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
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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
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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
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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
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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 107TCID50/0.1mL 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 Faculty of Veterinary Medicine Universiti Putra Malaysia (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 Universiti Putra Malaysia 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 Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Abdul Rahman Omar, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Aini Ideris, PhD Professor
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Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)
HASANAH MOHD GHAZALI, PhD
Professor and Dean School of Graduate Studies Universiti Putra Malaysia 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 3.3.2 Chorio-allantoic Membrane for UPM04019 66 3.3.3 Chorio-allantoic Membrane for UPM0081 67 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 Materials and Methods 92 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 Materials and Methods 137 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 (TCID
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
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
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attenuated vvIBDV and control group
5.4 Experiment 2: body weight (g) of chickens in the inoculated attenuated vvIBDV and control group
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 attenuated vvIBDV and control group
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 attenuated vvIBDV and control group
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
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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
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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)
Sigma) was removed. The cells were rinsed twice with warm 37oC sterile PBS,
pH7.2. An amount of 5 mL of warm antibiotic-trypsin versene (ATV) (Appendix
A) solution was added to chelate the cells. When the cells started to detach, the
ATV was poured away and the cell culture flask was shaken vigorously to
62
complete cell detachment. The cells were then resuspended in sufficient amount
of RPMI growth medium containing 10% (v/v) fetal bovine serum (FBS), and 1%
(v/v) antibiotic-antimycotic. This allowed preparation of two or three 25 cm2 cell
culture flasks from the original one of 25 cm2. The new flasks were incubated at
37oC in CO2 incubator (Thermo Forma, USA) and observed daily until the
confluent monolayers of cells were formed, usually within 2-3 days. Upon
formation of monolayers, the old media were replaced with fresh RPMI
maintenance medium with the same content as the growth medium, except that
FBS was reduced to 1% (v/v) (Appendix A).
3.2.7 Infections of Vero Cell and DF-1 Cells Monolayer
Three IBDV isolates (UPM94273, UPM04019 and UPM0081) were used to
infect the healthy, semi-confluent monolayers of Vero cells and DF-1 cells with
concentration of seed cells were 3 x 106 and 1.5 x 105 cells/mL, respectively.
The growth medium of the flasks was removed and the monolayers were
washed twice with prewarmed sterilized PBS. 0.1 mL of vvIBDV inoculums was
dispensed over the each monolayer, the inoculums was spread uniformly over
each monolayer and flasks were incubated at 37oC for I hour with intermittent
rotation to allow the virus to adsorb on the surface of Vero cells and DF-1 cells.
Five mL of sterilized pre warmed maintenance medium (Appendix A) was added
in each flask and incubated them at 37oC in 5% CO2. The monolayers were
63
examined twice daily under inverted microscope for cytopathogenic effects
(CPEs).
3.2.8 Harvesting of Virus
If the CPEs were found, infected cells were harvested. The virus infected cells
and culture medium were repeated frozen and thawed three times. After clarified
by low-speed centrifugation at 3,000 rpm for 20 minutes at 4oC, the supernatant
fluids were harvested and passed through a 0.45 µm filter, aliquot, and stored at
-80oC or used as IBDV inoculums inoculated in subsequent cell cultures for
further passages.
3.2.9 Adaptation and Attenuation
The passage 1 (P1) virus was inoculated again to fresh, healthy and semi
confluent monolayers of Vero cells and DF-1 cells using the same technique for
CPEs. The virus was harvested by three freeze-thaw cycles, clarified and
labeled as passage 2 (P2). In this way IBDV was adapted to Vero cells or DF-1
cells when the CPEs were clear and consistent. The adapted virus was serially
passage until it became attenuated (non-pathogenic).
64
3.2.10 Tissue Culture Infective Dose 50 (TCID50)
Titration of the virus was achieved by observation of the CPEs. The two
passages of 6th and 9th in DF-1 cells and the three passages of 10th, 15th and
20th in Vero cells were frozen and thawed for three times before they were
clarified at 4000 rpm for 20 minutes at 4oC. The supernatant was collected and
serial ten fold dilutions from this supernatant were prepared in PBS. Vero cells
and DF-1 cells monolayer were prepared in 96-well tissue culture microtitration
plate. Each dilution of virus (100 µl) was inoculated in each well of first row,
leaving the last two well as negative control. The plate was incubated at 37oC in
5% CO2 for 5 days, the plate was examined twice daily for CPEs. The virus titer
was determined by Reed-Muench method (1938). This method evaluates an
endpoint where 50% of the cell cultures were infected. The cell culture infectious
dose affecting 50% of the cultures (TCID50) was calculated using a formula that
takes into account the accumulated percentage of infected cultures (Appendix
B).
3.2.11 IBDV Identification and Confirmation
The growth of IBDV on Vero cells and DF-1 cells monolayers was identified
through its CPEs. The characteristics changes in the infected monolayers were
carefully noted after each passage. The time for the appearance of CPEs of the
65
adapted and attenuated virus on Vero cells and DF-1 cells was also recorded.
The virus of each passage was obtained from the infected tissue culture filtrate
and subjected to indirect immunoperoxidase (IIPS).
3.2.12 Indirect Immunoperoxidase Staining Test
The indirect immunoperoxidase test (IIP) was done according to the method of
Guvenc et al., 2004. The infected Vero cell and DF-1 cells were fixed with cold
methanol: acetone (50:50 v/v) for 5 minutes. The glass slides were then
immersed in 1% H2O2 in absolute methanol for 30 minutes. The PBS was then
added to the glass slide for 15 minutes. The glass slides were then air dried.
The hyper immune serum kindly provided by Prof. Dr. M. Hair-Bejo was diluted
1:1000 with PBS and added to the glass slide incubated for 1 hour in room
temperature. The glass slides were then washed 3 times with PBS for 5 minutes
each. The rabbit anti-chicken IgG-HRP conjugated secondary antibody (Bio-
Red, USA) was then added to the glass slides (1: 1000) and incubated for 1
hour at room temperature. DAB substrate solution (DAB reagent set, Invitrogen,
USA) was then added to the glass slides and incubated for 10 minutes in a dark
room. The slides were mounted with buffer glycerol and examined under light
microscope.
66
3.3 Results
3.3.1 Chorio-allantoic Membrane for UPM94273
There was 60% mortality of the embryo in CAM within 7 day post infection (pi) in
passage one and two. The CAM was mildly congested while the embryos were
haemorrhagic. On the third passage, 80% mortality occurred within day 7 pi. At
this passage, the embryos were severely hemorrhagic and oedematous (Table
3.1, Figure 3.1).
3.3.2 Chorio-allantoic Membrane for UPM04019
There was 100% mortality in the embryo in CAM within 7 day pi following
passage one and two. The CAM was severely congested while the embryos
were haemorrhagic. On the third passage, mortality occurred at day 5 pi. At this
stage, the embryos were severely hemorrhagic and oedematous (Table 3.1,
Figure 3.1).
67
3.3.3 Chorio-allantoic Membrane for UPM0081
There was 100% mortality in the embryo in CAM within 5 day pi in the passage
one, two and three. The CAM was severely congested. The dead embryos
showed severe petechial to ecchymotic hemorrhages (Table 3.1, Figure 3.1).
68
Figure 3.1a: (A): Uninfected control embryonated SPF chicken eggs. (B): UPM94273 dead embryo with severe haemorrhage
69
Figure 3.1b: (C): UPM04019 dead embryo with severe haemorrhage. (D): UPM0081 the embryo infected showed severe petechial to ecchymotic haemorrhage (arrows)
70
Table 3.1: Mortality of SPF embryonated eggs following vvIBDV inoculation into CAM route
vvIBDV strains
Passage
No. of SPF eggs
Cumulative mortality (Days post inoculation)
Total no. of SPF egg viable
Mortality (%)
1
2
3
4
5
6
7
1
5
0a/5b
0/5
1/5
1/5
2/5
2/5
3/5
2
60 UPM94273
2
5
0/5
1/5
1/5
2/5
2/5
2/5
3/5
2
60
3
5
0/5
2/5
2/5
3/5
3/5
3/5
4/5
1
80
1
5
0/5
2/5
2/5
3/5
4/5
4/5
5/5
0
100 UPM04019
2
5
0/5
2/5
3/5
3/5
3/5
4/5
5/5
0
100
3
5
0/5
2/5
4/5
4/5
5/5
5/5
5/5
0
100
1
5
0/5
1/5
3/5
3/5
5/5
5/5
5/5
0
100 UPM0081
2
5
0/5
2/5
2/5
4/5
5/5
5/5
5/5
0
100
3
5
0/5
2/5
3/5
4/5
5/5
5/5
5/5
0
100
a: Total number of eggs dead b: Total number of eggs inoculated
71
3.3.4 IBDV Replication and Adaptation in Vero Cell Line
The CPE was not observed in the first six passages for UPM94273 and
UPM04019. However, the UPM0081 was successfully adapted at 4th passage
with the formation of visible CPEs and attenuated in Vero cells at the 20th
passage. With UPM0081, at 4th and 5th passages, there was complete CPEs
up to 15 and 14 days pi, respectively (Figure 3.2), while at 6th, 9th, and 12th
passages, CPE was observed on 8, 7 and 6 days pi, respectively (Figure 3.3),
and at 13th to 20th passages, the CPE was observed at 4 days pi. (Figure 3.4)
(Table 3.2). The CPE in Vero cells was characterized by aggregates of tiny
round refractive cells that later spread to the entire cell sheet. These abnormal
cells later detached from the surface to leave empty areas in the cell culture.
3.3.5 IBDV Replication and Adaptation in DF 1 Cell Line
The CPE was not observed in the first six passages of UPM94273 and
UPM04019 in DF1 cell line. Again, UPM0081 showed CPE at the third to the
ninth passages. The CPE at the third passage started with rounding and
clumping of few cells at 3 days pi which later progressed to enhanced rounding
and clumping of cells at 5 days pi (Figure 3.5). At 4th passage, the CPE was
similar to those of the 3rd passage, but more concentrated with granular
cytoplasm. At 5th passage however, there was cellular degeneration and
detachment from the surface monolayer by 3 days pi, (Figure 3.6). At 4 days pi,
there was rounding and clumping of large number of cells with intense
72
cytoplasmic granulation and detachment of cells with few cells floating in the
media. At 6th to 9th passages, the CPE were characterized by degenerative and
more detachment of cells at 3 days pi (Figure 3.7, Table 3.3).
73
Table 3.2: Percentage of CPE monolayer Vero cells following UPM0081vvIBDV inoculation
a: Total number of CPE b: Total number of plates inoculated – The cells were harvested.
74
Table 3.3: Percentage of CPE monolayer DF-1 cells following vvIBDV inoculation
vvIBDV passage
No. of
monolayers
plates
CPEs (Days post inoculation)
Total no. of CPE/
plate
CPE
(%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1
4
0a/4b
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0
0
2
4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0
0
3
4
0/4
0/4
0/4
0/4
4/4
–
–
–
–
–
–
–
–
–
–
4
100
4
4
0/4
0/4
0/4
4/4
–
–
–
–
–
–
–
–
–
–
–
4
100
5
4
0/4
0/4
0/4
4/4
–
–
–
–
–
–
–
–
–
–
–
4
100
6
4
0/4
0/4
4/4
–
–
–
–
–
–
–
–
–
–
–
–
4
100
7
4
0/4
0/4
4/4
–
–
–
–
–
–
–
–
–
–
–
–
4
100
8
4
0/4
0/4
4/4
–
–
–
–
–
–
–
–
–
–
–
–
4
100
9
4
0/4
0/4
4/4
–
–
–
–
–
–
–
–
–
–
–
–
4
100
a: Total number of CPE b: Total number of plates inoculated – The cells were harvested.
75
3.3.6 IBDV Titration (TCID50/mL)
As shown in Table 3.4, the TCID50 of UPM0081 strain on Vero cell and DF1 cell
lines ranged from 103.5 to 107.4 and 101.5 to 102.5, respectively. The IBDV titre
continued to increase from P6 to P20 (Appendix B).
Table3.4: Virus titer determined by Tissue Culture Infective Dose50 (TCID50)
vvIBDV Passages
TCID50 on the Vero cells and DF-1 cell line
Vero cells
DF-1 cells
P6 103.5 101.5 P9 104.5 102.5
P10 104.7 ND P15 106.7 ND P20 107.4 ND
ND (Not done) 3.3.7 IBDV Identification through Indirect Immunoperoxidase Staining (IIPS) Test
In IIPS technique, the cytoplasm of the IBDV infected Vero cells and DF-1 cells
with CPE were positively stained light brown (Figures 3.8 and 3.9).
76
Figure 3.2: (A) Uninfected control Vero cells monolayer. (B) Cytopathic effect by UPM0081 isolate at 4th passage at day 15 pi. The arrow shows cell rounding and aggregation. 10 x. Bar = 200 µm
77
Figure 3.3: (A) Vero cell monolayer at 6th passage at day 8 pi (B). Vero cell monolayer at passage 12th at day 6 pi. The arrows show cell rounding and aggregate in clumps and granulation in cytoplasm. 10x. Bar = 200 µm
78
Figure 3.4: (A) A Vero cell monolayer at passage 13th at day 3 pi (B). A Vero cell monolayer at 20th passage at day 4 pi. The arrows show detachment of cells from the substrate with the eventual destruction of the entire monolayer. 10 x. Bar = 200 µm
79
Figure 3.5: (A) Uninfected control DF-1 cells monolayer. (B) Cytopathic effect of UPM0081 isolate of the 3rd passage at day 5 pi. The arrow shows cell rounding and clumping. 10 x. Bar = 200 µm
80
Figure 3.6: (A) DF-1 monolayer of the 4th passage at day 5 pi. Affected cells are more concentrated with granular cytoplasm. (B) DF-1 cells at 5th passage at day 4 pi. The arrow shows detachment of cells from the substrate (B). 10 x. Bar = 200 µm
81
Figure 3.7: (A) DF-1 cell monolayer at 6th passage at day 3 pi. (B) DF-1 cell monolayer at 9th passage at day 3 pi. The arrow shows degenerate cells and more detachment of cells from the substrate. 10 x. Bar = 200 µm
82
Figure 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 at day 2 pi. The arrow shows the presence of specific intracytoplasmic brownish colouration. 20 x. Bar = 100 µm
83
Figure 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 4th passage at day 2 pi. The arrow shows the presence of specific intracytoplasmic brownish colouration. 20 x. Bar = 100 µm
84
3.4 Discussion
This study was initiated to adapt and propagate local vvIBDV isolates in two cell
lines namely Vero cells and DF-1 cells. The three Malaysia isolates of vvIBDV
namely the UPM94273, UPM04019 and UPM0081 were first adapted through
serial passage in SPF embryonated eggs and the embryo showed characteristic
lesions of oedema, distension of the abdomen, generalized congestion and
haemorrhages of the body. The CAM inoculated with UPM04190 and UPM0081
isolates resulted in 100% embryonic death at passage three, while UPM94273
resulted in 50% to 80% embryonic death during three passages. These lesions
were consistent with other studies of vvIBDV (Hair-Bejo et al., 1995a;
Yamaguchi et al., 1996a), while the pathogenicity in the embryo suggests the
virulence of these isolates.
The embryonated egg adapted UPM0081 strain was successfully adapted to
grow in Vero cells after four passages and in DF-1 cells after three passages,
while the other failed to adapt on Vero cell and DF-1 cells after six passages.
The failure of some of the vvIBDV (UPM94273 and UPM04019) to adapt is
consistent with the findings of some investigators that it is usually difficult to
adapt and grow pathogenic bursal derived field strains in vitro as some wild type
strains had been reported to fail to adapt to mamallian cell culture, despite
increased serial blind passages (McFerran et al., 1980; Kibenge and McKenna,
1992; Hassan et al., 1996). The poor adaptation rates of vvIBDV to CEF have
85
been reported previously (Lee and Lukert, 1986). The possible reason for this
failure is not certain as some workers suggested a receptor theory which is still
very much under investigation (Zhu et al., 2008).
The growth pattern of UPM0081 in Vero cells was clear and consistent. It
showed CPEs of IBDV at the fourth passage onward up to 20th passage
(Ahasan et al., 2002), but slightly varied from findings of Rasool and Hussain
(2006) where the vvIBDV was adapted to Vero cell line after the third
passage.This observation was sharply in contrast to that of Tsai and Saif (1992)
and Hassan and Saif (1996) who showed CPE at second passage in BGM-70
cell line (baby monkey kidney cell line). The difference might be due to the cell
culture passage level of the virus strains used or variation in sensitivity of cell
culture to different strains.
The difference between primary cell culture and secondary cell culture to adapt
vvIBDV had been reported by many researchers. Some scientists were able to
detect CPEs in CEF only on 5th passage onwards (Pahar and Rai, 1997; Mathur
et al., 1999; Gupta et al., 2001), while in this study, CPE was evident in DF1 (a
cell line derived from EV-0 embryo) from the third passage with UPM0081 as
other vvIBDV isolates failed to adapt. This findings is in line with reports of other
workers (Wang et al., 2009) with IBDV Gt strain in CEF and DF-1 cells where
CPE was observed clearly at 48 hours pi. and became more obvious at 60
hours pi.
86
The classical method of detecting the replication of virus in the cell culture using
the cytopathic effect was augmented in this study with indirect
immunoperoxidase and the viral antigen was observed as brownish
intracytoplasmic granules in both Vero cells and DF-1 cells, These findings is
similar to the report of Guvenc et al., (2004).
Of note is the low titer obtained from DF-1 cells when compared to Vero cell line
as infectious virus titer in Vero cell line is 100 times more than that obtained with
DF-1 at passage 9. This observation is in sharp constrast with that of Lukert et
al., (1974) where approximately equal titers were obtained in Vero and CEK cell
line, but it similar to the report of Kibenge et al, (1988) where Vero cell line gave
180 times more than the total virus titer in CEF culture.The possible reason for
this discrepancy may be related to species difference in the origin of the two cell
lines and the receptors for attachment and replication (Kim et al., 2006). It has
been reported that DF1 uses a molecule that contain chicken heat shock protein
(cHsp90α) (Lin et al., 2007), while Vero cells utilise RAVP2 and RVVP2
receptors (Yip et al., 2007). However, the knowledge of these receptors or
coreceptors in IBDV is very poor. The variation in the mitotic and metabolic
rates of the individual cell lines can also be adduced for the difference. It is
pertinent to note that Vero cell line, BGM-70 cell line were from the kidney of
green monkey and baby monkey, respectively which strongly suggest an
inherent ability of kidney cells to permit the growth of the virus. It will be
87
interesting therefore to investigate more on the tropism of the virus in live
animals.
It is known that secondary cell lines have several advantages over the primary
cell cultures as the cost of maintenance of secondary cell line is lower than
production of CEF from SPF chicken embryos. And also, primary cell cultures
have short life span as a continuous cell line could afford continuous source of
cells by simple protocol when compared to primary cell cultures, which is time
consuming and laborious, In addition, some primary avian cell culture may
contain exogenous avian virus not found in secondary cell culture (van den
Berg, 2000).
