ORIGINAL PAPER

Ultrastructural and molecular characterizationof 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 inhabitingthe colon of swine, rodents, horses, nonhuman primates andhumans. In association with disease triggered by otherinfectious agents, B. coli may become a pathogenic oppor-tunist. This study describes the isolation, cultivation,morphological as well as molecular characterization ofB. coli isolated from the large intestine of a pig in thePhilippines. Based on scanning and transmission electronmicroscopy, this protozoon presents a dense ciliation in theoral structure and somatic cilia that are arranged in a moretransverse field. Oral and somatic monokinetids wereidentified in the cortex of the organism. The presence ofheterokaryotic nuclear condition is evident, and the cellbody of the ciliate shows numerous mucocysts, several foodvacuoles, mitochondria, endoplasmic reticulum, and con-tractile vacuoles. Polymerase chain reaction and phylo-genetic analysis based on the small subunit ribosomal RNAgene were performed in order to compare our isolate withother previously reported B. coli isolates. The full-lengthsequence of the SSU rRNA gene of the isolate showed 99%

similarity to other B. coli isolates reported in the GenBank.Phylogenetic analysis revealed that the isolate clustered withpreviously reported B. coli isolates from gorillas, pig, andostrich. To date, no studies on the ultrastructure andphylogeny of B. coli isolated in the Philippines have beenreported. Results from this study may serve as a baselinedata for further ultrastructural and phylogenetic studies onthis organism. This study also suggests that morphologicalcharacteristics along with molecular identification are essen-tial for validating and identifying species of Balantidium.

Introduction

Balantidium coli is considered as the largest protozoon andthe only parasitic ciliate known to infect humans (Solaymani-Mohammadi and Petri 2006). The parasite is often seenin 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 diseasetriggered by other infectious agents, B. coli may become apathogenic opportunist (Headley et al. 2008). As opportu-nistic organisms, the trophozoites of B. coli tend to becomeinvasive and penetrate the linings of the mucosa and sub-mucosa of the damaged intestine and within the lymphoidtissue of affected hosts, from which they travel throughoutthe rest of the body (Cho et al. 2006; Headley et al. 2008).In the intestinal lumen of swine and humans, encystmentoccurs and the large cysts transmit the infection throughcontaminated food or water (Schmidt and Roberts 2004).

Balantidiasis, an infectious disease caused by the organismB. coli, is a zoonotic disease. The infection is acquiredby humans via fecal-oral route. Humans infected with theorganism may appear asymptomatic, as does the pig, itsnormal host, or may develop dysentery similar to that of

M. L. Nilles-Bije :W. L. Rivera (*)Institute of Biology, College of Science,University of the Philippines, Diliman,Quezon City 1101, Philippinese-mail: wlrivera@science.upd.edu.ph

M. L. Nilles-BijeInstitute of Clinical Laboratory Sciences, Silliman University,Dumaguete City, Negros Oriental 6200, Philippines

W. L. RiveraMolecular Protozoology Laboratory,Natural Sciences Research Institute,University of the Philippines, Diliman,Quezon City 1101, Philippines

Parasitol Res (2010) 106:387–394DOI 10.1007/s00436-009-1673-9

amebiasis (Schuster and Ramirez-Avila 2008). Althoughthis disease in humans is rare, it is most prevalent in areaswhere pigs are raised and slaughtered (Anargyrou et al.2003). Infection with B. coli affecting the swine oftenpresents nonspecific symptoms and is most often asymp-tomatic. Yet these asymptomatic pigs serving as the majorhosts of the parasite are capable of transmitting the disease.Such mode of transmission highlights the significance ofpublic health interventions.

In this study, B. coli isolated from the large intestine of apig in the Philippines is described based on scanning(SEM) and transmission electron microscopy (TEM). Thefull-length small subunit ribosomal RNA (SSU rRNA) genesequence was also obtained and was used in constructing aphylogenetic tree that determined its position relative topreviously reported B. coli isolates. To date, no studies onthe ultrastructure and phylogeny of B. coli isolated in thePhilippines have been reported. Results of this study mayserve as a baseline data for further ultrastructural andphylogenetic studies on this organism.

