Summary of doctoral thesis: Isolation of bacillus subtilis and its application on the prevention of intestinal diseases in chicken

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).

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: Central library of Can Tho University National library of Vietnam LIST OF PUBLICATION RELATED TO THE THESIS 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 (2015) 5562 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 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 19th Federation of Asian Veterinary Associations Congress, Ho Chi Minh City, September 6-9th, 2016 Vietnam National University-Ho Chi Minh city Press, page 143147 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 (2016), 26-32 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 Le Thi Hai Yen and Nguyen Duc Hien, 2017 Isolation and characterization of probiotic Bacillus subtilis VL28 on chicken farms in Vietnam Proceedings of 33rd World Veterinary Congress, Incheon – Korea, August 27-31, 2016 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 gastrointestinal 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 - Scope: the strains of Bacillus subtilis were isolated form soil and chicken feces in 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 (107 CFU/g) with the dosage of 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 different sites in a poultry farm Fecal samples: fresh fecal samples were taken from the floor of chicken farm Added samples were taken from 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/ 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 50oC 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 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 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 50oC, 55oC and 60oC for 24 hours When it finished, bacteria growth ability was checked 3.2.2 Antibiotic susceptibilities (with 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 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 Sertaỗ et al (2014) such as culture B subtilis on a straight line on SA, kept at 37oC for 24 hours, and cultured pathogenic bacteria (Salmonella, E.coli) on the lines that were perpendicular to the growing bacteria, then were kept at 37oC 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 105 CFU/mL, 106 CFU/mL and 107 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 37oC 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 107 CFU/mL After that, adding mL of suspension into 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, 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 37oC for 24 hours, then cultured in 100 mL TSB, nurtured at 37oC 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 108 CFU/mL (0.5 McFarland turbidity) Then mL of cell suspension was mixture regularly for 10 seconds Adherence capacity of cells of the same strain was identified for 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 = hour At: OD600 of the suspension at times t = 1, 2, 3, and 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 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 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 37oC for 24 hours, and then adjusted with density of 108 CFU/mL Epithelial intestine of chicken were cut into segments with cm in length, then were put in buffer solution PBS for 30 minutes at 4oC Samples needed checking, were kept at 37oC 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 1x109 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 80oC, 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 3.3.1 Subjects and material The subjects of 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 x m The superficies of each pen were 2m2 and separated into 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 treatments: among them, treatments correspondent with supplementation of B subtilis and one control without B subtilis supplements Each treatment consisted of 30 chicken and repeated times Table 3.3: Schema of experiment performance Parameters T1 107 60 Chicken, day-old B subtilis supplement, CFU/g (*) Supplement, g/kg