Toxoplasma gondii archetypes II and III are mildly virulent, yet virulence of variant strains is largely unknown. While lineage II dominates in humans in Europe, lineage III strains are present in various intermediate hosts. In Serbia, lineage III represents 24% of the population structure and occurs most frequently in domestic animals, implying a significant presence in the human food web. In this study, the virulence of four genetically distinct lineage III variants was assessed in vivo and in vitro. In vivo, two strains were shown to be intermediately virulent and two mildly virulent, with cumulative mortalities of 69.4%, 38.8%, 10.7%, and 6.8%, respectively. The strain with the highest mortality has previously been isolated in Europe and may be endemic; the strain with the lowest mortality matches ToxoDB#54, while the remaining two represent novel genotypes. Identical alleles were detected at ROP5, ROP16, ROP18, and GRA15. A set of in vitro analyses revealed proliferation and plaque formation as virulence factors. Higher levels of expression of ENO2 in intermediately virulent strains point to enhanced metabolism as the underlying mechanism. The results suggest that metabolic attenuation, and possibly stage conversion, may be delayed in virulent strains.

In Europe, Toxoplasma gondii lineage II is dominant, and ToxoDB#1 the most frequently occurring genotype. The abundance of lineage III genotypes varies geographically and lineage I are rare, yet present in several regions of the continent. Data on the T. gondii population structure in southeastern Europe (SEE) are scarce, yet necessary to appreciate the diversity of the species in Europe. To help fill this gap, we genotyped 67 strains from nine species of intermediate hosts in Serbia by MnPCR-RFLP, determined the population structure, and identified the genotypes using ToxoDB. A neighbor-joining tree was also constructed from the isolates genotyped on nine loci. While 42% of the total genotype population consisted of ToxoDB#1 and ToxoDB#2, variant genotypes of both lineages comprised 46% of the population in wildlife and 28% in domestic animals and humans. One genotype of Africa 4 lineage was detected in a human sample. Interestingly, the findings include one lineage III variant and one II/III recombinant isolate with intercontinental distribution, which appear to be moderately related to South American genotypes. Based on these findings, SEE is a region of underappreciated T. gondii genetic diversity and possible strain exchange between Europe and Africa.

Toxoplasma gondii is an obligate intracellular parasite existing in three infectious life stages—tachyzoites, bradyzoites, and sporozoites. Rupture of tissue cysts and re-conversion of bradyzoites to tachyzoites leads to reactivated toxoplasmosis (RT) in an immunocompromised host. The aim of this study was to apply ImageJ software for analysis of T. gondii brain cysts obtained from a newly established in vivo model of RT. Mice chronically infected with T. gondii (BGD1 and BGD26 strains) were treated with cyclophosphamide and hydrocortisone (experimental group—EG) or left untreated as infection controls (ICs). RT in mice was confirmed by qPCR (PCR+); mice remaining chronically infected were PCR−. A total of 90 images of cysts were analyzed for fractal dimension (FD), lacunarity (L), diameter (D), circularity (C), and packing density (PD). Circularity was significantly higher in PCR+ compared to IC mice (p < 0.05 for BGD1, p < 0.001 for the BGD26 strain). A significant negative correlation between D and PD was observed only in IC for the BGD1 strain (ρ = −0.384, p = 0.048), while fractal parameters were stable. Significantly higher D, C, and PD and lower lacunarity, L, were noticed in the BGD1 compared to the more aggressive BGD26 strain. In conclusion, these results demonstrate the complexity of structural alterations of T. gondii cysts in an immunocompromised host and emphasize the application potential of ImageJ in the experimental models of toxoplasmosis.