Research & Development For Effective Avian Probiotics

Brian B. Oakley, Associate ProfessorMicrobial EcologyWestern Health Sciences UniversityCollege of Veterinary Medicine Pomona, CA

Poultry microbiology and its importance for nutrition and food safety

~ 9,000,000,000 broilers processed per yr in U.S.

~ 40,000,000 tons of feed per yr

Carbon footprint ~450,000,000,000 car miles yr-1

Major source of protein & foodborne illness

Microbiome of birds and production system as ‘super-organism’

Both Model and System

Renaissance of Microbial Ecology

Cellular and Molecular Gastroenterology and Hepatology 2015 1, 28-40DOI: (10.1016/j.jcmgh.2014.11.004) Copyright © 2015 The Authors

Renaissance of Microbial Ecology DNA sequencing costs are decreasing faster than

Moore’s law:

NGS Output

Pre-processed dataset

Reformat, parse, build data tables,

map OTUs to taxonomy

OTU/Taxonomy

Assignment

Statistical & Graphical summaries

>seq1 AACATCGTC>seq2 AACAGCCTA>seq3 AATATCGTA

UCLUST, USEARCHSILVA, PERL

R

mothur

seconds -minutes

PERL

PERL, QIIME

Bioinformatics gives the keys to the kingdom

seconds -minutes

Huse et al. 2007 Gen BiolQuince et al. 2009 Nat MetKunin et al. 2010 EM

Schloss et al. 2009 AEMOakley et al. 2012 ISMEJ

Pipeline built from generic building blocks

Last-generation Food Safety and Animal Health:

Oakley et al. 2012. DMID Rinttila et al. 2004. JAM

Pathogen detection and mitigation:

Next-generation management:

What do natural communities look like?

Why do pathogens persist?

Can we leverage this knowledge to displace pathogens and improve performance?

Can we manage the microbiota to achieve food safety and performance goals?

:

Community changes significantly as bird grows

Temporal differences > feed additives

What do natural communities look like?

Oakley et al. 2015 BMC Vet Res.

Degrees of Freedom

Sum of Squares

MeanSquares

F.Model

R2

Pr(>F)

Time

1

3.457

3.457

8.986

0.0912

0.0001

Treatment

3

1.137

0.379

0.985

0.0300

0.4935

Time:Treatment

3

1.019

0.340

0.883

0.0269

0.8455

Residuals

84

32.310

0.385

0.8520

Total

91

37.922

1.0000

Community changes significantly as bird grows

What do natural communities look like?

Oakley et al. 2015 BMC Vet Res.

Oakley and Kogut 2016

What do natural communities look like?

Cecum

Feces

Significant temporal and spatial differences

1 wk 3 wk 6 wk

What do natural communities look like?Significant temporal and spatial differences

Can we use recurring patterns to reconstitute a ‘normal flora’?

Oakley et al. 2015 BMC Vet Res.

What do natural communities look like?

Next-generation management:

What do natural communities look like?

Why do pathogens persist?

Can we leverage this knowledge to displace pathogens and improve performance?

Can we manage the microbiota to achieve food safety and performance goals?

:

• Analytical approach to explore taxon and metabolic pathway co-occurrence patterns

• Large NGS data set

• Allow inferences about physiology, ecological niche, and biotic interactions

• Networks reflect ecological relationships

Network Analysis

Network AnalysisOTU Level:

Network AnalysisOTU Level:

Can start to infer metabolic interdependencies, spatial and chemical niches…

Common themes of acetate consumption, butyrate production, oxic-anoxic interfaces,

Oakley et al. PLoS ONE 2013.

Why does Campylobacter persist? What is it’s ecological niche in the chicken??

Month Neg Pos1 2 42 8 43 2 84 10 65 2 96 3 17 6 48 5 39 2 3

10 2 211 2 312 2 3

GI microbiome of Campylobacter-positive versus negative birds

What is ecological niche of Campylobacter?

Significantly over-represented taxa (two examples):

P

Complex carbs

Oligosaccharides +ATP + NADH

Phosphoenolpyruvate + ATP + NADH

Low H2 High H2NADH -> NAD+ + H+ + 2e-

NADH + pyruvate -> ethanol, lactate, NAD+

Acetate

Butryrate

succinate

propionate

Major end products of fermentation by Alistipes, Rikenella

What is ecological niche of Campylobacter?

