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. 2017;24:48.
doi: 10.1051/parasite/2017047. Epub 2017 Dec 1.

Immunoproteomic Analysis of the Protein Repertoire of Unsporulated Eimeria Tenella Oocysts

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Free PMC article

Immunoproteomic Analysis of the Protein Repertoire of Unsporulated Eimeria Tenella Oocysts

Zhenchao Zhang et al. Parasite. .
Free PMC article

Abstract

The apicomplexan protozoans Eimeria spp. cause coccidioses, the most common intestinal diseases in chickens. Coccidiosis is associated with significant animal welfare issues and has a high economic impact on the poultry industry. Lack of a full understanding of immunogenic molecules and their precise functions involved in the Eimeria life cycles may limit development of effective vaccines and drug therapies. In this study, immunoproteomic approaches were used to define the antigenic protein repertoire from the total proteins of unsporulated Eimeria tenella oocysts. Approximately 101 protein spots were recognized in sera from chickens infected experimentally with E. tenella. Forty-six spots of unsporulated oocysts were excised from preparative gels and identified by matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF-MS) and MALDI-TOF/TOF-MS. For unsporulated oocysts, 13 known proteins of E. tenella and 17 homologous proteins to other apicomplexan or protozoan parasites were identified using the 'Mascot' server. The remaining proteins were searched against the E. tenella protein sequence database using the 'Mascot in-house' search engine (version 2.1) in automated mode, and 12 unknown proteins were identified. The amino acid sequences of the unknown proteins were searched using BLAST against non-redundant sequence databases (NCBI), and 9 homologous proteins in unsporulated oocyst were found homologous to proteins of other apicomplexan parasites. These findings may provide useful evidence for understanding parasite biology, pathogenesis, immunogenicity and immune evasion mechanisms of E. tenella.

Les protozoaires Apicomplexa Eimeria spp. causent les coccidioses, les maladies intestinales les plus communes chez les poulets. La coccidiose est un problème majeur de bien-être animal et a un fort impact économique sur l'industrie de la volaille. Le manque de compréhension complète des molécules immunogènes et de leurs fonctions précises impliquées dans les cycles de vie d'Eimeria peut limiter le développement de vaccins efficaces et de pharmacothérapies. Dans cette étude, des approches immunoprotéomiques ont été utilisées pour définir le répertoire des protéines antigéniques à partir des protéines totales des oocystes non sporulés d'Eimeria tenella. Environ 101 taches protéiques ont été reconnues dans des sérums de poulets infectés expérimentalement par E. tenella. Quarante-six taches d'oocystes non sporulés ont été excisées à partir de gels et identifiées par MALDI-TOF-MS et MALDI-TOF/TOF-MS par désorption laser assistée par matrice. Pour les oocystes non sporulés, 13 protéines connues d'E. tenella et 17 protéines homologues à d'autres parasites Apicomplexa ou protozoaires ont été identifiées en utilisant le serveur “Mascot”. Les protéines restantes ont été recherchées dans la base de données de séquences protéiques d'E. tenella en utilisant le moteur de recherche “Mascot in-house” (version 2.1) en mode automatisé, et 12 protéines inconnues ont été identifiées. Les séquences d'acides aminés des protéines inconnues ont été recherchées en utilisant BLAST contre des bases de données de séquences non redondantes (NCBI), et 9 protéines homologues des oocystes non-sporulés ont été trouvées homologues à des protéines d'autres parasites Apicomplexa. Ces résultats peuvent fournir des éléments utiles pour comprendre la biologie parasitaire, la pathogenèse, l'immunogénicité et les mécanismes d'évasion immunitaire d'E. tenella.

Figures

Figure 1
Figure 1
Identification of proteins of unsporulated E. tenella oocysts by 2-D gel electrophoresis. Isoelectro-focusing was performed with 600 μg protein for 50 kVh using a pI 3-10 strip. SDS-PAGE was performed on a 12.5% gel and then stained with CBB. Numbers are used to mark the spots analyzed.
Figure 1
Figure 1
Identification of proteins of unsporulated E. tenella oocysts by 2-D gel electrophoresis. Isoelectro-focusing was performed with 600 μg protein for 50 kVh using a pI 3-10 strip. SDS-PAGE was performed on a 12.5% gel and then stained with CBB. Numbers are used to mark the spots analyzed.
Figure 2
Figure 2
Immunoblot profiling of unsporulated E. tenella oocysts using chicken anti-E. tenella serum.
Figure 2
Figure 2
Immunoblot profiling of unsporulated E. tenella oocysts using chicken anti-E. tenella serum.
Figure 3
Figure 3
Spot 27's amino acid sequence homologous to Cryptosporidium hominis proteasome searched against the E. tenella genome using the ‘Mascot in-house’ server. A: E. tenella amino acid sequences obtained by MALDI-TOF-MS were searched using BLAST against the NCBI database. Deduced amino acid sequences on Spot 27 were aligned with the deduced sequence from the E. tenella genome Contig 12511: complements (12140…13529) are in bold red script. B: Alignment of the predicted amino acid sequence from Spot 27 (A) with the C. hominis proteasome subunit. The alignments of sequences are in bold red script.
Figure 3
Figure 3
Spot 27's amino acid sequence homologous to Cryptosporidium hominis proteasome searched against the E. tenella genome using the ‘Mascot in-house’ server. A: E. tenella amino acid sequences obtained by MALDI-TOF-MS were searched using BLAST against the NCBI database. Deduced amino acid sequences on Spot 27 were aligned with the deduced sequence from the E. tenella genome Contig 12511: complements (12140…13529) are in bold red script. B: Alignment of the predicted amino acid sequence from Spot 27 (A) with the C. hominis proteasome subunit. The alignments of sequences are in bold red script.

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