Abstract
Background:
Coccidiosis is an economically important disease in poultry caused by Eimeria species.

Aim:
The aim of this study was to design, express, and evaluate a recombinant 19kDa sporozoite protein (E19kDa) as a vaccine candidate against mixed Eimeria infection in broiler chickens.

Methods:
The study used bioinformatic analysis, protein expression, immunization trials, and immune response evaluation. The protein properties were predicted using ExPASy ProtParam, VaxiJen, and Protein-Sol servers. Gene 838 (Accession No. PV347147) was cloned into a pET-28b plasmid and expressed in E. coli BL21(DE3). IPTG induction levels were optimized, and expression was confirmed via qPCR. The protein was purified using Ni-NTA chromatography and validated by SDS-PAGE. Twenty Ross 308 chickens were divided into negative control, positive control (infected), and immunized challenge groups. Birds in the vaccine group received two subcutaneous doses of E19kDa with aluminum hydroxide adjuvant. The challenge was done using a mixed Eimeria oocyst suspension (1×10⁴ oocysts per species). Immunity was assessed through live weight, oocyst counts, white blood cell counts, and IgY levels.

Results:
The results showed high expression of E19kDa at 1 mM IPTG. SDS-PAGE confirmed the purified protein at ~29kDa. Immunized birds had better weight gain, lower oocyst counts (10,245 vs. 22,755), and increased WBCs and IgY titers compared to infected controls. ELISA confirmed a peak antibody response on day 28 post-vaccination. The vaccine significantly reduced clinical symptoms and mortality.

Conclusion:
This study highlights the potential of the recombinant E19kDa protein as a promising vaccine candidate against coccidiosis. The approach combining bioinformatics, molecular expression, and immunological testing offers a cost-effective solution for the disease.

Key words: Adjuvant, ELISA, Expression, Immune response, Oocyst count