First Report on the Occurrence and Subtypes of Blastocystis in Pigs in Poland Using Sequence-Tagged-Site PCR and Barcode Region Sequencing

Blastocystis is an enteric microorganism commonly found in humans and animals worldwide. Its pathogenic role in humans and transmission patterns has not been fully explained. However, nine subtypes (ST1–8, ST12) are considered as potentially zoonotic. Studies from various regions of the world show that pigs are mainly infected with ST5. Although pigs are important farmed animals in Poland, the question of Blastocystis infection in these animals has not yet been investigated. Herein, 149 pig stool samples from 10 Polish pig farms were analyzed using sequence-tagged-site PCR and barcode region sequencing. The percentage of samples in which Blastocystis was identified using each method separately was similar: 38.25% and 37.58%, respectively. However, the percentage of positive results obtained by combining both methods was 46.97%, which means that, depending on the method used, the number of undetected samples varied between 8.72% and 9.39%. This shows the methodological limitations of up-to-date molecular approaches commonly used in Blastocystis research. A moderate infection rate (44.4–50%) observed in different pig age groups with a vital predominance of ST5 (94.28%) in every age group shows that pigs are a likely natural host of ST5. A small percentage of mixed infections, namely ST5/ST1 (5.26%), ST5/ST3 (1.75%), and ST3/ST1 (1.75%), was observed only in animals of older age, suggesting that ST3 and ST1 can be acquired by pigs during contact with humans. This study provides the first data on the prevalence and Blastocystis subtypes (STs) distribution in pigs in Poland. The results also highlight the need for the development of new methods capable of detecting highly genetically diverse Blastocystis isolates and mixed infections.


Introduction
Blastocystis is a common enteric eukaryotic microorganism distributed worldwide, colonizing probably more than one billion people and a wide variety of mammals (wild, farm, and companion animals) as well as birds and insects [1][2][3]. Blastocystis pathogenicity and importance to public health is controversial since the organism is found in both asymptomatic and symptomatic individuals [4]. In the latter group, Blastocystis has been linked to gastrointestinal disorders (such as nausea, abdominal

Results
After testing 149 pig stool samples for Blastocystis using two PCR research methods, the results obtained by each method were not completely consistent. Blastocystis DNA was detected in 57 samples (38.25%) tested with STS primers and 56 (37.58%) samples tested with barcode region analysis. In 43 samples (28.85%), Blastocystis was detected consistently with both methods used, whereas in 14 samples (9.39%), Blastocystis was found using only STS PCR, and in 13 (8.72%) using only "barcode method". After adding up the results obtained with one or both of these methods, the actual number of Blastocystis-positive samples was 70 (46.97%). There were no statistical differences neither between the results obtained by two methods used in our study nor between each of these methods and the results obtained by the combination of both (p > 0.05). In 79 samples (53.02%), Blastocystis was not detected with any method (Figure 1). Pathogens 2020, 9, x FOR PEER REVIEW 3 of 14 actual number of Blastocystis-positive samples was 70 (46.97%). There were no statistical differences neither between the results obtained by two methods used in our study nor between each of these methods and the results obtained by the combination of both (p > 0.05). In 79 samples (53.02%), Blastocystis was not detected with any method (Figure 1).

Results of Barcode Region Analysis
Out of 66 samples that gave the product using barcode PCR, in 56 of them, obtained sequences corresponded to Blastocystis. Analysis of barcode region PCR products revealed ST5 in 52 out of 56 samples (92.85%) and ST1 in two of 56 samples (3.57%). The DNA sequences of these samples showed mostly high similarity (97-100%) to Blastocystis sequences deposited in GenBank. Sequences of the remaining two out of 56 samples (3.57%) (which gave no product with STS primers) had 87% and 76% similarity with Blastocystis sequences but not with a specific subtype. The sequence query facility recognized ST17 as the closest match to these sequences. However, due to low-quality of the DNA products the allocation remains highly uncertain. Hence, we classified these samples as Blastocystis ST indeterminate. In four cases the obtained sequences instead to Blastocystis corresponded to fungi of the genus Galactomyces, Picchia, and Yarrowia, while in six cases, the sequences were unreadable due to sequences overlapping and numerous gaps in the alignment. In all samples in which mixed infection was detected by STS primers, direct sequencing of the barcode region allowed to identify only one subtype: ST1 in a mixed sample of ST1/ST3, and ST5 in samples containing ST5/ST1 and ST5/ST3. In the case of 27 samples, the methods used gave inconsistent results. Details are presented in Table 1 and Figure 1.
In summary, analysis of the results of all 70 positive samples obtained with one of the two methods described above, or the combination of both, showed ST5 in 94.28% (66/70) of Blastocystis positive samples, ST1 in 7.14% (5/70), and ST3 in 2.85% (2/70). In two of 70 samples (2.85%), subtypes could not be identified.