Materials and methods

Isolation and cultivation of Balantidium coli

Fecal samples from the large intestine of a pig slaughtered inabattoir in Bais City, Negros Oriental, Philippines, werecollected and washed with Ringer's solution (consisting ofNaCl, KCl, CaCl2, and NaHCO3). The pig's intestinal organswere opened in a collecting vessel, flooded with Ringer'ssolution, and were emptied to remove the fecal contents(Glaser and Coria 1935). Fresh wet mounts were carried outto detect trophozoites of B. coli using a light microscopeviewed at ×100 and ×400 magnifications. Sample prepara-tion was done following the protocol of Klaas (1974) withsome modifications. Briefly, Balantidium-positive fecal con-tents were emulsified and mixed thoroughly with Ringer'ssolution. The samples were strained through several layers ofgauze and were transferred to 15 ml centrifuge tubes forcentrifugation at 15 g for 5 min. Samples were washed withRinger's solution three to five times or until the supernatantfluid was clear. The sediment was then resuspended in asmall amount of Ringer's solution after the final wash.Approximately 1 ml of the sediment was inoculated in aliquid medium of Ringer's solution previously warmed to37°C. Isolation of B. coli by limiting dilution was done in a96-well microtiter plate containing Ringer's solution. Motiletrophozoites of B. coli were picked out from the liquidmedium, washed with Ringer's solution using a micropipetteand were positively identified on a clean glass slide under alight microscope. The B. coli isolate was routinely cultivatedin a liquid medium of Ringer's solution supplemented with

10% heat-inactivated horse serum and to which a sprinkle ofheat-sterilized rice starch was added (Glaser and Coria1935). The isolate was maintained in culture at roomtemperature 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 fixedwith 2.5% glutaraldehyde. After several washes, the cellswere secondarily fixed with 1% OsO4, dehydrated in aseries of ethanol solutions, substituted with isoamyl acetateand then critical-point-dried using CO2. Samples werecoated with gold and observed in Hitachi S-510 scanningelectron microscope (SEM).

Transmission electron microscopy

B. coli cells were subjected to centrifugation at 2,000×g for5 min and were fixed with 2.5% glutaraldehyde. Ciliates werewashed three times with phosphate-buffered saline (PBS) toremove excess fixative and were post-fixed in 1% OsO4. Thesamples were washed with PBS, dehydrated in a gradedseries of acetone solutions, and gradually impregnated inEpon resin with heat polymerization. Semi-thin surveysections were cut with glass knives stained with toluidineblue and were used to orientate sections. Ultra-thin sectionsmounted on uncoated copper grids were stained with uranylacetate and lead citrate and viewed in a JEOL JEM TEM1010 electron microscope.

DNA extraction, polymerase chain reaction,and DNA sequencing

B. coli cells from culture tubes were pooled and subjected togenomic DNA extraction using the Chelex method (Walsh etal. 1991). Polymerase chain reaction (PCR) of the SSUrRNA genes was performed using Go Taq® Hot StartPolymerase Kit (Promega) and oligonucleotide primers,Euk A (5′-AACCTGGTTGATCCTGCCAGT-3′), and EukB (5′-TGATCCTTCTGCAGGTTCACCTAC-3′) (Medlin etal. 1988). The PCR assay was carried out in ASTEC PC707Programmable Thermal Controller consisting of 35 cycleswith the following stringent parameters: 30 s denaturationat 94°C, 45 s annealing at 50°C, and 130 s extension at72°C. An initial denaturation step consisting of incubationat 94°C for 130 s and final extension step consisting ofincubation at 72°C for 7 min were also included. Theresulting products were separated by gel electrophoresis ona 1.5% agarose gel stained with ethidium bromide. PCRproducts were purified using magnetic beads. DNAsequencing was performed using the same primers in ABIPRISM Big Dye® Terminator v1.1 Cycle Sequencing Kit

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(Applied Biosystems, Foster City, CA) on an automatedsequencer (ABI PRISM 3100 model; Applied Biosystems).

