Parasitol Res (2010) 106:387–394
DOI 10.1007/s00436-009-1673-9
ORIGINAL PAPER
Ultrastructural and molecular characterization
of Balantidium coli isolated in the Philippines
Ma. Lourdes Nilles-Bije & Windell L. Rivera
Received: 28 September 2009 / Accepted: 21 October 2009 / Published online: 10 November 2009
# Springer-Verlag 2009
Abstract Balantidium coli is a ciliated protozoon inhabiting
the colon of swine, rodents, horses, nonhuman primates and
humans. In association with disease triggered by other
infectious agents, B. coli may become a pathogenic oppor-
tunist. This study describes the isolation, cultivation,
morphological as well as molecular characterization of
B. coli isolated from the large intestine of a pig in the
Philippines. Based on scanning and transmission electron
microscopy, this protozoon presents a dense ciliation in the
oral structure and somatic cilia that are arranged in a more
transverse field. Oral and somatic monokinetids were
identified in the cortex of the organism. The presence of
heterokaryotic nuclear condition is evident, and the cell
body of the ciliate shows numerous mucocysts, several food
vacuoles, mitochondria, endoplasmic reticulum, and con-
tractile vacuoles. Polymerase chain reaction and phylo-
genetic analysis based on the small subunit ribosomal RNA
gene were performed in order to compare our isolate with
other previously reported B. coli isolates. The full-length
sequence of the SSU rRNA gene of the isolate showed 99%
M. L. Nilles-Bije : W. L. Rivera (*)
Institute of Biology, College of Science,
University of the Philippines, Diliman,
Quezon City 1101, Philippines
e-mail: wlrivera@science.upd.edu.ph
M. L. Nilles-Bije
Institute of Clinical Laboratory Sciences, Silliman University,
Dumaguete City, Negros Oriental 6200, Philippines
W. L. Rivera
Molecular Protozoology Laboratory,
Natural Sciences Research Institute,
University of the Philippines, Diliman,
Quezon City 1101, Philippines
similarity to other B. coli isolates reported in the GenBank.
Phylogenetic analysis revealed that the isolate clustered with
previously reported B. coli isolates from gorillas, pig, and
ostrich. To date, no studies on the ultrastructure and
phylogeny of B. coli isolated in the Philippines have been
reported. Results from this study may serve as a baseline
data for further ultrastructural and phylogenetic studies on
this organism. This study also suggests that morphological
characteristics along with molecular identification are essen-
tial for validating and identifying species of Balantidium.
Introduction
Balantidium coli is considered as the largest protozoon and
the only parasitic ciliate known to infect humans (Solaymani-
Mohammadi and Petri 2006). The parasite is often seen
in the lumen of the cecum and large intestine of swine,
nonhuman primates, and humans as a commensal organism
(Cho et al. 2006). However, in association with disease
triggered by other infectious agents, B. coli may become a
pathogenic opportunist (Headley et al. 2008). As opportu-
nistic organisms, the trophozoites of B. coli tend to become
invasive and penetrate the linings of the mucosa and sub-
mucosa of the damaged intestine and within the lymphoid
tissue of affected hosts, from which they travel throughout
the rest of the body (Cho et al. 2006; Headley et al. 2008).
In the intestinal lumen of swine and humans, encystment
occurs and the large cysts transmit the infection through
contaminated food or water (Schmidt and Roberts 2004).
Balantidiasis, an infectious disease caused by the organism
B. coli, is a zoonotic disease. The infection is acquired
by humans via fecal-oral route. Humans infected with the
organism may appear asymptomatic, as does the pig, its
normal host, or may develop dysentery similar to that of
388
amebiasis (Schuster and Ramirez-Avila 2008). Although
this disease in humans is rare, it is most prevalent in areas
where pigs are raised and slaughtered (Anargyrou et al.
2003). Infection with B. coli affecting the swine often
presents nonspecific symptoms and is most often asymp-
tomatic. Yet these asymptomatic pigs serving as the major
hosts of the parasite are capable of transmitting the disease.
Such mode of transmission highlights the significance of
public health interventions.
In this study, B. coli isolated from the large intestine of a
pig in the Philippines is described based on scanning
(SEM) and transmission electron microscopy (TEM). The
full-length small subunit ribosomal RNA (SSU rRNA) gene
sequence was also obtained and was used in constructing a
phylogenetic tree that determined its position relative to
previously reported B. coli isolates. To date, no studies on
the ultrastructure and phylogeny of B. coli isolated in the
Philippines have been reported. Results of this study may
serve as a baseline data for further ultrastructural and
phylogenetic studies on this organism.
