Parasitol Res (2011) 109:205–212
DOI 10.1007/s00436-010-2244-9
ORIGINAL PAPER
Development of a new PCR protocol to detect and subtype
Blastocystis spp. from humans and animals
Mónica Santín & María Teresa Gómez-Muñoz &
Gloria Solano-Aguilar & Ronald Fayer
Received: 30 September 2010 / Accepted: 14 December 2010 / Published online: 6 January 2011
# Springer-Verlag (outside the USA) 2011
Abstract Blastocystis spp. is commonly found in the feces
of humans worldwide. Infection has been reported as
asymptomatic, acute symptomatic, and chronic symptomatic.
This wide range of responses to infection could be related to
the genetic diversity of morphologically indistinguishable
specimens obtained from infected hosts. The former name
Blastocystis hominis is now reported as Blastocystis spp.
because of its genetic diversity. Blastocystis is recognized as
a complex of subtypes that have not been fully characterized
as independent species. The finding of Blastocystis spp. in
feces from several animal species suggests a zoonotic
potential. Based on conserved regions of published nucleo-
tide SSU rDNA sequences from all Blastocystis subtypes
found in GenBank, a PCR and sequencing protocol was
developed. The ~500 bp SSU rDNA gene fragment
amplified by this PCR is highly sensitive compared with
M. Santín (*) : R. Fayer
Environmental Microbial and Food Safety Laboratory,
Animal and Natural Resources Institute, Agricultural Research
Service, United States Department of Agriculture,
Building 173, BARC-East, 10300 Baltimore Avenue,
Beltsville, MD 20705, USA
e-mail: monica.santin-duran@ars.usda.gov
M. T. Gómez-Muñoz
Departamento de Sanidad Animal, Facultad de Veterinaria,
Universidad Complutense de Madrid,
Avenida Puerta de Hierro S/N,
28040 Madrid, Spain
G. Solano-Aguilar
United States Department of Agriculture, Beltsville Nutrition
Research Center, Diet Genomics and Immunology Laboratory,
Agricultural Research Service,
Building 307C, BARC-East, 10300 Baltimore Avenue,
Beltsville, MD 20705, USA
published primers and contains highly variable regions that
allow phylogenetic analysis of Blastocystis. These primers
were used to detect and subtype Blastocystis spp. specimens
from naturally infected humans, primates, cattle, pigs, and
chickens. Based on these findings, application of this method
can elucidate the complexity of this heterogeneous genus and
its role in human and animal disease, as well as its zoonotic
potential.
Introduction
Blastocystis is one of the most common human intestinal
parasites found in developed and developing countries (Tan
2004). This ubiquitous and enigmatic protozoan parasite
has also been identified in a wide range of animals. The
taxonomic status of Blastocystis has remained elusive until
recently, when small subunit ribosomal DNA (SSU rDNA)
phylogeny (Silberman et al. 1996), as well as a combined
analysis of eight molecules (Arisue et al. 2002) demon-
strated that it is a stramenopile. This eukaryotic group
includes unicellular and multicellular protists, including
brown algae, diatoms, chrysophytes, water molds, and
slime nets (Patterson 1994), and is a branch of the new
higher level taxon Chromalveolata (Adl et al. 2005).
Blastocystis is pleomorphic, presenting such a variety of
forms, even within a monoculture, that identification of
specific stages is problematic (Tan 2008). The difficulty in
identifying Blastocystis in fecal specimens has resulted in
confusion and misinterpretation regarding its life cycle, host
specificity, and pathogenicity. The pathogenic potential of
Blastocystis is controversial with numerous conflicting
reports regarding its ability to cause disease (Vogelberg
et al. 2010; Yakoob et al. 2010). Blastocystis has been found
206
not only in individuals with gastrointestinal symptoms and
skin rash but also in apparently healthy and asymptomatic
individuals (Boorom et al. 2008; Dominguez-Marquez et al.
2009). Transmission of this parasite is also uncertain. It is
generally accepted that transmission involves ingestion of
fecal matter following hand-to-hand, hand-to-food, or drink-
ing contaminated water (Leelayoova et al. 2008; Stark et al.
2007; Tanizaki et al. 2005; Yoshikawa et al. 2000, 2004,
2009). Although specimens collected from infected humans
and animals have been morphologically indistinguishable,
the application of molecular methods has shown remarkable
genetic diversity among specimens from both human and
animals (Arisue et al. 2003; Noel et al. 2003; Stensvold et al.
2009a). Recently, a consensus terminology for subtypes (ST)
