Characteristics Of A Precocious Line Of E. Acervulina And Construction Of Its Subtractive CDNA Library

Avian coccidiosis is the major parasitic disease of poultry infected by Eimeria spp. It inflicts severe economic losses on the poultry industry. At present, conventional disease control strategies have relied on prophylactic medication and immunization with live vaccines. Due to the emergence of drug resistant parasite and the danger of live vaccines etc, Novel approaches are urgently needed to study to control coccidiosis. At present, identification important parasite antigen genes are crucial for the design of novel control approaches.To find differences of characteristics between precocious and parent strains of E. acervulina, 18 passages were selected by serial through chickens apply to a method as described by Jeffers. Analyzed differences between the two strains according to the sizes of sporulated oocysts, reproduction and pathogenicity.In order to analysis and identify differentially expressed genes from precocious and parent strains of E. acervulina, sporulated oocysts of two strains were used as the driver and the tester, respectively. Two subtractive cDNA libraries were constructed using the suppression subtractive hybridization (SSH) technique. PCR amplification revealed that the two subtractive cDNA libraries of precocious strains and parent strains contained approximately 97% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from each of two subtractive cDNA libraries. twenty unique sequences(ESTs) were found from the subtractive cDNA library of precocious strains of E.acervulina, nine ESTs shared significant identity with the former, including hypothetical protein of Toxoplasma gondii, hypothetical protein of Cryptosporidium muris, Eimeria acervulina serpin, Eimeria acervulina merozoite surface antigen, poly(A) polymerase of Toxoplasma gondii, etc. Twenty one unique sequences(ESTs) were found from the subtractive cDNA library of parent strains of E.acervulina, seven ESTs shared significant identity with the former, including hypothetical protein of Toxoplasma gondii, hypothetical protein of Plasmodium falciparum, Protein F and A of Enterobacteria phage, eukaryotic translation initiation factor 4 of Homo sapiens, and so on. Four differentially expressed genes obtained from two subtractive cDNA libraries conformed by Real-time PCR, respectively, which demonstrated that these genes were indeed differentially expressed. These results have provided the foundation for selecting differentially expressed genes of precocious and parent strains of E.acervulina and further studying new approaches to control coccidiosis.