DOI: 10.

2478/s11686-014-0256-9
© W. Stefański Institute of Parasitology, PAS
Acta Parasitologica, 2014, 59(3), 448–453; ISSN 1230-2821

Molecular detection and characterization

of Theileria species in the Philippines
Lawrence P. Belotindos1, Jonathan V. Lazaro1, Marvin A. Villanueva2 and Claro N. Mingala2,3,4*
1
College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz 3120, Nueva Ecija, Philippines;
2
Animal Health Unit, Philippine Carabao Center National Headquarters and Gene Pool, Science City of Muñoz 3120, Nueva Ecija, Philippines;
3
Institute of Graduate Studies, Department of Animal Science, Central Luzon State University, Science City of Muñoz 3120, Nueva Ecija,
Philippines; 4Scientific Career System, National Academy of Science and Technology, Department of Science and Technology,
Taguig City 1631, Metro Manila, Philippines

Abstract
Theileriosis is a tick-borne disease of domestic and wild animals that cause devastating economic loss in livestock in tropical
and subtropical regions. Theileriosis is not yet documented in the Philippines as compared to babesiosis and anaplasmosis
which are considered major tick-borne diseases that infect livestock in the country and contribute major losses to the livestock
industry. The study was aimed to detect Theileria sp. at genus level in blood samples of cattle using polymerase chain reaction
(PCR) assay. Specifically, it determined the phylogenetic relationship of Theileria species affecting cattle in the Philippines to
other Theileria sp. registered in the GenBank. A total of 292 blood samples of cattle that were collected from various provinces
were used. Theileria sp. was detected in 43/292 from the cattle blood samples using PCR assay targeting the major piroplasm
surface protein (MPSP) gene. DNA sequence showed high similarity (90–99%) among the reported Theileria sp. isolates in the
GenBank and the Philippine isolates of Theileria. Phylogenetic tree construction using nucleotide sequence classified the Philip-
pine isolates of Theileria as benign. However, nucleotide polymorphism was observed in the new isolate based on nucleotide
sequence alignment. It revealed that the new isolate can be a new species of Theileria.

Keywords
Theileria spp.; Cattle; PCR; MPSP; Philippines

Introduction et al. 2010). As a result, serious economic losses in the live-

Theileriosis is a tick-borne disease of domestic and wild
animals. It is one of the most economically devastating dis-
eases of livestock in tropical and subtropical regions (Uilen-
berg 1995, Inci et al. 2009). At least five species of Theileria
(T. parva, T. annulata, T. taurotragi, T. velifera and mem-
bers of the T. sergenti/orientalis/buffeli group) have been
found to infect cattle. From the five species infecting cattle,
T. annulata and T. parva were pathogenic species. The par-
asites infect cattle during tick feeding which rapidly invade
mononuclear leukocytes and later erythrocytes in the piro-
plasm stage (Cox 1982, d’Oliveira et al. 1995, Parthiban et
al. 2010) and may cause general lymphadenopathy, fever,
anorexia, anemia, jaundice, weight loosed and eventually
death if untreated in susceptible animals. Most affected are
calves, stressed animals, or immunosupressed adult cattle as
well as non-indigenous breeds (Jang et al. 2003, Khukhuu
stock industry can subsequently arise (Tajima et al. 1998,
Khukhuu et al. 2010).
Detection of Theileria infection by polymerase chain re-
action (PCR) method is more sensitive and accurate compared
with serological tests and microscopic detection of piroplas-
mic forms (d’Oliveira et al. 1995, Almeria et al. 2001,
Damanli et al. 2005, Hoghooghi et al. 2011). Molecular de-
tection and classification of the Theileria sp. in a herd with or
without clinical signs of theileriosis, is able to highlight the
areas at risk from Theileria infection and may thus enable the
most suitable implementation of control measures which can
then give a considerable boost to the maintenance of herd
health and improve the productivity, profitability and welfare
of the animal and also improve the income of the smallholder
livestock owners.
The impact of tick-borne diseases in the Philippines has
not been as severe as elsewhere due to the inherent resistance

