the role of protein and amino acid nutrition in controlling clostridium perfringens in the ...

217 Pages·2012·2.19 MB·English

THE ROLE OF PROTEIN AND AMINO ACID NUTRITION IN
CONTROLLING CLOSTRIDIUM PERFRINGENS IN THE
GASTROINTESTINAL TRACT OF BROILER CHICKENS

A Thesis Submitted to the College of
Graduate Studies and Research
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
in the Department of Animal and Poultry Science
University of Saskatchewan
Saskatoon, Saskatchewan, Canada

by
Gemunu Prasanna Widyaratne

© Copyright Gemunu Prasanna Widyaratne, November 2012. All Rights Reserved.
PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a Postgraduate
degree from the University of Saskatchewan, I agree that the Libraries of this University
may make it freely available for inspection. I further agree that permission for copying
of this thesis in any manner, in whole or in part, for scholarly purposes may be granted
by the professor or professors who supervised my thesis work or, in their absence, by the
Head of the Department or the Dean of the College in which my thesis work was done.
It is understood that any copying or publication or use of this thesis or parts thereof for
financial gain shall not be allowed without my written permission. It is also understood
that due recognition shall be given to me and to the University of Saskatchewan in any
scholarly use which may be made of any material in my thesis.
Requests for permission to copy or to make other use of material in this thesis in
whole or part should be addressed to:
Head of the Department of Animal and Poultry Science
University of Saskatchewan
Saskatoon SK, Canada, S7N 5A8

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ABSTRACT
A series of experiments were conducted to examine the effect of dietary protein and
amino acids (AA) on intestinal Clostridium (C.) perfringens proliferation and necrotic
enteritis (NE) in broiler chickens.
The effects of dietary protein level and protein digestibility on the growth
performance and carcass characteristics of broilers were evaluated first. Low protein
(LoPro) diets supported growth performance equal to high protein (HiPro) diets when
highly digestible protein supplements were incorporated. Birds fed HiPro yielded more
breast meat than those fed LoPro while birds fed diets with highly digestible (HiDig)
protein supplements accumulated more abdominal fat than those fed diets containing
low digestible (LoDig) protein ingredients.
The next experiment focused on the development of a feasible experimental
model to induce NE experimentally in broilers to investigate potential dietary
approaches. Different modes of challenge (oral gavage, in-feed and in-water) with
different doses of C. perfringens inoculums (2 or 4 ml) were tested using
microbiological, pathological and hematological parameters. The findings suggested the
possibility to applly any of the evaluated treatments to induce the disease in broilers
raised on litter.
The next experiment was conducted to examine the effects of dietary protein
level and digestibility on the growth performance and intestinal ecosystem of C.
perfringens challenged and unchallenged broilers. The results of this experiment again
revealed the ability of LoPro diets to maintain the growth performance of broilers as
with HiPro diets, when HiDig protein supplements were included. The findings further
demonstrated significant reduction in the intestinal C. perfringens numbers and severity
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of intestinal lesions of infected broilers when LoPro diets and HiDig protein
supplements were fed.
The last experiment was performed to assess the methionine requirement for
optimum growth in broiler chickens fed diets supplemented with either DL-methionine
(DLM) or 2-hydroxy-4-methylthiobutanoic acid (HMB) under C. perfringens infected
and uninfected situations. None of the methionine sources affected the growth of C.
perfringens in the broiler GIT, however, infected birds needed more dietary methionine
to achieve maximum growth when DLM, but not HMB, was included in the diet.
Based on the overall findings it can be concluded that the dietary approaches
tested in this project may support our efforts in the development of nutritional strategies
to minimize the impact of C. perfringens on broiler production in an antibiotic free
livestock industry.

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ACKNOWLEDMENTS
I would first and foremost like to express my sincere gratitude and appreciation to my
supervisor Dr. M. D. Drew, for all his advice, guidance and encouragement throughout
the program. I am greatly thankful to Dr. A. G. Van Kessel, who took the time out to
help me when I was in need. I would also like to thank Drs. B. Laarveld, H. L. Classen,
M. Chirino-Trejo, and P. Leterme for serving on my advisory committee, and Dr. D. R.
Korver for serving as my external examiner.
I would like to acknowledge the Department of Animal and Poultry Science and
College of Graduate Studies and Research for generous scholarships I received during
my program. None of this research would have been possible without the financial
support for this project from Natural Sciences and Engineering Research Council of
Canada (NSERC) and Evonik-Degussa GmbH. I am very grateful for their involvement.
I wish to express my sincere thanks to Monique Burmester, Paula Mason and all
other employees at the Animal Care Unit where I conducted all my animal trials. I
would also like to acknowledge the excellent technical assistance given to me by Jason
Marshall. Thanks to my fellow graduate students for their substantial cooperation,
support and friendship over the past few years.
I would like to express my sincerest gratitude to my parents, Eddie and
Somalatha Widyaratne for having sown in me the principles of discipline, respect and
admiration for life. Finally but most importantly, I would like to thank my wonderful
wife Dushmanthi, loving daughter Amaya and cute son Dinel. Thank you for your love,
support and patience! Dushmanthi, you encouraged and convinced me to accept
challenges I contemplated I would never succeed, and I too will always be there to
support you in whatever targets you set out to accomplished.
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DEDICATION
With a great respect and heartfelt gratitude, this work is dedicated to my school Rahula
College, Matara – Sri Lanka, and all my past teachers.

