The chicken intestinal microbiome

Paul Wigley@DrWigley

wigley@Liverpool.ac.uk

What I am going to talk about

• An introduction to the chicken intestinal microbiome

• The microbiome in commercial production

• Unknowns and undcertainties

• Interventions to reduce infection and promote health and welfare

The chicken intestinal microbiome is

important in…….

• Nutrition & productivity

• Immune development/education

• Disease

• Health

• Carriage of (zoonotic) pathogens

• …and much more

What we know about the

chicken intestinal

microbiome

• Made up of around 500 bacterial taxa

• A big range of proteobacteria and archaebacteria species

• Hugely variable

• Differs between intestinal compartments

• Caeca have the greatest numbers (c1011 CFU/g) and diversity

• Small intestine c109 cfu/g

• Excess amounts of gamma-proteobacteria(enterobacteriacae) can be associated with poor gut health

• Can be modulated to improve health and/or productivity

Some key points…

The microbiome is not ‘one intestinal microbiome’

There is substantial inter and intra-bird variation between intestinal niches

Embryonic microbiomes are , almost certainly, sample contamination

It’s not just bacteria-the mycome and virome are poorly defined

The microbiome is very dynamic in structure especially in first 3 weeks of life

Analysing the faecal or caecal microbiome is not necessarily informative of the small intestine and digestion/feed utilisation

Defining 'good' or 'bad' bacteria or dysbacteriosis is not simple

Beta Diversity in the Chicken GutThe caecum and ileum have very different microbiomes

PCoA plot of weighted

Unifrac distance of

samples taken from the

caecum and ileum

between 0 and 42

days post hatch.

What Taxa Are Different Between caeca and ileum?

In most species the pioneer microbiome is maternally-derived

• Transfer during birth and at feeding in mammals

• Horizontal transmission from faecalmaterial in all species

• Transfer via placenta or in ovo(despite some reports) is unlikely

• No detectable intestinal bacterial population in chicken embryos sampled aseptically

And commercial production uses systems far removed from the jungle

But commercial birds don’t meet mum

• Modern hatcheries rear fertilised eggs to hatch which are then placed on farms for rearing

• High levels of hygiene and biosecurity

• Chicks have no contact with hens and unlike other species do not get their pioneer microbiome from mother

• Much vaccination take place in hatchery-spray or gel drop delivery to chicks or in ovo in the US

• So like human C-section babies , do commercial chicks get a ‘bad’ microbiome

Heatmap

Dendrogram of ASV

abundance in the

caecum between 0

and 42 days post

hatch

The developing broiler chicken microbiome

Taxa Plot showing relative

abundance at the genus level in

the caecal lumen (L) and mucus

(M) of 3 broiler breeds (A, B and

C) between 0 and 42 days post

hatch.

The microbiome & immune system

Clear role for microbiome in development of repertoire of the adaptive immune system

Role in development of regulatory and sentinel (Th17) immunity in the gut is less clear

Could the late/humansised pioneer microbiome lead to a delay or failure in proper development?

Potential consequences?

• Potential acquisition of a ‘humanised’ microbiome from hatchery workers

• Lack of diversity early in life-poor development of gut and mucosal immunity

• Contribution to poor gut health and infection susceptibility?

• Common members of early microbiome are often cause of early mortality in broilers (Escherichia coli & Enterococcus spp.)

Digging deeper

Host genetics have some effect on microbiome-but less than the external environment

Limitations of 16S-shotgun metagenomics

paint a fuller picture

• Samples of 24 broiler birds

• 283 novel species

• 42 novel genera

• Much greater richness of clostrdiales than ‘snaphot’ studies

• 16S does not show mycome/virome

Culture bias

• Culture in various conditions probably only reveals 10-20% of taxa in caeca

• Most species isolated are ‘usual suspects’-well characterised as easy to grow

• The more challenging bugs may be those of interest

• Need for improved/novel culture method to support genomics MALDI-TOF identification of species recovered from

caecal content of ROSS 308 broiler

Probiotics and CE products are cultured

…but what if the important bugs are hard/impossible to grow?

Antimicrobial usage• Antimicrobials are used across the world as

therapeutics, prophylactic drugs and growth promoters (AGP)

• High use considered driver for resistance especially in foodborne pathogens

• AGP banned in EU 2006 and decreased use in North America

• As therapeutics often given at flock level

• Increased antibiotic stewardship in EU

• In UK the British Poultry Council guidelines have decreased use by over 70%

• Need for alternatives-including pathogen control (NE, colibaccilosis, foodborne pathogens)

Probiotics and competitive exclusion products are touted as controls for food borne pathogens and antimicrobial-free growth promoters……

• Safe• Economic• Easy to administer via feed or water• May help establish a beneficial

microbiome• Positive effect on gut health • Can increase productivity

So can we make microbiome-based interventions to control pathogens effectively?

What’s a pathogen?What’s a commensal?

Competitive exclusion-principles and mechanisms

• Competitive exclusion-ecological term where one organism excludes another occupying same niche

• Described in chicken by Rantala & Nurmi 1973

• One bacterial species/combination of species can:

-Occupy physical a physical niche preventing colonisation-Out compete for nutritional resources-Produce metabolites (e.g SCFA) that inhibit other species-produce compounds like bacteriocins that inhibit -stimulate immunity

• Most effective exclusion is often by closely related organisms-e.g. between Salmonella serovars

Body of evidence of success

But does this translate to the farm?