of feed Number of experiment preparation Number of experiment days Treatment T2 T3 1 106 5x105 5 7 60 60 Ctr 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 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) fourth reation was Amylase There were 91 positive and 78 negative that were be rejected The spores B subtilis were known with capacity of growing at 50oC (Cowan and Steel, 2003) The results showed among 91 isolates,49 isolates could grow at 50oC Finally, cellulase reation was done in order to identify hydrolysis property for cellulose of bacteria Among 49 isolates being investigated, there were 29 isolates positive with cellulase They were identified continously by the kit API CH50B for strains 4.1.2.2 Identification by kit API CH50B After verifying biochemical properties, we found 29 strains that were satisfied with B subtilis properties Among 29 strains identified by kit API, 23 strains were identified as B subtilis/B amyloliquefaciens with accuracy ranging around 90.7% - 99.9%; the remains belonged to B licheniformis 23 strains above were carried out for gene sequence in order to confirm correctly B subtilis 4.1.2.3 Identification by PCR and the method of 16S rRNA partial sequence The results of electrophoresis PCR of 23 strains of bacteria showed successful amplification of the gene with the size of 500bp (Figure 4.4) M ST10 DT11 DT26 DT29 DT30 KG12 KG22 KG29 KG36 C 1500 bp 1500 bp M 1500 bp M CT11 AG07 AG17 AG19 AG60 VL16 ST06 ST08 C VL05 VL28 VL41 KG09 AG27 AG49 C Figure 4.4 Results of PCR of the strains of B subtilis in our study (M: standard scales 100bp; ST10, DT11,…: PCR of 23 strains of bacteria were carried out; C: controls negative) The 16S rRNA partial sequence After gene partial sequence and analysis with software BLAST compared to results in NCBI, there were 21 among 23 strains identified as B subtilis with high level of consistency (99%-100%), the rests were B amyloliquefaciens In short, from 296 bacteria isolates after being carried out the selection procedure by biochemical reactions and identification by the kit API and finally with the 16S rRNA partial sequence, there were 21 strains of bacteria identified 11 as B subtilis All of them were done with the selection procedures for probiotics in order to achieve the best one that had potential for the prevention of digestive diseases in chicken 4.2 Results of probiotic properties investigation 4.2.1 Temperature tolerance According to Sottnik (2002) poutry have body temperature around o 41.5 C, which is higher than that of mammals, therefore the probiotic candidates for poultry breeding should be able to grow at the temperatures that are higher than normal surrounding (30-37oC) Table 4.6 Results of investigation of the strains B subtilis’s temperature tolerance Strains KG36, AG19, VL05 AG07, AG27, AG49, AG60, VL16, VL28, VL41, ST08, DT30, KG09, KG12, KG22 CT 11, AG17, ST06, ST10, DT29, KG29 Total Number of strains Investigated temperature 50oC 55oC 60oC + + + 12 + + - + - - 21 21 15 Remark : + Able to live in media at investigated temperature - Unable to live in media at investigated temperature The results in Table 4.6 showed that all of 21 strains of bacteria could grow at the temperature of 50oC Therefore, the 21 strains of B subtilis that were isolated had tolerance of heat, and were sastified completely when were administered into poultry’s body In short, in the temperature’norm, all of 21 strains of investigated B subtilis could be chosen as probiotics 4.2.2 Susceptibility/ Resistance to antibiotic Susceptible capacity of probiotics are considered as one of the key norm for selection criteria of probiotic bacteria (Hummel et al., 2007) According to Gueimonde et al (2013), if probiotic bacteria are still sensitive to antibiotics, they will be safe in biological views, because they not contain plasmid and antibiotic –resistant gene 12 Figure 4.6 Susceptibility levels of 21 strains of B subtilis Figure 4.6 showed that 21 strains of B subtilis were sensitive to all of antibiotics with high percentage from 100% for the strains sensitive to erythromycin, enrofloxacin, doxycycline, norfloxacin, sulfadimidin