evaluación y selección de esporas de bacillus subtilis en...

Guillermo Tellez M.V.Z., MC., Ph.D.

University of Arkansas

Department of Poultry Science

[email protected]

Evaluación y selección de esporas de Bacillus

subtilis en la absorción de nutrientes, homeostasis fisiológica/inmunológica y control de

enteropatógenos

mailto:[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 micro-

structural 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/

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 iron-deficiency 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 fat-soluble 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/














• 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

Versus

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.

Enzyme Production

Intestinal Viscosity

Digestibility

Intestinal Microbial Balance

Germination in the GIT

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 spore-forming 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)

Cellulase Protease Lipase Phytase Xylanase

Evaluation of bacteriocin like compounds from 35 Bacillus spp. DFM candidates (mm of inhibition zone Overlay)

E. coli

S. Enteritidis

C. perfringens 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

Ingredients

Corn 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

Total 100.00 100.00 100.00 100.0 100.00

In vitro digestion methodology simulating the GIT of poultry

Crop storage simulation

Proventricular digestion

Intestinal digestion

40oC – 19 RPM 5 g – diet + 10 ml 0.03

M HCL pH measured (5.2) Tubes incubated for 30

min

30000 U – Pepsin and 2.5 ml of 1.5 M HCL

pH range (1.4 – 1.9) Incubation for 45 min

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



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)

Different high NSP poultry diets

Digesta viscosity

In vitro digestion methodology

http://www2.ca.uky.edu/

Clostridium perfringens proliferation

REDUCTION

• Crop storage simulation

• Proventricular

digestion • Intestinal

digestion

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

Ingredients

Corn 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

Total 100.00 100.00 100.00 100.0 100.00

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

*PBS only *TSB only *TSB + supernatants

*With the addition of thioglycolate ( 0.5 mg/mL)

4 h @ 40oC

200 RPM

24 h @ 40oC

24 h @ 40oC

4 h @ 40oC

200 RPM

Evaluation of in vitro viscosity of different diets with or without inclusion of DFM-Sporulin®

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.

a

Clostridium perfringens Log10 CFU

Corn-soybean base diet

0

1

2

3

4

5

6

7

8

PBS TSB+Thio TSB + diet TSB + DFM

b

a a a

Log10

Inoculum used 4 x 105 CFU of C. perfringens and 1 x 109 CFU of DFM N=5 replicates per treatment; P < 0.05 †

0

1

2

3

4

5

6

7


c

b b

a

Log10


Wheat-soybean base diet


0

1

2

3

4

5

6

7

8


Log10


Barley-soybean base diet

c b

d

a


0

1

2

3

4

5

6

7

8


Log10


Rye-soybean base diet

c

b b

a


0

1

2

3

4

5

6

7

8



Oats-soybean base diet

c

b b a

Log10


NON STARCH POLYSACCHARIDES (NSP)

• Cellulase

• Xylanase

• Protease

• Lipase

• Phytase

Viscosity was significantly reduced in high NSPs diets treated with the DFM in comparison to control diets

“Bacteriocins like” compounds

C. Perfringens proliferation was significantly reduced in high NSPs diets treated with the DFM in comparison to control diets

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

based diet

Grower Rye

based diet

Ingredients (%)

Rye 58.24 61.81

Soybean meal 31.16 26.93

Poultry fat 6.29 7.00

Dicalcium phosphate 1.79 1.66

Calcium carbonate 1.05 1.04

Salt 0.38 0.57

DL-Methionine 0.35 0.30

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

Calculated

analyses

Starter Rye

based diet

Grower Rye

based diet

ME (kcal/kg) 2850 2909

Crude protein (%) 22.38 20.79

Lysine (%) 1.32 1.19

Methionine (%) 0.64 0.58

Calcium (%) 0.90 0.86

Phosphorus (%) 0.45 0.42

Sodium (%) 0.16 0.23

Potassium (%) 0.88 0.82

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

Obtained supernatants and placed on ice until analysis

Viscosity samples were measured at least by duplicate at 40oC using a cone/plate viscometer

Centrifuged at 11000 x g for 5 minutes

Intestinal contents from duodenum to cloaca (Exp 1 and 2)

Intestinal contents from duodenum to Meckel’s diverticulum (Exp 3)

Bone parameters

www.instron.com.es

www.ibmi.in

Right Tibia

Breaking Strength

Left tibia

Bone Composition

% Ash, %Ca and %P Instron 4502

Tensile strength test system

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

a-bSuperscripts within columns indicate significant difference at p <0.05

Parameters Control Bacillus-DFM

Starter (0-7d)

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

Grower (7-28d)

BW (g) 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

Overall (0-28d)

BW (g) 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

106 spores/g of feed – Bacillus-DFM 8 replicates of 20 chickens per treatment (n=160 group)

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

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

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.

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

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

Starter (0-7d)

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

Grower (7-28d)

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

Overall (0-28d)

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

106 spores/g of feed – Commercial –DFM or Bacillus-DFM 8 replicates of 20 chickens per treatment (n=160 group)

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

Starter (0-7d)

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

Grower (7-28d)

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

Overall (0-28d)

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

106 spores/g of feed – Commercial –DFM or Bacillus-DFM 8 replicates of 20 chickens per treatment (n=160 group)

B. subtilis protect intestinal epithelial cells against injury and maintain intestinal homoeostasis

Moore et al., 2014

• Incrase IL-10

• B. subtilis quorum-sensing 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 IL-10) 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!

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