Use of Pre and Probiotics in Broilers

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Use of Pre- and Probiotics in Broilers

• Todd J. Applegate J.A. PattersonPurdue
  UniversityV. Klose University of Natural
  Resources Applied Life Sciences (BOKU), Vienna,
  Austria

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"The right to search for truth implies also a
duty one must notconceal any part of what one
has recognized to be true."        Albert
Einstein, 1879-1955
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"The right to search for truth implies also a
duty one must notconceal any part of what one
has recognized to be true."        Albert
Einstein, 1879-1955
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Definition of Probiotics

• microbial food supplements that beneficially
  affect the host by improving its intestinal
  microbial balance. Gibson and Roberfroid
  (1995)
• live microorganisms which when administered in
  adequate amounts confer a health benefit on the
  host. WHO/FAO (2001)
• food or food supplements containing defined
  microorganisms in sufficient numbers to reach the
  gut in viable status resulting in positive health
  effects after consumption. Wassenaar and Klein
  (2008)

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Definition of Probiotics

• microbial food supplements that beneficially
  affect the host by improving its intestinal
  microbial balance. Gibson and Roberfroid
  (1995)
• live microorganisms which when administered in
  adequate amounts confer a health benefit on the
  host. WHO/FAO (2001)
• food or food supplements containing defined
  microorganisms in sufficient numbers to reach the
  gut in viable status resulting in positive health
  effects after consumption. Wassenaar and Klein
  (2008)

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Competitive Exclusion-Concept (Nurmi and
Rantala, Nature 241, 1973)
Feeding of adult microflora (intestinal contents
/ feces) protects newly hatched chicks from
Salmonella infections via establishment of an
improved intestinal microflora
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• Undefined CE products from European perspective
• Lack of characterization
• Pathogenic subspecies
• No information about antibiotic resistance genes
• Lack of quality control measures
• No approval in EU

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Probiotics - Registration

• US GRAS system (established history of safe use
  (before 1958) or positive safety evaluation
• EU Qualified Presumption of Safety (QPS)
• Non-QPS strains full safety assessment
• VERSUS Competitive Exclusion (CE) products
• Still allowed in certain countries

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Safety testing for non-QPS (EU)

• 90 day oral toxicity (100 rats)
• Acute dermal irritation / corrosion (6 rabbits)
• Skin sensitization (15 guinea pigs)
• Mouse Micronucleus Assay (60 mice)
• Ames test for genotoxicity (in vitro)
• Chromosome Aberration (in vitro)
• Tolerance test in target species (100X dose)
• Lack of virulence determinants
• Minimum inhibitory concentration across spectrum
  of antibiotics (with plausible genetic basis of
  resistance determined)

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QPS System Result.

• Individual registration of each strain
• Predominance of single strain products (only 12
  strains approved to date)
• Bacillus cereus
• B. licheniformis
• B. subtilis
• Enterococcus faecium
• Pediococcus acidalactici
• Lactobacillus farciminis
• L. rhamnosus
• L. casei
• L. plantarum
• Streptococcus infantarius
• Saccharomyces cerevisiae

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Strain Selection (Dunne et al., 1999)

• Be of origin to the animal.
• Demonstrate non-pathogenic behavior.
• Exhibit resistance to technological processes
  (i.e. viability and activity in delivery
  vehicles).
• Prove resistant to gastric acid and bile.
• Adhere to gut epithelial tissue.
• Persist, albeit for short periods, in the GIT
• Produce antimicrobial substances.
• Modulate immune responses.
• Have the ability to influence metabolic
  activities (e.g., cholesterol assimilation,
  lactase activity, vitamin production).

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? different mechanisms?
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Broiler Microflora
Lu et al, 2003
16S rDNA library
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Broiler Microflora
Lu et al, 2003
16S rDNA library
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Broiler Ileal bacteria(16SrDNA clone libraires
T-RFLP)
Lu et al., 2008
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In vitro Screening

• Isolation and characterization of bacteria
• Evaluation and selection of strain able to
  exclude pathogens
• Risk assessment investigation for registration
• pH reduction capabilities / bacteriocin production

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Inhibition results
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Inhibition results of CE5-strains
5 strain mix
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Adhesion of probiotics to intestinal cells in
vitro

• to examine isolated strains from chicken with
  regard to their adhesion ability to Caco-2 and
  HT-29 intestinal cells in vitro.

