Development and Evaluation of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Diagnosis of Babesia ovis Infection in Sheep

Habibi, Gholamreza; Afshari, Asghar; Fathi, Saeed; Karimi, Gholamreza; Shahedi, Amin

doi:10.66224/ari.2026.372293.4060

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Diagnosis of Babesia ovis Infection in Sheep

Articles in Press

Document Type : Original Articles

Authors

¹ Department, Razi Vaccine and Serum Research Institute

² Parasite vaccine Research and production Department of Razi institute

³ Parasitology Department of Razi institute

10.66224/ari.2026.372293.4060

Abstract

Background: Babesiosis causes significant economic losses in tropical regions like Iran. While microscopy misses subclinical carriers and PCR is costly for routine use, crude antigen ELISA offers a practical, affordable alternative for resource-limited laboratories.
Objectives: This study aimed to develop and optimize an indirect enzyme-linked immunosorbent assay (iELISA) for Babesia ovis using crude antigens. We also monitored the temporal correlation between parasitemia and serological responses in an experimentally infected sheep.
Methods: Crude B. ovis antigen was prepared from infected sheep erythrocytes. The iELISA was optimized via checkerboard titration (antigen: 2 µg/mL; serum dilution: 1:100; HRP-conjugated anti-sheep IgG: 1:25,000). Diagnostic performance was evaluated using 20 positive and 32 confirmed negative sera. A healthy lamb was experimentally infected with cryopreserved B. ovis-infected blood. Monthly sampling included EDTA blood for PCR, serum for iELISA, and Giemsa-stained smears.
Results: The iELISA demonstrated 85% sensitivity, 84% specificity, 85% accuracy, 77% positive predictive value (PPV), and 90% negative predictive value (NPV). In the infected lamb, fever and acute parasitemia appeared on day 10 post-infection and resolved after imidocarb treatment. On day 12, microscopy, PCR, and iELISA were positive. During the carrier phase, microscopy was negative, while PCR and iELISA remained positive. By day 270, both microscopy and PCR were negative, but iELISA remained positive, indicating a persistent humoral immune response.
Conclusion: The developed crude antigen-based iELISA serves as a cost-effective tool for initial serological screening of B. ovis in sheep. With a high NPV (90.0%), it is particularly effective for screening, with PCR reserved for confirmation.

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