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Molecular Epidemiology and Pathogenicity of the Very Virulent Infectious Bursal
Disease Pathotype in United States Poultry
Dissertation
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Simone Tricia Stoute, D.V.M.
Graduate Program in Comparative and Veterinary Medicine
The Ohio State University
2012
Dissertation Committee:
Daral J. Jackwood, Advisor
Chang-Won Lee
Richard D. Slemons
Copyright by
Simone Tricia Stoute
2011
Abstract
Infectious bursal disease (IBD) was first recognized in 1962 and is presently one
of the most economically significant immunosuppressive diseases affecting most poultry
producing regions worldwide. The most severe form of the disease in terms of severity
of clinical signs, lesions and mortality rate is the very virulent (vv) IBD. This very
virulent pathotype can result in mortality rates as high as 100% in fully susceptible
flocks. While classic and variant strains of the virus are endemic in the U.S., there were
no reports of vvIBDV in poultry in the U.S. until 2009. Subsequent to the emergence of
vvIBDV in U.S. poultry, several IBDV reassortant viruses have been detected from
commercial and backyard chicken flocks within the U.S. These reassortant viruses have
all been isolated from commercial or backyard chicken flocks in the State of California.
Reverse transcriptase- polymerase chain reaction (RT-PCR) tests and sequencing on
bursal tissues collected from field cases originating from California poultry have detected
multiple vvIBDV reassortants. These include interserotypic reassortants with the genome
segment A matching the vvIBDV pathotype and genome segment B characteristic of a
serotype 2 IBDV (ID: K669, CA-K758, CA-D495). An interserotypic reassortant (ID:
D2712) with a serotype 2 segment A and serotype 1 segment B was also identified in 5
week old commercial turkeys in 2012. A vvIBDV reassortant with genome segment A
homologous to vvIBDV and genome segment B that aligned with serotype 1 classic
ii
IBDV strains from Australia was isolated (ID: 7741) in backyard brown leghorns in
2010.
The first investigation described in chapter 2 assessed the pathogenicity induced
by co-infection with very virulent infectious bursal disease virus (vvIBDV: rB strain) and
U.S. endemic IBDV pathotype in specific pathogenic free leghorns. The severity of
disease in co-challenged birds was compared to the disease induced by infection with the
vvIBDV (rB strain) alone. Four wk old and 6 wk old birds were challenged oronasally
with 105EID50 vv (rB strain) and 105EID50 of either a standard classic IBDV (STC
strain), subclinical variant IBDV (Delaware-E strain), subclinical variant IBDV (T1
strain) or avirulent serotype 2 IBDV (OH strain). The severity of disease was assessed
by comparing the 5-day mortality rates, severity of bursal lesions (mean bursal lesion
scores) and mean bursal body weights (BBW) in each of the challenged groups. A
mortality rate of 100% was observed in both 4 wk and 6 wk old birds inoculated with
only the vvIBDV (rB) strain. A relative reduction in the mortality and severity of disease
was observed in all groups co-challenged with rB and the second less virulent pathotypes
(STC, Del-E, T1, OH) in both 4 wk and 6 wk old birds. Co-challenge with rB and the
Del-E, T1 and OH strains resulted in a more significant decrease in mortality compared
to challenge with the two antigenically similar pathogenic strains, rB and STC. Viral
interference between the different pathotypes was hypothesized as the etiology of the
reduction in severity of disease in co-challenged birds.
iii
The third chapter investigated the pathogenicity of genome reassortant vvIBDV in 4 wk
old SPF leghorns and 1, 2, 3, and 4 wk old maternally immune broilers. The virulence of
California reassortants K669, 7741 and D2712 was compared with that of other IBDV
pathotypes circulating in the U.S. The phylogenetic relationship of emerging reassortant
strains with other endemic and foreign IBDV strains was also evaluated. Interserotypic
reassortant vvIBDV (K669) had a genome segment A matching the vvIBDV pathotype
and segment B matching serotype 2 IBDV. The K669 isolated was initially recovered
from 28 day old commercial broilers from Northern California in April 2011. Another
interserotypic reassortant (D2712) with a serotype 2 segment A and serotype 1 segment B
that aligned phylogenetically with classic IBDV was isolated from commercial California
turkeys in January 2012. In addition, a vvIBDV reassortant (7741) with a vvIBDV
genome segment A and a segment B that aligned phylogenetically with classic IBDV
strains isolated from Australia (AJ878641, AJ878682, AJ878639) was identified in
commercial layers from California in august 2010. In vivo virulence of reassortant
vvIBDV: 7741, K669 and D2712 was assessed relative to the virulence of other U.S.
endemic IBDV pathotypes: vv (rB strain), standard classic (cv) (STC strain) and
subclinical variant (sc) (Delaware E strain). Assessment of mortality, morbidity in 4 wk
old SPF leghorns indicated that reassortant vvIBDV (K669 and 7741) were less
pathogenic than the vvIBDV (rB strain) but more pathogenic than the standard classic
IBDV. In spite of this there were no significant differences in the severity of bursal
lesions in SPF groups inoculated with K669, D2712, rB, STC or Del-E. In vivo
pathogenicity of IBDV pathotypes was also assessed in maternally immune broilers at 1,
iv
2, 3 and 4 wks of age. No mortality was observed in maternally immune broilers
inoculated with any of the IBDV pathotypes at 1, 2, 3 and 4 wks of age. At 4 wks of age
there were no significant differences in the severity of bursal lesions and lymphocyte
depletion in the rB, K669 and STC groups. The severity of lesions in the 7741 and Del-E
inoculate groups was significantly lower than the rB, K669 and STC groups at 4 wks of
age. RT-PCR results and bursal lesions in broilers challenged at 2 wks of age indicate
that reassortant K669 was able to break through maternal immunity earlier than any of
the other challenge IBDV strains. The interserotypic reassortant D2712 was not
recovered 7 days PI from bursas from the SPF or maternally immune broiler groups
challenged with D2712 virus.
v
Dedicated to my mother Pamela Stoute, my husband Zaheer Mohammed, and two
children, Christian and Lauren Mohammed
vi
Acknowledgements
My sincere gratitude goes to my advisor and mentor Dr Daral Jackwood for giving me
the opportunity to work in his lab. His guidance, unwavering support and patience have
been instrumental in the completion of my research. I would also like to thank the other
members of my dissertation committee: Dr Richard Slemons, and Dr Chang-Won Lee for
their guidance and support.
I would like to thank my mother, Pamela Stoute for her unwavering love and support. I
will always be indebted to her for all the sacrifices she made so that I could pursue my
dreams. I am grateful to my husband who stood by me throughout my studies, and for his
love and care of our children Christian and Lauren Mohammed. I am inspired by his
selflessness in putting his dreams on hold so that I could pursue my own. I am always
thankful for being blessed with a supportive family and I acknowledge that without the
Best family my personal and academic achievements would probably not have been
possible. I would also like to acknowledge the 4 major mentors in my life: Dr Gabriel
Brown, Dr Bruce Charlton, Dr Arthur Bickford and Dr Daral Jackwood. I thank you
sincerely for all that you have taught me and for believing in me.
vii
Vita
1999-2004 ...................................................... D.V.M. The University of the West Indies,
School of Veterinary Medicine, Trinidad
2004-2005 ...................................................... Small Animal Veterinarian. Jones Animal
Clinic and Hospital, Trinidad
2005-2007 ...................................................... Poultry Medicine Teaching Assistant. The
University of the West Indies, Trinidad
2007-2009 ...................................................... Avian Pathology and Diagnostics Resident.
The University of California, Davis
2009 to present .............................................. Diplomate of the American College of
Poultry Veterinarians
2009-2012 ………………………………… Graduate Research Associate. PhD
Candidate. The Ohio State University,
FAHRP/OARDC
viii
Publications
1. Daral J. Jackwood, Beate M. Crossley, Simone T. Stoute, Susan Sommer-Wagner,
and Bruce R. Charlton. Diversity of Genome segment B from Infectious Bursal
Disease Viruses in the United States. Avian diseases. 56(1) 165-172. 2012.
2. Daral J. Jackwood, Susan E. Sommer-Wagner, Beate M. Crossley, Simone T.
Stoute, Peter R. Woolcock, and Bruce R. Charlton Identification and
pathogenicity of a natural reassortant between a very virulent serotype 1
infectious bursal disease virus (IBDV) and a serotype 2 IBDV. Virology
420(2):98-105. 2011.
3. Adesiyun AA, Fosgate GT, Seebaransingh R, Brown G, Stoute ST, StewartJohnson A. Virulence of Brucella abortus isolated from cattle and water buffalo.
Tropical Animal Health Production. 43(1): 13-6. 2011.
4. Jackwood, DJ, Sommer-Wagner SE, Stoute ST, Woolcock PR, Crossley BM,
Hietala SK, Charlton BR. Characteristics of a very virulent infectious bursal
disease virus from California. Avian Diseases. 53 (4): 592-600. 2009.
5. Simone T. Stoute, Daral J. Jackwood, Susan E. Sommer-Wagner, George L.
Cooper, Mark L. Anderson, Peter R. Woolcock, Arthur A. Bickford, Gabriel C.
Senties-Cue, and Bruce R. Charlton. The Diagnosis of Very Virulent Infectious
Bursal Disease in California Pullets. Avian Diseases. 53 (2): 321-326. 2009.
ix
6. Simone T. Stoute, Bruce R. Charlton, Arthur A. Bickford, and Mark C. Bland.
Respiratory Tract Trichomoniasis in Breeder Squab Candidates in Northern
California. Avian Diseases. 53 (1): 139-142. 2009.
7. Simone T. Stoute, A. A. Bickford, B. R. Charlton and R. L. Walker. Mycotic
Pododermatitis and Mycotic Pneumonia in Commercial Turkey Poults in
Northern California. Journal Veterinary Diagnostic Investigation. Vol. 21(4): 554557. 2009.
8. Simone T. Stoute, M. Bland and B. R. Charlton. A field comparison of farm
production parameters in beak trimmed layers versus non beak trimmed layers in
Northern California. Proceedings of 57th Western Poultry Conference. 52-54.
2008.
9. Abiodun Adesiyun, Simone T. Stoute, Berenice David. Pre-processed bovine milk
quality in Trinidad: Prevalence and characteristics of bacterial pathogens and
occurrence of antimicrobial residues in milk from collection centers. Food
Control.18: 312-320. 2007.0
Fields of Study
MAJOR FIELD: Comparative and Veterinary MedicineMolecular biology of Infectious Bursal Disease Virus
x
Table of Contents
Abstract ............................................................................................................................................ ii
Dedication ....................................................................................................................................... vi
Acknowledgements ........................................................................................................................ vii
Vita................................................................................................................................................ viii
Publications ..................................................................................................................................... ix
Fields of Study ................................................................................................................................. x
Table of Contents……………………………………………………………………………………………………………………….xi
List of Tables ................................................................................................................................ xiv
List of Figures ................................................................................................................................ xv
Chapter 1- Literature Review: Molecular Virology of the Very Virulent Infectious Bursal
Disease Virus .................................................................................................................................. 1
1.1 History and Epidemiology of vvIBDV .................................................................................. 2
1.2 Introduction ............................................................................................................................ 3
1.3 IBDV Classification ............................................................................................................... 4
1.3.1 IBDV Antigenic Variation .................................................................................................. 4
1.4 Chemical Composition and Replication of IBDV ................................................................. 5
1.4.1 Proteomics of Segment A of IBDV .................................................................................... 6
1.4.2 Molecular Characteristic of VP2 of IBDV ......................................................................... 7
xi
1.4.3 Proteomics of Segment B of IBDV................................................................................... 99
1.4.4 Phylogenetic Characterization of Segment B of IBDV .................................................... 10
1.5 Molecular Determinants of Pathogenicity of IBDV ............................................................ 12
1.6 IBDV Clinical Disease ......................................................................................................... 15
1.7 Isolation of IBDV ................................................................................................................ 16
1.8 IBDV Immunization ............................................................................................................ 18
1.9 References ............................................................................................................................ 21
Chapter 2 - Pathogenicity Associated with Co-infection with Very Virulent Infectious
Bursal Disease Virus (vvIBDV) and U.S. Endemic IBDV ........................................................ 29
2.1 Summary .............................................................................................................................. 30
2.2 Introduction .......................................................................................................................... 31
2.3 Materials and Methods......................................................................................................... 33
2.4 Results .................................................................................................................................. 38
2.5 Discussion ............................................................................................................................ 41
2.6 References ............................................................................................................................ 52
Chapter 3 - Molecular and Pathogenic Investigation of Reassortant Very Virulent
Infectious Bursal Disease Viruses in California. ................................................................... 64
3.1 Summary .............................................................................................................................. 65
3.2 Introduction .......................................................................................................................... 68
3.3 Materials and Methods......................................................................................................... 71
3.4 Results .................................................................................................................................. 78
xii
3.5 Discussion ............................................................................................................................ 89
3.7 Endnotes............................................................................................................................... 96
3.8 References .......................................................................................................................... 124
Bibliography ............................................................................................................................... 135
xiii
List of Tables
Table 2.1 Study design for experimental groups co-challenged with rB along with a
second IBDV pathotype compared to challenge with rB alone at 4 wks (1a-1f) and 6 wks
of age (2a-2f)..................................................................................................................... 46
Table 2.2 Pathology observed with 4 wk old SPF chickens co-challenged with vvIBDV
(rB) and a second IBDV pathotype compared to vvIBDV (rB) ....................................... 47
Table 2.3 Pathology observed with 6 wk old SPF chickens co-challenged with vvIBDV
(rB) along with a second IBDV pathotype. ...................................................................... 48
Table 3.1 Experiment 1: Pathogenicity investigation of reassortant IBDV in 4 wk SPF
chickens............................................................................................................................. 98
Table 3.2. Experiment 2: Pathogenicity in Maternally Immune Boilers Inoculated with
IBDV ................................................................................................................................. 99
Table 3.3. Experiment 2: Geometric mean titers (GMT) obtained from Viral
Neutralization (VN) tests using standard classic S706 and Del-E 8903 IBDV strains and
ELISA tests. .................................................................................................................... 101
xiv
List of Figures
Figure 2.1. Mortality 5 days post challenge in 4 wk and 6wk old SPF chickens
simultaneously inoculated with 105 EID50 vvIBDV (rB) and 105EID50 of either STC, DelE, T1 or OH strains of IBDV compared to mortality in chickens inoculated with vvIBDV
(rB) alone. ......................................................................................................................... 49
Figure 2.2. Restriction enzyme digestion of the amplified 743 bp fragment of segment A
of IBDV. The visible 424 bp and 172 bp bands from BstNI digestion of the segment A
fragment from groups 1a,1b,1c, and 1d are located in lanes 1, 2, 3 and 4 respectively.
The 100 bp ladder is located in lane 5. The visible 362 bp and 229 bp bands from MboI
enzyme digestion of segment A from groups 1a,1b,1c, and 1d are located in lanes 6, 7, 8
and 9 respectively. Digested fragments were run on a 2% metaphor agar gel. ................ 50
Figure 2.3. Restriction enzyme digestion using BamHI on the amplified 722 bp fragment
of segment B of IBDV. The 655 bp fragment from groups 1a, 1b, 1c, 1d, 1e are visible in
lanes 2, 3, 4, 5 and 6 respectively. Digested fragments were run on a 2% metaphor agar
gel. The 100 bp ladder is located in lane 1. This banding pattern is synonymous with
BamHI digestion of the 722 bp VP1 encoding region of vvIBDV strains........................ 51
Figure 3.1. Experiment 1: Mortality observed 7 days post challenge with different
strains of IBDV inoculated into 4 wk old SPF chickens ................................................ 102
Figure 3.2. Experiment 1: Mean BBW ratios calculated in IBDV challenge groups. ... 103
xv
Figure 3.3. Experiment 2: Geometric mean titers (GMT) in Maternally Immune Broilers
calculated with Viral Neutralization (VN) against Classic S706 Winterfield IBDV Strain
and Variant Del-E 8903 Strain and ELISA tests .......................................................... 1044
Figure 3.4. Experiment 2: Mean bursal lesions scores in maternally immune broilers
challenged between 1-4 wks of age. ............................................................................... 105
Figure 3.5. Experiment 2: Mean BBW ratios in Maternally Immune Broilers Challenged
with Different IBDV Strains at 1 wk of age. .................................................................. 106
Figure 3.6. Experiment 2: Mean BBW ratios in Maternally Immune Broilers Challenged
with Different IBDV Strains at 2 wk of age. .................................................................. 107
Figure 3.7. Experiment 2: Mean BBW ratios in Maternally Immune Broilers Challenged
with Different IBDV Strains at 3 wk of age ................................................................... 108
Figure 3.8. Experiment 2: Mean BBW ratios in Maternally Immune Broilers Challenged
with Different IBDV Strains at 4 wk of age. .................................................................. 109
Figure 3.9. Bursal lesion scores (BLS) graded 0-4. Microscopic appearance of the
varying severity of lymphocyte depletion based on the scoring system used. Score of ‘0’
indicated an absence of lesions; 1: <25% lymphocyte depletion in bursas; 2: 25-50%
lymphocyte depletion in bursas; 3: 50-75% lymphocyte depletion in bursas; 4: >75%
lymphocyte depletion in bursas. ..................................................................................... 110
Figure 3.10. Experiment 1. Hemorrhages on the mucosa of the proventriculus from rB,
K669 and 7741 inoculated SPF birds.............................................................................. 111
Figure 3.11. Picture A: Clinical signs of vvIBDV (rB strain) 48 hours post challenge of
SPF leghorn from experiment 2. Prostration, ruffled feathers and closing of eyes
xvi
observed. Picture B: Macroscopic appearance of bursa from vvIBDV (rB strain)
challenged SPF bird that succumbed to infection 3 days post challenge. Picture C:
Microscopic appearance of the bursa of Fabricius from picture B; lymphocyte depletion
and stromal hemorrhage observed. ................................................................................. 112
Figure 3.12. Microscopic appearance of bursa of Fabricius from SPF leghorn challenged
with reassortant 7741 4 days PI. Lymphocyte depletion and numerous cystic follicles
observed. Mag. x 4 .......................................................................................................... 113
Figure 3.13. Microscopic appearance of bursa of Fabricius from SPF leghorn challenged
with STC strain. Lymphocyte depletion, cystic follicles and interfollicular edema and
inflammation observed. Mag x 4 .................................................................................... 114
Figure 3.14. Microscopic appearance of bursa of Fabricius from SPF leghorn challenged
with variant Del-E. Significant lymphocyte depletion observed without significant
hemorrhage. Mag x 4 ...................................................................................................... 115
Figure 3.15. Microscopic appearance of bursa of Fabricius from SPF leghorn challenged
with reassortant K669. Severe stromal hemorrhage and extensive lymphocyte necrosis in
bursal follicles. Mag x 4.................................................................................................. 116
Figure 3.16. Experiment 1. Inoculation of SPF leghorns with D2712. Picture A:
hemorrhage (arrow) observed in the lamina propria of the cecal tonsils. Picture B:
pyogranuloma observed in cecal tonsil section. Mag x4 ................................................ 117
Figure 3.17. Experiment 1. Inoculation of SPF leghorns with D2712. Picture A:
Interfollicular inflammation (arrow). Picture B: Mild interfollicular hemorrhage (arrow).
