A new high-resolution next-generation sequencing (NGS)-based method was established to type and genetically-differentiate (i.e. subtype) closely related European type II Toxoplasma gondii strains.

T. gondii field isolates were collected from different parts of Europe and assessed by whole-genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. T. gondii isolates and T. gondii DNA present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples.

Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two non-archetypal and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited similar analytical sensitivity as MS typing.

The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among clonal European type II T. gondii strains. Furthermore, with available WGS data, additional target regions can be added to the method to potentially increase typing resolution.

Acknowledgements: This research was part of TOXOSOURCES, supported by funding from the European Union"s Horizon 2020 Research and Innovation program under grant agreement No. 773830: One Health European Joint Programme