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Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand
Accepted Manuscript Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand
Ruenruetai Udonsom, Rapeepan Prasertbun, Aongart Mahittikorn, Hirotake Mori, Tanasak Changbunjong, Chalit Komalamisra, Ai-rada Pintong, Yaowalark Sukthana, Supaluk Popruk PII: DOI: Reference:
S1567-1348(18)30480-5 doi:10.1016/j.meegid.2018.07.007 MEEGID 3579
To appear in:
Infection, Genetics and Evolution
Received date: Revised date: Accepted date:
19 April 2018 1 July 2018 4 July 2018
Please cite this article as: Ruenruetai Udonsom, Rapeepan Prasertbun, Aongart Mahittikorn, Hirotake Mori, Tanasak Changbunjong, Chalit Komalamisra, Ai-rada Pintong, Yaowalark Sukthana, Supaluk Popruk , Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand. Meegid (2018), doi:10.1016/j.meegid.2018.07.007
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ACCEPTED MANUSCRIPT Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand Ruenruetai Udonsoma, Rapeepan Prasertbuna, Aongart Mahittikorna, Hirotake Moria, Tanasak Changbunjongb,c, Chalit Komalamisrad, Ai-rada Pintonge, Yaowalark Sukthanaa, Supaluk Popruka Department of Protozoology, Faculty of Tropical Medicine, Mahidol University,
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a
Department of Pre-clinical and Applied Animal Science, Faculty of Veterinary Science,
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b
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Ratchawithi Road, Bangkok 10400, Thailand
Mahidol University, Nakhon Pathom 73170, Thailand
The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic
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c
Animals, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170,
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Thailand
Mahidol- Bangkok School of Tropical Medicine, Faculty of Tropical Medicine, Mahidol
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University, Ratchawithi Road, Bangkok 10400, Thailand e
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Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University,
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Ratchawithi Road, Bangkok 10400, Thailand
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Running Title: Blastocystis in cattle, goats, and pigs of Thailand *Corresponding author: Asst. Prof. Supaluk Popruk Department of Protozoology, Faculty of Tropical Medicine, Mahidol University 420/6 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand Phone: +66 (0) 23549100-4 Fax: +66 (0) 2643 5601 Email: [email protected]
ACCEPTED MANUSCRIPT Abstract Blastocystis spp. are common gastrointestinal parasites of humans and a variety of animals, with various host-specific subtypes. The aim of this study was to determine the prevalence of Blastocystis infection and identify subtypes in humans and domestic animals. One hundred and thirteen stool samples were collected from pigs, goats, and cattle in
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Ayutthaya Province (AP; central Thailand) and 218 stool samples were collected from pigs,
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dogs, cats, chickens, and humans in Kanchanaburi Province (KP; western Thailand).
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Blastocystis was detected by nested PCR targeting the SSU rRNA gene. Subtypes were identified by DNA sequencing, and phylogenetic analysis was conducted. The overall
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prevalence of Blastocystis in animals was 76.1% (86/113) and 11.88% (12/101) in AP and KP, respectively, whereas the prevalence in humans was 12.82% (15/117) in KP. The
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prevalence of Blastocystis in the AP pigs was 4.4 times higher than the KP pigs (87.88% vs 20.37%; p < 0.05). Blastocystis subtype 5 (ST5; zoonotic subtype) was the most abundant in
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pigs in both areas while Blastocystis ST10 and ST12 were most frequently found in cattle and
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goats. Blastocystis ST3, followed by ST2 and ST1 were predominantly found in humans in this study. However, this parasite could be transmitted from humans to pigs or pigs to
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humans, as well as amongst cattle and goats. Blastocystis ST10, ST12, and ST14 were found in cattle and goats in this study. Subtyping is necessary to determine host susceptibility,
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which may differ across hosts and areas, or multiple host types may have the same subtype. Further research is necessary to understand the relationship between hosts and Blastocystis STs Keywords: Blastocystis; zoonotic subtype; pigs; domestic animals
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1.
Introduction In 1911, Alexieff named Blastocystis sp. “Blastocystis enterocora,” and proposed that it
was yeast in the gastrointestinal tract of animals (Alexeieff, 1911). In the following year, Brumpt named the organism “Blastocystis hominis,” which was isolated from human stool
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(Brumpt, 1912) and was subsequently recognized worldwide. To date, the taxonomy of
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Blastocystis is complicated and remains controversial. Molecular techniques have been used to classify this organism as a Stramenopile, a complex and heterogeneous evolutionary
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assemblage of heterotrophic and photosynthetic protozoa (Silberman et al., 1996; Tan, 2008);
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however, Blastocystis does not have the typical features of a Stramenopile (Stensvold and Clark, 2016). It is defined mainly by the results of gene sequence comparisons, rather than by
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any visible characteristics. Blastocystis is commonly found in humans and a variety of animals including non-human primates, birds, and other mammals (Vaisusuk et al., 2017).
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Therefore, Blastocystis species can be named according to their host origins: for example, B.
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ratti in rats, B. galli in chickens, B. lapemi in sea snakes, and B. hominis in humans (Noel et al., 2005). B. hominis has also been found to exhibit differences in serology, isoenzymes, and
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karyotyping (Stenzel and Boreham, 1996). Molecular studies of Blastocystis have revealed that multiple species in the genus
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Blastocystis are the same organism, and studies have shown the similarity in DNA sequences. To date, researchers use a classification called the subtype number, instead of the multiple names of the species to differentiate the organism. At least 17 subtypes (STs) of Blastocystis have been reported in humans and animals using the small subunit rRNA gene (SSU rRNA) (Alfellani et al., 2013b). ST1–ST9 have been found in humans (Rene et al., 2009; Souppart et al., 2010) as well as animals (except ST9 found in human only), while ST10–ST17 are
ACCEPTED MANUSCRIPT subtypes specific to animals (Alfellani et al., 2013b). Thus there exists a risk of zoonotic transmission of Blastocystis ST1–ST8 to humans. Limited information exists concerning subtyping of Blastocystis in animals in Thailand. This study was conducted to identify the prevalence and subtype of Blastocystis in domestic animals and humans in central and western parts of Thailand. The results of subtype
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identification from this study can be used to assess the risk of zoonotic transmission in these
Materials and methods
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2.
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areas, as well as identify the reservoirs of Blastocystis.
This study was a cross-sectional study.
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2.1. Specimen collection
Central part of Thailand: Ayutthaya Province (AP)
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A total of 113 stool specimens were collected from 38 goats, 42 cattle, and 33 pigs in seven small private farms from December 2017 to January 2018. The study protocol was
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approved by the Ethics Committee at the Faculty of Tropical Medicine-Animal Care and Use
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Committee, Mahidol University (FTM-ACUC 006/2017). Western part of Thailand: Kanchanaburi Province (KP)
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A total of 218 stool specimens were collected from 54 pigs in four small private farms, and 117 humans, 13 dogs, 11 cats, and 23 chickens in communities near or in the
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farms, located along the Thai-Myanmar border, from December 2014 to March 2015. The study protocol was approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (MUTM 2014-051-01) and the Animal Care and Use Committee, Mahidol University (FTM-ACUC 006/2014). The type of animals selected were chosen according to whether that type of animal had close contact with humans in those areas. Fresh stool samples were collected either directly from the rectum of pigs kept in individual cages, or from the animal defecating (a
ACCEPTED MANUSCRIPT sample was taken from the middle of the stool, using a plastic spoon). One hundred and seventeen human stool samples were taken from pig handlers and individuals who live near pig farms. All stool samples were kept under cool conditions during transport to the Department of Protozoology, Faculty of Tropical Medicine, Mahidol University and preserved at −80℃ until DNA was extracted.
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2.2. DNA extraction and nested PCR of animal stool samples
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DNA was extracted from the stool samples using a commercially available DNA
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extraction kit (PSPⓇ Spin Stool Kit, Stratec Molecular, Berlin, Germany), according to the manufacturer’s instructions. The small subunit ribosomal RNA (SSU rRNA) gene was
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amplified from the DNA extracted from the stool samples using nested PCR. The PCR products were about 1,100 base pairs in length and used to characterize their STs (ST1– The
first
primer
set
was:
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ST17).
forward
GGGATCCTGATCCTTCCGCAGGTTCACCTAC-3ʹ)
and
primer
reverse
primer
RD3 RD5
(5ʹ(5ʹ-
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GGAAGCTTATCTGGTTGATCCTGCCAGTA-3ʹ) (Clark, 1997). The second set of primers was: forward primer BLF1 (5ʹ-GGAGGTAGTGACAATAAATC-3ʹ) and reverse primer BLR2 (5ʹ-ACTAGGAATTCCTCGTTCATG-3ʹ) (Wong et al., 2008). The PCR products
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were detected with 1.5% agarose gel electrophoresis, stained with ethidium bromide and
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visualized under a transilluminator. 2.3. DNA sequencing and phylogenetic analysis All amplified products were sequenced using the secondary PCR primer on an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The Blastocystis subtypes from each sample were identified by comparison of the homology of nucleotide sequences of positive samples with the Blastocystis sequences published in GenBank, using BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). All raw nucleotide sequences and published sequences of the various known Blastocystis subtypes (Table 1) from GenBank
ACCEPTED MANUSCRIPT were aligned and edited manually using BioEdit v.7.2.5 Software (Ibis Biosciences, Carlsbad, CA, USA). The sequences were then subsequently analyzed using Mega 6 software (Tamura et al., 2013) and the phylogenetic tree was constructed using the maximum likelihood (ML) method. Proteromonas lacertae was used as the outgroup. Branch reliability was assessed using bootstrap analysis (1000 replicates). Fifty representative nucleotide sequences were
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deposited in the GenBank database under the following accession numbers from MH104950
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to MH104999
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2.4. Statistical analysis
Fisher Exact tests were used to compare the prevalence of Blastocystis in the
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different groups. A p value of less than 0.05 was considered statistically significant. SPSS for Windows 18.0 (SPSS Inc.) was used for the data analysis.
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3. Results
The overall prevalence of Blastocystis infection in animals was 76.1% (86/113) and
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11.88% (12/101) in central (AP) and western (KP) parts of Thailand, respectively (Table 2). All infected AP cattle and goats had non-zoonotic Blastocystis STs; (ST10, ST12, and ST14). The prevalence of Blastocystis in pigs of both areas was high but it was significantly higher in
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the AP pigs as compared with the KP pigs, as shown Table 3. Moreover, there was a
KP area.
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significantly higher prevalence of a zoonotic ST in animals in the AP as compared with the
Most of the pigs in the AP and KP were Blastocystis ST5-positive (28/33 and 10/54 respectively), with the exception of one Blastocystis ST1 in each area. There were three Blastocystis STs observed in KP humans and the predominant subtype was Blastocystis ST3, followed by ST2, and ST1. However, there was a high number of suspected subtypes (suspected ST10, ST14, ST12 or 10, ST14 or 10 and suspected ST14 or 12) in AP cattle and goats. The sequences of these samples had relatively low similarity to existing STs and the
ACCEPTED MANUSCRIPT percent identities were less than 97% to 80%. Therefore, the true subtypes of these samples were not identified. We performed a phylogenetic analysis of Blastocystis-positive samples compared with the 17 reference subtype sequences in Genbank. Most of the Blastocystis ST5-positive samples from AP and KP pigs were closely related to animal-derived sequences in GenBank.
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There was one exception: one sequence of the Blastocystis ST5-positive samples from AP
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pigs was also closely related to human-derived sequences in GenBank, as shown in Fig 1.