Lymphocyte depletion was not a significant finding in bursas. Mag x4 ........................ 118
xvii
Figure 3.18. Inoculation of 9 day old embryo on right with reassortant K669 for viral
titration in embroyated eggs procedue. Five days PI, stunting of embryo observed. ..... 119
Figure 3.19. Liver in (A) taken 5 days PI from chicken embryo inoculated via the CAM
with reassortant IBDV strain D2712. The greenish discoloration of liver evident (arrow).
Chicken embryo tissues harvested from inoculated embryos were positive for D2712 by
real time RT-PCR. The two livers from (B) were from the un-inoculated controls. ...... 120
Figure 3.20. Phylogenetic analysis of partial genome segment A nucleotide sequences of
IBDV used in experiment 1 and IBDV from around the world. The neighbor-joining
method with up to 1000 bootstrap replicates was used................................................... 121
Figure 3.21. Phylogenetic analysis of partial genome segment B nucleotide sequences of
IBDV used in experiment 1 and IBDV from around the world. The neighbor-joining
method with up to 1000 bootstrap replicates was used................................................... 122
xviii
Chapter 1- Literature Review
Molecular Virology of the Very Virulent Infectious Bursal Disease Virus
1
1.1 History and Epidemiology of vvIBDV
The very virulent pathotype of infectious bursal disease virus (vvIBDV)
represents one of the most economically significant immunosuppressive diseases in the
international poultry industry. In addition to the spiking mortality and mortality induced
by acute infection with vvIBDV, birds surviving infection frequently exhibit an increased
susceptibility to opportunistic infections. The vvIBDV emerged from Holland (DV86
strain) then Belgium in 1986 and has since rapidly spread throughout most of Europe
[107]. In the early 1990’s vvIBDV rapidly spread throughout Asia, Africa, and South
America [78,107] and more recently the United States [57,102]. These viruses have been
identified on almost every continent but have not been identified in Australia and New
Zealand. Since its emergence, the virus has spread throughout most continents and by
1995, 80% of the member countries of the Office International Epizooties (OIE) reported
the occurence of vvIBDV [107]. The first isolates of vvIBDV (rA and rB strains) in the
United States was recovered in December 2008 from 2 commercial layer farms in
California [102]. These cases resulted in 26% and 24% mortality in 11 and 14 wk old
brown leghorns respectively [102]. The rA and rB strains isolated from the U.S. in 2008
were identical across the hypervaiable VP2 region to the UK 661, OKYM and Harbin
vvIBDV strains. Phylogenetic analysis of the proximal end of genome segment B from
position 337-855 was a 98.1% match between rA, rB and the other vvIBDV examined
compared to only an 88% match with classic IBDV segment B strains [57,102].The rA
2
and rB strains resulted in mortality ranging from 91-100% in SPF chickens [57,102].
Since then, several cases of vvIBDV and reassortant vvIBDV have been detected from
commercial and backyard avian species (chickens, turkeys, chukars) from California
[56]. As the virus continues to spread worldwide, there is an ongoing challenge to come
up with effective vaccine protocols that provide adequate immune protection against the
very virulent and antigenically variant pathotypes. The emergence of increasingly
pathogenic strains and novel molecular variants with relatively high stability in the
environment places high demands on epidemiological surveillance and preventive
medicine programs. Hence fundamental research into the immunology, pathobiology,
detection and control of vvIBDV are required to counteract the potentially devastating
economic effects of the virus.
1.2 Introduction
Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal
disease (IBD) also frequently referred to as “Gumboro disease”. IBD was first recognized
in 1962 in the Gumboro Delaware region. In the 1980’s there was an emergence of a
hypervirulent strain of IBDV (vvIBDV) in broilers in Europe that was capable of
overcoming vaccine immunity that was previously efficacious against the endemic IBDV
strains [107].
The severity and economic impact of the disease is influenced by a combination of
factors such as age and breed of flock, levels of maternally derived antibodies, virus
strain, vaccination protocol utilized, presence of concurrent pathogens as well as
environmental and management factors [78]. IBDV has a predilection for the bursa of
3
Fabricius where virulent IBDV strains target N-glycosylated membrane protein receptors
on actively dividing and differentiating IgM-bearing B-lymphocytes [24].
1.3 IBDV Classification
The virus belongs to the genus Avibirnavirus of the Birnaviridae family. IBD
viruses are single-shelled, non enveloped and icosahedral with a diameter ranging from
55-64 nm [24,34,85]. Of the 2 distinct serotypes of IBDV distinguishable by viral
neutralization and cross protection studies, only serotype 1 strains are pathogenic in
chickens. While serotype 2 strains are capable of infecting both chicken and turkey
species, clinical disease is not usually associated with serotype 2 IBDV [1,41,51]. Studies
on the serotype 2 OH strain found that the virus was non pathogenic in chickens and
turkeys but was pathogenic in chicken embryos after being passaged 5 times [1].
Serotype 1 strains naturally range in virulence and severity of immunosupression.
They can be further classified as avirulent, subclinical (sc), classical virulent (cv;
sometimes called standard), antigenic variant and very virulent (vv) pathotypes [26,108].
The vvIBDV designation is usually reserved for viruses causing high morbidity and
mortality.
1.3.1 IBDV Antigenic Variation
The lack of proof reading capability and relatively high mutation rate of RNA
polymerases of RNA viruses result in the emergence of quasispecies and genetic
diversification brought about by antigenic drift. This influences the emergence of
4
antigenic variants capable of escaping vaccine induced antibody cross protection.
Antigenic shift bought about by reassortment of the bisegmented IBDV genome also
leads to variation in pathogenic subtypes. Cross neutralization tests indicate that vvIBDV
are antigenically similar but not identical to classic viruses. Investigations using
monoclonal antibody panels have identified a modified epitope that exists on vvIBDV
strains that correspond to a mutation at position 222 in the first hydrophilic peak of VP2
[28]. While vvIBDV with more extensive epitope changes have also been identified [27]
their antigenic and epidemiology significance are considered questionable [107].
Sera of chickens vaccinated against classical strains have a restricted ability to
cross neutralize variant strains of IBDV [42]. Hence variants can cause disease in the face
of immunity against classical strains. Variants have been reported from North, Central
and South America [46] as well as in Australia, Spain and France [92]. Antigenic changes
observed in variant viruses have been attributed to a major antigenic shift involving
several amino acid changes in the variable region of viral protein 2 (VP2). Analyses of
field isolates of variant and classical strains of IBDV by antigen capture enzyme linked
immunosorbent assays (AC-ELSIA) typically utilize a panel of monoclonal antibodies
(MAbs) (e.g. MAbs 10, 57, 67, R63, B69) [99,100]. Viral cross neutralization tests (VN)
use polyclonal antibodies to distinguish between serotypes 1 and 2. The 2 serotypes
cannot be distinguished based on ELISA or fluorescent antibody (FA) tests [40].
Immunization against serotype 2 does not confer protection against serotype 1 strains
[51].
5
1.4 Chemical Composition and Replication of IBDV
The IBDV genome consists of 2 segments of double-stranded (ds) RNA,
designated as A and B [7,24,50]. The complete nucleotide sequences of the 2 segments
have been established and sequence data are available in Genbank for many IBDV
strains. The genome of IBDV encodes for 5 structural proteins designated VP1, VP2,
VP3, VP4, and VP5 [7,24]. The coding regions of both segment A and segment B are
flanked by short untranslated sequences on the 5’ and 3’end [82]. The genome of IBDV
remains protected within the virion throughout the virus replication cycle and viral
enzymes regulate several enzymatic activities related to replication such as transcription
[21,22]. The virus becomes transcriptionally activated in the presence of nucleotides and
non-polyadenylated mRNA are extruded through the pores of the capsid. It is presently
unknown if the mRNA’s are capped [101].
1.4.1 Proteomics of Segment A of IBDV
The larger segment A (3.3 kbp) contains 2 open reading frames (ORF). The larger
ORF encodes for a 109 kDa precursor polyprotein: pVP2-VP4-VP3 [5]. Autocleavage of
the VP4 at its’ N and C termini in the polyprotein facilitates the release of the outer and
inner structural capsid viral proteins designated pVP2 (48 kDa) and VP3 (32-35 kDa)
respectively as well as the putative viral protease, VP4 (24 kDa) [91]. The VP4 protein
utilizes an unusual serine-lysine (Ser-652 and Lys-692) catalytic dydad [65]. VP4
protease regulates the maturation of pVP2 by progressive trimming at its C terminus into
the mature VP2 and 4 small peptides on the surface of the virion [65]. It has been
6
hypothesized that these peptides may play a role in destabilization of the cell membrane
to facilitate virion entry [18]. Further maturation of pVP2 is thought to occur after viral
particle assembly and only the larger protein pVP2 can be demonstrated in infected cells
[19]. The final processing of pVP2 to VP2 is controlled by the correct scaffolding of the
VP3 inner capsid protein. VP3 interacts with the C-terminal end of the precursor pVP2,
the viral dsRNA and also with VP1 to regulate morphogenesis of the virus particle
[19,71]. VP3 has also been shown to elicit non neutralizing and non protective antibodies
whereas VP2 is an essential immunogen for the virus [8]. The smaller ORF on segment
A encodes for a small, non structural protein, VP5 (17-21 kDa) and it precedes and
partially overlaps the larger ORF [12] The exact function of VP5 is unknown but it
accumulates on the host plasma membrane and is thought to be involved in viral egress
and dissemination as well as anti-apoptosis in the early stages of infection [70,72,81].
Under conditions of high multiplicity of infection (MOI) excessive amounts of pVP2 and
a series of aberrant polypeptides can form incomplete particles (defective interfering
particles) [78].
1.4.2 Molecular Characteristic of VP2 of IBDV
IBDV has a typical laevo icosahedral capsid symmetry with triangulation number
T=13 [22]. The single layer capsid is unfenestrated and composed entirely of VP2
trimers, hence this is the only viral protein recognized by neutralizing antibodies [22].
The variable VP2 gene which encodes the major host protective antigens has been the
focus of most epidemiological investigations since this portion of the genome consists of
7
relatively conserved sequence regions unique to vvIBDV. The VP2 has 2 antigenic
domains; one conformation independent and the other conformation dependent. The
conformation independent domain elicits non neutralizing and non protective antibodies.
In contrast, the conformation dependent antigenic domain located on the mid third of the
VP2 gene elicits both neutralizing and passively protective antibodies. Some of the
epitopes located on both domains of VP2 are group specific while others are strain
specific [8].
VP2 is folded into 3 distinct domains, designated as the base (B), shell (S) and
projection (P) [22]. It is hypothesized that a domain in the VP2 regulates the sealing of
the interior and projecting domain of the virus [22]. Based on sequence alignments, the B
and S domains are formed by the relatively well conserved N and C terminal stretches of
VP2 while the P domain is formed from the more variable region of VP2 (aa: 206-330).
The S domain is central to the organization of the molecule. All 3 domains of VP2
participate in trimer contacts [22]. Amino acid variability between the 2 serotypes as well
among the pathogenic serotypes is primarily attributed to variation in the P domain. The
outermost part of the P domain is made up of 2 major hydrophilic peaks A (aa 212-224)
and B (aa 314-325) which are constituted from Loops PBC and PHI respectively. Deletion
studies indicate that these loops are critical for viral neutralization and are part of the
epitopes defined by neutralizing monoclonal antibodies hence variation in these regions
result in changes in reactivity against monoclonal antibodies. In addition, reverse genetics
has shown that a few amino acids in VP2 located in the most exposed loops at the top of
domain P at positions 253 (loop PDE) and 284 (loop PFG) also play important roles in viral
8
adaptation in cell cultures and virulence [10,22,66,70,80]. These 2 positions are located
on VP2 minor hydrophilic peaks 1 and 2: aa 248-253 and 279-290 respectively. These
amino acids in the hydrophilic peaks have side chains that point outward and are not
involved in interactions between subunits that stabilize the virion or in contacts important
for the folding of VP2. These amino acids are located in the most exposed part of VP2
exposed on the most external surface of the virus particle. It has been hypothesized that
the effect on pathogenicity is due to direct interaction of these residues with target cell
receptors [22].
Letzel et al. performed substitutions at positions 222, 318, 321, 323 and 330 along
the backbone of a D78 segment to identify residues involved with different antigenic
patterns. They concluded that single and multiple amino acid substitutions in loops PBC
and PHI control the reactivity against monoclonal antibodies. It was found that these 5
residues deeply modulate immunoreactivity [66]. Mutations created by reverse genetics
also showed that 3 residues in the PHI loop at positions 318, 321 and 323 are important
parameters involved in replication rates in cell culture. Other residues may not be critical
to reactivity with monoclonal antibodies but may facilitate binding. One such residue is
A323 which is thought to facilitate recognition of the epitope [66].
1.4.3 Proteomics of Segment B of IBDV
The smaller genome segment B (2.8 kbp) encodes for VP1 (90 kDa) which has
RNA dependent RNA polymerase activity and is responsible for genomic replication and
mRNA synthesis [79]. VP1 is covalently linked to the 5’ ends of the genomic RNA
9
segments and is also present as free protein in the viral capsid [78,101]. Birnavirus VP1
proteins form a distinct subgroup of RNA dependent RNA polymerases that lack a GDD
motif (Glycine-Aspartic acid-Aspartic acid) [98]. Phylogenetic analyses indicate that
segment B nucleotide sequences of vvIBDV strains form a distinct cluster [38,119] and it
has been suggested that these strains might have derived segment B by genetic
reassortment from an unidentified source [78].
The VP1 gene can be divided into 3 segments; the N-terminus (aa 1-167), the
central polymerase domain (aa 186- 658) and the C terminus (aa 659-878) [98]. The
central polymerase domain contain all the structural motifs characteristic of an RNA
dependent RNA polymerases, the N terminus functions in protein priming and it is
hypothesized that the C terminus may prevent back primed RNA synthesis during protein
priming. RNA polymerase activity could be demonstrated without pretreatment of the
virus. This indicates that viral transcription and replication occur following cell
penetration without uncoating of the virus. It has been suggested that nucleic acid
replication occurs by a strand displacement mechanism.
1.4.4 Phylogenetic Characterization of Segment B of IBDV
Phylogenetic analyses indicate that genome segment A and segment B evolved
independently, thus suggesting that reassortment may have played a role in the
emergence of the vvIBDV pathotype [36]. The emergence of vvIBDV is thought to have
10
occurred from a reassortant event between a segment B from an unknown reservoir and a
vv segment A that emerged at least 20 years before its expansion. At present it is
unknown weather this reservoir was from a wild bird or other animal species. Wild birds
have been suggested as a possible source of the virus based on the serological detection
of IBDV in wild bird species [32].
Yu and coworkers reported 3 branches of segment B based on phylogenetic
analysis of 38 strains of very virulent, classic, variant and attenuated strains [120]. Strains
in Branch I belong to non vvIBDVs and Branches II and III belong to vvIBDVs. Yu et al.
sequenced and analyzed segment B from 7 vvIBDV isolates from commercial broilers
and layers in China [120]. Interestingly it was found that 8 amino acids were conserved
among the 7 isolates: 4V, 61I, 145T, 287A, 508K, 511S, 646S and 687P in phylogenetic
Branches II and III [120]. Of the 8 conserved sequences found by Yu et al., 3 were in the
N terminus, 4 in the central domain and 1 in the C terminus. While it was hypothesized
that these 8 amino acids may contribute to increased virulence by enhancement of VP1
activity, further investigation using reversed genetics needs to be performed. Yu et al.
also found 5 amino acids (146D, 242E, 390M, 562P and 695R) conserved only in the
phylogenetic Branch III strains but not Branch II strains of vvIBDV isolates. These aa
changes might be linked to Segment B, Branch III evolutionary markers as opposed to
virulence factors as both Branch II and III are vvIBDVs. Also conserved in all vvIBDVs
were 55T and 63A in the 5’-UTRs and 2786C in the 3’-UTRs. Based on computational
studies it was suggested that the 55T might be functionally important to vvIBDV and that
the stable 5’-UTR secondary structure might be important for IBDV existence [120].
11
Jackwood et al. analyzed the segment B sequences from 67 IBDV strains isolated
in the U.S between 2002-2011. Phylogenetic analysis indicated that there were at least 4
different genetic lineages of segment B [47]. The vvIBDV were clustered within one
lineage with the other 3 branches containing segment B from non vvIBDV strains [47].
Among the non vvIBDV strains, one of the segment B lineages contained classic variant
and vaccine strains, a second contained an Australian segment B lineage and the last
contained a serotype 2 lineage. Interestingly, IBDV strains from California were present
within all 4 of the lineages. It was also found that genetic mutations were distributed
uniformly across the 5’ end of genome segment B. A high mutation frequency was noted
in the triplet of amino acids at position 145, 146 and 147. All the vvIBDV examined by
Jackwood and coworkers had the triplet amino acid motif TDN at positions 145, 146 and
147 [47].
1.5 Molecular Determinants of Pathogenicity of IBDV
Molecular epidemiological studies indicate that vvIBDV strains belong to the
same genetic lineage with a high degree of genetic and antigenic homogeneity. Studies
suggest that these strains evolved from a common ancestor emerging from a single
unique event and then subsequently evolved independently in Europe and Asia
[46,107,119]. Analysis of strains of vvIBDV indicate that they form a cluster and are
more closely related to classic IBDV than to other lineages [119]. Comparison of the
amino acid sequences of the very virulent and classical isolates of IBDV have
demonstrated that there are usually specific substitutions within the variable region of
12
VP2 of vvIBDV. Most vvIBDV have 4 typical amino acids in the VP2 region: A222,
I256, I294, and S299 [26]. The majority of vvIBDV are SspI positive and have the
molecular group 6 RFLP pattern [46]. Group and non neutralizing serotype specific
antibodies are mainly located on VP3 [78]. Typical amino acid markers for attenuation
are 253H and 284T [111].
