Dr. Tom Rehberger - Early Colonizing Bacteria in the GI Tract - Lessons from Poultry and Strategies for Swine
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Early Colonizing Bacteria in the GI Tract - Lessons from Poultry and Strategies for Swine - Dr. Tom Rehberger, President, Agro BioSciences, from the 2016 Allen D. Leman Swine Conference, September 17-20, 2016, St. Paul, Minnesota, USA. More presentations at http://www.swinecast.com/2016-leman-swine-conference-material
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Dr. Tom Rehberger - Early Colonizing Bacteria in the GI Tract - Lessons from Poultry and Strategies for Swine
- 1. + Early colonizing bacteria in the GI tract: lessons from poultry and strategies for swine Agro BioSciences, Inc
- 2. 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
- 3. 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
- 4. CLINICAL SUBCLINICAL A Bird’s Life – Early Stage 0 Broilers and Turkeys Brood Hatchery Immune Development Gastrointestinal Development Microbiological Challenges • Pathogenic E. coli • Toxigenic Clostridium Initial gut health Microbiological Challenges Clostridium – necrotic enteritis Ammonia Vertical Transmission of Pathogens
- 5. GI tracts a b c…j 10 birds/flock Extract gDNA Isolate predominant bacterial types (MRS, Chrom agar) Isolate gDNA- 5 isolates/bird Perform TRFLP Perform RAPDs Early Colonizing Bacteria- poultry Create dendrograms Product Development 16s DNA seq Identify predominant genotypes Serve as a baseline for perturbations
- 6. 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
- 7. 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
- 8. 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- 25 wks Jeanetta Kesner- 28 wks Mary Barb- 35 wks Victor Carr- 39 wks Eleanor Keplinger- 59 wks Brent Hartzler- 61 wks Samples not plated
- 9. 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- 59 wks Brent Hartzler- 61 wks LAB Plate Counts- Complex 2
- 10. 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
- 11. APEC GI Microbiology • No apparent transfer of the APEC populations from hen to day-of hatch APEC Counts- Complex 2 Hens 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- 59 wks Brent Hartzler- 61 wks
- 12. GI tracts a b c 10 birds/flock Extract gDNA Isolate predominant bacterial types Isolate gDNA- 5 isolates/bird Perform TRFLP Perform RAPDs Early Colonizing Bacteria Project Create dendrograms Product Development Direct selection process Identify predominant genotypes Serve as a baseline for perturbations 16s DNA seq
- 13. bird Early Colonizing Bacteria- Day-of Hatch and Hens Isolates within each bird are often identical or closely related- homogeneity within a bird Isolates from different birds within the same flock are often not identical- some variability in a floc Some isolates are shared between birds in different flocks- evidence of a core microbiome Hen isolates appear to be different from day-of hatch isolates RAPD CE GEL Images of LAB Isolate- Complex 1 Flock
- 14. 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 bird Isolates from different birds within the same flock are often not identical- variability within a flock Some isolates are shared between birds in different flocks- some evidence of a core microbiome? RAPD CE GEL Images of MRS Isolates
- 15. GI tracts a b c 10 birds/flock Extract gDNA Isolate predominant bacterial types Isolate gDNA- 10 isolates/bird Perform TRFLP Perform RAPDs Early Colonizing Bacteria Project Create dendrograms Product Development Identify predominant genotypes Serve as a baseline for perturbations 16s DNA seq
- 16. Bird DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH Hen DOH Hen DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH DOH Hen Hen DOH DOH DOH DOH Hen Hen Hen Hen Hen Hen Hen Hen Hen Hen Hen Hen Hen Hen BreederFlock 2 84 72 2 94 36 94 2 36 36 36 36 36 72 84 84 94 2 94 84 36 80 80 80 80 80 GlenisHousman 94 AlanKoogler 80 2 84 80 94 84 72 94 80 2 2 72 84 72 2 2 80 2 2 72 36 80 72 36 36 72 94 94 84 2 84 94 72 84 84 94 84 72 36 RonFreed RonFreed 72 2 80 36 LarryDepoy AlanKoogler LarryDepoy GlenisHousman CharlesMartin LarryDepoy GlenisHousman RexSours RonFreed CharlesMartin RexSours CharlesMartin AlanKoogler RexSours Lactobacillus johnsonii Enterococci Lactobacillus salivarius Lactobacillus crispatus Lactobacillus casei Pediococci TRFLP Analysis of Lactic Acid Bacteria Complex 1 Day-of hatch birds (blue), hens (yellow)