Results of Barcode Region Analysis
Out of 66 samples that gave the product using barcode PCR, in 56 of them, obtained sequences corresponded to Blastocystis. Analysis of barcode region PCR products revealed ST5 in 52 out of 56 samples (92.85%) and ST1 in two of 56 samples (3.57%). The DNA sequences of these samples showed mostly high similarity (97-100%) to Blastocystis sequences deposited in GenBank. Sequences of the remaining two out of 56 samples (3.57%) (which gave no product with STS primers) had 87% and 76% similarity with Blastocystis sequences but not with a specific subtype. The sequence query facility recognized ST17 as the closest match to these sequences. However, due to low-quality of the DNA products the allocation remains highly uncertain. Hence, we classified these samples as Blastocystis ST indeterminate. In four cases the obtained sequences instead to Blastocystis corresponded to fungi of the genus Galactomyces, Picchia, and Yarrowia, while in six cases, the sequences were unreadable due to sequences overlapping and numerous gaps in the alignment. In all samples in which mixed infection was detected by STS primers, direct sequencing of the barcode region allowed to identify only one subtype: ST1 in a mixed sample of ST1/ST3, and ST5 in samples containing ST5/ST1 and ST5/ST3. In the case of 27 samples, the methods used gave inconsistent results. Details are presented in Table 1 and Figure 1.
In summary, analysis of the results of all 70 positive samples obtained with one of the two methods described above, or the combination of both, showed ST5 in 94.28% (66/70) of Blastocystis positive samples, ST1 in 7.14% (5/70), and ST3 in 2.85% (2/70). In two of 70 samples (2.85%), subtypes could not be identified.

