INTESTINAL PARASITISM IN WORKING HORSES AND ASSOCIATED ZOONOTIC RISKS IN LOWLANDS OF NEPAL

The presence of intestinal parasites influences equines' well-being and working performance. However, the scenario of parasitism in working horses in the lowlands of Nepal is yet to be explored. The present study aimed to reveal the prevalence and diversity of intestinal parasites (protozoa and helminths) and to list the zoonotic species in working horses in the lowlands of Nepal. Fresh fecal samples (N=102) from horses were collected at two locations (Chitwan and Birgunj) in the lowlands of Terai and were transferred to the research laboratory. Coproscopy was carried out via direct wet mount, formalin ethyl acetate (FEA) sedimentation, saturated salt flotation, and acid-fast staining techniques. Coproscopy revealed an overall prevalence rate of 90.2% (92/102) with 15 known diverse species of parasites (Protozoa: 5 and Helminths: 10) and an unknown coccidian, out of which eight possess zoonotic potential. The prevalence and diversity of intestinal parasites were higher in adult than in young animals (90.7%; 15 spp. vs. 88.9%; 11 spp.) The overall prevalence of helminths was double that of protozoa (89.2% vs. 43.1%). Furthermore, polyparasitism was much more prevalent than monoparasitism (85.3% vs 4.9%). Co-infection with two parasite species (37%) was higher in young horses. In comparison, triplet infection (34%) was higher in adults, and a maximum concurrency of up to six species of parasites at a time was recorded. Following it, the differences in the prevalence rate of parasites based on the predictor of risks, like sex, grazing, domestication type, nature of the floor, and medication practices, were statistically significant. Working horses in the lowlands of Terai harbored a significant variety of intestinal parasites with important prevalence. Since eight of the reported parasitic species were zoonotic, infected horses pose a zoonotic risk to the owners. Therefore, timely deworming, pasture management, and reduction in working pressure are highly recommended.


INTRODUCTION
Horses (Equus caballus), Family: Equidae, Taxonomic serial number 180691 (www.itis.gov),are oddtoed domesticated mammals.They are well-known for their sturdy body complexion, highly adaptive running and social behavior (1).Horses have been a part of the Nepalese civilization for many years.Even though multi-purpose animals, horses are mainly used as a means of transportation (horse-driven carts, riding, goods carrying) in the Himalayan, Hilly, and Terai regions of Nepal.According to recent Livestock Statistics data, 54,864 horse heads are found in Nepal, and about 11% of their population is concentrated in the lowlands of Terai and inner Terai (2).The insufficient breeding centers, inffective conservation campaigns, poor nutrition, and modernization are threats for declining of the horse population (3).In this context, the role of gastrointestinal parasites (GIPs) in the health status and survival has not been evaluated and addressed so far.Similar to other terrestrial herbivores, horses are expected to harbor complex macroparasite communities in their GI tract, which is one of the common causes of diseases in equids.Various parasitic diseases, including GIPs have been chronologically reviewed in horses (4).The main GIPs of horses include worms, like redworms (Strongylus group), threadworms (Strongyloides spp.), roundworm (Parascaris equorum), tapeworms (Anoplocephala spp.), pinworms (Oxyuris equi), lungworms (Dictyocaulus arnfieldi) and bots (Gasterophilus intestinalis) (5).Although each parasite species infects and impacts differently, its burden directly impairs the growth and development, metabolism, nutrition absorption, working and reproductive performances (6,7).Notable pathogenic symptoms of GIPs are the loss of appetite, anemia, rough hair coat, tail rubbing, coughing, debilitation, diarrhea, and various types of colic leading to the death of these equids (5,8).Besides, horses are also known to carry zoonotic parasites (9,10) and because they fulfill the multipurpose roles as working animals, pets and livestock, their close association with humans increases the risk of transmission of parasitic infections.Interestingly, they naturally harbor more than 56 zoonotic pathogens including parasites, like Blastocystis, Cryptosporidium, Entamoeba, Giardia, Toxoplasma gondii, Echinococcus, Fasciola hepatica, Trichinella, and Trichostrongylus (9-11), and several studies (12-15) claimed for the clinical presence, or associated pathogenesis, as well as the fatal consequences caused by these parasites in humans globally.To date, very few research studies have been carried out in Nepal concerning intestinal parasitism in horses, and these prevalence studies are confined to rural settings in the Himalayan and hilly regions, like Mustang and Rukum districts (16,17).However, the status of parasitism in recently modernized and subtropical to tropical lowlands of Terai and inner Terai with working pressure is yet to be evaluated and discussed.In this research, we aimed to study the prevalence of protozoan and helminth parasite species in working horses at two different locations in the lowlands of Nepal and their possible zoonotic significance and risk factors for the human hosts.

