Download characterization of infectious bursal disease viruses

CHARACTERIZATION OF INFECTIOUS BURSAL DISEASE VIRUSES
ISOLATED FROM COMMERCIAL CHICKENS
by
Jacqueline Marie Harris
A thesis submitted to the Faculty of the University of Delaware in partial
fulfillment of the requirements for the Degree of Master of Science in Animal and
Food Sciences
Spring 2010
Copyright 2010 Jacqueline Marie Harris
All Rights Reserved
i
CHARACTERIZATION OF INFECTIOUS BURSAL DISEASE VIRUSES
ISOLATED FROM COMMERCIAL CHICKENS
by
Jacqueline Marie Harris
Approved:_______________________________________________________________
Jack Gelb, Jr., Ph.D.
Professor in charge of thesis on behalf of the Advisory Committee
Approved:_______________________________________________________________
Jack Gelb, Jr., Ph.D.
Chairperson of the Department of Animal and Food Sciences
Approved:_______________________________________________________________
Robin W. Morgan, Ph.D.
Dean of the College of Agriculture and Natural Resources
Approved:_______________________________________________________________
Debra Hess Norris, M.S.
Vice Provost for Graduate and Professional Education
ii
ACKNOWLEDGEMENTS
First and foremost, I would like to thank my advisor, Dr. Jack Gelb, Jr., for his
continual guidance, encouragement, and academic support throughout my graduate
education. I would like to give special thanks to the members of my graduate committee
for their excellent advice and contributions during my research project, especially Dr.
Daral Jackwood for his VP2 sequence analysis and Dr. Egbert Mundt for his monoclonal
antibody testing. I am deeply indepted to Brian Ladman whose valuable knowledge and
assistance enriched my growth as a student and researcher. I am truly grateful for all of
his patience and support throughout my graduate career.
In addition, I would like to thank Ruud Hein’s Intervet/Schering Plough lab
group, Bruce Kingham, Joanne Kramer, Dr. Conrad Pope, Marcy Troeber, Dr. Daniel
Bautista’s UD Lasher Lab group, Lauren Preskenis, Cindy Boettger, and Miguel Ruano
for their time and contributions to the success and completion of this research project.
I would like to express my sincerest gratitude to my family and friends for
their unconditional love and encouragement. Their unwavering support and confidence in
my abilities is what has shaped me into the person I am today. Most of all, I would like to
thank my mom, Donna Harris, whose faith, wisdom, support, and understanding has
given me the courage to face any challenge and the strength to succeed. I could not have
done this without you.
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TABLE OF CONTENTS
LIST OF TABLES………………………………………………………………………vi
LIST OF FIGURES……………………………………………………………………...ix
ABSTRACT.......................................................................................................................x
Chapter
1
INTRODUCTION………………………………………………………...…...1
2
REVIEW OF THE LITERATURE…………………………………………....6
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11
2.12
3
History of Infectious Bursal Disease Virus…………………………….6
Etiology of IBDV……………………………………………………....8
Chemical Composition of IBDV……………………………………….9
Viral Proteins of IBDV………………………………………………..10
Pathogenesis and Virus Replication of IBDV………………………...12
Transmission of IBDV………………………………………………..17
Incubation Period, Clinical Signs, and Mortality……………………..17
Gross Lesions…………………………………………………………18
Microscopic Lesions…………………………………………………..20
Antigenic Variation of IBDV Strains………………………………....22
Prevention and Control of IBDV……………………………………...24
Clinical and Differential Diagnosis of IBDV…………………………28
MATERIALS AND METHODS………………………………………..……35
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
3.10
3.11
3.12
Chickens…………………………………………………………...….35
Chicken Embryos…………………………………………………..…35
Viruses………………………………………………………………...35
Virus Isolation………………………………………………………...36
Tissues and Histopathology…………………………………………...37
Viral RNA Extraction…………………………………………………38
Reverse Transcription – Polymerase Chain Reaction
(RT-PCR)……………………………………………………………..38
Genome Sequencing and Analysis……………………………………40
Cultivation of Seed Stocks……………………………………...…….41
Antiserum Production…………………………………………………42
Virus Titration……………………………………………………...…43
Monoclonal Antibody Characterization………………………………44
iv
3.13
3.14
3.15
3.16
3.17
3.18
4
RESULTS…………………………………………………………………….59
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
5
Vaccines…………………………………………………………........45
Cell Cultures…………………………………………………………..46
Virus-neutralization…………………………………………………...46
Challenge of Immunity………………………………………………..48
Progeny Challenge……………………………………………………49
Bursa to Body Weight Calculations and Statistical Analysis……..…..50
VP2 Sequence Analysis…………………………………………….....59
Propagation and Pathology of Selected Viruses for Seed
Stocks…………………………………………………………………60
Monoclonal Antibody Testing………………………………………...61
Monoclonal Antibody Epitope Mapping……………………………...62
Virus Neutralization in Cell Culture………………………………….63
Active Immunity – Challenge with 89/03…………………………….64
Active Immunity – Challenge with PBG98…………………………..66
Passive Immunity – Progeny Challenge……………………………....68
DISCUSSION………………………………………………………………...88
CONCLUSION………………………………………………………………………….94
APPENDIX A. Epitope Profiles of Infectious Bursal Disease Viruses
Recognized by Monoclonal Antibodies Used in Antigen
Capture Enzyme-linked Immunosorbent Assays…………………..…..96
APPENDIX B. Standard Amino Acid Abbreviations and Side Chain
Properties Table……………………………………………………...…97
REFERENCES………………………………………………………………………......98
v
LIST OF TABLES
3.1. Accession numbers and broiler flock information for IBDV
isolation attempts………………………………………………………………...51
3.2. IBDV RT-PCR results from screening of the bursal homogenates
from 3- and 4-week-old commercial broiler chickens raised on
12 farms in the Delmarva Peninsula region…………………………...................52
3.3. Propagation of Selected Field Viruses for Seed Stocks…………………………….53
3.4. Monoclonal antibody (MAb) reactivity patterns of infectious
bursal disease virus field isolates using VP2 transfection and
immunofluorescence assays……………………………………………………..54
3.5. Monoclonal antibody (MAb) reactivity patterns of infectious
bursal disease virus field isolates and IBDV reference strains
using whole virus/ELISA ssays………………………………………………….55
3.6. Vaccination-challenge of SPF leghorns using Delaware variant
vaccine 89/03…………………………………………………………………….56
3.7 Vaccination-challenge of SPF leghorns using classic vaccine
PBG98…………………………………………………………………………...57
3.8. Progeny challenge using progeny of 18-week-old SPF leghorn
breeder chickens vaccinated with a licensed inactivated IBDV
vaccine containing classic and variant strains, as well as, a
strain of reovirus and challenged with IBDV field isolate 5038
and reference strain Delaware variant E…………………………………………58
4.1. Percent identity values of protein sequences of the variable
region of VP2 protein of IBDV field strains isolated from
Delmarva broiler farms and reference strains Delaware
variant E, STC, GLS, AL-2, and RS593 as determined by
multiple sequence alignment using the CLUSTAL W method……………….…69
vi
4.2. Correlation between predicted amino acid substitutions and
monoclonal antibody (MAb) 10 reactivity patterns for
passage 1 seed stock samples of IBDV field isolates and
IBDV reference strains…………………………………………………………..70
4.3. Correlation between predicted amino acid substitutions and
monoclonal antibody (MAb) 57 reactivity patterns for
passage 1 seed stock samples of IBDV field isolates and
IBDV reference strains…………………………………………………………..71
4.4. Correlation between predicted amino acid substitutions and
monoclonal antibody (MAb) 63 reactivity patterns for
passage 1 seed stock samples of IBDV field isolates and
IBDV reference strains…………………………………………………………..72
4.5. Correlation between predicted amino acid substitutions and
monoclonal antibody (MAb) 67 reactivity patterns for
passage 1 seed stock samples of IBDV field isolates and
IBDV reference strains…………………………………………………………..73
4.6. Correlation between predicted amino acid substitutions and
monoclonal antibody (MAb) 69 reactivity patterns for
passage 1 seed stock samples of IBDV field isolates and
IBDV reference strains…………………………………………………………..74
4.7. Virus-neutralization (VN) serum antibody titers of SPF
leghorn chickens against IBDV strains 89/03 and PBG98
as measured by beta VN assays in chicken embryo
fibroblast cells…………………………………………………………………...75
4.8. Percent protection determined by bursa to body weight ratios
of one-day-old SPF leghorn chickens subcutaneously
vaccinated with IBDV strain 89/03 and challenged at 21 days
post-vaccination via the intraocular route with IBDV field
isolates and reference strains STC and Delaware variant E……………………..76
4.9. Microscopic pathology of the bursa of Fabricius of SPF leghorn
chickens vaccinated with IBDV strain 89/03 and challenged
with IBDV field isolates 4813, 4947, 4955, 5038, and 5041
and IBDV reference strains STC and Delaware variant E………………………77
vii
4.10. Percent protection determined by bursa to body weight ratios
of one-day-old SPF leghorn chickens subcutaneously
vaccinated with IBDV strain PBG98 and challenged at 21 days
post-vaccination via the intraocular route with IBDV field
isolates and reference strains STC and Delaware variant E……………………..78
4.11. Microscopic pathology of the bursa of Fabricius of SPF leghorn
chickens vaccinated with IBDV strain PBG98 and challenged
with IBDV field isolates 4813, 4947, 4955, 5038, and 5041
and IBDV reference strains STC and Delaware variant E………………………79
4.12. Percent protection determined by bursa to body weight ratios
of progeny of 18-week-old SPF leghorn breeder chickens
vaccinated with a licensed inactivated IBDV vaccine containing
classic and variant strains, as well as, a strain of reovirus
and challenged with field isolate 5038 and reference strain
Delaware variant E at 10 days of age……………………………………………80
4.13. Percent protection determined by bursa to body weight ratios
of progeny of 18-week-old SPF leghorn breeder chickens
vaccinated with a licensed inactivated IBDV vaccine containing
classic and variant strains, as well as, a strain of reovirus
and challenged with field isolate 5038 and reference strain
Delaware variant E at 17 days of age……………………………………………81
4.14. Microscopic pathology of the bursa of Fabricius of progeny
from 18-week-old SPF leghorn breeder chickens vaccinated
with a licensed inactivated IBDV vaccine containing classic
and variant strains, as well as, a strain of reovirus and
challenged with field isolate 5038 and reference strain
Delaware variant E………………………………………………………………82
viii
LIST OF FIGURES
4.1. VP2 phylogenetic analysis of IBDV field isolates and reference
strains constructed using Molecular Evolutionary Genetics
Analysis (MEGA) software version 4.0 (Tamura et al., 2007)………………….83
4.2. Deduced amino acid sequences of the variable region of VP2
protein of IBDV field isolates and reference strains Delaware
variant E, STC, GLS, AL-2, and RS593. Major (Azad et al.,
1985; Bayliss et al., 1990) and minor (van den Berg et al., 1996;
Jackwood et al., 2006) hydrophilic peaks are indicated…………………………85
4.2. Serum antibody geometric mean titers (GMTs) of 89/03-vaccinated
and non-vaccinated SPF leghorn chickens before and after
challenge with IBDV field isolates and reference strains as
measured by ELISA……………………………………………………………..86
4.3. Serum antibody geometric mean titers (GMTs) of PBG98-vaccinated
and non-vaccinated SPF leghorn chickens before and after
challenge with IBDV field isolates and reference strains as
measured by ELISA……………………………………………………………..87
ix
ABSTRACT
Infectious bursal disease virus (IBDV) field isolates recovered in 2007 from
Delmarva Peninsula broiler farms with a history of poor performance were characterized.
The isolates originated from 3- and 4-week-old broilers having only passive breeder
vaccinations. A 743 base pair fragment of the VP2 coding region of each of the field
isolates was amplified by reverse transcriptase-polymerase chain reaction. VP2
sequencing and phylogenetic analysis showed that all of the isolates distributed into six
different clades representing serotype 1 Delaware variant viruses.
Two active vaccination-challenge experiments were performed to test the
antigenic and immunogenic properties of the IBDV field isolates and examine their
ability to break through immunity to a Delaware variant vaccine 89/03 and a classic
vaccine PBG98. Five field isolates representative of each molecular clade were used as
challenge viruses in three-week-old specific pathogen free (SPF) White Leghorn chickens
vaccinated with the Delaware variant and classic vaccines. The five field isolates induced
variant-like gross lesions in all non-vaccinated SPF chicks. However, a single
subcutaneous injection of either the 89/03 or PBG98 live, attenuated vaccine was
sufficient to protect chickens against challenge with the five field isolates.
Virus neutralization (VN) using antiserum produced against the five field isolates
showed no difference in neutralization of the 89/03 and PBG98 strains. This similarity in
x
neutralization indicated that the two cell-culture-adapted strains may not differ enough on
the basis of antigenicity to reveal a difference between the five IBDV field isolates.
The relatedness of the field isolates to other known variant IBDV strains was
evaluated using VP2 sequence analysis and two monoclonal antibody (MAb)
methodologies, transfection and immunofluorescence and whole virus, with an antigen
capture enzyme linked immunosorbent assay (AC-ELISA). Isolate 4813 was determined
to be most related to Delaware variant E (Del E) based on a 99.5% identity at the amino
acid level and its reactivity with monoclonal antibodies (MAbs) 63 and 67. Isolates 4947
and 4955 were determined to be most related to variant vaccine strain, RS593, based on a
98.1% identity at the amino acid level and similar MAb reactivity patterns. Isolate 5041
was found to be most antigenically similar to the GLS variant by the whole virus/ELISA
MAb test, but only 96.8% related at the amino acid level. Isolate 5038 failed to react with
MAbs used in either AC-ELISA experiment, which indicated that it may differ
antigenically from the IBDV reference strains tested.
Genomic characterization and comparison of the variable region of VP2 of the
five field isolates revealed amino acid substitutions in the four hydrophilic peaks
previously identified as being important for antigenic variation and the binding of
neutralizing antibodies. However, most of the VP2 sequence changes observed for the
five field isolates closely resembled those of other characterized variant strains.
A passive immunity progeny challenge was performed to assess passive IBDV
immunity in leghorn chicken progeny with maternal antibodies to IBDV. Ten-day-old
chicks challenged with isolate 5038 were less protected than chicks challenged with Del
xi
E. This finding indicated that 5038 was able to break through maternally-derived
immunity earlier than Del E.
The findings of this study indicated that field isolates 4813, 4947, 4955, and 5041
shared sequence and antigenic similarities with Delaware variant strains of IBDV. The
isolation of these viruses from commercial Delmarva broilers at 3-4 weeks of age is not
surprising as maternal antibodies to the virus normally are sufficiently low to permit
infection. On the other hand, solate 5038 appeared to be antigenically different than
IBDV reference strains Del E, STC, GLS, RS593, and AL-2 based on MAb testing and
antigenically different from Del E based on progeny challenge findings and warrants
further investigation.
xii
Chapter 1
INTRODUCTION
Infectious Bursal Disease (IBD) is caused by an acute, highly contagious
Birnavirus that results in mortality and immunosuppression of young chickens (1). Since
its original isolation in Gumboro, Delaware, the disease has inflicted profound economic
losses on the poultry industry worldwide (2). Infectious bursal disease virus (IBDV) and
other Birnaviruses are single-shelled, non-enveloped viruses that contain a bi-segmented,
double-stranded RNA genome (3, 4, 5, 6, 7, 8). The VP2 protein is the major protective
antigen of IBDV that contains specific epitopes responsible for inducing neutralizing
antibody responses (9, 10, 11). Amino acid changes that occur within the variable region
of VP2 can lead to variations in antibody recognition, as well as, antigenicity,
immunogenicity, virulence, and tissue tropism of IBDV strains (11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21). Two serotypes of IBDV have been identified; however, only serotype
I viruses are naturally pathogenic to chickens (22, 23, 24). Serotype I strains are
classified as classic, variant or very virulent IBDV strains and differ in their virulent,
antigenic, and pathogenic properties (22, 25, 26, 27).
1
The cloacal bursa is the target organ of IBDV infections; however, IBD viral
replication also occurs in other lymphoid structures including the spleen, thymus,
Harderian gland, and cecal tonsils (28, 29, 30, 31, 32). The virus preferentially affects
actively proliferating and differentiating B lymphocytes, which leads to an age-dependent
immunosuppression (33). Chicks infected less than one-week of age suffer severe and
permanent B-cell immunosuppression (31, 34, 35, 36, 37). The immunosuppressive
effects of IBDV infections not only enhance the chicken’s susceptibility to secondary
opportunistic infections such as gangrenous dermatitis, chicken anemia agent, inclusion
body hepatitis, respiratory diseases, and E. coli infections among others, but frequently
interfere with effective immune responses to vaccination (38, 39, 40, 41, 42, 43, 44).
During 2008, the value of broiler chickens produced in the Delmarva poultry
industry, alone, was over $2.1 billion (45). Therefore, minimizing immunosuppression
and its significant health and economic impacts is an ongoing challenge for broiler
companies. The primary method used for controlling IBD in the poultry industry is
vaccination (46, 47). The most common strategy is for hyperimmunized breeder flocks to
confer high titers of maternal antibodies to progeny chicks (37). Priming vaccinations in
the form of live-attenuated vaccines given at two to four weeks of age are followed by an
inactivated oil-emulsion vaccination, eight to fourteen weeks later. This boosts immunity
in breeder flocks and conveys a longer-lasting immunity to offspring. As a result, chicks
are protected from early immunosuppressive IBDV infections (46, 48, 49, 50, 51, 52, 53).
Because of differences in the effectiveness of breeder vaccination programs, broilers may
be vaccinated in an effort to gain active immunity against IBDV. However, proper timing
2
is critical to avoid possible neutralization of the vaccine virus by persistent maternal
antibodies (46, 49, 54, 55).
Common epitopes between IBDV pathotypes were demonstrated with the
production of IBDV variant-derived vaccines. Classic strain-derived vaccines offered
limited protection against variant IBDVs, whereas variant vaccines protected against
classic strains, as well as, homologous and heterologous variant strains (56, 57, 58). The
ability of IBDVs to cross-protect has played an important role in the development of
improved vaccines. Both classic and variant strains are often incorporated into
commercially available live and killed vaccines, so as to broaden the range of antigenic
subtypes and elicit a heightened immune response. However, the dose and strains of both
the vaccine and challenge viruses modulate the degree of protection afforded by these
vaccines (17, 58, 59). Differences in maternal antibody titers and vaccination route, as
well as, coexisting infections and the high mutation rate of RNA viruses, can strongly
influence when and if novel variant strains arise within the poultry industry (39, 47, 59).
