dr. tom rehberger - early colonizing bacteria in the gi tract - lessons from poultry and strategies...
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Early colonizing bacteria in the GI tract: lessons from poultry and strategies for swine
Agro BioSciences, Inc
Role of GI Colonizing Bacteria Immune development and immune function
• Early colonizing bacteria play a role in immune development Critical in-• acquired immune system (B and T cells)• innate immune system (mucin chemistry)
• Lactic acid bacteria on the epithelial surface help to develop a competent immune system
• process surface antigens• stimulates migration of immune cells to GI tissue• bind toll-like receptors- quiescence which helps to develop an appropriate
immune response reduce inflammation
Microbial homeostasis • Microbiota makes up the complex community that acts as barrier to infection
Competitive exclusion
Nutritional processing• Microbiota processes complex feed ingredients- more readily absorbed • Processes phytochemicals (polyphenols)- improves function and bioavailability
Early Colonizing Bacteria Project
Project Objectives
Phase 1• Isolate and characterize the early colonizing bacterial communities in turkey poults
and broiler chicks • Identify the core microbiome and characterize key isolates
Phase 2• Determine functional role of core microbiome isolates• Determine if they have potential in preventing disease and improving performance
CLINICAL
SUBCLINICAL
A Bird’s Life – Early Stage
0
Broilers and Turkeys
Brood
Hatchery
Immune DevelopmentGastrointestinal Development
Microbiological Challenges• Pathogenic E. coli • Toxigenic Clostridium
Initial gut health
Microbiological ChallengesClostridium – necrotic enteritis
Ammonia
Vertical Transmissionof Pathogens
GI tracts
a
b
c…j
10 birds/flock
Extract
gDNA
Isolate predominantbacterial types (MRS, Chrom agar)
Isolate gDNA- 5 isolates/bird
Perform TRFLP
Perform RAPDs
Early Colonizing Bacteria- poultry
Createdendrograms
Product Development
16s DNA seq
Identify predominant genotypesServe as a baseline for perturbations
Hen GI Microbiology
Breeder flock# and bird
• All birds had detectable (>100 cfu/g) lactic acid bacterial populations• 16 of the 18 birds had LAB populations > 1 x 106 cfu/g
LAB Plate Counts- Complex 1
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Flock# 2 Flock# 36 Flock# 72 Flock# 80 Flock# 84 Flock# 94
Day-of Hatch GI Microbiology
Breeder flock# and bird
• All birds had detectable (>100 cfu/g) lactic acid bacterial populations• 35 out of 60 birds had LAB populations > 1 x 106 cfu/g
LAB Plate Counts- Complex 1
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9
Flock #2 Flock #36 Flock #72 Flock #80 Flock #84 Flock #94
Hen GI Microbiology
Breeder flock and bird
• All birds had detectable lactic acid bacterial populations >100 cfu/g• All birds had LAB populations > 1 x 106 cfu/g
LAB Plate Counts- Complex 2
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Little Capon Farm- 25wks
Jeanetta Kesner- 28wks
Mary Barb- 35 wks Victor Carr- 39 wks Eleanor Keplinger- 59wks
Brent Hartzler- 61 wks
Samples not plated
Day-of Hatch GI Microbiology
Breeder flock and bird
• Only 12 of the 60 (20%) birds had detectable lactic acid bacterial populations >100 cfu/g
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 7 9
Smith Farm -house 1,2 Harterman Mary Barb- 35 wks Victor Carr- 39 wks Eleanor Keplinger- 59wks
Brent Hartzler- 61 wks
LAB Plate Counts- Complex 2
APEC GI Microbiology
• Not all trends match but potential transfer of the APEC populations from hen to day-of hatch
APEC Counts- Complex 1
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Flock# 2 Flock# 36 Flock# 72 Flock# 80 Flock# 84 Flock# 94
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Flock #2 Flock #36 Flock #72 Flock #80 Flock #84 Flock #94
Hens
Day-of Hatch
APEC GI Microbiology
• No apparent transfer of the APEC populations from hen to day-of hatch
APEC Counts- Complex 2Hens
Day-of Hatch
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Little Capon Farm- 25 wks Jeanetta Kesner- 28 wks Mary Barb- 35 wks Victor Carr- 39 wks Eleanor Keplinger- 59 wks Brent Hartzler- 61 wks
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Smith Farm Harterman Mary Barb- 35 wks Victor Carr- 39 wks Eleanor Keplinger- 59wks
Brent Hartzler- 61wks
GI tracts
a
b
c
10 birds/flock
Extract
gDNA
Isolate predominantbacterial types
Isolate gDNA- 5 isolates/bird
Perform TRFLP
Perform RAPDs
Early Colonizing Bacteria Project
Createdendrograms
Product Development
Direct selection processIdentify predominant genotypes
Serve as a baseline for perturbations
16s DNA seq
bird
Early Colonizing Bacteria- Day-of Hatch and Hens
Isolates within each bird are often identical or closely related- homogeneity within a birdIsolates from different birds within the same flock are often not identical- some variability in a flocSome isolates are shared between birds in different flocks- evidence of a core microbiomeHen isolates appear to be different from day-of hatch isolates
RAPD CE GEL Images of LAB Isolate- Complex 1 Flock
1 2 3 1 2 3 3 1 2 3 1 2 3
Flock
bird
Early Colonizing Bacteria Project
Isolates within each bird are often identical or closely related- homogeneity within a birdIsolates from different birds within the same flock are often not identical- variability within a flockSome isolates are shared between birds in different flocks- some evidence of a core microbiome?