H2 sinks:

SRBs: H2 + SO42- -> H2S Acetogens: H2 + CO2 -> acetate Methanogens: H2 + CO2 -> CH4 Uptake hydrogenases (over-represented in

Campylobacter, Helicobacter, & Megamonas)

Sargeant et al. 2014, PLoSONE

What is ecological niche of Campylobacter?

Conclusion: Campylobacter may actually be mutualist, not commensal!

Next-generation management:

What do natural communities look like?

Why do pathogens persist?

Can we leverage this knowledge to displace pathogens and improve performance?

Can we manage the microbiota to achieve food safety and performance goals?

:

Next-generation management:

Des

ired

stat

e

What is required to traverse alternate stable states?

Timely challenge: Define desired state!!

…And understand why. Ditto for undesired.

LRFI HRFI

LRFI HRFI

Genetics vs. microflora

Reciprocal transplant of fecal material from adults to chicks:

Plus uninoculated controls...

Can we manage the microbiota?

FemalesGenetics vs. microflora

Can we manage the microbiota?

Early stage inoculum significantly improves weight gain

Can we manage the microbiota?

Early stage inoculum significantly improves feed efficiency

Can we manage the microbiota?

Different cecal communities at 6 weeks

Can we manage the microbiota?

Can FMT ‘lay the golden egg’?

14 d1 d

5x

• Community composition• Metagenome• Host Transcriptome• Immune response (M1 vs. M2)• AME• Pathogen load• Growth

FMT+

FMT-

Different cecal communities at 6 weeks

Strain Identification

Microbiome datasets are amenable to high-throughput data mining to identify potentially desirable & undesirable taxa.

Statistical associations of particular taxa with pro- and anti-inflammatory cytokines…

Strain Identification

Oakley and Kogut 2016

Microbiome datasets can guide targeted cultivation of potentially novel strains from chicken G-I tract:

Novel strains shown in red genome sequenced April 2015

Strain Identification

Outer ring = consensus of cut sites

Fragment sizes and order reflect true arrangement in genome

Provides true barcode of genome

Provides template for assembly of genome sequence reads

Characterization of new isolates Genome Sequencing Optical Mapping

Strain characterization

CONCLUSIONS

Managing the microbiota can improve performance and food safety

Next-generation sequencing is a transformative tool

Proper management starts with understanding natural communities

‘Efficacy-first’ inversion of traditional strain-centric approach may be valuable

CONCLUSIONS

Food Animals = Tabula Rasa + Microbiome

Acknowledgements

USDA ARS Athens• Bruce Seal• Johnna Garrish• Mark Berrang• Nelson Cox• Eric Line• Jeff Buhr• Seung-Chul Yoon• Rick Meinersmann• Cesar Morales• Susan Brooks• Jessica Johnson (SCSU summer student)• Raja Chalghoumi (Fulbright fellow)

CDC Atlanta• Vladimir Loparev• Rebecca Lindsey

UGA• Sammy Aggrey• Steve Collett• Eldin Talundzic

SEPRL• Laszlo Zak• Michael Day

U.S. Poultry & Egg AssociationUSDA NIFA 1433 Formula FundsWesternU competitive Intramural

ARS College Station• Mike Kogut

Western University• Kris Irizzary• Yvonne Drechsler• Santiago Aguilar• Ella Richardson• Edith Martinez

Research & Development For Effective Avian ProbioticsSlide Number 2Slide Number 3Renaissance of Microbial Ecology Bioinformatics gives the keys to the kingdomLast-generation Food Safety and Animal Health:Next-generation management:Community changes significantly as bird growsCommunity changes significantly as bird growsSignificant temporal and spatial differencesSignificant temporal and spatial differencesSlide Number 12Next-generation management:Network AnalysisNetwork AnalysisNetwork AnalysisGI microbiome of Campylobacter-positive versus negative birdsWhat is ecological niche of Campylobacter?What is ecological niche of Campylobacter?What is ecological niche of Campylobacter?Next-generation management:Next-generation management:Slide Number 23Slide Number 24Slide Number 25Can we manage the microbiota?Can we manage the microbiota?Can FMT ‘lay the golden egg’?Slide Number 29Slide Number 30Slide Number 31Slide Number 32CONCLUSIONSSlide Number 34Acknowledgements