PREDISPOSING EFFECT OF THE MYCOTOXINS DEOXYNIVALENOL AND FUMONISINS ON NECROTIC ENTERITIS IN BROILER CHICKENS Gunther Antonissen Dissertation submitted in fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Veterinary Science 2015 Promoters Prof. dr. S. Croubels Prof. dr. A. Martel Prof. dr. F. Van Immerseel Department of Pharmacology, Toxicology and Biochemistry Department of Pathology, Bacteriology and Avian Diseases Faculty of Veterinary Medicine Ghent University Gunther Antonissen (2015) Predisposing effect of the mycotoxins deoxynivalenol and fumonisins on necrotic enteritis in broiler chickens Cover: Julie Muyle Print: University Press, Zelzate This thesis was funded by a PhD fellowship from Biomin GmbH, Herzogenburg, Austria The author and promoters give the permission to consult and to copy parts of this work for personal use only. Every other use is subject to copyright laws. Permission to reproduce any material should be obtained from the author. www.mytox.be “Learn how to see. Realize that everything connects to everything else.” Leonardo da Vinci TABLE OF CONTENTS LIST OF ABBREVIATIONS ................................................................................................. 1 GENERAL INTRODUCTION ............................................................................................... 7 1 Broiler chickens .................................................................................................................. 9 1.1 The gastrointestinal tract of broiler chickens ................................................................ 9 2 Mycotoxins ........................................................................................................................ 11 2.1 The mycotoxin problem .............................................................................................. 11 2.2 Deoxynivalenol and Fumonisins ................................................................................ 15 2.2.1 Chemical structure ............................................................................................... 15 2.2.2 Toxicokinetics ..................................................................................................... 17 2.2.3 Toxicodynamics .................................................................................................. 19 2.2.4 Impact on broiler performance ............................................................................ 22 2.2.5 Effect of DON and FBs at the gastrointestinal level ........................................... 23 2.3 Mycotoxin management ............................................................................................. 30 2.4 The impact of Fusarium mycotoxins on animal host susceptibility to infectious diseases ............................................................................................................................ 31 3 Necrotic enteritis in broiler chickens .............................................................................. 38 3.1 Etiology ...................................................................................................................... 38 3.2 Pathogenesis ................................................................................................................ 39 3.2.1 Toxin production ................................................................................................. 39 3.2.2 Single strain dominance ...................................................................................... 40 3.2.3 Adhesion to the intestinal epithelium .................................................................. 41 3.2.4 Predisposing factors............................................................................................. 41 3.3 Clinical signs ............................................................................................................... 44 3.4 Pathological lesions .................................................................................................... 44 3.4.1 Macroscopic observations ................................................................................... 44 3.4.2 Microscopic observations .................................................................................... 45 3.5 Control of necrotic enteritis ........................................................................................ 46 SCIENTIFIC AIMS ............................................................................................................... 47 EXPERIMENTAL STUDIES ............................................................................................... 51 CHAPTER 1 The predisposing effect of deoxynivalenol on necrotic enteritis in broiler chickens 53 CHAPTER 2 The predisposing effect of fumonisins on necrotic enteritis in broiler chickens ....... 