Identification and Characterization of Eimeria tenella Apical Membrane Antigen-1 (AMA1)

Abstract

Apical membrane antigen-1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. In this study, a full-length cDNA of AMA1 was identified from Eimeria tenella (Et) using expressed sequence tag and the rapid amplification of cDNA ends technique. EtAMA1 had an open reading frame of 1608 bp encoding a protein of 535 amino acids. Quantitative real-time PCR analysis revealed that EtAMA1 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second-generation merozoites). The ectodomain sequence was expressed as recombinant EtAMA1 (rEtAMA1) and rabbit polyclonal antibodies raised against the rEtAMA1 recognized a 58-kDa native parasite protein by Western Blotting and had a potent inhibitory effect on parasite invasion, decreasing it by approximately 70%. Immunofluorescence analysis and immunohistochemistry analysis showed EtAMA1 might play an important role in sporozoite invasion and development.

Population, genetic, and antigenic diversity of the apicomplexanEimeria tenellaand their relevance to vaccine development

“…In contrast, application of Fu and Li's D* and F* tests indicated low levels of balancing selection. Combined, these findings suggest a low level of balancing selection within specific, possibly epitope-coding, regions that is consistent with the incomplete nature of the immune protection attributable to AMA1 revealed when tested as a recombinant protein or DNA vaccine (14,26). The relatively short and apparently self-limiting in vivo Eimeria lifecycle also is likely to reduce the magnitude of antigen-specific immune selection.…”

“…As recombinant or vectored subunit anticoccidial vaccines become technically more feasible, the regional occurrence of each Eimeria species and the extent of vaccine-targeted antigenic diversity assume greater relevance (5,14). Under experimental conditions, studies with clonal reference strains of Eimeria have found antigens such as AMA1 to induce good levels of immunoprotection (14,26). AMA1 also is an effective vaccine candidate in related apicomplexans including Neospora caninum, T. gondii, and several Plasmodium species (27)(28)(29)(30), but its value for Plasmodium has been undermined by high levels of naturally occurring antigenic diversity (31,32).…”

“…1). The nature of the immune response stimulated by AMA1 is not currently clear, although T cells and IFN-γ are key components of the anti-E. tenella immune response (43), and antirecombinant E. tenella AMA1 antibodies have been shown to inhibit the invasion of host cells by sporozoites (26). Analysis of E. tenella AMA1-coding sequences for signatures of selection revealed limited and conflicting evidence.…”

Molecular characterization and functional analysis of subunit 7 of eukaryotic initiation factor 3 from Eimeria tenella

“…We also demonstrated the importance of EteIF3s7 in host cell invasion in invasion inhibition experiments. In our previous study, we showed that some anti-protein specific polyclonal antibodies reduced E. tenella sporozoites invasion in vitro (Han et al, 2013;Jiang et al, 2012b). In our current study, the reduced capacity of sporozoites preincubated with an anti-rEteIF3s7 polyclonal antibody to invade DF-1 cells demonstrated the importance of EteIF3s7 in host cell invasion.…”

Molecular characterization and protective efficacy of silent information regulator 2A from Eimeria tenella

“…The PCR primers for EtSIR2A were as follows: 5′-AAA AGA AAC TTC CTC CCA-3′and 5′-AAT CCT GTC TCC TCC AAA-3′. Three housekeeping genes of E. tenella , 18S rDNA, β-actin and gapdh , were used as reference genes for the purposes of normalization [26–28]. The qPCR reaction mixture (20 μl) contained 10 μl SYBR® Premix Ex Taq™ (2×) (TaKaRa), 1 μl (0.2 μM) of each primer, 1 μl of cDNA template and 7 μl RNase-free distilled H 2 O.…”