The cellular receptor of IBDV is not known but it has been suggested that
serotypes 1 and 2 utilize several cellular receptors on different cell types. Studies to date
suggest that virulence is determined by multiple molecular determinants, some but not all
of which have already been elucidated [57,78]. Reassortant serotype1/serotype2 IBDV
indicate that segment A determines tropism for the bursa of Fabricius and segment B is
involved in the efficiency of virus replication and hence influences virulence [10].
Evidence that VP2 is not the sole determinant of virulence was demonstrated by Boot and
coworkers using a fowlpox-based reverse genetics system to demonstrate that VP2 is not
the sole determinant of pathogenicity of the very virulent phenotype [10]. In vitro and in
vivo analysis of rescued segment reassorted IBDVs indicate that virulence factors are not
only located on segment A but also on segment B encoding the RNA directed RNA
polymerase of VP1 [9,63]. Jackwood et al. also reported the first naturally occurring
interserotypic reassortant IBDV with a segment A homologous with vvIBDV and a
segment B matching serotype 2 IBDV. These reassortants initially recovered from
California chickens also exhibited a reduction in pathogenicity in challenged chickens
compared to the vvIBDV pathotype [56].
13
The efficiency of interaction between VP1 and VP3 may also influence
pathogenicity as this interaction may influence the assembly of viral particles [19,72,78].
Recent evidence suggests that the accumulation of the non structural protein VP5 on the
cell membrane of infected cells is thought to be involved in viral release and induction of
apoptosis by the formation of pores in the cell membrane of infected cells [118]. In
contrast to this, VP5 has been reported to hinder apoptosis in the early stages of viral
infection by interfering with the capases and NF-KB pathways [70]. While VP5 is not
present in the virion [11] and is not essential for viral replication, recent reports suggest
that it may play a role in the very virulent pathotype and adaptive capacity of the virus
[118]. It has also been reported that exchange of the VP5 gene between a cell culture
adapted serotype 1 IBDV and a serotype 2 IBDV resulted in a reduction in the viral
replication and cytotoxicity in chicken embryo fibroblasts in the serotype 1 IBDV [96].
Research indicates that VP5 is not responsible for the cell tropism and pathogenicity of
vvIBDV in cell cultures since exchange of VP5 between vvIBDV and attenuated serotype
1 IBDV does not alter pathogenicity and replication [81]. The difference in pathogenicity
between serotype 1 and 2 strains was shown to not be attributable to the VP5 gene or the
non coding 3’ and 5’ regions of segment A or the N terminus of VP2 [95,96]. A lack of
expression of VP5 does not alter intracellular replication but it does hinder release of
viral progeny from infected cells. Data suggests that VP5 might be used as a cell surface
marker for the detection of IBDV infected cells by flow cytometry analysis [118].
.
1.6 IBDV Clinical Disease
14
The vvIBDVs tend to be characterized by higher mortality rates than classical
virulent strains, with mortality rates as high as 100% in SPF flocks, 60% layers and 30%
in broilers [107-109]. There are also reports of vvIBDV strains that cause much lower
mortality in the field [102]. The variability in mortality reported for vvIBDV, especially
with field cases might be due to the influence of factors such as age resistance, presence
of concurrent diseases and level of immune protection provided by vaccination protocols.
Kim et al. reported mortality ranging from 0.3-9.4% in 3 vvIBDV field isolates in Korea
with the characteristic amino acids at the variable VP2 region [62]. While mortality
ranging from 26-34% was reported from pullets infected with field isolates in California
[102].
While the period of greatest susceptibility is reportedly 3-6 wks of age, IBDV has
the potential to infect and cause disease in older chicken flocks before the onset of sexual
maturity and age related regression of the bursa of Fabricius [30,57,67,102]. Birds
younger than 3 weeks frequently exhibit an age related resistance to clinical disease but
can still develop subclinical disease and significant immune suppression [30]. After an
incubation period of 2-3 days, clinical signs of IBD include vent picking, vent feathers
soiled with blood, whitish watery diarrhea, ruffled feathers, anorexia, dehydration,
prostration and eventually death [102]. Mortality rates usually peak in flocks at about one
week post infection. Typical necropsy lesions include petechial and ecchymotic
hemorrhages of the pectoral and thigh muscles. Affected bursas are initially enlarged and
edematous within 3-5 days of infection before progressive atrophy occurs. Enlarged
bursas frequently are covered by a yellow gelatinous transudate. Yellow caseous cores
15
are occasional observed in the lumen of infected bursas [68]. Bursal histopathology
includes petechial and ecchymotic hemorrhages, lymphofollicular depletion and necrosis
as well as heterophilic inflammation. Variant strains of IBDV such as Delaware E are
characterized by bursal atrophy and involution of bursal follicles without a significant
inflammatory response. Additional lesions include mottling of the kidneys and renal
congestion with tubular necrosis which may be secondary to dehydration [102].
Lymphocyte depletion may also be observed in the thymus and spleen. Mucosal
hemorrhages in the cecal tonsils and junction of the proventriculus and gizzard are more
frequently associated with the more virulent strains of IBDV [68,102,116]. It has been
reported that lesions in non bursal organs are correlated with viral antigen distribution
[104]. While variant pathotypes typically result in subclinical disease with an absence of
overt clinical signs, immunosuppression secondary to marked bursal atrophy frequently
occurs [12].
1.7 Isolation of IBDV
The bursa of Fabricius is the organ of choice for recovery of IBDV. While other
organs such as the spleen may contain virus the concentration is typically low in non
bursal organs. The embryonating egg is considered to be the most sensitive substrate for
isolation of IBDV. The chorioallantoic membrane is the route of choice for viral
inoculation in 9-11 day old embryos. Pathogenic viruses such as the classic and vvIBDV
strains induce embryo mortality within 3-5 days post inoculation while variant strains
16
produce negligible mortality. Lesions attributable to classic viruses include carcass
congestion, petechial and ecchymotic hemorrhages (on cerebrum, toes and feather tracts),
necrotic livers, necrosis of livers and necrotic foci on spleens. Lesions induced in
embryos inoculated with variant strains of IBDV include stunting, necrotic and bile
stained livers, splenomegaly and cerebral and abdominal edema.
While embryo inoculation is best for initial isolation of IBDV, many strains of
IBDV have been adapted to grow in cell culture hence facilitating quantification by
plaque assays or microtitre techniques. In addition to culture in chicken cell lines such as
chicken embryo fibroblasts (CEF) [74], IBDV has been grown in duck and turkey
embryo cells [76] and mammalian cell lines derived from rabbit kidneys (RK-13), and
monkey kidney cells (BGM-70, Vero) [43,75]. The BGM-70 cell line has been reported
to be successful in supporting several strains of serotype 1 and 2 IBDV [43]. Several
blind passages are sometimes required before cytopathic effects are observed. The
vvIBDV is difficult to adapt to cell cultures. Adaptation of the vvIBDV to cell culture is
frequently associated with attenuation of the virus and a reduction in the ability of the
virus to replicate in the bursa of Fabricius [111].
The agar gel precipitation test (AGP) can be used for detection and quantitation of
group specific antigen. The viral neutralization test (VN) is more sensitive than AGP and
can be implemented in cross neutralization tests to serotype IBDV strains using
polyclonal antibodies [17,99,100]. Viral neutralization titers using VN tests can vary
widely depending on the indicator virus used in the test due to the range of antigenic
differences among IBDV isolates. Immunocytochemistry, and direct and indirect
17
immunofluorescence have been used for detection of IBDV antigen in tissues [2]. ELISA
test kits are also available for IBDV antibody quantitation and has the adavantage of
providing rapid results relative to VN tests [4].
In addition to the above mentioned tests, molecular analysis and in vivo
pathogenicity studies are needed to monitor evolutionary changes and the emergence of
quasispecies in the field viruses. The use of reverse transcriptase polymerase chain
reaction tests (RT-PCR) including multiplex RT-PCR has been instrumental in the
detection and characterization of strains of IBDV [58,77]. Real time PCR assays using
probes specific to serotype 1 vv and non vv strains and serotype 2 viruses are available.
RT-PCR assays are also available to detect segment B of IBDV [58].
1.8 IBDV Immunization
Despite the close antigenic relationship to classical serotype 1 strains, vvIBDVs
have the ability to break through antibody titers induced by IBDV vaccine protocols
based on classical strains [108]. Humoral immunity plays a critical role in protection of
chicks by the passive transfer of maternal antibodies in the first weeks of life. Progeny of
breeder flocks vaccinated with live vaccines are typically protected for 1-3 weeks while
boosting the immune response with killed, oil adjuvanted vaccines can provide extended
protection for 4-5weeks of age [73]. The use of antibody-flock profiling is frequently
used in the field in order to properly time vaccine administration in young chicks as
18
maternally derived antibodies could inhibit an active immune response. Effective
vaccination protocols are targeted at the immunization of flocks during the window of
susceptibility when maternal antibodies wane and IBDV antibody titers are insufficient to
provide adequate protection. Multiple vaccinations are often done in order to
accommodate birds with varying levels of maternal antibodies. Recent concepts in
vaccination explore the use of in ovo vaccination at 18 days of incubation with an
attenuated IBDV vaccine used either alone or in combination with anti-IBDV antibody to
form an immune complex [84].
Live vaccines are classified according to virulence as mild, mild intermediate,
intermediate and intermediate plus (‘hot’) strains. Most intermediate vaccines are
insufficient in providing adequate protection again vvIBDV, especially under conditions
of high infection pressure. Hence vaccine protocols incorporating the use of intermediate
plus vaccines may reduce mortality rates in the field from vvIBDV. Notably, while
intermediate plus vaccines are capable of breaking through maternal antibody titers as
high as 1:500, they can potentially induce bursal atrophy and immunosuppression
[78,84,123]. Live vaccines also have the potential to cause complications due to the
instability in their antigenic and pathogenic characteristics.
Tissue culture adaptation of IBDV is associated with viral attenuation. Research is
currently underway into the use of genetically engineered vaccines against IBD. These
include the use of subunit vaccines using vectors such as baculovirus, Semliki Forest
virus and yeast expressing the VP2 antigen of IBDV. Recombinant vaccine virus vectors
expressing IBDV antigens include fowl pox virus, chicken embryo lethal orphan virus
19
(CELO), Marek’s disease virus and herpes virus of turkeys (HVT). Zhou et al. reported
an efficacy of 87% against virulent IBD in commercial chickens using of a recombinant
Marek’s disease virus (rMDV1) vector expressing IBDV-VP2 antigens. Compared with
the commercial live vaccine this recombinant vaccine was considered advantageous due
to its stability, safety, absence of side effects in the bursa of Fabricius and the likely
persistence of protection throughout the life of the chicken [14,23,94,97,105].
While recombinant virus vaccines have been reported to confer protection in SPF
flocks against standard and variant strains, the efficacy of these vaccines against the
vvIBDV pathotype is variable [83]. Control efforts are complicated by the fact that IBDV
is highly contagious and relatively resistant to inactivation. A combination of effective
biosecurity measures and a vaccine protocol tailored to the flock are important in
minimizing the immunosuppressive and clinical effects of IBD.
20
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25
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26
66. P. S. Shwed, P. Dobos, L. Cameron, V. N. Vakharia, and R. Duncan, "Birnavirus
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27
77. R. W. Winterfield, A. M. Fadly, and A. A. Bickford, "Infectivity and distribution
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Dis. 47, 186-192 (2003).
28
Chapter 2 - Pathogenicity Associated with Co-infection with Very Virulent
Infectious Bursal Disease Virus (vvIBDV) and U.S. Endemic IBDV
29
2.1 Summary
The aim of this investigation was to assess the pathogenicity induced by co-infection
with very virulent infectious bursal disease virus (vvIBDV: rB strain) and U.S. endemic
IBDV pathotypes in specific pathogenic free leghorns. The severity of disease in cochallenged birds was compared to the disease induced by infection with the vvIBDV (rB
strain) alone. Four wk old and 6 wk old birds were challenged oronasally with 105EID50
vv (rB strain) and 105EID50 of either a standard classic IBDV (STC strain), subclinical
variant IBDV (Delaware-E strain), subclinical variant IBDV (T1 strain) or avirulent
serotype 2 IBDV (OH strain). The severity of disease was assessed by comparing the 5day mortality rates, severity of bursal lesions (mean bursal lesion scores) and mean bursal
body weights (BBW) in each of the challenged groups. A mortality rate of 100% was
observed in both 4 wk and 6 wk old birds inoculated with only the vvIBDV (rB) strain. A
relative reduction in the mortality and severity of disease was observed in all groups cochallenged with rB and the second less virulent pathotypes (STC, Del-E, T1, OH) in both
4 wk and 6 wk old birds. Co-challenge with rB and the Del-E, T1 and OH strains resulted
in a more significant decrease in mortality compared to challenge with the two
antigenically similar pathogenic strains, rB and STC. Viral interference between the
different pathotypes is hypothesized as the etiology of the reduction in severity of disease
in co-challenged birds.
30
2.2 Introduction
Infectious bursal disease virus (IBDV) is an immunosuppressive virus targeting
primarily B-lymphocytes in the bursa of Fabricius of chickens. IBDV are non enveloped
bisegmented RNA viruses belonging to the Avibirnavirus genus within the Birnaviridae
family. A range of pathogenicity is observed with IBDV with the most virulent strains
being designated as ‘very virulent’ infectious bursal disease viruses. Of the 2 distinct
serotypes of IBDV distinguishable by virus neutralization and cross protection studies,
only serotype 1 strains are pathogenic in chickens. Serotype 1 strains naturally range in
virulence and severity of immunosupression. IBDV strains are generally subdivided as
avirulent, subclinical (sc), classical virulent (cv; also referred to as standard), and very
virulent (vv) pathotypes [56].
Criteria for designation of IBDV strains as the vvIBDV pathotype are based on a
combination of molecular, antigenic and phenotypic characteristics [110]. Phylogenetic
analyses indicate that vvIBDV strains evolved from a common lineage and exhibit a high
degree of genetic and antigenic homogeneity [26,28,119,121] . The vvIBDV designation
is usually reserved for viruses causing higher mortality than classic strains. Mortality can
range between 40-100% in fully susceptible SPF chickens, 60% in layers and 30% in
broilers [57,108,109]. Some, but not all of the genetic determinants of pathogenicity have
been elucidated. Both segments A and B of the IBDV genome are thought to influence
pathogenicity [9,31,36]. Highly conserved sequences have been observed in the
hypervariable (hv) region of VP2 of vvIBDV strains. Four amino acids in the hvVP2 at
positions 222 (Ala), 256 (Ile) and 294 (Ile) and 299 (Ser) are highly correlated with
31
vvIBDV strains [6,110]. Using monoclonal antibody panels vvIBDV strains tested were
antigenically similar to classic serotype 1 strains with only minor variations noted
[57,106].
The vvIBDV pathotype was first reported in Belgium in the late 1980’s and has
since rapidly spread throughout Europe, Middle East, Asia and South America. Previous
to the emergence vvIBDV, the less virulent strains of IBDV were well controlled by
vaccination and mortality of less than 2% was typically observed in the field. The vv
pathotype of IBDV (rA and rB strains) was first isolated in the U.S. in California
commercial poultry in December 2008 [102]. The rA and rB vvIBDV resulted in spiking
mortality of 26% and 34% respectively on affected layer flocks. Despite ongoing
continuous surveillance for vvIBDV in California poultry, detection of vvIBDV has only
been sporadic since 2008 (personal communications: California Animal Health and Food
Safety; Dr Beate Crossley). This low incidence is contradictory to the typical explosive
expansion and establishment of the vvIBDV strain in other geographical regions. The
purpose of this investigation was to determine if co-infection with other endemic IBDV
pathotypes could alter the severity of clinical disease typically associated with the
vvIBDV pathotype. This in vivo challenge study investigated the pathogenicity
associated with co-infection of SPF chickens with vvIBDV (rB strain) and non vvIBDV
pathotype either: standard classic (STC strain), subclinical Delaware E (Del-E), variant
strain (T1) or avirulent serotype 2 Ohio strain (OH) in 4 wk and 6 wk old leghorns.
32
2.3 Materials and Methods
Viruses. The vvIBDV (rB strain) was isolated from commercial pullets in
California from the first reported case of vvIBDV in the U.S. in December 2008 [57,102].
The T1 subclinical antigenic variant was isolated in 2006 in Georgia, USA [54] . The
standard classic (STC) strain, avirulent serotype 2 OH strain and subclinical variants DelE and T1 were previously described [54]. These viruses were selected for co-challenge
with vvIBDV (rB strain) based on their endemicity in US poultry flocks.
Experimental design. The 50% egg infectious dose (EID50) for viruses used for
challenge was determined by viral titrations in 9-day-old embryonated SPF chicken eggs
via the chorioallantoic membrane (CAM) with 0.1ml of bursal homogenate as previously
described [57]. SPF leghorns 4 wk of age were divided into 5 challenge groups 1a, 1b, 1c,
1d, 1e and one uninoculated control group 1f (Table 2.1). Groups 1a-1d were
simultaneously inoculated with 0.l ml 105EID50 of rB and 0.l ml 105 EID50 a second
IBDV pathotype mixed within a single 1ml syringe and administered via the oronasal
route. Group 1a was challenged with rB and STC, group 1b received rB and Del-E, group
1c received rB and T1, and group 1d received rB and OH. Birds from group 1e were
challenged with 0.l ml 105 EID50 of rB strain without the addition of a second IBDV.
Five birds were allotted to each group except for group 1e which consisted of 10 birds.
The experiment was repeated in 6 wk old SPF birds divided into 5 challenge groups (2a2e) and 1 control group 2f. Experimental protocol was similar to that used for the 4 wk
old birds except that 3 birds were placed in the rB group 2e and 3 birds were placed in the
control group 2f (Table 2.1). Birds within each experimental group were separated and
33
housed in environmentally controlled Horsfall® isolation units and given feed and water
ad libitum.
Birds were monitored daily and morbidity and mortality were recorded. At 5 days
post challenge, birds from all experimental groups were humanely euthanized and
necropsied. At necropsy, gross lesions were recorded along with bursa and body weights
from euthanized birds. The bursa/body weight ratios were calculated from euthanized
birds only and not birds that died during the course of the experiment since dehydration
of carcasses from birds that died would have altered the validity of the results. Tissue
sections from the bursa, spleen, proventriculus and cecal tonsils were taken from all birds
for histopathology. Bursa tissue sections from all birds were also taken for molecular
detection of IBDV.