trimethoprim, to gentamycin (24% sensitive, 57% immediate, 19% resistant), neomycin (14% sensitive, 57% immediate, 29% resistant), oxytetracyline (33% sensitive, 33% immediate, 33% resistant), the lowest sensitivity was in the case of colistin with only 5% sensitive One thing should be noticed that there were until 95% strains of B subtilis in this investigation resistant to colistin, probably because colistin was used very frenquently in the treatment of digestive infectious disease VL28 Figure 4.7 Results of antibiogram of B subtilis AG27 and VL28 From the investigation of 21 isolated strains B subtilis we recognized that more than 50% of the strains were still sensitive to several antibiotics compared to strains in controls These results revealed that all of 21 investigated strains B subtilis were satisfied with the norm of antibiotic susceptibility and had the potential to be a probiotic 13 4.2.3 Capacity for extracellular enzyme induction 4.2.3.1 Induction capacity for enzyme namely amylase, protease and lipase The results of investigation of induction of enzyme capacity were presented in Table 4.7 Table 4.7: Induction of extracellular enzyme capacity of 21 strains B subtilis Bacteria Amylase Protease Lipase Control + + Control + + AG27, AG60, VL05, VL28, VL41, + + + DT29, KG09, KG12, KG22, KG36 CT11, AG07, AG17, AG19, AG49, VL16, ST06, ST08, ST10, DT30, + + KG29 Remark (+): able to produce enzyme, (-): unable to produce enzyme According to Parsons (2004) extracellular enzymes such as amylase, protease, and lipase play important roles in food digestion and facilitate food absorption Therefore, in order to become a probiotic, the strain B subtilis have to be able to produce many kinds of enzyme The results above showed that in 21 investigated strains of B subtilis, only 10 strains (AG27, AG60, VL05, VL28, VL41, DT29, KG09, KG12, KG22, KG36) could be able to produce all of kinds of enzymes, then these 10 strains were checked for the capacity against pathogenic bacteria 4.2.4 Capacity against pathogenic bacteria 4.2.4.1 Capacity against pathogenic bacteria of B subtilis by cross streak method The results in Table 4.9 showed that 10 strains of B subtilis had capacity against pathogenic bacteria in different degrees Table 4.9: Anti-bacterial distance of B subtilis by cross streak method Bacteria ĐC1 ĐC2 AG27 AG60 VL05 VL28 VL41 KG12 E coli 5.47c 6.10c 8.00b 6.00c 6.00c 10.00a 6.00c - Anti-bacterial distance (mm) S enterica Staphylococcus Streptococcus 6.27c 7.00bc 7.00bc 6.23c 12.00a 10.00a 10.00b c bc 6.00 7.00 4.00d b b 10.00 8.00 6.00c a a 13.00 10.00 12.00a b a 10.00 10.00 9.00b 10.00a 4.00d 14 KG22 KG09 KG36 DT29 SEM P a,b,c,d 4.00d 6.00c 0.184 0.00 6.00c 10.00b 9.00b 0.275 0.00 6.00c 6.00c 8.00b 0.281 0,00 0.167 0.00 the values with different superscripts in the same row were statistically different (P < 0.01) From the results of cross streak method, strains of B subtilis namely AG27, AG60, VL05 and VL28 revealed anti-bacterial activity against all of pathogenic bacteria, and they had strongly anti-bacterial property and would be investigated by direct resistance method in order to check and compare anti-bacterial activities at different densities of bacteria 4.2.4.2 Resistance capacity of B subtilis to E coli and S enterica by direct resistance method E coli The results of direct resistance of strains B subtilis (AG27, AG60, VL05 and VL28) to E coli at different concentration namely 105 CFU/ mL, 106 CFU/ mL, 107 CFU/ mL showed in Table 4.10 Table 4.10: The results of direct resistance of B subtilis AG27, AG60, VL05 and VL28 to E coli a,b,c,d Isolated bacteria Resistance diameter (mm) Cocentration E.coli E coli E coli Strain (CFU/mL) (105 CFU/mL) (106 CFU/mL) (107 CFU/mL) AG27 14.43bc 14.00a 12.07b c c AG60 14.20 11.00 10.33c 105 a b VL05 15.53 13.00 10.27c VL28 15.27ab 14.00a 13.50a SEM 0.204 0.166 0.214 P 0.005 0.00 0.00 AG27 16.57a 16.00b 12.30b AG60 15.23b 12.00d 12.57b 106 VL05 16.57a 14.00c 11.23c a a VL28 17.43 17.00 16.83a SEM 0.251 0.180 0.178 P 0.002 0.00 0.00 AG27 18.57ab 16.00b 14.23bc AG60 17.40b 14.00c 13.57c 