Controlling non-host specific Salmonella in poultry …...Controlling non-host specific Salmonella...
Transcript of Controlling non-host specific Salmonella in poultry …...Controlling non-host specific Salmonella...
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Controlling non-host specific Salmonella in
poultry using vaccination and feed additives
Prof. Dr. Ir. Filip Van Immerseel
Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820
Merelbeke, Belgium
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Non-host specific Salmonella:
what is the problem ?
• Zoonosis: transmission to humans
• One of the most important foodborne pathogens
worldwide
• Animals carry the bacteria, do not get ill
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EU prevalence !
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Human Salmonella prevalence EU - 2006
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Human Salmonella prevalence EU - 2006
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ENTERITIDIS worldwide pandemic !
2005 – data from WHO
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Human prevalance
BELGIUM
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Where is Enteritidis coming from?
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Where is Enteritidis coming from?
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Origin of US Salmonella Enteritidis outbreaks
Outbreaks of Salmonella enterica serotype Enteritidis infection in the United States,
with confirmed vehicle, by egg status, 1985–2003 (Braden, 2006)
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Laying hens
• High Salmonella prevalence
• Enteritidis most prevalent serotype
• Transmission to eggs:
specifically Enteritidis !
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EU mean 30.8%
High Salmonella flock prevalence, wide
variation in member state prevalence
Report of the Task Force on Zoonoses Data Collection on the Analysis of the baseline study on
the prevalence of Salmonella in holdings of laying hen flocks of Gallus gallus, The EFSA Journal
(2007) 97.
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Salmonella Enteritidis flock prevalence
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Enteritidis
Infantis
Typhimurium
Mbandaka
Livingstone
Hadar
Others
LAYING HENS
EGGS
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Salmonella Enteritidis predominantly
contaminates eggs !!!
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Laying hen flocks
FOCUS should be on controlling Salmonella
serotype ENTERITIDIS
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Routes of egg contamination
• Outer shell contamination
Fecal contamination !
REDUCE SHEDDING !
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Routes of egg contamination
• Internal egg contamination
– Eggshell and membrane penetration
– Reproductive tract colonization
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Internal egg contamination
Reproductive tract colonization
Depending on colonization place: incorporation in egg white, yolk
or albumen
PREVENT SYSTEMIC SPREAD !
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Prevention/control in layers
• 1 serotype to focus on
• Reduce shedding
• Decrease systemic spread
VACCINATION !!!
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antibody containing egg powder
Vaccination
active immunisation
live vaccines
inactivated vaccines
passive immunisation
maternal transferred antibodies
Vaccination of laying hens
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What is the ideal vaccine strain ?
• a high degree of protection against systemic and intestinal infection
•
• against a variety of important serovars (serogroups)
• adequate attenuation for poultry, other animal species, humans and the environment as well as animal welfare issues
• the vaccines should not affect growth of the animal
• vaccine strains should not be resistant to antibiotics
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What is the ideal vaccine strain ?
• vaccines should be easy to administer and need to have markers facilitating the differentiation from Salmonella wild-type strains
• application of vaccines should not interfere with Salmonella
detection methods
• humoral antibody response after vaccination should be distinguishably from a Salmonella wild-type response to allow the use of serological detection methods
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Inactivated vaccines
• a vaccine made from an infectious agent that has been inactivated or killed in some way
• parenteral administration to the single animal
• induction of (mostly) high antibody titres (detectable in
blood and yolk)
• correlation between antibody titre and „level of protection“ ?
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Inactivated vaccines
• administration of Salmonella-inactivated vaccines to
laying hens during production is possible
– Not allowed in registration files
– Drop in egg production
• Safe with regard to spread of vaccine strain
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EXAMPLE : SALENVAC
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Example : Nobilis Salenvac
• Description
– Inactivated vaccine poultry vaccine
– Contains formalin killed cells of Salmonella Enteritidis phage type 4
– Aluminium hydroxide gel as adjuvant
• Indication
– Induces active and passive immunity against S. Enteritidis in layers and breeders
– Aimed at reduction of Salmonella in eggs and poultry meat
• Administration
– Intramuscular injection into the leg or breast muscle
– The breast muscle is recommended for broiler breeders to avoid the risk of leg injuries
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Example : Nobilis Salenvac
• Vaccination schedule
– Normal vaccination schedule:
Twice 1 single dose of 0.5ml
Age : 10-12 weeks first, 14-18 weeks second administration
– High risk of early infection:
Triple dose
Age :0.1 ml at day-old, 0.5 ml 4 weeks, booster dose 14-18 weeks
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Example : Nobilis Salenvac
• Protection
– Protection develops after 2nd vaccination
– Full protection about 2 weeks after completion of regime
– Last vaccination should therefore be done at least 2 weeks prior
to transfer, because infection is likely to occur during or directly
after transfer.