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4. Discussion
Human-to-human transmission of Blastocystis infection seems likely in this study
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because infected persons who had close contact with others had the same subtypes; Blastocystis ST2 and Blastocystis ST3 in KP. Infected persons can easily spread the parasite
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to others if asymptomatic or if they have poor personal hygiene. Blastocystis ST2 is commonly reported in humans in Bolivia, Brazil, Columbia, Cambodia, and Malaysia, as
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shown in Table 4 (Malheiros et al., 2011; Noradilah et al., 2017; Ramirez et al., 2014;
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Ramirez et al., 2016; Wang et al., 2014). Blastocystis ST3 is the subtype most frequently observed in humans (Wang et al., 2014). Table 4 presents the overview of subtype
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distributions of Blastocystis in various countries. There are different subtypes demonstrated in different hosts or the same hosts or multiple hosts present in the same ST. In this study,
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animal-to-animal transmission of Blastocystis was demonstrated in pigs, cattle, and goats; Blastocystis ST5 in pigs, Blastocystis ST10, 12, and 14 in cattle and goats. Blastocystis ST5 was the predominant subtype in AP and KP pigs and highly prevalent. Therefore, transmission from pigs to humans might occur in those areas if humans exhibit poor personal hygiene, close contact with pigs, or possess a poor waste management system. Blastocystis ST5 has been detected in pigs in Australia, Cambodia, and Denmark (Stensvold et al., 2009; Wang et al., 2014). In KP, no Blastocystis ST5 was detected in humans. Therefore, pigs are
ACCEPTED MANUSCRIPT not likely a source of zoonotic Blastocystis in the KP area. However, there was one sequence from an AP pig that showed a potential relationship to human-derived sequences in GenBank. Palasuwan et al. (2016) reported a prevalence of 5.9% in humans living along the Chao Phraya River, Ayutthaya Province (AP), and Blastocystis ST3 was the predominant subtype, followed by ST2 and ST6 (Palasuwan et al., 2016). Our study area in AP overlapped
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with the study area in Palasuwan et al. (2016). Although pigs showed a high prevalence of
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Blastocystis ST5 in the AP area in this study, it was not detected in any human samples from
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the previous study.
One Blastocystis ST1-infected pig was found in both the AP and KP areas, and two
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human samples in KP also had this subtype. Blastocystis ST1 may be zoonotic, from animals such as pigs, cattle, and monkeys, and was the most common subtype in the Libyan
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population as well (Alfellani et al., 2013a; Noel et al., 2005; Tan, 2008). In Thailand, this subtype was reported in pigs (Thathaisong et al., 2003); however, there was a low prevalence
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of Blastocystis ST1 in AP and KP pigs in this study. Therefore, it seems unlikely that they are
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a zoonotic source of Blastocystis ST1 infection in this area. Blastocystis ST10 was the predominant subtype found in cattle and goats in this study,
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and this subtype has been commonly found in cattle from China, Denmark, and the USA (Santin et al., 2011; Song et al., 2017b; Stensvold et al., 2009; Zhu et al., 2017). Blastocystis
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ST12 was commonly found in cattle and goats in the present study as well. A previous study reported that this subtype was detected in humans in Bolivia who had close contact with infected animals (sheep and llamas) (Ramirez et al., 2016). Therefore, animals might be a possible source of Blastocystis. This subtype was also detected in giraffes and gray kangaroos (Parkar et al., 2010; Roberts et al., 2013). Blastocystis ST14 was detected in AP cattle and goats in this study, and this subtype was detected in camels from Libya, cattle from China and Libya (Alfellani et al., 2013b; Zhu et al., 2017), and cattle in China (Wang et al., 2018).
ACCEPTED MANUSCRIPT Different animal hosts and different areas might present the same Blastocystis ST. A wide host range of Blastocystis STs have been demonstrated worldwide (Table 4). 5.
Conclusions Blastocystis ST5 was highly prevalent in AP and KP pigs but was not detected in any
human samples from the studied area. However, it should be emphasized that pigs easily
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transmit this parasite through the fecal-oral route and it can spread rapidly. Therefore, it is
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important to control Blastocystis infection in infected pigs as well as other animals and
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humans. In addition, we recommend good waste management of infected animal and human stools as a means to control the infected cases in a restricted area. Further studies will be
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needed to identify the hosts of existing Blastocystis STs (ST1–ST17) as well as new Blastocystis STs in different hosts and different areas. This information can help to increase
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our understanding of host-parasite relationships. Acknowledgments
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We would like to thank all the participants and staff of the Faculty of Tropical
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Medicine, Mahidol University for their help in collecting stool samples. We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript and and Mr.
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Pichanan Arunyik for helping a graphical design of abstract. Conflict of interest
Funding
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We declare that we have no conflicts of interest.
This study was supported by a grant from Thanat-Molee Khoman Foundation and the Faculty of Tropical Medicine, Mahidol University, Thailand.
ACCEPTED MANUSCRIPT References Abe, N., 2004. Molecular and phylogenetic analysis of Blastocystis isolates from various hosts. Vet. Parasitol. 120, 235–242. Alexeieff, A., 1911. Sur la nature des formations dites “kystes de Trichomonas intestinalis.” C. R. Soc. Biol. 71, 296–298.
PT
Alfellani, M.A., Stensvold, C.R., Vidal-Lapiedra, A., Onuoha, E.S., Fagbenro-Beyioku, A.F.,
potential implications. Acta Trop. 126, 11–18.
RI
Clark, C.G., 2013a. Variable geographic distribution of Blastocystis subtypes and its
SC
Alfellani, M.A., Taner-Mulla, D., Jacob, A.S., Imeede, C.A., Yoshikawa, H., Stensvold, C.R.,
NU
Clark, C.G., 2013b. Genetic diversity of blastocystis in livestock and zoo animals. Protist. 164, 497–509.
MA
Arisue, N., Hashimoto, T., Yoshikawa, H., 2003. Sequence heterogeneity of the small subunit ribosomal RNA genes among blastocystis isolates. Parasitology. 126, 1–9.
D
Belleza, M.L., Reyes, J.C., Tongol-Rivera, P.N., Rivera, W.L., 2016. Subtype analysis of
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Blastocystis sp. isolates from human and canine hosts in an urban community in the Philippines. Parasitol. Int. 65, 291–294.
725–730.
CE
Brumpt, E., 1912. Blastocystis hominis n. sp. et formes voisines. Bull. Soc. Pathol. Exot. 5,
AC
Casero, R.D., Mongi, F., Sanchez, A., Ramirez, J.D., 2015. Blastocystis and urticaria: Examination of subtypes and morphotypes in an unusual clinical manifestation. Acta Trop. 148, 156–161. Cian, A., El Safadi, D., Osman, M., Moriniere, R., Gantois, N., Benamrouz-Vanneste, S., Delgado-Viscogliosi, P., Guyot, K., Li, L.L., Monchy, S., Noel, C., Poirier, P., Nourrisson, C., Wawrzyniak, I., Delbac, F., Bosc, S., Chabe, M., Petit, T., Certad, G., Viscogliosi, E., 2017. Molecular epidemiology of Blastocystis sp. in various animal
ACCEPTED MANUSCRIPT groups from two French zoos and evaluation of potential zoonotic risk. PLoS One. 12, e0169659. Clark, C.G., 1997. Extensive genetic diversity in Blastocystis hominis. Mol. Biochem. Parasitol. 87, 79–83. Das, R., Khalil, S., Mirdha, B.R., Makharia, G.K., Dattagupta, S., Chaudhry, R., 2016.
PT
Molecular characterization and subtyping of Blastocystis species in irritable bowel
RI
syndrome patients from North India. PLoS One. 11, e0147055.
SC
Dominguez-Marquez, M.V., Guna, R., Munoz, C., Gomez-Munoz, M.T., Borras, R., 2009. High prevalence of subtype 4 among isolates of Blastocystis hominis from
NU
symptomatic patients of a health district of Valencia (Spain). Parasitol. Res. 105, 949– 955.
MA
Forsell, J., Granlund, M., Samuelsson, L., Koskiniemi, S., Edebro, H., Evengard, B., 2016. High occurrence of Blastocystis sp. subtypes 1-3 and Giardia intestinalis assemblage
D
B among patients in Zanzibar, Tanzania. Parasit. Vectors. 9, 370.
PT E
Forsell, J., Granlund, M., Stensvold, C.R., Clark, C.G., Evengard, B., 2012. Subtype analysis of Blastocystis isolates in Swedish patients. Eur. J. Clin. Microbiol. Infect. Dis. 31,
CE
1689–1696.
Fouad, S.A., Basyoni, M.M., Fahmy, R.A., Kobaisi, M.H., 2011. The pathogenic role of
AC
different Blastocystis hominis genotypes isolated from patients with irritable bowel syndrome. Arab J. Gastroenterol. 12, 194–200. Jantermtor, S., Pinlaor, P., Sawadpanich, K., Pinlaor, S., Sangka, A., Wilailuckana, C., Wongsena, W., Yoshikawa, H., 2013. Subtype identification of Blastocystis spp. isolated from patients in a major hospital in northeastern Thailand. Parasitol. Res. 112, 1781–1786.
ACCEPTED MANUSCRIPT Lee, I.L., Tan, T.C., Tan, P.C., Nanthiney, D.R., Biraj, M.K., Surendra, K.M., Suresh, K.G., 2012. Predominance of Blastocystis sp. subtype 4 in rural communities, Nepal. Parasitol. Res. 110, 1553–1562. Leelayoova, S., Siripattanapipong, S., Thathaisong, U., Naaglor, T., Taamasri, P., Piyaraj, P., Mungthin, M., 2008. Drinking water: a possible source of Blastocystis spp. subtype 1
PT
infection in schoolchildren of a rural community in central Thailand. Am. J. Trop.
RI
Med. Hyg. 79, 401–406.
SC
Malheiros, A.F., Stensvold, C.R., Clark, C.G., Braga, G.B., Shaw, J.J., 2011. Short report: Molecular characterization of Blastocystis obtained from members of the indigenous
NU
Tapirape ethnic group from the Brazilian Amazon region, Brazil. Am. J. Trop. Med. Hyg. 85, 1050–1053.
MA
Meloni, D., Sanciu, G., Poirier, P., El Alaoui, H., Chabe, M., Delhaes, L., Dei-Cas, E., Delbac, F., Luigi Fiori, P., Di Cave, D., Viscogliosi, E., 2011. Molecular subtyping of
D
Blastocystis sp. isolates from symptomatic patients in Italy. Parasitol. Res. 109, 613–
PT E
619.
Menounos, P.G., Spanakos, G., Tegos, N., Vassalos, C.M., Papadopoulou, C., Vakalis, N.C.,
CE
2008. Direct detection of Blastocystis sp. in human faecal samples and subtype assignment using single strand conformational polymorphism and sequencing. Mol.
AC
Cell Probes. 22, 24–29. Moosavi, A., Haghighi, A., Mojarad, E.N., Zayeri, F., Alebouyeh, M., Khazan, H., Kazemi, B., Zali, M.R., 2012. Genetic variability of Blastocystis sp. isolated from symptomatic and asymptomatic individuals in Iran. Parasitol. Res. 111, 2311–2315. Nithyamathi, K., Chandramathi, S., Kumar, S., 2016. Predominance of Blastocystis sp. infection among school children in Peninsular Malaysia. PLoS One. 11, e0136709.
ACCEPTED MANUSCRIPT Noel, C., Dufernez, F., Gerbod, D., Edgcomb, V.P., Delgado-Viscogliosi, P., Ho, L.C., Singh, M., Wintjens, R., Sogin, M.L., Capron, M., Pierce, R., Zenner, L., Viscogliosi, E., 2005. Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. J. Clin. Microbiol. 43, 348–355.
