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Molecular epidemiology of Blastocystis in pigs and their in-contact humans in Southeast Queensland, Australia, and Cambodia
Accepted Manuscript Title: Molecular epidemiology of Blastocystis in pigs and their in-contact humans in Southeast Queensland, Australia, and Cambodia Author: Wenqi Wang Helen Owen Rebecca J. Traub Leigh Cuttell Tawin Inpankaew Helle Bielefeldt-Ohmann PII: DOI: Reference:
S0304-4017(14)00215-5 http://dx.doi.org/doi:10.1016/j.vetpar.2014.04.006 VETPAR 7213
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
25-9-2013 10-3-2014 5-4-2014
Please cite this article as: Wang, W., Owen, H., Traub, R.J., Cuttell, L., Inpankaew, T., Bielefeldt-Ohmann, H.,Molecular epidemiology of Blastocystis in pigs and their in-contact humans in Southeast Queensland, Australia, and Cambodia, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.04.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title:
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Molecular epidemiology of Blastocystis in pigs and their in-contact humans in Southeast
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Queensland, Australia, and Cambodia
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Authors:
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Wenqi Wang*a, Helen Owena, Rebecca J. Trauba, b, Leigh Cuttella, Tawin Inpankaew c, d,
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Helle Bielefeldt-Ohmanna, b
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Affiliations:
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a
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Australia
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b
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4067, Australia
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c
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University of Copenhagen, Denmark
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Bangkok,
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Thailand
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Australian Infectious Diseases Centre, The University of Queensland, St. Lucia, Queensland
Department of Veterinary Disease Biology, Faculty of Health and Medical Science,
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School of Veterinary Science, The University of Queensland, Gatton, Queensland 4343,
Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University,
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* Corresponding author. Tel: + 61 (7) 5460 1834; Fax: + 61 (7) 5460 1922; Email address:
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[email protected] (Wenqi Wang)
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Abstract
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Blastocystis, an intestinal protist commonly found in humans and animals worldwide, has
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been implicated by some as a causative agent in irritable bowel syndrome in humans. In pigs,
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infection with Blastocystis is commonly reported, with most pigs shown to harbour subtypes
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(ST) 1 or 5, suggesting that these animals are potentially natural hosts for Blastocystis.
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Although ST5 is considered rare in humans, it has been reported to be a potential zoonosis
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from pigs in rural China. To test these hypotheses, we conducted molecular analysis of faecal
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samples from pigs and in-contact humans from commercial intensive piggeries in Southeast
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Queensland (SEQ), Australia, and a village in rural Cambodia. The prevalence of Blastocystis
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in SEQ and Cambodian pigs was 76.7% and 45.2%, respectively, with all positive pigs
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harbouring ST5. It appears likely that pigs are natural hosts of Blastocystis with a high
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prevalence of ST5 that is presumably the pig-adapted ST in these regions. Amongst the SEQ
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piggery staff, 83.3% were Blastocystis carriers in contrast to only 55.2% of Cambodian
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villagers. The predominant STs found in humans were STs 1, 2 (Cambodia only) and 3.
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Interestingly, ST5 which is usually rare in humans was present in the SEQ piggery staff but
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not in the Cambodian villagers. We conclude that in intensive piggeries, close contact
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between pigs and their handlers may increase the risks of zoonotic transmission of
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Blastocystis.
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Keywords:
Blastocystis; Pig; Epidemiology; Zoonosis
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1. Introduction
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Blastocystis is an intestinal protist that is commonly found worldwide in a diverse range of
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species including humans, non-human primates (NHPs), other mammals and birds (Alfellani
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et al., 2013c; Stensvold et al., 2009). Blastocystis is genetically polymorphic and molecular
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analysis of the small subunit ribosomal RNA (SSU rRNA) gene has enabled division of this
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organism into 17 distinct subtypes (STs) found in a wide range of species (Alfellani et al.,
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2013c; Fayer et al., 2012; Parkar et al., 2010; Stensvold et al., 2009). Some STs show
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significant intra-ST genetic variability (Scicluna et al., 2006; Stensvold et al., 2012;
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Yoshikawa et al., 2009). Humans have been shown to carry STs 1-9 with ST3 the most
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frequently reported. Significant geographical variation in the prevalence and relative
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proportion of STs is known to exist, with other common STs including 1, 2 and 4 (Alfellani
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et al., 2013b). Faecal oral transmission either by direct contact or water-borne transmission is
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currently the most accepted mode of transmission (Lee et al., 2012b; Leelayoova et al., 2004;
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Leelayoova et al., 2008). Previous studies have isolated identical STs of Blastocystis from
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humans and their in-contact animals including pigs (Lee et al., 2012a; Lee et al., 2012b;
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Nagel et al., 2012; Parkar et al., 2007; Parkar et al., 2010; Stensvold et al., 2009; Yan et al.,
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2007), suggesting potential zoonotic transmission of Blastocystis.
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Blastocystis has been reported in pigs in some countries, with most pigs harbouring ST5 or
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ST1 and occasionally ST2 or ST3 (Alfellani et al., 2013c; Navarro et al., 2008; Thathaisong
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et al., 2003; Yan et al., 2007). In Valencia, Spain, Navarro et al. (2008) found ST1 to be the
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most prevalent among intensively reared pigs and, to a lesser extent, ST2. ST5 has also been
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identified in cattle, other livestock and captive apes (Alfellani et al., 2013b; Stensvold et al.,
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2009), but rarely in humans. The potential of pigs to act as zoonotic reservoirs of Blastocystis
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was demonstrated by Yan et al. (2007) in which two human ST5 isolates had restriction
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fragment length polymorphism (RFLP) patterns of the SSU rDNA were either identical or
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similar to those of 16 pigs living in the same rural area in China.
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The objective of this study was to ascertain if pigs are suitable animal models for studying
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Blastocystis infection by investigating if they are indeed natural hosts of Blastocystis as
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suggested by previous reports. Our selection criteria for a ‘natural host’ was based on (i) a
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host which commonly harbour Blastocystis (i.e.at a high prevalence) and (ii) of a
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predominant ST or STs. The second objective was to investigate the zoonotic potential of
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porcine Blastocystis by testing and genetically comparing Blastocystis recovered from pigs
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and their in-contact humans (i.e. piggery staff and villagers).
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Preceding studies investigating Blastocystis infection in pigs have been limited by their
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relatively small sample size and lack of molecular identification of Blastocystis ST (Abe et
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al., 2002; Navarro et al., 2008; Yan et al., 2007). In this study we genetically characterise the
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STs of Blastocystis in pigs and their in-contact humans in two different settings (intensive
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versus small holder piggeries) in Southeast Queensland (SEQ), Australia, and a rural
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Cambodian village.
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2.
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2.1 Sampling
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2.1.1
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Single faecal samples were collected from 240 pigs and 36 piggery staff from 18 commercial
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piggeries in SEQ between February 2012 and February 2013 and stored in 2.5% potassium
Materials and methods
Southeast Queensland (SEQ)
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dichromate prior to DNA extraction. The faecal samples were collected from approximately 10-25
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pigs at various stages of production (piglets, weaners, growers and sows) per piggery.
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2.1.2
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Single faecal samples were collected in May 2012 from 73 pigs and 210 villagers from 41
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households in Dong village, Preah Vihear province, Cambodia and stored in 2.5% potassium
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dichromate prior to DNA extraction. The majority of the households sampled (29/41) owned
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pigs and up to 0-4 pig and 1-9 human samples were collected per household.
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2.1.3
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The SEQ segment of this project was approved by the University of Queensland Animal
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Ethics Committee and Medical Research Ethics Committee with approval no.
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Ethical approval
ANRFA/472/11 and 2012000069, respectively. Sample collection from the Cambodian
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village was made possible by collaboration between the University of Copenhagen, Denmark,
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Department of Fisheries Post-Harvest Technologies and Quality Control, Cambodia, and the
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National Center for Parasitology, Entomology and Malaria control, Cambodia. The study
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protocol was approved by the National Ethics Committee Health Research, Ministry of
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Health in Cambodia. All persons were informed on the purpose of the study. Written
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informed consent was obtained from all individuals prior to enrolment.
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2.2
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2.2.1
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Faeces stored in 2.5% potassium dichromate was washed twice in 1× phosphate-buffered
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saline and centrifuged before the sediment was used for DNA extraction. DNA was extracted
Molecular analysis DNA extraction
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using the QIAamp DNA Stool Mini Kit (Qiagen, Germany) with minor alterations as per
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Wang et al. (2013).
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2.2.2
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Samples were first tested with a previously published nested PCR primer set and conditions
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that amplified an 1100 bp region of the Blastocystis SSU rRNA gene (Clark, 1997; Wong et
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al., 2008) (Table 1). This method was chosen since it provides a large fragment for
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phylogenetic analysis, and as it is a pan-Blastocystis technique, it allows for identification of
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known and unknown STs of Blastocystis. Each 25µl PCR reaction was run using
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approximately 50 ng of DNA and the conditions were as per Clark (1997) and Wong et al.
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(2008).
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2.2.3 Phylogenetic analysis
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All positive PCR products were purified using the PureLink Genomic DNA Mini Kit (Life
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Technologies Corporation, New York, USA) according to the manufacturer’s protocol.
