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A Survey for Zoonotic and Other Gastrointestinal Parasites in Pig in Bali Province, Indonesia

Pigs have potentially to transmit zoonotic gastrointestinal parasite disease both caused by protozoa and worm. The aim of this study was to identify gastrointestinal parasites that were potentially zoonotic in pigs in the province of Bali. A total of 100 fresh feces samples was collected from several pig farms in Bali, from Badung and Tabanan districts, each consisted of 50 samples. Pig feces samples were examined for the presence of eggs worms, cysts and oocysts for protozoa based on the morphology and size. Identification for protozoa and worms used native, sedimentation and sucrose flotation methods. Parameters measured were sex, feed and cage management. The result showed that the characteristic parameters for pigs in both district were generally female. Cage management for raising pigs mostly used group cage. Feed that provided in both district mostly used bran and concentrate. All of 100 pig feces samples that examined positive for parasites. There were 8 types of gastrointestinal parasites that have been identified. Four types of protozoa found were Entamoeba sp. (99%), Balantidium sp. (79%), Eimeria sp. (78%), Blastocystis sp. (69%) and four types of worms were Ascaris sp. (20%), Trichuris sp. (20%), Strongyloides sp. (19%), and Oesophagostomum sp. (8%). All pigs were infected with two or more parasites. The prevalence of parasitic gastrointestinal infections was different for each district, six genera (Entamoeba sp., Balantidium sp., Blastocystis sp., Eimeria sp., Oesophagostomum sp. and Trichuris sp.) were higher found in Tabanan district and the two genera (Ascaris sp. and Strongyloides sp.) were higher in Badung district. Oesophagostomum sp. was only found to infect pigs in Tabanan district. The conclusion is gastrointestinal parasites that found in pigs at Badung and Tabanan district Bali Province mostly have zoonotic potential.


INTRODUCTION
Pigs are one of the commodities in the livestock sector, which has great potential to be developed in the recent decades. The pig population in Indonesia continues to increase along with the increasing number of large-scale pig farms and individual pig farmers. One of the regions in Indonesia where most people raise pigs is in Bali Province. Bali Provincial Livestock Service 1 reports that the total pig population in 2016 reached 803,517. In Bali Province, pigs are an important commodity and most people in Bali maintain pigs as their primary and secondary business. In addition, pigs also play an important role in fulfilling daily food needs and as a complement to religious ceremonies. 2 Generally, pigs in Bali are traditionally raised with low nutritional value and poor hygiene. This condition make pigs are more vulnerable to various diseases and has potential to spread the diseases. 3 The existence of the diseases can cause considerable economic losses for pig farmers. Losses include a decrease in production due to inhibition of livestock growth and increase medical costs. 4 One of the diseases that can infect pig is gastrointestinal parasites. Economic losses caused by gastrointestinal parasites were significant, but farmers may not realize it because the symptoms tend to be subclinical and pigs may still look healthy. 5 Gastrointestinal parasites in pigs are protozoa and worms. The types of protozoa that can infect gastrointestinal tract of pigs include Entamoeba sp.; Balantidium sp.; Eimeria sp.; and Isospora sp. 6 Recent study by Yoshikawa et al.,7 in East Nusa Tenggara found the presence of protozoa Blastocystis sp. as much as 87.1%. Research about Blastocystis sp. in pigs in Bali Province, previously have not been reported. According to Suryastini et al., 8  Some gastrointestinal parasites in pigs have potentially to transmit zoonotic diseases to human. According to Schar et al., 9 there are five gastrointestinal parasites that can be detected in pigs with zoonotic potential, were Ascaris sp., Trichuris sp., Capillaria spp., Balantidium coli and Entamoeba sp. In addition, Wang et al., 10 stated that Blastocystis sp. in pigs also had zoonotic potentially. It will certainly have an impact on the animal welfare as well as pig farmers and surrounding communities close to the farm area. Therefore, the aims of this study was to determine zoonotic and other gastrointestinal parasites in pig at Bali province, Indonesia based on fecal examination and discuss their zoonotic potential.