RAPD CE GEL Images of MRS Isolates
GI tracts
a
b
c
10 birds/flock
Extract
gDNA
Isolate predominantbacterial types
Isolate gDNA- 10 isolates/bird
Perform TRFLP
Perform RAPDs
Early Colonizing Bacteria Project
Createdendrograms
Product Development
Identify predominant genotypesServe as a baseline for perturbations
16s DNA seq
B
ird
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B
reeder Flock
284722943694236363636367284849429484368080808080Glenis H
ousman
94Alan Koogler
8028480948472948022728472228022723680723636729494842849472848494847236Ron Freed
Ron Freed
7228036Larry Depoy
Alan Koogler
Larry Depoy
Glenis H
ousman
Charles M
artin
Larry Depoy
Glenis H
ousman
Rex Sours
Ron Freed
Charles M
artin
Rex Sours
Charles M
artin
Alan Koogler
Rex Sours
Lactobacillus johnsoniiEnterococci
Lactobacillus salivarius
Lactobacillus crispatus
Lactobacillus casei
Pediococci
TRFLP Analysis of Lactic Acid Bacteria
Complex 1 Day-of hatch birds (blue), hens (yellow)
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B
reeder Flock
Brent H
artzler
Brent H
artzler
Harterm
an
Sm
ith Farm 1&
2
Sm
ith Farm 1&
2
Eleanor K
eplinger
Sm
ith Farm 1&
2
Sm
ith Farm 1&
2
Victor C
arr
Eleanor K
eplinger
Sm
ith Farm 1&
2
Harterm
an
Harterm
an
Eleanor K
eplinger
Sm
ith Farm 1&
2
Harterm
an
Brent H
artzler
Sm
ith Farm 1&
2
Victor C
arr
Brent H
artzler
Harterm
an
Brent H
artzler
Victor C
arr
Sm
ith Farm 1&
2
Mary B
arb
Victor C
arr
Mary B
arb
Sm
ith Farm 1&
2
Victor C
arr
Victor C
arr
Sm
ith Farm 1&
2
Mary B
arb
Sm
ith Farm 1&
2
Harterm
an
Mary B
arb
Harterm
an
Brent H
artzler
Harterm
an
Mary B
arb
Mary B
arb
Mary B
arb
Eleanor K
eplinger
Eleanor K
eplinger
Eleanor K
eplinger
Brent H
artzler
Sm
ith Farm 1&
2
Brent H
artzler
Eleanor K
eplinger
Victor C
arr
Eleanor K
eplinger
Harterm
an
Victor C
arr
Mary B
arb
Brent H
artzler
Harterm
an
Sm
ith Farm 1&
2
Eleanor K
eplinger
Victor C
arr
Victor C
arr
Harterm
an
Brent H
artzler
Victor C
arr
Eleanor K
eplinger
Brent H
artzler
Brent H
artzler
Brent H
artzler
Mary B
arb
Harterm
an
Mary B
arb
Harterm
an
Victor C
arr
Mary B
arb
Mary B
arb
Eleanor K
eplinger
Mary B
arb
Victor C
arr
Mary B
arb
Harterm
an
Eleanor K
eplinger
Sm
ith Farm 1&
2
Victor C
arr
Brent H
artzler
Mary B
arb
Brent H
artzler
Eleanor K
eplinger
Victor C
arr
Mary B
arb
Victor C
arr
Eleanor K
eplinger
Mary B
arb
Brent H
artzler
Lactobacillus johnsoniiEnterococci
Lactobacillus salivarius
Lactobacillus crispatus
Lactobacillus casei
Pediococci
TRFLP Analysis of Lactic Acid Bacteria
Complex 2 Day-of hatch birds (blue), hens (yellow)
Early Colonizing Bacteria Project
Project Objectives
Phase 1• Isolate and characterize the early colonizing bacterial communities in turkey poults
and broiler chicks • Identify the core bacterial communities and isolates
Phase 2• Determine functional role of core community isolates• Determine if they have potential in preventing disease and improving performance
Fold Change in Gene Expression of MIP2, TNF-α, and IL-6 in Response to LPS and Bacterial Strains Relative to Unstimulated
Control Cells – IEC6 Cell Expression
311
1657
168
30
50
100
150
200
250
300
350
ΔCT LPS ΔCT 1E1 ΔCT 1E1+LPS