Histopathology. Tissue sections from bursa, spleen and proventriculus were
fixed in 10% neutral buffered formalin, sectioned at 4μm, stained with hematoxylin and
eosin and examined by light microscopy. The severity of lymphocyte depletion in bursal
follicles was quantified by assigning bursal lesion scores (BLS) ranging from 0 to 4.
Score of ‘0’ indicated an absence of lesions; 1: <25% lymphocyte depletion in bursas; 2:
25-50% lymphocyte depletion in bursas; 3: 50-75% lymphocyte depletion in bursas; 4:
>75% lymphocyte depletion in bursas. Mean BLS were calculated for each experimental
group and statistically significant differences among groups were determined using
GraphPad InStat version 3.1 statistics software. Data was analyzed using One-way
Analysis of Variance (ANOVA) and Bartlett’s test for homogeneity of variances and
34
Tukey-Kramer Multiple Comparisons tests. Statistical differences between groups were
measured at P < 0.01.
Statistical analysis. The bursa/body weight (BBW) ratios were calculated from
measurements made from euthanized birds at necropsy. BBW ratio = bursal weight (g)/
body weight (g) x 1000. The mean BBW ratio was calculated for each group. Statistically
significant differences among groups were determined using GraphPad InStat version 3.1
statistics software. ANOVA and Bartlett’s test for homogeneity of variances and TukeyKramer Multiple Comparisons tests were used. Statistical differences between groups
were measured at P < 0.01.
RNA extraction from bursas. Bursal tissues collected at necropsy were
homogenized using an equal volume (v/v) of TNE buffer (10mM Tris-HCl, pH 8.0,
100mM NaCl, 1mM ethylene diamine tetra acetic acid) added to bursal samples in Viral
Transport Medium (VTM, CAHFS-Davis; containing 9.6 g Minimal Essential Medium,
20 ml 1M Hepes buffer, 3.57 g NaHCO3, ACS grade, 250 mg gentamicin, 2500 μg
amphotericin B in 1 liter triple distilled H2O). A 300 μl volume was used to extract viral
RNA with Trizol (Invitrogen, Corp. Carlsbad, CA) using a standard procedure previously
described [59]. The RNA extracted from each sample was suspended in a 35 μl volume
of 90% dimethyl sulfoxide (DMSO; Sigma Chemical Co.) and used for reverse
transcriptase-polymerase chain reaction (RT-PCR) assays.
Real time RT-PCR amplification. Real time RT-PCR assays were performed
using a Roche® LightCycler 480 instrument (Roche Diagnostics Corp., Indianapolis, IN)
35
and the AgPath-IDTM One-Step RT-PCR kit (Ambion; Applied Biosystems, Foster City,
CA).
A Taq-Man® real time RT-PCR assay was used for the detection of a portion of
genome segment A of serotype 1 IBDV strains (14). Briefly, 1.0 μl of RNA extracted
from bursas was used for the assay. A 743 bp region of the hypervariable region of viral
protein 2 (hvVP2) from nucleotides 737-1479 was amplified using primers 743-F (5’GCCCAGAGTCTACACCAT-3’) and 743-R (5’-CCCGGATTATGTCTTTGA-3’). Two
Taq-Man® probes (Applied Biosystems, Foster City California) were used to identify and
differentiate the amplified RT-PCR products. One probe was specific to vvIBDV (FAM5’-CTCAGCTAATATCGATGC-3’) and the other probe was specific for non vvIBDV
(VIC-5’-CAGCCAACATTGATGC-3’).
cDNA synthesis and pre-denaturation was
performed at 48 C for 10 min and by 95 C for 10 min. This was followed by 40 cycles of
PCR at 95 C (denaturation) for 15 sec, 58 C (annealing) for 90 sec and 72 C (extension)
for 90 sec. After completion of the 40 cycles, a final extension at 72 C for 7 min was
performed.
For detection of serotype 2 segment A, the hvVP2 was amplified in a real time
RT-PCR assay using primers S2-F (5’-AAGTTGATGGCCACGTGCG-3’) and S2-2 (5’CTCGGATTATGTCCTTAAAACC-3’). These primers amplify a 778 bp region of the
serotype 2 VP2 gene from nucleotide 686 to 1,463 and do not amplify genome segment A
of serotype 1 IBDV. The amplified serotype 2, segment A product was detected using
Taq-Man® probe FAM-5’-GTCGCAACTCATCCCAAGT-3’ (Applied Biosystems,
Foster City California). The cDNA synthesis and pre-denaturation was conducted at 48 C
36
for 10 min and 95 C for 10 min. This was followed by 40 cycles of PCR at 95 C
(denaturation) for 15 sec, 58 C (annealing) for 90 sec and 72 C (extension) for 90 sec.
After completion of the 40 cycles, a final extension at 72 C for 7 min was performed.
A real time RT-PCR was also performed for partial sequence amplification of a
722 bp region of segment B of both serotype 1 and serotype 2 IBDV using primers B168AF
(5’-CATAAAGCCTACAGCTGGAC-3’)
and
B-889R
(5’-
GTCCACTTGATGACTTGAGG-3’). The amplified segment B product was detected
using Taq-Man® probe FAM-5’-TACTCAAGCAGATGAT-3’ (Applied Biosystems,
Foster City California). cDNA synthesis and pre-denaturation was conducted at 48 C for
10 min and
95 C for 10 min. This was followed by 40 cycles of PCR at 95 C
(denaturation) for 15 sec, 57 C (annealing) for 90 sec and 72 C (extension) for 90 sec.
After completion of the 40 cycles, a final extension at 72 C for 7 min was performed.
Restriction fragment length polymorphism (RFLP). The RFLP analysis was
performed along the RT-PCR and sequencing in order to determine if dual IBDV strains
were present in samples. Restriction fragment length polymorphism analysis was
performed on the PCR amplified 743 bp fragment of hvVP2 and the 722 bp fragment of
segment B. Segment A PCR products were digested using restriction enzymes BstNI and
MboI (Promega, Madison WI). Segment B PCR products were digested using BamHI and
EcoRI (Promega, Madison WI) as previously described [45]. Restriction enzyme digested
DNA was visualized on 2.0 % Metaphor® agarose gel stained with ethidium bromide.
37
Nucleotide sequence and predicted amino acid analysis. The RT-PCR products
were prepared for sequencing using a PCR Clean-Up System (Promega Corp., Madison,
WI). Sanger sequencing was conducted at the University of Wisconsin, Biotechnology
Center, DNA Sequence Laboratory (Madison, WI). Nucleotide sequence results were
downloaded using Chromas (Technelysium Pty. Ltd., Queensland, Australia). Sequence
analysis and Clustal W alignments were conducted using Accelrys Gene v2.5 software
(Accelrys, San Diego, CA).
2.4 Results
Pathogenicity in SPF chickens. The mean mortality observed with groups 1e and 2e
challenged with only the vvIBDV rB strain was 100% in both the 4 wk and 6 wk old
inoculated birds. Mortality was 80% (4/5) and 40% (2/5) in groups co-challenged with rB
and the STC strain at 4 wk and 6 wk of age, respectively. Mortality was lowest in groups
co-challenged with rB and the subclinical variant strains. Co-challenge with rB and Del-E
strains resulted in mortality of 40% (2/5) and 20% (1/5) in 4 wk and 6wk old birds
respectively. Co-challenge with rB and T1 strains induced mortalities of 0% (0/5) and
40% (2/5) in 4 wk and 6 wk old birds respectively. Mortalities of 40% (2/5) and 60%
(3/5) were recorded in birds co-challenged with rB and serotype 2 OH strains at 4 wk and
6 wks of age, respectively. No mortality was observed in any of the control groups at 4
wk and 6 wks of age (Table 2.2; Table 2.3).
Clinical signs observed in birds that succumbed to infection included depression,
inappetence, ruffled feathers and diarrhea. There were no statistical differences among
38
BBWs in any of the 5 groups (1a-1e; 2a-2e) at 4 wk and 6 wks of age. In addition there
was no statistical difference between the BBW from any of the virus challenge groups
compared to the control groups. Macroscopic bursal lesions observed included yellow
caseous cores, bursal edema with petechial hemorrhages on the mucosa and submucosa
in a few birds at 4 and 6 wks of age. No correlation was noted between the different viral
challenge groups and the severity of gross lesions observed in 4 wk and 6 wk old birds.
No gross or microscopic lesions were observed in any of the tissues of the control birds at
4 and 6 wks of age.
The mean BLS was significantly higher in the rB challenged group 1e compared to
groups co-challenged with rB and a second IBDV strain (1a-1d) in 4 wk old birds. There
was no significant difference in mean BLS in any of the viral challenge groups (2a-2e) at
6 wks of age. The BLS was 0 in both control groups 1f and 2f and no microscopic
abnormalities were observed.
Microscopic bursal lesions observed in all viral challenge groups (1a-1e; 2a-2e)
included lymphocyte depletion, inflammatory cell infiltration and stromal edema.
Microscopic hemorrhages were observed in a few bursal tissue sections in both 4 wk and
6 wk old birds. Lymphocyte depletion in the white pulp of the spleen was observed from
a few birds from vv/STC, vv/T1 and vv/OH groups in 4 wk old birds and from the
vv/STC groups in the 6 wk old birds. Mild hemorrhage was observed in the mucosa of
the proventriculus in a few sections from vv/STC, vv/Del-E and vv/OH groups in 4 wk
old birds and from vv/STC and vv/OH groups in the 6 wk old birds.
39
Molecular detection of IBDV pathotypes. The real time RT-PCR assay using the
probe specific to segment A of vvIBDV strains was positive in all 5 IBDV viral challenge
groups in 4 wk and 6 wk old birds. All 6 groups were negative for non vvIBDV segment
A using the real time RT-PCR assay in both 4 wk and 6 wk old birds. The real time RTPCR assay with the segment A serotype 2 probe was also negative in all 6 challenge
groups in both 4 wk and 6 wk old birds. The real time RT-PCR and segment B IBDV
probe was positive in both 4 wk and 6 wk old birds. This probe detects both serotype 1
and serotype 2 of IBDV. All real time RT-PCR assays were negative for IBDV in the 4
wk and 6 wk old control groups.
Sequencing. The PCR amplified segment A products from groups challenged with rB
alone (1e, 2e) as well as groups challenged with rB and a second IBDV strain (1a-1d; 2a2d) matched the 743 bp fragment of the hvVP2 of rB (vvIBDV: GQ221683) in both 4 wk
and 6 wk old birds. The real time PCR assays targeting the 722 bp fragment of segment B
was homologous with the partial segment B sequence of rB (GQ221685) in all viral
challenge groups (1a-1e; 2a-2e). Non vvIBDV sequences were not detected in any of the
experimental groups in 4 wk and 6 wk old birds.
RFLP analysis. The 743bp amplicon of segment A was digested using BstNI into 3
bands of 424 bp, 172 bp and 119 bp for all IBDV challenged groups (1a-1e; 2a-2e). MBoI
restriction enzyme digested the segment A amplicon into 362 bp, and 229 bp fragments
for all IBDV challenged groups (1a-1e; 2a-2e) (Fig.1). The segment A RFLP patterns
using BstNI and MboI are synonymous with those seen with digestion of the 743 bp
fragment of VP2 of vvIBDV.
40
The 722 bp amplified fragment of segment B was digested using BamHI into a 655
bp visible band for all treatment groups for all IBDV challenged groups (1a-1e; 2a-2e)
(Fig. 2.3). The smaller 67 bp band was not visible on the gel. This pattern is synonymous
with BamHI digestion of the 722 VP1 fragment of vvIBDV.
2.5 Discussion
A major objective of this study was to determine if co-challenge with endemic
IBDV pathotypes would alter the severity of clinical disease typically associated with
vvIBDV infection. The standard classic STC, variant Del-E, variant T1 and avirulent OH
strains were chosen for co-challenge with the rB strain based on their endemicity in
chickens in the United States. The severity of disease was assessed by evaluating the
mortality rates, BBW ratios and the severity of bursal lesions which was quantified using
the mean BLS.
The T1 strain was initially isolated from chickens in Georgia, U.S.A. The T1
variant was reported as a subclinical strain of IBDV capable of causing bursal lesions in
chickens with maternal immunity against both classic and variant Del-E IBDV strains
[54]. The T1 variant induced bursal atrophy and mild bursal inflammation in challenged
chickens but no mortality or morbidity was reported in chickens [54]. The variant Del-E
typically induces bursal atrophy and lymphocyte depletion but no mortality or clinical
disease in chickens [117]. The standard classic strain (STC) typically induces mortality
between 0-30% in SPF chickens [57,117]. Serotype 2 Ohio (OH) strains have been
41
previously described as avirulent in chickens [41,51]. While serotype 2 strains of IBDV
can be infectious, they are typically non pathogenic in chickens [6]. In addition, there is
no antibody cross protection between serotype 1 and serotype 2 strains [8]. The exact
mechanisms by which IBDV pathotypes induce mortality and disease have not been fully
elucidated.
The 100% mortality observed in 4 wk and 6 wk old SPF chickens challenged with
vvIBDV (rB strain) correlated with mortality reported by other investigators [57,102].
Jackwood et al. reported mortality between 91-100% in 4 wk SPF chickens challenged
with 102.0- 105.5EID50 of rB strain of vvIBDV [57]. Mortality between 90-100% was also
reported in SPF flocks inoculated with vvIBDV strains originating from Europe [16,108].
In this experiment, the mortality observed in SPF chickens simultaneously
challenged with vvIBDV (rB strain) and either the classic STC, variant (Del-E; T1) or
serotype 2 OH was less than the mortality observed in groups challenged with rB alone.
In addition co-challenge with rB and subclinical variants (T1, Del-E) results in a more
significant reduction in mortality compared to mortality induced by co-challenge with the
two antigenically similar pathogenic strains, rB and STC. This may be because of the
cumulative effect of having two pathogenic strains compared to challenge with a
combination of pathogenic (rB) and subclinical (Del-E; T1) or avirulent (OH) strains of
IBDV. In spite of the reduction in mortality in the dually challenged birds, bursal lesions
and lymphocyte depletion were still significant in all viral challenged groups in both the 4
wk and 6 wk old birds. The non bursal systemic lesions observed in the proventriculus,
cecal tonsils and spleen in some of the challenge groups are not unexpected since
42
pathogenicity of IBDV strains have been correlated with viral distribution in non bursal
lymphopoietic and hematopoietic organs [20,108].
The mean BLS was highest in groups challenged with rB alone in both 4 wk and 6
wk old birds. In 6 wk old birds the mean BLS (3.9) was highest in the group challenged
with rB alone. This BLS was not statistically different from the other IBDV cochallenged groups 2a-2d and is most likely attributed to the small sample size in the
groups challenged with rB alone. No statistical differences were noted in BBW in any of
the challenge groups in 4 wk and 6 wk old birds. This may partially be because by 5 days
post infection bursal edema and enlargement is beginning to subside and the bursa
frequently returns to its normal weight before atrophy occurs [20].
By evaluating the mortality rates and BLS it was hypothesized that co-infection
with rB and either STC, Del-E, T1 or OH strains reduced the severity of disease
compared to infection with rB alone. The PCR, RFLP and sequencing tests detected the
presence of only the rB strain by 5 days post challenge in all viral challenge groups (1a1e; 2a-2e) in both 4 wk and 6 wk old birds. Hence based on molecular results, it can be
inferred that the vvIBDV (rB strain) was able to outcompete the less virulent pathoytpes
STC, Del-E, T1 and OH by 5 days post challenge.
The results of this current investigation suggests that viral competition between
vvIBDV and endemic serotype 1 STC, Del-E, or T1 strains can reduce the severity of
disease in fully susceptible chickens. Similarly, this study indicated that simultaneous
challenge with serotype 1 (rB strain) and serotype 2 (OH strain) results in a reduction in
mortality in fully susceptible chickens. The latter observation is interesting since serotype
43
2 IBDV do not replicate in chicken lymphoid cells [86]. While serotype 2 IBDV do not
replicate in bursal lymphoid cells, binding studies show that they are still capable of
binding to lymphoid cells located in the bursa [86]. Saturation and competition
experiments have shown that receptor sites common to both serotype 1 and 2 are present
on lymphoid cells [86]. This suggests that viral interference between the rB and OH
strains may be occurring at the extracellular level and competition for B-lymphocyte cell
surface receptors may be a mechanism for the reduced mortality observed in the vv and
OH challenged birds.
In the co-challenged birds it is also possible that the stimulation of non-specific
immunity may be responsible for inducing some degree of protection against vvIBDV.
The stimulation of humoral immunity and antibody production against IBDV is not
expected to have a significant influence on host protection at 5 days post challenge.
Hence the antigenic similarities between rB and STC and induction of B-cell mediated
immunity are not believed to play a role in the decrease in disease severity in this
experiment.
Viral interference related to co-infection with multiple strains of IBDV has also
been reported by other researchers. A reduction in mortality was reported by Jackwood
when 4 wk old SPF chicks were simultaneously inoculated with T1 and vvIBDV (rB)
[44]. In vivo viral interference has also been reported with pathogenic and mild IBDV
strains given 24 hr apart [3]. The viral competition between the co-infecting IBDV
pathotypes may be influenced by factors such competition for cell surface receptors and
intracellular biosynthetic machinery required for viral replication [3,44,115]. Host factors
44
such as stimulation of non-specific immunity such as cytokine production may also act to
decrease the severity of pathology induced. It is also possible that the presence of the
non vvIBDV pathotypes may be stimulating the infected cells to produce interferon
proteins which can render uninfected cells resistant to viral infection.
In conclusion, this study supports the theory that coinfection with endemic IBDV
strains may be a significant factor influencing the relatively low mortality currently
associated with vvIBDV infection in the field in California. This makes it more plausible
that vvIBDV can be present but unrecognized in commercial poultry flocks for prolonged
periods. In addition the mean BLS scores of greater than 3 in the co-challenged groups
indicate that bursal injury and the secondary immune suppression can still be significant
factors in birds infected with multiple IBDV pathotypes. The ability of vvIBDV to
outcompete the endemic viruses could be a potential factor influencing the ability of
vvIBDV to become endemic and to become established in new regions.
These factors
emphasize the need for continued active surveillance in the field. Further research is also
needed to determine the efficacy of vaccination and cross protection between vvIBDV
and antigenically similar endemic classic IBDV.