Discussion
To the best of our knowledge, this is the first study providing information about Blastocystis prevalence and subtypes in pigs in Poland. Blastocystis has been reported as a common gut eukaryote in domestic pigs in several geographical regions of the world with the prevalence ranging from 7.5% to 100% [24].
In our study, the prevalence of Blastocystis in pigs was 46.97% which is similar to that obtained (also by PCR methods) in Spain (44.5%), Japan (44.1%), and Cambodia (45.2%), but lower than in most reports from Asian countries where infection rate often exceeds 70%, and in some of these countries it can even reach 100% [11,[24][25][26]38,39]. These differences may be affected by the age of animals tested, the season of sampling for testing, the manner of animal keeping or environmental or sanitary conditions [25,39]. In comparison to abundant data from Asia on the prevalence of Blastocystis in pigs, data from European countries are scarce and highly diverse. In contrast to the mentioned study from Spain showing 44.5% of Blastocystis carriage in pigs, Alfelani et al. [11] reported 28.5% of infected pigs in the UK while Suli et al. [40] reported 81.2% in Serbia. These varied results do not allow a conclusion about the real Blastocystis prevalence in pigs in Europe and indicate the need for further research in this field.
Some authors noted the age of animals as a factor that could affect their susceptibility to Blastocystis infection. Pakandl et al. [41] noticed for the first time that colonization of Blastocystis had already begun in three-day-old piglets, reaching 90% in one-week-old piglets, and persisted at slightly varying levels (84-93%) for the rest of the animals' life. The highest prevalence of Blastocystis in the youngest animals was observed by Navarro et al. [39] who explained this by the fact that very young animals without prior contact with Blastocystis are more susceptible to infection since they lack their sufficient immune response. On the contrary, the lowest prevalence observed in the youngest group of pigs was explained by effective immune protection due to the presence of maternal antibodies [42]. In our study, there was no significant difference between the age groups. The assessment of Blastocystis prevalence in pig age groups based on results obtained by different authors is not fully possible since age groups are defined differently in different studies.
To date, Blastocystis ST1-ST3 and ST5-ST7 have been reported in pigs worldwide with a definite predominance of ST5 followed by ST1 and ST3 [15,26,27,[42][43][44]. ST10 was also reported, but only in one piglet in one study only [24]. Recently, ST15 was also identified in pig faecal samples [45]. In our study, ST5 was the most frequent subtype identified. However, apart from detecting ST5 and ST1, barcode region analysis also revealed two Blastocystis-positive samples which subtypes could not be determined probably because of the poor quality of the PCR products. It cannot be excluded that they contained Blastocystis subtypes other than ST1-ST7, which would explain the negative results obtained for these samples using STS primers. In addition to ST5 and ST1, ST3 was detected in two samples tested using STS primers. However, this subtype was present exclusively in a mixed infection together with ST5 or ST1. The ST3 was not detected in these samples using barcode method because direct sequencing of PCR product in mixed infections allows for detection of only the subtype whose DNA is predominant in the sample (here ST5 or ST1) [46]. For this reason, and because of the RD5 primer's cross-reactivity with species other than Blastocystis [37], in three samples, sequencing revealed fungi of the genus Galactomyces, Pichia, and Yarrowia that are often present in the stool. Their detection, however, does not exclude the presence of Blastocystis in these samples, since ST5 was found in these samples using STS primers.
Despite our considerable effort, we failed to obtain PCR products in eleven samples using STS primers, although barcode region sequencing confirmed the presence of Blastocystis ST5 in these samples. Similar discrepancies about ST1-ST3 were observed by Stensvold [47]. STS primers were designed based on sequence data generated from randomly amplified polymorphic DNAs and still little is known about genetic diversity both between and within STS targets [46,48,49]. Therefore, it cannot be excluded that PCR products were not obtained because STS primers were not fully complementary to the binding sites in our DNA templates.
This plight has also occurred in the opposite direction, i.e., eight samples identified as Blastocystis ST5 with STS primers were negative with barcode PCR. Possibly, the worse quality of DNA templates of these samples caused that shorter DNA fragments (approximately~300 bp) were successfully amplified (with STS primers) while amplification of the longer~600 bp region failed, or it could be a sensitivity issue. Similar problems were encountered by Yoshikawa [50] when some samples positive with STS primers were negative in PCR with a 1.1 kbp SSU rDNA fragment.
Ultimately, the results of the study showed that, although the percentage of samples in which Blastocystis was identified using each method separately was almost identical, some samples determined as positive by one method remained undetected by the other one and vice versa. This shows that the use of anyone of these commonly adopted approaches separately may potentially lead to false-negative results in some samples. In our study, the underestimation of positive samples was 8.72% with STS PCR and 9.39% with barcode PCR and sequencing concerning the actual number of samples containing Blastocystis (Figure 1). However, the differences were not statistically significant (p > 0.05). This inconsistency in the results obtained using each method also highlighted what a challenge is to design Blastocystis specific primers [46,51].