In late 2007, thirty-three field isolates were obtained from Delmarva Peninsula
broiler farms that had a history of poor performance. Poor performing farms typically
exhibit clinical or subclinical disease shown by high mortality, morbidity, or
condemnations at processing (i.e. air sacculitis, septicemia/toxemia, Leucosis). Belownormal body weight, poor feed conversion, and immunosuppression are also
characteristics of these problem farms. Disease monitoring of these farms has been used
to track the incidence of Runting and Stunting Syndrome and immunosuppressive
diseases in a company with the “Delmarva syndrome,” which is expressed as a
3
combination of Gangrenous Dermatitis, Respiratory complex, Inclusion Body Hepatitis,
and/or RSS. This 28-day health monitoring study involved a weekly sampling of broilers
(1-4 weeks of age) from 12 different farms within the Delmarva area. Isolates taken from
grossly affected birds were chosen for this study based upon preliminary histopathology,
virus isolation, serology, and bursa to body weight ratios.
This research project was designed to characterize strains of recently isolated IBD
viruses. Fifteen out of the thirty-three isolates recovered from 3- and 4-week-old broilers
were found positive through virus isolation, histopathology, and RT-PCR and designated
for further investigation (Table 3.2). The variable region of the VP2 protein of the chosen
isolates was compared with that of existing classic and variant strains of IBDV. An
evolutionary phylogenetic tree demonstrated that none of the field isolates were related to
the classic strain of IBD, as all of the field isolates clustered with the Delaware variant E
strain (Figure 4.1). The chosen isolates were distributed into six individual clades (Figure
4.1 and Table 3.3). From each clade, a representative isolate was selected for virus
propagation and seed stock production of bursa of Fabricius origin using SPF white
leghorn chickens (Table 3.3).
The specific objectives of this research are to sequence the encoding region of
VP2 protein from IBDV field isolates, perform a comparative sequence analysis to assess
genetic characteristics, and select viruses for further characterization; to perform
monoclonal antibody testing on chosen isolates to determine their reactivity pattern
compared to IBDV classic and variant reference strains; to determine virus-neutralizing
antibody titers for IBDV field isolates against IBDV strains 8903 and PBG98; to evaluate
4
the cross protection elicited by commercial IBDV vaccines against challenge with each of
the new field isolates; to determine the protection afforded by maternal antibodies against
challenge with a field isolate and reference strain of IBDV.
5
Chapter 2
REVIEW OF THE LITERATURE
2.1 History of Infectious Bursal Disease Virus
Infectious bursal disease (IBD) was first recognized as a distinct clinical entity in
1957 (2). A. S. Cosgrove initially described the malady as “avian nephrosis” on account
of the tubular degenerative lesions found in the kidneys of infected broiler chickens. The
syndrome adopted the name “Gumboro disease” since the first outbreaks occurred in and
around the area of Gumboro, Delaware, USA. Predominant signs of illness included
trembling, ruffled feathers, watery diarrhea, anorexia, depression, severe prostration, and
death. In addition, hemorrhages in the thigh and leg muscles, increased mucus in the
intestine, liver lobe infarction, renal damage, and enlargement of the bursa of Fabricius
were lesions commonly observed at necropsy (2). Early studies suggested that the
causative agent was a nephropathogenic strain of infectious bronchitis virus due to
similar gross changes observed in the kidney by Winterfield and Hitchner (60).
Subsequent studies (42, 61), however, revealed that IBV immunized birds could still be
infected with the “infectious bursal agent” (IBA) and develop changes in their cloacal
bursas specific for the disease. Following successful isolation of IBA in embryonated
6
chicken eggs (62), Hitchner (63) proposed that the disease be termed “infectious bursal
disease” due to its pathognomonic bursa lesions.
The immunosuppressive effects of infectious bursal disease virus (IBDV)
infections were first disclosed by Allen et al. (34). In 1980, a second serotype was
reported (22). These factors, along with the high tendency for IBD infections to recur in
successive flocks, emphasized the need for stringent measures of prevention and control.
Prior to 1984, spread of both the clinical and subclinical forms of the disease was
satisfactorily controlled by vaccination programs. However, in 1984 and 1985, a
significant increase in mortality, condemnations, and vaccine failures were reported in
the Delmarva Peninsula broiler growing area (64, 65). These newly emergent viruses
were capable of breaking through maternal immunity against classic strains of IBDV (25,
56, 57, 65). In vivo reciprocal cross-challenge tests showed that unlike classic or standard
strains of IBDV, the field isolates caused rapid atrophy and minimal inflammation of the
cloacal bursa when inoculated into susceptible SPF leghorns (56). Studies suggested that
a major antigenic shift in serotype I viruses had occurred in the field (26, 66). The IBDV
field isolates were characterized as antigenic “variants” of serotype 1 IBDV, while the
older serotype 1 viruses discovered prior to these newly emergent viruses were called
classic strains of IBDV (65). Currently in the United States, clinical cases are rarely
reported and these variant strains are the predominant viruses circulating in the field (47).
Outbreaks of very virulent IBDV (vvIBDV) were first reported in Europe in
1987-1988 (67, 68, 69). Highly virulent IBDV (vvIBDV) infections are characterized by
a peracute onset of severe clinical disease and high mortality (67, 68, 70, 71). Although
7
these new serotype 1 viruses demonstrate increased virulence in their ability to break
through the existing level of maternal immunity; they are antigenically similar to the
classic strains of IBDV (68, 72, 73). Strains of vvIBDV have rapidly disseminated to
every poultry-producing country, except Canada, Mexico, Australia, and New Zealand
(69, 74, 75, 76, 77).
2.2 Etiology of IBDV
Infectious bursal disease virus (IBDV) is classified as a member of the
Birnaviridae family (1). The family includes 3 genera: Aquabirnavirus whose type
species is infectious pancreatic necrosis virus (IPNV), which infects fish, mollusks, and
crustaceans; Avibirnavirus whose type species is infectious bursal disease virus (IBDV),
which infects birds; and Entomobirnavirus whose type species is Drosophila X virus
(DXV), which infects insects (78). Viruses in this family possess bi-segmented, doublestranded RNA (dsRNA) genomes, which are packaged into single-shelled, non-enveloped
virions (3, 79). The capsid shell exhibits icosahedral symmetry composed of 32
capsomeres and a diameter ranging from 55 to 65 nm (6, 8, 80). Its structure is based on a
T = 13 lattice composed of trimeric subunits. Cryoelectron microscopy and image
processing analysis showed that the outer surface of the viral capsid is made up of 260
trimeric VP2 clusters, while the inner surface is composed of 200 Y-shaped trimeric VP3
structures (81, 82).
8
2.3 Chemical Composition of IBDV
Infectious bursal disease virus has a sedimentation rate of 460S in sucrose
gradients (83, 84). The buoyant density of IBDV in caesium chloride gradients ranges
from 1.31-1.34 g/ml for complete viral particles and below 1.33 g/ml for incomplete viral
particles (7, 8, 84, 85, 86, 87, 88, 89).
Infectious bursal disease virus is very stable and can persist indefinitely in poultry
houses despite thorough routine cleaning and disinfection (90). The virus is resistant to
chloroform and ether treatments, and it remains unaffected after a 5 hour incubation at
56°C and pH 2, although, it can be inactivated at pH 12. Exposure to 0.5% phenol and
0.125% thimerosal for 1 hour at 30°C had no effect, while contact with 0.5% formalin for
6 hours markedly reduced virus infectivity (91). Landgraf et al. (92) demonstrated that
IBDV is able to withstand 30 minutes at 60°C, but not 70°C and it is killed after 10
minutes in 0.5% chloramine disinfectant. In addition, invert soaps with 0.05% sodium
hydroxide can inactivate or strongly inhibit the virus (93). Due to the economics of
commercial poultry production, which involve the re-use of litter and short time intervals
between flocks, as well as the virus’s resistance toward heat and several physical and
chemical agents, IBDV survives in poultry houses for long periods (94).
The genome of IBDV is comprised of two segments of dsRNA as demonstrated
by polyacrylamide gel electrophoresis (79, 83). The larger segment, A, is 3261
nucleotides long and contains two open reading frames (ORF). The first ORF, preceding
and partially overlapping ORF2, encodes a 17 kDa non-structural protein, known as VP5
(95). The second ORF encodes a 110 kDa precursor polyprotein (NH3-VPX-VP4-VP3-
9
COOH), which is co-translationally processed to yield the protein capsid precursor VPX
or pVP2 (48 kDa), and proteins VP4 (28 kDa) and VP3 (32 kDa) (96). The pVP2 is
further processed by serial cleavages near its carboxy-terminus into mature VP2 (41 kDa)
and four peptides which remain associated with the virion (9, 97). VP2 and VP3 are the
major structural proteins of the virion, whereas VP4 is a virus-encoded protease (9, 98,
99). The pVP2 initially undergoes VP4-mediated processing events to yield shorter pVP2
polypeptides (100). Recently, Irigoyen et al. (100) proposed that VP2 Asp-431 is
responsible for catalyzing the last pVP2 to VP2 proteolytic event that occurs during
capsid maturation. Genomic segment B, which is 2827 nucleotides long, encodes VP1, a
97-kDa RNA dependent-RNA-polymerase (RdRp) (101). In both genomic segments,
short 5’ and 3’ terminal sequences (79 to 111 nucleotides long) flank the coding regions
(102). The 3’ untranslated regions of both the A and B segment have the potential to form
stem and loop secondary structures that may be essential for RNA replication (103).
2.4 Viral Proteins of IBDV
In virions, VP1 exists as both a covalently bound protein at the 5’ ends of the
genomic dsRNA strands, as well as, a free polypeptide (4, 104). It is responsible for viral
RNA replication following cellular infection and mRNA synthesis (105). It has also been
reported that VP1 may play an important role in IBDV virulence (106). The catalytic
motifs of polymerases from birnaviruses are arranged in a permuted order in the
sequence (107). As a result, the structure of the birnavirus polymerase VP1 adopts a
10
unique active site topology that has not been previously found with other RNA and DNA
polymerases (107).
VP2 (441 amino acids [aa]) is the main capsid protein, constituting 51% of the
viral proteins in serotype 1 viruses (1). It is the host-protective antigen, as it contains
serotype- and strain-specific epitopes responsible for inducing neutralizing antibodies (9,
10, 11, 108). It has also been reported to be an apoptotic inducer in mammalian cells, but
not in chicken embryo cells (109). VP2 is highly hydrophobic and folded into three
distinct domains termed the base (B), shell (S), and projection (P) (20, 110, 111). The B
and S domains are comprised of the conserved N- and C-terminal sequences of VP2,
while the P domain contains the conformation-dependent central variable region of VP2
(aa 206 to 350) (13, 73, 112). The significance of protein conformation for VP2
interaction with monoclonal antibodies has been affirmed by several studies where
denaturing conditions using Western immunoblotting prevented reactivity (21, 66, 73,
108). Within the central variable region are four stretches of hydrophilic amino acids that
are more prone to antigenically significant amino acid changes (12, 13, 15). These areas,
known as major hydrophilic peaks A (aa 212-224) and B (aa 314-324) and minor
hydrophilic peaks 1 (aa 249-254) and 2 (aa 279-290) (9, 15, 112, 113, 114, 115) reside in
the most exposed parts of the P domain (20).
The VP3 protein contains both conformational-independent, group-specific
epitopes (common in both serotypes) and serotype-specific epitopes (116, 117) that elicit
non-neutralizing and non-protective antibodies (11, 108). This multifunctional protein
11
interacts with VP1, VP2, and with genomic double-stranded RNA and plays a pivotal
role in virus assembly and morphogenesis (100, 118, 119, 120, 121).
VP4 is described as a minor, non-structural viral protease. Using a catalytic
serine-lysine (Ser-652 and Lys-692) dyad conserved among bacterial Lon proteases, VP4
is responsible for the proteolytic processing of the precursor polyprotein (122, 123, 124).
Sanchez and Rodriguez (124) identified two cleavage sites, 511LAA513 and 754MAA756,
that are important for the processing of the pVP2–VP4 and VP4–VP3 precursors,
respectfully. In addition, the self-assembling VP4 protease gives rise to specific
microtubules (type II tubules), which accumulate within infected cells; however, they are
not components of the mature virion (125).
VP5 is a highly basic, cysteine-rich protein that is conserved among all serotype 1
IBDV strains (126). Lombardo et al. (127) identified it as a class II transmembrane
protein with a cytoplasmic N-terminal domain and an extracellular C-terminal domain.
The VP5 protein is believed to play a role in viral egress, as well as, possess antiapoptotic function at the early stages of IBDV infection (127, 128, 129). Although it is
not essential for viral replication in cell culture (129), it does function in in vitro
pathogenesis and dissemination (126, 127, 128).
2.5 Pathogenesis and Virus Replication of IBDV
Two known serotypes of IBDV, designated serotypes I and II, have been
described (22, 89). The natural hosts of IBDV are the chicken and the turkey. Serotype I
viruses affect every breed of chicken, but the most severe clinical signs and lesions and
12
the highest mortality rate have been observed in white leghorns (47). Turkeys, ducks, and
ostriches can be naturally and experimentally infected with IBDV serotypes I and II, as
evidenced by serological response and isolation; however, the infections are apathogenic
(22, 89, 94, 130, 131, 132, 133, 134, 135, 136). Several other avian species including
rooks, wild pheasants, crows, gulls, and falcons, were reported to be susceptible to
infection or to possess antibodies against IBDV (137, 138). Yet, only serotype I viruses
replicate in lymphoid cells and are pathogenic to chickens (23, 24, 89, 139). Pathotypes
of serotype I strains are classified in increasing order of virulence as mild, intermediate
(serotype I vaccines), variant virulent, classical virulent, and very virulent or hypervirulent (75). The exhibition of clinical signs and gross and microscopic lesions is
contingent upon the virus pathotype and chicken immune status (31).
While viral replication initially occurs in the intestine, the main target organ for
pathogenic serotype I IBDV is the bursa of Fabricius, as it is the source for B
lymphocytes in avian species (28, 140). This was demonstrated by experiments in which
bursectomized chickens failed to develop clinical IBD, despite having IBDV infections
lethal for non-bursectomized chickens (141). In vivo and in vitro studies have
demonstrated that actively dividing, surface immunoglobulin M-bearing (IgM+) B
lymphocytes (B-cells) are the target cell for IBDV cytolytic infection (31, 141, 142, 143).
Moreover, the virus seems to affect immature B-cells to a greater degree than mature B
cells (33, 144). Throughout the juvenile life of the bursa, IgM+ bearing cells continuously
migrate to the bursa where they acquire the characteristics of mature, immunocompetent
B cells. Postbursal cells are seeded out to peripheral lymphoid tissues until involution of
13
the bursa occurs with the onset of oviposition (145, 146). The period of greatest
susceptibility to clinical IBDV infections is between 3 and 6 weeks after hatching (37,
147). The exact cellular receptor for IBDV attachment has not been identified; however,
it has been suggested that IBDV serotypes I and II may use multiple receptors, serotypespecific or common to both serotypes, on different cell types (47, 140). The mechanism
of viral RNA synthesis has not been clearly determined (47, 148). Genome-linked
proteins have been described, indicating IBDV replicates its nucleic acid by a semiconservative mechanism (105, 148, 149, 150).
Following host entry via oral infection or inhalation, IBDV initiates replication in
lymphocytes and macrophages of the gut-associated lymphoid tissues (GALT) such as
the cecum, duodenum, and jejunum. This stage of viral replication marks the primary
viremia. Within 5 hours post-infection, viral antigen reaches the liver, where it is trapped
and phagocytized by resident macrophages (Kupffer cells). Virus then enters the
bloodstream where it is distributed to other tissues including the bursa of Fabricius. By
13 hours post-infection, most bursal follicles are positive for virus and extensive necrosis
of immature B-cells in the medullary and cortical regions of the bursal follicles ensues
(28, 151, 152). By 16 hours post-infection, a second massive viremia occurs with
secondary replication in other B-lymphocyte-containing tissues including the spleen,
thymus, Harderian gland, and cecal tonsils (29, 30, 31, 32). Clinical signs and death may
result from the acute phase (7-10 days) of IBD. Factors such as pathogenicity and
virulence of a strain, as well as, the chicken’s age, breed, and immune status can
influence the outcome and severity of infection (37, 153).
14
Virus replication during the acute lytic phase results in a dramatic reduction in
circulating IgM+ cells and a prolonged suppression of the primary antibody response
(152, 153, 154, 155, 156). Secondary antibody responses are unaffected (157). In
chickens that survive the acute disease, virus replication subsides, and almost all bursal
follicles become repopulated with IgM+ B cells. The primary antibody response is
gradually restored to near normal levels (152). The destruction of Ig-producing B cells by
IBDV may be one of the main inhibitors of humoral immunity; however, the involvement
of other mechanisms such as altered antigen-presenting and helper T cell functions has
been proposed (153).
Depletion of B-cells in the bursa is accompanied by an influx of activated CD4+
and CD8+ T-lymphocytes (T-cells) (151, 153, 158, 159). By the seventh day of infection,
CD8+ cells are the predominant infiltrating T-cell. T-cells are resistant to infection and
replication of IBDV. However, IBDV infection can severely decrease the in vitro
proliferative response of T cells to mitogens, indicating that cellular immune responses
are also compromised (153). Evidence suggests that T cells may modulate IBDV
immunopathogenesis by restricting IBDV replication in the bursa in the early stage of the
disease (5 days post-infection). Through their release of cytokines and cytotoxic effects,
T-cells may enhance bursal tissue destruction, suppress immunity, and delay recovery of
bursa follicles. At the same time, T-cells may promote clearance of IBDV (153, 160,
161).
Along with its direct cytopathic effect on IgM+ cells, IBDV can infect cells of the
monocyte-macrophage lineage (141, 152, 162, 163, 164, 165, 166, 167, 168, 169, 170).
