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

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.

Discovery of Antigens and Cellular Mechanisms in the Protozoan Parasite Sarcocystis aucheniae Using Immunoproteomics

“…This type of study could not, however, be carried out for S. masoni, another SAC parasite that forms microscopic cysts in striated and cardiac muscles, due to the unavailability of genomic sequence data [18]. Although the antigenicity of the identified B-cell epitopes and their lack of cross-reactivity need experimental confirmation, the information provided in this study can be highly useful for the future development of peptide-based serological diagnostic methods, as has been described for T. gondii [28]. In addition, those B-cell epitopes with high antigenicity and water solubility but that showed significant conservation with proteins of other coccidia could be used for the development of peptide-based vaccination approaches.…”

Serotyping, a challenging approach for Toxoplasma gondii typing

“…Another peptide from GRA6 locus, named GRA6 Africa 1/b was described for Africa 1 lineage ( 40 ). Peptide GRA6 Africa 1/b is derived from an insertion sequences found in genotype III and Africa 1 genotype.…”

“…This epitope differs between genotype III and Africa 1 at position 206 involving a glycine to arginine substitution. This peptide had a sensitivity of 100% for Africa 1/b genotype, but cross-reacted with genotype II and III ( 40 ).…”

“…The serotype II profile is significantly more frequent in sera from Europeans, independent of clinical presentation of the disease ( Table 5 ) ( 26 , 28 , 30 , 40 – 43 ). In USA, serotype II was also associated with congenital toxoplasmosis ( 43 ).…”