To product large quantities of virus for adaptation, attenuation and vaccination
development, it is important that the virus grows to high titers in the in vitro
tissue culture systems. The UPM0081 strain produced higher titers when
propagated in Vero cells than in DF-1 cell line, the possible reason for this
observation is not clear.
It was concluded that only UPM0081 vvIBDV strain was successfully adapted
and attenuated in the two continuous cell lines, while the other strains
(UPM04190 and UPM94273) failed. The virus was first propagated in SPF
embryonated chicken eggs for three times prior to inoculation into the cell lines.
The UPM0081 vvIBDV strain yielded higher titer in Vero cells than DF-1 cells.
88
Thus, the propagation and attenuation of UPM0081 strain in Vero cells line has
high potential to be used for future IBD vaccines development.
CHAPTER 4
MOLECULAR CHARACTERISATION OF THE ADAPTED AND ATTENUATED vvIBDV ISOLATE
4.1 Introduction
Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus
belonging to the genus Avibirnavirus within the family Birnaviridae (Kibenge et
al., 1988). There are two distinct serotypes of IBDV (McFerran et al., 1980). The
Serotype 1 strains cause immunosuppression as well as an acute fatal disease
of young chickens while Serotype 2 viruses are avirulent for chickens. Serotype
1 viruses are further categorized as classical virulent, antigenic variant and very
virulent strains depending on their pathogenicity and/or antigenicity. Non-
enveloped icosahedral IBDV particles contain the bi-segmented dsRNA
genome. The major open reading frame (ORF) in the larger genome segment A
encodes a polyprotein which is co-translationally and autocatalytically cleaved
into the two structural viral proteins VP2 and VP3, and a viral protease VP4
(Hudson et al., 1986). A second ORF in the segment A encodes a nonstructural
protein, VP5 (Muller and Becht, 1982). The smaller segment B encodes the
VP1, which is an RNA-dependent RNA polymerase (RdRp) (Spies et al., 1987).
The PCR has been used to detect and characterise IBDV (Spies et al., 1987;
Wu et al., 1992). The PCR uses two oligonucleotide primers to hybridize the
90
segments of the template DNA, allowing DNA polymerase to produce more
DNA by copying the template DNA lying between the 3 ends of primers. The
oligonucleotides hybridize specifically to the target sequence and in the
presence of DNA polymerase coupled with excess deoxynucleotide
triphosphates, prime new DNA synthesis (Wu et al., 1992). IBDV being a double
stranded RNA virus, needs a reverse transcription (RT) in the PCR for IBD to
synthesize cDNA.
The hyper variable (hv) region in VP2 is the most investigated area of the IBDV
genome. It is an extreme sequence which encodes the immune dominant viral
epitopes (Becht et al., 1988). This epitope region contains only a 145 amino
acid (aa) fragment spanning from aa 206 to 350, which comprises the central hv
portion of the major viral capsid structure protein VP2. Within this region lies two
hydrophilic peaks, the first peak is from aa 212 to 224 and the second peak is
from aa 314 to 324 (Fahey et al., 1989; Bayliss et al., 1990; Brown et al., 1994).
Specific amino acid changes within the hv region and an adjacent downstream
serine-rich heptapeptide sequence (SWSASGS) and amino acid residues 326 to
332 have been proposed as potential sites responsible for virus attenuation or
antigenic determination (Heine et al., 1991; Vakharia et al., 1994; Yamaguchi et
al., 1996b; Dormitorio et al., 1997). In addition, several amino acid residue
changes at the VP2 gene can be used to differentiate the very virulent (vv),
attenuated (at), variant (va) and classical (ca) strains of IBDV. The amino acid
residue changes at P222A (proline to alanine), V256I (valine to isoleosine) and
91
L294I (leucine to isoleosine) can be used as a marker for vvIBDV, G254S
(glycine to serine) and Q249K (glutamine to lysine) for vaIBDV, while amino acid
changes at D279N (aspartic to aspargine) and A284T (alanine to threonine) has
been demonstrated for atIBDV (Yamaguchi et al., 1996b; Cao et al., 1998).
Sequence analysis of the structural protein VP2 of the isolate was focused
especially on the region between residue 206 and 360 which is referred to as
very variable region (Dormitorio et al., 1997; Lin et al., 1993).
The passage of the virus in vitro has been associated with attenuation of
virulence as evidenced by a reduction in the ability of the virus to induce bursal
lesions (Yamaguchi et al., 1996a). Thus, attenuated vvIBDV can be produce by
in vitro with extended serial passages. The Vero cells-adapted strains showed
considerable reduction in virulence (Hyuk and Soo, 2004), but the possible
genomic changes for the attenuation of vvIBDV in different cell lines have not
received sufficient attention especially with local isolates of vvIBDV.
Therefore, the objectives of the study were:
1. to amplify, clone and sequence the hypervariable region of VP2 gene
(643-bp) of the UPM0081 IBDV isolate adapted and attenuated in Vero
cells and DF-1
2. to determine the molecular characteristic of the adapted and attenuated
IBDV in the cell lines
92
4.2 Materials and Methods
4.2.1 Sample Preparation
The 5, 7, 8, 9, 10, 15, and 20 passages of UPM0081 vvIBDV isolate in Vero
cells and the 5 and 7 passages in DF-1 cells were choosen as the sample for
reverse transcriptase-polymerase chain reaction (RT-PCR). The virus infected
cells from the tissue culture flasks were repeatedly frozen and thawed three
times. After being clarified by low-speed centrifugation at 3000 rpm for 20
minutes at 4°C (Beckman, USA), the resultant supernatant fluids were
harvested and further clarified with centrifugation at 10,000 rpm for 30 minutes
at 4°C. The collected supernatant was passed through a 0.45 µm filter before
stored or used for RNA extraction.
4.2.2 RNA Extraction
The viral RNA was extracted by using Trizol reagent according to the
manufacturer‟s instruction (Gibco BRL, Life Technologies, USA). The partial
sample with the volume of 250 µl was mixed with 750 µl Trizol Reagent solution
in a 1.5 mL Eppendorf tube and incubated at room temperature (RT) for 5
minutes. Two hundred µl of chloroform was then added into the sample mixture.
The sample mixture was then covered tightly and shaken vigorously for 15
second, followed by incubation at RT for 15 minutes. The mixture was then
centrifuged at 12, 000g (Centrifuge 5417R, Eppendorf, USA) for 20 minutes at
93
4°C which separated the sample into three phases: the upper aqueous phase,
whitish interphase and the lower red phenol-chloroform phase. Only the upper
aqueous phase was pippetted slowly and genteelly into a new eppendorf tube
and 800 µl of isopropanol was added and left to stand in RT for 20 minutes. The
sample was then centrifuged at 12, 000g for 15 minutes at 4°C to pellet the
dsRNA. The supernatant was removed and the dsRNA pellet was then
resuspended in 1 mL of 75% ethanol and followed by centrifugation at 12, 000g
for 15 minutes at 4°C. The supernatant was discarded and 1 mL of 100%
ethanol was added to the pellet, re-suspended and centrifuged at 12, 000g for
15 minutes at 4°C. The pellet was then air dry. The pellet was then dissolved in
10 µl diethlpyrocarbonate (DEPC) treated deionized double-distilled water
(ddH2O). The RNA solution if not used immediately was kept in –80°C (Wu and
Lin, 1992).
4.2.3 Determination of RNA Concentration
The concentration and purity of extracted RNA was determined by using
spectrophotometer according to the method described by Sambrook et al.
(1989). The optical density (OD) of RNA was measured at its maximum
absorbance wavelength of 260nm. Once the OD unit was equivalent to 40 µl/mL
of dsRNA, the extracted RNA was read at wavelength of 260nm and 280
whereby the ratio of 260nm to 280nm estimates the purity of RNA. The
acceptable absorbance ratio of 260/280OD was in the range of 1.8-2.0.
94
4.2.4 Primer Design
One set primers designated as P1 and P2 (Liu et al., 1994) were used for RT-
PCR amplification.The P1 and P2 primers were used for amplification of the
hypervariable region of VP2 gene of the IBDV isolates as shown in (Table 4.1).
Table4.1: Primers used to amplify the HPVR VP2 gene
Primer Sequences Position (5‟ to 3‟)a
Product
(size)
P1
TCA CCG TCC TCA GCT TAC
622-639
HPVR
(643 bp) P2
TCA GGA TTT GGG ATC AGC
1247-1264
4.2.5 Reverse Transcription and PCR Reaction
The following protocol was according to Fiza et al. (2006). The genome of IBDV
was denatured by adding (1 µl) dimethyl sulfoxide (DMSO), reverse and forward
primers (1 µl) and (4 µl) DEPC water in heating block for 5 minutes at 95°C and
immediately placed on ice for 2-3 minutes. Reverse transcriptase was carried
out at 42°C for 1 hour in a total of 20 µl volume containing 9 µl of denatured
RNA, 4 µl MgCl2, 5 unit of AMV, 4 µl of reaction buffer, 2 µl of dNTP, and 0.5 µl
Rnasin. After heating to 99°C for I minute, 5 µl of cDNA was added into PCR
reaction. This mixture contained 1 µl of 10mM dNTP, 4 µl of MgCl2, 6 µl of PCR
95
buffer, (1µl) Reverse and Forward Primer, 5 µl cDNA, (31.5 µl) water and (0.5
µl) Taq Polymerase. PCR was performed in the thermocycler (MJ, BioRad,
USA) using the following temperature profile for 35 cycle 94°C for 1 minute,
52°C for 1 minute, 72°C for 2 minutes and then the reaction was allowed to go
at 72°C for 10 minutes.
4.2.6 Gel Electrophoresis and Ethidium Bromide Staining
The following protocol was according to Fiza et al. (2006). Agarose gel
electrophoresis was done using a 7 x 10 cm Mini-Sub cell GT gel
electrophoresis apparatus (Bio-Rad, USA). The 1% agarose gel was prepared
by adding 1g of agarose powder in 100 mL TAE (Tris-acetate-EDTA) buffer
(Appendix A) and boiled until the powder dissolved. The agarose solution was
cooled down to about 60°C before pouring onto a mini gel tray that had been
placed with an 8 wells comb and bordered with the gel casting gates within the
gel electrophoresis apparatus. The gel was allowed to solidify at room
temperature and gently the comb and gates were removed. TAE buffer was
pour onto the electrophoresis apparatus and submerged the solidified gel. DNA
ladder (100bp) (Promega, USA) was used as DNA marker. The DNA ladder
marker was prepared by mixing 10 µl of marker and 2 µl of 6x loading dye
solution. Other wells were loaded with 10 µl of RT-PCR products that had been
pre-mixed with 2 µl of loading dye. Gel electrophoresis was run at 80V for 35
min.
96
After electrophoresis was completed, the gel was transferred from the
electrophoresis apparatus and submerged into ethidium bromide staining
solution for 15-30 minutes. The gel was de-stained with distiled water for 15
minutes and placed onto the UV transluminator to visualize the nucleic acids
bands.
4.2.7 Purification of RT-PCR Products
Gel purification system from GeneAll® (GeneAll®, Seoul, Korea) or “Gel SV”
(GelSV®, Seoul, Korea) was used to extract the DNA from the gel slice. In order
to obtain good quality of DNA, the handling time on the transilluminator was kept
to the minimum. The purification of the PCR products from the gel was
performed according to the manufacturers‟ protocol (GeneAll®, Seoul, Korea).
The gel slice was transferred to a new 1.5 mL tube and weighed. Three volumes
(µl) of GB buffer were added for each volume (mg) of gel and the mixture was
incubated in a 50°C water bath until the gel melted. The tube was vortexed
every 3 minutes to facilitate the dissolution of gel. The mixture was transferred
on to a SV centrifuge column and was centrifuged for 1 minute after which the
pass-through was discarded. Then five hundred µl of GB buffer was added to
the column and centrifuged for 30 seconds and the pass-through discarded.
Seven hundred µl of NW buffer was added and centrifuged twice for 30 seconds
and discarded. The column was then transferred to a new tube and 30 µl of EB
buffer was added to the column; the column was left to stand on the tube for 1
97
minute and then centrifuged for 1.5 minutes. The tube containing the desired
DNA was stored at -80°C.
4.2.8 Molecular Cloning of Amplified Products and Analysis of Recombinant Plasmid
The PCR products were directly cloned into PCR®2.1-TOPO® vector using the
TOPO TA Cloning ® kit (Invitrogen, USA) by referring to its protocol. TOPO®
cloning reaction solution was set up by mixing 4 µl of fresh PCR product with 1
µl of salt solution and 1 µl of TOPO® vector. The reaction solution was gently
mixed, incubated for 5 minutes at room temperature and placed on ice. An
aliquot of 2 µl of the reaction solution was mixed with a vial of thawed One
Shot® Escherichia coli cells and incubated on ice for 5 minutes. The cells were
heat-shocked at 42ºC for 30 seconds without shaking and then immediately
incubated on ice for 1 minute. The cells were added with 250 µl of LB broth and
shook horizontally (200 rpm) at 37ºC for 1 hour. The transformed cells were
spread on Ampicillin-added selective LB plates in a volume of 10-50 µl per plate.
The plates were then incubated at 37ºC in inverted position for at least 12 hours
to allow the transformed cells to grow.
Based on blue white selection, 10 white colonies were picked and subcultured
onto another selective LB plates. At the same time, these colonies were also
being screened for positive transformation using PCR. Each PCR reaction
mixture consisted of 1 µl of 10x PCR buffer, 1µl of 25 Mm MgCl2, 1µl of 10dNTP,
98
0.5 µl of each P1 and P2 primers, 0.5 µl of Taq polymerase, 6.5 µl of ddH2O,
and a dot of the picked colony. The PCR program was set to 94ºC for 3 minutes
(for 1 cycle) and followed by 35 cycles of the following: 94ºC for 1 minute, 48ºC
for 1 minute and 72ºC for 10 minutes. Positive clone was identified by the
presence of band of interest after gel electrophoresis. A dot of the positive clone
was added into 4 mL of ampicillin-added LB broth and incubating in 37ºC with
horizontal shaking 200 rpm for about 12 hours. The culture clone, if not used
immediately was mixed with sterial glycerol (0.85 mL culture to 0.15 mL
glycerol) and stored at -80 ºC.
4.2.9 Plasmid Extraction and Purification
Plasmids from the positive clones were extracted and purified using Gene All
Kit® (Fragment, SV, Seoul, Korea) following manufacturer recommendations.
Briefly, the overnight culture of positive clone was first pelleted and all medium
was removed. The pellet was re-suspended in 250 µl of RNase-added cell
suspension buffer (Buffer S1) until homogenous. This was followed by adding
250 µl of cell lysis solution (Buffer S2) and gently mixed by inverting the capped
tube five times and incubated at RT for 5 minutes. Immediately after the
incubation 350 µl of neutralization buffer (Buffer S3) was added and the mixture
was centrifuged at 12, 000 g for 10 minutes. The supernatant was loaded onto a
spin column and centrifuged at 12, 000 g for 1 minute. The flow-through was
discharged. The cartridge was washed by adding 700 µl of wash buffer (Buffer
99
PW) onto the cartridge and centrifuged at 12, 000 g for 1 min. The cartridge was
re-centrifuged to remove residual wash buffer. The plasmid was eluted from the
cartridge by adding 50 µl of EB buffer, incubated at room temperature for 1
minute and centrifuged at 12, 000 g for 1 min. The purity and concentration of
the plasmid was determined using spectrophotometry and the plasmid was
stored at -20oC if not used immediately.
4.2.10 Sequence Assembly and Analysis Using Bioinformatics Software
The sequencing data were initially aligned and processed by using the basic
BLAST (Basic Local Alignment Search Tool) search programme of National
Table 4. 2: IBDV isolates used in the sequence analyses
Isolate
Strain
A
Accession
Country
B
References
UPM0081
VV
AY520910
Malaysia
Tan et al., 2004
UPM94268 VV AY333088 Malaysia Tan et al., 2004
UPM92/04 VV AF262030 Malaysia Hoque et al., 2001
UPM4/230 VV AY520911 Malaysia Tan et al., 2004
UPM94/274 VV AF527039 Malaysia Phong et al., 2003
B00/73 VV AY520909 Malaysia Tan et al., 2004
UK661 VV X92760 United Kingdom Brown and Skinner, 1996 Tasek94 VV AF322444 Indonesia Rudd et al., 2002
OKYM VV D49706 Japan Yamaguchi et al.,1996
HK-46 VV AF051838 Hong Kong Cao et al., 1998
Ehime 91 VV AB024076 Japan Tsukamoto et al., 1999
SH 95 VV AY 134874 China Sun et al., 2003
SH 92 VV AF533670 Korea Kim et al., 2004
CT CA AJ310158 France Lejal et al., 2000
Kal2001 CA AY311479 Egypt EL-Zeedt et al., 2003
GLS VA M97346 USA Vakharia et al., 1994
Variant A VA M64285 USA Lana et al., 1992
E/DEL VA X54858 USA Vakharia et al., 1992
UPM0081T10 AT* FJ824699 Malaysia Mohammed et al., 2009
UPM0081T15 AT* FJ898322 Malaysia Mohammed et al., 2009
UPM0081T20 AT* FJ898321 Malaysia Mohammed et al., 2009
Soroa AT AF140705 Cuba Femandez-Arias et al.,1988
OKYMT AT AJ427340 Japan Yamaguchi et al., 1996
PBG98 AT D00868 United Kingdom Spies et al., 1989
D78 AT AF499929 USA Hudson et al., 2003
AmerVH9907 AT AJ577092 Vietnam Ghazi et al., 2003
OH Serotype 2 M66722 USA Kibenge et al., 2008
Note: Strain represented IBDV strains: VV = very virulent; CA = classical; VA = variant; AT = attenuated. Country was the place of isolation. AT* indicated the strain identity as determined in this study.
101
4.2.11 Phylogenetic Tree Construction
Phylogenetic tree was constructed based on the hypervariable region of the
nucleotide sequence from position 516 to 1058 (Bayliss et al., 1990) and
deduced amino acid from 173 to 386 of the VP2 gene. Twenty four published
IBDV isolates were included in this study to investigate the evolutionary
relationships among the isolates. Sequences were aligned using ClustalX soft
were version 1.83. Translation to amino acid sequences were done using
biology work bench (version 3.2). The tree views were edited using Tree View X
version (http://evolgen.biol.metrou.ac.ip/TE/TE_man.html).
