Biosafety of the plasmid pcDNA3-1E of Eimeria acervulina in chicken.

To evaluate the biosafety of the plasmid pcDNA3-1E of Eimeria acervulina in chicken, two-week-old chickens were injected intramuscularly with the plasmid pcDNA3-1E at dose of 50 μg/chicken. At the 15 days post-injection, the tissue samples were collected, the total DNA was extracted, and the 3-1E gene was amplified by PCR. Genomic DNA was first purified away from free plasmid using gel electrophoresis, and then assayed for integrated plasmid using PCR amplification of the 3-1E gene. Simultaneously, the environmental dejection samples were collected, the total bacterial DNA was extracted and then transfer of the pcDNA3-1E gene was detected by PCR amplification of the 3-1E gene. Two-week-old chickens were injected intramuscularly with the plasmid pcDNA3-1E with three dosage groups of 100 μg, 500 μg and 2500 μg/chicken for 14 days respectively, and with physiological saline at dose of 2500 μL/chicken as control group for acute toxicity test. A target band of 583 bp was obtained by PCR with chicken genomic DNA as template. If the chicken genomic DNA was purified, no target band could be obtained. It showed that the recombinant plasmid pcDNA3-1E existed in tissues, and no genomic integration of DNA plasmid was detected in the immunized chickens. No target band was found by PCR with environmental dejection bacteria genomic DNA as template. It showed that integration and transfer phenomenon did not exist in environment. The acute toxicity results showed the typical clinical symptoms did not occur in the inoculated chickens, the blood biochemical indices and viscera configuration were not affected significantly in the inoculated group and control group (P>0.05). The results showed that the plasmid pcDNA3-1E was safe and suitable for chicken clinical trials.