The effects of Bacillus spp. use as a dietary probiotic in weaning piglets

Background: This study was conducted to evaluate the effects of supplementation with Bacillus-based probiotic (Bacillus subtilis ATCC 6051a, 1.6x109CFU/ml) on the performance of weaned piglets. Methods: A total of 60 piglets, 30 days±3 days of age, with an initial body weight (BW) of 8.41±0.92 kg were allotted randomly to 3 homogeneous groups (C, E1 and E2, 6 pens/10 piglets, 2 replicates/group) with a supplement of additive (Bs) based on 1% (E1), respectively 3% (E2)/kg feed. The feed was administered ad libitum in the flour form, 2 meals/day. Microbiological analysis and chemical composition of compound feed, the growth performances and diarrhoea incidence were assayed as well. Results: The addition of Bs didn’t influence significantly the BW or average daily gain (ADG). After 16 testing days, ADG was 1.13 times higher in E1 compared with C and with 1.04 times higher compared with E2. The feed intake (ADFI) increased with 1.08-fold in C diet compared with E1 diet. Feed efficiency diet was 1.22 times higher in E1 than C and with 1.05 higher than E2 diet. Conclusions: Addition of Bs-1% decreased diarrhoea incidence (P>0.05) by 7.6% vs. C and 3.8% vs. Bs-3%. 23.4% piglets had soft faeces, 43.75% score 2 (mild diarrhoea) and 32.81% score 3 (severe diarrhoea), no significant difference was noticed between the group (P˂0.05).

. Probiotics are implied in reducing the number of pathogenic bacteria from the animal gastrointestinal tract (GIT), maintaining and stabilizing a healthy microbiota as a preventive measure during the critical period of weaning [3]. Several probiotic products may contain a single strain or multi-strain from bacterial species -Bacillus Weaning is a difficult period for piglets due to their incomplete development of the enzymatic system and factors generating stress [11,34]. Piglets in the weaning crisis must adjust to a solid diet as contrasting to the liquid diet provided by the sow and their endogenous enzymes will take the same several days to correct it [12]. Early weaning removes piglets from a digestible feed source, and the gastrointestinal tract (GIT) complications are more susceptible to install it [13].
Besides, Bacillus spp. was reported to be a possible antibiotic alternative for animals [14]. At the end of the study, the piglets were maintained in the Experimental Farm IBNA Balotesti, Romania, until they recorded the weight of 25 ± 3 kg. After that, the animals were sold to the beneficiaries.

Microbiological analysis of feed
Compounds feed were analyzed microbiologically in the biotechnological laboratory of INCBNA Balotesti. Procedures of sample examination were following the protocol described in STAS 6953-81 (total number of fungi, NTF), SR 13178-1 (total number of germs, TNG), SR 13178-2 (total number of coliforms), SR 13178-2 (E. coli),, SR 5 colony-forming units per gram of sample (CFU/g).

Source of microbial feed additive
The microbial feed additive used in the current study was based on Bacillus subtilis ATCC 6051a. The strain's probiotic properties were analyzed in vitro, in another study [5].
Bacillus subtilis ATCC 6051a strain was grown in nutritive agar medium (Merck), at 37°C, 24h under shaking agitation (150 rpm) and aerobic conditions. The culture strain was prepared in culture form, every two days and stored in a fridge at 4°C in a sterile bottle. The optical density (OD) of culture was measured and resuspended with physiological saline sterile, for adjusted to 1.6 x 10 9 CFU/ml/kg feed viable spores. Each day, the supplement was mixed manually with the portion of the basal diet, which was previous calculated for piglets feeding.