4.3 Results 4.3.1 Amplification of the hypervariable Region of VP2 Gene
The dsRNA from the 5, 7, 8, 9, 10, 15 and 20 passages of UPM0081 vvIBDV in
Vero cells namely as UPM0081T5, UPM0081T7, UPM0081T8, UPM0081T9,
UPM0081T10, UPM0081T15, and UPM0081T20 respectively and from the 5
and 7 passages in DF-1 cells namely as UPM0081D5 and UPM0081D7,
respectively were extracted and synthesized to cDNA. The hypervariable region
of VP2 gene was successfully amplified. All the amplified cDNA showed
identical mobility on a 1% agarose gel. The PCR amplification was done by
using primer P1-P2 generate specific DNA band of 643 bp (Figures, 4.1 and
SH/95 as well as SH/92 which belong to the vvIBDV subgroup 1 branch;
UPM0081T10, UPM0081T15, and UPM0081T20 which were more closely
related to another group that had Soroa, OKYMT. PBG98, D78 and Amer
VH9907 isolates which are regarded as typical attenuated and ca IBDV strains,
such as CT and KAL2001 subgroup 2 branch, and finally subgroup 3 branch
which consists of vaIBDV strain such as GLS, Variant A and E/DEL.
107
Figure 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).
Figure 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).
108
Figure 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).
Figure 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).
109
Table 4.3: Number of nucleotide differences in HPVR of VP2 gene between IBDV isolates Passage Isolate Strain No.
Diff Parent isolate
Isolate Strain N. Diff
UPM0081T20
vs.
UPM0081
VV
29
UPM0081
vs.
UPM0081
VV
ID
UPM0081T20 vs. UPM94268 VV 30 UPM0081 vs. UPM94268 VV 1 UPM0081T20 vs. UPM92/04 VV 30 UPM0081 vs. UPM92/04 VV 1 UPM0081T20 vs. UPM4/230 VV 33 UPM0081 vs. UPM4/230 VV 4 UPM0081T20 vs. UPM94/274 VV 31 UPM0081 vs. UPM94/274 VV 4 UPM0081T20 vs. B00/73 VV 36 UPM0081 vs. B00/73 VV 7 UPM0081T20 vs. UK661 VV 31 UPM0081 vs. UK661 VV 6 UPM0081T20 vs. Task94 VV 35 UPM0081 vs. Task94 VV 9 UPM0081T20 vs. OKYM VV 33 UPM0081 vs. OKYM VV 6 UPM0081T20 vs. HK-46 VV 34 UPM0081 vs. HK-46 VV 7 UPM0081T20 vs. Ehime 91 VV 34 UPM0081 vs. Ehime 91 VV 7 UPM0081T20 vs. SH 95 VV 34 UPM0081 vs. SH 95 VV 11 UPM0081T20 vs. SH 92 VV 30 UPM0081 vs. SH 92 VV 5 UPM0081T20 vs. CT CA 13 UPM0081 vs. CT CA 40 UPM0081T20 vs. Kal2001 CA 14 UPM0081 vs. Kal2001 CA 39 UPM0081T20 vs. GLS VA 27 UPM0081 vs. GLS VA 39 UPM0081T20 vs. Variant A VA 32 UPM0081 vs. Variant A VA 39 UPM0081T20 vs. E/DEL VA 29 UPM0081 vs. E/DEL VA 39 UPM0081T20 vs. UPM0081T10 AT 0 UPM0081 vs. UPM0081T10 AT 29 UPM0081T20 vs. UPM0081T15 AT 0 UPM0081 vs. UPM0081T15 AT 29 UPM0081T20 vs. UPM0081T20 AT 0 UPM0081 vs. UPM0081T20 AT 29 UPM0081T20 vs. Soroa AT 13 UPM0081 vs. Soroa AT 40 UPM0081T20 vs. OKYMT AT 17 UPM0081 vs. OKYMT AT 42 UPM0081T20 vs. PBG98 AT 13 UPM0081 vs. PBG98 AT 42 UPM0081T20 vs. D78 AT 15 UPM0081 vs. D78 AT 39 UPM0081T20 vs. AmerVH9907 AT 15 UPM0081 vs. AmerVH9907 AT 40 UPM0081T20 vs. OH Serotype
2 157 UPM0081 vs. OH Serotype
2 168
Note:“No. Diff” is the nucleotide differences between passage (UPM0081T20) and parent isolate (UPM0081). All the three passages (UPM0081T10, UPM0081T15 and UPM0081T20) were similar and closely related to atIBDV publish gene bank by having 13-17 nucleotides difference, while parent isolate having high nucleotides difference 29-42 nucleotides. Strain represented IBDV strains: VV = very virulent; CA = classical; VA = variant; AT = attenuated
110
Table 4.4: Sequence identity matrix of VP2 genes nucleotides of IBDV isolates
Passage Isolate Strain Seq. Iden
Parent isolate
Isolate Strain Seq. Iden
UPM0081T20
vs.
UPM0081
VV
0.954
UPM0081
vs.
UPM0081
VV
ID
UPM0081T20 vs. UPM94268 VV 0.953 UPM0081 vs. UPM94268 VV 0.998 UPM0081T20 vs. UPM92/04 VV 0.953 UPM0081 vs. UPM92/04 VV 0.998 UPM0081T20 vs. UPM4/230 VV 0.948 UPM0081 vs. UPM4/230 VV 0.993 UPM0081T20 vs. UPM94/273 VV 0.951 UPM0081 vs. UPM94/273 VV 0.993 UPM0081T20 vs. B00/73 VV 0.949 UPM0081 vs. B00/73 VV 0.989 UPM0081T20 vs. UK661 VV 0.951 UPM0081 vs. UK661 VV 0.990 UPM0081T20 vs. Task94 VV 0.954 UPM0081 vs. Task94 VV 0.986 UPM0081T20 vs. OKYM VV 0.948 UPM0081 vs. OKYM VV 0.990 UPM0081T20 vs. HK-46 VV 0.947 UPM0081 vs. HK-46 VV 0.989 UPM0081T20 vs. Ehime 91 VV 0.947 UPM0081 vs. Ehime 91 VV 0.982 UPM0081T20 vs. SH 95 VV 0.947 UPM0081 vs. SH 95 VV 0.982 UPM0081T20 vs. SH 92 VV 0.953 UPM0081 vs. SH 92 VV 0.992 UPM0081T20 vs. CT CA 0.979 UPM0081 vs. CT CA 0.937 UPM0081T20 vs. Kal2001 CA 0.978 UPM0081 vs. Kal2001 CA 0.939 UPM0081T20 vs. GLS VA 0.958 UPM0081 vs. GLS VA 0.939 UPM0081T20 vs. Variant A VA 0.950 UPM0081 vs. Variant A VA 0.939 UPM0081T20 vs. E/DEL VA 0.954 UPM0081 vs. E/DEL VA 0.939 UPM0081T20 vs. UPM0081T10 AT 1.000 UPM0081 vs. UPM0081T10 AT 0.959 UPM0081T20 vs. UPM0081T15 AT 1.000 UPM0081 vs. UPM0081T15 AT 0.959 UPM0081T20 vs. UPM0081T20 AT ID UPM0081 vs. UPM0081T20 AT 0.959 UPM0081T20 vs. Soroa AT 0.979 UPM0081 vs. Soroa AT 0.939 UPM0081T20 vs. OKYMT AT 0.973 UPM0081 vs. OKYMT AT 0.943 UPM0081T20 vs. PBG98 AT 0.979 UPM0081 vs. PBG98 AT 0.943 UPM0081T20 vs. D78 AT 0.976 UPM0081 vs. D78 AT 0.939 UPM0081T20 vs. AmerVH9907 AT 0.976 UPM0081 vs. AmerVH9907 AT 0.937 UPM0081T20 vs. OH Seroty
pe 2 0.736 UPM0081 vs. OH Serot
ype 2 0.739
Note: “Seq. Iden” is the sequence nucleotide identity matrix between passage (UPM0081T20) and parent isolate (UPM0081). All the three passages (UPM0081T10, UPM0081T15 and UPM0081T20) were similar and closely related to atIBDV publish gene bank by having 97% sequence identity matrix, while parent isolate having low sequence identity matrix 93-95%. Strain represented IBDV strains: VV = very virulent; CA = classical; VA = variant; AT = attenuated
111
Table 4.5: Summary of the proposed molecular markers (amino acid residues) of UPM0081T10, UPM0081T15 and UPM0081T20 atIBDV isolates with other published IBDV strains. IBD Isolate
Strain
222
242
249
253
256
279
284
294
326
330
UPM0081
VV
A
I
Q
Q
I
D
A
I
S
S
UPM94268 VV A I Q Q I D A I S S UPM92/04 VV A I Q Q I D A I S S UPM4/230 VV A I Q Q I D A I S S UPM94/274 VV A I Q Q I D A I S S B00/73 VV A I Q Q I D A I S S UK661 VV A I Q Q I D A I S S Task94 VV A I Q Q I D A I S S OKYM VV A I Q Q I D A I S S HK-46 VV A I Q Q I D A I S S Ehime 91 VV A I Q Q I D A I S S SH 95 VV A I Q Q I D A I S S SH 92 VV A I Q Q I N A I S S CT CA P V Q H V N T L S R Kal2001 CA P V R H V N T L S R GLS VA T V K H V N T L S S Variant A VA Q V K Q V N A L S S E/DEL VA T V K Q V N A L S S UPM0081T10 AT P V R H V N T L L R UPM0081T15 AT P V R H V N T L L R UPM0081T20 AT P V R H V N T L L R Soroa AT P V R H V N T L S R OKYMT AT P V Q H V N T L S K PBG98 AT P V R H V N T L S R D78 AT P V Q H V N T L L R AmerVH9907 AT P V Q H V N T L S R OH Serotype2 P V S I I T T N I V
Note: The proposed molecular markers for atIBDV which were Pro [222], Val [242], Arg [249], His [253], Val [256], Asn [279], Thr [284], Leu [294], Leu [326] and Arg [330] were found in the HPVR of VP2 gene of these three isolates (Kwon and Kim, 2004; Wang, et al., 2004). Strain represented IBDV strains: VV = very virulent; CA = classical; VA = variant; AT = attenuated
112
Table 4.6: Number of amino acids differences in HPVR of VP2 gene between IBDV isolates
Passage Isolate Strain No. Diff
Parent isolate
Isolate Strain N. Diff
UPM0081T20
vs.
UPM0081
VV
10
UPM0081
vs.
UPM0081
VV
ID
UPM0081T20 vs. UPM94268 VV 11 UPM0081 vs. UPM94268 VV 1 UPM0081T20 vs. UPM92/04 VV 11 UPM0081 vs. UPM92/04 VV 1 UPM0081T20 vs. UPM4/230 VV 13 UPM0081 vs. UPM4/230 VV 3 UPM0081T20 vs. UPM94/273 VV 13 UPM0081 vs. UPM94/273 VV 3 UPM0081T20 vs. B00/73 VV 12 UPM0081 vs. B00/73 VV 2 UPM0081T20 vs. UK661 VV 11 UPM0081 vs. UK661 VV 1 UPM0081T20 vs. Task94 VV 12 UPM0081 vs. Task94 VV 3 UPM0081T20 vs. OKYM VV 11 UPM0081 vs. OKYM VV 1 UPM0081T20 vs. HK-46 VV 11 UPM0081 vs. HK-46 VV 1 UPM0081T20 vs. Ehime 91 VV 11 UPM0081 vs. Ehime 91 VV 1 UPM0081T20 vs. SH 95 VV 12 UPM0081 vs. SH 95 VV 2 UPM0081T20 vs. SH 92 VV 10 UPM0081 vs. SH 92 VV 2 UPM0081T20 vs. CT CA 4 UPM0081 vs. CT CA 12 UPM0081T20 vs. Kal2001 CA 5 UPM0081 vs. Kal2001 CA 11 UPM0081T20 vs. GLS VA 9 UPM0081 vs. GLS VA 13 UPM0081T20 vs. Variant A VA 11 UPM0081 vs. Variant A VA 11 UPM0081T20 vs. E/DEL VA 13 UPM0081 vs. E/DEL VA 13 UPM0081T20 vs. UPM0081T10 AT 0 UPM0081 vs. UPM0081T10 AT 10 UPM0081T20 vs. UPM0081T15 AT 0 UPM0081 vs. UPM0081T15 AT 10 UPM0081T20 vs. UPM0081T20 AT ID UPM0081 vs. UPM0081T20 AT 10 UPM0081T20 vs. Soroa AT 4 UPM0081 vs. Soroa AT 12 UPM0081T20 vs. OKYMT AT 7 UPM0081 vs. OKYMT AT 12 UPM0081T20 vs. PBG98 AT 4 UPM0081 vs. PBG98 AT 14 UPM0081T20 vs. D78 AT 5 UPM0081 vs. D78 AT 11 UPM0081T20 vs. AmerVH9907 AT 6 UPM0081 vs. AmerVH9907 AT 12 UPM0081T20 vs. OH Serotype
2 145 UPM0081 vs. OH Serotype
2 147
Note:“ No. Diff” is the amino acid differences between passage (UPM0081T20) and parant strain (UPM0081). All the three passages (UPM0081T10, UPM0081T15 and UPM0081T20) were similar and closely related to atIBDV publish gene bank by having 4-7 amino acid difference, while parent isolate had high amino acid difference 10-14 amino acid. Strain represented IBDV strains: VV = very virulent; CA = classical; VA = variant; AT = attenuated
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Table 4.7: Sequence identity matrix of VP2 genes amino acids of IBDV isolates
Passage Isolate Strain Seq. Iden
Parent isolate
Isolate Strain Seq. Iden
UPM0081T20
vs.
UPM0081
VV
0.953
UPM0081
vs.
B0081
VV
ID
UPM0081T20 vs. UPM94268 VV 0.948 UPM0081 vs. UPM94268 VV 0.995 UPM0081T20 vs. UPM92/04 VV 0.948 UPM0081 vs. UPM92/04 VV 0.995 UPM0081T20 vs. UPM4/230 VV 0.939 UPM0081 vs. UPM4/230 VV 0.985 UPM0081T20 vs. UPM94/273 VV 0.939 UPM0081 vs. UPM94/273 VV 0.985 UPM0081T20 vs. B00/73 VV 0.943 UPM0081 vs. B00/73 VV 0.990 UPM0081T20 vs. UK661 VV 0.948 UPM0081 vs. UK661 VV 0.995 UPM0081T20 vs. Task94 VV 0.943 UPM0081 vs. Task94 VV 0.985 UPM0081T20 vs. OKYM VV 0.948 UPM0081 vs. OKYM VV 0.995 UPM0081T20 vs. HK-46 VV 0.948 UPM0081 vs. HK-46 VV 0.995 UPM0081T20 vs. Ehime 91 VV 0.948 UPM0081 vs. Ehime 91 VV 0.995 UPM0081T20 vs. SH 95 VV 0.943 UPM0081 vs. SH 95 VV 0.990 UPM0081T20 vs. SH 92 VV 0.953 UPM0081 vs. SH 92 VV 0.990 UPM0081T20 vs. CT CA 0.981 UPM0081 vs. CT CA 0.943 UPM0081T20 vs. Kal2001 CA 0.976 UPM0081 vs. Kal2001 CA 0.948 UPM0081T20 vs. GLS VA 0.957 UPM0081 vs. GLS VA 0.939 UPM0081T20 vs. Variant A VA 0.948 UPM0081 vs. Variant A VA 0.948 UPM0081T20 vs. E/DEL VA 0.939 UPM0081 vs. E/DEL VA 0.939 UPM0081T20 vs. UPM0081T10 AT 1.000 UPM0081 vs. UPM0081T10 AT 0.953 UPM0081T20 vs. UPM0081T15 AT 1.000 UPM0081 vs. UPM0081T15 AT 0.953 UPM0081T20 vs. UPM0081T20 AT ID UPM0081 vs. UPM0081T20 AT 0.953 UPM0081T20 vs. Soroa AT 0.981 UPM0081 vs. Soroa AT 0.943 UPM0081T20 vs. OKYMT AT 0.967 UPM0081 vs. OKYMT AT 0.943 UPM0081T20 vs. PBG98 AT 0.981 UPM0081 vs. PBG98 AT 0.943 UPM0081T20 vs. D78 AT 0.967 UPM0081 vs. D78 AT 0.948 UPM0081T20 vs. AmerVH9907 AT 0.971 UPM0081 vs. AmerVH9907 AT 0.943 UPM0081T20 vs. OH Serotype
2 0.325 UPM0081 vs. OH Serotype
2 0.326
Note: “Seq. Iden” is the sequence amino acid identity matrix between passage (UPM0081T20) and parent isolate (UPM0081). All the three passages (UPM0081T10, UPM0081T15 and UPM0081T20) were similar and closely related to atIBDV publish gene bank by having 96-98% sequence identity matrix, while parent isolate having low sequence identity matrix 94-95%. Strain represented IBDV strains: VV = very virulent; CA = classical; VA = variant; AT = attenuated
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Figure 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.
115
Figure 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. .
116
Figure 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.
117
Figure 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.
118
Figure 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.
119
Figure 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.
120
Figure 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.
121
Figure 4.6: Amino acid sequence aligment of UPM0081T10, UPM0081T15 and UPM0081T20 IBDV passages Note: Deduced amino acid sequence of HPVR of VP2 from residue 173-386 (numbering of the segment A polyprotein of Bayliss et al., 1990) of the UPM0081T10, UPM0081T15 and UPM0081T20 IBDV passages and other published IBDV strains. Major hydrophilic domain (Peak A and B) and minor hydrophilic domains were boxed by black line. The serine rich heptapeptide was shown by underline.
122
Figure 4.6: Amino acid sequence aligment of UPM0081T10, UPM0081T15 and UPM0081T20 IBDV passages Note: Deduced amino acid sequence of HPVR of VP2 from residue 173-386 (numbering of the segment A polyprotein of Bayliss et al., 1990) of the UPM0081T10, UPM0081T15 and UPM0081T20 IBDV passages and other published IBDV strains. Major hydrophilic domain (Peak A and B) and minor hydrophilic domains were boxed by black line. The serine rich heptapeptide was shown by underline.
123
Figure 4.6: Amino acid sequence aligment of UPM0081T10, UPM0081T15 and UPM0081T20 IBDV passages Note: Deduced amino acid sequence of HPVR of VP2 from residue 173-386 (numbering of the segment A polyprotein of Bayliss et al., 1990) of the UPM0081T10, UPM0081T15 and UPM0081T20 IBDV passages and other published IBDV strains. Major hydrophilic domain (Peak A and B) and minor hydrophilic domains were boxed by black line. The serine rich heptapeptide was shown by underline.
124
.
Figure 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.
125
Figure 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.
Figure 4.9: Sequence nucleotide difference of VP2 genes of IBDV isolates Note: Twenty seven IBDV isolates were compared: vvIBDV(UPM94268, UPM92/04, UPM4/230, UPM94/273, B00/73, UK661, Tasek.94. OKYM, HK46, Ehime 91, SH/95 and SH/92), caIBDV (CT and KAL2001), variant (GLS, Variant A and E//DEL), attenuated (UPM0081T10, UPM0081T15, UPM0081T20, Soroa, KTI/99. PBG98, D78 and AmerVH9907), and a virulent serotype 2 (OH). Note that the three atIBDV isolates in this study (UPM0081T10, UPM0081T15 and UPM0081T20) were had 13 to 17 nucleotide difference when compare with publish atIBDV, while 29 to 36 with vvIBDV.
