Evaluación y selección de esporas de Bacillus
subtilis en la absorción de nutrientes, homeostasis
fisiológica/inmunológica y control de
enteropatógenos
Guillermo Tellez M.V.Z., MC., Ph.D.
University of Arkansas
Department of Poultry Science
[email protected]
Gut health research has its origin in human health
programs
• Where nutritional interventions,
such as probiotics and prebiotics,
are used to ameliorate conditions
such as inflammatory bowel
• Today, gut health is a major topic
for research not only in humans
but also in animals.
• It is now generally conceded that
maintenance or enhancement of
‘gut health’ is far more complex
than just the modulation of the
gut microflora through probiotics
or prebiotics.
This is not surprising considering that the gut
• Harbors more than 800
different species of bacteria
• Produces over 20 different
hormones
• Digests and absorbs the vast
majority of nutrients
• And accounts for 20% of body
energy expenditure.
• It is also the largest immune
organ in the body
Anything that affects the health of the gut will
undoubtedly influence the animal as a whole
Consequently, ‘gut health’ is
highly complex and encompasses:
• The macro and microstructural integrity of the gut
• The balance of the microflora
• And the status of the immune
system.
Further complexity arises from their interactions and
the resulting changes
• In gene expression, and
possibly, endocrine
regulation.
• This, in turn, may affect
the way nutrients are
partitioned and utilized
for organ development,
tissue growth and
immune system
maturation
Today, a newly hatched chick increases its body weight by
25% overnight and 5000% by 5 weeks, to 2kg.
This astonishing performance of
the modern chicken comes from:
(a) Intensive selection for
growth rate;
(b) Meticulous attention to
health and husbandry;
(c)
And advances in feed
formulation, matching the
nutrient contents of the feed
with the nutrient
requirements of the bird.
As the growth period is progressively shortened and
feed efficiency continuously improved
• The health care and
nutrition of the bird are
becoming more demanding.
• This makes it more
important to pay attention
to the minute changes that
occur in the gut, which are
often overlooked because
the damage is subtle and
usually characterized by
microscopic changes in the
mucosal layer.
http://laurenbeatssugar.blogspot.com/
As the Largest and Most Important Barrier
Against the External Environment
Maintains its selective
barrier function through
the formation of
complex protein-protein
networks that
mechanically link
adjacent cells and seal
the intercellular space.
One of the Basic Properties of GALT is
Oral Tolerance
• To harmless components of
microbiota and diet.
• Inappropriate
immunological reactions
against food proteins, such
as gluten, can lead to the
breakdown of oral tolerance
and the development of
intestinal immune
disorders.
• Is a chronic immune-mediated
enteropathy of small intestine
that is triggered by dietary
wheat gluten, or related rye
and barley proteins in
genetically susceptible
individuals.
• The clinical and pathological
spectrum of CD is
heterogeneous and there is no
current rodent model that
reproduces all aspects of
human celiac disease
Patients display intestinal
barrier dysfunction and
altered tight junction
protein expression allowing
abnormal penetration of
gluten-related peptides and
enteric microbes, which
could amplify any
subsequent immune
response
Clinical presentation of CD
can vary from a classical
malabsorption syndrome
to gastrointestinal
manifestations (similar to
irritable bowel syndrome)
or extra intestinal
presentations
(osteoporosis, and irondeficiency anemia).
• As in humans, in
commercial broiler
chickens, the composition
of the diet also greatly
influence digestibility and
gut health
• Specifically, rye-based
diets versus traditional
corn-based diets as a
source of energy.
The Inclusion of Rye in Poultry Diets
Has been fraught with
problems, principally
related to the production
of sticky droppings, mal
absorption syndrome,
elevated feed conversion
and intestinal bacterial
overgrowth
The Endosperm Cell Wall of Rye,
Wheat, or Barley
• Is comprised mainly of highly
branched arabinoxylans which
increase the viscosity of the
digesta.
