Veterinary Research Forum

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
Subjects
1.     Jacob SS, Sengupta PP, Paramanandham K, et al. Bovine babesiosis: an insight into the global perspective on the disease distribution by systematic review and meta-analysis. Vet Parasitol 2020; 283: 109136. doi: 10.1016/j.vetpar.2020.109136.
2.     Young KM, Corrin T, Wilhelm B, et al. Zoonotic Babesia: a scoping review of the global evidence. PloS One 2019; 14(12): e0226781. doi: 10.1371/journal. pone.0226781.
3.     Jerzak M, Gandurski A, Tokaj M, et al. Advances in Babesia vaccine development: an Overview. Pathogens 2023; 12(2): 300. doi: 10.3390/pathogens12020300.
4.     Ozubek S, Bastos RG, Alzan HF, et al. Bovine babesiosis in Turkey: impact, current gaps, and opportunities for intervention. Pathogens 2020; 9(12): 1041. doi: 10.3390/pathogens9121041.
5.     Moura ACDB, Filho ES, Barbosa EM, et al. Comparative analysis of PCR, real-time PCR and LAMP techniques in the diagnosis of Trypanosoma vivax infection in naturally infected buffaloes and cattle in the Brazilian amazon. Pak Vet J 2024; 44(1): 123-128.
6.     Hakimi H, Yamagishi J, Kawazu SI, et al. Advances in understanding red blood cell modifications by Babesia. PLoS Pathog 2022; 18(9): e1010770. doi: 10.1371/ journal.ppat.1010770.
7.     Khan SS, Ahmed H, Afzal MS, et al. Epidemiology, distribution and identification of ticks on livestock in Pakistan. Int J Environ Res Public Health 2022; 19(5): 3024. doi: 10.3390/ijerph19053024.
8.     Ola-Fadunsin SD, Sharma RSK, Abdullah DA, et al. The molecular prevalence, distribution and risk factors associated with Babesia bigemina infection in Peninsular Malaysia. Ticks Tick Born Dis 2021; 12(3): 101653. doi: 10.1016/j.ttbdis.2021.101653.
9.     Atif FA, Nazir MU, Roheen T, et al. Antitheilerial efficacy of juglone, buparvaquone and oxytetracycline against tropical theileriosis in naturally infected crossbred cattle. Pak Vet J 2024; 44(1): 129-134.
10. Schnittger L, Rodriguez AE, Florin-Christensen M, et al. Babesia: a world emerging. Infect Genet Evol 2012; 12(8): 1788-1809.
11. Abbas RZ, Khan AMA, Mares MM, et al. Adulticidal, larvicidal, and repellent potential of ethyl acetate extract of Moringa oleifera against Rhipicephalusmicroplus cattle ticks. Kafkas Univ Vet Fak Derg 2024; 30(5): 719-727.
12. Sayed RH, Elsaady SA, Shasha FA, et al. Diagnosis of Pasteurella multocida and Mannheimia haemolytica infections in cattle using lateral flow immunochromatographic assay. Int J Vet Sci 2023; 12(5): 646-651.
13. Kamyingkird K, Yangtara S, Desquesnes M, et al. Seroprevalence of Babesia caballi and Theileria equi in horses and mules from Northern Thailand. J Protozool Res 2014; 24: 11-17.
14. Clausen C, Schou K, Kirkeby C, et al. Communicating spatial variation in tick-borne pathogen prevalence through a website based on national surveillance data. In Proceedings: 3rd Conference on Neglected Vectors and Vector-Borne Diseases. Zaragoza, Spain 2016; 82-83.
15. Mero WMS, Issa AR, Arif SH. Epidemiology and the types of isolated echinococcal cysts from sheep and cattle slaughtered at Zakho abattoirs, Zakho District. Kurdistan Region, Iraq. Continental Vet J 2023; 3(2): 94-102.
16. Torianyk II, Tymchenko OM, Ostapets MO, et al. Use of polymerase chain reaction in verification and differential diagnosis of babesiosis pathogens. Regul Mech Biosyst 2020; 11(4): 563-567.
17. El-Ashker M, Hotzel H, Gwida M, et al. Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray. Vet Parasitol 2015; 207(3-4): 329-334.
18. Mohanta UK, Chikufenji B, Galon EM, et al. Molecular detection and phylogenetic analyses of Babesia spp. and Theileria spp. in livestock in Bangladesh. Microorganisms 2023; 11(6): 1563. doi: 10.3390/microorganisms11061563.