Figure 4.10 : Sequence nucleotide identity matrix of VP2 genes of IBDV isolates Note: Twenty seven IBDV isolates were compared: vvIBDV(UPM94268, UPM92/04, UPM4/230, UPM94/273, B00/73, UK661, Tasek.94. OKYM, HK46, Ehime 91, SH/95 and SH/92), caIBDV (CT and KAL2001), variant (GLS, Variant A and E//DEL), attenuated (UPM0081T10, UPM0081T15, UPM0081T20, Soroa, KTI/99. PBG98, D78 and AmerVH9907), and a virulent serotype 2 (OH). Note that the three atIBDV isolates in this study (UPM0081T10, UPM0081T15 and UPM0081T20) where identical (97%) to other atIBDV while The degree of identity was down to (95%) when compared with very virulent strains.
Figure 4.11: Sequence nucleotide difference of VP2 genes of IBDV isolates Note: Twenty seven IBDV isolates were compared: vvIBDV(UPM94268, UPM92/04, UPM4/230, UPM94/273, B00/73, UK661, Tasek.94. OKYM, HK46, Ehime 91, SH/95 and SH/92), caIBDV (CT and KAL2001), variant (GLS, Variant A and E//DEL), attenuated (UPM0081T10, UPM0081T15, UPM0081T20, Soroa, KTI/99. PBG98, D78 and AmerVH9907), and a virulent serotype 2 (OH). Note that the three atIBDV isolates in this study (UPM0081T10, UPM0081T15 and UPM0081T20) were had 4 to7 amino acid difference when compare with publish atIBDV, while 10 to 13 with vaIBDV.
Figure 4.12: Sequence amino acid identity matrix of VP2 genes of IBDV isolates Note: Twenty seven IBDV isolates were compared: vvIBDV(UPM94268, UPM92/04, UPM4/230, UPM94/273, B00/73, UK661, Tasek.94. OKYM, HK46, Ehime 91, SH/95 and SH/92), caIBDV (CT and KAL2001), variant (GLS, Variant A and E//DEL), attenuated (UPM0081T10, UPM0081T15, UPM0081T20, Soroa, KTI/99. PBG98, D78 and AmerVH9907), and a virulent serotype 2 (OH). Note that the three atIBDV isolates in this study (UPM0081T10, UPM0081T15 and UPM0081T20) where identical (98%) to other atIBDV while the degree of identity was down to (95%) when compared with very virulent strains.
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4.4 Discussion
The hypervariable region (HPVR) of VP2 is among the most studied parts of the
IBDV genome. The nucleotide and amino acid sequence data obtained for
different IBDV isolates showed this region to be the most diverse region of the
viral genome (Bayliss et al., 1990). The HPVR has also been shown to play a
key role in host immunity by forming a conformational epitopes that is the major
target to which host virus neutralizing antibodies are directed (Fahey et al.,
1989; Eterradossi et al., 1998). The domain composing this epitope is composed
of a collation of hydrophobic amino acids which are flanked on either side by two
hydrophilic peaks (aa 212-224 and aa314-324) (Lana et al., 1992). Substitution
of amino acid within the HPVR, particularly within the hydrophilic peaks, has
previously been associated with changes in viral antigenicity (Jackwood and
Saif, 1987; Ismail et al., 1990; Snyder 1990).
Various studies have demonstrated that passages of virulent viruses in
embryonated egg or tissue culture have decreased virulence and effective
immunogenicity (Yamaguchi et al., 1996b). In this study, the differences in
genetic composition between parental IBDV strains, UPM0081 and two
mammalian cell line (Vero cells and DF-1) passages culture derivatives as it
relates to the nucleotide and amino acid sequences of the VP2 variable region
were determined.
131
Sequence analyses of selected passages showed that there were no apparent
changes in the VP2 in two passages, Vero and DF-1 cells (UPM0081T5,
UPM008T7, UPM0081D5 and UPM0081D7). A few nucleotide changes in the
VP2 gene with no resultant amino acid substitution by the (UPM0081T8)
passage level, did not significantly affect the homology of amino acid sequence
with parental vvIBDV UPM0081 strains as the homolgy remained at 99%. The
result of the sequencing in (UPM0081T9) passage showed the first mutation in
amino acid sequence at position 222 (A to P) in the major hydrophilic peak A
VP2 gene. The change at this position has been reported to be useful in
predicting the antigenic or pathotypic characteristics of IBDV as A at position
222 has been associated with very virulent viruses, while P has been associated
with attenuated viruses which was the case in this study (Cao et al., 1998).
Further changes in the VP2 gene also occurred in UPM0081T10, UPM0081T15,
and UPM0081T20 passages as detected in the minor hydrophilic peak 1 and
minor hydrophilic peak 2 at position 242 (I to V), 249 (Q to R), 253 (Q to H), 256
(I to V), 279 (D to N) , 284 (A to T) and 294 (I to L). The amino acid substitutions
at positions 279 (D to N) and 284 (A to T) are commonly found in the attenuation
strain as observed in this study (Yamaguchi et al., 1996b). This substitution has
been reported when some highly virulent strains were attenuated and adapted to
CEF (Yamaguchi et al., 1996b; Lim et al., 1999).
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Some researchers also demonstrated the importance of VP2 in both the cellular
tropism and pathogenicity of IBDV but the alteration was that of two specific
amino acid 253 (Q to H) and 284 (A to T) within the VP2 protein which resulted
in both adaptations to tissue culture and attenuation in SPF chickens (Mundt
1999; Brandt et al., 2001; van Loon et al., 2002).
From the various reports, it is likely that amino acid 284 plays a central role in
infection of tissue culture (Yamaguchi et al., 1996b; Cao et al., 1998). However,
other amino acids are also important for adaptation. The probable mechanism
that resulted in the additional exchange of amino acid 253 (Q to H) which in turn
enhanced the amount of obtained virus after passaging needed to be
understood in order to explain IBDV adaptation, virus attachment and cell
tropism. It has been suggested that the three determinants (253, 279 and 284)
are located in the most exposed loops of projection domain of VP2, therefore
alternations of three residues of VP2 provides the virus an ability to penetrate
cell culture and grow to give high titer (Boot et al., 2000; Liu and Vakharia, 2004;
Coulibaly et al., 2005).
The importance of the change in the heptapeptide region in UPM0081T10,
UPM0081T15 and UPM0081T20, which changed from SWSASGS (residue 326-
332) in parental IBDV strain (UPM0081) to LWSARGS in cell culture adapted
IBDV strain is not certain. However the amino acid at position 330 (S) in parental
133
IBDV has been reported to be associated with virulence (Mundit, 1999), hence
the sequences of amino acids in the serine-rich heptapeptide may be useful in
the determination of the virulence of IBDV. It could also be useful to study the
effect of the type of tissue culture, and the passage level on the sequences of
amino acids in the serine-rich heptapeptide of IBDV.
The phylogenetic tree analysis was constructed based on nucleotide (residue
516-1158) and amino acid (173-386) by Clustal X method. The analysis showed
that (UPM0081T10, UPM0081T15 and UPM0081T20) and atIBDV strains (D78
and AmerVH9907) are in a branch in one group and they are similar to other
atIBDV.
It was concluded that the UPM0081T10, UPM0081T15 and UPM0081T20 were
successfully adapted and attenuated in Vero cells as atIBDV strain. The
phylogenetic analysis showed that Malaysian atIBDV isolates shared a common
origin with other attenuated isolates. The most favorable mutation among IBDV
passages used in this study was found in the HPVR of VP2 gene with amino
acid substitutions occurring between 253 (Q to H) and 256 (I to V) and also 279
(D to N) and 284 (A to T). This mutations observed could play a key role in the
adaptation and attenuation of Malaysian field vvIBDV strain (UPM0081) at
different passage levels to Vero cells. The attenuated isolates could in turn be
possible candidates for development live and inactivated IBD vaccines.
CHAPTER 5
PATHOGENICITY AND IMMUNOGENCITY OF THE ATTENUATED vvIBDV IN SPF CHICKENS
5.1 Introduction
Infectious bursal disease virus (IBDV) causes a highly contagious
immunosuppressive disease in young chickens, which is characterized by the
depletion of the lymphoid follicles of the bursa of Fabricius (Cosgrove, 1962;
Hirai et al., 1981; Okoye et al., 1984). Infection caused by other etiologic agents
such as IBDV infection has been reported to diminish the chicken‟s ability to
respond to vaccination (Cho et al., 1970; Allan et al., 1972; Faragher et al.,
1974; Giambrone et al., 1977).
Infectious bursal disease (IBD) manifests in two forms clinical and subclinical
which is age related. The clinical form of the disease occurred in chickens
between 3 to 6 weeks of age and is responsible for losses due to impaired
growth and death and carcass condemnation from skeletal muscle hemorrhages
(Fadley, 1983). Chicken infected with the classical IBDV (caIBDV) when less
than 3 weeks of age do not exhibit clinical infection, but develop subclinical
infection characterized by microscopic lesions in the bursa of Fabricius and
immunosuppression (Edwards, 1981). The bulk of the economic losses are
associated with subclinical IBDV infection due to the consequent severe and
prolonged immunosuppression (Ivanyi and Morris, 1976).
135
IBDV is a lymphotropic pathogen with a special predilection for differentiating
cells in the bursa of Fabricius. IBDV infection induces B-cell apoptosis, necrosis,
and bursal atrophy with a concomitant suppression of the humoral response
(Sivanandan and Maheswaran, 1980). The damage to the bursa varies with
some caIBDV strains which induced severe inflammatory response (Benton et
al., 1967; Tanimura et al., 1995) and other strains do result in bursal atrophy
with little or no inflammation (Allan, et al., 1972; Faragher et al., 1974; Tanimura
et al., 1995).
No treatment is available against IBDV infection as most poultry industry uses
different preventive programmes in order to minimize the effects of virus
exposure. These programmes include biosecurity, breeder flock vaccination
and/or progeny vaccination (Lucio and Hitchner, 1980). Although biosecurity is
an important step in the prevention of early exposure to infectious agent, IBDV is
very resistant to inactivation which results in its persistence in poultry houses for
long periods. Therefore, the poultry industry rely more on passive or active
immunity for the control and prevention of IBDV infection. The time of
vaccination in breeders and/or progeny is critical for the success since maternal
antibody can interfere with progeny vaccination (Wood et al., 1981).
The vaccination of chicken with IBD vaccine derived from the classical strains of
the virus had been successful until 1988 with the emergence of very virulent
form of IBDV (vvIBDV) in Europe and variant strains (vaIBDV) in the United
136
States which had been reported to induced massive mortalities in vaccinated
poultry flocks (Chettle el al., 1989; Rautenshlein et al., 2005). Numerous IBD
commercial vaccines are available and these vaccines have been subjectively
classified as mild, intermediate and intermediate plus (hot) IBD vaccines (Lukert
and Saif, 2003) based on the level of the attenuation of the vaccine virus
employed. The vaIBDV isolates had been reported to vary pathologically and
serologically from the caIBDV strains and they have been reported to have
different neutralizing epitopes which may account for the vaccine failure
observed in the field. It is therefore imperative that vaccines made from local
strains which is antigenically close to that of the wild-type viruses that the
present in the bird‟s environment will provide better protection (Jackwood,
2005). Apart from the antigenic closeness, literature abounds on the effect of
cell culture adaptation and passage level on pathogenicity and immunogenicity
of IBD vaccine. The adaptation and attenuation of IBDV in BGM (grivet monkey
kidney cell line) has been well described and the virus loss its pathogenicity at
more than 30 passages in the cell line (Tsai and Saif, 1992; Hassan and Saif,
1996). The replication of the attenuated virus in host had also been a subject of
debate as the titre induced by these attenuated IBD vaccines in some reports
had been low and some were not protective (Tsai and Saif, 1992). This study
therefore seek to evaluate the pathogenicity and immunogenicity of a Malaysian
isolate of vvIBDV (UPM0081) which was attenuated, propagated and adapted
in Vero cell line with the view of understanading the probable effect of the host
137
system on the immunogenicity, antigenicity and pathogenicity of the virus at
different passages.
The objectives of this study therefore were:
1. to determine the pathogenicity and immunogenicity of the attenuated
Malaysian isolate of vvIBDV (UPM0081) in Vero cells and in specific-
pathogen- free (SPF) chickens.
2. to determine the efficacy of the attenuated vvIBDV in SPF chickens
5.2 Materials and Methods
5.2.1 Chickens
One-day-old SPF chicks were housed in isolation units at the Experimental
House, Faculty of Veterinary Medicine, Universiti Putra Malaysia and reared
under standard conditions. Clean drinking water and commercial feed were
provided to the chicks.
138
5.2.2 Selection of IBDV Isolates
A local isolate of vvIBDV serotype 1 identified as UPM0081 strain with an
accession number of AY520910 was first passaged 3 times in 9 to 11-day-old
SPF embryonated chicks eggs prior to adaptation and attenuation in Vero cells
on passages 10, 15 and 20 as previously described (Section 3.2.9). The virus
titer on passages 10, 15 and 20 were 104.7, 106.7 and 107.4 TCID50/ 0.1 mL,
respectively (Section 3.2.10).
5.2.3 Adaptation of IBDV to Embryonated SPF Eggs
As previously described in (Section 3.2.3)
5.2.4 Tissue Culture Infective Dose 50 (TCID50
)
As previously described in (Section 3.2.10)
5.2.5 Experimental Design
Two separate experiments were conducted. The first was the preliminary study
to determine the pathogenicity and immunogenicity of the two vvIBDV
attenuated passages 10 and 15 in SPF chicks and the second to determine the
139
pathogenicity and immunogenicity of the two vvIBDV attenuated passaged 15
and 20 in SPF chicks.
5.2.6 Experiment 1
Eighteen, 42-day-old SPF chickens were allocated randomly into 3 groups
namely groups A, B and C with 8, 8 and 2 chickens in each group, respectively.
The chickens in group A were inoculated orally with the attenuated P10 IBDV
isolate (UPM0081T10) at a dose of 104.7 TCID50/ 0.1 mL. The chickens in group
B were inoculated with the attenuated P15 isolate (UPM0081T15) at a dose of
106.7 TCID50/ 0.1 mL while, the group C acted as the control group and remained
uninoculated. The chicken in each group were kept in different isolation unit and
were monitored daily of any abnormality and clinical signs. At day 14 post
inoculation (pi), the survived chickens were sacrificed. Blood samples and body
weight were collected. The gross lesions were recorded and bursa of Fabricius
was weight for evaluation of bursa to body weight ratio. Samples of bursa of
Fabricius were collected and fixed in 10% buffered formalin for histopathology
examination.
140
5.2.7 Experiment 2
One hundred and twenty five SPF chickens of 21-day-old were allocated
randomly into three main groups namely as group 1 (passage 15), group 2
(passage 20) and group 3 (control) with 40 chickens per group except for group
3 (45 chickens). The group 1 was further divided into three sub groups namely
subgroup 1 (a): 30 chickens which served as the sacrificed group. They were
inoculated orally (0.1 mL) with IBDV isolate (UPM0081T15) at a dose of 106.7
TCID50/ 0.1 mL. Subgroup 1 (b): five chickens were inoculated with same dose
orally and served as the positive challenge group and subgroup 1 (c): five
chickens were inoculated with the same dose and served as the mortality group.
The group 2 chickens were also divided into three subgroups. Subgroup 2 (a):
30 chickens which served as sacrificed group were inoculated orally (0.1mL)
with IBDV isolate (UPM0081T20) at a dose of 107.4 TCID50/ 0.1 mL. Subgroup 2
(b): five chickens were inoculated with the same dose orally and served as the
positive challenge group and subgroup 2 (c): five chickens were inoculated with
the same dose orally and served as the mortality group. The group 3 was
subdivided into three subgroups. Subgroup 3 (a): 35 chickens served as
sacrificed group. Subgroup 3 (b): five chickens were served as the positive
challenge group (uninoculated challenged) and subgroup 3 (c): five chickens
were left without inoculation and without challenged. The chickens were
inoculated with the IBDV at 21-day-old and challenged with field isolate of
141
vvIBDV (UPM0081) with a titer of 107.8 EID50/0.1mL at 35-day old. Five chickens
from subgroup 3 (a) were sacrificed at day 0 before IBDV inoculation. The
chickens from all subgroups 1 (a), 2 (a) and 3 (a) were sacrificed at days
1,3,5,7,10 and 14 post inoculation (pi) and 21 days in subgroups 1 (b), 2 (b),3
(b), 1(c), 2(c) and 3(c) or 1 week post vvIBDV challenged (Table 5.1). At
necropsy, blood samples were collected for detection of IBD antibody. The body
weight, bursa weight, bursa to body weight ratio and gross lesions of the
chickens were recorded. The bursa of Fabricius were collected and fixed into
10% buffered formalin for histology and the other part was for detection of IBDV
using (RT–PCR).
Table 5.1: Groups of SPF chickens inoculated with attenuated vvIBDV passage 15 and 20 and challenged with vvIBDV at day 14 post inoculation.
Definition
Time of sampling ( Days pi)
Time of sampling ( Days pc)
No. of chickens
0 1 3 5 7 10 14
7
1 / (P15)
(a) Attenuated vvIBDV inoculated (sacrificed)
– 5 5 5 5 5 5
30
(b) Attenuated vvIBDV inoculated (challenge)
– – – – – – –
5
5
c) Attenuated vvIBDV inoculated (% of mortality)
– – – – – – –
5
5
2 / (P20)
( a) Attenuated vvIBDV inoculated (sacrificed)
– 5 5 5 5 5 5
30
(b) Attenuated vvIBDV inoculated (challenge)
– – – – – – –
5
5
c) Attenuated vvIBDV inoculated (% of mortality)
– – – – – – –
5
5
3 / (control)
(a) Sacrificed without inoculation
5 5 5 5 5 5 5
35
(b) Without inoculation and vvIBDV challenge
– – – – – – –
5
5
(c) Without inoculation and without challenge (% of mortality)
– – – – – – –
5
5
142
5.2.8 IBD Challenge
The attenuated vvIBDV inoculated and control challenge groups were
challenged at 14 day post inoculation with a titer of 107.8 EID50/0.1mL of vvIBDV
(UPM0081) via the orally route and kept under strict observation for clinical
signs, gross lesions up to 7 days post challenged.
5.2.9 Histopathology
According to the method of Tanimura et al. (1995). Samples collected from the
experimentally infected SPF chickens were fixed in 10% buffered formalin for at
least 24 hours to be further processed for histopathological examination. The
bursa was trimmed to the thickness of 0.5 cm followed by dehydration through
grade series of alcohol and xylene in the automatic tissues processor (Leica
5500, Germany). The tissues were then embedded in paraffin wax, cut into 4
µm sections and mounted on glass slide. They were dewaxed and stained with
haematoxylin and eosin (H&E) (Lillie, 1965) (Appendix C), and examined under
light microscope to observe histological changes.
