Abstract
Enteric diseases caused by Salmonella are prevalent in poultry farming. With the forbiddance of antibiotics in feedstuff industry, Bacillus subtilis (B. subtilis) preparation as antibiotic alternatives against Salmonella infection has gained increasing attention recently. However, the protection modes of B. subtilis against Salmonella infection in broilers are strain-specific. In this study, probiotic B. subtilis LF11 significantly reduced diarrhea and mortality of broilers caused by Salmonella braenderup (S. braenderup) in spite of no inhibition effect on it in vitro. Here, the intestinal epithelial cells NCM460 were incubated to explore the protection of B. subtilis LF11 on intestinal epithelium against Salmonella. The results revealed that B. subtilis LF11 showed obvious exclusion activity with the decrease of adhesion and invasion of S. braenderup to NCM460 cells, accordingly with the increase of NCM460 cell survival compared with S. braenderup challenge alone. Meanwhile, RT-PCR and Western blot proved that the gene transcription and expression levels of four tight junction proteins in NCM 460 cells were upregulated, which was further confirmed by immunofluorescence observation. Besides, B. subtilis LF11 downregulated the gene transcription levels of the proinflammatory cytokines IL-6, IL-8, and TNF-α induced by S. braenderup H9812. ELISA analysis also verified that B. subtilis LF11 reduced the IL-8 production significantly. In general, B. subtilis LF11 has the ability to protect the intestinal epithelium against Salmonella infection by reducing the Salmonella adhesion and invasion, enhancing the intestinal barrier and attenuating the enterocyte inflammatory responses, and has the potential as probiotics to prevent enteric diseases in broilers.
Highlights
Intestinal barrier as the first line of defense between the host and the luminal environment plays a key role on the absorption of nutrients and adequate containment of microorganisms and molecules, which consists of mucus layer, intestinal epithelium, and intraepithelial immune cells (Wlodarska and Finlay, 2010; Sanchez de Medina et al, 2014; Awad et al, 2017)
B. subtilis LF11 was isolated from corn silage previously (Li, 2019); S. braenderup H9812 and other indicator strains Escherichia coli (E. coli) ATCC 25922, Listeria monocytogenes (L. monocytogenes) ATCC 19114, L. monocytogenes ATCC 19115, Staphylococcus aureus (S. aureus) ATCC 27217, Streptococcus agalactiae (S. agalactiae) ATCC 13813, Micrococcus luteus (M. luteus) 2016, Bacillus cereus (B. cereus) SDMCC 050292, Enterococcus faecalis (E. faecalis) SDMCC 050338, Streptococcus lutetiensis (S. lutetiensis) SDMCC 050401, and Streptococcus infantarius (S. infantarius) SDMCC 050384 were stored in our laboratory and cultured according to methods described previously (Parveen Rani et al, 2016)
When NCM460 cells were preincubated with B. subtilis LF11 and were infected by S. braenderup H9812, IL-8 production significantly decreased by 60.7% (p < 0.01), which reduced by 23.9% in the case of the coincubation with B. subtilis LF11 and S. braenderup H9812 (Figure 5B). These results suggested that B. subtilis LF11 attenuated significantly the release of proinflammatory cytokines induced by S. braenderup H9812, and the exclusion activity of B. subtilis LF11 was stronger than the competition activity
Summary
Intestinal barrier as the first line of defense between the host and the luminal environment plays a key role on the absorption of nutrients and adequate containment of microorganisms and molecules, which consists of mucus layer, intestinal epithelium, and intraepithelial immune cells (Wlodarska and Finlay, 2010; Sanchez de Medina et al, 2014; Awad et al, 2017). B. subtilis Protects IECs Against Salmonella intestinal barrier results in the permeability increasement and/or bacterial translocation, which in turn induces various enteric diseases (Natividad and Verdu, 2013; Sanchez de Medina et al, 2014). Salmonella infection involves normally the attachment to epithelial cells, the disruption of intestinal barrier, and internalization into lamina propria, and leads to the inflammatory responses and even induces diarrhea and death in broilers (Bruno et al, 2009; Gut et al, 2018; Wang et al, 2018). Using of antibiotics as a traditional way to prevent and treat enteric diseases has led to the evolution of resistant strains of pathogenic bacteria (Nami et al, 2015; Lei et al, 2020). The forbiddance of antibiotic growth promoters facilitates the development of the functional feed additives as potential alternatives for antibiotics to avoid the incidence of bacterial resistance (Oh et al, 2017)
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