Materials and methods
Isolation and cultivation of Balantidium coli
Fecal samples from the large intestine of a pig slaughtered in
abattoir in Bais City, Negros Oriental, Philippines, were
collected and washed with Ringer's solution (consisting of
NaCl, KCl, CaCl2, and NaHCO3). The pig's intestinal organs
were opened in a collecting vessel, flooded with Ringer's
solution, and were emptied to remove the fecal contents
(Glaser and Coria 1935). Fresh wet mounts were carried out
to detect trophozoites of B. coli using a light microscope
viewed at ×100 and ×400 magnifications. Sample prepara-
tion was done following the protocol of Klaas (1974) with
some modifications. Briefly, Balantidium-positive fecal con-
tents were emulsified and mixed thoroughly with Ringer's
solution. The samples were strained through several layers of
gauze and were transferred to 15 ml centrifuge tubes for
centrifugation at 15 g for 5 min. Samples were washed with
Ringer's solution three to five times or until the supernatant
fluid was clear. The sediment was then resuspended in a
small amount of Ringer's solution after the final wash.
Approximately 1 ml of the sediment was inoculated in a
liquid medium of Ringer's solution previously warmed to
37°C. Isolation of B. coli by limiting dilution was done in a
96-well microtiter plate containing Ringer's solution. Motile
trophozoites of B. coli were picked out from the liquid
medium, washed with Ringer's solution using a micropipette
and were positively identified on a clean glass slide under a
light microscope. The B. coli isolate was routinely cultivated
in a liquid medium of Ringer's solution supplemented with
Parasitol Res (2010) 106:387–394
10% heat-inactivated horse serum and to which a sprinkle of
heat-sterilized rice starch was added (Glaser and Coria
1935). The isolate was maintained in culture at room
temperature with subcultures done subsequently every 24 h.
Scanning electron microscopy
Motile trophozoites of B. coli were collected by centrifu-
gation at 2,000×g for 5 min. The final pellets were fixed
with 2.5% glutaraldehyde. After several washes, the cells
were secondarily fixed with 1% OsO4, dehydrated in a
series of ethanol solutions, substituted with isoamyl acetate
and then critical-point-dried using CO2. Samples were
coated with gold and observed in Hitachi S-510 scanning
electron microscope (SEM).
Transmission electron microscopy
B. coli cells were subjected to centrifugation at 2,000×g for
5 min and were fixed with 2.5% glutaraldehyde. Ciliates were
washed three times with phosphate-buffered saline (PBS) to
remove excess fixative and were post-fixed in 1% OsO4. The
samples were washed with PBS, dehydrated in a graded
series of acetone solutions, and gradually impregnated in
Epon resin with heat polymerization. Semi-thin survey
sections were cut with glass knives stained with toluidine
blue and were used to orientate sections. Ultra-thin sections
mounted on uncoated copper grids were stained with uranyl
acetate and lead citrate and viewed in a JEOL JEM TEM
1010 electron microscope.
DNA extraction, polymerase chain reaction,
and DNA sequencing
B. coli cells from culture tubes were pooled and subjected to
genomic DNA extraction using the Chelex method (Walsh et
al. 1991). Polymerase chain reaction (PCR) of the SSU
rRNA genes was performed using Go Taq® Hot Start
Polymerase Kit (Promega) and oligonucleotide primers,
Euk A (5′-AACCTGGTTGATCCTGCCAGT-3′), and Euk
B (5′-TGATCCTTCTGCAGGTTCACCTAC-3′) (Medlin et
al. 1988). The PCR assay was carried out in ASTEC PC707
Programmable Thermal Controller consisting of 35 cycles
with the following stringent parameters: 30 s denaturation
at 94°C, 45 s annealing at 50°C, and 130 s extension at
72°C. An initial denaturation step consisting of incubation
at 94°C for 130 s and final extension step consisting of
incubation at 72°C for 7 min were also included. The
resulting products were separated by gel electrophoresis on
a 1.5% agarose gel stained with ethidium bromide. PCR
products were purified using magnetic beads. DNA
sequencing was performed using the same primers in ABI
PRISM Big Dye® Terminator v1.1 Cycle Sequencing Kit
Parasitol Res (2010) 106:387–394
(Applied Biosystems, Foster City, CA) on an automated
sequencer (ABI PRISM 3100 model; Applied Biosystems).