PT
Noel, C., Peyronnet, C., Gerbod, D., Edgcomb, V.P., Delgado-Viscogliosi, P., Sogin, M.L.,
RI
Capron, M., Viscogliosi, E., Zenner, L., 2003. Phylogenetic analysis of Blastocystis
SC
isolates from different hosts based on the comparison of small-subunit rRNA gene sequences. Mol. Biochem. Parasitol. 126, 119–123.
NU
Noradilah, S.A., Moktar, N., Anuar, T.S., Lee, I.L., Salleh, F.M., Manap, S., Mohtar, N., Azrul, S.M., Abdullah, W.O., Nordin, A., Abdullah, S.R., 2017. Molecular
MA
epidemiology of blastocystosis in Malaysia: does seasonal variation play an important role in determining the distribution and risk factors of Blastocystis subtype infections
D
in the Aboriginal community? Parasit. Vectors. 10, 360.
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Palasuwan, A., Palasuwan, D., Mahittikorn, A., Chiabchalard, R., Combes, V., Popruk, S., 2016. Subtype distribution of Blastocystis in communities along the Chao Phraya
CE
River, Thailand. Korean J. Parasitol. 54, 455–460. Parkar, U., Traub, R.J., Vitali, S., Elliot, A., Levecke, B., Robertson, I., Geurden, T., Steele,
AC
J., Drake, B., Thompson, R.C., 2010. Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers. Vet. Parasitol. 169, 8–17. Pipatsatitpong, D., Leelayoova, S., Mungthin, M., Aunpad, R., Naaglor, T., Rangsin, R., 2015. Prevalence and risk factors for Blastocystis infection among children and caregivers in a child care center, Bangkok, Thailand. Am. J. Trop. Med. Hyg. 93, 310–315.
ACCEPTED MANUSCRIPT Poirier, P., Wawrzyniak, I., Albert, A., El Alaoui, H., Delbac, F., Livrelli, V., 2011. Development and evaluation of a real-time PCR assay for detection and quantification of blastocystis parasites in human stool samples: prospective study of patients with hematological malignancies. J. Clin. Microbiol. 49, 975–983. Popruk, S., Udonsom, R., Koompapong, K., Mahittikorn, A., Kusolsuk, T., Ruangsittichai, J.,
PT
Palasuwan, A., 2015. Subtype distribution of Blastocystis in Thai-Myanmar border,
RI
Thailand. Korean J. Parasitol. 53, 13–19.
SC
Ramirez, J.D., Florez, C., Olivera, M., Bernal, M.C., Giraldo, J.C., 2017. Blastocystis subtyping and its association with intestinal parasites in children from different
NU
geographical regions of Colombia. PLoS One. 12, e0172586. Ramirez, J.D., Sanchez, A., Hernandez, C., Florez, C., Bernal, M.C., Giraldo, J.C., Reyes, P.,
MA
Lopez, M.C., Garcia, L., Cooper, P.J., Vicuna, Y., Mongi, F., Casero, R.D., 2016. Geographic distribution of human Blastocystis subtypes in South America. Infect.
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Genet. Evol. 41, 32–35.
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Ramirez, J.D., Sanchez, L.V., Bautista, D.C., Corredor, A.F., Florez, A.C., Stensvold, C.R., 2014. Blastocystis subtypes detected in humans and animals from Colombia. Infect.
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Genet. Evol. 22, 223–228.
Rene, B.A., Stensvold, C.R., Badsberg, J.H., Nielsen, H.V., 2009. Subtype analysis of
AC
Blastocystis isolates from Blastocystis cyst excreting patients. Am. J. Trop. Med. Hyg. 80, 588–592.
Rivera, W.L., 2008. Phylogenetic analysis of Blastocystis isolates from animal and human hosts in the Philippines. Vet. Parasitol. 156, 178–182. Roberts, T., Stark, D., Harkness, J., Ellis, J., 2013. Subtype distribution of Blastocystis isolates from a variety of animals from New South Wales, Australia. Vet. Parasitol. 196, 85–89.
ACCEPTED MANUSCRIPT Ruaux, C.G., Stang, B.V., 2014. Prevalence of blastocystis in shelter-resident and clientowned companion animals in the US Pacific Northwest. PLoS One. 9, e107496. Sanpool, O., Laoraksawong, P., Janwan, P., Intapan, P.M., Sawanyawisuth, K., Thanchomnang, T., Changtrakul, Y., Maleewong, W., 2015. Genetic subtypes of Blastocystis isolated from Thai hospitalized patients in Northeastern Thailand.
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Southeast Asian J. Trop. Med. Public Health. 46, 184–190.
RI
Santin, M., Gomez-Munoz, M.T., Solano-Aguilar, G., Fayer, R., 2011. Development of a
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new PCR protocol to detect and subtype Blastocystis spp. from humans and animals. Parasitol. Res. 109, 205–212.
NU
Scanlan, P.D., Knight, R., Song, S.J., Ackermann, G., Cotter, P.D., 2016. Prevalence and genetic diversity of Blastocystis in family units living in the United States. Infect.
MA
Genet. Evol. 45, 95–97.
Silberman, J.D., Sogin, M.L., Leipe, D.D., Clark, C.G., 1996. Human parasite finds
D
taxonomic home. Nature. 380, 398.
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Song, J.K., Hu, R.S., Fan, X.C., Wang, S.S., Zhang, H.J., Zhao, G.H., 2017a. Molecular characterization of Blastocystis from pigs in Shaanxi province of China. Acta Trop.
CE
173, 130–135.
Song, J.K., Yin, Y.L., Yuan, Y.J., Tang, H., Ren, G.J., Zhang, H.J., Li, Z.X., Zhang, Y.M.,
AC
Zhao, G.H., 2017b. First genotyping of Blastocystis sp. in dairy, meat, and cashmere goats in northwestern China. Acta Trop. 176, 277–282. Souppart, L., Moussa, H., Cian, A., Sanciu, G., Poirier, P., El Alaoui, H., Delbac, F., Boorom, K., Delhaes, L., Dei-Cas, E., Viscogliosi, E., 2010. Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt. Parasitol. Res. 106, 505– 511.
ACCEPTED MANUSCRIPT Stensvold, C.R., Alfellani, M.A., Norskov-Lauritsen, S., Prip, K., Victory, E.L., Maddox, C., Nielsen, H.V., Clark, C.G., 2009. Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new subtype. Int. J. Parasitol. 39, 473–479. Stensvold, C.R., Christiansen, D.B., Olsen, K.E., Nielsen, H.V., 2011a. Blastocystis sp.
PT
subtype 4 is common in Danish Blastocystis-positive patients presenting with acute
RI
diarrhea. Am. J. Trop .Med. Hyg. 84, 883–885.
SC
Stensvold, C.R., Clark, C.G., 2016. Current status of Blastocystis: a personal view. Parasitol. Int. 65, 763–771.
NU
Stensvold, C.R., Nielsen, S.D., Badsberg, J.H., Engberg, J., Friis-Moller, N., Nielsen, S.S., Nielsen, H.V., Friis-Moller, A., 2011b. The prevalence and clinical significance of
MA
intestinal parasites in HIV-infected patients in Denmark. Scand. J. Infect. Dis. 43, 129–135.
D
Stenzel, D.J., Boreham, P.F., 1996. Blastocystis hominis revisited. Clin. Microbiol. Rev. 9,
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563–584.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular
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Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Tan, K.S., 2008. New insights on classification, identification, and clinical relevance of
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Blastocystis spp. Clin. Microbiol. Rev. 21, 639–665. Thathaisong, U., Siripattanapipong, S., Mungthin, M., Pipatsatitpong, D., Tan-ariya, P., Naaglor, T., Leelayoova, S., 2013. Identification of Blastocystis subtype 1 variants in the Home for Girls, Bangkok, Thailand. Am. J. Trop. Med. Hyg. 88, 352–358. Thathaisong, U., Worapong, J., Mungthin, M., Tan-Ariya, P., Viputtigul, K., Sudatis, A., Noonai, A., Leelayoova, S., 2003. Blastocystis isolates from a pig and a horse are closely related to Blastocystis hominis. J. Clin. Microbiol. 41, 967–975.
ACCEPTED MANUSCRIPT Vaisusuk, K., Saijuntha, W., Sedlak, S., Thanchomnang, T., Pilap, W., Suksavate, W., Stensvold, C.R., Tantrawatpan, C., 2017. Blastocystis subtypes detected in long-tailed macaques in Thailand-Further evidence of cryptic host specificity. Acta Trop. Villalobos, G., Orozco-Mosqueda, G.E., Lopez-Perez, M., Lopez-Escamilla, E., CordobaAguilar, A., Rangel-Gamboa, L., Olivo-Diaz, A., Romero-Valdovinos, M., Maravilla,
PT
P., Martinez-Hernandez, F., 2014. Suitability of internal transcribed spacers (ITS) as
RI
markers for the population genetic structure of Blastocystis spp. Parasit. Vectors. 7,
SC
461.
Villegas-Gomez, I., Martinez-Hernandez, F., Urrea-Quezada, A., Gonzalez-Diaz, M.,
NU
Durazo, M., Hernandez, J., Orozco-Mosqueda, G.E., Villalobos, G., Maravilla, P., Valenzuela, O., 2016. Comparison of the genetic variability of Blastocystis subtypes
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between human carriers from two contrasting climatic regions of Mexico. Infect. Genet. Evol. 44, 334–340.
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Wang, J., Gong, B., Yang, F., Zhang, W., Zheng, Y., Liu, A., 2018. Subtype distribution and
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genetic characterizations of Blastocystis in pigs, cattle, sheep and goats in northeastern China's Heilongjiang Province. Infect. Genet. Evol. 57, 171–176.
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Wang, W., Cuttell, L., Bielefeldt-Ohmann, H., Inpankaew, T., Owen, H., Traub, R.J., 2013. Diversity of Blastocystis subtypes in dogs in different geographical settings. Parasit.
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Vectors. 6, 215.
Wang, W., Owen, H., Traub, R.J., Cuttell, L., Inpankaew, T., Bielefeldt-Ohmann, H., 2014. Molecular epidemiology of Blastocystis in pigs and their in-contact humans in Southeast Queensland, Australia, and Cambodia. Vet. Parasitol. 203, 264–269. Wong, K.H., Ng, G.C., Lin, R.T., Yoshikawa, H., Taylor, M.B., Tan, K.S., 2008. Predominance of subtype 3 among Blastocystis isolates from a major hospital in Singapore. Parasitol. Res. 102, 663–670.
ACCEPTED MANUSCRIPT Yoshikawa, H., Tokoro, M., Nagamoto, T., Arayama, S., Asih, P.B., Rozi, I.E., Syafruddin, D., 2016. Molecular survey of Blastocystis sp. from humans and associated animals in an Indonesian community with poor hygiene. Parasitol. Int. 65, 780–784. Yoshikawa, H., Wu, Z., Kimata, I., Iseki, M., Ali, I.K., Hossain, M.B., Zaman, V., Haque, R., Takahashi, Y., 2004. Polymerase chain reaction-based genotype classification among
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human Blastocystis hominis populations isolated from different countries. Parasitol.
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Res. 92, 22–29.
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Zhao, G.H., Hu, X.F., Liu, T.L., Hu, R.S., Yu, Z.Q., Yang, W.B., Wu, Y.L., Yu, S.K., Song, J.K., 2017. Molecular characterization of Blastocystis sp. in captive wild animals in
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Qinling Mountains. Parasitol. Res. 116, 2327–2333.
Zhu, W., Tao, W., Gong, B., Yang, H., Li, Y., Song, M., Lu, Y., Li, W., 2017. First report of
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Blastocystis infections in cattle in China. Vet. Parasitol. 246, 38–42.
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Table 1 GenBank references for the Blastocystis ST1–ST17 sequences used to construct a phylogenetic tree Accession no.
