Veterinary Parasitology 120 (2004) 235–242

Short communication
Molecular and phylogenetic analysis of Blastocystis
isolates from various hosts
Niichiro Abe∗
Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences,
Tennoji-ku, Osaka 543-0026, Japan

Accepted 23 January 2004

Abstract
Blastocystis hominis is a protozoan parasite found in humans. B. hominis-like organisms have
been found in a variety of animals, but have been called Blastocystis sp. because the isolates from
animals were indistinguishable from B. hominis morphologically. Recent molecular studies show
that some isolates from animals have genetic similarity with B. hominis. However, it has been
unclear whether the isolates from animals have zoonotic potential or not. In the present study, the
SSUrDNA of 19 Blastocystis isolates from these animals was sequenced in its entirety, and the
phylogenetic relationship among isolates from humans and animals was clarified using available
nucleotide sequences of the same locus. Phylogenetic analysis showed that the 19 isolates analyzed
in the present study could be classified into seven groups (I–VII): Group I consisted of the isolates
from humans, primates, cattle, pigs and birds; Group II of the isolates from humans and primates;
Group III of the isolates from humans, cattle and pigs; Group IV of the isolates from primates,
birds and rodents; Group V of the isolates from cattle and pigs; Groups VI and VII of the isolates
from humans and birds. These results indicate that many of the isolates harboring in animals have
zoonotic potential, or have cross-transmissibility among heterogeneous hosts.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Blastocystis; Genetic diversity; Molecular phylogeny; Small subunit ribosomal RNA; Zoonosis

1. Introduction

Blastocystis hominis is a common protozoan parasite in the human intestinal tract. Recent
molecular studies indicate that B. hominis is not a single species, but composed of genetically
distinct but morphologically identical genotypes (Clark, 1997; Yoshikawa et al., 1998,
2000). On the other hand, B. hominis-like organisms have been found in a variety of animals
∗ Tel.: +81-6-6771-8331; fax: +81-6-6772-0676.

E-mail address: n.abe@iphes.city.osaka.jp (N. Abe).

0304-4017/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2004.01.003
236 N. Abe / Veterinary Parasitology 120 (2004) 235–242

(Yamada et al., 1987; König and Müller, 1997; Abe et al., 2002), but have been called
Blastocystis sp. because the isolates from animals were indistinguishable from B. hominis
by light and electron microscopy. However, recently, nine isolates, one from a pig, three from
rodents, four from birds and one from a monkey were suggested to have zoonotic potential
based on a comparison of small subunit ribosomal RNA gene (SSUrDNA) sequences (Arisue
et al., 2003; Nöel et al., 2003).
Epidemiological studies on Blastocystis infection in animals have shown a relatively high
incidence in cattle, pigs, primates and birds (Yamada et al., 1987; König and Müller, 1997;
Abe et al., 2002); however, phylogenetic relationships between B. hominis genotypes and
the isolates from these animals have been unclear because only a few such isolates have
been analyzed phylogenetically. In addition, there have been no reports concerning the
phylogenetic analysis of isolates from cattle. Recently, isolates from cattle, pigs, primates
and birds were characterized genetically using diagnostic PCR with subtype-specific primer
sets and restriction fragment length polymorphism (RFLP) analysis of SSUrDNA, and some
were shown to have genetic similarity with the multiple genotypes of B. hominis (Abe et al.,
2003a,b,c). However, it has been unclear whether these isolates are zoonotic or not, because
diagnostic PCR and RFLP analysis of SSUrDNA only provided preliminary information
on genetic relatedness between human and animals Blastocystis isolates. Therefore, in the
present study the SSUrDNA of Blastocystis isolates from cattle, pigs, primates and birds
was sequenced completely, and the phylogenetic relationship with B. hominis genotypes
was clarified to show the zoonotic potential of the isolates.

