PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: 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. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: 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 atypical 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 a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

Anses INRAE Ecole Nationale Vétérinaire d'Alfort Laboratoire de Santé Animale BIPAR Maisons Alfort France

Bavarian Health and Food Safety Authority Erlangen Germany

Central European Institute of Technology University of Veterinary Sciences Brno Brno Czech Republic

Centre National de Référence Toxoplasmose Centre Hospitalier Universitaire Dupuytren Limoges France

Centre of Epidemiology and Microbiology National Institute of Public Health Prague Czech Republic

Chemisches und Veterinäruntersuchungsamt Westfalen Standort Arnsberg Arnsberg Germany

Department of Analysis and Diagnostics Norwegian Veterinary Institute Ås Norway

Department of Animal Health Welfare and Food Safety Norwegian Veterinary Institute Tromsø Norway

Department of Microbiology National Veterinary Institute Uppsala Sweden

Department of Parasitology and Invasive Diseases National Veterinary Research Institute Pulawy Poland

Dutch Wildlife Health Centre Pathology Division Department of Pathobiology Faculty of Veterinary Medicine University of Utrecht Utrecht The Netherlands

Faculty of Veterinary Medicine University of Veterinary Sciences Brno Brno Czech Republic

Friedrich Loeffler Institut Federal Research Institute for Animal Health Institute of Diagnostic Virology Greifswald Insel Riems Germany

Friedrich Loeffler Institut Federal Research Institute for Animal Health Institute of Epidemiology Greifswald Insel Riems Germany

German Federal Institute for Risk Assessment Department for Biological Safety Berlin Germany

IDEXX Laboratories Kornwestheim Germany

Infectious Disease Preparedness Statens Serum Institut Copenhagen Denmark

Inserm U1094 IRD U270 Univ Limoges CHU Limoges EpiMaCT Epidemiology of chronic diseases in tropical zone Institute of Epidemiology and Tropical Neurology OmegaHealth Limoges France

Institute of Parasitology Vetsuisse Faculty University of Bern Bern Switzerland

Italian National Institute of Health Rome Italy

Landeslabor Berlin Brandenburg Frankfurt Germany

Parasitology Laboratory Instituto Nacional de Investigação Agrária e Veterinária Oeiras Portugal

SALUVET Animal Health Department Faculty of Veterinary Sciences Complutense University of Madrid Madrid Spain

University of Agricultural Sciences and Veterinary Medicine Cluj Napoca Faculty of Veterinary Medicine Department of Parasitology and Parasitic Diseases Cluj Napoca Romania

University of Agricultural Sciences and Veterinary Medicine Cluj Napoca Romania

Veterinary Research Institute Hellenic Agricultural Organisation DIMITRA Thessaloniki Greece

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A combination of GRA3, GRA6 and GRA7 peptides offer a useful tool for serotyping type II and III Toxoplasma gondii infections in sheep and pigs