Phylogenetic analysis
Sequences were uploaded into the Basic Local Alignment
Search Tool program to search for the most similar
reference sequences. These were then aligned and removed
of ambiguous nucleotide positions along with reference
sequences. The trees, rooted to a sequence of Dasytricha
ruminantium, were constructed using the GTR+Γ model of
the model-based neighbor-joining (NJ) as well as the non-
model based maximum parsimony (MP). D. ruminantium
was chosen as an outgroup in view of its close relationship
with Balantidium in a previous phylogenetic study (Strüder-
Kypke et al. 2006). The program PAUP* v. 4.0b10
(Swofford 2000) was used for NJ and MP analyses with
1,000 bootstrap replicates. Clusters in the phylogenetic trees
were considered valid if these had bootstrap support values
of greater than 50% from NJ and MP analyses. The trees were
viewed using the version 1.6.6 of TreeView (Page 1996).
GenBank references
The SSU rRNA gene sequence of the isolate generated in
this study was deposited in GenBank as accession no.
GQ903678. The reference sequences used in the construc-
tion of the phylogenetic tree are listed in Table 1.
Results
Isolation and cultivation of B. coli
Several attempts were made to clone B. coli by isolating a
single cell through limiting dilution; however, the results
were unsuccessful. Our findings showed that a sufficient
number of B. coli cells must be present in the initial
inoculation in order to produce successful subcultures.
Positive cultures containing B. coli, survived for 14 days
with subcultures done every 24 h. Cultures of the isolate
observed at 48 h without subcultures resulted to an
Table 1 Genbank reference sequences used in the construction of the phylogenetic tree
Species/isolate Location/source
Balantidium coli Gorilla gorilla
Balantidium coli Sus scrofa (wild pig) from Spain
Balantidium coli Struthio camelus (ostrich) from Spain
Balantidium coli BC34706 Gorilla gorilla from Limbe Wildlife Center, Cameroon
Balantidium entozoon Italy
Dasytricha ruminantium Guelph, Canada
389
increased bacterial density and the protozoa gradually
decreased in number. The cultures grew well at room
temperature. Figure 1 shows the schematic image of the
isolate as seen under a light microscope. General structures
such as cilia, vestibulum, macronucleus, and contractile
vacuoles are clearly seen.
Electron microscopy
Using SEM, the surface arrangement of the oral and
somatic cilia of B. coli was determined. The oral apparatus,
surrounded by radiating field of oral cilia was found at or
near surface of the body, located apically. A depression was
also observed on the oral apparatus or cell mouth of the
organism (Fig. 2a). The somatic cilia which covered most
of the body surface were arranged in a more transverse
field, extending to the oral and the caudal region, located at
the base of the organism. The cell surface was filled with
interkinetal ridges that were found between adjacent cilia
(Fig. 2b and b).
Examinations under TEM revealed the internal structure
of the organism. The cell body was rounded posteriorly and
narrowing anteriorly. Scattered in the cytoplasm were the
nuclei and several food vacuoles of the organism. Contrac-
tile vacuoles located in the anterior, mid-body, and posterior
of the ciliate which function for osmoregulation were also
present in the cytoplasm (Fig. 3a).
The anterior portion of the cell body harbored the oral
apparatus of the trophozoite. Ultra-thin section of the
isolate showed the apparent oral apparatus surrounded by
oral cilia (Fig. 3b). In contrast, situated in the posterior part,
was the caudal region of the isolate. The region was lined
with longitudinal rows of cilia. Found in this region was the
cytopyge, which functions as the anus of the ciliate. Food
vacuoles with ingested bacteria and food particles were also
found near the cytopyge of the trophozoite (Fig. 3b).
Transverse section of the kineties in the buccal cavity of
the trophozoite showed that several fibrils which appeared
as cross striations in the electron micrograph were found
along with the kinetosomes. The kinetosomes or basal
bodies of the oral cilia were seen as a singular component,
appearing as oral monokinetids (Fig. 4a).
Genbank accession no.