Study Area
This study was conducted in two district in Bali: Badung and Tabanan districts. In Badung district Indonesian Journal of Tropical and Infectious Disease, Vol. 8 No. 1 January-April 2020: 55-66 samples were taken in North Kuta sub-district, and in Tabanan district samples were taken in Baturiti sub-district. Geographically, Badung district located between 08˚14'20" -08˚50'48'' South latitude and 115˚05'00" -115˚26'16" East longitude. North Kuta sub-district has an area of 33.86 km 2 with an altitude of 0-65 meters above sea level. North Kuta sub-district was located in the lowlands close to urban areas. Geographically, Tabanan district located between 08˚14'30" -08˚30'07" South latitude and 114˚54'52" East longitude. Baturiti sub-district has an area of 99.17 km 2 with an altitude of 465-2082 meters above sea level. Baturiti sub-district was located in the highlands of rural areas. Dark blue colour is Badung district and pink colour is Tabanan district (Figure 1). A total of 100 pig fecal samples were taken randomly, from Badung and Tabanan districts consisted each 50 samples. Samples collection were conducted from 15 -22 January 2018.

Feces Samples Collection
Feces samples from several pig farmers are taken directly using gloves from the ground and after defecation and accompanied by a veterinarian from the local livestock department. All feces samples were collected in urine steril container and were preserved in 2.0% potassium dichromate for protozoa examinations and 10% formalin for helminths examination, then stored in cool box for transportation. For each animal was recorded with different code. Parameters included sex, feed and cage management.

Examination of Feces Samples
Samples were observed at Veterinary Parasitology Laboratory, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya Indonesia. Samples were examined for eggs worm, cyst and oocyst for protozoa. Identification for protozoa and egg worm using native, sedimentation and sucrose flotation methods. Feces were diluted with aquadest and then filtered. For native examination, the feces sample is stirred first using a stirring rod and then a small portion of feces sample is taken and placed on the object glass and the lid uses a cover glass after that check under the microscope 400x magnification. For sediment examination, filtrate were centrifugation at 1.500 rpm for 5 minutes (by centrifuge HC 1180T 8 HOLE WITH TIMER, China), then removed supernatant. This step repeated until 3 times. Take the sediment slowly and place it on the object glass then cover with a cover glass. The remaining sediment was added with sucrose solution until complete 12 mL to be centrifuged at 1.500 rpm for 10 min in a 15 mL plastic tube. Floated was added sucrose solution until mouth of tube and was covered by a cover glass. After 5 min, cover glass was transferred to object glass, and the eggs of worm were observed at 100x magnification and the cysts and oocysts of protozoa were observed at 400x magnification for identification by light microscopy. Identification for both protozoa and worm were based on the morphology and size of the eggs, cysts or oocysts. 11,12,13

RESULTS AND DISCUSSION
A total of 100 pig feces samples from Badung and Tabanan districts Bali province, Indonesia were identified. Information from each pig characteristics were provided in ( Table  1). Table 1 shows that the majority of the pig population in Tabanan and Badung districts are female and feed given to almost pigs is bran and concentrate. In Badung district some pigs were fed by leftovers from the kitchen while in Tabanan district some pigs were fed using plant origin ingredients, banana stems and taro leaves. The cage management in both districts mostly pig farmers are using group cages.
The results of identification indicate that the pigs in Bali are infected by 8 genera of parasites: Entamoeba sp., Balantidium sp., Eimeria sp., Blastocystis sp., Strongyloides sp., Trichuris sp., Ascaris sp. and Oesophagostomum sp. The morphological of the gastrointestinal parasites found in pigs in Bali Province are described in Figure 2.
