virulence of European type III and type II x III-recombinant natural clones. Mouse virulence of European type III and type II x III-recombinant natural clones of Toxoplasma gondii is not per se linked to the ROP18 and ROP5 genotype

Background: Toxoplasma gondii has a predominantly clonal population structure in North America and Europe, with types II and III prevailing in Europe. Surprisingly, sexual recombination among T. gondii clonal lineages seems to be rare. Nevertheless, recombinant T. gondii type II x III oocysts were isolated from a single naturally infected cat in a previous study. The present study aimed to compare recombinant clones from this source regarding their genotypes and mouse virulence phenotypes. Particular focus was on previously described polymorphic virulence factors. Methods: Acute virulence characteristics of the clones were assessed in BALB/c mice. All clones were typed by microsatellite and whole genome sequences (WGS) obtained by Illumina sequencing. The alleles of essential virulence factors, ROP5, ROP18, ROP16, and GRA15, were classified as virulent/non-virulent by WGS, and mRNA expression levels of these genes were assessed in J-774A.1 macrophages. In addition, Interleukin IL-10, IL-12, and TNFα mRNA levels were assessed after macrophages infection with or without IFN-γ stimulation. Results: Typing and WGS revealed four type II x III tachyzoite clones and one type III clone. All five clones possessed the virulent allele of ROP5. Only two of the three mouse virulent clones had the virulent ROP18 allele and were the only ones expressing high levels of ROP18 mRNA. Surprisingly, one of the clones with low ROP18 mRNA expression (i.e., the type III clone) was highly mouse virulent, while the remaining clones displayed intermediate virulence. The mouse virulent type III clone showed the highest ROP5 mRNA expression. However, neither the ROP16 and GRA15 mRNA expression profiles nor the interleukin expression profiles observed in in- vitro inoculated J-774A.1 macrophages could explain the differences in mouse virulence. Conclusion: While the mouse virulence of type II x III-recombinant clones could be explained by the presence of ROP18 expression, the high virulence of the type III clone remained unexplained. The expression of other virulence-associated genes (including ROP5), alone or in combination, might have contributed to the unexpected high virulence of a T. gondii type III clone.