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Some experimental data
• Woodward data !
• Clifton-Hadley data !
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Example : autologous vaccines
Isolate a local strain of Salmonella spp. from a poultry
house or animals
+
Kill culture by heat, formalin, etc. and add adjuvants
produce a specific inactivated vaccine for a particular
poultry farm
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Live vaccines
• oral administration via drinking water (natural route of
„infection“)
• induction of a local intestinal humoral and cellular immune response, production of secretory IgA
• weaker serological antibody response
• because of the excretion of live Salmonella vaccine
strains after administration, no use during egg production
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Example : Nobilis SG 9R
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Example : Nobilis SG 9R
• Description
– Live attenuated vaccine
– Based on S. Gallinarum strain 9R (rough strain)
• Indication
– Active immunisation of layers against S. Gallinarum
and S. Enteritidis
• Administration and vaccination schedule
– Subcutaneous in the neck at week 6 and week 14-16
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Example : AviPro Salmonella VacE
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Example : AviPro Salmonella VacE
• Description
– Live attenuated Salmonella vaccine
– Contains metabolic drift mutant bacteria of S. Enteritidis
Sm24/Rif12/SSq
• Indication
– Long-lasting protection in layers
• Administration
– Oral via drinking water
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Example : AviPro Salmonella VacE
• Vaccination schedule
– At day-old, 4 weeks, 16 weeks
• Protection
– Protection develops a few days post-vaccination
– Protection throughout the production cycle (at least until 60 weeks of age)
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Some experimental data
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Some experimental data
• 4 groups of animals :
– Non-vaccinated
– AviPro Salmonella VacE day 1, week 4 and 16
– AviPro Salmonella VacT day 1, week 4 and 16
– AviPro Salmonella VacE/T day 1, week 4 and 16
• Challenge infection intravenous with 10exp7 cfu of a Salmonella
Enteritidis field strain
• Sampling eggs for 3 weeks
• Euthanasia 3 weeks post-infection : bacteriological analysis of internal
organs
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Some experimental data
Reduction of internal organ colonization, including reproductive
tissues, even after iv infection !
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Some experimental data
Reduced egg contamination !
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EU legislation
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EU legislation
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EU legislation
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Salmonella Enteritidis flock prevalence
VA
CC
INA
TIO
N !!
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Salmonella food poisoning
in humans in Belgium
Start vaccination
of laying hens
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Result of laying hen vaccination
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Broilers
• A variety of serotypes
• Also Enteritidis and Typhimurium
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Salmonella prevalence in broiler flocks
EU mean
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Serotypes data EFSA 2006
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X
X
X
X
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Reduce prevalence !!
Prevalence Salmonella (Enteritidis + Typhimurium)
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Controlling Salmonella on meat
• Good cooking principles
• Decontamination meat
• Slaugterhouse hygiene
• Primary production measures: prevent entry of
Salmonella to slaugterhouse
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Controlling Salmonella in primary production
• Difficulties:
– Young slaughter age
– Susceptibility immediate post-hatch
– Variation of serotypes
– Housing type
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Controlling Salmonella in primary production
• Difficulties:
– Young slaughter age
– Susceptibility immediate post-hatch
– Variation of serotypes
– Housing typeClassical vaccination
not useful !
