Molecular Identification and Survey of Trichomonad Species in Pigs in Shanxi Province, North China

Simple Summary Three trichomonad species (Tritrichomonas foetus, Tetratrichomonas buttreyi, and Pentatrichomonas hominis) may affect pig production. Moreover, P. hominis is a zoonotic species. In the current investigation, 362 fecal samples collected from pigs in three representative counties in Shanxi Province, North China, were molecularly tested for the three trichomonad species. The results showed that T. buttreyi was detected as the most common species, with a prevalence of 49.72%. Its prevalence was associated with region and age. Only one sample was tested positive for T. foetus, and this isolate showed minor allelic variations compared with those reported previously. For T. buttreyi, eight distinct sequence types were obtained, and only two displayed 100% nucleotide homology with the corresponding sequences reported previously. For the first time, we reported the prevalence and genetic characterization of T. foetus and T. buttreyi in pigs in Shanxi Province, North China, which provides baseline information for planning control strategies. Abstract Several trichomonad species have already been identified in pigs, and their pathogenic potential may not be ruled out. To date, however, no information is available regarding the prevalence of trichomonads in pigs in Shanxi Province, North China. In the present study, a total of 362 fecal samples collected from pigs in three representative counties (Qi, Jishan, and Shanyin) in this province were examined for Tetratrichomonas buttreyi, Tritrichomonas foetus, and Pentatrichomonas hominis using a nested polymerase chain reaction (PCR) with primers targeting the small subunit ribosomal RNA (SSU rRNA) gene. The overall prevalence of T. buttreyi was 49.72%, and region and age were found to be significantly associated with T. buttreyi infection, respectively. Only one pig fecal sample from Qi County was found to be positive for T. foetus, and all samples were negative for P. hominis. Molecular evolutionary analysis revealed that some T. buttreyi isolates showed complete genetic identity with those reported previously, and some T. buttreyi isolates and one T. foetus isolate showed minor allelic variations compared with those reported previously. This is the report of the molecular epidemiology of T. foetus and T. buttreyi in pigs in Shanxi Province, North China. These findings not only enrich the knowledge on the distribution of these trichomonad species in pigs in China but also provide baseline information for planning future research and control strategies.

Tetratrichomonas buttreyi, originally found in pigs, was reported to be commensal to both cattle and pigs [8].However, the potential pathogenicity of T. buttreyi is possible, because a previous report indicated that the excessive multiplication of trichomonads could cause pathogenic effects [9].
Pentatrichomonas hominis is a trichomonad species reported in humans and important economic animals including pigs [10][11][12].Though P. hominis was originally thought to be a commensal organism, it has recently been detected in the feces of cats and dogs with diarrhea [13,14].Hence, the pathogenic potential of P. hominis in pigs may not be ruled out.Moreover, a previous study reported that P. hominis may be strongly linked with human gastrointestinal cancers [15].
Knowledge regarding the prevalence of the three trichomonad species mentioned above (T.foetus, T. buttreyi, and P. hominis) is of great importance from both human and veterinary medicine standpoints.To date, the prevalence of T. foetus, T. buttreyi, and P. hominis in pigs in two Chinese provinces has been investigated [10,16].The pig industry plays an important role in animal husbandry in Shanxi Province, North China.However, no information is currently available regarding the infection status of the three trichomonad species in pigs in this province.Thus, the prevalence of T. foetus, T. buttreyi, and P. hominis in pigs in Shanxi Province, North China was determined in the present study, followed by sequencing and phylogenetic analysis.

Sample Collection and DNA Extraction
In November 2020, a total of 362 fresh fecal samples were collected from pigs in the following counties: Shanyin (n = 183), Qi (n = 68), and Jishan (n = 111).The sampling locations included the northern, central, and southern parts of Shanxi Province, respectively.Along with fecal sample collection, age information was recorded for each animal from faceto-face interviews with the breeders.The fecal samples were transported immediately to the laboratory using an airtight foam box containing ice packs and then kept in a refrigerator at −20 • C until further use.Extraction of genomic DNA from 0.2 g of each fecal sample was carried out by using the EZNA Stool DNA extraction kit (Omega Bio-tek Inc., Norcross, GA, USA) according to the manufacturer's specifications, and the obtained genomic DNA samples were kept frozen at −20 • C until further use as templates for polymerase chain reaction (PCR) amplification.

