RNA Sequencing Analysis Of Chicken Cecum Tissues Following E. Tenella Infection And Coccidiosis Evaluation Of Jinghai Yellow Chicken Cross-breeding System Parents

Avian coccidiosis is a disease caused by protozoan parasites that occupies the chicken intestines, and the main feature of it is high mortality and hemorrhagic colitis. Chicks at 3 weeks to 2 months of age are most susceptible to be infected, and the incidence of E. tenella is up to 50%-70%, causing tremendous harm to poultry industry. In this study, on the one hand in order to find the key genes and pathways associated with chicken E. tenella resistance, Jinghai yellow chickens were used as test animals, and at 30 days of age they were infected with high-dose E. tenella, using RNA-seq technology to analysis genome expression levels of cecal tissue in infected chickens and non-infected chickens at eight days post infection, several of the differentially expressed genes were validated by qRT-PCR, and differentially expressed genes were carried out GO, KEGG enrichment analysis. On the other hand in order to ascertain E. tenella resistance of Jinghai yellow chicken cross-breeding system parents, three tests of cross-breeding system parents by high-dose E. tenella infection, coccidiosis vaccine immunity and high-dose E. tenella infection after vaccination were carried out, using survival ratio, relatively body weight gain, oocyst shedding number, cecum score, anticoccidial index (ACI), and nitric oxide (NO), catalase (CAT), y interferon (IFN-y) and other 10 plasma parameters to evaluate E. tenella coccidiosis and immune effects of cross-breeding system parental BB, FF, WW. The research results were as follows:1. RNA-seq analysis results indicated that:5477 differential expressed genes were detected as significant (Padj< 0.05) between the infected group and uninfected group. Among them,2942 genes were up-regulated and 2535 genes were down-regulated. Ten genes were randomly selected for qRT-PCR to validate the reliability of RNA-seq analysis results. The fold changes results of selected genes by qRT-PCR were basically consistent with that by RNA-Seq, which indicated that the RNA-seq analysis result was reliable.2. Gene ontology and KEGG pathway analysis indicated that modulated genes (differential expressed genes) were mainly involved in lipid metabolism, cell communication, inflammatory response, immunity/defense, responses to various stimuli, blood vessel development, signal transduction. Chickens mustered complex cecal molecular and immunological responses in response to E. tenella infection, which included pathways involved in focal adhesion, cytokine-cytokine receptor interaction, peroxisome, ECM-receptor interaction and peroxisome proliferatoractivated receptor (PPAR) signaling. The key differential expressed genes in above pathways played important roles in anticoccidial process, such as FAK, TGFB2, TGFB3, IL-6, IL-15, CD36.3. Jinghai yellow chicken cross-breeding system parental BB, FF and WW were infected by high-dose E. tenella at 30 days old. All evaluation parameters were detected at eight days post infection. The ACI value of infected group of WW, BB and FF commercial lines were 143.1, 142.6 and 119.3, respectively. The ACI value of WW was the highest and the ACI value of FF was the lowest. WW and BB were anticoccidial inefficient in response to coccidiosis, and the FF was invalid in response to coccidiosis. ACI value was significant associated with immune parameters of catalase (CAT), superoxide dismutase (SOD), interleukin-17 (IL-17) and IFN-y (P<0.05). The concentration of nitric oxide, interleukin-12, interleukin-17, SOD in BB infected group were significantly (P<0.05) higher than that in FF infected group. Comprehensive analysis showed that:E. tenella resistance of Jinghai yellow chicken hybrids matching system parental BB was significantly higher than FF chickens, slightly higher than WW chickens.4. Jinghai yellow chicken cross-breeding system parental BB and FF were immuned with coccidium vaccine. All evaluation parameters were detected at eight days post infection. The CAT, SOD, IL-17 levels in BB vaccine group were significantly higher than that in FF vaccine group (P<0.05); the CAT, SOD levels in BB vaccine group were significantly higher than that in control group (P<0.05); the CAT and GSH-Px levels in FF vaccine group were significantly higher than that in control group (P<0.05). Comprehensive analysis results showed that the immune effect of BB vaccine group was superior to that of FF vaccine group, and coccidiosis resistance of BB chickens was superior to that of FF chickens.5. Jinghai yellow chicken cross-breeding system parental BB and FF were immuned with two coccidium vaccine which were 500 live E. tenella sporulated oocysts and standard coccidiosis vaccine at 9 days old. Chickens were infected with high-dose E. tenella 24 days later. All evaluation parameters were detected at eight days post infection. The clinical manifestations, cecum score, relatively body weight gain, and ACI values were measured. The results showed that BB was superior to FF line in coccidiosis resistance. And the vaccinated group 1 was superior to vaccinated group 2. The CAT, SOD levels in vaccinated group 1 were significantly higher than that in vaccinated group 2 in two populations (P<0.05). In cross-breeding system parental BB, the IFN-y levels in vaccinated group 1 were significantly higher than that in vaccinated group 2 (P<0.05). In cross-breeding system parental FF, the B-C levels in vaccinated group 1 were significantly higher than that in vaccinated group 2 (P<0.05). Comprehensive analysis showed that the immune effect of vaccinated group 1 was superior to vaccinated group 2, the E. tenella resistance of BB was superior to FF.