45
Table 2.1. Study design for experimental groups co-challenged with rB along with a
second IBDV pathotype compared to challenge with rB alone at 4 wks (1a-1f) and 6 wks
of age (2a-2f)
Experimental Group Challenge virus 1 (105 EID50) Challenge virus 2 (105 EID50)
1a, 2a
vv (rB strain)
Standard classic (STC strain)
1b, 2b
vv (rB strain)
Variant (Del E)
1c, 2c
vv (rB strain)
Variant (T1)
1d, 2d
vv (rB strain)
Serotype 2 (OH strain)
1e, 2e
vv (rB strain)
-
-
-
1f, 2f (controls)
46
Table 2.2. Pathology observed with 4 wk old SPF chickens co-challenged with vvIBDV
(rB) and a second IBDV pathotype compared to vvIBDV (rB)
Trt Gp (4 wks)
IBDV challenge
virusesA
Mortality
Mean BBWB± SD Mean BLS ± SDC
1a
vv rB/STC
4/5
6.9
3.4 ± 0.55a
1b
vv rB/Del-E
2/5
4.67 ± 0.95a
3.2 ± 0.45a
1c
vv rB/T1
0/5
6.60 ± 2.15a
3.0 ± 0.00a
1d
vv rB/OH
2/5
5.65 ± 1.30a
3.2 ± 0.45a
1e
vv rB
10/10
ND
4.0 ± 0.00b
1f (-ve control)
control
0/5
5.62 ± 1.01a
0.0 ± 0.00c
A
SPF chickens in treatment groups 1a-1e inoculated with 105 EID50 vvIBDV (rB) and 105
EID50 of a second IBDV subtype via the oronasal route.
B
Bursa: body weight (BBW) ratio = bursa wt (g)/ body wt (g) X 1000. Mean BBW ratio
calculated in birds surviving viral challenge and presented as mean BBW ± standard
deviation (SD). ND = statistical significance not determined because the sample size was
too small (due to 100% mortality). Lower case letters indicate statistical differences
among the groups (P<0.01).
C
Severity of bursal histopathological lesions was graded on a scale of 0-4 with mean
lesion scores calculated for each challenge group. Score of ‘0’ indicated an absence of
lesions; 1: <25% lymphocyte depletion in bursas; 2: 25-50% lymphocyte depletion in
bursas; 3: 50-75% lymphocyte depletion in bursas; 4: >75% lymphocyte depletion in
bursas. Lower case letters indicate statistical differences among the groups (P<0.01).
47
Table 2.3. Pathology observed with 6 wk old SPF chickens co-challenged with vvIBDV
(rB) along with a second IBDV pathotype.
Mortality
Mean BBWB±
SD
Mean BLS ±
SDC
Trt Gp (6 wks)
IBDV challenge
virusesA
2a
vv rB/STC
2/5
6.27 ± 2.56a
3.2 ± 0.45a
2b
vv rB/Del-E
1/5
3.99 ± 0.74a
3.2 ± 0.45a
2c
vv rB/T1
2/5
3.63 ± 1.08a
3.0 ± 0.00a
2d
vv rB/OH
3/5
3.91 ± 0.66a
3.6 ± 0.55a
2e
vv rB
3/3
7.07 ± 1.30
3.9 ± 0.38a
2f (-ve control)
control
0/3
4.50 ± 0.56a
0.0 ± 0.00b
A
SPF chickens in treatment groups 2a-2e inoculated with 105 EID50 vvIBDV (rB) and 105
EID50 of a second IBDV subtype via the oronasal route.
B
Bursa: body weight (BBW) ratio = bursa wt (g)/ body wt (g) X 1000. Mean BBW ratio
calculated in birds surviving viral challenge and presented as mean BBW ± standard
deviation (SD). Lower case letters indicate statistical differences among the groups
(P<0.01).
C
Severity of bursal histopathological lesions was graded on a scale of 0-4 with mean
lesion scores calculated for each challenge group. Score of ‘0’ indicated an absence of
lesions; 1: <25% lymphocyte depletion in bursas; 2: 25-50% lymphocyte depletion in
bursas; 3: 50-75% lymphocyte depletion in bursas; 4: >75% lymphocyte depletion in
bursas. Lower case letters indicate statistical differences among the groups (P<0.01).
48
120
Mortality (%)
100
80
Mortality (%) 4wk old
60
Mortality (%) 6wk old
40
20
0
rB/STC rB/delE
rB/T1
rB/OH
rB
control
IBDV challenge group
Figure 2.1. Mortality 5 days post challenge in 4 wk and 6wk old SPF chickens
simultaneously inoculated with 105 EID50 vvIBDV (rB) and 105EID50 of either STC, DelE, T1 or OH strains of IBDV compared to mortality in chickens inoculated with vvIBDV
(rB) alone.
49
1
2
3
4
5
6
7
8
9
424 bp
362 bp
229 bp
172 bp
Figure 2.2. Restriction enzyme digestion of the amplified 743 bp fragment of segment A
of IBDV. The visible 424 bp and 172 bp bands from BstNI digestion of the segment A
fragment from groups 1a, 1b, 1c, and 1d are located in lanes 1, 2, 3 and 4 respectively.
The 100 bp ladder is located in lane 5. The visible 362 bp and 229 bp bands from MboI
enzyme digestion of segment A from groups 1a,1b,1c, and 1d are located in lanes 6, 7, 8
and 9 respectively. Digested fragments were run on a 2% metaphor agar gel.
50
1
2
3
4
5
6
655 bp
Figure 2.3. Restriction enzyme digestion using BamHI on the amplified 722 bp fragment
of segment B of IBDV. The 655 bp fragment from groups 1a, 1b, 1c, 1d, 1e are visible in
lanes 2, 3, 4, 5 and 6 respectively. Digested fragments were run on a 2% metaphor agar
gel. The 100 bp ladder is located in lane 1. This banding pattern is synonymous with
BamHI digestion of the 722 bp VP1 encoding region of vvIBDV strains.
51
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63
Chapter 3 - Molecular and Pathogenic Investigation of Reassortant Very Virulent
Infectious Bursal Disease Viruses in California.
64
3.1 Summary
The very virulent pathotype of infectious bursal disease virus (vvIBDV) was first
identified in commercial layers in California, U.S.A. in December 2008. Subsequent to
this, several field cases of vvIBDV and reassortant vvIBDV have been diagnosed in
commercial and backyard poultry within this state. The aim of this investigation was to
characterize and compare the pathogenicity of reassortant IBDV strains isolated from
California poultry. The phylogenetic relationship of emerging reassortant strains with
other endemic and foreign IBDV strains was also evaluated.
An interserotypic
reassortant vvIBDV (K669) with genome segment A matching the vvIBDV pathotype
and segment B matching serotype 2 IBDV was isolated from commercial broilers from
Northern California. Another interserotypic reassortant (D2712) with a serotype 2
segment A and serotype 1 segment B that aligned phylogenetically with classic IBDV
was isolated from commercial California turkeys. In addition, a vvIBDV reassortant
(7741) with a vvIBDV genome segment A and a segment B that aligned phylogenetically
with classic IBDV strains isolated from Australia (AJ878641, AJ878682, AJ878639)
was identified in commercial layers from California. In vivo virulence of reassortant
vvIBDV: 7741, K669 and D2712 was assessed relative to the virulence of other U.S.
endemic IBDV pathotypes: vv (rB strain), standard classic (cv) (STC strain) and
subclinical variant (sc) (Delaware E strain). Assessment of mortality and morbidity in 4
wk old SPF leghorns indicated that reassortant vvIBDV (K669 and 7741) were less
65
pathogenic than the vvIBDV (rB strain) but more pathogenic than the standard classic
IBDV. No morbidity, mortality or pathological lesions were associated with challenge
with interserotypic reassortant D2712 in SPF or maternally immune chickens. In spite of
this there were no significant differences in the severity of bursal lesions in SPF groups
inoculated with K669, 7741, rB, STC or Del-E. In vivo pathogenicity of IBDV
pathotypes was also assessed in maternally immune broilers at 1, 2, 3 and 4 wks of age.
No mortality was observed in maternally immune broilers inoculated with any of the
IBDV pathotypes at 1, 2, 3 and 4 wks of age. At 4 wks of age there were no significant
differences in the severity of bursal lesions and lymphocyte depletion in the rB, K669 and
STC groups. The severity of lesions in the 7741 and Del-E inoculated groups was
significantly lower than the rB, K669 and STC groups at 4 wks of age. RT-PCR results
and bursal lesions in broilers challenged at 2 wks of age indicate that reassortant K669
was able to break through maternal immunity earlier than any of the other challenge
IBDV strains. The interserotypic reassortant D2712 was not recovered from bursas from
the SPF or maternally immune broiler groups challenged with D2712 virus.
66
Key Words: reassortant, very virulent infectious bursal disease, California, pathogenicity
Abbreviations: BBW = bursa/body weight ratio;BLS = bursal lesion scores; CAM =
chorioallantoic membrane; DMSO = dimethyl sulfoxide; EID50 = 50% egg infectious
dose; GMT = geometric mean titer; hvVP2 = hypervariable region of viral protein 2; IBD
= infectious bursal disease; NJ = Neighbor-Joining; RT-PCR = reverse transcriptasepolymerase chain reaction; SPF = specific pathogen free; SPF = specific pathogen free;
TNE buffer = 10mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM ethylene diamine tetra
acetic acid; UPGMA = unweighted pair group with arithmetic mean; vvIBDV = very
virulent infectious bursal disease virus
67
3.2 Introduction
Infectious bursal disease (IBD) was initially identified in the U.S.A. in Delaware
in 1962. The severity of disease observed in the field is influenced by a combination of
factors including IBDV strain(s), age and breed of flock, vaccination protocol being used,
presence of concurrent pathogens and environmental and management practices. IBDV
has a predilection for the B lymphocytes in the bursa of Fabricius where virulent strains
cause lymphocyte depletion. This typically results in immune suppression which
predisposes surviving birds to opportunistic pathogens. The very virulent (vv) pathotype
was first described in Europe in the 1980’s and has since rapidly spread across Asia and
to most major poultry producing regions worldwide [16,107,108]. The vvIBDV was first
officially reported in U.S. poultry in 2009 [57,102]. Since then several additional cases of
vvIBDV and reassortant vvIBDV have been isolated within the state [47,56].
IBDV are bisegmented, non enveloped, icosahedral RNA viruses belonging to the
Avibirnavirus genus within the Birnaviridae family. Segment A is 3261 nucleotides and
has two open reading frames. The larger ORF encodes for a 110-kDa precursor
polyprotein: pVP2-VP4-VP3. Autocleavage of VP4 facilitates the release of the outer and
inner structural capsid viral proteins designated VP2 and VP3 respectively as well as the
viral protease, VP4 [5,37]. The smaller overlapping ORF on segment A encodes for a 17kDa nonstructural protein VP5 which is thought to influence apoptosis and viral release
[72,112,118]. The smaller genome segment B is 2827 nucleotides and encodes for the
RNA directed RNA polymerase designated VP1 which is responsible for genome
68
replication and mRNA synthesis [71]. The VP5 protein is covalently linked to the 5’ ends
of the genomic RNA segments [101]
IBDV strains are classified as either serotype 1 or serotype 2 strains. Serotype 2
strains are generally avirulent in both chicken and turkeys. A range in pathotypes exists
for serotype 1 strains and they are frequently classified as subclinical (sc), classic (cv) or
very virulent (vv) viruses. Since some but not all of the genetic determinants of
pathogenicity are known, classification of strains as vvIBDV is currently based on a
combination of molecular, pathogenic and antigenic criteria [110]. The most virulent
strains of IBDV are characterized by very high mortality and morbidity with mortality
rates as high as 100% reported in fully susceptible birds, 60% in layer and 40% in
broilers [57,102,107,108]. Antigenic IBDV variation can be determined by the use of
selected panels of monoclonal antibodies. Neutralizing antibodies bind within a minimal
region of the hypervariable region (hv) of VP2 between amino acids 206-350 [6]. Reports
indicate that vvIBDV strains are antigenically similar to classic IBDV but modified
epitopes on vvIBDV have been identified with the use of neutralizing monoclonal
antibodies [28,29]. Molecular characteristics of both genome segment A and segment B
are thought to influence virulence.
Based on VP2 sequence alignments, amino acid
residues 222 (Ala), 256 (Ile), 294 (Ile) and 299 (Ser) are highly correlated with the vv
pathotype [107,110].
Molecular epidemiological studies indicate that vvIBDV strains belong to the
same genetic lineage with a high degree of genetic and antigenic homogeneity. Studies
suggest that these strains evolved from a common ancestor emerging from a single
69
unique event and then subsequently evolved independently in Europe and Asia
[36,39,64,119]. Phylogenetic analysis of vvIBDV isolates indicates that there is a strong
co-evolution of genome segment A and genome segment B [36]. In spite of this, natural
reassortment of the virus has been reported to occur especially in regions where the
parent strains have been established for a significant number of years [36,56].
Phylogenetic analyses indicate that segment B nucleotide sequences of vvIBDV strains
form a distinct cluster and that these strains might have derived their segment B by
genetic reassortment from an unidentified source [38,39,78,101]. The bisegmented
genome of IBDV facilitates both homologous and non homologous reassortment.
Heterogenicity in IBDV viral RNA also occurs by substitution mutations which result in
antigenic drift and recombination which results in antigenic shift. Hence IBDV exists as a
population of genetic variants characterized by one or multiple quasispecies. This
facilitates evolution of the virus and confers viral adaptive potential [35,53].
Since 2008 several cases of vvIBDV and reassortant IBDV has been identified in
the State of California, U.S. [56,57,102]. The first natural interserotypic vvIBDV
reassortants identified in the U.S. were reported in 2011[56]. These reassortants were
reported to have segment A sequences matching the vvIBDV genotype and segment B
matching serotype 2 IBDV [56]. A recent reassortant (D2712 strain) recovered from
commercial turkeys in California has a segment A matching serotype 2 IBDV and
segment B matching serotype 1 IBDV. Due to the lack of a complete list of validated
pathogenicity markers, in vivo pathogenicity testing remains a valuable method of
assessing the pathogenicity of these emerging reassortant viruses. This investigation
70
assessed the pathogenicity of reassortant IBDV isolated from commercial and backyard
avian species in California. Specific pathogenic free (SPF) and maternally immune
broilers were challenged with reassortant IBDV strains: K669, D2712 and 7741 as well
as endemic very virulent (vv: rB strain), classic virulence (cv: STC strain) and subclinical
(sc: Delaware E strain) IBDV. In order to assess the influence of decreasing level of
maternal antibodies against classic and variant IBDV, the maternally immune broilers
were challenges at 1, 2, 3 and 4 wks of age. In addition, the phylogenetic relationship
between both segment A and segment B of the California reassortant IBDVs were
compared with other strains of IBDV described worldwide.
3.3 Materials and Methods
Viruses. The vvIBDV (rB) and reassortant vvIBDV (K669, 7741, D2712) isolates
were obtained from clinical cases submitted to the California Animal Health and Food
Safety (CAHFS) laboratory. Viral isolates were subsequently sent to the Food Animal
Health Research Program (FAHRP) for further characterization. The 7741 reassortant
vvIBDV was isolated in August 2010 from 8-10 wk old backyard brown leghorns from
Southern California. The K669 reassortant vvIBDV isolate was identified in April 2011
from 28 day old broilers from a commercial farm in Northern California experiencing
higher than normal mortality. Reassortant vvIBDV isolate D2712 was recovered from
commercial 5 wk old turkeys from California. The D2712 isolate was from a commercial
turkey necropsy case in California. The bursas and intestines from the field case were
negative for IBDV by real time PCR but egg passage of intestinal tissue from the case
resulted in the recovery of IBDV in the allantoic fluid 5days post inoculation. Genomic
71
analysis of D2712 indicated the segment A matched serotype 2 IBDV and the segment B
matched serotype 1 IBDV. The rB isolate of vvIBDV was obtained from the initial case
of vvIBDV reported in N. California poultry in December 2008 [57,102]. IBDV viral
isolates: standard classic strain (STC), and variant Delaware E (Del-E) originated from
IBDV strains circulating in the U.S. and have been previously described [54].
In vivo experiments. All birds used in challenge experiments 1 and 2 were
housed in Horsfall® isolation units and provided with feed and water ad libitum. The
mean egg infectious dose (EID50) for IBDV isolates was determined by viral titrations in
9-day-old embryonated SPF chicken eggs via the chorioallantoic membrane (CAM) with
0.1ml of bursal homogenate as previously described [57].
Experiment 1: In vivo challenge in SPF leghorns. The pathogenicity of
vvIBDV reassortants K669, 7741 and D2712 relative to vvIBDV (rB), cvIBDV (STC),
scIBDV (Del-E) was assessed in 4 wk old SPF leghorn chickens (Charles River
Laboratories, North Franklin, CT). Birds were separated into 6 treatment groups and 1
control group with 10 birds placed in each group. Birds within each group, except for the
controls were inoculated with either: K669, 7741, D2712, rB, STC or Del-E at 105 EID50
in a 0.1 ml volume by the oronasal route as previously described [57]. Birds were
monitored twice daily and morbidity and mortality within each group were recorded. At 7
days post challenge, surviving birds were euthanized and necropsy was performed on all
birds in each treatment group. At necropsy, gross lesions were recorded along with the
bursa and body weights from euthanized birds. Tissue sections from the bursa, thymus,
spleen and cecal tonsils were taken from all birds for histopathology. Bursal sections
72
from all birds were also harvested for subsequent molecular detection and
characterization of IBDV.
Experiment 2: In vivo challenge in maternally immune broilers. Broilers with
maternal immunity against IBD, coccidiosis, Pasteurella multocida, fowl pox, avian
encephalomyelitis (AE), infectious bronchitis, Newcastle disease, avian reovirus,
Marek’s disease, infectious laryngotracheitis (ILT) and chicken anemia virus (CAV)
were used in the challenge experiments. Vaccination of parent breeder flocks against
IBDV was done using standard classic Bursine®-2, variant Delaware-E (Del-E), variant
Delaware-A (Del-A) and classic D78 vaccines given between 1-18 wks of age. The
maternally immune broiler progeny used in experiment 2 were unvaccinated. One wk old
maternally immune broilers were subdivided into 6 IBDV challenge groups that were
oronasally administered 0.1 ml 105EID50 of either STC, Del-E, rB, K669, 7741 or D2712
IBDV and an uninoculated control group. Eight birds were placed into each challenge
group and 5 birds in the control group. This challenge experiment was repeated in 2, 3
and 4 wk old maternally immune broilers with 5 birds placed within each group.