107 VL05 19.07ab 16.00b 15.17b VL28 20.20a 19.00a 17.17a SEM 0.449 0.194 0.301 P 0.014 0.00 0.00 the values with different superscripts in the same row were statistically different (P < 0.01) 15 The results revealed that all of strains AG27, AG60, VL05 and VL28 had anti-bacterial activities to E coli at the concentration of 105 CFU/mL, 106 CFU/mL and 107 CFU/mL It means that all of strains of B subtilis mentioned have the potential in prevention and treatment of diseases due to E coli in poultry S enterica Table 4.11: Results of direct resistance of B subtilis AG27, AG60, VL05 and VL28 to S enterica Isolated bacteria Concentration (CFU/mL) 10 106 107 a,b,c,d d Strain AG27 AG60 VL05 VL28 SEM P AG27 AG60 VL05 VL28 SEM P AG27 AG60 VL05 VL28 SEM P Resistant Diameter (mm) S enterica (105 CFU/mL) 20.23a 19.33a 19.17a 20.17a 0.331 0.109 22.83a 20.50a 21.17a 22.2a 0.676 0.143 25.33a 21.57b 23.17ab 25.83a 0.702 0.009 S enterica (106 CFU/mL) 20.00a 16.00b 15.00b 19.00a 0.255 0.00 21.00a 17.00b 18.00b 20.00a 0.303 0.00 25.00a 21.00b 20.00b 24.00a 0.338 0.00 S enterica (107 CFU/mL) 15.50a 12.30b 11.03b 16.83a 0.302 0.00 16.50b 13.90c 12.93c 18.50a 0.352 0.000 17.67b 15.50c 14.33c 20.17a 0.323 0.00 the values with different superscript in the same row were statistically different (P < 0.01) Table 4.11 showed all of strains of B.subtilis had anti-bacterial activities against S enterica, among them AG27 and VL28 expressed resistance activities much more at the investigated concentration 4.2.5 Gastric acid and bile salts tolerance In chicken, gastric pH (proventricular and gizzard) varied in the range of 2.5-3.5 (Gauthier, 2002) With the first concentration of B subtilis that was modified at 4.5x106 CFU/mL, we investigated gastric acid and bile salts tolerance at pH 4, 3, and pH in simulated gastric acid and bile salts The followings were the results of investigation at pH 16 Table 4.16: Investigation of acid and bile salts at pH Strains ĐC1 ĐC2 AG27 AG60 VL05 VL 28 SEM P a,b,c,d mn log CFU/mL d 0.00 4.76c 5.43b 0.00d 0.00d 6.62a 0.027 0.00 Amount of bacteria and % survival at different time 10 mns 30 mns 60 mns 90 mns % 72 82 99 log CFU/mL d 0.00 4.69c 5.39b 0.00d 0.00d 6.58a 0.017 0.00 % 70 81 99 log CFU/mL d 0.00 4.58c 5.31b 0.00d 0.00d 6.54a 0.035 0.00 % 69 80 98 log CFU/mL d 0.00 4.24c 5.21b 0.00d 0.00d 6.48a 0.062 0.00 % 64 78 97 log CFU/mL d 0.00 3.67c 5.19b 0.00d 0.00d 6.46a 0.037 0.00 % 55 78 97 the values with different superscript in the same row were statistically different kê (P < 0.01) C1: control strain B subtilis (ATCC ® 19659™) C2 : control strain (products in the market) Table 4.16 showed AG60, VL05 and control could not definitely be tolerant at pH 2, at the time of mn, concentration of AG60, VL05 and control1 reduced towards In the meantime, AG27 and VL28 had quite good tolerence, VL28 still maintained survival rate that was almost unchanged during 90 minutes of investigation (99% at mn and 97% after 90 mn), AG27 maintained 82% at mn and remained 78% at 90 mns These two strains were really potential candidates so they were checked for the other norms of probiotics 4.2.6 Adherence capacity 4.2.6.1 Auto-adherence capacity The results showed that percentage of adherence between B subtilis strains had tendency to increase progressively by time After hours kept at room temperature, adherence capacities were nearly double compared to those at hour (Table 4.17) Table 4.17: Investigation of auto-adherence Strain bacteria C1 C2 AG27 VL28 SEM P a,b Adherence capacities at different time (%) hour hours hours hours 43.9a 62.5a 66.4 72.2 30.2ab 57.8ab 65.0 66.1 24.3b 62.1a 63.3 69.8 36.8ab 40.9b 61.9 74.5 3.51 4.02 2.11 2.13 0.021 0.016 0.508 0.106 hours 73.8ab 76.7ab 70.2b 82.0a 2.13 0,024 the values with different superscript in the same row were statistically different (P < 0.01) 17 4.2.6.2 Adherence capacity to pathogenic bacteria The results of Table 4.18 revealed that all of strains AG27, VL28, and strain controls had adherence capacity with E coli and S enterica However the percentage of adherence capacity of VL28 was higher than that of AG27 and strain control