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Unidirectional DNA sequencing was performed with the reverse primer of the secondary
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PCR with an Applied Biosystems 3130/3130xl Genetic Analyzer. The DNA sequences were
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first analysed with Finch TV v 1.4.0 (Geospiza Inc., Seattle, WA, USA) and subsequently
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compared with published sequences from GenBank (National Center for Biotechnology
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Information) using BLAST 2.2.9 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). These sequences
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were then aligned with previously published sequences of the SSU rRNA gene of the various
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Blastocystis STs, which were sourced from GenBank using BioEdit v 7.1.3.0 software (Ibis
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Biosciences, Carlsbad, CA, USA). The sequences were subsequently analysed using Mega
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4.1 software (The Biodesign Institute, Tempe, AZ, USA) to construct a neighbour joining
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tree for subtyping of the Blastocystis isolates. Proteromonas lacertae (U37108) was used as
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an out-group.
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PCR amplification with nested PCR
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The ST1, ST3 and ST5 sequences from pigs and humans were compared using multiple
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sequence alignment by Mega 4.1 software (The Biodesign Institute, Tempe, AZ, USA).
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2.2.4 Development of subtype –specific (ST-specific) primers
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Pan-subtype targeted PCRs can result in preferential amplification of predominant STs over
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others in mixed infections. Therefore, we developed and applied ST-specific primers for STs
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1, 2, 3 and 5 (Table 1) based on the STs identified in the humans and pigs using nested PCR.
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Application of ST-specific PCRs aimed to investigate whether greater than single ST
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infections were present in each host and to increase the probability of determining whether
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cross-transmission of Blastocystis was occurring between pigs and humans in the same
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geographical setting. ST-specific primers for STs 1, 3 and 5 were designed based on the
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“barcode” region of the Blastocystis SSU rRNA gene, while the ST-specific primers of ST2
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were designed from the non-barcode region of the SSU rRNA (see acknowledgment). The
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respective primers were tested against STs 1 - 6 DNA, respectively to ensure the absence of
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cross-reactivity. The following protocol was followed 1) All human samples were tested
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with ST5-specific primers, regardless of Blastocystis status 2) porcine samples with suspected
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mixed infections (characterised by overlapping double peaks at single nucleotide
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polymorphisms) were tested with ST-specific 1, 3 and 5 primers (Table 2). In addition,
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Cambodian pigs were also tested with ST-specific 2 primers as this ST was also found in
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Cambodian villagers, but not in SEQ staff. Lastly, porcine samples diagnosed with STs 1 or 3
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(Table 2) were tested with ST-specific 5 primers to ensure mixed infectious with ST5 were
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not overlooked, as this was the predominant ST found in both pig populations. When an
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“aberrant” ST was found in pigs or humans with ST-specific primers (i.e. ST5 in humans and
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STs 1 or 3 in pigs), the PCR products were sequenced and compared with sequences of the
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same ST from the other host (humans/pigs).
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PCR reaction mixtures for ST-specific primers were similar consisting of 50 ng of faecal
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genomic DNA, 1.0 units of HotStar Taq Plus DNA Polymerase (Qiagen, Germany) and 10
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pmol of each forward and reverse primer. The MgCl2 concentration was optimised for each
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primer pair and ranged from 0.5 to 1.5 mmol/L. Nuclease free water was added to adjust the
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final reaction volume to 25 µl. Positive controls for each ST and a negative control (2 µl of
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distilled water) were included in each PCR run. Primer sequences and cycling conditions are
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presented in Table 1.
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2.2.5 Control testing of samples
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As part of internal process control, we tested all samples that were negative using the
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Blastocystis-specific nested PCR for the presence of amplifiable DNA and to rule out
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potential PCR inhibition. These samples were tested using published universal primers that
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amplify a 140 bp fragment of the 18S ribosomal RNA gene from eukaryotic DNA (Fajardo et
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al., 2006). PCR cycling conditions were modified from a real-time PCR protocol to a
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conventional protocol using an annealing temperature of 60°C as recommended.
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2.2.6 Statistical analysis
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The percentage prevalence of each test group and 95% confidence intervals were calculated
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using Epitools epidemiological calculators (AusVet Animal Health Services and Australian
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Biosecurity Cooperative Research Centre for Emerging Infectious Disease,
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http://epitools.ausvet.com.au). Chi-squared (χ²) tests were used to compare the Blastocystis
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prevalence between the Cambodian and SEQ pigs and humans respectively. A P-value of less
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than 0.05 was considered statistically significant. The statistical software used was
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Calculation for the Chi-Square test: An interactive calculation tool for chi-square tests of
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goodness of fit and independence (Preacher, 2001).
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The prevalence of Blastocystis in both populations of pigs was high (SEQ 76.7%; Cambodia
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45.2%) (Table 2), but was significantly higher in the SEQ pigs than in the Cambodian pigs (p
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<0.05; Table 3). All SEQ piggeries (18/18) and majority (24/29) of the pig-rearing
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households in the Cambodian village contained pigs that were positive for Blastocystis. All
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infected SEQ and Cambodian pigs harboured ST5 (Table 4). This included 17 (7.1%) SEQ
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pigs with mixed infections of ST5 and ST1 and/or ST3, or ST5 and unknown mixed STs
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While no mixed infections (with STs 1, 2, 3 and 5) were detected in the Cambodian pigs
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(Table 4).
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The high Blastocystis prevalence, in addition to the predominance of ST5 in both pig
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populations, corroborated the results of previous studies (Navarro et al., 2008; Yan et al.,
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2007) which suggest that pigs are likely to be natural hosts for Blastocystis ST5. All
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Blastocystis-positive pigs regardless of geographical setting harboured ST5, the majority as
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single infections. This is in agreement with previous smaller scale studies (Alfellani et al.,
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2013c; Stensvold et al., 2009; Yan et al., 2007; Yoshikawa et al., 2004), including a study in
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Vietnam that reported 12/12 Blastocystis-positive pigs tested for ST5. In China 16 pigs were
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identified as harbouring ST5 only (Yan et al., 2007). In contrast, a large scale study
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conducted in Valencia, Spain, found ST1 the predominant ST in intensively raised pigs
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(Navarro et al., 2008). This variation of predominant STs (STs 1 or 5) found in pigs could be
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attributed to geographical ST variation as observed in non-human primates (NHPs) (Alfellani
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et al., 2013a). Here, investigators identified STs 1, 2 and 3 infections in NHPs as being
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independent of geographical association, while ST8 was primarily observed in species native
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to Asia or South America (Alfellani et al., 2013a).
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Results and discussion
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SEQ pigs had a significantly higher prevalence of Blastocystis (76.7%) compared to pigs
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from Cambodia (45.2%). A possible explanation could be that the intensively raised SEQ
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pigs have a significantly higher rate of contact with other pigs compared to the Cambodian
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pigs that are raised singly or in small herds of up to four pigs in a small fenced off area
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adjacent to village houses. Consequently, the risk of faecal-oral transmission of Blastocystis
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is greater in the SEQ pigs as compared to the Cambodia pigs.
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In this study, the prevalence of Blastocystis in SEQ intensive piggery staff (83.3%) was
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significantly higher than Cambodian villagers (55.2%) (p <0.05; Table 3, 5). Although the
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prevalence of Blastocystis in humans is generally higher in developing than developed
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countries, prevalence can also vary within countries among different subpopulations (Tan,
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2008). The disparity in Blastocystis prevalence between SEQ piggery staff and Cambodian
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villagers may be a reflection of animal handlers (piggery staff) being at higher risk of
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acquiring zoonotic infections (Driscoll, 2006; Mahdi and Ali, 2002; Murdoch, 2007),
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including Blastocystis (Parkar et al., 2010; Rajah Salim et al., 1999). Amongst the
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Cambodian villagers, only a negligible difference was observed between the prevalence of
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Blastocystis amongst the pig-rearing villagers (55.5%) and non pig-rearing villagers (54.7%),
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which is likely owing to primarily anthroponotic transmission. The differences in the
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prevalence of STs 1, 2 and 3 between pig-rearing and non-pig rearing villagers were not
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statistically significant.
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Subtypes 3 and 1 were most commonly detected in all humans tested. This was followed by
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ST5 in 13.9% (5/36) of the SEQ piggery staff and ST2 in 23.3% (27/116) of the Cambodian
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villagers (Table 5). Although 2 SEQ staff were found to carry mixed infections of STs 5 and
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3, neither ST5 nor mixed infections (with STs 1, 2, 3 and 5) were detected in the Cambodian
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villagers (Table 5). ST distribution studies conducted in various geographic regions have
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demonstrated substantial geographical variation in STs between populations (Alfellani et al.,
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2013b). In addition, they have also shown that humans may carry STs 1-9 with ST3 being the
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most prevalent followed by STs 1, 2 and 4 and that ST2 is commonly reported in certain
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areas such as Brazil, Europe and Central Asia (Alfellani et al., 2013b). Our results are in
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agreement with these findings and endorse the presence of ST2 as common in humans in
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rural Cambodia. In our study, all human samples were tested with the pan-ST nested PCR
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and the ST5 specific primers only and these identified STs 1, 2, 3 and 5. Other STs such as
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STs 4, 6, 7 and 8 have also been reported in Australia and/or Southeast Asia (Alfellani et al.,
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2013b; Nagel et al., 2012), however these additional STs were not specifically tested for with
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ST-specific primers in this study and their presence could therefore not be excluded.
