The Distribution Offour Eimeria DNA Vaccines In Organic Tissues And Its Release To The Environment

To evaluate the biosafety of the Eimeria DNA vaccines pVAX1.0-TA4-IL-2, pVAX1.0-NA4-IL-2, pVAX1.0-EM8-IL-2, pVAX1.0-LDH-IL-2, the following trials have been fulfilled.According to the minimum age1single dose of target animal safety testing requirements, the experimental animals were divided into a vaccinated group and a control group, each chicken was boosted with25μg of the vaccine at the age of1day, intramuscularly inoculated while the negative control recieved only PBS. Another category of animals treated differently was also included in the evaluation programme, this time the chichen recieved3doses on a weekly basis, as follows:2groups of chicken experimental and a negative control, the experimental group was inoculated intramuscularly with25μg of the vaccine per chicken at7,14and21days respectively, meanwhile the negative control were inoculated only with PBS.Validation of the vaccine was also investigated by monitoring the stability and stiffness of the product after being produced on different times and stored for a while, alongside with this, dose optimization tests were also carried-out to determine the applicable range of doses. Experimental chickens were divided and treated according to the production batch:the first batch, the second batch and the third batch beside a non-vaccinated control group, a total of3groups were injected with75μg per chicken intramuscularly at14days each batch; The biosafety was evaluated by means of observation on the development of immune responses in the immunized chickens, such as mental status, foraging, drinking water, intestinal discharges and the death of chickens, and the details were recorded. Starting from1month aftre the intiation of the vaccination procedure and then at1.5months,2months,3months and4months, samples were collected from the vaccine treated chickens; including blood for cell count, tissues form the heart, liver, spleen, kidney, intestines beside tissues form muscle injection site.In accordance with plasmid DNA vaccine gene sequences, primers were designed and synthesized and utilized by PCR method for the detection of Kana gene; TA4gene; NA4gene; EM8gene and LDH gene, to determine distribution and retention time of the plasmid or other parts of gene fragments in the visceral tissues and whether they were integrated into animal tissue cell chromosome. The histopathological changes in the heart, liver, spleen, kidney, intestine and injection site in the muscles were also observed by organ paraffin sectioning.To detect wether these vaccines were environmently friendly or not,1week before vaccinating the chickens, a routine check for any DNA frgments in the chicken premises was carried out involving surrounding water. Then, every2weeks the surrounding water and experimental chicken droppings were sampled and examined using PCR technique to detect any of these genes:Kana gene; TA4gene; NA4gene; EM8gene and LDH gene, thereby judging plasmid transfer and plasmid DNA is whether the main metabolites were emitted into the environment.According to other non-target animal safety testing requirements, using experimental mice, injected with25μg, at3weeks of age were vaccinated one leg muscles. After1to3months samples were collected regularly including blood as well as tissues from the heart, liver, spleen, kidney and muscle injection site. In accordance with the PCR method to determine plasmid in non-target animal organs and tissue distribution and retention time, and whether the integration into the chromosome; preparation of paraffin slice observation of plasmid DNA can cause a target animal pathological injury.The results showed that the DNA vaccines did not induce any abnormal clinical responses in the. immunized chickens and mice. The five genes didn’t transfer into the normal intestinal flora, showing that this DNA vaccine didn’t excrete into the surroundings through intestinal flora and droppings. The plasmid could exist in heart, liver, spleen, kidney and the injected tissues at least for two months. The plasmid DNA didn’t integrate with chromosome DNA. Also, there was no pathological effect observed in the main organs.In one word, the DNA vaccine had no problems on biosafety, according to the requirement of Rules of Management on Agricultural Transgenic organision.