of Blastocystis was developed on the basis of SSU-rDNA
gene analysis and nine subtypes were established and
designated as ST1 to ST9 (Stensvold et al. 2007). More
recently, a tenth subtype (ST10) was described from both
primates and ungulates in Denmark (Stensvold et al. 2009a).
It is likely that the confusion regarding Blastocystis
pathogenicity can be related to diagnostic limitations and
differences in the virulence of different subtypes (Stensvold
et al. 2009b, c). Blastocystis has low host specificity and is
considered a potential zoonotic pathogen, because infections
in humans have been associated with contact with primates,
pigs, and poultry (Li et al. 2007; Noel et al. 2005; Stensvold
et al. 2009a, b; Yoshikawa et al. 2009). The subtype ST-2
was shared between local rhesus monkeys and children in
Nepal, and therefore, monkeys were considered as a possible
source of Blastocystis ST-2 infection of humans (Yoshikawa
et al. 2009). Similarly, Parkar et al. (2007) found ST-1 in
zoo-keepers and primates at the Perth Zoo. Pig ownership
was found to be a risk factor for Blastocystis in humans in
China (Li et al. 2007). In a study conducted in China, the
same subtype ST-5 was observed in 16 pigs, as well as in
three humans living in the same rural area (Yan et al., 2007).
The present study was undertaken to design, test, and
select primers that target a region of the SSU rDNA gene
that would facilitate detection and identification of subtypes
of Blastocystis in human and animal feces. The ultimate
goal is to use this new assay to increase our knowledge of
subtypes regarding transmission routes, host specificity,
zoonotic significance, and association with disease.
Material and methods
Sources of specimens, parasite purification, and DNA
extraction
Thirty-six Blastocystis specimens from humans and animals
were included in the present study (Table 1). Thirty-five
Parasitol Res (2011) 109:205–212
fecal specimens containing Blastocystis were obtained from
naturally infected hosts. One specimen was obtained from
the American Type Culture Collection (ATCC # 50608D).
All samples of animal origin, except for those from the
chicken, were preliminarily identified as positive by micros-
copy. For all the human samples PCR was used to screen the
samples. Blastocystis in animal feces was concentrated by
CsCl centrifugation as previously described (Santín et al.
2004). Total DNA was extracted from each CsCl-
concentrated fecal sample using a modification of the
DNeasy Tissue Kit (Qiagen, Valencia, CA). A 50-μl
suspension of concentrated Blastocystis stages was sus-
pended in 180 μl of ATL buffer (supplied by the
manufacturer) and vortexed. To this suspension, 20 μl of
proteinase K (20 mg/ml) was added and the mixture was
incubated overnight at 55 C. The next day, 200 μl of AL
buffer were added and purification proceeded as per
manufacturer's instructions. The nucleic acids were eluted
in 100 μl of AE buffer.
Total DNA from human stools was extracted using the
QIAamp DNA stool minikit (Qiagen, Valencia, CA).
Briefly, 1 g of homogenized fecal sample was thawed,
weighed, and resuspended with lysis buffer. The suspension
was heated to 95 C to increase DNA yield, to remove
inhibitors, and to increase proteinase K digestion before
DNA was bound to a column, washed, and eluted in TE
buffer.
Gene amplification and sequencing
To amplify a fragment of the SSU rDNA gene from various
Blastocystis specimens, a PCR protocol was developed,
using primers complementary to conserved regions of
published nucleotide SSU rDNA sequences of Blastocystis
downloaded from GenBank (Table 2). A specific set of
primers were designed based upon multiple sequence
alignment of the SSU rDNA gene. The primers forward
Blast 505–532 (5′ GGA GGT AGT GAC AAT AAA TC 3′)
(previously used by Böhm-Gloning et al. 1997) and reverse
Blast 998–1017 (5′ TGC TTT CGC ACT TGT TCA TC 3′)
amplifies a ca. 500 (479)-bp fragment, containing a variable
region that allows subtyping of Blastocystis specimens. The
locations of the primers based on reference nucleotide
sequence U51151 are at nucleotide positions 445–464 and
905–924. Each 50-μl PCR mixture contained 1× PCR
buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 U Taq
(Qbiogene, Irvine, CA), 2.5 μl BSA (0.1 g/10 ml), and
1 μM of each primer. A total of 35 cycles, each consisting
of 95 C for 30 s, 54 C for 30 s, and 72 C for 30 s, was
performed; an initial pre-heat step at 95 C for 4 min and
a final extension step at 72 C for 5 min were also
included.
Parasitol Res (2011) 109:205–212 207

