In Vivo and In Vitro Virulence Analysis of Four Genetically Distinct Toxoplasma gondii Lineage III Isolates

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.