NE in broilers is caused by C. perfringens type A or C, which shows necrotic lesions in the broilers intestinal mucosa, thus reducing the feed conversion rate and causing serious economic losses in the broiler industry. C. butyricum, as a probiotic, can maintain the growth performance of broilers [21]. The supplement of C. butyricum in feed can promote growth performance and increase feed utilization [41, 45]. In this study, AGD decreased and FCR increased significantly after NE infection. After supplementation, C. butyricum could inhibit the decrease of AGD and the increase of FCR caused by C. perfringens-induced NE, thus confirming previous results.
The intestinal tissue morphology, villus height, and crypt depth are important indicators for the measurement of intestinal health, injury, and recovery. The nutrient absorption in the intestine increased with the increase of intestinal epithelial transport and the height of villus [28]. Dietary supplementation with probiotics such as Lactobacillus acidophilus and C. butyricum enhanced the restoration of intestinal morphology, the increase of villus height, and the decrease of crypt depth [7, 22, 46]. In this study, the NE broilers showed severe hemorrhage in the small intestinal tissue, proliferation of inflammatory cells, shorting of villus, and increase of crypt depth. By comparison, all the selected indices were clearly improved in the broilers fed with C. butyricum supplemented diet, suggesting that C. butyricum was beneficial to NE broilers to maintain the intestinal function.
IgA is the main component of the intestinal mucosal barrier and plays a role in intestinal protection. Previous studies have reported that C. butyricum can lead to reduction serum IgA after the Escherichia coli K88 infection [45]. Moreover, C. butyricum can promote the secretion of IgA in broilers [41, 42]. In the present study, we have shown that C. butyricum can inhibit the secretion of the IgA caused by NE infection (NECB1 and NECB2), thus supporting Huang’s results [18]. These results show that C. butyricum plays a role in intestinal mucosal immune protection after pathogen infection.
The endotoxin produced by LPS is related to tight junction proteins and intestinal permeability [16, 31], and it is an indicator for evaluating the integrity and function of the intestinal barrier [22]. Our results showed that the serum endotoxin was increased in NE broilers but was not increased in NE broilers fed with C. butyricum added diet, suggesting that C. butyricum aided in the maintenance of the intestinal function in NE broilers. These results are consistent with previous reports [22, 46].
Tight junction proteins are very important for the integrity and function of the epithelial barrier. They maintain the diffusion barrier and seal the intercellular space. OCLN is a TJ protein that acts as an adhesion molecule between cells to maintain and regulate the barrier function of TJs. CLDNs also play important role in regulating cellular signal transduction and paracellular transport in the intestinal epithelium [20]. ZO proteins are located on the surface of the inner cytoplasmic membrane. ZO-1 is important, because it connects TJ proteins and the actin backbone [24]. Alpha-toxin of C. perfringens can damage the intestinal mucosal barrier [27], and the expression of TJ proteins such as CLDN-1, CLDN-3, OCLN, and ZO-1 after NE decreased to varying degrees compared with the control group [33, 39, 44]. NE leads to mRNA expression increase in the pore-forming TJ protein CLDN-2 [44].Similar to CLDN-2, CLDN-5 is a pore-forming protein. In the present study, we observed that CLDN-5 increased with C. perfringens-induced NE but with supplementation of C. butyricum, the mRNA expression of CLDN-5 decreased. This showed that the addition of C. butyricum removed the decreasing tendencies of CLDN-1, CLDN-3, OCLN, and ZO-1 expression because of NE, thus confirming the results of previous studies [18, 33]. MUCs interact with IgA and various growth factors to maintain the relatively stable intestinal environment [14]. MUCs have potential binding sites for pathogenic microorganisms and colonization of some bacteria could induce MUCs expression change [36]. After broilers develop NE, the expression of MUCs such as MUC2 and MUC5AC changed [13, 38]. Butyric acid could increase the mRNA expression of MUC2 inhibited by NE [33]. Our data are in agreement with these results. Furthermore, MUCs could provide nutrition for the proliferation of C. perfringens [4]. Although C. perfringens may be present in the normal flora of healthy broilers but it is generally believed that a large number of C. perfringens proliferate in the intestinal tract, causing NE in chickens. In healthy birds, C. perfringens is present in the range of 102–104 CFU/g intestinal contents, but in NE, the concentration of C. perfringens increased to 107–109 CFU/g [32]. In our result, the C. perfringens plc increased as a result of NE, but the supplementation of probiotic inhibited the increase in plc expression. Similar results were reported by Xu [40] and Huang [18]. This finding indicates that C. butyricum can reduce the proliferation rate of C. perfringens and intestinal barrier damage caused by NE.