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Comparison of the 606 bp and 403 bp nested PCR product regions of the SSU rRNA of ST1
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andST3 from pigs and humans respectively, revealed 100% sequence identity for all three
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STs regardless of host and geographical location. Comparison of a 477 bp region of the
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nested PCR product of the SSU rDNA of ST5 also revealed 100% identity, with the
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exception of a single nucleotide polymorphism (with either adenine or thymine) at 398 bp
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(GenBank accession no. KJ082062) that was shown to be independent of host and
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geographical location. This suggests that the SEQ pigs and piggery staff in this study carried
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very similar Blastocystis STs 1, 3 and 5, which may have been transmitted between these
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hosts within their environment.
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Despite ST5 being rarely reported in humans, 13.9% (5/36) of SEQ piggery staff harboured
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identical STs to in-contact pigs, suggesting that these piggery workers may have acquired the
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infection from intensively raised pigs. Piggery staff members are repeatedly exposed to large
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amounts of pig faeces during cleaning of high density pig pens. High pressured water hoses
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cause aerosolisation of faeces and potentially increase risk of faecal-oral transmission. In
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contrast, Cambodian villagers that live in close proximity with the pigs they rear did not
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harbour ST5 despite a Blastocystis prevalence of 45.2% in pigs. This is likely due to the less
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intensive nature of pig rearing in Cambodian villages, where each household rears a
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maximum of 4 pigs confined to small areas adjacent to or beneath the house. To further
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investigate this hypothesis, future work should include testing a greater number of piggery
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staff as well as a control group of piggery staff that do not have direct contact with pigs (e.g.
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administrative staff) together with non-piggery workers from the same geographical area.
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Comparison of the SEQ human and pig Blastocystis isolates characterised in this study using
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a MLST approach would add further evidence for the parasite’s potentially zoonotic abilities;
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however, this approach has not yet been developed for ST5.
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A minority of SEQ pigs were found to harbour mixed infections with ST5 and ST1 (n=9)
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and/or ST3 (n=9), which were not observed in Cambodian pigs using both nested and ST-
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specific PCR. Comparison of the pig and human ST1 and ST3 sequences showed 100%
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identity, respectively. Subtype 3 has been reported in pigs but is uncommon (Abe et al., 2003;
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Stensvold et al., 2009; Yoshikawa et al., 2004), whilst ST1 is common in pigs in certain
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subpopulations such as commercial piggeries in Valencia, Spain and pigs kept at an army
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base in Chonburi, Thailand (Navarro et al., 2008; Thathaisong et al., 2003). Given that ST5
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was the predominant ST in pigs from SEQ and that the piggery staff predominantly
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harboured STs 1 or 3, there is a possibility that the SEQ pigs harbouring STs 1 or 3 may have
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acquired the Blastocystis infection from human handlers. In other words, this could be a
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reverse zoonosis, though MLST of isolates would be required to further confirm this
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hypothesis.
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4 Conclusion
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The results of the study support the hypothesis that pigs are natural hosts of Blastocystis due
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to the high prevalence in different geographical localities and predominant infection with ST5
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which has been proposed as a pig-adapted ST. There was a concurrent high prevalence of
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Blastocystis in both SEQ piggery staff and Cambodian villagers with STs 1 and 3 being the
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most predominant STs in both regions. In addition, ST2 was detected in the Cambodian
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villagers. These findings could reflect geographical variation or isolation among certain STs.
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The study also indicated the proposed pig-adapted ST5 may be a zoonosis as 13.9% of SEQ
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piggery staff harboured ST5 which is otherwise considered rare in humans. Piggery staff are
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most likely to have acquired ST5 infection through close contact with pig faeces while
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carrying out daily routines. In addition, the presence of STs 1 and/or 3 in a minority of SEQ
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pigs in addition to piggery staff could indicate reverse zoonotic transmission.
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Acknowledgments
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This project has been supported by the William Peter Richards Trust Award Grant (WW) and
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The University of Queensland New Staff Start-up Grant (HO). Special thanks to Paul Noone,
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piggery staff, staff in the School of Veterinary Science especially Dr Kit Parke and the UQ
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Veterinary Clinical Studies Centre for their assistance in sample collection and processing.
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The Cambodian samples were collected as part of Dr. Tawin Inpankaew’s PhD project that is
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funded by the University of Copenhagen, Denmark. We would also like to acknowledge staff
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of The National Center for Parasitology, Entomology and Malaria Control, Cambodia, Mr
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Wissanuwat Chimnoi and Ms Supanun Boonchob, staff of The faculty of Veterinary
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Medicine, Kasetsart University, Thailand, Dr Chhay Somany, deputy director of the
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provincial health department at Preah Vihear province and all the participants for their
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assistance in sample collection from Dong village in Cambodia. Lastly, many thanks to Dr.
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Christen Rune Stensvold for kindly giving us access to the STS 1, 2 and 3 primers that he
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designed.
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Conflict of interest statement 13
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The authors declare that they have no competing interests.
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References
309
Abe, N., Wu, Z., Yoshikawa, H., 2003. Zoonotic genotypes of Blastocystis hominis detected in cattle and pigs by PCR with diagnostic primers and restriction fragment length
311
polymorphism analysis of the small subunit ribosomal RNA gene. Parasitol Res 90,
312
124-128.
cr
313
ip t
310
Alfellani, M.A., Jacob, A.S., Perea, N.O., Krecek, R.C., Taner-Mulla, D., Verweij, J.J., Levecke, B., Tannich, E., Clark, C.G., Stensvold, C.R., 2013a. Diversity and
315
distribution of Blastocystis sp. subtypes in non-human primates. Parasitology 140,
316
966-971.
an
317
us
314
Alfellani, M.A., Stensvold, C.R., Vidal-Lapiedra, A., Onuoha, E.S., Fagbenro-Beyioku, A.F., Clark, C.G., 2013b. Variable geographic distribution of Blastocystis subtypes and its
319
potential implications. Acta Trop 126, 11-18.
d
Alfellani, M.A., Taner-Mulla, D., Jacob, A.S., Imeede, C.A., Yoshikawa, H., Stensvold, C.R.,
te
320
M
318
Clark, C.G., 2013c. Genetic diversity of Blastocystis in livestock and zoo animals.
322
Protist 164, 497-509.
323
Ac ce p
321
Clark, C.G., 1997. Extensive genetic diversity in Blastocystis hominis. Mol Biochem
324 325
Parasitol 87, 79-83.
Driscoll, T., 2006. Work-related infectious and parasitic diseases Australia. Australian Safety and Compensation Council, Australian Government. Available from
326
http://www.safeworkaustralia.gov.au/sites/SWA/about/Publications/Documents/415/
327 328
Workrelated_Infectious_parastitic_disease_Australia.pdf. Last accessed on 18th
329
September 2013.
330
Fajardo, V., Gonzalez, I., Lopez-Calleja, I., Martin, I., Hernandez, P.E., Garcia, T., Martin, R., 2006. PCR-RFLP authentication of meats from red deer (Cervus elaphus), fallow
331
15
Page 15 of 23
332
deer (Dama dama), roe deer (Capreolus capreolus), cattle (Bos taurus), sheep (Ovis
333
aries), and goat (Capra hircus). J Agric Food Chem 54, 1144-1150. Fayer, R., Santin, M., Macarisin, D., 2012. Detection of concurrent infection of dairy cattle
335
with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and
336
microscopic methods. Parasitol Res 111, 1349-1355.Lee, L.I., Chye, T.T.,
337
Karmacharya, B.M., Govind, S.K., 2012a. Blastocystis sp.: waterborne zoonotic
338
organism, a possibility? Parasit Vectors 5, 130-134.
cr
us
339
ip t
334
Lee, L.l., Tan, T.C., Tan, P.C., Nanthiney, D.R., Biraj, M.K., Surendra, K.M., Suresh, K.G., 2012b. Predominance of Blastocystis sp. subtype 4 in rural communities, Nepal.
341
Parasitol Res 110, 1553-1562.
Leelayoova, S., Rangsin, R., Taamasri, P., Naaglor, T., Thathaisong, U., Mungthin, M., 2004.
M
342
an
340
Evidence of waterborne transmission of Blastocystis hominis. Am J Trop Med Hyg
344
70, 658-662.
d
343
Leelayoova, S., Siripattanapipong, S., Thathaisong, U., Naaglor, T., Taamasri, P., Piyaraj, P.,
346
Mungthin, M., 2008. Drinking water: a possible source of Blastocystis spp. subtype 1
347
infection in schoolchildren of a rural community in central Thailand. Am J Trop Med
Ac ce p
te
345
Hyg 79, 401-406.
348 349
Mahdi, N.K., Ali, N.H., 2002. Cryptosporidiosis among animal handlers and their livestock in Basrah, Iraq. East Afr Med J 79, 550-553.
350 351
Murdoch, B., 2007. Zoonoses - Animal diseases that may also affect humans. Department of Environmental and Primary Industries, Victoria, Australia. Available from
352 353
http://www.dpi.vic.gov.au/agriculture/pests-diseases-and-weeds/animal-
354
diseases/zoonoses/zoonoses-animal-diseases-that-may-also-affect-humans. Last
355
accessed on 18th September 2013.
16
Page 16 of 23
356
Nagel, R., Cuttell, L., Stensvold, C.R., Mills, P.C., Bielefeldt-Ohmann, H., Traub, R.J., 2012.
