Plasmid DNA could be delivered into Eimeria maxima unsporulated oocyst with gene gun system

Abstract

Eimerian coccidia are the most common parasitic organisms infecting chickens. The feasibility of genetic manipulation of these parasites via electroporation is proven, but this method is cumbersome and time consuming. Here we report our endeavour to develop a rapid and simple transfection method by gene gun. Tungsten particles coated with plasmid DNA encoding enhanced yellow fluorescent protein (EYFP) were used for the bombardment of Eimeria maxima unsporulated oocysts. Seven Mpa (1015 psi) helium pressure, 65 mm target distance and -0.098 Mpa (24.8″ Hg) chamber vacuum were the optimised parameters for bombardment. After sporulation, the bombarded oocysts were inoculated into chickens, and the progeny oocysts were checked under fluorescent microscope and subjected to genomic DNA extraction, which was used either for polymerase chain reaction (PCR) amplification or plasmid rescue assay. Although the expression of EYFP was not observed, the gene was amplified from both genomic DNA and the rescued plasmid, suggesting that the plasmid DNA existed in the form of episome. These results are encouraging for the genetic processing of the sporogony stage of eimerian parasites.