Phylogenetic analysis

Sequences were uploaded into the Basic Local AlignmentSearch Tool program to search for the most similarreference sequences. These were then aligned and removedof ambiguous nucleotide positions along with referencesequences. The trees, rooted to a sequence of Dasytricharuminantium, were constructed using the GTR+Γ model ofthe model-based neighbor-joining (NJ) as well as the non-model based maximum parsimony (MP). D. ruminantiumwas chosen as an outgroup in view of its close relationshipwith 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 with1,000 bootstrap replicates. Clusters in the phylogenetic treeswere considered valid if these had bootstrap support valuesof greater than 50% from NJ andMP analyses. The trees wereviewed using the version 1.6.6 of TreeView (Page 1996).

GenBank references

The SSU rRNA gene sequence of the isolate generated inthis 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 asingle cell through limiting dilution; however, the resultswere unsuccessful. Our findings showed that a sufficientnumber of B. coli cells must be present in the initialinoculation in order to produce successful subcultures.Positive cultures containing B. coli, survived for 14 dayswith subcultures done every 24 h. Cultures of the isolateobserved at 48 h without subcultures resulted to an

increased bacterial density and the protozoa graduallydecreased in number. The cultures grew well at roomtemperature. Figure 1 shows the schematic image of theisolate as seen under a light microscope. General structuressuch as cilia, vestibulum, macronucleus, and contractilevacuoles are clearly seen.

Electron microscopy

Using SEM, the surface arrangement of the oral andsomatic cilia of B. coli was determined. The oral apparatus,surrounded by radiating field of oral cilia was found at ornear surface of the body, located apically. A depression wasalso observed on the oral apparatus or cell mouth of theorganism (Fig. 2a). The somatic cilia which covered mostof the body surface were arranged in a more transversefield, extending to the oral and the caudal region, located atthe base of the organism. The cell surface was filled withinterkinetal ridges that were found between adjacent cilia(Fig. 2b and b).

Examinations under TEM revealed the internal structureof the organism. The cell body was rounded posteriorly andnarrowing anteriorly. Scattered in the cytoplasm were thenuclei and several food vacuoles of the organism. Contrac-tile vacuoles located in the anterior, mid-body, and posteriorof the ciliate which function for osmoregulation were alsopresent in the cytoplasm (Fig. 3a).

The anterior portion of the cell body harbored the oralapparatus of the trophozoite. Ultra-thin section of theisolate showed the apparent oral apparatus surrounded byoral cilia (Fig. 3b). In contrast, situated in the posterior part,was the caudal region of the isolate. The region was linedwith longitudinal rows of cilia. Found in this region was thecytopyge, which functions as the anus of the ciliate. Foodvacuoles with ingested bacteria and food particles were alsofound near the cytopyge of the trophozoite (Fig. 3b).

Transverse section of the kineties in the buccal cavity ofthe trophozoite showed that several fibrils which appearedas cross striations in the electron micrograph were foundalong with the kinetosomes. The kinetosomes or basalbodies of the oral cilia were seen as a singular component,appearing as oral monokinetids (Fig. 4a).

Table 1 Genbank reference sequences used in the construction of the phylogenetic tree

Species/isolate Location/source Genbank accession no.

Balantidium coli Gorilla gorilla AF029763

Balantidium coli Sus scrofa (wild pig) from Spain AM982722

Balantidium coli Struthio camelus (ostrich) from Spain AM982723

Balantidium coli BC34706 Gorilla gorilla from Limbe Wildlife Center, Cameroon EU680309

Balantidium entozoon Italy EU581716

Dasytricha ruminantium Guelph, Canada U57769

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A higher magnification of the cell periphery exhibitedthe cortical plasma or the cortex of B. coli. The plasma-lemma, belonging to the pellicle of the organism along withits associated structures comprised the cortex of theorganism (Fig. 4b). Located immediately beneath the cellmembrane or plasmalemma were alveoli. This alveolarsystem was seen as flattened vacuoles lined by the outeralveolar membrane. In addition to these alveoli werenumerous mucocysts. The membrane bound mucocysts ofB. coli apparently were docked just below the plasmamembrane. These mucocysts, the only type of extrusomesfound in the organism were seen in their resting state aspolyhedral crystalline bodies (Fig. 4b).