15
IBDV-infected macrophages may play a role in virus dissemination from the gut to the
bursa and other peripheral organs (166). In addition, activated macrophages may secrete
chemotactic, proinflammatory, and other immunoregulatory cytokines that recruit
heterophils, T cells, and macrophages to the site of infection. Elevated levels of these
inflammatory mediators and chemokines during the acute phase can enhance local
inflammation and tissue destruction (141, 152, 162, 163, 164, 166, 167, 168, 169, 170,
171).
The lack of inflammation observed with variant IBDV infections supports the
theory that apoptosis, in addition to necrosis, is responsible for IBDV-induced cellular
destruction and immunosuppression (172, 173, 174, 175). Apoptosis, or programmed cell
death, occurs in both physiological and pathological conditions and is characterized by
several morphological changes including cell shrinkage, chromatin condensation, and
chromosomal DNA fragmentation with no inflammation. IBDV induces apoptosis in
chicken peripheral bursal lymphocytes, chicken embryos, chicken embryo fibroblasts,
and Vero cells (172, 176, 177). Moreover, apoptosis has been reported in lymphoid cells
and tissues of young chickens and embryos experimentally infected with pathogenic
serotype I IBDV strains (173, 174, 175, 178, 179, 180, 181). Two viral proteins of IBDV,
VP2 and VP5, are suspected to play a role in the induction of apoptosis (109, 126, 182).
Apoptosis has been detected in antigen-negative cells in the vicinity of productively
infected cells, suggesting that indirect mechanisms may also be involved in this process
(174, 175, 183).
16
2.6 Transmission of IBDV
IBDV is highly contagious and ubiquitous. The disease can be spread by direct
and indirect contact between infected and susceptible flocks. Once infected with IBDV,
chickens are capable of shedding the virus in the feces for as long as 16 days (184). There
is no evidence to suggest that IBDV is spread via transovarial transmission (47). Benton
et al. (91) reported that poultry houses which previously harbored infected flocks
remained infective for at least 122 days and that fomites (water, feed, droppings)
contaminated with IBDV contribute to viral dissemination (91). No specific vectors or
reservoirs of IBDV have been established, but the virus has been isolated from mosquitos
(Aedes vexans), rats, and lesser mealworms (Alphitobius diaperinus) (47, 185, 186, 187,
188).
2.7 Incubation Period, Clinical Signs, and Mortality
IBDV has a short incubation period of 2 to 3 days and the infection generally lasts
5 to 7 days. One of the earliest signs of IBDV infection is the tendency for birds to
engage in vent picking. Clinical signs are described as acute onset of depression,
trembling, white or watery diarrhea, anorexia, prostration, ruffled feathers, vent feathers
soiled with urates, and hemorrhages in pectoral and thigh muscles. In severe cases, birds
become dehydrated, and in the terminal stages subnormal temperatures and death ensue
(2, 31). Naive chickens between 3 and 6 weeks of age are most susceptible to the clinical
form of IBD, which causes impaired growth, immunosuppression, and mortality (43, 90,
189). Clinical signs are mainly characteristic of IBDV serotype I classic strains (65). In
17
fully susceptible flocks, mortality associated with classic strain infections may range
from 1-60%, with high morbidity of up to 100% (47, 113, 147). Variant IBDV strains do
not produce overt clinical signs, but cause immunosuppression, and may cause mortality
due to secondary opportunistic infections in immune compromised birds (37, 47, 190). In
contrast, vvIBDV strains cause mortality of 50-60% in laying hens, 25-30% in broilers,
and 90-100% in susceptible SPF leghorns (37, 67, 68, 70).
Susceptible chickens younger than three weeks of age may not exhibit clinical
signs, but develop subclinical infections. This results in a decreased humoral antibody
response due to B lymphocyte depletion in the cloacal bursa (184), and a severe and
prolonged immunosuppression (34). The most significant economic losses result from
subclinical infections. This form of IBD infection greatly enhances the chicken’s
susceptibility to sequelae such as gangrenous dermatitis, chicken anemia virus, inclusion
body hepatitis, respiratory diseases, and bacterial infections (38, 39, 40, 41, 43, 191, 192).
Moreover, it frequently interferes with effective immune responses to vaccination against
Newcastle disease, Marek’s disease, and infectious bronchitis (34, 35, 42, 193).
2.8 Gross Lesions
Gross lesions observed in birds that succumb to IBDV infection include
dehydration of the breast and leg musculature, darkened discoloration of the pectoral
muscles, occasional hemorrhages in the leg, thigh, and pectoral muscles, increased mucus
in the intestine, and renal changes. The gross appearance of the kidneys may appear
normal in birds that are necropsied during the course of infection. In birds that die or are
18
in advanced stages of the disease, kidneys frequently show swelling and pallor with
heavy accumulation of urates in the tubules and ureters (47).
The bursa of Fabricius is the predominant lymphoid organ affected by IBDV.
Infections with classic strains of IBDV cause inflammation and hypertrophy of the bursa
as early as day 3 post-infection. By day 4, the bursa is double its original size and weight
due to edema and hyperemia. By day 5, the bursa returns to its normal weight, but
continues to atrophy until reaching one-third or less of its original weight following day 8
post-infection (47). In contrast, variant strains of IBDV typically cause a rapid atrophy,
mucosal edema, and firmness of the bursa in the absence of inflammation (31, 65, 194).
Only one variant isolate (IN) has been reported to cause bursal inflammation (195). By
day 2 or 3 post-infection, a gelatinous yellowish transudate covers the serosal surface of
the bursa and longitudinal striations become visible. The bursa’s normal white color
shifts to cream and then, in some cases, gray during and following the period of atrophy.
In addition, necrotic foci and petechial or ecchymotic hemorrhages on the mucosal
surface may be observed in infected bursas.
Moderate to severe splenomegaly with small gray foci uniformly distributed on
the surface has been reported (47, 196). Occasionally, petechial hemorrhages will occur
in the mucosa at the juncture of the proventriculus and gizzard (2, 47). Compared to
moderately pathogenic IBDV strains, vvIBDV strains induce similar bursal lesions, but
cause more severe damages to the cecal tonsils, thymus, spleen, and bone marrow (14,
47, 165).
19
2.9 Microscopic Lesions
IBDV infections produce microscopic lesions primarily in the lymphoid tissues
(i.e. cloacal bursa, spleen, thymus, cecal tonsils, and Hardarian gland). Pathologic
observations of experimental cases were reported by Helmboldt and Garner (29) and
Cheville (196). Degeneration and necrosis of B lymphocytes in the medullary region of
the bursal follicles is apparent within one day of exposure. Depleted lymphocytes are
quickly replaced by heterophils, pyknotic debris, and hyperplastic reticuloendothelial
(RE) cells. By day 3 or 4 post-infection, IBDV-associated lesions are visible within all
bursal follicles. At this time, infections with classic IBDV strains have caused an
inflammatory response marked by severe edema, heterophil infiltration, and hyperemia in
the bursa. Inflammation diminishes by day 4 post-infection (PI), and as necrotic debris is
cleared by phagocytosis, cystic cavities develop in the medullary areas of the lymphoid
follicles. Necrosis and phagocytosis of heterophils and plasma cells occur within the
follicle, as well as, in the interfollicular connective tissue. In addition, a fibroplasia in the
interfollicular connective tissue may appear and the surface epithelium of the bursa
becomes involuted and abnormal (36, 196). Proliferation of the bursal epithelial layer
generates a glandular structure of columnar epithelial cells that contains globules of
mucin. During this stage of the infection, scattered foci of repopulating lymphocytes were
observed; however, these did not develop into healthy follicles (29, 197). Microscopic
lesions caused by variant strains are characterized by extensive follicular lymphoid
depletion and rapid plical atrophy of the cloacal bursa in the absence of an inflammatory
response (31, 197).
20
In the early stages of infection, the spleen exhibits hyperplasia of
reticuloendothelial cells surrounding the adenoid sheath arteries. By day 3 PI, diffuse
lymphoid necrosis occurs in the germinal centers and around the periarteriolar and
periellipsoid lymphatic sheaths. Cell populations in the spleen rapidly recover and the
germinal follicles sustain no permanent damage (29, 197).
During the acute phase of the infection, the thymus undergoes a marked atrophy
and widespread apoptosis of cortical lymphocytes. However, within a few days of
infection, the lesions are overcome and the thymus is restored to its normal state (181).
These lesions have not been associated with virus replication in thymic cells (31, 147).
IBDV-induced damage to the cecal tonsils may involve acute heterophilic inflammation
and lymphocyte depletion with regeneration on day 5 PI (29). Tanimura and Sharma
(151) reported that antigen-positive cells mainly localized to the germinal centers of the
cecal tonsils.
Infection with IBDV causes severe plasma cell depletion in the Harderian gland
and prevents the normal infiltration of plasma cells into the gland. This reduction is shortlived and plasma cell counts are restored in approximately 14 days PI (47, 198, 199).
Lesions characterized by large casts of homogeneous material infiltrated with heterophils
have been reported in the kidneys; however, they are minimal and believed to be nonspecific (29, 47, 200). Slight perivascular infiltration of monocytes may be observed in
the liver (200). There is evidence that IBDV replication also occurs in the bone marrow
(165, 197).
21
2.10 Antigenic Variation of IBDV Strains
Historically, mutations in the IBDV genome have impacted antibody recognition
and led to variations in antigenicity, immunogenicity, virulence, and tropism of
circulating infectious bursal disease virus (IBDV) strains (11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21). Therefore, continuous surveillance, along with rapid identification and
characterization of new IBDV isolates and comparison with previously described viruses
is of vital importance (30, 75, 201). The molecular basis for these emerging antigenic
differences was traced to antigenic domains of the VP2 protein of IBDV (11). The viral
capsid protein, VP2, is the major host protective immunogen, as it is the only viral
protein responsible for the induction of neutralizing antibodies and for serotype
specificity (9, 73, 75, 108, 202). Neutralizing monoclonal antibodies have been shown to
bind within a central variable region between amino acids (aa) 206 and 350 (AccI-SpeI
fragment) of the VP2 protein. This region is composed of hydrophobic aa, flanked by two
short hydrophilic peaks, A and B (aa residues 212-224 and 314-324, respectively) (9, 12,
112, 115). Subsequently, two smaller hydrophilic peaks, 1 and 2 (aa 249-254 and aa 279290, respectively) were also identified (113, 115). Amino acid changes correlated with
antigenic variation are typically found within these peaks (12, 13, 15, 17, 112, 203, 204,
205).
Antigenic variation, including the existence of a second serotype, were first
introduced by McFerran et al. (22), who reported a lack of in vitro reciprocal virus
neutralization between serotype I and serotype II IBDV strains. Serotype I viruses cause
immunologic disease in chickens, while serotype II strains only induce sub-acute
22
infections in turkeys (66). Moreover, serotype II viruses do not protect against challenge
with serotype I viruses (23, 58, 206). Heterogeneity in regards to antigenicity,
pathogenicity, virulence, and immunosuppressive potential was also demonstrated among
serotype I strains in virus neutralization (VN) and in vivo reciprocal cross-challenge tests
(22, 25, 26, 58, 64, 65). Jackwood and Saif (26) identified six different subtypes among
thirteen serotype I strains in a reciprocal VN study using polyclonal antisera, supporting
the existence of antigenic diversity among IBDV commercial vaccine strains and field
strains. To help elucidate antigenic variations responsible for vaccination failures, Snyder
et al. (207, 208) designed a select panel of monoclonal antibodies (MAbs) (e.g. 8, 179,
B69, R63, and 10) for differentiation between classic strains (e.g. D78) and several
subtypes of the variant strains. All classic IBDV viruses and vaccines isolated prior to
1985 were efficiently neutralized by MAb R63 and B69 (e.g. STC, D78); whereas, IBDV
variants lack either the B69 site (e.g. Delaware variant E (DelE)) or both the B69 and
R63 sites (e.g. GLS) (208, 209). Due to the fact that these Mab-defined neutralization
sites previously found on all classic strains were absent from new field isolates, it was
predicted that a major antigenic shift in serotype I field viruses had occurred in the U.S
(30, 66, 208, 209). Neutralizing MAbs including MAbs 8, 179, R63, B69, 57, 10 and 67
and non-neutralizing MAbs such as MAbs B29 and BK9 are commonly used for
antigenic analyses of IBDV field isolates by antigen capture enzyme-linked
immunosorbent assay (AC-ELISA) (21, 15, 66, 72, 202, 205, 207, 208, 209, 210, 211,
212). For example, the RS593 variant is defined by MAbs 8, 67, and 179, while the AL2
variant is defined by MAbs 8, 57, 67, and 179 (213). Australian variants showing little
23
similarity to U.S. variants at both the antigenic and genetic level were similarly identified
(211). Currently, serotype I IBDV viruses are antigenically grouped as classic (also
known as standard) and variant strains based on virus neutralization (47, 212).
In contrast, the vvIBDV strains are considered to be the most antigenically similar
to classic IBDV strains, despite their enhanced pathogenic properties and ability to
circumvent maternal immunity induced by classical, mild IBDV vaccines (51, 55, 67, 68,
69, 72, 214). Previous findings have suggested that vvIBDVs may be evolving; however,
further investigation is needed to determine the biological significance of these antigenic
differences (205, 215, 216).
2.11 Prevention and Control of IBDV
Infectious bursal disease virus is both highly contagious and very resistant to
inactivation, which accounts for its persistent survival on poultry farms, despite
disinfection (37, 47, 91). Therefore, even with strict biosecurity programs (e.g. ‘down
time’ between broods, all-in/all-out production, cleaning and disinfection of the premises
and equipment), vaccination is especially important to reduce the incidence and impact of
IBD in the poultry industry (37, 47). Traditionally, breeder flocks are hyperimmunized
with live and killed vaccines in order to confer high titers of maternal antibodies to their
progeny (37). This passive immunity protects chicks against early immunosuppressive
infections for 1 to 3 weeks; however, protection may be extended to 4 or 5 weeks by
boosting the immunity in breeders with oil-adjuvanted vaccines (47, 48, 49, 50, 51, 52,
53). In addition, young broiler chicks are sometimes actively immunized before the total
24
waning of maternal antibodies. Maternal antibody titers may vary considerably due to
differences in breeder vaccination programs, the age of the breeder flocks supplying
progeny, and normal variation between hen titers in the same flock. This makes the
timing of broiler vaccination in relation to waning maternal antibodies critical, to prevent
persistent maternal antibodies from potentially neutralizing the vaccine virus (46, 49, 54,
55). Skeeles et al. (217) reported that maternal antibody titers must fall below 1:64 before
broiler chicks can be effectively vaccinated with a live-attenuated IBDV strain.
Serological monitoring of antibody levels in a breeder flock or its progeny is usually
necessary to determine the proper time to vaccinate (51).
Prior to 1985, both clinical and subclinical forms of IBD were controlled by
vaccination programs utilizing classic strains. However, in 1984 and 1985 vaccination
failures were reported from the Delmarva poultry-producing area (64, 65). The IBDV
field isolates, characterized as “variant” strains were capable of producing bursal lesions
in vaccinated birds, despite the presence of maternal antibodies to IBDV (25, 56, 57, 65,
66). Common epitopes between IBDV pathotypes were reported with subsequent
production of IBDV variant-derived vaccines. Live and killed vaccines made from classic
strains offered limited protection against variant IBDVs, whereas those made from
variant strains protected against classic strains, as well as, homologous and heterologous
variant strains (56, 57, 58). The ability of IBDVs to cross-protect has played an important
role in the development of improved vaccines. Commercially available live and killed
vaccines have since been reformulated to include both classic and variant virus strains, so
as to broaden the range of antigenic subtypes and elicit a heightened immune response
25
(17, 58, 59). However, vaccine efficacy highly depends on the dose and strains of the
vaccine and challenge viruses, as well as, the route of administration, the appropriate
vaccination time, and the levels of maternal antibodies (58, 218). Newly emerging IBDV
strains capable of circumventing maternally-derived and active immunity induced by
commercial IBDV vaccines have been reported (17).
Classical live attenuated vaccines may induce broad, lifelong protection, but they
also carry residual pathogenicity and the potential to revert to virulence (37, 75, 219,
220). Several live-attenuated virus vaccines that differ according to their virulence and
antigenic characteristics are available commercially. With regard to virulence, vaccine
strains are characterized as mild, mild intermediate, intermediate, intermediate plus, or
“hot,” depending on the degree of attenuation (47). Mild strains are predominantly used
for breeder vaccinations, but due to their extreme sensitivity to homologous maternal
antibody interference, they are normally administered between four and eight weeks of
age when maternal antibodies have waned (37). Intermediate vaccines are frequently
administered to broilers and pullets (221). In addition, intermediate vaccines may be
administered by nebulisation to day-old broiler chicks who possess low levels of maternal
antibodies at hatch (37). Less attenuated (hot) strains are known to cause histological
lesions in SPF chickens and may provoke immunosuppression (37). Live-attenuated
vaccines are administered via drinking water application or nebulisation between the ages
of 7 days and 2 or 3 weeks (37, 47).
Killed-virus vaccines in an oil adjuvant are often used to boost levels of maternal
antibodies and confer longer-lasting immunity in breeder hens. The duration and
26
uniformity of this immunity may be influenced by the concentration and antigenic
specificity of the vaccine strain (37). These vaccines are not ideal for stimulating a
primary antibody response; therefore, they tend to be most effective in chicks that have
been “primed” with a live virus vaccine or naturally infected through field exposure to
IBDV (47, 52, 53, 220, 222, 223). Currently, many oil-adjuvant vaccines contain both
classic and variant IBDV strains (47). These vaccines do not protect against vvIBDV
strains (51, 223). Killed-virus vaccines are administered by subcutaneous or
intramuscular injection at sixteen to twenty weeks of age (37).
A more recent development in IBD control is the use of in ovo vaccination, which
involves inoculating 18-day-old chicken embryos with live IBD vaccine, either alone or
complexed in vitro with anti-IBDV antibodies (224, 225). This method helps to avoid
interference by maternal antibodies, while effectively initiating a primary antibody
response, and is efficacious against IBDV for the duration of the growing period (224,
226, 227).
Expression of IBDV proteins in yeast (228, 229) and the baculovirus system (230,
231, 232, 233, 234, 235) have been studied for use as subunit vaccines. VP2 protein of
IBDV expressed in a baculovirus system was tested in commercial broiler breeders and
immunity was conferred to their progeny (235). A Pichia pastoris yeast-expressed
recombinant VP2 has been used as an oil-adjuvanted vaccine in broilers in Israel (236).
