| In this study, considering the high similarity of pig to human in the characteristics of anatomy, histology, physiology and nutrition metabolism, especially the digestive system, a human flora-associated (HFA) piglet model colonized with human intestinal flora was first established. The effects of human commensal bacteria on the gastrointestinal morphology, the proinflammatory cytokines in the serum and the local mucosal immunocompetent cells were studied. The possible mechanism on the effects of the predominant intestinal bacteria on the gastrointestinal mucosal immunity in germ-free piglet was explored. On the basis of the establishment of this model, the effects of the Fructo-oligosaccharides prebiotics on the intestinal mucosal immune in human flora-associated piglets were studied. The results are as follows:1 Establishment of human flora-associated (HFA) pigletIn order to successfully establish a HFA piglet model, the piglet variety, the source of fecal suspension, feeding and management conditions were discussed through five independent experiments in this study. Guizhou pig was selected in the first two experiments, Meishan, a Chinese indigenous breed, was selected in the last three experiments. The piglets obtained by sterile caesarean section were passed through a disinfectant delivery window into the gnotobiotic barrier system. The experiment was accomplished in the gnotobiotic barrier system.9,13,10,12 and 8 of the sterile were respectively administrated in the five experiments. The piglets were orally fed with the human fecal suspension at 12h after the birth and under the condition that the piglets were confirmed in good health. The fecal suspension of a healthy 10-year-old boy was used in the first three experiments, and the fecal suspension of a healthy adult man was used in the last two experiments. In the first 3d, the piglet was fed with 1 ml of fecal suspension per day; during 4-10 day-old, the piglet was fed with 1 ml of fecal suspension every other day. In the process of feeding and management, all the piglets were artificially fed with milk powder before weaning, and fed with infant rice after weaning. The room temperature was controlled well. All piglets were timely supplied with iron, vitamins and trace elements. The mental state and growth performance of piglets was observed, and the survival rate of piglets was analyzed in the different periods. The results showed that Meishan pig had a better growth performance and a higher survival rate comparing to the Guizhou pig. Meishan pigs fed with the fecal suspension of a healthy adult man showed better growth performance and higher survival rate in comparison with that fed with the fecal suspension of healthy boy,. The results suggest that Meishan pigs and fecal suspension of healthy adult man were more benefit to establish the HFA piglet model.2 Effects of human intestinal commensal bacteria on the gastrointestinal morphology and proinflammatory cytokines in the serum in HFA PigletThe newborn germ-free piglets, obtained by caesarean section, were weighed and orally inoculated with a human and a porcine fecal suspension, and artificially fed to establish a human flora-associated (HFA) group (n=8) and pig flora-associated (PFA) group (n=8), respectively. All pigs were weighed and slaughtered at six weeks. The serum was collected from blood by centrifuge. Tissue samples of intestine and stomach were collected and placed in Bouin's fixative. The gastrointestinal morphology was stained with Hematoxylin-eosin (H/E). The level of serum pro-inflammatory cytokines (TNF-a and IL-1β) was detected by ELISA kit. The results were as follows:1) Both groups of pigs performed well but HFA pigs had a somewhat better daily weight gain (p< 0.05).2) The jejunal villus height and crypt depth in the HFA pigs were significantly higher than PFA pigs (p< 0.05).3) When comparing with PFA pigs, the concentration of serum cytokine TNF-a was significantly lower (p< 0.05). The concentration of IL-1βwas lowest in all pigs and could not be detected. These results showed that the human intestinal symbiotic bacteria colonized in the intestine of pigs stimulated the development of the jejunum, promoted the growth of piglets, and played an important role on maintaining host health.3 Effects of human intestinal commensal bacteria on the level of the gastrointestinal local mucosal immunity in HFA PigletTissue samples of gastric fundus, gastric body, gastric pylorus, duodenum, jejunum, ileum and colon were collected from the human flora-associated (HFA) group (n=8) and pig flora-associated (PFA) group (n=8), respectively. Some samples were snap frozen in liquid nitrogen and stored at-80℃, the others were placed in Bouin's fixative. Tissue samples were studied by histochemistry and immunohistochemistry methods for detection of the immunocompetent cells in gastrointestinal mucosa. The results were as follows:1) The number of intraepithelial lymphocytes in jejunum was lower (p< 0.01), but the number of goblet cells containing neutral mucins was significantly higher in HFA pigs than PFA pigs (p< 0.05). No differences were observed in the total number of goblet cells and the number of mast cells.2) In