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ManagementVaccination
Feed additives
PRIMARY PRODUCTION
MEASURES
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ManagementVaccination
Feed additives
PRIMARY PRODUCTION
MEASURES
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Feed additives for use in broilers
• Antibiotics
• Competitive exclusion products
• Probiotics
• Prebiotics
• Short and medium-chain fatty acids
• Plant extracts - botanicals
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Antibiotics – growth promoters
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Antibiotics for Salmonella control
Reasons : antibiotic resistance, induction of carrier animals
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EFSA, 2006
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Competitive exclusion (CE) products
• freeze-dried products derived from healthy, pathogen-free birds
• It prevents colonization of the gastro-intestinal tract of poultry by pathogenic (harmful) bacteria
• occupying all of the ecological niches in the gut of the birds and so denying pathogenic bacteria space to establish themselves, SCFA production, …
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CE products - application
• Day-old chicks
• Poultry after treatment with antibiotics, creating an
imbalance in the normal gut flora
• Birds that are stressed due to relocation, laying or
moulting.
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Example : Aviguard• White free flowing powder that is easily dispersible in water
• Storage temperature 2°C to 8°C
• Contains over 200 normal avian gut flora micro-organisms
• Shelf life from manufacture is 18 months
• The product is packaged in foil laminate sachets sufficient for 2000, 5000 or 10,000 chicks
• Product can be applied by spray treatment or drinking water application
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Example : Aviguard
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Example : Aviguard
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Competitive exclusion (CE) products
• Disadvantage:
– Microbiota undefined
• Possibility of pathogens in products
• Possibility of antimicrobial resistance gene
tranmission
Not very commonly used !!
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Probiotics
• live microbial feed supplements which beneficially affect
the host animal by improving its intestinal microbial
balance
• Defined products
• On the market :
– Mainly lactobacilli
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Protection much less pronounced compared to CE products !
Trying multi-species defined products ??
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Prebiotics
• non-digestible feed ingredients that beneficially affect the
host by selectively stimulating the growth and/or activity
of one or a limited number of bacterial species already
resident in the colon, and thus attempt to improve host
health
• Mono-, di-, oligo- or ploysaccharides
• Natural or synthetic
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Prebiotic examples : mannose / MOS
Type I fimbriae
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Salmonella
Host cell
inflammatory
cytokine
response
Invasion
AdherenceEntry
Prebiotic examples : mannose / MOS
Mannose will
block adhesion !
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Prebiotic examples : mannose / MOS
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Prebiotic examples : mannose / MOS
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Prebiotic examples: many more …
• Beta-glucans : would induce immunity
• FOS (fructo-oligosaccharides), inulin : bifidogenic
• GOS
• Guar gum
• Many, many more ….
• Working action ? Benefit ?
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Acidic compounds
• Short-chain fatty acids (SCFA)
– Formic, acetic, propionic, butyric acid
• Medium-chain fatty acids (MCFA)
– Caproic, carpic, caprylic acid
• Combination of acids
– Example : butyric and capric acid
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SCFA to control Salmonella
• Drinking water additives :
– Effects limited to water itself and crop
• Feed additives :
– Effects can be long-lasting dependent on formulation
(powder, coating)
Resorption
Effect in crop
Gradual release
Effect in whole
GI tract
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In vivo trial
Five groups of 20 chickens :
CTRL FORMIC ACETIC PROPIONIC BUTYRIC
Infection at day 5 with 5.10³ cfu S. Enteritidis
Euthanasia at day 8
Bacteriological analysis of caeca, liver and spleen : titration on BGA
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Caecal colonization
0
1
2
3
4
5
6
7
8
9
CTRL FOR ACE BUT PROP
treatment
me
an
lo
g c
fu/g
a
a
b
c
ac
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CTRL
(n=20)
ACE
(n=20)
PROP
(n=20)
BUT
(n=20)
Negative 0* 0 0 0
x < 102 cfu/g 6 1 8 11
10² < x < 10³ cfu/g 0 1 1 2
103 < x < 104 cfu/g 0 0 1 1
