Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes
Abstract Background Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for...
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| Format: | Article |
| Language: | English |
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BMC
2023-11-01
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| Series: | Parasites & Vectors |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13071-023-06045-z |
| _version_ | 1827635407990292480 |
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| author | Believe Ahedor Davaajav Otgonsuren Atambekova Zhyldyz Azirwan Guswanto Noel Muthoni Mumbi Ngigi Maria Fátima Rodríguez Valinotti Hemal Kothalawala Nizanantha Kalaichelvan Seekkuge Susil Priyantha Silva Hemali Kothalawala Tomás Javier Acosta Thillaiampalam Sivakumar Naoaki Yokoyama |
| author_facet | Believe Ahedor Davaajav Otgonsuren Atambekova Zhyldyz Azirwan Guswanto Noel Muthoni Mumbi Ngigi Maria Fátima Rodríguez Valinotti Hemal Kothalawala Nizanantha Kalaichelvan Seekkuge Susil Priyantha Silva Hemali Kothalawala Tomás Javier Acosta Thillaiampalam Sivakumar Naoaki Yokoyama |
| author_sort | Believe Ahedor |
| collection | DOAJ |
| description | Abstract Background Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype. Methods A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T . equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically. Results Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays. Conclusions The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes. Graphical abstract |
| first_indexed | 2024-03-09T15:26:22Z |
| format | Article |
| id | doaj.art-718c0e0b1e594231ae080947c0284437 |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-03-09T15:26:22Z |
| publishDate | 2023-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Parasites & Vectors |
| spelling | doaj.art-718c0e0b1e594231ae080947c02844372023-11-26T12:29:45ZengBMCParasites & Vectors1756-33052023-11-0116111010.1186/s13071-023-06045-zDevelopment and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypesBelieve Ahedor0Davaajav Otgonsuren1Atambekova Zhyldyz2Azirwan Guswanto3Noel Muthoni Mumbi Ngigi4Maria Fátima Rodríguez Valinotti5Hemal Kothalawala6Nizanantha Kalaichelvan7Seekkuge Susil Priyantha Silva8Hemali Kothalawala9Tomás Javier Acosta10Thillaiampalam Sivakumar11Naoaki Yokoyama12National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary MedicineNational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary MedicineNational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary MedicineNational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary MedicineNational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary MedicineCentro de Diagnostico VeterinarioVeterinary Research InstituteDepartment of Farm Animal Production and Health, Faculty of Veterinary Medicine and Animal Science, University of PeradeniyaDepartment of Animal Production and HealthDepartment of Animal Production and HealthUniversidad Nacional de CanendiyuNational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary MedicineNational Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary MedicineAbstract Background Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype. Methods A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T . equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically. Results Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays. Conclusions The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes. Graphical abstracthttps://doi.org/10.1186/s13071-023-06045-zEquine piroplasmosisGenotypePolymerase chain reactionSpecificityTheileria equi |
| spellingShingle | Believe Ahedor Davaajav Otgonsuren Atambekova Zhyldyz Azirwan Guswanto Noel Muthoni Mumbi Ngigi Maria Fátima Rodríguez Valinotti Hemal Kothalawala Nizanantha Kalaichelvan Seekkuge Susil Priyantha Silva Hemali Kothalawala Tomás Javier Acosta Thillaiampalam Sivakumar Naoaki Yokoyama Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes Parasites & Vectors Equine piroplasmosis Genotype Polymerase chain reaction Specificity Theileria equi |
| title | Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes |
| title_full | Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes |
| title_fullStr | Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes |
| title_full_unstemmed | Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes |
| title_short | Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes |
| title_sort | development and evaluation of specific polymerase chain reaction assays for detecting theileria equi genotypes |
| topic | Equine piroplasmosis Genotype Polymerase chain reaction Specificity Theileria equi |
| url | https://doi.org/10.1186/s13071-023-06045-z |
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