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A new PCR-RFLP method for detection of Anaplasma marginale based on 16S rRNA
1Veterinary Department of Isfahan Research Center for Agriculture and Natural Resources, Isfahan, Iran. vnoaman@yahoo.com
Abstract:
Species of the genus Anaplasma (Rickettsiales: Anaplasmataceae) are obligate intracellular tick borne pathogens. Three species of Anaplasma that infect cattle and sheep (A. marginale, A. centrale and A. ovis) are well recognized. Of these erythrocytic Anaplasma, A. marginale can cause diseases in the livestock with high economical losses. Species-specific PCR based on 16S rRNA gene is commonly used for detection of Anaplasma species but can not differentiate A. marginale, A. centrale and A. ovis because of sequence similarity. In this study DNA extraction was performed on 50 blood samples with presence of Anaplasma spp. in marginal point of erythrocytes in their blood smears. The extracted DNA from blood cells was analyzed by PCR and PCR-RFLP using primers derived from 16S rRNA gene and restriction endonuclease Bst1107 I. The restriction endonuclease Bst1107I only recognizes the sequence (GTATAC) in corresponding PCR product of A. marginale and cut it. The nucleotide sequence of the A. marginale 16S rRNA gene was determined and compared with the sequences of A. marginale in GenBank. The 16S rRNA of A. marginale in Iran was completely similar to the related sequence deposited in GenBank at accession number of M60313. In the present study we propose a new PCR-restriction fragment length polymorphism analysis (RFLP) method based on 16S rRNA gene for specific detection of A. marginale.
Insights
This study introduces a new PCR-restriction fragment length polymorphism (RFLP) method for accurately detecting Anaplasma marginale in livestock. This technique improves upon existing PCR methods by specifically identifying A. marginale, a significant cause of economic loss.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Parasitology
Background:
- Anaplasma species are tick-borne pathogens affecting livestock, with Anaplasma marginale causing significant economic losses.
- Current species-specific PCR methods targeting the 16S rRNA gene struggle to differentiate closely related Anaplasma species like A. marginale, A. centrale, and A. ovis due to sequence similarity.
Purpose of the Study:
- To develop and validate a novel molecular diagnostic method for the specific detection of Anaplasma marginale.
- To address the limitations of existing PCR techniques in differentiating Anaplasma species in cattle and sheep.
Main Methods:
- DNA extraction from 50 Anaplasma-infected cattle and sheep blood samples.
- Analysis using Polymerase Chain Reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) with primers targeting the 16S rRNA gene.
- Application of the restriction endonuclease Bst1107I, which specifically recognizes and cuts the PCR product of A. marginale.
Main Results:
- The PCR-RFLP method using Bst1107I successfully differentiated A. marginale from other Anaplasma species.
- The 16S rRNA gene sequence of the Iranian A. marginale isolates showed complete similarity to the GenBank reference sequence (accession number M60313).
Conclusions:
- A new, specific PCR-restriction fragment length polymorphism (RFLP) assay based on the 16S rRNA gene has been developed for the precise identification of Anaplasma marginale.
- This method offers improved diagnostic capability for Anaplasma infections in livestock, aiding in disease management and economic loss mitigation.
