1. No category

1
2
Dahlem
3
4
5
Beispielbild
Food Safety
and Quality
Jürgen Zentek
Institute of Animal Nutrition,
Nutrition Freie Universität Berlin
Food safety
y
• Establishing expert working groups
• FOODSEG aims at supporting the European
Commission
• To organise symposia, to integrate experts
• To disseminate research results
• Researchers exchange programme
Crops
Humans
Animals
7
Feed
8
Salmonella prevalence in the EU
Pigs
Poultry
n Isolates
4504
S. Typhimurium
S
S. Derby
S. Rissen
S 4,12:i:S.
4 12 i
47,3
47
3
10,8
3,6
33
3,3
S. Enteritidis
S. 1,4,5,12:i:S. 4,5,12:i:S. Infantis
S. London
S. Anatum
Other serovars
2,8
2,5
2,2
1,8
,
1,6
1,3
22 8
22,8
S. Enteritidis
S
S. Typhimurium
S. Infantis
S Paratyphi
S.
P t hi b
var. Java
S. Virchow
S. Hadar
S. Livingstone
S. Mbandaka
S. Senftenberg
S. Bredeney
Other serovars
Feed
5888
%
37 6
37,6
7,2
6,3
49
4,9
599
S. Mbandaka
S
S. Senftenberg
S. Agona
S Rissen
S.
Ri
12,9
12
9
10,7
8,3
80
8,0
3,1
3,0
2,9
2,2
,
1,7
1,4
29 6
29,6
S. Lexington
S. Tennessee
S. Havana
S. Infantis
S. Typhimurium
S. Livingstone
Other serovars
6,5
4,8
4,7
4,7
,
3,2
3,2
33 1
33,1
(EFSA 2007; EFSA 2009)
9
Salmonella prevalence in feedstuffs
2002
2003
2004
2005
2006
%
Fish meal
2,1
1,6
1,1
0,4
1,9
Meat and bone meal
2,9
0,5
1,7
1,3
2,3
Cereals
12
1,2
10
1,0
07
0,7
05
0,5
03
0,3
Plant proteins
5,3
4,8
5,7
4,3
2,5
(EFSA 2007)
10
Feed as source for Salmonella
• Depends on the epidemiological situation:
- Countries with low prevalence:
- Feed is one of the most important sources
- Countries with high prevalence:
- Feed is of minor importance
11
Salmonella cleanup costlier than expected
The estimated cost of cleaning up piggeries and
poultry farms from a salmonella outbreak last spring
continues to rise. According to estimates by insurance
firms, the final bill will exceed 20 million euros. Some
80 farms and production areas were contaminated by
tainted fodder from Raisio Feed...
Feed
http://yle.fi/uutiset/news/2009/10/salmonella_cleanup_costlier_than_expected_1092429.html
12
Prevention of Salmonella entry
• Prevent primary contamination
- Heat
H t treatment
t t
t
- Organic acids
• Avoid recontamination
- Feed mills: air flow
flow, acid treatment
- Farm, hygiene, storage
• Salmonella control programmes
- Combination of management and hygiene
13
Salmonella analysis
y
in crushing
g plants
p
Oliveira et al. 2010
14
Microbiota
• Nutritional factors for gut health in poultry
1 Digestion:
1.
Di
ti
-
Fermentation of indegstible feed compounds
-
Release of metabolic products
2. Health:
-
C l i ti resistance
Colonization
i t
-
Stimulation of the immune system
15
Microbiota
• „Manipulation“ of intestinal microbiota by dietetic factors
- Performance
- Stab
Stabilized
ed health
ea t
- Reduced risk for zoonoses: Salmonella, Campylobacter
• Alternative to antibiotic growth promoters?
