aviFORUM-CARNE 2019: Ponencia Richard Ducatelle

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