Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immunoassay in horses from São Paulo State, Brazil.
Abstract: The erythrocytic-stage surface protein, Equi Merozoite Antigen 1 (EMA-1), is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding the entire EMA-1 of Theileria equi Jaboticabal strain was cloned and expressed in Escherichia coli as a histidine-tagged protein (His6-EMA1). The expressed EMA-1 reacted with specific antibodies in Western blot and had an apparent molecular mass of 34 kDa which was largely consistent with its theoretical value. The nucleotide sequence of the EMA-1 gene of Jaboticabal strain was comparatively analyzed with other published sequences. The results indicated a high degree of homology with EMA-1 genes of all other strains isolated from various countries. The recombinant purified His6-EMA1 protein was tested in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies anti-T. equi in horses. The ELISA clearly differentiated T. equi-infected from Babesia caballi-infected horse sera or normal horse sera. Field serum samples collected from horses in the State of São Paulo, Southeastern Brazil, were examined for the diagnosis of T. equi infection by ELISA. Of 170 samples analyzed, 95.88% (163/170) were positive for T. equi infection. These results suggest that the His6-EMA1 protein expressed in E. coli could be a reliable immunodiagnostic antigen for ELISA test and that T. equi infection is a serious concern in the State of São Paulo, Brazil.
Publication Date: 2011-03-29 PubMed ID: 21439233DOI: 10.1590/s1984-29612011000100011Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The researchers produced a recombinant protein known as EMA-1 to diagnose Theileria equi, a horse disease. This diagnostic tool was tested in horses in São Paulo State, Brazil, revealing a high infection rate.
Understanding the Research
- This study focuses on equine piroplasmosis, which is a disease in horses caused by Theileria equi. The team sought to design a reliable diagnostic method for this disease.
- Equi Merozoite Antigen 1 (EMA-1), an erythrocytic-stage surface protein, was chosen to develop this diagnostic. EMA-1 was cloned from the Theileria equi Jaboticabal strain and then expressed in the Escherichia coli bacterium as a histidine-tagged protein (His6-EMA1).
Key Findings
- The expressed EMA-1 reacted with specific antibodies when tested with Western blot, a common method for detecting specific proteins.
- Its molecular mass primarily agreed with the expected value, validating the successful production of EMA-1.
- The EMA-1 gene showed a high degree of homology when compared with published sequences, indicating a strong similarity to EMA-1 genes from various other strains isolated globally.
- The purified His6-EMA1 protein was then used in an enzyme-linked immunosorbent assay (ELISA) to detect anti-T. equi antibodies in horses.
- It was found that the ELISA successfully distinguished between equi-infected horses and Babesia caballi-infected horses or healthy horses, demonstrating the reliability of this diagnostic tool.
- When field serum samples from São Paulo were tested with ELISA, 95.88% (163/170) were found to be positive for T. equi infection, indicating a widespread presence of the disease in this region.
Implications of the Study
- The successful expression of the His6-EMA1 protein in E. coli suggests this method could be used in the future to produce immunodiagnostic antigens reliably.
- The ELISA test developed in the study can effectively differentiate between T. equi and Babesia caballi infections in horses, which could drastically improve diagnostic accuracy for equine piroplasmosis.
- The high prevalence of T. equi infection found in São Paulo underscores the need for effective diagnostic methods and possibly, urgent interventions to manage and control the disease.
Cite This Article
APA
Baldani CD, Hilario E, Nakaghi AC, Bertolini MC, Machado RZ.
(2011).
Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immunoassay in horses from São Paulo State, Brazil.
Rev Bras Parasitol Vet, 20(1), 54-60.
https://doi.org/10.1590/s1984-29612011000100011 Publication
Researcher Affiliations
- Departamento de Medicina e Cirurgia Veterinaria, Instituto de Veterinaria, Universidade Federal Rural do Rio de Janeiro, Brazil.
MeSH Terms
- Animals
- Brazil
- Horse Diseases / diagnosis
- Horse Diseases / immunology
- Horses
- Immunoenzyme Techniques
- Protozoan Proteins / analysis
- Protozoan Proteins / biosynthesis
- Recombinant Proteins / analysis
- Recombinant Proteins / biosynthesis
- Theileriasis / diagnosis
- Theileriasis / immunology
Citations
This article has been cited 3 times.- Yang G, Zhou B, Chen K, Hu Z, Guo W, Wang X, Du C. Diagnostic Performance of Competitive ELISA and Western Blot Methods for the Detection of Antibodies against Theileria equi and Babesia caballi. Microorganisms 2022 Dec 21;11(1).
- Yang G, Chen K, Guo W, Hu Z, Qi T, Liu D, Wang Y, Du C, Wang X. Development of a Test Card Based on Colloidal Gold Immunochromatographic Strips for Rapid Detection of Antibodies against Theileria equi and Babesia caballi. Microbiol Spectr 2022 Feb 23;10(1):e0241121.
- Tirosh-Levy S, Gottlieb Y, Fry LM, Knowles DP, Steinman A. Twenty Years of Equine Piroplasmosis Research: Global Distribution, Molecular Diagnosis, and Phylogeny. Pathogens 2020 Nov 8;9(11).
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