*Corresponding author: cnmingala@hotmail.com

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Characterization of Theileria species in the Philippines
of local breeds. The two diseases of major concern to Philip-
pine authorities are babesiosis caused by Babesia bigemina,
B. bovis and B. major (Bock et al. 2004) and anaplasmosis
caused by Anaplasma marginale and A. centrale (Ybañez
et al. 2013), collectively producing tick fever transmitted
by Rhipicephalus (Boophilus) microplus. Rhipicephalus (Boo-
philus) microplus is known to transmit Theileria parasites in
South Asian countries but in the Philippines there no study
that this tick species transmit Theileria parasites.
Theileriosis is not yet documented in the Philippines as
compared to babesiosis and anaplasmosis, thus, the present
study was aimed to detect Theileria sp. at gene level in blood
samples of cattle using PCR. Specifically, it determined phy-
logenetic relationship of Theileria sp. affecting cattle in the
Philippines to other Theileria species registered in the Gen-
Bank and the strain of theileriosis in cattle.
Materials and Methods
Sample collection and DNA extraction
A total of 292 blood samples of cattle that were collected in cat-
tle farms in Laguna (147 samples) and Pangasinan (40 samples)
in Luzon region and Cebu (77 samples) and Bohol (28 samples)
in Visayas region in the Philippines. The samples were collected
for a month and stored till all samples were collected. The
300 µl blood samples were placed in 1.5 ml microcentrifuge
tube for DNA extraction. Samples were stored at 4°C.
DNA was extracted from the whole blood samples using
Wizard Genomic DNA extraction kit (Promega, USA) ac-
cording to the manufacturer’s instructions. Extracted DNA
was placed in 1.5 microcentrifuge tube and was stored at 4°C
until use.
Amplification of Bovine β-Actin and Theileria sp. major
piroplasm surface protein (MPSP) genes
DNA extract was confirmed by amplifying bovine β-actin en-
coding gene (housekeeping gene) to assured that the genomic
DNA was extracted from all the samples. Amplification was
carried out in a total volume of 20 μl containing 13.3 μl dou-
ble distilled water (DDW), 2.0 µl of 10× PCR buffer (10 mM
Tris-HCl (pH 9.0), 50 mM KCl, 1.25 mM MgCl2), 1.6 µl of
dNTPs (2.5 mM each, Takara, Japan), 0.5 μl each of 10 pmol
of each primers (β-actin Forward – 5’CGC ACC ACC GGC
ATC GTG AT’3; β-actin Reverse – 5’TCC AGG GCC ACG
TAG CAG AG’3) primers (Tajima et al. 1998) , 0.1 µl of 5U
of Taq polymerase (Takara, Japan) and 2.0 μl of DNA tem-
plate. The PCR included an initial denaturation for five min at
94°C, 35 cycles of denaturing step for 30 sec at 94°C, anneal-
ing step for 30 sec at 55°C, and extension step for 30 sec for
72°C and a final extension step for five min at 72°C. The am-
plified sequence size was 227 bp. A negative control contain-
ing DDW instead of DNA template was used.
449
Only positive samples to β-actin were used for the detec-
tion of Theileria sp. Primers were designed from conserved
sequences among the MPSP genes of Theileria sp. registered
in the Genbank using Pimer-BLAST tool of National Center
for Biotechnology Information (NCBI). Amplification by PCR
in a 20 μl reaction volume, containing 13.3 μl double distilled
water (DDW), 2.0 μl 10× PCR Buffer (10 mM Tris-HCl (pH
9.0), 50 mM KCl, 1.25 mM MgCl2; Takara, Japan), 1.6 µl of
dNTPs (2.5 mM each; Takara, Japan), 0.5 μl each of 10 pmol
of each primers (Ts1(F) 5’ CAC TCC AAC AGT CGC CCA
CAG AC’3; Ts2(R) 5’ CAG CGC TGA GGA CGG CAA
GTG’3), 0.1 µl of 5U of Taq polymerase (Takara, Japan) and
2.0 μl of DNA template. PCR condition included an initial de-
naturation at 94°C for five min, 40 cycles of denaturing step
at 94°C for one min, annealing step at 58°C for one min, and
extension step at 72°C for one min followed by an additional
extension step at 72°C for 7 min. The amplified sequence size
was 560 bp. A negative control containing DDW instead of
DNA template was used. As positive controls, DNA isolates
from trial experiments was cloned and confirmed by se-
quencing were used. The amplified PCR products were visu-
alized on 2% agarose gel stained with ethidium bromide under
UV illuminator.
Molecular cloning and sequencing of Theileria sp. MPSP
gene
From 43 positive samples for Theileria only highly bright and
thick band was selected for DNA sequencing which was done
in the Research Center for Zoonoses Control, Hokkaido Uni-
versity in Japan and Macrogen Inc, in Seoul, South Korea. The
amplified band corresponding to the size of the target gene
were excised from the gel and purified. The purified gene frag-
ments were ligated into the pGEM-T easy vector and trans-
formed into competent cells. In each experiment, 8–10
plasmid clones containing the target gene were sequenced
using automated DNA sequencer.
Data analysis
Sequence data analysis was performed using the Basic Local
Alignment Search Tool (BLAST) search of the National Cen-
ter for Biotechnology Information (NCBI) for homology
analyses of Theileria MPSP gene. The sequences were edited
using CLUSTALX program. Phylogenetic analysis was per-
formed using MEGA4.
Results and Discussion
Molecular detection of Theileria sp.
Pre-diagnostic PCR assay was employed to determine the
quality of the extracted DNA sample used in the study using
bovine β-actin primers (F/R). All blood samples used in the
study were positive (100%) to bovine β-actin which indicated
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450 Lawrence P. Belotindos et al.

