A parasitological survey and the molecular detection of Entamoeba species in pigs of East Java, Indonesia

Background and Aim: In several countries, two Entamoeba porcine species, Entamoeba suis and Entamoeba polecki (subtype 1 and 3), have been detected using molecular methods and identified pathogenicity associated with enteritis. However, globally, Entamoeba infection prevalence in pigs is extremely limited. This study aimed to coprologically and genetically examine pig parasites to estimate prevalence of Entamoeba in three pig farms in East Java, Indonesia. Materials and Methods: Hundred porcine fecal samples (Landrace) were collected from three East Javan farms in well-known swine industry regions. Fecal samples were examined under a microscope after sugar-flotation centrifugation, and molecular species and subtype identification were performed using polymerase chain reaction (PCR) and primer pairs targeting small-subunit ribosomal RNA. Results: Microscopy examinations identified parasites in 89/100 fecal samples; Entamoeba spp. cysts were the most frequent in these samples. Polymerase chain reaction showed that 58 samples were comprised of mixed Entamoeba suis and Entamoeba polecki, 22 E. suis alone, and nine E. polecki alone infections. Epolec F6–Epolec R6 primers successfully amplified E. polecki ST1–4 subtypes, while Epolecki 1–Epolecki 2 amplified only the E. polecki ST1 subtype. Entamoeba polecki ST1-specific primers successfully detected the ST1 subtype in 19/67 E. polecki positive samples. Conclusion: Entamoeba spp. prevalence in Indonesian pigs was previously shown to be high. On coprological examination of East Javan pigs, we detected high Entamoeba spp. levels, in which we genetically identified as E. suis (80.0%), E. polecki (67.0%), and E. polecki ST1 (19%).


Introduction
Entamoeba genus parasites are typically found in many vertebrate species, including humans and livestock [1], while several species, which infect pigs include Entamoeba suis, Entamoeba polecki, Entamoeba histolytica, and Escherichia coli [2][3][4]. The parasitic life cycle generally comprises two steps; trophozoites which represent motile and proliferative stages and cysts which represent environmental stages, but some species are excluded as they lack encysts [2,3]. Cysts are shed in host feces and are resistant to disinfectants, thereby providing new host infection sources through oral routes. Conventionally, species classification within the genus was based on derived hosts and morphological data, such as zoites size or the number of nuclei in mature cysts [5]. Recently, to resolve difficulties related to morphological similarities, molecular analyses have been extensively used to distinguish species and genotypes [5][6][7][8].
In the genus, most Entamoeba spp. are believed to be harmless; however, some species such as E. histolytica in humans and animals, and Entamoeba invadens in reptiles, are highly virulent [1,9].
To date, two Entamoeba spp., E. suis and E. polecki, have been reported in pigs [10,11], although E. histolytica has been shown to infect mini pigs [12] experimentally. Several Entamoeba species exhibit zoonotic potential and include E. polecki, E. histolytica, and E. coli [3][4][5]. Entamoeba suis predominantly infects pigs, while E. polecki has been detected in multiple hosts, including humans and pigs. Sequence analysis of small-subunit ribosomal RNA (SSU rRNA) further subclassified E. polecki into four genetic subtypes (ST1-4) [5,13]. ST1 was found in pigs and humans; ST3 in pigs, humans, and birds; and ST2 and ST4 in humans and primates [5]. Previously, the infection and disease characteristics of E. suis and E. polecki in pigs have not been reported. However, species/genotypes infecting pigs have been implicated in severe lesions associated with enteritis [7,8]. Entamoeba suis invades the lamina propria and causes hemorrhagic colitis [14], while E. polecki induces refractory proliferative enteritis, which causes lethal lesions when combined with Lawsonia intracellularis or Salmonella enterica serovar Typhimurium [7,8,15,16]. Although these reports come from Japanese pigs, it was reported that coinfection with E. polecki (unknown subtype) and Brachyspira hyodysenteriae was associated with necrotizing typhlocolitis in a Spanish pig with severe diarrhea [17]. Globally, Entamoeba infections in pigs are extremely limited and parasitic pathogenicity cannot be fully assessed based on the aforementioned data. However, a surveillance program in Tangerang, West Java, Indonesia, identified a high porcine Entamoeba spp. prevalence; 81.1% E. suis, and 18.4% and 17.3% E. polecki ST1 and ST3, respectively [4]. These findings potentially suggest that some Indonesian pigs are frequently infected with these parasites, although no other data are available.
This study aimed to coprologically and genetically examine pig parasites to estimate prevalence of Entamoeba in three pig farms in East Java, Indonesia. We used several molecular methods (polymerase chain reaction [PCR], sequencing, and phylogenetic tree analysis) using SSU rRNA as a marker. We also detected other coinfection gastrointestinal parasites.