Table 1 Blastocystis isolates used in the study with information on hosts, locations, subtypes, and Genbank accession numbers

Isolate Host Location ST GenBank accession number

H-10 Human Colombia ST-1 HQ641595

H-14 Human Colombia ST-1 HQ641596
H-33 Human Colombia ST-1 HQ641597
H-42 Human Colombia ST-1 HQ641598
H-7 Human Colombia ST-2 (4 different nucleotide sequences) HQ641599-HQ641602
H-11 Human Colombia ST-2 (2 different nucleotide sequences) HQ641603- HQ641604
H-12 Human Colombia ST-2 HQ641605
H-29 Human Colombia ST-3 (5 different nucleotide sequences) HQ641606- HQ641610
H-30 Human Colombia ST-3 HQ641611
H-41 Human Colombia ST-3 HQ641612
H-57 Human Colombia ST-3 HQ641613
H-5 Human Colombia ST-1 (1 nucleotide sequence) HQ641614-HQ641620
ST-2 (6 nucleotide sequences)
H-1 Human Spain ST-4 HQ641621
ATTC 50608D Human USA ST-4 HQ641622
C-3066 Cattle USA ST-10 HQ641623
C-3067 Cattle USA ST-10 HQ641624
C-3069 Cattle USA ST-10 HQ641625
C-3070 Cattle USA ST-10 HQ641626
C-3071 Cattle USA ST-10 HQ641627
C-3072 Cattle USA ST-10 HQ641628
C-3073 Cattle USA ST-10 HQ641629
P-5 Swine USA ST-5 HQ641630
P-6 Swine USA ST-5 HQ641631
P-8 Swine USA ST-5 HQ641632
P-12 Swine USA ST-5 HQ641633
P-13 Swine USA ST-5 HQ641634
P-20 Swine USA ST-5 HQ641635
P-18 C Swine Spain ST-5 HQ641636
PR-1 Primate (Hapalemur aureus) Spain ST-1 (5 nucleotide sequences) HQ641637-HQ641641
PR-4 Primate (Cercopithecus hamlyni) Spain ST-1 (1 nucleotide sequence) HQ641642-HQ641651
ST-2 (2 nucleotide sequences)
ST-3 (7 nucleotide sequences)
PR-5 Primate (Lemur catta) Spain ST-4 HQ641652
PR-9 Primate (Mandrillus leucophaeus) Spain ST-3 HQ641653
PR-11 Primate (Gorilla gorilla) Spain ST-2 (2 nucleotide sequences) HQ641654-HQ641655
PR-13 Primate (Cercocebus atys) Spain ST-3 HQ641656
PR-14 Primate (Cercocebus neglectus) Spain ST-3 HQ641657
Ch-1 Chicken Spain ST-6 (2 nucleotide sequences) HQ641658-HQ641661
ST-7 (2 nucleotide sequences)

PCR products were analyzed on 1% agarose gel,
visualized by ethidium bromide staining, and purified
with Exonuclease I/Shrimp Alkaline Phosphatase (Exo-
SAP-IT™) (USB Corporation, Cleveland, Ohio). Purified
products were sequenced in both directions. The same
PCR primers were used in 10-μl reactions, Big Dye™
chemistries, and an ABI3100 sequencer analyzer (Applied
Biosystems, Foster City, CA).
DNA from other organisms, Cryptosporidium parvum
Beltsville isolate, Giardia duodenalis Assemblage A isolate
WB, Giardia duodenalis Assemblage E isolate Beltsville,
Enterocytozoon bieneusi genotype PtEb IX isolate 64,
208 Parasitol Res (2011) 109:205–212

