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[Cloning, expression, molecular characterization of the MSP5 protein from PR1 strain of Anaplasma marginale and its
Elizabete Regina M Marana1, Flora S Kano, Josy C Vicentini
1Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina - UEL, Campus Universitário, Rod. Celso Garcia Cid, PR 445, km 380, CP 6001, CEP 86051-990 Londrina, PR, Brasil.
Abstract:
A competitive enzyme-linked immunosorbent test using the PR1 recombinant major surface protein 5 (rMSP5-PR1-ELISA) of Anaplasma marginale was standardized and validated using sera from anaplasmosis free and endemic regions. The sequencing of the msp5 gene of PR1 isolate showed 98% of identity with the Florida and Saint Maries isolates, 97% with Brazil (Pernambuco) and Havana isolates; and 91% with A. centrale. The cELISA-PR1 test was compared to IFI and cELISA-USA. For the standardization and validation of the cELISA-PR1, 380 bovine sera were used, whereas 245 truly positives and 135 truly negatives sera tested by the cELISA-USA. In the standardization of the cELISA-PR1 135 negative and 148 positive bovine sera were used. The cELISA-PR1 and IFI tests showed 100 and 99.3% specificity, 100 and 98%, sensibility, and a kappa coefficient of 0.993 and 0.978, respectively. For test validation, 245 bovine sera from an anaplasmosis endemic area were analyzed by the cELISA-PR1 and IFI, which showed 96.7 and 69.1% specificity, 98.9 e 96.3% sensibility and kappa coefficient of 0.956 and 0.699, respectively. These results indicate that the cELISA-PR1, likewise the cELISA-USA, could sensitively and specifically detect cattle naturally infected with A. marginale and would be recommended for epidemiological studies, eradications program, and regulation of international cattle movement, while IFI, which presented lower specificity should not be used in situations that demand more specific diagnosis.
Insights
A new competitive enzyme-linked immunosorbent assay (cELISA-PR1) effectively detects Anaplasma marginale in cattle. This highly sensitive and specific test is recommended for epidemiological studies and disease control programs.
Area of Science:
- Veterinary Immunology
- Molecular Diagnostics
- Bovine Infectious Diseases
Background:
- Anaplasma marginale causes significant economic losses in cattle worldwide.
- Accurate diagnostic tools are crucial for managing bovine anaplasmosis.
- Existing diagnostic methods like Indirect Fluorescent Antibody test (IFI) may lack sufficient specificity.
Purpose of the Study:
- To standardize and validate a competitive enzyme-linked immunosorbent assay (cELISA-PR1) for detecting Anaplasma marginale.
- To compare the performance of cELISA-PR1 against established diagnostic tests (IFI and cELISA-USA).
- To assess the utility of cELISA-PR1 in epidemiological studies and disease control.
Main Methods:
- Standardization and validation of cELISA-PR1 using 380 bovine sera from endemic and free regions.
- Genetic sequencing of the msp5 gene from the PR1 isolate for comparison with other isolates.
- Comparative analysis of cELISA-PR1 with IFI and cELISA-USA using sera tested by cELISA-USA.
Main Results:
- The cELISA-PR1 demonstrated high sensitivity (98-100%) and specificity (99.3-100%) during standardization and validation.
- The PR1 isolate's msp5 gene showed high sequence identity (91-98%) with other Anaplasma marginale isolates.
- cELISA-PR1 exhibited superior specificity compared to IFI, especially in validation tests (96.7% vs 69.1%).
Conclusions:
- The cELISA-PR1 is a sensitive and specific diagnostic tool for detecting cattle infected with Anaplasma marginale.
- cELISA-PR1 is suitable for epidemiological surveillance, eradication programs, and regulating international cattle movement.
- IFI is less recommended for specific diagnoses due to its lower specificity.