• Elevated viscosity reduces
digestibility and performance
by interfering with the
movement of particles and
solutes across the intestinal
lumen, interfering with
digestion
• Favoring intestinal bacterial
overgrowth and leaky gut
Feeding Cereals High in NSP
Leads to increased feed
conversion ratios and lower
body-weight gain in chickens:
1. Increased digesta viscosity
2. Thickening of the mucous
layer on the intestinal
mucosa
3. Epithelial cell apoptosis
4. And significant T-cell
infiltration into the
mucosa (Dysbacteriosis)
NSP Increassig Digesta Viscosity Rresponsible
for Poor Digestibility
• Interfering with the movement
of particles and solutes across
the intestinal lumen
• Preventing the access of
digestive enzymes
• Reducing intestinal absorption
of sodium and calcium
• Reduction of conjugated bile
acid, affecting fat
emulsification and fat
digestibility
In Addition High NSP Diets
Have also been associated
with NE, a multi-factorial
disease caused by C.
perfringens that is
probably the most
important bacterial
disease in terms of
economic implications in
broiler chickens
Increase Intestinal Viscosity
In our two experiments,
the viscosity of the gut in
chickens fed with rye was
so extreme, the
supernatant being more
semi-solid than fluid, that
it alone could be directly
responsible for the poor
performance
Bone Mineralization
The significant reduction in
bone mineralization also
confirmed previous studies
that have shown that rye
utilization in poultry or
gluten intolerance in
humans, are also associated
with mal absorption of
minerals, lipids and fatsoluble vitamins and with
deterioration of bone
mineralization
Bacterial Translocation
The altered intestinal
permeability due to
changes in tight junction
proteins may be the
pathological mechanism
underlying BT of gut flora
from the intestinal lumen
to the liver which is a
precursor to systemic
bacterial infections
http://scdlifestyle.com/2010/03/the-scd-diet-and-leaky-gut-syndrome/
Versus
• In the present study, broilers
fed with a rye-based diet
showed an increase in
viscosity, elevated BT, and
increased serum FITC-d.
• These changes were
associated with significant
bacterial overgrowth when
compared with chickens that
received a corn based diet
Similarly FITC-d
• Is a large molecule (3-5 kDa)
which does not usually leak
through the intact GIT
• However, when
conditions disrupt the tight
junctions between
epithelial cells, the FITC-d
molecule can
enter circulation as
demonstrated by an
increase in trans-mucosal
permeability
Enzimas Comerciales
Since poultry has little or
no intrinsic enzymes
capable of hydrolyzing
NSP, exogenous xylanases
as additives are used in an
attempt to reduce this
anti-nutritive factors
Alternative grains:
Ingredient
NSP
g/kg DM
Corn
97
Wheat
119
Rye
152
Barley
186
Oats
232
Knudsen (1997)
Bedford (2002)
www.thepoultrysite.com
Bacillus spp.
 Germination
in the GIT
Enzyme Production
Intestinal Viscosity
Digestibility
Intestinal Microbial Balance
All of them?
Bacterial spores are dormant life forms which can exist in a desiccated
and dehydrated state indefinitely.
• Russell H. et al. report the
isolation and growth of a
previously unrecognized sporeforming bacterium (Bacillus sp.)
from a 250 million-year-old salt
crystal.
• Complete gene sequences of the
16S ribosomal DNA show that
the organism is part of the
lineage of B. marismortui
NATURE, Vol. 407, 19 OCTOBER 2000
Producción de Esporas en Biorreactores
•
•
•
•
105 espores mL-1 (19),
108 espores mL-1 (20),
109 espores mL-1 (21, 22)
El más álto reporte es
7.4 109 espores mL-1.
Biotechnol. Prog. 2005, 21, 1026-1031
Mayores logros después de 8 años de
investigación
• La producción de
esporeas mediante la
utilización de un
método de
fermentación sólido
• 1011 esporas/gramo
Previously: relative enzyme activity
Latorre et al. 2014
31 candidates
(preselected from
>1,000,000 screened)
TSB overnight (37oC)
3 Candidates were then washed 3
times (2000 g for 15 min)
Lipase
Cellulase
Protease
Phytase
Xylanase
Evaluation of bacteriocin like compounds from 35 Bacillus spp.
