The effect of infection with Toxoplasma gondii strains on the IFN-g production and parasitic load in tissues of experimentally infected pigs

Abstract

Objectives: Toxoplasma gondii is an intracellular parasite of humans and animals. The infection with this pathogen causes severe diseases in humans and has an important economic impact in domestic animals. One of the main sources of infection in humans is the consumption of raw or undercooked meat. The objective of this study was to determine the influence of subsequent infections of pigs with different T. gondii strains on the parasitic load of tissues, and to study the accompanying immune response. Parasitic load after infection with different T. gondii strains The parasitic load in tissues of experimentally infected pigs was determined after a consecutive infection with two T. gondii strains: Gangji (type I/II) and LR (type II). Seronegative 6-weeks old pigs were simultaneously inoculated with 6000 tissue cysts of the IPB-Gangji strain (group 1, n=3) or the IPB-LR strain (group 2, n=3). Sixty days later both groups were re-infected with 6000 tissue cysts of the two respective strains (group 1: Gangji/LR and group 2: LR/Gangji). A third group of animals (n=3) were inoculated with 6000 tissue cysts of the IPB-Gangji strain at day 60 PI (group 3: Gangji). The animals (group 1, 2 and 3) were euthanized to determine the parasitic load by qPCR in the following tissues: brain, heart, spleen, skeletal muscles and diaphragm. In all groups brain and heart samples showed the highest parasitic load. In group 1 (Gangji/LR) most skeletal muscles tested contained parasitic DNA, while in group 2 (LR/Gangji) only few muscle samples tested positive. Moreover, the amount of DNA found in these muscles was remarkably lower than in positive tissues of group 1. Group 3 (Gangji ½ t) showed a very high and a moderate parasitic load in brain and two muscle samples, respectively. Interestingly, these positive tissues contained less parasite-derived DNA than the same tissues of group 1 but more than those of group 2, suggesting a clearance (effect of IPB-Gangji infection on IPB-LR-infection). Involvement of T cells in the immune response: The production of IFN-g by T lymphocytes was determined by qPCR. Hereto, PBMCs were isolated from blood samples taken every 14 days, and subsequently cultured for 48 hours in the presence of total lysate antigen (TLA) or ConA as a positive control. IFN-g detection showed an increased production in all infected animals compared to control animals, which correlated with the inoculation time point,and was independent from the inoculation strain. This elevated production did not reach the maximum level in group 3, as observed in group 1 and 2. The involvement of different T-cell subpopulations in the cellular response after infection with two strains of T. gondii was analysed by flow cytometry. Hereto, two groups of 6-weeks old pigs were inoculated with 6000 tissue cysts of the IPB-Gangji strain (group 4, n=3) or the IPB-LR strain (group 5, n=3). The PBMCs were isolated and cultured as described above. The T-cell populations (CD3+, CD4+, CD8+) and the intracellular IFN-g production were detected by flow cytometry. Infection by both strains induced IFN-g production, which was positively correlated with the time point of the inoculation and was brought about by CD3+CD4+ and CD3+CD8+ proliferating T lymphocytes. In group 4 and 5 mainly the CD3+CD8hi T-cells and, to a lesser extent, the CD3+CD4+ population produced IFN-g, whereas the CD3+CD8int showed the fewest IFN-g positive cells. Conclusions: This study and previous data suggest that the IPB-Gangji strain can induce a clearance of the infected tissues, irrespective of the strain of the initial inoculation. The cellular response to parasitic infection was demonstrated by the time-dependent and strain-independent increased production of IFN-g by mainly CD3+CD8hi T cells