Thesis

MOLECULAR CHARACTERISATION, ATTENUATION AND INACTIVATION
OF VERY VIRULENT INFECTIOUS BURSAL DISEASE VIRUS FOR THE
DEVELOPMENT OF TISSUE CULTURE-BASED VACCINES
By
MAJED H. MOHAMMED
Thesis Submitted to the School of Graduate Studies, Universiti Putra
Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of
Philosophy
July 2010

2
DEDICATED WITH LOVE AND GRATITUDE
TO:
MY DEAREST (THE SPIRIT OF MY FATHER), MOTHER, WIFE (MAYADA),
TWO LOVELY SONS (ALI AND MOHAMMED)
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in
fulfilment of the requirement for the degree of Doctor of Philosophy

3
MOLECULAR CHARACTERISATION, ATTENUATION AND INACTIVATION
OF VERY VIRULENT INFECTIOUS BURSAL DISEASE VIRUS FOR THE
DEVELOPMENT OF TISSUE-CULTURE BASED VACCINES
By
MAJED H. MOHAMMED
July 2010
Chairman: Profesor Dr. Mohd Hair Bin Bejo, PhD
Faculty: Veterinary Medicine
Infectious bursal disease (IBD), an economically important infectious viral
disease of poultry, is caused by IBD virus (IBDV) belonging to Avibirnavirus
genus of Birnaviridae family. The disease causes considerable mortality and
immunosuppression. Emergence of very virulent IBDV (vvIBDV) strains in
different parts of the world in late 1980‟s including Malaysia in 1991, have
demanded further research efforts in understanding the added complexicity of
the disease process and the means to control and prevent outbreaks of the
disease. Treatment of IBD is of no value and the disease can only be controlled
and prevented by proper vaccination programme and biosecurity. It was the
objectives of the study to determine the molecular characteristics and effects of
attenuation and inactivation of Malaysian field isolates of vvIBDV for tissue
culture based IBD vaccines development. Three IBDV isolates identified as
UPM04190, UPM94273 and UPM0081 with an accession number of AY791998,
AF527039 and EF208038, respectively were propagated in specific-pathogenic-
free (SPF) embryonated chickens egg via chorioallontoic membrane (CAM) for

4
three times and infected onto two types of continuous cell line namely the DF-1
and Vero cell lines. The UPM0081 vvIBDV isolate successfully infected these
cells while the other vvIBDV isolates failed. The virus was passaged serially 20
and 9 times in Vero cells and DF-1 cell lines, respectively. The cytopathic effects
(CPEs) were observed and virus from each passage was confirmed through
indirect immunoperoxidase staining test. The UPM0081 was adapted to Vero
cells and DF-1 cells line in fourth and third passage, respectively.
The molecular characteristics of the virus at different passages in Vero cells and
two passages in DF-1 cell line were characterized by using reverse transcriptase
polymerase chain reaction (RT-PCR). The nucleotide base sequence of a 643
bp fragment of genome segment A containing the partial coding sequence of
VP2 and the entire hyper-variable region were determined. No apparent
changes by sequence analysis of selected passage in VP2 gene at passage 5
(UPM0081T5) and passage 7 (UPM0081T7) in Vero cells and DF-1 cell line.
One amino acid substitution change occurred in passage 8 (UPM0081T8) and
passage 9 (UPM0081T9): 222 (A to P). Further changes in the VP2 gene were
recorded in passage 10 (UPM0081T10), passage 15 (UPM0081T15), and
passage 20 (UPM0081T20) 222: (A to P), 242 (I to V), 253 (Q to H), 256 (I to V),
279: (D to N), 284: (A to T), 294 (I to L), 326 (S to L), and 330 (S to R). Amino
acid substitution at positions 279 (D to N) and 284 (A to T) were commonly
found in the attenuated IBDV strains.

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The pathogenicity and immunogenicity properties of the UPM0081 vvIBDV
passages 10, 15 and 20 isolates on Vero cells were evaluated in this study. The
results revealed that only UPM0081T10 was still pathogenic to SPF chickens. It
caused clinical signs, gross lesions, 25% mortality and histological changes in
bursa of Fabricius. Neither clinical signs nor gross lesions were observed in the
SPF chickens inoculated with either UPM0081T15 or UPM0081T20. Efficacy
test demonstrated that both UPM0081T15 and UPM0081T20 could provide
100% protection in highly susceptible SPF chickens when challenged with
vvIBDV (UPM0081) at virus titer of 107.8
ELD50/0.1 mL per chicken.
The UPM0081T15 and UPM0081T20 IBDV isolates were inactivated using
either Binary ethyleneimine (BEI) or Electrolysed water-Catholyte-Anolyte
(ECA). Complete inactivation of UPM0081T15 with titer of 106.7
TCID50/0.1 mL
and UPM0081T20 with titer of 107.4
TCID50/0.1ml occurred after 24 hours with
either BEI or ECA. The inactivated viruse suspension and an equal volume of
Freund‟s incomplete adjuvant were mixed together (water-in-oil) emulsion and
injected subcutaneously into 42-day-old SPF chickens to determine the safety
and immunogenicity of the inoculum. Neither clinical signs nor gross lesions
were observed in both groups of chickens before and after vvIBDV challenged.
High and protective level IBD antibody titer was recorded more in BEI than ECA
groups at 2 weeks post infection and 2 weeks post challenged. The study
showed that both the inactivated UPM0081T15 and UPM0081T20 either in BEI
or ECA was safe and could provide 100% protection against vvIBDV challenged

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with titer of 107.8
EID50/ 0.1 mL, while that of ECA could not protect fully SPF
chicken against bursal lesion.
In conclusion, vvIBDV UPM0081 was successfully adapted and attenuated in
continuous cell line (Vero cells) after fifteen and twenty passages. The
attenuated and inactivatted local vvIBDV named UPM0081T15 and
UPM0081T20 conferred full protection to the immunized SPF chickens against
vvIBDV.
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia
sebagai memenuhi keperluan untuk ijazah Doktor Falsafah
PENCIRIAN SECARA MOLEKUL, PELEMAHAN DAN INAKTIVASI VIRUS
PENYAKIT BERJANGKIT BURSA YANG AMAT VIRULEN UNTUK
PEMBANGUNAN VAKSIN YANG BERASASKAN KULTUR TISU

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Oleh
MAJED H. MOHAMMED
July 2010
Pengerusi: Profesor Dr. Mohd Hair Bejo, PhD
Fakulti: Perubatan Veterinar
Wabak penyakit infeksi bursa (IBD) adalah sejenis jangkitan virus yang menular
di kalangan ayam dan menjejas kepentingan ekonomi. Penyakit ini adalah
disebabkan oleh virus penyakit bursa berjangkit (IBDV) yang tergolong dalam
genus Avibirnavirus dari keluarga Birnaviridae. IBD menyebabkan kadar
kematian yang tinggi serta boleh melemahkan imun dan daya tahan untuk
melawan penyakit. Kehadiran strain yang amat virulen IBDV (vvIBDV) di serata
dunia pada penghujung tahun 1980an, termasuk di negara Malaysia dalam
tahun 1991 telah meningkatkan keperluan kajian penyelidikan demi memahami
proses jangkitan yang kompleks serta mengenalpasti kaedah untuk mengawal
dan mencegah penyakit ini. Rawatan perubatan tidak akan memberi kesan
kecuali dengan kaedah vaksinasi serta biosekuriti. Objektif penyelidikan ini
adalah untuk membuat pencirian di peringkat molekul serta mengesan kesan
sampingan daripada proses pelemahan vvIBDV di kalangan isolat IBDV dari
Malaysia dalam sel kultur untuk tujuan perkembangan vvIBDV vaksin. Tiga
IBDV asingan tempatan yang dinamakan UPM04190, UPM94237 and
UPM0081 dengan nombor perolehan AY791998, AF527039 and EF208038
telah di biak ke dalam telur ayam spesifik-pathogen-bebas (SPF) melalui
disuntikan ke dalam membran korioalontoik (CAM) sebanyak tiga kali serta telah

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suntik ke dalam dua jenis sel kultur jenis berurutan iaitu sel DF-1 dan sel Vero.
Isolat UPM0081 vvIBDV telah berjaya menyebabkan jangkitan di dalam sel
tersebut manakala asingan yang lain gagal disesuaikan ke dalam sel kultur.
Virus tersebut telah di pasage sebanyak dua puluh kali di dalam sel Vero dan
sebanyak sembilan kali di dalam sel DF-1. Kesan sitopatik (CPEs) telah
dikesan dan setiap virus dari setiap pasage telah dikenal pasti melalui ujian
imunoperoxidase tidak terus. UPM0081 telah diadaptasi ke dalam sel Vero pada
pasage yang ke empat dan di dalam sel DF-1 pada pasage yang ke tiga.
Pencirian molekul virus pada waktu yang berbeza di dalam sel Vero dan dua
pasage di dalam sel DF-1 telah dikaji melalui tindak balas transkripsi balik reaksi
rangkaian polimerasi (RT-PCR). Rangkaian nukleotida pada kedudukan 643 bp
dalam genom segmen A mempunyai separa kodon gen protein virus 2 (VP2)
dan juga seluruh bahagian variable tinggi telah dikesan. Analisis jujukan
menunjukkan beberapa pasage di dalam gen VP2 gene pada pasage 5
(UPM0081T5) dan juga pasage 7 (UPM0081T7) di dalam sel Vero dan DF-1
tidak menunjukkan sebarang perubahan. Seterusnya satu perubahan
melibatkan penukaran asid amino telah berlaku di dalam pasage 8
(UPM0081T8) dan juga pasage 9 (UPM0081T9) 222 (A to P). Perubahan
seterusnya di dalam gen VP2 telah dikesan di dalam pasage 10 (UPM0081T10),
15 (UPM0081T15), 20 (UPM0081T20): 222 (A to P), 242 (I to V), 253 (Q to H),
256 (I to V), 279 (D to N), 284 (A to T), 294 (I to L), 326 (S to L) dan juga 330 (S
to R). Perubahan asid amino pada kedudukan 279 (D to N) dan 284 (A to T)
kerap di kesan dalam IBDV strain yang lemah.

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Kepatogenan dan keimunan UPM0081 vvIBDV pasage 10, 15 and 20 isolat
dalam sel Vero telah dikaji dalam kajian ini. Keputusan kajian menunjukkan
bahawa hanya UPM0081T10 masih menampilkan cirri-ciri patogenisitinya di
dalam ayam SPF. Jangkitan dengan strain ini mengakibatkan kesan klinikal
termasuk pembentukan lesi, 25% kematian dan juga perubahan patologi di
dalam bursa Fabricius. Walaubagaimanapun, pemerhatian yang sama tidak
berlaku dengan strain UPM0081T15 ataupun UPM0081T20.
Ujian keberkesanaan telah menunjukkan bahawa UPM0081T15 dan juga
UPM0081T20 boleh memberi 100% perlindungan ke atas ayam SPF yang
sangat sesuai menerima jangkitan apabila disuntik dengan vvIBDV (UPM0081)
mengunakan virus titer 107.8
ELD50/0.1 mL untuk setiap ayam.
UPM0081T15 dan UPM0081T20 IBDV isolat telah dibunuh dengan
mengunakan Binary ethyleneimine (BEI) atau Electrolysed water-Catholyte-
Anolyte (ECA). UPM0081T15 dengan virus titer 106.5
TCID50/0.1 mL dan
UPM0081T20 dengan virus titer 107
TCID50/0.1mL telah dikesan mati
sepenuhnya seawal 24 jam dengan menggunakan BEI atau ECA. Virus yang
telah dibunuh berserta adjuvan Freund‟s tidak lengkap dalam kuantiti yang
sama telah di campurkan dan disuntik di bawah kulit ayam SPF berumur 42 hari
ke dalam ayam SPF untuk menguji kepatogenan dan keimunan inokulum.
Kedua dua kumpulan ayam tidak menunjukkan sebarang perubahan klinikal
selepas infeksi dengan vvIBDV. Kadar antibodi yang tinggi dan melindung

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telah direkod dengan mengunakan BEI berbanding ECA pada minggu ke dua
selepas suntikan dan minggu ke dua selepas infeksi dengan vvIBDV.
Kajian penyelidikan ini menunjukkan bahawa UPM0081T15 dan UPM0081T20
yang telah dibunuh dengan mengunakkan BEI ataupun ECA adalah selamat
dan boleh menyebabkan 100% perlindungan terhadap vvIBDV dengan
menggunakan virus titer 107.1
EID50/ 0.1 mL, manakala ECA tidak dapat
memberi perlindungan yang sepenuhnya di dalam ayam SPF chicken daripada
bursal lesi di bursa Fabricius.
Kesimpulannya, vvIBDV UPM0081 telah berjaya disesuaikan dan dilemahkan
di dalam sel jenis berurutan (sel Vero) selepas lima belas hingga dua puluh
pasage. Virus vvIBDV daripada asingan tempatan ini yang lemah dan telah
dimatikan dan dinamakan sebagai UPM0081T15 dan UPM0081T20 boleh
memberi perlindungan sepenuhnya kepada ayam SPF terhadap jangkitan
vvIBDV.
ACKNOWLEDGEMENT
All praise for Almighty Allah, Lord of all creations Who has granted me
His blessings throughout my life and backed me up to luxuriate in
the researches of this study.

