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DOI: 10.

© 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*
College of Veterinary Science and Medicine, Central Luzon State University, Science City of Muñoz 3120, Nueva Ecija, Philippines;
Animal Health Unit, Philippine Carabao Center National Headquarters and Gene Pool, Science City of Muñoz 3120, Nueva Ecija, Philippines;
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

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.

Theileria spp.; Cattle; PCR; MPSP; Philippines

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

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

*Corresponding author:

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Characterization of Theileria species in the Philippines 449

of local breeds. The two diseases of major concern to Philip- Only positive samples to β-actin were used for the detec-
pine authorities are babesiosis caused by Babesia bigemina, tion of Theileria sp. Primers were designed from conserved
B. bovis and B. major (Bock et al. 2004) and anaplasmosis sequences among the MPSP genes of Theileria sp. registered
caused by Anaplasma marginale and A. centrale (Ybañez in the Genbank using Pimer-BLAST tool of National Center
et al. 2013), collectively producing tick fever transmitted for Biotechnology Information (NCBI). Amplification by PCR
by Rhipicephalus (Boophilus) microplus. Rhipicephalus (Boo- in a 20 μl reaction volume, containing 13.3 μl double distilled
philus) microplus is known to transmit Theileria parasites in water (DDW), 2.0 μl 10× PCR Buffer (10 mM Tris-HCl (pH
South Asian countries but in the Philippines there no study 9.0), 50 mM KCl, 1.25 mM MgCl2; Takara, Japan), 1.6 µl of
that this tick species transmit Theileria parasites. dNTPs (2.5 mM each; Takara, Japan), 0.5 μl each of 10 pmol
Theileriosis is not yet documented in the Philippines as of each primers (Ts1(F) 5’ CAC TCC AAC AGT CGC CCA
compared to babesiosis and anaplasmosis, thus, the present CAG AC’3; Ts2(R) 5’ CAG CGC TGA GGA CGG CAA
study was aimed to detect Theileria sp. at gene level in blood GTG’3), 0.1 µl of 5U of Taq polymerase (Takara, Japan) and
samples of cattle using PCR. Specifically, it determined phy- 2.0 μl of DNA template. PCR condition included an initial de-
logenetic relationship of Theileria sp. affecting cattle in the naturation at 94°C for five min, 40 cycles of denaturing step
Philippines to other Theileria species registered in the Gen- at 94°C for one min, annealing step at 58°C for one min, and
Bank and the strain of theileriosis in cattle. 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
Materials and Methods DNA template was used. As positive controls, DNA isolates
from trial experiments was cloned and confirmed by se-
Sample collection and DNA extraction quencing were used. The amplified PCR products were visu-
alized on 2% agarose gel stained with ethidium bromide under
A total of 292 blood samples of cattle that were collected in cat- UV illuminator.
tle farms in Laguna (147 samples) and Pangasinan (40 samples)
in Luzon region and Cebu (77 samples) and Bohol (28 samples) Molecular cloning and sequencing of Theileria sp. MPSP
in Visayas region in the Philippines. The samples were collected gene
for a month and stored till all samples were collected. The
300 µl blood samples were placed in 1.5 ml microcentrifuge From 43 positive samples for Theileria only highly bright and
tube for DNA extraction. Samples were stored at 4°C. thick band was selected for DNA sequencing which was done
DNA was extracted from the whole blood samples using in the Research Center for Zoonoses Control, Hokkaido Uni-
Wizard Genomic DNA extraction kit (Promega, USA) ac- versity in Japan and Macrogen Inc, in Seoul, South Korea. The
cording to the manufacturer’s instructions. Extracted DNA amplified band corresponding to the size of the target gene
was placed in 1.5 microcentrifuge tube and was stored at 4°C were excised from the gel and purified. The purified gene frag-
until use. ments were ligated into the pGEM-T easy vector and trans-
formed into competent cells. In each experiment, 8–10
Amplification of Bovine β-Actin and Theileria sp. major plasmid clones containing the target gene were sequenced
piroplasm surface protein (MPSP) genes using automated DNA sequencer.