HT-29
Caco-2
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Adhesion to intestinal cells
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Reduction of pH
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Reduction of pH
Result Improved reduction of pH using a
combination of selected CE-strains (synergistic)
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Where do probiotic strains go?Molecular
tracking

• Plasmid encoded fluorescent protein gene placed
  w/ inducible promoter (Geoffroy et al., 2000)
• DNA label
• Silent Mutations (w/o affecting AA sequence of
  gene product) (Malinen et al., 2001)
• .... Great for research (wild type vs.
  modified).not so great registering / selling a
  GMO in certain markets
• Plasmid profiling, ribotyping, random amplified
  polymorphic DNA, DNA fingerprinting via PFGE
• . Limited in complex microbial environments
  (i.e. the GUT)

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Where do probiotic strains go?Molecular
tracking

• Genome-based tracking techniques can ID specific
  sequences among highly similar genomes
• Suppression subtractive hybridization (SSH)
• Differential display PCR
• Representational difference analysis
• Microarray

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Necrotic enteritis
Normal
Normal healthy intestinal villi
Enteritis shortened blunted villiless surface
area but what else
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GIT Responses to Insult

• a) Peristalsis increases
• b) Epithelial turnover (changes to
  proliferation, apoptosis, differentiation of
  cells towards immune response) c) Mucin
  productiond) Paracellular barrier Tight
  Junctions (osmotic diarrhea)e) Facilitation of
  commensal flora through secretionsf) Unfamiliar
  response Acute phase response
• g) Acquired response

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Probiotic (multi-strain) Influence on Immunity
(Dalloul Lillehoj, 2005)

• During Emeria acervulina challenge
• Improved serum and intestinal markers if immunity
• 4X fewer oocysts shed
• Improvements to intestinal structure

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Multistrain Probiotic Intestinal measures (7
days after coccidial vaccine challenge)
Teichmann, Applegate, Reisinger, Mohnl,
unpublished
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Why do we need to know where the strains go?
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Lactobacillus plantarum Influence on Human
Duodenal Mucosa RNA
Upregulated
van Baarlen et al., 2009
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Lactobacillus plantarum Influence on Human
Duodenal Mucosa RNA
Upregulated
Upregulated
van Baarlen et al., 2009
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Lactobacillus plantarum Influence on Human
Duodenal Mucosa RNA
Upregulated
Upregulated
van Baarlen et al., 2009
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(No Transcript)
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Cost of Immunity Acute Phase Response (feed
intake 14-22 days)
a
abP lt 0.05
b
b
b
Jiang et al., 2009
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Cost of Immunity Acute Phase Response (feed
intake)
a
abcP lt 0.05
ab
a
b
b
c
c
b
a
b
b
b
Jiang et al., 2009
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Cost of Immunity Acute Phase Response (Weight
gain)
P 0.001
Jiang et al., 2009
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Cost of Immunity Acute Phase Response (Weight
gain)
P 0.068
P 0.001
Probiotic supplementation reduced the cost of
immune response by 40
Jiang et al., 2009
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Immunological Response

• Quantity Quality
• Holistic Effect on Bird

Feed intake vs. Control
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Prebiotics

• Non-digestible food ingredients that
  beneficially affect the host by selectively
  stimulating the growth and/or activity of
  intestinal bacteria that may improve host health
• Synbiotics
• Combinations of prebiotics and probiotics
• Gibson and Roberfroid, 1995

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Prebiotics

• Fructooligosaccharides
• Oligofructose
• Inulin
• STOC
• Sucrose thermal
• oligosaccharide caramel
• GOS
• Stachyose, Raffinose
• TOS
• Transgalactooligosaccharides

• Lactulose
• Lactitol
• Lactose
• ? Galactooligosaccharides
• Xylooligosaccharides

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Do pre- /or pro-biotics affect pathogens???

• Approach 1
• Change in a) presence / absence b)
  concentration
• Approach 2
• Change in virulence (e.g. hilA from Salmonella)
• Approach 3
• Translocation (e.g. Salmonella in spleen)

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Ileal Loop/Salmonella Attachment Assay
1 h, 37 C 20 CO2
Ileal segmenta) flushed, b) homogenized, c)
serially diluted d) enumerated for S.
typhimurium on LB-kan
Kanamycin-resistant S. typhimurium
10-cm section of ileum
10-cm section of ileum
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Influence of diet on ileal susceptibility to
Salmonella Enteriditis attachment
Burkholder, Patterson Applegate, unpublished
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Conclusions

• Knowns
• Positively influence performance (not in all
  cases but neither do AGPs)
• Modulate Immunity
• Suppression of pathogens (broad array of
  capability(ies)

• Unknowns
• Where do they go in the GIT ( what life stage
  are they)
• Holistic view of immune modulation
• Synergy in vivo
• Limitations
• Tracking tools
• Registration hurdles

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What Should be done with Present Days
Information?

• Probiotics today.are NOT a commodity (i.e.
  products are unique)
• Specific Pathogen Exclusion on farm (e.g.
  Salmonella)..but what happens when put in
  contaminated crates to go to plant?
• Immunological enhancement
• Need to answer physiological questions
• What life stage
• Where are they living
• What interaction occurs with host what is
  hosts response