Host
Subtype
Reference
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
U51151 AB070989 AB091241 AF538349 AB107961 JQ665860 JQ665867 AB070987 AB107969 EF200010 EU445487 AB070988 AB107963 AB107965 EU445494 AY244620 U51152 AB070998 AB107964 AB107966 EF468654 EU445485 JQ665850 AB070991 AY135412 AB107970 AB107971 AF408425 KC148207 KX234638 KX234639 KX352006 GU256900 KY823301 GU256935 KY823303 KC148210 EU427512 KC148208
human human chicken horse pig human human human pig-tailed macaque human pig human pig cattle human human guinea pig pig pig cattle human chicken human human duck lemur pheasant human camel goat bird fish elephant lechwe quokka
1 1 1 1 1 1 1 2 2 2 2 3 3 3 3 4 4 5 5 5 5 6 6 7 7 8 8 9 10 10 10 10 11 12 13 14 15 16 17
(Silberman et al., 1996) (Arisue et al., 2003) (Arisue et al., 2003) (Thathaisong et al., 2003) (Abe, 2004) (Thathaisong et al., 2013) (Thathaisong et al., 2013) (Arisue et al., 2003) (Abe, 2004) (Leelayoova et al., 2008) (Rivera, 2008) (Arisue et al., 2003) (Abe, 2004) (Abe, 2004) (Rivera, 2008) (Yoshikawa et al., 2004) (Silberman et al., 1996) (Arisue et al., 2003) (Abe, 2004) (Abe, 2004) Yan unpublished (Rivera, 2008) (Thathaisong et al., 2013) (Arisue et al., 2003) (Noel et al., 2003) (Abe, 2004) (Abe, 2004) (Yoshikawa et al., 2004) (Alfellani et al., 2013b) Noradilah unpublished Noradilah unpublished Noradilah unpublished (Parkar et al., 2010) (Zhao et al., 2017) (Parkar et al., 2010) (Zhao et al., 2017) (Alfellani et al., 2013b) Yoshikawa unpublished (Alfellani et al., 2013b)
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camel red kangaroo Libya gundi
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wild ass
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No.
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Table 2 Frequency of Blastocystis infection subtypes in stool samples from animals and humans in two provinces
ST 2
ST 3
ST 5
ST 10
ST 12
ST 14
Suspec ted ST10
1 1
-
-
28 28
2 2 4
4 6 10
3 3
6 10 16
-
-
1 -
-
-
-
1 2
6
7
10 -
-
3 1 2
6 6
8 1 7
10 10 -
-
Suspec ted ST12 or 10
9 9
-
-
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1 2 3
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-
Suspec ted ST14
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ST 1
Suspec ted ST14 or 10
-
Suspec ted ST14 or 12
3 3
8 1 9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
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Central part AP: Cattle (n=42) Goats (n=38) Pigs (n=33) Total Western part KP: Dogs (n=13) Cats (n=11) Chickens (n=23) Pigs (n= 54) Humans (n=117) Total - anima ls - huma ns
No. of observed subtype
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Study area
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AP = Ayutthaya Province, KP = Kanchanaburi Province
Table 3 Prevalence of Blastocystis infection in animals by study area
No. of Blastocystis positive samples/ No. of samples examined
No. of zoonotic Blastocystis subtype samples/ No. of samples examined (prevalence %)
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29/33 11/54 (87.88%) (20.37%)
p-value* AP KP <0.00001 Animals 29/113 12/101 (cat, (25.66%) (11.88%) cattle, chicken, dogs, goats and pigs) <0.00001
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Pigs
KP 1/47 (2.13%)
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AP 57/80 Animals (71.25%) (cat, cattle, chicken, dogs, goats)
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*Fisher Exact test for the animal (cat, cattle, chicken, dogs, goats) and pig prevalence comparison between Kanchanaburi Province (KP) and Ayutthaya Province (AP): p < 0.05.
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** Fisher Exact test for the prevalence of zoonotic Blastocystis subtype (ST) comparison in animals between Kanchanaburi Province (KP) and Ayutthaya Province (AP): p < 0.05.
ACCEPTED MANUSCRIPT Table 4 Subtype distribution of Blastocystis in Thailand and worldwide
Host
References
Subtype No. of subtype
Countri es
1
2
3
4
5
6
7
8
10
1 2
1 3
14
1 6
Mixed subtyp es
Unkno wn subtyp e
68
10
4
49
5
humans
65
11
41
3
Bolivia
humans
40
Brazil
humans
66
27
1 0 1 3 2 1
humans
22
2
7
8
humans
12
4
3
4
humans
125
70
4 0
15
birds
47
cattle marsupia ls dogs
20 25
rodents nonhuman primates humans
3 2
181
82
humans
256
55
Ecuador
humans
25
3
Mexico
humans
47
24
humans
80
30
8
3
1
8
4 7
2 1
(Casero et al., 2015) (Ramirez et al., 2016) (Ramirez et al., 2016) (Malheiros et al., 2011) (Ramirez et al., 2016) (Santin et al., 2011) (Ramirez et al., 2014)
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12
2
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11
4 7
2 5
15
1 5 3
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2
4 7 5 0
33
7
4
7
14 2 21
2
5
2
9
8
42
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1 1
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Columb ia
12
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humans
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Argenti na
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American continent
1
1 1
2
Peru
humans
13
12
1
USA
cattle
7
pigs humans
6 10
2
dogs
4
2
2
cats
7
1
4
humans
25
1
humans
22
9
non-
8
7
(Ramirez et al., 2016) (Ramirez et al., 2017) (Ramirez et al., 2016) (Villalobos et al., 2014) (VillegasGomez et al., 2016) (Ramirez et al., 2016) (Santin et al., 2011)
6 3
5
(Scanlan et al., 2016) (Ruaux and Stang, 2014) 2
European continent Denmar k
4
1
1 9
11
2
2
2
2
2
(Stensvold et al., 2011a) (Stensvold et al., 2011b) (Stensvold
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1
1
3 4
nonhuman primates carnivore s Artiodact yla humans
48
15
9
8
4
1
1
2
12
3
Greece
humans
45
9
6
27
1
Italy
humans
30
2
5
13
6
Spain
human
51
1
2
7
1
1
3
1
Sweden
nonhuman primates humans
63
10
9
30
UK
humans
16
2
9
1 3 1
nonhuman primates sheep humans
70
7
1 1
22
1
1 271
34
2 6
1 11 4
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1 7 4
1 7
3 2
humans
110
15
Libya
humans
38
19
Liberia Nigeria Tanzani a
humans humans humans
30 23 92
2
7 10 36
7
2 7
2 8
8
1
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1
(Yoshikaw a et al., 2004) (Menounos et al., 2008) (Meloni et al., 2011) (Domingue z-Marquez et al., 2009) (Santin et al., 2011)
4
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1
1
(Forsell et al., 2012) (Stensvold et al., 2009)
4
8
2
49
3
2
4 8
8 5
1
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14
5
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Egypt
(Poirier et al., 2011) (Cian et al., 2017)
2
1
African continent
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22 1 1
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German y
24
17
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France
3 3
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human primates pigs cattle sheep deer dogs humans
1 3
1
4
3 3
1 3
5
(Alfellani et al., 2013a)
10
(Fouad et al., 2011) (Alfellani et al., 2013a)
15
8 9 27
3 3
5 1 (Forsell et al., 2016)
Asian continent Cambod ia China
humans
116
33
pigs pigs
33 419
15
45
2
20 39 7 1
9
31
6
cattle
54
goats
458
India
pigs cattle sheep humans
6 14 6 65
1 6
59
Iran
humans
100
21
25
Nepal
humans
63
2
1
11
1
(Wang et al., 2014)
13 1 41
10
29 2
12 3
10 3
2 1
(Song et al., 2017a) (Zhu et al., 2017) (Song et al., 2017b) (Wang et al., 2018)
6 2 1
21
1
33
41
8
(Das et al., 2016) (Moosavi et al., 2012) (Lee et al.,
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pigs chickens rodents humans
73 8 9 186
44
1 3
10 1
9 1 3
humans
191
63
2 7
98
3
humans
195
19
1
53
5
dogs humans chickens monkeys pigs humans
23 5 1 4 2 9
1 1
2
2 4
2
humans
13
monkeys
154
34
humans
20
4
humans
25
2
3
20
humans
77
24
1 1
41
humans
56
12
humans
58
55
humans pigs horses
153 20 1
13 8 20 1
dogs
2
2
humans pigs
30 184
11 4
2012) (Yoshikaw a et al., 2016)
47 59 8 7
1
89
27
6
10
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6
1 1 1
1
1
11
4 8
72
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7 1
12
2
2
SC
2 1 2
1
32
2
1
1 0
2
7
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29 14
Australian continent Australi a
2
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Singapo re Thailan d
40
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Philippi nes
118
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Malaysi a
humans
2
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Indonesi a
16 4
3 16 7
6
9
(Nithyamat hi et al., 2016) (Noradilah et al., 2017) (Belleza et al., 2016) (Rivera, 2008)
(Wong et al., 2008) (Palasuwan et al., 2016) (Vaisusuk et al., 2017) (Sanpool et al., 2015) (Pipatsatitp ong et al., 2015) (Popruk et al., 2015) (Jantermtor et al., 2013) (Thathaiso ng et al., 2013) (Thathaiso ng et al., 2003)
(Wang et al., 2013) (Wang et al., 2014)
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ST5; KPPEK1-52 ST5; KPPEK1-53 ST5; KPPEK1-51 ST5; KPPEK1-10 ST5; KPPEK1-9 ST5; KPPEK1-8 ST5; KPPEK1-7 53 ST5; KPPEK1-6 ST5; KPPEK1-3 ST5; AYP 33 ST5; AYP 32 61 ST5; AYP 31 ST5; AYP 30 ST5; AYP 29 ST5; AYP 23 ST5; AYP 1 88 ST5; AYP 13 77 ST5; AYP 14 ST5; AYP 28 95 ST5 AB107964; pig ST5 AB070998; pig 69 ST5 AB107966; cattle ST5 EF468654; human 98 ST5; AYP 25 ST2; KPH 43.1 ST2; KPH 44.1 77 56 ST2; KPH 42.1 ST2; KPH 18.1 100 ST2 AB070987; human ST2 AB107969; monkey ST2 EF200010; human 57 ST2 EU445487; pig 69 ST1 AB091241; chicken 99 73 ST1 AB107961; pig ST1 JQ665867; human ST1 AB070989; human 98 ST1; KPH 28.2 ST1 AF538349; horse 45 49 ST1; AYP 24 ST1 JQ665860; human 5 ST1 U51151; human ST1; KPH 47.1 ST3 AB107965; cattle ST3; KPD 50 ST3; KPH 2.2 100 ST3 AB070988; human ST3 AB107963; pig 39 ST3 EU445494; human ST3; KPH 22.2 ST3; KPH 24.2 ST3; KPH 24.3 Proteromonas lacertae
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Fig 1. Molecular phylogenetic analysis using maximum likelihood. The evolutionary history was inferred using maximum likelihood, based on the Tamura 3parameter model. KPPEK = pig in Kanchanaburi Province; AYP = pig in Ayutthaya Province; KPH = human in Kanchanaburi Province
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Graphical abstract
ACCEPTED MANUSCRIPT Highlights
High percentages of Blastocystis infection in AP and KP pigs (29/33, 87.88%; 11/54, 20.37% respectively), cattle (21/42, 50%), goat (36/38, 94.74%) and humans (15/117, 12.82%) and this study was the first report of Blastocystis infection in cattle and goats in Thailand.
Blastocystis ST5 was the predominant subtype in both AP (Ayutthaya Province) and
studied areas, in this study.
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Pigs may be a natural host of Blastocystis, in this study.