357
Blastocystis subtypes in symptomatic and asymptomatic family members and pets and
358
response to therapy. Intern Med J 42, 1187-1195. Navarro, C., Dominguez-Marquez, M.V., Garijo-Toledo, M.M., Vega-Garcia, S., Fernandez-
ip t
359
Barredo, S., Perez-Gracia, M.T., Garcia, A., Borras, R., Gomez-Munoz, M.T., 2008.
361
High prevalence of Blastocystis sp. in pigs reared under intensive growing systems:
362
frequency of ribotypes and associated risk factors. Vet Parasitol 153, 347-358.
us
363
cr
360
Parkar, U., Traub, R.J., Kumar, S., Mungthin, M., Vitali, S., Leelayoova, S., Morris, K., Thompson, R.C., 2007. Direct characterization of Blastocystis from faeces by PCR
365
and evidence of zoonotic potential. Parasitology 134, 359-367. Parkar, U., Traub, R.J., Vitali, S., Elliot, A., Levecke, B., Robertson, I., Geurden, T., Steele,
M
366
an
364
J., Drake, B., Thompson, R.C., 2010. Molecular characterization of Blastocystis
368
isolates from zoo animals and their animal-keepers. Vet Parasitol 169, 8-17. Rajah Salim, H., Suresh Kumar, G., Vellayan, S., Mak, J.W., Khairul Anuar, A., Init, I.,
te
369
d
367
Vennila, G.D., Saminathan, R., Ramakrishnan, K., 1999. Blastocystis in animal
371
handlers. Parasitol Res 85, 1032-1033.
372
Ac ce p
370
Scicluna, S.M., Tawari, B., Clark, C.G., 2006. DNA barcoding of Blastocystis. Protist 157, 77-85.
373 374
Stensvold, C.R., Alfellani, M., Clark, C.G., 2012. Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infect Genet Evol 12, 263-273.
375 376
Stensvold, C.R., Alfellani, M.A., Norskov-Lauritsen, S., Prip, K., Victory, E.L., Maddox, C.,
377
Nielsen, H.V., Clark, C.G., 2009. Subtype distribution of Blastocystis isolates from
378
synanthropic and zoo animals and identification of a new subtype. Int J Parasitol 39,
379
473-479.
17
Page 17 of 23
380
Tan, K.S., 2008. New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev 21, 639-665.
381
Thathaisong, U., Worapong, J., Mungthin, M., Tan-Ariya, P., Viputtigul, K., Sudatis, A.,
383
Noonai, A., Leelayoova, S., 2003. Blastocystis isolates from a pig and a horse are
384
closely related to Blastocystis hominis. J Clin Microbiol 41, 967-975.
ip t
382
Wang, W., Cuttell, L., Bielefeldt-Ohmann, H., Inpankaew, T., Owen, H., Traub, R.J., 2013.
386
Diversity of Blastocystis subtypes in dogs in different geographical settings. Parasit
387
Vectors 6, 215.
us
Wong, K.H., Ng, G.C., Lin, R.T., Yoshikawa, H., Taylor, M.B., Tan, K.S., 2008.
an
388
cr
385
Predominance of subtype 3 among Blastocystis isolates from a major hospital in
390
Singapore. Parasitol Res 102, 663-670.
391
M
389
Yan, Y., Su, S., Ye, J., Lai, X., Lai, R., Liao, H., Chen, G., Zhang, R., Hou, Z., Luo, X., 2007. Blastocystis sp. subtype 5: a possibly zoonotic genotype. Parasitol Res 101,
393
1527-1532.
te
394
Yoshikawa, H., Abe, N., Wu, Z., 2004. PCR-based identification of zoonotic isolates of Blastocystis from mammals and birds. Microbiology 150, 1147-1151.
Ac ce p
395 396
d
392
Yoshikawa, H., Wu, Z., Pandey, K., Pandey, B.D., Sherchand, J.B., Yanagi, T., Kanbara, H., 2009. Molecular characterization of Blastocystis isolates from children and rhesus
397
monkeys in Kathmandu, Nepal. Vet Parasitol 160, 295-300.
398
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1
Table 1. PCR primers for amplification of partial fragments of the Blastocystis small subunit
2
ribosomal RNA (SSU rRNA) used to genetically characterise SEQ and Cambodian
3
Blastocystis isolates
ip t
Primer / Primer Name and Sequence (5’ to 3’)
Product Size
Nested PCR–primary set,
cr
Annealing Temperature (°C)
RD3 GGGATCCTGATCCTTCCGCAGGTTCACCTAC
1800 bp
RD5 GGAAGCTTATCTGGTTGATCCTGCCAGTA
us
(65°C; Clark (1997))
Nested PCR – secondary set,
using 1ul of PCR product from
Forward GGAGGTAGTGACAATAAATC
an
(49°C; Wong et al. (2008);
Reverse ACTAGGAATTCCTCGTTCATG
STS 2
STS 3
d
Forward GCTCGTAGTTGAAGTGAGGGAGTG 203-204 bp
Reverse ATCGAACACGAAAGCCTTCAATC Forward GATTGAGAACAACGTACAAACC
(58°C)
Reverse ATGAACCAAACCAATCAAATAC
STS 5
Forward GTAAATGACCAATTCTTATTAC
(56°C)
336 bp
Reverse CTCAATCCAATTGCAAGAC
Ac ce p
(65°C)
Forward TAATACATGAGAAAGTCCTCTGG
te
(57°C)
M
primary step.) STS 1
1100 bp
339-341 bp
106 bp
Reverse GTGGGATGAAGTATATGATG
4 5
Page 19 of 23
5
Table 2. Prevalence of Blastocystis infection in SEQ and Cambodian pig and human samples
6
using the nested PCR primer set and subtype characterisation by sequence analysis. SEQ pigs
Cambodian
SEQ piggery
Cambodian
(n=240)
pigs
staff
villagers (n=210)
(n=73)
(n=36)
76.7% (184)
45.2% (33)
83.3% (30)
ST 1
4
0
11
ST 2
0
0
ST 3
4
0
ST 5
167
20
ST infections
9
13
(SMSI)*
us
33
0
27
16
45
an
M
Suspected mixed
55.2% (116)
cr
% positive
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Infection
3
0
0
11
*Mixed ST infections were suspected when double peaks were observed during sequence
8
analysis.
te
Ac ce p
9
d
7
Page 20 of 23
9 10
Table 3. Statistical significance of difference between Blastocystis prevalence of SEQ and
11
Cambodian pigs and humans respectively.
χ² test 95% Confidence Interval
cr
Pigs
ip t
Prevalence with Group tested
SEQ
184/240 (76.7%; 71.3, 82.0)
χ²=9.58, df=1,
•
Cambodian
33/73 (45.2%; 33.8, 56.6)
p-value=0.00196
an
Humans
us
•
SEQ
30/36 (83.3%; 71.2, 95.5)
χ²=10.1, df=1,
•
Cambodian
116/210 (55.2%; 48.5, 62.0)
p-value=0.0015
M
•
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d
12
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12
Table 4. Blastocystis prevalence and subtype distributions in SEQ and Cambodian pigs
13
(combined results of nested and ST-specific primers 1, 2, 3 and 5 PCR) Cambodian pigs
(n=240)
(n= 73)
% positive
76.7% (184)
45.2% (33)
5 only
167
1 and 5
6
2
0
3 and 5
4
cr
ip t
SEQ pigs
an
us
20
M
Subtype
1, 3 and 5
0 0
3
0
4
13
d
ST5+unknown mixed STs
0
Note: All samples were tested with the nested PCR, but only samples with ST1 (4) or ST3 (4)
15
infections (identified with nested PCR) or SMSI (9) were tested with ST-specific primers 1,
16
2, 3 and/or 5. As such, we are unable to rule out SMSI with other unidentified STs.
17
Test results of ST-specific 1, 2, 3 and/or 5 primers showed that: 1) all 4 ST1 and 4 ST3
18
infections (nested PCR) had mixed infections with ST5, 2) out of 9 SMSI (Table 2), 2 had
19
mixed infections of STs 1/5, 3 with mixed infections of STs 1/3/5 and 4 had mixed infections
20
of STs 5 and other unidentified STs. We cannot exclude that these SMSIs involved other STs
21
that were not tested using additional ST-specific primers.
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22
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22
Table 5. Blastocystis prevalence and subtype distribution in SEQ piggery staff and
23
Cambodian villagers (combined results of nested and ST-specific primers 1, 2, 3 and 5 PCR). Subtype
Cambodian villagers
(n=36)
(n=210)
% positive
83.3% (30)
55.2% (116)
1 only
11
2 only
0
3 only
14
5 only
3
3 and 5
2 0
cr us an
27 45 0 0 11
d
(using nested PCR)
33
M
SMSI
ip t
SEQ staff
Note: The human samples were all tested with the nested PCR and ST5-specific primers only.
25
Eleven of the SMSI observed in the Cambodian human mixed infections detected using
26
nested PCR were not further investigated. Test results of ST-specific 5 primers showed that 2
27
SEQ staff with ST3 infections (nested PCR) had mixed infections with ST5 and none of the
28
Cambodian villagers had ST5.