Fig. 1. PCR amplification of the Theileria major piroplasm surface protein (MPSP) genes. Sample PCR products (10 µl from each sample)
were electrophoresed on a 2% agarose gel stained with ethidium bromide showing amplified target of 560 bp from the MPSP gene. M – 100
bp size DNA marker (Intron, 100 ng/µl); Lane 1–23 – DNA samples; Lane 9, 18 – positive samples

that the extracted DNA samples were of good quality. Ex-
tracted DNA must be of good quality for molecular analysis
particularly in epidemiological survey of pathogen of veteri-
nary importance (Ferreira et al. 2010). Bovine β-actin is so
ubiquitous that it is considered a house-keeping protein and
historically been used as an external standard for Western blot-
ting assay (Karakozova et al. 2006). Fig. 1 shows the analy-
sis of the PCR amplification for the detection of Theileria sp.
by using newly designed primers (Ts1/Ts2) set in 2% agarose
gel electrophoresis.
The newly designed primers (Ts1/Ts2) which successfully
optimized and amplified MPSP genes with the product size of
560 bp. Furthermore, DNA was extracted and subjected for
DNA sequence and the result confirmed the amplified DNA
was Theileria sp.
Using PCR assay, the MPSP gene was positively detected
using the newly designed primers (Ts1/Ts2). The detection rate
of Theileria sp. was 14.7% (43/292) by PCR targeting MPSP
gene. Positive samples were distributed throughout the four
provinces in the Philippines as showed in Table I. PCR method
provides accurate and useful means of detecting infections be-
fore the appearance of clinical signs compared to the serolog-
ical and direct microscopy (d’Oliveira et al. 1995, Almeria et
al. 2001, Damanli et al. 2005, Hoghooghi et al. 2011). Ac-
cording to the study Parthiban et al. (2010), a utilization PCR
technique had high sensitivity which can amplify even small
amount of Theileria parasites. The clinical application of this
technique could help small livestock producers to avoid eco-
nomic damage caused by bovine theileriosis (Shimizu et al.
2000, Ota et al. 2009).
DNA sequence and phylogenetic analysis
DNA sequence of the Philippine isolates of Theileria MPSP
gene was submitted in GeneBank with the accession number
AB753031. The nucleotide sequence of MPSP gene were
analysed using the Basic Local Alignment Search Tool
(BLAST) search of the National Center for Biotechnology In-
formation (NCBI) for homology analyses of Theileria MPSP
gene. Sequence showed high similarity (90–99%) between the
Philippine isolates of Theileria MPSP gene sequence and
Theileria sergenti/buffeli/orientalis group registered in the
GenBank indicating the isolate was a benign species of Thei-
leria. Phylogenetic findings showed that the Philippine iso-