Table 2 Reference sequences

for each Blastocystis subtype ST GenBank Host Location Reference
included in this study with their
GenBank accession number, ST-1 U51151 Human USA Silberman et al. 1996
host, locations and reference AB107962 Human Japan Abe (2004)
AB070993 Chicken Japan Arisue et al. (2003)
AB070989 Human Japan Arisue et al. (2003)
ST-2 AB070997 Primate Japan Arisue et al. (2003)
AB107969 Primate Japan Abe (2004)
AB070987 Human Japan Arisue et al. (2003)
ST-3 AB091234 Human Japan Arisue et al. (2003)
AB107963 Pig Japan Abe (2004)
AB070992 Human Japan Arisue et al. (2003)
ST-4 U26177 Guinea pig USA Leipe et al. (1996)
AB071000 Rat Japan Arisue et al. (2003)
AY244620 Human Germany Yoshikawa et al. (2004)
ST-5 AB070998 Pig Japan Arisue et al. (2003)
AB107964 Pig Japan Abe (2004)
AB070999 Pig Japan Arisue et al. (2003)
ST-6 AY135411 Turkey France Noel et al. (2003)
AB107972 Bird Japan Abe (2004)
AB070990 Human Japan Arisue et al. (2003)
ST-7 AY590109 Human France Noel et al. (2003)
AF408427 Human Japan Arisue et al. (2003)
AB070996 Quail Japan Arisue et al. (2003)
AY135409 Chicken France Noel et al. (2003)
AB107973 Bird Japan Abe (2004)
ST-8 AB107971 Bird Japan Abe (2004)
AB107970 Primate Japan Abe (2004)
ST-9 AF408425 Human Japan Yoshikawa et al. (2004)
AF408426 Human Japan Yoshikawa et al. (2004)
ST-10 FM164412 Cattle Denmark Stensvold et al. (Stensvold et al. 2009a, b, c)
FM164413 Cattle Denmark Stensvold et al. (2009a, b, c)