Ethical approval
The research protocol was reviewed by our Local Animal Care and Use Committee (Ethics Clearance No. 1.KE.105.08.2021) under the guidance of the Ethical Clearance Commission Faculty of Veterinary Medicine, Universitas Airlangga, Indonesia.

Study period and location
The study was conducted from November to January 2021. The samples were collected from three pig farms in East Java, Indonesia. The samples were processed at the Laboratory of Biomolecular, Faculty of Veterinary Medicine, Universitas Airlangga.

Fecal sampling
Hundred porcine fecal samples (Landrace) were collected from three well-known swine industry regions in East Java; Farm A, Mojokerto; Farm B, Malang; and Farm C, Tulungagung. These farms were selected as they were the largest managed farms in each area (approximately 6000 pigs at Farm A, 10,600 at Farm B, and 14,400 at Farm C). At Farm A, 68 fecal samples (from 3 to 6-month-old animals) were collected immediately after defecation at a slaughterhouse. Fecal samples were randomly collected at the other farms: Farm B; 4 from <3-month-old, 7 from 3 to 6-month-old, and 5 from >6-month-old animals; and at Farm C, 1 from <3-month-old, 12 from 3 to 6-month-old, and 3 from >6-month-old animals. At all farms, 10-20 post weaned piglets were housed in pig pens on sawdust or soil floors until 3 months old. Pigs > 3 months old were reared individually in cages. Pigs were treated monthly for nematodes using anthelmintics such as albendazole and mebendazole. Animals showed no clinical symptoms, including diarrhea during sampling (91 normal and nine soft feces samples). Pig ages during sampling were recorded. Feces were stored in plastic bags at 4°C until examination.

Parasitological examination
Fecal samples were examined under microscopy after sugar-flotation centrifugation according to a modified method [18][19][20]. Briefly, 5-10 g fecal sample was diluted in distilled water and filtered through gauze. After centrifugation, a sugar solution (specific gravity = 1.2) was added to the sediment and the samples were centrifuged. Parasites floating on the sugar solution surface were recovered using a Pasteur pipette and washed in distilled water. Finally, purified parasites were resuspended in 1 mL phosphate-buffered saline and stored at 4°C. Next, a 15 μL aliquot of parasite solution was placed onto a glass slide and smears were examined under a light microscope (Olympus, Japan) to enumerate parasites [20,21]. Samples positive for Entamoeba cysts underwent molecular analyses.

Bioinformatics
To confirm sequencing outputs, the E. polecki amplicon from a Mojokerto sample (MJK_B28) was purified using a QIAquick PCR Purification Kit ( Qiagen, Germany) and subjected to double-directional sequencing using PCR forward and reverse primers of E. polecki ST1 on an ABI PRISM 310 Genetic Analyzer ( Applied Biosystems, USA). Sequencing was verified by inverting sequencing results from the reverse primer and aligning them to the forward primer sequence in the Clone Manager Professional 9 program (Version 9 for Windows, Scientific and Educational Software; http://www. scied.com) and comparing with a GenBank reference sequence (E. polecki pig, accession No. LC230018).
To identify closely related DNA sequences, the MJK_B28 sequence was used as a query in a National Center for Biotechnology Information nucleotide BLAST search; (http://www.ncbi.nlm.nih. gov>blast). The MJK_B28 sequence and all query hits with identities of >90% to the original (MJK_ B28 sequence) were included in the dataset. Dataset sequences were then multiply aligned in ClustalW2 (http://www.ebi.ac.uk) [24] and a phylogram was generated using the neighbor-joining method [25] in MEGA6 [26].

Results
Microscopy examinations identified parasites in 89/100 fecal samples (Table-2). Entamoeba spp. cysts were the most frequently observed cysts, although samples may have included other organisms similar to cysts as iodine staining was not performed. The average      (Figures-1a-c).
Entamoeba polecki Mojokerto (MJK_B28) alignment to gene bank sequence data (LC230016 and LC230018 accession numbers) showed 96% identity (Figure-2). Phylogenetic tree analysis showed that E. polecki from Mojokerto (MJK_B28) was identified in the E. polecki ST1 group and was relatively close to E. polecki in humans (FR686383) and E. polecki in pigs from other countries (MK801429, AF149913, MK801460, LC230016, and LC082305 accession numbers). This analysis also used out groups (less closely related to the in-group) with several related sequences, such as several Entamoeba species in humans (Entamoeba gingivalis, E. histolytica, and Entamoeba dispar), Entamoeba ranarum (frog), Entamoeba invader (snake), and E. coli (human) (Figure- . Several related sequences, but with significantly less identity, were used as outgroups. Sequences were multiply aligned in ClustalW2 [24]. The phylogram was assembled using the neighbor-joining method [25]. Entamoeba spp. subtypes are indicated and bootstrap values from 1000 replications are shown for each branch. The scale bar indicates a phylogenetic distance of 0.05 nucleotide substitutions/site.