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I would like to express my heartiest gratitude and appreciation to my supervisor,
Professor Dr. Mohd Hair Bejo for providing his invaluable advice, constant
guidance, encouragement and incitement that has stimulated me to accomplish
my PhD research. I want to thank him for granting me a generous opportunity to
work in his laboratory as a graduate student. His honest advice, patience,
thorough guidance and calm demeanor has steered my research towards
success. He challenged me to set my bench mark even higher and to look for
solutions to problems rather than focus on the problem. I have learned to have
confidence in myself and in my work as a result. And I would like to thank him
for his never ending support he had for me during my long journey of doctorate
study program. He was the brother, the friend, and even sometimes the father
who I lost before being my research advisor, reconstruct my whole life by
teaching me the true meaning of doing my best for anything encountered, and to
set goals more aggressive and ambitious.
Thank you professor.
I would like to express my sincere thanks and appreciation to Professor Datin
Paduka Dr. Aini Ideris, and Professor Dr. Abdul Rahman Omar, my co-
supervisors for their constructive instructions, proper guidance and motivation
throughout my study period.
One more time I would like to thank gratefully each of Mr. Saipuzaman Ali, Mr.
Mohd Kamaruddin and Mrs. Siti Khadijah the laboratory staffs. And also to all

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my friends in the laboratory for always being willing to render assistance
throughout the course of my study.
I would also like to thank Universiti Putra Malaysia for the award of the graduate
research fellowship (GRF) which has supported me during my study.
I have no words to express gratitude to my family in Iraq, (the spirit of my father)
and my mother who always encouraged me to obtain higher education, special
thanks to my brother, sister, my wife and all other family members for their moral
support and countless prayers throughout the course of my life.
May Allah give them a long, prosperous and happy life (Aa‟meen)
I certify that an Examination Committee met on 6th
July 2010 to conduct the final
examination of Majed H. Mohammed on his Doctor of Philosophy thesis entitled
“Molecular Characterisation, Attenuation and Inactivation of Very Virulent
Infectious Bursal Disease Virus for the Development of Tissue-Culture Based
Vaccines” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act
1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The
Committee recommends that the candidate be awarded the relevant degree.
Members of the Examination Committee are as follows:
RASEDEE @ MAT BIN ABDULLAH, PhD
Professor,
Faculty of Veterinary Medicine,
Universiti Putra Malaysia.
(Chairman)

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SITI SURI ARSHAD, PhD
Associate Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
JASNI BIN SABRI, PhD
Associate Professor
(Member)
EMDADUL HAQUE CHOWDHURY, PhD
Professor,
Department of Pathology
Faculty of Veterinary Science
Bangladesh Agriculture Science
2202 Mymensingh
Bangladesh
___________________________________
HASANAH MOHD GHAZALI, PhD
Professor and Dean
School of Graduate Studies
Date:
This thesis submitted to the Senate of Universiti Putra Malaysia has been
accepted as fulfilment of the requirement for the degree of Doctor of Philosophy.
The members of the Supervisory Committee were as follows:
Mohd Hair Bejo, PhD
Professor
(Chairman)
Abdul Rahman Omar, PhD
Professor
(Member)
Aini Ideris, PhD
Professor

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(Member)
HASANAH MOHD GHAZALI, PhD
Professor and Dean
School of Graduate Studies
Date: 12 August 2010
DECLARATION
I declare that the thesis is my original work except for quotations and citations
which have been duly acknowledged. I also declare that it has not been
previously, and is not concurrently, submitted for any other degree at Universiti
Putra Malaysia or at any other institution.
MAJED H. MOHAMMED
Date: 6 July 2010

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TABLE OF CONTENTS
Page
DEDICATION ii
ABSTRACT iii
ABSTRAK vii
ACKNOWLEDGEMENTS xi
APPROVAL xiii
DECLARATION
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
CHAPTER
xv
xxi
xxiv
xxix
1 INTRODUCTION 1
2 LITERATURE REVIEW
2.1 Infectious Bursal Disease 9
2.1.1 Clinical Signs and Gross Lesions 11
2.1.2 Histopathology 14
2.1.3 Pathogenesis 16
2.1.4 Immunosuppression 18
2.1.5 Epidemiology of IBD 20
2.1.6 Transmission 21
2.2 Infectious Bursal Disease Virus 22
2.2.1 IBDV Genome 23
2.2.2 IBDV Proteins 25
2.2.3 Antigenic and Virulence Variation 27
2.3 Isolation Adaptation and Attenuation of IBDV 31
2.3.1 Chicken Embryos 31
2.3.2 Cell Culture 32
2.4 General Information on the Immune System 36
2.4.1 Innate Immunity 37
2.4.2 Adaptive Immunity 38
2.4.3 Humoral (B-cell mediated) Immunity 38
2.4.4 Cell-mediated (T-cell mediated)Immunity 39
2.4.5 Relationship between B-and-T-cells 40
2.4.6 Effect of IBDV on innate immunity 40
2.4.7 Effect of IBDV on humoral immunity 40
2.4.8 Effect of IBDV on cellular immunity 41
2.5 Vaccination 42
2.5.1 Live Virus Vaccines 43

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2.5.2 Inactivated of Virus 46
2.5.3 Recombinant and DNA Vaccine 48
2.5.4 Anti-viral Drugs 51
3 ADAPTATION AND ATTENUATION OF vvIBDV ISOLATES
IN TISSUE CULTURE FOR DEVELOPMENT OF VACCINES
53
3.1 Introduction 53
3.2 Materials and Methods 57
3.2.1 IBDV Isolates 57
3.2.2 IBDV Inoculums Preparation 58
3.2.3 Propagation of Viruses in SPF Embryonated Chicken
Eggs via Chorioallantoic Membrane
58
3.2.4 Adaptation, Replication and Attenuation of vvIBDV in
Cell Culture
60
Vero Cell Line 60
DF-1 Cell Line 60
3.2.5 Resuscitation of Frozen Cell Line 61
3.2.6 Sub Culturing of Adherent Monolayer 61
3.2.7 Infection of Vero Cell and DF-1 Cells Monolayer 62
3.2.8 Harvesting of Virus 63
3.2.9 Adaptation and Attenuation 63
3.2.10 Tissue Culture Infective Dose 50 (TCID50) 64
3.2.11 IBDV Identification and Confirmation 64
3.2.12 Indirect Immunoperoxidase Staining Test 65
3.3 Rusults 66
3.3.1 Chorio-allantoic Membrane for UPM94372 66
3.3.4 IBDV Replication and Adaptation in Vero Cell Line 70
3.3.5 IBDV Replication and Adaptation in DF 1 Cell Line 70
3.3.6 IBDV Titration (TCID50/ml) 75
3.3.7 IBDV Identification though Indirect Immunoperoxidase
Staining (IIPS) Test
75
3.4 Discussion 84
4 MOLECULAR CHARACTERIZATION OF THE ADAPTED
AND ATTENUATTED vvIBDV ISOLATE
89
4.1 Introduction 89
4.2.1 Sample Preparation 92
4.2.2 RNA Extraction 92
4.2.3 Determination of RNA Concentration 93
4.2.4 Primer Design 94
4.2.5 Reverse Transcription and PCR Reaction 94
4.2.6 Gel Electrophoresis and Ethidium Bromide Staining 95
4.2.7 Purification of RT-PCR Products 96
4.2.8 Molecular Cloning of Amplified Products and Analysis 97

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of Recombinant Plasmid
4.2.9 Plasmid Extraction and Purification 98
4.2.10 Sequence Assembly and analysis Using
Bioinformatics Software
99
4.2.11 Phylogenetic Tree Construction 101
4.3 Results 101
4.3.1 Amplification of the Hypervariable Region of VP2
Gene
101
4.3.2 PCR Analysis of Recombinant Colonies 102
4.3.3 Nucleotide Sequence Analysis 102
4.3.4 Amino Acid Sequence Analysis 104
4.3.5 Phylogenetic Analysis 105
4.4 Discussion 130
5 PATHOGENICITY AND IMMUNOGENCITY OF THE
ATTENUATED vvIBDV IN SPF CHICKENS
134
5.1 Introduction 134
5.2.1 Chickens 137
5.2.2 Selection of IBDV Isolates 138
5.2.3 Adaptation of IBDV to Embryonated SPF Eggs 138
5.2.4 Tissue Culture Infective Dose 50 (TCID50
) 138
5.2.5 Experimental Design 138
5.2.6 Experiment 1 139
5.2.7 Experiment 2 140
5.2.8 IBD Challenge 142
5.2.9 Histopathology 142
5.2.10 Histopathological Lesion Scoring 143
5.2.11 Collection of Samples for Serological Test 143
5.2.12 Antibody Assay 144
5.2.13 Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR)
144
5.2.14 Statistical Analysis 145
5.3 Rusults 145
5.3.1 Clinical Signs 145
Experiment 1 145
Experiment 2 146
5.3.2 Body Weight 149
Experiment 1 149
Experiment 2 150
5.3.3 Bursa Weight 151
Experiment 1 151
Experiment 2 152
5.3.4 Bursa to Body Weight Ratio 153
Experiment 1 153
Experiment 2 154

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5.3.5 Gross Pathology 155
Experiment 1 155
Experiment 2 156
5.3.6 Histopathological Changes and Lesion Scoring 163
Experiment 1 163
Experiment 2 164
5.3.7 Enzyme Linked Immunosorbent Assay (ELISA) 181
Experiment 1 181
Experiment 2 181
5.3.8 Detection of the Virus or Viral RNA using RT-PCR 182
5.4 Discussion 183
6 SAFETY AND IMMUNOGENICITY OF THE
INACTIVATED ATTENUATED vvIBDV IN SPF CHICKENS
187
6.1 Introduction 187
6.2.1 Virus and Cells 191
6.2.2 Harvesting of Virus 191
6.2.3 Tissue Culture Infective Dose 50 (TCID50
) 192
6.2.4 Inactivation of vv IBDV 192
Binary ethylenmine (BEI) Treatment 192
Electrolysed water-Catholyte-Anolyte (ECA)
Treatment
193
6.2.5 Determination of Time Required to Inactivate Virus 193
6.2.6 Perparation of Killed- Virus Oil Emulsion 194
6.2.7 Experimental Design 194
6.2.8 Microscopic Examination and Lesion Score 195
6.2.9 Determination of ELISA Titer Against Inactivated
IBDV Vaccine
196
6.2.10 Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR)
196
6.2.11 Statistical analysis 197
6.3 Results 197
6.3.1 Inactivation of the Virus Attenuated vvIBDV 197
6.3.2 Clinical Signs 198
6.3.3 Body Weight 200
6.3.4 Bursa Weight 201
6.3.5 Bursa to Body Weight Ratio (1x10-3
) 202
6.3.6 Gross Lesions 203
6.3.7 Histological Lesions Score 207
6.3.8 Antibody Titer (ELISA) 212
6.3.9 Detection of the Virus or Viral RNA using RT-PCR 213
6.4 Discussion 214
7 GENERAL DISCUSSION, CONCLUSION AND 220

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RECOMENDATION FOR FUTURE RESEARCH
7.1 General Discussion 220
7.2 Conclusion 226
7.3 Recommendation for Further Research 228
BIBLOGRAPHY 230
APPENDICES 260
BIODATA OF STUDENT 271
LIST OF PUBLICATIONS 273

