Role of cytokines and major histocompatibility complex restriction in mouse resistance to infection with a natural recombinant strain (type I-III) of Toxoplasma gondii

Abstract

Herein we characterized various genetic markers and the biological behavior of a natural recombinant strain of Toxoplasma gondii (P-Br). From nine genetic markers analyzed, three (B1, ROP1, and SAG1) and three (cS10-A6, GRA6, and SAG3) markers belong to parasites from the type I and type III lineages, respectively. The SAG2 and L363 loci were shown to be type I-III chimera alleles. The cB2l-4 microsatellite marker showed a unique haplotype. The P-Br strain presented low virulence in the acute phase of infection and was cystogenic during the chronic infection. The interleukin 12/gamma interferon axis and inducible nitric oxide synthase were main determinants of resistance during the acute infection with the P-Br strain. As opposed to infection with the type II strain of T. gondii (ME-49), peroral infection with the P-Br strain led only to a light inflammatory infiltrate and no major lesions in the intestine of the C57BL/6 mice. In addition, the BALB/c (resistant to ME-49) and C57BL/6 (susceptible to ME-49) mice were shown, respectively, to be more susceptible and more resistant to cyst formation and toxoplasmic encephalitis when infected with the P-Br strain. Further, the C57BL/KsJ and DBA2/J congenic strains containing major histocompatibility complex (MHC) haplotype "d" were more resistant than the parental strains (C57BL/6 and DBA1/J), when infected with the ME-49 but not with the P-Br strain. Together, our results indicate that resistance to cyst formation and toxoplasmic encephalitis induced during infection with P-Br is not primarily controlled by the MHC haplotype d, as previously reported for type II strains of T. gondii.

FIG. 1.

The coding region of the SAG1 (GenBank accession no. AY187278) gene from strain P-Br presented 99.9, 97, and 97% homology with SAG1 from RH, ME-49, and VEG, respectively. The coding region of the SAG2 gene (GenBank accession no. AY187279) from P-Br presented 100, 98, and 99% homology with SAG2 from RH, ME-49, and VEG, respectively. The coding region of the SAG3 gene (GenBank accession no. AY187280) from P-Br presented 100, 98, and 99% homology with SAG1 from RH, ME-49, and VEG, respectively. Underlined letters indicate natural nucleotide mutations of type I, type II, and type III standard strains in relation to the sequence of the same DNA from the P-Br strain.

FIG. 2.

Survival of female BALB/c, C3H/He, and C57BL/6 mice after peroral infection with four cysts of either strain P-Br or ME-49 of T. gondii. Infection with P-Br resulted in low (3 of 35 mice), high (22 of 27 mice), and high (41 in 54 mice) survival in C3H/HeJ, BALB/c and C57BL/6 mice, respectively. Infection with ME-49 resulted in low (5 of 22), moderate (9 of 16), and high (28 of 32) survival in C57BL/6, C3H/He, and BALB/c mice, respectively. The experiment was repeated three to five times and provided similar results. The presented results are pooled from the experiments for each parasite and mouse lineage combination. All the survivors were infected, as confirmed by the presence of cysts in the brain. The significance of survival rate differences between lineages infected with the same strain and of differences between strains in terms of behavior in the same mouse lineage was determined by the Kruskal-Wallis test (P < 0.05) 15, 30, and 45 days postinfection. This analysis indicates that the survival curve of C3H/He mice was statistically different from those of C57BL/6 and BALB/c mice infected with P-Br, that the survival curve of C57BL/6 mice was statistically different from those of C3H/He and BALB/c mice infected with the ME-49 strain, and that the survival curves of C3H/He and C57BL/6 mice were statistically significant when mice infected with P-Br and ME-49 were compared.

FIG. 3.

Histological changes in the ilea of C57BL/6 mice infected perorally with T. gondii. Animals were infected with 4 (A and B) or 20 (C and D) cysts of either the ME-49 (left) or P-Br (right), and histological analyses of small intestine were performed at 7 days postinfection. The villi clearly visible in panels B and D are almost indiscernible in panels A and C due to inflammatory infiltration and initial stages of necrosis in mice that were infected with ME-49. Six sections of the entire length of small intestine from each mouse were examined. Five mice were used for each group. Slides were stained with hematoxylin and eosin. Magnification, ×60.

FIG. 4.

Production of IFN-γ, IL-10, IL-12, TNF-α, and RNI by spleen cells from BALB/c, C3H/He, and C57BL/6 mice after peroral infection with four cysts of either the P-Br or ME-49 strain of T. gondii. Cytokine and nitrite levels were evaluated 7 and 15 days after inoculation of the parasite. The values shown are the mean of three animals per data points. The experiment was repeated twice and provided similar results.

FIG. 5.

Survival of female BALB/c, C57BL/6, and C57BL/KsJ (A) and DBA1/J and DBA2/J (B) mice after peroral infection with four cysts of either strain P-Br or ME-49 of T. gondii. Infection with P-Br resulted in high survival of BALB/c (22 of 27), C57BL/6 (41 of 54), and C57BL/KsJ (6 of 8) mice, respectively. Infection with P-Br resulted in moderate survival of DBA1/J (11 of 22) and high survival of DBA2/J (11 of 15) mice. Infection with ME-49 resulted in low and high survival of C57BL/6 (5 of 22) and C57BL/KsJ (7 of 7) mice, as well as high survival of BALB/c (28 of 32) mice, respectively. Infection with ME-49 resulted in high and low survival of DBA2/J (9 in 11) and DBA1/J (0 in 15) mice, respectively. All the survivors were infected, as confirmed by the presence of cysts in the brain. The experiment was repeated three times and provided similar results. The results presented are the pooled data of three experiments. The significance of survival differences between lineages infected with the same strain and of differences between strains in terms of parasite behavior in the same mouse lineage was determined by the Kruskal-Wallis test (P < 0.05) in 15, 30, and 45 days postinfection. This analysis indicate that the survival curve of C57BL/6 mice was statistically different from those of C57BL/KsJ and BALB/c mice infected with the ME-49 strain, that the survival curve of DBA1/J mice was statistically different from that of DBA2/J mice infected with ME-49 strain, and that the survival curves of C57BL/6 mice were statistically significant when mice infected with P-Br and ME-49 were compared.