Discussion
This is the first report showing Entamoeba spp. cyst percentages in pig feces. Average and maximum numbers were not found to be differences among pig ages; however, a thorough statistical analysis was not performed due to low study animal numbers. The majority of feces samples were normal and pigs exhibited no clinical symptoms. During E. histolytica infection, cysts are generally found in the stool, while trophozoites are typically found in watery or dysenteric feces concomitant with clinical symptoms [27]. Furthermore, species-specific immunity during primary infection with Entamoeba spp., such as E. histolytica, may have key resistance roles against reinfection [28,29]. In our study, cysts identified in formed stool, with no clinical signs in animals, may have reflected acquired immunity following the previous infections; however, further investigations with more samples, especially from younger animals (e.g., < 3 months old), are required to elucidate parasite pathogenicity, especially during initial infections.
To date, Entamoeba spp. have been detected in fecal specimens (ranging from a few to approximately 10) in several countries, for example, Indonesia, Sweden, the United Kingdom, and Germany [5,10,30]. Recently, in more than 500 pigs in China, E. suis, E. polecki ST1, and ST3 were identified by Li et al. [31] in 13.0%, 45.2%, and 34.1% of samples, respectively, while Ji et al. [3] observed that 0.8%, 38.2%, and 10.0% of samples were positive, respectively. In West Java, the prevalence of E. suis, E. polecki ST1, and ST3 in 196 fecal samples was 81.1%, and 18.4%, and 17.3%, respectively [4]. In our study, the Mojokerto amplicon, using species-specific E. polecki ST1 primers, showed 19% (19/100) positivity. The MJK_B28 sample was confirmed by sequencing and a phylogenetic tree identified 96% homology with E. polecki ST1 in humans (FR 686383 and LC 230016) ( Figures-2 and 3). These are a new finding of mixed infection with E. suis and E. polecki ST1, and coinfection with Eimeria spp., Isospora suis, Trichuris suis, and Ascaris suum (Table-2), in Mojokerto, East Java, Indonesia. According to Stensvold et al. [5], E. polecki is a protozoan parasite in the digestive tract that attacks pigs, monkeys, primates, and birds and has zoonotic potential. Smallsubunit ribosomal RNA sequence analysis resulted in the further subclassification of E. polecki into four genetic subtypes (ST1-4) [3,4,7]. ST1 is found in pigs and humans, the most frequently reported zoonoses are ST1 and ST3, while ST2 and ST4 are specific subtypes in humans and non-human primates [5]. Our results were consistent with this report and confirmed high E. suis prevalence in Indonesian pigs, although surveillance of more pigs over a wider geographical area is required in future studies.
Other parasites, including Eimeria spp. oocysts or Cystoisospora suis, were identified in 24 samples, while A. suum and T. suis eggs were identified in 11 and 21 samples, respectively. All samples contained mixed Entamoeba spp. infections. Although few reports focusing on gastrointestinal parasites in Indonesian pigs have been published, Eimeria spp. or C. suis, T. suis, and A. suum prevalence is reported as 42.2%, 7.8%, and 11.8% in healthy pigs, and 38.6%, 52.3%, and 9.1% in dead pigs, respectively, in Central Papua (no Entamoeba spp. descriptions are provided) [32]. In another West Javan study, Eimeria spp. or C. suis, T. suis, and A. suum prevalence was reported as 79.1%, 4.6%, and 1.0%, respectively [4,33]. These findings suggest that coccidian prevalence, including Eimeria spp. or C. suis, is high in Indonesian pigs, with E. suis the most prevalent.
Gastrointestinal parasite infections are transmitted through fecal-oral routes and may be associated with farm management systems [34]. In our study, 10-20 piglets were reared in the same pen, suggesting that parasite transmission and initial infections had easily occurred. In addition, we conducted our study during the rainy season; therefore, parasite spread may have occurred more through contaminated rainwater. In future studies, we will compare parasite prevalence between rainy and dry seasons to understand the impact of different climate conditions. Because porcine Entamoeba spp. virulence remains largely uncharacterized, larger studies on younger or preweaned piglets are required to fully understand parasitic prevalence and clarify pathogenicity and associated effects on porcine productivity during breeding.

Conclusion
Entamoeba spp. prevalence in Indonesian pigs is too high. We detected high Entamoeba spp. levels during coprological examinations in East Javan pigs. Parasites were genetically identified as E. suis (80.0%), E. polecki (67.0%), and E. polecki ST1 (19%). Previously, a high E. suis prevalence was reported in Indonesian pigs; therefore, our results are congruent with these findings, although our sample numbers were low. In addition, we detected E. polecki ST1, which is a potentially zoonotic protozoan. In future studies, more samples are required to evaluate parasite pathogenicity, especially during initial infection stages in younger animals.