104 < x < 105 cfu/g 3 0 2 3
105 < x < 106 cfu/g 2 2 1 3
106 < x < 107 cfu/g 8 3 3 0
More than 107 cfu/g 1 13 4 0
Number of animals colonizing the caeca at day 8 of life
* Number of animals
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In vivo trial – butyric acid
Five groups of 20 chickens :
CTRL POWDER COATED COMBI
butyric acid 0.63g/kg
Infection at day 5 with 5.10³ cfu S. Enteritidis
Euthanasia at day 8
Bacteriological analysis of caeca, liver and spleen : titration on BGA
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Colonization
0
2
4
6
8
10
12
14
16
CTRL POWDER COATED COMBI
num
be
r of
anim
als
(n =
25)
Low
Intermediate
High
Caecal colonization
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CTRL
(n=25)
POWDER
(n=25)
COATED
(n=25)
COMBI
(n=25)
Negative 0* 0 2 1
x < 102 cfu/g 6 8 12 8
10² < x < 104 cfu/g 2 1 3 2
104 < x < 105 cfu/g 3 1 0 6
105 < x < 106 cfu/g 4 3 4 5
More than 106 cfu/g 10 12 4 3
Number of animals colonizing the caeca at day 8 of life
* Number of animals
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Broiler experiment – seeder model
Two groups of 40 chickens :
CTRL POWDER COATED COMBI
butyric acid 0.63g/kg
Infection of 15 animals at day 5 with 5.10³ cfu S. Enteritidis
Weekly cloacal swabs
Euthanasia at day 42
Bacteriological analysis of caeca, liver and spleen : titration on BGA
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Broiler experiment – seeder model
0
10
20
30
40
50
60
70
80
90
100
0 6 12 18 24 30 36 42
age
% p
os
CTRL
BUT
excretion
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Butyric acid
• Also ‘fuel’ for epithelial cells
– Longer villi
– More absorptive surface
– Faster repair mucosal damage
Better performance
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Medium-chain fatty acids
Control MCFA
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Vaccination of broilers ?
• Passive immunisation – maternal antibodies
• Active vaccination ?
– Colonization - inhibition
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Vaccination of broilers ?
• Laying hens : live and killed vaccines available
one serotype predominating (Enteritidis)
• Broilers : some specific problems with vaccination :
– Young slaughter age (6 weeks)
– Immature immune system
– The induction of a specific antibody response takes about 2 weeks
– Protective mucosal immunity at the intestinal level is difficult to achieve
Requirements :
A) Confer resistance in the immediate post-hatch period
B) Maintain long-term protective effects
C) Protect against multiple serotypes
Classical vaccination not applicable !
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Colonization-inhibition (CI)
• Oral administration of Salmonella organisms to newly
hatched chickens confers, within 24hrs of inoculation, a
high degree of resistance against challenge infection
with another Salmonella strain of the same serotype
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Wild type strain
Caecal colonization
log
cfu
/g c
ec
um
age (days)
Wild type strain(inoculated day of hatch)
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Wild type strain as ‘CI’ strain
Wild type strain
Wild type ‘CI’ strain (76Sa88)
(inoculated day of hatch)
Wild type challenge strain (147)
(inoculated 24h later)
log
cfu
/g c
ec
um
age (days)
Caecal colonization
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∆hilA strain as ‘CI’ strain in seeder model
0
20
40
60
80
100
120
0 5 10 15 20 25 30 35 40 45
days
% p
os c
loa
ca
sw
ab
s
Wild type strain
∆hilA strain as ‘CI’ strain
(inoculated day of hatch)
Wild type challenge strain (147)
(inoculated 24h later)
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∆hilA strain as ‘CI’ strain in seeder model
0
20
40
60
80
100
120
0 5 10 15 20 25 30 35 40 45
days
% p
os c
loa
ca
sw
ab
s
Wild type strain
∆hilA strain as ‘CI’ strain
(inoculated day of hatch)
Wild type challenge strain (147)
(inoculated 24h later)
Conclusion: Reduced shedding up to slaughter age
in seeder bird model !!!
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Colonization-inhibition using live attenuated
vaccine strains
• Still at an experimental scale
• Applicable for broilers provided that :
– Vaccine strains are safe
– Vaccine strains are cleared at slaugter age
0
20
40
60
80
100
120
0 5 10 15 20 25 30 35 40 45
days
% p
os c
loa
ca
sw
ab
s
Still need additional
attenuations !
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Conclusion for broiler feed additives
• Will not completely eliminate Salmonella
• Have to be used in an overall control plan
– Hygiene, biosecurity, management, ….
• Can contribute to decreased shedding and colonization
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Overall conclusion
• Tools are available to decrease shedding,
colonization, meat and egg contamination
• Multiple control strategies should be used
in combination to be effective
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Thanks for your attention !