Role of intestinal integrity In the prevention of Necrotic Enteritis Richard Ducatelle, Annatachja Degrande, Evelyne Dierick & Filip Van immerseel Dept Pathology, Bacteriology and Avian Medicine
Ban on antibiotic growth promoters (AGP) in EU (01-01-2006) •
Health problems in broiler flocks
•
Sudden increase in necrotic enteritis
Necrotic enteritis
•
Enteric disease, widespread in broilers –
•
•
Global economic loss: 2 billion US$/ year
Causative agent: Clostridium perfringens Age-specific onset: 2-4 weeks post-hatch –
Maternal antibodies decline
Necrotic enteritis Clinical necrotic enteritis
•
High mortality peaks
•
Confluent mucosal necrosis in the small intestine
Necrotic enteritis Sub-clinical necrotic enteritis
• Decreased performance, no mortality • Multifocal necrosis in the small intestine • (Cholangio)hepatitis • Economic impact: 1500 US$ / flock (Skinner et al., 2010) + condemnations
Etiology Clostridium perfringens
•
Gram-positive, anaerobe rod, spore forming
•
Ubiquitous in the environment
•
Auxotrophic for 13 amino acids
•
Toxinotype
Alpha toxin
Beta toxin
Epsilon toxin +
A
+
B
+
+
C
+
+
D
+
E
+
Iota toxin
+ +
Onset of necrotic enteritis
+ not necessarily!!
PREDISPOSING FACTORS
Predisposing factors
• Coccidiosis • Feed • Others - Hygiene / shed management - Stocking density - Immunosuppression
feed ingredients • Wheat (vs corn)
Riddell and Kong, 1992
• Barley (vs corn)
Kaldhusdahl and Hofshagen, 1992
• Fishmeal (vs soy)
Truscott and Al-Sheikhly, 1977
• DDGS 20%
Barekatian et al., 2013
Dysbiosis
Gut leakage
Paracellular Pathway lumen apical
basal
Propria mucosae
Paracellular Pathway lumen apical
basal
Propria mucosae
Experimental in vivo model for sub-clinical NE d16 Gumboro vaccine Feed + fishmeal (30%)
d17
d18
d19
d20
d21
d22
d23
d24
x
x
x
x
x
x
x
x
x
x x
x
x
x
10-fold dose of Paracox-5 Oral inoculation with C. perfringens strain 56 Autopsy
x x
x
40-70% of infected birds present necrotic lesions Gholamiandehkordi et al., 2007
Experimental in vivo model for sub-clinical NE Necrotic lesions
Predisposing factors
feed ingredients quality d16 Gumboro vaccine Feed + fishmeal (30%)
d17
d18
d19
d20
d21
d22
d23
d24
x
x
x
x
x
x
x
x
x
x x
x
x
x
*10-fold dose of Paracox-5 *Oral inoculation with C. perfringens strain 56 Autopsy
x x
x
DON contaminated feed x x x x x x x x x
Timbermont et al., 2010
feed ingredients quality
ü
Mycotoxin contamination feed: multi-mycotoxin LC-MS/MS DON (µg/kg feed)
Blank - diet
DON - diet
starter diet
75 ± 22
3761 ± 1100
grower diet
83 ± 24
4281 ± 1300
finisher diet
100 ± 29
4384 ± 1300
*± SD
Percentage of birds with necrotic lesions
feed ingredients quality 50 45 40 35 30 25 20 15 10 5 0
Blank DON Blank DON + Cp + Cp
DON induces oxidative stress ANALYSIS qRT-PCR à jejunal zinc and methionine transporters
zinc transporter (ZNT) 1: ↓ DON, FBs and DON+FBs diet à regulating intracellular level of zinc (= anti-oxidant)
(Antonissen et al., 2016)
19
ionophore anticoccidials in-feed from day 1
ionophore anticoccidial
concentration
lasalocid
75 ppm
salinomycin
70 ppm
maduramicin
5 ppm
narasin
70 ppm
narasin + nicarbazin
50 ppm + 50 ppm
Lanckriet et al., 2010
ionophore anticoccidials Percentage of birds with necrotic lesions over the 3 sampling days 60%
N-M, IC: non-medicated, infected control
50%
N-M, UIC: non-medicated, uninfected control
40%
30%
P <0.007 20%
10%
0% lasalocid
Lanckriet et al., 2010
salinomycin
maduramicin
narasin
narasin + nicarbazin
N-M, IC
N-M, UIC
21
fasting d16 Gumboro vaccine Feed + fishmeal (30%)
d17
d18
d19
d20
d21
d22
d23
d24
x
x
x
x
x
x
x
x
x
x x
x
x
x
10-fold dose of Paracox-5 Oral inoculation with C. perfringens strain 56
x x
x
Autopsy Fasting for 12h/day x x x x
Tsiouris et al. 2010
fasting
2,5
Lesion score
2
1,5
1
0,5
0
N NF C CF N = control NF= control + fasting for 12h on days 16, 17, 18, 19 C = challenge Cp + Paracox 10X CF= challenge Cp + Paracox 10X + fasting for 12h on days 16, 17, 18, 19 Tsiouris et al. 2010
ionophore anticoccidials d16 Gumboro vaccine Feed + fishmeal (30%)
d17
d18
d19
d20
d21
d22
d23
d24
x
x
x
x
x
x
x
x
x
x x
x
x
x
10-fold dose of Paracox-5 Oral inoculation with C. perfringens strain 56 Autopsy
x x
x
In feed ionophore x x x x x x x x x
Lanckriet et al., 2010
Stocking density 3,5
c
3
Lesion score
2,5
b 2 1,5 1
a
1 = 15 chickens / m2 2 = 30 chickens / m2 3 = 15 chickens / m2 + C. perfringens 4 = 30 chickens / m2 + C. perfringens
Tsiouris et al. 2015
a
0,5 0 1
2
3
4