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LIST OF TABLES
Table Page
3.1 Mortality of SPF embryonated eggs following vvIBDV inoculation
into CAM route
70
3.2 Percentage of CPE monolayer Vero cells following UPM0081
vvIBDV inoculation
73
3.3 Percentage of CPE monolayer DF-1 cells following vvIBDV
inoculation
74
3.4 Virus titer determined by tissue culture Infective Dose 50 (TCID50) 75
4.1 Primers used to amplify the HPVR VP2 gene 94
4.2 IBDV isolates used in the sequence analyses 100
4.3 Number of nucleotide differences in HPVR of VP2 gene between
IBDV isolate
109
4.4 Sequence identity matrix of VP2 genes nucleotides of IBDV
isolates
110
4.5 Summary of the proposed molecular markers (amino acid
residues) of UPM0081T10, UPM0081T15 and UPM0081T20
atIBDV isolates with other published IBDV strains
111
4.6 Number of amino acids differences in HPVR of VP2 gene
between IBDV isolates
112
4.7 Sequence identity matrix of VP2 genes amino acids of IBDV
isolates
113
5.1 Groups of SPF chickens inoculated with attenuated vvIBDV
passage 15 and 20 and challenged with vvIBDV at day 14 post
inoculation
141
5.2 Rate of mortality and the percentage of protection based on the
number of chickens that survived at day 7 post challenged
149
5.3 Experiment 1: body, bursa, bursa to body weight ratio (1 x 103
),
lesion scoring and ELISA titer of SPF chicken inoculated
175

21
attenuated vvIBDV and control group
5.4 Experiment 2: body weight (g) of chickens in the inoculated
176
5.5 Experiment 2: body, bursa, bursa to body weight ratio (1 x 103
)
and lesion scoring of SPF chicken inoculated attenuated vvIBDV
and uninoculated challenge group
177
5.6 experiment 2: body, bursa, bursa to body weight ratio (1 x 103
)
and lesion scoring of SPF chicken inoculated attenuated vvIBDV
and control group
177
5.7 Experiment 2: bursa weight (g) of chickens in the inoculated
178
5.8 Experiment 2: bursa to body weight ratio (1 x 103
) of chickens in
the inoculated attenuated vvIBDV and control group
179
5.9 Experiment 2: lesions scoring of chickens in the inoculated
180
5.10 Antibody titers (mean titer ± standard deviation) to IBD
determined by ELISA in the attenuated vvIBDV inoculated groups
182
6.1 Different time interval to inject SPF embryonated eggs by two
kinds of killed vvIBDV (BEI and ECA)
193
6.2 Different groups of chickens inoculated with two types of
inactivated vvIBDV (BEI and ECA) and the control group
195
6.3 Mortality of SPF embryonated eggs following inoculation (BEI and
ECA) into CAM route
197
6.4 Efficacy of the inactivated attenuated vvIBDV (UPM0081) in SPF
chickens
200
6.5 Body weight of chickens in the inactivated attenuated vvIBDV
inoculated and control group at 2 weeks post challenged
201
6.6 Bursa weight of chickens in the inactivated attenuated vvIBDV
inoculated and control group at 2 weeks post challenge
202
6.7 Bursa to body weight ratio of chickens in the inactivated
attenuated vvIBDV inoculated and control group at 2 weeks post
challenged
203
6.8 Lesion score of chickens in the inactivated attenuated vvIBDV 212

22
inoculated and control group at 2 weeks post challenge
6.9 Antibody titers to IBDV determined by ELISA in the inactivated
attenuated vvIBDV inoculated and uninoculated groups after two
weeks of post inoculated and two weeks post challenged
213

23
LIST OF FIGURE
Figure Page
3.1a
3.1b
(A):Uninfected control embryonated SPF chicken eggs. (B): UPM94273
dead embryo with severe haemorrhage
(C): UPM04019 dead embryo with severe haemorrhage. (D): UPM0081
the embryo infected showed severe petechial to ecchymotic
haemorrhage (arrows)
68
69
3.2 (A) Uninfected control Vero cells monolayer. (B) Cytopathic effect of
UPM0081 isolate in 4th
passage days 15 pi. The arrows show cell
rounding and aggregation. 10 x . Bar = 200 µm
76
3.3 (A) Vero cell monolayer in 6th
passage days 8 pi (B). Vero cell
monolayer in passage 12th
, days 6 pi. The arrows shows cell rounding
and aggregate in clumps and granulated in cytoplasm. 10x. Bar = 200
µm
77
3.4 (A) Vero cell monolayer in passage 13th
, day 3 pi (B). Vero cell
monolayer in 20th
passage days 4 pi. The detachment of cells from the
substrate, with the eventual destruction of the entire monolayer. 10 x.
Bar = 200 µm
78
3.5 (A) Uninfected control DF-1 cells monolayer. (B) Cytopathic effect of
UPM0081 isolate in 3rd
passage days 5 pi. The arrow shows cell
rounding and clumping. 10 x. Bar = 200 µm
79
3.6 (A) DF-1 monolayer in 4th
passage day 5 pi, affected cells were more
concentrated with granular cytoplasm (B). DF-1 cells passage 5th
day 4
pi, the arrow shows detachment of cells from the substrate (B). 10 x.
Bar = 200 µm
80
3.7 (A) DF-1 cell monolayer in passage 6th
, day 3 pi (B). DF-1 cell
monolayer in 9th passage, days 3 pi the arrow shows degenerated
cells and more detachment of cells from the substrate. 10 x. Bar = 200
µm
81
3.8 Identification of IBD antigens in Vero cells culture using infected cell
cultures stained with HRP-conjugated antibody. (A) Uninfected control
Vero cells. (B) Vero cells infected with UPM0081 at 20th
passage days
2 pi. Note specific intracytoplasmic brownish colouration. 10 x. Bar =
200 µm
82
3.9 Identification of IBD antigens in DF-1 cells culture using infected cell
cultures stained with HRP-conjugated antibody. (A) Uninfected control
DF-1. (B) DF-1 infected with UPM0081 at passage 4 day 2 pi. Note
83

24
specific intracytoplasmic brownish colouration. 10x. Bar = 200 µm
4.1 Hypervariable region (643pb) of IBDV VP2 genes Lane 1- Negative
control; Lane 2 positive UPM0081D5; Lane 3 positive UPM0081D7;
Lane 4 positive UPM0081T5 and Lane 5 positive UPM0081T7; M- 100
bp DNA marker (Promega, USA)
107
4.2 Hypervariable region (643pb) of IBDV VP2 genes Lane 1- positive
UPM0081T8; Lane 2 positive UPM0081T9; Lane 3 positive
UPM0081T10; Lane 4 positive UPM0081T15 and Lane 5 positive
UPM0081T20; Lane 6- Negative control; M- 100 bp DNA marker
(Promega, USA)
107
4.3 PCR screening on white colonies amplification of IBDV genes Lane 1,
2 and 3 white colonies positive for VP2 gene passages (UPM0081D5,
UPM0081T5 and UPM0081T7 respectively; Lane 4 Negative control;
M- 100 bp DNA marker (Promega, USA)
108
4.4 PCR screening on white colonies amplification of IBDV genes Lane
1,2,3,4,5,6 and 7 white colonies positive for VP2 gene passages
(UPM0081D7, UPM0081T8, UPM0081T9, UPM0081T10,
UPM0081T15 and UPM0081T20 respectively; Lane 7 Negative control;
M- 100 bp DNA marker (Promega, USA)
108
4.5 Nucleotide sequences of HPVR of VP2 from nucleotide 516-1158
(numbering of Bayliss et al., 1999) of the UPM0081T10, UPM0081T15
and UPM0081T20 IBDV passages compared with other published
IBDV strains. A dot indicated position where the sequence is identical
to others
114
4.6 Amino acid sequence aligment of UPM0081T10, UPM0081T15 and
UPM0081T20 IBDV passages
121
4.7 Phylogenetic tree based on nucleotide sequence of HPVR of VP2 gene
of IBDV isolates, displaying relationship of UPM0081T10,
UPM0081T15 and UPM0001T20 passages and other published
atIBDV strains
124
4.8 Phylogenetic tree based on amino acids sequence of HPVR of VP2
gene of IBDV isolates, displaying relationship of UPM0081T10,
UPM0081T15 and UPM0001T20 passages and other published
atIBDV strains
125
4.9 Sequence nucleotide difference of VP2 genes of IBDV isolates 126
4.10 Sequence nucleotide identity matrix of VP2 genes of IBDV isolates 127

25
4.11 Sequence nucleotide difference of VP2 genes of IBDV isolates 128
4.12 Sequence amino acid identity matrix of VP2 genes of IBDV isolates 129
5.1a
5.1b
Experiment 1 (preliminary study): bursa of Fabricius in SPF chickens
day 14 pi. (A) Group C: normal. (B) Group A: Passage 10 bursa of
Fabricius with mild to moderate odema with yellowish gelatinous
material (arrow)
Experiment 1 (preliminary study): bursa of Fabricius in SPF chickens
day 14 pc. (C) Group B: passage 15 normal
157
158
5.2a
5.2b
Experimental 2 (challenged groups): bursa of Fabricius in SPF
chickens day 7pc. (A) Group1 (b): passage 15 normal. (B) Group 2(b):
passage 20 normal.
Experimental 2 (challenged groups): bursa of Fabricius in SPF
chickens. (C) Group 3(b): control positive severee haemorrhages day 4
pc.
159
160
5.3a
5.3b
Experiment 2 (challenged groups): proventriculus and gizzard in SPF
chickens day 7pc. (A) Group 1 (b): passage 15 normal (B) Group 2 (b):
passage 20 normal
Experiment 2 (challenged groups): proventriculus and gizzard in SPF
chickens day 7pc. (C) Group 3 (b): control positive hemorrhage on the
mucosa of the proventriculus at the junction with the gizzard (arrow).
161
162
5.4a
5.4b
Experiment 1 (preliminary study): day 14 pi. bursa of Fabricius (A)
Control group: No lesions were observed lesion score of 0 (B) Group B:
Normal, large active follicles consist of lymphoid cells (arrow) lesion
score of 0 (. HE, 10x. Bar = 200µm.
Experiment 1 (preliminary study): bursa of Fabricius. (C) Group A:
Oedematous bursa with degeneration, necrosis (arrow) and infiltration
of inflammatory cells (arrow), follicular cyst (arrow) in the medulla,
lesion score of 5 at day 2 pi. (D) Group A: More severe lymphoid
necrosis (arrow) in the mudella, lesion score of five at day 5 pi. HE,
20x. Bar = 100 µm
167
168
5.5a
5.5b
Experiment 2 (sacrificed groups): day 7 pi. bursa of Fabricius. (A)
Group 1(a): Mild degeneration and necrosis of the follicles (arrow)
lesion score of 1 (B) Group 2(a) Mild degeneration and necrosis of the
follicles (arrow) lesion score of 1. HE, 10x. Bar = 200µm
Experiment 2 (sacrificed groups): day 7 pi. bursa of Fabricius. (C)
Group 3(a): very clear cortex and medulla packed with healthy follicles,
lesion score of 0. HE, 10x. Bar = 200µm
169
170

26
5.6a
5.6b
Experiment 2 (challenged groups): day 7 pc. bursa of Fabricius. (A)
Group 1(b): Mild degeneration and necrosis of the follicles (arrow),
lesion score of 1 (B) Group 2(b) Mild degeneration and necrosis of the
follicles (arrow) lesion score of 1 HE, 10x. Bar = 200µ
Experiment 2 (challenged groups): day 7 pc. (C) Group 3(b): Depletion
of bursa follicles with cysts contains cell debris with fibrinous exudates
at medulla follicle (arrow), the interstitial connective tissues were
obvious, edematous and infiltrated with inflammatory cells (arrow),
lesion score of 5. HE, 20x. Bar = 100µm
171
172
5.7a
5.7b
Experiment 2 bursa of Fabricius (mortality groups): day 7 pc. (A) Group
1(c): Mild lymphoid deplesion (arrow), lesion score of 1 (B) Group 2(c):
Mild lymphoid deplesion (arrow), lesion score of 1. HE, 10x. Bar =
200µm
Experiment 2 (mortality groups): day 7 pc. bursa of Fabricius. (C)
Group 3(c): Mild lymphoid deplesion (arrow), lesion score of 1. HE,
10x. Bar = 200µm
173
174
5.8 Hypervariable region (643pb) amplification of IBDV VP2 genes. Lane 1
Day 1; Lane 2 Day 3; Lane 3 Day 5 Day ; Lane 4 Day 7; Lane 5 Day
183
10; Lane 6 Day 14; Lane 7 Day 21; and Lane 8 Negative control; M-
100 bp DNA marker (Promega, USA)
6.1a
6.1b
bursa of Fabricius (BF) in SPF chickens. (A) Group C1: normal (B)
Group C2: severee haemorrhagic
bursa of Fabricius (BF) in SPF chickens. (C) Group BEIP15: normal (D)
Group BEIP20: normal.
205
206
6.2 bursa of Fabricius (A) Group C1 (Control negative): Apparently normal
lymphoid follicles, lesion score of 0 (B) Group C2 (Control positive):
lesion score of 5, day 2 pi, severe follicular necrosis with cyst formation
on the follicles (arrow) and infiltration of inflammatory cells and oedema
fluid at interstitial space (arrow). HE, 20x. Bar = 100µm
209
6.3 bursa of Fabricius day 14 pc. (A) Group BEIP15: Mild degeneration
and necrosis of the follicles (arrow), lesion score of 1 (B) Group
BEIP20: Mild degeneration and necrosis of the follicles (arrow), lesion
score of 1. HE, 10x. Bar = 200µm
210
6.4 bursa of Fabricius day 14 pc. (A) Group ECAP15: Mild to moderate
lymphoid necrosis (arrow), lesion score of 1.5. (B) Group ECAP20: Mild
to moderate lymphoid necrosis (arrow), lesion score of 1.5. HE, 10x.
Bar = 200µm
211