19. Looi ML, Zakaria H, Osman J, et al. Quantity and quality assessment of DNA extracted from saliva and blood. Clin Lab 2012; 58(3-4): 307-312.
20. Desjardins P, Conklin D. NanoDrop microvolume quantitation of nucleic acids. J Vis Exp 2010; 45: 2565. doi: 10.3791/2565.
21. Sivakumar T, Kothalawala H, Abeyratne S, et al. A PCR-based survey of selected Babesia and Theileria parasites in cattle in Sri Lanka. Vet Parasitol 2012; 190(1-2): 263-267.
22. Akcakavak G, Karataş O, Tuzcu N, et al. Determination of apoptosis, necroptosis and autophagy markers by real-time PCR in naturally infected pneumonic pasteurellosis caused by Pasteurella multocida and Mannheimia haemolytica in cattle. Pak Vet J 2024; 44: 483-489.
23. Swords WE. Chemical transformation of E. coli. Methods Mol Biol 2003; 235: 49-53.
24. Sambrook J, Russell W. The condensed protocols from molecular cloning: a laboratory manual. Cold Spring Harbor Lab. Press, Cold Spring Harbor, 2006.
25. Kemmer S, Bang S, Rosenblatt M, et al. BlotIt-optimal alignment of Western blot and qPCR experiments. PloS One 2022; 17(8): e0264295. doi: 10.1371/journal. pone.0264295.
26. Malik MA, Subhani MI, Alvi MA, et al. High genetic variability in full-length cox2 and nad6 genes of Echinococcus granulosus sensu stricto and Echinococcus ortleppi recovered from cattle. Pak Vet J 2024; 44: 148-154.
27. Ahmed Z, Ali A, Waqas M, et al. Molecular identification and characterization of piroplasms infecting cattle in Azad Kashmir. Pak Vet J 2024; 44(3): 611-618.
28. Zaheer H, Rashid I, Akbar H, et al. Expiry of a completely splenectomised calf in post-operative period due to mixed piroplasm infection: a case report. Ann Parasitol 2020; 66(4): 599-606.
29. Ali KN, Marif HF. Babesiosis in cattle. Int J Agric 2023; 3: 114-121.
30. Toure A, Sanogo M, Sghiri A, et al. Diagnostic accuracy of an indirect Enzyme Linked Immunosorbent Assay (iELISA) for screening of Babesia bovis in cattle from West Africa. Life (Basel) 2023; 13(1): 203. doi: 10.3390/life13010203.
31. Haider A, Hussain K, Mares MM, et al. In vitro and in vivo anthelmintic activity of Nicotiana tabacum against Haemonchus placei in cattle. Pak Vet J 2024; 44(3): 745-750.
32. Bayer PM, Fabian B, Hübl W. Immunofluorescence assays (IFA) and enzyme-linked immunosorbent assays (ELISA) in autoimmune disease diagnostics- -technique, benefits, limitations and applications. Scand J Clin Lab Invest Suppl 2001; 235: 68-76.
33. Kebzai F, Ashraf K, Rehman MU, et al. Molecular analysis and associated risk factors of Theileria annulata in cattle from various zones of Balochistan, Pakistan. Kafkas Univ Vet Fak Derg 2024; 30(1): 15-21.
34. AbouLaila M, Terkawi M, Menshawy S, et al. Identification, characterization and protective effects of the apical membrane antigen-1 (AMA-1) homolog from the Argentina strain of Babesia bigemina. Ann Clin Investig 2019;1(1): 1001.
35. Dominguez M, Zabal O, Wilkowsky S, et al. Use of a monoclonal antibody against Babesia bovis merozoite surface antigen‐2c for the development of a competitive ELISA test. Ann N Y Acad Sci 2004; 1026: 165-170.
36. Molloy JB, Bowles PM, Jeston PJ, et al. Development of an enzyme-linked immunosorbent assay for detection of antibodies to Babesia bigemina in cattle. Parasitol Res 1998; 84(4): 651-656.
37. Rajesh V, Elamaran M, Vidya S, et al. Plasmodium vivax: genetic diversity of the apical membrane antigen-1 (AMA-1) in isolates from India. Exp Parasitol 2007; 116(3): 252-256.
Veterinary Research Forum
Volume 17, Issue 6
June 2026
Pages 399-405

  • Receive Date 10 July 2025
  • Revise Date 07 September 2025
  • Accept Date 11 October 2025

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