Effects Of Dietary Manganese Sources And Levels On The Immune Responses Of Broilers Following Oral Salmonella Typhimurium

This study included two experiments.The objective of the first experiment was to investigate the effects of high dietary manganese(Mn)on the immune responses of broilers following oral Salmonella typhimurium(S.typhimurium)inoculation,and to identify the important process regulated by Mn in these immune responses.The aim of the second experiment was to determine the influences of different Mn sources and level on the Mn-regulated immune processes in broilers following oral S.typhimurium inoculation,and these potential mechanisms.Experiment 1:effects of high dietary Mn on the immune responses of broilers following oral S.typhimurium inoculationA total of 144 1-day-old male broilers(Cobb 500)were randomly divided into two groups with 6 replicates each treatment(20 birds per replicate cage).The two treatments included a basal diet(containing 20.04 mg Mn/kg)plus an additional 40(the control group)or 400 mg Mn/kg(the H-Mn group).The broilers were fed with the designed diet for 7 days,and then one half of broilers in each replicate cage were orally inoculated with 5 × 107 CFUs of S.typhimurium(ATCC#14028)or phosphate-buffered saline(PBS).Peripheral blood,spleens,cecal tonsils,and bursa of Fabricius were collected from,Salmonella-inoculated and-noninoculated broilers(n =6)2 days post-inoculation(2 DPI)and 7 days post-inoculation(7 DPI).Peripheral blood lymphocyte subpopulations were determined by flowcytometry.The mRNA abundance of genes was determined by quantitative real-time polymerase chain reaction.Salmonella counts were higher(P<0.05)in the H-Mn group than those in the control group at 2 DPI in cecal contents of Salmonella-inoculated broilers.High dietary Mn increased(P<0.05)CD3+CD4+ and CD3+CD8+ T cells percentages in the peripheral blood of Salmonella-inoculated broilers at 2 DPI.Salmonella inoculation increased(P<0.05)interleukin(IL)-6 mRNA expression in spleens and bursa of· Fabricius at 2 DPI and increased(P<0.05)IL-1β and IL-6 mRNA expression in cecal tonsils at 7 DPI in the H-Mn group.These changes were not observed in the control group.High dietary Mn increased(P<0.05)interferon-,y(IFN-y)in spleens and decreased(P<0.05)IFN-y and IL-12 mRNA expression in cecal tonsils of Salmonella-inoculated broilers at 2 DPI.High dietary Mn decreased(P<0.05)IL-17 mRNA expression in the bursa of Fabricius at 7 DPI,but increased(P<0.05)this expression in cecal tonsils at 2 and 7 DPI in Salmonella-inoculated broilers.These results suggested that dietary Mn level promoted the inflammation reaction,and affected the T helper(Th)1-cytokines reaction in spleens and cecal tonsils,and Thl7-mediated immunity in cecal tonsils and the bursa of Fabricius of broilers following oral S.typhimurium inoculation.Experiment 2:dietary Mn sources and levels influenced the immune responses of broilers following oral S.typhimurium and these potential mechanismsThis exprement was conducted in 2 X 2 X 3 factorial designment with 2 Mn sources(Mn-methionine complex(MnMet)or Mn sulfate(MnSO4),2 stress condition(Salmonella-inoculation or-noninoculation),and 3 Mn levels(a basal diet plus additional 0(Control),40 or 100 mg Mn/kg),and two Mn source shared one control group.Thus,a total of 600 1-day-old male broilers(Cobb 500)were randomly divided into 10 treatment groups with 6 replicates each group(10 birds per replicate cage).After feeding the designed diets for 6 days,Salmonella-tredted broilers were orally inoculated with 4 x 108 cfu of S.typhimurium(SC0906)or equal volume of PBS.One bird was sacrificed at 2 DPI and 7 DPI in each replicate.The results:(1)Salmonella counts in cecal contents were higher(P<0.05)in Salmoenlla-inocualted broilers fed with the basal diet supplemented with 100 mg Mn/kg than those broilers fed with 40 mg Mn/kg at 2 DPI.MnMet complex decreased(P<0.05)the number of Salmonella in cecal contents of Salmonella-inoculation broilers as compared with Mn sulfate at 2 DPI.(2)At 2 DPI,plasma IL-1 concentration was higher(P<0.05)in the MnMet-100 group than that in the MnMet-40 and MnSO4-100 groups.As compared with control and 40 mg Mn/kg supplemental groups,addition of 100 mg Mn/kg had a trend to increase plasma IL-6 concentration(P<0.1).MnMet complex increased(P<0.05)plasma IL-1 and IL-6 concentrations at 2 DPI,whereas decreased(P<0.05)these indices at 7 DPI as compared with Mn sulfate.At 2 DPI,Mn supplementation decreased(P<0.05)the mRNA expression of IL-1β and IL-6 in spleens and bursa of Fabricius as compared with control.However,TNF-a mRNA abundance in bursa of Fabricius was higher(P<0.05)in the MnSO4-100 group than that in the MnSO4-40 group at 2 DPI.At 7 DPI,Mn supplementation downrgulated TNF-α mRNA level in cecal tonsils of broilers as compared with control.The IL-1 and IL-6 mRNA abundance were lower(P<0.05)in the MnMet-100 group than those in the MnMet-40 group at 7 DPI,but no significant differences were observed in these indices between MnS4-40 and MnSO4-100 groups.At 2 DPI,MnSOD mRNA expression increased(P<0.05)with dietary Mn level increasing in spleens.The NF-κB mRNA abundance in spleens was higher(P<0.05)in the MnSO4-100 group than that in the MnSO4-40 group at 7 DPI,but no significant differences were observed between MnMet-100 and MnMet-40 groups.At 7 DPI,Mn supplementation increased(P<0.05)IL-12 and IFN-γ mRNA levels in spleens of broilers as compared with control.MnMet complex uprcgulated(P<0.05)NF-κB and TRAF6 mRNA expression in bursa of Fabricius as compared with Mn sulfate at 2 DPI.(3)With dietary Mn level increasing,MnSOD activity in spleens and thymus increased(P<0.05),concentration of H2O2 in the mitochondria of spleens increased(P<0.05),while concentrations of ROS and NO in the mitochondria of thymus and bursa of Fabricius decreased(P<0.05).MnMet complex decreased(P<0.05)MnSOD activity in spleens and bursa of Fabricius as compared with Mn sulfate.These results suggested that Mn might regulate the inflammation reaction in broilers following oral S.typhimurium inoculation,through influencing ROS,H2O2 and NO production mediated by regulating MnSOD gene transcription and MnSOD activity.In summary,Mn could regulate the transcription and release of proinflammatory cytokines in broilers following oral S.typhimurium inoculation,through influencing H2O2/NF-κB pathway medieated by regulating of MnSOD transcription and activity.