Table 4.18 Investigation of adherence capacity to E coli and S enterica Time 0h hrs a,b,c,d Strains ĐC ĐC AG 27 VL28 SEM P C1 CC AG 27 VL28 SEM P Adherence rate to pathogenic bacteria (%) E coli S enterica 8.60b 25.75a b 10.14 12.62c c 4.76 16.15bc 24.82a 20.79ab 0.627 1.37 0.00 0.00 26.30c 59.51b 41.35b 46.14c c 19.33 36.40d a 60.98 65.64a 2.56 1.22 0.000 0.001 the values with different superscript in the same row were statistically different (P < 0.01) 4.2.6.3 Adherence capacity to epithelial intestines Adherence capacity to strains AG27 and VL28 were observed through the slide of micro epithelial intestine’s chicken, HE colorations Observation of the slide with optical microscopy at magnification 400 times and 1000 times were showed in Figure 4.21 Figure 4.21 Adherence capacity of strains VL28 (left) and AG27(right) in epithelial intestine (400X and 1000X) 18 The results showed that strains B subtilis had adherence capacity to epithelial cells of intestines After verifying adherence properties of two strains VL28 and AG27, we found that they had high auro-adherence capacity as well as adherence to epithelial cells of intestines However, in AG25, that property with pathogenic bacteria was very low Although AG27 had many beneficial probiotic properties, it showed less anti-bacterial activities to pathogenic invasing chicken’s digestive tract Therefore we only used VL28 to investigate the ability to live in chicken’s intestines 4.2.7 Capacity to live within chicken’s gastro-intestinal tract The results of investigating the capacity to live within the gastro-intestinal tract of chicken were manifested with the concentrations of the B subtilis spores VL28 and their survival rates recorded at the different times in table 4.19 Table 4.19 Capacity to live of B subtilis VL28 within the gastro-intestinal tract of chicken Concentration and % spores at different times 24 hrs 48 hrs 72 hrs 96 hrs 120 hrs Samples log % log % log % log % log % CFU/g CFU/g CFU/g CFU/g CFU/g 4.40 49 3.96 44 3.28 36 2.85 32 2.59 29 S intestines 4.32 48 3.61 40 3.1 34 2.8 31 2.5 28 Caecum 44 3.32 37 3.07 34 2.69 30 2.26 25 L intestines 3.94 Remark: First dosage of B subtilis VL28 for drinking 109 CFU/mL, mL each chicken Table 4.19 showed that the spores of B subtilis VL28 could exist within the gastrointestinal tract of chicken, and concentration decreased progressively with time In short, 21 B subtilis strains were obtained after the process of isolation, selection and identification After investigating probiotic properties of 21 B subtilis strains, we did choose B subtilis strain VL28 as it sensitive to several antibiotics, had temperature tolerance, had capacity to produce enzymes namely amylase, protease and lipase Moreover, it had high antibacterial activities to E coli and S enterica, tolerance to gastric acid and bile salts, had high adherence property and could live within the digestive tract of poultry We carried out experiments in chicken in order to identify the effective dosage as well as protective abilities for digestive disease in B subtilis VL28 19 4.3 Experiment results, evaluating effectiveness products in chicken 4.3.1 Results of Experiment 1: Identifying the dosage of using B subtilis Experiment and with the treatment T1 having the supplement B subtilis VL28 in food with the dosage of 107 CFU/g (5 g/kg feed), T2 with the dosage of 106 CFU/g (5 g/kg feed), T3 with 5x105 CFU/g (5 g/kg feed), Control only given feed used in chicken farms The results of observing the growth in weight, food consumption and food conversion ratio (FCR) in chicken during weeks experiment were presented in Table 4.20 Table 4.20: Growth in weight, food consumption and FCR in chicken during weeks experiment Parameters Period 1-28 days W at beginning, g/ch W at 28 days, g/ch Weight growth, g/ch F consumption, g/ch FCR Period 29-56 days W at 56 days, g/ch Weight growth, g/ch F consumption, g/ch FCR Period 1-56 days Weight growth, g/ch F consumption, g/ch FCR a,b,c,d Treatment T2 T3 SEM P 69.43 69.55 69.57 69.50 423.63 392.35 381.77 369.2 354.20a 322.80b 312.20c 299.70d 653.70 671.20 669.60 695.00 