comparison with PFA pigs, the areas of IgA producing cells in the gastric pylorus (p< 0.05) and jejunum (p< 0.01) and the areas of IgG producing cells in the duodenum, jejunum and ileum (p< 0.01) were significantly higher in HFA pigs. The areas of IgG producing cells in the three part of stomach were increased, but there was no significant difference.3) There was a significant increase (p< 0.01) in the area of CD4+ T cells of the jejunum within HFA group in comparison with PFA group.4) The areas of MHC class II expressing cells were significantly higher in the HFA pigs than PFA pigs, respectively in the duodenum (p< 0.01) and colon (p< 0.05).5) A similar distribution tendency of IgA and IgG producing cells, CD4+ T cells and MHC class II expressing cells was observed in pigs of two groups. These results showed that the human intestinal symbiotic bacteria stimulated the function of the local intestinal mucosal immunity in germ-free piglets, but didn't change the distribution characteristics of mucosal immunocompetent cells in different part of intestine.4 Research on the composition of the predominant intestinal bacteria in HFA pigletThe fecal sample was collected from the human flora-associated (HFA) group (n=8) and pig flora-associated (PFA) group (n=8), respectively. Microbacteria molecular ecosystem method was used to detect the quantity of the four predominant bacteria, such as Bacteroides spp., Clostridium leptum cluster, Bifidobacteriua Spp. and lactic acid bacteria (LAB). The results were as follows:1) The predominant flora such as Bacteroides spp., Clostridium leptum cluster, Bifidobacteriua Spp. and LAB was detected in the fecal samples if HFA and PFA piglets. The quantity of Bacteroides spp. and Clostridium leptum cluster was significantly higher in human fecal sample than that in HFA piglet, but the quantity of LAB was significantly lower (p< 0.01), no significant difference was observed in Bifidobacteriua Spp.. The quantity of Bacteroides spp. was significantly lower in pig fecal sample than that in PFA piglet (p< 0.01), but there was no significant difference in Clostridium leptum cluster, Bifidobacteriua Spp. and LAB.2) A significant increase of the amount of the Bifidobacteria spp. was observed in HFA pigs compared with PFA ones (p< 0.01), and the composition of Bifidobacteria spp. was different between the two groups piglet. However, the amounts of the other three microbial populations (Bacteroides spp., Clostridium leptum cluster and LAB) in the feces showed no significant differences between the two groups. These results confirmed that the composition of human gut microbes and the quantity increase of Bifidobacteria spp. stimulated the activation of intestinal mucosal immunocytes and the secreting of nature antibody, so that to promote the level of host intestinal mucosal immunity. The results suggest that commensal bacteria have great effects on the intestinal health and development.5 Effects of Fructo-oligosaccharides on the intestinal mucosal immunocompetent cells in HFA pigletThe fifteen newborn piglets obtained by caesarean operation were weighed and randomly divided into three groups. Piglets in SPF (Specific Pathogen Free, control group) group were fed with kalium phosphate buffer (contain 10% glycerine). Piglets in HFA (human flora-associated) group were fed with human bacteria, all piglets were fed with 1 ml of fecal suspension per day in the first 3d, then fed with 1 ml of fecal suspension every other day during 4-10 day-old. Piglets in FOS (Fructo-oligosaccharides) group were fed with fructo-oligosaccharides (0.5g/kg weight) besides the human fecal suspension in the whole feeding period. All piglets were reared in a barrier system for 45 days under germ free condition, and then they were weighed and killed. Tissue samples from the duodenum, jejunum, ileum and colon were collected. The intestinal mucosal immunocompetent cells were studied by the histochemical and immunohistochemical methods. The results were as follows:1) The daily weight gain of piglet in FOS group was higher than that in HFA group. 2) The number of IgA producing cells in the jejunum was significantly higher in FOS group than that in HFA group (p< 0.05), the number of IgG producing cells in the duodenum was significantly lower (p< 0.01); no significant differences were detected in the numbers of small intestinal intraepithelial lymphocytes and mast cells between the two groups.3) When comparing with control group (SPF group), a significant increase of the number of IgA producing cells was observed in the ileum within the HFA and FOS group (p< 0.01), however, the number of IgG producing cells distributed in the duodenum was significantly higher in piglets of HFA group as compared to piglets of control group (p< 0.01).4) The most IgA and IgG producing cells was located in the duodenum, and the number of IgA and IgG producing cells progressively decreased along with the jejunum, ileum and colon. The results suggested that the fructo-oligosaccharides supplied a protection on the intestinal epithelium, in some extent, also promoted the level of local intestinal mucosal immunity of HFA piglets.
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