Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella

Abstract

The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

Poultry Coccidiosis: Design and Interpretation of Vaccine Studies

“…Chapman and Shirley (102) used a modified version of the former protocol and assigned cut-offs for drug sensitivity; >160 = sensitive, partially resistant = 120-160 and <120 = resistant. Some vaccine studies have used ACI as a method to evaluate vaccine efficacy and assigned different cut-off scores for efficacy with some variation between studies; an ACI of <160 is generally considered to be ineffective (103)(104)(105)(106)(107). However, whether this adapted scoring method is overly simplistic to evaluate the efficacy and usefulness of vaccines in the field has yet to be determined.…”

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.…”

Eimeria tenella Translation Initiation Factor eIF-5A That Interacts With Calcium-Dependent Protein Kinase 4 Is Involved in Host Cell Invasion

“…Signal peptides were predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/). Transmembrane motifs were determined using TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/), and protein motifs were predicted using Motif Scan (http://hits.isb-sib.ch/ cgi-bin/motif_scan) (Zhai et al, 2016).…”