Bacillus subtilis DSM 32315 (GutCare®) restores microbiota composition an promote performance in broilers under necrotic enteritis infection

Overall, feeding B. subtilis DSM 32315 significantly improved feed efficiency and growth performance under enteric challenge conditions;

Furthermore, dietary supplementation of B. subtilis DSM 32315 had beneficial effect attenuating the changes caused by the C. perfringens infection by increasing the frequency of Ruminococcus and unclassified members of the family Ruminococcaceae and partially restored the frequency of Bacteroides to a similar value as that of the uninfected group.

Clostridium perfringens is a normal inhabitant of the gastrointestinal tract of healthy birds that may cause necrotic enteritis (NE) after a sudden change in the microbiota or after intestinal mucosa damage caused by coccidiosis (Willians, 2005). The sudden increase in the mortality in the flock is one of the observed signs in a NE outbreak, and the dead birds will often present gross lesions in the small intestine. These intestinal lesions can also cause lower feed efficiency and consequently compromise the performance of birds. Due to these problems, the cost of NE worldwide was estimated to US $6 billion annually (Wade and Keyburn, 2015). A common practise to prevent NE outbreaks is to use antibiotics; however, concerns over antibiotic resistance led to regional bans in order to reduce antibiotic growth promoters (AGP) use in poultry feed. Consequently, the use of probiotics has received special attention as potential alternatives for AGP since they may provide similar improvement in performance. GutCare^® is a Bacillus-based probiotic containing the strain Bacillus subtilis DSM 32315 and has a positive effect on a healthy gut balance. Recent studies demonstrated that B. subtilis DSM 32315 is effective against C. perfringens infection and contributes to ameliorate the negative impact of NE (Facts & Figures n° 15152; Facts & Figures n° 15153; Facts & Figures n° 15161). Since, enteric challenges (such as NE) are associated to sudden changes in gut microbiota, this study was conducted to evaluate the effects of the dietary supplementation of GutCare^® on performance and intestinal microbiota of broiler chickens induced to necrotic enteritis by C. perfringens inoculation. This trial was conducted at the Southern Poultry Research by Greg Mathis and in collaboration with Dr. Applegate from University of Georgia, United States.

One thousand two-hundred day-old male chicks (Cobb 500) with an average initial weight of 42.0 ± 0.1 g were randomly assigned to one of the three dietary treatments with eight pen replicates of 50 birds each. Birds were spray vaccinated with Coccivac on day of hatch and were raised on built up litter to increase the challenge. The basal diets composed mainly by corn-soybean meal formulated to meet Evonik amino acids recommendations (AMINOChick^® 2.0) for starter (day 1-14), grower (day 15-28), and finisher (day 29-42) phases with diets provided in crumbles (starter) or as pellets (grower and finisher). The treatments consisted of an 1) uninfected group (basal diet without probiotic); 2) infected group (basal diet without probiotic and C. perfringens infection); and 3) infection + 500 g/ton GutCare^® (1 x 10⁶ CFU of B. subtilis DSM 32315/g of feed). The infection model consisted in the administration of C. Perfringens (1.0 X 10^8-9 CFU/g) mixed in the feed on days 18, 19, and 20. Feed and water were provided ad libitum during the period.

Birds and feed were weighed weekly by pen to evaluate body weight gain (BW gain), feed intake (FI), and feed conversion ratio (FCR). Cause, date and weight of dead birds were recorded on daily basis. Three birds from each pen were randomly selected and sacrificed on day 21 to evaluate intestinal lesions. The evaluation of the intestinal lesions considered the following criteria: 0=normal intestines, 1=slight mucus covering and loss of tone, 2=severe necrotizing enteritis, and 3=extreme necrotizing enteritis with presence of blood in the lumen. Two birds from each pen were also selected and sacrificed on day 21 to collect digesta samples from ileum and cecum for microbiome analysis. Microbiota was determined using Next Generations Sequencing (NGS). All sequence data processing was performed using QIIME v. 1.9.1 software package. The frequency of the main bacterial groups observed was submitted to a non-parametric one-way ANOVA (Kruskal-Wallis test) followed by Dunn test. The data regarding the performance was analyzed by ANOVA using SAS 9.4 (P ≤ 0.05), and the means compared by Student-Newman-Keuls test.