2. Materials and methods

As shown in Table 1, the SSUrDNA of 19 isolates from cattle, pigs, primates and birds
was sequenced in its entirety. The subtypes or ribodemes of 12 isolates (CJ99-344, 353, 363,
PJ99-154, 172, 162, MJ99-424, 123, 568, 116, 130, BJ99-310) had been identified previ-
ously by diagnostic PCR with subtype-specific primer sets and RFLP analysis of SSUrDNA
(Abe et al., 2003a,b,c), however, neither the subtypes nor the ribodemes of the other six
isolates (CJ99-284, 350, PJ99-148, 188, MJ99-132, BJ99-319) except BJ99-569 were deter-
mined because these isolates were not amplified with subtype-specific primer sets, and the
RFLP profiles did not correspond to any from ribodemes reported previously (Clark, 1997).
The extraction of genomic DNA from each isolate and the PCR amplification of the
complete SSUrDNA were performed following the methods reported previously (Abe et al.,
2003a,b,c). Each PCR product was purified with a Qiagen Gel Extraction Kit (Qiagen
GmbH, Hilden, Germany) following the manufacturer’s directions.
DNA sequencing was performed using an ABI PRISM BigDye Terminator Cycle Se-
quencing FS Ready Reaction kit (PE Applied Biosystems, Foster City, CA) on an automated
sequencer (ABI PRISM 310 model; PE Applied Biosystems, Foster City, CA). The complete
SSUrDNA was sequenced using the following six primers: SR1F (5 -GCT TAT CTG GTT
GAT CCT GCC AGT AGT-3 ), F70 (5 -TGT TCA AAG CAG RCG TTT GY-3 ), ABBH1
(5 -GCT GGA GGC AAT AAC AGG TCT G-3 ), B6 (5 -TAG CCA TCT CTC AGG CTC
CCT CTC-3 ), B71 (5 -GGT GCC CTT CCG TCA ATT CC-3 ), and SR1R (5 -TGA TCC
TTC CGC AGG TTC ACC TA-3 ). SR1F and SR1R primers were developed previously
 N. Abe / Veterinary Parasitology 120 (2004) 235–242 237

Table 1
Blastocystis isolates examined in the present study
Isolates Original animal species Subtypes Ribodemes References Accession
of each isolate numbersa
Cattle
CJ99-344 Bos taurus 1 1 Abe et al. (2003c) –b
CJ99-353 B. taurus 3 2 Abe et al. (2003c) –b
CJ99-363 B. taurus 3 2 Abe et al. (2003c) AB107965
CJ99-284 B. taurus ND Urc Abe et al. (2003c) AB107966
CJ99-350 B. taurus ND Urc Abe et al. (2003c) –b
Pigs
PJ99-154 Sus domesticus 1 1 Abe et al. (2003c) AB107961
PJ99-172 S. domesticus 1 1 Abe et al. (2003c) AB107962
PJ99-162 S. domesticus 3 2 Abe et al. (2003c) AB107963
PJ99-148 S. domesticus ND Urc Abe et al. (2003c) –b
PJ99-188 S. domesticus ND Urc Abe et al. (2003c) AB107964
Primates
MJ99-424 Pongo pygmaeus 1 1 Abe et al. (2003b) AB107967
MJ99-123 Mandrillus leucophaeus Variantd Ure Abe et al. (2003b) –b
MJ99-568 Cercopithecus aethiops Variantd Ure Abe et al. (2003b) AB107968
MJ99-116 Macaca nemestrina ND 6 Abe et al. (2003b) AB107969
MJ99-130 M. silenus ND 6 Abe et al. (2003b) –b
MJ99-132 Varecia variegata ND Urc Abe et al. (2003b) AB107970
Birds
BJ99-310 Bambusicola thoracica 4 UR Abe et al. (2003a) AB107972
BJ99-319 Argusianus argus ND Urc Abe et al. (2003a) AB107971
BJ99-569 Anser cygnoides 2 UR Present study AB107973
ND: not determined; UR: unknown rebodemes.
a Accession numbers are assigned to the complete SSUrDNA sequences obtained in the present study.
b The sequences of SSUrDNA from the six isolates, CJ99-344, CJ99-353, CJ99-350 and PJ99-148, MJ99-123,

and MJ99-130, are identical to those from the isolates, PJ99-154, PJ99-162, PJ99-188, MJ99-568, and MJ99-116,
respectively.
c Showed the same RFLP profiles among six isolates.
d Both isolates were found to be different variant of subtype 1 each other.
e Showed the same RFLP profiles between the isolates MJ99-123 and MJ99-568.

