Neospora (N.) caninum is an obligate intracellular protozoan parasite of economic importance for the dairy industry due to its abortifacient nature. This study aimed to develop an N. caninum IgG ELISA Kit (N) based on the recombinant antigen (rNcSRS2) to diagnose bovine neosporosis. We cloned C terminal region comprising of 1054 base pairs of NcSRS2 in expression vector pET-28α and expressed it in the prokaryotic expression system (BL21, DE3). The expressed protein was purified using Nickel-affinity-columns and was characterized by immunoblotting. Purified 0.5µg/mL of rNcSRS2 was used to coat flat-bottomed microtiter polystyrene plate to develop the ELISA kit (kit N) for diagnosis of bovine neosporosis. To this end, 400 cattle sera were screened through our newly developed kit N. Out of 400 samples, 72 (34 positive and 38 negative) were selected to be tested with commercially available N. caninum Antibody Test Kit, cELISA (E) in comparison with kit N. Out of the 34 samples tested positive with kit N, 33 were also found positive in gold standard (kit E), while all the 38 samples tested negative in kit N were also found negative with gold kit E. Thus, the sensitivity of the kit N was found as 100% and the specificity as 97.4% (95% C.I: 97.277-97.595) and the cut-off value was 0.54. Overall prevalence of neosporosis in three districts of Punjab was 8.5% (34/400; 95% CI: 5.77-11.23). The district wise prevalence of neosporosis in cattle herds in Lahore, Sheikhupura and Narowal was 10.5% (22/210; 95% CI: 6.33-14.63), 4.5% (05/112; 95% CI: 0.64-8.29) and 9.0% (7/78; 95% CI: 2.63-15.32), respectively. The promising sensitivity and specificity of this format of ELISA allows for designing larger scale and more reliable sero-epidemiological surveys of bovine neosporosis across diverse cattle herds in different regions of Pakistan.

To Cite This Article: Shabir S, Younus M, Rahman S, Khan S, Abbasi AM, Hussain N, Younas M, Rashid MI, Suleman M and Akbar H, 2026. Development of rNcSRS2 IgG ELISA kit for serological investigation of bovine neosporosis at dairy farms of Punjab, Pakistan. Pak Vet J, 46(2): 321-328. http://dx.doi.org/10.29261/pakvetj/2026.024