Growth rate
Dierick et al. accepted
Growth rate
Dierick et al. accepted
Growth rate
Severe lesions cause weight loss Dierick et al. accepted
Growth rate
Fast growing birds have More severe lesions Dierick et al. accepted
Fast growing birds Slow growing birds
cold stress d16 Gumboro vaccine Feed + fishmeal (30%)
d17
d18
d19
d20
d21
d22
d23
d24
x
x
x
x
x
x
x
x
x
x x
x
x
x
10-fold dose of Paracox-5 Oral inoculation with C. perfringens strain 56
x x
x
Autopsy 15°C for 12h x x x x x
Tsiouris et al. 2010
cold stress 4,5 4
*
Lesion score
3,5 3 2,5 2 1,5 1 0,5 0
N NS C CS N = control NS= control + cold stress 15°C for 12h on days 16, 17, 18, 19, 20 C = challenge Cp + Paracox 10X CS= challenge Cp + Paracox 10X + cold stress 15°C for 12h on days 16, 17, 18, 19, 20 Tsiouris et al. 2010
heat stress 40 35 30
Percentage of birds
25 20 15 10 5 0 1
2
3
4
5
6
Lesion score
Clostridium perfringens Clostridium perfringens + repeated acute heat stress (35°C, 12h, 5x ) Tsiouris et al., 2018
> 30° C (Geraert et al., 1996)
Heat stress
Reduced feed intake Altered post-absorptive carbohydrate, lipid and protein metabolism Reduced anti-oxidant capacity Increased susceptibility to disease Hypoxia and reduced blood supply to intestinal epithelium Increased intestinal permeability Endotoxaemia and inflammation Inhibition of immune response Suppression of circulating white blood cells Increased heterophil to lymphocyte ratio
(Li X. et al., 2014; Habibian M. et al., 2014)
Heat stress > 30° C (Geraert et al., 1996) Reduced feed intake Altered post-absorptive carbohydrate, lipid and protein metabolism Reduced anti-oxidant capacity Increased susceptibility to disease Hypoxia and reduced blood supply to intestinal epithelium Increased intestinal permeability Endotoxaemia and inflammation Inhibition of immune response Suppression of circulating white blood cells Increased heterophil to lymphocyte ratio (Li X. et al., 2014; Habibian M. et al., 2014)
Heat stress > 30° C (Geraert et al., 1996) Reduced feed intake Altered post-absorptive carbohydrate, lipid and protein metabolism Reduced anti-oxidant capacity Increased susceptibility to disease Hypoxia and reduced blood supply to intestinal epithelium Increased intestinal permeability Endotoxaemia and inflammation Inhibition of immune response Suppression of circulating white blood cells Increased heterophil to lymphocyte ratio (Li X. et al., 2014; Habibian M. et al., 2014)
The vicious circle of tight junction barrier disruption
(Al-Sadi et al., 2009)
Ileal Transepithelial a Electrical Resistance (TER) 12 h ACUTE HEAT STRESS 220 199.5 z
180.7yz
TER, Ω/cm2
180 136.5 y
140
138.8 y
b Column1 TN Control
PFTN Control Column2c d HS Control Column3 ®Zne Availa Column4
100
60 a b c d e
SwGr - 231
yz
Transepithelial Electrical Resistance, higher number indicates barrier function Thermal Neutral Control: 120 ppm Zn from ZnSO4; Ad libitum intake Pair-fed Thermal Neutral Control: 120 ppm Zn from ZnSO4 Heat Stress Control: 120 ppm Zn from ZnSO4 Availa-Zn: 60 ppm Zn from ZnSO4 and 60 ppm Zn from Availa-Zn zinc amino acid complex; Heat stressed LSmeans lacking a common superscript letter differ, P < 0.05
Conclusions: 1. Coccidiosis 2. Excess protein in feed 3. mycotoxins 4. Crowding 5. Fast growth rate 6. Heat stress
Gut leakage
feed ingredients
Feedstuff (%) Wheat Rye Soybean meal (48) Soybeans Rapeseed meal Animal fat Soy oil Vitamin + trace (vitamix) CaCO3 Di-Ca-phosphate NaCl Na-bicarbonate L-Lys-HCl DL-Methonine L-threonine Phytase
Starter diet
Grower diet
Finisher diet
49.29 5.00 29.37 7.50 2.00 2.50 1.00 1.000 0.820 0.650 0.264 0.104 0.160 0.256 0.071 0.020
55.62 5.00 23.16 7.50 2.00 2.60 1.00 1.000 0.908 0.361 0.226 0.157 0.175 0.208 0.064 0.020
59.00 5.00 20.11 7.50 2.00 2.70 1.00 1.000 0.826 0.107 0.268 0.101 0.154 0.167 0.049 0.020
+ 60 ppm ZnSO4 (control group) or 60 ppm zinc-aminoacid complexes (AvailaÂŽZn, Zinpro Corporation, Minnesota, USA)
Effect on villus morphology • Jejunum samples day 10 *
600
500
ila Zn A va VL
Zn SO
4
400
VL
Villuslengte (µM)
700
Effect on performance: FCR
Feed conversion ratio
1.300
* 1.200
1.100
1.000
ZnSO4 (D0-10)
Availa-Zn (D0-10)
ZnAA (D0-10)
Malondialdehyde in plasma MDA D10 **
18 16 14
va ila Zn A
4
12
Zn SO
MDA (nmol/mL)
20
Conclusions: 1. Coccidiosis 2. Excess protein in feed 3. mycotoxins 4. Crowding
Oxidative stress to the enterocytes
5. Fast growth rate 6. Heat stress
Gut leakage Clostridium XXXXX