Effect of Feeding Bacillus subtilis Spores to Broilers Challenged with Salmonella enterica serovar Heidelberg Brazilian Strain UFPR1 on Performance, Immune Response, and Gut Health

Abstract

Salmonellosis is a poultry industry and public health concern worldwide. Recently, Salmonella enterica serovar Heidelberg (SH) has been reported in broilers in Brazil. The effect of feeding a blend of three strains of Bacillus subtilis (PRO) was studied in broilers orally challenged (10⁷ CFU/chick) or not with a SH isolated in south of Brazil (UFPR1 strain). Twelve male Cobb 500 broilers per pen were randomly assigned to six treatments in a 3 × 2 factorial experiment where PRO was added at 0, 250, or 500 g/ton of broiler feed and fed to either SH-challenged (SH Control, SH + PRO 250, and SH + PRO 500) or non-challenged birds (Control, PRO 250, and PRO 500). Broiler performance, histologic alterations in intestinal morphology, Salmonella quantification and immune cells counts in liver (macrophages, T CD4+ and T CD8+) were analyzed. Changes in the intestinal microbiota of broilers were also studied by metagenomics for Control, SH Control, SH + PRO 250, and SH + PRO 500 only. Feeding PRO at 250 or 500 g/ton reduced SH counts and incidence in liver and cecum at 21 days of age. It was observed that PRO groups increased the macrophage mobilization to the liver in SH-challenged birds (P < 0.05) but reduced these cells in the liver of non-challenged birds, showing an interesting immune cell dynamics effect. PRO at 250 g/ton did not affect gut histology, but improved animal performance (P < 0.05) while PRO at 500/ton did not affect animal performance but increased histologic alteration related to activation of the defense response in the ileum in SH challenged birds compared to control birds (P < 0.05). SH + PRO 500 group presented a more diverse cecal microbiota (Shannon-Wiener index; P < 0.05) compared to Control and SH Control groups; while SH + PRO 250 had greater ileal richness (JackkNife index) compared to Control (P < 0.05). PRO was effective in reducing Salmonella colonization in liver and cecum when fed at 250 or 500 g/ton to broilers inoculated with SH strain UFPR1. PRO promotes positive alterations in performance (at 250 g/ton), immune modulatory effect in the gastrointestinal tract, SH reduction, and intestinal microbiota modulation.

Keywords: 16S; gut health; gut microbiome; immunity; poultry; probiotic; salmonellosis.

Figure 1

Salmonella sp. quantification. (A) Salmonella sp counts (Log CFU/g) in liver and cecum at 7 days of age (4 days after inoculation) in treatments SH Control, SH + PRO 250, and SH + PRO 500 according to adapted methodology by Cox et al. (21). (B) Salmonella sp. counts (Log CFU/g) in liver and cecum at 21 days of age in treatments SH Control, SH + PRO 250, and SH + PRO 500 according to adapted methodology by Cox et al. (21). (C) Relative abundance using metagenomics analysis in ceca at 21 days of age in treatments SH Control, SH + PRO 250, and SH + PRO 500. Non-challenged groups (Control, PRO 250, and PRO 500) were negative for Salmonella in both methodologies. Different letters indicate significant differences at P < 0.05 at Kruskal–Wallis.

Figure 2

Histological alterations in liver (A,B) and ileum (C,D) according to I See Inside (ISI) scoring methodology (100×). (A) Liver from SH Control, presenting score 3 of hydropic degeneration (HD) at 7 days of age. (B) Liver from SH + PRO 500, normal hepatocytes at 7 days of age. (C) Ileum from SH Control, villi with scores zero for epithelial thickness (ET) and lamina propria thickness (LPT) at 21 days of age. (D) Ileum from SH + PRO 250 with score 2 for epithelial thickness (ET), score 2 for proliferation of goblet cells (PGCs) and score 2 for LPT with inflammatory cells infiltration (ICI) at 21 days of age.

Figure 3

Rarefaction plot from ileal (A) and cecal (B) microbiota of groups Control, SH Control, SH + PRO 250, and SH + PRO 500. *P < 0.05. **P = 0.08. Rarefaction analysis suggested that the number of sequences from all experimental samples were enough to uncover major Operational Taxonomic Units (OTUs).

Figure 4

Ileal and cecal diversity (Shannon–Wiener), evenness (Hill), and richness (JackkNife) index of groups Control, SH Control, SH + PRO 250, and SH + PRO 500. *p < 0.05. **p = 0.08.

Figure 5

Relative abundance of bacteria population in ileal microbiota (A) and cecal microbiota (B) of groups Control, SH Control, SH + PRO 250, and SH + PRO 500 at 21 days of age, analyzed by sequencing using Illumina MiSeq System.

Figure 6

Relative abundance of distinct groups at the deepest level identified in ileum (A) and cecum (B) of groups Control, SH Control, SH + PRO 250, and SH + PRO 500. A yellower color depicts a greater bacterial abundance to up to 46.2% in ileum (A) abundance and up to 20.2% in cecum (B) abundance. Groups with abundance less than 1% were not considered. U.m., unidentified members. * Indicate significant differences at P < 0.05.