Encephalitozoon hellem ATTC# 50451, Encephalitozoon
cuniculi ATTC# 50602, and Encephalitozoon intestinalis
(DNA was extracted from spores originally isolated from an
AIDS patient and grown in culture provided by Dr.
Elizabeth Didier, Tulane Regional Primate Research Center,
Covington, Louisiana), was also tested to assess the
specificity of the primers.
The nucleotide sequences obtained in this study have
been deposited in GenBank under accession numbers
HQ641595-HQ641661.
Cloning
When mixed infection within a specimen was suspected
from the sequence traces, the PCR products of SSU
rDNA were cloned using the TOPO TA cloning kit
(Invitrogen Corp., Carlsbad, CA) and transformants were
selected from each specimen and screened by PCR, and
sequenced in both directions using M13 forward and
reverse primers. Up to ten clones from each specimen
were sequenced.
Phylogenetic analysis
All sequences (excluding vector and primer sites) were
subjected to BLAST searches in the GenBank database to
confirm specimens as Blastocystis spp. Subtype terminology
for Blastocystis was used according to Stensvold et al.
(2007). Nucleotide sequences for this study, as well as type
sequences obtained from GenBank from each of the ten
subtypes currently recognized, were aligned using the Clustal
W algorithm in the Megalign module (DNASTAR Inc.,
Madison, WI). Clustal W determines that when a gap is
inserted, it can be removed only by editing, so final
alignment adjustments were made manually to remove
artificial gaps. Phylogenetic and molecular evolutionary
Parasitol Res (2011) 109:205–212
analyses were made using MEGA software version 4
(Tamura et al. 2007). Phylogenetic inference was by the
neighbor-joining (NJ) method of Saitou and Nei (1987).
Genetic distance was calculated with the Kimura 2-parameter
model. Branch reliability was assessed using bootstrap
analysis (1,000 replicates).
Results
A PCR amplicon of approximately 500 bp was successfully
generated from all 36 specimens included in the study. All
PCR amplicons were sequenced to determine the subtypes
following the subtype classification designated by Stensvold
et al. (2007). Direct subtyping was accomplished for 28
specimens, detecting ST-1 in four human specimens, ST-2 in
one human specimen, ST-3 in three humans and three
primate specimens, ST-4 in two human and one primate
specimens, ST-5 in seven pig specimens, and ST-10 in seven
cattle specimens (Table 1). However, in eight specimens
(H-5, H-7, H-11, H-29, PR-1, PR-4, PR-11, and Ch-1),
mixed infection within a specimen were suspected from
the sequence traces, and those samples were subjected to
cloning. Heterogeneity in nucleotide sequences were
observed among clones (Table 1). In five specimens, three
human (H-7, H-11, and H-29) and two primate (PR-1 and
PR-11) all sequences for each single specimen were located
within an independent clade (ST-1, ST-2, or ST-3). The
heterogeneous sequences from one primate specimen (PR-1)
were all located within ST-1; from three specimens, two
human (H-7 and H-11), and one primate (PR-11), within ST-
2 clade; and from one human specimen (H-29) within ST-3
clade. However, in three specimens, one human (H-5),
one primate (PR-4), and one chicken (Ch-1), nucleotide
sequences were located in different ST clades. Human
specimen H-5 nucleotide sequences were located in two ST
clades, ST-1, and ST-2; primate specimen PR-4 nucleotide
sequences were located in three, ST-1, ST-2, and ST-3; and
the chicken specimen CH-1 nucleotide sequences were
located in two clades ST-6 and ST-7.
A phylogenetic analysis of all sequences obtained in
this study was carried out together with reference
sequences (Table 2) obtained from GenBank (Fig. 1).
The two reference sequences from subtype 10 (ST-10)
were excluded from the construction of the phylogenetic
tree, since the overlapping region with sequences from our
study was not long enough for FM164413, and sequence
FM164412 did not overlap with our nucleotide sequence.
However, when a BLAST search was conducted including
only the area that overlaps among our cattle nucleotide
sequences and FM164413 (240 bp that includes nucleo-
tides from position 365 to 604 from reference nucleotide
sequence FM164413), the closest match (similarity of
209
98.3%) was FM164413. These data confirm the identifi-
cation of sequence specimens analyzed from cattle as
ST-10. The phylogenetic tree revealed the presence of ten
different clades that corresponded exactly to the ST 1 to
10, indicating that the sequence patterns of the variable
regions amplified correlated well with the phylogenetic
tree-based grouping.
The sensitivity of the primers using this PCR protocol
was determined by serial dilution of Blastocystis DNA. As
little as 0.0001 ng of Blastocystis DNA (data not shown)
could be detected. The specificity of these primers was
tested against samples of DNA known to be positive for C.
parvum, G. duodenalis, E. bieneusi, E. hellem, E. cuniculi,
and E. intestinalis, and the PCR assay did not cross-react
with any of them (data not shown).
Discussion
Blastocystis is a common intestinal parasite of humans and
animals. Diagnosis is difficult, relying on light microscopy
of fecal smears, which has low sensitivity; or on culture
methods that are more sensitive, but time-consuming and
not available in most diagnostic laboratories. In contrast,
PCR has been found to be a rapid and highly sensitive tool
for the identification of many parasites, essential for
detecting genetic variation between organisms that are
morphologically indistinguishable, such as genotypes of
Giardia and species of Cryptosporidium. Several methods,
including arbitrarily primed PCR and subtype-specific
sequence-tagged site (STS) primers, have been developed
and used in studies to detect genetic variations among
Blastocystis specimens (Arisue et al. 2003; Yoshikawa et al.
2003). In the present study, a pair of PCR primers was
successfully developed for the identification and subtyping
using direct sequencing of Blastocystis. The specificity of
these primers was confirmed by the successful amplifica-
tion of DNA from all Blastocystis specimens included in
the study and their inability to amplify DNA from other
protist parasites. The assay developed in this study was
compared with previous published protocols that used PCR
for subtyping of Blastocystis. Our PCR protocol has the
advantage of greater sensitivity than the PCR method used
by Stensvold et al. (2006, 2009a, b, c). PCRs described by
Stensvold et al. (2006) and (2009a) were able to detect
0.01 ng and 0.001 ng of Blastocystis DNA, respectively.
PCR by Parkar et al. (2007) has the same sensitivity as our
PCR, but our PCR is a one-step PCR, whereas the PCR
used by Parkar et al. is a nested PCR. The possibility of
contamination increases when using a nested PCR. The
PCR method used by Menounos et al. (2008) is more
sensitive than our PCR; they were able to detect 0.00001 ng
of Blastocystis DNA. However, the disadvantage of using
210 Parasitol Res (2011) 109:205–212