27
6.5 Hypervariable region (643pb) amplification of IBDV VP2 genes. (1)
BEIP15 negative (2) BEIP20 negative (3) ECAP15 negative (4)
ECAP20 negative (5) C2 posative. (M) 100 bp DNA marker (Promega,
USA).
214

28
LIST OF ABBREVIATIONS
ATV Antibiotic-trypsin versene
AA Amino acid sequences
AGID Agar gel immunodiffusion
AGPT Agar gel precipitation test
atIBDV Attenuated strain of infectious bursal disease virus
BEI Binary ethylenimine
BF Bursa of Fabricius
bp Base pair
CAM Chorioallantoic membrane
caIBDV Classical strain of infectious bursal disease virus
cDNA Complementary deoxyribonucleic acid
CEF Chicken embryo fibroblast
CMI Cell-mediated immunity
DAB Diaminobenzidine tetrahydrochloride
ddH2O Deionized double-distilled water
DMSO Dimethylsulphoxide
DNA Deoxyribonucleic acid
dNTP Deoxynucleoside triphosphate
dsDNA Double-stranded DNA
ECA Electrolysed water-Catholyte-Anolyte
EID50 Embryo effective dose fifty
ELISA Enzyme-linked immunosorbent assay
FBS Fetal bovine serum
HE Haematoxylin-and-eosin
HPVR Hypervariable region
IBD Infectious bursal disease
IBDV Infectious bursal disease virus
IPNV Infectious pancreatic necrosis virus
IPS Immunoperoxidase staining technique

29
IPTG Isopropyl-ß-D-thiogalactosidase
kb Kilobase pair
kD Kilo Dalton
LB Luria-Bertani
Min Minute
NaCl Sodium chloride
nt Nucleotide
OD Optical density
OIE Office international des epizooties
ORF Open reading frame
PBS Phosphate-buffered saline
pH Hydrogen ion exponent
pi post infection
% Percentage
PCR Polymerase chain reaction
RNA Ribonucleic acid
rpm Revolution per minute
RT-PCR Reverse transcriptase-polymerase chain reaction
RT Room temperature
SPF Specific-pathogen-free
TAE Tris-acetate-EDTA
UPM Universiti Putra Malaysia
vaIBDV Variant strain of infectious bursal disease virus
Vero Green Monkey Kidney
vvIBDV Very virulent strain of infectious bursal disease virus
w/v Weight per volume
X-gal 5-bromo-4-choro-3-indolyl-ß-D-galactopyranoside
µg Microgram
µl Microliter
µm Micrometer

30
Amino Acid Single/Three Letter Amino Acid Code
Alanine A Ala
Arginine R Arg
Asparagine N Asn
Aspartic Acid D Asp
Glutamine Q Gln
Glutamic Acid E Glu
Glycine G Gly
Isoleucine I IIe
Leucine L Leu
Lycine K Lys
Methionine M Met
Phenylalanine F Phe
Proline P Pro
Serine S Ser
Threonine T Thr
Thyptophan W Trp
Valine V Val

31
CHAPTER 1
INTRODUCTION
Infectious bursal disease virus (IBDV) also called Gumboro disease after the
geographic location in the state of Delaware where the first outbreak occurred,
causes immunosuppression in young chickens and an acute disease in chickens
between 3 to 6 weeks old (Ramm et al., 1991). The disease is endemic with
95% presence as reported by of the Office of International Epizooties (OIE)
member countries, in spite of intensive vaccination and biosafety practices (van
den Berg., 2000). This indicates that the current control measures of this virus
are not very effective.
The disease causes economic losses due to increase susceptibility to other
pathogens (bacterial, viral and protozoan) and decrease vaccination efficacy.
Impaired growth and death are also common and the mortality rates do vary
from insignificant levels to 100%, depending on the strain involved in the
outbreak (Lasher and Shane, 1994).
The target organ of IBDV is the bursa of Fabricius, which is a specific reservoir
for B lymphocyte cells in avian species. The severity of the disease has been
reported to be directly related to the number of susceptible cells present in the
bursa. Therefore, the age range of chickens susceptible to IBDV infection is
between 3 to 6 weeks, when the bursa of Fabricius is at its maximum

32
development. Massive growth of the virus in the bursal cells causes cellular
destruction and the subsequent dissemination of the virus causes disease and
death.
Infection with IBDV often results in immunosuppression (Allan et al., 1972). The
immunosuppressive effects with classical IBDV (caIBDV) appears to be more
pronounced if the virus exposure occurs within the first 2-3 weeks of age of the
chickens as the degree of immunosuppression varies, depending on the
virulence of the virus and time of infection. Immunosuppression may be
accompanied by overt clinical or subclinical outbreaks of infectious bursal
disease (IBD). In this case, the humoral immune response is clearly depressed,
but transient depression of the cellular immune response occurs (Confer et al.,
1981).
IBDV is a member of the genus Avibirnavirus in the family Birnaviridae. The
member of this family contain a genome consists of two segments of double-
stranded RNA (dsRNA), designated A and B (Dobos et al., 1979; Muller et al.,
1979b), with icosahedral symmetry and a diameter of about 60nm (Hirai and
Shimakura, 1974). The virus has five proteins recognized as VP1 to VP5. The
smaller RNA segment known as segment B of the genome, with a length of
about 2.8 kb encodes for VP1, which is a 90-kD multifunctional protein with
polymerase and capping enzyme activities (Spies et al., 1987). The larger
segment A with a length of about 3.2 kb encodes for VP2, VP3, VP4 and VP5.
The VP2 and VP3 are the major proteins of the virions constituting 51% and

33
40%, respectively of the total proteins and contain the major neutralizing
epitopes. The VP2 has the serotype specific epitope and VP3 has a group
specific antigen. VP4 is a minor protein involved in the processing of the
precursor polyprotein (Fahey et al., 1989).
Many IBDV has been characterized molecularly using the hypervariable region
which is located in VP2 of IBDV genome (Brown et al., 1994). This region
encodes the main host protective immunogen polypeptides of the virus (Azad et
al., 1987; Becht et al., 1988; Fehey et al., 1989). It consists of 145 amino acids
from amino acid positions 206 to 350 and within this region there are two
hydrophilic peaks. The first peak (peak A) is from amino acid positions 212 to
224 and the second peak (peak B) is from amino acid positions 314 to 324
(Bayliss et al., 1990; Heine et al., 1991; Brown et al., 1994). Specific amino acid
changes within hypervariable region and serine heptapeptide motif sequence
(SWSASGS), which is adjacent to peak B, are potential sites responsible for
virus attenuation or antigenic determination (Heine et al., 1991; Yamaguchi et
al., 1996b). In addition, several amino acid molecules change at the
hypervariable domain of the VP2 gene which has been used to differentiate the
virus into very virulent, attenuated, variant and classical strains. The amino acid
residue changes at 222 (P to A), 242 (V to I), 253 (H to Q), 256 (V to I), 294 (L to
I), and 299 (N to S), are the markers for vvIBD (Cao et al; 1998; Brown et al.,
1994; Rudd et al., 2002) while the marker for variant strain are at 245 (G to S)
and 249 (Q to K) and that of attenuated strains are at 279 (D to N), and 284 (A
to T) (Yamaguchi et al., 1996b; Cao et al., 1998).

34
The cloacal bursa and spleen are the tissue of choice for the isolation of IBDV,
but the bursa is the most common tissue chosen to isolate IBDV. Other organs
contain the virus, but at a lower concentration and probably only because of the
viremia (Lukert and Saif, 2003). The chorioallontoic membrane (CAM) of 9-11
days old embryos was also the most sensitive route for isolation of the virus
(Hitchner, 1970). The IBDV do infect and grow in various primary cell culture of
avian origin like chicken embryo kidney (CEK), chicken embryo bursa (CEB) and
chicken embryo fibroblast (CEF) cells (Raymond and Hill, 1979; Yamaguchi et
al., 1996a).
Mammalian continuous cell lines had also been reported to be susceptible to
IBDV and these include RK-13 derived from rabbit kidney (Rinaldi et al., 1972),
Vero cells derived from adult African green monkey kidney (Leonard, 1974;
Jackwood et al., 1987; Kibenge et al., 1988; Peilin et al., 1997; Ahasan et al.,
2002) BGM-70 derived from baby grivet monkey kidney (Jackwood et al., 1987),
MA-104 derived from foetal rhesus monkey (Jackwood et al., 1987), and OK
derived from ovine kidney (Kibenge and Mckenna, 1992).
The use of these continuous cell lines of mammalian origin has been found to
have advantages over the use of primary cell culture of avian origin. Continuous
cell lines are easier to handle and maintain compared to primary cell culture,
and are free from vertically transmitted extraneous viruses (Hassan et al., 1996).
Its usage will be timely for laboratories that have limited or no access to specific
pathogen free (SPF) eggs or chicks. Thus, if higher virus titer could be obtained

35
from continuous cell lines, it will be valuable and economical to adopt the cell
lines to grow IBDV.
Conventional immunizations with live and killed vaccine are the principle
methods for control of IBD in chickens. Live virus vaccines are generally derived
from the serial passages in embryonated eggs or tissue culture (van den Berg,
2000). The degree of attenuation of the vaccine strains can be classified as mild,
intermediate and hot depending on the its ability to cause the varying degree of
histological lesions. Although serotype 1 vaccine strains cause no mortality, its
use still cause different degrees of bursal lesions that range from mild to
moderate or even severe (van den Berg, 2000). The higher the virulence of the
vaccine virus strain, the more severe damage of the bursal lymphocytes resulted
(Kelemen et al., 2000). Nonetheless, as it should be, the lesion caused by the
vaccine strain is less severe than the field strain (Rosales et al., 1989a).
The major problem with active immunization of maternally immune chickens is
ability to determine the proper time of vaccination that allows for adequate
replication of the vaccine virus and at the same time protects young chicken
from disease. The time of vaccination varies with the level of maternal
antibodies, route of vaccination and virulence of the vaccine virus. For a
successful vaccination program, factors like environmental stresses,
management and flock profiling for the presence of maternal antibodies should
be taken into account (Lukert and Saif, 2003). Inactivated vaccines are usually
used in the breeder hens for them to pass down high, uniform, and persistent

36
antibody titres to the progeny (Cullen and Wyeth, 1976; Wyeth and Cullen,
1978; Wyeth and Cullen, 1979; Guittet et al., 1992). For the vaccination to be
effective, the hens must be previously vaccinated with a live virus or had been
exposed to the virus in the farm. Inactivated vaccines are administered to the
layers through subcutaneous or intramuscular routes at sixteen to twenty week
old. In this way, the chicks will have the protective maternal antibodies up to
thirty days (Wyeth and Cullen, 1979; Box, 1989; van den Berg and Meulemans,
1991; Wyeth et al., 1992). However, the chicks will not be protected from the
challenge of the highly pathogenic IBDV strains at later age (Wyeth and Cullen,
1979; Van den Berg and Meulemans, 1991).
Inactivated vaccine is usually prepared from the bursal homogenates of infected
chicks, or from viral cultures on embryonated eggs or tissue culture, where the
virus is inactivated by formaldehyde and various alkylating agents like
Binaryethylenimine (BEL), betapropiolactone and prepared as the oil emulsions
(van den Berg, 2000). Killed virus vaccines in oil adjuvant are used to boost and
prolong immunity in breeder flocks, but they are not practical and desirable for
inducing a primary response in young chicken (Lukert and Saif, 2003). Oil-
adjuvant vaccines are most effective in chicken that have been primed with live
virus either in the form of vaccine or field exposure to the virus (Wyeth and
Cullen, 1979).
To date, several types of IBD vaccines were imported for use in Malaysia. They
include live attenuated and killed vaccines. The evaluation on the safety and

37
efficacy of the imported IBD vaccines for local used available in the market
commercially demonstrated that most of the vaccines studied were consider to
be unsafe and not effective to confer full protection against the vvIBDV
challenged. The failure of those IBD vaccines to induce IBD antibody titer had
been previously reported (Hair-Bejo et al., 1995a; 1995b). Despite the
vaccination program adopted, frequent outbreaks of IBD do occur from time to
time. The worst was the emergence of a new highly pathogenic strain of
(vvIBDV) which complicates the immunization programme of the disease.
Differences in the antigenicity between the vaccine and field viruses have been
recognised as one of the major reason for vaccination failure.
This antigenic variation has been reported to be present among the recent field
strains of the virus (Jackwood, 2005) and this could be attributed to the failure of
protection by the existing vaccines.
In the present study, it is believed that attempt to develop local live attenuated
and killed vaccines in tissue culture, has opened great opportunity to a great and
potential for the control of IBDV infection and its associated immune
suppression. The use of local vaccine has helped to control IBD in regional
regions where outbreaks were not controlled by commercially available vaccines
(Hair-Bejo, et al., 1995b).

