Necrotic enteritis (NE) causes huge economic losses to the poultry industry. Probiotics are used as potential alternatives to antibiotics to prevent NE. It is known that Clostridium butyricum can act as a probiotic that can prevent infection. However, whether or not it exerts a beneficial effect on NE in chickens remains elusive. Therefore, we investigated the impact of C. butyricum on immune response and intestinal microbiota during the development of NE in chickens, including experimental stages with basal diets, high-fishmeal-supplementation diets, and Clostridium perfringens challenge. Chickens were divided into two groups from day 1 to day 20: one group had its diet supplemented with C. butyricum supplementation and one did not. At day 20, the chickens were divided into four groups: C. perfringens challenged and unchallenged chickens with and without C. butyricum supplementation. All groups were fed a basal diet for 13 days and thereafter a basal diet with 50% fishmeal from day 14 to 24. Chickens were infected with C. perfringens from day 21 to 23. At days 13, 20 and 24, samples were collected for analysis of the relative expression of immune response and intestinal mucosa barrier-related genes and intestinal microbes. The results show that C. butyricum can inhibit the increase in IL-17A gene expression and the reduction in Claudin-1 gene induced-expression caused by C. perfringens challenge. Moreover, C. butyricum was found to increase the expression of anti-inflammatory IL-10 in infected chickens. Although C. butyricum was found to have a significant beneficial effect on the structure of intestinal bacteria in the basal diet groups and decrease the abundance of C. perfringens in the gut, it did not significantly affect the occurrence of intestinal lesions and did not significantly correct the shift in gut bacterial composition post C. perfringens infection. In conclusion, although C. butyricum promotes the expression of anti-inflammatory and tight junction protein genes and inhibits pro-inflammatory genes in C. perfringens-challenged chickens, it is not adequate to improve the structure of intestinal microbiota in NE chickens. Therefore, more effective schemes of C. butyricum supplementation to prevent and treat NE in chickens need to be identified.

Necrotic enteritis (NE) caused by Clostridium perfringens is responsible for huge financial losses in the poultry industry annually. A diet highly supplemented with fishmeal is one factor predisposing chickens to the development of clinical NE. However, the effects of fishmeal-rich diets on the gut microbiota and immune response in chickens with C. perfringens challenge over the long-term are not well-understood. Here, a chicken NE model was established in which chickens were fed high fishmeal diet and subsequently infected with C. perfringens (FM/CP). Two control groups of chickens, one that was not infected and had a high fishmeal feeding (FM) and another group only infected with C. perfringens with basic diets (CP), were used as comparators. We analyzed the gut microbiota and immune response of the three groups at the age of 20, 24 [1 day post-infection (dpi)] and 30 days (7 dpi) using 16S rDNA sequencing and real-time PCR, respectively. We found that the composition of the gut microbiota had significant shifted in both the CP and FM/CP groups, although the CP group did not have intestinal lesions. The structure of the gut microbiota in C. perfringens-challenged chickens, independent of a high fishmeal diet, had the tendency to return to their non-infection state if the chickens no longer received C. perfringens challenge. Gut microbiota variation with time in challenged chickens with high fishmeal diet feeding was superimposed upon that of non-infected chickens with high fishmeal feeding. For the immune response, the relative expression of IL-8 in the ileum was significantly higher in infected chickens independent of high fishmeal feeding than in non-infected chickens. However, the expression of alpha 1-acid glycoprotein (AGP) and serum amyloid A (SAA) genes in chicken liver were significantly increased in FM/CP compared to the other groups. In conclusion, high fishmeal feeding induced significant changes to the structure of chicken gut microbiota over time and such changes provided an opening for C. perfringens infection to progress to NE. The relative expression of AGP and SAA in liver tissue may be used as diagnostic biomarkers for poultry NE but such an indication requires further investigation.

The severe side effects of chemosynthetic anti-diarrhea drugs have created an interest in low-toxic alternative plant-derived compounds. FengLiao consists of Polygonum hydropiper Linn. and Daphniphyllum calycinum Bench., and is widely used in China to treat diarrhea due to low levels of toxicity. In this study, the effects of FengLiao were analyzed in a castor oil-induced diarrhea model, using the anti-diarrhea drug, loperamide, as the positive control. The effects were evaluated using stool characteristics and the expression levels of various diarrhea-related factors in the jejunum and liver, as well as changes in the microbiota of the jejunum. The symptoms of diarrhea and stool consistency were improved through FengLiao and loperamide treatment. Furthermore, FengLiao down-regulated alpha 1-acid glycoprotein (AGP) and C-reactive protein (CRP) levels, and up-regulated transferrin (TRF) mRNA levels in the liver, and down-regulated Aquaporin 3 (AQP3) and Na+/H+ exchanger isoform 8 (NHE8) expression in the epithelial cells of the jejunum. It also increased the relative abundance of Bifidobacterium, Aerococcus, Corynebacterium_1 and Pseudomonas, and lowered the Firmicutes/Bacteroidetes (F/B) ratio, which maintained the balance between immunity and intestinal health. Taken together, FengLiao alleviated castor oil-induced diarrhea by altering gut microbiota, and levels of jejunum epithelial transport proteins and acute phase proteins.

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