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).
Trang 1MINISTRY 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
Trang 2THE 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
Trang 3LIST 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
Trang 4Chapter 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
Trang 5- 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
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
Trang 6Fecal 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
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
Trang 7Bacteria 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
Trang 8bacteria, then were kept at 37C 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
Trang 9regularly 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
Trang 103.3.1 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
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)
Trang 11Gaining 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
(-)
Infectious chicken , days old
S enterica (1), CFU/mL
18 7.5x104
18 7.5x104
18 7.5x104
18 7.5x104
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
Trang 12- 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 (-)
Infectious chicken, days old
E coli (1), CFU/mL
18 5x106
18 5x106
18 5x106
18 5x106
(1) E coli isolated from sick chicken. (3), (4) Pattison, 2008
(2) Useful dosage of B subtilis chosen in experiment1 (10 7 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
Trang 13Chapter 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
isolates
Results Positive Negative
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
Trang 14fourth 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
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
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)