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Article

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

by
Jae-Won Byun
1,
Jung-Hyun Park
2,
Bo-Youn Moon
1,
Kichan Lee
1,
Wan-Kyu Lee
2,
Dongmi Kwak
3 and
Seung-Hun Lee
2,*
1
Animal and Plant Quarantine Agency, Gimcheon 39660, Korea
2
College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Korea
3
College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea
*
Author to whom correspondence should be addressed.
Animals 2021, 11(9), 2659; https://doi.org/10.3390/ani11092659
Submission received: 9 August 2021 / Revised: 4 September 2021 / Accepted: 8 September 2021 / Published: 10 September 2021
(This article belongs to the Special Issue Epizootiology of Farm Animal Diseases)
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Abstract

:

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.

1. 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.

2. Materials and Methods

2.1. 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.

2.2. 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].

2.3. 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].

2.4. PCR-RFLP

To differentiate B. coli variant A from variant B, a PCR-RFLP technique was developed using the restriction enzymes ApoI and PflMI. The reference sequences of B. coli variant A (JQ073359) and variant B (JQ073358) were obtained from the GenBank database. In silico PCR-RFLP predicted that ApoI cut B. coli variant A into 227-, 135-, 70-, 70-, and 35-bp fragments and variant B into 293- and 235-bp fragments. PflMI had no cut site in B. coli variant A and 312- and 216-bp fragments in B. coli variant B (Table 1).
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.

2.5. 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.

3. Results

3.1. 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).

3.2. 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).

3.3. 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, in cases of samples infected by a single B. coli variant, all the clones contained the corresponding variant only.

4. Discussion

B. coli is distributed worldwide, and its main host is pigs [7]. Previous studies have reported the prevalence of B. coli in domestic pigs in different countries including 46.4% and 84.1% in Japan (155/334 and 212/252, respectively), 93.0% in India (93/100), 16.8% in China (94/560), 40.0% in Bangladesh (44/110), 1.6%, in Turkey (4/238), 51.5% in Nigeria (207/402), 64.1% in Kenya (196/306), 0.7% in Germany (2/287), 61.6% in Italy (149/242), and 60.9% in Brazil (236/387) [8,21,22,23,24,25]. The prevalence in domestic pigs is highly variable, ranging from 0.7% to 93.0%, and might be affected by the breeding system, hygiene conditions, season, diagnostic method, and climate. Even in microscopic examinations, performance can vary according to techniques, materials, and the investigator’s experience [7,26]. As shown in this study, molecular methods generally show higher sensitivity than microscopic examinations. Previous studies were mainly based on the microscopic method; therefore, the true prevalence in those studies may have been underestimated.
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.
Different risk factors have been suggested for B. coli infection including both host and environmental factors [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]. Well-designed 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.

5. 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.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/ani11092659/s1, Figure S1: Microscopic identification of Balantioides coli in pig feces. (A) Without staining, showing a bean-shaped macronucleus (arrow). (B) Lugol’s iodine staining showing the cyst wall (arrowhead). Bar = 50 μm.

Author Contributions

Conceptualization, J.-W.B. and S.-H.L.; Methodology, D.K. and S.-H.L.; Formal analysis, J.-W.B., J.-H.P., W.-K.L., D.K. and S.-H.L.; Investigation, J.-W.B., B.-Y.M., K.L. and S.-H.L.; Resources, J.-W.B., J.-H.P., B.-Y.M., K.L., W.-K.L. and S.-H.L.; Data curation, J.-W.B., J.-H.P. and S.-H.L.; Writing—original draft preparation, J.-W.B.; Writing—review and editing, W.-K.L., D.K. and S.-H.L.; Supervision, S.-H.L.; Funding acquisition, J.-W.B., W.-K.L. and S.-H.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a research grant from the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry, and Fisheries (IPET) through the Agriculture, Food, and Rural Affairs Convergence Technologies Program for Educating Creative Global Leader, funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (grant number: 320005-4); the program (B-1543069-2021-22-03) of the Animal and Plant Quarantine Agency (APQA) and Ministry of Agriculture, Food and Rural Affairs (MAFRA); and the “Regional Innovation Strategy (RIS)” through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (MOE).

Institutional Review Board Statement

Ethical review and approval were waived for this study as the study focused on post-slaughter samples. Animal contact during antemortem examination was minimal, consistent with routine physical examination procedure, and conducted under the supervision of a licensed veterinarian.

Data Availability Statement

The data presented in this study are contained within this article and all sequences obtained in this study were submitted to the GenBank Database (Accession Nos. MZ676825-MZ676859).