16
Microbiota
• Manipulation of the intestinal microbiota in chicken
- Feed composition
- Technology
ec o ogy
- Feed additives
17
Nutrition and microbiota
• Feed structure and particle size
- Coarse feed structure
- More
o e fermentation
e e tat o in tthe
e ccrop
op
- More lactic acid
- Higher secretory activity of the gizzard
- Less bacteria in the small intestine
intestine, reduction of
salmonella colonization
Svihus 2006
18
Organic
g
acids
RCOOH
Bacterial Cell
Schädigung der
DNA- Damage
DNA-Feinstruktur
DNA
Feinstruktur
RCOO -
H+
pH ↓
H+
Erhöhter
ATP consumption
ATPATP-Verbrauch
DNA
ATP
P+ADP
Stö
Störung
d
der
Protein synthesis
Proteinsysnthese
Ribosome
Ribosom
Enzyme
Beeinträchtigte
ATP-regeneration
ATP-Regeneration
Enzyme functions
19
Nutrition and microbiota
• Organic acids
- Na-Butyrate
- 500,
500 1000,
1000 2000 mg/kg
/k
- Performance ↑
- Feed conversion improved
Hu und Guo 2007
- Fumaric/formic acid
- Growth rate ↑
- Microbiota changes in the ileum and caecum
Brzoska 2007
20
Nutrition and microbiota
• Organic acids
- Caprylic acid
- 0,35,
0 35 0
0,7,
7 1
1,4
4 and
d2
2,8%
8%
- Infection with Campylobacter jejuni on
day 3 after hatching
- 0,7 und 1,4 % caprylic acid reduced
Campylobacter significantly (3-4
logarithm
g
steps)
p )
Santos et al. 2008a, b
21
Nutrition and microbiota
• Probiotics
- Lactobacillus spp.
spp
- Performance, digestibility ↑, immune response after
vaccination↑ (Apata 2008)
- Lower Salmonella concentrations, higher phagocytosis
activity
ti it (Higgins et al. 2007; Vicente et al. 2008)
- Lower Salmonella concentrations in Turkeys (Grimes et al. 2008)
- No effect on C. jejuni Infection (Stern et al. 2008)
22
Probiotics: effectiveness
• Effectiveness depends on
- Application of a sufficient number of living
(?)
microorganisms
- High survival rate
- Product
- GI-tract
GI tract
23
Probiotics: GI-tract
• Passage through/colonisation of the GI-tract
GI tract
- Factors:
- pH in the stomach
- Enzymes: stomach and small intestine
- Bile
- Self regulating / hostile micro-ecology
micro ecology
24
Interactions – „Quorum
Q
Sensing“
g
Bioaktive
Peptide
Pigmente
Stressproteine
Oberflächenaktive
Substanzen
Kurzkettige
Fettsäuren
Lektine
Enzyme
Antimikrobielle
Substanzen
gasförmige
Moleküle
Lipopolysaccharide
Aminosäuren
Nukleinsäuren,
miRNA
Kohlenhydrate
Hormonähnliche
Substanzen
Vitamine (z.B.
Biotin, Folsäure)
Amine
mod. Shenderov 2011
25
Mode of action of probiotics
p
Growth inhibition, exclusion
Immune modulation
Adherence inhibition
Growth inhibtion
− Pathogens
− Harmful bacteria
Growth stimulation
− Bifidobacteria
− Lactobacilli
− Adherence inhibition
− Growth inhibtion
Probiotic
Lactate
Lactate utilizing bacteria
−
−
−
−
−
−
pH ↓
Epithelial cell growth ↑
Colonic blood flow ↑
Motility modification
Absorption of water, minerals ↑
Mucus production ↑
Short chain fatty acids
Mod. OHASHI and USHIDA 2009
26
Results: E. faecium in sow feces of control vs. EF group
g p
Lena Martin
Results by Ingo Starke
27
Probiotic strain in piglets
pg
E. faecium (NCIMB 10415) day 14
E. faecium (NCIMB 10415) day 35
