Bacillus amyloliquefaciens CECT 5940 (Ecobiol®) alone or in combination with antibiotic growth promoters improve performance in broilers under enteric pathogen challenge

Coccidiosis is endemic in the commercial broiler industry and is caused by the development and reproduction of Eimeria species, which can infects a specific area of the gut and cause tissue damage in the intestinal epithelial cells (Ritzi et al., 2014; Ritzi et al., 2016). The disruption of the intestinal epithelial cells leads to the impaired absorption of nutrients and may open a way to other enteric diseases such as necrotic enteritis caused by the abundant growth of Clostridium perfringens bacteria (Awad, 2017). In poultry production, it is common to use coccidiostasts to control Eimeria spp and antibiotics to control pathogenic bacteria, avoiding further development of enteric diseases. Antibiotic growth promoters (AGP) have been used to control pathogenic bacteria and to improve performance and health, however, concerns over antibiotic resistance led to regional bans in order to reduce AGPs use in poultry feed. Therefore, the use of probiotics has received special attention, as a replacement of AGP or even act jointly (In countries were AGPs in feed is still allowed) to help decrease the dietary supplementation of the AGP. Probiotics are living microorganisms provided in the feed, which are able to reduce pathogen load by various mechanisms of actions thus enhancing health and improve nutrient absorption in the host. Ecobiol® is a probiotic product containing Bacillus amyloliquefaciens CECT 5940 strain, which is a naturally occurring strain with fast growing characteristics and high tolerance to gastric and bile secretions. B. amyloliquefaciens CECT 5940 is able to improve feed conversion and reduce pathogenic bacteria in the intestine (Facts & Figures n°15160). Considering the benefits of the probiotic for gut health and its potential to replace AGPs, it was hypothesized that Bacillus amyloliquefaciens CECT 5940 can improve performance of broilers by reducing the negative impacts of the infection and has potential to replace AGPs or act synergistically when fed with AGPs. Therefore, this study was conducted to evaluate Bacillus amyloliquefaciens CECT 5940 (Ecobiol®) alone or in combination with AGPs in the performance of broilers under enteric pathogen challenge. This trial was conducted by Dr. Nilva Kazue Sakomura at “São Paulo State University (UNESP)” in Brazil.

One thousand five-hundred thirty day-old male chicks (Cobb 500) with an average initial weight of 50.2 ± 0.1 g were randomly assigned to one of the five dietary treatments with nine pen replicates of 34 birds each. The basal diets composed by corn-soybean-meat and bone meal were formulated to meet Evonik amino acids recommendations (AMINOChick® 2.0) for starter (day 1-13), growerI(day 14-21), grower II (day 22-35) and finisher (day 36-42) phases with all diets provided in mash form. The treatments consisted of a non-challenged group receiving a basal diet without feed additives; Challenged group receiving a basal diet without feed additive; Challenge + 0.05 g/kg BMD (Bacitracin methylene disalicylate); Challenge + 1g/kg Ecobiol® (1 x 10⁶ CFU/g of Bacillus amyloliquefaciens CECT 5940); and Challenge + 0.05g/kg BMD + 1g/kg Ecobiol®. All birds were challenged by oral inoculation on day 17 with 1mL of Eimeriamaxima (5.0 X 10⁴ CFU/g) and Clostridium Perfringens (2.5 X 10⁶ CFU/g) on days 18, 19, and 20. Feed and water were provided ad libitum during the experimental period. 

Body weight and feed intake were recorded at days 21, 35, and 42 of the experimental period for calculation of weight gain and feed conversion ratio. Cause, date and weight of dead birds were recorded daily. Score of litter moisture (0 = Dry friable; 1 = Dry with very fine texture; 2 = Sticky on compression or crumbles; 3 = Clod on compression; 4 = Wet; 5 = Drops of water come out on compression), fecal (0 = Solid with white cap; 1 = Bulckyand moist; 2 = Watery; 3 = No consistency, watery, mucus, undigested feed, sloughed mucosa, foamy) and cecal droppings (0 = Black to brown and firm, 1 = Lack consistence and foamy; 2 = watery and foamy; 3 = Foamy like mousse, spread, liquid) were evaluated at day 28. Two birds from each pen were randomly selected and sacrificed on days 21 and 28 to evaluate general intestinal health and to collect digesta samples from ileum and cecum for further analysis. The score evaluation of the intestinal health considered the presence (1) or absence (0) of the following parameters: ballooning, Muscle tone, thin or fragile, abnormal content (excessive slime, water, gas, greasy aspect or mixture of these), swollen or red, sloughed mucosa or presence of undigested feed. The contents of the ileum and cecum were collected and stored at −20°C for Clostridium perfringens counts. Quantitative PCR targeting the 16S rRNA genes of C.perfringens was used in the quantification. Body weight uniformity, footpad lesions (0 = none; 1 = mild; 2 = moderate; 3 = Severe; 4 = extreme), hock burn (0 = none; 1 = mild; 2 = moderate; 3 = Severe; 4 = extreme), carcass and breast meat yields were evaluated on days 42. Treatments were statistically analyzed using one-way ANOVA procedure in SAS v9.4 software followed by mean comparison with SNK (Student-Newman-Keuls) test at 5% of probability or non-parametric Kruskal-Wallis test followed by Dunn-Bonferroni test at 5% probability for score frequency.

