Vet World   Vol.18   July-2025  Article - 9 

Development and validation of a recombinant Rap1-based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in Kazakhstan

Background and Aim: Bovine babesiosis, caused by Babesia bovis, poses significant economic challenges to Kazakhstan’s cattle industry. Early and accurate detection is crucial for interrupting transmission cycles, particularly in regions lacking advanced diagnostic infrastructure. This study aimed to develop a rapid lateral flow immunoassay (LFIA) using a recombinant C-terminal fragment of the recombinant rhoptry-associated protein 1 (rRap1) antigen for the serodiagnosis of bovine babesiosis.

Materials and Methods: A C-terminal fragment (amino acids 345–480) of the B. bovis Rap1 gene was codon optimized and expressed in Escherichia coli. The recombinant protein was purified using metal-affinity chromatography and validated through sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and nanoflow liquid chromatography-tandem mass spectrometry. A diagnostic evaluation was performed using enzyme-linked immunosorbent assay (ELISA) and LFIA on sera from 102 uninfected and 15 infected cattle, all of which had been pre-tested using polymerase chain reaction. Colloidal gold-protein G conjugates were prepared for LFIA, and test conditions were optimized for antigen concentration and serum dilution. Assay performance was compared with previously published LFIAs.

Results: A 21-kDa rRap1 protein was successfully expressed and demonstrated high specificity to positive control sera. ELISA and LFIA both detected antibodies in 13 of 15 infected samples (sensitivity 86.6%). Specificity was 90.1% for ELISA and 88.2% for LFIA. Receiver operating characteristic analysis showed an area under the curve of 0.83, and Cohen’s Kappa indicated fair-to-moderate agreement between ELISA and LFIA. The LFIA exhibited comparable performance to assays based on merozoite surface antigen 1 or spherical body protein antigens, marking the first successful use of a B. bovis Rap1 C-terminal fragment for LFIA-based field diagnostics in Kazakhstan.

Conclusion: The developed rRap1-based LFIA is a promising, field-deployable diagnostic tool for bovine babesiosis, offering rapid results without the need for laboratory equipment. Despite slightly lower sensitivity than ELISA, its simplicity, cost-effectiveness, and specificity support its use in large-scale epidemiological surveillance. Further validation in diverse field conditions and cattle populations is recommended to refine sensitivity and broaden applicability.

Keywords: Babesia bovis, bovine babesiosis, enzyme-linked immunosorbent assay, lateral flow immunoassay, rapid diagnostics, recombinant rhoptry-associated protein 1, serodiagnosis.

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How to cite this article: Mukantayev K, Adish Z, Kanayev D, Tokhtarova L, Abirbekov B, Abduraimov Y, Rsaliyev A, and Tursunov K (2025) Development and validation of a recombinant Rap1-based lateral flow immunoassay for rapid serodiagnosis of bovine babesiosis in Kazakhstan, Veterinary World, 18(7):1881-1890.

Received: 20-03-2025   Accepted: 06-06-2025   Published online: 11-07-2025

Corresponding author: Kanat Tursunov    E-mail: kanat_tka@mail.ru

DOI: 10.14202/vetworld.2025.1881-1890

Copyright: Mukantayev, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.