The cilia of B. coli originated from the kinetosomes orbasal bodies. The surface of the cilium was enclosed by amembrane found to be continuous with the plasmamembranecovering the outer surface of the cell. The plasmalemma wasunderlaid by another membrane which formed alveolarspaces surrounding the kinetid (Fig. 4c). Located inside ofthe cytoplasm was an axoneme, surrounded by longperipheral and central fibrils. Longitudinal thin sectionshowed that the interior of kinetosome which anchored theaxoneme is frequently separated by a septum and that thefibrils in the center and the axoneme frequently end at around darkened structure, the axosome (Fig. 4c). Rows ofcilia arising from single rows of subsurface kinetosomes andtheir associated fibrils were also found in the cortex of theisolated B. coli, thus, the ciliate was shown to have uniformrows of monokinetid somatic cilia covering the surface ofthe organism’s cell body (Fig. 4c and d).

Among the cytoplasmic organelles surrounding thenuclei of B. coli were mitochondrion, endoplasmic reticu-lum, contractile vacuoles, and several food vacuoles. Themitochondrion of the organism was a double-membraneorganelle. Found inside of the mitochondrion, were cristaethat were seen as internal membranous tubules (Fig. 4e).

Along with the ribosomes, is the endoplasmic reticulumof B. coli which appeared as flat, extending membranoustubules. Small membrane bound vesicles are also found atthe periphery of the endoplasmic reticulum of the isolate(Fig. 4f).

Molecular characterization

PCR of the SSU rRNA genes of B. coli isolate generatedDNA fragments of the size ∼1.5 kb as determined byagarose gel electrophoresis (data not shown). These frag-

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

shown. Bar, 10 μm. b Magnified somatic cilia (ci) and interkinetalridges (ir). Bar, 1 μm

Fig. 1 Schematic illustration of the Philippine B. coli isolate showingthe general structures as seen under the light microscope: vestibulum(vb), macronucleus (man), food particles (fp) and contractile vacuoles(cv). Bar, 15 μm

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ments were excised and purified for DNA sequencing. Thelength of the SSU rDNA sequence obtained from the B. coliisolate was 1,543 bp.

The Philippine B. coli isolate was found to be 99% similarto the four B. coli sequences reported in the GenBank, with98% coverage. These reference isolates were collected froma 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 isolaterelative to reference B. coli isolates and the outgroup,D. ruminantium, was determined based on the constructedphylogenetic tree. It is apparent that the Philippine B. coliisolate clustered with reference B. coli isolates and separatefrom D. ruminantium and another related species ofBalantidium, B. entozoon (Fig. 5).

Discussion

B. coli is regarded as a common commensal parasite of thelarge intestine of pigs and has been reported in camels,horses, rodents, nonhuman primates, and humans (Headleyet al. 2008). Human cases of B. coli infection have beenfound in individuals who had contact with pigs (Esteban etal. 1998), pig excreta (Sharma and Harding 2003), and foodor water contaminated with the parasite (Esteban et al.1998). B. coli has been reported in immunocompromisedpatients causing pulmonary infection (Anargyrou et al.2003) and dysentery (Yazar et al. 2004).

In this study, B. coli was isolated from the large intestineof a pig. Efforts made to produce cloned cells of B. coliwere unsuccessful. Our findings are in agreement with thereport of Levine (1939) who also failed to obtain pure lines

after several attempts of isolating a single cell of B. coli. Inour study, it was noted that a considerable number of B. colicells 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 onlyif 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 ofthe protozoa is slow compared to the bacteria present in thecultures, and that these bacteria were able to utilize theconstituents of the growth medium or release inhibitingsubstances detrimental to the protozoa long enough beforethe 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, whetherderived from swine or human hosts, is difficult to maintainin cell cultures for any length of time and that this wasprobably due to certain bacteria and their metabolic productsthat are harmful to the growth of the ciliate and that shortinterval transplantation is necessary to keep the organismsunder cultivation (Glaser and Coria 1935). Certain variablesare involved in the cultivation of B. coli in vitro. Amongthese are the growth medium, pH (optimal range, 5.4–8) ofthe culture medium, associated undefined bacterial flora, andstrain differences of the parasites (Schuster and Ramirez-Avila 2008).

