Identification of Zoonotic Balantioides coli in Pigs by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Its Distribution in Korea

Simple Summary Balantioides coli is a protozoan parasite that can infect humans, and its main reservoir is pigs. Recent studies suggested that one of B. coli variants, named variant A, has zoonotic potential. Previous studies have reported B. coli infection in pigs in different countries; however, the prevalence of the zoonotic variant is limited due to a lack of molecular information. In this study, we developed a molecular technique-based method that could differentiate B. coli variant A from B without sequence analysis. Using the method, 174/188 (94.6%) pig fecal samples collected in domestic pigs in Korea were positive for B. coli, and of the samples, 62 (33.7%) were the zoonotic variant. To our knowledge, this is the first study to develop a method to differentiate B. coli variants A and B without sequence analysis and to assess the molecular epidemiology of B. coli in pigs. Abstract Balantioides coli is a zoonotic protozoan parasite whose main reservoir is pigs. Recent studies have shown that B. coli variant A but not B has zoonotic potential. While B. coli infection has been reported in different animals and countries, the prevalence of the zoonotic variant is limited due to a lack of molecular information. Therefore, this study investigated the prevalence of B. coli in domestic pigs in Korea and assessed its zoonotic potential. A total of 188 pig fecal samples were collected from slaughterhouses in Korea. B. coli was identified by microscopy and molecular methods. B. coli was identified in 79 (42.9%) and 174 (94.6%) samples by microscopy and polymerase chain reaction (PCR), respectively. This study also developed a PCR-restriction fragment length polymorphism (PCR-RFLP) method to differentiate B. coli variant A from B without sequence analysis. Using this method, 62 (33.7%) and 160 (87.0%) samples were positive for variants A and B, respectively, and 48 (26.1%) samples were co-infected with both variants. Sequence and phylogenetic analyses showed a high genetic diversity of B. coli in pigs in Korea. To our knowledge, this is the first study to develop a method to differentiate B. coli variants A and B without sequence analysis and to assess the molecular epidemiology of B. coli in pigs. Continuous monitoring of zoonotic B. coli in pigs should be performed as pigs are the main source of human balantidiasis.


Introduction
Balantioides coli, the only ciliate protozoan parasite, can infect humans. The main reservoir of B. coli is pigs; however, it also infects other animals including non-human primates, cattle, buffalo, sheep, goats, rodents, and birds [1][2][3][4][5]. With its broad host range, B. coli is distributed worldwide, especially in tropical and subtropical areas. B. coli was first named Paramecium coli in 1857 (reviewed by [2]) and was transferred to the genus Balantidium in 1863 (reviewed by [2]). Recent advances in molecular techniques have revealed genetic differences between B. coli and other Balantidium spp., and B. coli has been moved to the genus Neobalantidium [4]. As Neobalantidium is synonymous with Balantiodies as proposed by Alexeieff in 1931, Balantioides was used as the correct genus name [6].
The transmission of B. coli to its host occurs via the fecal-oral route, and B. coli parasitizes the large intestine, cecum, and colon of its hosts [2,7]. Humans and animals are infected by ingesting B. coli cysts directly or indirectly through contaminated food and water. Infection does not generally cause clinical symptoms in immunocompetent animals or humans. However, in immunocompromised hosts such as patients with AIDS or co-infection with other pathogens, B. coli causes diarrhea, malnutrition, and other gastrointestinal symptoms [2,8]. While few clinical cases have been reported in animals, cases of human balantidiasis have been reported with dysentery as the main symptom [2,5].
There is no standardized diagnostic method for B. coli and general coproscopic examination methods based on floatation or sedimentation are used [7]. Due to its distinctive size and morphological characteristics, the diagnosis of B. coli is straightforward. However, microscope-based diagnosis has disadvantages such as low sensitivity, inability to evaluate genetic characteristics, and difficulty in differentiating morphologically similar pathogens (e.g., Buxtonella spp.) [2,8].
Recent advancements in molecular techniques have allowed for the investigation of different molecular characteristics of B. coli. Ponce-Gordo et al. [1] performed molecular analysis of B. coli based on the 18S-rRNA-ITS1-5.8S-rRNA-ITS2 regions (hereafter, ITS region) and reported at least two genetic variants; namely, variants A and B. Of these, B. coli variant A is considered to be zoonotic; however, studies on its association with animal species and distribution are insufficient due to a lack of molecular information [9]. Previous PCR-based studies have identified genetic variants of B. coli using sequence analysis with cloning, which requires significant labor, time, and cost [1,8]. To evaluate zoonotic B. coli in many samples from different regions, more precise, labor-, time-, and cost-efficient methods need to be developed. PCR-restriction fragment length polymorphism (PCR-RFLP) is one of the most commonly used tools to analyze the molecular characteristics of pathogens. It has been used for various purposes including species differentiation, pathogenicity prediction, and genotypic analysis [10][11][12].
Previous studies have reported the presence of B. coli infection in humans and pigs in Korea, some of which were related to clinical cases [13][14][15][16]. However, the studies diagnosed B. coli infection based on microscopic examination without molecular analysis. Therefore, molecular information on B. coli in domestic pigs and humans in Korea is lacking and the zoonotic potential of B. coli in Korea is unknown. Considering reports of human balantidiasis and the presence of B. coli in animals in Korea, the distribution and prevalence of zoonotic B. coli requires evaluation.
Therefore, the purpose of this study was two-fold. First, we developed a PCR-RFLP method to differentiate B. coli variants A and B. Second, we investigated the prevalence of B. coli and its genetic variants in pigs in Korea and molecularly characterized them.

