Isolation of bacillus subtilis and its application on the prevention of intestinal diseases in chicken

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Specialization: Pathology and treatment of animals Code: 62 64 01 02 LE THI HAI YEN ISOLATION OF BACILLUS SUBTILIS AND ITS APPLICATION ON THE PREVENTION OF INTESTINAL DISEASES IN CHICKEN Can Tho, 2018 THE STUDY WAS COMPLETED AT CAN THO UNIVERSITY Scientific supervisor: Assoc. Prof. Doctor NGUYEN DUC HIEN The thesis was defended at the university examination committee At.., Cantho University At.. hour ., on date..month... year Reviewer 1: Reviewer 2: Reviewer 3: The dissertation is available in Libraries: 1. Central library of Can Tho University. 2. National library of Vietnam. LIST OF PUBLICATION RELATED TO THE THESIS 1. Le Thi Hai Yen and Nguyen Duc Hien, 2015. Bacillus subtilis isolated as a probiotic from soil and feces on chicken farms in Can Tho City. Veterinary Sciences and Techniques. ISSN 1859-4751. Vol. 6 (2015) 55- 62 2. Le Thi Hai Yen and Nguyen Duc Hien, 2015. Study on the probiotic properties of Bacillus spp. strains isolated from poultry farms at Cantho City. Proceedings of National Conference on Animal & Veterinary Science. ISBN 978-604-60-2019-6. Agriculture publishing house, page 485-491 3. Le Thi Hai Yen and Nguyen Duc Hien, 2016. Isolation and identification of Bacillus subtilis isolated from soil and feces on chicken farms in the Mekong delta, Vietnam. Proceedings of the 19 th Federation of Asian Veterinary Associations Congress, Ho Chi Minh City, September 6-9 th , 2016. Vietnam National University-Ho Chi Minh city Press, page 143- 147 4. Le Thi Hai Yen and Nguyen Duc Hien, 2016. Evaluation of the probiotic properties of Bacillus subtilis strains isolated from Mekong delta. ISSN 1859-2333. Can Tho University Journal of Science. Vol 2 (2016), 26-32 5. Le Thi Hai Yen and Nguyen Duc Hien, 2017. Assessment of gastric acid, bile salt tolerance and aggregation ability of Bacillus subtilis AG27 and VL28. Proceedings of National Conference on Animal & Veterinary Science. ISBN 978-604-60-2492-7. Agriculture publishing house, page 341-346 6. Le Thi Hai Yen and Nguyen Duc Hien, 2017. Isolation and characterization of probiotic Bacillus subtilis VL28 on chicken farms in Vietnam. Proceedings of 33 rd World Veterinary Congress, Incheon – Korea, August 27-31, 2016. 7. Le,T.H.Y. and Nguyen,D.H., 2017. Bacillus subtilis strain VL28 16S ribosomal RNA gene, partial sequence. GenBank: KY346980.1 https://www.ncbi.nlm.nih.gov/nuccore/KY346980 Chapter I: INTRODUCTION 1.1. Necessity In recent years, the strong development of poultry breeding has brought great values to economo-social benefits. However, it also results in many concerns. One of the concerning issue is the overuse of antibiotics in the prevention from diseases and in the stimulating growth. In consequence, antibiotic resistant bacteria are increasing in nature and that affects significantly the use of antibiotics for infectious diseases in human beings. Therefore, the majority of developed countries did limit the use of antibiotics in breeding. In order to replace them in breeding, scientists put forward different solutions, one of them is using probiotic- useful microorganisms in gastro- intestinal activities and, broadly speaking, in improving health. Bacillus subtilis are universal bacteria that are present in nature, and they are almost not harmful to human being as well as to several kinds of animals but resistant strongly to several physical and chemical factors. As a result, they were selected and chosen as probiotics for human beings and breeding animals in industrial models. However, B. subtilis have the diversity in biological properties, so all of them could not be used as probiotic and just some strains of probiotic could be suitable and effective for certain animals. The thesis “Isolation of Bacillus subtilis and its applications on the prevention of intestinal diseases in chicken” was carried out in order to find out the alternatives that could replace antibiotics in breeding, to increase productivity and effectiveness in industrial chicken breeding and to reduce the risks of antibiotic resistant bacteria spreading in nature. 1.2. The objectives At least one Bacillus subtilis strain was isolated in some provinces in the Mekong Delta, having probiotic properties such as: ability to produce digestive enzymes (amylase, protease, lipase); resistance to digestive juice (gastric juice and bile acid), ability to adhere to intestinal mucus and resistance to some pathogenic bacteria (S. enterica, E. coli). - The effective dosages of that probiotic isolate were identified to prevent the intestinal diseases in chicken caused by E. coli and Salmonella. 