1.85c 2.08b 2.14b 2.32a 0.09 1.09 1.14 9.39 0.03 0.000 0.000 0.000 0.079 0.000 1061.7 638.1a 1721b 2.69b 970.5 962.0 582.6b 556.3c 1776b 1745b 3.05a 3.14a 938.0 590.8b 1944a 3.29a 5.24 4.844 34.80 0.02 0.000 0.000 0.007 0.001 992.2a 2370c 2.39c 900.9b 868.5c 2587b 2548bc 2.87b 2.94b 892.5b 2851a 3.19a 5.245 45.83 0.054 0.000 0.001 0.001 T1 Control the values with different superscript in the same row were statistically different (P < 0.01) W: weight, F: Feed, FCR: Feed conversion rate, ch: chicken The results form Table 4.20 showed that supplementation of B subtilis VL28 (107 CFU/g) in treatment had the role of facilitating the growth in weight in chicken not only in the early period (1-28 days), but also still benefical to the late one (29-56 days), therefore, the growth in weight for all treatment reached the maximum values (992.2g), increased by 11.17% and food consumption (2370g) decreased by 6.87% Furthermore, FCR was recorded with the minimum values (2.39), decreased by 25% compared to those of controls This experiment demonstrated that with the dosage of supplement 0.5% B subtilis VL28 with the concentration of 107 CFU/g into food was benefical to chicken development from to 56 days old From the results above, the experiment showed that preventive capacities of probiotic, when 20 chicken were infective with S enterica and E coli, were effective with the supplement of 0.5% B subtilis VL28 and the concentration of 107 CFU/g in food 4.3.2 Results of Experiment 2: Evaluating protective capacities of probiotic compared to antibiotics in chicken that were infectious with S enterica Experiment 2, after causing S enterica infection in chicken at the 18th day old, we supplemented B subtilis VL28 (Treatment 1=T1) in feed in order to compare with other treatments with antibiotics The results of experiments during the experiment showed in the table below: Table 4.21: Mortality rate, growth in weight and FCR in chicken during weeks in experiment Parameters Period 1-14 days W at the beginning, g/ch Weight growth, g/ch F consumption, g/con FCR Mortality rate , % Period 15-28 days Weight growth, g/ch F consumption, g/ch FCR Mortality rate , % Period 29-56 days Weight growth , g F consumption, g FCR Mortality rate , % T1 T2 Treatment T3 C (+) C (-) SEM P 68.71 69.40 68.42 129.7a 125,1bc 127.5ab 248.9bc 255.7ab 258.7a 1.92b 2.02a 2.03a 2.2 3.3 4.4 68.32 124.7c 247.2c 1.98a 2.2 67.69 125.4bc 248.9bc 1.99a 2.2 0.41 0.59 1.78 0.01 0.98 0.118 0.000 0.004 0.000 0.452 252.8a 234.4ab 239.2ab 548.2ab 572.2a 588.6a 2.17c 2.44b 2.46b b b 1.1 5.7 7.0b 122.2c 450.7c 3.69a 72.7a 226.0b 508.0b 2.25bc 1.1b 4.66 8.88 0.06 1.959 0.00 0.00 0.00 0.00 542.3a 1877a 3.46c 1.11b 307.5d 1480c 4.81a 20.37a 518.2b 1738b 3.35c 2.30b 5.04 27.98 0.06 2.00 0.00 0.00 0.00 0.00 493.5c 496.8bc 1956a 1999a 3.96b 4.02b b 4.8 7.4b a,b,c the values with different superscript in the same row were statistically (P < 0.01) T1: Supplementation of B subtilis VL28 107 CFU/g (5 g/kg food) T2: Supplementation of oxytetracyclin 50 mg/kg food T3: Supplementation of enrofloxacin 15 mg/kg food C (+): Causing infection of S enterica without treatment, C (-): No causing infection Period from 1-14 days old in this period, chicken were not infectious Data from Table 4.21 showed that there were not statistically different among the treatments above However, regarding the growth in weight parameter, Treatment showed the growth in weight was higher, achieving 129.7g, that was statistically different from other treatment Period from 15-28 days old: After being caused S enterica infection in the 18th day, in the C (+), chicken began to show sick signs and dead after days 21 of infection, the number of dead chicken peaked after 5-7 days of infection In the 10th day, the C (+) had only 5-7 chicken left /30 chicken at the beginning The treatment T1, T2, T3 and C (-) had mortality rate of 1.1%; 5.7%, 7.0% and 1.1% respectively, there were not statistically different among all the treatments above From the table above, we found that B subtilis VL28, oxytetracylin and enrofloxacin showed their effectiveness in treatment of S