| Chicken coccidiosis is an infection which leads to huge economic losses in poultry and is caused by intracellular apicomplexan parasites belonging to the genus Eimeria. Various strategies such as prophylaxis with anti-coccidial drugs, selection of disease recalcitrant chicken strains, live and attenuated vaccines and augmentation of innate immunity are actively applied to control coccidiosis. Each of these methods has advantages and some disadvantages in controlling coccidiosis. To address the current limitations of live vaccines, there is a dire need to enhance the immunogenicity of Eimeria sp., by means of adjuvants for effective prevention of coccidiosis. Cytokines have remarkable effects on the immunogenicity of antigens and play a pivotal role in shaping the nature of immune responses. Interleukin 4 (IL-4), for instance, importantly activates peripheral CD4+T cells and stimulates antibody production against gastrointestinal parasites. The present study was therefore designed to investigate the adjuvant effect of chicken interleukin-4 (ChIL-4) in enhancing the immunogenicity of E. mitis and, further, exploitation of transgenic E. mitis(tE.mitis) expressing ChIL-4 as a novel vaccine vector to control coccidiosis. For this purpose, a stable tE. mitis strain population incorporated with the chicken interleukin-4 (IL-4) was obtained by pyrimethamine selection. For the development of tE. mitis, wild type E. mitis (wE.mitis) sporozoites were nucleofected with a double cassette vector carrying chicken IL-4 and enhanced yellow fluorescent protein gene (EYFP) fused to DHFR mutant protein gene. Nucleofected E. mitis sporozoites were inoculated into primary chicken kidney cells (PCKCs) and chickens via the cloacal route for in-vitro and in-vivo investigation, respectively. The recovered fluorescent oocysts were sorted by fluorescence activated cell sorting (FACS) and tE. mitis oocysts were then passaged for seven generations successively for the establishment of a stable tE. mitis line. Stability of the transfected E. mitis was observed by fluorescent microscopy and its genomic DNA was confirmed by PCR and plasmid rescue technique. To evaluate and compare the performance of tE. mitis with its parental strain, analysis of tE. mitis oocyst shedding in feces, humoral immune response, hematological aberrations and weight gain index was recorded. The fecundity potential of tE. mitis was significantly reduced by six fold when compared to the wild type parent strains. The tE. mitis oocyst dejection peak was delayed by 22 hours as compared to the parental strain. The humoral immune response demonstrated that serum IgY titers significantly escalated in the tE. mitis treated group as compared to its wild type counterpart. Transgenic EmiChIL-4 immunized birds excreted fewer oocysts as compared to wild type vaccinated chicken following challenge with the wild type parental E. mitis strain, indicating that ChIL-4 incorporation may have enhanced the immunogenicity of the tE. mitis strain. The hematological index showed a positive trend in birds vaccinated with tE. mitis as compared to those infected with parental wE. mitis strain. The total leukocyte count, including eosinophils, heterophils and lymphocytes decreaed in birds inoculated with wE. mitis as compared to birds in control group, and tE. mitis. Improvement in the body weight of experimental chicks infected with tE. mitis oocysts was recorded as compared to those infected with tE. mitis oocysts. Overall, these findings indicate that tE. mitis expressing ChIL-4 functions well as an adjuvant, conferring reduced fertility and enhanced immunogenicity of the tE. mitis. This discrimination may be therefore applied as a potential vaccine vector to control coccidiosis as well as pathogens from which antigens are expressed by tE. mitis in future. Nevertheless, a detailed investigation on its biological safety as well as its immunogenicity is essentially required before its final usage as a vaccine vector. |
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