Study On The Infection Status Of Intestinal Parasites In Chickens From Partial Regions Of Shaanxi Province And Nucleic Acid Vaccines Against Cryptosporidium

Cryptosporidium are parasitic intracellular protozoan parasites of medical and veterinary importance that can inhabit epithelial cells andcause diarrhea and gastroenteritis of a wide range of vertebrate hosts.To investigate the infection status of intestinal parasites in chickens in Shaanxi province, a total of 336 fecal samples collected from 18 chicken farms in six regions were examined for the presence of eggs/oocyts by the saturation salt solution flotation method and Sheather’s sugar flotation technique in this study. The Eimeria spp. oocysts, Cryptosporidium oocysts, Heterakis gallinarum eggs and Ascaridia galli eggs were detected, with infection rates of 44.94%, 0.89%, 25.30%, and 1.19%, respectively. Then, Cryptosporidium oocysts were identified using the nested-PCR based on the 18 S rRNA gene. The result showed that all three Cryptosporidium samples were belonged to Cryptosporidium baileyi.In this study, so as to explore AOX(mitochondrion) and TSP6(microneme) genes of Cryptosporidium parvum the protective effect to Cryptosporidium baileyi, two DNA fragments of protective antigen genes were amplified by PCR with primers according to GenBank and inserted into pGEM-T Easy vector for constructing pGEM-T-AOX and pGEMT-TSP6 respectively. Then the two DNA fragments were cloned into pVAX1 vector using recombinant DNA technology and obtained the two nucleic acid vaccines: pVAX1-AOX and pVAX1-TSP6. Chickens were injected intramuscularly with pVAX1, pVAX1-AOX, pVAX1-TSP6 and pVAX1-AOX+pVAX1-TSP6 respectively, and changes of humoral and cellular levels were elevated by the immunological methods. The results showed that the level of specific antibody IgG in sera, secretion of cytokines IFN-γ and lymphocyte proliferation were all obviously improved in immune groups. The immune protective experiments showed that oocysts shedding became reduced obviously and the duration time of shedding oocysts became shorten in immune groups. The results showed that DNA vaccines of pVAX1-AOX and pVAX1-TSP6 have a better immune protective effect to the infection of Cryptosporidium.