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TABLE OF CONTENT

PERMISSION TO USE ................................................................................................................. I
ABSTRACT .................................................................................................................................. II
ACKNOWLEDMENTS ............................................................................................................. IV
DEDICATION .............................................................................................................................. V
TABLE OF CONTENT .............................................................................................................. VI
LIST OF TABLES ....................................................................................................................... X
LIST OF FIGURES .................................................................................................................. XII
1. INTRODUCTION ..................................................................................................................... 1
2. LITERATURE REVIEW ......................................................................................................... 3
2.1. Necrotic enteritis in broiler chickens....................................................................... 3
2.1.1. The disease ........................................................................................................... 3
2.1.2. The pathogen ........................................................................................................ 4
2.1.3. Pathogenesis ......................................................................................................... 6
2.1.4. Clostridium perfringens virulence factors ........................................................... 9
2.2. Experimental induction of necrotic enteritis ........................................................ 14
2.3. Clostidium perfringens and broiler growth performance .................................... 16
2.4. Control using antibiotics ........................................................................................ 17
2.5. Alternative strategies .............................................................................................. 19
2.6. Predisposing factors ................................................................................................ 20
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2.6.1. Cereal type ......................................................................................................... 21
2.6.2. Exogenous enzymes ........................................................................................... 25
2.6.3. Non-nutritional ingredients ................................................................................ 28
2.6.5. Feed processing and feeding .............................................................................. 30
2.7. Dietary protein, amino acids and necrotic enteritis ............................................. 31
2.7.1. Low protein diets................................................................................................ 35
2.7.2. High protein diets ............................................................................................... 40
2.7.3. Amino acid digestibility and availability ........................................................... 42
2.8. Digestible amino acid values in broiler ration formulation ................................ 43
2.9. Summary .................................................................................................................. 44
3. EFFECTS OF PROTEIN LEVEL AND DIGESTIBILITY ON THE GROWTH
AND CARCASS CHARACTERISTICS OF BROILER CHICKENS .................................. 47
3.1. Abstract .................................................................................................................... 47
3.2. Introduction ............................................................................................................. 48
3.3. Materials and methods ........................................................................................... 49
3.3.1. Experiment 1 ...................................................................................................... 50
3.3.2. Experiment 2 ...................................................................................................... 51
3.3.3. Chemical analysis............................................................................................... 59
3.3.4. Calculations and statistical Analysis .................................................................. 59
3.4. Results ...................................................................................................................... 61
3.5. Discussion ................................................................................................................. 70
4. EVALUATION OF MICROBIOLOGICAL, PATHOLOGICAL AND
HEMATOLOGICAL PARAMETERS IN AN EXPERIMENTAL DISEASE
MODEL FOR NECROTIC ENTERITIS IN BROILER CHICKENS RAISED ON
LITTER ........................................................................................................................................ 76
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4.1. Abstract .................................................................................................................... 76
4.2. Introduction ............................................................................................................. 77
4.3. Materials and methods ........................................................................................... 79
4.3.1. Birds and management ....................................................................................... 79
4.3.2. Feed and feeding ................................................................................................ 80
4.3.3. Preparation of innoculum ................................................................................... 80
4.3.4. Experimental design and challenge procedure ................................................... 85
4.3.5. Sample and data collection................................................................................. 87
4.3.6. Whole-blood chemiluminescence assay ............................................................ 88
4.3.7. Pathological examination ................................................................................... 89
4.3.8. Genomic DNA extraction and purification ........................................................ 89
4.3.9. Bacterial enumeration ........................................................................................ 92
3.3.10. Statistical analysis ............................................................................................ 94
4.5. Results ...................................................................................................................... 94
4.6. Discussion ............................................................................................................... 100
5. EFFECTS OF DIETARY PROTEIN LEVEL AND PROTEIN DIGESTIBILITY
ON THE GROWTH PERFORMANCE AND INTESTINAL ECOSYSTEM OF
BROILER CHICKENS INFECTED WITH CLOSTRIDIUM PERFRINGENS ................ 111
5.1. Abstract .................................................................................................................. 111
5.2. Introduction ........................................................................................................... 112
5.3. Materials and methods ......................................................................................... 115
5.3.1. Birds and management ..................................................................................... 115
5.3.2. Experimental design ......................................................................................... 116
5.3.3. C. perfringens challenge procedure ................................................................. 116
5.3.4. Sample and data collection............................................................................... 117
5.3.5. Pathological examination ................................................................................. 117
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5.3.6. Genomic DNA extraction and purification ...................................................... 118
5.3.7. Bacterial enumeration ...................................................................................... 118
5.3.8. Volatile fatty acid and ammonia analyses ........................................................ 118
5.3.9. Statistical analysis ............................................................................................ 119
5.4. Results .................................................................................................................... 120
5.5. Discussion ............................................................................................................... 133
6. ORAL CLOSTRIDIUM PERFRINGENS CHALLENGE AFFECTS THE
METHIONINE REQUIREMENTS OF BROILER CHICKENS ........................................ 144
6.1 Abstract ................................................................................................................... 144
6.2. Introduction ........................................................................................................... 145
6.3. Materials and methods ......................................................................................... 147
6.3.1. Birds and management ..................................................................................... 147
6.3.2. Dietary treatments ............................................................................................ 148
6.3.3. C. perfringens challenge procedure ................................................................. 152
6.3.4. Experimental design ......................................................................................... 153
6.3.5. Sample and data collection............................................................................... 153
6.3.6. Pathological examination ................................................................................. 153
6.3.7. Genomic DNA extraction and purification ...................................................... 154
6.3.8. Bacterial enumeration ...................................................................................... 154
6.3.9. Statistical analysis ............................................................................................ 154
6.4. Results .................................................................................................................... 154
6.5. Discussion ............................................................................................................... 156
7. GENERAL DISCUSSION ................................................................................................... 164
8. REFERENCES ...................................................................................................................... 169

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