Moreover, live recombinant viral vaccines expressing the VP2 protein such as fowlpox
virus, herpes virus of turkey (HVT), and Newcastle disease virus have been reported
(237, 238, 239, 240, 241); however, only one recombinant vaccine derived from HVT has
27
been licensed to date (242). Such technologies could allow discrimination between
antibodies induced by vaccines (anti-VP2 only) and those induced by infection (anti-VP2
and VP3), which may enhance field monitoring of IBDV (75).
2.12 Clinical and Differential Diagnosis of IBDV
Diagnosis of acute clinical forms of IBD is made according to disease evolution
(rapid onset followed by recovery in 5-7 days) and post-mortem examination of gross or
microscopic lesions of the bursa of Fabricius (47). Distinct clinical changes in the size
and color of the bursa will help differentiate all acute IBD cases from conditions
commonly mistaken for IBD such as avian coccidiosis, chicken infectious anaemia,
mycotoxicoses, and certain nephropathogenic forms of infectious bronchitis (37, 47, 60).
In contrast, diagnosis of subclinical and immunosuppressive forms of IBD is made
retrospectively by histopathology of the atrophied bursa (47). Due to the
immunosuppressive nature of IBDV, its existence within a flock may be manifested by an
increased susceptibility to conditions such as gangrenous dermatitis, inclusion body
hepatitis, Marek’s disease, infectious laryngotracheitis, and salmonellosis and
colibacillosis (38, 40, 43, 191, 193, 243). However, the histologic response associated
with each of these conditions is typically different from that of IBD (244).
IBDV is most commonly isolated from cloacal bursa tissues as the bursa is the
primary target organ for viral infection and contains higher concentrations of virus than
other non-bursal lymphoid tissues and organs. Propagation of both virulent and vaccine
IBDV can be performed in 9-to-11-day-old fertile specific pathogen free chicken eggs
28
(47, 244, 245). The most sensitive route of inoculation is via the chorioallantoic
membrane (CAM); however, embryos inoculated via the allantoic sac and yolk sac can
also be infected (63). CAM inoculation with classic IBDV strains produces embryo
mortality in 3 to 5 days post-inoculation. Gross embryo lesions may include cutaneous
congestion, edematous distension of the abdomen, petechial hemorrhages along the
feather tracts and occasional hemorrhages in the toe joints and cerebral area, necrosis of
the liver, pallor of the heart, congestion and some necrosis of the kidneys, extreme
congestion of the lungs, and pallor of the spleen with occasional small necrotic foci.
Plaques are seldomly present on the CAM, but small surface hemorrhages have been
observed (47, 244, 246). In contrast, variant IBDV strains do not cause embryo mortality
and rarely produce congestion or hemorrhages. Embryo lesions characteristic of variant
IBDV infection include cerebral and abdominal edema, stunting, off-white or creamcolored skin and feathers, necrosis and bile stasis of the liver, and splenomegaly (25, 47,
244). McFerran et al. (22) reported that three out of seven wild type IBDV isolates
recovered from infected bursae failed to replicate in chicken embryo fibroblast (CEF)
cells, but propagated in embryonating eggs. Very virulent IBDV strains are also
propagated in embryonated chicken eggs, producing severe lesions and high mortality at
low virus doses (229, 244).
Many IBDV isolates have been successfully adapted to cell cultures of chicken
embryo origin, including chicken embryo bursal cells, kidney cells, and fibroblast cells
(47, 133, 244, 247, 248). Moreover, IBDV has been grown in cell lines including turkey
and duck embryo cells (133), mammalian cell lines derived from rabbit kidneys (RK-13)
29
(249), Vero cells (6, 250, 251), derived from African green monkey kidneys; BGM-70
cells (251), derived from givet monkey kidneys; and MA-104 cells (251) from fetal
rhesus monkey kidneys. Although the virus may be adapted to cell culture, isolation and
propagation of some IBDV field strains may be difficult with this technique (22, 244,
252, 253, 254). Initial isolation of IBDV is best accomplished via embryo inoculation
(244). However, studies have demonstrated that IBDV pathogenicity and
immunogenicity is reduced following propagation in embryos and cell cultures (190,
255).
Direct and indirect immunofluorescence assays, immunocytochemistry, or direct
examination by electron microscopy are useful tools for isolation and identification of
IBDV in infected tissues (22, 47, 256). Rapid detection and typing of IBD field cases
may be accomplished using nucleic acid probes and antigen-capture enzyme
immunoassays (207, 257, 258, 259). Conventional reverse transcription (RT) followed by
the polymerase chain reaction (PCR) is also commonly used for detection of IBDV
serotypes, and less regularly, for differentiating IBDV subtypes (27, 260, 261, 262, 263).
A need for more rapid methods of characterizing the VP2 variable region in different
subtypes and strains of IBDV led to recent developments in molecular diagnostic
techniques based on RT-PCR technology (212). Initial approaches involved the use of
restriction enzymes (RE) (e.g. BstNI, MboI, and SspI) for the restriction fragment length
polymorphism (RFLP) analysis of RT-PCR products (263, 264, 265, 266, 267, 268, 269,
270, 271, 272). Designed to detect unique banding patterns associated with antigenic
variation within the VP2 encoding region, these methods were beneficial for placing
30
IBDV strains into molecular groups (13, 212, 244, 263, 266, 267). However, these
techniques later proved to be unreliable due to inconsistent correlations between RFLP
patterns of RT-PCR products and IBDV antigenic subtypes (212, 244, 273). Recent
developments of the RT-PCR technique include the use of multiplex RT-PCR or
quantitative real-time RT-PCR methods and DNA sequencing of RT-PCR products (212,
274, 275, 276, 277). Quantitative real-time RT-PCR methods allow for rapid, sensitive,
and accurate detection and differentiation of IBDV subtypes (classic, variant, and very
virulent) by subtype-specific primers and also provide a quantitative measure of the viral
load in test samples from poultry flocks (212, 274, 278, 279, 280).
Nucleotide and amino acid sequencing of the entire VP2 encoding region of many
IBDV field isolates is frequently used for comparison with previous strains and
phylogenetic analysis (14, 15, 59, 194, 215, 281). These comparative analyses are highly
useful for identifying putative amino acids within the central variable region that are
responsible for molecular and phenotypic variations among IBDV subtypes and strains
(21, 59, 73, 201, 211, 282, 283). Both variant and very virulent IBD viruses have
distinctive nucleotide and amino acid substitutions, relative to the classic subtype, that
can be rapidly detected by DNA sequencing of RT-PCR products (212). For example,
residues at positions 253, 279, and 284 of the VP2 protein sequence have been reported
to impact tissue culture infectivity (284, 285), virulence, and pathogenic phenotype (16,
18) of IBDV strains. Amino acids that significantly affect antigenicity are at position 222
and within the hydrophilic peak B region aa 314-324 (205, 286). Moreover, the
hydrophilic peak B region is involved in the formation of neutralizing epitopes (12, 59,
31
287). A serine-rich heptapeptide, S-W-S-A-S-G-S, (residues 326 to 332) is conserved in
various vvIBDV strains isolated in Japan, as well as, antigenic virulent variants isolated
in the U.S., and may influence virulence and pathogenicity. Mildly pathogenic and tissueculture-adapted strains contain fewer serine residues in this heptapeptide (12, 264, 285,
288, 289, 290).
Serological detection methods including the enzyme-linked immunosorbent assay
(ELISA) test, the agar gel precipitin (AGP) test, and the virus neutralization (VN) test are
often employed for confirmatory diagnosis of IBDV (47, 144, 212, 291). The AGP test
can be used for the rapid detection of IBDV group-specific soluble antigens or IBDV
antibodies in convalescent birds. While simple to perform, low sensitivity, the inability to
detect serotypic differences, and non-quantitative results are serious limitations (47, 212,
244). In contrast, the VN test is a more sensitive antibody detection tool and can be used
for both antibody quantitation and differentiation of IBDV serotypes and subtypes (23,
26, 206, 212, 292, 293). The ability of serum to neutralize a reference virus is determined
and a neutralization endpoint titer (VN titer) is expressed as the reciprocal of the highest
dilution of serum that prevents cytopathic effects (47, 212). VN tests are often employed
for IBDV identification in embryos and tissue culture after its adaptation to these host
systems and for assessment of antigenic and immunogenic changes (94, 190, 211, 212,
294, 295). VN tests serve to compare virus strains by evaluating the ability of antiserum
raised against one IBDV strain to neutralize a heterologous strain (212). However, only
IBDV strains that efficiently replicate in embryos or cell culture can be used in the VN
32
assay. Attempts to adapt the virus to a host may result in antigenic and pathological
changes to the virus that may render VN results as suspect (190, 212, 255)
Marquardt et al. (144) first described the indirect ELISA test for IBDV
antibodies, but it failed to differentiate between antiserum to IBDV serotypes I and II (24,
206). Moreover, many commercial ELISA kits detect antibodies to both serotypes and
vary considerably in their sensitivity and specificity (47, 212, 296, 297). The antigen
capture ELISA (AC-ELISA) system using monoclonal antibodies (MAbs) was developed
for characterizing the antigenic properties and relatedness of IBDV strains (66, 72, 202,
208, 210). Commonly used to measure IBDV serum antibody titers, this sensitive and
rapid method allows for differentiation of classic, variant, and vvIBDV subtypes (66,
113, 202, 205, 211, 298). These titers are useful for evaluating the flock immunity level
during an outbreak, as well as, the efficacy of a vaccination program (46, 47). Polyclonal
antibodies with AC-ELISA assays are more likely to be used for general screening of
tissue samples for IBDV (94, 244). The disadvantages to using such ELISA tests are the
extensive preparation of the MAbs, the cultivation of IBDV in CEF or chicken bursae,
and the fact that results do not always correlate with VN test findings (212).
In vivo cross-protection studies, vaccination-challenge studies, and progeny
challenge studies are frequently performed for assessment of IBDV vaccine efficacy and
to determine the pathogenicity and antigenic phenotypes of IBDV strains (26, 58, 194).
More recently, an IBDV reverse genetics system was implemented to introduce selected
amino acid changes into the VP2 encoding region of the classic IBDV strain D78 in order
to assess antigenic determinants of IBDV (21, 201, 299). This process combined with
33
nucleotide and amino acid sequencing and MAb reactivity patterns may provide a more
comprehensive analysis of IBDV strains for better diagnosis and vaccination program
design (300).
34
Chapter 3
MATERIALS AND METHODS
3.1 Chickens
Fertile specific pathogen free (SPF) white leghorn chicken eggs were obtained
from SPAFAS, Charles River Laboratories (Norwich, Connecticut). Eggs came from a
single breeder flock and were incubated at 37°C for 3 weeks prior to hatching. At oneday of age, the chicks were placed into negative-pressure, gloveport Horsfall isolator
units with diptanks in the Charles C. Allen Laboratory at the University of Delaware,
Newark, DE. Feed and water were provided ad libitum.
3.2 Chicken embryos
Fertile SPF chicken eggs were purchased from Sunrise Farms (Catskill, NY). The
eggs were incubated at 37°C for 9 to 11 days prior to inoculation with infectious bursal
disease virus (IBDV) for virus isolation and virus titration.
3.3 Viruses
IBDV reference strains STC and Delaware variant E (DelE) are maintained as
seed stocks at the University of Delaware. Thirty-three IBDV field isolates (Table 3.1)
were obtained from three- and four-week old commercial broiler chickens raised on the
35
Delmarva Peninsula. Five cloacal bursas from each commercial broiler submission found
to be IBD positive by histopathology and virus isolation were thawed at room
temperature and suspended in 5-7mL of tryptose phosphate broth (TPB) (Thermo Fischer
Scientific, Lenexa, KS) supplemented with antibiotics (Penicillin G 10,000 IU/mL,
Streptomycin Sulfate 10,000 μg/mL, and 25 μg/mL of Amphotercin) (MP Biomedicals,
LLC, Solon, OH). Bursas were homogenized using Tenbroeck tissue grinders and 1mL
aliquots were stored at -80°C. Subsequent RT-PCR was performed using original virus
stocks. Fifteen of the thirty-three field isolates found to be positive by histopathology,
virus isolation, and RT-PCR were designated for VP2 sequencing. IBDV variant vaccine
89/03 (Intervet-Schering Plough Animal Health, Millsboro, DE) and the classic PBG98
strain (Lohmann Animal Health, Harrisonburg, VA) of IBDV were propagated in chicken
embryo fibroblast (CEF) cells. At 48 hours post-inoculation (PI), cells and supernatant
fluids were harvested and aliquoted into 2.0 ml cryogenic vials to serve as viral stocks.
3.4 Virus Isolation
Frozen bursal samples were thawed and prepared as 20% weight to volume (w/v)
suspensions in TPB supplemented with antibiotics (Penicillin G 10,000 IU/mL,
Streptomycin Sulfate 10,000 μg/mL, and 25 μg/mL of Amphotercin) (MP Biomedicals,
LLC, Solon, OH) (244). Using Tenbroeck tissue grinders, the samples were homogenized
and frozen and thawed three times. Homogenates were centrifuged at 1000 rpm for 10
minutes and the supernatant fluid was collected. Ten-day-old embryonated SPAFAS eggs
were inoculated with 0.1mL of the supernatant fluid via the dropped chorioallantoic
36
membrane (CAM) route and incubated at 37°C. Eggs were candled daily for one week
and mortality was recorded. At 7 days post-inoculation (PI), the eggs were chilled at 4°C
for 24 hours. Embryos were examined for gross IBD lesions. Characteristic embryo
lesions of classic IBDV include early mortality (3-5 days PI), liver necrosis, hemorrhage,
a pale heart, edematous extension of the abdomen, and a pale spleen (25). Characteristic
lesions induced by variant IBDV strains include cream-colored, edematous embryos with
bile stasis of the liver, liver necrosis, splenomegaly and limited mortality (25). Any
mortality within the first 24 hours post-inoculation was considered non-specific and the
eggs were discarded.
3.5 Tissues and Histopathology
Tissue samples collected from commercial broiler flocks and from experimental
SPF leghorn chickens inoculated with control and field isolates were fixed in 10%
volume to volume (v/v) neutral buffered formalin (Richard-Allan Scientific, Kalamazoo,
MI). Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained
with hematoxylin and eosin (190, 255). Tissues were microscopically evaluated on the
basis of the extent of necrosis and degeneration of follicular lymphocytes, the presence of
plical, follicle, and muscle wall edema, hyperemia, and heterophilic inflammation and
infiltration (226). Cloacal bursa lesions were scored based on the following system: 1 =
normal, no IBDV related lesions; 2 = mild, scattered IBDV related cell depletion in a few
follicles; 3 = moderate, 1/3 to 1/2 of the follicles with IBDV related atrophy or depletion
of follicular lymphocytes; and 4 = severe atrophy of all the follicles, inflammation, and
37
acute necrosis (301). Bursal scores of 1 and 2 were considered protected and scores of 3
and 4 were considered unprotected.
Thymus tissues were evaluated based on the degree of cortical lymphocyte loss,
thymocyte apoptosis, and atrophy of thymic cells. Spleens were assessed for increased
germinal center formation, as well as, lymphoid depletion in the germinal centers and
around the periellipsoid and periarteriolar lymphoid sheath (197).
3.6 Viral RNA Extraction
Field isolates producing microscopic pathology consistent with IBDV infection
were designated for molecular testing. Bursal homogenates were treated in equal v/v
ratios of phenol/chloroform with isoamyl alcohol (Fischer Scientific, Fair Lawn, NJ).
Samples were vortexed and centrifuged at 17,000 rcf for 10 minutes. The supernatant was
removed and used in the Qiagen RNeasy Mini kit (QIAGEN Operon, Valencia, CA) to
extract total RNA, as per the manufacturer’s instructions.
3.7 Reverse Transcription – Polymerase Chain Reaction (RT-PCR)
IBDV RT-PCR screening of the bursal homogenates was conducted using a
modified version of the GeneAmp RNA PCR kit (Perkin Elmer, Roche Molecular
Systems, Branchburg, NJ) protocol. Duplicate reactions were run for each isolate.
Denaturation of dsRNA was performed using 4μL of 90% Dimethylsulfoxide (ATCC,
Manassas, VA), 2μL of random hexamers, and 4μL of dsRNA extracted from IBDV
isolates. Complementary DNA (cDNA) was synthesized from viral RNA in the same
38
tube by the addition of 32μL of a reverse transcriptase master mix containing 4μL of
MgCl, 2μL of 10x PCR buffer, 8μL of DXTP mixture, 1μL of RNase inhibitor, and 1μL
of reverse transcriptase. Twenty-one microliters of sample were removed and placed in a
200μL PCR tube to serve as a duplicate PCR reaction. Seventy-eight microliters of a
PCR master mix containing 4μL of MgCl, 8μL of 10x PCR buffer, 65.5μL of DEPC
treated water, and 0.5μL of Taq polymerase, were added to each tube. Two microliters of
the forward primer 5’-GCCCAGAGTCTACACCAT-3’ and reverse primer 5’CCCGGATTATGTCTTTGA-3’ (59) set were also added to each tube (Eurofins MWG
Operon, Huntsville, AL). RT and PCR were conducted using the AB Applied Biosystems
2720 Thermal Cycler and the PE Applied Biosystems GeneAmp PCR System 9700
Version 2.25. Thermal cycling was performed in three sequential phases. The cycle
parameters for the denaturation phase of dsRNA consisted of one 94°C hold for 7
minutes. The RT phase involved one 42°C hold for 42 minutes, followed by a 99.9°C
hold for 5 minutes. The PCR cycle parameters were as follows: one 94°C hold for 2
minutes; 35 cycles at three temperatures (respectively): 95°C for 40 seconds, 53°C for 40
seconds, and 69°C for 50 seconds; one 72°C hold for 7 minutes, followed by a 4°C hold.
PCR products were electrophoresed in 1.8% GenePure LE Agarose (ISC BioExpress,
Kaysville, UT) containing ethidium bromide (25ng/mL) (MP Biomedicals, Inc., Solon,
OH) and observed under UV light. Bands were excised from the gel and DNA was
extracted according to the IllustraTM GFX PCR DNA and Gel Band Purification Kit, as
per the manufacturer’s instructions (GE Healthcare UK Limited, Buckinghamshire, UK).
