Gastrointestinal parasites of indigenous pigs (Sus domesticus) in south‐central Nepal

Abstract Background Intestinal parasites have a significant impact on productivity of pigs. Additionally, presence of zoonotic parasites in pig faeces used as fertilizer and ingestion of raw or undercooked pork products originated from parasite‐infested pigs pose a risk to human health. Objectives The aim of the study was to estimate the prevalence and diversity of gastrointestinal (GI) parasites in indigenous pigs (Sus domesticus) maintained under traditional rearing system in Nepal. Methods Fresh faecal samples (n = 100) were collected from the pigs of varying age and sex maintained in 18 small‐scale farms in south‐central Nepal. Samples were processed using various standard methods and examined for parasite eggs, cysts or oocysts. Results Prevalence of GI parasites in indigenous pigs was 91%, comprising of 14 different genera of protozoans and helminths. Male pigs generally had a higher (97.5%) prevalence of GI parasites than females (87%). While 90% of the suckling and weaner piglets were positive for the GI parasites, all growers and 85% the adult pigs were infected with the parasites. Entamoeba spp. were the primary protozoans in all age groups. Strongyloides sp. was more prevalent helminths in suckling and weaner piglets, whereas Ascarid spp. were higher in both growers and adults. Triplet infection was higher (33.3%) in suckling and weaner piglets, while quadruplet and pentuplet infections were higher (p < .05) among growers (46.7%) and adults (30%), respectively. Conclusions The indigenous pigs harbour a higher prevalence and greater diversity of GI parasites. GI parasitism varies by sex and age of the pigs.


| INTRODUC TI ON
Pigs are small, adaptable, rapidly growing and multiparous livestock species reared globally for their meat value. Their manure can also be used to produce biogas and as a soil fertilizer. For a long time, pig husbandry practices were restricted to very few ethnic communities in Nepal due to religious constraints and misconceptions of pork meat (Nidup et al., 2010). However, with increased urbanization, and cross-culture experiences, people's perception regarding pig rearing and pork consumption has changed in recent years. That might be why the annual demand of pork in Nepal was reported to increase by 10% (Thapa, 2018), and the pork was in the 4th position of all the meat consumed across the country (Pant, 2017).
The Nepalese pig industry is relatively small, as represented by its Bangur, and others (Anonymous, Unknown). It is usually common in Nepal to raise the indigenous pigs under traditional farm management (Paudel et al., 2014). Their feed mainly includes kitchen waste, garbage, roots, green forages and locally available grains like ricebran, maize, husks, and others (Nidup et al. 2010). In contrast, exotic breeds of pigs have been imported in Nepal since 1957 AD to upgrade the native pigs via crossbreeding. They include Tamworth, Saddleback, Fauyen, Landrace, Hampshire, Duroc and The Large White (Yorkshire White; Anonymous, Unknown) and are raised primarily by urban and commercial farmers with special care and feeds (Paudel et al., 2014). Whatever the farming breed and management practices adopted, the pork industry is believed to play a substantial role in meeting the food security and boosting the economy of Nepalese farmers.
While pig farming in Nepal supports the sustainable livelihood of farmers, it is still facing many challenges regarding productivity, profitability and sustainability. Some of the existing challenges are lack of sufficient meat-processing factories and clean slaughterhouses, high feeding cost, poor market linkage, lack of adequate breeding farms, presence of primitive breeds (genetic threats), primitive model of farming practices and parasitic diseases (Gatenby & Chemjong, 1992;Paudel et al., 2014;Nidup et al., 2010). Among parasitic diseases, the gastrointestinal (GI) parasites are responsible for a substantial loss on the efficiency of pig production by competing directly for nutrients required for optimum growth and reproduction or by causing tissue injuries (lesions), leading to condemnation of organs during meat inspection, poor feed conversion, diarrhoea and dehydration or even death of the animals.