DFM candidates (mm of inhibition zone Overlay)
E. coli
C. perfringens
S. Enteritidis
C. difficile
Ingredients (%) of the diets used for in vitro digestion with or without
inclusion of DFM-Sporulin®
Item
Corn-based diet
Wheat-based diet
Barley-based diet
Rye-based diet
Oat-based diet
55.80
0.0
0.0
0.0
0.0
Wheat
0.0
65.69
0.0
0.0
0.0
Barley
0.0
0.0
62
0.0
0.0
Rye
0.0
0.0
0.0
61.3
0.0
Oats
0.0
0.0
0.0
0.0
61.3
Soybean meal
36.94
26.55
29.30
30.0
30.0
Vegetable oil
3.32
3.72
5.0
5.0
5.0
Dicalcium phosphate
1.7
1.86
1.7
1.7
1.7
Calcium carbonate
1.0
0.91
0.8
0.8
0.8
Salt
0.30
0.30
0.30
0.30
0.30
DL-Methionine
0.25
0.26
0.26
0.26
0.26
Vitamin premix
0.3
0.3
0.3
0.3
0.3
L-Lysine HCl
0.10
0.16
0.16
0.16
0.16
Choline chloride 60 %
0.10
0.10
0.10
0.10
0.10
Mineral premix
0.25
0.25
0.25
0.25
0.25
100.00
100.00
100.00
100.0
100.00
Ingredients
Corn
Total
In vitro digestion methodology simulating the
GIT of poultry
Crop storage
simulation
 40oC – 19 RPM
 5 g – diet + 10 ml 0.03
M HCL
 pH measured (5.2)
 Tubes incubated for 30
min
Vegetative and
spore count
Proventricular
digestion
 30000 U – Pepsin and
2.5 ml of 1.5 M HCL
 pH range (1.4 – 1.9)
 Incubation for 45 min
Vegetative and
spore count
 68.3 mg of 8 x
Pancreatin in 3.25 ml of
1.0 M NaHCO3
 pH range (6.3 – 6.7)
 Incubation for 2 h
Vegetative and
spore count
Intestinal
digestion
Previously: In vitro digestion methodology simulating
the GIT of poultry using different poultry diets
with/without inclusion of Bacillus-DFM candidate
Latorre et al. 2014; Tellez et al. 2014
Bacillus-DFM
candidate
(3 isolates)
In vitro digestion methodology
Different high NSP poultry diets
REDUCTION
Digesta
viscosity
Clostridium
perfringens
proliferation
•
Crop storage
simulation
•
Proventricular
digestion
•
Intestinal
digestion
http://www2.ca.uky.edu/
Ingredients (%) of the diets used for in vitro digestion with or without
inclusion of DFM-Sporulin®
Item
Corn-based diet
Wheat-based diet
Barley-based diet
Rye-based diet
Oat-based diet
55.80
0.0
0.0
0.0
0.0
Wheat
0.0
65.69
0.0
0.0
0.0
Barley
0.0
0.0
62
0.0
0.0
Rye
0.0
0.0
0.0
61.3
0.0
Oats
0.0
0.0
0.0
0.0
61.3
Soybean meal
36.94
26.55
29.30
30.0
30.0
Vegetable oil
3.32
3.72
5.0
5.0
5.0
Dicalcium phosphate
1.7
1.86
1.7
1.7
1.7
Calcium carbonate
1.0
0.91
0.8
0.8
0.8
Salt
0.30
0.30
0.30
0.30
0.30
DL-Methionine
0.25
0.26
0.26
0.26
0.26
Vitamin premix
0.3
0.3
0.3
0.3
0.3
L-Lysine HCl
0.10
0.16
0.16
0.16
0.16
Choline chloride 60 %
0.10
0.10
0.10
0.10
0.10
Mineral premix
0.25
0.25
0.25
0.25
0.25
100.00
100.00
100.00
100.0
100.00
Ingredients
Corn
Total
In vitro digestion using 5 different poultry diets
with/without inclusion of DFM-Sporulin®
Total 3:15 h
Centrifuged for 30 min @ 2000 g
Obtained
Supernatants
Supernatants
Supernatants from each diet with and with out DFM (1:2)
and individually spiked with C. perfringens
to an initial concentration of 105 cfu/mL
4h
@ 40oC
200 RPM
4h
@ 40oC
200 RPM
*PBS only
*TSB only
24 h
@ 40oC
*TSB + supernatants
24 h
@ 40oC
*With the addition of thioglycolate ( 0.5 mg/mL)
Evaluation of in vitro viscosity of different diets with
or without inclusion of DFM-Sporulin®
a
a-b (P<0.05).