38
The objectives of this study were:
1. to adapt and attenuate vvIBDV isolates in tissue cultures for development of
vaccines.
2. to determine the molecular characteristic of the adapted and attenuated
vvIBDV isolate.
3. to determine the pathogenicity and immunogenicity of the attenuated vvIBDV
in SPF chickens
4. to determine the safety and immunogenicity of the inactivated attenuated
vvIBDV in SPF chickens

CHAPTER 2
LITERATURE REVIEW
2.1 Infectious Bursal Disease
Infectious bursal disease (IBD) is a highly contagious viral disease of young
chickens characterised by destruction of the lymphoid cells in the bursa of
Fabricius. Other lymphoid organs are also affected but to a lesser degree
(Cheville, 1967; Lukert and Saif, 1997). The disease in a fully susceptible
chicken flock, occurs at 3 to 6 weeks of age and the economic impact of the
disease are manifold which includs losses due to morbidity and mortality.
Immunosuppression experienced by the surviving chickens could exacerbate
infections with other disease agents coupled with reduced chicken‟s ability to
respond to vaccination. The economic impact of the disease is influenced by
pathogenicity of the virus, susceptibility of the flock, presence of other prevalent
pathogens, the environment and poor management practices (Saif, 1998).
The causative agent for IBD is a bisegmented, double stranded RNA virus that
belongs to the family Birnavirideae of the genus Avibirnavirus (Dobos et al.,
1979; Muller et al., 1979b). Two distinct serotypes have been recognized.
Pathogenic strains are grouped in serotype 1 viruses while serotype 2 strains
are non-pathogenic.

40
Until 1987, the virus strains were of low virulence causing less then 2% mortality
and the disease was satisfactorily controlled by vaccination. But in 1986, an
outbreaks of IBD were reported, despite vaccination with a classical strain of
IBD vaccine (Jackwood and Saif, 1987). In 1987, very virulent IBDV (vvIBDV)
was isolated in Holland and Belgium (Chettel et al., 1989; van den Berg, 2000).
The mortality rate associated with vvIBDV infection in 3 to 14 weeks old
replacement pullet had been reported to reach 70% while that of broiler flocks
was 30% mortality (van den Berg and Meulamans, 1991). The pathogenic
disease attributed to this strain had spread worldwide including in Malaysia
(Hair-Bejo, 1992), China (Gaudry, 1993), Indonesia (Rudd et al., 2002), Russia
(Shcherbakova et al., 1998) and Japan (Nunoya et al., 1992). The vvIBDV
strains are characterised by severe damage of the bursa and higher mortality
rate in susceptible flocks. These vvIBDV strains, are antigenically similar to the
classical but can established infection in chicken with antibody levels that are
protective against classical strains. The emergence of the vvIBDV has
complicated the immunization programmes against the disease. Early
vaccination may result in failure due to the interference by the maternally
derived antibody (MDA), while delay may cause field virus infection. Therefore
vvIBDV have become an economically important pathogen in the poultry
industries worldwide (Yamaguchi et al., 1997; Chen et al., 1998; Eterradosi et
al., 1998).

41
2.1.1 Clinical Signs and Gross Lesions
The incubation period of IBD range from 2-4 days. The infection of susceptible
broiler or layer pullet flocks is characterized by acute onset of depression.
Chickens are disinclined to move and peck at their vents (Cosgrove, 1962). In
acute outbreaks, the chicks appear sleepy and have a reduce food intake.
Terminally, birds may show sternal or lateral recumbency with coarse tremor
(Lasher and Shane, 1994). White or watery diarrhea, solid vent feathers and
vent pecking are seen. The feathers are ruffled, the birds have an unsteady gait
and may become prostrate and trembling prior to death (Cosgrove, 1962;
Chettle et al., 1989; Hair-Bejo, 1993; Lasher and Shane, 1994; Lukert and Saif,
1997).
The short duration of clinical signs and mortality pattern are considered to be of
diagnostic significance in IBD (Lasher and Shane, 1994). Affected flocks
showed depression for 5-7 days during which mortality rises rapidly for the first
two days then declines sharply as clinical normality returns (Parkhurst, 1964).
There is usually 100% morbidity, but the mortality varies depending on the virus
strains.
Clinical signs alone are not sufficient to make a diagnosis, but when combined
with gross lesions, it is possible to arrive at a preliminary diagnosis (Saif, 1998).
Changes in lymphoid organs are typical of the disease. The bursa of Fabricius,
which is the main target of the virus, undergoes major changes beginning at 3

42
days post infection post. Infection (pi). It increases in size reaching twice the
normal size by 4 days pi followed by atrophy, and reaching one third of its
original weight by 8 days pi (Saif, 1998).
By day 2 or 3 pi, the bursa usually has a gelatinous yellowish transudate
covering the serosal surface. Longitudinal striations became prominent and the
color changed from white to creamy. The transudate disappeared as the bursa
returned to its normal size and the organs turned gray during the period of
atrophy (Lukert and Saif, 2003).
The tissue distribution and severity of lesions is dependent on the subtype and
pathogenicity of the virus (Rosenberger and Cloud., 1986). Infected birds are
dehydrated and have darkened discoloration of pectoral muscles. Hemorrhages
occur in thigh and pectoral muscles and are also reported from the mucosa at
the proventriculus-gizzard junction and on the serosal surface and the bursa
(Hanson, 1962). Extensive hemorrhages could be seen on the entire bursa.
There is increased mucus in the intestine and renal changes are observed in
diseased birds which had been attributed to dehydration (Lukert, and Saif,
2003). The kidneys, tubules and ureters are so distended and filled with urates
that they appeared white (Cosgrove, 1962).
Pathologic changes in the spleen and thymus were less prominent than those of
the bursa (Cosgrove, 1962; Inoue et al., 1994). The spleen might be slightly
enlarged and usually had small gray foci uniformly dispersed on the surface

43
(Inoue et al., 1994). Lesions in these organs are noticed at the same time as the
changes occurred in the bursa. These lesions resolved within 1 or 2 days of
appearance (Helmboldt and Garner, 1964).
The vvIBDV infections are characterized by severe clinical signs, high mortality,
and a sharp death curve followed by rapid recovery. The vvIBDV strains have
the same clinical signs and incubation period of 4 days as classical viruses
(caIBDV) but the acute phase is exacerbated (van den Berg, 2000). The vvIBDV
strains cause more severe lesions in the cecal tonsils, thymus, spleen and bone
marrow and a greater decrease in thymic weight index as compared to the
(caIBDV) strains but, bursal lesions are similar. It has been shown that the
pathogenicity of field strains of IBDV correlated with lesion production in non-
bursal lymphoid organs. The results also suggest that pathogenicity of IBDV
may be associated with virus antigen distribution in non-bursal lymphoid organs
(Tanimura et al., 1995).
Chickens affected by the variant IBDV (vaIBDV) are characterized by severe
bursal atrophy and immunosuppression (Lukert and Saif, 1997) without showing
the inflammation induced symptoms associated with the infection of caIBDV
(Sharma et al., 1989). Attenuated strains have been adapted to chick embryo
fibroblast (CEF) cells or other cell lines. These strains do not cause disease in
chickens, and therefore some of them are being used as live vaccines (Lim et
al., 1999).

44
2.1.2 Histopathology
Histolopathologic changes occur in the bursa, spleen, thymus, Harderian gland
and cecal tonsils. The first obvious lesion of infection occurs in the bursa of
Fabricius and it is the most severely affected organ. Degeneration and necrosis
of individual lymphocytes in the medullary region of the bursa occur as early as
1 day post infection. Lymphocyte degeneration is accompanied by nuclear
pyknosis and formation of lipid droplets in the cytoplasm (Cheville, 1967).
Degenerating lymphocytes are surrounded by macrophages. Lymphocytes are
replaced by heterophils, pyknotic debri, and hyperplastic reticuloendothelial
cells.
By 3 or 4 days post infection, all lymphocytes would have been affected. At this
point of time the bursal weight increases due to edema, hyperemia, and
accumulation of heterophils. As the inflammatory reaction subsides, cystic
cavities appear in the medullary region of the bursal follicles. Necrosis and
phagocytosis of the heterophils take place and fibroplasia occurs in the inter-
follicular connective tissue (Helmboldt and Garner, 1964; Cheville, 1967; Lukert
and Saif, 2003). The proliferation of the bursal epithelial layer occurs producing
glandular structures of columnar epithelial cells containing globules of mucin.
Follicular regeneration and repopulation of follicles with the lymphocytes occur
but healthy follicles are not formed during the observed time span of 18 days
(Helmboldt and Garner, 1964).

45
The spleen shows hyperplasia of the reticuloendothelial cells around the
adenoid sheath arteries during the early stages of infection. Lymphoid necrosis
occurs in the peri-arteriolar lymphoid sheath by 3 days pi. The spleen recovers
shortly without any sustainable damage to the germinal follicles (Cheville, 1967;
Lukert and Saif, 2003).
Changes in thymus and cecal tonsils appear shortly after infection and include
areas of lymphoid necrosis and hyperplasia of the reticular and epithelial
components in the medullary region of thymic follicles (Cheville, 1967). The
damage is less extensive than in the bursa and is quickly repaired by 12 days pi
(Cheville, 1967).
The Harderian gland is reported to be severely affected by the virus in 1 day old
chickens (Survashe et al., 1979). Normally, the gland is populated with plasma
cells as the chicken ages but the infection prevents this infiltration. Harderian
gland of the chickens infected at 1 day of age has 5-10 folds fewer plasma cells
than those of uninfected chickens from 1-7 weeks of age (Dohms et al., 1981).
However, lymphoid follicles and heterophil populations in the Harderian gland
are not affected by IBDV infection, nor could necrotic or degenerative changes
be found in the acini or excretory ducts.
In contrast, the broilers infected at 3 weeks of age have a 51% reduction in
plasma cell content at 5-14 days pi (Dohms et al., 1981). Plasma cell numbers
reduction was temporary and the levels became normal after 14 days. Histologic

46
lesions appearing in the kidneys were nonspecific and resulted from dehydration
(Helmboldt and Garner, 1964). The liver had some slight perivascular infiltration
of monocytes (Peters, 1967).
2.1.3 Pathogenesis
Pathogenesis is the process through which the virus cause injury to the host
leading to mortality, disease or immunosuppression. The different pathotypes of
IBDV have different degree of pathogenicity, virulence and antigen distribution in
different organs (Lukert and Hitchner, 1984). The natural infection is usually via
the oral route accompanied by the gut associated lymphoid cells (Becht, 1980).
Following oral inoculation of IBDV in susceptible birds, the virus replicate
primarily in the macrophage and lymphoid cells of the gut-associated lymphoid
tissue during 4 to 6 hours pi (Kaufer and Weiss, 1976) and leads to primary
viremia. Then virus travels to liver via portal vein and localized in the bursa of
Fabricius as the target organ via blood stream where IBDV replication occur at
13 hour post inoculation (Muller et al., 1979a). After massive replication in the
follicle of the bursa of Fabricius, the virus will be released into the blood as
secondary viremia. This will be followed by virus replication and destruction to
another organ such as cecal tonsil, spleen, bone marrow, gut associated
lymphoid tissue and also replication in bursa of Fabricius (Muller et al., 1979a;
Becht, 1980). Consequently, clinical sign and mortality occur within 48 to 72
hours (Kaufer and Weiss, 1976). The cause of death in clinical IBD is mainly due