143
5.2.10 Histopathological Lesion Scoring
The histological scoring of bursa of Fabricius was performed based on the
previously described methods (Hair-Bejo et al., 2000). Briefly, bursa of Fabricius
lesions were scored in a scale of 0 to 5 based on the presence of lymphoid cell
necrosis, degeneration, oedema, hetrophil infiltration, and follicular cysts
formation. The score of 0 represented no lesion observed, 1 for mild, 2 for mild
to moderate, 3 for moderate, 4 for moderate to severe and 5 for severe lesions
which were divided into chronic and acute forms (Appendix D).
5.2.11 Collection of Samples for Serological Test
The blood samples were collected to assess the immunity prior and after the
attenuated vvIBDV inoculation at days 0, 1, 3, 5, 7, 10, 14 and 21 post
inoculation (pi). The blood was collected from different groups of chickens via
heart or wing vein using 2 and 5 mL sterile syringe and poured into test tubes
kept in slanting position to collect the serum. The samples were stored at -80oC
till used.
144
5.2.12 Antibody Assay
The technique of the test was followed as described by Howie and Thorsen,
(1981), and was conducted by One Point Health Company using precoated
ELISA kit (BioChek, UK). Briefly, the test samples were diluted to five hundred
fold (1:500) dilution. The diluted samples (100 µl) were dispensed into the
appropriate 96-well plates coated with IBDV viral antigens. The plates were
incubated for 30 min at room temperature (RT) and were washed with 300 µl of
ddH2O 5 times at the end of incubation period followed by addition of 100 µl of
Anti-chicken: alkaline phosphates conjugated into each well. The plate were
allowed to incubate at RT for 30 min and washed 5 times again before adding
100 µl of p-Nitrophenyl phosphate (PNPP) substrate solution into each test well
which was then incubated for 15 min at RT. Finally, 100 µl of stop solution was
added into each well to stop the reaction and the absorbance were read at 405
All the chickens in this group did not show any clinical signs of IBDV infection in
the first day pi. At day 2 pi, some of the chickens exhibited typical clinical signs
of IBD including anorexia, severe depression and ruffled feathers. Two out of
eight (25%) chickens died at day 2 and 4 pi.
146
Group B: Passage 15
No clinical signs and mortality were observed in any bird throughout the
experiment.
Group C: Control Group
No clinical signs and mortality were observed in any bird throughout the
experiment.
5.3.1.2 Experiment 2
Passage 15
Group 1(a): Attenuated vvIBDV Inoculation (Sacrificed)
No clinical signs were observed in this group throughout the experiment.
Group 1(b): Attenuated vvIBDV Inoculation (vvIBDV challenge)
All the chickens did not show any abnormal IBD clinical signs and were 100%
protected against vvIBDV challenged (Table 5.2).
147
Group 1(c): Attenuated vvIBDV Inoculation (% of mortality)
No abnormal clinical signs of IBD were recorded throughout the experiment.
Passage 20
Group 2(a): Attenuated vvIBDV Inoculation (Sacrificed)
No abnormal clinical signs were observed in the chicken throughout the
experiment.
Group 2(b): Attenuated vvIBDV Inoculation (vvIBDV challenged)
No abnormal clinical signs were observed and the chickens were 100%
protected against vvIBDV challenged (Table 5.2).
Group 2(c): Attenuated vvIBDV Inoculation (% of mortality)
No abnormal clinical signs of IBD were recorded throughout the experiment.
148
Control
Group 3(a): Non Attenuated vvIBDV Inoculated (Sacrificed)
No clinical signs were observed in any of the chicken throughout the experiment.
Group 3(b): Uninoculated and vvIBDV Challenge (vvIBDV challenge)
No abnormal clinical signs were observed during the first day of the experiment.
At day 2 pi almost all of the chickens exhibited typical clinical signs of IBD
including, anorexia, sever depression and ruffled feathers. However, no diarrhea
and urate were observed. Five out of five chickens (100%) died at day 5 post
challenged (Table 5.2).
Group 3(c): Non Attenuated vvIBDV Inoculation (%of mortality)
No abnormal clinical signs were observed in any chickens throughout the
experiment.
149
Table 5.2: Rate of mortality and the percentage of protection based on the number of chickens that survived at day 7 post challenged
Challenged groups
Total accumulative death
Number of death/total number of chicken
% of % of
Mortality Protection
Days (Post challenged)
1 2 3 4 5 6 7
Group 1(b): Attenuated vvIBDV inoculation and vvIBDV challenge
0/5 0/5 0/5 0/5 0/5 0/5 0/5
0% 100%
Group 2(b): Attenuated vvIBDV inoculation and vvIBDV challenge
0/5 0/5 0/5 0/5 0/5 0/5 0/5
0% 100%
Group 3(b): Without inoculation and vvIBDV challenge
0/5 2/5 2/5 3/5 5/5 5/5 5/5
100% 0%
All chickens that survived from vvIBDV challenged showed no significant clinical abnormality observed at day 7 post-challenged. 5.3.2 Body Weight 5.3.2.1 Experiment 1 Groups A, B and C
There were no difference in the body weight of chickens between groups B
(867.5 ± 46.5g) and C (875.0 ± 35.3g), while mean body weight of chickens
group A (695.0 ± 102.5g) were significant lower (p<0.05) than the group B
(Table 5.3).
150
5.3.2.2 Experiment 2
Groups 1(a), 2(a) and 3(a)
Body weight of chickens from the group 1(a) increased significantly (p<0.05)
from day old (82.6 ± 3.7g) to day 14 (351.0 ± 6.5g). The body weight of the
group 2(a) also had significant increased (p<0.05) from day 5 (137.0 ± 8.3g) to
day 14 (355.4 ± 7.7g) (Table 5.4). The body weight of the chickens from the
group 3(a) showed significant increased (p<0.05) from day 1 (81.6 ± 2.1g) to
day 14 (357.0 ± 5.7g). There was no significant difference (p>0.05) in the body
weights of the chickens among groups 1(a), 2(a) and 3(a), respectively
throughout the experiment (Table 5.4).
Groups 1(b), 2(b) and 3(b)
No significant differences (p>0.05) in body weight were observed between
groups 1(b) (478.0 ± 5.7g) and 2(b) (480.0 ± 6.1g) at day 7 post challenged. The
mean body weights of chickens group 3(b) (441.0 ± 13.8g) was significantly
(p<0.05) lower than those of chickens attenuated vvIBDV inoculated with
passage 15 and 20 and challenged with vvIBDV in groups 1(b) and 2(b)
respectively (Table 5.5).
151
Groups 1(c), 2(c) and 3(c)
There were no significant difference (p>0.05) in the body weight of chicken in all
groups 1(c) 468.0 ± 29.2g, 2(c) 478.2 ± 29.4g and 3(c) 478.8 ± 29.0g at day 7
post challenged (Table 5.6).
5.3.3 Bursa Weight 5.3.3.1 Experiment 1
Groups A, B and C
No difference was observed in the bursa weight of chickens in groups B (2.8 ±
0.2g) and C (2.9 ± 0.1g) at day 14 post inoculation. The chickens in group A (1.5
± 0.2g) which were significantly lower (p<0.05) than the group B (Table 5.3).
152
5.3.3.2 Experiment 2
Groups1 (a), 2(a) and 3(a)
The chickens in group 1(a) had a significant increased (p<0.05) bursal weight
from day 1 (0.14 ± 0.04g) to day 14 (1.87 ± 0.06g). The bursa weight in the
groups 2 (a) and 3 (a) were also increased from day 1 (0.13 ± 0.03g) and (0.14
± 0.02g) to day 14 (1.86 ± 0.06g) and (1.88 ± 0.05g), respectively. There was no
significant difference (p>0.05) in bursal weight among these groups throughout
the experiment (Table 5.7).
Groups 1(b), 2(b) and 3(b)
The mean bursal weight (0.4 ± 0.1g) of the group 3(b), birds was significantly
lower (p<0.05) than those of the attenuated vvIBDV inoculated challenged birds
in both groups 1(b) (2.5 ± 0.1) and 2(b) (2.5 ± 0.1), while there was no significant
difference (p>0.05) between the groups 1(b) and 2(b) (Table 5.5).
153
Groups 1(c), 2(c) and 3(c)
There were no significant difference (p>0.05) in the bursa weight of chicken in
all groups 1(c) 2.34 ± 0.1g, 2(c) 2.42 ± 0.1g and 3(c) 2.48 ± 0.1g at day 7 post
challenged (Table 5.6).
5.3.4 Bursa to Body Weight Ratio 5.3.4.1 Experiment 1
Groups A, B and C (1x103)
No difference was observed in the bursa to body weight ratio of chickens in
groups B (3.2 ± 0.4) and C (3.2 ± 0.1) at day 14 post inoculation. The chickens
in group A (2.1 ± 0.4) were significantly lower (p<0.05) than the group B (Table
5.3).
154
5.3.4.2 Experiment 2
Groups1 (a), 2(a) and 3(a) (1x103)
The bursa to body weight ratio gradually increased (p<0.05) from day 1 (1.7 ±
0.4) to day 10 (5.0 ± 0.2) in group 1(a) and remained stable (p>0.05) from day
10 (5.0 ± 0.2) to day 14 (5.1 ± 0.1). The ratio also increased from day 1 (1.6 ±
0.4) to day 10 (5.1 ± 0.1) in group 2(a) and also remain stable (p>0.05) from day
10 (5.1 ± 0.1) to day 14 (5.1 ± 0.1). The ratio in group 3(a) also followed similar
pattern with increased from day 1 (1.6 ± 0.2) to day 10 (5.0 ± 0.1) and similar
stability in values (p>0.05) from day 10 (5.0 ± 0.1) to day 14 (5.2 ± 0.1) (Table
5.8). There was no significant (p>0.05) difference among group 1(a), group 2(a)
and group 3(a) throughout the experiment (Table 5.8).
Groups1 (b), 2(b) and 3(b) (1x103)
There was no significant (p>0.05) differences in the bursa body weight ratio
between two groups 1(b) (5.2 ± 0.2) and 2(b) (5.1 ± 0.1) while the ratio of
chickens non inoculated and challenged with vvIBDV group 3(b) (0.9 ± 0.1) was
significantly (p<0.05) lower than those of chickens inoculated with passage 15
and 20 and challenged with vvIBDV in groups 1(b) and 2(b), respectively
(Table 5.5).
155
Groups 1(c), 2(c) and 3(c) (1x103)
There were no significant difference (p>0.05) in the bursa weight of chicken in
all groups 1(c) (5.0 ± 0.1), 2(c) (5.1 ± 0.1) and 3(c) (5.2 ± 0.1g) at day 7 post
challenged (Table 5.6).
5.3.5 Gross Pathology 5.3.5.1 Experiment 1
Groups A, B and C
The bursa of Fabricius (BF) remained normal in groups B and C throughout the
experiment. However in group A, there was mild petechial haemorrhage in the
breast muscle at day 3 pi. At day 4 pi, the BF showed mild to moderate oedema
and necrosis with yellowish gelatinous material (Figures 5.1a and b).
156
5.3.5.2 Experiment 2
Groups 1(a), 2(a) and 3(a)
All sacrificed chickens did not show any gross lesions throughout the
experimental period
Groups 1(b), 2(b) and 3(b)
The BF and other organs remained normal in the attenuated vvIBDV inoculated
and challenged with vvIBDV in the groups 1(b) and 2 (b). However in
uninoculated and challenged group 3(b), at 2-4 days post challenged, the BF
was swollen (inflamed) and appeared odematous with severe hemorrhage
(Figures 5.2a and b), Hemorrhages of the proventricular mucosa and at the
junction with the gizzard was recorded (Figures 5.3a and b).
Groups 1(c), 2(c) and 3(c)
All sacrificed chickens did not show any gross lesions at day 7 pc.
157
Figure 5.1a: Experiment 1 (preliminary study): bursa of Fabricius in SPF chickens at day 14 pi. (A) Group C: Control normal. (B) Group A: Passage 10 bursa of Fabricius with mild to moderate odema with yellowish gelatinous material (arrow)
158
Figure 5.1b: Experiment 1 (preliminary study): bursa of Fabricius in SPF chickens at day 14 pi. (C) Group B: passage 15 normal
159
Figure 5.2a: Experimental 2 (challenged groups): bursa of Fabricius in SPF chickens at day 7pc. (A) Group1 (b): passage 15 normal. (B) Group 2(b): passage 20 normal.
160
Figure 5.2b: Experimental 2 (challenged groups): bursa of Fabricius in SPF chickens. (C) Group 3(b): control positive with severe haemorrhages at day 4 pc
161
Figure 5.3a: Experiment 2 (challenged groups): proventriculus and gizzard in SPF chickens at day 7pc. (A) Group 1 (b): passage 15 normal (B) Group 2 (b): passage 20 normal
162
Figure 5.3b: Experiment 2 (challenged groups): proventriculus and gizzard in SPF (C) Group 3 (b): control positive hemorrhage on the mucosa of the proventriculus at the junction with the gizzard (arrows) at day 4 pc
163
5.3.6 Histopathological Changes and Lesion Scoring
5.3.6.1 Experiment 1
Group A
The histopathological changes were severe in the bursa of Fabricius. The
changes were hyperemia, haemorrhage and oedema, followed by lymphoid
depletion, bursal atrophy and cyst formation. The lesion score was 4.66 ± 0.4
(Figures 5.4b, Table 5.3).
Group B
There was no visible histological lesion in bursa of Fabricius and the lesion
score was (0.4 ± 0.1) ( Figure 5.4a, Table 5.3).
Group C
The bursa follicles were apparently normal with
lesion score of 0.5 ± 0.07 (Figure 5.4a).
164
5.3.6.2 Experiment 2
Groups 1(a), 2(a) and 3(a)
Groups 1(a)
The bursa of Fabricius was characterized by normal to mild tissue reaction.
There was no significant difference (p>0.05) in the lesion score from day 1 (0.34
± 0.54) to day 14 pi (0.42 ± 0.08) throughout the experiment (Table 5.9, Figure
5.5a).
Group 2(a)
Histopathological lesions in bursa of Fabricius remained unchanged (p>0.05)
from day 1 (0.34 ± 0.05) to day 14 (0.38 ± 0.14) throughout the experiment
(Table 5.9, Figure 5.5a).
Group 3(a)
No observable lesions were present in the bursa of Fabricius (p>0.05) from day
1 (0.32 ± 0.44) to day 14 (0.36 ± 0.08). There was no significant difference
(p>0.05) in the lesion score between groups 1(a), 2 (a) and 3 (a) throughout the
experiment (Figures 5.5b, Table 5.9).
165
Groups 1(b), 2(b) and 3(b)
Groups 1(b)
The bursa of Fabricius was characterized by normal to mild tissue reaction with
active follicles with lesion scoring of 0.8 ± 0.2 (Figure 5.6a, Table 5.5).
Group 2(b)
The bursa of Fabricius was characterized by normal to mild tissue reaction with
active follicles (Figure 5.6a). There was no significant difference (p>0.05) in the
lesion score between groups 2(b) (0.9 ± 0.1) and 1(b) (0.8 ± 0.2) (Table 5.5).
Group 3(b)
The histopathological feature was remarkable. There was severe tissue reaction
as many lymphoid follicles appeared heavily depleted of lymphoid cells. There
was also lymphoid cells aggregation in the cortex, of some follicles while cellular
vacuolation and cyst still existed in some follicles. The interstitial space was
infiltrated with inflammatory cells. The lesion score (4.5 ± 0.5) was significantly
(p<0.05) higher than those of chickens in groups 1(b) and 2(b) respectively
(Table 5.5, Figures 5.6b).
166
Groups 1(c), 2(c) and 3(c)
Groups 1(c)
All chickens in this group showed normal to mild bursitis with lesion score of 0.4
± 0.1 (Table 5.6, Figure 5.7a).
Groups 2(c)
The bursa of Fabricius was characterized by normal to mild tissue reaction with
lesion score of 0.4 ± 0.1 (Table 5.6, Figure 5.7a).
Groups 3(c)
All chickens in this group showed normal to mild bursitis with lesion score of 0.3
± 0.1. There was no significant difference (p>0.05) in the lesion score among
groups 1(c ), 2(c) and 3(c) (Table 5.6, Figure 5.7b).