Chickens from each group were euthanized and necropsied 7 days post challenge. At
necropsy bursa and body weights were recorded. Tissue sections from bursa, spleen,
proventriculus and cecal tonsils of each bird were saved for histology. Bursa tissue
sections were also saved for molecular diagnostics.
Serology. Serum samples were collected from the brachial vein from 10-15
randomly selected broiler controls from experiment 2 at one-day of age, and at 1, 2, 3 and
4 wk of age. In vitro viral neutralization (VN) tests and enzyme-linked immunosorbent
73
assay (ELISA) were performed using a procedure previously described [25]. VN assays
were used to calculate geometric mean titers (GMT) at each age group using BGM-70
cells and a constant virus-varying antibody method. Sera samples were diluted 2 fold
starting at 1:100 dilution and 102 median tissue culture infectious doses of viral antigen
were used in the assay. Tissue culture adapted classic S706 Winterfield strain (Merial
Select, Inc., Gainsville, GA) and variant Del-E 8903 (Nobilis, Intervet International B.V.)
were used for IBDV antigens in the VN assays. Results were compared with GMT using
IBDV ELISA kits (IDEXX® Laboratories, Inc., Westbrook, ME) on serum samples.
ELISA antibody titers were also evaluated against avian reovirus, chicken anemia virus,
Mycoplasma gallisepticum and Mycoplasma synoviae.
Histopathology.
Tissues sections from experiment 1 and 2 were taken for
histopathological examination. Tissues were fixed in 10% neutral buffered formalin,
sectioned at 4μm, stained with hematoxylin and eosin and examined by light microscopy.
The severity of lymphocyte depletion in bursal follicles was quantified by assigning
bursal lesion scores (BLS) ranging from 0 to 4. Score of ‘0’ indicated an absence of
lesions; 1: <25% lymphocyte depletion in bursas; 2: 25-50% lymphocyte depletion in
bursas; 3: 50-75% lymphocyte depletion in bursas; 4: >75% lymphocyte depletion in
bursas (Figure 3.9).
Statistical analysis. The bursa/body weight (BBW) ratios from each group in
experiments 1 and 2 were calculated from measurements made from euthanized birds at
necropsy. BBW ratio = bursal weight (g)/ body weight (g) x 1000. The mean BBW ratio
from each group as well as the mean BLS from each group was calculated. Statistically
74
significant differences among groups were determined using GraphPad InStat version 3.1
statistics software. One-way Analysis of Variance (ANOVA) and Bartlett’s test for
homogeneity of variances along with Bonferroni or Dunnett Multiple Comparisons Post
Tests were performed. Statistical differences between groups were measured at P < 0.01.
Detection and characterization of IBDV. Bursal tissue homogenization was
done using an equal volume (v/v) of TNE buffer (10mM Tris-HCl, pH 8.0, 100mM NaCl,
1mM ethylene diamine tetra acetic acid) added to bursal samples in viral transport media
(VTM). A 300 μl volume was used to extract viral RNA with Trizol (Invitrogen, Corp.
Carlsbad, CA) using a standard procedure previously described [59]. The RNA extracted
from each sample was suspended in a 35 μl volume of 90% dimethyl sulfoxide (DMSO;
Sigma Chemical Co.) and used for reverse transcriptase-polymerase chain reaction (RTPCR) assays.
Real time RT-PCR amplification. Real time RT-PCR assays were performed
using a Roche LightCycler 480 instrument (Roche Diagnostics Corp., Indianapolis, IN)
and the AgPath-IDTM One- RT-PCR kit (Ambion; Applied Biosystems, Foster City, CA).
A real time RT-PCR assay was used for the detection of a portion of genome
segment A of serotype 1 IBDV strains [55]. Briefly, a 1.0 μl volume of viral RNA
extracted from bursas was used for the assay. A 743 bp region of the hypervariable region
of viral protein 2 (hvVP2) from nucleotides 737-1479 was amplified using primers 743-F
(5’-GCCCAGAGTCTACACCAT-3’) and 743-R (5’-CCCGGATTATGTCTTTGA-3’).
Two Taq-Man® probes (Applied Biosystems, Foster City California) were used to
75
identify and differentiate the amplified RT-PCR products. One probe was specific to
vvIBDV (FAM-5’-CTCAGCTAATATCGATGC-3’) and the other probe was specific for
non vvIBDV that are endemic to California (VIC-5’-CAGCCAACATTGATGC-3’).
cDNA synthesis and pre-denaturation was performed at 48 C for 10 min and by 95 C for
10 min. This was followed by 40 cycles of PCR at 95 C (denaturation) for 15 sec, 58 C
(annealing) for 90 sec and 72 C (extension) for 90 sec. After completion of the 40 cycles,
a final extension at 72 C for 7 min was performed.
For detection of serotype 2 segment A, the hvVP2 was amplified in a real time
RT-PCR assay using primers S2-F (5’-AAGTTGATGGCCACGTGCG-3’) and S2-2 (5’CTCGGATTATGTCCTTAAAACC-3’). These primers amplify a 778 bp region of the
serotype 2 VP2 gene from nucleotide 686 to 1,463 and do not amplify genome segment A
of serotype 1 IBDV. The amplified serotype 2, segment A product was detected using
Taq-Man® probe FAM-5’-GTCGCAACTCATCCCAAGT-3’ (Applied Biosystems,
Foster City California). The cDNA synthesis and pre-denaturation was conducted at 48 C
for 10 min and 95 C for 10 min. This was followed by 40 cycles of PCR at 95 C
(denaturation) for 15 sec, 58 C (annealing) for 90 sec and 72 C (extension) for 90 sec.
After completion of the 40 cycles, a final extension at 72 C for 7 min was performed.
A real time RT-PCR was also performed for partial sequence amplification of a
722 bp region of segment B of both serotype 1 and serotype 2 IBDV using primers B168AF
(5’-CATAAAGCCTACAGCTGGAC-3’)
and
B-889R
(5’-
TCCACTTGATGACTTGAGG-3’). The amplified segment B product was detected
using Taq-Man® probe FAM-5’-TACTCAAGCAGATGAT-3’ (Applied Biosystems,
76
Foster City California). cDNA synthesis and pre-denaturation was conducted at 48 C for
10 min and
95 C for 10 min. This was followed by 40 cycles of PCR at 95 C
(denaturation) for 15 sec, 57 C (annealing) for 90 sec and 72 C (extension) for 90 sec.
After completion of the 40 cycles, a final extension at 72 C for 7 min was performed.
RT-PCR was also conducted to obtain full length genome sequences of segment
A and B of reassortant IBDV 7741 and K669. The amplifications were performed in a
thermal cycler PCR System2720 (Gene Amp, Applied Biosystems, Perkin-Elmer,
Carlsbad, CA) using a SuperScript® III One-Step RT-PCR System with Platinum® Taq
High Fidelity (InvitrogenTM, Carlsbad CA). Briefly a 1.0 μl volume of RNA in DMSO
was used for the assay. Full length segment A was amplified using primers SA1-5’AGGATACGATGGGTCTGAC-3’ and DJA2- 5’-AGGGGACCCGCGAACGGATCC.
Full
length
segment
B
GGATACGATGGGTCTGACC-3’
was
amplified
and
using
primers
DJB1-5’-
DJB2-5’-GGGGGCCCCCGCAGGCG-3’.
cDNA synthesis and pre-denaturation was performed using 1 cycle of 55 C for 30 min
then 94 C for 2 min. This was followed by 40 cycles of PCR amplification at 95 C
(denaturation) for 15 sec, 58 C (annealing) for 30 sec and 68 C (extension) for 210 sec.
Final extension was performed at 68 C for 5 min.
Nucleotide sequence and predicted amino acid analysis. The RT-PCR products
obtained from each group were prepared for sequencing using a PCR Clean-Up System
(Promega Corp., Madison, WI). Full length genome sequences of segment A and segment
B for 7741 and K669 viruses using primers and procedures previously described [59].
Two additional primers pairs were included for complete assembly of segment B; primer
77
pair
710F-5’-
ATCCTCTAAAACTTGGGTA-3’
and
1438R-5’-
CCTCTGGTGAGGATGTAGTA-3’ and SB1437-5’-GTACTACATCCTCACCAGA-3’
and SB2210-5’-ACTGGCTTGTTCAGTTCGG-3’. Cycle sequencing was conducted at
the University of Wisconsin, Biotechnology Center, DNA Sequence Laboratory
(Madison, WI). Nucleotide sequence results were downloaded using Chromas
(Technelysium Pty. Ltd., Queensland, Australia). Sequence analysis and Clustal W
alignments were conducted using Accelrys Gene v2.5 software (Accelrys, San Diego,
CA).
Phylogenetic analysis. Phylogenetic and molecular evolutionary analysis of
segment A and segment B of reassortants with IBDV strains worldwide was conducted
using MEGA v5.05 software [103]. Phylogenetic trees were prepared using the neighborjoining (NJ) distance method with 1000 bootstrap replicates.
3.4 Results
Experiment 1: Pathogenicity of IBDV strains in SPF leghorns. The virulence
of the California reassortants K669, 7741, D2712 was compared to vvIBDV (rB),
cvIBDV (STC) and scIBDV (Del-E) pathotypes (Table 3.1). At 7 days post challenge, the
mortality in SPF chickens inoculated with reassortants K669 and 7741 was 70% and 50%
respectively. The mortality recorded with both reassortant IBDV treatment groups (K669
and 7741) was less than that of the rB inoculated group (90%) but higher than the group
inoculated with STC (40%) (Figure 3.1). No mortality or morbidity was noted in the
78
D2712, Del-E or control groups. Birds from all groups that succumbed to infection within
the 7 days post challenge displayed typical clinical signs of IBDV infection including
depression, prostration, ruffled feathers and diarrhea (Figure 3.11).
Macroscopically, bursas from treatment groups: K669, 7741, STC, Del-E and rB
were generally atrophied compared to the non inoculated controls. Bursas from D2712
inoculated birds were normal in size and dimensions but petechial hemorrhages were
observed macroscopically on the mucosal surface of 6/9 bursas. Bursas from SPF birds
challenged with scIBDV (Del-E) were consistently more atrophied than any of the other
groups. The occurrence and severity of bursal hemorrhages was a less consistent finding
among the different treatment groups. Mucosal and serosal hemorrhages were present
occasionally (<50%) in bursas from the STC, rB, K669 and 7741 groups and ranged from
diffuse petechial hemorrhages to extensively hemorrhagic reddened bursas. Diffuse
petechial hemorrhages were observed in only 1/10 bursas from the Del-E challenged
group. Non bursal lesions were also present in birds from the STC, rB, K669, 7741
groups. Birds from these groups occasionally exhibited petechial and ecchymotic
hemorrhages on the skeletal muscles typically distributed on the pectoral and thigh
muscles. Petechial hemorrhages were also occasionally present on the mucosa of the
proventriculi from rB and reassortant groups K669 and 7741. While in most cases these
hemorrhages were focused at the junction of the proventriculus and gizzard, some were
diffusely distributed throughout the proventricular mucosa. Macroscopic lesions were not
observed in control birds and non bursal lesions were not observed in birds challenged
with the Del-E or D2712 strain.
79
Statistical analysis was performed on BBW calculated at necropsy for the STC,
Del-E, and reassortants D2712, K669, 7741 and control groups (Table 3.1; Figure 3.2).
The rB group was not included in the statistical analysis of mean BBW since only 1 bird
survived viral challenge hence sample size was inadequate for analysis. There was no
significant differences among the mean BBW ratios of the IBDV challenge groups: 7741,
K669 and STC. The mean BBW ratio of the Del-E group was significantly smaller that
all other treatment groups as well as the control group. Conversely, the mean BBW ratio
of the control group was significantly larger than for treatment groups K669, 7741, STC
and Del-E. There was no significant difference between the mean BBW ratios of the
control group and the D2712 inoculated group.
Statistically, there was no significant difference in the severity of bursal
histological lesions (BLS) among the 5 treatment groups: 7741, K669, rB, STC and DelE at day 7 post challenge and these groups scored significantly higher BLS than the
control group. While there was mild stromal hemorrhage and inflammation in 4/10 bursas
from the D2712 group, no lymphocyte depletion was observed microscopically in any of
the bursal tissue sections. In addition, there were no significant differences between the
mean BLS in the control and D2712 groups. No microscopic lesions were observed in
bursas or tissue sections from the control group. Diffuse lymphoid depletion and necrosis
of all or most of the bursal follicles was a consistent microscopic finding in the 5
treatment groups: 7741, K669, rB, STC and Del-E. The presence and severity of bursal
hemorrhage was a less consistent finding microscopically in the viral challenge groups
but some degree of bursal hemorrhage was present in at least one bursa of each of the
80
treatment groups. Lymphoid depletion and necrosis was observed in the white pulp of all
splenic sections from vvIBDV challenged birds. Splenic changes were a less consistent
finding in treatment groups 7741, K669, rB, STC and Del-E and ranged from mild to
moderate lymphoid depletion in a few tissue sections. No abnormalities were observed in
splenic sections from D2712 inoculated birds. Necrosis and hemorrhage were observed in
the lamina propria of the cecal tonsils in a few sections from vvIBDV challenged birds.
Typhlitis with pyogranulomatous inflammation and mucosal hemorrhages were observed
in some cecal tonsil sections from D2712 inoculated birds. No cecal tonsil lesions were
observed in the 7741, K669, STC and Del-E groups.
Experiment 1: Molecular analysis of segment A. Full length nucleotide and
amino acid identity was determined for reassortants, K669 and 7741. Partial sequencing
of genome segment A and B from reassortant D2712 was also performed. Genome
segment A and B of 7741, D2712 and K669 were compared to other IBV strains in
Genbank including California vvIBDV (rB strain) and previously reported California
interserotypic reassortants CA-D495 and CA-K785 [56,57,102]. Both CA-D495 and CAK785 IBDV sequences were obtained from isolates from the first reported naturally
occurring interserotypic IBDV from California in 2011 [56]. CA-D495 and CA-K785
were reported to have genome segment A that aligned with vvIBDV rB strain and
segment B that aligned with serotype 2 IBDV.
Reassortant K669 segment A was recovered from the bursas of the K669
challenge group using the real time PCR and vvIBDV segment A probe. Sequencing of
the segment A amplicon was homologous with the K669 isolate used in the challenge
81
inoculum. Pylogenetic analysis using the NJ method with up to 1000 bootstrap replicates
indicated that reassortant K669 most closely matched vvIBDV isolates from California
(Figure 3.17). Comparison of the hvVP2 nucleotide sequences of segment A of K669
(JN585293) was 99.9% (701 bp/702 bp) homologous with vvIBDV rB strain
(GQ221683) isolated in 2008 from California [57,102]. Full length comparison of
segment A of reassortant K669 was 99.9% (3057 bp/3060 bp) identical to the segment A
of CA-D495 (JF907703) and 98.5% (3125 bp/3172 bp) identical to reassortant CA-K785
(JF907702). Reassortants K669, CA-D495 and CA-K785 were 100% homologous (734
bp/743 bp) identical across the hvVP2 region. K669 translated amino acid sequence was
99.9% (1060 aa/1061 aa) identical to CA-D495 with 1 amino acid substitution observed
across the full segment A. Reassortant K669 segment A translated amino acid sequence
was 99.4% (1049 aa/1055aa) identical to amino acid sequence of segment A of CAK785.
Full length nucleotide sequence of segment A of K669 was a 97.9% (3111
bp/3178 bp) match to vvIBDV; UK661 (NC004178) isolated from Europe. In comparison
to non vv pathotypes, reassortant K669 was only a 94.6% (648 bp/ 685 bp) match with
segment A of STC (D00499) and a 94.2% (645 bp/ 685 bp) match with Del-E
(AF133904) across the hvVP2 region of segment A. Reassortant K669 was only a 78.6%
(541 bp/ 688 bp) nucleotide match with serotype 2 OH (U30818) strain across the hvVP2
of segment A. Full length comparison of segment A of reassortant K669 was 97.5%
(3105 bp/ 3184 bp) identical to segment A of reassortant 7741. Reassortants K669 and
7741 were a 97.3% match (666 bp/684 bp) across the hvVP2 region. Both reassortants
82
K669 and 7741 had the amino acids: A222, I242, I256, I294 and S299 typically observed
with segment A vvIBDV.
Reassortant 7741 segment A was recovered from the bursas of the 7741 challenge
group using the real time PCR and vvIBDV segment A probe. Sequencing of the segment
A amplicon was homologous with the 7741 isolate used in the challenge inoculum.
Phylogenetic analysis of 7741 with segment A from other IBDV strains around the world
was done using the neighor-joining method with 1000 bootstrap replicates (Figure 3.17).
Segment A of 7741 most closely aligned with the segment A of vvIBDV and vvIBDV
reassortants isolated from California (Figure 3.17). Reassortant 7741 (JQ403646) was
97.6% (685 bp/702 bp) identical to California vvIBDV rB isolate and 97.4% (684 bp/ 702
bp) identical to the European vvIBDV isolate NC 004178 across the hvVP2 region.
Reassortant 7741 also aligned closely with interserotypic reassortants CA-D495 and CAK785 across the hvVP2; Isolate 7741 exhibited 98.8% (3135 bp/ 3172 bp) nucleotide
homology with CA-785 and 97.4% (2982 bp/3060 bp) homology with CA-495
throughout the length of segment A. Comparing the hvVP2 region, reassortant 7741 was
a 97.7% (695 bp/711 bp) nucleotide match with CA-785 and a 97.2% (691 bp/711 bp)
match with CA-495. In comparison with other IBDV pathotypes used in experiment 1,
7741 was 94.3% (662 bp/702 bp) homologous with STC, 93.9% (659 bp/702 bp)
homologous with Del-E and only 77.6% (554 bp/714 bp) homologous with serotype 2
OH across the hvVP2 region.
The translated amino acid sequences of 7741 exhibited a high degree of
homology with the vvIBDV (rB) and other vvIBDV reassortants K669, CA-785 and CA83
495. The amino acid sequence of segment A of 7741 was identical (234 aa/234 aa) to rB
and reassortants CA-785, CA-495 and K669 across the hvVP2. A comparison of the full
length of the translated amino acid sequences of segment A, 7741 was a 99.5% (1054 aa/
1054 aa) match with CA-785, a 99.3% (1013 aa/1020 aa) match with CA-495 and a
99.3%(1053 aa/1060 aa) match with K669.