73 CHAPTER 3 The impact of deoxynivalenol and fumonisins on the intestinal barrier.................... 99 3.1 Impact of deoxynivalenol and fumonisins on intestinal micro-environment ............. 99 3.2 Impact of chronic exposure to deoxynivalenol on the absorption of fumonisins .... 127 DISCUSSION ....................................................................................................................... 141 REFERENCES ..................................................................................................................... 159 SUMMARY ........................................................................................................................... 199 SAMENVATTING ............................................................................................................... 207 CURRICULUM VITAE ...................................................................................................... 215 BIBLIOGRAPHY ................................................................................................................ 219 DANKWOORD .................................................................................................................... 231 LIST OF ABBREVIATIONS 3ADON 3-acetyldeoxynivalenol 15ADON 15-acetyldeoxynivalenol 5-HIAA 5-hydroxyindoleacetic acid 5-HT 5-hydroxytryptamine 1-P 1-phosphate αHF alpha-hemolysin ABA aminobenzoic agent ACN acetonitrile ADME absorption, distribution, metabolism and excretion AFs aflatoxins AFB1 aflatoxin B1 AGP antibiotic growth promoters AIMD apparent ileal methionine digestibility AMP aerobic mesophilic bacteria APEC avian pathogenic Escherichia coli AUC0-t area under the plasma concentration-time curve from time 0 to t AXOS arabinoxylan oligosaccharides BLASTN nucleotide basic local alignment search tool β-PFT β-pore forming toxin BW body weight BWG body weight gain CaCo human colon adenocarcinoma cell line CKK cholecystokinin CCLi1 chemokines C-C motif ligand 1 inflammatory Cl total body clearance CLDN3 claudin 3 CLDN4 claudin 4 CLDN5 claudin 5 Cl/F clearance divided by the absolute oral bioavailability Cmax maximal plasma concentration Ct treshold cycle values CXCLi1 chemokine C-X-C motif ligand 1 inflammatory 1 CXCLi2 chemokine C-X-C motif ligand 2 inflammatory CYP450 cytochrome P450 DGGE denaturing gradient gel electrophoresis DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate DOM-1 de-epoxy-DON DON deoxynivalenol DP declustering potential dsDNA double-stranded DNA ECMM extracellular matrix molecules ELEM equine leukoencephalomalacia ERK1/2 extracellular signal regulated protein kinase 1 and 2 ETEC enterotoxigenic Escherichia coli EU European Union EUR euro F oral bioavailability FBs fumonisins FB1 fumonisin B1 FB2 fumonisin B2 FB3 fumonisin B3 FCR feed conversion ratio FHB Fusarium Head Blight Disease FOS fructo-oligosaccharides FRC feed conversion rate Fuc fucose Gal galactose GalNac N-acetylgalactosamine GlcNac N-acetylglucosamine GLUT glucose transporter protein GSH glutathione Hck hematopoietic cell kinase HFB1 hydrolyzed FB1 HIF-1α hypoxia inducible factor 1 subunit alpha HMOX heme-oxygenase 2 HPLC-FL high performance liquid chromatography with fluorescence detection Hsp heat shock protein HT-29 human colonic and rectal adenocarcinoma cell line HT-29 16E human colonic goblet cell line IARC International Agency for Research on Cancer IEC intestinal epithelial cell IFN-Ɣ interferon-γ Ig immunoglobulin IgA immunoglobulin A IL interleukin IPEC porcine IEC IS internal standard Isc current IV intravenous JNK1/2 c-Jun N-terminal kinase 1 and 2 kel elimination rate constant LAB lactic acid bacteria LC-MS/MS liquid chromatography-tandem mass spectrometry LD50 lethal dose for 50% of the subjects LOAEL lowest-observed-adverse-effect level LOD limit of detection LOQ limit of quantification Man mannose MAPKs mitogen-activated protein kinases MCP-1 monocyte chemo-attractant protein-1 MDA malondialdehyde MDR1 multi-drug resistance protein 1 – P-glycoprotein MOS mannan-oligosaccharides MRP2 multi-drug resistance-associated protein 2 mRNA messenger ribonucleic acid MRT mean residence time MS mass spectrometry MUC1 intestinal membrane-associated mucin MUC2 secretory mucin 3 MUC3 secretory mucin NE necrotic enteritis NELoc1 major pathogenicity locus NetB NE β-like toxin NeuAc N-acetyl-neuraminic acid NIV nivalenol NSPs non-starch polysaccharides OPA ortho-phthalaldehyde OTA ochratoxin A OTU operational taxonomic unit PAM porcine alveolar macrophage cell line pBD1 porcine β-defensin 1 pBD2 porcine β-defensin 2 PBS phosphate buffered saline PCR polymerase chain reaction PD potential difference pHFB1 partially hydrolyzed FB1 PKR RNA-activated protein kinase