*
Viscosity was measured after 3 h and 15 min of in vitro digestion at 40oC, the data reported is
the mean of 5 replicates per diet per treatment.
Clostridium perfringens Log10 CFU
Corn-soybean base diet
Log10
8
a
a
a
TSB+Thio
TSB + diet
TSB + DFM
7
6
5
b
4
3
2
1
0
PBS
Inoculum used 4 x 105 CFU of C. perfringens and 1 x 109 CFU of DFM
N=5 replicates per treatment; P < 0.05
Clostridium perfringens Log10 CFU
Wheat-soybean base diet
Log10
7
a
6
b
b
5
c
4
3
2
1
0
PBS
TSB+Thio
TSB + diet
TSB + DFM
Inoculum used 4 x 105 CFU of C. perfringens and 1 x 109 CFU of DFM
N=5 replicates per treatment; P < 0.05
Clostridium perfringens Log10 CFU
Barley-soybean base diet
Log10
8
a
7
b
c
6
5
4
d
3
2
1
0
PBS
TSB+Thio
TSB + diet
TSB + DFM
Inoculum used 4 x 105 CFU of C. perfringens and 1 x 109 CFU of DFM
N=5 replicates per treatment; P < 0.05
Clostridium perfringens Log10 CFU
Rye-soybean base diet
Log10
8
a
7
6
5
b
b
c
4
3
2
1
0
PBS
TSB+Thio
TSB + diet
TSB + DFM
Inoculum used 4 x 105 CFU of C. perfringens and 1 x 109 CFU of DFM
N=5 replicates per treatment; P < 0.05
Clostridium perfringens Log10 CFU
Oats-soybean base diet
Log10
8
7
b
6
5
a
b
c
4
3
2
1
0
PBS
TSB+Thio
TSB + diet
TSB + DFM
Inoculum used 4 x 105 CFU of C. perfringens and 1 x 109 CFU of DFM
N=5 replicates per treatment; P < 0.05
Viscosity was significantly reduced in high NSPs
diets treated with the DFM in comparison to control diets
• Cellulase
• Xylanase
• Protease
• Lipase
• Phytase
NON STARCH POLYSACCHARIDES (NSP)
C. Perfringens proliferation was significantly reduced in high NSPs
diets treated with the DFM in comparison to control diets
“Bacteriocins like” compounds
Ingredients (%) of the diets used for in vivo studies with or without inclusion
of candidate Bacillus-DFM candidate for experiment 1, 2 and 3
Item
Starter Rye
Grower Rye
Calculated
Starter Rye
Grower Rye
based diet
based diet
analyses
based diet
based diet
ME (kcal/kg)
2850
2909
Ingredients (%)
Rye
58.24
61.81
Crude protein (%)
22.38
20.79
Soybean meal
31.16
26.93
Lysine (%)
1.32
1.19
Poultry fat
6.29
7.00
Methionine (%)
0.64
0.58
Dicalcium phosphate
1.79
1.66
Calcium (%)
0.90
0.86
Calcium carbonate
1.05
1.04
Phosphorus (%)
0.45
0.42
Salt
0.38
0.57
Sodium (%)
0.16
0.23
DL-Methionine
0.35
0.30
Potassium (%)
0.88
0.82
Vitamin premix
0.20
0.20
L-Lysine HCl
0.22
0.20
Choline chloride 60 %
0.10
0.10
Mineral premix
0.10
0.10
Threonine
0.08
0.06
Selenium
0.02
0.02
Ethoxyquin
0.02
0.02
Total
100
100
http://msue.anr.msu.edu/
Bacterial translocation
Right half of liver
Weighed and
Homogenized
1:4 wt/vol dilutions Saline
Tenfold serial dilutions
– sterile 96 well plate
MacConkey agar plate
Viscosity measurements
 Intestinal contents from duodenum to cloaca (Exp 1 and 2)
 Intestinal contents from duodenum to Meckel’s
diverticulum (Exp 3)
 Centrifuged at 11000 x g for 5 minutes
 Obtained supernatants and
placed on ice until analysis
 Viscosity samples were measured at least by duplicate at
40oC using a cone/plate viscometer
Bone parameters
Right Tibia
Left tibia
Breaking
Strength
Bone
Composition
Instron 4502
Tensile strength test system
% Ash, %Ca and %P
www.instron.com.es
www.ibmi.in
Evaluation of in vitro viscosity of different diets with
or without inclusion of Bacillus-DFM candidates
a-b (P<0.05).