47
to circulatory failure as a result of severe hemorrhages (Hair-Bejo, 1993).
Severe dehydration owing to diarrhea and reduce water intake could also lead to
circulatory failure and death (Hair-Bejo, 1993).
Haemorrhage in IBDV infected chicken can be due to impairment of the clotting
mechanism due to destruction of thrombocyte (Skeeles et al., 1980) and
depletion of haemolytic component (Skeelas et al., 1980). In addition
haemorrhages can also be the result of formation of immune complexes
culminating to an Arthus reaction.
Microscopic lesion particularly in the bursa of Fabricius is similar to an Arthus
reaction, which is caused by deposition of antigen antibody complement
complexes which in turn induces production of chemotactic factors,
haemorrhages and leukocytes infiltration (Skeeles et al., 1979). Two week old
chicks showed less circulating complement than 8 weeks old chicks and did not
show the Arthus reaction (Skeelas et al., 1979). In addition, IBDV infected
chickens showed prolonged clotting time, which has consequently induced
hemorrhagic lesions in the birds (Skeeles et al., 1979).
The target organ of IBDV is the bursa of Fabricius at its maximum development.
Orally inoculated IBDV in bursectomized and non-bursectomized birds showed
that the replication of the virus occurred in the gut-associated lymphoid tissues
(Muller et al., 1979a; Kaufer and Weiss, 1980) and the second replication, in the

48
bursa of Fabricius that is responsible for the high titer of the virus and also for
clinical signs and mortality.
IBDV has a predilection for actively dividing immunoglobulin G and M bearing
cells (Hirai et al., 1981). This makes the B lymphocytes to be the main cells
affected by the virus. Since the maturation B lymphocyte occurs in the bursa of
Fabricius, this organ and the lymphocytes are the most affected during infection.
Therefore infected chicken became deficient in the production of optimum levels
of antibodies against divers antigen (Faragher et al., 1974; Giambrone et al.,
1977).
2.1.4 Immunosuppression
IBDV infected chickens are immunosuppressed and susceptible to other avian
pathogens, such as Mycoplasma gallisepticum (Nunoya et al.,1995),
Staphylococcus aureus (Santivatr et al., 1981; McNamee, 2000), Escherichia
coli (Igbokwe et al., 1996), Eimeria tenella (Giambrone et al., 1977; Anderson et
al.,1977), Newcastle disease virus (NDV) (Almassy and Kakuk, 1976; Westbury,
1978; Rosales et al., 1989b), chicken anaemia virus (CAV) (Yuasa et al.,1980;
Rosenberger and Cloud, 1989), reovirus (Moradian et al., 1990), Marek‟s
disease virus (Cho, 1970), infectious laryngotracheitis virus (Rosenberger and
Gelb, 1978), infectious bronchitis virus (IBV) (Winterfield et al., 1978; Pejkovski
et al., 1979), and adenovirus infection (Fadley et al., 1976). Simultaneous
infections by IBV and IBDV usually lead to secondary infection of the respiratory

49
tract caused by E.coli (Naqi et al., 2001). IBDV-infected chickens also failed to
response to anti-coccidial drug treatments during the coccidiosis outbreak and
this result in high mortality (McDougald et al., 1979).
IBDV induced immunosuppression may be due to the direct destruction of B
lymphocytes (Ramm et al., 1991; Saif, 1998), and possibly the elimination of
crucial elements within the bursal microenvironment (Ramm et al., 1991).
Infection of day-old SPF chicks with a virulent IBDV strain reduced the antibody
production against IBV in tears as well as in serum (Thompson et al., 1997;
Gelb, et al., 1998). Studies showed that immunosuppression caused by IBDV
infection could last for at least up to six weeks of age (Wyeth, 1975; Giambrone,
1979; Lucio and Hitchner, 1980). The most severe and longest-lasting
immunosuppression occurs when day-old chicks are infected with IBDV (Allan et
al., 1972; Faragher et al., 1974; Sharma et al., 1989). Fortunately this is
uncommon in the field because chicks usually have high maternal antibody (van
den Berg, 2000). However, at two to three weeks when maternal antibody
wanes, then the infection is likely to occur (van den Berg, 2000). In situations
where the bursa of Fabricius of young birds were destructed, this has been
shown to affect the effectiveness of the subsequent vaccination programmes
(Giambrone et al., 1976).

50
2.1.5 Epidemiology of IBD
The actual distribution of IBD around the world is difficult to ascertain because of
the subclinical nature of the disease. The first outbreak due to the classical IBDV
(caIBDV) occurred in 1957 in the US town of Gumboro and was initially
described as avian nephrosis (Cosgrove, 1962). It was characterized by flock
morbidity of 10-25% and mortality averaging 5% (Lasher and Shane, 1994). The
disease later discovered in 1971 in India (Mohanty et al., 1971), 1973 in Japan
(Hirai et al., 1974), 1974 in Australia, (Firth, 1974), and 1974 in United Kingdom
(Edwards, 1981). The prevalence of clinical IBD was reduced following the
introduction of live vaccines from 1966 onwards (Edgar and Cho, 1965).
In 1983, antigenic variant IBDV (vaIBDV) was reported in the USA (Jackwood
and Sommer, 1999), in China (Cao et al., 1998) and in Australia (Sapats and
Ignjatovic, 2000). Chickens vaccinated with caIBDV vaccines were not protected
against these new „variant strains‟ and they succumbed to immunosuppressive
form of the disease (Ture et al., 1993; Vakhaira et al., 1994).
The vvIBDV strains, a newly evolved strain associated with very high mortality
were first observed in Europe in the late 1980s (Chettle et al., 1989; van den
Berg et al., 1991). To date, vvIBDV infections have been documented in Europe
(Chettle et al., 1989; Pitcovski et al., 1998), Asia (Japan) (Nunoya et al., 1992;
Lin et al., 1993), China (Cao et al, 1998), Malaysia (Hair-Bejo, 1992) and Africa

51
(Zierenberg et al., 2000). Until now, none of vvIBDV was reported in United
States, Australia, Canada and New Zealand (Sapat and Ignijatovic, 2000).
It was hypothesized that the initial outbreaks of IBD in the USA arose by
mutation of an Aquabirnavirus such as infectious pancreatic necrosis virus
(IPNV) (Lasher and Shane, 1994), there is no published evidence that IBDV
serotype 1 existed in turkey flocks prior to 2003, although an earlier report
suggested that turkeys might be infected with IBDV serotype 1 and 2 (McNulty
et al., 1979). The latest report by Owoade et al (2004) showed that turkeys
should be considered to be susceptible to vvIBDV infection.
2.1.6 Transmission
IBD has been an economically significant, widely distributed condition affecting
flocks of chickens. The causal virus is transmitted laterally by direct and indirect
contact between infected and susceptible flocks (Lasher and Shane, 1994), but
not transmitted vertically by transovarian route (Lukert and Saif, 1997). Indirect
transmission of virus most probably occurs on fomites (feed, clothing and litter)
or through air (Benton et al., 1967). There is no evidence of egg transmission of
the virus and no carrier state has been detected in chickens (Saif, 1998).
Infected chickens shed IBDV at one day after infection and can transmit the
virus for at least 14 days (Vindervogel et al., 1976), but not exceeding 16 days
(Benton et al., 1967; Winterfield et al., 1972; Lasher and Shane, 1994).

52
Operation of multi-age broiler and pullet replacement farms, defects in
biosecurity or proximity of farms to road used to transport poultry may contribute
to high prevalence of infection (Lasher and Shane, 1994). The virus can remain
viable for up to 60 days in poultry house litter (Vinervogel et al., 1976). In
addition, rodent, wild birds and insects including mites may be playing an
important role in transmission of IBDV (Brady, 1970). Beside, the lesser meal
worm was recognized as a carrier and the virus has been isolated from
mosquitoes and evidence of infection in rats has been reported but there is no
indication that either species is a reservoir for the virus (Saif, 1998). In contrast
Pages-Mante, et al (2004) show that the possibility that dog could eventually be
carrier of IBDV after eating infected chicken either voluntarily or accidentally.
2.2 Infectious Bursal Disease Virus
The etiological agent of the disease is infectious bursal disease virus (IBDV)
belonging to the family Birnaviridae of the genus Avibirnavirus. The genus name
Birnavirus was proposed to describe viruses with 2 segments of double stranded
RNA. Other viruses included in this group are infectious pancreatic necrotic virus
(IPNV) of fish, tellina virus, oyster virus, blotched snakehead virus (BSVN) (Da
Costa et al., 2003) and crab virus of bivalve mollusks belonging to
Aquabirnavirus while Drosophila X virus belongs to genus Entomobirnavirus. All
of these contain two segments of double stranded RNA surrounded by a single
protein capsid of icosahedral symmetry (Dobos et al., 1979)

53
2.2.1 IBDV Genome
IBDV contains a genome composed of two segments and double stranded RNA
(dsRNA), designated A and B (Dobose et al., 1979; Mundt and Muller, 1995).
The dsRNA genome is enclosed within a non-enveloped icosahedral capsid
approximately 60nm in diameter (Mundt and Muller, 1995). The larger segment
A (3.4kb) contain two open reading frames (ORFs) of 3,039 pb and 438 pb,
which partially overlap at 5‟ end of the genome (Bayliss et al., 1990; Mundt et
al., 1995). The larger ORF encodes a 110KDa precursor polyprotein (NH2-VP2-
VP4-VP3-COOH) which is autocatalytically cleaved by cis-acting viral protein
VP4 into three proteins designated precursor VP2(pVP2)(48kDa), VP3(23KDa).,
and VP4 (28KDa) (Sanchez and Rodriguez 1999; Lejal et al., 2000; Birgham et
al., 2002). The pVP2 is further processed into VP2 (38KDa) during maturation of
the viral particle (Sanchez and Rodriguez., 1999; Lejal et al., 2000; Birgham et
al., 2002). VP2 the major structural protein of the viral capsid, carries highly
conformational epitopes responsible for the induction of neutralising antibodies
that confer protective immunity (Azad et al., 1987; Becht et al., 1988; Jagadish
et al., 1988). VP3 is the second structural protein of the viral capsid, recognized
by non-neutralising antibodies that often cross-react with both serotypes
(Hudson et al., 1986; Bottcher et al., 1997). The smaller ORF of segment A
encodes VP5 (17KDa), a 145 amino acid non structural protein of unknown
function (Mundt et al., 1995). VP5 has been shown for viral replication and
infection, but plays an important role in the release of viral progeny from infected
cells which are important for its pathogenicity (Mundt et al., 1995; Mundt et al.,

54
1997). The smaller segment B (2.8kb) encodes VP1 (90KDa), RNA-dependent
RNA polymerase (RdRp) with capping enzyme activities (Mundt et al., 1995).
Several attempt to elucidate the residues responsible for the pathogenicity of
IBDV has identified conserved amino acid substitutions throughout both genome
segments (Lejal et al., 2000). Development and application of reverse system
for IBDV has shown that neither the non-coding regions (NCRs), nor residue
within VP1 or the N terminus of VP2 is responsible to increase pathogenicity of
IBDV (Mundt and Vakharia, 1996; Yao et al., 1998). These results suggest that
virulence determinants reside within the VP2, VP4 and/or VP3 proteins.
Comparison of the deduced amino acid sequence of the large segment of IBDV
strains showed that the most amino acid change occurs in the central
hypervariable region between residues 206 and 350 of VP2 protein (Bayliss et
al., 1990). VP2 has been shown to be the variable region which encodes the
neutralisation antigenic epitope (Chen et al., 1998). This region is highly
conformation dependent, and it is constituted by hydrophobic fragment flanked
by hydrophilic peaks (Fahey et al., 1989; Fahey et al., 1991; van den Berg et al.,
1996). VP2 of the virus is shown to be responsible for increased apoptosis in a
variety of different mammalian cell lines (Fernendez Aries et al., 1997).