enterica, mortality rate of all the treatment 1, 2, were by far lower than that of control (+) Food consumption and FCR in the treatments were similar, that proved L28 had anti-bacterial activities equivalent to those of antibiotics Sick chicken were operated, then obtained lesions were described as : swollen and congestive liver, swollen gall bladder, hemorrhage of intestine and proventricular, such signs of lesions were compatible to lesions caused by S entercica (Fig 4.26) Swollen and congestive liver and spleen Swollen gall bladder Congestive intestines Hemorrhage of proventricular Figure 4.26 Chicken’s lesions in experiments that causing S enterica infection The results of gene partial sequence did identify that the gene order of isolated bacteria was identification 100% with S enterica Period from 29-56 days: mortality rates of treatment 1, treatment 2, treatment and control (-) were statistically different, but all of them were significantly different from that of control (+) That meant the treatment with probiotic and antibiotics could cure disease; mortality rates decreased, and the weight growth were higher compared to control (+) Evaluating the whole experiment (1-56 days), we recognized that treatment had capacity of treating infectious disease due to S enterica, as the effectiveness was proved with the following parameters: mortality rates, growth in weight during experiment, food consumption and FCR The treatment with B subtilis VL28 was more effective than those with antibiotics as the mortality rates, FCR and food consumption were lower but the growth in weight was better than treatments with antibiotics 4.3.3 Results of experiment 3: Evaluating protective capacity of probiotic compared to antibiotics in chicken that were infectious with E coli 22 In these experiments, B subtilis VL28 was added in chicken feedd and we caused E coli infection in chicken at 18 days old in order to compare to the treatments with antibiotics namely enrofloxacin and oxytetracyclin In the beginning of the experiment, mortality rates and food consumption were not different among the treatments above However, in treatment 1, the growth in weight and FCR showed improvement, its weight growth was highest (130.3g) that difference was statistically significant compared to the rests: treatment (126.2g) and treatment (124.8g) control (+) 123.5g and control (-) 124.2g In addition, FCR of treatment was lowest NT1 (1.89), that difference was statistically significant compared to the rests (Table 4.25) Table 4.25: Mortality rate, growth in weight and FCR in chicken during weeks in experiment Parameter Period 1-14 days Mortality rate , % Weight growth, g FC, g FCR Period 15-28 days Mortality rate , % Weight growth , g FC, g FCR Period 29-56 days Mortality rate , % Weight growth , g FC, g FCR a,b Treatment T3 C (+) SEM P 2.22 124.2bc 246.6 1.97a 0.96 0.527 200 0.018 0.74 0.00 0.04 0.00 35.25a 119.1b 477.5b 4.01a 1.11b 238.2a 521.8ab 2.19b 1.95 0.00 4.28 0.00 11.35 0.004 0.037 0.00 6.57 283.3d 1258c 4.44a 4.48 480.8c 1666b 3.47c 2.01 0.356 4.826 0.00 22.73 0.00 0.035 0.00 T1 T2 2.22 130.3a 246.6 1.89b 3.33 126.2b 255.2 2.02a 3.33 124.8bc 251.7 2.02a 2.22 123.5c 247.1 2.00a 4.56b 246.9a 551.9a 2.24b 5.75b 242.2a 544.4a 2.25b 8.05b 240.6a 552.5a 2.30b 1.15 545.6a 1791a 3.28d 6.04 513.1b 1816a 3.54c 6.18 502.7bc 1864a 3.71b C(-) the values with different superscript in the same row were statistically (P < 0.01) T1: supplementation of B subtilis VL28 107 CFU/g (5 g/kg feed) NT2: supplementation of oxytetracyclin 50 mg/kg feed NT3: supplementation of enrofloxacin 15 mg/kg feed C (+): Causing E.coli infection without treatment, C (-): no causing infection The results of the 1-14 days period showed that using probiotic B subtilis VL28 helped the growth in weight and improved clearly FCR These results were compatible with those of Gao et al., (2017), in this study, B subtilis helped to improve the growth in weight but did not improve food consumption 23 Period from 15-28 days After causing E coli infection in the 18th day, in the control (+) chicken became sick and dead in the 5th day after infection