E. faecium (NCIMB 10415) day 56
28
Incidence of postweaning diarrhea:
Strain specificity, application initiation, procedure and dose
Incidence off diarrhea
a (%)
100
80
p = 0.05
60
p < 0.1
p < 0.01
0 01
40
20
0
Control
n
B. cereus
var. toyoi
o
p
q
E. faecium NCIMB 10415
i l t
n Sow & piglet o Sow & piglet p All piglets
q Weaner
Sows:
108 cfu/kg
109 cfu/kg
No probiotics
No probiotics
Nursing:
109 cfu/kg
108 cfu/kg
109 cfu/day
No probiotics
Weaner:
109 cfu/kg
108 cfu/kg
109 cfu/kg
109 cfu/kg
Simon 2008
29
Results E. faecium trial
Quantitative monitoring
Q
g of bacteria in sow feces
No persistent changes visible for most bacterial groups
–Exception: Lactobacilli
Lactobacilli
12
Control
Probiotic
*
11
log cell number/ g wett weight
•
*
10
*
9
8
7
6
5
4
-28
-21
-14
-7
0
7
14
time [d]
Project A1 (Vahjen and Starke)
30
Results E. faecium trial
Qualitative monitoring of bacteria in sow feces (DGGE)
•
Reduction of bacterial diversity in sow feces post partum
•
Reduction of bacterial „uniformity“ (evenness) throughout the trial
Evenness
S hannon diversity index
1.0
*
2.4
*
*
*
*
2.2
*
*
2.0
*
0.8
band numb
ber
band numbe
er
*
0.9
*
1.8
1.6
0.7
0.6
1.4
0.5
1.2
1.0
0.4
-28
-21
-14
-7
0
tim e [d]
7
14
21
28
-28
Control
Probiotic
-21
-14
-7
0
7
14
21
28
time [d]
=> Qualitative bacterial changes take place in the sow
=>
> Decreased diversity due to quantitative increase of
dominating species?
Project A1 (Vahjen and Starke)
31
Bifidobacteria in piglets
pg
Bifidobacteria at day 14
Bifidobacteria at day 28
32
Isolation frequency of E. coli serogroups in piglets (terminal colon;14. – 56. d)
Exp. 2
Exp.
p 1
E. faecium NCIMB 10415 (to Sows and piglets )
Relative Is
R
solation frequen
ncy (%)
B. cereus var. toyoi (to Sows and piglets )
100% = Control group
100
80
60
40
20
0
ND
β-hämo.
O8
ND
O108
O139
ND, not detectable
ND
O141:K85
ND
O147
Scharek et al., 2005
Tedin, 2005
33
Less diarrhoea by
y probiotics
p
•
Prevention of diarrhea in piglets
- Bacillus subtilis ((Bhandari et al. 2008))
- Bacillus subtilis und B. licheniformis (Alexopoulos et al. 2004)
- Enterococcus
E t
faecium
f
i
(T
(Taras
ett al.
l 2006
2006; Z
Zeyner und
dB
Boldt
ldt 2006)
- Lactobacillus sobrius (Konstantinov et al. 2008)
- Lactobacillus rhamnosus (Zhang et al. 2010)
- Pediococcus acidilactici und Saccharomyces
y
cerevisiae boulardii
(Lessard et al. 2009)
- Escherichia coli Nissle (Trebichavsky et al. 2010)
34
Probiotics: antagonisms
g
• Theory
Th
- Probiotics occupy
- Epithelial receptors
- Ecological niches
- Antagonize/displace other
bacteria including pathogens
- Affect immune function
http://www.bu.edu/bostonia/fall09/disease/disease.jpg
35
Probiotics: antagonisms
g
• Adhesion of probiotic and enteropathogenic bacteria to
isolated porcine intestinal mucus
Treatment of intestinal mucus:
−B. lactis BB12
−L.
L rhamnosus LGG
−
−
−
Probiotics
adhesion of tested
pathogens ↓
Strain differences
Location differences
Enteropathogens
Collado et al. 2007
36
Probiotics and p
pathogenic
g
bacteria
•
Bacillus cereus in sows and piglets
- Salmonella infection: Faecal shedding ↘
Tedin et al. in prep.
