Effects Of Bacillus On Intestinal T Cells Immune Responses In F4R~-Weaned Piglets Challenged With F4~+Escherichia Coli

Piglet diarrhea seriously threatens the healthy development to pig industry. F4ab/ac+ Enterotoxigenic Escherichia coli (ETEC) is a major cause of weanling piglet diarrhea induced by Escherichia coli. Although breeding of Fab/ac4 receptor-negative pigs may prevent F4ab/ac+ E. coli-induced post-weaning diarrhea in newly weaned pigs, the underlying immunity is poorly understood. Selected probiotic mixtures can enhance regulatory T (Treg) cell function, maintain the intestinal immune homeostasis, hence, probiotic represent a promising alternative to antibiotics for controlling enteric infections. Bacillus subtilis and Bacillus licheniformis had been used in human and livestock decades for regulation of innate and adaptive immune responses. But the underlying immunity between Bacillus and intestinal mucosa is poorly understood.In this study.a total of 32 F4ab/acR crossbred (Landrace × Large White) piglets of mixed gender, selected from 8 different litters, weaned at 21 days of age were divided into four groups: each group received a different treatment, as follows:(1) control (CONT) group (oral administration of sterile physiological saline); (2) ETEC group (oral administration of sterile physiological saline and oral challenge with E. coli); (3) LDBE group (oral administration of low-dose probiotic mixture [3.9 × 107 CFU/mL] and oral challenge with E. coli); (4) HDBE group (oral administration of high-dose probiotic mixture [7.8 × 107 CFU/mL] and oral challenge with E. coli). At 9:00 h each day on days 1 through 7, pigs in the CONT and ETEC groups were administered 10 mL of sterile physiological saline orally, whereas pigs in the LDBE and HDBE groups received an equal volume of low-dose (3.9 × 107 CFU/mL, once daily) or high-dose (7.8 × 107 CFU/mL, once daily) probiotic mixture solution, respectively, via oral administration. At 9: 00 h on day 8, pigs in the ETEC, LDBE and HDBE groups were administered 10 mL of E. coli culture (109 CFU/mL) orally, whereas pigs in the CONT group received the same volume of sterile physiological saline. The probiotic groups did not received any more probiotics after E. coli challenge. Piglets were euthanized on day 15. The jejunal and ileal enteritis were assessed by H&E straining; T help cells related gene expressions in the intestines was assessed by real-time PCR; The Treg cells subsets within PB and GALT were analyzed by four-color flow cytometry; Blood samples were collected from the jugular vein and serum concentrations of IL-10, IL-17A, IgA and TNF-awere detected by porcine-specific commercially available ELISA kits.Administration of BLS-mix increased the percentage of Foxp3-IL-10+ Treg cells but not of Foxp3+IL-10+ Treg cells among peripheral blood CD4+ T cells. A low dose of BLS-mix feeding resulted in increased expression of mRNAs for IL-6, TNF-α, IL-10, and the transcription factors Foxp3 and T-bet in the jejunum. Administration of either a low or high dose BLS-mix also led to an increase in the percentage of CD4+Foxp3+ Treg cells among intraepithelial lymphocytes and CD4+IL-10+ T cells in the small intestinal Peyer’s patches and the lamina propria of F4ab/acR pigs following F4ab/ac+ E. coli challenge. The increased number of IL-10-producing CD4+ T cells was attributed to an increase in the proportion of Foxp3 IL-10+ rather than Foxp3+IL-10+ Treg cells.Our data indicate that oral inoculation of newly weaned F4 ab/acR pigs with F4ab/ac+ E. coli results in enteritis and excessive systemic inflammatory responses, and that oral administration of either a low or high dose of BLS-mix leads to an increase in the proportion of Foxp3-IL-10+ but not of Foxp3+IL-10+ Treg cells both in the blood as well as the intestinal mucosa of F4 ab/acR- pigs following F4ab/ac+ E. coli challenge. Our data indicate that the induction of IL-10-producing Tr1 cells by BLS-mix cannot account for protecting newly weaned F4ab/acR- pigs from F4ab/ac+ E. coli infection, and that excessive generation of CD4+IL-10+ Treg cells during intestinal inflammation that is caused by an enteric pathogen might prohibit clearance of the pathogen.