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KP (Kanchanaburi Province) pigs and this ST may be the pig-adapted ST in the
Ruenruetai Udonsom, Rapeepan Prasertbun, Aongart Mahittikorn, Hirotake Mori, Tanasak Changbunjong, Chalit Komalamisra, Ai-rada Pintong, Yaowalark Sukthana, Supaluk Popruk PII: DOI: Reference:
S1567-1348(18)30480-5 doi:10.1016/j.meegid.2018.07.007 MEEGID 3579
To appear in:
Infection, Genetics and Evolution
Received date: Revised date: Accepted date:
19 April 2018 1 July 2018 4 July 2018
Please cite this article as: Ruenruetai Udonsom, Rapeepan Prasertbun, Aongart Mahittikorn, Hirotake Mori, Tanasak Changbunjong, Chalit Komalamisra, Ai-rada Pintong, Yaowalark Sukthana, Supaluk Popruk , Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand. Meegid (2018), doi:10.1016/j.meegid.2018.07.007
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ACCEPTED MANUSCRIPT Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand Ruenruetai Udonsoma, Rapeepan Prasertbuna, Aongart Mahittikorna, Hirotake Moria, Tanasak Changbunjongb,c, Chalit Komalamisrad, Ai-rada Pintonge, Yaowalark Sukthanaa, Supaluk Popruka Department of Protozoology, Faculty of Tropical Medicine, Mahidol University,
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a
Department of Pre-clinical and Applied Animal Science, Faculty of Veterinary Science,
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b
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Ratchawithi Road, Bangkok 10400, Thailand
Mahidol University, Nakhon Pathom 73170, Thailand
The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic
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c
Animals, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom 73170,
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Thailand
Mahidol- Bangkok School of Tropical Medicine, Faculty of Tropical Medicine, Mahidol
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University, Ratchawithi Road, Bangkok 10400, Thailand e
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Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University,
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Ratchawithi Road, Bangkok 10400, Thailand
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Running Title: Blastocystis in cattle, goats, and pigs of Thailand *Corresponding author: Asst. Prof. Supaluk Popruk Department of Protozoology, Faculty of Tropical Medicine, Mahidol University 420/6 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand Phone: +66 (0) 23549100-4 Fax: +66 (0) 2643 5601 Email: [email protected]
ACCEPTED MANUSCRIPT Abstract Blastocystis spp. are common gastrointestinal parasites of humans and a variety of animals, with various host-specific subtypes. The aim of this study was to determine the prevalence of Blastocystis infection and identify subtypes in humans and domestic animals. One hundred and thirteen stool samples were collected from pigs, goats, and cattle in
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Ayutthaya Province (AP; central Thailand) and 218 stool samples were collected from pigs,
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dogs, cats, chickens, and humans in Kanchanaburi Province (KP; western Thailand).
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Blastocystis was detected by nested PCR targeting the SSU rRNA gene. Subtypes were identified by DNA sequencing, and phylogenetic analysis was conducted. The overall
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prevalence of Blastocystis in animals was 76.1% (86/113) and 11.88% (12/101) in AP and KP, respectively, whereas the prevalence in humans was 12.82% (15/117) in KP. The
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prevalence of Blastocystis in the AP pigs was 4.4 times higher than the KP pigs (87.88% vs 20.37%; p < 0.05). Blastocystis subtype 5 (ST5; zoonotic subtype) was the most abundant in
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pigs in both areas while Blastocystis ST10 and ST12 were most frequently found in cattle and
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goats. Blastocystis ST3, followed by ST2 and ST1 were predominantly found in humans in this study. However, this parasite could be transmitted from humans to pigs or pigs to
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humans, as well as amongst cattle and goats. Blastocystis ST10, ST12, and ST14 were found in cattle and goats in this study. Subtyping is necessary to determine host susceptibility,
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which may differ across hosts and areas, or multiple host types may have the same subtype. Further research is necessary to understand the relationship between hosts and Blastocystis STs Keywords: Blastocystis; zoonotic subtype; pigs; domestic animals
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1.
Introduction In 1911, Alexieff named Blastocystis sp. “Blastocystis enterocora,” and proposed that it
was yeast in the gastrointestinal tract of animals (Alexeieff, 1911). In the following year, Brumpt named the organism “Blastocystis hominis,” which was isolated from human stool
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(Brumpt, 1912) and was subsequently recognized worldwide. To date, the taxonomy of
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Blastocystis is complicated and remains controversial. Molecular techniques have been used to classify this organism as a Stramenopile, a complex and heterogeneous evolutionary
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assemblage of heterotrophic and photosynthetic protozoa (Silberman et al., 1996; Tan, 2008);
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however, Blastocystis does not have the typical features of a Stramenopile (Stensvold and Clark, 2016). It is defined mainly by the results of gene sequence comparisons, rather than by
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any visible characteristics. Blastocystis is commonly found in humans and a variety of animals including non-human primates, birds, and other mammals (Vaisusuk et al., 2017).
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Therefore, Blastocystis species can be named according to their host origins: for example, B.
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ratti in rats, B. galli in chickens, B. lapemi in sea snakes, and B. hominis in humans (Noel et al., 2005). B. hominis has also been found to exhibit differences in serology, isoenzymes, and
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karyotyping (Stenzel and Boreham, 1996). Molecular studies of Blastocystis have revealed that multiple species in the genus
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Blastocystis are the same organism, and studies have shown the similarity in DNA sequences. To date, researchers use a classification called the subtype number, instead of the multiple names of the species to differentiate the organism. At least 17 subtypes (STs) of Blastocystis have been reported in humans and animals using the small subunit rRNA gene (SSU rRNA) (Alfellani et al., 2013b). ST1–ST9 have been found in humans (Rene et al., 2009; Souppart et al., 2010) as well as animals (except ST9 found in human only), while ST10–ST17 are
ACCEPTED MANUSCRIPT subtypes specific to animals (Alfellani et al., 2013b). Thus there exists a risk of zoonotic transmission of Blastocystis ST1–ST8 to humans. Limited information exists concerning subtyping of Blastocystis in animals in Thailand. This study was conducted to identify the prevalence and subtype of Blastocystis in domestic animals and humans in central and western parts of Thailand. The results of subtype
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identification from this study can be used to assess the risk of zoonotic transmission in these
Materials and methods
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2.
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areas, as well as identify the reservoirs of Blastocystis.
This study was a cross-sectional study.
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2.1. Specimen collection
Central part of Thailand: Ayutthaya Province (AP)
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A total of 113 stool specimens were collected from 38 goats, 42 cattle, and 33 pigs in seven small private farms from December 2017 to January 2018. The study protocol was
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approved by the Ethics Committee at the Faculty of Tropical Medicine-Animal Care and Use
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Committee, Mahidol University (FTM-ACUC 006/2017). Western part of Thailand: Kanchanaburi Province (KP)
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A total of 218 stool specimens were collected from 54 pigs in four small private farms, and 117 humans, 13 dogs, 11 cats, and 23 chickens in communities near or in the
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farms, located along the Thai-Myanmar border, from December 2014 to March 2015. The study protocol was approved by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (MUTM 2014-051-01) and the Animal Care and Use Committee, Mahidol University (FTM-ACUC 006/2014). The type of animals selected were chosen according to whether that type of animal had close contact with humans in those areas. Fresh stool samples were collected either directly from the rectum of pigs kept in individual cages, or from the animal defecating (a
ACCEPTED MANUSCRIPT sample was taken from the middle of the stool, using a plastic spoon). One hundred and seventeen human stool samples were taken from pig handlers and individuals who live near pig farms. All stool samples were kept under cool conditions during transport to the Department of Protozoology, Faculty of Tropical Medicine, Mahidol University and preserved at −80℃ until DNA was extracted.
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2.2. DNA extraction and nested PCR of animal stool samples
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DNA was extracted from the stool samples using a commercially available DNA
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extraction kit (PSPⓇ Spin Stool Kit, Stratec Molecular, Berlin, Germany), according to the manufacturer’s instructions. The small subunit ribosomal RNA (SSU rRNA) gene was
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amplified from the DNA extracted from the stool samples using nested PCR. The PCR products were about 1,100 base pairs in length and used to characterize their STs (ST1– The
first
primer
set
was:
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ST17).
forward
GGGATCCTGATCCTTCCGCAGGTTCACCTAC-3ʹ)
and
primer
reverse
primer
RD3 RD5
(5ʹ(5ʹ-
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GGAAGCTTATCTGGTTGATCCTGCCAGTA-3ʹ) (Clark, 1997). The second set of primers was: forward primer BLF1 (5ʹ-GGAGGTAGTGACAATAAATC-3ʹ) and reverse primer BLR2 (5ʹ-ACTAGGAATTCCTCGTTCATG-3ʹ) (Wong et al., 2008). The PCR products
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were detected with 1.5% agarose gel electrophoresis, stained with ethidium bromide and
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visualized under a transilluminator. 2.3. DNA sequencing and phylogenetic analysis All amplified products were sequenced using the secondary PCR primer on an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The Blastocystis subtypes from each sample were identified by comparison of the homology of nucleotide sequences of positive samples with the Blastocystis sequences published in GenBank, using BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi). All raw nucleotide sequences and published sequences of the various known Blastocystis subtypes (Table 1) from GenBank
ACCEPTED MANUSCRIPT were aligned and edited manually using BioEdit v.7.2.5 Software (Ibis Biosciences, Carlsbad, CA, USA). The sequences were then subsequently analyzed using Mega 6 software (Tamura et al., 2013) and the phylogenetic tree was constructed using the maximum likelihood (ML) method. Proteromonas lacertae was used as the outgroup. Branch reliability was assessed using bootstrap analysis (1000 replicates). Fifty representative nucleotide sequences were
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deposited in the GenBank database under the following accession numbers from MH104950
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to MH104999
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2.4. Statistical analysis
Fisher Exact tests were used to compare the prevalence of Blastocystis in the
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different groups. A p value of less than 0.05 was considered statistically significant. SPSS for Windows 18.0 (SPSS Inc.) was used for the data analysis.
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3. Results
The overall prevalence of Blastocystis infection in animals was 76.1% (86/113) and
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11.88% (12/101) in central (AP) and western (KP) parts of Thailand, respectively (Table 2). All infected AP cattle and goats had non-zoonotic Blastocystis STs; (ST10, ST12, and ST14). The prevalence of Blastocystis in pigs of both areas was high but it was significantly higher in
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the AP pigs as compared with the KP pigs, as shown Table 3. Moreover, there was a
KP area.
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significantly higher prevalence of a zoonotic ST in animals in the AP as compared with the
Most of the pigs in the AP and KP were Blastocystis ST5-positive (28/33 and 10/54 respectively), with the exception of one Blastocystis ST1 in each area. There were three Blastocystis STs observed in KP humans and the predominant subtype was Blastocystis ST3, followed by ST2, and ST1. However, there was a high number of suspected subtypes (suspected ST10, ST14, ST12 or 10, ST14 or 10 and suspected ST14 or 12) in AP cattle and goats. The sequences of these samples had relatively low similarity to existing STs and the
ACCEPTED MANUSCRIPT percent identities were less than 97% to 80%. Therefore, the true subtypes of these samples were not identified. We performed a phylogenetic analysis of Blastocystis-positive samples compared with the 17 reference subtype sequences in Genbank. Most of the Blastocystis ST5-positive samples from AP and KP pigs were closely related to animal-derived sequences in GenBank.
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There was one exception: one sequence of the Blastocystis ST5-positive samples from AP
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pigs was also closely related to human-derived sequences in GenBank, as shown in Fig 1.