39
DNA was resuspended in 50μL of elution buffer and quantitated using the NanoDrop
1000 Spectrophotometer (Thermo Scientific, Wilmington, DE).
3.8 Genome Sequencing and Analysis
Genome sequencing was performed at the University of Delaware Sequencing
and Genotyping Center, Newark, DE. Forward primer 5’-GCCCAGAGTCTACACCAT3’ and reverse primer 5’-CCCGGAATTATGTCTTTGA-3’ specific for the VP2
encoding region (59) was provided at 2pm/μL (Eurofins MWG Operon, Huntsville, AL).
Comparative analysis of IBDV VP2 nucleotide and protein sequences was conducted by
creating a CLUSTAL W alignment (MegAlign; Version 8.0.2; DNAStar, Inc., Madison,
WI). Amino acid identity values were determined based on amino acid sequence data of
fifteen IBDV infected bursal homogenates, IBDV variant strains, DelE (Delmarva
Peninsula, University of Delaware), GLS (Delmarva Peninsula, University of Maryland),
and AL-2 (Perdue Farms Inc., Vangs Farm, NC), variant vaccine strain, RS593 (Foster
Farms Inc., CA), and classic challenge strain, STC (USDA-APHIS, Ames, IA) (Table
4.1). Deduced amino acid sequences from the fifteen RT-PCR positive IBDV samples
(Table 3.2) were analyzed by Dr. Daral Jackwood (The Ohio State University).
Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 (302) and the
unweighted pair-group method using arithmetic averages (UPGMA) method were used to
conduct a VP2 phylogenetic analysis (303). The phylogenetic tree was evaluated for
evolutionary relationships between the 14 field isolates and existing classic and variant
strains.
40
3.9 Cultivation of Seed Stocks
Seed stocks were made for six IBDV field isolates (DMV/4813/07,
DMV/4947/07, DMV/4955/07, DMV/5036/07, DMV/5038/07, DMV/5041/07) and two
IBDV reference strains (DelE and STC) (Table 3.3). Seventy-one, 1-day-old SPF leghorn
chickens were divided into eight groups of seven birds each and one negative control
group of fifteen birds. Birds were placed into negative-pressure, Horsfall isolator units in
the Allen Laboratory at the University of Delaware, Newark, DE and provided food and
water ad libitum. Groups were challenged on separate days to avoid cross-contamination.
For each isolate, seven chickens were inoculated via ocular and intranasal routes with
250μL of viral inoculum prepared from bursal homogenates as previously described.
Chickens were evaluated daily for the development of clinical signs and mortality. Three
days post-inoculation (PI), five birds and one negative control bird were euthanized via
cervical dislocation. Birds were assessed for gross IBDV-specific lesions and cloacal
bursas were removed.
From each harvested bursa, a small section of tissue was cut and placed in 10%
volume to volume (v/v) neutral buffered formalin (Richard-Allan Scientific, Kalamazoo,
MI) for histopathology. Remaining bursal tissues were pooled according to virus
treatment and homogenates were prepared as 20% weight to volume (w/v) suspensions in
TPB containing antibiotics (Penicillin G 10,000 IU/mL, Streptomycin Sulfate 10,000
μg/mL, and 25 μg/mL of Amphotercin) (MP Biomedicals, LLC, Solon, OH). Bursas were
homogenized using a sterile electric tissue grinder (PRO Scientific Inc.) and subjected to
1 freeze-thaw cycle. Tubes were incubated at room temperature for 1 hour and
41
centrifuged at 4°C for 30 to 40 minutes at 2500 rpm to pellet cellular debris. Supernatant
was aliquoted into sterile 2 mL cryogenic vials (Fisher Scientific, Fair Lawn, NJ) to serve
as virus seed stocks and stored at -80°C. Viral material from a single aliquot was
streaked onto a nutrient agar plate to screen for bacterial contamination. The following
day, plates were examined for bacterial growth and results were recorded.
Viral RNA was extracted from the seed stock preparations, amplified by RT-PCR,
and sequenced, as previously described. Nucleotide and amino acid sequence results for
each virus strain were compared with the initial sequence data to determine if mutations
had occurred. In addition, each viral seed stock was tested for potential contamination
with infectious bronchitis virus (IBV) (304, 305), reovirus (306), Newcastle disease virus
(NDV) (307) using RT-PCR, and adenovirus (308) using PCR.
3.10 Antiserum Production
During the production of seed stocks, two birds were retained in each treatment
group for serum antibody production. At 4 weeks PI, blood was collected via cardiac
puncture. Clotted blood samples were incubated at room temperature for 1 to 2 hours and
were then placed at 4°C for 24 hours. Samples were centrifuged at 1000 rpm for 15
minutes and the serum was harvested and pooled according to virus treatment. Sera were
incubated at 56°C for 30 minutes to inactivate complement. Stocks of antisera were
maintained in aliquots in sterile 2 mL cryogenic vials and stored at -20°C.
Serum was tested for the presence of IBDV specific antibodies using the agar gel
precipitin (AGP) test (38). Agar was prepared by dissolving 1 gram of 1% Bacto agar
42
(Becto, Dickinson and Company, Sparks, MD) and 8 grams of NaCl (Fisher Scientific,
Fair Lawn, NJ) in 100 mL of distilled water by heating. Ten to twelve mL of melted agar
were poured into 60mm x 15mm plates. After solidifying at room temperature for 12
hours, seven 5 mm holes were punched into the gel. For each plate, National Veterinary
Services Laboratory (NVSL) IBD Antiserum (APHIS, Ames, IA) was loaded in each of
the three alternate outer wells. Antisera from the field isolates were placed in the
remaining three alternate outer wells. The center well contained NVSL IBD Antigen
(APHIS, Ames, IA). Each of the wells was loaded with 12μL of material four times,
allowing for adequate absorption between each loading. Plates were incubated at room
temperature and observed for precipitin lines of identity consistent with positive control
samples for up to 72 hours. Results were recorded daily.
3.11 Virus Titration
Ten-fold, serial dilutions (10-1 to 10-7) were prepared in TPB containing
antibiotics (Penicillin G 5000 IU/mL, Streptomycin Sulfate 5000 μg/mL, and 12.5 μg/mL
of Amphotercin) and incubated for 1 hour at room temperature. Ten to eleven day old
embryonated chicken eggs were inoculated via the chorioallantoic membrane route with
0.1 mL of virus dilution per egg. Five embryos were inoculated for each dilution, and
candled daily. Any mortality within the first 24 hours was discarded, and subsequent
mortality was held at 4°C. At one week PI, living embryos were chilled at 4°C for 24
hours. The following day, embryos were examined for gross IBDV lesions. Virus titers
43
were calculated for each IBDV seed stock by the Reed and Müench method (309) and
expressed as embryo infectious dose (EID50/mL).
3.12 Monoclonal Antibody Characterization
Bursal-derived seed stocks were analyzed by Dr. Egbert Mundt, at the University
of Georgia, using a panel of IBDV-specific monoclonal antibodies (MAbs) 57, 63, 67,
69, and 10 by an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) (72,
207, 208) as described by Icard et al. (201). Polyclonal rabbit VP1 antiserum served as a
control for transfection and monoclonal antibody (MAb) tests were performed in
duplicate (21, 122). With this system, the VP2 encoding region of the IBDV field isolate
is cloned into an IBDV backbone (D78) and transfected into cell culture along with other
viral elements. Successful cotransfection of cRNA, as indicated by VP1 fluorescence,
results in IBDV infection of the cells. Cells are reacted with the panel of IBDV-specific
MAbs and processed for immunofluorescence.VP2 epitopes of the IBDV field isolates
are determined by a positive or negative reactivity pattern and compared to those of
IBDV reference strains (Table 3.4) (201).
MAb testing using a whole virus and ELISA assay was performed by the Poultry
Service Laboratory of Intervet/Schering-Plough Animal Health as described by Snyder et
al. (66) and Intervet-Schering Plough Animal Health. In this system, the whole viral
genome of the IBDV field isolate is reacted directly with the panel of monoclonal
antibodies. Included in this panel were IBDV-specific MAbs 10, 57, 63, 67, and 69.
MAbs B29, 8, and BK9 were used as positive controls. MAb testing was performed on
44
the original bursal homogenates from field cases, as well as, the bursal-derived seed
stocks (Intervet-Schering Plough Animal Health).
To determine the binding ability of each field virus to the specific monoclonal
antibodies, absorbance data was used to calculate a MAbX/MAb B29 binding index,
where MAbX is the specific MAb that was tested and MAb B29 reacts with an epitope
found in all IBDV (positive control). The MAb index for each IBDV field virus was
calculated based on the following formula: Absorbance of virus antigen against MAb X –
Absorbance of MAb X normal bursa / Absorbance of the virus antigen against MAb B29
– Absorbance of MAb B29 normal bursa. A value that was greater or equal to 0.7 was
expressed as positive (+). A value that was between 0.3 and 0.69 demonstrated moderate
to low binding activity. A value that was equal to or less than 0.29 was expressed as
negative (-) (Table 3.5) (310).
As a quality control and for comparison, six control samples were run
simultaneously. These included the previously characterized variant viruses, DelE (56,
57) GLS (66), and AL-2 (310), one variant vaccine strain, RS593 (Intervet, Inc.,
Millsboro, DE), one classic strain, STC (APHIS, Ames, IA), and normal bursa as a
negative control (Table 3.5).
3.13 Vaccines
Two commercially available live vaccine strains, 89/03 (Intervet-Schering Plough
Animal Health, Millsboro, DE) and PBG98 (Lohmann Animal Health, Harrisonburg,
VA) were obtained. Vaccine 89/03 contains an attenuated IBDV serotype 1 variant strain
45
(Delaware variant E) that elicits cross-protection against Delaware variant E, STC, GLS,
and RS593 strains of IBDV (311). PBG98 contains an attenuated IBDV serotype 1
classic strain (312).
3.14 Cell Cultures
Chicken embryo fibroblast (CEF) cells were prepared from 9- to 11-day-old
fertile SPF chicken eggs (Sunrise Farms, Catskill, NY) as previously described (313). A
10ml volume of 2x trypsin was added to tissue fragments prior to trypsinization for 7
minutes in a 37°C shaker cabinet. Cells were collected in a mesh filtered flask containing
5% fetal bovine serum (FBS) (Hyclone Laboratories, Inc, Logan, UT). Remaining tissue
fragments were re-trypsinized for 5 minutes in 10ml of 2x trypsin. Collected cells were
centrifuged for 10 minutes at 1000 rpm and trypsin solution was discarded. Pelleted cells
were resuspended in medium 199 (M199) and F10 medium (Hyclone Laboratories, Inc,
Logan, UT) supplemented with antibiotics (Penicillin G 100 IU/mL, Streptomycin
Sulfate 100 μg/mL, and 0.25 μg/mL of Amphotercin) (MP Biomedicals, LLC, Solon,
OH) and 5% FBS at a 1:500 dilution.
3.15 Virus-neutralization
Virus-neutralization (VN) antibody titers were determined for IBDV field isolates
4813, 4947, 4955, 5038, 5041 against IBDV strains 89/03 and PBG98. Beta-VN assays
(constant-virus diluted-serum) were performed using viral stocks diluted in cell-culture
medium to contain 100 TCID50. To confirm that 100 TCID50 per 0.1ml was used, back-
46
titrations of the viruses were performed with each virus neutralization test. Antiserum
collected from birds inoculated with IBDV field isolates was diluted in cell culture
media. After an initial 1:100 dilution, serial two-fold dilutions (1:100 to 1:12800) were
prepared in 96-well flat-bottomed microplates (Costar, Cambridge, MA) in duplicate.
Each well contained 25µL of diluted serum, to which was added 25µL of virus
suspension containing 100 TCID50 per 0.1ml. Back titrations of the viruses using known
negative control serum from SPF leghorn chickens (SPAFAS Charles River Laboratories,
Norwich, CT) were performed to verify that the test virus dose contained 100 TCID50.
Plates were incubated for 1 hour at 37°C before 100µl of CEF cells diluted 1:500 was
added to each well. Cell cultures were incubated at 37°C in 5% CO2 atmosphere and
examined at 48 hours PI. Cell culture media was discarded and 100 ul of 10% volume to
volume (v/v) neutral buffered formalin (Richard-Allan Scientific, Kalamazoo, MI) were
added to the wells of the microtiter dishes. Cells were fixed for 5 minutes and formalin
was discarded. Cells were stained with 250 ul of 0.05% Crystal Violet (CV) (Fischer
Scientific, Fair Lawn, NJ) in distilled water for 30 minutes and CV was discarded.
Microtiter dishes were washed 2 times with tap water and drained for 2-3 minutes. Cell
cultures were scored as positive or negative for cytopathogenic effect (CPE). Virusneutralizing antibody titers were determined based on the reciprocal of the highest
dilution of serum that neutralized 100 TCID50 of IBDV (244). A four-fold difference in
neutralization of the viruses was considered to be significant (J. Gelb, Jr., personal
communication, March 11, 2010).
47
3.16 Challenge of Immunity
IBDV vaccine 89/03 was diluted in TPB containing antibiotics (Penicillin G 5000
IU/mL, Streptomycin Sulfate 5000 μg/mL, and 12.5 μg/mL of Amphotercin) according to
the manufacturer’s instructions. Seven groups of fifteen SPF leghorn chickens per group
were vaccinated at one day of age. Each chicken was subcutaneously injected in the back
of the neck with 0.2 ml of the vaccine. Seven groups of five birds per group served as
unvaccinated challenge controls, and they were subcutaneously injected in the back of the
neck with 0.2 ml of TPB. Unvaccinated challenge controls and IBDV vaccinated
chickens were raised separately until challenge. At 21 days of age, unvaccinated
challenge controls and IBDV vaccinated chickens were re-housed in the same isolation
unit according to the specific challenge virus to be administered (Table 3.6). Blood
samples were collected from 10 birds per group and serum was harvested, as previously
described. Each treatment group was challenged via eye drop with 102.0 TCID50/0.1 ml of
the following IBDV field isolates: 4813p1; 4947p1; 4955p1; 5038p1; and 5041p1.
Reference strains, STC and Delaware variant E (Del E), served as positive controls. One
group of fifteen birds was not challenged and served as non-vaccinated, non-challenged
controls. At 28 days of age (7 days post-challenge), all birds were bled, euthanized, and
weighed. Tissues (cloacal bursa, thymus, spleen) were removed from each bird, and
cloacal bursas were weighed and assessed for IBDV gross lesions. Sections of each tissue
were placed in 10% volume to volume (v/v) neutral buffered formalin solution (RichardAllan Scientific, Kalamazoo, MI) for histological evaluation. Birds with gross or
microscopic bursa lesions and bursa to body weight indices of less than two standard
48
deviations below the mean of the negative control group were considered unprotected
against challenge. IBDV-specific antibody levels in serum were evaluated prior to
challenge at 21 days of age and at 7 days post-challenge by the IBDV Antibody Test Kit
(IDEXX Laboratories Inc., Westbrook, ME), as per the manufacturers’ instructions.
A second vaccine challenge study was performed with PBG98 (Table 3.7).
3.17 Progeny Challenge
A progeny challenge study was performed in collaboration with
Intervet/Schering-Plough Animal Health, Millsboro, DE. Three groups of 18-week-old
SPF leghorn breeders were vaccinated with a licensed inactivated IBDV vaccine
containing classic and variant strains, as well as, a strain of reovirus (Intervet/ScheringPlough Animal Health) (Table 3.8). Each leghorn breeder was given a single
intramuscular (IM) injection of vaccine (0.5mL per bird) in the breast muscle. No live
priming vaccination was administered. Progeny chicks were hatched and housed in
filtered-air negative-pressure isolation units. Two groups of SPF (IBDV antibody
negative) leghorn chickens served as non-vaccinated challenge controls, while a third
served as non-vaccinated, non-challenged controls. Progeny and SPF controls were
challenged via the intraocular route with 102.0 EID50 per bird at 10 and 17 days of age
with IBDV field isolate 5038 and IBDV reference strain Del E. Isolate 5038 was selected
for the progeny challenge trial based on its lack of binding with neutralizing monoclonal
antibodies (Tables 3.4 and 3.5). At 7 days post-challenge, all birds were euthanized, and
bursa and body weights were measured. Tissue sections of each bursa were placed in
49
10% volume to volume (v/v) neutral buffered formalin solution (Richard-Allan
Scientific, Kalamazoo, MI) for histological evaluation. Birds with microscopic bursa
lesions and bursa to body weight indexes of less than two standard deviations below the
mean of the negative control group were considered unprotected against challenge.
3.18 Bursa to Body Weight Calculations and Statistical Analysis
A bursa of Fabricius/body weight ratio for each bird was calculated according to
the following formula: bursa weight in grams X 1000/body weight in grams (31). The
geometric mean and standard deviation of bursa/body weight ratios was determined for
each treatment group and the negative control group. A percent protection score was
determined based on the percentage of birds within a treatment group that were protected
(314). The significance of differences in percentages was analyzed by two-tailed chisquare tests (P < 0.05).
50
Table 3.1. Accession numbers and broiler flock information for IBDV isolation
attempts.
Accession
#
07-4810
07-4811
07-4813
07-4814
07-4815
07-4816
07-4901
07-4903
07-4906
07-4907
07-4908
07-4909
07-4910
07-4911
07-4912
07-4913
07-4947
07-4948
07-4950
07-4952
07-4955
07-4956
07-4957
07-5032
07-5033
07-5034
07-5036
07-5037
07-5038
07-5040
07-5041
07-5042
07-5043
07-4810
Positive
Negative
Negative
Positive
Negative
Negative
Negative
Positive
Positive
Negative
Positive
Negative
Negative
Negative
Virus
Isolation (VI)
N/AA
N/A
IBDV
N/A
N/A
IBDV
N/A
N/A
N/A
IBDV
IBDV
N/A
None
N/A
N/A
N/A
Positive
Negative
Negative
Positive
Positive
Positive
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Negative
IBDV
N/A
N/A
IBDV
IBDV
IBDV
N/A
IBDV
IBDV
IBDV
None
IBDV
IBDV
IBDV
IBDV
None
IBDV
N/A
Grower Name
Submitted
Histology
Atlanta
Ball Farm
Schmick
Lynn McColley
Lynn McColley
Charles Moore
Lowry Farm
Dennis Shockley
Dennis Shockley
Bilden
Bilden
Charles Moore
Alan Ralph
Bruce Clark
Atlanta
Foster Farm
Oak Hall
Oak Hall
Ball Farm
Schmick
Charles Moore
Schmick
McColley
Bilden
Bilden
Charles Moore
Bruce Clark
Dennis Shockley
Dennis Shockley
Lowery
Foster
Oak Hall
Oak Hall
Atlanta
8/7/2007
8/7/2007
8/7/2007
8/7/2007
8/7/2007
8/7/2007
8/9/2007
8/9/2007
8/9/2007
8/9/2007
8/9/2007
8/9/2007
8/9/2007
8/9/2007
8/9/2007
8/9/2007
8/10/2007
8/10/2007
8/10/2007
8/10/2007
8/10/2007
8/10/2007
8/10/2007
8/14/2007
8/14/2007
8/14/2007
8/14/2007
8/14/2007
8/14/2007
8/14/2007
8/14/2007
8/14/2007
8/14/2007
8/7/2007
Negative
Negative
A
VI not performed.