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S0304-4017(14)00215-5 http://dx.doi.org/doi:10.1016/j.vetpar.2014.04.006 VETPAR 7213
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
25-9-2013 10-3-2014 5-4-2014
Please cite this article as: Wang, W., Owen, H., Traub, R.J., Cuttell, L., Inpankaew, T., Bielefeldt-Ohmann, H.,Molecular epidemiology of Blastocystis in pigs and their in-contact humans in Southeast Queensland, Australia, and Cambodia, Veterinary Parasitology (2014), http://dx.doi.org/10.1016/j.vetpar.2014.04.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Title:
2
Molecular epidemiology of Blastocystis in pigs and their in-contact humans in Southeast
3
Queensland, Australia, and Cambodia
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4 5
Authors:
7
Wenqi Wang*a, Helen Owena, Rebecca J. Trauba, b, Leigh Cuttella, Tawin Inpankaew c, d,
8
Helle Bielefeldt-Ohmanna, b
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an
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Affiliations:
11
a
12
Australia
13
b
14
4067, Australia
15
c
16
University of Copenhagen, Denmark
17
d
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Bangkok,
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Thailand
M
te
d
Australian Infectious Diseases Centre, The University of Queensland, St. Lucia, Queensland
Department of Veterinary Disease Biology, Faculty of Health and Medical Science,
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School of Veterinary Science, The University of Queensland, Gatton, Queensland 4343,
Department of Parasitology, Faculty of Veterinary Medicine, Kasetsart University,
21
* Corresponding author. Tel: + 61 (7) 5460 1834; Fax: + 61 (7) 5460 1922; Email address:
22
[email protected] (Wenqi Wang)
23
1
Page 1 of 23
23
Abstract
25
Blastocystis, an intestinal protist commonly found in humans and animals worldwide, has
26
been implicated by some as a causative agent in irritable bowel syndrome in humans. In pigs,
27
infection with Blastocystis is commonly reported, with most pigs shown to harbour subtypes
28
(ST) 1 or 5, suggesting that these animals are potentially natural hosts for Blastocystis.
29
Although ST5 is considered rare in humans, it has been reported to be a potential zoonosis
30
from pigs in rural China. To test these hypotheses, we conducted molecular analysis of faecal
31
samples from pigs and in-contact humans from commercial intensive piggeries in Southeast
32
Queensland (SEQ), Australia, and a village in rural Cambodia. The prevalence of Blastocystis
33
in SEQ and Cambodian pigs was 76.7% and 45.2%, respectively, with all positive pigs
34
harbouring ST5. It appears likely that pigs are natural hosts of Blastocystis with a high
35
prevalence of ST5 that is presumably the pig-adapted ST in these regions. Amongst the SEQ
36
piggery staff, 83.3% were Blastocystis carriers in contrast to only 55.2% of Cambodian
37
villagers. The predominant STs found in humans were STs 1, 2 (Cambodia only) and 3.
38
Interestingly, ST5 which is usually rare in humans was present in the SEQ piggery staff but
39
not in the Cambodian villagers. We conclude that in intensive piggeries, close contact
40
between pigs and their handlers may increase the risks of zoonotic transmission of
41
Blastocystis.
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Keywords:
Blastocystis; Pig; Epidemiology; Zoonosis
44
2
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1. Introduction
46
Blastocystis is an intestinal protist that is commonly found worldwide in a diverse range of
47
species including humans, non-human primates (NHPs), other mammals and birds (Alfellani
48
et al., 2013c; Stensvold et al., 2009). Blastocystis is genetically polymorphic and molecular
49
analysis of the small subunit ribosomal RNA (SSU rRNA) gene has enabled division of this
50
organism into 17 distinct subtypes (STs) found in a wide range of species (Alfellani et al.,
51
2013c; Fayer et al., 2012; Parkar et al., 2010; Stensvold et al., 2009). Some STs show
52
significant intra-ST genetic variability (Scicluna et al., 2006; Stensvold et al., 2012;
53
Yoshikawa et al., 2009). Humans have been shown to carry STs 1-9 with ST3 the most
54
frequently reported. Significant geographical variation in the prevalence and relative
55
proportion of STs is known to exist, with other common STs including 1, 2 and 4 (Alfellani
56
et al., 2013b). Faecal oral transmission either by direct contact or water-borne transmission is
57
currently the most accepted mode of transmission (Lee et al., 2012b; Leelayoova et al., 2004;
58
Leelayoova et al., 2008). Previous studies have isolated identical STs of Blastocystis from
59
humans and their in-contact animals including pigs (Lee et al., 2012a; Lee et al., 2012b;
60
Nagel et al., 2012; Parkar et al., 2007; Parkar et al., 2010; Stensvold et al., 2009; Yan et al.,
61
2007), suggesting potential zoonotic transmission of Blastocystis.
62
Blastocystis has been reported in pigs in some countries, with most pigs harbouring ST5 or
63
ST1 and occasionally ST2 or ST3 (Alfellani et al., 2013c; Navarro et al., 2008; Thathaisong
64
et al., 2003; Yan et al., 2007). In Valencia, Spain, Navarro et al. (2008) found ST1 to be the
65
most prevalent among intensively reared pigs and, to a lesser extent, ST2. ST5 has also been
66
identified in cattle, other livestock and captive apes (Alfellani et al., 2013b; Stensvold et al.,
67
2009), but rarely in humans. The potential of pigs to act as zoonotic reservoirs of Blastocystis
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was demonstrated by Yan et al. (2007) in which two human ST5 isolates had restriction
69
fragment length polymorphism (RFLP) patterns of the SSU rDNA were either identical or
70
similar to those of 16 pigs living in the same rural area in China.
71
The objective of this study was to ascertain if pigs are suitable animal models for studying
72
Blastocystis infection by investigating if they are indeed natural hosts of Blastocystis as
73
suggested by previous reports. Our selection criteria for a ‘natural host’ was based on (i) a
74
host which commonly harbour Blastocystis (i.e.at a high prevalence) and (ii) of a
75
predominant ST or STs. The second objective was to investigate the zoonotic potential of
76
porcine Blastocystis by testing and genetically comparing Blastocystis recovered from pigs
77
and their in-contact humans (i.e. piggery staff and villagers).
78
Preceding studies investigating Blastocystis infection in pigs have been limited by their
79
relatively small sample size and lack of molecular identification of Blastocystis ST (Abe et
80
al., 2002; Navarro et al., 2008; Yan et al., 2007). In this study we genetically characterise the
81
STs of Blastocystis in pigs and their in-contact humans in two different settings (intensive
82
versus small holder piggeries) in Southeast Queensland (SEQ), Australia, and a rural
83
Cambodian village.
84
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85
2.
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2.1 Sampling
87
2.1.1
88
Single faecal samples were collected from 240 pigs and 36 piggery staff from 18 commercial
89
piggeries in SEQ between February 2012 and February 2013 and stored in 2.5% potassium
Materials and methods
Southeast Queensland (SEQ)
4
Page 4 of 23
90
dichromate prior to DNA extraction. The faecal samples were collected from approximately 10-25
91
pigs at various stages of production (piglets, weaners, growers and sows) per piggery.
92
2.1.2
93
Single faecal samples were collected in May 2012 from 73 pigs and 210 villagers from 41
94
households in Dong village, Preah Vihear province, Cambodia and stored in 2.5% potassium
95
dichromate prior to DNA extraction. The majority of the households sampled (29/41) owned
96
pigs and up to 0-4 pig and 1-9 human samples were collected per household.
97
2.1.3
98
The SEQ segment of this project was approved by the University of Queensland Animal
99
Ethics Committee and Medical Research Ethics Committee with approval no.
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Cambodia
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Ethical approval
ANRFA/472/11 and 2012000069, respectively. Sample collection from the Cambodian
101
village was made possible by collaboration between the University of Copenhagen, Denmark,
102
Department of Fisheries Post-Harvest Technologies and Quality Control, Cambodia, and the
103
National Center for Parasitology, Entomology and Malaria control, Cambodia. The study
104
protocol was approved by the National Ethics Committee Health Research, Ministry of
105
Health in Cambodia. All persons were informed on the purpose of the study. Written
106
informed consent was obtained from all individuals prior to enrolment.
107
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2.2
109
2.2.1
110
Faeces stored in 2.5% potassium dichromate was washed twice in 1× phosphate-buffered
111
saline and centrifuged before the sediment was used for DNA extraction. DNA was extracted
Molecular analysis DNA extraction
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112
using the QIAamp DNA Stool Mini Kit (Qiagen, Germany) with minor alterations as per
113
Wang et al. (2013).
114
2.2.2
115
Samples were first tested with a previously published nested PCR primer set and conditions
116
that amplified an 1100 bp region of the Blastocystis SSU rRNA gene (Clark, 1997; Wong et
117
al., 2008) (Table 1). This method was chosen since it provides a large fragment for
118
phylogenetic analysis, and as it is a pan-Blastocystis technique, it allows for identification of
119
known and unknown STs of Blastocystis. Each 25µl PCR reaction was run using
120
approximately 50 ng of DNA and the conditions were as per Clark (1997) and Wong et al.
121
(2008).
122
2.2.3 Phylogenetic analysis
123
All positive PCR products were purified using the PureLink Genomic DNA Mini Kit (Life
124
Technologies Corporation, New York, USA) according to the manufacturer’s protocol.