1.3. Subjects and scope - Subjects: Bacillus subtilis strains 2 - Scope: the strains of Bacillus subtilis were isolated form soil and chicken feces in 7 provinces belonging to the Mekong Delta namely: Can Tho, Hau Giang, An Giang, Vinh Long, Dong Thap, Soc Trang, Kien Giang. 1.4. Novel aspects - This is the first research did select the local B. subtilis strain that showed effectiveness in the prevention of intestinal diseases in chicken in Mekong Delta, especially the diseases were caused by S. enterica and E. coli. - This is the scientific research on the isolation and selection of probiotic bacteria systematically based on International Standards. - Twenty one B. subtilis isolates were correctly identified from fecal and soil samples in Mekong Delta regions. Among them, B. subtilis VL28 was chosen to use as a probiotic strain for supplementation in chicken feed. - B. subtilis VL28 (10 7 CFU/g) with the dosage of 5 g/kg of chicken feed was able to replace antibiotics in treatment of intestinal diseases against S. enterica and E. coli. Also, it could increase chicken growth and reduce food conversion rate (FCR) compared with the control. - The 16S rRNA partial sequence of the new strain B. subtilis VL 28 was approved by NCBI Genbank with access code KY346980. 1.5. Applicability in practice This study has high practical values in production of probiotics in order to prevent and treat intestinal diseases in poultry, as well as to increase poultry performance and reduce the overuse of antibiotics as stimulating growth substances. Also, it helps to provide clean poultry meat resources without antibiotic residues, and as a result it will protect community health. Chapter III. MATERIALS AND METHODS 3.1.Content 1: Isolation B. subtilis strains - B. subtilis were isolaled by traditional methods: based on colony charatetistics and observation under microscopy, gram-stain technique and biochemical reactions. - Accurate identification of strains by kit API 50 CHB. - Accurate expertise of species and strains of Bacillus isolates by using the method of 16S rRNA partial sequence Sampling Soil samples: Soils were taken in the surface layer with 4-5 cm in depth. Added samples were obtained from 5 different sites in a poultry farm. 3 Fecal samples: fresh fecal samples were taken from the floor of chicken farm. Added samples were taken from 5 different sites in the chicken farm (four from each corner and one from the center). The amounts of each soil or fecal sample were about 30-50 g. The samples were put in sterile plastic bags (polypropylene). After taking the samples, the label was noted with site and time, and then they were preserved in the cool box and were sent to laboratory. Sample sizes Sampling was based on the non-probability sampling methods 20 samples for each province (10 soil samples + 10 fecal samples). Total: 140 samples/ 7 provinces, cities in the Mekong Delta. Sample preparation Taking 10 g of sample + 90 mL sterile physiological saline serum in triangular jar and shaking regularly. Heating at 80ºC for 20-25 minutes to select bacteria that would be Bacillus spp. (Eman, 2013). Bacteria isolation After heating, samples were diluted and spread in TSA media, at 30ºC for 24 hours. After maintaining, we chose separate and different colonies and cultured, isolates combining with observation, recognization the shape of bacteria under microscopy in order to achieve the uniform ones. The biochemical properties of B. subtilis were checked based on the method of Cowan and Steel (2004) with some modifications. Biochemical reactions include: lecithinase (-), catalase (+), VP (+), amylase (+), able to grow at 50 o C and cellulase (+) and they were done in mentioned order to screen bacteria belonging to B. subtilis. The satisfied strains then were identified by the kit API 50 CHB, in case that the results showed they were B. subtilis, they were done with the 16S RNA partial sequence to confirm again. After DNA extraction of bacteria, PCR amplification of target sequences using universal primers for a segment of 16S rRNA (Saminathan and Narayanan, 2015) that had the following order: 27F: (5'-AGAGTTTGATCMTGGCTCAG-3') 1492R: (5'-TACGGYTACCTTGTTACGACTT-3'27F) Procedure for isolation and verification B. subtilis 4 Bacteria isolates uniform ↓ Test Lecithinase: (-) ↓ Test Catalase: (+) ↓ Test VP: (+) ↓ Test Amylase: (+) ↓ Able to grow at 50ºC ↓ Test Cellulase: (+) ↓ API CH50B ↓ 16S rRNA partial sequence Figure 3.1. Schema of screening, identification of B. subtilis 3.2. Content 2: Selection of B. subtilis that have probiotic properties There are 7 norms : 3.2.1.Temperature tolerance. The temperature of bacteria was investigated by the method of Barbosa et al. (2000) with modification. The culture of bacteria needed checking on the agar plate TSA and kept at 50 o C, 55 o C and 60 o C for 24 hours. When it finished, bacteria growth ability was checked. 