ΔCT 1037 ΔCT 1037+LPS
Fold Change Gene Expression: MIP2(BA)
728
16 63
230
20
100200300400500600700800
ΔCT LPS ΔCT 1E1 ΔCT 1E1+LPS
ΔCT 1037 ΔCT 1037+LPS
Fold Change Gene Expression: TNFa(BA)
452
1569
186
30
100
200
300
400
500
ΔCT LPS ΔCT 1E1 ΔCT 1E1+LPS
ΔCT 1037 ΔCT 1037+LPS
Fold Change Gene Expression: IL6(BA)
GUT START - Trial 2
Experimental Design
Company: Broiler Company – SE United States, 1.5 M birds/wk
Application: Spray cabinet
Product: GUT START formulation 2 –90% Lactobacillus salivarius, L. plantarum10% Bacillus subtilis (two strains)
Samples: hatchery alternated GUT START and No GUT START (control) every week for 12 weeks
GITsAge Time
Points Treatments Reps
30 x 2 x 2 x 2 = 240 total GITs(10 birds from
3 flocks)(DOH and 7
day)(GUT START and No GUT START)
GUT START Trial 2
Results
0
1
2
3
4
5
DOH 7 d
Log 10
(CFU
/g)
GUT START Control
APEC Levels
a
b
c
ab
Significance by one-way ANOVA (p≤0.05)
GUT START Trial 2
Results7 d - APEC levels by Log
0%10%20%30%40%50%60%70%80%
<2 2-3 3-4 4-5 5-6 6-7 >7Log CFU/g category
GUT START Control
Percent of 7 day old birds whose APEC levels fit into each quantitative category
GUT START Trial 2
Results – Performance Data
Feed Conversion and average weight gain – TBD (sorting data)
7 day mortality -
Weekly coefficient of variability (CV)
GUT START’s calculated value was > 4:1 ROI
GUT START 1.57%Control 1.65%
GUT START 10.28Control 11.28
4.8% decrease
8.8% decrease
Project ObjectivesPhase 1• Understand the diversity of lactic acid bacteria within the gastrointestinal
tract of pigs- GI section, pigs, source (sow flow)-site, company• Identify the key microbial isolates of the core microbiome- correlate to
performance and health
Phase 2• Identify the function of the key microbial isolates of the microbiome• Determine if they have potential in preventing disease and improving
performance
Samples- pars esophageal, jejunum, ileum
Analysis- Microbiology: enumeration of lactic acid bacteria, E. coli, Salmonella Microbial Ecology: TRFLP analysis by section- total bacteria, LAB
Early Colonizing Bacteria Project- Swine
SUBCLINICAL
A Pig’s Life – Early Stage
Immune DevelopmentGastrointestinal Development
Microbiological Challenges• Pathogenic E. coli • Toxigenic Clostridium
CLINICAL
Birth Wean
Microbiological Succession• nutrient changes• stress – social changes
Pre-wean Nursery
GI tractsPars esophagus
JejunumIleum
4-5 Pigs / Sow Flow
Extract
gDNA
Isolate predominantbacterial types (MRS, Chrom agars)
MRS isolates- 10 isolates/section/pigE. coli isolates- 5 isolates/section/pig
Perform TRFLP
Perform RAPDs
Createdendrograms
Product Development
16s DNA seq
TRFLP primers-Total Bacteria (3 enzymes)LAB (3 enzymes)
Identify predominant genotypes
Microbial Terroir Program – Early Colonizing Bacteria
Full Microbial Succession Sampling
16S Taxonomic Identification of MRS derived isolates by Age
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
3 10 20 23 34 48
Day of Age
Enterococcaceae
Bacillaceae
Enterobacteriaceae
Flavobacteriaceae
Streptococcaceae
Lactobacillaceae
16S Taxonomic Identification of MRS derived isolates by Age
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