The results of the performance are presented in Tables 1. Feed intake was higher in the challenged group without feed additives compared to the unchallenged group and the group receiving Ecobiol® (P < 0.05), but no significant differences were observed when compared to the groups receiving BMD or its combination with Ecobiol® (P > 0.05). Feed conversion ratio and weight gain were not significantly different between the treatments at 21 days (P > 0.05), which was the day the right after the challenge. On the other hand, feed intake and weight gain were significantly lower in the challenged groups compared to the unchallenged group at 35 and 42 days. The birds undergoing the enteric pathogen challenge had significantly better feed conversion ratio when fed Ecobiol®, BMD or their combination compared to those that did not receive any feed additive (P < 0.05), but the improvement was not enough to provide the same FCR as observed in birds of the unchallenged group at 35 and 42 days. Mortality was not significantly different between the treatments (P > 0.05).

Generally, in subclinical challenge conditions the birds receiving probiotics can recover the performance after some time and present similar performance as the non-challenged birds due to health improvement and better nutrient utilization. Despite the observed improvement in FCR with the use of feed additives, the absence of coccidiostats might have created a huge impact on broiler’s performance and they could not achieve the same performance as the unchallenged group.

The results of the uniformity, carcass and breast meat yields are presented in Tables 2. Uniformity in the body weight, carcass and breast meat yields were improved in the challenged groups receiving either Ecobiol®, BMD or their combination compared to the challenged group without any feed additive (P < 0.05) and were not statistically different from the unchallenged group (P > 0.05). This might indicate a better utilization of nutrient for lean tissue deposition since the body weight of birds receiving feed additives at 42 (Table 1) was not as good as in the unchallenged group, but provided comparable carcass and breast meat yield values.

The results of the score evaluation are presented in Tables 3. The average scores for cecal droppings and hock burn was not significantly different between treatments (P > 0.05). The challenged groups receiving Ecobiol® or the combination with BMD presented lower wet litter score compared to the challenged group without feed additives or the challenged group receiving only BMD (P < 0.05). Studies indicated that BMD supplementation decreased villi height (Miles et al., 2006; Koltes et al., 2017) and this might have contributed to impaired litter quality in the current study. The fecal score in the challenged group was higher compared to the unchallenged group and the groups receiving Ecobiol®, BMD or their combination (P < 0.05). The wet litter and fecal dropping scores might be related to the observed footpad dermatitis score, being higher in the challenged group without feed additive compared to the other treatments (P < 0.05).

The results of the PCR analysis are presented in Table 4. The rt-qPCR analysis indicated that either Ecobiol®, BMD or their combination were able to reduce the numbers of clostridium perfringens in the ileum at 21 and 28 days. However, clostridium perfringens in cecum on days 21 (P = 0.060) or at 28 (P = 0.076) were tending to be lower in birds fed Ecobiol®, BMD or their combination compared to the challenged un-supplemented group. 

The observations of the intestinal health score are presented in Table 5. The presence of abnormal content (excessive slime, water, gas, greasy aspect or mixture of these) was similar between the challenged groups at 21 days. However, there is a significant reduction in the frequency at 28 days in birds receiving either Ecobiol®, BMD or their combination (P < 0.05). There was no significant difference between treatments at 21 days for ballooning, thin mucosa, swollen intestine or sloughed mucosa (P > 0.05). At 28 days of age, the challenged groups receiving Ecobiol®, BMD or their combination, presented significantly lower frequency for abnormal content and swollen intestine compared to the challenged group without feed additives (P < 0.05). Except for the challenged group receiving just BMD, there was a significantly lower frequency for ballooning in the treatments receiving Ecobiol® or its combination with BMD (P < 0.05) compared to the group without feed additives.

The main observations from necropsy as presented in Table 5 are common characteristics in dysbacteriosis and they are related to Infectious agents such as Clostridium perfringens, which may exacerbate Eimeria infections (Chapman et al., 2002; De Gussem, 2007). Therefore, it is clear that the reduction of C. perfringens presented in Table 4 is one of the main factors resulting in the observed lower frequency of abnormal content, ballooning and inflammation in the gut at 28 days of age.

In conclusion, the dietary supplementation of Ecobiol® can replace AGPs totally or partially, since feeding Ecobiol® alone provided comparable results to feeding BMD or its combination regarding feed conversion, reduction of C. perfringens in ileum, uniformity, carcass and breast meat yield during an enteric pathogen challenge. Furthermore, these improvements in the performance are attributable to better intestinal health scores and wet litter scores.