3.2.2. Antibiotic susceptibilities (with 9 antibiotics available and frequent use for poultry namely: erythromycin, gentamycin, neomycin, oxytetracyclin, doxycycline, colistin, sulfadimidin - trimethoprim, norfloxacin, enrofloxacin). The antibiotic susceptibilities of bacteria were identified by the method of antibiotic disk diffusion according to the guidelines of Clinical and Laboratory Standards Institute - CLSI, 2015 (Wayne, 2015). 3.2.3. Induction of digestive enzyme capacity (amylase, protease, lipase): The investigated bacteria were qualified and quantified then bacteria that had the induction capacity of all 3 enzymes above were chosen. 3.2.4. Capacity against pathogenic bacteria Capacity against pathogenic bacteria was investigated by cross streak method. It was investigated in Starch agar media (SA). The procedures were followed by the method of Sertaç et al. (2014) such as culture B. subtilis on a straight line on SA, kept at 37 o C for 24 hours, and cultured pathogenic bacteria (Salmonella, E.coli) on the lines that were perpendicular to the growing 5 bacteria, then were kept at 37 o C for 24 hours. Capacity against bacteria was measured by the distance of antibacterial sites expressed by mm according to Hutt et al. (2006). Experiment the capacity against bacteria by the method of direct resistance: that was performed with the method of Moore et al. (2013): pathogenic bacteria and B. subtilis were activated in TSB media and were kept in suitable temperature for 24 hours. Suspension of B. subtilis was modified to order to achieve the concentration of 10 5 CFU/mL, 10 6 CFU/mL and 10 7 CFU/mL that were correspondent with those of pathogenic bacteria and used to check the resistance capacity. Putting 100 µL suspension of pathogenic bacteria on agar plate and using a glass spreader to spread it evenly on the agar surface; then putting 10 µL suspension of B. subtilis correspondent with the different concentration investigated on the surface of agar plate that had pathogenic bacteria, kept at 37 o C for 24 hours. Resistance capacity was measured with the diameter of inhibition of pathogenic bacteria and expressed by mm. Evaluation of resistance capacity was done by the method of Sumathi and Reetha (2012). 3.2.5. Acid and bile salt tolerance. It was done by the method of Corcoran et al. (2005) and Dunne (2001). B. subtilis was streaked on DSM, for 24 - 48 hours at 30ºC. Then it caused the suspension inducing bacteria in buffer solution PBS pH 7.2, and diluted to achieve the density of bacteria in suspension at 10 7 CFU/mL. After that, adding 1 mL of suspension into 9 mL of simulate gastric solution containing Glucose (3.5 g/L), NaCl (2.05 g/L), KH2PO4 (0.6 g/L), CaCl2 (0.11 g/L), and KCl (0.37 g/L) with titration at pH 2, 3, 4, 5 by HCl 1M and filted by filter membrance 0.2 μm. Then Pepsin (13.3 mg/L) and bile juice (0.05 g/L) were put in primary solution before the experiment was carried ou. Regular mixed and kept mixture solution at 37ºC in shake machine for 90 minutes, at the same time checked the density of bacteria at 0; 10; 30; 60, 90 minutes. The survival percentage of B. subtilis was calculated. 3.2.6. Adherence ability - Autoaggregation: that was done by the method of Del Re et al. (2003) with modification following the description of Kos et al. (2003). Bacteria activation should be checked in TSA media, kept at 37 o C for 24 hours, then cultured in 100 mL TSB, nurtured at 37 o C for 18 hours. Bacterial biomass obtained after culture were cleaned twice and made suspension in Phosphate buffered saline (PBS) so that the concentration of bacteria was about 10 8 CFU/mL (0.5 McFarland turbidity). Then 4 mL of cell suspension was mixture 6 regularly for 10 seconds. Adherence capacity of cells of the same strain was identified for 5 hours at room temperature. After every hour, taking 0.1 mL solution floating on the surface in the different test tube containing 3.9 mL PBS and identified optical density of solution with wavelengths at 600 nm (OD600). The results were calculated based on the formula below: Adherence capacity (%) = (Ao - At)/Ao × 100 Ao: OD600 of the suspension at time t = 0 hour At: OD600 of the suspension at times t = 1, 2, 3, 4 and 5 hours - Adherence ability between different strains: it was identified by the method Kos et al. (2003) with modification following the description of Anwar et al. (2014). Sample preparation methods were similar to the method mentioned above,however isolates were done with 2 experiment bacteria namely E. coli and S. enterica. Adherence capacity between different strains was calculated based on the formula below: Adherence capacity between different strains (%) = [(Ax + Ay)/2 - A(x+y)]/[(Ax+Ay)/2]×100 Ax as OD600 of bacteria x in control tube Ay as OD600 of bacteria y in control tube A(x+y) as OD600 of the mixture of 2 strains x and y - Adherence capacity to epithelial intestine. It was carried out by the method of Piatek et al. (2012): B. subtilis strains were duplicated in 20 mL of NB solution, kept at 37 o C for 24 hours, and then adjusted with density of 10 8 CFU/mL. Epithelial intestine of chicken were cut into segments with 1 cm in length, then were put in buffer solution PBS for 30 minutes at 4 o C. Samples needed checking, were kept at 37 o C for 30 minutes. Bacteria solution was rejected, samples were fixed in formalin and made for tissue slides. 