Effects Of Bacillus Cereus PAS38 On Immune Related Differential Expression Genes Of Spleen In Broilers

To investigate the immunomodulatory effects of Bacillus cereus PAS38 on spleen of broiler,120 one-day-old white feather broilers were randomly divided into 4 groups with 3replicates per group.The CNTL group was fed the basal diet,without inoculated any vac-cine.The PB group fed the basal diet with the B.cereus PAS38 preparation 1.0×10⁶ CFU/g,without inoculated any vaccine.The VAC group was fed the basal diet and was inoculated by the attenuated vaccine of Newcastle disease virus on the 7^th day and the 21^st day.The PBVAC group fed the basal diet with the B.cereus PAS38 preparation 1.0×10⁶ CFU/g,and was inoculated by the attenuated vaccine of Newcastle disease virus on the 7^th day and the 21^stday.On the28^th day and the 42^nd day,their growth performance,number of intes-tinal flora,and serum antibody titer were measured.On the 42^nd day,the relative weights and the tissue structures of immune organs were observed,and the differentially expressed genes（DEGs）were screened using RNA-seq.Finally,q RT-PCR technique was used to verify the reliability of RNA-seq data.The test results and conclusions are as follows:1.On the 42^nd day,the body weight of the broiler chickens in the PB group was sig-nificantly higher than that in the CNTL group（P<0.05）.The net weight gain of the PBVAC group was significantly higher than that of the VAC group（P<0.05）in the period from the28^th day to the 42^nd day.The net weight gain of broiler chickens in the PB group was significantly higher than that in CNTL group（P<0.05）in the period from the7^th day to the 42^nd day.For feed conversion ratio,the PB and PBVAC groups were higher than the CNTL group to a certain extent.All of these results indicated that B.cereus PAS38 can promote the growth of broilers.2.On the 28^th day,the number of E.coli in ileal of in the PBVAC group broiler was significantly lower than that in the CNTL group（P<0.05）.In the cecum,the number of E.coli in the PB group and the PBVAC group were significantly lower than that in both of the CNTL group and the VAC group（P<0.01）.On the 42^nd day,the addition of B.cereus PAS38 could extremely significantly（P<0.01）reduce the number of E.coli and increase the amount of Lactobacilli in the ileum and cecum.These results indicated that the addi-tion of B.cereus PAS38 can significantly reduce the number of E.coli and increase the number of Lactobacillus in the broiler intestine.3.The PB,VAC and PBVAC group had improved the relative weight of immune or-gans compared with the CNTL group,but the difference was not significant.Immune or-gan histological observation revealed that the immune organs of the four groups were normal and no disease.Compared with the CNTL group,the lymphocyte density in the immune organs became larger of the other three groups.On the 28^th day,the serum anti-body titer of the PBVAC group was significantly higher than that of the VAC group（P<0.05）,and on the 42^nd day,that only showed a little increase trend.These results sug-gested that B.cereus PAS38 can promote the growth and development of immune organs of broilers to a certain extent,and regulate the chickens’humoral immune function.4.A total of 12 samples were sequenced by RNA-seq.85.30 Gb clean data was ob-tained with about 6.41Gb clean data for each sample.The clean reads of each sample was mapped to the reference genome（Gallus Gallus）respectively.The efficiency of alignment ranged from 61.15%to 65.41%.Through gene annotation,most of the 16398 genes were annotated to the NR database,14699 genes were annotated in the GO database,and 9454genes were annotated in KEGG database with 244 KEGG pathways.5.Biological replicates varied in each group.The biological replicates of PB group were higher than the control group,and the PBVAC group showed greater variability compared to the VAC group,indicating that addition of B.cereus PAS38 can stabilize the gene expression of the broiler’s spleen,and amplified the body’s immune response after vaccination.The DEGs of CNTL vs PB,CNTL vs VAC,CNTL vs PBVAC,PB vs VAC,PB vs PBVAC and VAC vs PBVAC were 105,20,994,56,55,245 respectively.Among them,the proportion of down-regulated expression was 77.88%,50%,63.51%,67.27%,65.45%and 52%respectively,and DEGs in―immune system process‖respectively ac-counted for 29.79%,36.84%,25.8%,48.21%,36.36%and 35.51%.The number of DEGs in VAC vs PBVAC was much higher than that in PB vs PBVAC,indicating that the gene expression pattern of spleen in the PBVAC group is closer to the PB group.Compared with the CNTL group,most of the DEGs in the PB and PBVAC groups were down-regulated.The immune-related DEGs like PROKR2,DMBT1,and CTGF were sig-nificantly down-regulated in the PB group,and were involved in the inflammatory re-sponse.The DEGs,NKL and GBP1,with antibacterial effect were upregulated suggesting that B.cereus PAS38 can regulate the immune response of the chickens by weakening the inflammatory response and enhancing the antibacterial function.In VAC vs PBVAC,most immune-related DEGs are involved in innate immunity.The DEGs Cl EC2E,LYSg,STX11,and Mx confirmed that.The probiotic Bacillus played an auxiliary role in vaccine effect in non-specific immunity.6.The four DEGs（CD8A,IRF1,Igsf1,and GBP1）randomly selected were verified by q RT-PCR withβ-Actin as reference gene.The results were similar to RNA-seq data,sug-gesting the reliability of the latter.In conclusion,all results showed that addition of B.cereus PAS38 in broiler diets can improve the growth performance of broilers,regulate the microecological environment of the intestine,promote the development and maturation of immune organs,regulate the immune function in the broiler spleen by attenuating inflammatory response and enhancing antibacterial capability,and play an auxiliary role in the Newcastle disease vaccine effect in non-specific immunity.