The results of the performance are presented in Tables 1. From 1 to 28 days, BW gain was higher in the uninfected group compared to the infected groups receiving or not B. subtilis DSM 32315 (P < 0.05). Feed conversion ratio (FCR_adj.) at 28 days was significantly better in the infected group receiving B. subtilis DSM 32315 compared to the infected group without probiotic, but it was significantly higher than the FCR observed in the uninfected group (P < 0.05). At 42 days, both BW gain and FCR_adj were significantly improved in the infected group receiving B. subtilis DSM 32315 compared to the infected group without probiotic; but not comparable to the uninfected group. Birds receiving probiotics normally presents a compensatory growth after a subclinical infection and the performance is comparable to the health birds; however, it seems that the infection model applied here was stronger than expected. Nevertheless, the improvement with B. subtilis DSM 32315 compared to the infected group without probiotic is evident and demonstrated that birds responds to probiotics even under harsh challenging conditions. Mortality in the infected group without probiotic was significantly higher than in the groups without infection and the infected group receiving probiotic (P < 0.05).

Table 1: Effect of Bacillus subtilis DSM 32315 on performance parameters in broilers under necrotic enteritis challenge.

A number of studies have shown significant improvement in FCR_adj by dietary supplementation of B. subtilis DSM 32315 over a period of 42 days (Facts & Figures n° 15136; Facts & Figures n° 15143; Facts & Figures n° 15152). The better FCR_adj observed might be associated to reduced villus shortening as demonstrated in previous study (Facts & Figures n° 15161), suggesting that B. subtilis DSM 32315 can help preserve the intestinal integrity, minimizing the negative impact of C. perfringens on performance. Consequently, probiotics supplementation can promote performance due to improved intestinal mucosa integrity and digestive function (Tellez et al., 2006).

The ileal digesta microbiota was dominated by bacteria belonging to the phylum Firmicutes (99.2 %), with a small proportion of Actinobacteria (0.5 %) and Bacteroides (0.1 %; data not shown). On a downstream level, main genera of bacteria observed in the ileal digesta microbiota were Lactobacillus, followed by Ruminococcus, Enterococcus, Streptococcus, and Blautia. The order Clostridiales was also representative in the ileal digesta. However, no statistical differences were observed on the relative frequency of these bacterial groups between treatments (Table 2).

Table 2: Relative abundance (%) of the main bacterial groups present in the ileal microbiota of broiler chickens supplemented with B. subtilis DSM 32315 and submitted to a necrotic enteritis infection model

Table 3: Relative abundance (%) of the main bacterial groups present in the ileal microbiota of broiler chickens supplemented with B. subtilis DSM 32315 and submitted to a necrotic enteritis infection model.

The relative abundance of the main groups observed in the cecal microbiota of the birds is shown in the Table 3. The cecal microbiota of the birds was dominated by Bacteroides, Lactobacillus, Ruminococcus, and member of the family Ruminococcaceae.

It was observed that the C. perfringens infection decreased the relative frequency of Ruminococcusand Ruminococcaceae (P = 0.01) and increased the relative frequency of Bacteroides (P = 0.01). However, supplementation of B. subtilis DSM 32315 increased the frequency of Ruminococcus and unclassified members of the family Ruminococcaceae and partially restored the frequency of Bacteroides to a similar value as that of the uninfected group. This is an interesting result, since bacteria from Ruminococcaceae families are known as one of the most important butyric acid-producing bacteria that are present in health birds (Onrust et al., 2015). When butyrate-producing bacteria are present in a sufficiently high concentration, the epithelial barrier integrity will be stronger and inflammatory reactions will be reduced (Lopetuso et al., 2013). Therefore, stabilization of the gut microflora B. subtilis DSM 32315 may have contributed to the significant reduction of the intestinal lesion score observed in this study (Figure 1).

Figure 1: Average lesion score observed after infection with C. Perfringens.

Previous study with birds’ infection with C. perfringens showed an increased lesion score and mortality, reaching up to 1.4 and 35 %, respectively, but the dietary inclusion of B. subtilis DSM 32315 significantly reduced lesion score and mortality to 0.8 and 15 %, respectively (Facts and Figures n°15141). In the present study, similar trend was observed when B. subtilis DSM 32315 significantly reduced lesion score and mortality to 0.5 and 11 %, respectively.

In conclusion, C. perfringens infection disturbed the balance of the ileal and cecal microbiota but the dietary supplementation of B. subtilis DSM 32315 (GutCare^®) kept the microbiota structure very similar to that of the uninfected birds. Additionally, there was no effect of the treatment groups on the microbiota composition of the ileum; the cecal microbiota, however, showed some changes in terms of its composition, but the dietary supplementation with B. subtilis DSM 32315 had beneficial effects in attenuating these changes. The stabilization of the gut microflora contributed to a better weight gain, feed conversion and decreased intestinal lesion score observed with the supplementation of B. subtilis DSM 32315 in the overall period.