Reassortant D2712 segment A and segment B were not recovered from the bursas
of the D2712 challenge group using the real time PCR and any of the IBDV probes
described in the methodology.
The hvVP2 of segment A and partial segment B
sequences of D2712 was obtained by egg passage of allanotic fluid obtained from egg
passage of intestinal tissue from the commercial turkey case. Real time RT-PCR of egg
passaged virus was positive using the segment A serotype 2 probes and segment B probe.
The nucleotide sequence of hvVP2 of segment A of D2712 (JX235359) most closely
matched serotype 2 OH strain (Figure 3.17). There was a 92.7% (637 bp/ 687 bp) match
between hvVP2 of D2712 and OH compared to only a 74% (511 bp/ 685 bp) match with
Del-E, 77.5% (531 bp/865 bp) match with STC, 77.1% (515 bp/668 bp) match with K669
and 73.5% (505 bp/867 bp) match with reassortant 7741.
Experiment 1: Molecular analysis of segment B. Reassortant K669 segment B
(JN411134) was recovered from the bursas of the K669 challenge group using the real
time PCR and IBDV segment B probe. Sequencing of the segment B amplicon was
homologous with the K669 isolate used in the challenge inoculum. The segment B of
K669 (JN411134) most closely aligned with the segment B of serotype 2 IBDV with a
97.6% (2671 bp/2769 bp) nucleotide match with the OH strain (U30819). Genome
84
segment B of K669 was a 99.8% (2690 bp/ 22712 bp) and a 97.9% (2723 bp/2780 bp)
match with the segment B of interserotypic reassortants CA-D495 (JF907704) and CA785 (JF907705) respectively. The segment B of K669 was less closely aligned with the
other IBDV strains used in experiment 1. The segment B of K669 exhibited only 94.1%
(2571 bp/2733 bp) homology with Del-E, 94.5% (682 bp/722 bp) homology with hvVP2
of STC and 89.2% (514 bp/ 576 bp) homology with hvVP2 of rB.
Reassortant 7741 segment B (JQ403647) was recovered from the bursas of the
7741 challenge group using the real time PCR and IBDV segment B probe. Sequencing
of the segment B amplicon was homologous with the 7741 isolate used in the challenge
inoculum. Phylogenetic analysis of segment B of 7741 and other IBDV isolates from
around the world was performed. Based on the NJ method with 1000 bootstrap replicates,
the segment B of 7741 most closely aligned with the segment B of Australian isolates
N1/99 (AJ878641) [93], isolate 002/73 (AJ878682) [5] and isolate V877 (AJ878639) [93]
classic viruses (Figure 3.18). Segment B of 7741 did not align strongly with the segment
B from STC, rB, Del-E, OH and reassortants CA-785 and CA-495 (Figure 3.18).
The segment B of D2712 (JX235358) recovered by PCR amplification of egg
passaged intestinal contents from the original commercial turkey case was sequenced.
Phylogenetic analysis of segment B using the NJ method with up to 1000 bootstrap
replicates indicated that D2712 most closely aligned with serotype 1 classic viruses
(Figure 3.18).
85
Experiment 2: Pathogenicity of IBDV strains in maternally immune broilers.
No mortality was observed in any of the 7 experimental challenge groups inoculated at
either 1, 2, 3 or 4 wks of age (Table 3.2). There were no significant differences in mean
BBW ratios among the different challenge groups in 1 and 2 wk old broilers (Figure 3.5
and 3.6). In 3 and 4 wk old broilers, the groups challenged with K669 had significantly
smaller mean BBW ratios. There were no statistical differences in mean BBW in any of
the other groups at 3 and 4 wks of age (Figure 3.7 and 3.8).
No bursal lesions were observed in any of the experimental groups at 1 wk of age
except for the D2712 inoculated birds. This correlated with a significantly higher BLS in
the D2712 group compared to the other groups at 1 wk of age. In bursas from the D2712
birds, mild lymphocyte depletion and inflammation in the interfollicular region was
observed in 5/8 sections. Necrotic debris and a granulomatous lesion were observed in
the mucosa of the cecal tonsils in 1 section from the D2712 group at 1 wk of age. No
other lesions were observed in the bursa, spleen, cecal tonsils or proventricular sections
in the STC, Del-E, rB, K669, 7741 and control groups at 1 wk of age.
In broilers challenged at 2 wks of age the mean BLS was significantly higher than
the control in groups challenged with K669 and D2712 (Figure 3.1). The mean BLS was
not significantly higher than the controls in the other groups challenged with STC, Del-E,
rB or 7741. Lymphocyte depletion ranging from moderate to severe was observed in all
bursas from the K669 inoculated birds at 2 wks of age. Mild lymphocyte depletion was
observed in 7/8 bursas in group inoculated with D2712. Mild lymphocyte depletion
(BLS:1) was observed in 1/8 bursas from the STC and rB groups in birds challenged at 2
86
wks of age. No other lesions were observed in the spleen, cecal tonsils and proventriculus
tissues in the Del-E, 7741, D2712, STC, K669 and control groups at 2 wks of age.
In broilers challenged at 3 wks of age the mean BLS was significantly higher than
the controls in the STC, rB, K669, 7741 groups. The mean BLS in groups challenged
with Del-E and D2712 were not significantly different from the control group in 3 wk old
birds. Macroscopically, edema and petechial hemorrhages were observed in 1/5 bursas
from the STC group and 3/5 bursas from the rB group in birds challenged at 3 wks of
age. Lymphocyte depletion in the white pulp of the splenic sections was observed in
groups inoculated with STC and rB and 7741. Mild hemorrhage was observed in a few
sections of the cecal tonsils in the 7741, D2712, and rB groups challenged at 3 wks of
age. No lesions were observed in the spleen, cecal tonsils and proventriculus of the DelE, K669, and control groups.
At 4 wks of age bursal lesions were observed in all of the broiler challenge
groups: STC, Del-E, rB, K669, 7741 and D2712. Macroscopically all bursas were yellow,
and friable with a gelatinous exudate in the STC and rB groups. All bursas were small,
yellow, friable with gelatinous exudate in the K669 groups challenged at 4 wks of age. In
7741 group, 1/5 bursas with yellow, friable with a gelatinous exudate and 4/5 bursas were
large, edematous and flaccid with a normal color. The mean BLS were significantly
higher than the controls in the STC, rB, 7741 and K669 groups inoculated at 4 wks of
age. Lymphocyte depletion was observed the white pulp of the spleen in some sections
from the 7741, K669, STC and rB groups challenged at 4 wks of age. Mild hemorrhage
in the mucosa of the provetriculus was observed in a few cecal tonsil sections from the
87
7741, Del-E, STC and rB groups. An abscess was also observed in one cecal tonsil
section from the Del-E group in the 4 wk old challenge. No abnormalities were observed
in the proventricular sections of any of the challenge groups at 1, 2, 3 and 4 wks of age.
Experiment 2: Molecular analysis in maternally immune broiler challenge.
All groups were negative for IBDV by real time PCR at 1 wk of age. Broilers challenged
with reassortant K669 at 2 wks of age were positive for IBDV using the real time PCR
with segment B probe and segment A vvIBDV probe. Broiler groups challenged with
STC, rB, K669 and 7741 at 3 wks of age were positive for IBDV by PCR testing. The rB,
K669 and 7741 were IBDV positive using the real time PCR with the segment B and vv
segment A probe. The STC group was positive using the real time PCR with the segment
B and non vv segment A probe. The groups challenged at 4 wks of age with either: STC,
Del-E, rB, 7741 or K669 were positive for IBDV by real time PCR. The Del-E group was
detected using the segment A probe and segment A non vv probe. All control groups and
groups challenged with D2712 were negative for IBDV by PCR (Table 3.2). Segment A
and segment B sequencing results from all IBDV positive groups correlated with the
IBDV sequences of the IBDV strain used for viral challenge.
Experiment 2: Serology. Using both ELISA and the VN assays with S706 and
Del-E antigen, the IBDV antibody titers exponentially decreased from day of age to 4
wks of age (Figure 3.3). At 1 day of age, the mean VN titers using the classic S706
antigen were similar to the ELISA titers. At 1 day of age, the VN titer determined using
the variant Del-E antigen was approximately half of the mean titer determined using the
ELISA assay and the VN with S706 antigen assay. At 1, 2 and 3 wks of age the ELISA
88
IBDV titers were higher that VN titers determined using both S706 antigen and Del-E
antigen at similar ages (Table 3.3). The mean antibody titer determined using VN and the
Del-E antigen was consistently lower than the titer calculated using the ELISA test and
VN S706 antigen. At 4 wks of age, no serum antibodies were detected using the VN
assays with either the S706 or Del-E antigen. At 4 wks of age very low antibody titers
(GMT 283) were detected using the ELISA test.
3.5 Discussion
The very virulent pathotype of IBDV (rA and rB strain) was first detected in the
U.S.A. in commercial pullets in Northern California in December 2008 [57,102]. The
first naturally occurring interserotypic reassortant IBDV (CA-495, CA-785) reported
were from commercial pullets and backyard chickens in Northern California. These
isolates were detected in 2009, 5 months after the initial isolation of vvIBDV in
California [56]. Since then several additional reassortants have been detected in poultry
flocks within the State. This paper investigates the phylogenetic relationship of both
segment A and segment B of reassortant IBDV strains D2712, 7741 and K669 from
California poultry compared with other IBDV strains worldwide. The pathogenicity of
these reassortants was assessed in 4 wk old SPF leghorns. In addition the ability of these
viruses to overcome maternal immunity was assessed by in vivo challenge in broilers
with passive immunity against classic and variant IBDV.
89
Confirmatory diagnosis and characterization of IBDV strains are typically based
on RT-PCR assays and partial sequencing of both segment A and segment B of the IBDV
genome from RT-PCR positive samples. Phylogenetics have shown that sequencing of
the VP2 of segment A and sequencing of the 5’ two-thirds of segment B produced
accurate phylogenetic information that was comparable to analysis of the complete
nucleotide sequences [64]. In experiment 2, the results of the IBDV RT-PCR assays were
highly correlated with the statistical analyses of mean bursal lesion scores (BLS). In most
cases, challenge groups testing positive for IBDV by RT-PCR also had statistically
significant higher BLS compared to the control groups. Correlation between PCR results
and bursal lesion scores was observed in all groups except in the case of the D2712
inoculated groups at 1 and 2 wks of age and the Del-E group at 4 wks of age. While mild
lymphocyte depletion was observed in 3/5 (BLS: 0.60) bursas and the PCR was positive
in the Del-E group at 4 wks of age, the BLS was not statistically different from the
control group. This lack of statistical significance may be a consequence of the sample
size and the fact that the bursal lesions observed at 4 wks were mild and ranged between
0 and 1 (data not shown).
Despite the significantly higher BLS and the detection of bursal lesions in D2712
inoculated broilers at 1 and 2 wks of age the PCR tests for both genome segment A and B
was consistently negative. This may indicate that the D2712 strain was not present or
present in too low quantity to be detected by PCR. It is also possible that the bursal
lesions in groups challenged with D2712 may have been caused by an etiological agent
other than IBDV. Contamination of the challenge inoculum with a bacteria or virus could
90
have resulted in the bursal injury observed microscopically. Hence the presence of bursal
injury in a few IBDV negative bursas challenged with D2712 in experiment 2 is not
surprising since lymphocyte depletion is not a pathogneumonic lesion of IBDV and other
etiological agents such as avian adenoviruses [33] have been associated with bursal
atrophy. Bacteriology and viral isolation techniques were not performed on bursas in
challenge experiments in this investigation. It is less likely that the lesions were caused
by an etiological agent present in the broiler flock before the time of bursal challenge
since no lesions were observed in the control birds. In spite of the high correlation
between bursal lesion scores and IBDV infection, the use of BLS to predict IBDV
infection was shown to be less sensitive than the PCR test. Statistical analyses of mean
BLS correlated with the PCR results 89% (25/28) of the time.
Similar negative PCR results were observed with D2712 challenge in broilers in
experiment 1 using SPF layers. No IBDV virus was recovered 7 days post inoculation of
SPF birds challenged with D2712 virus. Challenge of 4 wk old commercial turkeys with
D2712 failed to detect virus by PCR in bursa and intestinal tissues at 5 days post
challenge (data not shown). Sequencing and phylogenetics were performed using the
D2712 isolate recovered from the small intestines of the commercial turkeys from the
initial case submission. The D2712 isolate had a segment A that aligned with serotype 2
IBDV while the segment B was genetically related to serotype 1 classic strains. Previous
in vitro and in vivo research has shown that the B-cell tropism of serotype 1 IBDV is
determined by the genome segment A of IBDV [122]. In addition, serotype 2 IBDV do
not replicate in lymphoid cells but can be propagated in chicken embryos [8,86].
91
Zierenberg et al. generated a reassortant IBDV with a serotype 2 segment A and a
pathogenic serotype 1 segment B by reverse genetics [122]. In vivo challenge of SPF
chickens with this interserotypic reassortant failed to demonstrate lesions at 5 and 15
days PI in the bursa, thymus, spleen or cecal tonsils. Attempts by PCR failed to
demonstrate the virus at 5 and 15 days PI in the bursa, thymus spleen or cecal tonsils
[122].
Results of both SPF and broiler challenges with D2712 further support the
findings that IBDV strains with a serotype 2 segment A are unable to replicate in the
bursa of Fabricius. Replication of the D2712 strain in the respiratory tissues of chickens
was not investigated in this case.
In experiment 1, the mortality induced by the reassortant viruses, K669 and 7741
was less than that induced by the very virulent rB strain but higher than the STC or Del-E
groups. Reassortants K669 and 7741 both have serotype 1 segment A matching the
vvIBDV genotype hence the ability of these strains to replicate within the bursa of
Fabricius is not surprising. Zierenberg et al. reported that replacing the segment B of
serotype 1 classic IBDV with a serotype 2 strain did not affect the B cell tropism of the
virus. The reduced virulence of reassortants with vv segment A and non vv segment B
supports the hypothesis that both segment A and segment B influence pathogenicity.
While the segment A determines the bursa tropism, segment B influences the efficiency
of viral replication. Hence it can be deduced that the reduced virulence of 7741 and K669
is due to a reduction in the efficiency of viral replication that is modulated by the
presence of non vv segment B. A similar reduction in pathogenicity was observed with
92
natural IBDV reassortants with serotype 1 segment A reported by Jackwood et al. [56]
and Le Nouen et al. [63].
While no mortality was observed in maternally immune broilers challenged at 1,
2, 3, and 4 wks of age, lymphocyte depletion was still observed in several of the IBDV
challenge groups. The reassortant K669 was able to overcome high levels of maternal
IBDV antibodies as early as 2 wks of age in experiment 2 and cause significant
lymphocyte depletion within bursal follicles. The segment A of reassortant K669 was
homologous with the vv genotype and segment B matched serotype 2 IBDV. The
antigenicity of IBDV strains are determined by epitopes within the VP2 of segment A. In
spite of the antigenic similarity reported between the classic and very virulent viruses,
experiment 2 suggests a lack of cross protection between the classic IBDV antibodies and
the reassortant K669 antigen. Further in vitro investigation using monoclonal antibody
panels and the K669 antigen are required to determine the degree of cross protection
between K669 and classic IBDV antibodies.
In experiment 2 the K669 strain was able to cause bursal lesions and was
detected by PCR as early as 2 wks of age. The K669 virus was able to break through
maternal antibodies earlier than the other IBDV pathotypes used for viral challenge
including the rB strain. This result is surprising since the segment A of K669 was a
99.9% (701 bp/702 bp) match and 100% amino acid match (234/234) with the vvIBDV
rB strain within the hVP2 region. While the majority of epitopes that elicit neutralizing
antibodies are located within the hvVP2 region, antiVP3 neutralizing monoclonal
antibodies have been described [90,114].
Further work using monoclonal antibody
93
panels will be useful to determine if K669 possesses any modified neutralizing epitopes.
In spite of the apparent ability of K669 to break through maternal immunity earlier than
the vvIBDV rB strain, mortality results in challenge experiment1 with 4 wk old SPF birds
indicate that K669 was less pathogenic than rB. A reduction in pathogenicity was also
reported by Jackwood et al. with interserotypic reassortants CA-495 and CA785 [56].
Interestingly the K669 segment A was 99.9% identical to segment A of CA-495 and 99.8
% identical to segment B of CA-495. The K669 isolate was recovered in commercial
broilers from Northern California in 2011 while the genetically related strain CA-495 was
recovered from backyard chickens on a different farm in the same geographical region in
2009.
In birds challenged at 3 wks of age, the maternal antibody titers are reduced to
non protective levels (ELISA GMT: 490) and this correlated with bursal lymphocyte
depletion and RT-PCR positive results in the STC, rB, K669, and 7741 challenge groups.
The Del-E variant virus was not detected until birds were challenged at 4 wks of age. An
interesting observation was the absence of mortality in 4 wk old maternally immune
broilers challenged with STC, rB, 7741 and K669, compared with the occurrence of
mortality in 4 wk old SPF birds challenged with these same strains. It is hypothesized that
the maternally immune broilers also had a better developed non specific immunity that
could be providing some protection in the broilers. In spite of this, significant lymphocyte
depletion and secondary immune suppression was still a factor in the broilers.
In experiment 1, the mean BBW ratios in groups challenged with 7741, K669, rB
STC and Del-E were all significantly reduced compared to the BBW ratios of the control
94
group. In experiment 1, the use of BBW ratios was a reliable indicator of bursal injury
and IBDV positive groups. In experiment 2, the use of statistical differences in BBW to
predict IBDV positive groups was unreliable in the majority of cases. In experiment 2,
the presence of maternal immunity is having a protective effect against bursal injury
hence the BBW and BLS do not reflect the dramatic changes as seen with the bursas of
SPF challenged birds. In experiment 2, the BBW ratios were only statistically different
from the controls in the K669 inoculated broilers at 3 and 4 wks of age. The lack of
correlation between the mean BBW ratios and RT-PCR in maternally immune broilers
suggests that BBW ratios are not sensitive enough to be a reliable indicator of bursal
disease and IBDV positive bursas in commercially reared broilers.
