Veterinary World, EISSN: 2231-0916 RESEARCH ARTICLE Available at www.veterinaryworld.org/Vol.13/December-2020/12.pdf Open Access Bacillus subtilis and lactic acid bacteria improve the growth performance and blood parameters and reduce Salmonella infection in broilers Nalisa Khochamit1 , Surasak Siripornadulsil1,2,3 , Peerapol Sukon4 and Wilailak Siripornadulsil1,2,3 1. Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand; 2. Research Center for Environmental and Hazardous Substance Management, Khon Kaen University, Khon Kaen 40002, Thailand; 3. Center of Excellence on Hazardous Substance Management (HSM), Patumwan, Bangkok, 10330 Thailand; 4. Research Group for Preventive Technology in Livestock, Department of Anatomy, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. Corresponding author: Wilailak Siripornadulsil, e-mail: [email protected] Co-authors: NK: [email protected], SS: [email protected], PS: [email protected] Received: 09-07-2020, Accepted: 02-11-2020, Published online: 16-12-2020 doi: www.doi.org/10.14202/vetworld.2020.2663-2672 How to cite this article: Khochamit N, Siripornadulsil S, Sukon P, Siripornadulsil W (2020) Bacillus subtilis and lactic acid bacteria improve the growth performance and blood parameters and reduce Salmonella infection in broilers, Veterinary World, 13(12): 2663-2672. Abstract Aim: The aim of the study was to determine the potentials and effects of Bacillus subtilis and lactic acid bacteria (LAB) as probiotics on broiler growth, health, and Salmonella infection. Materials and Methods: To evaluate the inoculum size applicable for broilers, 1-day-old broilers were orally fed fresh cultures of single strains and a B. subtilis KKU213/Pediococcus pentosaceus NP6 mixture at 108 and 1012 colony-forming unit (CFUs)/mL/chick. The body weight gain (BWG), Salmonella contamination level and total Bacillus and LAB abundances in the crop and intestine were measured. Subsequently, 1-day-old broilers were orally fed of KKU213, CH403, and Pediococcus acidilactici SH8 at 1010 CFUs/mL, followed by inulin. After 35 days, the BWG, Bacillus and LAB abundances in the cecum, blood parameters, and KKU213 colonization were assessed. Results: The broilers fed single strains or KKU213+NP6 exhibited a higher BWG and a higher crop LAB abundance than the controls (p<0.05). Probiotic feeding decreased the intestinal Salmonella abundance and correspondingly increased the LAB abundance. The broilers fed the mixed culture (KKU213+CH403+SH8) followed by prebiotics showed lower mortality, higher blood high-density lipoprotein levels, and lower blood uric acid levels than the controls (p<0.0004). Probiotic feeding significantly increased the Bacillus and LAB counts (p<0.05). A CE330 isolate obtained from the cecum after 35 days of KKU213 feeding was closely related to B. subtilis KKU213. Conclusion: B. subtilis KKU213 is a potent probiotic strain that can survive, colonize and reduce Salmonella infection in broilers and improve their growth and health. This strain, combined with different LAB can act synergistically in the gut and promote broiler growth. Keywords: broiler, Bacillus subtilis, lactic acid bacteria, probiotics. Introduction products [2]. The use of probiotics as alternatives to anti- Broilers (Gallus gallus domesticus) are the most biotics in the poultry industry has become an increasingly economically important widespread domestic animals popular due to the global trend in agriculture of banning for poultry meat production [1]. Farmers often use the use of in-feed antibiotics as growth promoters and antibiotics to enhance growth and prevent pathogens can potentially minimize enteric diseases [3,4]. in broilers, and one important example is Salmonella Probiotics are live microorganisms that benefit infection, a major foodborne illness called salmonellosis. the health of the host by improving its nutritional and intestinal microbial balance [5,6]. The microflora in the Among the pathogenic Salmonella serovars, Salmonella gastrointestinal (GI) tract of poultry might be modified enterica Enteritidis and Typhimurium have been impli- by probiotics, which can play a role in the competitive cated in foodborne gastroenteritis worldwide. Antibiotics exclusion (CE) of pathogens, including Salmonella. CE are mostly recognized as harmful chemicals and lead to is one of the modes of action through which a benefi- increases in antibiotic-resistant bacteria, an imbalance cial probiotic protects against pathogen infection in the of the microflora in the gut, and drug residues in food intestinal epithelial cells of animals. However, whether Copyright: Khochamit, et al. Open Access. This article is effective CE through the use of a single strain or a mix- distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ ture of probiotics results in activity, such as immuno- by/4.0/), which permits unrestricted use, distribution, and modulation, adhesion site competition, and antimicro- reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the bial agent production, by one or multiple strains in the Creative Commons license, and indicate if changes were made. GI tract of the host remains unclear [7]. The inhibition The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data of Salmonella infection by lactic acid bacteria (LAB) made available in this article, unless otherwise stated. in intestinal epithelial cells has been documented [8,9]. Veterinary World, EISSN: 2231-0916 2663 Available at www.veterinaryworld.org/Vol.13/December-2020/12.pdf Table-1: Antibacterial activity against Salmonella enterica strains isolated from contaminated chicken meat of the lyophilized cell-free supernatant of Pediococcus pentosaceus NP6 heated at 100°C for 15 min or at 121°C for 15 min or treated with pepsin or trypsin. Salmonella serovar Inhibition zone (mm) Control 100°C, 121°C, 1 mg/mL 1 mg/mL SEM p-value 15 min 15 min Pepsin, 3 h Trypsin, 3 h Salmonella Agona 1935 9.69 8.75 9.38 10.13 9.50 0.76 0.78 Salmonella Amsterdam 1936 9.38 8.88 9.38 10.25 9.13 0.33 0.09 Salmonella Braenderup 10.75 9.75 11.50 10.38 9.75 0.82 0.53 Salmonella Bareilly 5387 10.63 9.00 9.75 11.00 10.00 0.45 0.05 Salmonella Brunei 916 9.13 8.63 9.00 9.50 9.25 0.65 0.91 Salmonella Emek 4759 9.75bc 9.25bc 11.50a 10.25ab 8.50c 0.31 <.0001 Salmonella Enteritidis 9.25 8.88 9.75 9.75 8.50 0.39 0.14 Salmonella Enteritidis 176-1 9.00 9.06 9.38 8.88 8.50 0.22 0.14 Salmonella Hvittingfoss 5382 10.00 9.56 8.75 8.75 9. 00 0.45 0.25 Salmonella Kentucky 10.38 9.94 10.75 9.00 9.00 0.47 0.06 Salmonella Mbandaka 5412 9.56 9.50 10.13 9.13 9.00 0.34 0.21 Salmonella Ohio 10.13 9.88 10.50 9.25 9.00 0.36 0.05 Salmonella Paratyphi 8486 12.38 11.93 10.00 10.50 10.75 0.60 0.06 Salmonella Singapore 5416 10.31 10.00 9.50 8.00 9.00 0.48 0.03 Salmonella Typhimurium 11.00 9.50 9.50 10.50 10.38 0.56 0.28 Salmonella Typhimurium 5313 8.88 8.94 9.75 8.63 8.63 0.37 0.24 Salmonella Urbana 5381 10.13 8.75 9.38 9.25 9.75 0.38 0.17 Salmonella Weltevreden 5383 11.06 9.63 10.25 10.25 9.75 0.42 0.18 Among bacterial species, several members of LAB and fermented fish and is an effective Salmonella inhibitor Bacillus, either as single or combined cultures, have in fish samples [13]; (ii) Pediococcus acidilactici SH8 been widely used as probiotics or microbial feed addi- (MF061302.1), which was isolated from fermented tives for chickens [10,11]. We have previously reported shrimp, can inhibit Gram-positive and Gram-negative the potential of Bacillus subtilis KKU213 and some bacterial pathogens and does not exert a negative impact LAB strains as potential probiotics in broilers [10,12]. on broilers [10]; and (iii) CH403, which was isolated The aims of this study were to determine the from the intestine of a domestic Thai fowl and can efficiency of selected bacteriocin-producing strains of inhibit a wide range of Gram-negative bacteria. All LAB B. subtilis KKU213 and LAB as probiotics that can strains were cultivated in de Man, Rogosa and Sharpe improve the growth, microbial gut community, and (MRS) broth under microaerobic conditions at 42°C for health of broiler and reduce Salmonella contamination 48 h. All 18 serovars of S. enterica were isolated from in these organisms. The effects of different numbers contaminated chicken meat and cultured by growing in of bacterial cells administered as a feed inoculum con- nutrient broth and swabbed on Salmonella Shigella (SS) sisting of single strains or a mixture were assessed. agar when tested for anti-Salmonella activity. Materials and Methods In vitro anti-Salmonella activity of P. pentosaceus NP6 P. pentosaceus NP6 was grown in MRS broth Ethical approval All animal experiments were conducted in accor- and then incubated at 37°C for 48 h. The cell-free dance with the guidelines and recommendations of supernatant (CFS) was centrifuged at 8000 rpm for µ the Institutional Animal Care and Use Committee and 30 min, filtered through a 0.22- m filter and lyo- philized. The protein concentration of CFS was mea- approved by the Animal Ethics Committee of Khon sured using the Quick StartTM Bradford Protein Assay Kaen University (approval no. AEKKU 22/2558). (Bio-Rad Laboratories, Hercules, USA). The CFS Study period and location was resuspended in 1 mL of ddH2O, heated at 100°C The animal experiments were carried out at the for 15 min and 121°C for 15 min, and treated with Department of Anatomy, Faculty of Veterinary Science, 10 mg/mL proteolytic enzymes, trypsin, and pepsin for Khon Kaen University, Thailand, in July-August 2018.