Collection of Pig Fecal Samples
From May to November 2020, 188 pig fecal samples from 32 farms were collected from slaughterhouses in Korea ( Figure 1). Information on the rearing regions and sample collection dates were recorded. All pigs were raised for meat production and were approximately six months of age. Fresh fecal samples were collected directly from the large intestine by dissection to avoid cross-or environmental contamination, transported to the College of Veterinary Medicine, Chungbuk National University, Korea, and stored at 4 • C.

Microscopical Identification of B. coli
The transported fecal samples were homogenized using a sterilized wooden stick, and 1 g and 200 mg of fecal samples were taken for microscopic examination and DNA extraction, respectively. Microscopic examination was performed using the fecal flotation technique with sodium nitrate, as previously described [17].

DNA Extraction, PCR, Cloning, and Sequencing
Genomic DNA was extracted from the fecal samples using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was kept at -20 °C until further analysis.
For molecular diagnosis, PCR targeting the ITS region of B. coli was performed as previously described [1]. In brief, PCR was performed using AccuPower ® HotStart PCR PreMix (Bioneer, Korea) containing 2 μL template DNA, 1 μL of each forward and reverse primer (0.5 μM final concentration), and distilled water to a final volume of 20 μL. The PCR conditions were as follows: initial denaturation for 5 min at 94 °C; 30 cycles of 1 min at 94 °C, 1 min at 56 °C, and 1 min at 72 °C; and final extension for 5 min at 72 °C. After verification of the PCR conditions using a positive control, PCR was performed without a positive control to avoid cross-contamination, with distilled water included as a negative control in each experiment. The expected amplicon sizes were 528-or 537-bp according to the B. coli variant. The PCR products were visualized by gel electrophoresis.
For sequencing and phylogenetic analysis, 11 samples were randomly selected and amplicons of the expected size were cut, purified, and cloned into E. coli DH5α using the pGEM-T easy vector system (Promega, Madison, WI, USA). At least three colonies per sample were selected and sequenced using a universal primer set (M13F and M13R) by Macrogen (Daejeon, Korea). The sequences obtained were aligned using MEGA 7.0. and the species were determined using a BLASTn search [18,19].

Microscopical Identification of B. coli
The transported fecal samples were homogenized using a sterilized wooden stick, and 1 g and 200 mg of fecal samples were taken for microscopic examination and DNA extraction, respectively. Microscopic examination was performed using the fecal flotation technique with sodium nitrate, as previously described [17].