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4. Discussion
Human-to-human transmission of Blastocystis infection seems likely in this study
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because infected persons who had close contact with others had the same subtypes; Blastocystis ST2 and Blastocystis ST3 in KP. Infected persons can easily spread the parasite
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to others if asymptomatic or if they have poor personal hygiene. Blastocystis ST2 is commonly reported in humans in Bolivia, Brazil, Columbia, Cambodia, and Malaysia, as
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shown in Table 4 (Malheiros et al., 2011; Noradilah et al., 2017; Ramirez et al., 2014;
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Ramirez et al., 2016; Wang et al., 2014). Blastocystis ST3 is the subtype most frequently observed in humans (Wang et al., 2014). Table 4 presents the overview of subtype
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distributions of Blastocystis in various countries. There are different subtypes demonstrated in different hosts or the same hosts or multiple hosts present in the same ST. In this study,
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animal-to-animal transmission of Blastocystis was demonstrated in pigs, cattle, and goats; Blastocystis ST5 in pigs, Blastocystis ST10, 12, and 14 in cattle and goats. Blastocystis ST5 was the predominant subtype in AP and KP pigs and highly prevalent. Therefore, transmission from pigs to humans might occur in those areas if humans exhibit poor personal hygiene, close contact with pigs, or possess a poor waste management system. Blastocystis ST5 has been detected in pigs in Australia, Cambodia, and Denmark (Stensvold et al., 2009; Wang et al., 2014). In KP, no Blastocystis ST5 was detected in humans. Therefore, pigs are
ACCEPTED MANUSCRIPT not likely a source of zoonotic Blastocystis in the KP area. However, there was one sequence from an AP pig that showed a potential relationship to human-derived sequences in GenBank. Palasuwan et al. (2016) reported a prevalence of 5.9% in humans living along the Chao Phraya River, Ayutthaya Province (AP), and Blastocystis ST3 was the predominant subtype, followed by ST2 and ST6 (Palasuwan et al., 2016). Our study area in AP overlapped
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with the study area in Palasuwan et al. (2016). Although pigs showed a high prevalence of
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Blastocystis ST5 in the AP area in this study, it was not detected in any human samples from
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the previous study.
One Blastocystis ST1-infected pig was found in both the AP and KP areas, and two
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human samples in KP also had this subtype. Blastocystis ST1 may be zoonotic, from animals such as pigs, cattle, and monkeys, and was the most common subtype in the Libyan
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population as well (Alfellani et al., 2013a; Noel et al., 2005; Tan, 2008). In Thailand, this subtype was reported in pigs (Thathaisong et al., 2003); however, there was a low prevalence
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of Blastocystis ST1 in AP and KP pigs in this study. Therefore, it seems unlikely that they are
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a zoonotic source of Blastocystis ST1 infection in this area. Blastocystis ST10 was the predominant subtype found in cattle and goats in this study,
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and this subtype has been commonly found in cattle from China, Denmark, and the USA (Santin et al., 2011; Song et al., 2017b; Stensvold et al., 2009; Zhu et al., 2017). Blastocystis
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ST12 was commonly found in cattle and goats in the present study as well. A previous study reported that this subtype was detected in humans in Bolivia who had close contact with infected animals (sheep and llamas) (Ramirez et al., 2016). Therefore, animals might be a possible source of Blastocystis. This subtype was also detected in giraffes and gray kangaroos (Parkar et al., 2010; Roberts et al., 2013). Blastocystis ST14 was detected in AP cattle and goats in this study, and this subtype was detected in camels from Libya, cattle from China and Libya (Alfellani et al., 2013b; Zhu et al., 2017), and cattle in China (Wang et al., 2018).
ACCEPTED MANUSCRIPT Different animal hosts and different areas might present the same Blastocystis ST. A wide host range of Blastocystis STs have been demonstrated worldwide (Table 4). 5.
Conclusions Blastocystis ST5 was highly prevalent in AP and KP pigs but was not detected in any
human samples from the studied area. However, it should be emphasized that pigs easily
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transmit this parasite through the fecal-oral route and it can spread rapidly. Therefore, it is
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important to control Blastocystis infection in infected pigs as well as other animals and
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humans. In addition, we recommend good waste management of infected animal and human stools as a means to control the infected cases in a restricted area. Further studies will be
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needed to identify the hosts of existing Blastocystis STs (ST1–ST17) as well as new Blastocystis STs in different hosts and different areas. This information can help to increase
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our understanding of host-parasite relationships. Acknowledgments
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We would like to thank all the participants and staff of the Faculty of Tropical
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Medicine, Mahidol University for their help in collecting stool samples. We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript and and Mr.
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Pichanan Arunyik for helping a graphical design of abstract. Conflict of interest
Funding
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We declare that we have no conflicts of interest.
This study was supported by a grant from Thanat-Molee Khoman Foundation and the Faculty of Tropical Medicine, Mahidol University, Thailand.
ACCEPTED MANUSCRIPT References Abe, N., 2004. Molecular and phylogenetic analysis of Blastocystis isolates from various hosts. Vet. Parasitol. 120, 235–242. Alexeieff, A., 1911. Sur la nature des formations dites “kystes de Trichomonas intestinalis.” C. R. Soc. Biol. 71, 296–298.
PT
Alfellani, M.A., Stensvold, C.R., Vidal-Lapiedra, A., Onuoha, E.S., Fagbenro-Beyioku, A.F.,
potential implications. Acta Trop. 126, 11–18.
RI
Clark, C.G., 2013a. Variable geographic distribution of Blastocystis subtypes and its
SC
Alfellani, M.A., Taner-Mulla, D., Jacob, A.S., Imeede, C.A., Yoshikawa, H., Stensvold, C.R.,
NU
Clark, C.G., 2013b. Genetic diversity of blastocystis in livestock and zoo animals. Protist. 164, 497–509.
MA
Arisue, N., Hashimoto, T., Yoshikawa, H., 2003. Sequence heterogeneity of the small subunit ribosomal RNA genes among blastocystis isolates. Parasitology. 126, 1–9.
D
Belleza, M.L., Reyes, J.C., Tongol-Rivera, P.N., Rivera, W.L., 2016. Subtype analysis of
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Blastocystis sp. isolates from human and canine hosts in an urban community in the Philippines. Parasitol. Int. 65, 291–294.
725–730.
CE
Brumpt, E., 1912. Blastocystis hominis n. sp. et formes voisines. Bull. Soc. Pathol. Exot. 5,
AC
Casero, R.D., Mongi, F., Sanchez, A., Ramirez, J.D., 2015. Blastocystis and urticaria: Examination of subtypes and morphotypes in an unusual clinical manifestation. Acta Trop. 148, 156–161. Cian, A., El Safadi, D., Osman, M., Moriniere, R., Gantois, N., Benamrouz-Vanneste, S., Delgado-Viscogliosi, P., Guyot, K., Li, L.L., Monchy, S., Noel, C., Poirier, P., Nourrisson, C., Wawrzyniak, I., Delbac, F., Bosc, S., Chabe, M., Petit, T., Certad, G., Viscogliosi, E., 2017. Molecular epidemiology of Blastocystis sp. in various animal
ACCEPTED MANUSCRIPT groups from two French zoos and evaluation of potential zoonotic risk. PLoS One. 12, e0169659. Clark, C.G., 1997. Extensive genetic diversity in Blastocystis hominis. Mol. Biochem. Parasitol. 87, 79–83. Das, R., Khalil, S., Mirdha, B.R., Makharia, G.K., Dattagupta, S., Chaudhry, R., 2016.
PT
Molecular characterization and subtyping of Blastocystis species in irritable bowel
RI
syndrome patients from North India. PLoS One. 11, e0147055.
SC
Dominguez-Marquez, M.V., Guna, R., Munoz, C., Gomez-Munoz, M.T., Borras, R., 2009. High prevalence of subtype 4 among isolates of Blastocystis hominis from
NU
symptomatic patients of a health district of Valencia (Spain). Parasitol. Res. 105, 949– 955.
MA
Forsell, J., Granlund, M., Samuelsson, L., Koskiniemi, S., Edebro, H., Evengard, B., 2016. High occurrence of Blastocystis sp. subtypes 1-3 and Giardia intestinalis assemblage
D
B among patients in Zanzibar, Tanzania. Parasit. Vectors. 9, 370.
PT E
Forsell, J., Granlund, M., Stensvold, C.R., Clark, C.G., Evengard, B., 2012. Subtype analysis of Blastocystis isolates in Swedish patients. Eur. J. Clin. Microbiol. Infect. Dis. 31,
CE
1689–1696.
Fouad, S.A., Basyoni, M.M., Fahmy, R.A., Kobaisi, M.H., 2011. The pathogenic role of
AC
different Blastocystis hominis genotypes isolated from patients with irritable bowel syndrome. Arab J. Gastroenterol. 12, 194–200. Jantermtor, S., Pinlaor, P., Sawadpanich, K., Pinlaor, S., Sangka, A., Wilailuckana, C., Wongsena, W., Yoshikawa, H., 2013. Subtype identification of Blastocystis spp. isolated from patients in a major hospital in northeastern Thailand. Parasitol. Res. 112, 1781–1786.
ACCEPTED MANUSCRIPT Lee, I.L., Tan, T.C., Tan, P.C., Nanthiney, D.R., Biraj, M.K., Surendra, K.M., Suresh, K.G., 2012. Predominance of Blastocystis sp. subtype 4 in rural communities, Nepal. Parasitol. Res. 110, 1553–1562. Leelayoova, S., Siripattanapipong, S., Thathaisong, U., Naaglor, T., Taamasri, P., Piyaraj, P., Mungthin, M., 2008. Drinking water: a possible source of Blastocystis spp. subtype 1
PT
infection in schoolchildren of a rural community in central Thailand. Am. J. Trop.
RI
Med. Hyg. 79, 401–406.
SC
Malheiros, A.F., Stensvold, C.R., Clark, C.G., Braga, G.B., Shaw, J.J., 2011. Short report: Molecular characterization of Blastocystis obtained from members of the indigenous
NU
Tapirape ethnic group from the Brazilian Amazon region, Brazil. Am. J. Trop. Med. Hyg. 85, 1050–1053.
MA
Meloni, D., Sanciu, G., Poirier, P., El Alaoui, H., Chabe, M., Delhaes, L., Dei-Cas, E., Delbac, F., Luigi Fiori, P., Di Cave, D., Viscogliosi, E., 2011. Molecular subtyping of
D
Blastocystis sp. isolates from symptomatic patients in Italy. Parasitol. Res. 109, 613–
PT E
619.
Menounos, P.G., Spanakos, G., Tegos, N., Vassalos, C.M., Papadopoulou, C., Vakalis, N.C.,
CE
2008. Direct detection of Blastocystis sp. in human faecal samples and subtype assignment using single strand conformational polymorphism and sequencing. Mol.
AC
Cell Probes. 22, 24–29. Moosavi, A., Haghighi, A., Mojarad, E.N., Zayeri, F., Alebouyeh, M., Khazan, H., Kazemi, B., Zali, M.R., 2012. Genetic variability of Blastocystis sp. isolated from symptomatic and asymptomatic individuals in Iran. Parasitol. Res. 111, 2311–2315. Nithyamathi, K., Chandramathi, S., Kumar, S., 2016. Predominance of Blastocystis sp. infection among school children in Peninsular Malaysia. PLoS One. 11, e0136709.
ACCEPTED MANUSCRIPT Noel, C., Dufernez, F., Gerbod, D., Edgcomb, V.P., Delgado-Viscogliosi, P., Ho, L.C., Singh, M., Wintjens, R., Sogin, M.L., Capron, M., Pierce, R., Zenner, L., Viscogliosi, E., 2005. Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. J. Clin. Microbiol. 43, 348–355.