3.2.7. Growth capacity in chicken’s intestine It was done by the method of Cartman et al. (2008): one -day chicken were given spores of B. subtilis with 0.1 mL bacterial suspension containing 1x10 9 CFU/mL. After that, at the time of 24, 48, 72, 96 and 120 hours, chicken were selected randomly, they were operated and taken samples from ileum, caecum and large intestines. Cleaning samples, managing with temperature at 80 o C, for 20 minutes to kill living cells and other bacteria. Then, spreading the samples to check the density of B. subtilis at different times mentioned above. Mean value was calculated by the number of spores/g of small intestines, caecum and large intestines. 3.3. Content 3: experiment, evaluation of probiotic products containing B. subtilis in chicken 7 3.3.1. Subjects and material The subjects of 3 experiments were B. subtilis strains that were isolated and selected in our study. Experiment chicken: on-day old chicken, belonging to Greenfeed GF168 and raised at Vemedim Corporation Food and vaccinations were done following the protocols of breeding company 3.3.2. Experiment chicken farms Chicken were raised in pens with the zinc frame walls in the size of 0.6 x 1 x 2 m. The superficies of each pen were 2m 2 and separated into 2 parts, each part containing 15 chickens. Containers for feed and drink were made for each part of farm. 3.3.3.Experiment1: Identifying the effective dosage of B. subtilis Aim: evaluating the safety and identifying the effective dosage of products for chicken. Experiment Performance: Experiments were performed based on random ways including 4 treatments: among them, 3 treatments correspondent with supplementation of B. subtilis and one control without B. subtilis supplements. Each treatment consisted of 30 chicken and repeated 3 times. Table 3.3: Schema of experiment performance Parameters Treatment T1 T2 T3 Ctr Chicken, day-old 1 1 1 1 B. subtilis supplement, CFU/g (*) 10 7 10 6 5x10 5 - Supplement, g/kg of feed 5 5 5 - Number of experiment preparation 7 7 7 7 Number of experiment days 60 60 60 60 Note: (*): reference dosage from the experiment of Knap et al. (2011) and Teo et al. (2006) Parameters for experiment monitoring Food intake: identifying by weighing given feed and redundant food every day of each treatment during the experiment. Number of dead chicken: reporting number of dead chicken every day and summing them up for every two weeks. Gaining weight: chicken were weighted at the beginning of experiment, then every 2 weeks they were weighted until the end of experiment, and then we calculated the weight of each treatment. Gaining weight (g) = Weight at the end (g) – Weight at the beginning (g). 8 Gaining weight for the whole Average gaining weight (g/day) = Number of experiment days All feed intake (g) Food conversion rate = All weight of chicken (g) 3.3.4.2. Experiment 2 Aim: to evaluate protective capacity of probiotic compared to antibiotic in chicken when they were infectious with S. enterica. Experiment performance: there were 5 branches in the random way based on the method of Knap et al. (2011). (1) Control (-): non infectious, normal feed. (2) Control (+): infectious by S. enterica, normal feed (3) Treatment 1: infectious by S. enterica, feed with the supplementation of B. subtilis 5 g/kg feed, eating continuously during the experiment. (4) Treatment 2: infectious by S. enterica, feed with the supplementation of oxytetracyclin 50 mg/kg food, 5 days continuously from the infectious day. (5) Treatment 3: infectious by S. enterica, feed with supplementation of enrofloxacin 15 mg/kg of food, 5 days continuously from the infectious day. Each treatment consisted of 30 chicken and repeated 3 times. Table 3.4: Schema of experiment 2 Parameters Treatment T1 T2 T3 C (+) Ctr (-) Experiment chicken, days old 1 1 1 1 1 Infectious chicken , days old S. enterica (1) , CFU/mL 18 7.5x10 4 18 7.5x10 4 18 7.5x10 4 18 7.5x10 4 - - B. subtilis suppl (2) , g/kg feed 5 - - - - Oxtetracyclin, mg/kg feed (3) - 50 - - - Enrofloxacin, mg/kg eedd (4) - - 15 - - Number of experiment days 60 60 60 60 60 (1) S. enterica isolated from sick chicken. (2) Useful dosage of B