AF029763
AM982722
AM982723
EU680309
EU581716
U57769
390
Fig. 1 Schematic illustration of the Philippine B. coli isolate showing
the general structures as seen under the light microscope: vestibulum
(vb), macronucleus (man), food particles (fp) and contractile vacuoles
(cv). Bar, 15 μm
A higher magnification of the cell periphery exhibited
the cortical plasma or the cortex of B. coli. The plasma-
lemma, belonging to the pellicle of the organism along with
its associated structures comprised the cortex of the
organism (Fig. 4b). Located immediately beneath the cell
membrane or plasmalemma were alveoli. This alveolar
system was seen as flattened vacuoles lined by the outer
alveolar membrane. In addition to these alveoli were
numerous mucocysts. The membrane bound mucocysts of
B. coli apparently were docked just below the plasma
membrane. These mucocysts, the only type of extrusomes
found in the organism were seen in their resting state as
polyhedral crystalline bodies (Fig. 4b).
Fig. 2 Scanning electron micrographs of the Philippine B. coli isolate.
a General view showing the oral apparatus (oa) and the caudal region
(cr). The interkinetal ridges (ir) along with its adjacent cilia (ci) are
Parasitol Res (2010) 106:387–394
The cilia of B. coli originated from the kinetosomes or
basal bodies. The surface of the cilium was enclosed by a
membrane found to be continuous with the plasma membrane
covering the outer surface of the cell. The plasmalemma was
underlaid by another membrane which formed alveolar
spaces surrounding the kinetid (Fig. 4c). Located inside of
the cytoplasm was an axoneme, surrounded by long
peripheral and central fibrils. Longitudinal thin section
showed that the interior of kinetosome which anchored the
axoneme is frequently separated by a septum and that the
fibrils in the center and the axoneme frequently end at a
round darkened structure, the axosome (Fig. 4c). Rows of
cilia arising from single rows of subsurface kinetosomes and
their associated fibrils were also found in the cortex of the
isolated B. coli, thus, the ciliate was shown to have uniform
rows of monokinetid somatic cilia covering the surface of
the organism’s cell body (Fig. 4c and d).
Among the cytoplasmic organelles surrounding the
nuclei of B. coli were mitochondrion, endoplasmic reticu-
lum, contractile vacuoles, and several food vacuoles. The
mitochondrion of the organism was a double-membrane
organelle. Found inside of the mitochondrion, were cristae
that were seen as internal membranous tubules (Fig. 4e).
Along with the ribosomes, is the endoplasmic reticulum
of B. coli which appeared as flat, extending membranous
tubules. Small membrane bound vesicles are also found at
the periphery of the endoplasmic reticulum of the isolate
(Fig. 4f).
Molecular characterization
PCR of the SSU rRNA genes of B. coli isolate generated
DNA fragments of the size ∼1.5 kb as determined by
agarose gel electrophoresis (data not shown). These frag-
shown. Bar, 10 μm. b Magnified somatic cilia (ci) and interkinetal
ridges (ir). Bar, 1 μm
Parasitol Res (2010) 106:387–394
Fig. 3 Transmission electron
micrographs of the Philippine
B. coli isolate. a Longitudinal
thin section showing the general
aspect. The conspicuous
macronucleus (man) and smaller
micronucleus (min) are seen.
Four contractile vacuoles (cv)
and several food vacuoles (fv)
are also observed. Bar, 6.25 μm.
b Ultra-thin section showing the
oral apparatus (oa) and caudal
region (cr). The large macro-
nucleus (man) is seen near the
middle portion of the cell body.
Micronucleus is no longer
visible. Bar, 5.71 μm
ments were excised and purified for DNA sequencing. The
length of the SSU rDNA sequence obtained from the B. coli
isolate was 1,543 bp.
The Philippine B. coli isolate was found to be 99% similar
to the four B. coli sequences reported in the GenBank, with
98% coverage. These reference isolates were collected from
a lowland gorilla (accession no. AF029763), a captive gorilla
(accession no. EU680309), a wild pig (accession no.
AM982722), and an ostrich (accession no. AM982723).
The phylogenetic position of the Philippine B. coli isolate
relative to reference B. coli isolates and the outgroup,
D. ruminantium, was determined based on the constructed
phylogenetic tree. It is apparent that the Philippine B. coli
isolate clustered with reference B. coli isolates and separate
from D. ruminantium and another related species of
Balantidium, B. entozoon (Fig. 5).