DNA Extraction, PCR, Cloning, and Sequencing
Genomic DNA was extracted from the fecal samples using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was kept at -20 • C until further analysis.
For molecular diagnosis, PCR targeting the ITS region of B. coli was performed as previously described [1]. In brief, PCR was performed using AccuPower ® HotStart PCR PreMix (Bioneer, Korea) containing 2 µL template DNA, 1 µL of each forward and reverse primer (0.5 µM final concentration), and distilled water to a final volume of 20 µL. The PCR conditions were as follows: initial denaturation for 5 min at 94 • C; 30 cycles of 1 min at 94 • C, 1 min at 56 • C, and 1 min at 72 • C; and final extension for 5 min at 72 • C. After verification of the PCR conditions using a positive control, PCR was performed without a positive control to avoid cross-contamination, with distilled water included as a negative control in each experiment. The expected amplicon sizes were 528-or 537-bp according to the B. coli variant. The PCR products were visualized by gel electrophoresis.
For sequencing and phylogenetic analysis, 11 samples were randomly selected and amplicons of the expected size were cut, purified, and cloned into E. coli DH5α using the pGEM-T easy vector system (Promega, Madison, WI, USA). At least three colonies per sample were selected and sequenced using a universal primer set (M13F and M13R) by Macrogen (Daejeon, Korea). The sequences obtained were aligned using MEGA 7.0. and the species were determined using a BLASTn search [18,19]. The PCR-RFLP reaction was performed as follows: 10 µL PCR products, five units restriction enzyme (New England Biolabs, Ipswich, MA, USA), 2 µL 10X buffer (New England Biolabs), and distilled water up to a total volume of 20 µL. The mixtures containing ApoI and PflMI were incubated at 50 • C and 37 • C, respectively, for one hour.

Sequence and Phylogenetic Analyses
For molecular characterization, sequence analysis was performed using DnaSP v6, while phylogenetic analysis was performed using MEGA 7.0 [20]. The phylogenetic tree was constructed using the maximum likelihood method, with verification of the bootstrap values by 500 bootstrap replications. The analysis also included sequences reported from other animal species and countries obtained from the GenBank database. Spathidium amphoriforme (AF223570) was included as an outgroup.

Prevalence of B. coli by Microscopy and PCR
Microscopy identified B. coli in 79 (42.9%) out of 184 fecal samples. All identified B. coli were in the cyst stage and not the trophozoite stage ( Figure S1). Through PCR, 174 (94.6%) out of 184 samples were positive for B. coli.
All the sampled regions showed a high prevalence, ranging from 75.0% to 100% (Table 2). In addition, all farms had at least one B. coli-infected pig (data not shown).

PCR-RFLP and Prevalence According to Variant
PCR-RFLP using ApoI and PflMI showed clear differentiation between B. coli variants A and B (Figure 2). Using PCR-RFLP, 62 (33.7%) and 160 (87.0%) samples were positive for variants A and B, respectively, and 48 (26.1%) samples were co-infected with both variants (Table 3).

Sequence and Phylogenetic Analyses
Of the 11 positive samples, 35 colonies were selected and sequences were successfully obtained. Sequence analysis showed 92.9-100% intraspecies identity. The sequences showed 63 polymorphic sites including in 19 and 42 positions in variants A and B, respectively. In addition, 33 haplotypes were identified, with 11 and 23 haplotypes in variants A and B, respectively. Phylogenetic analysis showed that both B. coli variants were present in pigs in Korea ( Figure 3). All sequences obtained in this study were submitted to the GenBank database (Accession Nos. MZ676825-MZ676859).
There was agreement between the results obtained by PCR-RFLP and sequencing. Not all variants were identified in some samples co-infected with B. coli variants; however,

