Prevalence and Genetic Identification of Three Entamoeba Species in Pigs in Southeastern China

Parasitic Entamoeba spp. can infect many classes of vertebrates including humans and pigs. Entamoeba suis and zoonotic Entamoeba polecki have been identified in pigs, and swine are implicated as potential reservoirs for Entamoeba histolytica. However, the prevalence of Entamoeba spp. in pigs in southeastern China has not been reported. In this study, 668 fecal samples collected from 6 different regions in Fujian Province, southeastern China, were analyzed to identify three Entamoeba species by nested PCR and sequencing analysis. The overall prevalence of Entamoeba spp. was 55.4% (370/668; 95% CI 51.6% to 59.2%), and the infection rate of E. polecki ST1 was the highest (302/668; 45.2%, 95% CI 41.4% to 49.0%), followed by E. polecki ST3 (228/668; 34.1%, 95% CI 30.5% to 37.7%) and E. suis (87/668; 13.0%, 95% CI 10.5% to 15.6%). E. histolytica was not detected in any samples. Moreover, the coinfection rate of E. polecki ST1 and ST3 was 25.1% (168/668; 95% CI 21.9% to 28.4%), the coinfection rate of E. polecki ST1 and E. suis was 3.7% (25/668; 95% CI 2.3% to 5.2%), the coinfection rate of E. polecki ST3 and E. suis was 0.3% (2/668), and the coinfection rate of E. polecki ST1, E. polecki ST3, and E. suis was 4.0% (27/668; 95% CI 2.5% to 5.5%). A representative sequence (MK347346) was identical to the sequence of E. suis (DQ286372). Two subtype-specific sequences (MK357717 and MK347347) were almost identical to the sequences of E. polecki ST1 (FR686383) and ST3 (AJ566411), respectively. This is the first study to survey the occurrence and to conduct molecular identification of three Entamoeba species in southeastern China. This is the first report regarding mixed infections with E. suis, E. polecki ST1, and E. polecki ST3 in China. More research studies are needed to better understand the transmission and zoonotic potential of Entamoeba spp.


Introduction
e genus Entamoeba comprises many free-living and parasitic species and can infect all classes of vertebrates and some invertebrates. Some Entamoeba species (e.g., E. histolytica, E. dispar, E. coli, E. moshkovskii, E. hartmanni, and E. polecki) have been identified in humans [1][2][3][4], and most are considered harmless, but some of these species still cause disease. Amoebiasis caused by E. histolytica is the third leading parasitic disease causing morbidity and mortality in humans, causing up to 50, 000 deaths per year, just behind malaria and schistosomiasis [4][5][6][7]. e disease is characterized as amebic colitis and liver abscess in humans and animals [3,8]. Although E. histolytica has not been detected in farmed pigs thus far, and the susceptibility of swine to E. histolytica infection was revealed only under experimental conditions, swine have been considered as potential reservoirs for E. histolytica [9][10][11][12].
Two species, E. suis [13] and E. polecki [14], have been identified in pigs. E. suis appears to be mostly restricted to pigs [2,3,15,16] and has been suggested to cause hemorrhagic colitis by breaking down the lamina propria [10]. Unlike E. suis, which infects pigs and potentially gorillas [17], E. polecki can infect many kinds of hosts, including humans, nonhuman primates, and pigs. e intraspecific variation of E. polecki was revealed by molecular analysis of the small-subunit ribosomal DNA, which showed that E. polecki could be divided into 4 subtypes (E. polecki ST1-ST4) [17,18]. All the subtypes have been found in humans, E. polecki ST1 and E. polecki ST3 have also been found in pigs, and E. polecki ST2 also exists in nonhuman primates [12,17], while human cases of E. polecki primarily involve E. polecki ST4 [19]. For a long time, E. polecki ST4 was only known from humans. Recently, however, ST4 was found in wild Celebes crested macaques (Macaca nigra) [20]. Although E. polecki is considered less pathogenic to humans or animals in the case of solo infection, coinfections with other pathogens, such as Lawsonia intracellularis, may increase the severity of the disease [3].
Swine husbandry plays an indispensable role in the animal husbandry in China. Because of the prosperity of swine husbandry and the high population density in China, the risk of exposure to zoonotic Entamoeba spp. is inevitable. However, the molecular epidemiology of Entamoeba spp. in pigs in southeastern China has not been reported. is study determined the prevalence of three Entamoeba species in pigs in southeastern China using molecular detection, determined the genetic identity of these Entamoeba species by phylogenetic analysis, and evaluated the zoonotic potential of Entamoeba spp.

Study Sampling.
A total of 668 fecal samples were collected from 6 regions in Fujian Province, southeastern China ( Figure 1). All specimens from pigs, including weaned piglets, suckling piglets, sow, boars, nursery pigs, and fattening pigs, were collected directly from each pig's rectum or were immediately collected from the ground after defecation by the pigs. Fecal samples were marked with the corresponding sex, developmental stage, and origin of the pigs and then stored at 4°C until DNA extraction (generally within 48 hours).

Isolation of Genomic DNA.
According to the manufacturer's instructions, genomic DNA was extracted from approximately 200 mg of each fecal samples using a Stool DNA kit (OMEGA D4015-02), and the DNA was stored at − 20°C until use.

