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; 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
2
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 Philippine 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 Theileriosis is a tick-borne disease of domestic and wild animals. It is one of the most economically devastating diseases of livestock in tropical and subtropical regions (Uilenberg 1995, Inci et al. 2009). At least five species of Theileria (T. parva, T. annulata, T. taurotragi, T. velifera and members 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 parasites infect cattle during tick feeding which rapidly invade mononuclear leukocytes and later erythrocytes in the piroplasm 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
et al. 2010). As a result, serious economic losses in the livestock industry can subsequently arise (Tajima et al. 1998, Khukhuu et al. 2010). Detection of Theileria infection by polymerase chain reaction (PCR) method is more sensitive and accurate compared with serological tests and microscopic detection of piroplasmic forms (d’Oliveira et al. 1995, Almeria et al. 2001, Damanli et al. 2005, Hoghooghi et al. 2011). Molecular detection 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: [email protected]
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Characterization of Theileria species in the Philippines
of local breeds. The two diseases of major concern to Philippine 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 (Boophilus) 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 phylogenetic relationship of Theileria sp. affecting cattle in the Philippines to other Theileria species registered in the GenBank 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 cattle 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) according 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 encoding 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 double 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 template. The PCR included an initial denaturation for five min at 94°C, 35 cycles of denaturing step for 30 sec at 94°C, annealing 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 amplified sequence size was 227 bp. A negative control containing DDW instead of DNA template was used.
Only positive samples to β-actin were used for the detection 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 denaturation 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 sequencing were used. The amplified PCR products were visualized 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 University 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 fragments were ligated into the pGEM-T easy vector and transformed 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 Center for Biotechnology Information (NCBI) for homology analyses of Theileria MPSP gene. The sequences were edited using CLUSTALX program. Phylogenetic analysis was performed 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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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. Extracted DNA must be of good quality for molecular analysis particularly in epidemiological survey of pathogen of veterinary 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 blotting assay (Karakozova et al. 2006). Fig. 1 shows the analysis 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 before 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). According 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 economic 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 Information (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 Theileria. 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 Luzon Visayas
Laguna Pangasinan Cebu Bohol Total
No. samples
PCR positive
Positive rate
147 40 77 28 292
20 16 6 1 43
13.6 40.0 7.8 3.6 14.7
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Characterization of Theileria species in the Philippines
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 (Chitose) which revealed similar phylogenetic relationships with high bootstrap value (Fig. 2). Nucleotide sequence analysis of MPSP gene of the Philippine isolates of Theileria was compared with Theileria sergenti/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 Theileria could be a new species of Theileria. To date, no Theileria infection had been reported in the Philippines [Bureau of Animal Industry, personal communication]. 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.
Conclusions 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. Nucleotide sequence homology of MPSP gene of Philippine isolate 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 phylogenetic tree with a high bootstrap support values. It is recommended that more isolates from other geographical regions
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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 genotyping 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 research. Also, appreciations are extended to Prof. Yasuhiko Suzuki and Dr. Chie Nakajima of the Research Center for Zoonoses Control, Hokkaido University, for their technical assistance.
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Received: September 24, 2013 Revised: April 7, 2014 Accepted for publication: April 8, 2014
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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; 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
2
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 Philippine 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 Theileriosis is a tick-borne disease of domestic and wild animals. It is one of the most economically devastating diseases of livestock in tropical and subtropical regions (Uilenberg 1995, Inci et al. 2009). At least five species of Theileria (T. parva, T. annulata, T. taurotragi, T. velifera and members 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 parasites infect cattle during tick feeding which rapidly invade mononuclear leukocytes and later erythrocytes in the piroplasm 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
et al. 2010). As a result, serious economic losses in the livestock industry can subsequently arise (Tajima et al. 1998, Khukhuu et al. 2010). Detection of Theileria infection by polymerase chain reaction (PCR) method is more sensitive and accurate compared with serological tests and microscopic detection of piroplasmic forms (d’Oliveira et al. 1995, Almeria et al. 2001, Damanli et al. 2005, Hoghooghi et al. 2011). Molecular detection 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: [email protected]
449
Characterization of Theileria species in the Philippines
of local breeds. The two diseases of major concern to Philippine 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 (Boophilus) 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 phylogenetic relationship of Theileria sp. affecting cattle in the Philippines to other Theileria species registered in the GenBank 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 cattle 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) according 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 encoding 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 double 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 template. The PCR included an initial denaturation for five min at 94°C, 35 cycles of denaturing step for 30 sec at 94°C, annealing 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 amplified sequence size was 227 bp. A negative control containing DDW instead of DNA template was used.
Only positive samples to β-actin were used for the detection 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 denaturation 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 sequencing were used. The amplified PCR products were visualized 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 University 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 fragments were ligated into the pGEM-T easy vector and transformed 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 Center for Biotechnology Information (NCBI) for homology analyses of Theileria MPSP gene. The sequences were edited using CLUSTALX program. Phylogenetic analysis was performed 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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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. Extracted DNA must be of good quality for molecular analysis particularly in epidemiological survey of pathogen of veterinary 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 blotting assay (Karakozova et al. 2006). Fig. 1 shows the analysis 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 before 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). According 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 economic 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 Information (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 Theileria. 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 Luzon Visayas
Laguna Pangasinan Cebu Bohol Total
No. samples
PCR positive
Positive rate
147 40 77 28 292
20 16 6 1 43
13.6 40.0 7.8 3.6 14.7
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Characterization of Theileria species in the Philippines
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 (Chitose) which revealed similar phylogenetic relationships with high bootstrap value (Fig. 2). Nucleotide sequence analysis of MPSP gene of the Philippine isolates of Theileria was compared with Theileria sergenti/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 Theileria could be a new species of Theileria. To date, no Theileria infection had been reported in the Philippines [Bureau of Animal Industry, personal communication]. 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.
Conclusions 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. Nucleotide sequence homology of MPSP gene of Philippine isolate 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 phylogenetic tree with a high bootstrap support values. It is recommended that more isolates from other geographical regions
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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 genotyping 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 research. Also, appreciations are extended to Prof. Yasuhiko Suzuki and Dr. Chie Nakajima of the Research Center for Zoonoses Control, Hokkaido University, for their technical assistance.
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