Study area
The study was conducted within Ratnanagar Municipality (27.62°N, 84.51°E) and Birgunj Metropolitan City (27.0°N, 84.52°E) in the lowlands of Nepal (Fig. 1).Ratnanagar Municipality lies in eastern Chitwan in Central Nepal and has a reasonably subtropical to tropical climate (maximum average temperature 21-35 °C and minimum average temperature 10-24 °C) (18).Chitwan has a low population of horses, and most of them are found in the Sauraha area, the main entrance to Chitwan National Park (CNP).Horses are used for pulling carts (Tanga & Baggi) carrying local and international tourists from the east-west highway (Sauraha Chowk) to the CNP entry point.Similarly, Birgunj Metropolitan City lies in southern Terai in the Parsa district and is also known as the Gateway of Nepal.It has a sub-tropical monsoon climate with a very hot and humid summer (maximum average temperature 21.8-35.8°C and minimum average temperature 9.9-25.8°C (Retrieved on 18th June 2023 from https://en.climate-data.org/asia/nepal/central-development-region/birganj-47722/).The horse carts have historically been the popular mode of transportation for the local people and outsiders during short visits or festive ceremonies in Terai.Therefore, despite modernization, the use of carts is a popular means for carrying people and goods from Birgunj to Raxaul, a border region in India and nearby locations or vice versa.

Sample collection, preservation, and examination
The fecal samples were collected from November 9, 2020 to April 15, 2021.All horses studied were the indigenous breed (mainly Terai Pony) working horses aged three months to 21 years.Based on their age, sampled horses were classified into two groups: young (≤3 years) and adults (> 3 years).A total of 102 fresh fecal samples (25 from Chitwan and 77 from Birgunj) were collected via non-invasive technique immediately, right after the defecation.The samples were preserved in 2.5% potassium dichromate solution in screw-capped disinfected vials and then transported to the research laboratories (Nepal Academy of Science and Technology, Lalitpur).Finally, the samples were stockpiled at 4°C in a refrigerator before the laboratory investigation.

Laboratory processing
The coproscopy was carried out following four different standard Ova and Parasite examination techniques, including direct wet mount, formalinethyl acetate (FEA) sedimentation, saturated salt (45% w/v NaCl) flotation, and acid-fast staining techniques based on the procedure previously documented in the literature (19-23).To prepare a direct wet smear, a small amount of the sample was taken in a glass slide, mixed with 1-2 drops of normal saline or iodine, and then observed under the microscope (40x).For FEA sedimentation, 2g of feces were mixed in 12 ml of normal saline (0.9%), filtered using a tea strainer into a conical centrifuge tube, and subsequently centrifuged with normal saline and the mixture of 4 ml of ethyl acetate and 10 ml of 10% formalin at (1200 rpm for 5 mins).The supernatant and debris plug were discarded, and a single drop of sediment was observed under the microscope (40x).Similarly, the sediment obtained after FEA sedimentation was used for smear preparation for acid-fast staining.After air-drying, the smear was fixed in absolute methanol for 2 min, flooded with carbol fuchsin stain for 15 min, destained with acid alcohol for one min, counterstained with malachite green for another minute and rinsed with tap water.
Finally, the smear was air-dried and examined under the microscope (100 x) with immersion oil.For the flotation technique, the sediment obtained after the first centrifugation was added to the saturated salt solution and centrifuged at (1200 rpm for 5 min).Without discarding the supernatant, flotation media was added further to fill the tube, and a coverslip was placed on its mouth.Finally, after 10 mins, the coverslip was picked up carefully and placed on a glass slide for microscopic examination (40x).

