Contributors: Friedrich-Loeffler-Institut (FLI); Universidad Complutense de Madrid = Complutense University of Madrid Madrid (UCM); University of Veterinary and Pharmaceutical Sciences Brno (VFU); Biologie moléculaire et immunologie parasitaires et fongiques (BIPAR); École nationale vétérinaire d'Alfort (ENVA)-Laboratoire de santé animale, sites de Maisons-Alfort et de Normandie; Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE); University of Agricultural Sciences and Veterinary Medicine Cluj Napoca = Universitatea de Științe Agricole și Medicină Veterinară Cluj-Napoca; INDEXX Laboratories, Kornwestheim, Germany; German Federal Institute for Risk Assessment Berlin (BfR); Ellinikos Georgikos OrGanismos-DIMITRA (ELGO -DIMITRA); National Veterinary Research Institute Pulawy, Pologne (NVRI); National Institute of Public Health Prague; Universität Bern = University of Bern = Université de Berne (UNIBE); Hôpital Dupuytren CHU Limoges; Institut d'Epidémiologie Neurologique et de Neurologie Tropicale; Epidémiologie des Maladies Chroniques en zone tropicale (EpiMaCT); Institut de Recherche pour le Développement (IRD)-CHU Limoges-Institut d'Epidémiologie Neurologique et de Neurologie Tropicale-Institut National de la Santé et de la Recherche Médicale (INSERM)-OmégaHealth (ΩHealth); Université de Limoges (UNILIM)-Université de Limoges (UNILIM); Neuroépidémiologie Tropicale (NET); CHU Limoges-Institut d'Epidémiologie Neurologique et de Neurologie Tropicale-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Génomique, Environnement, Immunité, Santé, Thérapeutique (GEIST); CHU Limoges; Istituto Superiore di Sanità = National Institute of Health (ISS); Landeslabor Berlin-Brandenburg; Bavarian Health and Food Safety Authority Oberschleißheim, Germany; National Veterinary Institute Uppsala (SVA); Norwegian Veterinary Institute Oslo (NVI); Instituto Nacional de Investigação Agrária e Veterinária = National Institute for Agrarian and Veterinary Research Oeiras, Portugal (INIAV); Universiteit Utrecht / Utrecht University Utrecht; Statens Serum Institut Copenhagen; Projekt DEAL; European Project: 773830,H2020-SFS-2017-1,COVRIN (a component of European Joint Programme One Health) (2021)
نبذة مختصرة : International audience ; 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.
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