The Philippine B. coli isolate presented morphologicalfeatures coincident with previous B. coli descriptions(Anargyrou et al. 2003; Sharma and Harding 2003; Yazaret al. 2004; Solaymani-Mohammadi and Petri 2006; Schusterand Ramirez-Avila 2008). The isolate also presented ultra-structural features similar to those described by Skotarczakand Kołodziejczyk (2005). Mucocysts described as single-membrane, polyhedral crystalline structures deposited

Fig. 3 Transmission electronmicrographs of the PhilippineB. coli isolate. a Longitudinalthin section showing the generalaspect. The conspicuousmacronucleus (man) and smallermicronucleus (min) are seen.Four contractile vacuoles (cv)and several food vacuoles (fv)are also observed. Bar, 6.25 μm.b Ultra-thin section showing theoral apparatus (oa) and caudalregion (cr). The large macro-nucleus (man) is seen near themiddle portion of the cell body.Micronucleus is no longervisible. Bar, 5.71 μm

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below the plasma membrane of the trophic ciliate were iden-tified. Balantidium along with other ciliates, Tetrahymena,Colpidium, Ophryoglena, Holophyra, and Didinium havesac-like extensions called mucocysts, below their pelliclewhose mucoid content is expelled upon stimulation (Grell1973). Endoplasmic reticulum appearing as flat, extendingmembranous tubules was also described in this study alongwith several scattered free ribosomes in the cytoplasm of theorganism. Golgi bodies were not seen in the cell body of the

isolate. This supports the findings of Skotarczak andKołodziejczyk (2005) that Golgi bodies were not found inB. coli and that some membranes of the endoplasmicreticulum and small vesicles assume a similar role to theGolgi complex. The mitochondrion of the protozoon wasreported in this study as a spheroidal double-membraneorganelle with internal membranous tubules. The tropho-zoites of the isolate are often found at the bottom of theculture tubes. Thus, the presence of the mitochondria in theprotozoon makes the organism a facultative anaerobe.Several food vacuoles with ingested bacteria and foodparticles were also described in this study. Contractilevacuoles were also found in the cell body of the isolate.These contractile vacuoles act as ion regulating organelles byexpelling excess ions that have accumulated from theenvironment (Anderson 1988). The cell body of the isolatewas entirely covered by uniform rows of somatic mono-kinetid cilia where ciliary rows arise from single rows ofsubsurface kinetosomes and their associated fibrils (Schusterand Ramirez-Avila 2008). The somatic cilia are arranged in amore transverse field extending to the oral and caudal regionwhere the cytopyge of the organism is located. The oralapparatus of the protozoon was densely ciliated andcomposed of simple oral monokinetid cilia. Hence, theresults of our ultrastructural analysis clearly placed theisolate under the genus Balantidium, based on the newtaxonomy recently revised by the committee of International

Fig. 4 Transmission electron micrographs of the Philippine B. coliisolate showing the detailed internal structures. a Horizontal sectionthrough kineties of B. coli showing the kinetosomes (ks) found in theoral apparatus of the ciliate. Bar, 0.57 μm. b Thin section showing thecortex. The surrounding plasma membrane (pm) and outer alveolarmembrane (oalm) lining the alveoli system (al alveolus) can be seen.Membrane-bound mucocysts (mu) are also observed below the plasmamembrane. Cilium in transverse section with its 9+2 representation isalso shown. Peripheral (pmt) and central (cmt) microtubules of theciliary base are also observed. Bar, 0.19 μm. c Longitudinal thinsection of the kinetids. The alveoli (al) system lined by the plasmamembrane (pm) is shown. Peripheral (pf) and central fibrils (cf)enclosing the axoneme (a) anchored by the kinetosomes (ks) areobserved. Septum (s) and axosome (ax) found at the junction ofthe 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 severalfree ribosomes (ri) scattered in the cytoplasm. Bar, 0.23 µm. f Thinsection showing the endoplasmic reticulum (er). Bar, 0.31 μm

R

0.01

Balantidium coli from an ostrich (Struthio camelus) AM982723

Balantidium coli isolate BC34706 from a captive gorilla (Gorilla gorilla) EU680309