PT
Noel, C., Peyronnet, C., Gerbod, D., Edgcomb, V.P., Delgado-Viscogliosi, P., Sogin, M.L.,
RI
Capron, M., Viscogliosi, E., Zenner, L., 2003. Phylogenetic analysis of Blastocystis
SC
isolates from different hosts based on the comparison of small-subunit rRNA gene sequences. Mol. Biochem. Parasitol. 126, 119–123.
NU
Noradilah, S.A., Moktar, N., Anuar, T.S., Lee, I.L., Salleh, F.M., Manap, S., Mohtar, N., Azrul, S.M., Abdullah, W.O., Nordin, A., Abdullah, S.R., 2017. Molecular
MA
epidemiology of blastocystosis in Malaysia: does seasonal variation play an important role in determining the distribution and risk factors of Blastocystis subtype infections
D
in the Aboriginal community? Parasit. Vectors. 10, 360.
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Palasuwan, A., Palasuwan, D., Mahittikorn, A., Chiabchalard, R., Combes, V., Popruk, S., 2016. Subtype distribution of Blastocystis in communities along the Chao Phraya
CE
River, Thailand. Korean J. Parasitol. 54, 455–460. Parkar, U., Traub, R.J., Vitali, S., Elliot, A., Levecke, B., Robertson, I., Geurden, T., Steele,
AC
J., Drake, B., Thompson, R.C., 2010. Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers. Vet. Parasitol. 169, 8–17. Pipatsatitpong, D., Leelayoova, S., Mungthin, M., Aunpad, R., Naaglor, T., Rangsin, R., 2015. Prevalence and risk factors for Blastocystis infection among children and caregivers in a child care center, Bangkok, Thailand. Am. J. Trop. Med. Hyg. 93, 310–315.
ACCEPTED MANUSCRIPT Poirier, P., Wawrzyniak, I., Albert, A., El Alaoui, H., Delbac, F., Livrelli, V., 2011. Development and evaluation of a real-time PCR assay for detection and quantification of blastocystis parasites in human stool samples: prospective study of patients with hematological malignancies. J. Clin. Microbiol. 49, 975–983. Popruk, S., Udonsom, R., Koompapong, K., Mahittikorn, A., Kusolsuk, T., Ruangsittichai, J.,
PT
Palasuwan, A., 2015. Subtype distribution of Blastocystis in Thai-Myanmar border,
RI
Thailand. Korean J. Parasitol. 53, 13–19.
SC
Ramirez, J.D., Florez, C., Olivera, M., Bernal, M.C., Giraldo, J.C., 2017. Blastocystis subtyping and its association with intestinal parasites in children from different
NU
geographical regions of Colombia. PLoS One. 12, e0172586. Ramirez, J.D., Sanchez, A., Hernandez, C., Florez, C., Bernal, M.C., Giraldo, J.C., Reyes, P.,
MA
Lopez, M.C., Garcia, L., Cooper, P.J., Vicuna, Y., Mongi, F., Casero, R.D., 2016. Geographic distribution of human Blastocystis subtypes in South America. Infect.
D
Genet. Evol. 41, 32–35.
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Ramirez, J.D., Sanchez, L.V., Bautista, D.C., Corredor, A.F., Florez, A.C., Stensvold, C.R., 2014. Blastocystis subtypes detected in humans and animals from Colombia. Infect.
CE
Genet. Evol. 22, 223–228.
Rene, B.A., Stensvold, C.R., Badsberg, J.H., Nielsen, H.V., 2009. Subtype analysis of
AC
Blastocystis isolates from Blastocystis cyst excreting patients. Am. J. Trop. Med. Hyg. 80, 588–592.
Rivera, W.L., 2008. Phylogenetic analysis of Blastocystis isolates from animal and human hosts in the Philippines. Vet. Parasitol. 156, 178–182. Roberts, T., Stark, D., Harkness, J., Ellis, J., 2013. Subtype distribution of Blastocystis isolates from a variety of animals from New South Wales, Australia. Vet. Parasitol. 196, 85–89.
ACCEPTED MANUSCRIPT Ruaux, C.G., Stang, B.V., 2014. Prevalence of blastocystis in shelter-resident and clientowned companion animals in the US Pacific Northwest. PLoS One. 9, e107496. Sanpool, O., Laoraksawong, P., Janwan, P., Intapan, P.M., Sawanyawisuth, K., Thanchomnang, T., Changtrakul, Y., Maleewong, W., 2015. Genetic subtypes of Blastocystis isolated from Thai hospitalized patients in Northeastern Thailand.
PT
Southeast Asian J. Trop. Med. Public Health. 46, 184–190.
RI
Santin, M., Gomez-Munoz, M.T., Solano-Aguilar, G., Fayer, R., 2011. Development of a
SC
new PCR protocol to detect and subtype Blastocystis spp. from humans and animals. Parasitol. Res. 109, 205–212.
NU
Scanlan, P.D., Knight, R., Song, S.J., Ackermann, G., Cotter, P.D., 2016. Prevalence and genetic diversity of Blastocystis in family units living in the United States. Infect.
MA
Genet. Evol. 45, 95–97.
Silberman, J.D., Sogin, M.L., Leipe, D.D., Clark, C.G., 1996. Human parasite finds
D
taxonomic home. Nature. 380, 398.
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Song, J.K., Hu, R.S., Fan, X.C., Wang, S.S., Zhang, H.J., Zhao, G.H., 2017a. Molecular characterization of Blastocystis from pigs in Shaanxi province of China. Acta Trop.
CE
173, 130–135.
Song, J.K., Yin, Y.L., Yuan, Y.J., Tang, H., Ren, G.J., Zhang, H.J., Li, Z.X., Zhang, Y.M.,
AC
Zhao, G.H., 2017b. First genotyping of Blastocystis sp. in dairy, meat, and cashmere goats in northwestern China. Acta Trop. 176, 277–282. Souppart, L., Moussa, H., Cian, A., Sanciu, G., Poirier, P., El Alaoui, H., Delbac, F., Boorom, K., Delhaes, L., Dei-Cas, E., Viscogliosi, E., 2010. Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt. Parasitol. Res. 106, 505– 511.
ACCEPTED MANUSCRIPT Stensvold, C.R., Alfellani, M.A., Norskov-Lauritsen, S., Prip, K., Victory, E.L., Maddox, C., Nielsen, H.V., Clark, C.G., 2009. Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new subtype. Int. J. Parasitol. 39, 473–479. Stensvold, C.R., Christiansen, D.B., Olsen, K.E., Nielsen, H.V., 2011a. Blastocystis sp.
PT
subtype 4 is common in Danish Blastocystis-positive patients presenting with acute
RI
diarrhea. Am. J. Trop .Med. Hyg. 84, 883–885.
SC
Stensvold, C.R., Clark, C.G., 2016. Current status of Blastocystis: a personal view. Parasitol. Int. 65, 763–771.
NU
Stensvold, C.R., Nielsen, S.D., Badsberg, J.H., Engberg, J., Friis-Moller, N., Nielsen, S.S., Nielsen, H.V., Friis-Moller, A., 2011b. The prevalence and clinical significance of
MA
intestinal parasites in HIV-infected patients in Denmark. Scand. J. Infect. Dis. 43, 129–135.
D
Stenzel, D.J., Boreham, P.F., 1996. Blastocystis hominis revisited. Clin. Microbiol. Rev. 9,
PT E
563–584.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular
CE
Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Tan, K.S., 2008. New insights on classification, identification, and clinical relevance of
AC
Blastocystis spp. Clin. Microbiol. Rev. 21, 639–665. Thathaisong, U., Siripattanapipong, S., Mungthin, M., Pipatsatitpong, D., Tan-ariya, P., Naaglor, T., Leelayoova, S., 2013. Identification of Blastocystis subtype 1 variants in the Home for Girls, Bangkok, Thailand. Am. J. Trop. Med. Hyg. 88, 352–358. Thathaisong, U., Worapong, J., Mungthin, M., Tan-Ariya, P., Viputtigul, K., Sudatis, A., Noonai, A., Leelayoova, S., 2003. Blastocystis isolates from a pig and a horse are closely related to Blastocystis hominis. J. Clin. Microbiol. 41, 967–975.
ACCEPTED MANUSCRIPT Vaisusuk, K., Saijuntha, W., Sedlak, S., Thanchomnang, T., Pilap, W., Suksavate, W., Stensvold, C.R., Tantrawatpan, C., 2017. Blastocystis subtypes detected in long-tailed macaques in Thailand-Further evidence of cryptic host specificity. Acta Trop. Villalobos, G., Orozco-Mosqueda, G.E., Lopez-Perez, M., Lopez-Escamilla, E., CordobaAguilar, A., Rangel-Gamboa, L., Olivo-Diaz, A., Romero-Valdovinos, M., Maravilla,
PT
P., Martinez-Hernandez, F., 2014. Suitability of internal transcribed spacers (ITS) as
RI
markers for the population genetic structure of Blastocystis spp. Parasit. Vectors. 7,
SC
461.
Villegas-Gomez, I., Martinez-Hernandez, F., Urrea-Quezada, A., Gonzalez-Diaz, M.,
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Durazo, M., Hernandez, J., Orozco-Mosqueda, G.E., Villalobos, G., Maravilla, P., Valenzuela, O., 2016. Comparison of the genetic variability of Blastocystis subtypes
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between human carriers from two contrasting climatic regions of Mexico. Infect. Genet. Evol. 44, 334–340.
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Wang, J., Gong, B., Yang, F., Zhang, W., Zheng, Y., Liu, A., 2018. Subtype distribution and
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genetic characterizations of Blastocystis in pigs, cattle, sheep and goats in northeastern China's Heilongjiang Province. Infect. Genet. Evol. 57, 171–176.
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Wang, W., Cuttell, L., Bielefeldt-Ohmann, H., Inpankaew, T., Owen, H., Traub, R.J., 2013. Diversity of Blastocystis subtypes in dogs in different geographical settings. Parasit.
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Vectors. 6, 215.
Wang, W., Owen, H., Traub, R.J., Cuttell, L., Inpankaew, T., Bielefeldt-Ohmann, H., 2014. Molecular epidemiology of Blastocystis in pigs and their in-contact humans in Southeast Queensland, Australia, and Cambodia. Vet. Parasitol. 203, 264–269. Wong, K.H., Ng, G.C., Lin, R.T., Yoshikawa, H., Taylor, M.B., Tan, K.S., 2008. Predominance of subtype 3 among Blastocystis isolates from a major hospital in Singapore. Parasitol. Res. 102, 663–670.
ACCEPTED MANUSCRIPT Yoshikawa, H., Tokoro, M., Nagamoto, T., Arayama, S., Asih, P.B., Rozi, I.E., Syafruddin, D., 2016. Molecular survey of Blastocystis sp. from humans and associated animals in an Indonesian community with poor hygiene. Parasitol. Int. 65, 780–784. Yoshikawa, H., Wu, Z., Kimata, I., Iseki, M., Ali, I.K., Hossain, M.B., Zaman, V., Haque, R., Takahashi, Y., 2004. Polymerase chain reaction-based genotype classification among
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human Blastocystis hominis populations isolated from different countries. Parasitol.
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Res. 92, 22–29.
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Zhao, G.H., Hu, X.F., Liu, T.L., Hu, R.S., Yu, Z.Q., Yang, W.B., Wu, Y.L., Yu, S.K., Song, J.K., 2017. Molecular characterization of Blastocystis sp. in captive wild animals in
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Qinling Mountains. Parasitol. Res. 116, 2327–2333.
Zhu, W., Tao, W., Gong, B., Yang, H., Li, Y., Song, M., Lu, Y., Li, W., 2017. First report of
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Blastocystis infections in cattle in China. Vet. Parasitol. 246, 38–42.
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Table 1 GenBank references for the Blastocystis ST1–ST17 sequences used to construct a phylogenetic tree Accession no.