PCR Analysis
The positivity of T. buttreyi, T. foetus and P. hominis was determined by nested PCR amplification of a fragment of the small subunit ribosomal RNA (SSU rRNA) gene, and the first-round PCR was performed using the same primer set (forward: 5 ′ -GCGCCTGAGAGATAGCGACTA-3 ′ ; reverse: 5 ′ -GGACCTGTTATTGCTACCCTCTTC-3 ′ ) with the following cycling conditions: initial denaturation at 95 • C for 10 min, followed by 30 cycles of 95 • C for 1 min, 60 • C for 1 min, 72 • C for 1 min, and a final extension step of 72 • C for 10 min [10].To identify T. foetus, the first-round PCR product was subjected to a second-round PCR reaction with inner primers (forward: 5 ′ -GGTTGTTTGTATAGGATTGC-3 ′ ; reverse: 5 ′ -TGCCCTCATAAAAGGACAA-3 ′ ) and cycle conditions as follows: an initial denaturation step at 95 • C for 10 min, followed by 30 cycles of 30 s at 95 • C, 30 s at 54 • C, 30 s at 72 • C, and a final 10 min at 72 • C. To identify T. buttreyi, the first-round PCR product was subjected to a second-round PCR reaction with inner primers (forward: 5 ′ -GTTTTTTCTCAGGCAGCAATG-3 ′ ; reverse: 5 ′ -GCAACCTAGAAACCTAGGCG-3 ′ ) and cycle conditions as follows: an initial denaturation step at 95 • C for 10 min, followed by 30 cycles of 45 s at 95 • C, 45 s at 60 • C, 45 s at 72 • C, and a final 10 min at 72 • C. To identify P. hominis, the first-round PCR product was subjected to a second-round PCR reaction with inner primers (forward: 5 ′ -TGTAAACGATGCCGACAGAG-3 ′ ; reverse: 5 ′ -CAACACTGAAGCCAATGCGAGG-3 ′ ) and cycle conditions as follows: an initial denaturation step at 95 • C for 10 min, followed by 30 cycles of 30 s at 95 • C, 30 s at 55 • C, 30 s at 72 • C, and a final 10 min at 72 • C.
The PCR reaction mixture (a final volume of 25 µL) was composed of 2.5 µL 10 × PCR buffer (Mg 2+ free), 0.2 mM of dNTP mixture, 1.5 mM of MgCl 2 , 0.125 µL of Ex Taq polymerase (5 U/µL) (TaKaRa, Dalian, China), 2 µL of genomic DNA (used as a DNA template in the primary PCR) or the primary PCR product (used as a DNA template in the secondary PCR), and 0.4 µM of each primer.After amplification, the sizes of the secondary PCR products were checked by migration on 1.5% agarose gel electrophoresis.

Sequencing and Phylogenetic Analysis
Positive PCR products were excised from the gels and purified, followed by Sanger sequencing in both directions by Sangon Biotech Co., Ltd.(Shanghai, China) using an Applied Biosystems™ 3730XL DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).The nucleotide sequences obtained in this study were edited and compared for similarity with those available in the archival database GenBank by using the Basic Local Alignment Search Tool (BLAST).A phylogenetic tree was constructed by using the neighborjoining (NJ) method described previously [16].Genetic distances were calculated in MEGA 7 software using the Kimura parameter-2 model [17], and the robustness of the findings was evaluated with 1000 bootstrap replicates [18].

Statistical Analysis
Chi-square analysis was performed to assess the relationships between parasitic infections and the different independent factors addressed in the present study using the software SPSS 26.0 (IBM, Chicago, IL, USA).The magnitude of the association was tested by calculating odds ratios (ORs) with 95% confidence intervals (95% CIs).A p-value less than 0.05 was considered to indicate a statistically significant result.

