Recombinant Dense Granule Protein (NcGRA4) Is a Novel Serological Marker for Neospora caninum Infection in Goats

Neosporosis caused by Neospora caninum is now recognised globally as one of the major causes of productivity losses in small and large ruminants. Its diagnosis is primarily based on serological techniques, and various serological tests have been developed utilising native-soluble antigens or live tachyzoites. However, these tests frequently cross-react with proteins from other protozoa. To overcome this problem, we produced a recombinant protein from a synthetic NcGRA4 gene and examined the ability to detect N. caninum infection in field samples using indirect enzyme-linked immunosorbent assay (iELISA). The technique demonstrated good specificity and sensitivity, and its use on serum samples from the field revealed that it may be used to effectively diagnose neosporosis in goats.Abstract: Neospora caninum is widely recognised as one of the most significant causes of abortion in cattle, with infections also occurring in sheep and goats. To prevent and control animal neosporosis, it is crucial to develop sensitive and specific methods for detecting N. caninum infection. Recently, several recombinant proteins have been utilised in serological assays for the diagnosis of neosporosis. In this study, we used commercial gene synthesis to produce dense granular antigen 4 (NcGRA4) recombinant protein. NcGRA4 plasmids were expressed in the Escherichia coli system and then purified. The purified recombinant protein was analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. To evaluate the diagnostic potential of recombinant NcGRA4 protein, we tested 214 serum samples from goat farms via indirect enzyme-linked immunosorbent assay (iELISA) and compared the results to those from the indirect fluorescent antibody test (IFAT). Western blotting analysis revealed a single NcGRA4 band with an expected molecular weight of 32 kDa. The specific IgG against N. caninum was detected in 34.1% and 35% of samples evaluated by NcGRA4 iELISA and IFAT, respectively. The sensitivity and specificity of the NcGRA4 iELISA were 71.6% and 86.3%, respectively, when compared with the results from IFAT. Our results demonstrate that a recombinant protein that can be used to detect animal neosporosis can be produced using a synthetic NcGRA4 gene. Overall, recombinant NcGRA4 shows promise as a sensitive and specific serological marker for identifying target IgG in goat samples.