In the recent decade, Blastocystis prevalence and subtypes in pigs have been studied mainly in countries of Southeast Asia [15,[24][25][26][27]42,43] and a lesser extent in Australia [25,51] and USA [52,53]. To date, the most common subtype reported in pigs in these regions was ST5. In our study, ST5 was also the most frequently identified subtype, which suggests that there is no geographic restriction in the occurrence of ST5 in pigs. However, more research from Europe as well as other world regions are needed to confirm this observation. Scarce data from Europe showed the predominance of ST5 in pigs in Denmark [16] while ST1 was the most prevalent subtype in France [54] and Spain [39]. These data are difficult to assess and compare since they are mostly from before 2013 and apply to small groups of animals (≤20). The largest examined group (from Spain) consisted of 395 pigs. However, only eight of 122 Blastocystis-positive samples were randomly selected for sequencing [39]. In turn, authors from Serbia [40] who examined a group of 48 animals did not determine Blastocystis subtypes but focused on the frequency of Blastocystis in this group.
Apart of pigs, ST5 was also identified in other livestock animals including cattle [11,16,55,56], goats [57,58], sheep [44,59], and poultry [60]. However, in these groups of animals, ST5 was less common than in pigs. On the other hand, some authors did not found ST5 in these animals [11,43,44,61]. This suggests that pigs are the main host of ST5 and probable source of Blastocystis infection for other livestock animals which are often kept together or have contact with each other on farms. The sporadic detection of ST6 and ST7 in pigs (subtypes mostly found in birds, including domestic birds [16,62]) can denote that these subtypes, although specific for birds, could have been acquired by pigs through faecal-oral route [26]. This shows the probability of easy transmission of Blastocystis between different animal species.
Humans have been shown to harbour ten Blastocystis STs (ST1-ST9, ST12) but the majority of infections (~90%) are caused by four subtypes with a predominance of ST3 followed by ST1, ST2, and ST4, with proportions varying by geographical region. To date, ST5 has been sporadically reported in humans [63]. A handful of reports suggest that humans could have acquired this subtype by contact with pigs [15,25,27,38]. On the other hand, Blastocystis transmission in the opposite direction, i.e., from humans to pigs, seems to occur as well [26,27]. The following examples best describe different variants of this probably two-way transmission.
In a study in pigs and their in-contact humans, performed by Wang et al. [25], all Blastocystis positive pigs had ST5. Additionally, ST1 and ST3 were found in a small percentage of samples, and exclusively in mixed infection with ST5. Inversely, piggery workers harboured mainly ST3 and ST1, although mixed infection with ST5/ST3 or ST5 alone was found in some of them. Moreover, a comparison of the sequences of ST1, ST3, and ST5 obtained from pigs and their in-contact humans revealed 100% identity in the case of ST1 and ST3, as well as almost 100% (with a single nucleotide polymorphism) in the case of ST5, suggesting possible cross-infection with these subtypes between pigs and their caregivers.
Similarly, Pintong et al. [27] noticed that some sequences of ST5 and ST1 found in pigs and people working on or living near pig farms were highly similar (98-99%) to each other as well as to sequences of ST5 and ST1 derived from humans, pigs and cattle, previously reported in GenBank, which suggest the possibility of multi-directional transmission of these subtypes between livestock animals and people.
In another study, apart from ST5 that was found in all pigs in the study group, the authors also found ST1 in 16.4% and ST2 in 11% of animals. These two subtypes were common in children living in an area where the study was conducted. Interestingly, none of the children harboured ST5 despite the poor hygiene conditions prevailing in this village [26]. However, Udonsom et al. [43] reported that people living around the pig farms did not host ST5, which was found in pigs, and vice-versa-ST1, ST2, or ST3, although present in these people, were not detected in pigs. When compared to the GenBank database, only one ST5 isolated from a pig was closely related to Blastocystis sequence derived from a human, which shows that transmission between humans and animals does not always occur.
These examples show that, although pigs seem to be a natural host for ST5, this subtype can adapt to a human host, and can be transmitted from pigs to humans, which may be favoured by poor sanitation, improper waste management in pig farms, lack of personal hygiene, or no access to clean potable water [25,27]. Although, as shown above, contact or sharing living space with pigs does not always result in such a transmission.
On the other hand, the presence of ST3 (which predominates in humans) and ST1 (also common in humans) in pigs indefinitely smaller quantities than ST5, and often in mixed infection together with ST5 (as took place in our own and some of the mentioned studies), suggests that pigs probably acquire these subtypes by contact with humans. The ability of Blastocystis to be transmitted from humans to pigs was noticed for the first time in 1993 by Pakandl et al. [64] who performed the successful experimental transfer of a human Blastocystis isolate (of unknown ST) to a gnotobiotic piglet (although only one of 16 piglets became infected).
An interesting observation in our study is that ST5 was the only subtype found in the youngest group of pigs (piglets up to four weeks old). In older groups, in addition to the presence of ST5, other Blastocystis subtypes were also detected either in mixed or homogenous infections. This suggests that older animals, i.e., those that have had longer contact with people, have acquired subtypes usually harboured by humans (Table 2).
To date, there is no Polish publication about the occurrence of Blastocystis in animals. Only a few conference reports are available regarding small groups of birds [65,66] and animals from the Wrocław Zoo [67]. Among them, ST5 was found in chickens and among zoo animals but the authors did not provide any information in what animal species. Livestock has not been examined so far. When it comes to humans, ST5 was not detected in any of the human samples tested in a few regions of Poland. However, no information is available on whether these people have had any contact with animals [30][31][32][33]68].

