A comparative study of serological tests used in the diagnosis of Toxoplasma gondii infection in small ruminants evidenced the importance of cross-reactions for harmonizing diagnostic performance

Toxoplasma gondii is a major foodborne zoonotic pathogen that can be transmitted through the consumption of raw or undercooked meat of small ruminants, among others. Serology has been suggested as an epidemiological indicator and several tests are available nowadays. However, there is no comparative study with the most used ones. Therefore, the objective of this study was to develop and validate two in-house tests (Western blot -TgSALUVET WB- and ELISA -TgSALUVET ELISA 2.0-) and perform a comparative study including such tests and four commercial ELISA kits (IDScreen®, PrioCHECK®, Pigtype® and IDEXX). First, a specific pattern of recognition of immunodominant antigens by TgSALUVET WB was determined with serum panels of noninfected sheep and sheep infected with T. gondii or Neospora caninum. Next, TgSALUVET WB was used as a reference to preliminary validate TgSALUVET ELISA 2.0 using sera from sheep and goats naturally infected with T. gondii. Then, the abovementioned sheep serum panels were analyzed by all tests and subjected to TG-ROC analyses and agreement tests, and cross-reactivity with the anti-N. caninum IgGs was studied. All the techniques were accurate enough for the cutoff values initially suggested with all serum panels (Se and Sp ≥ 94%), except for PrioCHECK®, which showed 83% Sp. However, a cutoff readjustment improved their diagnostic performance. Additionally, cross-reactions between anti-N. caninum antibodies and T. gondii antigens were detected with all tests. Thus, a second cutoff readjustment was carried out and the use of both readjusted cutoff values is recommended to obtain comparable data and avoid false-positive results. •A restrictive criterion of positivity was defined for an in-house Western blot test.•Updated diagnostic performance of commonly used serological tests is provided.•Cross-reactivity with anti-N. caninum IgGs was found in all tests.•Readjusted cutoffs are suggested to harmonize data and avoid false-positive results.