A new method for purification ofEimeria tenella merozoites

Xie, Meng; Gilbert, Jeffrey M.; Fuller, Alberta L.; McDougald, L. R.

doi:10.1007/bf00932562

Cited by 31 publications

(14 citation statements)

References 16 publications

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“…from chickens inoculated with 8.0 × 10 4 sporulated oocysts per bird. Isolation was carried out as previously described [30]. Isolated sporozoites and second-generation merozoites were stored in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella

Wang¹,

Huang²,

Dong³

et al. 2016

PLoS ONE

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Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. The basis of cell invasion is not completely understood, but some key molecules of host cell invasion have been discovered. This paper investigated the characteristics of calcium-dependent protein kinase 4 (EtCDPK4), a critical molecule in E. tenella invasion of host cells. A full-length EtCDPK4 cDNA was identified from E. tenella using rapid amplification of cDNA ends. EtCDPK4 had an open reading frame of 1803 bp encoding a protein of 600 amino acids. Quantitative real-time PCR and western blotting were used to explore differences in EtCDPK4 transcription and translation in four developmental stages of E. tenella. EtCDPK4 was expressed at higher levels in sporozoites, but translation was higher in second-generation merozoites. In vitro invasion inhibition assays explored whether EtCDPK4 was involved in invasion of DF-1 cells by E. tenella sporozoites. Polyclonal antibodies against recombinant EtCDPK4 (rEtCDPK4) inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored EtCDPK4 distribution during parasite development after E. tenella sporozoite invasion of DF-1 cells in vitro. The results showed that EtCDPK4 might be important in sporozoite invasion and development. To analyze EtCDPK4 functional domains according to the structural characteristics of EtCDPK4 and study the kinase activity of rEtCDPK4, an in vitro phosphorylation system was established. We verified that rEtCDPK4 was a protein kinase that was completely dependent on Ca2+ for enzyme activity. Specific inhibitors of rEtCDPK4 activity were screened by kinase activity in vitro. Some specific inhibitors were applied to assays of DF-1 cell invasion by E. tenella sporozoites to confirm that the inhibitors functioned in vitro. W-7, H-7, H-89, and myristoylated peptide inhibited DF-1 invasion by E. tenella sporozoites. The experimental results showed that EtCDPK4 may be involved in E. tenella invasion of chicken cecal epithelial cells.

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Section: Methodsmentioning

confidence: 99%

Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella

Wang¹,

Huang²,

Dong³

et al. 2016

PLoS ONE

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“…from birds that had been inoculated with 1×10 5 SO/bird. Isolation was carried out as previously described (Xie et al 1990). Isolated SZ and sMZ were stored in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

Identification and partial characterization of a serine protease inhibitor (serpin) of Eimeria tenella

et al. 2011

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Serine protease inhibitors (serpins) mediate many biological processes, including immune responses to pathogenic infection. In this study, a member of the serpin superfamily was identified from the common poultry parasite Eimeria tenella by expressed sequence tag analysis and the rapid amplification of cDNA ends technique. The full-length cDNA was 1,918 bp and had an open reading frame of 1,248 bp encoding a polypeptide of 415 amino acids with the theoretical isoelectric point of 5.26 and predicted molecular weight of 45.5 kDa. Real-time quantitative PCR analysis revealed that the serpin gene was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites). The sequence encoding the mature protein was amplified by PCR, cloned into the pET28(a) vector, and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was prepared and used to determine invasion inhibition capacity and localization; the results suggested that the serpin may play an important role in invasion and survival of the sporoziotes in the host.

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“…from chickens that had been inoculated with 1×10 5 sporulated oocysts per bird. Isolation was carried out as previously described [32]. Isolated sporozoites and merozoites were stored in liquid nitrogen until required.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2012

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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.

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A new method for purification ofEimeria tenella merozoites

Cited by 31 publications

References 16 publications

Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella

Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella

Identification and partial characterization of a serine protease inhibitor (serpin) of Eimeria tenella

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

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