DNA Sequence Analysis
The sequence of the T. foetus SSU rRNA gene obtained in the present study has been deposited in GenBank with the accession number: PP256583.Sequence comparisons using BLAST showed that the sequence obtained in the present study had a high degree of sequence similarity (99.78%) with those of T. foetus from pigs (MK801504, KX833160, and KM205209), cattle (AY055799), and cats (OR033176 and AF466749).
T. buttreyi-positive samples were sequenced, and eight distinct sequence types exhibiting 98.56%-99.84%sequence identity were obtained (Figure 2).These sequences are publicly available at GenBank (accession numbers PP256574-PP256581).One sequence (PP256575) showed 100% identity with those of T. buttreyi from pigs (KX833156 and KM205212) and cattle (MK880285) from different areas of China, as well as pigs from the Philippines (JX565058) and Germany (MK801506), and horses (AY886859) from the Czech Republic.One sequence (PP256577) was identical to those of T. buttreyi from wild boars (AY886865) and Hanuman langur (HQ149978) from the Czech Republic, as well as pigs (JX565053) from the Philippines.Of the remaining six sequences, each was nearly identical to one of the sequences derived from pigs (KX833155, KX833156, and KX833158) in Anhui Province, China.

DNA Sequence Analysis
The sequence of the T. foetus SSU rRNA gene obtained in the present study has been deposited in GenBank with the accession number: PP256583.Sequence comparisons using BLAST showed that the sequence obtained in the present study had a high degree of sequence similarity (99.78%) with those of T. foetus from pigs (MK801504, KX833160, and KM205209), cattle (AY055799), and cats (OR033176 and AF466749).
T. buttreyi-positive samples were sequenced, and eight distinct sequence types exhibiting 98.56%-99.84%sequence identity were obtained (Figure 2).These sequences are publicly available at GenBank (accession numbers PP256574-PP256581).One sequence (PP256575) showed 100% identity with those of T. buttreyi from pigs (KX833156 and KM205212) and cattle (MK880285) from different areas of China, as well as pigs from the Philippines (JX565058) and Germany (MK801506), and horses (AY886859) from the Czech Republic.One sequence (PP256577) was identical to those of T. buttreyi from wild boars (AY886865) and Hanuman langur (HQ149978) from the Czech Republic, as well as pigs (JX565053) from the Philippines.Of the remaining six sequences, each was nearly identical to one of the sequences derived from pigs (KX833155, KX833156, and KX833158) in Anhui Province, China.
Though some SSU rRNA sequences obtained in the present study (such as PP256574 and PP256576) did not show 100% identity with the reference sequences in GenBank, the phylogenetic tree confirmed without doubt that all these sequences belonged to T. buttreyi based on the NJ method (Figure 3).Though some SSU rRNA sequences obtained in the present study (such as PP256574 and PP256576) did not show 100% identity with the reference sequences in GenBank, the phylogenetic tree confirmed without doubt that all these sequences belonged to T. buttreyi based on the NJ method (Figure 3).

Discussion
Of the techniques developed for the diagnosis of T. foetus infection (fecal culture, direct fecal smear examination, and single-tube nested PCR), the single-tube nested PCR showed higher sensitivity and specificity [19].Meanwhile, a previous study using nested PCR amplification of a fragment of the SSU rRNA gene successfully distinguished and identified the trichomonads isolated from pigs to species level [10].Hence, we molecularly