PCR Amplification of Entamoeba spp.
e extracted fecal genomic DNA was used to determine the species/subtypes of Entamoeba spp. by nested PCR targeting the small-subunit ribosomal RNA (SSU rRNA) gene. e first set of primers, E-1 and E-2, and the second set of primers, EH-1 and EH-2, were used to detect E. histolytica [1]. e first round of nested PCR used primers 764-RD3, and the second round of nested PCR used primers 764-765, to identify E. suis [15]. e primary PCR for identifying E. polecki was performed using primer set Epolec F6-Epolec R6, and then the secondary PCR for subtype-specific characterization of E. polecki used primers Epolecki 1-Epolecki 2 (ST1) and EpST3F1-EpST3R2 (ST3) [2,12].
An amplification reaction volume of 25  e obtained sequences were analyzed using the BLAST program at the NCBI website. Mega 7.0 (http://www.megasoftware.net/) software was used to perform phylogenetic analyses by the neighbor-joining method with the Kimura-2 parameter model. Bootstrap analysis with 1000 replicates was used to assess the robustness of cluster formation.

Data
Analysis. SPSS 22.0 (IBM Corp., New York, USA) was used to analyze the data.
e associations between infection rates of different sampling areas and the associations between infection rates of different developmental stages of pigs were explored using the chi-square test. Differences were considered statistically significant when P < 0.05. Analysis of the infection rates of Entamoeba spp. in different sampling areas showed that there were regional differences (χ 2 � 167.453, P < 0.05), with the rates being much lower in Putian and Longyan than in other regions.

Distribution of Entamoeba spp. in Different Developmental Stages of Swine.
e detailed data of distribution of Entamoeba spp. are shown in Table 2. Analysis of the infection rates of Entamoeba spp. in different developmental stages showed that there was a developmental stage predisposition to infection with Entamoeba spp. (χ 2 � 50.362, P < 0.05), with the rates being much lower in suckling pigs than in other developmental stages.

Phylogenetic and Sequencing Analysis of Entamoeba spp.
e positive product of E histolytica was not amplified by nested PCR in all samples. Representative sequences were submitted to GenBank under accession numbers MK347346 (E. suis), MK347347 (E. polecki ST3), and MK357717 (E. polecki ST1). Meanwhile, the three representative sequences displayed 100% sequence identity to other obtained sequences of PCR-positive samples of E. suis and E. polecki ST1 and ST3 in this study. e sequence of E. suis (MK347346) was identical to the sequence isolated from pigs (DQ286372). e representative sequences of E. polecki ST1 (MK357717) and E. polecki ST3 (MK347347) were almost identical to reference sequences of E. polecki ST1 (AF149913) and E. polecki ST3 (LC067574), respectively, and compared with the corresponding reference sequences, each current sequence has 1 different substitution. We chose known sequences [12] to build the phylogenetic tree of the E. polecki subtypes detected in the current study, and the results showed that MK357717 shared a common clade with AF149913 (E. polecki ST1) and MK347347 shared a common clade with LC067574 (E. polecki ST3) (Figure 2).

Discussion
Traditional microscopic examination is the most commonly used clinical diagnostic tool for examining the presence of Entamoeba organisms in fresh or fixed stool samples [1,7,21,22]. However, several distinct Entamoeba spp. with similar morphological characteristics (for instance, the E. dispar, a nonpathogenic species, is morphologically identical to E. histolytica) cannot be distinguished by microscopic examination alone [1,2,4,7]. erefore, accurate identification of species/subtypes of Entamoeba was performed with molecular tools including PCR and nucleotide sequencing [1,7,12,19,23,24].
e phylogenetic analysis indicated that the isolates from the samples for E. polecki were E. polecki ST1 and E. polecki ST3. Infection with E. polecki ST1 was the most common (45.2%) in the present study, which was consistent with the observations reported in Indonesia, Vietnam, and eastern China [12,20,29]. Mixed infections, including infection with       histolytica was not observed in farmed pigs in this study, which was consistent with the previous research [10][11][12].
Traditionally, E. suis was considered to be mostly restricted to pigs [2,3,15,16]. However, the sequence of an Entamoeba isolated from a gorilla (FR686456) was similar to the sequence of E. suis (DQ286372) with one substitution [17], so whether E. suis only infects pigs should be verified by more studies.
e results show that pig infection with Entamoeba spp. was related to the sampling areas and the developmental stages of swine (P < 0.05), but this is not in agreement with the observation made in pigs by Li et al.
(there was no age predisposition in pigs) [12]. erefore, more research studies are needed to confirm whether sampling area and types of swine are risk factors for Entamoeba spp. infection. ere were only detected three Entamoeba species (E. histolytica, E. suis, and E. polecki ST1 and E. polecki ST3) in this study, and more research studies are needed to determine prevalence and genetic identification of other species/subtypes in pigs in China in the future.

Conclusion
e present study conducted a prevalence survey and molecular identification of three Entamoeba species in pigs in southeastern China. e overall infection rate of Entamoeba spp. was 55.4%. E. suis and zoonotic E. polecki ST1 and E. polecki ST3 have been found in pigs. us, further attention should be paid to the risk of the transmission of Entamoeba spp. between animal reservoirs and humans. e statistical analysis (SPSS) suggested that sampling areas and developmental stages of swine are associated with swine infection with three Entamoeba species. is is the first report of mixed infections with E. suis, E. polecki ST1, and E. polecki ST3 in China.

Data Availability
e data used to support the findings of this study are included within the article.