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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
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The chicken intestinal microbiome is
important in…….
• Nutrition & productivity
• Immune development/education
• Disease
• Health
• Carriage of (zoonotic) pathogens
• …and much more
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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
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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
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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.
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What Taxa Are Different Between caeca and ileum?
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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
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And commercial production uses systems far removed from the jungle
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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
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Heatmap
Dendrogram of ASV
abundance in the
caecum between 0
and 42 days post
hatch
The developing broiler chicken microbiome
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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.
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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?
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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.)
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Digging deeper
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Host genetics have some effect on microbiome-but less than the external environment
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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
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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
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Probiotics and CE products are cultured
…but what if the important bugs are hard/impossible to grow?
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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)
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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
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So can we make microbiome-based interventions to control pathogens effectively?
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What’s a pathogen?What’s a commensal?
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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
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Body of evidence of success
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But does this translate to the farm?
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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
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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
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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)
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UK FSA retail chicken
survey 2014-2017
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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
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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?
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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
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CMT reduces C. jejuni
Transmission in seeder
experiments
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Reduction of intestinal burden @ 15 days post challenge (36do)
CM
T
Avi
guar
d
Int.
contr
ol
Ext
. cont
rol
0
2
4
6
8
10
12
Lo
g10 C
FU
/g
**
***
***
CM
T
Avi
guard
Int.
contr
ol
Ext
. contr
ol 0
2
4
6
8
10
12
Lo
g10 C
FU
/g
****
****
****
**
**
Caecal C. jejuni load Ileal C. jejuni load
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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
T
Ext
. Contr
ol
CM
T
Ext
. Contr
ol 0
2
4
6
8
10
12
14
Log C
FU
/g
Experiment 3 Experiment 4
**** ****
Experiment 2
Experiment 3
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6 311 4
51
5
8
19
41
2
11
96
71
69
375
305
65
2
2
1
1
1
117
7
14
4
9
16
4
12
12
11
1
0
50
100
150
200
250
300
350
400
450
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
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16S sequencing of aviguard v transplant material
Aviguard Inoc. CMT Inoc.
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Modulation of the microbiotaCMT Treated Ext. Control Int. Control Aviguard Treated CMT Treated Ext. Control Int. Control Aviguard Treated
Class Family
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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.
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Spraying caecal material onto eggs
before hatch leads to increased diversity in
the early chick microbiome-BUT
only spore-formers
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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
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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)