| Bacillus is widely used in the livestock industry. The probiotic Bacillus amyloliquefaciens SC06(Ba) was isolated from soil and kept at China Center for Type Culture Collection (CCTCC No:M2012280), In previous study, we found that dietary supplementation with the probiotic Ba could improve the growth performance and decrease the diarrhea incidence in the weaned piglets. Based on the study above, the mechanism of the probiotc Ba regulating the gut immunity will be further explored. In addition, this study was designed to evaluate the effect of fattening pig diets containing the probiotic Ba as alternative antibiotic (kitasamycin), main contents as follows:(1) The probiotc Ba mediated enterotoxigenic Escherichia coli (ETEC)-induced pro-inflammatory responses and MAPKs:Porcine intestinal epithelial cell line (IPEC-1) formed an epithelial monolayer and the microvillus structures after12days incubation, These results have indicated that IPEC-1cells have successfully differentiated. We introduced the differentiated IPEC-1cells and observed the significant up-regulation expression of pro-inflammatory mediators after ETEC treatment (108cfu/ml), The probiotic Ba could effectively down-regulate IL-6, IL-la and TNF-a production in the differentiated IPEC-1cells with ETEC treatment, These data indicated that Ba inhibited ETEC-induced pro-inflammatory responses in the differentiated IPEC-1cells. Furthermore, the probiotic Ba attenuated ETEC-induced activation of the MAPKs pathways in differentiated IPEC-1cells. The activation level of the ERK1/2, JNK and p38was significantly decreased in the differentiated IPEC-1cells pre-incubated with Ba for24h. In sum, the probiotic Ba could inhibit ETEC-induced pro-inflammatory responses by suppression of MAPKs signaling pathways in intestinal epithelial cells.(2) Probiotic Bacillus amyloliquefaciens SC06mediated M1macrophage polarization in bone marrow-derived macrophages:Oral administration of Bacillus amyloliquefaciens SC06could decrease bacterial translocation in C57BL/6mice. Furthermore, dramatically increase in the percentage and number of macrophages were observed in the spleen of Ba-treated mice compared with the control and0.85%NaCl group (P<0.05). In contrast, there have been no statistically changes in the splenic CD4+T CD8+T and B cells. Meanwhile, no dramatically changes of IgG and IgM levels were found in intestinal mucosa among the groups. We were interested to know whether probiotic Ba could induce macrophages polarization. Real-time fluorescence PCR analysis demonstrated that the expression of IL-1β, iNOS, TNF-α and IL-6genes for M1macrophages was significantly increased at1.5h after probiotic Ba treatments compared to the probiotic Ba-free treatment (P<0.01), whereas the expression of M2macrophage marker genes (Arg1, Fizz1, MR, Ym1) was decreased (P<0.05). Furthermore, the phagocytic activity was dramatically increased in the Ba-treated BMDMs using a FITC-dextran endocytosis assay. These data indicated that probiotic Ba facilitated polarization of M1macrophages and enhanced its phagocytic capacity. Moreover, the Ba also mediated the autophagy in bone marrow-derived macrophages. The results provided the new insight that how probiotic Ba reduce the diarrhea and clear intracellular pathogens in weaned piglets.(3) The effects of dietary supplemented Ba replacement with kitasamycin on growth performance and gut flora in the fattening pigs:Adding Ba to swine diets increased ADG (P<0.05) and ADFI (P<0.05) compared to control fed pigs. Compared with the control fed pigs, villus height was significantly increased (P<0.05), crypt depth ratio (V:C) have no significant change (P<0.05). Roche454sequencing data showed that the dominant bacteria of the cecum were the Firmicutes and Bacteroides in fattening pigs. Bacteroides dominated a large portion in the cecum and the Fusobacterium accounted for a small proportion. Cecum of the fattening pigs main contained the Firmicutes, Bacillus, Clostridium and Actinobacteria. Bacillus accounted for the major proportion of Firmicutes in the antibiotic group, antibiotics+Ba and Ba treatment group were67.98%,82.13%and84.48%and the proportion of the Erysipelotrichi holded13.9%,6.77%and6.15%, respectively. Taken together, the Ba could promote growth performance and improve the intestine health, as well as regulate microbial ecological balance in the fattening pigs.(4) Dietary supplemented Ba alleviated fed diets containing kitasamycin inducing liver injury in fattening pigs:Most of serum biochemical parameters were not different in the three dietary treatments and were within the normal physiologic concentrations. Interestingly, pigs fed diets containing kitasamycon with no Ba had higher glutamic pyruvic transaminase (ALT) and glutamic oxalacetic transaminease (AST) content than the other two treatments (P<0.05), especially the ALT43.8IU/L which was over the normal physiological range. Therefore, these data indicated that the Ba might prevent fed diets containing kitasamycin inducing liver damage. The most abnormality seen was damage to mitochondria and endoplasmic reticulum (ER) including the endoplasmic reticulum dilated and vesiculated, mitochondria swell, cristae vague in fed diets containing antibiotic kitasamycin. Dramatically increase in mitochondrial diameter was found in fed diets containing antibiotic kitasamycin compared with fed diets containing Ba (P<0.05). The average number of ER vacuoles per field in fed diets containing antibiotic kitasamycin was notably higher than that fed diet containing Ba groups (P<0.05). These results suggested that normal ER and mitochondria were restored when fattening pigs were fed with probiotic Ba-supplemented diet. Probiotic Ba may prevent fed diets containing kitasamycin induced-liver injury by attenuated ER and mitochondria damage.The results provide the implications for elucidating the mechanism that the probiotic Ba regulates the immune response, and also supplying the technological and theoretical guidance for application of the probiotic Ba in livestock and feed industry. |
PDF Full Text Request