(Yoshikawa et al., 2000), but the other four primers were newly designed to prime conserved
regions of SSUrDNA sequences obtained from two B. hominis strains, Nand II (GenBank
No. U51151) and NIH-1295-1 (GenBank No. U51152), using MacVector ver 7.0 (Oxford
Molecular Ltd.).
Sequences obtained from the 19 Blastocystis isolates in the present study were aligned
with the 25 nucleotide sequences available for B. hominis or Blastocystis sp. (Table 2)
using Clustal-X (version 1.63b; December 1997). These 25 isolates have been sequenced
previously along the entire (Arisue et al., 2003) or partial (Nöel et al., 2003) length of
the SSUrDNA. Since the nucleotide sequences of the SSUrDNA reported by Nöel et al.
(2003) were incomplete, 1663 alignable sites among the 44 isolates were selected and
used for the phylogenetic analysis. Evolutionary distance between different isolates was
calculated using the Kimura 2-parameter method, and phylogenetic trees were constructed
238 N. Abe / Veterinary Parasitology 120 (2004) 235–242

Table 2
Details of the previously characterized isolates used in the present study
Isolates Species Hosts Countries Accession numbers

HJ96A-29 B. hominis Human Japan AB070989

HJ96A-26 B. hominis Human Japan AB070988
HJ97-2 B. hominis Human Japan AB070991
HV93-13 B. hominis Human Japan AB070986
HJ96-1 B. hominis Human Japan AB070987
HJ96AS-1 B. hominis Human Japan AB070990
HJ00-4 B. hominis Human Japan AF408425
Nand II B. hominis Human USA U51151
HT98-1 B. hominis Human Thailand AB070992
B B. hominis Human Singapore AF408427
–a B. hominis Human France AY135402b
JM92-2 Blastocystis sp. Japanese monkey Japan AB070997
SY94-3 Blastocystis sp. Pig Japan AB070998
SY94-7 Blastocystis sp. Pig Japan AB070999
–a Blastocystis sp. Pig Czech Republic AY135403b
CK86-1 B. hominis Chicken Japan AB070993
CK92-4 Blastocystis sp. Chicken Japan AB070994
–a Blastocystis sp. Chicken France AY135410b
QQ93-3 Blastocystis sp. Japanese quail Japan AB070995
QQ98-4 Blastocystis sp. Japanese quail Japan AB070996
–a Blastocystis sp. Duck France AY135412b
–a Blastocystis sp. Turkey France AY135411b
NIH-1295-1 B. hominis Guinea-pig USA U51152
RN94-9 Blastocystis sp. Brown Norway rat Japan AB071000
–a Blastocystis sp. Rat France AY135408b
a Isolates were not named.
b Partial sequences (1693–1763 bp) of SSUrDNA.

using the neighbor-joining algorithm (Saitou and Nei, 1987). Branch reliability was assessed
using bootstrap analyses (1000 replicates). The SSUrDNA sequences of the Blastocystis
isolates, obtained in the present study, have been deposited in GenBank under accession
Nos. AB107961–AB107973. Since the six isolates CJ99-344, CJ99-353, CJ99-350 and
PJ99-148, MJ99-123, and MJ99-130 were identical in SSUrDNA sequence to PJ99-154,
PJ99-162, PJ99-188, MJ99-568, and MJ99-116, respectively, these isolates from cattle,
pigs, and primates were not deposited in GenBank (Table 1).