*
Viscosity was measured after 3 h and 15 min of in vitro digestion at 40oC, the data reported is
the mean of 5 replicates per diet per treatment.
Evaluation of body weight, feed intake and feed conversion ration in broiler
chickens consuming a Rye-soybean diet with or without dietary inclusion of
Bacillus-DFM candidates
Parameters
Control
Bacillus-DFM
BW (g)
95.90 ± 3.21a
101.93 ± 3.12a
FI (g)
118.05 ± 2.78a
116.33 ± 2.29a
FCR
1.23 ± 0.03a
1.15 ± 0.02a
708.25 ± 6.22b
728.20 ± 6.18a
FI (g)
1701.62 ± 17.13a
1694.10 ± 16.77a
FCR
2.40 ± 0.02a
2.33 ± 0.02b
804.13 ± 7.30b
830.13 ± 6.43a
FI (g)
1820.02 ± 17.83a
1810.44 ± 17.03a
FCR
2.26 ± 0.03a
2.18 ± 0.02b
Starter (0-7d)
106 spores/g of feed –
Bacillus-DFM
Grower (7-28d)
BW (g)
8 replicates of 20
chickens per treatment
(n=160 group)
Overall (0-28d)
BW (g)
a-bSuperscripts within
columns indicate significant difference at p <0.05
Evaluation of intestinal viscosity, bacterial translocation and bone parameters
in 28d-old broiler chickens fed with a Rye-based diet with/without a selected
Bacillus-DFM candidates.
Diet
Intestinal viscosity
( cP)
Bacterial translocation
(Log10 CFU)
Rye-Soybean
96.16 ± 2.95a
1.35 ± 0.45a
Rye-Soybean + DFM
61.52 ± 2.34b
0.27 ± 0.27b
a-bSuperscripts within
columns indicate significant difference at p <0.05
Intestinal viscosity is expressed in centipoise from 8 chickens / group
Liver bacterial translocation from 8 chickens/group
Breaking
strength
(kg/mm2)
Tibia
diameter
(mm)
Total ash
(%)
Ca
(%)
P
(%)
Rye-Soybean
22.15 ± 0.93b
5.47 ± 0.08a
44.87 ± 0.95b
18.48 ± 0.27b
9.15 ± 0.12b
Rye-Soybean
+ DFM
26.51 ± 1.68a
5.58 ± 0.20a
55.01 ± 0.61a
29.48 ± 0.27a
15.15 ± 0.13a
Diet
Tibias from 8 chickens / group were collected to evaluate bone parameters. Data is expressed as Mean ± SE.
a-b Superscripts within columns indicate significant difference at p<0.05.