Discussion
B. coli is regarded as a common commensal parasite of the
large intestine of pigs and has been reported in camels,
horses, rodents, nonhuman primates, and humans (Headley
et al. 2008). Human cases of B. coli infection have been
found in individuals who had contact with pigs (Esteban et
al. 1998), pig excreta (Sharma and Harding 2003), and food
or water contaminated with the parasite (Esteban et al.
1998). B. coli has been reported in immunocompromised
patients causing pulmonary infection (Anargyrou et al.
2003) and dysentery (Yazar et al. 2004).
In this study, B. coli was isolated from the large intestine
of a pig. Efforts made to produce cloned cells of B. coli
were unsuccessful. Our findings are in agreement with the
report of Levine (1939) who also failed to obtain pure lines
391
after several attempts of isolating a single cell of B. coli. In
our study, it was noted that a considerable number of B. coli
cells must be present to initiate successful subcultures.
Thus, results of our study agreed with those of Levine
(1939) that the protozoa would survive in cell cultures only
if sufficient number of B. coli cells were present at the start,
to ensure favorable results in subculturing the organism.
This perhaps is due to the fact that the fission rate of
the protozoa is slow compared to the bacteria present in the
cultures, and that these bacteria were able to utilize the
constituents of the growth medium or release inhibiting
substances detrimental to the protozoa long enough before
the propagation of these trophic ciliates has progressed.
Cultivations of B. coli were often made in xenic culti-
vation media (Clark and Diamond 2002). B. coli, whether
derived from swine or human hosts, is difficult to maintain
in cell cultures for any length of time and that this was
probably due to certain bacteria and their metabolic products
that are harmful to the growth of the ciliate and that short
interval transplantation is necessary to keep the organisms
under cultivation (Glaser and Coria 1935). Certain variables
are involved in the cultivation of B. coli in vitro. Among
these are the growth medium, pH (optimal range, 5.4–8) of
the culture medium, associated undefined bacterial flora, and
strain differences of the parasites (Schuster and Ramirez-
Avila 2008).
The Philippine B. coli isolate presented morphological
features coincident with previous B. coli descriptions
(Anargyrou et al. 2003; Sharma and Harding 2003; Yazar
et al. 2004; Solaymani-Mohammadi and Petri 2006; Schuster
and Ramirez-Avila 2008). The isolate also presented ultra-
structural features similar to those described by Skotarczak
and Kołodziejczyk (2005). Mucocysts described as single-
membrane, polyhedral crystalline structures deposited
392 Parasitol Res (2010) 106:387–394
 Parasitol Res (2010) 106:387–394 393

RFig. 4 Transmission electron micrographs of the Philippine B. coli
isolate showing the detailed internal structures. a Horizontal section
through kineties of B. coli showing the kinetosomes (ks) found in the
oral apparatus of the ciliate. Bar, 0.57 μm. b Thin section showing the
cortex. The surrounding plasma membrane (pm) and outer alveolar
membrane (oalm) lining the alveoli system (al alveolus) can be seen.
Membrane-bound mucocysts (mu) are also observed below the plasma
membrane. Cilium in transverse section with its 9+2 representation is
also shown. Peripheral (pmt) and central (cmt) microtubules of the
ciliary base are also observed. Bar, 0.19 μm. c Longitudinal thin
section of the kinetids. The alveoli (al) system lined by the plasma
membrane (pm) is shown. Peripheral (pf) and central fibrils (cf)
enclosing the axoneme (a) anchored by the kinetosomes (ks) are
observed. Septum (s) and axosome (ax) found at the junction of
the kinetosomes and axonemes are also present. Bar, 0.23 μm.
d Horizontal thin section showing the somatic monokinetids. Bar,
0.23 µm. e Ultra-thin section showing the mitochondrion with several
free ribosomes (ri) scattered in the cytoplasm. Bar, 0.23 µm. f Thin
section showing the endoplasmic reticulum (er). Bar, 0.31 μm
below the plasma membrane of the trophic ciliate were iden-
tified. Balantidium along with other ciliates, Tetrahymena,
Colpidium, Ophryoglena, Holophyra, and Didinium have
sac-like extensions called mucocysts, below their pellicle
whose mucoid content is expelled upon stimulation (Grell
1973). Endoplasmic reticulum appearing as flat, extending
membranous tubules was also described in this study along
with several scattered free ribosomes in the cytoplasm of the
organism. Golgi bodies were not seen in the cell body of the
isolate. This supports the findings of Skotarczak and
Kołodziejczyk (2005) that Golgi bodies were not found in
B. coli and that some membranes of the endoplasmic
reticulum and small vesicles assume a similar role to the
Golgi complex. The mitochondrion of the protozoon was
reported in this study as a spheroidal double-membrane
organelle with internal membranous tubules. The tropho-
zoites of the isolate are often found at the bottom of the
culture tubes. Thus, the presence of the mitochondria in the
protozoon makes the organism a facultative anaerobe.
