Development of apical membrane antigen-1 based ELISA kit for the diagnosis of Babesia infection in cattle

Mahmood, Muhammad Shahid; Rafique, Azhar; Aslam, Faiza; Hafeez, Mian Abdul; Aslam, Bilal; Aljasir, Sulaiman Fahad

doi:10.30466/vrf.2025.2065465.4829

Development of apical membrane antigen-1 based ELISA kit for the diagnosis of Babesia infection in cattle

Document Type : Original Article

Authors

Muhammad Shahid Mahmood ¹

Azhar Rafique ²

Faiza Aslam ³

Mian Abdul Hafeez ⁴

Bilal Aslam ⁵

Sulaiman Fahad Aljasir ⁵

¹ Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan

² Department of Zoology, Faculty of Life Sciences, Government College University, Faisalabad, Pakistan

³ Livestock and Dairy Development Department, Government of Punjab, Punjab, Pakistan

⁴ Department of Parasitology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore, Pakistan

⁵ Department of Veterinary Preventive Medicine, College of Veterinary Medicine, Qassim University, Buraydah, Saudi Arabia

https://doi.org/10.30466/vrf.2025.2065465.4829

Abstract

The current finding reports the development of immunocapture-enzyme linked immunosorbent assay (iELISA) with recombinant apical membrane antigen-1 (rAMA-1) to identify an infection of Babesia in naturally infected cattle. The 48.00 kDa protein-encoding rAMA-1 gene was cloned into the pET-28a (+) expression vector and expressed in Escherichia coli. The resulting congregate protein was refined under native settings. Towards the evaluation of the diagnostic potential of AMA-1 as a sero-diagnostic reagent, a panel of sera samples from Babesia infected cattle and uninfected sera, as well as Babesia positive samples with other species, including B. bigemina, B. divergens, B. major, B. occultans, were utilized. Additionally, the efficacy of rAMA-1-based serological assays was compared with commercially available kits using 200 samples taken from cattle suspected of babesiosis. The results demonstrated that the iELISA using rAMA-1 exhibited a diagnostic sensitivity of 88.89%, when compared to the commercially available ELISA kit as a reference test. The specificity of this assay was 76.66%. These findings suggest that the iELISA employing rAMA-1 can be utilized on large-scale epidemiological surveys and clinical detection of Babesia infection in cattle.

Keywords

Apical membrane antigen-1 gene

Babesia

Domestic cattle

ELISA