Animals and diet
The biological test was conducted on 60 piglets Topigs hybrid [♀ Large White × Hybrid (Large White × Pietrain) × ♂ Talent, mainly Duroc] weaned at 30 days ± 3 days of ages, the average initial body weight of 8.41 ± 0.92 kg. The piglets were randomly allotted to 3 groups distributed in 6 pens with 10 piglets, two replicates for each group: a control group (C) and 2 experimental groups with a supplement of Bacillus subtilis (Bs) bacterial strain based on 1% (E1, Bs-1%), respectively 3% (E2, Bs-3%). The minimum bacterial concentration of Bs was 1.6 x 10 9 UFC/ml/kg feed. Pens measured 2.40 m × 1.80 m x 0.800 m, each with slatted plastic flooring. All pens had one self-feeder and a nipple drinker. Ventilation was delivered by a mechanical system with automatic adjustments. The room temperature was approximately 25°C. The experiment lasted 16 days. The feed was administered ad libitum in the flour form, two meals/day with permanent access to water. The intake and refusal of feed were recorded daily. The feed structure is shown in Table 1.
Animals from C group were fed with the classical diet without Bs-feed additive. The groups Bs-1% and Bs-3% were received the same concentration of Bacillus subtilis ATCC 6051a, but in different levels of feed addition. The animals were supervised daily to identify the piglets with diarrhoea severity (%). The faeces of every animal were examined visually. A subjective scoring system was used to determine the severity of diarrhoea, ranging from 1 to 3: 1 soft faeces; 2 mild diarrhoea, 3: severe diarrhoea. The incidence of diarrhoea was expressed as the average number of days with diarrhoea related to the total monitoring days (seven days) [11].
Throughout the experimental period we supervised the following parameters: body weight (BW), average daily feed intake (g feed/piglet/day, ADFI), average daily gain (g/piglet/day, ADG), feed conversion ratio (g feed/g gain, G: F).

Statistical analysis
The data were analyzed with SPSS Statistics version 20.0 (2011) and were expressed as the mean and standard error of the mean (SEM). A probability value of P≤0.05 was considered to be statistically significant and tendencies were noted under conditions of 0.05<P<0.10.

Raw material microbiology
The results of the bacteriological examination of the raw materials used in piglet diet are presented in Table 2. The number of aerobic bacteria did not reach 10 7 for TNG, 10 3 for coliforms, respectively 10 2 for E. coli. The presence of Salmonella spp.
according to EN Regulation, is essential to be excluded in 25 g feed materials.
Furthermore, TNG defines how many aerobic, mesophilic microorganism colonies, such as bacteria, yeast, and mold fungi will grow for 24-48 hours on an agar nutritive plate at 37°C. Mycological analyses of raw materials were shown in Table 3 and the contamination rate is in normal value, did not exceed 10 3 CFU/ml.
Based on the results of microbiological analyses, the raw materials present a low level of contaminationand can be used in piglets' diet.

Growth performance
The growth performance may be linked to diarrhoea incidence, and the diarrhoea appearance of piglets was noted daily in this study. The piglets were weighed individually at the beginning of the experiment (30d ± 3) and on day 16. The feed offered and refused was weighed daily.
In this study, we noticed some differences between groups caused by treatment. At the beginning of testing the animal had not any sign of illness and were kept in similar environmental conditions. Table 4 shows the bio-productive performances obtained by supplemented feed with a bacterial preparation-based Bacillus subtilis.
The addition of Bs did not influence significantly the body weight (BW) or average daily gain (ADG). After 16 testing days, the ADG was 1.13 times higher in the Bs-1% group compared to the C group and 1.04 times higher vs. Bs-3% group respectively.
The feed intake (ADFI) increased 1.08-fold in C diet than the Bs-1% diet. Feed efficiency was 1.22 times higher when the diet was supplemented with Bs-1% compared to C diet and 1.05 times vs. Bs-3%. The values of BW, ADG, G/F are similar with the literature results [15] at the addition of Bacillus-based probiotic when a concentration of 2 x 10 9 CFU/kg feed was used, but no significant results were found between treatments.
Addition of Bs-1% vs. Bs-3% improved BW of piglets weaning in the 1st and 3rd week, but the values were insignificant (P>0.05).

Diarrhoea incidence
In our study, we did not notice a serious digestive disorder. Higher effectiveness had 1% dietary supplement of Bs (Figure 1). Addition of Bs-1% decreased diarrhoea incidence by 7.6% compared to the C group (P>0.05) and by 3.8% vs. Bs-3% group. 23.4% piglets had soft faeces, 43.75% had score 2 (mild diarrhoea) and 32.81% had score 3 severe diarrhoea; however, no significant difference was noticed between groups.
In the current study, some piglets in all treatment's groups were affected by diarrhoea, the mild score being predominantly. Piglets fed diets supplemented with Bs-1% had lower diarrhoea frequency than C and Bs-3% group, but no differences were noted (P>0.05). The addition of B. subtillis ATCC 6051a in piglets feed did not result in any improvement in the diarrhoea frequency.

Discussion
Weaning is a worrying period in the life cycle of pigs, which is associated with changes in diet, gut environment and gut morphology, and thus may result in low growth percentage, high diarrhoea incidence and imbalanced intestinal microecology [16]. The weaning period is a critical time in whole piglet's life, because the young pigs are separated by the sow, resulting in an abrupt change in diet, a move to a new environment and mixing with unfamiliar animals, generally younger [17,18]. All raw materials used in the diet must to be safe and suitable for their utilization in animal nutrition. Generally, raw materials are recognized as safe (GRAS) or otherwise deemed acceptable for use in feed [18]. The composition of feedstuff and feeding management are critical issues that influence the health state of piglets after weaning [19]. The animal feed can be exposed to numerous factors (biological, chemical, physical and other agents), which can affect the animal status health and, indirectly, human [18]. Previous studies with supplementation of Bacillus-based probiotic had a significant effect on ADG and ADFI of weaned piglets, reducing the F: G [19,20].
In our study, soybean meal was included 13% (w/w) as main protein resource for animal production [21] and the addition of Bacillus subtilis can ferment it, as affirmed Kiers et al. [24], due to the hydrolysis of protein to amino acids and peptides [22]. Bacillus subtilis strain concentration is in concordance with EFSA Guide,,Panel on Additives and products or substances used in Animal Feed" which affirms that the minimum inclusion level in piglets feeding stuffs is 1 x 10 9 CFU/kg feed [23].
The effect of a probiotic could be affected by strain composition and inclusion levels, but rendering to the results of previous reports, it should be noted that the positive effects of probiotics on growth performance were always observed in the early period after weaning [24].
The dramatic fluctuations in GIT after weaning and the gut needs time to adapt to the new feed, may cause a gastrointestinal infection, mostly colibacillosis diarrhoea, which producing an extensive morbidity and/or mortality (around 17% of piglets born in Europe) in the most serious of affected cases [25].
Bacillus spp. is not a principal member of the normal animal microbiota and could not colonize the intestine for long periods; it consumes oxygen rapidly and reduces the pH value, which this parameter favors Lactobacilli number and inhibits pathogens like E. coli and Salmonella spp. [27]. As a Gram-positive bacterium and the ability to form endospores, Bacillus subtilis can endure the high temperatures of animal feed pelleting and also, to be stable for long time storage [26].
Post-weaning diarrhoea (PWD) is a condition in weaned piglets characterized by the frequent ejection of soggy faces during the first 2 weeks after weaning and continues to represent one of the major economic problems for the pig industry worldwide [27]. Several studies with the addition of B. subtilis as DFM products may be a means for GIT health by beneficial bacteria stability, reducing diarrhoea incidence and enhancing the growth performance [28,29].
The results from previous studies [30,31] found that Bacillus supplementation reduced the incidence and severity of diarrhoea in weaning piglets.

Conclusions
The results of this study indicate that dietary supplementation with Bacillus subtilis ATCC 6051a (1.6 x 10 9 UFC/kg feed), did not influence significantly the body weight gain, feed intake while feed conversion ratio in the weaned piglets.
The results suggest that the addition of Bacillus-as DFM did not ameliorate the incidence of diarrhoea of piglets during experimental design. Consent for publication ,,Not applicable".

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.