Acknowledgments

The authors thank the veterinary officers and staff for their assistance during the sample collection.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Animals 11 02659 g001 550
Figure 1. Map of Korea. The sites of pigs reared are indicated in gray. GG, Gyeonggi-do; GW, Gangwon-do; CN, Chungcheongnam-do; CB, Chungcheongbuk-do; GB, Gyeongsangbuk-do; GN, Gyeongsangnam-do; JB, Jeollabuk-do; JN, Jeollanam-do; JJ, Jeju-do.
Figure 1. Map of Korea. The sites of pigs reared are indicated in gray. GG, Gyeonggi-do; GW, Gangwon-do; CN, Chungcheongnam-do; CB, Chungcheongbuk-do; GB, Gyeongsangbuk-do; GN, Gyeongsangnam-do; JB, Jeollabuk-do; JN, Jeollanam-do; JJ, Jeju-do.
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Animals 11 02659 g002 550
Figure 2. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) results of Balantioides coli variants A and B with restriction enzyme digests by ApoI and PflMI. M, marker; 1, B. coli variant A (537-bp); 2, B. coli variant B (528-bp); 3, B. coli variant A treated with ApoI (227, 135, 70, 35-bp); 4, B. coli variant B treated with ApoI (293, 235-bp); 5, B. coli variant A treated with PflMI (537-bp); 6, B. coli variant B treated with PflMI (312, 216-bp). Note that the 35-bp fragment is not visible.
Figure 2. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) results of Balantioides coli variants A and B with restriction enzyme digests by ApoI and PflMI. M, marker; 1, B. coli variant A (537-bp); 2, B. coli variant B (528-bp); 3, B. coli variant A treated with ApoI (227, 135, 70, 35-bp); 4, B. coli variant B treated with ApoI (293, 235-bp); 5, B. coli variant A treated with PflMI (537-bp); 6, B. coli variant B treated with PflMI (312, 216-bp). Note that the 35-bp fragment is not visible.
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Animals 11 02659 g003 550
Figure 3. Phylogenetic analysis of Balantioides coli in pigs in Korea. The tree was constructed using MEGA 7.0 and the maximum-likelihood method with 500 replications. The sequences analyzed in this tree were obtained from the GenBank database and are described with their accession numbers, hosts, and isolated countries. The sequences identified in this study are indicated with arrows. Spathidium amphoriforme (AF223570) is included as an outgroup. Bootstrap values less than 40 were omitted.
Figure 3. Phylogenetic analysis of Balantioides coli in pigs in Korea. The tree was constructed using MEGA 7.0 and the maximum-likelihood method with 500 replications. The sequences analyzed in this tree were obtained from the GenBank database and are described with their accession numbers, hosts, and isolated countries. The sequences identified in this study are indicated with arrows. Spathidium amphoriforme (AF223570) is included as an outgroup. Bootstrap values less than 40 were omitted.
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Table
Table 1. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) results of Balantioides coli variants A and B digested with restriction enzymes ApoI and PflMI.
Table 1. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) results of Balantioides coli variants A and B digested with restriction enzymes ApoI and PflMI.
B. coliPCR
Product (bp)		Restriction Enzyme (bp)
ApoIPflMI
Variant A537227, 135, 70, 70, 35537
Variant B528293, 235312, 216
Table
Table 2. Prevalence of zoonotic Balantioides coli in pigs in Korea according to province.
Table 2. Prevalence of zoonotic Balantioides coli in pigs in Korea according to province.
ProvinceNo. of SamplesNo. of Positive (%)
MicroscopyPCRB. coli Variant A
Gyeonggi-do4723 (48.9)41 (87.2)14 (34.1)
Chungcheongnam-do85 (62.5)6 (75.0)0 (0.0)
Gyeongsangbuk-do4514 (31.1)45 (100)24 (53.3)
Gyeongsangnam-do6021 (35.0)58 (96.7)14 (24.1)
Jeollabuk-do1611 (68.8)16 (100)8 (50.0)
Jeollanam-do85 (62.5)8 (100)2 (25.0)
Total18479 (42.9)174 (94.6)62 (33.7)
Table
Table 3. Proportions of Balantioides coli genetic variants in pigs in Korea.
Table 3. Proportions of Balantioides coli genetic variants in pigs in Korea.
B. coli InfectionNo. of Samples (%)
Co-infection (variant A + variant B)48 (26.1)
Single infection (variant A)14 (7.6)
Single infection (variant B)112 (60.9)
Non10 (5.4)
Total184
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Byun, J.-W.; Park, J.-H.; Moon, B.-Y.; Lee, K.; Lee, W.-K.; Kwak, D.; Lee, S.-H. Identification of Zoonotic Balantioides coli in Pigs by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Its Distribution in Korea. Animals 2021, 11, 2659. https://doi.org/10.3390/ani11092659

AMA Style

Byun J-W, Park J-H, Moon B-Y, Lee K, Lee W-K, Kwak D, Lee S-H. Identification of Zoonotic Balantioides coli in Pigs by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Its Distribution in Korea. Animals. 2021; 11(9):2659. https://doi.org/10.3390/ani11092659

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Byun, Jae-Won, Jung-Hyun Park, Bo-Youn Moon, Kichan Lee, Wan-Kyu Lee, Dongmi Kwak, and Seung-Hun Lee. 2021. "Identification of Zoonotic Balantioides coli in Pigs by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Its Distribution in Korea" Animals 11, no. 9: 2659. https://doi.org/10.3390/ani11092659

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