Development of a new serotyping ELISA for Toxoplasma gondii type II, type III and Africa 1 lineages using in silico peptide discovery methods, well categorized feline and human outbreak serum samples

Hüseyin Can,Ayşegül Aksoy Gökmen,Cemal Ün,Mervenur Güvendi,Sedef Erkunt Alak,Aytül Gül,Aurélien Mercier,Selçuk Kaya,Aysu Değirmenci Döşkaya,Ahmet Efe Köseoğlu,Muhammet Karakavuk,Tuğba Karakavuk,Ceren Gül,Mert Döşkaya,Adnan Yüksel Gürüz

doi:10.1186/s12879-022-07088-w

Hüseyin Can, Ayşegül Aksoy Gökmen + Show 13 more

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https://doi.org/10.1186/s12879-022-07088-w

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BackgroundDiscovery of new Toxoplasma gondii serotyping epitopes is important due to reports showing the influence of genotype on the severity of toxoplasmosis. In Turkey, genotypes belonging to type II, type III and Africa 1 lineages were mainly detected. The present study focused on to find out epitopes with high discriminative capacity to serotype these genotypes using well characterized strains isolated from Turkey.MethodsTo meet this objective, GRA6 and GRA7 genes were sequenced from strains belonging to the type II, III and Africa 1 lineages, and B cell epitopes inside these sequences were predicted by Bcepred and additional docking analysis was performed with B cell receptor. Based on these analyses, 22 peptides harboring lineage specific epitopes were synthesized. Then, the serotyping potency of these peptides was tested using peptide ELISA and well categorized serum samples collected from stray cats infected with genotypes of the different lineages type II (n:9), III (n:1) and Africa 1 (n:1). As a result of peptide-ELISA, a serotyping schema was constructed with peptides that show high discriminative capacity and this assay was validated by sera collected from humans after an outbreak (n:30) and mother/newborn pair sera (n:3). Later, the validated serotyping schema was used to serotype a larger group of human (n:38) and cat (n:24) sera.ResultsAmong 22 peptides, GRA6II/c, GRA7III/d, and GRA6 Africa 1/b epitopes have shown discriminative capacity. During the validation of peptide-ELISA, the serotype of toxoplasmosis outbreak and mother/newborn cases were detected to be serotype II. Moreover, the analyses in a larger group showed that serotype II was prevalent in humans and stray cats.ConclusionsOverall, the results showed that the serotyping schema could be successfully used to serotype T. gondii infections caused by type II, III and Africa 1 genotype.

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Discovery of new Toxoplasma gondii serotyping epitopes is important due to reports showing the influence of genotype on the severity of toxoplasmosis
Polymorphisms Among T. gondii isolates from lineages type II, III and Africa 1 genotype, polymorphisms in GRA6 protein were detected at 20 different amino acid positions (Table 1)
Polymorphisms in GRA7 protein were detected at 23 different amino acid positions (Table 2)

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Discovery of new Toxoplasma gondii serotyping epitopes is important due to reports showing the influence of genotype on the severity of toxoplasmosis. Three major clonal lineages called as type I, II and III were well identified using several genetic markers including isoenzymes, single nucleotide polymorphisms and microsatellites [6] In addition to these classical clonal lineages, a greater genetic diversity of Toxoplasma has been described associated with other divergent or recombinant lineages and occasionally some so-called atypical strains [6, 7]. Africa 1 lineage has been detected in domestic cats and in two different human congenital toxoplasmosis cases [3, 8]. A recent study performed in France detected cases of severe toxoplasmosis associated with strains belonging to the Africa 1 lineage in immunocompetent patients [16]

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