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) 5562
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 143147
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
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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 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
T1
1
107
5
7

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
1
106
5x105
5
5
7
7
60
60

Ctr
1
7
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

Experiment chicken, days old
Infectious chicken , days old
S. enterica (1), CFU/mL
B. subtilis suppl (2), g/kg feed
Oxtetracyclin, mg/kg feed (3)
Enrofloxacin, mg/kg eedd (4)
Number of experiment days

Treatment
T3

T1

T2

1
18
7.5x104
5
60

1

18
7.5x104
50
60

1
18
7.5x104
15
60

C (+)
1
18
7.5x104
60

Ctr
(-)
1
60

(1)

S. enterica isolated from sick chicken.
Useful dosage of B. subtilis chosen in experiment1 (107 CFU/g)
(3), (4)
Pattison, 2008
(2)

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
Experiment chicken, days old

Infectious chicken, days old
E. coli (1), CFU/mL
B. subtilis supplement (2), g/kg feed
Oxytetracyclin, mg/kg feed (3)
Enrofloxacin, mg/kg feed (4)
Number of days with supplement
Number of experiment day

T1
1
18
5x106
5
60
60

T2
1
18
5x106
50
5
60

Treatment
T3
C (+)
1
1
18

18
5x106 5x106
15
5
60

60

C (-)
1
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

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
3

Investigated temperature
50oC

55oC
60oC
+
+
+

12

+

+

-

6

+

-

-

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.

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 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

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, 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

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 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
Concentration
(CFU/mL)

10

5

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 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
Strains
ĐC1
ĐC2
AG27
AG60
VL05
VL 28
SEM
P
a,b,c,d

0 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 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
bacteria
C1
C2
AG27
VL28
SEM
P
a,b

Adherence capacities at different time (%)
1 hour
2 hours
3 hours
4 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

5 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 4 strains AG27, VL28, and 2
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 2 strain control.
Table 4.18. Investigation of adherence capacity to E. coli and S. enterica

Time

0h

5 hrs

a,b,c,d

Strains
ĐC 1
ĐC 2
AG 27
VL28
SEM
P
C1
CC 2
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 2 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 2 strains VL28 (left) and AG27(right)
in epithelial intestine (400X and 1000X)

18


The results showed that 2 strains B. subtilis had adherence capacity to
epithelial cells of intestines. After verifying 3 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, 1 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 7 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 3
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 1 and 4 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 8
weeks experiment were presented in Table 4.20.
Table 4.20: Growth in weight, food consumption and FCR in chicken during 8
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 1 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 1 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 1 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 8
weeks in experiment 2
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 1 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 3 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, 3 were by far lower than that of
control (+). Food consumption and FCR in the 3 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 3 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 1 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 2 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