Host
Subtype
Reference
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
U51151 AB070989 AB091241 AF538349 AB107961 JQ665860 JQ665867 AB070987 AB107969 EF200010 EU445487 AB070988 AB107963 AB107965 EU445494 AY244620 U51152 AB070998 AB107964 AB107966 EF468654 EU445485 JQ665850 AB070991 AY135412 AB107970 AB107971 AF408425 KC148207 KX234638 KX234639 KX352006 GU256900 KY823301 GU256935 KY823303 KC148210 EU427512 KC148208
human human chicken horse pig human human human pig-tailed macaque human pig human pig cattle human human guinea pig pig pig cattle human chicken human human duck lemur pheasant human camel goat bird fish elephant lechwe quokka
1 1 1 1 1 1 1 2 2 2 2 3 3 3 3 4 4 5 5 5 5 6 6 7 7 8 8 9 10 10 10 10 11 12 13 14 15 16 17
(Silberman et al., 1996) (Arisue et al., 2003) (Arisue et al., 2003) (Thathaisong et al., 2003) (Abe, 2004) (Thathaisong et al., 2013) (Thathaisong et al., 2013) (Arisue et al., 2003) (Abe, 2004) (Leelayoova et al., 2008) (Rivera, 2008) (Arisue et al., 2003) (Abe, 2004) (Abe, 2004) (Rivera, 2008) (Yoshikawa et al., 2004) (Silberman et al., 1996) (Arisue et al., 2003) (Abe, 2004) (Abe, 2004) Yan unpublished (Rivera, 2008) (Thathaisong et al., 2013) (Arisue et al., 2003) (Noel et al., 2003) (Abe, 2004) (Abe, 2004) (Yoshikawa et al., 2004) (Alfellani et al., 2013b) Noradilah unpublished Noradilah unpublished Noradilah unpublished (Parkar et al., 2010) (Zhao et al., 2017) (Parkar et al., 2010) (Zhao et al., 2017) (Alfellani et al., 2013b) Yoshikawa unpublished (Alfellani et al., 2013b)
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camel red kangaroo Libya gundi
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wild ass
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No.
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Table 2 Frequency of Blastocystis infection subtypes in stool samples from animals and humans in two provinces
ST 2
ST 3
ST 5
ST 10
ST 12
ST 14
Suspec ted ST10
1 1
-
-
28 28
2 2 4
4 6 10
3 3
6 10 16
-
-
1 -
-
-
-
1 2
6
7
10 -
-
3 1 2
6 6
8 1 7
10 10 -
-
Suspec ted ST12 or 10
9 9
-
-
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Suspec ted ST14
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ST 1
Suspec ted ST14 or 10
-
Suspec ted ST14 or 12
3 3
8 1 9
-
-
-
-
-
-
-
-
-
-
-
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-
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Central part AP: Cattle (n=42) Goats (n=38) Pigs (n=33) Total Western part KP: Dogs (n=13) Cats (n=11) Chickens (n=23) Pigs (n= 54) Humans (n=117) Total - anima ls - huma ns
No. of observed subtype
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Study area
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AP = Ayutthaya Province, KP = Kanchanaburi Province
Table 3 Prevalence of Blastocystis infection in animals by study area
No. of Blastocystis positive samples/ No. of samples examined
No. of zoonotic Blastocystis subtype samples/ No. of samples examined (prevalence %)
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29/33 11/54 (87.88%) (20.37%)
p-value* AP KP <0.00001 Animals 29/113 12/101 (cat, (25.66%) (11.88%) cattle, chicken, dogs, goats and pigs) <0.00001
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KP 1/47 (2.13%)
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AP 57/80 Animals (71.25%) (cat, cattle, chicken, dogs, goats)
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*Fisher Exact test for the animal (cat, cattle, chicken, dogs, goats) and pig prevalence comparison between Kanchanaburi Province (KP) and Ayutthaya Province (AP): p < 0.05.
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** Fisher Exact test for the prevalence of zoonotic Blastocystis subtype (ST) comparison in animals between Kanchanaburi Province (KP) and Ayutthaya Province (AP): p < 0.05.
ACCEPTED MANUSCRIPT Table 4 Subtype distribution of Blastocystis in Thailand and worldwide
Host
References
Subtype No. of subtype
Countri es
1
2
3
4
5
6
7
8
10
1 2
1 3
14
1 6
Mixed subtyp es
Unkno wn subtyp e
68
10
4
49
5
humans
65
11
41
3
Bolivia
humans
40
Brazil
humans
66
27
1 0 1 3 2 1
humans
22
2
7
8
humans
12
4
3
4
humans
125
70
4 0
15
birds
47
cattle marsupia ls dogs
20 25
rodents nonhuman primates humans
3 2
181
82
humans
256
55
Ecuador
humans
25
3
Mexico
humans
47
24
humans
80
30
8
3
1
8
4 7
2 1
(Casero et al., 2015) (Ramirez et al., 2016) (Ramirez et al., 2016) (Malheiros et al., 2011) (Ramirez et al., 2016) (Santin et al., 2011) (Ramirez et al., 2014)
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12
2
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11
4 7
2 5
15
1 5 3
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2
4 7 5 0
33
7
4
7
14 2 21
2
5
2
9
8
42
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1 1
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12
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humans
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Argenti na
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1
1 1
2
Peru
humans
13
12
1
USA
cattle
7
pigs humans
6 10
2
dogs
4
2
2
cats
7
1
4
humans
25
1
humans
22
9
non-
8
7
(Ramirez et al., 2016) (Ramirez et al., 2017) (Ramirez et al., 2016) (Villalobos et al., 2014) (VillegasGomez et al., 2016) (Ramirez et al., 2016) (Santin et al., 2011)
6 3
5
(Scanlan et al., 2016) (Ruaux and Stang, 2014) 2
European continent Denmar k
4
1
1 9
11
2
2
2
2
2
(Stensvold et al., 2011a) (Stensvold et al., 2011b) (Stensvold
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1
1
3 4
nonhuman primates carnivore s Artiodact yla humans
48
15
9
8
4
1
1
2
12
3
Greece
humans
45
9
6
27
1
Italy
humans
30
2
5
13
6
Spain
human
51
1
2
7
1
1
3
1
Sweden
nonhuman primates humans
63
10
9
30
UK
humans
16
2
9
1 3 1
nonhuman primates sheep humans
70
7
1 1
22
1
1 271
34
2 6
1 11 4
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1 7 4
1 7
3 2
humans
110
15
Libya
humans
38
19
Liberia Nigeria Tanzani a
humans humans humans
30 23 92
2
7 10 36
7
2 7
2 8
8
1
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1
(Yoshikaw a et al., 2004) (Menounos et al., 2008) (Meloni et al., 2011) (Domingue z-Marquez et al., 2009) (Santin et al., 2011)
4
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1
1
(Forsell et al., 2012) (Stensvold et al., 2009)
4
8
2
49
3
2
4 8
8 5
1
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14
5
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Egypt
(Poirier et al., 2011) (Cian et al., 2017)
2
1
African continent
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22 1 1
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German y
24
17
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France
3 3
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human primates pigs cattle sheep deer dogs humans
1 3
1
4
3 3
1 3
5
(Alfellani et al., 2013a)
10
(Fouad et al., 2011) (Alfellani et al., 2013a)
15
8 9 27
3 3
5 1 (Forsell et al., 2016)
Asian continent Cambod ia China
humans
116
33
pigs pigs
33 419
15
45
2
20 39 7 1
9
31
6
cattle
54
goats
458
India
pigs cattle sheep humans
6 14 6 65
1 6
59
Iran
humans
100
21
25
Nepal
humans
63
2
1
11
1
(Wang et al., 2014)
13 1 41
10
29 2
12 3
10 3
2 1
(Song et al., 2017a) (Zhu et al., 2017) (Song et al., 2017b) (Wang et al., 2018)
6 2 1
21
1
33
41
8
(Das et al., 2016) (Moosavi et al., 2012) (Lee et al.,
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pigs chickens rodents humans
73 8 9 186
44
1 3
10 1
9 1 3
humans
191
63
2 7
98
3
humans
195
19
1
53
5
dogs humans chickens monkeys pigs humans
23 5 1 4 2 9
1 1
2
2 4
2
humans
13
monkeys
154
34
humans
20
4
humans
25
2
3
20
humans
77
24
1 1
41
humans
56
12
humans
58
55
humans pigs horses
153 20 1
13 8 20 1
dogs
2
2
humans pigs
30 184
11 4
2012) (Yoshikaw a et al., 2016)
47 59 8 7
1
89
27
6
10
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6
1 1 1
1
1
11
4 8
72
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7 1
12
2
2
SC
2 1 2
1
32
2
1
1 0
2
7
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29 14
Australian continent Australi a
2
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Singapo re Thailan d
40
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Philippi nes
118
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Malaysi a
humans
2
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Indonesi a
16 4
3 16 7
6
9
(Nithyamat hi et al., 2016) (Noradilah et al., 2017) (Belleza et al., 2016) (Rivera, 2008)
(Wong et al., 2008) (Palasuwan et al., 2016) (Vaisusuk et al., 2017) (Sanpool et al., 2015) (Pipatsatitp ong et al., 2015) (Popruk et al., 2015) (Jantermtor et al., 2013) (Thathaiso ng et al., 2013) (Thathaiso ng et al., 2003)
(Wang et al., 2013) (Wang et al., 2014)
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ST5; KPPEK1-52 ST5; KPPEK1-53 ST5; KPPEK1-51 ST5; KPPEK1-10 ST5; KPPEK1-9 ST5; KPPEK1-8 ST5; KPPEK1-7 53 ST5; KPPEK1-6 ST5; KPPEK1-3 ST5; AYP 33 ST5; AYP 32 61 ST5; AYP 31 ST5; AYP 30 ST5; AYP 29 ST5; AYP 23 ST5; AYP 1 88 ST5; AYP 13 77 ST5; AYP 14 ST5; AYP 28 95 ST5 AB107964; pig ST5 AB070998; pig 69 ST5 AB107966; cattle ST5 EF468654; human 98 ST5; AYP 25 ST2; KPH 43.1 ST2; KPH 44.1 77 56 ST2; KPH 42.1 ST2; KPH 18.1 100 ST2 AB070987; human ST2 AB107969; monkey ST2 EF200010; human 57 ST2 EU445487; pig 69 ST1 AB091241; chicken 99 73 ST1 AB107961; pig ST1 JQ665867; human ST1 AB070989; human 98 ST1; KPH 28.2 ST1 AF538349; horse 45 49 ST1; AYP 24 ST1 JQ665860; human 5 ST1 U51151; human ST1; KPH 47.1 ST3 AB107965; cattle ST3; KPD 50 ST3; KPH 2.2 100 ST3 AB070988; human ST3 AB107963; pig 39 ST3 EU445494; human ST3; KPH 22.2 ST3; KPH 24.2 ST3; KPH 24.3 Proteromonas lacertae
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0.02
Fig 1. Molecular phylogenetic analysis using maximum likelihood. The evolutionary history was inferred using maximum likelihood, based on the Tamura 3parameter model. KPPEK = pig in Kanchanaburi Province; AYP = pig in Ayutthaya Province; KPH = human in Kanchanaburi Province
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Graphical abstract
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High percentages of Blastocystis infection in AP and KP pigs (29/33, 87.88%; 11/54, 20.37% respectively), cattle (21/42, 50%), goat (36/38, 94.74%) and humans (15/117, 12.82%) and this study was the first report of Blastocystis infection in cattle and goats in Thailand.
Blastocystis ST5 was the predominant subtype in both AP (Ayutthaya Province) and
studied areas, in this study.
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Pigs may be a natural host of Blastocystis, in this study.
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KP (Kanchanaburi Province) pigs and this ST may be the pig-adapted ST in the