Diarrhea of piglets has been a major problem in the pig industry, which can lead to high mortality of piglets in china. RV is one of the main viral pathogens that cause diarrhea and death in piglets. Many experiments have found that RV can decrease growth performance, induce diarrhea, and impair immune function in children and young animals [22–24], which was consistent with the results of this study. In addition, our study also showed that RV infection increased RV-Ab levels in serum and jejunal mucosa of weaned pigs, and induced the decreasing cell viability and the increasing generation of NSP4 in IPEC-J2 cells. These results demonstrated that the in vivo and in vitro models of RV infection in weaned piglets and IPEC-J2 cells were successfully implemented, respectively.
In this study, dietary LNT supplementation could reduce F/G ratio of weaned pigs, which is consistent with previous studies . And we also found that LNT relieved the effect of RV infection on ADG and F/G ratio of weaned pigs. The digestion, absorption and utilization of nutrients are important factors that affect growth performance, which, to some extent, can be evaluated by nutrient digestibility and blood urea nitrogen level. The previous study has shown that dietary LNT supplementation could increase the nutrient digestibility of pigs , which is consistent with the results of our study. Moreover, in the present study, supplementing LNT in diets might also alleviate the increasing SUN level in piglets with or without RV infection. Thus, it is possible that LNT promoting the growth of piglets should be relative with the increasing digestion, absorption and utilization of nutrients.
The gut health, especially integrity of intestinal epithelial cells, is very important to the digestion and absorption function. Our previous study has shown that dietary LNT supplementation improved the morphology of jejunal mucosa in the normal and RV-infected weaned pigs . Moreover, in present study, we also found that LNT polysaccharide enhanced the viability of IPEC-J2 cells, and alleviated the negative effect of RV challenge on the viability of IPEC-J2 cells.
Concentrations of LNT polysaccharide ranged from 1.5 to 24.0 mg/L were chosen in our study, which are in accordance with the levels probably encountered in the gastrointestinal tract of piglets after feed intake in animal breeding experiments. Based on the water content in the digesta of small intestine and the recommending dose of LNT product (purity of polysaccharide is 30%) in feeds, the concentrations of LNT product should be 4.5–72.0 mg/L. Finally, the effective dose of LNT polysaccharide (purity of polysaccharide is 95%) in cell experiments was about 1.5–24.0 mg/L.
Immune function can withstand the pathogen, which is very pivotal to maintain gut health of animals and human. Many researches have reported that LNT can regulate immune function [27–29]. Immunoglobulin levels are the important index of humoral immunity . In many in vivo and in vitro studies, LNT can regulate the immunoglobulin levels [31–34]. This study reported that dietary LNT supplementation could increase serum IgA, IgM and IgG levels in weaned piglets, and further increase IgM level of serum and RV-Ab level of serum and jejunal mucosa in RV-infected piglets. Besides these, we also found that LNT administration restored the effect of jejunal mucosal sIgA level, which is considered as the important content of intestinal immunologic barrier [35, 36]. These demonstrate that LNT improved the growth and health, at least partially, via increasing the humoral immunity of body.
As a kind of type I interferon, IFN-β can stimulate the productions of antiviral protein in cells via up-regulating expressions of some genes (such as ISG-15), which reduces virus replication. Then, it was considered as antiviral agents . Under some disease condition (i.e. human immunodeficiency virus, HIV), LNT induces interferon secretion [38, 39]. The current study also showed that, under RV infection condition, LNT administration further increased the IFN-β level in the jejunal mucosa of weaned pigs, and the IFN-β and ISG-15 mRNA expressions in IPEC-J2 cells. Thus, dietary LNT supplementation alleviating RV-induced diarrhea could be associated with IFN-β-increasing antivirus capacity.
When RV invades gut and cells via pathogen-associated molecular pattern (PAMPs), these PAMPs are sensed by pattern recognition receptors (PRRs) of host . These PRRs, such as Toll-like receptors (TLRs), RIG-I-like receptors, are expressed on many cells (including immune cells, epithelial cells) [41, 42]. Following PAMPs’ stimulation, host immunity (PRRs and their related signaling pathways) will be activated, and lead to the transcription and synthesis of many cytokines (especially, type I interferon). This will efficiently inhibit pathogen invasion . In our study, RV infection, to some extent, up-regulated TLR3, RIG-I, MDA5 and MAVS mRNA expressions in IPEC-J2 cells, which may have induced the anti-viral immunity of host. Additionally, we also found that, in RV-infected IPEC-J2 cells, LNT polysaccharide further stimulated RIG-I, MDA5 and MAVS mRNA expressions, but did not affect TLR3 mRNA expression. These also showed that LNT polysaccharide increased anti-viral function of type I interferon via regulating RIG-I/MDA5/MAVS pathways, which could be not associated with TLR3 and its relative pathways.
Host defense peptides (HDPs) also are an important part of innate immunity, which are the barriers for preventing invasion of pathogens . This process is derived from HDPs regulating inflammatory cytokines and responses via some signaling pathways, such as Toll-like receptors 4, mitogen-activated protein kinase and nuclear factor-kappa B signaling pathways [45–48]. And the HBD-2 mRNA expression in pulmonary epithelial cells could be induced by lentinan in a concentration- and time-dependent manner . The current study showed that RV infection decreased the mRNA expressions of pBD1, pBD2 and pBD3 in jejunal mucosa of piglets and IPEC-J2 cells, but LNT administration relieved the effect of RV infection on pBD1, pBD2 and pBD3 mRNA expressions in jejunal mucosa of piglets, and pBD2 and pBD3 mRNA expressions in IPEC-J2 cells. About the different results between in vivo and in vitro trials, it was possibly derived from LNT contents and HDPs’ generation mechanisms. Thus, LNT improving immune function of RV-infected host could be due to the stimulation of HDPs in intestinal mucosa.