Gene expression of pro-inflammatory (IL-8, IL-18, TNF-α, and IFN-γ) and anti-inflammatory (IL-10) cytokines in the duodenum of broiler chickens exposed to lipopolysaccharides from Escherichia coli and Bacillus subtilis

Abstract

Background and aim: Intestinal infections are associated with Gram-negative bacteria like Escherichia coli. When eliminated by treatments during replication, E. coli release lipopolysaccharides (LPS) that can activate the intestinal immune system and increase the expression of cytokines, such as interleukin (IL)-8, IL-18, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), by the intestinal epithelium under pathological conditions. This study aimed to evaluate the addition of Bacillus subtilis to the duodenal gene expression of pro-inflammatory and anti-inflammatory cytokines in broilers exposed to LPS from E. coli.

Materials and methods: RNA was extracted using the Zymo Research total RNA commercial kit, according to the manufacturer's specifications, from the intestinal tissue of the duodenum previously resuspended in the lysis buffer of the kit. The expression of the cytokines of interest was measured using the QuantiNova SYBR green real-time polymerase chain reaction kit (Qiagen). Transcript quantification was performed by the ΔΔC(t) method using glyceraldehyde 3-phosphate dehydrogenase as a normalizing constitutive gene.

Results: For the measurement of pro-inflammatory (IL-8, IL-18, TNF-α, and IFN-γ) and anti-inflammatory (IL-10) cytokines, there was no statistically significant difference (p > 0.05) between the basal diet and the diet with antibiotic (avilamycin). There was a statistical difference (p < 0.05) between diets with LPS. The diet with B. subtilis presented the lowest expression; the results differed on each sampling day (days 14, 28, and 42).

Conclusion: A decrease in the expression of pro-inflammatory cytokines (IL-8, IL-18, TNF-α, and IFN-γ) and an increase in IL-10 (anti-inflammatory) was observed; in this way, a balance of the inflammatory response to bacterial infection is achieved, suggesting that the use of B. subtilis as an additive in a broiler diet has a similar effect to that produced with antibiotic growth promoter.

Keywords: Escherichia coli; antibiotic; enteritis; lipopolysaccharides.

Figure-1

Expression of ARNm of interleukin-8 between each diet of the experiment and different sampling days. Basal diet (DB): Commercial diet without the addition of antimicrobial, lipopolysaccharides (LPS) and Bacillus subtilis. Diet 2 (D2): DB plus the addition of antimicrobial (avilamycin, 10 ppm). Diet 3 (D3): DB plus the addition of B. subtilis at a rate of 50 ppm of food. Diet 4 (D4): DB plus the addition of 1.0 μg of LPS/g in the feed. Diet 5 (D5): DB plus the addition of 1.0 μg of LPS/g in the feed and in the antimicrobial. Diet 6 (D6): DB plus the addition of 1.0 μg of LPS/g in the feed and Bacillus subtilis at a rate of 50 ppm of feed. ^{A, B, C, D, E} means with different superscripts are statistically different (p < 0.05). Means with a common superscript (variable under study) do not differ statistically (p > 0.05).

Figure-2

ARNm of tumor necrosis factor-alpha expression between each experiment diet and different sampling days. Basal diet (DB): Commercial diet without the addition of antimicrobial, lipopolysaccharides (LPS), and Bacillus subtilis. Diet 2 (D2): DB plus the addition of antimicrobial (avilamycin, 10 ppm). Diet 3 (D3): DB plus the addition of B. subtilis at a rate of 50 ppm of food. Diet 4 (D4): DB plus the addition of 1.0 μg of LPS/g in the feed. Diet 5 (D5): DB plus the addition of 1.0 μg of LPS/g in the feed and in the antimicrobial. Diet 6 (D6): DB plus the addition of 1.0 μg of LPS/g in the feed and B. subtilis at a rate of 50 ppm of feed. ^{A, B, C, D, E} means with different superscripts are statistically different (p < 0.05). Means with a common superscript (variable under study) do not differ statistically (p > 0.05).

Figura-3

Expression of ARNm of interleukin-18 between each experiment diet and different sampling days. Basal diet (DB): Commercial diet without the addition of antimicrobial, lipopolysaccharides (LPS) and Bacillus subtilis. Diet 2 (D2): DB plus the addition of antimicrobial (avilamycin, 10ppm). Diet 3 (D3): DB plus the addition of B. subtilis at a rate of 50 ppm of food. Diet 4 (D4): DB plus the addition of 1.0 μg of LPS/g in the feed. Diet 5 (D5): DB plus the addition of 1.0 μg of LPS/g in the feed and in the antimicrobial. Diet 6 (D6): DB plus the addition of 1.0 μg of LPS/g in the feed and B. subtilis at a rate of 50 ppm of feed. ^{A, B, C, D, E} means with different superscripts are statistically different (p < 0.05). Means with a common superscript (variable under study) do not differ statistically (p > 0.05).

Figure-4

Expression of ARNm interferon-gamma between each experiment diet and different sampling days. Basal diet (DB): Commercial diet without the addition of antimicrobial, lipopolysaccharides (LPS), and Bacillus subtilis. Diet 2 (D2): DB plus the addition of antimicrobial (avilamycin, 10ppm). Diet 3 (D3): DB plus the addition of B. subtilis at a rate of 50 ppm of food. Diet 4 (D4): DB plus the addition of 1.0 μg of LPS/g in the feed. Diet 5 (D5): DB plus the addition of 1.0 μg of LPS/g in the feed and in the antimicrobial. Diet 6 (D6): DB plus the addition of 1.0 μg of LPS/g in the feed and B. subtilis at a rate of 50 ppm of feed. ^{A, B, C, D, E} means with different superscripts are statistically different (p < 0.05). Means with a common superscript (variable under study) do not differ statistically (p > 0.05).

Figure-5

Expression of ARNm of interleukin-10 between each diet of the experiment and different sampling days. Basal diet (DB): Commercial diet without the addition of antimicrobial, lipopolysaccharides (LPS), and Bacillus subtilis. Diet 2 (D2): DB plus the addition of antimicrobial (avilamycin, 10 ppm). Diet 3 (D3): DB plus the addition of B. subtilis at a rate of 50 ppm of food. Diet 4 (D4): DB plus the addition of 1.0 μg of LPS/g in the feed. Diet 5 (D5): DB plus the addition of 1.0 μg of LPS/g in the feed and in the antimicrobial. Diet 6 (D6): DB plus the addition of 1.0 μg of LPS/g in the feed and Bacillus subtilis at a rate of 50 ppm of feed. ^{A, B, C, D, E} means with different superscripts are statistically different (p < 0.05). Means with a common superscript (variable under study) do not differ statistically (p > 0.05).