Prevention And Treatment Of Coccidiosis With Herbal Medicine And The DNA Vaccine

Avian coccidiosis is an important disease that widely spreads with a high incidence in chicken farms and is often found in chicks at 15 to 50 days old. If not effectively controlled, the prevalence of infection will be high up to 50-70% with a mortality of 20-30% or 80% in severe cases. Statistical analysis indicated that the annual cost caused by the disease is estimated at ?00 million in the world. Current control of coccidosis mainly depends on synthetic chemicals or antibiotics. The durable use of the chemical drugs in the poultry industry easily leads to the resistant strains as well as the drug residues in the chickens and their products which are harmful to humans. Therefore, more and more research has shifted to non-antibiotic approaches such as medicinal herbs and vaccines. Salmonella typhimurium, one of the invasive bacterial species, can be attenuated without loss of invasiveness. Some of attenuated S. typhimurium strains have been reported being used as delivery vectors for foreign genes of bacterial or viral sources to the host tissues such as spleen and lymph node where they are expressed to induce specific immune responses. No related research has been found in this; country. The main purposes of this study were 1) to isolate and identify coccidia spp. in chicken farms in Hangzhou in order to provide a basis for the prevention and treatment of the disease in this region; 2) to evaluate a herbal complex in prevention and treatment of coccidiosis and 3) to construct the prokaryotic and eukaryotic expression vectors for 5401 gene and evaluate its immunogenicity and feasibility of the use of S. typhimurium as a delivery vector.Investigation was carried out of the coccidian infection in chickens in Hangzhou regioa The main species of the pathogens were E. tenella, E. necatrix and E. maxima. A liquid and a powder made from a herbal complex were studied for their anticoccidial effects against E. tenella. Results indicated that the liquid had a good anticoccidian action against E. tenella with an anticocidian index (ACI) of 180.8; the herbal powder had an anticoccidian action against E. tenella with an ACI of 178.8.Gene 5401 in E. tenella ZJ strain was amplified using RT-PCR. according to the sequence synthesized by Danforth. An EcoR I was ligated to the upper and Sal I to the lower of the primer for cleavage. The PCR product was around 900bp which was ligated into pGEM-T vector, forming E. coli DH-5 a competent cell. Determination of the nucleotide sequence in the positive clone indicated that the full-length of 5401 gene was 864bp in E. tenella ZJ strain, being an ORF. Compared to the 5401 sequence reported by Danforth, the bases in two regions were mutated with a homology of 99.8% for the nucleotide sequence or99.3% for the amino acid sequence. The positive 5401 ORF was subcloned into an E. coli vector pET-30a, forming an expression vector pET-30a-5401 to produce a fusion protein. The result indicated that the gene 5401 in pET-30a was induced by IPTG to express protein in E. coli BL21 (DE3), a high expression level was found at 6h, and the expressed protein was about 26.3% of total bacterial protein. Determined by SDS-PAGE and Western-blot, the fusion 5401 protein was about 66.2 kDa instead of 31.0 kDa, suggesting it might be in a dimer.E. tenella ZJ strain 5401 gene was cloned into a eukaryotic expression plasmid pcDNA3, and the recombinant eukaryotic plasmid was transformed by electroporation into the attenuated S. typhimurium strain ZJ111, resulting in ZJlll/pcDNA3-5401 which was then used to infect the Vero cells. A positive signal was detected by DIG in DNA extracted from the cells. A 31 kDa band of the 5401 protein was identified by SDS-PAGE and Western-blotting, suggesting that the attenuated S. typhimuriumtfiat present 5401 gene to Vero cells expressing protein.The 3-day-old chicks were vaccinated by oral administration of ZJlll/pcDNA3-5401 as a DNA vaccine and a booster was performed after 2 weeks. Neither death nor side effects were found. At 4 weeks after the secondary immunization, t...