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.
Highlights
Chicken coccidiosis is a protozoal disease caused by infection with several Eimeria species and leads to high annual economical losses in the poultry industry due to high morbidity, reduction of body weight, and treatment costs [1]
By analysis of the sequence, the open reading frame (ORF) was deduced to encode a polypeptide of 407 amino acids with a calculated molecular mass of 46.04 kDa and a theoretical isoelectric point of 9.12
SignalP program analysis revealed that the Nterminus of EtCHP559 contained a signal peptide of 25 aa, with a hypothetical cleavage site located between alanine and aspartic acid
Summary
Chicken coccidiosis is a protozoal disease caused by infection with several Eimeria species and leads to high annual economical losses in the poultry industry due to high morbidity, reduction of body weight, and treatment costs [1]. The control of Eimeria infection is still based mainly on anticoccidial drugs and live vaccines. These measures have been restricted by some reasons, for example, the consumer attention to food safety, the rise of drug resistance, and the high production expenses etc. Eimeria are classified in the phylum Apicomplexa, which contains obligate intracellular parasites including medical and veterinary pathogens such as Plasmodium, Toxoplasma, Cryptosporidium, Neospora and Sarcocystis. These protozoan parasites are characterized by a peculiar organelle complex located at the apical end [4]. The levels of gene expression among these stages often differ greatly and this increased the difficulty of developing the cost-effective subunit vaccines
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