Maike Joeres (HGW-Insel Riems / DE), Dr. med. vet. Pavlo Maksimov (HGW-Insel Riems / DE), Dr. rer. nat. Dirk Höper (HGW-Insel Riems / DE), Sten Calvelage (HGW-Insel Riems / DE), Mercedes Fernández-Escobar (Madrid / ES), Prof., Dr. Rafael Calero-Bernal (Madrid / ES), Prof., Dr. Bretislav Koudela (Brno / CZ), Prof., Dr. Radu Blaga (Maisons-Alfort / FR), Dr. Majda Globokar Vrhovec (Kornwestheim / DE), Dr. Kaya Stollberg (Berlin / DE), Dr. Nadja Bier (Berlin / DE), Prof., Dr. Smaro Sotiraki (Thessaloniki / GR), Dr. Jacek Sroka (Pulawy / PL), Dr. Weronika Piotrowska (Pulawy / PL), Prof., Dr. Petr Kodym (Prague / CZ), Dr. Walter Basso (Bern / CH), Prof. Dr. Franz J. Conraths (HGW-Insel Riems / DE), Prof., Dr. Aurélien Mercier (Limoges / FR), Lokman Galal (Limoges / FR), Prof., Dr. Marie-Laure Dardé (Limoges / FR), Anamaria Balea (Cluj-Napoca / RO), Dr. Furio Spano (Rome / IT), Prof., Dr. Simone Caccio (Rome / IT), Prof., Dr. Luis Miguel Ortega-Mora (Madrid / ES), Dr. Pikka Jokelainen (Copenhagen / DK), Dr. Gereon Schares (HGW-Insel Riems / DE)
Abstract text
Introduction
Toxoplasma gondii has a clonal population structure in Europe, where the type II clonal lineage prevails. A genotyping method based on 15 microsatellite (MS) regions represents the current reference standard.
Objectives
We aimed to establish a highly discriminatory next-generation sequencing (NGS)-based typing method for T. gondii to differentiate closely-related type II strains, to trace back infection sources and monitor for recombinant and emerging T. gondii strains.
Materials & methods
Toxoplasma gondii field isolates (n=110) were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference), different 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 in order to establish a multilocus Ion AmpliSeq-based typing method. The isolates analysed by WGS were typed with the novel method, and the sensitivity of the method was tested with serially diluted reference DNA samples.
Results
Type I, II and III clonal lineages were clearly separated by the 18 HPR Ion AmpliSeq typing technique. Two recombinant strains were correctly identified as type II x III strains. Among the type II strains, the method could differentiate a larger number of haplotypes compared to MS typing. The numbers and identities of SNPs were identical in outbreak isolates, indicating a common source. Furthermore, almost all SNPs identified by the method corresponded to those that were expected based on WGS. By testing serially diluted DNA samples, the technique exhibited similar analytical sensitivity as MS typing.
Conclusion
The results of our study show that reliable typing of T. gondii strains using an NGS-based multilocus typing method is feasible. The developed 18 HPR-based Ion AmpliSeq method has a high typing resolution, which appears promising for tracing infection sources in outbreaks and for detecting recombinant strains.
Funding: This work was done as part of TOXOSOURCES project, supported by funding from the European Union"s Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme.