ARTICLES

 

Effects of Bacillus spp. as a supplemental probiotic in diets for weaned piglets

 

 

M. Dumitru^I; M. Hăbeanu^I; I. Sorescu^{I, II, #}; C. Tabuc^I

^INational Research Development Institute for Biology and Animal Nutrition (IBNA), Bucharest, No. 1, Balotesti, Ilfov, 077015, Romania
^IIThe Institute of Diagnostic and Animal Health, Bacteriology Laboratory, Street Dr. Staicovici, No. 63, District 5, 050557, Bucharest, Romania

 

 

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ABSTRACT

This article evaluated the effects of supplemental probiotic Bacillus subtilis (Bs) ATCC 6051a (1.6x10⁹ cfu/mL) in diets for weaned piglets on their performance and on the occurrence of diarrhoea. Sixty piglets, 30 ±3 days old with initial bodyweight of 8.41±0.92 kg, were allotted randomly to six pens of ten piglets. There were two replicates of each treatment, namely a control diet (C), a diet supplemented with 1% Bs (E1), and a diet supplemented with 3% Bs (E2). Feed was provided ad libitum as flour in two meals per day. Feed materials were examined for total numbers of fungi, aerobic mesophilic bacteria (TNG), Coliforms, Escherichia coli and Salmonella spp. The addition of Bs did not influence (P >0.05) bodyweight (BW) or average daily weight gain (ADWG). However, across the experimental period ADWG was greater in E2 and E1 than in C (>1.12 and 1.08 times compared with C). Feed intake (ADFI) by pigs fed C was greater than pigs fed E1 and E2. Feed efficiency was higher in E1 and E2 than the C diet. Addition of 1% Bs decreased (P<0.05) diarrhoea occurrence around 8% compared with C, and 4% compared with 3% Bs. A total of 23.4% of the piglets produced soft faeces. Diarrhoea scores of 2 (mild diarrhoea) and 3 (severe diarrhoea) were observed in 43.75% and 32.81% of the pigs. No differences (P >0.05) were detected between the treatments. The results suggested that E1 could positively affect growth performance and mitigate the occurrence of diarrhoea.

Keywords: feed additive, microbiology, performance, weaned pig, weaning diarrhoea

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Introduction

Weaning is a difficult period for piglets owing to incomplete development of their enzymatic systems and digestive disturbances (Dlamini et al., 2017), with these factors generating stress (Lee et al., 2014; Habeanu et al., 2015). At weaning, piglets must adjust to a solid diet instead of the milk provided by the sows, and their endogenous enzymatic system requires several days to adjust (Guevarra et al., 2019). Early weaning removes piglets from an easily digested feed source and the gastrointestinal tract (GIT) is susceptible to adverse consequences as a result (Taylor & Roese, 2006). A managerial challenge in weaning piglets is to reduce the incidence of digestive disorders. The first sign of gastrointestinal disorder is diarrhoea (Mach et al., 2015; Nowland et al., 2019). Antibiotics have long been used in feeding piglets to maintain their digestive health, but routine feeding of antibiotics has led to problematic levels of microbial resistance.

Probiotics are non-pathogenic live organisms, which, when administered in sufficient amounts, produce beneficial effects on the health of the host (FAO, 2001). Among other effects, probiotics may reduce the number of pathogenic bacteria in the GIT, and maintain a stable population of intestinal microbiota (Yirga, 2015; Dumitru et al., 2020a). Thus, probiotics may be beneficial to piglets around the time of weaning (Corcionivoschi et al., 2010). Probiotic products can contain a single or multiple strains of the bacterial species Bacillus (B. cereus var. toyoi, B. licheniformis, B. subtilis), Enterococcus (E. faecium), Lactobacillus (L. acidophilus, L. casei, L. farciminis, L. plantarum, L. rhamnosus), Pediococcus (P. acidilactici), Streptococcus (S. infantarius), and microscopic fungi such as yeasts Saccharomyces cerevisiae and Kluyveromyces (FAO, 2016; Dumitru et al., 2018; Sorescu et al., 2019). Known as a strict aerobe (Hu et al., 2015), Bacillus was noticed for its advantages because it is spore forming (Kim et al., 2019). In addition, Bacillus spp. tolerates the low pH in the stomach, is resistant to bile salts, and has thermo-stability during processing and long-term storage of feed (Ragul et al., 2017). Studies that used Bacillus spp. as a direct-fed microbial supplement (Leser et al., 2008) reported favourable results in swine diet with beneficial effects on growth and feed efficiency (Wang et al., 2011; Link et al., 2016). In addition, Bacillus spp. was reported to be a possible antimicrobial growth promoter and alternative to antibiotics for animals (Bedford et al., 2014).

Therefore, this study was designed to investigate the efficacy of Bacillus subtilis ATCC 6051a as a probiotic by studying its effects on growth performance and the incidence of diarrhoea when fed to weanling piglets.

 

Materials and Methods

The animals were provided by INCDBNA Balotesti Experimental Farm, Romania. The experiment was carried out according to the protocol approved by the Ethical Committee of the National Research-Development Institute for Animal Nutrition and Biology Balotesti (INCDBNA), Romania (Habeanu et al., 2015). The procedures were in agreement with Council Directive 2010/63/EU legislation for the protection of animals used for scientific purposes. No antibiotic was supplied to the animals during the experiment.

Piglets used in the experiment were a hybrid of Topigs germplasm TN Talent x (Large White χ (Large White χ Pietrain). At the end of the study, the piglets were maintained at the Experimental Farm IBNA Balotesti, Romania, until they reached a bodyweight of 25 ± 3 kg, when they were sold.

The crude protein (CP) of the diet was determined using a semi-automatic classical Kjeldahl method with a Tecator Kjeltek auto analyser 1030 (FOSS - Tecator AB, Hoganas, Sweden). The fat was established as ether extract (EE) by continuous extraction in solvent with a Soxhlet apparatus. The crude fibre (CF) was determined with the classical semi-automatic Fibertec-Tecator method (FOSS - Tecator AB, Hoganas, Sweden) and the ash content was determined by incineration at 550 °C until the sample reached a constant mass. The nitrogen-free extract (NFE) was calculated with the formula:

Metabolizable energy (ME) was calculated with the regression equation developed by the Oskar Kellner Institute of Animal Nutrition:

where: dCP, dEE, dCF and dNFE are digestible CP, EE, CF and NFE.

Microbiological examination of the samples followed the protocols described in the Romanian standards STAS 6953-81, namely SR 13178-1: total number of fungi (TNF) SR 13178-2: total number of germs (TNG), SR 13178-2: total number of Coliforms) (E. coli), and SR 12824: Salmonella spp. Plates were incubated aerobically. Results were expressed as the average of three dilutions as logarithm (base 10) colony-forming units per gram of sample (cfu/g).

The microbial feed additive in the current study was based on Bacillus subtilis ATCC 6051a (BsJ.The Bs strain was grown in nutritive medium (Merck KGaA, Darmstadt, Germany), at 37 °C for 24 hours under shaking agitation (150 rpm) and aerobic conditions. The culture strain was prepared in liquid form every two days and stored at 4 °C in a sterile bottle. The optical density of the culture was measured and re-suspended in sterile physiological saline (0.85%, w/v) to a concentration of 1.6 x 10⁹ cfu/mL/g^-1 feed viable spore. Each day the supplement was mixed manually with the basal diet for the piglets.

Growth performance was evaluated with 60 Topigs piglets weaned at 30 ± 3 days old with an average initial bodyweight of 8.41 ± 0.92 kg. The piglets were divided randomly into three groups distributed in six pens of ten piglets, two replicates for each group, namely the control group (C) and two experimental groups each receiving a supplement of Bs strain at 1% (E1) and 3% (E2) level. The minimum bacterial concentration of Bs was 1.6 x 10⁹ cfu/mL/g¹ feed. The pens measured 4.3 m², each with slatted plastic flooring. Each pen had one self-feeder and a nipple-type drinking fountain. Ventilation was delivered by a mechanical system with automatic adjustments. The room temperature was approximately 25 °C. The experiment lasted 16 days, although generally after weaning, the mortality rate because of digestive disorders is highest in the first seven days (Habeanu et al., 2015). The feed (Table 1) was provided in flour form twice daily according to appetite and water was provided ad libitum. The intake and refusals of feed were recorded daily. Animals from C group were fed the control diet without Bs supplementation. In E1 and E2 the concentrate portion of the ration was unchanged, but different levels of supplement were used.

The animals were monitored daily and the severity of diarrhoea was recorded. The faeces of every animal were examined visually in the morning at 08h00 after the piglets had been fed. A subjective scoring system was used to determine the severity of diarrhoea, ranging from 1 to 3, namely 1: soft faeces; 2: mild diarrhoea, 3: severe diarrhoea, which was monitored by the same evaluator. The incidence of diarrhoea was expressed as the average number of days with diarrhoea related to the total monitoring days (Habeanu et al., 2015). Throughout the experiment, final bodyweight (FBW), average daily feed intake (ADFI) (g feed/piglet/day), ADWG (g/piglet/day), and feed conversion ratio (FCR) (g feed/g gain) were also recorded.

The data were fit to a general linear model using SPSS version 20.0 (IBM Corp., Armonk, New York, USA). The results were expressed as mean and standard error of mean. The diets were considered fixed factors. The effects were considered significant at P-value < 0.05, and were regarded as a trend when 0.05 < P < 0.10. If a fixed effect was significant, the means were compared with the Tukey post hoc test. Pearson correlation coefficients were used to assess relationships among the traits.

 

Results and Discussion

At the beginning of testing, the animals had not exhibited any signs of illness and were kept in similar environmental conditions. The results of the bacteriological examination of the raw materials in the diets are described in Table 2.

The number of aerobic bacteria did not exceed 10⁷ cfu/g for TNG, 10³ cfu/g for Coliforms, and 10² cfu/g for E. coli. According to EN regulation Salmonella spp. must be excluded in 25 g feed materials. The total number of germs defines how many aerobic, mesophilic microorganism colonies such as bacteria, yeast and mould fungi will grow for 24--48 hours on agar nutritive at 37 °C. The mycological status of the raw materials indicated that contamination was within the normal range, that is, not exceeding 10³ cfu/g (Table 3). Based on these results, the raw materials used in this study had low levels of contamination and could be used in the diets for piglets.

No significant effects were noticed between groups (P >0.10) in bio-productive performances from the addition of probiotic bacteria, although numerically greater values were registered for E1 and E2 throughout the experiment (Table 4). The values of FBW, ADWG and feed efficiency were similar to those of the literature on the addition of Bacillus-based probiotic when a concentration of 2 x 10⁹ cfu/g¹ feed was used (Upadhaya et al., 2015).

In this study, no serious digestive disorders were observed and differences among the treatments were not significant (Figure 1). Some piglets in each group were affected by diarrhoea, with the mild score being predominant. E1 decreased diarrhoea incidence around 8% compared with C group and 4% compared with E2. A total of 23.4% piglets had soft faeces, 43.75% had score 2 (mild diarrhoea), and 32.81% had score 3 (severe diarrhoea). In the present study, GP may be linked to diarrhoea occurrence, which was noted daily. Some numerical differences between groups might be associated with the probiotic treatment. However, some studies have found that the use of probiotics could significantly improve intestinal health and promote growth of weanling piglets (Liu et al., 2018).

Weaning is a worrying period in the life cycle of pigs because it is associated with changes in diet, environment and gut morphology, and may result in low growth percentage, high diarrhoea incidence and imbalanced intestinal ecology (Giang et al., 2012). Hu et al. (2014) supposed that B. subtiíis strain could balance piglet microbiota by stimulating beneficial bacteria, improving health of the GIT, diminishing diarrhoea incidence and enhancing GP. In addition to separation from their dam at weaning, several other factors can influence the piglets' lives including mixing with other litters in an unfamiliar environment and changing their diet from easily digestible milk to less digestible solid ingredients (Lalles, 2007; Giang et al., 2012; Pluske et al., 2018).

Animal feed can be exposed to numerous factors (biological, chemical, physical and other agents), which can affect their health status, and, indirectly, that of human beings (Giang et al., 2012). The microbiological quality of feed is important in the health status of animals (Kwiatek, 2011). Feed is considered one of the main carriers of bacteria pathogens in animal production (Fink-Gremmels, 2012). Additionally, the use of raw materials in feed must be safe for and appropriate to animal nutrition. Generally, raw materials are recognized as safe or considered acceptable for use in feed (Pluske et al., 2018). Feed composition and feeding handling are critical issues that influence the health status of piglets after weaning (Nuntapaitoon et al., 2018). In this study, analyses of raw materials did not find microbial contamination exceeding the maximum allowable cfu/g (Table 2). For example, total number of microbes per gram provides an indication of feed quality that results from production processes and sanitary conditions during plant growth, harvest, storage and transport (Kwiatek, 2011). Contamination of the feed with E. coli and Salmonella spp. bacteria are considered indicators during the feed production process (Kelley & Walker, 1999). Generally, plants are the principal source of fungi and contamination may take place during storage (Kwiatek, 2011). Like bacteria, the presence of moulds in feeds implies a risk to animal health arising from ingredients of animal origin.

In the current study, 13% (w/w) soybean meal was included as the main source of protein for animal production (Lallès, 2008). The addition of Bs as a direct-fed microbial product could ferment the soybean meal through hydrolysis of protein to amino acids and peptides (Dong et al., 2014; Kiers et al., 2003). The vegetative cells of Bacillus could secrete extracellular products (Bernardeau et al., 2017) as enzymes (carbohydrase, lipase, protease, etc.) that are involved in the degradation of anti-nutritive fractions from feed (Asmare, 2014). For example, the Bacillus group can produce extracellular enzymes as protease (Degering et al., 2010), which are involved in the process of digestion with an improvement in the GP of animal (Kaewtapee et al., 2017).

In the current study, the addition of Bs to piglet feed was in accordance with European Food Safety Authority Guide (2021) which affirms that the minimum inclusion level in feedstuffs for piglets is 1 x 10⁹ cfu/g^-1 feed (Yuan et al., 2017). The presence of spores as bacterium protection enable Bacillus spp. to withstand environmental conditions such as pH, bile salts, temperature, radiation, pressure, and chemical agents, and factors that can destroy the vegetative form (Bernardeau et al., 2017). Dumitru et al. (2019; 2018a) reported that B. subtiíis ATCC 6051a tolerated well GIT conditions such as low pH and bile salts, making it a commensal bacterium for animals that ingest it (Vasques, 2016). The Bacillus subtiíis ATCC 6051a strain also tolerated 80 °C temperature for two hours which allows its inclusion in animal diets even if it is ground and pelleted (Dumitru et al., 2019). However, the results of supplementation of feed with Bacillus spp. are variable. Chen et al. (2013) reported that the supplementation of B.subtilis var. natto improved the BW and ADFI of geese in a concentration of 10⁸ cfu/kg feed and AWDG of broilers (Zhang et al., 2012). Other researchers reported that the supplementation of B. subtilis to diet improved the growth performance of pigs (Alexopoulos et al., 2004; Wang et al., 2011; Lee et al., 2014). Bacillus-based probiotic had significant effects on ADWG and ADFI of weaned piglets by reducing the feed conversion ratio (Nuntapaitoon et al., 2018; Liao & Nyachoti, 2017). Bacillus spp. supplementation had a significant effect on performance parameters, nutrient digestibility, and faecal microbiota in pig diets (Cheng & Kim, 2019; Balasubramanian et al., 2016). For example, the addition of two levels of Bacillus íicheniformis (1.6 x 10⁹ cfu and 4.8 x 10⁹ cfu) in piglet feed improved BW and AWDG significantly when a high concentration was added (Dumitru et al., 2020b). The variations in the results of these studies could be attributed to factors such as diet composition, feed form and their interaction with other bacterial strains used as dietary additives (Chesson, 1994).