F I G U R E 1 Map of the study area
showing the locations of sample collection | 3 ADHIKARI et Al.

| Study area
The study was conducted from June to September 2019 in Shaktikhor area of Kalika Municipality (251-1,003 m above sea level, a.s.l.) in the Chitwan district of Nepal ( Figure 1). The area lies in the southcentral part of Nepal and is approximately 182 km away from the capital city Kathmandu. The region experiences a tropical to subtropical climate with an annual mean temperature of 29.3°C in summer and 9.4°C in winter. The average yearly precipitation in the area is 199 mm (Adhikari et al., 2020). The indigenous/ethnic people living in the study area prefer traditional small-scale pig rearing. Most of the household rears two to ten locally available indigenous breed Sus domesticus ("Chwanche" in the Nepali language; Figure 2) under scavenging management (Pant, 2017)  into suckling and weaner (piglets <4 months), growers (4-8 months), and adults (>8 months) groups as previously described (Sharma et al., 2020). The inclusion criteria of the survey farms were accessibility of the farms and voluntary participation of the pig farmers.

| Sample collection
Approximately 10 g of fresh faecal samples were non-invasively collected from 100 pigs (one sample per animal) of different ages and sexes owned by 18 smallholder farmers. Briefly, the topper layer of the stool that has not touched the ground immediately after defecation was collected with gloved hands. Utmost care was taken to avoid contamination of the samples. The samples were then placed into 20 ml screw-cap sterile vials containing 2.5% weight/volume (w/v) potassium dichromate. To avoid duplicating the samples, the sampled pigs were marked with identifiers. The samples were immediately transported to the Animal Research Laboratory (ARL) of the Nepal Academy of Science and Technology (NAST) for analysis. Upon receipt at the ARL, the samples were stored at 4°C until further processing.

(b) (a)
An adult female pig in wet soil inside a pen.
(c) Pigs in the wood-built pen.
(d) A grower feeding on cattle and buffalo dung.
filtered through a tea strainer into a 50 ml beaker using 43 ml of floatation fluid made up of 45% NaCl. Then, 0.15 ml of the filtrate was placed into each depth of the McMaster slide using a pipette, and the slide was examined using a 100× total magnification under a compound microscope. All oocysts or eggs in both chambers were counted, and their sum was multiplied by 100. Finally, the resulting product was divided by 2 to calculate the epg or opg.

| Data analysis
Data were expressed as a number of parasite positive samples (frequency) and prevalence in terms of percentage using the Microsoft

Excel 2016 for Windows (Microsoft Corporation). The GraphPad
Prism Software Windows v5.00 (GraphPad Software, Inc.) was used to perform statistical analysis of the data. The chi-square (χ 2 ) test was used to compare the differences in the prevalence of GI parasites by sex and age of the pigs. For all analyses, p < .05 (at 95% confidence interval) was considered statistically significant.