Evaluation of body weight, feed intake and feed conversion ration in broiler
chickens consuming a Barley/Corn(15%)/Soybean diet with or without dietary
inclusion of Bacillus-DFM candidates
Parameters
Control
Commercial-DFM
Bacillus-DFM
BW (g)
101.6 ± 3.65a
98.0 ± 2.22a
103.8 ± 3.26a
FI (g)
135.5 ± 6.70a
131.5 ± 5.16a
131.4 ± 4.48a
FCR
1.33 ± 0.054a
1.34 ± 0.051a
1.27 ± 0.052a
BW (g)
1235.1 ± 13.01b
1242.7 ± 16.72b
1291.6 ± 14.43a
FI (g)
2092.9 ± 20.82a
2015.8 ± 34.79a
2046.1 ± 23.50a
FCR
1.69 ± 0.011a
1.62 ± 0.016b
1.58 ± 0.015b
BW (g)
1336.7 ± 13.53b
1340.7 ± 17.60b
1395.4 ± 17.02a
FI (g)
2228.4 ± 23.07a
2177.5 ± 34.82a
2147.3 ± 23.62a
FCR
1.66 ± 0.011a
1.59 ± 0.013b
1.55 ± 0.017c
Starter (0-7d)
106 spores/g of feed –
Commercial –DFM or
Bacillus-DFM
8 replicates of 20
chickens per treatment
(n=160 group)
Grower (7-28d)
Overall (0-28d)
a-bSuperscripts within columns indicate significant difference
at p <0.05
Evaluation of body weight, feed intake and feed conversion ration in broiler
chickens consuming a Corn/DDGS (8%)/Soybean diet with or without dietary
inclusion of Bacillus-DFM candidates
Parameters
Control
Commercial-DFM
Bacillus-DFM
BW (g)
150.6 ± 3.21a
149.3 ± 1.18a
148.8 ± 1.15a
FI (g)
177.0 ± 6.39a
164.3 ± 5.40a
175.1 ± 7.31a
FCR
1.17 ± 0.02a
1.10 ± 0.03a
1.18 ± 0.04a
BW (g)
1286.4 ± 13.38b
1297.0 ± 13.71ab
1335.3 ± 14.09a
FI (g)
2081.8 ± 19.75a
2064.1 ± 23.35a
2052.3 ± 20.84a
FCR
1.62 ± 0.01a
1.59 ± 0.02a
1.53 ± 0.01b
BW (g)
1437.0 ± 14.48b
1446.3 ± 13.54ab
1484.0 ± 14.51a
FI (g)
2212.6 ± 19.92a
2193.4 ± 23.01a
2182.6 ± 19.58a
FCR
1.54 ± 0.01a
1.52 ± 0.01a
1.47 ± 0.01b
Starter (0-7d)
106 spores/g of feed –
Commercial –DFM or
Bacillus-DFM
8 replicates of 20
chickens per treatment
(n=160 group)
Grower (7-28d)
Overall (0-28d)
a-bSuperscripts within
columns indicate significant difference at p <0.05
B. subtilis protect intestinal epithelial cells against
injury and maintain intestinal homoeostasis
Moore et al., 2014
• Incrase IL-10
• B. subtilis quorumsensing molecule induces
heat shock proteins,
which protect intestinal
epithelial cells against
injury and loss of barrier
function
• Reduce bacterial
translocation
Bacillus coagulans anti-inflammatory effects
(Jensen et al. 2010)
• Inhibition of reactive oxygen
species (ROS), reduced
polymorphonuclear (PMN)
cell
• Chemotactic migration in
response to IL-8 and
leukotriene B4
• Production of the Th2
cytokines (IL-4, IL-6 and IL10) and the inhibition of IL-2
Together, they Represent a Step Towards the Application of
Nutrigenomics in the Context of a Chicken Model.
The incorporation of one or
more 'omics' techniques (in
particular, assessment of the
microbiome) will provide a
better understanding of how
dietary food components
can affect physiological
functions and the
fundamental cellular and
molecular mechanisms
implicated in the digestive
process of high NSP diets in
chickens.
Screening of Bacillus spp DFM Candidates
Significantly reduced both
Viscosity, BT, C. perfringens
Proliferation and
Improve Performance in
Corn or High NSP diets
M54 Isolation,screening and identification of Bacillus spp.as direct fed
microbial (DFM) candidates for aflatoxin B1 biodegradation
Galarza-Seeber R., J. Latorre, B. Hargis and G. Tellez
Bacillus Isolates
16 S Identification
Candidate 1
B. amyloliquefaciens
Candidate 2
B. megaterium
Candidate 3
B. subtilis
68
Muchas Gracias!
[email protected]