51
Age
(weeks)
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
3
Table 3.2. IBDV RT-PCR results from screening of the bursal homogenates from 3- and
4-week-old commercial broiler chickens raised on 12 farms in the Delmarva Peninsula
region.
Accession #
07-4813
07-4816
07-4947
07-4952
07-4955
07-4956
07-5032
07-5033
07-5034
07-5036
07-5037
07-5038
07-5040
07-5041
07-5042
07-5043
07-4907
07-4908
07-4910
Bursal
Histology
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Virus
Isolation
IBDV
IBDV
IBDV
IBDV
IBDV
IBDV
IBDV
IBDV
IBDV
None
IBDV
IBDV
IBDV
IBDV
None
IBDV
IBDV
IBDV
None
52
Age (weeks)
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
3
3
3
RT-PCR
Result
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Negative
Negative
Negative
Negative
Table 3.3. Propagation of Selected Field Viruses for Seed StocksA.
IBDV Strain
Isolator Group
N/A
Neg. Control
Negative Control
N/A
Clade 1
4813
1
Yes
Clade 2
5038
2
Yes
Clade 3
4947
3
Yes
Clade 4
Clade 5
4955
4
Yes
5036
5
No
Clade 6
5041
6
Yes
Classic Reference strain
STC
7
Yes
Variant Reference strain
DelE
8
Yes
53
Virus Clade
Viable Virus
ReisolatedB
A
One IBDV field isolate from each of the six clades identified in the phylogenetic tree was randomly selected for propagation
in SPF leghorn chickens. Classic challenge virus, STC, and Delaware variant E (DelE) served as reference strains.
B
Confirmed by histopathological examination.
53
Table 3.4. Monoclonal antibody (MAb) reactivity patterns of infectious bursal disease virus field isolates using VP2
transfection and immunofluorescence assaysA.
MAb and dilution tested
54
10
57
63
67
69
VP1 hisE
IsolateBC
4813p1
(1:50)D
-
(1:50)
-
(1:400)
+
(1:50)
+
(1:50)
-
(1:200)
+
4813p2
-
-
+
+
-
+
4947p1
-
-
-
-
-
+
4947p2
4955p1
-
-
-
-
-
+
-
-
-
+
-
+
4955p2
-
-
-
+
-
+
5038p1
-
-
-
-
-
+
5038p2
-
-
-
-
-
+
5041p1
-
-
-
-
-
+
5041p2
-
-
-
-
-
+
A
Performed by Egbert Mundt, University of Georgia.
Bursal-derived seed stocks of each field isolate were used in the AC-ELISA.
C
Two plasmids were constructed for each field isolate; p1 and p2.
D
MAbs diluted in phosphate buffered saline.
E
Rabbit VP1 antiserum (122) used as a control for transfection.
B
54
Table 3.5. Monoclonal antibody (MAb) reactivity patterns of infectious bursal disease virus field isolates and IBDV reference
strains using whole virus/ELISA assaysA.
MAb
55
Isolate
4813origA
4813p1B
4947orig
4947p1
4955orig
4955p1
5038orig
5038p1
5041orig
5041p1
STCC
DelED
GLSD
RS593D
AL2D
Negative
Control
10
+
+
-
57
+
+
+
63
+
+
+
+
-
67
+
+
+
+
+
+
+
69
+
-
B29
+
+
+
+
+
+
+
+
+
+
+
+
8
+
+
+
+
+
+
+
+
+
+
+
BK9
+
+
+
+
+
-
-
-
-
-
-
-
-
-
A
Performed by Intervet/Schering-Plough Animal Health, Millsboro, DE.
Original and p1 (single passage seed stocks in SPF chickens) bursal homogenates of each field isolate were used in the ACELISA.
C
Classic virus STC (USDA-APHIS, Ames, IA).
D
Variant viruses Delaware Variant E (Delmarva Peninsula, University of DE), GLS (Delmarva Peninsula, University of MD),
RS593 (ISPAH, Millsboro, DE) and AL-2 (Perdue Farms Inc., Vangs Farm, NC).
B
55
Table 3.6. Vaccination-challenge of SPF leghorns using Delaware variant vaccine 89/03.
56
Vaccine
Vaccination Age
No. Birds
Challenge Virus
Challenge TiterC (EID50/bird)
89/03A
1 dayB
15
4813p1
102.0
89/03
1 day
15
4947p1
102.0
89/03
1 day
15
4955p1
102.0
89/03
1 day
15
5038p1
102.0
89/03
1 day
15
5041p1
102.0
89/03
1 day
15
STC
102.0
89/03
None
1 day
15
Del E
102.0
n/a
5
4813p1
102.0
None
n/a
5
4947p1
102.0
None
n/a
5
4955p1
102.0
None
n/a
5
5038p1
102.0
None
n/a
5
5041p1
102.0
None
n/a
5
STC
102.0
None
n/a
5
Del E
102.0
None
n/a
15
None
n/a
A
Intervet/Schering Plough Animal Health, Millsboro, DE
0.2 mL per bird via subcutaneous injection.
C
0.1 mL per bird via the intraocular route at 21 days of age.
B
56
Table 3.7. Vaccination-challenge of SPF leghorns using classic vaccine PBG98.
57
Vaccine
Vaccination Age
No. Birds
Challenge Virus
Challenge TiterC (EID50/bird)
PBG98A
1 dayB
15
4813p1
102.0
PBG98
1 day
15
4947p1
102.0
PBG98
1 day
15
4955p1
102.0
PBG98
1 day
15
5038p1
102.0
PBG98
1 day
15
5041p1
102.0
PBG98
PBG98
1 day
15
STC
102.0
1 day
15
Del E
102.0
None
n/a
5
4813p1
102.0
None
n/a
5
4947p1
102.0
None
n/a
5
4955p1
102.0
None
n/a
5
5038p1
102.0
None
n/a
5
5041p1
102.0
None
n/a
5
STC
102.0
None
n/a
5
Del E
102.0
None
n/a
15
None
n/a
A
Lohmann Animal Health, Harrisonburg, VA
0.2 mL per bird via subcutaneous injection.
C
0.1 mL per bird via intraocular route at 21 days of age.
B
57
Table 3.8. Progeny challenge using progeny of 18-week-old SPF leghorn breeder chickens vaccinated with a licensed
inactivated IBDV vaccine containing classic and variant strains, as well as, a strain of reovirus and challenged with IBDV
field isolate 5038 and reference strain Delaware variant E.
58
Vaccine
Age of Hens at
VaccinationB
Age of Hens
at Lay
No. Birds
Challenge Virus
Challenge TiterC (EID50/bird)
AA
18 weeks
34 weeks
12
5038p1
102.0
A
18 weeks
34 weeks
12
Del E
102.0
A
18 weeks
34 weeks
12
none
n/a
Non-vaccinated
n/a
34 weeks
12
5038p1
102.0
Non-vaccinated
n/a
34 weeks
12
Del E
102.0
Non-vaccinated
n/a
34 weeks
12
none
n/a
A
Intervet/Schering Plough Animal Health, Millsboro, DE
0.5 mL per bird by intramuscular injection.
C
0.1 mL per bird via the intraocular route.
B
58
Chapter 4
RESULTS
4.1 VP2 Sequence Analysis
To assess genetic characteristics of IBDV field isolates and identify specific
viruses for further characterization, VP2 sequencing and comparative analysis was
performed. A primer pair that amplifies a 743 base pair fragment of the VP2 encoding
region was used to sequence the variable regions of IBDV field isolates and five IBDV
reference strains. Fifteen deduced amino acid sequences of the major structural VP2
protein were obtained from RT-PCR positive samples (Table 3.2). Percent amino acid
sequence identity values among the field isolates, reference strains, STC (USDA-APHIS,
Ames, IA), Delaware Variant E (Delmarva Peninsula, University of DE), GLS (Delmarva
Peninsula, University of MD), AL-2 (Perdue Farms Inc., Vangs Farm, NC) and variant
vaccine strain, RS593 (Intervet/Schering-Plough Animal Health, Millsboro, DE) are
shown in Table 4.1. Field isolates showed less than a 6% divergence from the classic
STC strain and less than a 3% divergence from the Delaware variant E strain. The
isolates showed less than a 5% divergence from GLS and less than a 4% divergence from
AL-2 and RS593 (Table 4.1). A VP2 phylogenetic tree was constructed by comparing
deduced amino acid sequences of the field isolates with known VP2 sequences contained
59
in an IBD database (Figure 4.1). All of the field isolates clustered under the Delaware
variant E strain and were distributed into six distinct clades (Figure 4.1 and Table 3.3).
Analysis of the VP2 sequences of IBDV field isolates and reference strains showed that
the majority of the amino acid substitutions were observed in major hydrophilic peaks A
(aa 210-225) and B (aa 312-325) and minor hydrophilic peaks 1 (aa 248-252) and 2 (aa
279-289) (Figure 4.2) (9, 15, 112, 113, 114, 115)
4.2 Propagation and Pathology of Selected Viruses for Seed Stocks
Five field isolates (4813, 4947, 4955, 5038, and 5041) representative of each of
the six clades determined in the phylogenetic analysis were selected for propagation in
SPF leghorn chickens. In addition, two IBDV reference strains, STC and Delaware
variant E (Del E) were used for the cultivation of seed stocks (Table 3.3). As a result of
STC infection, clinical signs included ruffled feathers, reddish, almond-shaped eyes,
watery diarrhea, and depression. Upon harvest at three days post-inoculation, cloacal
bursas appeared slightly enlarged, round or oval-shaped, cream-colored, and contained
longitudinal striations and a gelatinous transudate around the serosal surface. Spleens
were normal in size and color. Birds infected with Del E and IBDV field isolates showed
minimal clinical signs; however, ruffled feathers and depression were observed with
some birds. Cloacal bursas were normal to small in size, firm, and displayed slight
mucosal edema. Slight splenomegaly was observed with some birds. Histological data
demonstrated that the two IBDV reference strains and five of the six propagated IBDV
field isolates were viable, and they were subsequently used for further analysis.
60
4.3 Monoclonal Antibody Testing
To characterize the antigenic properties of the IBDV field isolates 4813, 4947,
4955, 5038, and 5041 and to determine their relatedness to classical and variant IBDV
strains, two antigen capture enzyme-linked immunosorbent assays (AC-ELISA) were
performed using IBDV-specific monoclonal antibodies (MAbs) (15, 21, 66, 114, 207).
MAb reactivity pattern results from the MAb test using a transfection and
immunofluorescence assay system (E. Mundt, University of Georgia) are presented in
Table 3.4. All of the isolates reacted with the polyclonal anti-VP1 rabbit serum,
indicating that cotransfection of cRNA was successful and the tests were valid (E. Mundt,
unpubl. data; 21). Epitope B69 was absent from the five field isolates, thereby
differentiating them from the classic strains of IBDV. Isolate 4813 reacted with MAb 63
and MAb 67, and isolate 4955 showed a positive reactivity to MAb 67. Isolates 4947,
5038, and 5041 displayed no reactivity with MAbs 10, 57, 63, 67, and 69.
MAb reactivity pattern results from the MAb test using a whole virus/ELISA
assay system (P. Lynch, Intervet/Schering-Plough Animal Health) are presented in Table
3.5. Original bursal samples obtained from commercial broilers of 4947, 4955, and 5041
isolates did not react with MAbs B29, 8, or BK9; however, bursal samples taken from
one passage (p1) of 4955 and 5041 in SPF chickens did react with B29 and 8 MAbs. The
p1 sample of 4947 only reacted with MAb B29. Epitope B69 was absent from the five
field isolates, again, differentiating them from the classic strains of IBDV. Isolate 4813
shared a similar reaction pattern with Delaware variant E (Del E) in its binding of MAbs
R63 and 67. Isolate 4947 showed a similar reaction pattern to RS593 in its binding of
61
MAbs B29 and 67. Isolate 4955 reacted with MAb 67 in both assays. In addition, MAbs
B29 and 8 reacted with isolate 4955, which together demonstrated its similarity to the
IBDV variant vaccine strain RS593. Isolate 5041 shared a similar reaction pattern to the
GLS variant in its binding of MAbs 57 and 10. Aside from a reaction with B29, 8, BK9,
and VP1 MAbs, IBDV field isolate 5038 displayed no reactivity with the other MAbs
used in the panel.
4.4 Monoclonal Antibody Epitope Mapping
Correlations between predicted amino acid substitutions and monoclonal antibody
(MAb) reactivity patterns were previously described by Vakharia et al. (15) and Sellers et
al. (114) (Tables 4.2 – 4.6). A glutamine at position 249 has been reported to be essential
for the binding of MAb 69 (Table 4.2). All five field isolates had a glutamine to lysine
substitution at this position and escaped the binding of neutralizing MAb 69. Amino acids
likely to be important in the binding of MAb 10 are threonine at position 286, glycine at
position 318, and aspartic acid at position 323 (Table 4.3). This is supported by the
positive reactivity with reference strains STC and GLS. Isolate 5038 and reference strain
RS593 differ by only one amino acid (i.e. isolate 5038 showed an aspartic acid to
glutamic acid substitution at position 323, and RS593 showed a threonine to isoleucine
substitution at position 286) and escaped the binding of MAb 10.
The reactivity of the MAb 57 epitope has been previously shown to depend on a
glutamic acid at position 321, which is supported by the positive reactivity with isolate
5041, and reference strains GLS and AL-2 (Table 4.4). Interestingly, isolate 5038
62
contained a glutamic acid residue at position 321, but did not react with MAb 57. This
suggests that recognition of the MAb 57 epitope may involve other critical amino acid
residues. A glutamic acid to alanine substitution at position 321 appears to inhibit the
binding of MAb 57 and allows for the binding of MAb 63, as previously reported by
Letzel et al. (21). This is supported by the positive reactivity with isolate 4813 and
reference strains Del E and STC. Isolates 4947 and 4955, and reference strain RS593
contained an alanine at position 321 and did not react with MAb 63 (Table 4.5). This
suggests that substitutions at other positions along the sequence may play a role in the
binding of MAb 63.
Three amino acids that appear to be essential for the binding of MAb 67 include
asparagine at position 213, isoleucine at position 286, and aspartic acid at position 318
(Table 4.6) (15). Isolate 4947 and reference strains RS593, and AL-2 reacted with Mab
67, despite variation in their residues at position 318. This suggests that substitutions at
position 318 may be less critical for the binding of MAb 67. Previous reports identified a
serine/threonine residue at position 222 as important for the binding of MAb 67 (Table
4.6) (21). However, isolates 5038 and 5041 and reference strain GLS contained
serine/threonine residues at position 222 and did not bind MAb 67.
4.5 Virus Neutralization in Cell Culture
The virus neutralizing (VN) antibody titers in serum from SPF leghorn chickens
infected with IBDV field isolates and reference strains against IBDV viruses 89/03 and
PBG98 are summarized in Table 4.7. All of the sera cross-neutralized the variant 89/03
63
strain and the classic PBG98 strain. A four-fold difference was considered to be
significant; however, with the exception of isolate 4813 and the two IBDV reference
strains (Del E and STC), no significant difference in neutralization of the two viruses was
observed.
4.6 Active Immunity – Challenge with 89/03 Vaccine
SPF leghorn chickens vaccinated with IBDV strain 89/03 and challenged with
IBDV field isolates 4813, 4947, 4955, 5038, and 5041 and reference strains STC and
Delaware variant E exhibited no clinical signs or mortality throughout the duration of the
study. At 2 days post-challenge, clinical signs characteristic of classic IBDV including
dehydration, depression, and ruffled feathers were observed in one non-vaccinated SPF
leghorn chicken infected with field isolate 4947. All other non-vaccinated, challenged
controls and non-vaccinated, non-challenged control birds were normal.
Mean bursa to body weight ratios were calculated and percent protection values
were determined based on a minimum protection level of two standard deviations less
than the mean of the negative control group (Table 4.8). All chickens vaccinated with
89/03 were completely protected against challenge with the five field isolates and
reference strains. Disease incidence observed in non-vaccinated, chickens challenged
with field isolates 4947, 5038, STC and Delaware variant E ranged from 20 to 40%. Nonvaccinated chicks challenged with isolates 4955 and 5041 showed 60% disease incidence,
while non-vaccinated chicks infected with isolate 4813 displayed 80% incidence. Bursal
64
atrophy was observed in all non-vaccinated, challenged chicks, whereas all bursas
removed from 89/03-vaccinated chicks were normal in size.
Figure 4.3 presents the geometric mean antibody titers (GMTs) for antisera
collected prior to challenge at 21 days of age as measured by ELISA. Non-vaccinated,
non-challenged control birds and non-vaccinated, challenged control birds had low
antibody titers (GMTs of 2 and 4, respectively), whereas, all birds vaccinated with 89/03
showed high antibody titers (GMTs ranging from 1485 to 2294). Antibody GMTs for
antisera collected 7 days post-challenge showed an increase in anti-IBDV ELISA titers in
both 89/03-vaccinated (GMTs ranging from 2156 to 3206) and non-vaccinated (GMTs
ranging from 2154 to 3178) chickens. Non-vaccinated, non-challenged control birds
tested seronegative for IBDV antibodies (Figure 4.3).
