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

Chia sẻ: Co Ti Thanh | Ngày: | Loại File: PDF | Số trang:28

Thêm vào BST

Báo xấu

37
lượt xem 3
download

Download Vui lòng tải xuống để xem tài liệu đầy đủ

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).

Chủ đề:

Bình luận(0) Đăng nhập để gửi bình luận!

Lưu

Nội dung Text: Summary of doctoral thesis: 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 19th Federation of Asian Veterinary Associations Congress, Ho Chi Minh City, September 6-9th, 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 33rd 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
- 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 (107 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. 2
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 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 3
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 50oC, 55oC and 60oC 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 37oC for 24 hours, and cultured pathogenic bacteria (Salmonella, E.coli) on the lines that were perpendicular to the growing 4
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 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 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 4 mL of cell suspension was mixture 5
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 37oC for 24 hours, and then adjusted with density of 108 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 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 6
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 2m2 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 (*) 107 106 5x105 - 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). 7
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 18 18 18 18 - S. enterica (1), CFU/mL 7.5x104 7.5x104 7.5x104 7.5x104 - 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. subtilis chosen in experiment1 (107 CFU/g) (3), (4) Pattison, 2008 Procedures: - Causing infection: 18 day-old chickens were given 0,5ml solution containing S. enterica with density of 5x104 CFU/mL (Knap et al., 2011). - Chicken were operated for investigation (5 chicken/ branch) on 21 and 42 days old, reporting lesions, taking samples form liver, spleen, frozing them then transferring to laboratory for checking of pathogenic bacteria. 8
- Recording chicken’s manifestation, mortality rate in all treatments. 3.3.4.3. Experiment 3: Aim: to evaluate protective capacity of probiotic in chicken compared to antibiotics when they were infectious with E. coli. Experiment performance: It was done with the method of Teo and Tan (2006): there were 5 treatments in the random way, among them there were two controls: control negative and control positive, in the others chicken were given antibiotics and B. subtilis: (1) Control (-): no infectious, normal feed. (2) Control (+): infectious by E. coli, normal feed. (3) Treatment 1: infectious by E. coli, supplemention of B. subtilis 5 g/kg feed, eating continuously during experiment. (4) Treatment 2: infectious by E. coli, supplemention of oxytetracyclin 50 mg/kg feed, 5 days continuously from the infectious day. (5) Treatment 3: infectious by E. coli, supplemention of enrofloxacin 15 mg/kg feed, 5 days continuously from the infectious day. Each treatment consisted of 30 chicken and repeated 3 times. Table 3.5: Schema of experiment 3 Parameters Treatment T1 T2 T3 C (+) C (-) Experiment chicken, days old 1 1 1 1 1 Infectious chicken, days old 18 18 18 18 - E. coli (1), CFU/mL 5x106 5x106 5x106 5x106 - B. subtilis supplement (2), g/kg feed 5 Oxytetracyclin, mg/kg feed (3) - 50 - - - Enrofloxacin, mg/kg feed (4) - - 15 - - Number of days with supplement 60 5 5 - - Number of experiment day 60 60 60 60 60 (1) E. coli isolated from sick chicken. (3), (4) Pattison, 2008 (2) Useful dosage of B. subtilis chosen in experiment1 (107 CFU/g) Procedure: - Causing infection: 18 day-old chicken were drunk with 0.5 mL solution containing E. coli with concentration of 5x106 CFU/mL (Teo and Tan, 2006). - Chicken were operated for investigation (5 chicken/ treatment) on the 21 and 42 days old, reporting lesions, taking samples form liver, spleen, frozen then transferring to laboratory for checking of pathogenic bacteria. - Recording chicken’s manifestations, mortality rate in all treatment. 3.4. Data analysis Original data from the experiment were managed with the software Microsoft Excel 2010, then were analyzed by using ANOVA and General Linear Model in Minitab 16.1 Software. 9
Chapter IV. RESULTS AND DISCUSSION 4.1. Isolation and identification B. subtilis 4.1.1. Results of isolation Bacillus spp. There were 70 soil and 70 fecal samples from chicken farms in 7 provinces. They were managed with heat, diluted, cultured and kept at 30ºC during 24 hours. After that, based on the shape of colonies, they were chosen separately. The culture conversion were done several times until they become uniform. The results showed that 296 bacteria isolates were rod-shaped, gram positive and able to produce spores after 48 hours of observation. The colonies of 296 bacteria isolates were separated by streak-plating bacterial cultures to isolate single colonies and reserved in nutrient broth solution containing 16% glycerol, preserved in deep freezers 86oC. 4.1.2. Results of identification of Bacillus spp. 4.1.2.1. Identification by biochemical reactions From 296 isolates, we checked biochemical norms with the following steps: negative with Lecithinase, positive with catalase, VP, amylase, cellulase and able to develop at 50oC (Cowan and Steel, 