Estimation of parasitic burden/severity of infection A 2-Cell McMaster Counting Slide (Hawksley and Sons
Ltd) was used to estimate the burden of helminthic infection.It was quantified by counting the number of eggs released per g of faeces (epg), according to manufacturer's instructions and as previously explained (24,25).

Parasitic identification
A light microscope (B-383PLi, OPTIKA) and ImageJ (National Institute of Health) software for microscopy and photo micrometry, respectively, were used.The identification of parasitic stages like trophozoites, cysts, oocysts, eggs, and larvae was carried out as previously explained (25-27).

Data analysis
The overall prevalence of parasites and individual parasites prevalence was calculated by dividing total positive samples or positively specified parasites by the total number of samples and then multiplying it by 100.Data were analyzed using GraphPad Prism (Prism 5 for Windows v.5).Chi-square and Fisher exact tests were used to evaluate the significance level between any variables.The p values less than 0.05 were considered statistically significant (95% confidence level).

DISCUSSION
In the present study, the overall prevalence rate of intestinal parasites in horses was 90.2% (92/102).This rate was lower than the findings from Rome (100%; n=10) and Libya (98%; n=50) (28) but higher than other studies from Nepal (81.9 % -84.76 %; n=105 (16,17), India (84%; n=100) (29), Ethiopia (63.73% -73.81%; n= 102-317) (30,31), and Colombia (87.5%; n=1004) (32).The results might be associated with methodological tools and techniques during sample collection, laboratory techniques, geo-climatic conditions, horse rearing, and health management practices.The subtropical-to-tropical geo-climatic condition, traditional rearing practices, irregular deworming and other veterinary checkups, and repeated animal exposure to contaminated water bodies and grazing sites are the underlying causes for the current high prevalence rates.The prevalence and diversity of helminths were higher than the protozoa, which agreed with the Libya report (28).Strongylids (74.5%) were the most dominant GIPs in the current equines.The rate was lower than reported in Rome (100%) (33) and Colombia (86.4%-89.4%)(32) but higher than reported in India (52.38%) (29) and Nepal (68.57%) (16).Based on morphology, equine strongylids include Strongylinae (large strongyles) and Cyathostominae (small strongyles) (26), and interestingly, they constituted more than 75% of the total parasites infecting horses globally (26).In this study, the larval culture to distinguish the species of strongylids was not performed; therefore, all the egg morphologies were considered "strongyle".Notably, horses never develop complete immunity against the strongylids;  .These consequences and intestinal parasitism are enhanced by partial or complete starvation and malnutrition (74) .Shedding eggs by different nematodes showed various results in young and adult horses.For example, Parascaris equorum showed the highest prevalence among young and strongyles showed the highest prevalence among adults, indicating differences between young and adult GI anatomy and physiology or other unknown factors.Most horses in the current study were working types; thus, high horse mobility may be associated with high egg shedding by many helminths, including Parascaris sp.(75).Furthermore, feed deprivation or malnutrition not only causes the declination of energy, stamina, and focus by adding strain but also leads to intestinal ulcers resulting from the repeated exposure of intestinal mucosa to high acidity (74,76).However, further equine immunology before and after carting would correctly answer this hypothesis.Moreover, the current result suggests a greater possibility that a higher load of strongyles, as suggested by fecal epg count and prevalence of Entamoeba spp., including other species in the current equines, may be associated to malnutrition and stress.Also, seasonal and other climate factors may determine the epg counts (75), while the current study lacked a seasonal context.In order to characterize the epidemiology of intestinal parasites, the One Health Approach should be applied including all associated sources of parasite infection in man and domestic animals, other than single equid hosts, like grazing, soil, and water sources.Another limitation of the current study is the lack of histopathological tests.Further studies must confirm the underlying association of polyparasitism in these hosts.