The generation of interserotypic reassortants in the field requires that the host
species of origin be co-infected with both serotype 1 and serotype 2 IBDV and that both
viruses are capable of replicating within the same host cell. It is unlikely that the
interserotypic reassortants recovered from field caseswere generated in lymphoid cells
since serotype 2 viruses do not typically replicate in lymphocytes [8]. In addition, it is
unlikely that the original host was a chicken since serotype 2 viruses have not been
recovered from chickens [48,51]. The likelihood of a turkey host of origin cannot be
discounted since both serotype 1 vvIBDV [87,89] and serotype 2 IBDV [15,49,52,76,86]
have been isolated from turkeys. While the replication kinetics of IBDV in other species
have not been thoroughly investigated, the generation of reassortant IBDV in a species
other than turkeys cannot be discounted since IBDV have been recovered from several
avian [60,61] and non avian species [88].
95
Despite the muti-segmented nature of the genome of IBDV, relatively few
naturally occurring reassortant IBDV have been reported based on evaluation of the
thousands of Genbank sequences [113]. This may be a reflection of the low frequency of
generation of IBDV reassortants in nature. Reassortant IBDV have been generated in
vitro by reverse genetics [69,122]. Hypothetically, the potential to generate reassortants
requires the prerequisite that the host cell be co-infected with at least 2 different IBDV
genome subtypes. While all the exact factors influencing the rate of generation of IBDV
reassortants in nature is unknown, it is most likely multifactorial and not only dependent
on having co-infection with multiple IBDV subtypes. Alteration of factors such as the
dosage and ratio of viruses given and the timing of exposure to viral pathotypes may
influence the ability to recover reassortants in vivo. The complex interaction between the
host, virus and environment may all be influencing the low rate of generation of
reassortants in nature. It has been suggested that the emergence of vvIBDV occurred
from a reassortant event [13,39,63,119], hence the emergence of increasing virulent
IBDV pathotypes and IBDV stains with epitope changes that escape neutralizing
antibodies are a real concern.
3.7 Endnotes
The nucleotide sequences of the reassortants reported in this paper have been submitted
to Genbank (Mail Stop K710, Los Alamos National Laboratories, Los Alamos, NM
87545). The GenBank/EMBL/DDBJ accession numbers for the full length sequences of
segment A and B of IBDV strain K669 are JN585293 and JN411134 respectively. The
accession numbers for the full length sequences of segment A and B of IBDV strain 7741
96
are JQ403646 and JQ403647 respectively. The accession numbers for the partial
sequences determined in this study are JQ619639 for the VP1 gene of the STC strain and
JQ619640 for the VP1 gene of the T1 strain. The accession numbers for the VP2 and
VP1
of
D2712
are
JX235359
respectively.
97
and
JX235358
Table 3.1. Experiment 1: Pathogenicity investigation of reassortant IBDV in 4 wk SPF chickens
IBDV challenge virusA
Mortality
Mean BBWB ± SD
Mean BLSC ± SD
Reassortant (7741)
5/10
3.14 ± 0.39a
3.14 ± 0.38a
Reassortant (K669)
7/10
3.44 ± 0.99a
3.50 ± 0.58a
Reassortant (D2712)
0/10
5.23 ± 1.24 c
0.00 ± 0.00b
vvIBDV (rB)
9/10
2.69 ND
3.43 ± 0.53a
cvIBDV (STC)
4/10
2.53 ± 0.92a
3.40 ± 0.52a
scIBDV (Del-E)
0/10
1.69 ± 0.28b
3.20 ± 0.41a
Control
0/10
5.41 ± 0.93c
0.00 ± 0.00b
98
A
Each challenged bird was inoculated with 105.0 EID50 in 0.1ml volume.
B
Mean bursa: body weight (BBW) ratio = bursa wt (g)/ body wt (g) X 1000. Mean BBW ratio calculated in birds surviving viral challenge and presented as mean
BBW ± standard deviation (SD). Lower case letters indicate statistical differences among the groups (P<0.01). ND = statistical significance not determined
because the sample size was too small.
C
Severity of bursal histopathological lesions was graded on a scale of 0-4 with mean lesion scores calculated for each challenge group. Score of ‘0’ indicated an
absence of lesions; 1: <25% lymphocyte depletion in bursas; 2: 25-50% lymphocyte depletion in bursas; 3: 50-75% lymphocyte depletion in bursas; 4: >75%
lymphocyte depletion in bursas. Lower case letters indicate statistical differences among the groups (P<0.01). The bursal lesion scores (BLS) for each group
presented as the mean BLS ± standard deviation (SD).
98
Table 3.2. Experiment 2: Pathogenicity in maternally immune boilers inoculated with
IBDV
Age
(wks)
IBDV
virusA
Mortality
Mean BBWC ±
SD
Mean BLSD ±
SD
PCR testE
B
1
STC
0/8
3.10 ± 0.70a
0.00 ± 0.00a
-
1
Del-E
0/8
2.32 ± 0.37a
0.00 ± 0.00a
-
1
rB
0/8
2.38 ± 0.57a
0.00 ± 0.00a
-
0/8
2.38 ± 0.81
a
0.00 ± 0.00
a
-
a
0.00 ± 0.00
a
-
1
K669
1
7741
0/8
2.34 ± 0.60
1
D2712
0/8
2.26 ± 0.54a
0.88 ± 0.83b
-
0/5
2.38 ± 0.55
a
0.00 ± 0.00
a
-
a
0.13 ± 0.35
a
-
1
Control
2
STC
0/8
2.40 ± 0.77
2
Del-E
0/8
2.05 ± 0.31a
0.00 ± 0.00a
-
2
rB
0/8
2.95 ± 0.85a
0.13 ± 0.35a
-
0/8
1.74 ± 0.65
a
b
+
a
a
-
2
K669
3.50 ± 0.76
2
7741
0/8
2.47 ± 0.59
2
D2712
0/8
2.31 ± 0.73a
1.00 ± 0.53b
-
0/5
2.72 ± 0.63
a
a
-
a
b
+
2
Control
0.00 ± 0.00
0.00 ± 0.00
3
STC
0/5
3.00 ± 1.31
2.60 ± 1.52
3
Del-E
0/5
2.82 ± 0.71a
0.40 ± 0.55a
-
3
3
rB
K669
0/5
0/5
2.44 ± 1.40a
0.78 ± 0.42b
3.20 ± 1.30b
3.60 ± 0.55b
+
+
3
7741
0/5
2.64 ± 0.69a
1.60 ± 0.89b
+
3
D2712
0/5
2.63 ± 0.64a
0.20 ± 0.45a
-
3
Control
0/5
2.47 ± 0.51a
0.00 ± 0.00a
-
a
b
4
4
STC
Del-E
0/5
0/5
2.02 ± 0.66
2.49 ± 0.22a
3.80 ± 0.45
0.60 ± 0.55a
+
+
4
rB
0/5
1.61 ± 0.23a
4.00 ± 0.00b
+
4
K669
0/5
1.15 ± 0.27b
4.00 ± 0.00b
+
0/5
2.92 ± 0.95
a
b
+
a
a
-
0.00 ± 0.00a
-
4
7741
4
D2712
0/5
3.33 ± 0.54
4
Control
0/5
2.12 ± 0.40a
A
Each IBDV challenged bird was oronasally inoculated with 10
B
Mortality rate 7 days post challenge.
99
1.80 ± 1.30
0.60 ± 0.55
5.0
EID50 in 0.1ml volume.
Continued
C
Bursa:body weight (BBW) ratio = bursa wt (g)/ body wt (g) X 1000. Mean BBW ratio calculated in birds
surviving viral challenge and presented as mean BBW ± standard deviation (SD). Lower case letters
indicate statistical differences among the groups (P<0.01).
D
Severity of bursal histopathological lesions was graded on a scale of 0-4 with mean lesion scores
calculated for each challenge group. Score of ‘0’ indicated an absence of lesions; 1: <25% lymphocyte
depletion in bursas; 2: 25-50% lymphocyte depletion in bursas; 3: 50-75% lymphocyte depletion in bursas;
4: >75% lymphocyte depletion in bursas. The bursal lesion scores (BLS) for each group presented as the
mean BLS ± standard deviation (SD). Dunnett Multiple Comparisons Test was used to compare all
treatment groups against the control group. Lower case letters indicate statistical differences compared to
control group (P<0.01).
E
The symbol ‘+’ indicates the bursal sample tested positive for IBDV and the strain used for inoculation
correlated with RT-PCR and sequencing results. The symbol ‘-’ indicates that the bursal sample tested
negative using the RT-PCR tests.
100
Table 3.3. Experiment 2: Geometric mean titers (GMT) obtained from viral neutralization
(VN)A tests using standard classic S706 and Del-E 8903 IBDV strains and ELISAB tests.
Geometric Mean Titers (GMT)
Age (wks)
1
VN- classic S706
11220
VN- Del-E
5522
ELISA
11116
7
3871
2245
6179
14
1277
569
1322
21
297
20
490
28
0
0
283
A
VN test was performed using a constant virus-varying antibody assay. Sera samples
were diluted two fold starting at 1:100 dilution and 102 median tissue culture infectious
doses of viral antigen. The titers for VN are the reciprocal of the last dilution that
neutralizes 50% of the cell culture wells.
B
The ELISA test used was the IDEXX IBD-XR (IDEXX® Laboratories, Inc. Gainsville,
GA)
101
Mortality (%)
Mortality (% ) Induced by IBDV strains inoculated in SPF
chickens
100
90
80
70
60
50
40
30
20
10
0
Mortality (%)
rB
K669
7741
STC
Del-E
Control
IBDV strain
Figure 3.1. Experiment 1: Mortality observed 7 days post challenge with different strains
of IBDV inoculated into 4 wk old SPF chickens
102
Mean BBW ratios of SPF birds challenged with different
IBDV strains
6
BBW ratio
5
4
3
Mean BBWB ± SD
2
1
0
Control
K669
rB
7741
STC
Del-E
IBDV Strains
Figure 3.2. Experiment 1: Mean BBW ratios calculated in IBDV challenge groups.
103
Geometric Mean Titers (GMT)
12000
10000
8000
VN- classic S706
6000
VN- Del_E
ELISA
4000
2000
0
1
7
14
21
28
Age (days)
Figure 3.3. Experiment 2: Geometric mean titers (GMT) in maternally immune broilers
calculated with viral neutralization (VN) against classic S706 Winterfield IBDV strain
and variant Del-E 8903 strain and ELISA tests
104
Mean BLS in Maternally Immune Broilers Challenged with Different Strains of IBDV
4.0
Mean Bursal Lesion Scores
3.5
3.0
Mean BLS_wk1
mean BLS_wk2
2.5
2.0
mean BLS_wk3
1.5
mean BLS_wk4
1.0
0.5
0.0
STC
Del-E
rB
K669
7741
D2712
Control
IBDV Challenge Strain
Figure 3.4. Experiment 2: Mean bursal lesions scores in maternally immune broilers
challenged between 1-4 wks of age.
105
3.5
Mean BBW ratio
3
2.5
2
Mean BBW_wk1
1.5
1
0.5
0
STC
Del-E
rB
K669
7741
D2712 Control
IBDV Challenge Group
Figure 3.5. Experiment 2: Mean BBW ratios in maternally immune broilers challenged
with different IBDV strains at 1 wk of age.
106
3.5
3
Mean BBW Ratio
2.5
2
1.5
1
Mean BBW_wk2
0.5
0
STC
Del-E
rB
K669
7741 D2712 Control
IBDV Challenge Group
Figure 3.6. Experiment 2: Mean BBW ratios in maternally immune broilers challenged
with different IBDV strains at 2 wk of age.
107
3.5
Mean BBW Ratio
3
2.5
2
Mean BBW_wk3
1.5
1
0.5
0
STC
Del-E
rB
K669
7741
D2712 Control
IBDV Challenge Group
Figure 3.7. Experiment 2: Mean BBW ratios in maternally immune broilers challenged
with different IBDV strains at 3 wk of age
108
Mean BBW Ratio
4
3.5
3
2.5
2
1.5
1
0.5
0
Mean BBW_wk4
STC
Del-E
rB
K669 7741 D2712 Control
IBDV Challenge Group
Figure 3.8. Experiment 2: Mean BBW ratios in maternally immune broilers challenged
with different IBDV strains at 4 wk of age.
109
0
3
2
1
4
Figure 3.9. Bursal lesion scores (BLS) graded 0-4. Microscopic appearance of the
varying severity of lymphocyte depletion based on the scoring system used is shown.
Score of ‘0’ indicated an absence of lesions; 1: <25% lymphocyte depletion in bursas; 2:
25-50% lymphocyte depletion in bursas; 3: 50-75% lymphocyte depletion in bursas; 4:
>75% lymphocyte depletion in bursas.
110
7741
rB
rB
K669
Figure 3.10. Experiment 1. Hemorrhages on the mucosa of the proventriculus from rB,
K669 and 7741 inoculated SPF birds.
111
A
B
C
Figure 3.11. Picture A: Clinical signs of vvIBDV (rB strain) 48 hours post challenge of
SPF leghorn from experiment 2. Prostration, ruffled feathers and closing of eyes
observed. Picture B: Macroscopic appearance of bursa from vvIBDV (rB strain)
challenged SPF bird that succumbed to infection 3 days post challenge. Picture C:
Microscopic appearance of the bursa of Fabricius from picture B; lymphocyte depletion
and stromal hemorrhage observed.
112
Figure 3.12. Microscopic appearance of bursa of Fabricius from SPF leghorn challenged
with reassortant 7741 4 days PI. Lymphocyte depletion and numerous cystic follicles
observed. Mag. x 4
113
Figure 3.13. Microscopic appearance of bursa of Fabricius from SPF leghorn challenged
with STC strain. Lymphocyte depletion, cystic follicles and interfollicular edema and
inflammation observed. Mag x 4
114
Figure 3.14. Microscopic appearance of bursa of Fabricius from SPF leghorn challenged
with variant Del-E. Significant lymphocyte depletion observed without significant
hemorrhage. Mag x 4
115
Figure 3.15. Microscopic appearance of bursa of Fabricius from SPF leghorn challenged
with reassortant K669. Severe stromal hemorrhage and extensive lymphocyte necrosis in
bursal follicles. Mag x 4
116
A
B
Figure 3.16. Experiment 1. Inoculation of SPF leghorns with D2712. Picture A:
hemorrhage (arrow) observed in the lamina propria of the cecal tonsils. Picture B:
pyogranuloma observed in cecal tonsil section. Mag x4
117
A
B
Figure 3.17. Experiment 1. Inoculation of SPF leghorns with D2712. Picture A:
Interfollicular inflammation (arrow). Picture B: Mild interfollicular hemorrhage (arrow).
Lymphocyte depletion was not a significant finding in bursas. Mag x4
118
Uninoculated
control
Embryo inoculated
with interserotypic
reassortant: K669
Figure 3.18. Inoculation of 9 day old embryo on right with reassortant K669 for viral
titration in embroyated eggs procedue. Five days PI, stunting of embryo observed.
119
A
B
Figure 3.19. Liver in (A) taken 5 days PI from chicken embryo inoculated via the CAM
with reassortant IBDV strain D2712. The greenish discoloration of liver evident (arrow).
Chicken embryo tissues harvested from inoculated embryos were positive for D2712 by
real time RT-PCR. The two livers from (B) were from the un-inoculated controls.
120
58
37
32
46
90
24
53
38
79
60
56
90
47
21
58
19
24
15
11
18
45
30
96
99
90
72
99
89
97
France_01 F10
VV Europe_ NC004178
VV_UK 012
Denmark014
Denmark01 5
Brazil01 B1
Spain01 S1
Venezuela_04VEN120
vvIBDV
Reassortant 7741
rA
rB
Reassortant CA-785
Reassortant CA-D495
Reassortant K669
Bolivia07B113
CA-K2296
STC
Classic IBDV
Spain 01 S8
Del-E_AF133904
USA05AK L17
USA05CA75
Variant IBDV
USA06CA521
Intervet D78
Mexico07M167
ViBursa
D2712
Serotype 2 IBDV
OH
Korea 97K9596
T1_AF281238
France 04F7
Dominican Rep 99DR4
Panama 06P121b
Figure 3.20. Phylogenetic analysis of partial genome segment A nucleotide sequences of
IBDV used in experiment 1 and IBDV from around the world. The neighbor-joining
method with up to 1000 bootstrap replicates was used.
121
AJ878655 BURSINE2
AJ878659 BURSINE
AJ878660 CEVAC IBDL
AJ878676 DEL E
AJ878643 F52-70
Variant
IBDV
AJ878644 CU1WT
AJ878675 VARIANT A
Intervet Bursavac
Intervet Univaxplus
AJ878657 228E
AJ878653 CT4-4
AJ878654 D78
AJ878656 BURS706
AJ878658 TADFORTE
AJ878651 78GSZ
Classic IBDV
AJ878652 80GA
AJ878677 GLS5
AJ878683 TY89
AJ878684 23 82
OH Serotype 2
Reassortant CA K669
Serotype 2 IBDV
Reassortant CA 495
Reassortant CA K785
AJ878671 HENAN
AJ878681 GX
AJ878642 05-5
AJ878640 06-95
AJ878641 N1-99
AJ878639 V877
AJ878682 002-73
AJ878638 88180
CA rA
CA rB
AJ878673 BD3
AJ878668 HK46
AJ878669 95072.2
AJ878670 96108
AJ878665 91184
vvIBDV
AJ878666 UK661
AJ878663 89299
AJ878662 849VB
AJ878650 00-40
AJ878674 99141
AJ878678 94432
AJ878680 99323
AJ878664 912383
AJ878672 99009
AJ878667 DV86
AJ878661 89163
AJ878649 93-35
AJ878679 91168
122
0.01
Continued
Figure 3.21. Phylogenetic analysis of partial genome segment B nucleotide sequences of
IBDV used in experiment 1 and IBDV from around the world. The neighbor-joining
method with up to 1000 bootstrap replicates was used.
123
3.8 References
1. G. A. Abdel-Alim and Y. M. Saif, "Pathogenicity of Embryo-Adapted Serotype 2 OH
Strain of Infectious Bursal Disease Virus in Chickens and Turkeys," Avian Dis.
46, 1001-1006 (2002).
2. G. M. Allan, M. S. McNulty, T. J. Connor, R. M. McCracken, and J. B. McFerran,
"Rapid diagnosis of infectious bursal disease infection by immunofloresence on
clinical material," Avian Pathol. 13, 419-427 (1984).
3. S. Ashraf, G. A. Abdel-Alim, M. Q. Al-Natour, and Y. M. Saif, "Interference Between
Mild and Pathogenic Strains of Infectious Bursal Disease Virus in Chickens,"
Avian Dis. 49, 99-103 (2005).
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