2003). The isolates that were not satisfied with these norms were rejected. Finally the isolates had 6 biochemical properties of B. subtilis were identified by the kit API CH50B. The results of biochemical checking showed in Table 4.2 Table 4.2: Bacteria screening results by biochemical tests Nomrs Number of Results isolates Positive Negative Lecithinase 296 66 230 Catalase 230 230 0 VP 230 169 61 Amylase 169 91 78 Lives at 50ºC 91 49 42 Cellulase 49 29 20 According the selection procedures, the first biochemical reaction was lecithinase. This key norm helped to eliminate Bacillus that had toxins. There were 66 among 296 isolates positive with lecithinase, so these 66 isolates were eliminated and not checked with the remaining norms. The second biochemical reations with catalase were carried out for the 230 remaining isolates. All of them were positive with catalase, it meant that all of them could grow in aerobe conditions. The following step was VP rection, it helped to eliminate the isolates that were negative as B. Subtilis were positive wih VP. As 169 positive among 230 isolates, 61 negative isolates were eliminated. The 10
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 6 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 M CT11 AG07 AG17 AG19 AG60 VL16 ST06 ST08 C 1500 bp 1500 bp M VL05 VL28 VL41 KG09 AG27 AG49 C Figure 4.4. Results of PCR of the strains of B. 1500 bp 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 Number Investigated temperature Strains of strains 50oC 55oC 60oC KG36, AG19, VL05 3 + + + AG07, AG27, AG49, AG60, VL16, VL28, VL41, ST08, 12 + + - DT30, KG09, KG12, KG22 CT 11, AG17, ST06, ST10, 6 + - - DT29, KG29 Total 21 21 15 3 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 do 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. Figure 4.7. Results of antibiogram of B. subtilis AG27 VL28 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 2 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 1 + + - Control 2 + + - 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 3 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 4 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 Anti-bacterial distance (mm) E. coli S. enterica Staphylococcus Streptococcus ĐC1 5.47c 6.27c 7.00bc - ĐC2 6.10c - 7.00bc 6.23c AG27 8.00b 12.00a 10.00a 10.00b AG60 6.00c 6.00 c 7.00 bc 4.00d VL05 6.00c 10.00 b 8.00 b 6.00c VL28 10.00a 13.00 a 10.00 a 12.00a VL41 6.00c 10.00 b 10.00 a - KG12 - 9.00b 10.00a 4.00d 14
KG22 4.00d 6.00c 6.00c - KG09 6.00c 10.00b - - KG36 - 9.00b 6.00c - DT29 - - 8.00b - SEM 0.184 0.275 0.281 0.167 P 0.00 0.00 0,00 0.00 a,b,c,d the values with different superscripts in the same row were statistically different (P < 0.01) From the results of cross streak method, 4 strains of B. subtilis namely AG27, AG60, VL05 and VL28 revealed anti-bacterial activity against all of 4 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 4 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 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 a,b,c,d the values with different superscripts in the same row were statistically different (P < 0.01) 15
The results revealed that all of 4 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 4 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 Resistant Diameter (mm) Concentration Strain S. enterica S. enterica S. enterica (CFU/mL) (105 CFU/mL) (106 CFU/mL) (107 CFU/mL) AG27 20.23a 20.00a 15.50a 5 AG60 19.33a 16.00b 12.30b 10 VL05 19.17a 15.00b 11.03b VL28 20.17a 19.00a 16.83a SEM 0.331 0.255 0.302 P 0.109 0.00 0.00 AG27 22.83a 21.00a 16.50b AG60 20.50a 17.00b 13.90c 106 VL05 21.17a 18.00b 12.93c VL28 22.2a 20.00a 18.50a SEM 0.676 0.303 0.352 P 0.143 0.00 0.000 AG27 25.33a 25.00a 17.67b AG60 21.57b 21.00b 15.50c 107 VL05 23.17ab 20.00b 14.33c VL28 25.83a 24.00a 20.17a SEM 0.702 0.338 0.323 P 0.009 0.00 0.00 a,b,c,d d the values with different superscript in the same row were statistically different (P < 0.01) Table 4.11 showed all of 4 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 2 in simulated gastric acid and bile salts. The followings were the results of investigation at pH 2 16
Table 4.16: Investigation of acid and bile salts at pH 2 Amount of bacteria and % survival at different time Strains 0 mn 10 mns 30 mns 60 mns 90 mns log log log log log % % % % % CFU/mL CFU/mL CFU/mL CFU/mL CFU/mL d d d d d ĐC1 0.00 - 0.00 - 0.00 - 0.00 - 0.00 - ĐC2 4.76c 72 4.69c 70 4.58c 69 4.24c 64 3.67c 55 AG27 5.43b 82 5.39b 81 5.31b 80 5.21b 78 5.19b 78 AG60 0.00d - 0.00d - 0.00d - 0.00d - 0.00d - VL05 0.00d - 0.00d - 0.00d - 0.00d - 0.00d - VL 28 6.62a 99 6.58a 99 6.54a 98 6.48a 97 6.46a 97 SEM 0.027 0.017 0.035 0.062 0.037 P 0.00 0.00 0.00 0.00 0.00 a,b,c,d 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 1 could not definitely be tolerant at pH 2, at the time of 0 mn, concentration of AG60, VL05 and control1 reduced towards 0. 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 0 mn and 97% after 90 mn), AG27 maintained 82% at 0 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 5 hours kept at room temperature, adherence capacities were nearly double compared to those at 1 hour (Table 4.17). Table 4.17: Investigation of auto-adherence Strain Adherence capacities at different time (%) bacteria 1 hour 2 hours 3 hours 4 hours 5 hours C1 43.9a 62.5a 66.4 72.2 73.8ab C2 30.2ab 57.8ab 65.0 66.1 76.7ab AG27 24.3b 62.1a 63.3 69.8 70.2b VL28 36.8ab 40.9b 61.9 74.5 82.0a SEM 3.51 4.02 2.11 2.13 2.13 P 0.021 0.016 0.508 0.106 0,024 a,b the values with different superscript in the same row were statistically different (P < 0.01) 17