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Detection of Theileria orientalis in Iran by semi-nested PCR
Peyman Ghaemi1, Nasser Hoghooghi-Rad, Parviz Shayan
1Department of Parasitology and Mycology, Science and Research Branch, Islamic Azad University, Tehran, Iran. vetiran@gmail.com
Insights
A new semi-nested PCR method successfully identified Theileria orientalis and Theileria annulata co-infections in native Iranian cattle. This molecular assay confirms T. orientalis presence in northern Iran, aiding in disease diagnosis.
Area of Science:
- Veterinary Parasitology
- Molecular Diagnostics
- Food Animal Health
Background:
- Theileria species are tick-borne protozoan parasites causing significant economic losses in cattle globally.
- Distinguishing between Theileria orientalis and Theileria annulata is crucial for effective disease management, especially when co-infections occur.
- Previous diagnostic methods lacked the specificity to differentiate these closely related Theileria species in mixed infections.
Purpose of the Study:
- To develop and validate a semi-nested PCR assay for the accurate identification and differentiation of Theileria orientalis and Theileria annulata in cattle.
- To investigate the prevalence of Theileria species, including co-infections, in native cattle populations in Golestan province, Iran.
- To provide a molecular tool for the first-time detection of Theileria orientalis in Iranian cattle.
Main Methods:
- Collected 160 blood samples from apparently healthy native cattle in Golestan province, Iran (2009-2011).
- Employed a semi-nested PCR strategy utilizing specific primer sets targeting the 18S rRNA gene for Theileria sp., T. orientalis, and T. annulata.
- Analyzed PCR products using 2% agarose gel electrophoresis to determine amplicon sizes and confirm species-specific infections.
Main Results:
- Initial PCR detected Theileria sp. in 13 out of 160 samples (8.12%).
- Semi-nested PCR identified T. orientalis in 9 of these 13 positive samples (5.62%) and T. annulata in 12 of the 13 samples (7.5%).
- This study provides the first molecular evidence of T. orientalis in native cattle in northern Iran and demonstrates the capability to detect concurrent infections.
Conclusions:
- The developed semi-nested PCR assay is effective for differentiating T. orientalis and T. annulata, including in cases of co-infection.
- This molecular diagnostic approach confirms the presence of T. orientalis in native cattle in northern Iran.
- The findings highlight the importance of molecular diagnostics for accurate Theileria species identification and epidemiological surveillance in cattle.
Abstract:
In order to identify and differentiate Theileria orientalis in cattle which may be infected with Theileria annulata simultaneously, a semi-nested PCR was performed. Thus, 160 blood samples were collected from apparently healthy native cattle in Golestan province of northern Iran, during 2009 to 2011. The Tbs-S/Tbs-A primer set derived from the 18S rRNA encoding gene was used for first PCR amplification, and the amplified sequence weight by this primer set for Theileria sp. was 426-430 bp. Then, DNA solution from purified PCR product was used for the semi-nested PCR analysis. The first PCR product amplified using T. orientalis primer set (To-S/Tbs-A) derived from the 18SrRNA encoding gene, and this specific primer weight was 235 bp. Also, the first PCR product amplified using T. annulata primer set (Ta-S/Tbs-A) derived from the 18SrRNA encoding gene and this specific primer weight was 193 bp. Having extracted DNA of each sample, using Tbs-S/Tbs-A primer set for PCR and analyzing the PCR products on the 2% agarose gel electrophoresis, 13 out of 160 blood samples (8.12%) were positive for Theileria sp. Meanwhile, performing semi-nested PCR with T. orientalis-specific primers, 9 out of 13 blood samples (5.62%) were positive and performing semi-nested PCR with T. annulata-specific primers, 12 out of 13 blood samples (7.5%) were also positive. This molecular assay approves the presence of T. orientalis in the native cattle of northern parts of Iran for the first time. In addition, this procedure will detect the concurrent infection of T. orientalis and T. annulata in the cattle too.