•
Enterococcus faecium in piglets
- Salmonella infection: Faecal shedding and organ colonization ↑, ±
Szabo et al. 2009, Janczyk et al. 2011
37
Probiotics and p
pathogenic
g
bacteria
• Bacillus sp. -> Incidence of diarrhea ↓
% Tie
ere mit D
Darrhöe
Kontrollgruppe
70
60
50
40
30
20
10
0
Probiotikagruppe
101
19
1
3
5
S l
Salmonella
ll Typhimurium
T hi
i
7
9
11 13 15 17 19 21 23 25 27 29
Tage post infectionem
Tedin et al. (2012)
38
Probiotics and p
pathogen
g
shedding
g
• Bacillus sp. -> Salmonella Typhimurium ↓
( )
(trials 1 and 2)
Number
rs of Salmone
ella
log S
Salmonel
la/g(LOG)
10
Control
Bacillus
8
6
4
2
0
0
1
2
3
4
Time post
infection
[Weeks]
Wochen
post
infectionem
Tedin et al. (2012)
39
Probiotics: Antagonisms
g
Lactobacillus sobrius attenuates ETEC-Infections in piglets (Bacterial
counts in the ileum digesta)
Kontrolle
L. sobrius
18,0
16,0
14,0
12,0
10,0
*
8,0
6,0
*
*
4,0
,
2,0
<3
0,0
Gesamtkeimzahl
L. sobrius-like
L. sobrius DSM 16698
Kons
stantinov
v et al. 2008
20 0
20,0
log/g
•
ETEC
40
Probiotics and viral colonization
• Piglets without or with E. faecium
- Differences in the shedding of some viruses
- Astrovirus
st o us -> co
control
to g
group
oup o
only
y
- Rotavirus -> shedded later and at lower levels in the
probiotic piglet group
Kreuzer et al. in prep.
41
Mode of action of probiotics
p
Growth inhibition, exclusion
Immune modulation
Adherence inhibition
Growth inhibtion
− Pathogens
− Harmful bacteria
Growth stimulation
− Bifidobacteria
− Lactobacilli
− Adherence inhibition
− Growth inhibtion
Probiotic
Lactate
Lactate utilizing bacteria
−
−
−
−
−
−
pH ↓
Epithelial cell growth ↑
Colonic blood flow ↑
Motility modification
Absorption of water, minerals ↑
Mucus production ↑
Short chain fatty acids
Mod. OHASHI and USHIDA 2009
42
Probiotics: immune system
y
• Theory
- Modulation of the intestinal immune response
- Gut associated immune system
- General immune system
43
Mode of action of probiotics
p
Growth inhibition, exclusion
Immune modulation
Adherence inhibition
Growth inhibtion
− Pathogens
− Harmful bacteria
Growth stimulation
− Bifidobacteria
− Lactobacilli
− Adherence inhibition
− Growth inhibtion
Probiotic
Lactate
Lactate utilizing bacteria
−
−
−
−
−
−
pH ↓
Epithelial cell growth ↑
Colonic blood flow ↑
Motility modification
Absorption of water, minerals ↑
Mucus production ↑
Short chain fatty acids
Mod. OHASHI and USHIDA 2009
44
Probiotics: GI-tract
• Metabolic effects in the intestinal tract
- Metabolic activity is difficult to verify
- O
Organic
ga c ac
acids,
ds, bacte
bacteriocins….