All of the feces samples that have been examined, overall positive for gastrointestinal parasites ( Table 2). It means all of pigs were infected with gastrointestinal parasites. The highest prevalence was Entamoeba sp. (99%) respectively, was Balantidium sp., Blastocystis sp., Eimeria sp., Oesophagostomum sp. and Trichuris sp.) were higher found in Tabanan district and the two genera (Ascaris sp. and Strongyloides sp.) were higher in Badung district. Oesophagostomum sp. was only found to infect pigs in Tabanan.
One pig could infected with two or more parasites, even, the pigs were infected with seven species of parasites. In detail, the mix infection was presented in Table 3. Almost all of mix infections involve Entamoeba sp. There is no single infection.
In Indonesia, especially in Bali Province, studies about gastrointestinal parasites have been widely reported. However, most of these studies focus on one type of parasite. There have not been many studies that discuss about mixed infection between protozoa and worms in each pig. From the results of this study showed that gastrointestinal parasites in pigs in Badung and Tabanan districts found several parasites that have zoonotic potential.   Protozoa are the most common parasites that infect pigs in both districts. The higher prevalence of protozoa is dominated by Entamoeba sp. (100%). This result was higher than the study by Suryawan et al., 14 , which stated that out of 102 faecal samples of pigs in Papua, 34.2% were infected with Entamoeba sp. Research by Agustina et al.,.15 in Bali Province found that the prevalence of Amoeba sp. in pig fecal samples as much as 82.4%. This is certainly a concern, because all pig samples examined in this study were 100% positive for Entamoeba sp.
Entamoeba sp. is a protozoa that can infect human and animals. According to Matsubayashi et al., 16 17 from samples of pigs and human on four pig farms in Colombia showed that pig faecal samples were positive for E. coli, human faecal samples were also positive for E. coli and E. hystolitica / dispar. The presence of E. coli species in pigs and humans in Colombia shows the possibility of zoonotic potential of these parasites, so further molecular identification needs to be done. However, study by Agustina et al., 18 about the incidence of Entamoebiasis in pigs in Bali Province showed negative PCR results on E. polecki, so the zoonotic potential needs to be studied further.
In this study, another protozoa found was Balantidium sp. The prevalence in this study was 83%. In Indonesia, the incidence of Balantidium sp. in pigs had been widely studied by Agustina et al. and Yuliari et al., .18,19 with a prevalence of 61.2%, and 36.4%, respectively. In Korea 6 was recorded the prevalence of Balantidium sp. in pigs was 64.7%, in China 20 was 22.79%, and in Cambodia 9 was 15.8%.
Balantidium sp. is a protozoa that can cause balantidiosis. Balantidiosis is a zoonotic disease that can infect human and animals through the world. Pigs are natural reservoir for Balantidium sp. Transmission of the disease by faecal-oral route. In pigs it is usually asymptomatic and these protozoa live in the lumen of the cecum and colon. Transmission between human and animals can occur as well as humans to humans. In human, the incidence of balantidiosis can be asymptomatic. Severe infection can cause diarrhea and abdominal discomfort. Balantidiosis can occur due to several factors, such as sanitation, climate conditions, and community culture. An important factor in the spread of disease to humans is the presence of infected pigs and careless disposal of animal waste. This often occurs in poor rural areas where people tend to live near their livestock, so the disease is easily spread. Some sectors that have a high risk of being infected by Balantidium sp. are veterinarians, animal handlers and butchers. 21,22 Eimeria sp. is a protozoa that can cause coccidiosis. The prevalence of Eimeria sp. in this study was 83%, higher than the study Yuliari et al., 19 in pigs in Papua, Indonesia, with an average prevalence was 68.2%. In Bali Province, the prevalence of Eimeria sp. in pigs was reported by Agustina et al., 15 as much as 54.8%. The incidence of coccidiosis in several countries has also been reported 13,20,23,24,25 with a prevalence 16.53%, 16.7%, 47%, 89.2% and 3%, respectively. Coccidiosis in young pigs can cause diarrhea and can be predispose to secondary infections by viruses or bacteria. In severe cases, pigs can become dehydrated with a 10-59% chance of death. Animals that have been repeatedly infected have no clinical symptoms, and can transmit to other animals and pollute the surrounding environment. 15 Research about Blastocystis sp. in pigs in Bali Province, previously have not been reported. In this study, the prevalence of Blastocystis sp. in pigs was 60%. In Indonesia, research on Blastocystis sp. was reported for the first time 7 and Blastocystis sp. was found in humans, pigs, chickens and rodents in the Winyapu area, Southwest Sumba District, East Nusa Tenggara Province, and evidenced by PCR methods. So far, there are 17 Blastocystis sp. subtypes that have been identified based on gen analysis of small subunits ribosomal RNA (SSU rRNA). 26 Humans can be infected by 9 subtypes (ST1-ST9). 27 In China 28 was reported that there were 3 zoonotic subtypes in pigs, namely ST1, ST3 and ST5, which showed that Blastocystis sp. in pigs could be zoonotic.