Fig. 1 Phylogenetic tree of 51 H-5.4 Colombia (ST-2)

H-12 Colombia (ST-2)
the SSU-rDNA gene sequences H-11.1 Colombia (ST-2)
of Blastocystis specimens was H-5.7 Colombia (ST-2)
64 H-7.1 Colombia (ST-2)
inferred using the neighbor- 70 PR-4.2 Spain (ST-2)
joining method. Reference ST-2 Human Japan (AB070987)

sequences from GenBank 93 PR-4.3 Spain (ST-2)

ST-2 Primate Japan (AB070997)
have the accession number PR-11.1 Spain (ST-2)
100
in parenthesis. Bootstrap ST-2 Primate Japan (AB107969)
PR-11.2 Spain (ST-2)
proportions (%) are attached H-11.2 Colombia (ST-2)
to the internal branches (1,000 99 H-5.1 Colombia (ST-2)
H-5.6 Colombia (ST-2)
replicates). Names of the 92
H-7.2 Colombia (ST-2)
specimens, host, and locations, 56
H-7.4 Colombia (ST-2)
H-5.2 Colombia (ST-2)
as well as the GenBank acces- 100
H-5.5 Colombia (ST-2)
sion numbers are shown in H-7.3 Colombia (ST-2)
ST-1 Human Japan (AB107962)
parenthesis. Bootstrap values 68 H-10 Colombia (ST-1)
of less than 50% are 95
ST-1 Human Japan (AB070989)

not shown H-14 Colombia (ST-1)

ST-1 Human Japan (U51151)
97 PR-1.1 Spain (ST-1)
77 PR-1.2 Spain (ST-1)
100
PR-4.1 Spain (ST-1)
74 ST-1 Chicken Japan (AB070993)
66 H-42 Colombia (ST-1)
83 PR-1.4 Spain (ST-1)
PR-1.5 Spain (ST-1)
95 H-33 Colombia (ST-1)
H-5.3 Colombia (ST-1)
59 PR-1.3 Spain (ST-1)
73 ST-5 Pig Japan (AB107964)
69 P-18C Spain (ST-5)
67
ST-5 Pig Japan (AB070998)
ST-5 Pig Japan (AB070999)
69 P-5 USA (ST-5)
100
P-6 USA (ST-5)

96
P-12 USA (ST-5)
P-13 USA (ST-5)
P-8 USA (ST-5)
65
P-20 USA (ST-5)
PR-4.5 Spain (ST-3)
PR-4.7 Spain (ST-3)
63
PR-4.8 Spain (ST-3)
PR-4.6 Spain (ST-3)
100
PR-14 Spain (ST 3)
99 53
PR-4.9 Spain (ST-3)
ST-3 Human Japan (AB070992)
PR-4.10 Spain (ST-3)
99
PR-9 Spain (ST-3)
92
PR-4.4 Spain (ST-3)
PR-13 Spain (ST-3)
H-29.4 Colombia (ST-3)
70
H-29.5 Colombia (ST-3)

90
H-41 Colombia (ST-3)
H-29.3 Colombia (ST-3)
67 ST-3 Pig Japan (AB107963)
H-29.1 Colombia (ST-3)
ST-3 Human Japan (AB091234)
H-57 Colombia (ST-3)
H-29.2 Colombia (ST-3)
60 H-30 Colombia (ST-3)
100 ST-9 Human Japan (AF408425)
ST-9 Human Japan (AF408426)

