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Vaccination of chickens with a chimeric DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 induces protective immunity against coccidiosis

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Abstract

A fusion DNA vaccine co-expressed Eimeria tenella TA4 and chicken IL-2 (chIL-2) was constructed and its efficacy against E. tenella challenge was observed. TA4 gene of E. tenella and chIL-2 gene were cloned into expression vector pcDNA3.1 and pcDNA4.0c in different forms, producing vaccines pcDNA3.1-TA4-IL-2, pcDNA3.1-TA4 and pcDNA4.0c-IL-2. The expression of aim genes in vivo was detected by RT-PCR and western blot. Animal experiment was carried out to evaluate the immune efficacy of the vaccines. Results indicated these DNA vaccines were successfully constructed and the antigen genes could be expressed effectively in vivo. The animal experimental results showed that DNA vaccines could obviously alleviate cecal lesions, body weight loss and increase oocyst decrease ratio. The ACI of pcDNA3.0-TA4-IL-2 group was 192, higher than that of pcDNA3.1-TA4 group. The results suggested that TA4 was an effective candidate antigen for vaccine and co-expression of cytokine with antigen was an alternative method to enhance DNA vaccine immunity.

Introduction

Eimeria tenella is an obligate intracellular apicomplexan (coccidian) parasite that infects chicken and causes a severe form of enteritis, resulting significant economic losses especially to the poultry industry. Current means of control rely primarily on the prophylactic use of coccidiostats included in the feed. The development of resistance to these drugs and the public demand for chemical-free agricultural practices has limited the application of anti-coccidial drugs and driven the need for development of anti-coccidial vaccines (Williams, 2002).

Immunity to avian coccidiosis is largely dependent on cellular immunity (Rose and Hesketh, 1982). It was reported that DNA vaccines could successfully induce protective cellular and humoral immune responses against coccidiosis (Song et al., 2001). Administration of cytokines, such as IFN-γ, IL-1, IL-2 and IL-15, could enhance host immune responses induced by DNA vaccines to E. tenella and E. acervulina (Kim et al., 1999, Song et al., 2001, Xu et al., 2006). TA4 antigen is located on the surface of E. tenella sporozoites (Brothers et al., 1988). Subcutaneous immunization of chickens with Escherichia coli-expressed TA4 revealed that the protein was immunogenic (Thomas et al., 2003).

Here, we reported the construction of a fusion DNA vaccine co-expressed E. tenella TA4 and chicken IL-2 (chIL-2) and its efficacy against coccidiosis.

Section snippets

Antiserum, plasmids, parasites and animals

Rabbit antiserum against recombinant TA4 protein, plasmids pET28a-TA4 containing the full length of TA4 gene and pMDT-18-IL-2 with the complete chIL-2 gene were provided by the Laboratory of Veterinary Molecular and Immunological Parasitology, Nanjing Agricultural University, China. Sporulated oocysts of E. tenella isolated from Jiangsu Province of China (JS) were stored in 2.5% potassium dichromate solution at 4 °C and passed through chickens at least every 3 months. New-hatched Chinese Yellow

Identification of DNA vaccines

After digestion with BamHI and EcoRI, recombinant plasmid pcDNA3.1b-TA4 produced a fragment of approximately 690 bp of TA4 gene. A fragment of approximately 450 bp of chIL-2 gene was resulted by the digested recombinant plasmid pcDNA4.0c-IL-2 with EcoRI and NotI. The digestion of recombinant plasmid pcDNA3.1b-TA4-IL-2 with BamHI and NotI produced a fragment of approximately 1140 bp, which is equal to molecular mass summation of TA4 gene and IL-2 gene, while digestion with BamHI and EcoRI produced

Discussion

Coccidiosis mainly causes intestinal epithelium lesions, reduction of body weight and feed efficiency. Severe mortality is not the major detriment caused by coccidiosis. In this research, although no chicken died from coccidial challenge in any group, severe intestinal lesions and body weight loss were indicative of active intestinal disease caused in the positive control groups.

Since DNA immunization is capable of inducing both antibody and cell-mediated immunity (Tang et al., 1992, Manickan

Acknowledgement

This work was supported by the National Hi-Tech Research and Development Program of China (863 program) under grant number 2006AA10A207.

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These authors equally contributed to this work.

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