3. Results

The complete SSUrDNA was successfully amplified in each Blastocystis isolate exam-
ined in the present study (data not shown), and the full-length sequence ranged from 1741
(BJ99-310) to 1796 bp (BJ99-569). The SSUrDNA sequences obtained in the present study
(19 isolates) were aligned with those from 25 isolates from humans and animals (Table 2).
The percentages of sequence similarity among the 44 isolates ranged from 84.7 to 100%.
The isolates which showed high similarity to each other were classified into seven groups
 N. Abe / Veterinary Parasitology 120 (2004) 235–242 239

(I–VII). Group I consisted of 10 isolates, from humans (Nand II, HJ96A-29), pigs (PJ99-172,
154, and an isolate reported by Nöel et al., 2003), cattle (CJ99-344), a chicken (CK86-1) and
primates (MJ99-424, 123 and 568), which ranged in similarity from 98.2 to 100%. Group
II consisted of four isolates, one from a human (HJ96-1) and three from primates (JM92-2,
MJ99-116 and 130), with 98.7 to 100% similarity. Group III consisted of seven isolates, from
humans (HT98-1, HJ96A-26, HV93-13 and an isolate reported by Nöel et al., 2003), cattle
(CJ99-353, 363) and a pig (PJ99-162), which ranged in similarity from 97.7 to 100%. Group
IV consisted of five isolates, from rodents (RN94-9, NIH-1295-1 and an isolate reported
by Nöel et al., 2003), a primate (MJ99-132) and a pheasant (BJ99-319), with similarity
ranging from 94.8 to 99.9%. Group V consisted of six isolates, from cattle (CJ99-284, 350)
and pigs (SY94-3, 7, PJ99-148 and 188), and the similarity was 99.5–100%. Group VI con-
sisted of six isolates, from humans (HJ96AS-1, HJ00-4), a chicken (CK92-4), a partridge
(BJ99-310), a quail (QQ93-3), and a turkey (Nöel et al., 2003) which ranged in similarity
from 96.2 to 97.2%. Group VII consisted of six isolates, from humans (B, HJ97-2), a quail
(QQ98-4), a goose (BJ99-569), a chicken (Nöel et al., 2003), and a duck (Nöel et al., 2003)
with 92.0–93.5% similarity. The relatedness of the isolates grouped by sequence similarity
is also reflected in the phylogenetic analysis. Namely, the isolates included in the same
group clustered with each other group with good bootstrap support, and therefore seven
independent monophyletic groups were constructed (Fig. 1). These monophyletic groups
corresponded exactly to Groups I–VII.

4. Discussion

In an epidemiological study of B. hominis among humans, animal handlers had signifi-

cantly higher rates of infection than individuals who did not work with animals (Salim et al.,
1999). Therefore, it had been suspected that some Blastocystis isolates from animals have
zoonotic potential. Recent study by Arisue et al. (2003) indicates that Blastocystis isolates
from humans and animals can be classified into seven groups (I–VII) on the basis of the
SSUrDNA sequence as shown in Fig. 1. Group I consist of isolates from humans and a bird,
Group II those from humans and a primate, Groups III–V those from only humans, rodents
and pigs, respectively, and Groups VI and VII those from both humans and birds (Arisue
et al., 2003). The isolate (NIH-1295-1) from a guinea-pig classified into Group IV, was
suggested to be zoonotic by RFLP analysis of SSUrDNA (Clark, 1997). Therefore, the five
groups (I, II, IV, VI and VII) are thought to be zoonotic (Arisue et al., 2003). In the present
study, it was newly suggested that some isolates from cattle, pigs, primates, as well as birds
were zoonotic in Group I. In addition, some isolates from cattle and pigs were included in
Group III which had consisted of only human isolates (B. hominis). Therefore, this group
also appears to be zoonotic. Isolates of Groups IV and V, originally only from rodents and
pigs, respectively, were found to have a wide range of host including a bird and a primate,
and cattle, respectively. In this study, phylogenetic tree was constructed adding six isolates
from European countries (Nöel et al., 2003), those added clustered with the isolates in all
groups (Fig. 1, indicated in boldfaced). Therefore, the isolates from humans or animals in
many countries appear to be classifiable into any of the groups shown in Fig. 1, but the
molecular analysis of human and animal isolates from many countries is necessary.
240 N. Abe / Veterinary Parasitology 120 (2004) 235–242

Fig. 1. Phylogenetic relationship of the Blastocystis isolates from humans and animals as inferred by
neighbor-joining analysis of complete SSUrDNA sequences. Bootstrap proportions (%) are indicated at each
branch except for the very shorts ones within each group. The partially sequenced six isolates collected in France
or the Czech Republic are indicated in boldfaced. Names of the isolates and accession numbers in GenBank are
shown in parentheses.