100
ST-6 Bird Japan (AB107972)
ST-6 Human Japan (AB070990)
100
Ch-1.2 Spain (ST-6)
89
79
Ch-1.1 Spain (ST-6)
89 ST-6 Turkey Japan (AY135411)
C-3067 USA (ST-10)
C-3069 USA (ST-10)
C-3071 USA (ST-10)
100
C-3073 USA (ST-10)
C-3066 USA (ST-10)
C-3070 USA (ST-10)
C-3072 USA (ST-10)
91
100 ST-8 Primate Japan (AB107970)
ST-8 Bird Japan (AB107971)
PR-5 Spain (ST-4)
98
64 ST-4 Guinea pig USA (U26177)
99 ST-4 Rat Japan (AB071000)
52 H-1 Spain (ST-4)

52
ST-4 Human Germany (AY244620)
50 ATTC 50608D USA (ST-4)
ST-7 Human France (AY135409)
99 ST-7 Bird Japan (AB107973)
100 Ch-1.4 Spain (ST-7)
99 ST-7 Human France (AY590109)
99
Ch-1.3 Spain (ST-7)
99 ST-7 Human Japan (AF408427)
87 ST-7 Quail Japan (AB070996)

0.02
Parasitol Res (2011) 109:205–212
the PCR method of Menounos et al. is that the amplicon
amplified by their PCR method is very short, only 260 bp.
Although this would not be a problem for diagnostic
purposes, it clearly limits the accuracy required for
subtyping.
Blastocystis has been found not only in humans but also
in a wide range of animals, such as nonhuman primates,
pigs, cattle, rodents, and birds (Abe 2004; Arisue et al.
2003; Navarro et al. 2008; Noel et al. 2005; Stensvold et al.
2009a). The mode of transmission is unclear but may be
associated with animal contact and with the ingestion of
food or water contaminated with cysts from reservoir hosts
(Al-Binali et al. 2006; Leelayoova et al. 2004, 2008; Parkar
et al. 2007; Salim et al. 1999; Suresh et al. 2005; Taamasri
et al. 2000). Blastocystis displays a broad genetic diversity
and probably comprises many species based on the
evolutionary distance observed among different subtypes,
which is comparable to or greater than that observed among
other stramenopile species (Noel et al. 2005). However, at
present, there is not enough information within the genus
Blastocystis except to indicate that ten subtypes exist.
Blastocystis has low host specificity, and previous studies
suggested its zoonotic potential. Some specimens from
animal origin have been regarded as zoonotic, based on
genotypic homology to human specimens (Arisue et al.
2003; Noel et al. 2003, 2005). Primers developed in the
present study were able to detect and subtype using direct
sequencing Blastocystis spp. specimens from naturally
infected cattle, pigs, humans, primates, and chickens,
identifying eight of the ten subtypes currently recognized,
and thereby, establishing its usefulness for the identification
and subtyping of Blastocystis specimens collected from
humans and animals.
The subtype distribution detected in this study reflects
the subtype distribution in different host reported in
previous reports (Stensvold et al. 2009a). All specimens
identified as ST-1, ST-2, ST-3, and ST-4 in the present
study were from human and primate specimens, whereas all
specimens from pigs were ST-5, and all cattle specimens
were ST-10. Although specimen PR-4.5 was identified as
ST-4 in the phylogenetic tree, it could represent a new
clade. The chicken specimen examined in this study was a
mixed infection with subtypes ST-6 and ST-7, both of
which ST are known to contain mostly bird specimens
(Stensvold et al. 2009a). Sequence heterogeneity was found
among different clones in eight specimens (H-5, H-7, H-11,
H-29, PR-1, PR-4, PR-11, and Ch-1). It is possible that the
heterogeneity is due to the presence of multi-copy genes of
SSU rDNA in the Blastocystis genome. However, clear
mixed infections with more than one ST were present in
three specimens: human specimen H-5, primate specimen
PR-4, and chicken specimen Ch-1. Two subtypes were
present in H-5 (ST-1 and ST-2) and in Ch-1 (ST-6 and
211
ST-7), and three subtypes were present in PR-4 (ST-1, ST-2,
and ST-3).
Pathogenicity of Blastocystis is a controversial matter
and additional research is needed to determine potential
differences in pathogenicity of Blastocystis infections
associated with different subtypes. Application of the
method developed and tested in the present study can
elucidate the complexity of this heterogeneous genus and
its role in human or animal diseases, as well as its zoonotic
potential.
Acknowledgment The authors thank Meghan Heffron for her
technical services in support of this study.
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