Discussion
Of the techniques developed for the diagnosis of T. foetus infection (fecal culture, direct fecal smear examination, and single-tube nested PCR), the single-tube nested PCR showed higher sensitivity and specificity [19].Meanwhile, a previous study using nested PCR amplification of a fragment of the SSU rRNA gene successfully distinguished and identified the trichomonads isolated from pigs to species level [10].Hence, we molecularly examined trichomonads in pig fecal samples in Shanxi Province in the present study.The results obtained represent the first molecular evidence for the existence of T. foetus and T. buttreyi infections in pigs in Shanxi Province, North China.
The prevalence of trichomonads detected in this study (50.00%) was higher than that reported for pigs in two Chinese provinces, Jilin (43.04%) and Anhui (47.40%) [10,16].Meanwhile, the infection rate of T. foetus in pigs observed in the present study (0.28%) was lower than that reported for pigs in other parts of the world, such as Poland (16.28%),Australia (64.50%), and Japan (56.25%) [20][21][22].Moreover, a statistically significant difference in the prevalence of T. buttreyi among age groups was observed in the present study, and pigs aged > 6 months had a 2.26 times higher risk of infection compared with those aged ≤4 months.In addition, the prevalence of T. buttreyi varied in different counties.The differences in the prevalence may result from various variables, such as the examination method used, sample size, seasonality, and geographical region.
A previous study showed that the prevalence of P. hominis, T. buttreyi, and T. foetus in pigs in Jilin Province, Northeast China, was 24.05%, 14.57%, and 12.03%, respectively [10].However, all pig fecal samples were tested negative for P. hominis in this study.Notably, our findings showed that T. buttreyi was the most common species, and only one sample was tested positive for T. foetus.The results were similar to those of a previous study in Anhui Province, in which T. buttreyi was detected as a predominant species, and a low prevalence of T. foetus was observed [16].
Two sequences obtained in the present study showed 100% identity with those of T. buttreyi isolates deposited previously in GenBank, regardless of the geographical locations, suggesting the presence of strong clonality among them.In addition, minor allelic variations were also present in the SSU rRNA sequences from the T. buttreyi isolates obtained in this study.The results were similar to those obtained in the previous studies [10,23], which mentioned that the minor genetic distinctness might be due to the expected variation within the multiple copies of the SSU rRNA genes in any given genome or due to adaptation to different hosts and surrounding environments.

Figure 1 .
Figure 1.Agarose gel electrophoresis of the PCR products.(A) Lane M: 1000 bp DNA ladder; lane PC: positive control; lane NC: negative control; lane 1-3: nested PCR amplification with primers specific for a 452-bp segment of the T. foetus SSU rRNA gene using the genomic DNA extracted from each pig fecal sample as a template.(B) Lane M: 1000 bp DNA ladder; lane PC: positive control; lane NC: negative control; lane 1-5: nested PCR amplification with primers specific for a 623-bp segment of the T. buttreyi SSU rRNA gene using the genomic DNA extracted from each pig fecal sample as a template.

Figure 1 .
Figure 1.Agarose gel electrophoresis of the PCR products.(A) Lane M: 1000 bp DNA ladder; lane PC: positive control; lane NC: negative control; lane 1-3: nested PCR amplification with primers specific for a 452-bp segment of the T. foetus SSU rRNA gene using the genomic DNA extracted from each pig fecal sample as a template.(B) Lane M: 1000 bp DNA ladder; lane PC: positive control; lane NC: negative control; lane 1-5: nested PCR amplification with primers specific for a 623-bp segment of the T. buttreyi SSU rRNA gene using the genomic DNA extracted from each pig fecal sample as a template.

Figure 2 .
Figure 2. Alignment of the SSU rRNA sequences of T. buttreyi isolates obtained in the present study (PP256574-PP256581) and that of a T. buttreyi isolate from a previous study (KM205212).Dots represent nucleotides identical to the consensus sequence, while dashes indicate base deletion.

Figure 2 . 8 Figure 3 .
Figure 2. Alignment of the SSU rRNA sequences of T. buttreyi isolates obtained in the present study (PP256574-PP256581) and that of a T. buttreyi isolate from a previous study (KM205212).Dots represent nucleotides identical to the consensus sequence, while dashes indicate base deletion.Vet.Sci.2024, 11, x FOR PEER REVIEW 6 of 8

Figure 3 .
Figure 3. Phylogenetic relationships among trichomonad isolates inferred with a neighbor-joining analysis based on the nucleotide sequences of the SSU rRNA gene, rooted using Barbulanympha ufalula as an outgroup.The SSU rRNA sequences generated in the present study are indicated by black triangles.

Table 1 .
The prevalence of Tetratrichomonas buttreyi in pigs in Shanxi Province, North China.

Table 1 .
The prevalence of Tetratrichomonas buttreyi in pigs in Shanxi Province, North China.