oc s
- E. faecium:
- Lactate in small intestine: tendency ↑
- Colon: Effects small
45
Probiotics: GI-tract
• Apparent ileal and total tract nutrient digestibility on day 54
of life in piglets fed either a control diet (CON) or 3.6 x 106
cfu/g Enterococcus faecium NCIMB 10415 (EF)
OM
CP
starch
Ca
P
CON
0.62 ± 0.13
0.77 ± 0.05
0.93 ± 0.08
0.68 ± 0.18
0.68 ± 0.10
EF
0 66 ± 0.08
0.66
0 08
0 77 ± 0.06
0.77
0 06
0 94 ± 0.03
0.94
0 03
0 62 ± 0.20
0.62
0 20
0 70 ± 0.08
0.70
0 08
0.553
0.997
0.786
0.685
0.793
CON
0.85 ± 0.03
0.84 ± 0.02
0.99 ± 0.04
0.62 ± 0.04
0.69 ± 0.03
EF
0.84 ± 0.03
0.82 ± 0.03
1.00 ± 0.02
0.53 ± 0.08
0.65 ± 0.04
0.800
0.283
0.141
0.057
0.166
Ileal
P-value
Total Tract
P-value
46
Mode of action of probiotics
p
• Enterococcus faecium NCIMB 10415: performance and
small intestinal digestive physiology in piglets
- Most intestinal changes were age-dependent
- Nutritional impact of Enterococcus faecium in piglets
- Feed conversion (↘)
- Jejunal villus length and crypt depth ±
- Intestinal lactase activity ↘
- SGLT1 expression, Na-coupled glucose transport ±
47
Epithelial
p
barier function
•
Some probiotics/conditioned culture medium:
- increased transepithelial electrical resistance in vitro
•
E. coli strain Nissle 1917:
-
•
upregulates zona-occludens 1 expression in apical tight junctions
Probiotic mixture vsl#3:
- maintained apical tight junction integrity in colonic epithelial cells in
Dss-induced
Dss
induced colitis
•
Heat-killed L. rhamnosus:
- protected
t t d against
i t mucosall b
barrier
i permeability
bilit d
defects
f t iin mice
i with
ith
Dss-induced colitis
Review by Gareau et al. 2010
48
Perspectives
p
for research on p
probiotics
•
Effects on:
- Microbiome/”virome”: diversity, functionality, individual patterns
- Host genotype interaction
- Pathogen effects on epithelium, role of specific surface-layer proteins
- Barrier function
- e.g. release of mucus
- Intercellular integrity of apical tight junctions
- Immune system, signaling effects
- Innate immune response
- Adaptive immune system
TLRs
DCs
T reg cells
49
Conclusion
P f
Performance
Food safety
Animal health
Economy
Efficient feed
utilization
50
Conclusion
• In the last years,
years many efforts have been made to
reduce Salmonella problems
• Efficient systems established in Europe
• Success rates are obvious, for instance:
- Salmonella cases in humans in EU (EFSA):
−2004: 195.947
−2008:
2008 133
133.258
258
51
Evening
gp
programme
g
• Bus transport to the city centre -> Alexanderplatz
• Restaurant: 20:00
52
Transport
p
from Alexanderplatz
p
to Seminaris
• Take U2 direction Wittenbergplatz
• Take U3 direction „Krumme Lanke“, leave the train at the
station „Dahlem
Dahlem Dorf“
• Now you are max. 5 min from the hotel Seminaris
• Buy a ticket „AB“ for 2,30 € (one way)
53
54
55
Mycotoxins
y
• Aflatoxins
• Liver toxicity
toxicity, carcinogenic
• Excretion via milk
Photos: J. Böhm, E. Razzazi
56
Aflatoxins
• Aspergillus flavus, parasiticus
Aflatoxin B1
• High
g temperature
p
and humidity
y
• Cotton seed, peanut products and others
Photos: J. Böhm, E. Razzazi
57
Analytical methods
• Methods for feed analysis
- Robustness
- Speed
- Accuracy
58
Aflatoxin B1 in feed (
(UK,, 1992))
Afl B1
Total
Gesamtzahl
45
40
40
Reiter, 2009
35
35
32
30
25
n
21
20
20
20
18
15
15
15
15
11
10
5
3
1
0
Corn
gluten
Rice
bran
Cotton
seed
Sun
flower
Palm
kernels
Soya
meal
0
Peas,
beans
Scudamore et al. 1997
59
Aflatoxin B1 in feed
Afl B1
Total
Gesamtzahl
600
536
500
400
n
311
300
200
122
100
0
109
97
80
54
Maize
37
0
3
Wheat
bran
Soya
meal
8
3
Corn
gluten
Complete
feed
9
Peanut
meal
19
71
3
Straw,
silage
27
Rice
bran
7
Other
materials
Binder et al. 2007
60