Optimization of the most widely used serological tests for a harmonized diagnosis of Toxoplasma gondii infection in domestic pigs

A combination of GRA3, GRA6 and GRA7 peptides offer a useful tool for serotyping type II and III Toxoplasma gondii infections in sheep and pigs

“…In pigs, on the other hand, no significant differences were observed when comparing the two different type II and two different type III strains beyond a slightly better prediction rate for GRA6-I/III-213 vs. GRA6-II-214 and GRA3-I/III-43 vs. GRA3-II-43 ratios for the ShSp1 type II and the ShSp24 type III infected piglets, respectively. Since the ShSp1 and CZ-Tiger belong to the same ToxoDB genotype #3, and the ShSp24 and CZ-Šimková belong to the same ToxoDB genotype #2 ( López-Ureña et al., 2023 ), similar reactivity patterns were expected. Nonetheless, as stated above, it is possible that these slight differences in prediction rates can be explained by the type of animal infected rather than the isolate itself, as piglets, which showed the better prediction rates, were infected with the ShSp1 and ShSp24 strains ( Largo-de la Torre et al., 2022 ).…”

“…Given the promising results obtained with sheep sera, we set out to investigate the usefulness of the same 10 peptides in pigs. A total of 40 samples from previous experimental infections performed in 20 piglets ( Largo-de la Torre et al., 2022 ) and 20 sows ( López-Ureña et al., 2023 ) were analyzed: 22 serum samples from 11 animals infected with the type II ShSp1 (n=10) and CZ-Tiger (n=12) strains, as well as 18 serum samples from 9 animals infected with the type III ShSp24 (n=10) and CZ- Šimková (n=8) strains ( Table 1 , Supplementary File 1 ).…”

“…Firstly, serum samples from seropositive pregnant sheep orally infected at day 90 of gestation with 10 or 1000 oocysts of the TgShSp1 (type II, ToxoDB #3), TgShSp16 (type II, ToxoDB #3), TgShSp24 (type III, ToxoDB #2) ( Fernández-Escobar et al., 2020 ) and ME49 (type I, ToxoDB #1) ( Sánchez-Sánchez et al., 2019 ) strains were collected at different times post-infection (pi). Similarly, serum samples from seropositive piglets obtained at 21 and 42 days after being orally infected with 1000 oocysts of the TgShSp1 and TgShSp24 strains (type II and III, ToxoDB #3 and #1, respectively) ( Largo-de la Torre et al., 2022 ), as well as serum samples from prepubertal sows orally infected with 400 oocysts of the CZ-Tiger (type II, ToxoDB #3) and CZ-Šimková (type III, ToxoDB #2) strains ( López-Ureña et al., 2023 ) were used. Further details on each individual sample regarding ID, antibody levels, days post-infection (dpi) or specific uses can be found in Supplementary File 1 .…”

“…Previous studies from our group have established laboratory developed ELISA protocols for testing sheep and pig serum samples against T. gondii infection ( Sánchez-Sánchez et al., 2019 ; Largo-de la Torre et al., 2022 ; López-Ureña et al., 2023 ; Vallejo et al., 2023 ). In the present work, however, we aimed at standardizing sheep and pig ELISAs using the best conditions with a whole tachyzoite soluble antigen, so as to better compare our results to those obtained in the most recent T. gondii serotyping work performed with mouse and human samples ( Arranz-Solís et al., 2021 ) while minimizing the background noise.…”

“…To develop the enzymatic reaction, ABTS was used as above, and the OD 405 measured until the positive control incubated with the soluble antigen reached values of 1.0-1.2, usually within 60-90 minutes, after which the reaction was stopped by addition of 100 μl per well of a 0.3N oxalic acid solution. Previously characterized positive and negative serum samples ( López-Ureña et al., 2023 ) were used in each plate, with positive/negative ratios ranging from 11 to 15.…”