Histopathological examination of the bursa of Fabricius of non-vaccinated SPF
leghorn chickens challenged with IBDV field isolates and reference strains revealed
lesions typical of IBDV (Table 4.9). All non-vaccinated challenge controls showed no
protection based on histopathology. All of the non-vaccinated, non-challenged controls
showed no disease incidence. SPF leghorn chickens vaccinated with 89/03 and
challenged with STC showed 94% protection. No other 89/03-vaccinated birds displayed
histological IBDV lesions in the bursa.
Microscopic evaluation of the thymus revealed significant lesions in one 89/03vaccinated SPF leghorn chicken challenged with isolate 4947, where there was diffuse,
moderate to severe cortical lymphocyte loss. Two non-vaccinated SPF leghorns infected
with STC also showed significant thymic lesions, with minimal to moderate cortical
65
lymphocyte loss with and without associated heterophilic infiltration. Microscopic
changes in the thymus were not observed in non-vaccinated, non-challenged control birds
or the remainder of 89/03-vaccinated and non-vaccinated, challenged birds. An increase
in the number of germinal centers in the spleen was observed in several 89/03-vaccinated
and non-vaccinated chicks; however, this finding was not considered a significant
microscopic lesion of IBDV infection.
4.7 Active Immunity – Challenge with PBG98
SPF leghorn chickens vaccinated with IBDV strain PBG98 and challenged with
IBDV field isolates 4813, 4947, 4955, 5038, and 5041 and reference strains STC and
Delaware variant E exhibited no clinical signs or mortality after virus challenge.
Similarly, non-vaccinated challenged controls and non-vaccinated, non-challenged
control birds displayed no clinical signs or mortality during the study. Percent protection
of SPF leghorn chickens vaccinated with PBG98 as determined by mean bursa to body
weight indexes ranged from 80 to 100% (Table 4.10). Non-vaccinated, challenged SPF
leghorns showed 100% disease incidence (Table 4.10). Bursal atrophy was observed in
all non-vaccinated, challenged chicks, whereas all bursas from PBG98-vaccinated chicks
were normal in size.
Figure 4.4 presents the geometric mean antibody titers (GMTs) for antisera
collected prior to challenge at 21 days of age as measured by ELISA. Non-vaccinated,
non-challenged control birds and non-vaccinated, challenged control birds had antibody
titers below the baseline of 18, whereas, all birds vaccinated with PBG98 showed
66
antibody titers ranging from 1934 to 4362. Antibody GMTs for antisera collected 7 days
post-challenge showed an increase in anti-IBDV ELISA titers in both PBG98-vaccinated
(GMTs ranging from 5610 to 9097) and non-vaccinated, challenged (GMTs ranging from
1335 to 3122) chickens. Non-vaccinated, non-challenged control birds tested
seronegative for IBDV antibodies (Figure 4.4).
Histopathological examination of the bursa of Fabricius of non-vaccinated SPF
leghorn chickens challenged with IBDV field isolates and reference strains revealed
lesions typical of IBDV (Table 4.11). Based on histopathology, all non-vaccinated,
challenged controls were unprotected. All of the non-vaccinated, non-challenged controls
showed 100% protection. Birds vaccinated with PBG98 displayed 60% protection against
challenge with 4813, 87% protection against challenge with 4947, 40% protection against
challenge with 4955, 67% protection against challenge with 5038, 73% protection against
challenge with 5041, 93% protection against challenge with STC, and 93% protection
against challenge with Del E.
Microscopic evaluation of the thymus revealed significant lesions in one nonvaccinated SPF leghorn chicken challenged with STC, where there was diffuse, moderate
cortical lymphocyte loss via apoptosis. In the same bird, diffuse ellipsoid degeneration in
the spleen was observed along with many foamy macrophages and plasma cells in the red
pump. Microscopic lesions in the thymus and spleen were not observed in nonvaccinated, non-challenged control birds or the remainder of PBG98-vaccinated and nonvaccinated, challenged birds.
67
4.8 Passive Immunity - Progeny Challenge
Percent protection of white leghorn progeny challenged on day 10 and day 17 as
determined by bursa to body weight indexes is displayed in Table 4.12 and Table 4.13,
respectively. Progeny challenged on day 10 showed greater protection against challenge
with Del E (64%) than those challenged with 5038 (33%). Day 10 SPF leghorn control
birds showed 100% disease incidence following challenge with both Del E and 5038.
Progeny challenged on day 17 displayed equal protection (42%) against challenge with
isolate 5038 and Del E. Day 17 SPF leghorn controls exhibited 92% disease incidence
following challenge with both Del E and 5038. Bursal atrophy was observed in all SPF
leghorn non-vaccinated, IBDV-challenged chicks following the day 10 and 17 challenge.
Isolate 5038 and Del E induced minimal to moderate (33-64%) bursal atrophy in progeny
challenged on day 10 and 17.
Results of histopathological examination of the bursa of Fabricius of progeny and
SPF leghorn chickens challenged with isolate 5038 and Del E are displayed in Table
4.14. Percent protection for day 10 challenge as determined by microscopic pathology
showed 42% protection in progeny challenged with Del E; whereas, no protection was
observed in progeny challenged with 5038. SPF leghorn non-vaccinated, IBDV challenge
control chickens showed 100% disease incidence following day 10 challenge with both
Del E and 5038. Percent protection for day 17 challenge showed minimal protection (8%)
in progeny challenged with 5038 and progeny challenged with Del E. SPF leghorn nonvaccinated, IBDV challenge control chickens showed 100% disease incidence following
day 17 challenge with both Del E and 5038.
68
Table 4.1. Percent identity values of protein sequences of the variable region of VP2 protein of IBDV field strains isolated
from Delmarva broiler farms and reference strains Delaware variant E, STC, GLS, AL-2, and RS593 as determined by
multiple sequence alignment using the CLUSTAL W methodA.
Percent Identity
69
Percent Divergence
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1.8
2.3
0.0
1.8
2.7
0.0
0.0
1.9
1.8
3.2
3.2
0.4
3.7
2.4
0.4
5.1
3.7
2.3
1.8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
98.2 97.8 100.0 98.2 97.3 100.0 100.0 98.1 98.2 96.9 96.9 99.6 96.4 97.7 99.6 95.1 96.4 97.8 98.2
97.8 98.2 100.0 95.5 98.1 98.2 100.0 99.1 95.1 95.1 97.8 95.5 97.7 97.8 94.2 95.5 96.9 98.2
2.3
97.8 97.8 96.0 97.7 97.8 97.7 97.8 96.0 96.0 97.3 95.5 100.0 97.3 94.2 96.4 97.3 98.2
1.8 2.3
98.2 97.3 100.0 100.0 98.1 98.2 96.9 96.9 99.6 96.4 97.7 99.6 95.1 96.4 97.8 98.2
0.0 2.3 1.8
95.5 98.1 98.2 100.0 99.1 95.1 95.1 97.8 95.5 97.7 97.8 94.2 95.5 96.9 98.2
4.6 4.2 2.7
4.6
97.2 97.3 95.4 95.5 96.9 96.9 97.3 95.1 95.8 97.3 93.7 96.0 96.4 96.0
1.9 2.4 0.0
1.9 2.8
100.0 98.1 98.1 97.2 97.2 99.5 96.8 97.7 99.5 95.4 96.8 97.7 98.1
1.8 2.3 0.0
1.8 2.7 0.0
98.1 98.2 96.9 96.9 99.6 96.4 97.7 99.6 95.1 96.4 97.8 98.2
0.0 2.4 1.9
0.0 4.8 1.9
1.9
99.1 95.4 95.4 97.7 95.8 97.7 97.7 94.4 95.8 96.8 98.1
0.9 2.3 1.8
0.9 4.6 1.9
1.8 0.9
95.1 95.1 97.8 95.5 97.7 97.8 94.2 95.5 96.9 99.1
5.1 4.2 3.2
5.1 3.2 2.8
3.2 4.8 5.1
100.0 96.9 96.4 96.3 97.3 95.1 98.7 96.9 96.0
5.1 4.2 3.2
5.1 3.2 2.8
3.2 4.8 5.1
0.0
96.9 96.4 96.3 97.3 95.1 98.7 96.9 96.0
2.3 2.7 0.4
2.3 2.7 0.5
0.4 2.4 2.3
3.2 3.2
96.4 97.2 99.1 94.6 96.4 97.8 97.8
4.6 4.6 3.7
4.6 5.1 3.3
3.7 4.3 4.6
3.7 3.7 3.7
95.8 96.9 94.6 96.9 97.3 96.0
2.4 0.0 2.4
2.4 4.3 2.4
2.4 2.4 2.4
3.8 3.8 2.8
4.3
97.2 94.4 96.8 97.2 98.1
2.3 2.7 0.4
2.3 2.7 0.5
0.4 2.4 2.3
2.7 2.7 0.9
3.2 2.8
95.1 96.9 98.2 97.8
6.1 6.1 5.1
6.1 6.6 4.8
5.1 5.8 6.1
5.1 5.1 5.6
5.6 5.8 5.1
96.0 94.6 95.1
4.6 3.7 3.7
4.6 4.2 3.3
3.7 4.3 4.6
1.4 1.4 3.7
3.2 3.3 3.2
4.2
97.3 96.4
3.2 2.7 2.3
3.2 3.7 2.4
2.3 3.3 3.2
3.2 3.2 2.3
2.7 2.8 1.8
5.6 2.7
97.3
1.8 1.8 1.8
1.8 4.2 1.9
1.8 1.9 0.9
4.2 4.2 2.3
4.2 1.9 2.3
5.1 3.7 2.7
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
A
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
DMV/4813/07
DMV/4816/07
DMV/4947/07
DMV/4952/07
DMV/4955/07
DMV/4956/07
DMV/5032/07
DMV/5033/07
DMV/5034/07
DMV/5036/07
DMV/5037/07
DMV/5038/07
DMV/5040/07
DMV/5041/07
DMV/5042/07
DELE
STC
GLS
AL-2
RS593
Protein sequences were generated using an ABI Prism 3130XL Genetic Analyzer (Applied Biosystems Inc., Foster City, CA)
and analyzed with MegAlign v8.0.2 software (DNAStar, Inc., Madison, WI).
69
Table 4.2. Correlation between predicted amino acid substitutions and monoclonal antibody (MAb) 69 reactivity patterns for
passage 1 seed stock samples of IBDV field isolates and IBDV reference strainsA.
Amino Acid Position
70
MAb 69
Transfection and
ImmunofluorescenceB
+
-
Whole Virus/
ELISAC
+
-
Virus
249
280
326
4813
4947
4955
5038
5041
STC
DelE
GLS
RS593
AL2
Lys
Lys
Lys
Lys
Lys
Gln
Lys
Lys
Lys
Lys
Asn
Asn
Asn
Asn
Thr
Asn
Asn
Asn
Asn
Asn
Ser
Ser
Ser
Ser
Thr
Ser
Ser
Ser
Ser
Ser
A
p1 sample is SPF bursal origin stock.
Performed by Egbert Mundt, University of Georgia.
C
Performed by Intervet/Schering-Plough Animal Health, Millsboro, DE.
B
70
Table 4.3. Correlation between predicted amino acid substitutions and monoclonal antibody (MAb) 10 reactivity patterns for
passage 1 seed stock samples of IBDV field isolates and IBDV reference strainsA.
MAb 10
Transfection and
ImmunofluorescenceB
71
+
+
-
Whole Virus/ELISAC
+
+
+
-
A
Virus
Amino Acid Position
286
318
323
4813
4947
4955
5038
5041
STC
DelE
GLS
RS593
AL2
Ile
Ile
Ile
Thr
Ile
Thr
Ile
Thr
Ile
Ile
Glu
Asp
Asp
Glu
Asp
Asp
Glu
Asp
Asp
Asp
p1 sample is SPF bursal origin stock.
Performed by Egbert Mundt, University of Georgia.
C
Performed by Intervet/Schering-Plough Animal Health, Millsboro, DE.
B
71
Asp
Asn
Asp
Gly
Ser
Gly
Asp
Gly
Gly
Asn
Table 4.4. Correlation between predicted amino acid substitutions and monoclonal antibody (MAb) 57 reactivity patterns for
passage 1 seed stock samples of IBDV field isolates and IBDV reference strainsA.
Amino Acid Position
MAb 57
72
Transfection and ImmunofluorescenceB
+
+
+
Whole Virus/ ELISAC
+
+
+
+
A
p1 sample is SPF bursal origin stock.
Performed by Egbert Mundt, University of Georgia.
C
Performed by Intervet/Schering-Plough Animal Health, Millsboro, DE.
B
72
Virus
321
4813
4947
4955
5038
5041
STC
DelE
GLS
RS593
AL2
Ala
Ala
Ala
Glu
Glu
Ala
Ala
Glu
Ala
Glu
Table 4.5. Correlation between predicted amino acid substitutions and monoclonal antibody (MAb) 63 reactivity patterns for
passage 1 seed stock samples of IBDV field isolates and IBDV reference strainsA.
MAb 63
Transfection and
ImmunofluorescenceB
73
+
+
+
-
Virus
Amino Acid Position
321
4813
4947
4955
5038
5041
STC
DelE
GLS
RS593
AL2
Ala
Ala
Ala
Glu
Glu
Ala
Ala
Glu
Ala
Glu
Whole Virus/ ELISAC
+
+
+
-
A
p1 sample is SPF bursal origin stock.
Performed by Egbert Mundt, University of Georgia.
C
Performed by Intervet/Schering-Plough Animal Health, Millsboro, DE.
B
73
Table 4.6. Correlation between predicted amino acid substitutions and monoclonal antibody (MAb) 67 reactivity patterns for
passage 1 seed stock samples of IBDV field isolates and IBDV reference strainsA.
Amino Acid Position
74
MAb 67
Transfection and
ImmunofluorescenceB
+
+
+
+
+
Whole Virus/ ELISAC
+
+
+
+
+
+
Virus
213
286
318
4813
4947
4955
5038
5041
STC
DelE
GLS
RS593
AL2
Asn
Asn
Asn
Asp
Asp
Asp
Asn
Asp
Asn
Asn
Ile
Ile
Ile
Thr
Ile
Thr
Ile
Thr
Ile
Ile
Asp
Asn
Asp
Gly
Ser
Gly
Asp
Gly
Gly
Asn
A
p1 is SPF bursal origin stock.
Performed by Egbert Mundt, University of Georgia.
C
Performed by Intervet/Schering-Plough Animal Health, Millsboro, DE.
B
74
Table 4.7. Virus-neutralization (VN) serum antibody titers of SPF leghorn chickens against IBDV strains 89/03 and PBG98 as
measured by beta VN assays in chicken embryo fibroblast cellsA.
IBD Virus
B
C
PBG98D
75
Antiserum
89/03
Negative Control
<100
<100
4813
>12800
3200
4947
1600-3200E
1600-3200E
4955
3200-6400E
1600
5038
5041
3200
800
1600
3200
STC
3200
>12800
>12800
6400
Delaware Variant E
A
Reciprocal of the highest dilution of serum that neutralized 100 TCID50 of IBDV.
Antiserum was harvested at 4 weeks post-inoculation from SPF leghorn chickens infected with IBDV field isolates and
reference strains.
C
Variant virus
D
Classic virus
E
Value range due to discrepancy between duplicates.
B
75
Table 4.8. Percent protection determined by bursa to body weight ratios of one-day-old SPF leghorn chickens
subcutaneously vaccinated with IBDV strain 89/03 and challenged at 21 days post-vaccination via the intraocular route with
IBDV field isolates and reference strains STC and Delaware variant E.
Challenge Virus
Negative Control
4813
4947
76
4955
5038
5041
STC
Delaware variant E
Treatment
n/a
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Percent
ProtectionB
100*
20
100
80
100*
40
100*
60
100*
40
100*
60
100*
60
A
B
Mean Bursa to Body Weight RatioA
4.98
5.31
1.33
4.69
1.89
5.50
1.39
5.62
1.59
5.31
1.62
5.08
1.55
5.60
1.69
Minimum protection level (Control Group Mean – 2 Standard Deviations)
Astericks indicate a significant difference by two-tailed chi-square analysis compared with non-vaccinated challenge
controls within the same treatment group (P < 0.05).
76
Table 4.9. Microscopic pathology of the bursa of Fabricius of SPF leghorn chickens vaccinated with IBDV strain 89/03 and
challenged with IBDV field isolates 4813, 4947, 4955, 5038, and 5041 and IBDV reference strains STC and Delaware
variant EA.
Bursal
lesion
scores
1
2
3
77
4
Histopathological
bursa of Fabricius
lesions
Normal
No IBDV related
lesions
Mild scattered IBDV
related cell depletion
in a few follicles
Moderate
1/3 to 1/2 of the
follicles with IBDV
related atrophy or
depletion of cells
Severe atrophy of all
follicles
Inflammation
Acute necrosis
4813
V
4947
NV
16/16
V
Challenge GroupsB
5038
4955
NV
16/16
V
NV
16/16
NV
16/16
V
STC
NV
6/6
V
Del E
NV
14/16
V
NV
16/16
1/16
1/16
5/5
5/5
5/5
A
B
V
5041
5/5
4/4
5/5
Birds were challenged via the intraocular route at 21 days of age and bursas were collected 7 days post-challenge.
V = Vaccinated
NV= Non-vaccinated
77
5/5
Table 4.10. Percent protection determined by bursa to body weight ratios of one-day-old SPF leghorn chickens
subcutaneously vaccinated with IBDV strain PBG98 and challenged at 21 days of age via the intraocular route with IBDV
field isolates and reference strains STC and Delaware variant E.
Challenge Virus
Negative Control
4813
4947
78
4955
5038
5041
STC
Delaware variant E
Treatment
n/a
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Vaccinated
Non-vaccinated
Percent ProtectionB
87*
0
100*
0
93*
0
87*
0
80*
0
100*
0
87*
0
A
B
Mean Bursa to Body Weight RatioA
6.08
5.68
1.14
4.69
1.25
5.55
1.51
5.57
1.57
5.17
1.50
5.74
1.77
5.00
1.60
Minimum protection level (Control Group Mean – 2 Standard Deviations)
Astericks indicate a significant difference by two-tailed chi-square analysis compared with non-vaccinated challenge controls
within the same treatment group (P < 0.05).
78
Table 4.11. Microscopic pathology of the bursa of Fabricius of SPF leghorn chickens vaccinated with IBDV strain PBG98
and challenged with IBDV field isolates 4813, 4947, 4955, 5038, and 5041 and IBDV reference strains STC and Delaware
variant EA.