167
Figure 5.4a: Experiment 1 (preliminary study) at 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 ( ), lesion score of 0. HE, 10x. Bar = 200µm
168
Figure 5.4b: Experiment 1 (preliminary study) bursa of Fabricius (C) Group A: Oedematous bursa with degeneration, necrosis ( ) and infiltration of inflammatory cells ( ), follicular cyst ( ) in the medulla, lesion score of 5 at day 2 pi. (D) Group A: More sever lymphoid necrosis ( ) in the medulla, lesion score of 5 at day 5 pi. HE, 20x. Bar = 100 µm
169
Figure 5.5a: Experiment 2 (sacrificed groups) day 7 pi. bursa of Fabricius (A) Group 1(a): Mild degeneration and necrosis of the follicles ( ), lesion score of 1. (B) Group 2(a): Mild degeneration and necrosis of the follicles ( ), lesion score of 1. HE, 10x. Bar = 200µm
170
Figure 5.5b: 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
171
Figure 5.6a: Experiment 2 (challenged groups) at day 7 pc. bursa of Fabricius (A) Group 1(b): Mild degeneration and necrosis of the follicles ( ), lesion score of 1. (B) Group 2(b): Mild degeneration and necrosis of the follicles ( ), lesion score of 1. HE, 10x. Bar = 200µm
172
Figure 5.6b: Experiment 2 (challenged groups) at day 7 pc. bursa of Fabricius (C) Group 3(b): Depletion of bursa follicles with cysts contains cell debris and fibrinous exudates at medulla follicle ( ), the interstitial connective tissues were obvious, edematous and infiltrated with inflammatory cells ( ), lesion score of 5. HE, 20x. Bar = 100µm
173
Figure 5.7a: Experiment 2: bursa of Fabricius (mortality groups) at day 7 pc. (A) Group 1(c): Mild lymphoid depletion ( ), lesion score of 1. (B) Group 2(c): Mild lymphoid depletion ( ), lesion score of 1. HE, 10x. Bar = 200µm
174
Figure 5.7b: Experiment 2 (mortality groups) at day 7 pc. bursa of Fabricius (C) Group 3(c): Mild lymphoid depletion ( ), lesion score of 1. HE, 10x. Bar = 200µm
175
Table 5.3: Experiment 1: body, bursa, bursa to body weight ratio (1 x 103), lesion scoring and ELISA titer of SPF chicken inoculated attenuated vvIBDV and control group
Group / passage
Weights, lesion scoring and ELISA titer at day 14 pi (Mean ± SD)
Body weight
Bursa weight
Bursa body weight
ratio (x103)
Lesion scoring
ELISA titer
A / P10
695.0 ± 102.5
a
(n = 6)
1.5 ± 0.2
a
(n = 6)
2.1 ± 0.4
a
(n = 6)
4.66 ± 0.4
a
(n = 6)
8174.2 ± 264.5
a
(n = 6)
B / p15
867.0 ± 46.5b
(n = 8)
2.8 ± 0.2
b
(n = 8)
3.2 ± 0.4
b
(n = 8)
0.47 ± 0.1
b
(n = 8)
2808.0 ± 413.0
b
(n = 8)
ab Values with different subscripts within column differ significantly at p<0.05 due to different passages (P10 and P15) n number of sacrificed chicken
176
Table 5.4: Experiment 2: body weight (g) of chickens in the inoculated attenuated vvIBDV and control group
Group / passage
Body weight (Mean ± SD g)
Time (Days pi)
1 3 5 7 10 14
1(a) / p15
82.6 ± 3.7
a
P
(n= 5)
104.0 ± 4.5
ab
P
(n= 5)
135.0 ± 7.9
b
P
(n= 5)
199.6 ±30.0
c
P
(n= 5)
300.0 ± 15.8
d
P
(n= 5)
351.0 ± 6.5
e
P
(n= 5)
2(a) / p20
82.8 ± 3.8
a
P
(n= 5)
105.8 ± 9.1
a
P
(n= 5)
137.0 ± 8.3
b
P
(n= 5)
202.2 ± 7.1
c
P
(n= 5)
305.2 ± 10.9
d
P
(n= 5)
355.4 ± 7.7
e
P
(n= 5)
3(a) / control
81.6 ± 2.1
a
P
(n= 5)
106.2 ± 5.0
b
P
(n= 5)
136.6 ±5.9
c
P
(n= 5)
208.0 ± 5.7
d
P
(n= 5)
306.6 ± 7.5
e
P
(n= 5)
357.0 ± 5.7
f
P
(n= 5)
ab Values with different subscripts within rows differ significantly at p<0.05 due to time effects pq Values with different subscripts within column differ significantly at p<0.05 due to different passages (P15 and P20) and control group n number of sacrificed chicken
177
Table 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
Group / passage
Weights and lesion scoring at day 7 pc. (Mean ± SD g)
Body weight
Bursa weight
Bursa body weight ratio
(x103)
Lesion scoring
1(b) / P15
478.0 ± 5.7
a
(n = 5)
2.5 ± 0.1
a
(n = 5)
5.2 ± 0.2
a
(n = 5)
0.8 ± 0.2
a
(n = 5)
2(b) / p20
480.0 ± 6.1
a
(n = 5)
2.5 ± 0.1
a
(n = 5)
5.1 ± 0.1
a
(n = 5)
0.9 ± 0.1
a
(n = 5)
3(b) uninoculated
challenge
441.0 ± 13.8
b
(n = 5)
0.4 ± 0.1
b
(n = 5)
0.9 ± 0.1
b
(n = 5)
4.5 ± 0.5
b
(n = 5)
ab Values with different subscripts within column differ significantly at p<0.05 due to different passages (P15 and P20) and control group n number of sacrificed chicken Table 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
Group / passage
Weights and lesion scoring at day 7pc. (Mean ± SD )
Body weight
Bursa weight
Bursa body weight ratio
(x103)
Lesion scoring
1 (c) / P15
468.0 ± 29.2a
(n = 5)
2.34 ± 0.1a
(n = 5)
5.0 ± 0.1a
(n = 5)
0.4 ± 0.1a
(n = 5)
2 (c) / P20
478.2± 29.4a
(n = 5)
2.42 ± 0.1a
(n = 5)
5.1 ± 0.1a
(n = 5)
0.4 ± 0.1a
(n = 5)
3 (c) / control
478.8 ± 29.0 a
(n = 5)
2.48 ± 0.1a
(n = 5)
5.2 ± 0.1a
(n = 5)
0.3 ± 0.1a
(n = 5)
ab Values with different subscripts within column differ significantly at p<0.05 due to different passages (P15 and P20) and control group n number of sacrificed chicken
178
Table 5.7: Experiment 2: bursa weight (g) of chickens in the inoculated attenuated vvIBDV and control group
Group / passage
Bursa weight (Mean ± SD g)
Time (Days pi)
1 3 5 7 10 14
1(a) / p15
0.14 ± 0.04
a
P
(n= 5)
0.29 ± 0.08
ab
P
(n= 5)
0.48 ± 0.04
b
P
(n= 5)
0.73 ± 0.06
c
P
(n= 5)
1.53 ± 0.05
d
P
(n= 5)
1.87 ± 0.06
e
P
(n= 5)
2(a) / p20
0.13 ± 0.03
a
P
(n= 5)
0.2 8 ± 0.03
ab
P
(n= 5)
0.37 ± 0.01
b
P
(n= 5)
0.72 ± 0.05
c
P
(n= 5)
1.56 ± 0.03
d
P
(n= 5)
1.86 ± 0.06
e
P
(n= 5)
3(a) / control
0.14 ± 0.02
a
P
(n= 5)
0.32 ± 0.04
b
P
(n= 5)
0.40 ± 0.04
b
P
(n= 5)
0.72 ± 0.51
c
P
(n= 5)
1.56 ± 0.06
d
P
(n= 5)
1.88 ± 0.05
e
P
(n= 5)
ab Values with different subscripts within rows differ significantly at p<0.05 due to time effects pq Values with different subscripts within column differ significantly at p<0.05 due to different passages (P15 and P20) and control group n number of sacrificed chicken
179
Table 5.8: Experiment 2: bursa to body weight ratio (1 x 103) of chickens in the inoculated attenuated vvIBDV and control group
Group / passage
Bursa to body weight ratio 1 x 10
3 (Mean ± SD)
Time (Days pi)
1 3 5 7 10 14
1(a) / p15
1.7 ± 0.4a
P
(n= 5)
2.8 ± 0.1b
P
(n= 5)
2.9 ± 0.3 bc
P
(n= 5)
3.4 ± 0.3c
P
(n= 5)
5.0 ± 0.2d
P
(n= 5)
5.1 ±0.1d
P
(n= 5)
2(a) / p20
1.6 ± 0.4a
P
(n= 5)
2.6 ± 0.1b
P
(n= 5)
2.7 ± 0.2b
P
(n= 5)
3.5 ± 0.3c
P
(n= 5)
5.1 ± 0.1d
P
(n= 5)
5.1 ± 0.1d
P
(n= 5)
3(a) / control
1.6 ± 0.2a
P
(n= 5)
2.8 ± 0.2b
P
(n= 5)
2.8 ± 0.3b
P
(n= 5)
3.4 ± 0.2c
P
(n= 5)
5.0 ± 0.1d
P
(n= 5)
5.2 ± 0.1d
P
(n= 5)
ab Values with different subscripts within rows differ significantly at p<0.05 due to time effects pq Values with different subscripts within column differ significantly at p<0.05 due to different passages (P15 and P20) and control group n number of sacrificed chicken
180
Table 5.9: Experiment 2: lesions scoring of chickens in the inoculated attenuated vvIBDV and control group
Group / passage
Lesion scoring (Mean ± SD g)
Time (Days pi)
1 3 5 7 10 14
1(a) / p15
0.34± 0.54a
P
(n= 5)
0.36 ± 0.08a
P
(n= 5)
0.38 ± 0.04a
P
(n= 5)
0.36 ± 0.05a
P
(n= 5)
0.36 ± 0.13a
P
(n= 5)
0.42 ± 0.08a
P
(n= 5)
2(a) / p20
0.34 ± 0.05a
P
(n= 5)
0.36 ± 0.08a
P
(n= 5)
0.40 ± 0.10a
P
(n= 5)
0.38 ± 0.08a
P
(n= 5)
0.44 ± 0.08a
P
(n= 5)
0.38 ± 0.14a
P
(n= 5)
3(a) / control
0.32 ± 0.44 a
P
(n= 5)
0.36 ± 0.08a
P
(n= 5)
0.36 ± 0.05a
P
(n= 5)
0.32 ± 0.13a
P
(n= 5)
0.34 ± 0.05a
P
(n= 5)
0.36 ± 0.08a
P
(n= 5)
ab Values with different subscripts within rows differ significantly at p<0.05 due to time effects pq Values with different subscripts within column differ significantly at p<0.05 due to different passages (P15 and P20) and control group n number of sacrificed chicken
At two weeks post inoculation, there was a significant difference in the mean
antibody titer (p<0.05) between group A (8174 ± 264) and B (2808 ± 413), while
no antibody was detected in the control group (Table 5.3).
5.3.7.2 Experiment 2
The IBDV antibody titers were detected in the both groups 1 (a) (2066 ± 2452)
and 2 (a) (4205 ± 4223) at day 5 post inoculation (pi). The antibody titers
increased at day 7 pi (8844 ± 18989) and (7663 ± 4244) in group 1(a) and 2(a),
respectively. The maximum antibody titers of 10849 ± 3448 and 11329 ± 2943
in group1 (a) and group 2(a), respectively were observed at 10 days pi. At day
14 pi the antibody titer decreased in both groups 8067 ± 5517 and 6169.4 ±
4013 in group 1(a) and 2(a), respectively. The lowest antibody titers were found
in the group 1(b) 3245 ± 347 and 2(b) 4735 ± 3244 at day 21 pi. There was no
significant difference (p>0.05) in antibody titers of the two groups 1 (a) and
group 1(b) from day 1 to day 21 pi. The control group had no detectable IBD
antibody titer throughout the entire experiment (Table 5.10).
182
Table 5.10: Antibody titers (mean titer ± standard deviation) to IBD determined by ELISA in the attenuated vvIBDV inoculated groups
(Days post inoculation)
ELISA titer (Mean ± SD)
Group / passage
1 (a) / p15
2(a) / p20
1
0 ± 0a p
0± 0a p
3
0 ± 0a p
0.± 0a p
5
2066 ± 2452b p
4205± 4223b p
7
8844 ± 18989c p
7663 ± 4244bc p
10
10849 ± 3448c p
11329 ± 2943c p
14
8067 ±5517c p
6169 ± 4013b p
21
3245 ± 347b p
4735 ±3244b p
ab Values with different subscripts within column differ significantly at p<0.05 pq Values with different subscripts within rows differ significantly at p<0.05
5.3.8 Detection of the Virus or Viral RNA using RT-PCR
The viral RNA was detected in the bursa homogenates from chickens inoculated
with attenuated vvIBDV passage 15 and 20 in group1 (a), 2 (a) and 3 (a) at day
1, 3,5,7,10,14 pi and group 1(c), 2(c) and 3(c) at day 21 pi using RT-PCR
(Figure 5.8).
183
Figure 5.8: Hypervariable region (643pb) amplification of IBDV VP2 genes. Lane 1 Day 1; Lane 2 Day 3; Lane 3 Day 5 ; Lane 4 Day 7; Lane 5 Day 10; Lane 6 Day 14; Lane 7 Day 21; and Lane 8 Negative control; M- 100 bp DNA marker (Promega, USA).
5.4 Discussion
In the present study, the Vero cells adapted vvIBDV strain (UPM0081) after 10,
15 and 20 passages were evaluated in SPF chicks for its pathogenicity and
immunogenicity. In the first experiment, the results of the pathogenicity test
indicated that P10 was still pathogenic as evident by the higher bursa lesion
score of 4 and this was in agreement with various investigators especially on
vaccine evaluation (Cursiefen et al., 1979; Muskett et al., 1979; Edwards et al.,
1982). Such vaccine candidate that induces bursal lesions were graded as
intermediate hot vaccines since they induce bursal damage and severe
immunosuppression that are indistinguishable from the field strains (Mazariegos
et al., 1990). The vaccine of this nature are useful to induce active immunity in
chicks with very high level of maternal immunity and in areas where IBD is a
184
serious challenge. The fact this attenuated virus induces bursal lesion also
agreed with the findings of Raue et al. (2004) that with lower level of attenuation
of IBDV, there is a considerably increase risk of reversion to virulence. In the
case of P10, it can be concluded that the 10 serial passage in Vero cell line is
not enough for sufficient loss of pathogenicity needed for vaccine production
(Rodriguez-Chavez et al. (2002). This observation further revealed the influence
of host system and passage level on the pathogenicity of this strain (Hassan
and Saif 1996).
In this study, the vvIBDV at P15 lost its pathogenicity in SPF chicken as evident
by lower bursal lesion score. This was also observed when the caIBDV passage
in BGM-70 or CEF cells resulted in loss of pathogenicity (Hassan and Saif,
1996) at 6 passages and loss of replication at 30 and 40 passages in BMG 70
cells (Tsai and Saif 1992). The loss of pathogenicity may be because there was
no optimal microenviroment and host factors that are necessary for efficient
virus replication in cells like Vero cells (Lange et al., 1987).
The examination of bursal histopathology, bursa body weight ratio and
percentage protection are common measures used to evaluate the virulence of
vvIBDV. In this study, these methods revealed that the P15 and P20 UPM0081
were not pathogenic as there was no significant difference in the bursa weight
and bursal body weight ratio of the chickens in inoculated groups and that of the
control group. This was not the case with the inoculated and challenged group
185
and non inoculated and challenged group as there was a significant difference
(p<0.05) in the bursa weight and bursal body weight ratio (Hassan and Saif
1996; Yamagoshi et al., 1996b; Rasool and Hussain, 2006). The
histopathological features and the bursa lesion score in inoculated groups with
Vero cell adapted, attenuated vvIBDV UPM0081 at P15 and P20 also concured
with the fact that the attenuated strain is non pathogenic and do confer 100%
protection to SPF chicks against vvIBDV field strain (Abdel-Alim and Saif
(2001).
The immunogenicity of P10 and P15 Malaysian vvIBDV isolate revealed that
significant antibody level were detected in serum samples from P10 inoculated
group than P15 inoculated group in experiment 1 after 14 days post inoculation,
and this is may be related to the level of attenuation and tropism of the
attenuated virus from P10 virus as it induced obvious clinical syndrome and
bursal damage which may account for the high level of antibody recorded. In
experiment 2 however, the antibody titers in both inoculated groups were
detected after 5 days post inoculation (Ashraf et al., 2005). The antibody titer
increased rapidly at day 7 post inoculation, and the maximum antibody titers
were found at day 10 post inoculation in both groups, these results suggest that
the induced antibody level in both inoculated groups is sufficient to protect the
chicks and this was substantiated by the 100% protection against vvIBDV
challenge. Although the IBD titres observed in this study was not compared with
that from embryo or bursal derived cell line adapted vvIBDV, the study clearly
186
showed that Vero cell attenuated vvIBDV in passages 15 and 20 do induce
sufficient antibody titre to efficiently result in 100% protection against field
vvIBDV. It was reported that chickens innoculated with cell culture derived IBDV
strains showed poorer immunity when compared with birds that received bursa
derived or embryo derived strains (Rodriguez-Chavez et al., 2002). In contrast,
the VN titres obtained in propagated vaccines in BGM70 and BF cells did not
differ significantly (Hassan and Saif, 1996).
The virus was detected in bursal tissue collected from chickens inoculated with
Vero cells adapted vvIBDV (P15 and P20) at 1, 3,5,7,10,14 and 21 days pi by
RT-PCR. These showed that the virus at this level of passage is replicating
actively in the bursal of Fabricius of inoculated chickens. This was also
observed by Abdel-Alim, (2000) where IBDV was detected in the bursal
homogenates of chicks inoculated with the cell culture adapted virus up to 21
days pi. This ability to replicate was reported to be lost in IBDV passaged in
BGM-70 30 to 40 times (Tsai and Saif 1992). This feature has not been
previously described in Vero attenuated vvIBDV.
It was concluded that the Malaysian vvIBDV (UPM0081) adapted and
attenuated in Vero cell line passage 15 and 20 could provide 100% protection to
SPF chickens from vvIBDV challenged. Hence, the attenuated strains
established in this study may provide a useful virus seed for vaccine
development in the future against vvIBDV field challenge.
CHAPTER 6
SAFETY AND IMMUNOGENICITY OF THE INACTIVATED ATTENUATED vvIBDV IN SPF CHICKENS
6.1 Introduction
Infectious bursal disease virus (IBDV) is the causative agent of a highly
contagious disease of young chickens. The virus infects and multiplies in
immature B lymphocyte found in the bursa of Fabricius, which can result in
immunosuppression (Faragher et al., 1974; Nick et al., 1976). Recent research
had indicated the emergence of vvIBDV with difference in virulence and the
antigenic variation associated with the strain has been the greatest obstacle for
successful control of this disease (van den Berg, 2000).
Presently, there is no treatment against IBDV infection and the effect of virus
exposure. The induction of active immunity by vaccine is still the effective
method of control, but this should include biosecurity measures (Lucio and
Hitchner, 1979; Baxendale and Luttiken, 1981). The preparation of an effective
vaccine is precluded by the isolation and characterisation of an appropriate
IBDV strains with subsequent attenuation before the preparation into live or
killed vaccines. The inactivation of antigen can be carried out by physical or
chemical agents. For physical methods, heat and radiation are involved while for
chemical reagents, beta propiolactone, formaldehyde, aziridines and other
188
derivatives had used (Bahnemann, 1990). Of these, formalin had been the
most popular before the advent of Binary ethylenimine (BEI), a type of alkylating
group compounds (aziridines) which reacts poorly with proteins and for this
reason, it does not alter the antigenic components of the virus. BEI has an
inactivation reaction that is more specific for the nucleic acid and has been
known to produce antigenically superior vaccine (Bahnemann, 1990). BEI has
been used to inactivate different viruses such as rabies (Larghi and Nebel,
1980), foot-and-mouth disease virus (Dilovski and Tekerlekov, 1983),
bluetongue virus (Stott et al., 1979), porcine parvovirus (Buonavoglia et al.,
1988), African horse sickness virus (Soliman et al., 1996) and Newcastle
disease virus (King, 1991). The use of this agent in preparation of IBD vaccine
are few in literature (Habib et al., 2006).
On the other hand, Electrolysed water-Catholyte-Anolyte (ECA) is an activated
solution with a highly oxidized anolyte solution which functions as a very fast
acting antimicrobial agent that destroys viruses and other microorganisms
(Anonymous, 1997). Studies have suggested that the hypochlorous acid in the
course of its production can penetrate microbial cell membranes and in turn
exert antimicrobial action through the oxidation of key metabolic systems
(Albrich et al., 1986; Barrette et al., 1989; Hurst et al., 1991). The use of this
agent in the inactivation of IBD viruses has not received adequate attention.
Since the purpose of vaccination is the production of a strong immune response
to the administered antigen, this feat often requires the addition of an adjuvant
189
(Bomford, 1998). Commonly, water in oil (W/O) emulsions is recommended for
small ruminants, bovine, poultry and fishes when long term immunity is required.
In the case of IBD, mineral oil based emulsions had been used as adjuvant in
order to protect birds against IBDV infection (Benjamin and Hitchner, 1978). The
W/O emulsions usually allow for the reduction of the vaccine dose or the antigen
concentration, which is important to make such vaccines to be cost effective
(Aucouturier et al., 2001).