Table I. Distribution of positive samples and positive rate of PCR results for the detection of Theileria MPSP
gene in cattle blood by collection sites in the Philippines

Collection site No. samples PCR positive Positive rate

Laguna 147 20 13.6
Luzon
Pangasinan 40 16 40.0
Cebu 77 6 7.8
Visayas
Bohol 28 1 3.6
Total 292 43 14.7
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Characterization of Theileria species in the Philippines 451

Fig. 2. Phylogenetic relationships of the Philippine isolate of Theileria in cattle based on nucleotide sequence of MPSP gene. The Philippine
isolate of Theileria MPSP gene sequence determined in this study are shown in box with asterisks (**). Accession number of each Theileria
sp. is indicated at the end of each branch. The length of the horizontal bar indicates the number of nucleotide substitution per site. Numbers
shown at the branch nodes indicate the bootstrap values

lates of Theileria was located in separated cluster in Type 1 Conclusions

(Chitose) which revealed similar phylogenetic relationships
with high bootstrap value (Fig. 2).
Nucleotide sequence analysis of MPSP gene of the Philip-
pine isolates of Theileria was compared with Theileria ser-
genti/buffeli/orientalis MPSP gene registered in the GenBank.
Two nucleotide polymorphisms were observed in loci 79 and
427 of the nucleotide sequence among 29 isolates in the study
(Fig. 3), which indicated that the Philippine isolates of Thei-
leria could be a new species of Theileria.
To date, no Theileria infection had been reported in the
Philippines [Bureau of Animal Industry, personal communi-
cation]. The study was successfully detected Theileria in
blood samples in cattle using PCR assay which indicated that
Theileria infection is already present in the country.
Using PCR as a diagnostic tool, detection of Theileria sp.
targeting the MPSP gene showed 15% infection rate among
the 292 apparently healthy cattle blood sample tested. Nu-
cleotide sequence homology of MPSP gene of Philippine iso-
late Theileria showed high similarity (90–99%) among the
reported Theileria sergenti/buffeli/orientalis isolates in the
GenBank.
Phylogenetic relationship of Theileria sp. affecting the
Philippines to other Theileria sp. registered in the GenBank
showed that the Philippine Theileria isolate belonged to the
benign or less virulent strains of Theileria based on its phylo-
genetic tree with a high bootstrap support values. It is recom-
mended that more isolates from other geographical regions
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452
Fig. 3. Nucleotide sequence analysis of Philippine Theileria MPSP gene. The sequence of major piroplasm surface protein (MPSP) gene of
Philippine Theileria isolate was aligned and compared with Theileria sergenti/buffeli/orientalis MPSP gene isolates registered in the
GenBank Nucleotide polymorphisms were observed in 79 and 427 loci
must be examined to provide more information for the geno-
typing of Philippine isolate Theileria.
Acknowledgments. This research was supported by the Philippine
Carabao Center. Thanks are extended to the management especially
Dr. Libertado C. Cruz, Executive Director, and the rest of staff of the
Animal Health Unit in providing the needed samples for this re-
search. Also, appreciations are extended to Prof. Yasuhiko Suzuki
and Dr. Chie Nakajima of the Research Center for Zoonoses Con-
trol, Hokkaido University, for their technical assistance.
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