Construction And Analysis Of Subtractive CDNA Libraries Of Differential Immunogenicity Strains Of Eimeria Maxima By Suppression Subtractive Hybridization

Avian coccodiosis is a serve parasitic disease for the poultry industry, caused by intracellular protazoa including several species of the coccidian. It inflict severe economic losses on the poultry industry. Coccidia infection results in extensive destruction of the duodenum epithelium accompanied by severe depression in body weight gain, reduced feed efficiency and intestinal shedding of parasite oocysts. Eimeria maxima is one of the most common species of coccidian occurring in flocks all over the world, and it is the most immunogenic of the seven species of Eimeria that infected the domestic chicken, and has also been demonstrated to be partial immunity to other species. As a result, E.maxima is almost included in all type of vaccines. Paradoxically, immunological differences within the strains of E.maxima are common, which led to a failed immunization. So the design and application of vaccine depend on the studies of strains isolated from the different regions, especially the analysis of differentially expressed genes in differential immunogenicity strains of E.maxima. SSH(Suppression subtractive hybridization) is a technology to find putative virulent genes based on suppresive PCR. It has been used in gaining new genes and molecule evolution. In order to clone and identify differentially expressed genes between differential immunogenicity strains of E.maxima and find the molecular mechanism relating to antigen variation in E.maxima, Nantong strain (NT) and Shanghai strain (SH) of E.maxima were tested for their ability to induce protection and cross-protection against their homologous or heterologous strains, assessed by weight gain, lesion score and reduction in oocyst production. Than, the two subtractive cDNA libraries of sporulated oocysts of NT strain and SH strain were constructed using the technique of SSH and were analyzed, respectively. 1. The cross-protection between NT and SH strains of E.maximaNT and SH strains of E.maxima were tested for their ability to induce protection and cross-protection against their homologous or heterologous strains, assessed by weight gain, lesion score and reduction in oocyst production. The results were follows as:①Immunized with a single dose of 0.25×104 oocysts and challenged with 10×104 oocysts after two weeks, there were no differences of weight gains among the groups challenged with homologous strains and the control (P>0.05), and the reduction of lesion score was 79.55 % (NT strains)and 88.46 %( SH strains), respectively. There were also no differences of weight gains between the groups immunized with NT strains and challenged with SH strains and the control (P>0.05), and the reduction of lesion score was 71.15 %. But there was significant differences of weight gains between the groups immunized with SH strains and challenged with NT strains and the control (P<0.05), and the reduction of lesion score was 36.36 %.②Immunized with a single dose of 100 oocysts and challenged with 100 oocysts after two weeks, NT and SH strains afforded 100 % and 97.56 % protection against challenge with homologous parasites, respectively. NT strain afforded 80.98 % protection against challenges with SH strain, and SH strain afforded 41.20 % protection against challenges with NT strain. Conclusions: SH and NT strains of E.maxima were highly immunogenic to homologous strains. NT strains has cross-protection to SH strains, and SH strains has no cross-protection to NT strains. The immunogenicity of the two strains were not changed with passage and conservation in laboratory.2. Construction and Analysis of Subtractive cDNA Libraries of NT and SH Strains of E.maxima by Suppression Subtractive HybridizationIn order to clone and identify differentially expressed genes in two immunologically dinstinct strains of Eimeria maxima, the two subtractive cDNA libraries of sporulated oocysts of NT and SH strains of E.maxima were constructed using the technique of suppression subtractive hybridization(SSH), respectively. PCR amplification revealed that the SH strain subtractive cDNA library and NT strain subtractive cDNA library contained approximated 91 % and 93 % recombinant clones, respectively. Plasmid inserts were PCR amplified and the lengths were 250 bp～1.0 kb. Fifty positive clones from each subtractive cDNA libraries were sequenced and analyzed in GenBank with BLAST online, respectively. A total of 36 useful sequences were abtained from two subtractive cDNA libraries, and four unique sequences were obtained from the SH strain subtractive cDNA libraries, three unique sequences were obtained from the NT strain subtractive cDNA libraries, respectively. Other sequences represented novel genes of E.maxima. These results would provided the foundation for cloning new genes of E.maxima and further studying molecular mechanism relating to antigen variation in E.maxima.