The effect of a probiotic could be affected by strain composition and inclusion levels, but positive effects of probiotics on growth performance were always observed in the early period after weaning (Kiers et al., 2003). Ahmed et al. (2014) reported that the inclusion of Bacillus in piglet feed increased ADWG and ADFI through 0 to 28 days after weaning (3.2 x 10⁶ cfu/g).

The faecal and intestinal microbial population of piglets with and without Bacillus subtilis ATCC 6051a was evaluated by Dumitru et al. (2020b). The results showed that Lactobacillus counts were higher in the ileum and faeces of piglets on the diet with Bs1% with a decrease in pathogenic bacteria such as Escherichia coli biotype ^-hemolytic, which is a major source of serious illnesses.

Fluctuations in the GIT microbiota after weaning and the time taken to adapt to the new feed may cause a gastrointestinal infection, mostly Coíibaciííosis diarrhoea, which produces extensive morbidity and mortality (around 17% of piglets born in Europe) in the most serious cases (Nam et al., 2012). The results of this study indicated that feeding E1 to piglets could balance diarrhoea incidence during weaning (Figure 1), with an effects that was more pronounced than for E2 probably because of variation in the response of individual animals Furthermore, a negative Pearson correlation was found between level of Bacillus and AWDG over 16 days (r = -0.41).

In the literature (Cai et al., 2015), it was stipulated that Bacillus spp. is not a principal member of normal animal microbiota and could not colonize the intestine for long periods. It consumes oxygen rapidly and reduces the intestinal pH which favours Lactobacilli and inhibits pathogens such as E. coli and Salmonella spp. As a gram-positive bacterium with the ability to form endospores, Bs can endure high temperatures of animal feed pelleting and remain stable for long-time storage (Rychen et al., 2018).

Post-weaning diarrhoea in piglets is characterized by the frequent ejection of soggy faeces during the first weeks after weaning and represents one of the major economic problems for the pig industry worldwide (Cai et al., 2015). The current data are in accord with several studies that refer to the addition of Bs products as a means of promoting GIT health with stable beneficial bacteria, and enhancing growth performance (Kritas et al., 2004; Sun & Kim, 2017; Wu et al., 2018) and reducing the incidence and severity of diarrhoea in weaning piglets (Baker et al., 2013; Pluske, 2013).

 

Conclusions

The beneficial effects of Bacillus subtiíis ATCC 6051 were not statistically significant. However, dietary supplementation with Bs might improve the health and growth of piglets in the weaning crisis. Experimentation with different concentrations of Bs is necessary to establish the beneficial effects of this supplemental probiotic on piglets during weaning.

 

Acknowledgements

This study was funded by the Romanian Ministry of Research and Innovation through sub-Program 1.2 -Institutional Performance, Program 1 - Developing National R & D, National Research and Development and Innovation Contract no. 17 PFE/17.10.2018 and Project 8PCCDI/2018 pc2.

Authors' Contributions

MD was responsible for conducting and monitoring the experiment, in vitro testing probiotic properties, which were presented in another study (Dumitru et al., 2018a), and preparing bacterial culture based on Bs. Mh was involved in the feeding trial and performed statistical analysis. IS and CT conducted sample collection and laboratory analysis. All authors read and approved the final manuscript.

Conflict of Interest Declaration

The authors declare that there is no conflict of interest.

 

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Received 10 April 2020
Accepted 14 April 2021
Published 17 September 2021

 

 

# Corresponding author: mihaela.dumitru22@yahoo.com