55
2.2.2 IBDV Proteins
Five viral proteins have been described in the IBDV virion namely VP1, VP2,
VP3, VP4 and VP5 (Nick et al., 1976). VP1, VP4 and VP5 are non-structural
viral proteins whereas VP2 and VP3 are structural viral proteins (Mundt et al.,
1995; Nagarajan and Kibenge, 1997).
VP1, a RNA dependent RNA polymerase of the IBDV, is present in small
amounts (3%) in the virion. It is 90 kDa in molecular weight (Lasher and Shane,
1994). VP1 is both a free polypeptide and a genome-linked protein (Muller and
Nitschke, 1987; Kibenge and Dharma, 1997). It plays a key role in the
encapsidation of the viral particles (Lombardo et al., 1999).
VP2 is a 454 amino acid long polypeptide that builds up the external virus capsid
(Kibenge et al., 1988). Expression or deletion studies have shown VP2 amino
acid positions 206 to 350 to represent a major conformational, neutralizing
antigenic domain (Azad et al., 1987). Most amino acid changes between IBDV
strains are clustered in this region, thus referred to as VP2 variable domain
(Bayliss et al., 1990). This domain is composed of hydrophobic amino acid
flanked by two hydrophilic peaks A and B, which span amino acid 210 to 225
and amino acid 312 to 324, respectively (Azad et al., 1987). Variations in IBDV
antigenicity have been shown to depend on changes in peaks A and B. Two
smaller hydrophilic areas of VP2 variable domain, amino acid 248 to 252 and
279 to 290 were recently reported to also influence IBDV antigenicity (van den

56
Berg et al., 1996). Only two mutations of the VP2 (Q253H and A284T) are
enough to attenuate a vvIBDV strains (UK661 isolate) and enabling it to grow in
cell culture (van Loon et al., 2002). VP2 is an important IBDV structure protein
as the antigenic site that is responsible for the induction of neutralizing
antibodies are centrally located on VP2 gene (Fahey et al., 1989; Becht et al.,
1988). Monoclonal antibodies (MAbs) had been successfully raised against VP2
and VP3 but only those reacting to VP2 have the ability to neutralize the virus
(Azad et al., 1987; Becht et al., 1988; Snyder et al., 1988). Thus, it was
suggested that the hyper variable region of the VP2 gene is responsible for the
virus antigenicity and the induction of host neutralizing antibodies (Schnitzler et
al., 1993). VP2 is also an apoptotic inducer where its expression in various
mammalian cell lines leads to apoptosis (Fernandez-Arias et al., 1997). In vivo
studies and molecular characterization suggest that some of the VP2 residues
may play a role in molecular determinants for the virulence, cell tropism and
pathogenic phenotype of vvIBDV (Brandt et al., 2001).
VP3 is a group specific antigen which is recognized by non-neutralising
antibodies. VP3 is 40% of the complete virion protein with 32 kDa molecular
weight (Becht et al., 1988; Oppling et al., 1991). It is responsible for the
structural integrity of the virion and has been identified as a major antigenic
component of the virus (Fahey et al., 1985). VP3 reacts with serotypes1 and 2
and perform as an intermediate, which interacts with both the VP2 and VP1, and
the formation of VP1-VP2 complexes is likely to be an important step in the
morphogenesis of IBDV particles (Lombardo et al., 1999).

57
VP4 is fourth viral protein with 28 kDa molecular weight. It is a non-structural
polypeptide, representing 6% of the viral protein. VP4 is involved in the
autoprocessing of the virus polyprotein producing VPa, VP3 and VP4 (Lasher
and Shane, 1994; van den Berg, 2000). The amino acids for this proteolytic
activity have been identified to be a serine lysine catalytic dyad (S652 and K
692) (Lejal et al., 2000).
VP5 is also nonstructural IBDV protein that has been identified in IBDV infected
cells. The VP5 is located at the second ORF on the segment A of the IBDV
genome which encodes polyprotein of 21 kDa molecular weight. This
polypeptide more probably has a regulatory function and may play a key role in
virus release and dissemination (Mundt et al., 1995; Lombardo et al., 2000).
2.2.3 Antigenic and Virulence Variation
IBDV is endemic throughout the world but several different antigenic and
pathogenic types exist in specific geographic locations. Two serotypes of IBDV
are recognized by the virus neutralization test. These two serotypes are
antigenically distinct (Mcferran et al., 1980). Serotype 1 viruses are pathogenic
to chickens and differ in their virulence (Winterfield et al., 1978). They cause
lesions in the bursa of Fabricius by lymphocytic depletion (Schroder et al., 2000)
whereas, serotype 2 viruses are avirulent to chickens and are isolated mainly
from turkeys (Ismail et al., 1988; Kibenge et al., 1991).

58
Serotype 1 viruses can be broadly divided into classic (ca), variant (va) and very
virulent (vv) IBDVs. Until 1987, the strains of virus were of low virulence and
were controlled by vaccination. Emergence of variant viruses was first reported
in USA in 1987. These viruses were reported to undergo an antigenic drift
against which the classical IBD vaccines were not protective (Jackwood and
Saif, 1987; Snyder et al., 1992).
Six antigenic subtypes of IBDV serotype 1 viruses have been identified by the
virus neutralization test (Jackwood and Saif, 1987). Variant viruses that were
found in the USA and Australia are different from the classic viruses in terms of
pathogenicity and immunogenicity. They overcome the immunity induced by
classic serotype 1 viruses and cause rapid bursal atrophy with minimal or no
inflammatory response (Mcferran et al., 1980; Jackwood and Saif, 1987; Hassan
and Saif, 1996).
Vaccination with one serotype 1 subtype did not ensure protection from
challenge with another subtype suggesting that variant viruses are antigenically
different from classical viruses (Mcferran et al., 1980; Jackwood and Saif, 1987;
Ismail and Saif, 1991; Hassan and Saif, 1996). Variant viruses present in the
USA and Australia are not closely related to each other (Sapats and Ignjatovic,
2000). Significant antigenic differences exist among serotype 1 strains as
detected by virus neutralization and this led to the grouping of the serotype 1
viruses into 6 subtypes (Hassan and Saif, 1996) hence virus neutralization test

59
proved to be serotype specific and could distinguish between the two serotypes
(Jackwood et al., 1982; Jackwood et al., 1985; Hassan and Saif 1996).
Serotype 2 viruses are immunologically distinct from serotype 1 viruses since
vaccination with serotype 2 (OH) viruses did not confer protection against
serotype 1. Cross protection studies indicated that the variant viruses were
different from other subtypes of serotype 1 IBDVs. Both serotype 1 and 2
viruses share common group antigens which could be detected by (AGPT),
flourescent antibody test and ELISA (Jackwood et al., 1982; Jackwood et al.,
1985; Jackwood and Saif 1987; Chettle et al., 1989). Capsid proteins VP2 and
VP3 contain epitopes that are responsible for group antigenicity (Becht et al.,
1988). The VP2 carries the serotype specific antigens responsible for the
induction of neutralizing protective antibodies (Azad et al., 1987; Becht et al.,
1988).
VP2 is the major host-protective immunogen of IBDV and it contains the
determinants responsible for antigenic variation (Fahey et al., 1989; Brown et
al., 1994; Vakharia et al., 1994). The antigenic site which is responsible for the
induction of neutralizing antibodies against IBDV, are centrally located on VP2
gene and known as hypervariable region (Azad et al., 1987; Becht et al., 1988).
The sequences of the major host-protective immunogen VP2 are highly
conserved except the central Accl-Spel (206-350) restriction fragment of
hypervariable region (Heine et al., 1991; Brown and Skinner, 1996).
Representing only 16% of segment A, this region displays the greatest amount

60
of amino acid sequence variations between the pathogenic serotype 1 strains
(Becht, 1980; Kibenge et al., 1990).
The HPVR encodes for the immunodominant viral epitopes (Becht et al., 1988;
Fahey et al., 1989) or neutralizing antigenic epitopes. There are at least three
distinct, non-overlapping and conformation-dependent epitopes (Azad et al.,
1987; Becht et al., 1988; Oppling et al., 1991). These epitopes are located at the
central variable region of the VP2 gene and is comprised of 145 amino acids
from amino acids 206-350. Within this region, there are two hydrophilic peaks
which is highly conformation dependent (Fahey et al., 1991; van den Berg et al.,
1991).
The first peak is from amino acid 212 to 224 where as the second peak is from
amino acids 314 to 324 (Fahey et al., 1989; Bayliss et al., 1990; Heine et al.,
1991; Schnitzler et al., 1993; Brown et al., 1994). Within the first hydrophilic
peak of the hydrophilic region, position 222 appears to play a crucial role in
epitope formation. In classical virus strain, proline (P) is found at position 222,
while glutamine (Q), therionine (T) or serine (S) are found in variant strains and
alanine (A) is found in vv strains (Vakharia et al., 1994; Dormitorio et al., 1997).
Minor mutations in the hydrophilic peaks can result in antigenic drift (Schnitzler
et al., 1993). The amino acid residue changes at the P222A (proline to alanine),
V256I (valine to isoleucine) and L294I (leucine to isoleucine) can be used as a
marker for vvIBDV, G254S (glycine to serine) and Q249K (glutamine to lysine)
for variant strains whereas, amino acid changes at D279N (aspartic acid to

61
aspargines) and A248T (alanine to theronine) are common among attenuated
strains (Yamaguchi et al., 1996b; Cao et al., 1998). The hydrophilic regions are
thought to play an important role for the formation and stabilization of the virus
neutralizing epitopes (Heine et al., 1991; Schnitzler et al., 1993; Vakharia et al.,
1994).
In addition, specific amino acid changes do occur within the HPVR, an adjacent
downstream serine-rich heptapeptide sequence (SWSASGS) which are located
after the second hydrophilic region, amino acid residue 326 to 332 have been
proposed as potential sites responsible for virus attenuation (Heine et al., 1991;
Vakharia et al., 1994; Yamaguchi et al., 1996b; Dormitorio et al., 1997) or
antigenic determinants associated with the virulence of IBDV (Brown et al.,
1994).
2.3 Isolation, Adaptation and Attenuation of IBD Virus
2.3.1 Chicken Embryos
Initially, most workers had difficulty in isolating of the virus in chicken embryos.
Landgraf et al. (1967) reported a typical experience using the allantoic sac route
of inoculation. Hitchner (1970) demonstrated that chorioallantoic membrane
(CAM) of 9-11 days old embryos was the most sensitive route of isolation of
IBDV. Hitchner (1970) observed that most mortality occurred between the 3rd
and 5th
days post inoculation as affected embryos had edematous distention of

62
the abdomen, petechiae and congestion of the skin and occasionally echymotic
hemorrhages in the toe joints and cerebrum.
2.3.2 Cell Culture
Many strains of IBDV have been adapted to primary cell culture of chicken
embryo origin and cytopathic effects have been observed. These cells include
chicken embryo kidney (CEK), chicken embryo bursa (CEB) and chicken
embryo fibroblast (CEF) cells (Lukert and Davis, 1974; McNulty et al., 1979).
Cell culture adapted IBDV grows in several mammalian continuous cell lines
such as RK-13 derived from rabbit kidney (Rinaldi et al, 1972), Vero cells
derived from adult African green monkey (Leonard, 1974; Lukert et al., 1975;
Jackwood et al., 1987), BGM-70 cells derived from baby grivet monkey kidney
and MA-104 cells derived from rhesus monkey kidney (Jackwood et al., 1987).
Continuous cell lines has been found to yield higher virus titers compared to
primary cell culture, thus are more suitable to use for vaccine production. Three
strains of serotype1 IBDV (SAL, D78, 2512), one of the serotype 2 (OH) and one
vaIBDV strain (Variant A) were grown in Vero and (CEF) cell culture. The latent
period in Vero cells ranged from 12-18 hours, which has longer than 4-6 hours
period observed in CEF cultures from strains SAL, D78 and OH. There was
more extensive maturation phase and higher yield of virus in Vero cells than in
CEF cultures. Total titers of theses viruses of 5.35 to 6.10 log10 TCID50/ mL in
CEF occurred 24-40 hours post infection (pi) although the CPEs were not seen

63
until 72 hours pi. By comparison, their total infectious virus titers of 6.85 to 8.35
log10 TCID50/mL in Vero cell occurred from 48 hours pi coinciding with
appearance of CPEs. The growth curve of variant A in Vero cells differed from
other viruses by showing steadily extracellular and cell associated virus titer
throughout the 72 hours observation period. Only very low titers of variant A
were obtained in CEF cultures and no growth curve in CEF was reported
(Kibenge et al., 1988).
Vero cell line was found to be more susceptible than ovine kidney (OK) cell line
for IBDV. Kibenge et al., (1992) used OK cell line, Vero cell line and CEF
culture to attempt IBDV isolation from 26 suspected samples. Virus was isolated
from 2 of 26, 3 of 26 and 3 of 25 samples on OK, Vero and CEF cultures,
respectively. However, in contrast to IBDV replication in Vero and CEF, isolated
virus was unable to induce serially sustained CPEs during successive passages
in OK cell line. The cytopathogenicity of chloroform un-treated virus passages
on OK cells was revived and maintained upon passages in Vero cells (Kibenge
et al., 1992). An initial single passage of suspected field material in OK cells
followed by further passages in Vero cells resulted in virus isolation from 6 of 26
samples which was a better recovery than when either cell line was used alone
or when CEF culture was used. Twenty of twenty six samples were originally
positive when examined by nucleic acid hybridization with radio-labeled IBDV-
cDNA, indicating that some of the samples that were negative upon virus
isolation using OK and Vero cells may have contained inactivated virus. When
two variant strains of IBDV, IN and E were serially passaged in BGM-70 cell line