CONCLUSION AND RECOMMENDATION
The existing horse population in lowland touristic areas of Nepal possesses a high prevalence and diversity of GIPs.These GIPs vary with age and sex.The grazing opportunity, access to outdoor water bodies, mixed domestication, muddy stable, and irregular deworming practices are the major contributing factors.GIPs in horses may have the potential of public health significant zoonoses, suggesting the necessity of the One Health Approach in parasite research.The latter means expanding the range of hosts as well as the nearby environment, including grazing ecosystem, soil, water sources, and stable.In this context, in vivo studies of equine parasites using different mammalian models will be helpful in identifying their zoonotic potentialities in humans.While stressed and malnourished equines may carry higher loads of parasites, the current study concludes that regular deworming, pasture management, improved and timely feeding, reduced strain and optimal rest can reduce GIPs.These measures, along with molecular taxonomy, will support the effective therapeutic and efficient management of the equine industry in Nepal.

FUNDING
The authors received no financial support for this article's research, authorship, and/or publication.Laboratory facilities were provided by the Nepal Academy of Science and Technology.

Table 1 .
GIPs of horses (N) = 102 from horses in lowland of Nepal.* p<0.05 (Fisher Exact Test), while comparing the rates between young and adult animals.RR: Relative Risk, CI: Confidence Interval.

Table 2 .
Assessment of risk factors among the horse population (N=102) p-values (Chi-Square Test/Fisher Exact Test.Risk Ratio: RR, Odds Ratio: OR, CI: Confidence Interval.*: <0.05, ns: not significant. horses (43), further molecular taxonomy would confirm the species of the current study.Notably, Anoplocepha perfoliata (20.6%) is the only cestode reported in this study.It was also reported from Libya (28) and Ethiopia (15.7%) (30).Its infection occurs via the oribatid mite ingestion while grazing on the contaminated pasture (44).In the context of trematodes, Gastrodiscus sp.(17.6%), a large intestinal amphistome, was also reported from Nepal (6.67%) (17) and India (7.14%) (29).prevalence of Entamoeba spp. in domestic buffaloes (47) and genetically confirmed E. bovis from wild water buffaloes (48) are circulating in the same ecological niches.For the first time, other protozoa like Iodamoeba sp.(2.9%) and Blastocystis sp.(3.9%) have been reported from horses in Nepal.Even though Iodamoeba sp.commonly infects humans, non-human primates, and swine, its occurrence in horses might be interesting.Hypothetically, we assumed its acquisition via crosstransmission from either humans or pigs because this parasite has already been reported in these hosts in the same geography (15,24).The presence of Blastocystis sp.(3.9%) was reported from Colombia (43.8%) and Thailand (12.5%).To date, 12 Blastocystis subtypes have been confirmed in horses, out of which seven have already been reported in humans and are potentially zoonotic (49).Another zoonotic parasite Cryptosporidium sp.(23.5%), was reported in the current horses.Similar reports are present in Libya (33%) (28) and Italy (37.8%) (50).Studies reported C. parvum, C. hominis, C. muris, C. horse genotype, C. tyzzeri, C. erinacei, and C.

Table 3 .
Concurrency of GIPs in young and adult horses population (N=102).The p-values were calculated using Fisher Exact Test.Risk Ratio: RR, Odds Ratio: OR, CI: Confidence Interval.*: <0.05, ns: not significant.
In this study, polyparasitism was dominant over monoparasitism.It is difficult to explain the effects of multiple infections as each co-infecting member induces various responses (65).These responses may be positive, negative, or neutral (66).For example, co-