[An indirect ELISA for the detection of Babesia caballi in equine animals].
Abstract: To clone and express BC48 gene of Babesia caballi, and to establish an indirect ELISA for the diagnosis of B. caballi in equine animals. Methods: The genomic DNA of B. caballi was extracted from the infected donkey blood. BC48 gene was amplified by PCR. The PCR product was cloned into expression plasmid pET28a, and expressed in E. coli BL21 with IPTG induction. The recombinant protein was purified by Ni-NTA affinity chro-matography and was used as a diagnostic antigen to establish an indirect ELISA. The reaction conditions of the indirect ELISA were optimized. Specificity and sensitivity of this method were evaluated. Results: BC48 gene of B. caballi was 1 272 bp. The recombinant protein was expressed in E. coli BL21 as a soluble protein with a molecular weight of about M, 46 000 under induction of IPTG. The concentration of purified protein was 12.98 mg/ml. The best conditions were obtained for the ELISA when the antigen concentration was 65 microg/ml with the serum dilution of 1:80. The protein specifically reacted with serum from donkey infected by B. caballi, but did not react with serum from donkey infected by Theileria equi (B. equi). Both ELISA and microscopy were applied to examine 17 donkeys in the field, 3 were positive by ELISA and 2 were found parasite-positive, respectively. Conclusions: The indirect ELISA method may be used to detect B. caballi infection in equine animals.
Publication Date: 2010-09-02 PubMed ID: 20806504
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- English Abstract
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research discusses the cloning and expression of the Babesia caballi BC48 gene in order to create an indirect ELISA for the diagnosis of B. caballi in equine animals. The study proved successful, with the ELISA showing specific reactivity to B. caballi infected serum and promising applicability for detection of the infection in horses and donkeys.
Methodology
- Babesia caballi, a pathogenic protozoan, was extracted from infected donkey blood, and the BC48 gene was subsequently amplified using PCR (Polymerase Chain Reaction).
- The amplified BC48 gene was inserted into an expression plasmid pET28a and expressed in the bacteria E. coli BL21.
- The expression was induced by IPTG (a molecule which induces protein expression).
- The recombinant protein was then purified using a method called Ni-NTA affinity chromatography and utilized as a diagnostic antigen to establish an indirect Enzyme-Linked Immunosorbent Assay (ELISA).
- The reaction conditions of the indirect ELISA were optimized with respect to the serum/antigen interaction.
- Testing was performed to gauge the specificity and sensitivity of this diagnostic method.
Results
- The BC48 gene, 1272 base pairs long, was successfully cloned and expressed in E. coli BL21. The expressed protein had a molecular weight of approximately 46,000 grams per mole.
- The protein was obtained at a concentration of 12.98 mg/ml after purification.
- Optimized conditions of the ELISA determined an antigen concentration of 65 micrograms/ml and serum dilution of 1:80.
- Serum from B. caballi-infected donkeys reacted specifically to the protein, indicating successful infection detection. However, there was no reaction when tested with serum from donkeys infected with Theileria equi (another protozoan), thereby proving the specificity of the diagnostic method.
- Using both ELISA and microscopic examination, 17 donkeys were tested in the field. Three were determined positive by ELISA and two were found parasite-positive upon microscopic inspection.
Conclusions
- The ELISA diagnostic method developed in this research could potentially be used for detection of B. caballi infection in equine animals such as donkeys and horses.
Cite This Article
APA
Gong ZL, Liu GY, Xie JR, Chai HP, Zhang LY, Li ZX, Tian ZC, Wang L, Liu JG.
(2010).
[An indirect ELISA for the detection of Babesia caballi in equine animals].
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 28(3), 200-204.
Publication
Researcher Affiliations
- Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
MeSH Terms
- Animals
- Babesia / cytology
- Babesia / immunology
- Babesia / isolation & purification
- Babesiosis / diagnosis
- Babesiosis / parasitology
- Babesiosis / veterinary
- Enzyme-Linked Immunosorbent Assay / methods
- Enzyme-Linked Immunosorbent Assay / veterinary
- Horse Diseases / diagnosis
- Horse Diseases / parasitology
- Horses
- Protozoan Proteins / isolation & purification
Citations
This article has been cited 2 times.- Wang M, Guo W, Igarashi I, Xuan X, Wang X, Xiang W, Jia H. Epidemiological investigation of equine piroplasmosis in China by enzyme-linked immunosorbent assays. J Vet Med Sci 2014 Apr;76(4):549-52.
- Ullah A, Geng M, Chen W, Zhu Q, Shi L, Zhang X, Akhtar MF, Wang C, Khan MZ. Effect of Parasitic Infections on Hematological Profile, Reproductive and Productive Performance in Equines. Animals (Basel) 2025 Nov 14;15(22).
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