The effect of Lactobacillus on the expression of porcine β-defensin-2 in the digestive tract of piglets

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Abstract

Some probiotic lactobacilli strains are known to confer health benefits to piglets by modulating its immune functions. This study investigates how intestinal mucosal immune responses in the digestive tract of neonatal piglets are influenced by Lactobacillus colonization. L. salivarius B1, isolated from the intestinal mucosa of a healthy piglet, was confirmed to possess excellent probiotic properties, such as strong adhesion to Caco-2 cells and high lactic acid production in vitro. It is hypothesized that early administration of L. salivarius B1 might facilitate colonization and promote intestinal mucosal immune responses in the piglet. The neonatal piglet was used as a model to investigate the in vivo colonization of L. salivarius and the effect on pBD-2 expression in response to L. salivarius B1. Results show that L. salivarius B1 colonizes duodenal mucosa early and upregulates the expression of pBD-2 in a non-inflammatory manner (p < 0.01). Levels of pBD-2 in saliva were observed to increase upon exposure of enterocytes to L. salivarius B1 and were tightly correlated with the administration of successive doses of L. salivarius B1. These findings provided further insight into the effects of probiotic Lactobacillus on the intestinal mucosal immune response of the neonatal piglet and suggest that pBD-2 upregulation may be in part responsible for the immune response induced by L. salivarius.

Introduction

Probiotic microorganisms confer a health benefit on the host when administered in adequate amounts (Vanderpool et al., 2008). The interaction between probiotics and enterocytes initiates immunomodulation in the host, and is responsible in part for the benefits conferred by probiotics (Collado et al., 2009, Delcenserie et al., 2008). Recent reports have shown that certain probiotics can enhance the function of the intestinal epithelial barrier by inducing the production of defensins (Schlee et al., 2007, Schlee et al., 2008).

Lactobacillus is prevalent in the intestinal tract in humans and animals. Several probiotic Lactobacillus strains confer health benefits to piglets by modulating their immune functions (Walsh et al., 2008, Wang et al., 2009). These health benefits are in part dependent on persistent Lactobacillus colonization of the gut mucosa (Ouwehand et al., 2002). Screening for properties such as adhesion to intestinal epithelial cells and lactic acid production can help in the identification of novel probiotic strains (Borchers et al., 2009, Collado et al., 2009). In addition, host specificity of the probiotic bacteria is also important to consider and recommended as one of the safe selection criteria (Bernardeau et al., 2008).

Studies have shown that microbial flora in the gastrointestinal tract influences the development of the immune system of piglets (Butler et al., 2000, Butler et al., 2009). Most studies have focused on studying the effects of probiotics on the immune system of weaning piglets. The gut microbiota may be more easily colonized in neonatal piglets than in those of health piglets with stable microbial populations(Mackie et al., 1999), and colonization in neonatal piglets has a long-term impact on gut community structure (Thompson et al., 2008). Therefore, the neonatal piglet is an appropriate model for investigating the effects of probiotcs on the intestinal mucosal immune response.

An innate immune response of neonatal piglets is the secretion of antimicrobial peptides (AMPs). AMPs known as defensins play an important role in the innate immune response of the intestine. More than ten β-defensins have been described in the pig. The β-defensins are expressed in the epithelial cells of the gastrointestinal tract as well as other organs exposed to the external environment (Sang et al., 2006). Previous studies have indicated that pBD-2 is expressed in the intestine (Veldhuizen et al., 2007) and promotes intestinal health in pigs (Veldhuizen et al., 2008). Some investigations have demonstrated that exposure to Lactobacillus can induce human β-defensin (hBD-2) production in enterocytes (Schlee et al., 2008).

In the present study, L. salivarius B1 was isolated from the duodenal mucosa of a healthy piglet and showed beneficial probiotic properties in vitro. To further investigate the effects of L. salivarius B1 in vivo, neonatal piglets were used as a model to investigate the expression of pBD-2 induced by L. salivarius B1 in the digestive tract of piglets.

Section snippets

Lactobacillus isolation and growth conditions

Lactobacillus strains were isolated from the duodenum samples of different pig breeds (30 days old) that were freshly slaughtered. The strains were cultured on modified Mann, Rogosa and Sharpe (MRS) medium (CaCO3 final concentration 0.75%, pH 5.8) and incubated at 37 °C for 24–36 h in 5% CO2. The isolated strains were grown on modified MRS agar plates. The lactic acid produced by the bacteria reacts with the CaCO3 in the medium and causes a clear plaque to appear around the colony. Lactic acid

Screening and identification of Lactobacillus in vitro

Thirty-six strains were isolated from the duodenum of Laiwu black piglets (Bn, n = 1…8), Landrace piglets (Cn, n = 1…9), Duroc piglets (Dn, n = 1…8) and mini piglets of Guizhou (Xn, n = 1…11). The strains were initially analyzed by microscopy and biochemical identification. PCR products amplified using primers specific for Lactobacillus confirmed the strains belonged to the genus Lactobacillus.

Lactic acid-production varied among the thirty-six isolates. Ten of thirty-six strains produced higher amounts

Discussion

Though the use of Lactobacillus to modulate the immune responses of piglets has been extensively reported (Konstantinov et al., 2008, Pieper et al., 2009, de LeBlanc Ade et al., 2010), it is important to continue screening for better and improved probiotic lactobacillus strains. L. salivarius can regulate the secretion of cytokines in vitro, though this property is strain dependent (Drago et al., 2010). The use of porcine L. salivarius clinically as a probiotic has been reported rarely, and it

Conflict of interest

All authors of this paper had no conflicts with other people or organizations.

Acknowledgments

This work was supported by a grant (30871858) from the National Science Grant of China and a grant (BE200830155) from the Support Program of Jiangsu province and a grant (GJJ10410) from Education Department of Jiangxi province, as well as helps by key laboratory animal biotechnology of Jiangxi province the ministry agriculture of China of Jiangxi Agricultural University and Liuhe Breeding Base, Jiangsu Province Academy of Agricultural Sciences.

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