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Abstract：
BACKGROUND: Discovery 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.
METHODS: To 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.
RESULTS: Among 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.
CONCLUSIONS: Overall, 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.
METHODS: To 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.
RESULTS: Among 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.
CONCLUSIONS: Overall, 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.
摘要：
背景：新的弓形虫血清分型表位的发现很重要，因为有报道显示基因型对弓形虫病的严重程度有影响。在土耳其,属于II型的基因型，主要检测到III型和非洲1谱系。本研究的重点是使用从土耳其分离的特征良好的菌株，找出具有高区分能力的表位，以对这些基因型进行血清型化。
方法：为了实现这一目标，GRA6和GRA7基因从属于II型的菌株中测序，III和非洲1血统，通过Bcepred预测这些序列中的B细胞表位，并用B细胞受体进行额外的对接分析。基于这些分析,合成了22种具有谱系特异性表位的肽。然后，使用肽ELISA和从感染不同谱系II型（n:9）基因型的流浪猫收集的良好分类的血清样品来测试这些肽的血清分型效力。III(n:1)和非洲1(n:1)。作为肽ELISA的结果，使用具有高区分能力的肽构建了血清分型方案，该测定通过爆发后从人类收集的血清（n：30）和母亲/新生儿对血清（n：3）进行了验证。稍后，经过验证的血清分型方案用于对更大的一组人(n:38)和猫(n:24)血清进行血清型。
结果：在22个肽中，GRA6II/c,GRA7III/d,和GRA6非洲1/b表位已显示出判别能力。在肽ELISA的验证过程中，弓形虫病爆发和母亲/新生儿病例的血清型被检测为血清型II.此外，在一个更大的群体中的分析表明，血清型II在人类和流浪猫中普遍存在。
结论：总体而言，结果表明，血清分型模式可以成功地用于II型弓形虫感染的血清型，Ⅲ和非洲1基因型。
方法：为了实现这一目标，GRA6和GRA7基因从属于II型的菌株中测序，III和非洲1血统，通过Bcepred预测这些序列中的B细胞表位，并用B细胞受体进行额外的对接分析。基于这些分析,合成了22种具有谱系特异性表位的肽。然后，使用肽ELISA和从感染不同谱系II型（n:9）基因型的流浪猫收集的良好分类的血清样品来测试这些肽的血清分型效力。III(n:1)和非洲1(n:1)。作为肽ELISA的结果，使用具有高区分能力的肽构建了血清分型方案，该测定通过爆发后从人类收集的血清（n：30）和母亲/新生儿对血清（n：3）进行了验证。稍后，经过验证的血清分型方案用于对更大的一组人(n:38)和猫(n:24)血清进行血清型。
结果：在22个肽中，GRA6II/c,GRA7III/d,和GRA6非洲1/b表位已显示出判别能力。在肽ELISA的验证过程中，弓形虫病爆发和母亲/新生儿病例的血清型被检测为血清型II.此外，在一个更大的群体中的分析表明，血清型II在人类和流浪猫中普遍存在。
结论：总体而言，结果表明，血清分型模式可以成功地用于II型弓形虫感染的血清型，Ⅲ和非洲1基因型。
