Archetypal type II and III Toxoplasma gondii oocysts induce different immune responses and clinical outcomes in experimentally infected piglets

Abstract

Livestock animals, such as swine, are an important source of Toxoplasma gondii in the human population. Currently, there is limited knowledge regarding the potential influence that the T. gondii genotype might exert on establishing infection in swine. Herein, we investigated the role of 2 T. gondii isolates, type II and III, representative of the genotypes circulating in Europe, in the immune responses and infection dynamics in piglets. Recently obtained oocysts (103) from the T. gondii field isolates TgShSp1 (type II, ToxoDB genotype #3) and TgShSp24 (type III, #2) were used for oral infection. Thirteen 50-day-old female piglets of the Landrace-Large White crossbreed were randomly allocated into three different groups: Group 1 (G1, n=5), inoculated with TgShSp1; Group 2 (G2, n=5), inoculated with TgShSp24; and Group 3 (G3, n=3), a non-infected control group. Clinical signs were monitored daily until 42 days post-infection (dpi) when piglets were euthanized. Blood samples were collected weekly to test the cellular immune response in parasite-stimulated peripheral blood and specific IgG, IgG1 and IgG2, responses in sera. Parasite distribution and burden were evaluated in target tissues using a mouse bioassay and quantitative RT−PCR (qPCR). Apathy and a moderate decrease in feed consumption were observed in G1 and G2 piglets between 5 and 8 dpi, coinciding with fever (>40°C). G2 piglets had higher temperatures for a longer duration. Using mouse bioassay and qPCR, the detection frequency was higher in G2 vs. G1, and the highest parasite burdens in target tissues were also found in G2. Seroconversion was detected at 14 dpi in both infected groups, but higher antibody levels were observed in G2 piglets. Cytokine analyses revealed the production of IL-8, IL-1β and IFN-ɤ from 7 dpi in both infected groups. Moreover, IL-12 was produced from 7 dpi in G1 and from 14 dpi in G2. Levels of IL-8 were higher in G2, but IL-1β, IL-12 and IFN-ɤ were higher in G1 at 14 dpi. This cytokine profile reveals a predominant proinflammatory response that could be involved in limiting T. gondii infection in piglets, although it is more efficient against TgShSp1 type II-driven infection.

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

“…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 ).…”

“…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 ).…”

“…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.…”

Limited value of current and new in silico predicted oocyst-specific proteins of Toxoplasma gondii for source-attributing serology

“…The experimental design is detailed in Figure 2. Three panels of sera from either experimentally infected pigs (panels 1 and 2) or sheep (panel 3), previously well-characterized by a battery of conventional serological tests (Largo-de la Torre et al, 2022;Loṕez-Ureña et al, 2023a;Loṕez-Ureña et al, 2023b; Supplementary Figure 2, and Supplementary Table 3), were used. First, a selection of pig and sheep sera were used as reference for the screening of the POIs by WB: three pigs infected with oocysts and two pìgs infected with tissue cysts, up to 3 weeks post-infection (wpi) (panel 1), ii) one non-infected pig and two pigs infected with oocysts, up to 42 days post-infection (dpi) (panel 2) and iii) one non-infected sheep and two sheep infected with oocysts, up to 21 dpi (panel 3).…”