Several factors that related to the emergence of Blastocystis sp. infection are lack of the environmental hygiene, poor community sanitation, socio-economic status and lifestyle. Blastocystis sp. can infect humans and some animals including pigs, cows, monkeys and chickens. Some zoonotic subtypes of these animals have been isolated, therefore, they can act as reservoir hosts. Transmission can occur from human to human, from human to animal and from animal to human by faecal-oral route. 29,30 In this study found various types of nematode worms namely Strongyloides sp., Trichuris sp., Ascaris sp. and Oesophagostomum sp. This result is also evidenced by the existence of investigations in Indonesia found various types of worms that often infect pigs. Study by Agustina 31 in pigs in Bali found Oesophagostomum sp. with the prevalence of 47.5%. In addition, research by Fendryanto et al., 3 on piglets in Bali found the prevalence of Ascaris sp., Trichuris sp. and Strongyloides sp. with the prevalence of 33.2%, 14.0% and 57.6%, respectively. In Poland, study by Wictor and Jarosz 32 noted the prevalence of worms in pigs was found Ascaris sp. Strongyloides ransomi (23.3%), Ascaris suum (13.3%) and Trichuris suis (6.6%). Research by Nonga and Paulo 34 in Tanzania showed that differences in the prevalence of gastrointestinal worms in some areas may arise due to differences in environmental conditions that are conducive to the parasite survival, the number of definitive hosts infected, type of feed and animal diet and the hosts immune system.
Strongyloides sp. is an important parasite that can be infected most of the suckling piglets. The worms predilection is in the small intestine. Common clinical symptoms that may occur are diarrhea followed by progressive dehydration. In severe infections, death usually occurs before piglets are between 10 and 14 days old, but if piglets can survive, dwarfism can occur. Recent research by Giang et al., 35 states that the type of Strongyloides sp. in pigs in Vietnam based on molecular identification is S. ransomi. S. ransomi has a similar morphology to S. papillosus, but in molecular analysis based on 18S rDNA, S. ransomi is close to S. venezuelensis. The zoonotic aspect and importance of Strongyloides sp. in veterinary medicine are discussed more detail in Thamsborg et al., 36 which states that until now S. ransomi in pigs has not been zoonotic, but there are other species such as S. stercoralis in dogs have zoonotic potential to humans.
Trichuris sp. is a type of worm that commonly infect pigs and live in the large intestine. Pigs are considered as the natural host of Trichuris sp, although primates and humans may be infected. Trichuris sp. infection can cause ulceration in the lining of the intestinal mucosa, damage to blood capillaries and secondary infections can occur by bacteria. Clinical symptoms in pigs include anorexia, slimy and bloody diarrhea, dehydration and death occur in severe cases. Trichuris sp. can survive for several years outside the hosts. So far, it is still a question of whether or not Trichuris sp. is zoonotic. According to Nejsum et al., 37 stated that the species Trichuris trichiura in humans can be found in pigs, but until now most worms did not survive. This shows that human crossinfection can occur with T. suis in pigs under experimental conditions.