- 18. 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
- 19. 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 16 57 168 3 0 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 2 0 100 200 300 400 500 600 700 800 ΔCT LPS ΔCT 1E1 ΔCT 1E1+LPS ΔCT 1037 ΔCT 1037+LPS Fold Change Gene Expression: TNFa(BA) 452 15 69 186 3 0 100 200 300 400 500 ΔCT LPS ΔCT 1E1 ΔCT 1E1+LPS ΔCT 1037 ΔCT 1037+LPS Fold Change Gene Expression: IL6(BA)
- 20. 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. plantarum 10% Bacillus subtilis (two strains) Samples: hatchery alternated GUT START and No GUT START (control) every week for 12 weeks GITs Age 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)
- 21. GUT START Trial 2 Results 0 1 2 3 4 5 DOH 7 d Log10(CFU/g) GUT START Control APEC Levels a b c ab Significance by one-way ANOVA (p≤0.05)
- 22. GUT START Trial 2 Results 7 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 >7 Log CFU/g category GUT START Control Percent of 7 day old birds whose APEC levels fit into each quantitative category
- 23. 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 10.28 Control 11.28 4.8% decrease 8.8% decrease
- 24. Project Objectives Phase 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
- 25. SUBCLINICAL A Pig’s Life – Early Stage Immune Development Gastrointestinal Development Microbiological Challenges • Pathogenic E. coli • Toxigenic Clostridium CLINICAL Birth Wean Microbiological Succession • nutrient changes • stress – social changes Pre-wean Nursery
- 26. GI tracts Pars esophagus Jejunum Ileum 4-5 Pigs / Sow Flow Extract gDNA Isolate predominant bacterial types (MRS, Chrom agars) MRS isolates- 10 isolates/section/pig E. coli isolates- 5 isolates/section/pig Perform TRFLP Perform RAPDs Create dendrograms 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
- 27. 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
- 28. 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 pentosaceus Klebsiella pnemoniae Streptococcus pocorum Streptococcus sp. Streptococcus alactolyticus Lactobacillus mucosae Bacillus sp. Streptococcus hyointestinalis Lactobacillus crispatus Escherichia coli Lactobacillus sp. Lactobacillus delbrueckii Lactobacillus salivarius Lactobacillus amylovorus Lactobacillus vaginalis Chryseobacterium spp. Lactobacillus johnsonii Lactobacillus reuteri Streptococcus gallolyticus
- 29. GI tracts Pars esophagus Jejunum Ileum 4-5 Pigs / Sow Flow Extract gDNA Isolate predominant bacterial types (MRS, Chrom agars) MRS isolates- 10 isolates/section/pig E. coli isolates- 5 isolates/section/pig Perform TRFLP Perform RAPDs Create dendrograms 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
- 30. 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 Analysis Total Bacteria: Effect of Site n=30 pigs
- 31. 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. crispatus Streptococcus Lactobacillus 540 145 165 160 224 153 524 175 123 420 169 587 186 228 Minor Peaks Microbial Community Analysis Lactic Acid Bacteria: Effect of Site n=30 pigs
- 32. 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 301 93 Bacillus Lactobacillus 193 74 502 557 148 156 173 84 524 476 144 Minor Peaks Microbial Community Analysis Total Bacteria: Effect of Age n= 25 pigs
- 33. 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. johnsonii L. crispatus Streptococcus Lactobacillus 540 145 165 160 224 153 524 175 123 420 169 587 186 228 Minor Peaks Microbial Community Analysis Lactic Acid Bacteria: Effect of Age n= 25 pigs
- 34. 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 Analysis Lactic Acid Bacteria: Site C x Age n= 5 pigs
- 35. Project Objectives Phase 1 • Understand the diversity of lactic acid bacteria within the gastrointestinal tract of pigs- GI section, pigs, source (sow flow)-site, company Diversity observed across sections, pigs and sites- impact of weaning • Identify the key microbial isolates of the core microbiome- correlate to performance and health Next 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