DNA extract was confirmed by amplifying bovine β-actin en- Data analysis
coding gene (housekeeping gene) to assured that the genomic
DNA was extracted from all the samples. Amplification was Sequence data analysis was performed using the Basic Local
carried out in a total volume of 20 μl containing 13.3 μl dou- Alignment Search Tool (BLAST) search of the National Cen-
ble distilled water (DDW), 2.0 µl of 10× PCR buffer (10 mM ter for Biotechnology Information (NCBI) for homology
Tris-HCl (pH 9.0), 50 mM KCl, 1.25 mM MgCl2), 1.6 µl of analyses of Theileria MPSP gene. The sequences were edited
dNTPs (2.5 mM each, Takara, Japan), 0.5 μl each of 10 pmol using CLUSTALX program. Phylogenetic analysis was per-
of each primers (β-actin Forward – 5’CGC ACC ACC GGC formed using MEGA4.
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 Results and Discussion
of Taq polymerase (Takara, Japan) and 2.0 μl of DNA tem-
plate. The PCR included an initial denaturation for five min at Molecular detection of Theileria sp.
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 Pre-diagnostic PCR assay was employed to determine the
72°C and a final extension step for five min at 72°C. The am- quality of the extracted DNA sample used in the study using
plified sequence size was 227 bp. A negative control contain- bovine β-actin primers (F/R). All blood samples used in the
ing DDW instead of DNA template was used. 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- ical and direct microscopy (d’Oliveira et al. 1995, Almeria et
tracted DNA must be of good quality for molecular analysis al. 2001, Damanli et al. 2005, Hoghooghi et al. 2011). Ac-
particularly in epidemiological survey of pathogen of veteri- cording to the study Parthiban et al. (2010), a utilization PCR
nary importance (Ferreira et al. 2010). Bovine β-actin is so technique had high sensitivity which can amplify even small
ubiquitous that it is considered a house-keeping protein and amount of Theileria parasites. The clinical application of this
historically been used as an external standard for Western blot- technique could help small livestock producers to avoid eco-
ting assay (Karakozova et al. 2006). Fig. 1 shows the analy- nomic damage caused by bovine theileriosis (Shimizu et al.
sis of the PCR amplification for the detection of Theileria sp. 2000, Ota et al. 2009).
by using newly designed primers (Ts1/Ts2) set in 2% agarose
gel electrophoresis. DNA sequence and phylogenetic analysis
The newly designed primers (Ts1/Ts2) which successfully
optimized and amplified MPSP genes with the product size of DNA sequence of the Philippine isolates of Theileria MPSP
560 bp. Furthermore, DNA was extracted and subjected for gene was submitted in GeneBank with the accession number
DNA sequence and the result confirmed the amplified DNA AB753031. The nucleotide sequence of MPSP gene were
was Theileria sp. analysed using the Basic Local Alignment Search Tool
Using PCR assay, the MPSP gene was positively detected (BLAST) search of the National Center for Biotechnology In-
using the newly designed primers (Ts1/Ts2). The detection rate formation (NCBI) for homology analyses of Theileria MPSP
of Theileria sp. was 14.7% (43/292) by PCR targeting MPSP gene. Sequence showed high similarity (90–99%) between the
gene. Positive samples were distributed throughout the four Philippine isolates of Theileria MPSP gene sequence and
provinces in the Philippines as showed in Table I. PCR method Theileria sergenti/buffeli/orientalis group registered in the
provides accurate and useful means of detecting infections be- GenBank indicating the isolate was a benign species of Thei-
fore the appearance of clinical signs compared to the serolog- 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
Pangasinan 40 16 40.0
Cebu 77 6 7.8
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). Using PCR as a diagnostic tool, detection of Theileria sp.
Nucleotide sequence analysis of MPSP gene of the Philip- targeting the MPSP gene showed 15% infection rate among
pine isolates of Theileria was compared with Theileria ser- the 292 apparently healthy cattle blood sample tested. Nu-
genti/buffeli/orientalis MPSP gene registered in the GenBank. cleotide sequence homology of MPSP gene of Philippine iso-
Two nucleotide polymorphisms were observed in loci 79 and late Theileria showed high similarity (90–99%) among the
427 of the nucleotide sequence among 29 isolates in the study reported Theileria sergenti/buffeli/orientalis isolates in the
(Fig. 3), which indicated that the Philippine isolates of Thei- GenBank.
leria could be a new species of Theileria. Phylogenetic relationship of Theileria sp. affecting the
To date, no Theileria infection had been reported in the Philippines to other Theileria sp. registered in the GenBank
Philippines [Bureau of Animal Industry, personal communi- showed that the Philippine Theileria isolate belonged to the
cation]. The study was successfully detected Theileria in benign or less virulent strains of Theileria based on its phylo-
blood samples in cattle using PCR assay which indicated that genetic tree with a high bootstrap support values. It is recom-
Theileria infection is already present in the country. mended that more isolates from other geographical regions
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452 Lawrence P. Belotindos et al.

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- Karahan M., Altay K. 2005. Prevalence and distribution of
typing of Philippine isolate Theileria. tropical theileriosis in Eastern Turkey. Veterinary Parasitol-
ogy, 127, 9–15. DOI:10.1016/j.vetpar.2004.08.006.
d’Oliveira C., Van M.A., Habela M., Jacquiet P., Jongejan F. 1995.
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Acknowledgments. This research was supported by the Philippine cattle by PCR. Journal of Clinical Microbiology, 33, 2665–
Carabao Center. Thanks are extended to the management especially 2669.
Dr. Libertado C. Cruz, Executive Director, and the rest of staff of the Ferreira E.C., Gontijo C.M., Cruz I., Melo M.N., Silva A.M. 2010.
Animal Health Unit in providing the needed samples for this re- Alternative PCR protocol using a single primer set for as-
search. Also, appreciations are extended to Prof. Yasuhiko Suzuki sessing DNA quality in several tissues from a large variety of
and Dr. Chie Nakajima of the Research Center for Zoonoses Con- mammalian species living in areas endemic for leishmania-
trol, Hokkaido University, for their technical assistance. sis. Memórias do Instituto Oswaldo Cruz, 105, 895–898. DOI:
Hoghooghi N., Ghaemi P., Shayan P., Eckert B., Sadr-Shirazi N. 2011.
Detection of native carrier cattle infected with Theileria annu-
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Received: September 24, 2013

Revised: April 7, 2014
Accepted for publication: April 8, 2014

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