Inactivated vaccines are most commonly used to vaccinate layer and breeder
birds prior to the laying period. Most inactivated vaccines are used to boost
immunity that has been provided by priming with live vaccines. Alternatively, the
inactivated vaccines that are expected to provide life-long immunity without prior
priming are used in breeders to confer immunity to progeny especially in
endemic IBD areas (van den Berg, 2000).
Freund‟s Complete Adjuvant (FCA), a mixture of a non-metabolizable oil, such
as a mineral oil, a surfactant (Arlacel A) and mycobacteria (M. tuberculosis ) has
been used for many years to enhance immunologic response to antigens, and
even today is considered to be one of the most effective adjuvants (Jackson and
Fox, 1995). However, despite FCA being an effective adjuvant, it has attending
problem and hazards associated with its use especially at the injection site
where it may results in a chronic inflammation response that may be severed
and painful, depending on the site as well as the quantity and quality of adjuvant
190
injected (Broderson, 1989). The inflammatory response may result in the
formation of chronic granulomas, sterile abscesses, and/or ulcerating tissue
necrosis (Stills and Bailey, 1991). FCA is also a potential hazard for laboratory
personnel as accidental self inoculation can result in tuberculin sensitization
followed by chronic local inflammation, which responds poorly to antibiotic
treatment. Accidental splashing of FCA in the eye can result in severe ocular
irritation and even blindness (Kleinman et al., 1993). Freund‟s Incomplete
Adjuvent (FIA) is similar to FCA but does not contain Mycobacterium. FIA is
frequently used to boost immune system after the use of live vaccines.
The objectives of the study were:
1. to determine the safety and immunogenicity of the attenuated and inactivated
Malaysian isolate of vvIBDV (UPM0081) in specific-pathogen-free (SPF)
chickens.
2. to determine the efficacy of the inactivated vvIBDV in SPF chickens.
191
6.2 Materials and Methods 6.2.1 Virus and Cells
Passage 15 (UPM0081T15) and passage 20 (UPM0081T20) of vvIBDV were
used as previously described (Section 3.2.6). Briefly Vero cell were grow in
polystyrene 150 cm2 (Nunc Easyflasks, Denmark) containing 30 mL RPMI
supplemented with 10% FBS. A confluent cell monolayer was infected with
vvIBDV (UPM0081) and the virus was allowed to adsorb for 60 minutes at 37oC
in 5% CO2 with intermittent rotation to allow the virus to adsorb on the surface of
Vero cells. After viral adsorption, the inoculums was removed and replaced with
30 mL RPMI containing 1% FBS and retained in an incubator at 37oC in 5%
CO2, for 4 days.
6.2.2 Harvesting of Virus
The Vero cells infected with the IBDV were harvested when cytopathic effect
(CPE) reached 90%. The IBDV-infected cells were centrifuged at 3000 rpm for
20 minutes at 4oC the resultant supernatant fluids were harvested, filtered
through a 0.45 µm filter (Sartorius, Germany), aliquot and stored at -80oC.
192
6.2.3 Tissue Culture Infective Dose 50 (TCID50
)
As previously described (Section 3.2.10)
6.2.4 Inactivation of vv IBDV
The inactivation of the two Vero cell adapted and attenuated vvIBDV at passage
15 (P15) and 20 (P20) were carried out using Binary ethylenimine (BEI) and
(C1), except in groups ECAP15 and ECAP20 (732.5 ± 53.8)g and (737.0 ±
65.0g), which were significantly lower (p<0.05) when compared with the groups
BEIP15, BEIP20 and C1, respectively (Table 6.5).
201
Table 6.5: Body weight of chickens in the inactivated attenuated vvIBDV inoculated and control group at 2 weeks post challenged
Body weight (Mean ± SDg)
Group / passage
BEI / P15
BEI / P20 ECA / P15 ECA / P20 C1 / CN C2 / CP
980.0 ± 86.4
a
(n=4)
967.5 ± 91.0
a
(n=4)
732.5 ± 53.8
b
(n=4)
737.5 ± 65.0
b
(n=4)
995.0 ± 42.0
a
(n=4)
NA
NA: Not available due to the death of chickens abValues with different superscripts within rows differ significantly at p<0.05 due to treatment effects n-number of chickens sacrificed
6.3.4 Bursa Weight
There was no significant difference (p>0.05) in the bursa weight of chickens
among groups C1 (3.07 ± 0.31g), BEIP15 (3.01 ± 0.22g), BEIP20 (2.90 ± 0.26g)
and ECAP15 (2.40 ± 0.35g), except for chickens in group ECAP20 (2.3 ± 0.42g)
which were significant lower (p<0.05) than the groups C1 and BEIP15 (Table
6.6).
202
Table 6.6: Bursa weight of chickens in the inactivated attenuated vvIBDV inoculated and control group at 2 weeks post challenge
Bursa weight (Mean ± SDg)
Group / passage
BEI / P15
BEI / P20 ECA / P15 ECA / P20 C1 / CN C2 / CP
3.01 ± 0.22
a
(n=4)
2.90 ± 0.26
ab
(n=4)
2.40 ± 0.35
ab
(n=4)
2.30 ± 0.42
b
(n=4)
3.07 ± 0.31
a
(n=4)
NA
NA: Not available due to the death of chickens abValues with different superscripts within rows differ significantly at p<0.05 due to treatment effects n-number of chickens sacrificed
6.3.5 Bursa to Body Weight Ratio (1x10-3)
There was no significant difference (p>0.05) between the groups BEIP15 (3.06
± 0.04) and BEIP20 (3.08 ± 0.46), when compared to the group C1 (3.09 ±
0.32). However the bursa to body weight ratio in the groups ECAP15 (2.07 ±
0.43) and ECAP20 (2.05 ± 0.17) were significantly (p<0.05) lower than groups
BEIP15, BEIP20 and C1 (Table 6.7).
203
Table 6.7: Bursa to body weight ratio of chickens in the inactivated attenuated vvIBDV inoculated and control group at 2 weeks post challenged
Bursa to body weight ratio (x 10
3)(Mean ± SDg)
Group / passage
BEI / P15
BEI / P20 ECA / P15 ECA / P20 C1 / CN C2 / CP
3.06 ± 0.04 a
(n=4)
3.08 ± 0.46 a
(n=4)
2.07 ± 0.43 b
(n=4)
2.05 ± 0.17 b
(n=4)
3.09 ± 0.32 a
(n=4)
NA
NA: Not available due to the death of chickens abValues with different superscripts within rows differ significantly at p<0.05 due to treatment effects n-number of chickens sacrificed
6.3.6 Gross Lesions
Group: C1 (Control Negative)
The chickens did not shown any gross lesions of IBD after two weeks post
challenged (Figure 6.1a)
Group: C2 (Control Positive)
Severe haemorrhagic, oedematous and necrosis of bursa of Fabricius were
observed in the dead chickens at days 2 and 4 post challenged (Figure 6.1a).
204
Group: BEI P15
The chickens did not shown any gross lesions of IBD after two weeks post
challenged (Figure 6.1b).
Group: BEI P20
The chickens did not shown any gross lesions of IBD after two weeks post
challenged (Figure 6.1b)
Group ECA P15
All chickens showed smaller bursal size after two weeks post challenged as
compared to the size of the BF pre vaccination.
Group ECA P20
All chickens showed smaller bursal size after two weeks post challenged as
compared to the size of the BF pre vaccination.
205
Figure 6.1a: bursa of Fabricius (BF) in SPF chickens. (A) Group C1: normal (B) Group C2: severe haemorrhages and oedema
206
Figure 6.1b: bursa of Fabricius (BF) in SPF chickens. (C) Group BEIP15: normal (D) Group BEIP20: normal
207
6.3.7 Histological Lesions Score
Group: C1
All chickens in this group showed normal to mild bursitis with lesion score of
0.45 ± 0.17 (Figure 6.2, Table 6.8).
Group: C2
Histopathological changes at day 2 post-challenged was severe lymphoid
depletion, lymphocytolysis, degeneration and formation of follicular cyst with
fibrin exudates in the medulla and mononuclear inflammatory cells, oedema in
the interstitial connective tissue with lesion score of 4.72 ± 0.32. (Figure 6.2,
Table 6.8).
Group: BEIP15
The bursa of Fabricius was characterized by normal to mild tissue reaction with
lesion score of 0.62 ± 0.09 (Figure 6.3, Table 6.8).
208
Group: BEIP20
All chickens in this group showed normal to mild bursitis with lesion score of
0.50 ± 0.81 (Figure 6.3, Table 6.8).
Group: ECAP15
All the chickens in this group showed mild to moderate degeneration and
necrosis of lymphoid cells in the some of lymphoid follicles especially in the
medulla with lesion score of 1.45 ± 0.12 (Figure 6.4, Table 6.8).
Group: ECAP20
All chickens in this group showed mild to moderate bursitis with lesion score of
1.35 ± 0.25 (Figures 6.4, Table 6.8)
209
Figure 6.2: bursa of Fabricius. (A) Group C1 (Control negative): Normal lymphoid follicles, lesion score of 0. (B) Group C2 (Control positive): lesion score of 5, day 2 pi, sever follicular necrosis with cyst formation in the follicles ( ) and infiltration of inflammatory cells and oedema fluid at interstitial space ( ). HE, 20x. Bar = 100µm
210
Figure 6.3: bursa of Fabricius at day 14 pc. (A) Group BEIP15: Mild degeneration and necrosis of the follicles ( ), lesion score of 1. (B) Group BEIP20: Mild degeneration and necrosis of the follicles ( ), lesion score of 1. HE, 10x. Bar = 200µm
211
Figure 6.4: bursa of Fabricius at day 14 pc. (A) Group ECAP15: Mild lymphoid cells necrosis ( ), lesion score of 1. (B) Group ECAP20: Mild cells lymphoid necrosis ( ), lesion score of 1. HE, 10x. Bar = 200µm
212
Table 6.8: Lesion score of chickens in the inactivated attenuated vvIBDV inoculated and control group at 2 weeks post challenge
Bursa lesion scoring (Mean ± SD)
Group / passage
B1 / P15
B2 / P20 E1 / P15 E2 / P20 C1 / CN C2 / CP
0.62 ± 0.09a
(n=4)
0.50 ± 0.81a
(n=4)
1.45 ± 0.12b
(n=4)
1.35 ± 0.25b
(n=4)
0.45 ± 0.17a
(n=4)
4.72 ± 0.32c
(n=4)
abcValues with different superscripts within rows differ significantly at p<0.05due to treatment effects n number of chickens sacrificed
6.3.8 Antibody Titer (ELISA)
There was no significant difference (p>0.05) between the mean antibody titer at
two weeks post inactivated attenuated vvIBDV inoculation in the two types of
(BEIP20), 12131 ± 1932 (ECAP15), except for chickens in group ECAP20 (6615
± 3886) which are significant lowered (p<0.05) when compared to BEIP15,
BEIP20 and ECAP15 groups. At 2 weeks post challenged there was no
significant difference (p>0.05) in mean titers in the three groups: 10262 ± 6405
(BEIP15), 8847 ± 4992 (BEIP20) and 6756 ± 1214 (ECAP15), except for
vaccinated group ECAP20 (3397 ± 1698) which had a significantly lowered titers
(p<0.05) when compared to the groups BEIP15 and BEIP20 (Table 6.9).
213
6.3.9 Detection of the Virus or Viral RNA using RT-PCR
No viral RNA was detected in the bursa homogenates from chickens inoculated
with inactivated vvIBDV in groups BEIP15, BEIP20, ECAP15 and ECAP20 at
day 14 pc (Figure 6.5).
Table 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.
Antibody titer ( Mean ± SD)
Time (Weeks pi & pc)
Group / passage
2 weeks pi
2weeks pc
BEIP15
12303 ± 4515a
p
10262 ± 6405a
p
BEIP20
13557 ± 2479a p
8847 ± 4992a
q
ECAP15
12131 ± 1932a p
6756 ± 1214ab
q
ECAP20
6615 ± 3886b p
3397 ± 1698b
q
CI
ND
ND
C2
ND
NA
ab Values with different subscripts within column differ significantly at p<0.05 pq Values with different supscripts within rows differ significantly at p<0.05
NA: Not available due to the death of chickens ND: Not detected
214
Figure 6.5: Hypervariable region (643pb) amplification of IBDV VP2 genes at day 14 pc. (1) BEIP15 negative (2) BEIP20 negative (3) ECAP15 negative (4) ECAP20 negative (5) C2 positive. (M) 100 bp DNA marker (Promega, USA).
6.4 Discussion
The study showed that UPM0081T15 and UPM0081T20 attenuated vvIBDV
were successfully inactivated by Binary ethylenimine (BEI) and Electrolysed
water-Catholyte-Anolyte (ECA) 24 hours after the treatment. The inactivated
vvIBDV conjugated with incomplete Freund‟s adjuvant could provide full
protection (100%) against vvIBDV (UPM0081) challenged. Inoculation of
inactivated IBDV could give complete protection with no obvious IBD clinical
signs, as it was reported previously (Maas et al., 2001).
215
The mean body weight of treatment groups (C1, BEIP15 and BEIP20) was not
significantly (p>0.05) different throughout the experimental study while that of
groups ECAP15 and ECAP20 was significant lower (p<0.05) when compared to
that of groups C1, BEIP15 and BEIP20 at week 2 post vaccination. The bursal
weight of the chicken followed similar pattern like the body weight except that of
group ECAP15 was significantly different from ECAP20 group, the exactly
reason to this is unknown.
As regards the bursa to body weight ratio, there was no significant difference
(p>0.05) in BEI treatment groups (B1, B2) and the control negative group (C1).
These results corroborated those reported by Hassan and Saif (1996) where
they also reported no significant difference in the bursa to body weight ratio of
chicken administered with BGM-70 attenuated and inactivated IN and STC
strains of IBDV and the control group. The chickens in groups (ECAP15 and
ECAP20) on the other hand had a significantly (p<0.05) lower bursa body
weight ratio when compared with (C1). This clearly showed the superiority of
BEI in effectively inactivating the vvIBDV and also in protecting the chicken
against bursal damage.
Histopathological assessment also revealed similar pattern as the bursa lesion
score was normal to mild in treatment groups C1, BEIP15 and BEIP20 groups
while that of ECAP15 and ECAP20 had mild to moderate.
216
On the basis of humoral immune response, the results showed that the
inactivated passage 15 UPM0081T15 and passage 20 UPM0081T20 of vvIBDV
used in all the treatment groups were immunogenic with increased in antibody
titers in all inoculated groups 2 weeks pi. It is evident that groups BEIP15,
BEIP20 and ECAP15 had no significant titres (p>0.05) while group ECAP20 was
significantly (p<0.05) lower 2 weeks pi (Jackwood, 2005; Habib et al., 2006)
while that with ECAP15 and ECAP20 were observed probably for the first time
and this shows that ECA has a potential as a inactivating and immunogenic
agent especially with IBD viruses. The possible mechanism of inactivation of
ECA as regards IBDV needed to be investigated while that of BEI and its use in
the inactivation of viruses for vaccine development abound in literature
(Buonavoglia et al., 1988; Bahnemann, 1990).
Post challenged titres measured by ELISA followed the same pattern with the
post inoculation titres except that the titres were lowered and this are quite
understandable as the field virus has been reported to mop up antibodies.
Again, it could be deduced from the post challenged titers that the BEI-
inactivated groups gave higher antibody titers than ECA inactivated groups
although ECAP15 group had better response than ECAP20. The probable
reason for the higher titers in ECAP15 group may not be acertain but it could be
related with the level of passage as ECAP15 had 15 passages when compared
to 20 passages before inactivation in ECAP20. This could best be said to be a
function of the available viral antigen in each group.
217
The result of this study also showed that a single dose of the inactivated
passage 15 UPM0081T15 and passage 20 UPM0081T20 of IBDV in both
groups (BEI and ECA) could give 100% protection against vvIBDV challenged,
which is in contrast with the report of 100 % protection obtained with the use of
two doses of killed IBD vaccines at a week interval in 3 weeks SPF chickens
(Hsieh et al., 2007).
Since the current facts showed that the humoral immune response plays the
principal role in defense against vvIBDV (Lukert and Saif, 1997), there may be a
need to study the cell mediated immune response and the effect of vaccine
inactivated by these substances (BEI and ECA) on the cellular mechanism since
T cells are also important in the protection against virulent IBDV (Rautenschlein
and Sharma, 2002).
In order to have a better understanding of the chemical inactivation of the IBDV,
the efficacy of these chemicals was studied by using two passages (P15 and
P20) of attenuated Vero adapted IBDV, the probable effect of the passages was
evident in ECA groups with P15 giving promising results in terms of immune
response pre- and post challenged. The results obtained with ECA group do not
totally showed that the ECA inactivated passage 15 UPM0081T15 and passage
20 UPM0081T20 of IBDV could not be useful as a vaccine, as it could be given
twice to enhance the immune titers in endemic IBD areas or in chicks with high
maternal antibodies. This sugestion needed to be verified before adoption.
218
The scope of this study did not cover the possible effect of the adjuvant
employed but it was assumed that the adjuvant also enhanced the humoral
response that was recorded and the use of tween-80 in the combination with
Freund‟s incomplete adjuvant (FIA) made the adjuvant less viscous and this
may have reduced the side effects associated with the adjuvant and the
possible alteration of the antigenicity of IBDV (Stone et al.,1978).
The chemical interactions between the BEI, ECA and incomplete Freund‟s
adjuvant needed to be investigated as this may give a clue to the difference in
the humoral and histopathological scores observed in the two treatment groups.
In this experiment, the possibility of replication of the inactivated virus was also
investigated by using RT-PCR. No viral genetic material was detected in the
bursa of Fabricius of the inoculated birds at two weeks post challenged,
indicating that the IBDV was inactivated and there was no replication of viral
RNA or inactivated virus in bursa of Fabricius. This further showed that the
reversal to virulence as in the case of attenuated live vaccine is not possible in
this case.
It was concluded that chickens inoculated once with inactivated (BEI or ECA)
attenuated vvIBDV UPM0081 in Vero cell at passages 15 and 20 could
adequately protect against vvIBDV challenged. It also revealed that antibody
specific to IBD detected by ELISA in BEI treated virus was higher than ECA
219
treated virus. Although both inoculated viruses (BEI or ECA) were able to
protect chicken against vvIBDV challanged, ECA treated virus could not provide
protection against bursal damage in challenged chickens.
CHAPTER 7
GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATIONS FOR FUTURE RESEARCH
7.1 General Discussion
Since the control of IBD is based mainly on vaccination, continuous efforts were
directed towards developing effective and economic vaccines against IBDV. The
work presented in this dissertation was initiated to investigate the adaptation
and attenuation local vvIBDV in tissue culture, and possible vaccine production
with thr improving the efficacy of local attenuated and inactivated vaccines
which are employed in the effective control of IBD in Malaysia, and world wide.
In this experiment, a total of three Malaysia vvIBDV field strains designated as
UPM04190, UPM94273 and UPM0081 with an accession number of AY791998,
AF527039 and EF208038, respectively were used in first set of investigations.