3 10 20 23 34 48
Day of Age
Enterococcus sp.Pediococcus pentosaceusKlebsiella pnemoniaeStreptococcus pocorumStreptococcus sp.Streptococcus alactolyticusLactobacillus mucosaeBacillus sp.Streptococcus hyointestinalisLactobacillus crispatusEscherichia coliLactobacillus sp.Lactobacillus delbrueckiiLactobacillus salivariusLactobacillus amylovorusLactobacillus vaginalisChryseobacterium spp.Lactobacillus johnsoniiLactobacillus reuteriStreptococcus gallolyticus
GI tractsPars esophagus
JejunumIleum
4-5 Pigs / Sow Flow
Extract
gDNA
Isolate predominantbacterial types (MRS, Chrom agars)
MRS isolates- 10 isolates/section/pigE. coli isolates- 5 isolates/section/pig
Perform TRFLP
Perform RAPDs
Createdendrograms
Product Development
16s DNA seq
TRFLP primers-Total Bacteria (3 enzymes)LAB (3 enzymes)
Identify predominant genotypes
Microbial Terroir Program – Early Colonizing Bacteria
Full Microbial Succession Sampling
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
A B C D E
Site - Total Bacteria
L. delbrueckii Enterobacteriaceae Clostridium 301 93
Bacillus Lactobacillus 193 74 502
557 148 156 173 84
524 Minor Peaks
Microbial Community AnalysisTotal Bacteria: Effect of Site
n=30 pigs
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
A B C D E
Site - LAB
L. delbrueckii Streptococcus L. reuteri B. subtilis group L. johnsonii L. crispatusStreptococcus Lactobacillus 540 145 165 160224 153 524 175 123 420169 587 186 228 Minor Peaks
Microbial Community AnalysisLactic Acid Bacteria: Effect of Site
n=30 pigs
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Day 3 Day 10 Day 20 Day 23 Day 34 Day 48
Age - Total Bacteria
L. delbrueckii Enterobacteriaceae Clostridium 30193 Bacillus Lactobacillus 19374 502 557 148156 173 84 524476 144 Minor Peaks
Microbial Community AnalysisTotal Bacteria: Effect of Age
n= 25 pigs
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Day 3 Day 10 Day 20 Day 23 Day 34 Day 48
Age - LAB
L. delbrueckii Streptococcus L. reuteri B. subtilis group L. johnsoniiL. crispatus Streptococcus Lactobacillus 540 145165 160 224 153 524175 123 420 169 587186 228 Minor Peaks
Microbial Community AnalysisLactic Acid Bacteria: Effect of Age
n= 25 pigs
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
3 10 20 23 34 48
Site C - LAB
L. delbrueckii Streptococcus L. reuteri B. subtilis group L. johnsonii
L. crispatus Streptococcus Lactobacillus 540 145
165 160 224 153 Minor Peaks
NHF Microbial Community AnalysisLactic Acid Bacteria: Site C x Age
n= 5 pigs
Project ObjectivesPhase 1• Understand the diversity of lactic acid bacteria within the gastrointestinal
tract of pigs- GI section, pigs, source (sow flow)-site, companyDiversity observed across sections, pigs and sites- impact of weaning
• Identify the key microbial isolates of the core microbiome- correlate to performance and healthNext steps- correlate microbial isolates to performance using multivariate statistical methods
Phase 2• Identify the function of the key microbial isolates of the microbiome• Determine if they have potential in preventing disease and improving
performance
Early Colonizing Bacteria Project- Swine