125
Unidirectional DNA sequencing was performed with the reverse primer of the secondary
126
PCR with an Applied Biosystems 3130/3130xl Genetic Analyzer. The DNA sequences were
127
first analysed with Finch TV v 1.4.0 (Geospiza Inc., Seattle, WA, USA) and subsequently
128
compared with published sequences from GenBank (National Center for Biotechnology
129
Information) using BLAST 2.2.9 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). These sequences
130
were then aligned with previously published sequences of the SSU rRNA gene of the various
131
Blastocystis STs, which were sourced from GenBank using BioEdit v 7.1.3.0 software (Ibis
132
Biosciences, Carlsbad, CA, USA). The sequences were subsequently analysed using Mega
133
4.1 software (The Biodesign Institute, Tempe, AZ, USA) to construct a neighbour joining
134
tree for subtyping of the Blastocystis isolates. Proteromonas lacertae (U37108) was used as
135
an out-group.
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PCR amplification with nested PCR
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Page 6 of 23
The ST1, ST3 and ST5 sequences from pigs and humans were compared using multiple
137
sequence alignment by Mega 4.1 software (The Biodesign Institute, Tempe, AZ, USA).
138
2.2.4 Development of subtype –specific (ST-specific) primers
139
Pan-subtype targeted PCRs can result in preferential amplification of predominant STs over
140
others in mixed infections. Therefore, we developed and applied ST-specific primers for STs
141
1, 2, 3 and 5 (Table 1) based on the STs identified in the humans and pigs using nested PCR.
142
Application of ST-specific PCRs aimed to investigate whether greater than single ST
143
infections were present in each host and to increase the probability of determining whether
144
cross-transmission of Blastocystis was occurring between pigs and humans in the same
145
geographical setting. ST-specific primers for STs 1, 3 and 5 were designed based on the
146
“barcode” region of the Blastocystis SSU rRNA gene, while the ST-specific primers of ST2
147
were designed from the non-barcode region of the SSU rRNA (see acknowledgment). The
148
respective primers were tested against STs 1 - 6 DNA, respectively to ensure the absence of
149
cross-reactivity. The following protocol was followed 1) All human samples were tested
150
with ST5-specific primers, regardless of Blastocystis status 2) porcine samples with suspected
151
mixed infections (characterised by overlapping double peaks at single nucleotide
152
polymorphisms) were tested with ST-specific 1, 3 and 5 primers (Table 2). In addition,
153
Cambodian pigs were also tested with ST-specific 2 primers as this ST was also found in
154
Cambodian villagers, but not in SEQ staff. Lastly, porcine samples diagnosed with STs 1 or 3
155
(Table 2) were tested with ST-specific 5 primers to ensure mixed infectious with ST5 were
156
not overlooked, as this was the predominant ST found in both pig populations. When an
157
“aberrant” ST was found in pigs or humans with ST-specific primers (i.e. ST5 in humans and
158
STs 1 or 3 in pigs), the PCR products were sequenced and compared with sequences of the
159
same ST from the other host (humans/pigs).
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PCR reaction mixtures for ST-specific primers were similar consisting of 50 ng of faecal
161
genomic DNA, 1.0 units of HotStar Taq Plus DNA Polymerase (Qiagen, Germany) and 10
162
pmol of each forward and reverse primer. The MgCl2 concentration was optimised for each
163
primer pair and ranged from 0.5 to 1.5 mmol/L. Nuclease free water was added to adjust the
164
final reaction volume to 25 µl. Positive controls for each ST and a negative control (2 µl of
165
distilled water) were included in each PCR run. Primer sequences and cycling conditions are
166
presented in Table 1.
167
2.2.5 Control testing of samples
168
As part of internal process control, we tested all samples that were negative using the
169
Blastocystis-specific nested PCR for the presence of amplifiable DNA and to rule out
170
potential PCR inhibition. These samples were tested using published universal primers that
171
amplify a 140 bp fragment of the 18S ribosomal RNA gene from eukaryotic DNA (Fajardo et
172
al., 2006). PCR cycling conditions were modified from a real-time PCR protocol to a
173
conventional protocol using an annealing temperature of 60°C as recommended.
174
2.2.6 Statistical analysis
175
The percentage prevalence of each test group and 95% confidence intervals were calculated
176
using Epitools epidemiological calculators (AusVet Animal Health Services and Australian
177
Biosecurity Cooperative Research Centre for Emerging Infectious Disease,
178
http://epitools.ausvet.com.au). Chi-squared (χ²) tests were used to compare the Blastocystis
179
prevalence between the Cambodian and SEQ pigs and humans respectively. A P-value of less
180
than 0.05 was considered statistically significant. The statistical software used was
181
Calculation for the Chi-Square test: An interactive calculation tool for chi-square tests of
182
goodness of fit and independence (Preacher, 2001).
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183 184
3
185
The prevalence of Blastocystis in both populations of pigs was high (SEQ 76.7%; Cambodia
186
45.2%) (Table 2), but was significantly higher in the SEQ pigs than in the Cambodian pigs (p
187
<0.05; Table 3). All SEQ piggeries (18/18) and majority (24/29) of the pig-rearing
188
households in the Cambodian village contained pigs that were positive for Blastocystis. All
189
infected SEQ and Cambodian pigs harboured ST5 (Table 4). This included 17 (7.1%) SEQ
190
pigs with mixed infections of ST5 and ST1 and/or ST3, or ST5 and unknown mixed STs
191
While no mixed infections (with STs 1, 2, 3 and 5) were detected in the Cambodian pigs
192
(Table 4).
193
The high Blastocystis prevalence, in addition to the predominance of ST5 in both pig
194
populations, corroborated the results of previous studies (Navarro et al., 2008; Yan et al.,
195
2007) which suggest that pigs are likely to be natural hosts for Blastocystis ST5. All
196
Blastocystis-positive pigs regardless of geographical setting harboured ST5, the majority as
197
single infections. This is in agreement with previous smaller scale studies (Alfellani et al.,
198
2013c; Stensvold et al., 2009; Yan et al., 2007; Yoshikawa et al., 2004), including a study in
199
Vietnam that reported 12/12 Blastocystis-positive pigs tested for ST5. In China 16 pigs were
200
identified as harbouring ST5 only (Yan et al., 2007). In contrast, a large scale study
201
conducted in Valencia, Spain, found ST1 the predominant ST in intensively raised pigs
202
(Navarro et al., 2008). This variation of predominant STs (STs 1 or 5) found in pigs could be
203
attributed to geographical ST variation as observed in non-human primates (NHPs) (Alfellani
204
et al., 2013a). Here, investigators identified STs 1, 2 and 3 infections in NHPs as being
205
independent of geographical association, while ST8 was primarily observed in species native
206
to Asia or South America (Alfellani et al., 2013a).
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Results and discussion
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Page 9 of 23
SEQ pigs had a significantly higher prevalence of Blastocystis (76.7%) compared to pigs
208
from Cambodia (45.2%). A possible explanation could be that the intensively raised SEQ
209
pigs have a significantly higher rate of contact with other pigs compared to the Cambodian
210
pigs that are raised singly or in small herds of up to four pigs in a small fenced off area
211
adjacent to village houses. Consequently, the risk of faecal-oral transmission of Blastocystis
212
is greater in the SEQ pigs as compared to the Cambodia pigs.
213
In this study, the prevalence of Blastocystis in SEQ intensive piggery staff (83.3%) was
214
significantly higher than Cambodian villagers (55.2%) (p <0.05; Table 3, 5). Although the
215
prevalence of Blastocystis in humans is generally higher in developing than developed
216
countries, prevalence can also vary within countries among different subpopulations (Tan,
217
2008). The disparity in Blastocystis prevalence between SEQ piggery staff and Cambodian
218
villagers may be a reflection of animal handlers (piggery staff) being at higher risk of
219
acquiring zoonotic infections (Driscoll, 2006; Mahdi and Ali, 2002; Murdoch, 2007),
220
including Blastocystis (Parkar et al., 2010; Rajah Salim et al., 1999). Amongst the
221
Cambodian villagers, only a negligible difference was observed between the prevalence of
222
Blastocystis amongst the pig-rearing villagers (55.5%) and non pig-rearing villagers (54.7%),
223
which is likely owing to primarily anthroponotic transmission. The differences in the
224
prevalence of STs 1, 2 and 3 between pig-rearing and non-pig rearing villagers were not
225
statistically significant.
226
Subtypes 3 and 1 were most commonly detected in all humans tested. This was followed by
227
ST5 in 13.9% (5/36) of the SEQ piggery staff and ST2 in 23.3% (27/116) of the Cambodian
228
villagers (Table 5). Although 2 SEQ staff were found to carry mixed infections of STs 5 and
229
3, neither ST5 nor mixed infections (with STs 1, 2, 3 and 5) were detected in the Cambodian
230
villagers (Table 5). ST distribution studies conducted in various geographic regions have
231
demonstrated substantial geographical variation in STs between populations (Alfellani et al.,
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Page 10 of 23
2013b). In addition, they have also shown that humans may carry STs 1-9 with ST3 being the
233
most prevalent followed by STs 1, 2 and 4 and that ST2 is commonly reported in certain
234
areas such as Brazil, Europe and Central Asia (Alfellani et al., 2013b). Our results are in
235
agreement with these findings and endorse the presence of ST2 as common in humans in
236
rural Cambodia. In our study, all human samples were tested with the pan-ST nested PCR
237
and the ST5 specific primers only and these identified STs 1, 2, 3 and 5. Other STs such as
238
STs 4, 6, 7 and 8 have also been reported in Australia and/or Southeast Asia (Alfellani et al.,
239
2013b; Nagel et al., 2012), however these additional STs were not specifically tested for with
240
ST-specific primers in this study and their presence could therefore not be excluded.