64
for 30 times and 40 times respectively, it resulted in loss of pathogenicity.
However, both viruses maintained their antigenicity and immunogenicity as
demonstrated by immunofluorescence and virus neutralization tests. When
inactivated preparation of both passaged viruses was inoculated in SPF chicken,
satisfactory protection was obtained (Tsai and Saif, 1992).
A variant IBDV strain 977 was passaged in cell culture, plaque purified and
attenuated by serial passages at a high multiplicity of infection in CEF. Cell
culture passaged virus caused less bursal atrophy and splenomegaly than did
the original isolate and retained immunogenicity (Bayyari et al., 1996).
Mohamed et al. (1996b) investigated the pathogenicity of bursa derived and
tissue culture attenuated classic (STC) and variant (IN) serotype 1 strains of
IBDV. The IN bursa derived virus caused bursal necrosis and atrophy earlier
than bursa derived STC virus. Both viruses lost their pathogenicity after four
passages in BGM-70. A statistically significant level (p<0.05) of protection was
observed in SPF chicken vaccinated with the attenuated IN virus used as a live
or inactivated vaccine followed by homologous (STC) challenged with bursa
derived virus (Hassan et al., 1996; Mohamed et al.,1996a).
Mohamed et al. (1996a) also investigated the effect of host system on the
pathogenicity, immunogenicity and antigenicity of IBDV. One classic (SAL) and
one variant (IN) strain of IBDV were passaged separately six times in three host
systems BGM-70 continuous cell line, CEF and embryonated chicken eggs.
Passages in BGM-70 cells or CEF resulted in loss of pathogenicity but virus

65
passaged in embryos maintained its pathogenicity (Mohamed et al., 1996a).
Although the CEF and Vero cells infected with IBDV exhibited the biochemical
features of apoptosis, agarose gel electrophoresis of DNA extracted from IBDV
infected cells revealed the characteristic laddering pattern of DNA fragmentation
which was more intense in infected CEF than Vero cells. The appearance of
apoptotic nucleosomal DNA fragments in IBDV infected CEF was independent
of virus replication and occurred at an early stage following an in vitro infection
(Tham and Moon, 1996).
Highly virulent the vvIBDV strains were adapted through serial passages in
embryonated eggs. The embryonated egg-adapted vvIBDV was successfully
adapted to grow CEF with CPEs. The embryonated egg and cell culture adapted
virus strains had significantly reduced pathogenicity and did not kill any young
chicken in experimental infection. The bursal lesions of the adapted strain-
infected chicken were similar to those observed in classic strain-infected
chicken. Cross virus neutralization analysis showed antigenic diversity between
the cell culture adapted vvIBDV and classical strains. Immunization with adapted
strains in chicken showed good protection against the infection of vvIBDV,
especially, in case of 3 days post-immunization challenged hence adapted virus
strains showed effective immunogenicity hence they appeared to provide a new
and effective live vaccine against vvIBDV (Yamaguchi et al., 1996b).
Yamaguchi et al. (1996b) studied the changes in the virus population during
serial passage in chicken and chicken embryo fibroblast cells. Two attenuated

66
infectious bursal disease virus used as commercial live vaccine were passage
five successive times in SPF chicken and CEF cell. Both attenuated strains
increased in virulence during the passage in susceptible chicken as evidenced
by the decrease in bursa to body weight ratio. A direct nucleotide sequence
analysis of the VP2 hypervariable domain amplified by RT-PCR revealed that
the nucleotide at position 890 (T) in both strains was (A) after the passage in
chicken. In addition, the nucleotide at position 890 (A) was T or C after the
subsequent passage in CEF cells. Because of the nucleotide differences, the
amino acid residue at position 235 (His) in both vaccines was Gln after the
passage in chicken, and the amino acid residue Gln was changed back to His
during subsequent passage in CEF cells. The digestion of the amplified
fragment with restriction endonuclease Stu 1 and Neo 1 which recognize the
sequence difference at position 890, showed the population of the virus that had
amino acid Gln at position 253 was gradually increase during the passage in
chicken. The population of the virus that had amino acid His at position 253 was
gradually increased during the subsequent passage in CEF cells.
2.4 General Information on the Immune System
The immune system is an important part of any live entity, protecting the host
from infections existing in the environment such as viruses, bacteria and
parasites and from other non-infectious foreign substance such as protein and
polysaccharide (Abbas, et al 2001; Calder and Kew, 2002). Bone marrow, lymph
nodes, spleen, and the thymus are essential elements of the immune response

67
of chicken to microorganism. The first is the innate (or natural) immunity and the
second is the adaptive (specific, acquired) immunity (Abbas et al., 2001).
2.4.1 Innate Immunity
The innate immune system is the initial level of immune response that combats
infections. Its properties are defined in the germ line. Innate immunity has no
memory property. It consists of anatomic, physiologic and phagocytic / endocytic
barriers and chemical protection such as gastric acid (Medzhitov and Janeway,
1997). These anatomic barriers are the first line of defence against invaders.
They include the skin and mucous membranes. Physiological barriers in innate
response, such as pH, temperature and oxygen tension limit microbial growth.
Phagocytic cells are critical in the defence against pathogens. Some primary cell
of the innate immunity system include phagocytic / endocytic barriers such as
(heterophils), monocytes and phagocytic macrophages. These cells have
specific receptors associated with common bacterial molecules. Monocytes and
lymphocytes can create and secrete cytokines which are non-immunoglobulin
Polypeptides, in response to interaction with a specific antigen (Ag), a non-
specific Ag or a non-specific soluble stimulus. Cytokines affect the magnitude of
inflammatory or immune responses. They regulate other cells of the immune
system. The secretion of cytokines may be triggered by the interaction of a
lymphocyte with its specific Ag but cytokines are not Ag-specific. Thus, they
bridge innate and adaptive immunities. Macrophages are important phagocytic
cells that participate in non specific and specific immunity. They can destroy

68
infected cells and ingested microbes and support other cells of the immune
system to generate an immune response (Abbas et al., 2001).
2.4.2 Adaptive Immunity
When the innate immune system cannot handle and destroy the encountered
pathogen, adaptive immunity is the next line of defence in its support. Acquired
immunity is very specific and has an immunologic memory. The immunologic
memory allows this specific immunity to remember the molecular features of a
pathogen that has been previously encountered and handled. Adaptive immunity
includes both humoral and cell-mediated immune response (Abbas et al., 2001).
2.4.3 Humoral (B cell-mediated) Immunity
Humoral immunity can combat certain infections through circulating antibodies
such as immunoglobulin (Ig) (Devereux, 2002). The antibodies are generated as
soon as a germ is encountered and remain in the immune system.
Immunoglobulin molecules are the cell surface receptor of B-lymphocytes
derived from the bursa of Fabricius in chicken. Antibodies in birds fall into three
major categories: IgM, IgG (also called IgY) and IgA. It has been observed that
mature B-cells, which have a single antigen specificity, travel towards different
lymphoid organs in order to properly interact with an antigen (Abbas et al.,
2001). The antibodies produced are usually incapable of struggling against

69
viruses and some types of bacteria intracellularly. However, they are powerful at
destroying extracellular pathogens.
2.4.4 Cell-mediated (T-cell mediated) Immunity
Cell- mediated immune response becomes active when the humoral immune
response is not capable of eliminating the antigen (Erf, 2004). T-lymphocytes
play an important role in the cell-mediated immune system and are capable of
handling and mitigating the risk of intracellular pathogens (Chen et al., 1991;
Devereux, 2002).
T-cell can recognize antigens through the T-cell receptor (TCR) and other
accessory adhesion molecules. All T-cells express the CD3 complex but T-cell
has discrete subpopulation, thus distinguishing them as cytotoxic or regulatory
T-cells. Cytotoxic cells eliminate mostly virus-infected and tumor cells, they are
inclined to express the CD8 complex, a specific molecule on their surface (Chan
et al., 1988; Janeway et al., 2001). Regulatory T-cells, also called T- helper cells
(Th) express the CD4 cell-surface molecules and play a major role in the
immune system (Astile et al., 1994). Such cells produce cytokines that are
needed for T- and B- cells to become active (Chan et al., 1988; Janeway et al.,
2001). These cytokines are capable of activating component of non-specific
immunity and thus enhance better functioning of the immune system. The Th-
cells are subdivided into type-1 T-helper cells (Th1) and type-2 T-helper cells
(Th2). The classification of regulatory T-cells is based on the profile of cytokines

70
produce and their function (Bottomly, 1988). Th1-cells an important role in cell
mediated immune response while Th2-cells participate in the induction of a
strong humoral immune response (Constant and Bottomly, 1997).
2.4.5 Relationship Between B- and T-cells
B-cells do not need antigen-presenting cells, because B-cells can bind directly
with antigens. However, they do need cytokines created by Th cells in order to
be completely active and become antibody-producing plasma cells (T-
dependent response). Consequently B-cells obtain support from Th-cells.
Nevertheless, it is known that there are certain antigens, such as T- independent
antigens, that activate B-cells irrespective of Th- cells (Abbas et al., 2001).
2.4.6 Effect of IBDV on Innate Immunity
IBDV modulates macrophage functions. There is indirect evidence that the in
vitro phagocytic activity of these cells may be compromised (Lam, 1998).
Macrophages are important cells in the immune system and the altered
functions of these cells may influence normal immune responsiveness in birds.
2.4.7 Effect of IBDV on Humoral Immunity
IBDV has an affinity for the immature B lymphocytes (Sivanandan and
Maheswaran, 1980) and actively dividing B lymphocytes thereby causing a

71
complete lysis of IgM bearing B cells which in turn result in the decrease in
circulating IgM cells. Infected chicken produces less level of antibodies against
the antigen (Kim et al., 1999). Only primary antibody responses are affected.
Secondary responses remain unaltered (Rosenberger et al 1994; Sharma et al.,
1989). IBDV induced humoral deficiency is reversible and overlaps with the
restoration of bursal morphology (Sharma et al., 2000).
Chickens infected with IBDV at 1 day of age were found to be completely
deficient in serum IgG and produced only a monomeric immunoglobulin M (IgM)
(Ivanyi, 1975). IgG levels varied depending on the age at the time of infection
(Hirai et al., 1981). The number of B cells in peripheral blood was reduced after
infection with IBDV, but T cells were not appreciably affected (Hirai et al., 1981;
Sivanandan and Maheswaran, 1980). The adverse effect on antibody responses
is due to the damage to the B cells in the bursa and the blood since the virus
has a predilection for actively dividing B cells as compared to the mature B cells
(Sivanandan and Maheswaran, 1980).
2.4.8 Effect of IBDV on Cellular Immunity
T-cells in spleen and peripheral circulation are affected during IBDV infection
(Confer et al., 1981; Sivanandan and Maheswaran, 1980; Kim et al., 1999). The
mitogenic inhibition of T cells occurred early, during the first 3 to 5 days of virus
exposure but later returned to normal levels. During the period of mitogenic
inhibition, T cells of IBDV infected chickens also failed to secrete IL-2 upon in

72
vitro stimulation with mitogens (Kim et al., 1999; Sharma and Fredericksen,
1987).
2.5 Vaccination
IBDV is highly infectious, very resistant in the environment and can persist in the
poultry houses after cleaning and disinfection. The virus is also resistant to ether
and chloroform. It is inactivated at pH 12.0 but unaffected at pH 2.0.
Consequently the virus can persist in the chicken houses for long periods
(Benton et al., 1967). Therefore, hygienic measures alone are not enough to
control this disease and vaccination is the principle method used for the control
of IBD in chicken (Kibenge et al., 1988).
The most common strategy followed to control IBD is by achieving passive
and/or active immunity in chickens (van den Berg, 2000). Passive immunity is
referred to the transfer of IBDV specific, neutralizing antibodies from
hyperimmunized parent flocks to their progeny (Sharma and Rosenberger,
1978). These maternally derived antibodies protect baby chick from early
immunosuppressive effect caused by IBDV. Passive immunity conferred to
progeny chicks normally last up to 21 days of age approximately. However, the
vaccination of parent breeders with an inactivated IBDV oil-emulsified vaccine
extends the range of maternal antibody protection up to 30-38 days of age
(Lucio and Hitchner, 1979; Baxendale and Lutticken, 1981; Lukert and Saif,
1997). Attempts have been made to confer passive protection by performing

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