Balantidium coli from a wild pig (Sus scrofa) AM982722

Balantidium coli, from a lowland gorilla (Gorilla gorilla) AF029763

Balantidium coli isolate from a pig in the Philippines

Balantidium entozoon EU581716

Dasytricha U57769

94/100

64/--

0.01

Balantidium coli from an ostrich (Struthio camelus) AM982723

Balantidium coli isolate BC34706 from a captive gorilla (Gorilla gorilla) EU680309

Balantidium coli from a wild pig (Sus scrofa) AM982722

Balantidium coli, from a lowland gorilla (Gorilla gorilla) AF029763

Balantidium coli isolate from a pig in the Philippines GQ903678

Balantidium entozoon EU581716

Dasytricha ruminantium

94/100

64/--

0.01

Balantidium coli from an ostrich (Struthio camelus) AM982723

Balantidium coli isolate BC34706 from a captive gorilla (Gorilla gorilla) EU680309

Balantidium coli from a wild pig (Sus scrofa) AM982722

Balantidium coli, from a lowland gorilla (Gorilla gorilla) AF029763

Balantidium coli isolate from a pig in the Philippines

Balantidium entozoon EU581716

Dasytricha U57769

94/100

64/--

0.01

Balantidium coli from an ostrich (Struthio camelus) AM982723

Balantidium coli isolate BC34706 from a captive gorilla (Gorilla gorilla) EU680309

Balantidium coli from a wild pig (Sus scrofa) AM982722

Balantidium coli, from a lowland gorilla (Gorilla gorilla) AF029763

Balantidium coli isolate from a pig in the Philippines GQ903678

Balantidium entozoon EU581716

Dasytricha ruminantium

94/100

64/--

Fig. 5 Consensus phylogenetictree of Balantidium coli isolatedfrom a pig in the Philippinesand from Balantidiumsp. sequences from Genbankbased on 1,250 unambiguouslyaligned base pairs of the SSUrDNA region. This tree isconstructed using the GTR+Γmodel of the model-basedneighbor-joining (NJ) methodand rooted to a sequence ofDasytricha ruminantium.Figures on the branchesrepresent percentage bootstrapsupport from NJ and thenonmodel-based maximumparsimony (MP) methods,respectively (bootstrap supportpercentages below 50% are notshown). The scale bar on thelower left side represents onesubstitution change per100 nucleotide positions

Parasitol Res (2010) 106:387–394 393

Society of Protistologists and allied groups (Schuster andRamirez-Avila 2008).

Based on the sequence analysis of the SSU rRNA gene,the isolate was identified as B. coli. Sequence alignmentshowed that the sequence of the Philippine isolate corre-spond to published B. coli sequences. The sequence of theisolate showed 99% similarity to homologous sequences ofother B. coli reported previously. Based on the phylogenetictree generated by neighbor-joining and maximum parsi-mony analyses, the Philippine B. coli isolate and the B. colireference isolates clustered together regardless of hostspecies, with good bootstrap support values.

A previous study suggested that morphological charac-teristics and host species as bases for the identification ofBalantidium species are insufficient (Ponce-Gordo et al.2008). The results of their morphological and geneticanalyses suggest that, B. struthionis, reportedly found inostriches, is a synonym of B. coli. This is consistent withthe findings of this study that B. coli isolated from the largeintestine of pig shared 99% homology to previous B. coliisolates suggesting the fact that this ciliate might have lowhost specificity.

On the bases of the ultrastructural characterization and thesequence of the SSU rRNA gene, the Balantidium that wasisolated from the large intestine of a pig is then defined asB. coli. This study confirms that host-specificity andmorphological features are not enough criteria in describingand identifying the species of Balantidium. We highlyrecommend the development of new a culture medium thatwould encourage propagation and longer survival of B. coliin culture.We also recommend that the criteria to validate andidentify species of Balantidium must be based on morpho-logical characteristics, genetic identification, and sequencecomparison of genes having taxonomic importance.

Acknowledgements This work was supported financially by a grantfrom the Faculty Development Committee of Silliman University. Wethank Neilfred G. Bije, Davin Edric V. Adao, and John Anthony D.L.Yason for their technical assistance. The experiments conducted in thisstudy comply with the current laws of the Philippines.

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