241
Comparison of the 606 bp and 403 bp nested PCR product regions of the SSU rRNA of ST1
242
andST3 from pigs and humans respectively, revealed 100% sequence identity for all three
243
STs regardless of host and geographical location. Comparison of a 477 bp region of the
244
nested PCR product of the SSU rDNA of ST5 also revealed 100% identity, with the
245
exception of a single nucleotide polymorphism (with either adenine or thymine) at 398 bp
246
(GenBank accession no. KJ082062) that was shown to be independent of host and
247
geographical location. This suggests that the SEQ pigs and piggery staff in this study carried
248
very similar Blastocystis STs 1, 3 and 5, which may have been transmitted between these
249
hosts within their environment.
250
Despite ST5 being rarely reported in humans, 13.9% (5/36) of SEQ piggery staff harboured
251
identical STs to in-contact pigs, suggesting that these piggery workers may have acquired the
252
infection from intensively raised pigs. Piggery staff members are repeatedly exposed to large
253
amounts of pig faeces during cleaning of high density pig pens. High pressured water hoses
254
cause aerosolisation of faeces and potentially increase risk of faecal-oral transmission. In
255
contrast, Cambodian villagers that live in close proximity with the pigs they rear did not
256
harbour ST5 despite a Blastocystis prevalence of 45.2% in pigs. This is likely due to the less
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Page 11 of 23
intensive nature of pig rearing in Cambodian villages, where each household rears a
258
maximum of 4 pigs confined to small areas adjacent to or beneath the house. To further
259
investigate this hypothesis, future work should include testing a greater number of piggery
260
staff as well as a control group of piggery staff that do not have direct contact with pigs (e.g.
261
administrative staff) together with non-piggery workers from the same geographical area.
262
Comparison of the SEQ human and pig Blastocystis isolates characterised in this study using
263
a MLST approach would add further evidence for the parasite’s potentially zoonotic abilities;
264
however, this approach has not yet been developed for ST5.
265
A minority of SEQ pigs were found to harbour mixed infections with ST5 and ST1 (n=9)
266
and/or ST3 (n=9), which were not observed in Cambodian pigs using both nested and ST-
267
specific PCR. Comparison of the pig and human ST1 and ST3 sequences showed 100%
268
identity, respectively. Subtype 3 has been reported in pigs but is uncommon (Abe et al., 2003;
269
Stensvold et al., 2009; Yoshikawa et al., 2004), whilst ST1 is common in pigs in certain
270
subpopulations such as commercial piggeries in Valencia, Spain and pigs kept at an army
271
base in Chonburi, Thailand (Navarro et al., 2008; Thathaisong et al., 2003). Given that ST5
272
was the predominant ST in pigs from SEQ and that the piggery staff predominantly
273
harboured STs 1 or 3, there is a possibility that the SEQ pigs harbouring STs 1 or 3 may have
274
acquired the Blastocystis infection from human handlers. In other words, this could be a
275
reverse zoonosis, though MLST of isolates would be required to further confirm this
276
hypothesis.
277
4 Conclusion
278
The results of the study support the hypothesis that pigs are natural hosts of Blastocystis due
279
to the high prevalence in different geographical localities and predominant infection with ST5
280
which has been proposed as a pig-adapted ST. There was a concurrent high prevalence of
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Blastocystis in both SEQ piggery staff and Cambodian villagers with STs 1 and 3 being the
282
most predominant STs in both regions. In addition, ST2 was detected in the Cambodian
283
villagers. These findings could reflect geographical variation or isolation among certain STs.
284
The study also indicated the proposed pig-adapted ST5 may be a zoonosis as 13.9% of SEQ
285
piggery staff harboured ST5 which is otherwise considered rare in humans. Piggery staff are
286
most likely to have acquired ST5 infection through close contact with pig faeces while
287
carrying out daily routines. In addition, the presence of STs 1 and/or 3 in a minority of SEQ
288
pigs in addition to piggery staff could indicate reverse zoonotic transmission.
289
Acknowledgments
290
This project has been supported by the William Peter Richards Trust Award Grant (WW) and
291
The University of Queensland New Staff Start-up Grant (HO). Special thanks to Paul Noone,
292
piggery staff, staff in the School of Veterinary Science especially Dr Kit Parke and the UQ
293
Veterinary Clinical Studies Centre for their assistance in sample collection and processing.
294
The Cambodian samples were collected as part of Dr. Tawin Inpankaew’s PhD project that is
295
funded by the University of Copenhagen, Denmark. We would also like to acknowledge staff
296
of The National Center for Parasitology, Entomology and Malaria Control, Cambodia, Mr
297
Wissanuwat Chimnoi and Ms Supanun Boonchob, staff of The faculty of Veterinary
298
Medicine, Kasetsart University, Thailand, Dr Chhay Somany, deputy director of the
299
provincial health department at Preah Vihear province and all the participants for their
300
assistance in sample collection from Dong village in Cambodia. Lastly, many thanks to Dr.
301
Christen Rune Stensvold for kindly giving us access to the STS 1, 2 and 3 primers that he
302
designed.
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303 304
Conflict of interest statement 13
Page 13 of 23
305
The authors declare that they have no competing interests.
306
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307 308
References
309
Abe, N., Wu, Z., Yoshikawa, H., 2003. Zoonotic genotypes of Blastocystis hominis detected in cattle and pigs by PCR with diagnostic primers and restriction fragment length
311
polymorphism analysis of the small subunit ribosomal RNA gene. Parasitol Res 90,
312
124-128.
cr
313
ip t
310
Alfellani, M.A., Jacob, A.S., Perea, N.O., Krecek, R.C., Taner-Mulla, D., Verweij, J.J., Levecke, B., Tannich, E., Clark, C.G., Stensvold, C.R., 2013a. Diversity and
315
distribution of Blastocystis sp. subtypes in non-human primates. Parasitology 140,
316
966-971.
an
317
us
314
Alfellani, M.A., Stensvold, C.R., Vidal-Lapiedra, A., Onuoha, E.S., Fagbenro-Beyioku, A.F., Clark, C.G., 2013b. Variable geographic distribution of Blastocystis subtypes and its
319
potential implications. Acta Trop 126, 11-18.
d
Alfellani, M.A., Taner-Mulla, D., Jacob, A.S., Imeede, C.A., Yoshikawa, H., Stensvold, C.R.,
te
320
M
318
Clark, C.G., 2013c. Genetic diversity of Blastocystis in livestock and zoo animals.
322
Protist 164, 497-509.
323
Ac ce p
321
Clark, C.G., 1997. Extensive genetic diversity in Blastocystis hominis. Mol Biochem
324 325
Parasitol 87, 79-83.
Driscoll, T., 2006. Work-related infectious and parasitic diseases Australia. Australian Safety and Compensation Council, Australian Government. Available from
326
http://www.safeworkaustralia.gov.au/sites/SWA/about/Publications/Documents/415/
327 328
Workrelated_Infectious_parastitic_disease_Australia.pdf. Last accessed on 18th
329
September 2013.
330
Fajardo, V., Gonzalez, I., Lopez-Calleja, I., Martin, I., Hernandez, P.E., Garcia, T., Martin, R., 2006. PCR-RFLP authentication of meats from red deer (Cervus elaphus), fallow
331
15
Page 15 of 23
332
deer (Dama dama), roe deer (Capreolus capreolus), cattle (Bos taurus), sheep (Ovis
333
aries), and goat (Capra hircus). J Agric Food Chem 54, 1144-1150. Fayer, R., Santin, M., Macarisin, D., 2012. Detection of concurrent infection of dairy cattle
335
with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and
336
microscopic methods. Parasitol Res 111, 1349-1355.Lee, L.I., Chye, T.T.,
337
Karmacharya, B.M., Govind, S.K., 2012a. Blastocystis sp.: waterborne zoonotic
338
organism, a possibility? Parasit Vectors 5, 130-134.
cr
us
339
ip t
334
Lee, L.l., Tan, T.C., Tan, P.C., Nanthiney, D.R., Biraj, M.K., Surendra, K.M., Suresh, K.G., 2012b. Predominance of Blastocystis sp. subtype 4 in rural communities, Nepal.
341
Parasitol Res 110, 1553-1562.
Leelayoova, S., Rangsin, R., Taamasri, P., Naaglor, T., Thathaisong, U., Mungthin, M., 2004.
M
342
an
340
Evidence of waterborne transmission of Blastocystis hominis. Am J Trop Med Hyg
344
70, 658-662.
d
343
Leelayoova, S., Siripattanapipong, S., Thathaisong, U., Naaglor, T., Taamasri, P., Piyaraj, P.,
346
Mungthin, M., 2008. Drinking water: a possible source of Blastocystis spp. subtype 1
347
infection in schoolchildren of a rural community in central Thailand. Am J Trop Med
Ac ce p
te
345
Hyg 79, 401-406.
348 349
Mahdi, N.K., Ali, N.H., 2002. Cryptosporidiosis among animal handlers and their livestock in Basrah, Iraq. East Afr Med J 79, 550-553.
350 351
Murdoch, B., 2007. Zoonoses - Animal diseases that may also affect humans. Department of Environmental and Primary Industries, Victoria, Australia. Available from
352 353
http://www.dpi.vic.gov.au/agriculture/pests-diseases-and-weeds/animal-
354
diseases/zoonoses/zoonoses-animal-diseases-that-may-also-affect-humans. Last
355
accessed on 18th September 2013.