Several food vacuoles with ingested bacteria and food
particles were also described in this study. Contractile
vacuoles were also found in the cell body of the isolate.
These contractile vacuoles act as ion regulating organelles by
expelling excess ions that have accumulated from the
environment (Anderson 1988). The cell body of the isolate
was entirely covered by uniform rows of somatic mono-
kinetid cilia where ciliary rows arise from single rows of
subsurface kinetosomes and their associated fibrils (Schuster
and Ramirez-Avila 2008). The somatic cilia are arranged in a
more transverse field extending to the oral and caudal region
where the cytopyge of the organism is located. The oral
apparatus of the protozoon was densely ciliated and
composed of simple oral monokinetid cilia. Hence, the
results of our ultrastructural analysis clearly placed the
isolate under the genus Balantidium, based on the new
taxonomy recently revised by the committee of International

Fig. 5 Consensus phylogenetic Balantidium coli from an ostrich (Struthio camelus) AM982723
tree of Balantidium coli isolated
from a pig in the Philippines
and from Balantidium
sp. sequences from Genbank
based on 1,250 unambiguously Balantidium coli isolate BC34706 from a captive gorilla (Gorilla gorilla) EU680309
aligned base pairs of the SSU
rDNA region. This tree is
constructed using the GTR+Γ
model of the model-based
neighbor-joining (NJ) method 64/-- Balantidium coli from a wild pig (Sus scrofa) AM982722
and rooted to a sequence of
Dasytricha ruminantium.
Figures on the branches
represent percentage bootstrap 94/100
Balantidium coli, from a lowland gorilla (Gorilla gorilla) AF029763
support from NJ and the
nonmodel-based maximum
parsimony (MP) methods,
respectively (bootstrap support
percentages below 50% are not Balantidium coli isolate from a pig in the Philippines GQ903678
shown). The scale bar on the
lower left side represents one
substitution change per
100 nucleotide positions
Balantidium entozoon EU581716

Dasytricha ruminantium U57769

0.01
394
Society of Protistologists and allied groups (Schuster and
Ramirez-Avila 2008).
Based on the sequence analysis of the SSU rRNA gene,
the isolate was identified as B. coli. Sequence alignment
showed that the sequence of the Philippine isolate corre-
spond to published B. coli sequences. The sequence of the
isolate showed 99% similarity to homologous sequences of
other B. coli reported previously. Based on the phylogenetic
tree generated by neighbor-joining and maximum parsi-
mony analyses, the Philippine B. coli isolate and the B. coli
reference isolates clustered together regardless of host
species, with good bootstrap support values.
A previous study suggested that morphological charac-
teristics and host species as bases for the identification of
Balantidium species are insufficient (Ponce-Gordo et al.
2008). The results of their morphological and genetic
analyses suggest that, B. struthionis, reportedly found in
ostriches, is a synonym of B. coli. This is consistent with
the findings of this study that B. coli isolated from the large
intestine of pig shared 99% homology to previous B. coli
isolates suggesting the fact that this ciliate might have low
host specificity.
On the bases of the ultrastructural characterization and the
sequence of the SSU rRNA gene, the Balantidium that was
isolated from the large intestine of a pig is then defined as
B. coli. This study confirms that host-specificity and
morphological features are not enough criteria in describing
and identifying the species of Balantidium. We highly
recommend the development of new a culture medium that
would encourage propagation and longer survival of B. coli
in culture. We also recommend that the criteria to validate and
identify species of Balantidium must be based on morpho-
logical characteristics, genetic identification, and sequence
comparison of genes having taxonomic importance.
Acknowledgements This work was supported financially by a grant
from the Faculty Development Committee of Silliman University. We
thank Neilfred G. Bije, Davin Edric V. Adao, and John Anthony D.L.
Yason for their technical assistance. The experiments conducted in this
study comply with the current laws of the Philippines.
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