Targeting interventions

Need to understand which taxa are

associated with good outcomes i.e. what are

protective-experimentally or

through large association studies

Where protection is need e.g. caeca for

Campylobacter, more small intestine for NE

Having live therapeutic interventions which actually reach and

colonise intestinal sites-so probably not current

probiotic strains

Safe and can be delivered

effectively/economically

Campylobacter –our main focus

• Most common cause of foodborne bacterial gastroenteritis

• Over 500,000 cases in UK annually

• Around 60-70% of retail chicken Campylobacter positive

• Limited effective control on farm or post slaughter

• No vaccine

• Probiotics and competitive exclusion not really effective

Probiotics have limited efficacy in robust challenge models

• Although probiotics are probably beneficial they are insufficient to stop Campylobacter colonisation

• May limit extra-intestinal spread

• May limit inflammation in gut and promote gut health

Feed additive incorporating Bacillus amyloliquifaciens reduces inflammation &

spread to liver but not gut colonisation in C. jejuni challenge (with DuPont Animal Nutrition)

UK FSA retail chicken

survey 2014-2017

Campylobacter lab confirmed UK cases 2012-2017

68701

66382 66716

5979758911

63304

40000

45000

50000

55000

60000

65000

70000

75000

2012 2013 2014 2015 2016 2017

Confirmed cases

So could a faecal or caecaltransplant approach be more effective?….

• In modern production chicks do not acquire a ‘natural’ chicken microbiome

• Could a transfer approach reduce pathogen carriage by exclusion, improved immune development or both?

• Would early transfer protect against C. jejuni colonisation or transmission when other approaches have not?

caecal transplantation in chickens to control Campylobacter

• Unlike other livestock chicks do not receive a ‘pioneer’ chicken microbiome from the mother-hatched and raised under clean conditions (Salmonella control)

• Early administration of a ‘healthy’ microbiome may help immune development and reduce pathogen carriage

• Transplant material harvested from ‘clean’ 8 week old broiler chickens, diluted and snap frozen

• Material fed to chicks within 4 hours of hatch

CMT reduces C. jejuni

Transmission in seeder

experiments

Reduction of intestinal burden @ 15 days post challenge (36do)

CM

T

Avi

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d

Int.

contr

ol

Ext

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0

2

4

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FU

/g

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CM

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Caecal C. jejuni load Ileal C. jejuni load

And is reproducible..

External

Control

CMT

2 dpi 5 dpi 8 dpi 12 dpi

3 dpi 5 dpi 7 dpi 10 dpi

External

Control

CMT

Shedding

detected

Shedding not

detected CM

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Ext

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ol 0

2

4

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Log C

FU

/g

Experiment 3 Experiment 4

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Experiment 2

Experiment 3

6 311 4

51

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41

2

11

96

71

69

375

305

65

2

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117

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7doa Aviguard. Aviguard Inoc. 7doa Internal control. CMT Inoc. 7doa CMT. 7doa Extrnal control.

Nu

mb

er

of

un

iqu

e A

SV s

eq

ue

nce

s

Verrucomicrobia

TM7

Tenericutes

Spirochaetes

Proteobacteria

Planctomycetes

OD1

NKB19

Fusobacteria

Firmicutes

Deferr ibacteres

Cyanobacteria

Bacteroidetes

Actinobacteria

Unclassified

FIGURE 7. MICROBIAL COMPOSITION OF CAECAL SAMPLES FROM 7 DAY OF AGE TREATMENT GROUPS, CMT INOCULUM AND AVIGUARD INOCULUM FOLLOWING 16S rRNA SEQUENCING. COMPOSITION IS DISPLAYED AS THE NUMBER OF ASV SEQUENCES PER TAXA AT FAMILY LEVEL.

Aviguardtreated chicks

Aviguard In house control

Transplant material

Transplanted chicks

Hatchery control

Caecal transplant massively increases numbers of taxa in caeca, thought not diversity compared to commercial microflora administered in same way

16S sequencing of aviguard v transplant material

Aviguard Inoc. CMT Inoc.

Modulation of the microbiotaCMT Treated Ext. Control Int. Control Aviguard Treated CMT Treated Ext. Control Int. Control Aviguard Treated

Class Family

Modulation of the microbiota CMT Treated Aviguard Treated

Internal Control External Control

• 7 d.p.h caecal content of CMT treated birds was characterized by a predominance of Ruminococcaceae

• Increases in Ruminococcaceaeaccompany an associated decrease in Enterobacteriaceae in CMT and Aviguard treated birds.

Spraying caecal material onto eggs

before hatch leads to increased diversity in

the early chick microbiome-BUT

only spore-formers

Poultry Smartphone App

• Basic pocket guide for infectious disease of the chicken

• Free

• Currently available for Android

• https://play.google.com/store/apps/details?id=uk.ac.liverpool.pocketguideforpoultrydiseases

Thanks • Amy Wedley

• Rachel Gilroy

• Sian Pottenger

• Peter Richards

• Gemma Wattret

• Lizeth Lacharme-Lora

• Sue Jopson

• Al Darby

• John Kenny

• DuPont Animal Nutrition

• Houghton Trust

• Julian Ketley & Natalie Barratt (Leicester)