Diagnostic approach for early Toxoplasma gondii infection in pregnant sheep based on IgM serology

IgM detection is commonly employed for early diagnosis in human congenital toxoplasmosis. In small ruminants, accurate methods are needed to identify early foetal death (8-13 days post-infection), which often occurs without IgG seroconversion and parasite detection in the foetuses. The study aimed to develop and validate an ELISA based on lyophilized Toxoplasma gondii tachyzoites to detect anti-T. gondii IgM antibodies (IgM-ELISA) for sheep sera following WOAH guidelines. Using IgM-Western Blot (IgM-WB) as a reference test, sera panels comprising 735 sera from sheep experimentally- and naturally-infected with either T. gondii or other abortifacient pathogens were used. The IgM-TgSALUVET-ELISA demonstrated successful analytical validation, showing excellent precision (CV < 5%), good accuracy (R² = 0.85-0.96) and analytical sensitivity (detection limit: DO = 0.1). While IgM-WB showed no cross-reactivity between T. gondii antigens and anti-Neospora caninum and anti-Chlamydia abortus antibodies, IgM ELISA yielded false positives. IgM kinetics analyses in T. gondii experimentally-infected sheep enabled early diagnosis capability of IgM-ELISA and IgM-WB, detecting IgM immunoglobulins at 10 and 13 dpi, aligning with the early abortion timeframe. The IgM-ELISA achieved 84% diagnostic sensitivity, 83.9% specificity, AUC = 0.88, and substantial agreement with IgM-WB (Kappa = 0.61). Field investigations based on paired and sequential IgM/IgG comparisons suggest the IgM detection is useful as a marker of recent T. gondii infection after excluding other abortifacient pathogens. However, prolonged anti-T. gondii IgM persistence may complicate the interpretation of results. In sum, the IgM-ELISA test showed good performance under the conditions tested, although potential interferences limit its use as a stand-alone assay. Therefore, a sequential diagnostic approach in the event of suspected foetal death during early T. gondii infection in a flock is proposed.