Clostridium Perfringens Alpha Toxin and Netb Toxin Derivatives As Vaccine Antigens for Chickens
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Clostridium perfringens alpha toxin and NetB toxin derivatives as vaccine antigens for chickens DORIEN MOT Dissertation submitted in fulfillment of the requirements for the degree of Doctor in Veterinary Sciences (PhD), Faculty of Veterinary Medicine, Ghent University, December 2016 Promotors Prof. Dr. Ir. F. Van Immerseel Prof. Dr. R. Ducatelle Faculty of Veterinary Medicine Department of Pathology, Bacteriology and Poultry Diseases Table of contents Table of contents ............................................................................................................................. 3 Abbreviation list ............................................................................................................................. 5 Symbols ........................................................................................................................................... 8 Chapter 1. General introduction ................................................................................................... 9 1.1. Necrotic enteritis in broiler chickens ........................................................................................... 11 1.2. Clostridium perfringens, the causative agent ............................................................................... 12 1.2.1. Alpha toxin .......................................................................................................................... 15 1.2.2. NetB .................................................................................................................................... 17 1.3. Clinical symptoms and pathogenesis ............................................................................................ 19 1.4. Antibody responses to C. perfringens antigens ............................................................................ 21 1.5. The rise of necrotic enteritis and the consequences ..................................................................... 22 1.6. Preventive and curative treatments for necrotic enteritis ............................................................. 23 1.7. Vaccination against necrotic enteritis in broiler chickens ............................................................ 24 1.7.1. An overview of vaccination studies against necrotic enteritis ........................................... 24 1.7.1.1. Live attenuated vaccines ................................................................................................ 24 1.7.1.2. Protein-based vaccines ................................................................................................... 25 1.7.1.2.1. Crude supernatant vaccines ................................................................................. 25 1.7.1.2.2. Alpha toxin .......................................................................................................... 26 1.7.1.2.3. NetB toxin ........................................................................................................... 28 1.7.1.2.4. Other proteins ...................................................................................................... 28 1.7.1.3. Attenuated live vectors expressing C. perfringens proteins ........................................... 29 Chapter 2. Scientific Aims ........................................................................................................... 47 3 Chapter 3. Experimental studies ................................................................................................. 51 3.1. Day-of-hatch vaccination is not protective against necrotic enteritis in broiler chickens .... 53 3.2. Identification of immunogenic Clostridium perfringens supernatant proteins of strain 23...73 3.3. Variable protection against experimental broiler necrotic enteritis after immunisation with the C-terminal fragment of Clostridium perfringens alpha-toxin and a non-toxic NetB variant . 85 Chapter 4. General Discussion .................................................................................................. 109 Summary ..................................................................................................................................... 127 Samenvatting .............................................................................................................................. 133 Curriculum Vitae ....................................................................................................................... 139 Bibliography ............................................................................................................................... 143 Dankwoord .................................................................................................................................. 147 4 List of abbreviations AGP’s antimicrobial growth promotors BHI brain heart infusion BCA bicinchoninic acid BLAST basic local alignment search tool BP base pair CFU colony forming units CN/DAB chloronaphthol- and diaminobenzidine-based CP Clostridium perfringens CPA alpha toxin CPE Clostridium perfringens enterotoxin DNA deoxyribonucleic acid EF-G elongation factor G EF-Tu elongation factor Tu ELISA enzyme-linked immunosorbent assay EU European Union FBA Fructose-1,6-biphosphate aldolase FPV Recombinant fowl poxvirus GAPDH glyceraldehyde-3-phosphate dehydrogenase GMO genetically modified organisms GST glutathione S-transferase HP hypothetical protein HRP horseradish peroxidase 5 HVT herpesvirus of turkey Ig immunoglobulin IM intramuscularly IPTG isopropyl-β-D-thiogalactopyranoside kDa Kilo Dalton LMH leghorn male hepatoma cell line MALDI TOF Matrix assisted laser desorption ionization time-of-flight MWCO molecular weight cut-off Naglu N-acetylglucosaminidase NCBI National center for biotechnology information NE necrotic enteritis NetB necrotic Enteritis Toxin B-like netB gene encoding NetB toxin Ni-NTA nitrilotriacetic acid bound Ni+2 ions OD optical density ON overnight ORF open reading frame PBS phosphate buffered saline PCR polymerase chain reaction PD prepacked disposable PES polyether sulfone PFO perfringolysin O PFT pore-forming toxin PFOR pyruvate:ferredoxin oxidoreductase Pgm phosphoglyceromutase 6 PVDF polyvinylidene difluoride rNetB recombinant NetB RT room temperature SC subcutaneous SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM standard error of the mean SN supernatant TB terrific broth TBS tris-buffered saline TCP total combining power tHP truncated hypothetical protein TMB tetramethylbenzidine TpeL toxin perfringens large UV-Vis ultraviolet-visible v/w volume/weight 7 Symbols > greater than TM trade mark α alpha β beta ε epsilon ι iota µ micro 8 Chapter 1 General introduction Chapter 1: General introduction General introduction 1.1. Necrotic enteritis in broiler chickens Necrotic enteritis is an important disease of broiler chickens, caused by the bacterium Clostridium perfringens. It is one of the gastrointestinal diseases in poultry that has gained worldwide importance during the last decade. Gastrointestinal diseases in broilers, including necrotic enteritis, viral enteritis, coccidiosis and syndromes such as dysbiosis and malabsorption, have become increasingly important worldwide for multiple reasons. First, high-density floor-housing supports easy spread of excreted gut pathogens (Guardia et al., 2011). Secondly, due to improvements in genetics, broilers have become incredibly efficient in converting feed into body mass and the gastro-intestinal tract of these animals is highly efficient in absorbing nutrients (Havenstein et al., 2003). Gut micro-organisms play an essential role in degradation of feed components. There is a complex interplay between gut bacteria and the gastro- intestinal mucosa, that can be beneficial or harmful for the host, depending on the microbial composition (Valeria et al., 2011). Nutritionists are also constantly looking for improving the limits of digestibility. As a side effect, gut health problems have arisen, related to shifts in enteric bacterial populations and bacterial overgrowth. Excess feed nutrients in the gut are used by facultative pathogenic micro-organisms, such as certain Clostridium perfringens (C. perfringens) strains. Shifts in enteric populations also lead to dysbacteriosis, a hitherto poorly defined syndrome causing inflammatory reactions in the gut resulting in poor performance and an increased feed conversion ratio (Teirlynck et al., 2011). It has also been shown that genetics play a role in the development of the intestine, which can affect the microbiota composition (Lumpkins et al., 2010). There is thus an interplay between host genetics and feed utilization that is important in either preventing or causing gastrointestinal problems. Another issue that is important in this context is the public and governmental pressure to reduce the use of antibiotics in broilers. The traditional antimicrobial growth promotors (AGP’s), used in the past not only to improve feed conversion ratios and body weight gain, but also to prophylactically control diseases such as necrotic enteritis, have been banned 11 Chapter 1: General introduction in the European Union in 2006. Consumers in other countries are also putting pressure on the poultry industry to rear animals without AGP’s. Therapeutic antibiotics are nowadays widely used for preventive and curative control of gastro-intestinal pathologies and their preventative use is heavily disputed. All of these factors have led to the emergence of necrotic enteritis in broilers, caused by chicken-adapted