Cytokines regulate cell growth and immune response and participate in inflammatory response. Among these cytokines, TGFB1 is related to mucosal immune tolerance. IL-10 is mainly secreted by T cells and is an important anti-inflammatory cytokine, and IL-10 has a barrier protective effect [30]. TNF-α is a key regulator of inflammation. IL-6 is an effective pro-inflammatory cytokine of Th1 cells, mainly secreted by intestinal epithelial cells [1]. We observed that at the instance of NE, the expression of anti-inflammatory factors TGFB1, IL-10, and pro-inflammatory cytokines TNF-α and IL-6 increased. However, supplementation of C. butyricum lowered the upregulation of TGFB1, IL-10, IL-6, and TNF-α observed in NE infection. Our findings support the increase in cytokine secretion after the addition of probiotics or food additives that can reduce inflammation after pathogens infection in broilers and pigs [6, 7, 12, 43]. This result shows that an increase of immune factors caused by NE owing to C. perfringens stimulates the inflammatory immune response in the intestine.
Ussing chamber is a useful tool for detecting the changes in the current and resistance of epithelial tissue by using electrodes to assess the integrity of the intestinal epithelial barrier. Evidence has been presented that after supplementing probiotic Yeast Saccharomyces boulardii in pig for 8 days, Gt remained unchanged, while Isc decreased, but Isc recovered after 16 days [29]. Gt (tissue conductance) is a sign of tissue integrity, while Isc (short-circuit current) is a sign of net ion migration activity in the intestine, and the decrease of Gt and Isc indicates the closure of ion channels [28]. Previous work had reported that after broilers were infected with Salmonella or Campylobacter, Gt decreased because of the closure of ion channels, and the decrease of Gt was related to the decrease of net charge transfer of epithelial cells and the decrease in Isc [2, 3]. Hence, the intestinal function of livestock and poultry is improved by probiotics. In the present study, Ussing chamber analysis showed that Gt and Isc were significantly decreased in NE broilers, suggesting that the integrity of the intestinal epithelial barrier declined. Interestingly, the supplementation of C. butyricum could reverse the decrease in the values of Isc and Gt, signifying that C. butyricum can potentially maintain the integrity of the intestinal epithelial barrier in NE broilers. These results were in accordance with the expression patterns of CLDN-1, MUC-2, OCLN, and ZO-1 as indicated by real-time PCR assay. However, the addition of organic acids dose had no effect on the intestinal barrier [28]. The differential values of Gt and Isc in this study maybe caused by the different dosages of C. butyricum. The flux of FD4 between the intestinal epithelium mainly occurred through the cell bypass pathway. The increased flux of FD4 reflects the increased paracellular permeability and impaired intestinal barrier, which is inversely proportional to OCLN expression level [5, 17, 44]. In the present study, when NE was induced by the C. perfringens infection, the flux of FD4 on the mucosal side increased, whereas the expression of OCLN decreased, indicating that the permeability of the intestine was increased, that is, the intestinal barrier was damaged. The addition of food additives such as L-arginine can inhibit the increase of FD4 flux caused by NE [44]. In the present study, the addition of C. butyricum showed the same result, indicating that C. butyricum can promote and maintain intestinal permeability of broilers.