meleagridis; moreover, a few of them are zoonotically significant (Abe et al., 2002;Jian et al., 2014;Lupo et al., 2008).Its risk of cross-transmission usually increases within the large population size and close contact of canids with other animals and humans.Similarly, human cases of Sarcocystis have been reported before (Fayer et al., 2015).Several species like Sarcocystis cruzi, Sarcocystis caninum, Sarcocystis ovicanis, Sarcocystis miescheriana, Sarcocystis bertrami, Sarcocystis fayeri, Sarcocystis hemionilatrantis and Sarcocystis svanai (Dubey, 2003;Dubey et al., 2006;Soulsby, 2012) have already been reported from canines.Since canids are predators and scavenge for food, consumption of raw meat/tissues and exposure to tainted faeces contribute to Sarcocystis.Another coccidian parasite, Hammondia/Neospora, can be critical for sympatric animals because of its causal association with abortion in cattle and other vertebrates (Dubey, 2003;Dubey et al., 2006).Notably, the presence of Entamoeba sp. and Giardia sp. in the current dog populations indicates that zoonotic possibilities in the nearby humans and animals with these protozoa should be further elaborated.
Interestingly, B. coli in the faecal samples indicated that dogs could be important sources of zoonosis or cross-transmission for humans, although ciliates are unusual and rarely infect the canine population (Chalmers, 2014).
Relating to the parasitic burden, current canid populations were reported to shed a moderate-to-higher number of eggs and oocyst (epg/opg) per gram in their faeces.The higher epg/opg count for parasites like Ancylostoma spp., T. canis, Trichuris spn2.and Cystoisospora spp. in the present study corresponds with the finding results from various geographies like Nigeria (Sowemimo, 2009), South Africa (Mukaratirwa & Singh, 2010) and Portugal (Cardoso et al., 2014).This indicates that feral dogs are prone to high burden of parasitic infection, which can be a sign of their frail GI health and intensified transmission risk.
Concerning the concurrency of the parasitic infection, co-infection with multiple species of parasites is higher than a single infection, and irrespective of age and sex, triplet infection was highly reported.
Polyparasitism generally occurs when one species of co-infected member facilitates the occurrence of another or when the host is exposed to common risk factors (Vaumourin et al., 2015).Furthermore, host characteristics/behaviour, host distribution and habitat use, the host's life history traits and the nature of the infecting parasites determine the occurrence of polyparasitism (Vaumourin et al., 2015).Although it may result in positive, negative or neutral interactions among the coinfecting members within the host (Hoarau et al., 2020), its synergistic effect can lead to enhanced virulence and disease severity.
A few limitations of the current study should be considered.
The foremost is the methodological constraint, which involves smear assessment and examination of single-spot faecal samples.Such methods might not be efficient enough to address the pre-patent infections and the intermittent shedding of the oocysts and eggs.Furthermore, it can also underrate the quantification of epg/opg burden, prevalence rate and polyparasitism.The species-level identification of eggs and oocysts of parasites would have been better with molecular methods.
Second, it is the cross-sectional nature of study, which might be inadequate to determine the precise reason behind the zoonosis observed in the study.Yet, we stringently assured the quality control during our field surveys and laboratory techniques that have produced the best results of canid GI parasites.

CONCLUSIONS AND RECOMMENDATIONS
The current study detailed the diversity of intestinal, protozoan and helminth parasites of free-ranging urban street dogs significant for zoonosis.Due to their scavenging nature, these dogs can act as reservoirs and primary hosts of many parasites.They shed the cysts, oocysts and eggs of the intestinal parasites into the environment openly.As a result, they can be critical sources of zoonosis in humans.Thus, it is a matter of concern for veterinarians and public health workers to design a strategic plan for deworming and vaccination to maintain their positive health and to check the possible risk of canidborne zoonoses.Similarly, an integrated approach of 'One Health' involving clinical, veterinary, environmental, ecological and socioeconomical factors must be considered to evaluate the possibility of cross-transmission/zoonosis.In addition, detailed histopathological and molecular studies will support the causal association of single or mixed infections in canids.

( 27 •
32′53.88″North, longitude 85 • 20′15.00″East), the third largest city within the country (Figure 1) with higher rate of urbanization in recent years.It is situated in the south-central part of the Kathmandu Valley, the country's capital in central Nepal.The city is well known for its rich cultural importance, particularly the ancient tradition, arts and crafts.The ancient monument 'Patan Durbar Square' listed by UNESCO as one of the World Heritage Site lies in this region.Lalitpur has a humid subtropical type of climate.The temperature is relatively high, with the highest annual range of 18-29 • C and the lowest range of (3-20 • C), even though it receives fairly distributed rainfall throughout the year ranging from 153 to 241 mm (Retrieved on 3 February 2022 from http://hikersbay.com/climate-conditions/nepal/ patan/climate-conditions-in-lalitpur.html?lang = en).According to Dog Survey Report 2015, 2793 dogs live freely on the streets of Lalitpur Metropolitan City (https://animalnepal.wordpress. ent techniques, including direct wet mount, formalin-ethyl acetate (FEA) sedimentation, saturated salt (45% w/v NaCl) flotation and F I G U R E 2 Feral dogs in human inhabitant areas.(A and B) Puppies in the garbage.(C) An adult licking water.(D) Feral dogs freely roaming.
Age and sex-wise prevalence of zoonotic and non-zoonotic parasites in urban street dogs (N = 332).
Burden (eggs/oocysts per gram, epg/opg) in the faecal samples with respect to age and sex of the dogs.