Ascaris sp. is disease that can cause ascariasis and commonly found in pigs. This typical worm species also found in wild pigs. If pig infected with a severe infection, intestinal obstruction can occur, loss of appetite, vomiting, jaundice and death. In the case of moderate infection can occur low appetite, low food efficiency and slow growth. Ascaris sp. is zoonotic and can infect humans and other mammals by consuming food or water contaminated by infective eggs. Ascaris sp. eggs in a dry environment can last 2 to 4 weeks, while in a humid and cold environment they can survive eight weeks and become an infective stage in the environment. After ingestion, eggs hatch into larvae through the intestinal wall, pass through the liver and migrate to the lungs, and adult worms have a predilection in the small intestine. 38 The occurrence of zoonosis Ascaris sp. has been reported 39 which identified 14 cases of ascariasis in humans in contact with pigs in Maine, USA. In addition, research conducted by Nejsum et al., 40 stated that ascariasis is a case of zoonosis in Denmark, where humans are in direct contact with pigs and pig feces.
In t hi s s t u d y, the p r e v a l e n c e of Oesophagostomum sp. only found in Tabanan district. Oesophagostomum sp. is known as a worm nodule that has predilection in the large intestine in cecum and colon. Oesophagostomum sp. worm infection occurs when pigs eat plants or foods that contaminated by infective larvae 31. Oesophagostomum sp. infection in pigs can cause lack of appetite, poor growth rates, easy secondary infection and can cause death 41 . So far there have been no studies that discuss the possibility of zoonosis of Oesophagostomum sp. in pigs.
In this study, all pig were infected with mixed parasite protozoa and worms. According to Tolistiawaty et al., 42 parasitic infections generally occur due to the weakness resistance of the animal to parasites. Mixed infections often occur, and making it difficult to know the specific symptoms that seen. Infection that occurs is usually caused by several types of worms in the intestine and other organs. The way of animal treatment also very influential on the incidence of gastrointestinal parasitic infections. This is supported by research from Supriadi et al., 43 which was stated that gastrointestinal parasitic infections in pigs can be caused by poor management. Poor cage sanitation is also a factor that increases the risk of parasitic infection and does not rule out the possibility of transmission to humans, especially for pig owners (zoonosis). In addition, according to Roesel et al., 44 stated that the most important factors associated with gastrointestinal parasitic infections in pigs are related to sanitation, especially cleaning of pig stool regularly from the cage and the use of disinfectants.
In Badung and Tabanan districts, most people use group cages to raising pigs. This type of maintenance system includes intensive maintenance where the pig is caged in a cage. According to Lai et al., 20 raised pigs traditionally have a higher prevalence of the disease, this is because intensive pig farming has better maintenance management. Although intensive maintenance implements better management, it seems that it cannot help reduce the incidence of disease infection effectively. The possibility of a parasitic infection occurs due to a lack of public awareness about the good sanitation, besides that habit from pigs by eat in soil contaminated with faeces can be predispose to infection. Research by Mutua et al., 45 stated that pig needs energy, amino acids, minerals, vitamins and water. These elements are needed for the process of growth, reproduction and lactation.

CONCLUSION
Gastrointestinal parasites that found in pigs in Badung and Tabanan districts Bali Province mostly have zoonotic potential. These parasites included Entamoeba sp., Balantidium sp., Eimeria sp., Blastocystis sp., Strongyloides sp., Trichuris sp., Ascaris sp. and Oesophagostomum sp. This study is expected to provide information to improve the hygiene and sanitation in terms of raising pigs, to provide a basis for further control and treatment in pigs that infected with gastrointestinal parasites as well as providing information about zoonotic potential that can arise.