B. hominis has been genotyped into subtypes 1–4 based on diagnostic PCR with subtype-
specific primer sets (Yoshikawa et al., 1998, 2000), or into ribodemes 1–7 based on RFLP
analysis of SSUrDNA (Clark, 1997). Yoshikawa et al. (2000) showed that subtypes 1 and
3 corresponded to ribodemes 1 and 2, respectively. The 19 isolates analyzed in the present
study have already been genotyped by both methods mentioned above (Table 1) (Abe et al.,
2003a,b,c). In the present phylogenetic analysis, the six isolates (PJ99-154, 172, CJ99-344,
 N. Abe / Veterinary Parasitology 120 (2004) 235–242 241

MJ99-123, 424, 568) found to be of subtype 1 (including the variant of subtype 1) or

ribodeme 1 were included in Group I (Fig. 1). Interestingly, CK86-1 and Nand II included in
the same group are also of subtype 1 or ribodeme 1 (Yoshikawa et al., 2000), and HJ96A-29
in the same group was found to be a variant of subtype 1. Therefore, Group I appears
to be the genotype that includes the isolates corresponding to subtype 1 (ribodeme 1) or
variants of subtype 1. Similarly, the isolates that correspond to ribodeme 6 (MJ99-116, 130),
subtype 3 (ribodeme 2) (CJ99-353, 363, PJ99-162), subtype 4 (BJ99-310), and subtype 2
(BJ99-569) (Table 1), were clustered with the isolates found to be of ribodeme 6 (JM92-2),
subtype 3 (ribodeme 2) (HJ96A-26, HV93-13), subtype 4 (HJ96AS-1), and subtype 2 (B),
respectively (Fig. 1) (Yoshikawa et al., 2000; Arisue et al., 2003). Therefore, the four groups
(II, III, VI and VII) also appear to be genotypes that include the isolates corresponding to
ribodeme 6, subtype 3 (ribodeme 2), subtype 4, and subtype 2, respectively. On the other
hand, six isolates (BJ99-319, MJ99-132, CJ99-284, 350, PJ99-148, and 188) showed the
same RFLP profiles (Table 1), but were divided into Groups IV (BJ99-319, MJ99-132) or
V (CJ99-284, 350, PJ99-148, 188) (Fig. 1). This result indicates that phylogenetic analysis
based on nucleotide sequences of SSUrDNA is superior to the RFLP analysis of SSUrDNA
for the classification of genetically similar isolates. Therefore, the present study shows that
the classification between “subtypes” and “groups” is correlated (subtype 1 or variant of
subtype 1 corresponds to Group I, subtype 2 to Group VII, subtype 3 to Group III, and
subtype 4 to Group VI, respectively), and diagnostic PCR using subtype-specific primer
sets is a useful tool for screening the groups of isolates. However, diagnostic primers specific
to the subtypes corresponding to Groups II, IV, and V have yet to be developed.
Although the morphological features of the isolates from various animals were similar
to those of B. hominis (Abe et al., 2002), and extensive karyotypic polymorphism has been
recognized among the isolates of B. hominis (Carbajal et al., 1997), several new species
of Blastocystis (B. anseri, B. galli and B. anatis from domestic birds, B. ratti from rats, B.
lemuri from primates) except for B. hominis in humans had been proposed based on host
origin, morphological characteristics or karyotypic patterns (Belova and Kostenko, 1990;
Belova, 1991, 1992; Chen et al., 1997; Krylov and Belova, 1997). Although the SSUrDNA
sequences of these isolates proposed as new species are not available currently, it is likely
that these isolates are genetically similar to B. hominis, and can be included into any of
the groups shown in the present or previous study (Arisue et al., 2003). If almost all the
isolates from humans and animals have zoonotic potential, or cross-transmissibility among
heterogeneous hosts is common in Blastocystis organisms, a speciation based on the host’s
name would be confusing. Therefore, the adequate classification of Blastocystis isolates
should be based on phylogenetic analysis using sequence information from many more
Blastocystis isolates from various hosts.

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