Stool Sampling and Processing
Between November 2017 and February 2018, 149 pig stool samples including 32 piglets (<4 weeks old), 18 weaners (1-3 months old), 54 porkers (3-9 months old) and 45 sows (>12 months old) were collected from 15 piggeries in the Pomerania Province. Then, ten samples were taken from each of 14 piggeries and nine samples from one piggery. One stool sample was taken from each animal pen.
Stool samples were collected from the ground after animal defecation (from the top of droppings to minimize contamination), placed into plastic stool collection containers, transported to the Department of Tropical Medicine and Epidemiology (Medical University of Gdańsk) under cool conditions, then suspended in 70% ethanol and stored at 4 • C until DNA extraction.

DNA Extraction and PCR Amplification
Immediately before DNA extraction, alcohol was washed off the stool samples with phosphate-buffered saline (PBS) two times and then one time with sterile distilled water by centrifugation at 2000× g for 10 min. Each time the supernatant was discarded carefully with a pipette.
Genomic DNA was extracted from washed samples using Genomic Mini AX Stool Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's instruction. All PCR templates were additionally treated with an Anti-Inhibitor Kit (A&A Biotechnology, Gdynia, Poland) to remove PCR inhibitors that could disturb reaction. Extracted DNAs were stored at −20 • C before PCR amplification.
For specific detection of Blastocystis two conventional PCR methods were performed: (i) PCR with subtype-specific sequence-tagged-site (STS) diagnostic primers described by Yoshikawa et al. [36] (Table 3) with the Blastocystis-specific BhRDr and the broad-eukaryote-specific RD5 primers described by Scicluna et al. [37] ( Table 3). The reaction mixture was as above. The PCR conditions were as follows: 4 min at 94 • C (initial denaturation) followed by 35 cycles of 15 s at 95 • C, 15 s at 60 • C, 30 s at 72 • C, and a final extension step of 5 min at 72 • C.
Negative (water instead of extracted DNA) and positive (a sample successfully sequenced for Blastocystis before this study) control was placed in each amplification batch. PCRs were conducted in a thermal cycler (Mastercycler, Eppendorf, Hamburg, Germany). The PCR products were electrophoresed on a 1.5% agarose gel (Sigma, St. Louis, MO, USA) at 150 V for 40 min with Tris-borate-EDTA buffer. Bands were visualized by Midori Green DNA Stain (Nippon Genetics Europe, Duren, Germany) using an ultraviolet gel documentation system Gel Doc-It (UVP LLC, Upland, CA, USA).

Barcode Sequence Analysis
Direct sequencing of barcode region PCR products was performed in both directions by standard procedure with the primers used for amplification. The obtained sequences were then analyzed using GeneStudio Pro Software (GeneStudio Inc., Suwanee, GA, USA) and compared with sequences available in GenBank using NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST). Barcode PCR results were considered positive if direct sequencing of the products confirmed Blastocystis. The chosen Blastocystis sequences identified in this study have been deposited in the GenBank database under accession numbers of MT373829 to MT373878. Blastocystis subtypes affiliation was determined using the sequence query facility at www.pubmlst.org/blastocystis recommended by Stensvold [47], which is under the consensus terminology for Blastocystis subtypes established in 2007 [69].

Statistical Analysis
Prevalence values (percentage of positive samples) are given with 95% confidence limits in parenthesis (CL95) or error bars on figures, calculated using bespoke software "PERCENTAGE CONFIDENCE LIMITS VS 13" (courtesy of Prof. F.S. Gilbert and Prof. J.M. Behnke, University of Nottingham) based on the tables of Rolf and Sokal [70,71]. Differences between age groups were analyzed using ANOVA. Differences between diagnostic methods were analyzed using Student's t-test. Descriptive analysis (percentages) was used to recognize Blastocystis STs distribution.

Conclusions
To the best of our knowledge, this study is the first to evaluate the colonization and Blastocystis subtypes' distribution in pigs in Poland. A moderate Blastocystis infection rate (44.4-50%) was observed in different age groups of pigs with a vital predominance of ST5 in every age group, suggesting that pigs are natural host of ST5. The observation that, in animals, mixed infections (ST5/ST1, ST3/ST1) appear with age may indicate that ST3 and ST1 could have been acquired by pigs during contact with humans. This shows clearly the need for further research in pigs and their in-contact humans from multiple world regions to understand the specificity and relationship of different Blastocystis STs within and between individual host groups. This will help to establish whether pigs are a reservoir of Blastocystis and determine their role in spreading this infection to humans.
The results also highlighted methodological limitations of up-to-date molecular approaches used commonly in Blastocystis identification, i.e., the possibility of missing some infections during diagnostics. Consequently, it also demonstrated the need to include in the assessment molecular methods less frequently used by researchers to identify Blastocystis in order to select and use a PCR method capable of detecting the highly genetically diverse Blastocystis isolates as well as mixed infections at one go.