| RE SULTS
A total of 91 faecal samples (91%) had one or more GI parasites.
Regarding age-wise distribution, we found a 90% prevalence of enteric parasites in suckling and weaner piglets, 100% in growers and 85% in adult pigs (Table 1). Entamoeba spp. were primary protozoa in all three age groups. Regarding helminths, Strongyloides sp. was higher in suckling and weaner piglets, whereas Ascarid spp.
were higher in both growers and adult pigs.
The overall prevalence of GI parasites was higher in male pigs (95.7%) than the females (87%), without statistical significance were from adults, whereas 6.7% samples were from growers. These parasites were found in only one sample of sucklings and weaners  ( Table S3). Age-and sex-wise distribution of polyparasitim in those pigs was statistically significant (p < .05; Table 2).
One of the reasons for the higher prevalence of GI parasites in this study could be due to the poor rearing condition of the pigs.
Many farmers in the study area were unaware of effective pigrearing and farm management practices. In Nepal, pig farmers usually do not pay much attention to the indigenous breeds compared with the crossbreds (Gatenby & Chemjong, 1992). The sampled indigenous pigs were fed inadequately and untimely and kept in wood-built and untidy pens with the porous floor ( Figure 2). These components generate higher moisture and attract mechanical vectors such as flies in the pens that may have contributed to the acquisition of diverse parasites.
The methodological variation could be another potential contributor to the higher prevalence of enteric parasites in the study. We have applied multiple techniques for faecal analysis, including direct wet mount, floatation, sedimentation, acid-fast staining and sporulation, which might have cumulatively contributed to higher detection rates of the enteric parasites. More importantly, pigs themselves are the natural reservoir of many GI parasites recorded in this study (Ji et al., 2019;Schuster & Ramirez-Avila, 2008). Indigenous breeds also naturally possess a high GI parasitic rate (Murthy et al., 2016), possibly contributing to a high prevalence rate in the faecal samples studied.
We found the highest parasitic prevalence (100%) in growers and the lowest (85%) in adult pigs. Similar results were also reported from Tanzania (Nonga & Paulo, 2015) and India (Sharma et al., 2020). The higher prevalence of enteric parasites in the growers might be due to their higher exposure to the outside environment. After completing the weaning period, they need to search for food themselves; thus, they forage on grasses in the open fields and get exposed to various parasites.
On the other hand, the lower prevalence of GI parasites in adult pigs could be due to the enhanced resistance and susceptibility to reinfection governed by an increased immunological memory (Brake, 2003).
The findings of a higher prevalence of GI parasites in males lie in agreement with other published reports (Dey et al., 2014;Sharma et al., 2020;Sowemimo et al., 2012). The lower GI parasitism in females in our study could be due to deworming practice performed by few farmers (field observation) for adult pregnant pigs in pre-farrowing condition (2 weeks before farrowing). Additionally, testosterone hormone, which acts as an immunosuppressant (Salvador et al., 1996) Barbosa et al., 2015). Various amoeba species have been previously reported in pigs, such as E. suis, E. histolytica and E. polecki (Ji et al., 2019;Mendoza-Gómez et al., 2015). In addition to the pathogenic species, non-pathogenic amoeba like E. coli and I.
We also assessed the polyparasitism of the GI parasites in pigs.
The current rate of 85% concurrency and the maximum number of faecal samples with triplet to pentuplet infections indicate dominant polyparasitism and high intensity of endoparasites in the pigs.
Multi-parasitism is associated with greater exploitation of the host defense mechanism (Schjørring & Koella, 2003) and may impact negatively (Vaumourin et al., 2015). For example, mixed intestinal infections, including Cryptosporidium sp., led to piglet death in Australia (Morgan et al., 1999). Similarly, cystoisosporiasis with any other parasitic or bacterial or viral infections leads to excessive mortality (Worliczek et al., 2007). The parasitic severity measured by epg or opg suggests that pigs were severely infected with various species of Eimeria, Cystoisospora, Ascaris, strongyle, Strongyloides, hookworm and Trichuris. Thus, the effects of mixed infections by all pathogen communities, rather than a single species, should be evaluated while assessing the severity of infections and the resulting pathologies (Serrano & Millán, 2014;Zhang et al., 2016).

| CON CLUS IONS
The indigenous pigs (S. domesticus) maintained under traditional rearing system in Nepal had a higher prevalence (91%) GI parasites (protozoans and helminths). GI parasitism varied by sex and age of the pigs. Our findings suggest that indigenous pigs under traditional management can harbour a wide variety of GI parasites with higher prevalence. Thus, periodic trainings on practices for healthy and sustainable pig husbandry should be conducted targeting rural pig farmers. Additionally, deworming practices could help them to achieve maximum productivity and reduce the risk of transmitting potential pig-borne zoonotic diseases.

ACK N OWLED G EM ENTS
The authors are thankful to Mr. Ganga Ram Regmi and Mr. Purna Ale of Third Pole Conservancy (Bhaktapur, Nepal) for their support in fields and preparing geographical information system (GIS) maps; Dr. Nirajan Bhattarai (Associate Professor, Agriculture and Forestry University, Rampur, Chitwan, Nepal) for his support in pig identification. We acknowledge the support of the authorities at Kalika

Municipality (Chitwan, Nepal) and Kalika Municipality Veterinary
Services by providing permission to conduct the study and the Animal Research Laboratory for providing laboratory studies.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest. Writing-review & editing.

E TH I C S A PPROVA L
The authors have adhered to ethical policies of the journal. We declare that the study was conducted on the faecal samples of naturally infected pigs and no experimental infection of the pigs was performed during the study. The fieldwork was conducted with permission from the Kalika Municipality (Chitwan, Nepal) and the Kalika Municipality Veterinary Services (Chitwan, Nepal) (Permission no. 07/2075/76).

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in the main text of the manuscript and the Supporting Information files.