16
Page 16 of 23
356
Nagel, R., Cuttell, L., Stensvold, C.R., Mills, P.C., Bielefeldt-Ohmann, H., Traub, R.J., 2012.
357
Blastocystis subtypes in symptomatic and asymptomatic family members and pets and
358
response to therapy. Intern Med J 42, 1187-1195. Navarro, C., Dominguez-Marquez, M.V., Garijo-Toledo, M.M., Vega-Garcia, S., Fernandez-
ip t
359
Barredo, S., Perez-Gracia, M.T., Garcia, A., Borras, R., Gomez-Munoz, M.T., 2008.
361
High prevalence of Blastocystis sp. in pigs reared under intensive growing systems:
362
frequency of ribotypes and associated risk factors. Vet Parasitol 153, 347-358.
us
363
cr
360
Parkar, U., Traub, R.J., Kumar, S., Mungthin, M., Vitali, S., Leelayoova, S., Morris, K., Thompson, R.C., 2007. Direct characterization of Blastocystis from faeces by PCR
365
and evidence of zoonotic potential. Parasitology 134, 359-367. Parkar, U., Traub, R.J., Vitali, S., Elliot, A., Levecke, B., Robertson, I., Geurden, T., Steele,
M
366
an
364
J., Drake, B., Thompson, R.C., 2010. Molecular characterization of Blastocystis
368
isolates from zoo animals and their animal-keepers. Vet Parasitol 169, 8-17. Rajah Salim, H., Suresh Kumar, G., Vellayan, S., Mak, J.W., Khairul Anuar, A., Init, I.,
te
369
d
367
Vennila, G.D., Saminathan, R., Ramakrishnan, K., 1999. Blastocystis in animal
371
handlers. Parasitol Res 85, 1032-1033.
372
Ac ce p
370
Scicluna, S.M., Tawari, B., Clark, C.G., 2006. DNA barcoding of Blastocystis. Protist 157, 77-85.
373 374
Stensvold, C.R., Alfellani, M., Clark, C.G., 2012. Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infect Genet Evol 12, 263-273.
375 376
Stensvold, C.R., Alfellani, M.A., Norskov-Lauritsen, S., Prip, K., Victory, E.L., Maddox, C.,
377
Nielsen, H.V., Clark, C.G., 2009. Subtype distribution of Blastocystis isolates from
378
synanthropic and zoo animals and identification of a new subtype. Int J Parasitol 39,
379
473-479.
17
Page 17 of 23
380
Tan, K.S., 2008. New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev 21, 639-665.
381
Thathaisong, U., Worapong, J., Mungthin, M., Tan-Ariya, P., Viputtigul, K., Sudatis, A.,
383
Noonai, A., Leelayoova, S., 2003. Blastocystis isolates from a pig and a horse are
384
closely related to Blastocystis hominis. J Clin Microbiol 41, 967-975.
ip t
382
Wang, W., Cuttell, L., Bielefeldt-Ohmann, H., Inpankaew, T., Owen, H., Traub, R.J., 2013.
386
Diversity of Blastocystis subtypes in dogs in different geographical settings. Parasit
387
Vectors 6, 215.
us
Wong, K.H., Ng, G.C., Lin, R.T., Yoshikawa, H., Taylor, M.B., Tan, K.S., 2008.
an
388
cr
385
Predominance of subtype 3 among Blastocystis isolates from a major hospital in
390
Singapore. Parasitol Res 102, 663-670.
391
M
389
Yan, Y., Su, S., Ye, J., Lai, X., Lai, R., Liao, H., Chen, G., Zhang, R., Hou, Z., Luo, X., 2007. Blastocystis sp. subtype 5: a possibly zoonotic genotype. Parasitol Res 101,
393
1527-1532.
te
394
Yoshikawa, H., Abe, N., Wu, Z., 2004. PCR-based identification of zoonotic isolates of Blastocystis from mammals and birds. Microbiology 150, 1147-1151.
Ac ce p
395 396
d
392
Yoshikawa, H., Wu, Z., Pandey, K., Pandey, B.D., Sherchand, J.B., Yanagi, T., Kanbara, H., 2009. Molecular characterization of Blastocystis isolates from children and rhesus
397
monkeys in Kathmandu, Nepal. Vet Parasitol 160, 295-300.
398
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1
Table 1. PCR primers for amplification of partial fragments of the Blastocystis small subunit
2
ribosomal RNA (SSU rRNA) used to genetically characterise SEQ and Cambodian
3
Blastocystis isolates
ip t
Primer / Primer Name and Sequence (5’ to 3’)
Product Size
Nested PCR–primary set,
cr
Annealing Temperature (°C)
RD3 GGGATCCTGATCCTTCCGCAGGTTCACCTAC
1800 bp
RD5 GGAAGCTTATCTGGTTGATCCTGCCAGTA
us
(65°C; Clark (1997))
Nested PCR – secondary set,
using 1ul of PCR product from
Forward GGAGGTAGTGACAATAAATC
an
(49°C; Wong et al. (2008);
Reverse ACTAGGAATTCCTCGTTCATG
STS 2
STS 3
d
Forward GCTCGTAGTTGAAGTGAGGGAGTG 203-204 bp
Reverse ATCGAACACGAAAGCCTTCAATC Forward GATTGAGAACAACGTACAAACC
(58°C)
Reverse ATGAACCAAACCAATCAAATAC
STS 5
Forward GTAAATGACCAATTCTTATTAC
(56°C)
336 bp
Reverse CTCAATCCAATTGCAAGAC
Ac ce p
(65°C)
Forward TAATACATGAGAAAGTCCTCTGG
te
(57°C)
M
primary step.) STS 1
1100 bp
339-341 bp
106 bp
Reverse GTGGGATGAAGTATATGATG
4 5
Page 19 of 23
5
Table 2. Prevalence of Blastocystis infection in SEQ and Cambodian pig and human samples
6
using the nested PCR primer set and subtype characterisation by sequence analysis. SEQ pigs
Cambodian
SEQ piggery
Cambodian
(n=240)
pigs
staff
villagers (n=210)
(n=73)
(n=36)
76.7% (184)
45.2% (33)
83.3% (30)
ST 1
4
0
11
ST 2
0
0
ST 3
4
0
ST 5
167
20
ST infections
9
13
(SMSI)*
us
33
0
27
16
45
an
M
Suspected mixed
55.2% (116)
cr
% positive
ip t
Infection
3
0
0
11
*Mixed ST infections were suspected when double peaks were observed during sequence
8
analysis.
te
Ac ce p
9
d
7
Page 20 of 23
9 10
Table 3. Statistical significance of difference between Blastocystis prevalence of SEQ and
11
Cambodian pigs and humans respectively.
χ² test 95% Confidence Interval
cr
Pigs
ip t
Prevalence with Group tested
SEQ
184/240 (76.7%; 71.3, 82.0)
χ²=9.58, df=1,
•
Cambodian
33/73 (45.2%; 33.8, 56.6)
p-value=0.00196
an
Humans
us
•
SEQ
30/36 (83.3%; 71.2, 95.5)
χ²=10.1, df=1,
•
Cambodian
116/210 (55.2%; 48.5, 62.0)
p-value=0.0015
M
•
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12
Page 21 of 23
12
Table 4. Blastocystis prevalence and subtype distributions in SEQ and Cambodian pigs
13
(combined results of nested and ST-specific primers 1, 2, 3 and 5 PCR) Cambodian pigs
(n=240)
(n= 73)
% positive
76.7% (184)
45.2% (33)
5 only
167
1 and 5
6
2
0
3 and 5
4
cr
ip t
SEQ pigs
an
us
20
M
Subtype
1, 3 and 5
0 0
3
0
4
13
d
ST5+unknown mixed STs
0
Note: All samples were tested with the nested PCR, but only samples with ST1 (4) or ST3 (4)
15
infections (identified with nested PCR) or SMSI (9) were tested with ST-specific primers 1,
16
2, 3 and/or 5. As such, we are unable to rule out SMSI with other unidentified STs.
17
Test results of ST-specific 1, 2, 3 and/or 5 primers showed that: 1) all 4 ST1 and 4 ST3
18
infections (nested PCR) had mixed infections with ST5, 2) out of 9 SMSI (Table 2), 2 had
19
mixed infections of STs 1/5, 3 with mixed infections of STs 1/3/5 and 4 had mixed infections
20
of STs 5 and other unidentified STs. We cannot exclude that these SMSIs involved other STs
21
that were not tested using additional ST-specific primers.
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22
Page 22 of 23
22
Table 5. Blastocystis prevalence and subtype distribution in SEQ piggery staff and
23
Cambodian villagers (combined results of nested and ST-specific primers 1, 2, 3 and 5 PCR). Subtype
Cambodian villagers
(n=36)
(n=210)
% positive
83.3% (30)
55.2% (116)
1 only
11
2 only
0
3 only
14
5 only
3
3 and 5
2 0
cr us an
27 45 0 0 11
d
(using nested PCR)
33
M
SMSI
ip t
SEQ staff
Note: The human samples were all tested with the nested PCR and ST5-specific primers only.
25
Eleven of the SMSI observed in the Cambodian human mixed infections detected using
26
nested PCR were not further investigated. Test results of ST-specific 5 primers showed that 2
27
SEQ staff with ST3 infections (nested PCR) had mixed infections with ST5 and none of the
28
Cambodian villagers had ST5.
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24
Page 23 of 23