and the number of dead chicken peaked during 5-10 days after infection, in the 10th day in the control (+) there were 15 chicken left/30 chicken Mortality rates in T1, T2, T3 were 4.56%, 5.75%, 8.05% respectively, this showed B subtilis VL28 and antibiotics namely enrofloxacin and oxytetracyclin could reduce mortality rates in E.coli infectious chicken so they could be used for treatment In the treatment with B subtilis VL28, mortality rates, the growth in weight, food consumption and FCR were similar to those in control (-) without disease Sick chicken were operated, then obtained lesions were described as: Pericarditis, Swollen liver and spleen, Granulomata in liver and spleen, Enteritis with bubbles (Fig 4.32) These lesions were compatible to those caused by E coli Pericarditis, Swollen liver and spleen Granulomata liver and spleen in Enteritis Enteritis with bubbles 4.32 Chicken’s lesions in the treatment with E coli infection Identifying suitable biochemical reactions showed the compatibility with biochemical properties of E coli The results of gene partial sequence did identify that the gene order of isolated bacteria was identification 100% with E coli Period from 29-56 days In the late period, mortality rates decreased in all of the treatments with infection, it meant that chicken recovered after infective period However, in the control (+), chicken were still affected by E coli This could explain although mortality rates decreased significantly compared to those in the early one, the growth in weight did not improve clearly compared to the early period, with FCR that was higher than in the other treatment We could recognize that using continuously probiotic B subtilis VL28 would bring benefits to chicken’s development Not only chicken are healthy but their immune systems were strengthened in order to resist to pathogenic bacteria 24 4.4 Register of gene partial sequence of B subtilis VL28 in Genbank From the results above, we did register the gene partial sequence of B subtilis VL28 in NCBI and the Genbank approved the 16S rRNA of B subtilis VL 28 with access code: KY346980 Available access to the website below: https://www.ncbi.nlm.nih.gov/nuccore/ky346980 Chapter V: CONCLUSIONS AND PROPOSALS 5.1 Conclusions - 21 strains of B subtilis were isolated and accurately verified gene partial sequence from soil and fecal chicken - B subtilis strain VL28 was selected and had beneficial probiotic properties for chicken: high sensitivity to several antibiotics, temperature tolerance, induction of enzyme capacity such as amylase, protease and lipase with high effectiveness, high resistant capacity against E coli and S enterica, tolerance to gastric acid and bile salts, high adherence capacity and stability in digestive tract in poultry - The study showed that the supplementation of B subtilis strain VL28 with concentration of 107 CFU/g at the dosage of g/kg feed was able to replace antibiotics in the prevention of digestive infectious diseases in chicken, simultaneously increased chicken growth and FCR in chicken 5.2 Proposals - Extending further studies on application of B subtilis strain VL28 in prevention and treatment for digestive infectious diseases such as Salmonellosis, Colibacillosis, Necrotic Enteritis … at fieldwork in industrial levels - Optimizing the procedure of fermentation for biomass of B subtilis VL28 with high densities and low costs - Optimizing the procedure of maintenance for B subtilis strain VL28 and creation of probiotic products to increase animal productivity and to provide the resource of clean meat without antibiotic residues that aims at community health protection 25 ... 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. .. food consumption 23 Period from 15-28 days After causing E coli infection in the 18th day, in the control (+) chicken became sick and dead in the 5th day after infection and the number of dead chicken. .. infectious diseases in chicken, simultaneously increased chicken growth and FCR in chicken 5.2 Proposals - Extending further studies on application of B subtilis strain VL28 in prevention and treatment