The inoculation of theses isolates into the embryonated chicken eggs was
observed to result in embryonic death and the lesions observed was similar to
those observed in other vvIBDV strains (Hair-Bejo et al.,1995a; Yamaguchi et
al., 1996a).
In this study, the adaptation and attenuation of a local strain, in two continuous
cell lines namely Vero cells and DF-1 cells was demonstrated. UPM0081 strain
was successfully adapted to grow in Vero cells after four passages and DF-1
221
cells after three passages, while the other isolates failed to adapt on Vero cells
and DF-1 cells after six passages. These findings further reinforced the view
that several wild strains are difficult to replicate and grow in vitro, and some, if
they eventually grow, often require several weeks and blind passages
(McFerran et al., 1980; Hassan et al., 1996). In this investigation, there was no
evidence of growth by CPEs in the first, second and third passages while at the
fourth, clear and constant CPEs of IBDV were found in Vero cells monolayer.
The CPEs involved aggregation of Vero cells, formation of round, refractile cells
and finally granulation and detach from the surface. In DF-1 cells, the first and
second passages of the UPM0081 strain did not produce any CPEs, while the
third gave clear and consistent CPEs of cell rounding, granulation and
detachment from the substrate. The growth of the virus in infected cell cultures
(Vero cells and DF-1 cells) was further confirmed with stained HRP-conjugated
antibody in IIPS. The infected cell cultures appeared as specific intracytoplasmic
brown colour when examine under light microscope.
When the virus titers obtained were compared in Vero and DF-1 cells, the result
showed that Vero cells yielded higher titers than DF-1 cells, and for this reason,
it was concluded that the Vero adapted attenuated virus should be further
studied for possible adoption as a candidate for an attenuated and inactivated
local vaccine development.
222
During the last few years, many molecular studies have detected genomic
changes that account for attenuation in tissue culture of IBDV. The comparison
of the local attenuated (at) IBDV strains obtained in this study, with other IBDV
strains was done by molecular methods since the molecular characterization of
IBDV isolates from different geographical regions and identification of
differences among them had helped in developing a correct and effective
vaccine (Pitcovski et al., 1998).
In this study, Vero cells and DF-1 cells adapted vvIBDV local isolates were
used. The viral RNA extraction was done using Trizol reagent. RT-PCR was
performed for all the RNA samples using P1 and P2 pair of primers specific to
HPVR of VP2 gene to confirm the presence and genetic mutation of IBDV in cell
line. The expected 643-bp was obtained for each passage and cloned in the
PCR 2.1- TOPO TA Cloning.
In this study, the first changes of amino acid residues, occurred at positions 222
(A to P) in passage 8 (UPM0081T8) and further mutation of amino acid residue
was observed at positions 242 (I to V), 249 (Q to R), 253 (Q to H), 256 (I to V),
279 (D to N) , 284 (A to T) and 294 (I to L), in passages 10, 15 and 20
(UPM0081T10, UPM0081T15, and UPM0081T20). These mutations were
similar to those reported for IBDV attenuated strains (Yamaguchi et al., 1996b).
223
It is interesting to note that all the three amino acid substitution at position 253
(Q to H), 279 (D to N) and 284 (A to T) are within the areas of the protein
predicted to be associated with antigenic variation, as well as back bone
flexibility. It is possible that these changes especially at residue (284) could
result in significant functional rearrangement of the virus capsid that is
advantageous for virus replication and attenuation in Vero cells.
In phylogenetic tree based on segment A, IBDV strains are usually distinctly
grouped into separate branches according to their serotype and virulence. In
this study, UPM0081T10, UPM0081T15 and UPM0081T20 IBDV passages
were closely related to atIBDV strains and they formed a subbranch with classic
attenuated (D78) and atIBDV AmerVH9907. Generally, many attenuated
vaccines had been successfully used to develop attenuated live vaccine as
evidenced by a reduction in the ability of the virus to induce bursal lesions
(Yamaguchi et al., 1996a).
Based on the pathogenicity and immunogenicity attenuated vvIBDV in SPF
chicken in this study, the first preliminary investigation revealed that P10
(UPM0081T10) was still pathogenic, as it caused mortality (25%) in SPF
chicken and bursa lesion score of 4 (Raue et al., 2004), while the P15
(UPM0081T15) virus was observed to have lost their pathogenicity after
passage in Vero cells as evident by no mortality, bursal lesion score of 0. These
224
findings are similar to that reported for other attenuated IBDV strains (Guittet et
al., 1992).
It should be noted that passaging in vitro of an IBDV isolate does not only
reduces strain virulence but also changes its antigenicity. Therefore, most of the
live attenuated vaccines available today have a different passage history and
they show a different level of virulence and antigenicity. Thus, it is believed that
an antigenically superior vaccine can best be obtained from vvIBDV strains
provided the attenuation is adequately achieved (Melchior and Melson 1989) as
observed in this study.
It was also noted that the inoculated passage 10 (UPM0081T10) had higher IBD
antibody titer than the passage 15 (UPM0081T15) at 2 weeks post inoculation.
This is may be due to the fact that the virus in passage 10 is still more
pathogenic and subsequently immunogenic than passage 15. The study
showed that passages 15 (UPM0081T15) and 20 (UPM0081T20) were safe and
could confer 100% protection against vvIBDV field challenged with no clinical
signs of IBD.
The two Vero cells adapted attenuated UPM0081T15 and UPM0081T20
vvIBDV also provided full protection from bursal lesion against the vvIBDV
challenged. These findings support the previous report by Ismail and Saif
225
(1991), in which two virulent strains were adapted and passaged in BGM cell
line (BGM-70).
Both viruses (UPM0081T15 and UPM0081T20) also produced titers detectable
after 5 days post inoculation which increased rapidly at day 7pi, and the
maximum antibody titers were found at day 10 pi in both groups, these results
suggest that the acquired serum antibody in both groups, was sufficient to fullly
protect the chickens (100%) against vvIBDV challenge.
The study went further to showed that viral antigen was detectable in the bursa
homogenates of SPF chickens up to 21 days using RT-PCR after inoculation of
two passages (UPM0081T15 and UPM0081T20) Vero adapted attenuated
isolates. This observation was similar to the findings of Abdel-Alim, (2001) who
also found detectable RNA in bursa haemogenates up to 21 days.
The pathogenecity and immunogenicity of two types of inactivated local vvIBDV
(UPM0081T15 and UPM0081T20) isolates were also evaluated in SPF
chickens. The two vaccine candidates were successfully inactivated by Binary
ethylenimine (BEI) and Electrolysed water-Catholyte-Anolyte (ECA), and they
were found to fully protected (100%) the SPF chickens against challenge
vvIBDV strain (UPM0081) challenged, throughout the period of the experimental
study. The BEI treated inactivated Vero adapted isolates fully protected the
birds against bursal damage when challenged with vvIBDV strain (UPM0081)
226
while the chicken vaccinated with ECA treated inactivated Vero adapted isolates
had mild to moderate bursal lesions. The findings also indicated that the killed
vaccines used (BEI and ECA) were capable of inducing satisfactory serologic
response as evident by the increased in antibody titers observed in all the
vaccinated birds, but BEI-inactivated vaccines gave higher antibody titers than
ECA inactivated vaccines.
In the present study, the possible influence of Freund‟s incomplete adjuvant
(FIA) water in oil (W/O) emulsions on the inactivated (BEI and ECA) IBDV
antigen was also explored. This study showed that the adjuvant combined with
the inactivating agents do gave satisfactory and protective humoral response in
BEI inactivated IBDV while the possible reason for the low titers in ECA
inactivated IBDV may not be unconnected with the combination.
7.2 Conclusion
This study has shown that the Malaysian vvIBDV strains UPM0081 strain was
successfully propagated and attenuated in Vero cells and DF-1 cell lines with
higher titers recorded in Vero cells, hence Vero cell line could be used as a
model to study the growth kinetic of the IBDV isolate.
Based on molecular techniques, the IBDV passages, UPM0081T10,
UPM0081T15 and UPM0081T20 were characterised as atIBDV. The sequence
227
analysis showed that most mutation among these isolates focused on the HPVR
of VP2 gene especially between 253 (Q to H), 256 (I to V), 279 (D to N) and 284
(A to T) and these mutations could be said to play an important role in the
adaptation Malaysian vvIBDV isolate (UPM0081) to Vero and DF-1 cell lines.
The phylogenetic analysis also showed that these cell line adapted isolates
were evolutionary closed to atIBDV strains.
It is also clear from the present findings that the inoculation of Malaysian
vvIBDV adapted and attenuated in Vero cell line passages 15 and 20 conferred
full protection to the immunized SPF chickens.
The water in oil emulsion Freund‟s Incomplete Adjuvant inactivated (BEI and
ECA) vaccine candidates also protected the chickens fully from vvIBDV
challenged. However, the antibody response to IBD detected by ELISA in group
with BEI was higher than the ECA group. Although the two groups were able to
protect SPFchickens from mortality and clinical signs of the disease, the group
with BEI fully protected against bursal lesion while the ECA group could not.
From this study, these possible vaccine candidates may be recommended for a
field trial to further evaluate the safety and efficacy of these possible vaccines
on a larger scale.
228
7.3 Recommendation for Further Research
1- To demonstrate the role of receptors in the adaptation and attenuation of
vvIBDV in Vero cells using monoclonal antibodies.
2- To investigate the use of chicken stem cell line in the adaptation and
attenuation of vvIBDV.
3- To further the investigation into the influence of serum or protein and insulin-
like growth factor I (IGF-I) on the ability of vvIBDV strains to infect and replicate
in cell culture.
4- To improve the quality of attenuated and inactivated vaccine candidates by
using different mechanisms of increasing vaccine virus concentration so as to
provide adequate protection by a single dose.
5- To study the cell mediated immune (CMI) response of chicken using the
attenuated and inactivated vaccines candidates.
6- To adopt the use of real time RT-PCR in the evaluation of vaccination
program in order to differentiate between wild IBDV and the spread of vaccine
229
virus in poultry flocks. It will also help to obtain the virus titers than the
qualitative result from RT-PCR.
7- To conduct field trail on the safety and efficacy of the live and inactivated
atIBDV seed virus for vaccine development.
230
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APPENDIX A
Buffer and Media
1. PBS (pH 7.4)
NACl 8.0 g KCl 0.2 g Na2HPO4 1.15 g KH2PO4 0.2 g The above chemicals were dissolved in 800 mL distilled water. The final volume of the buffer was then brought up to 1000 mL after adjusted the pH to 7.4. The buffer was autoclaved at 121 ºC for 15 minutes at 151 bs pressure. 3. Antibiotic-Antimycotic (100X) Liquid Penicillin G (sodium salt) 10,000 units/mL Streptomycin Sulfate 10,000 ug/mL Amphotericin B 25 ug/mL 0.85% Saline 100 mL 4. Growth Medium RPMI 1640 (Gibco) 10 g FBS 100 mL NaHCO3 0.58 g Distilled Water 1 litre Add 1 mL of antibiotic-antimycotic. Sterile by filtration using 0.22 µm pore size filter and store at 4 ºC. 5. Maintenance Medium RPMI 1640 (GIBCO) 10 g TPB 10 mL FBS 10 mL NaHCO3 3.7 g Distilled Water 1 litre
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Add 1 mL of antibiotic-antimycotic. Sterile by filtration using 0.22 µm pore size filter and store at 4 ºC. Note:
For the growth and maintenance medium, 1 mL of each antibiotic-antimycotic must be added. Then warm it to 37ºC before use.
FBS must be inactivated at 56 ºC for 1 hour to inactivate the non-specific inhibitor before it was used in maintenance media in case of cell culture inoculation with particular virus.
6. Antibiotic-Trypsin-Versine (ATV)
Trypsin 0.5 g Versine (EDTA) 0.2 g
NaCl 8 g KCl 0.4 g Dextrose 1 g NaHCO3 0.58 g Penicillin 2 x 100 units Streptomycin 100 mg Phenol Red 0.02 g The above are made up to 1000 mL with distilled water and the resulting solution is sterilized by 0.22 µm pore size filter and store at 4 ºC.
7. TAE Buffer (10x stock)
40 mM Tris-base 48.4 g 20 mM Glacial acetic-acid 11.4 mL 0.5 M EDTA 20 mL In 1000 mL of distilled water.
TCID50 = Dilution where CPE > 50% + 0.45 = 7 + 0.45 = 7.45 / 100 µl One unit 107.4 TCID50/0.1 mL
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APPENDIX C
Chemicals for Histopathology
1. 10% Buffered Formalin (Luna, 1968) 37-40% formalin 100 mL Distilled water 900 mL Sodium phosphate monobasic 4 mL Sodium phosphate dibasic 6.5 mL (anhydrous) 2. Haematoxylin and Eosin Staining (H & E) i. Harris Hematoxylin
Distilled water 200 mL Hematoxylin crystal 1 g Ammonium or potassium sulfate 20 g Mercuric oxide (red) 0.5 g The hematoxylin is dissolved in the alcohol, whilst the ammonium is dissolved in water with the aid of heat. Both the hematoxylin and the alum solution are combined and boiled. The addition of mercuric oxide make the solution takes a dark purple colour. Filter the solution before using or storing.
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APPENDIX D
LESION SCORING Table G: Lesion scoring for bursa of Fabricius
Lesion Scoring Description
0 (Normal) Normal or undetectable lesion
1 (Mild) Mild degeneration and necrosis especially at the medullary region of lymphoid.
2 (Mild to Moderate) Mild to moderate degeneration and necrosis of lymphoid cells in some lymphoid follicles especially in the medulla. Interstitial connective tissue becomes oedematous and filled with inflammatory cells.
3 (Moderate) Moderate necrotized follicles involving both the cortex and medulla. Pyknotic nuclei were scattered in follicles. The interstitial space was obvious and present of heterophils, macrophages and a few erythrocytes and fibroblasts. Epithelial lining was thickened and vacuolated in some areas.
4 (Moderate to severe) Moderate to severe depletion of lymphoid cells. Lymphoid cells aggregation was found in the cortex of some follicles. Necrotic cells and cysts were present in some follicles especially in the medulla. The interstitial space was infiltrated and pack with fibrinous connective tissues. The intra and extra follicular areas might be hyperaemic and haemorrhagic. Epithellium was thickened, corrugated and vacuolated in some areas.
5 (Severe) Severe follicular necrosis and degeneration involving both cortex and medulla. Follicular cysts with fibrinous exudates and cell debris were frequently observed. The interstitial connective tissue was obvious, oedematous and infiltrated with mild to moderate inflammatory cells. The epithelial lining of the bursa was thickened and vacuolated
Or
Moderate to severe atrophy of the bursa follicles with cellular degeneration and necrosis. Cysts might be present in some follicles. The interstitial connective tissue as obvious and infiltrated with fibroblast and inflammatory cells
271
BIODATA OF AUTHOR
The author, Majed Hameed Mohammed, was born in 21st of April 1965, in Baghdad,
Iraq. He was the eldest son in his family of five. He is married and has got 2 children.
He did his primary education in Baghdad from 1971 to 1977. The author continued his
secondary school education at AL-Mansoor secondary school and he finished his final
public examination successfully on 1983 with an average grade 82%.
On October, 1983, the author furthered his study in Bachelor Science of Veterinary
Medicine in Faculty of Veterinary Medicine, Baghdad University. After finishing the
degree he graduated on 1st of July 1989 with final average of (77) and he ranked (3) in a
class numeration of 185 students.
Immediately after completion the bachelor degree, he worked as a researcher assistant
at Pathology and Poultry Department Laboratory, Faculty of Veterinary Medicine,
Baghdad University. He spent 6 months during his service as a research assistant and
later joined to the ranks of military army to perform his compulsory service in his
country. Then after one year, he enrolled as a full-time candidate pursuing his study in
Master Science program in field of poultry diseases at the same Faculty of Veterinary
Medicine on 1991.
On 1993, the author was awarded a Master‟s degree in poultry diseases with standard
very good for submission of thesis titled “The effect of Gumboro disease on the
incidence of airsacculitis”. After completion all the requirements for M.Sc. degree, the
272
author has got a job as a lecturer on 1993 in the same Department of Poultry Disease,
Veterinary Medicine.
The author engaged to work as a part time in the field of Veterinary supervision of
poultry farms and as a veterinarian at a center of agricultural research and animal
production.
On 2006, the author got an offer letter to persue his Ph.D. programme at Faculty of
Veterinary Medicine, Universiti Putra Malaysia in the field of Pathology. Upon
completing his Ph.D. the author plans to embark on an academic career and further
play an active role ensha‟allah through post doctorate research programme.
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LIST OF PUBLICATIONS
PATENT Novel attenuated and inactivated tissue culture adapted infectious bursal disease vaccine against very verulent infectious bursal disease virus Infection, (submitted to innovation and commercialization center UPM for patent submission) JOURNALS 1. Mohammed, M.H., Hair-Bejo, M., Omar, A.R. and Aini, I. (2010). Adaptation, attenuation and molecular characterisation of very virulent infectious bursal disease virus of Malaysia isolate in Vero cells. Acta Vet. Hungarica (to be submitted). 2. Mohammed, M.H., Hair-Bejo, M., Omar, A.R. and Aini, I. (2010). Pathogenicity and Immunogenicity of very virulent infectious bursal disease virus adapted in Vero cells in Malaysia. Acta Vet. Scandinavica (to be submitted). IN PROCEEDING 1. Mohammed, M.H., Hair-Bejo, M., Omar, A.R. and Aini, I. (2008). Adaptation and propagation of very virulent infectious bursal disease virus of Malaysian isolate in mammalian cell line. In: Proceeding of 29th Malaysian Society of Animal Production (MSAP) Annual Conference, 25-27 May 2008, Bayview Beach Resort, Penang. P.36 2. Mohammed, M.H., Hair-Bejo, M., Omar, A.R. and Aini, I. (2008). Adaptation and propadation of very virulent infectious bursal disease virus of Malaysian isolate in DF-1 cell line. In Proceeding of 20th Veterinary Association Malaysia (VAM) congress, 15th -17thAugust 2008, Equatorial Hotel Bangi-Putrajaya. P. 51 3. Mohammed, M.H., Hair-Bejo, M., Omar, A.R. and Aini, I. (2009). Efficacy of inactivated very virulent infectios bursal disease virus isolate of Malaysia in specific pathogenic free chickens.In Proceeding of 21th Veterinary Association Malaysia (VAM) congress, 7-9th August 2009, the Legend, Water Chalets Port Dickson, Negeri Sembilan. P.20 GENBANK
1. Mohammed, M.H., Hair-Bejo, M., Omar, A.R. and Aini, I (2009). Accession number FJ824699 (IBD UPM0081T10 VP2 gene). In: GenBank – http://www.ncbi.nlm.nih.gov/ 2. Mohammed, M.H., Hair-Bejo, M., Omar, A.R. and Aini, I (2009). Accession number FJ898322 (IBD UPM0081T15 VP2 gene). In: GenBank – http://www.ncbi.nlm.nih.gov/