Sequence and Phylogenetic Analyses
Of the 11 positive samples, 35 colonies were selected and sequences were successfully obtained. Sequence analysis showed 92.9-100% intraspecies identity. The sequences showed 63 polymorphic sites including in 19 and 42 positions in variants A and B, respectively. In addition, 33 haplotypes were identified, with 11 and 23 haplotypes in variants A and B, respectively. Phylogenetic analysis showed that both B. coli variants were present in pigs in Korea (Figure 3). All sequences obtained in this study were submitted to the GenBank database (Accession Nos. MZ676825-MZ676859).
There was agreement between the results obtained by PCR-RFLP and sequencing. Not all variants were identified in some samples co-infected with B. coli variants; however, in cases of samples infected by a single B. coli variant, all the clones contained the corresponding variant only.
Few studies have evaluated B. coli in pigs in Korea. To our knowledge, only two studies have investigated the prevalence of B. coli in pigs by microscopy, with reported prevalences of 79.4% (108/136) and 66.6% (263/395) from samples collected in Chungcheongnam-do and Chungju city, respectively [13,27]. However, due to the lack of molecular information, the zoonotic potential of B. coli has not been evaluated.
Even before applying molecular techniques to B. coli, the role of pigs in the transmission of B. coli to humans is well known [28]. In developing countries, human balantidiasis is mainly caused by poor sanitation systems and ingestion of B. coli-contaminated food and water [28][29][30]. Molecular techniques make it possible to evaluate the role of animals and the zoonotic potential of B. coli according to their molecular characteristics. Ponce-Gordo et al. first suggested the zoonotic potential of B. coli variant A, which was identified in Bolivian patients and pigs [9]. Subsequent studies investigated the presence of B. coli variant A in animals from other countries. To date, B. coli variant A has been identified only in humans, non-human primates (gorillas and chimpanzees), guinea pigs, ostriches, and pigs [3,4,8,9]. Of these, B. coli from guinea pig was identical to the human-genotype, suggesting the importance of animals in the transmission of human balantidiasis [3].
Previous studies identified B. coli variant A based on sequence analysis after cloning; however, it is not an appropriate method to evaluate the true prevalence of each B. coli variant. As Ponce revealed, both variants A and B can exist in a single B. coli cell, and the results obtained by cloning are affected by probability [9]. To overcome this limitation, this study developed PCR-RFLP to evaluate the true prevalence of both B. coli variants in pigs.
This study is the first to show the molecular prevalence of B. coli variants A and B in pigs in Korea, with a predominance of variant B. This result is consistent with those of previous studies in China, which reported a higher prevalence of B. coli variant B compared to variant A [8,31]. To date, few studies have investigated B. coli based on molecular techniques, and the distribution of zoonotic B. coli worldwide is uncertain. More studies should be conducted with molecular information on B. coli variants in different countries.
Cases of human balantidiasis have been reported sporadically in Korea, with clinical signs varying from asymptomatic to dysentery [14,16,32,33]. Human balantidiasis has also been reported in other countries including Europe, Canada, and the U.S. [34][35][36][37]. The main clinical signs of balantidiasis are gastrointestinal disorders including diarrhea and abdominal pain [2]; however, infection and clinical signs are not confined to the gastrointestinal system. Previous studies have reported B. coli infection in different organs including the lungs, liver, genitourinary tract, cervical cord, brain, ascitic fluid, and eyes [2,14,16,32,33]. Unfortunately, these studies were mainly based on microscopic examination, histopathologic diagnosis; thus, the genetic variants causing human balantidiasis have not been identified. A recent study reported a case of human balantidiasis with dysentery among workers on pig farms in China [38]. Although the study did not also confirm the genetic variant, the findings demonstrated the importance of pigs as a source of B. coli infection in humans.  [2,7]. Sex and age are the most common host-related risk factors associated with the disease. To our knowledge, there is no consensus on risk factors for B. coli infection. For example, different studies have reported different prevalence according to age group, some of which were statistically significant, while others were not [8,24,[39][40][41]. In addition, the results of risk factor analysis for sex are contradictory [24,40,41]. Welldesigned and controlled studies are required for risk factor analysis.
B. coli is generally considered a non-pathogenic or opportunistic pathogen in animals and humans because of its high prevalence and low clinical cases. Comorbidities such as bacterial and viral infections may be related to the onset of clinical symptoms [2]. A recent study showed that B. coli infection alters gut microbiota by increasing Escherichia-Shigella and Campylobacter and decreasing Ruminococcaceae and Clostridiaceae [42], and the authors stated that B. coli needs to be considered as a pathogenic or opportunistic pathogenic parasite [42].
The results of the sequence and phylogenetic analyses in this study demonstrated the high level of genetic diversity of B. coli in pigs in Korea. Variants A and B are currently considered the main classification criteria in B. coli; however, Ponce-Gordo et al. classified the variants in more detail including A0, A1, A2, B0, and B1 [9]. Previous studies have unsuccessfully attempted to identify an association between genetic variants and animal species [3,4,9]; however, the accumulation of molecular information from different animals using different genetic markers may be helpful.

Conclusions
To our knowledge, this is the first study to develop a method to identify the genetic variants of B. coli in pigs without sequence analysis and to investigate the molecular epidemiology of B. coli in pigs. The results of this study showed the high prevalence of B. coli in pigs in Korea and the predominance of genetic variant B. Moreover, B. coli variant A, which has zoonotic potential, is widely distributed in Korea. Although the pathogenicity of B. coli in pigs is not critical, pigs are the main source of human balantidiasis. Therefore, molecular-based investigation of zoonotic B. coli in pigs in different countries is required.