One of the first consequence of the genetic selection of today broiler chickens, is the tremendous increase of feed intake. Indeed, the gut has to deal with a huge amount of feed every day, challenging not only the digestive capacities but more generally gut functioning. Due to the high feed intake, the often poor quality of the raw materials, the environmental challenges, including pathogens and toxins, the intestine is in a constant inflammation state. Thus, the first line of defense, namely the intestinal immune system, is key to maintain gut health and consequently, plays an important role in animal performance. Disturbances of the immune homeostasis, due to stresses and life events, leads to an inflammatory response.
Antibiotic Growth Promoters (AGPs) have long been used to help the animals to support the challenges of the production and have shown rather consistent effects on performance. However, with the development of antimicrobial resistance, AGPs have been phased out of feeds and farmers are looking for efficient alternatives. The search for alternatives to AGPs has long been hampered by the poor understanding of the mode of action of AGPs. Indeed, several mechanisms have been proposed to explain the effect of AGPs as growth enhancers. First, the inhibition of microbiota development reduces microbial use of nutrients and growth-depressing metabolites such as ammonia and bile degradation products. Second, the reduction of endemic subclinical infection lowers the metabolic costs of the immune system. Third, the development of thinner intestinal wall increases nutrient absorption. However, all these mechanisms are based on the hypothesis that the intestinal microflora depresses animal growth, either directly or indirectly, and that the mechanism of AGPs is based on their antibiotic properties. It has also been reported that antibiotics may act on the host itself as well. Indeed, they have been shown to have anti-inflammatory effects through reduction of cytokine production, decrease of reactive oxygen species, effects on phagocytosis... (Niewold, 2007, 2015). These anti-inflammatory effects have a huge consequence on nutrient use for maintenance and this may explain largely the growth permitting effect and the consistency of the effects observed with antibiotics, such as bacitracin.
In order to develop alternative to AGPs with consistent performance, it might be relevant to include, during the selection process, the evaluation of the anti-inflammatory properties of probiotics, as these properties are strain specific. In order to develop a consistent probiotic, the effects of a new Bacillus subtilis strain on inflammation have been investigated in comparison with existing commercially available B. subtilis strains.
A model to evaluate intestinal inflammation
Human intestinal epithelial cells (Caco-2 cells) were used to evaluate the ability of different B. subtilis strains to prevent inflammation in stimulated and non-stimulated conditions. A 14-day differentiated Caco-2 cells monolayer, established in a Transwell system, was exposed overnight on its apical side to vegetative cells of each B. subtilis strains (B. subtilis 29784, and two commercially available B. subtilis strains, Bs A and Bs B), grown in LB medium (10 7 CFU/well), or to epigallocatechin gallate (EGCG, 100 µM) known for its anti-inflammatory properties and used here as a positive control. Trans Epithelial Resistance (TER) and interleukin IL-8 production were then monitored as indicators of intestinal permeability and inflammation, respectively.
As expected, TER was not affected by IL-1 and was increased by EGCG. Interestingly, the 3 B. subtilis strains had different impact on TER. Whereas Bs A decreased TER, Bs B had no effect and the new B. subtilis 29784 increased it. IL-1 induced inflammation as shown by an increase in IL-8 production. All strains tested were able to significantly reduce IL-8 level. In these conditions, however, the new B. subtilis was the only strain able to fully reduce the inflammatory response, as shown by the equal level of IL-8 secreted by the IL-1-stimulated cells treated with B. subtilis 29784, as well as EGCG, and the non-stimulated cells (data not shown).
Figure 1: TER variation in 14-d Caco-2 cells under normal or stimulated conditions
Inflammation response to better screen the consistent probiotic
Our results clearly show that different B. subtilis strains can have different levels of efficacy in modulation of inflammatory response and intestinal permeability. The new B. subtilis both enhances intestinal barrier and reduces intestinal inflammatory status, it could therefore be a good candidate to enhance consistently gut health and animal performance. To validate that this anti-inflammatory properties improve the consistency of response, several broiler trials were performed either in standard breeding conditions or in challenged conditions where the effect of the new Bacillus subtilis strain was tested in comparison with a commercial available product (Devillard et al. 2016). The diets were similar between the studies and based on corn-soybean meal, but they differed by the presence or absence of animal by-products and of phytase. Noticeably, the strains did not show the same efficacy to improve performance data. B. subtilis 29784 significantly improved the performance in the 3 studies with an average improvement of 3.8% on BWG and 3.2% on FCR.
The competitor product was able to improve significantly BWG and/or FCR in only one of the studies (average improvement in the 3 studies: 1.7% for BWG and -2.2% for FCR). Considering the response to probiotics, it appears that the level of performance was not the driver but rather the level of stimulation of the gut functioning. Indeed high performing birds having a rather high feed intake appeared to have a strong response to supplementation by this new Bacillus subtilis strain.
In addition, the consistency of the response has been verified considering different probiotic batches of production. Table 1 shows the consistency through 4 different production batches of this new strain of Bacillus subtilis reaching an average: +3.3%.
Moreover, challenging the birds with necrotic enteritis causative agent (Clostridium perfringens) induced significantly better performance (Rhayat et al. 2016)
Table 1: consistency of the growth improvement with 4 different production batches of the same new strain of Bacillus subtilis (corn-soybean meal based diets, coccidiostats, no AGP, phytase, male Ross PM3 up to 42 days)
Antibiotic Growth Promoters have long been used but controversy on their mode of action still remains. They might effectively act as antimicrobial, reducing intestinal microbial population or metabolism, but they can also have significant effect on the host through reduction of mucosal inflammation. When searching for alternatives, such as probiotics, investigating the potential to reduce gut inflammation is important to improve consistency of the response. Our recent results clearly show that different B. subtilis strains can have different levels of efficacy in modulating inflammation and this should be taken into account to help selecting effective strains.
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Article made possible through the contribution of Rhayat L. et al.