Challenge GroupsB
Bursal
lesion
scores
1
2
79
3
4
Histopathological
bursa of
Fabricius lesions
Normal
No IBDV related
lesions
Mild scattered
IBDV related cell
depletion in a
few follicles
Moderate
1/3 to 1/2 of the
follicles with
IBDV related
atrophy or
depletion of cells
Severe atrophy of
all follicles
Inflammation
Acute necrosis
4813
V
4947
NV
V
4955
NV
V
5038
NV
9/15
12/15
6/15
10/15
3/15
1/15
4/15
2/15
2/15
1/15
1/15
2/15
5/5
1/15
5/5
3/15
5/5
A
B
V
2/15
5041
NV
V
STC
NV
11/15
V
Del E
NV
14/15
14/15
1/15
1/15
NV
2/15
5/5
2/5
5/5
5/5
Birds were challenged via the intraocular route at 21 days of age and bursas were collected 7 days post-challenge.
V = Vaccinated
NV= Non-vaccinated
79
V
5/5
Table 4.12. Percent protection determined by bursa to body weight ratios of progeny of 18-week-old SPF leghorn breeder
chickens vaccinated with a licensed inactivated IBDV vaccine containing classic and variant strains, as well as, a strain of
reovirus and challenged with field isolate 5038 and reference strain Delaware variant E at 10 days of age.
80
Vaccine
Challenge Virus
Percent Protection
Mean Bursa to Body Weight
RatioA
A
5038
33*
2.53
A
Del E
64
2.95
A
None
100
4.22
Non-vaccinated
Non-vaccinated
5038
0
1.01
Del E
0
1.22
Non-vaccinated
None
100
4.00
B
A
B
Minimum protection level (Control Group Mean – 2 Standard Deviations).
Astericks indicate a significant difference by two-tailed chi-square analysis compared with non-vaccinated challenge controls
within the same treatment group (P < 0.05).
80
Table 4.13. Percent protection determined by bursa to body weight ratios of progeny of 18-week-old SPF leghorn breeder
chickens vaccinated with a licensed inactivated IBDV vaccine containing classic and variant strains, as well as, a strain of
reovirus and challenged with field isolate 5038 and Delaware variant E at 17 days of age.
81
Vaccine
A
A
A
Challenge Virus
5038
Del E
None
Percent Protection
42
42
100
Mean Bursa to Body Weight
RatioA
2.93
2.36
4.28
Non-vaccinated
5038
8
1.33
Non-vaccinated
Del E
8
1.43
Non-vaccinated
None
100
3.76
B
A
B
Minimum protection level (Control Group Mean – 2 Standard Deviations).
Astericks indicate a significant difference by two-tailed chi-square analysis compared with non-vaccinated challenge controls
within the same treatment group (P < 0.05).
81
Table 4.14. Microscopic pathology of the bursa of Fabricius of progeny from 18-week-old SPF leghorn breeder chickens
vaccinated with a licensed inactivated IBDV vaccine containing classic and variant strains, as well as, a strain of reovirus
and challenged with field isolate 5038 and Delaware variant EA.
Bursal
lesion
scores
1
2
82
3
4
Histopathological
bursa of Fabricius
lesions
Normal
No IBDV related
lesions
Mild scattered IBDV
related cell depletion in
a few follicles
Moderate
1/3 to 1/2 of the follicles
with IBDV related
atrophy or depletion of
cells
Severe atrophy of all
follicles
Inflammation
Acute necrosis
Day 10 Progeny Group ChallengeB
5038
Del E
Neg Control
V
NV
V
NV
3/12
V
NV
V
11/11
13/13
1/12
NV
V
NV
1/12
V
NV
11/11
11/11
2/12
1/12
11/12
1/12
12/12
6/12
1/12
12/12
10/12
A
B
Day 17 Progeny Group ChallengeB
5038
Del E
Neg Control
12/12
11/12
12/12
Birds were challenged via the intraocular route at 10 and 17 days of age and bursas were collected 7 days post-challenge.
V = Vaccinated
NV = Non-vaccinated
82
Del E
Clade 1
Clade 2
Figure 4.1. VP2 phylogenetic analysis of IBDV field isolates and reference strains
constructed using Molecular Evolutionary Genetics Analysis software version 4.0 (302)
and the unweighted pair-group method using arithmetic averages method (303).
83
Clade 3
Clade 4
Clade 5
Clade 6
STC
Figure 4.1 continued.
84
DELE
DMV/4813/07
DMV/4947/07
DMV/4955/07
DMV/5038/07
DMV/5041/07
STC
GLS
RS593
AL-2
Minor Hydrophilic peak 1
(aa 249-254)
Major Hydrophilic peak A (aa 212-224)
210
A
.
.
.
.
.
.
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A
.
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D
.
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N
.
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D
D
D
D
.
.
Y
.
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Q
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F
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Y
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Q
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T
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P
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F
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S
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A
.
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N
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T
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S
.
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.
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V
.
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I
.
Q
.
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.
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.
S
N
N
N
.
.
G
.
N
.
L
.
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.
V
.
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L
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G
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A
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T
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I
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Y
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L
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F
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I
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G
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F
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D
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G
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T
.
S
.
S
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S
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A
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T
E
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M
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A
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290
DELE
DMV/4813/07
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DMV/5041/07
STC
GLS
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330
DELE
DMV/4813/07
DMV/4947/07
DMV/4955/07
DMV/5038/07
DMV/5041/07
STC
GLS
RS593
AL-2
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Figure 4.2. Deduced amino acid sequences of the variable region of VP2 protein of
IBDV field isolates and reference strains Delaware variant E, STC, GLS, AL-2, and
RS593. Major (9, 112, 315) and minor (113, 115) hydrophilic peaks are indicated.
85
V
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Pre-Challenge
Post-Challenge
10000
9000
8000
7000
GMTs
6000
5000
4000
3000
86
2000
1000
0
Vacc Non- Vacc Non- Vacc Non- Vacc Non- Vacc Non- Vacc Non- Vacc NonVacc
Vacc
Vacc
Vacc
Vacc
Vacc
Vacc
Neg
Control
4813
4947
4955
5038
5041
STC
Del E
Challenge Viruses
Figure 4.3. Serum antibody geometric mean titers (GMTs) of 89/03-vaccinated and non-vaccinated SPF leghorn
chickens before and after challenge with IBDV field isolates and reference strains as measured by ELISA.
86
Pre-Challenge
Post-Challenge
10000
9000
8000
7000
GMTs
6000
5000
4000
87
3000
2000
1000
0
Vacc
Neg
Control
Non- Vacc
Vacc
4813
Non- Vacc Non- Vacc
Vacc
Vacc
4947
4955
Non- Vacc
Vacc
5038
Non- Vacc Non- Vacc
Vacc
Vacc
5041
STC
NonVacc
Del E
Challenge Viruses
Figure 4.4. Serum antibody geometric mean titers (GMTs) of PBG98-vaccinated and non-vaccinated SPF leghorn
chickens before and after challenge with IBDV field isolates and reference strains as measured by ELISA.
87
Chapter 5
DISCUSSION
Infectious bursal disease virus is among the most important pathogens of
poultry, because it replicates and damages tissues of the immune system (94, 316).
Permanent immunosuppression results from infection of chickens less than one-week of
age (141). The virus is ubiquitous in poultry production systems because of its unusually
resistant physicochemical characteristics combined with the infrequent clean-out and
disinfection of poultry houses (94, 316). Although IBDV vaccination is regularly
practiced in the industry, selection of novel antigenic types has occurred (25, 56, 57, 64,
201). Thus, ongoing surveillance for new IBDV isolates is important so that new isolates
may be antigenically characterized and evaluated for the ability to break through
immunity provided by current vaccines.
Thirty-three IBDV field isolates were recovered from 3- and 4-week-old
broilers, located on 12 different farms on the Delmarva Peninsula. Flocks raised on these
farms had a history of poor performance due to recurrent cases of gangrenous dermatitis,
inclusion body hepatitis, runting and stunting syndrome, and respiratory complex. Fifteen
of the isolates found to be IBDV positive by histopathology, virus isolation, and RT-PCR
were used for sequencing of the variable region of VP2. VP2 phylogenetic analysis of the
deduced amino acid sequences showed that the isolates distributed into six different
88
clades, all representing serotype 1 Delaware variant viruses. Five field isolates
representative of the six molecular clades were selected for further analysis.
The characterization of the Delmarva isolates showed that one of the five field
isolates, 5038, was antigenically different from reference strains STC, Del E, GLS,
RS593, and AL-2 based on monoclonal antibody (MAb) reactivity patterns. This was
surprising because, in both of the active vaccination-challenge studies, vaccinated
leghorn chickens exhibited solid immunity against the 5038 virus. Virus neutralization
tests correlated with these findings in that 5038 antiserum neutralized 100 TCID50 of both
the 89/03 virus and the PBG98 virus. In contrast to the active vaccination-challenge
studies, a passive immunity progeny challenge indicated that 5038 was able to break
through maternally-derived immunity earlier than Del E due to its antigenic difference.
Use of the amino acid sequence in combination with the antigenic profile as
determined by MAb reactivity has been shown to be highly informative in the antigenic
characterization of field isolates (201). Genomic characterization and comparison of the
variable region of VP2 of 5038 showed that the sequences at amino acid positions 249K
and 254S were similar to other variant strains (16, 17, 194, 286, 317, 318). Residues at
positions 253, 279, and 284 of the VP2 protein sequence have been reported to impact
virulence and pathogenic phenotype (16, 319), while residues at position 222 and within
the hydrophilic peak B region (aa 314-324) have been found to affect antigenicity (205,
286). In addition, amino acid positions 318 and 322 are thought to play a role in the
pathogenicity of IBDV (17). Interestingly, the VP2 sequence of 5038 at amino acid
positions 222T, 253Q, 279N, 284A, and 322G, was also similar to other characterized
89
variant strains (16, 17, 194, 201, 286, 317, 318). The deduced amino acid sequence
showed that 5038 shared one amino acid substitution, 294I, that is unique to vvIBDV
strains (14, 215, 318); however, none of the other genetic elements common to vvIBDV
were present. Unfortunately, VP2 sequence analysis did not allow the pinpointing of the
exact mutations responsible for the lack of MAb binding with 5038. Studies involving
selection and comparative nucleotide sequencing of escape mutants may help to
determine the genetic basis of this unique reaction pattern with the MAbs.
In this study, we also demonstrated that four of the five field viruses, 4813,
4947, 4955, and 5041, were antigenically similar to several well-characterized IBDV
strains (i.e. Delaware variant E, RS593, GLS). Genomic characterization of the variable
region of VP2 of the four isolates showed that the sequence at amino acid positions
222T/S, 249K, 253Q, 254S/N, 279N, 284A, and 322G/E was similar to other
characterized variant strains (16, 17, 194, 201, 286, 317, 318). Position 318 was the most
mutated with four different amino acids, aspartic acid, asparagine, glycine, and serine.
Jackwood et al. (17) reported similar findings from a sequence comparison of 25 IBDV
strains placed in the same or different molecular groups by RT/PCR-RFLP. It is possible
that the 318 mutation played a role in the emergence of these four field isolates.
However, active vaccination-challenge studies indicated that a single dose of either
classic or variant live, attenuated vaccine was sufficient to protect maternal antibody free
chicks challenged at 3 weeks of age.
The fact that isolate 5038 appeared to be antigenically different from STC, Del
E, RS593, GLS, and AL-2 by MAb testing and antigenically different from Del E by the
90
progeny challenge warrants further investigation. In the past, genetic mutation in the
variable region of VP2 of the IBDV genome has enabled the virus to evade immunity, as
well as, exhibit variations in antigenicity, virulence, cell tropism, and pathogenic
phenotype (16, 17, 58, 263, 320). While SPF leghorn chickens serve as an acceptable
model for in vivo IBDV challenge studies, variation in susceptibility to IBDV infection
has been observed among different chicken breeds (321, 322). Thus, experimentation
using broiler chickens would provide a more accurate overview about the situation in the
field. The next step will be to examine the ability of 5038 to break through maternal
immunity to commercial IBDV classic and variant vaccines in broiler chickens. If this
virus once again proves to be antigenically different from the vaccines used in in vivo
testing, then the issue of vaccine strain selection may be raised. On the other hand, if little
to no vaccine breakthrough is observed, then the MAbs used in the antigen capture
ELISA tests may need to be re-evaluated for their usefullness against IBDV strains
currently circulating in the field.
A hypothesis to explain the apparent vaccine breakthrough of field isolates
4813, 4947, 4955, and 5041, is that these viruses could be the result of factors such as
poor vaccine delivery or the natural waning of maternal antibodies rather than antigenic
drift (59, 283). The Delmarva broilers from which these IBDV field isolates originated
only received passive breeder vaccinations, which normally afford protection for up to 23 weeks of age. Therefore, it is not surprising that the broilers were susceptible to
infection at 3 and 4 weeks of age. Active immunization of broiler progeny would extend
the life of IBDV protection (323); however, high maternal antibodies can sometimes
91
neutralize the vaccine virus and delay or prevent stimulation of a protective immune
response (51, 324). Variation in the natural waning of maternal antibodies makes it
difficult to determine the optimal timing for broiler vaccinations (46, 47, 283, 325, 326,
327). Consequently, active immunization is not always a practical solution to vaccination
failures.
As shown by this study, the vaccine response of optimally vaccinated birds is
not always a true picture of the situation in the field. Highly industrialized poultryproducing systems such as those located on the Delmarva Peninsula involve high density
poultry houses and hatcheries with litter that may be removed perhaps every three years.
Such conditions can have a significant effect on stress during rearing, nutrition, and the
prevalence and control of major poultry diseases. Moreover, clean-out and disinfection is
not always handled properly and, at times, may not be effective at eliminating highly
resistant viruses such as IBDV (46, 59, 321, 325). In addition, inconsistencies of vaccine
dosage, the indirect targeting of tissues that generate immunity, unequal distribution, and
variability in water quality are common problems that accompany vaccination methods
used in large scale poultry operations (325).
Although current vaccination programs and strict sanitary procedures help to
minimize the occurrence of IBD outbreaks, field cases continue to emerge among broiler
flocks (273, 318, 328, 329). The immunosuppressive nature of IBDV makes this virus a
major economic concern to the poultry industry as it not only contributes to vaccination
failures (26, 318), poor broiler performance, and increased condemnations at processing,
but it frequently results in an increase of secondary infections (46, 115, 153, 330, 331). It
92
is not clear whether IBDV was the underlying cause for the recurring poor performance
of the Delmarva poultry flocks involved in this study. However, IBDV-associated
immunosuppression may have played a role in the prevalence of gangrenous dermatitis,
inclusion body hepatitis, runting and stunting syndrome, and respiratory complex on
these problem farms. We observed by molecular characterization and in vivo studies that
field isolate 5038 may differ antigenically from vaccine strains currently used in the field.
For this reason, continuous surveillance is necessary to identify, isolate, and characterize
what may be new IBDV strains circulating within the poultry industry.
93
CONCLUSION
Fifteen infectious bursal disease virus field isolates were recovered from 3-4 week old
commercial broiler chickens raised on the Delmarva Peninsula in 2007. The
viruses were identified by histopathology, virus isolation, and RT-PCR. VP2
phylogenetic analysis identified all of the isolates as serotype 1 Delaware variant
viruses.
Five of the field isolates distributed into six clades and were selected for further study.
The five isolates, 4813, 4947, 4955, 5038, and 5041 produced gross and microscopic
pathology of the bursa of Fabricius consistent with Delaware variant infection.
Active immunization of specific-pathogen-free leghorn chickens with highly attenuated
Delaware variant 89/03 and classic PBG98 live vaccines produced solid immunity
upon challenge with each of the field isolates.
Based on the antigenic similarity of field isolates 4813, 4947, 4955, and 5041 to wellcharacterized strains of IBDV, it is highly probable that the broilers from which
these isolates originated became infected due to waned maternal antibodies.
Therefore, in order to improve the performance of these problem farms in the
Delmarva Peninsula, broiler vaccination programs for IBDV may need to be
implemented.
94
Isolate 5038 was found to be antigenically different from STC, Del E, GLS, RS593, and
AL2 based on its inability to react with monoclonal antibodies. In vivo testing
showed that 5038 was antigenically different from Del E based on its ability to
break through maternally-derived immunity earlier than Del E challenge in 10-dayold SPF leghorn chickens.
On the other hand, in both active vaccination-challenge studies, vaccinated birds
exhibited solid immunity against 5038, and genomic characterization and
comparison of the variable region of VP2 did not indicate specific mutations that
may be responsible for the lack of monoclonal antibody binding. It is possible that
the MAbs used in the antigen capture ELISA tests are not entirely effective for
IBDV strains currently circulating in the field. For a more comprehensive analysis,
new MAbs may need to be produced.
VP2 sequencing and phylogenetic analysis may be useful molecular tools for identifying
differences among strains, but they do not always predict how the viruses will
behave biologically.
95
APPENDIX A
Epitope profiles of infectious bursal disease viruses recognized by monoclonal
antibodies used in antigen capture enzyme-linked immunosorbent assays.
Ruud Hein (Intervet/Schering Plough Animal Health)
96
APPENDIX B
Standard Amino Acid Abbreviations and Side Chain Properties Table
Amino Acid
3-Letter
1-Letter
Side chain
polarity
Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
Ala
Arg
Asn
Asp
Cys
Glu
Gln
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
A
R
N
D
C
E
Q
G
H
I
L
K
M
F
P
S
T
W
Y
V
nonpolar
polar
polar
polar
nonpolar
polar
polar
nonpolar
polar
nonpolar
nonpolar
polar
nonpolar
nonpolar
nonpolar
polar
polar
nonpolar
polar
nonpolar
http://en.wikipedia.org/wiki/Amino_acid
97
Side chain
charge
(pH 7)
neutral
positive
neutral
negative
neutral
negative
neutral
neutral
positive
neutral
neutral
positive
neutral
neutral
neutral
neutral
neutral
neutral
neutral
neutral
Hydropathy
index
1.8
-4.5
-3.5
-3.5
2.5
-3.5
-3.5
-0.4
-3.2
4.5
3.8
-3.9
1.9
2.8
-1.6
-0.8
-0.7
-0.9
-1.3
4.2
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