Download persistence, distribution and immunopathogenesis of infectious

PERSISTENCE, DISTRIBUTION AND IMMUNOPATHOGENESIS OF
INFECTIOUS BURSAL DISEASE VIRUS IN CHICKENS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Abdul Rauf M.Sc.
Graduate Program in Veterinary Preventive Medicine
The Ohio State University
2011
Dissertation Committee:
Dr. Y. M. Saif (Advisor)
Dr. Daral J Jackwood
Dr. Chang Won Lee
Dr. Gireesh Rajashekara
Dr. Renukaradhya J. Gourapura
Copyrighted by
Abdul Rauf
2011
ABSTRACT
There have been questions regarding the persistence of infectious bursal disease virus
(IBDV) in processed meat destined for export. A series of experiments were designed to
determine the persistence, distribution and quantification of IBDV strains in lymphoid
and non-lymphoid tissues of specific pathogen free (SPF) chickens and commercial
broiler chickens. Five separate experiments were conducted using 2 and 4 weeks old SPF
chickens and 2 weeks old commercial broilers having maternally derived antibodies. Four
different types of lymphoid and seven different non-lymphoid tissues were sampled from
individual chickens in each experiment. Viral RNA (vRNA) was extracted from tissues
and subjected to real time RT-PCR. The virus strains were detected the longest in bursal
tissues followed by spleen, thymus and bone marrow. In non-lymphoid tissues, both of
the strains were detected for the longest period in the caecum followed by liver, kidney,
pancreas, lungs, thigh muscles and breast muscles. Upon infection with the standard
challenge (STC) strain, a serotype 1 classic virus, the vRNA was detected in bursa of SPF
chickens up to 8 weeks post-inoculation (PI) and in commercial broiler chickens the virus
was detected up to 6 weeks PI. In addition, we were able to isolate the STC strain up to 4
and 3 weeks PI from the bursal homogenates of SPF and commercial chickens,
respectively. The Indiana (IN) strain, a serotype 1 variant virus, vRNA was detected up to
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5 weeks PI in SPF and up to 3 weeks PI in commercial chickens in bursal tissue. The IN
strain was isolated up to 10 days post-inoculation (DPI) from bursal tissues collected
from both SPF and commercial chickens. In commercial broilers the STC was detected at
DPI 3 in thigh muscles and only one breast muscle sample was RT-PCR positive at DPI
14. The IN strain was not detected in thigh muscles while it was detected in breast
muscles up to two weeks post-inoculation (PI) in commercial chickens. Whereas in SPF
chickens, the vRNA of STC and IN strains were detected in thigh muscles up to 4 and 2
weeks PI and in breast muscles up to 3 and 2 weeks PI, respectively. This is the first
detailed report on the persistence and distribution of classic and variant strains of IBDV
in different tissues of SPF and commercial chickens. The study indicated that the
persistence and distribution of IBDV in non-lymphoid tissues, specifically, thigh muscles
and breast muscles is significantly different between SPF and commercial chickens.
Although the virus can persist in the bursa of SPF chickens for 4 weeks, it is very
unlikely that the infectious virus will be present in the processed meat. In addition, the
RT-PCR results are not sufficient to indicate the presence of the infectious virus.
A study was undertaken to better understand the innate immune response against IBDV.
In this study 3 weeks old SPF chickens were inoculated intraocularly with STC and
Indiana (IN) strains representing either classic IBDV (cIBDV) or variant IBDV (vIBDV),
respectively. Bursal lesions, macrophages and T cells infiltration were quantified. We
examined the differential expression of virus-induced innate and proinflamatory
cytokines, chemokines and toll like receptors (TLRs) and their adaptor molecules. These
findings indicate that cIBDV- and vIBDV-infection mediated the differential induction of
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proinflammatory cytokines, innate cytokines and upregulated the expression of TLRs as
well as their adaptor molecules. This information will be important in understanding the
immune responses against IBDV infection in chickens.
In the last part we studied the adaptive immune responses of IBDV in chickens. The
IBDV actively replicates in B cells and causes severe bursal damage. Generally, T cells
are refractory to infection with IBDV but are known to promote viral clearance.
However, the mechanisms of T cell mediated viral clearance are not well understood. In
this study, we evaluated the molecular mechanisms of cytotoxic T cell responses in the
pathogenesis of IBDV in chickens. Infection of chickens with IBDV was accompanied
by the infiltration of CD4+ and CD8+ T cells in the bursa. There was an upregulation in
the gene expression of important cytolytic molecules; perforin (PFN), granzyme-A
(Gzm-A), DNA repair and apoptotic proteins; high mobility proteins group (HMG) and
poly (ADP-ribose) polymerase (PARP) in the bursa of Fabricius (BF), whereas
expression of NK (natural killer) lysin was downregulated. Importantly, PFN producing
CD4+ and CD8+ T cells were also detected in the bursa of IBDV-infected chickens by
immunohistochemistry. The Th1 cytokines, IL-2 and IFN-γ expression was also strongly
upregulated, suggesting the activation of T cells. The findings of this study highlight the
mechanisms of IBD pathogenesis and the role of cytotoxic T cells in the clearance of
virus-infected cells.
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I dedicate this effort to my parents Mr. and Mrs. Abdul Malik Khattak and to all
of my Teachers
v
ACKNOWLEDGEMENTS
I wish to express my special gratitude to my advisor Dr. Yehia M. Saif for his support,
intellectual guidance, encouragement, and great patience throughout my graduate
program. Without his sincere support, guidance, and help, this work would not have been
possible. His pleasant, kind and generous personality shaped my thoughts to be an
optimistic and look at the brighter aspects of my endeavors. I am forever grateful to him.
I wish also to thank my other graduate committee members, Dr. Daral J. Jackwood, Dr.
Chang Won Lee, Dr. Gireesh Rajashekara and Dr. Renukaradhya J. Gourapura for their
assistance and invaluable advice.
I would like to express my sincere appreciation to Dr. Mahesh Khatri for his valuable
contribution to my work.
I am thankful to Dr. Kwonil Jung and Dr. Alexander Rodriguez-Palaci for their technical
support, their invaluable advice and help during my graduate program.
I am very grateful to Dr. Juliette Hanson, Gregory Myers, Kingsly Berlin and Todd Root
for their support with animal studies. I want to thank my classmates, and colleagues
Maria Murgia, and former members of Dr. Y.M Saif lab Dr.Hadi Yassine and Dr. Y.
Tang, for their generous support and help. I also want to especially acknowledge Hannah
Gehman, Robin Weimer and Megan Strother. I sincerely appreciate what they did for me
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during my time here. Their kind, generous, considerate, and helpful personalities will be
remembered forever.
Finally, I want to thank my loving parents Mr. and Mrs. Abdul Malik Khattak, my
brother Abdul Salam Khattak , I know that I owe their immeasurable debt of gratitude,
and I could not compensate it in my life. Without their everlasting and unwavering love,
encouragement, faith in me, my dream would not have come true. Very special thanks to
my great mom for her understanding, support and encouragement. I would like to express
my appreciation for my loving sisters Imrana Malik and Farzana Malik.
Finally I owe my greatest appreciation to my wife Naheed, her valuable suggestions
made my life much easier than I thought. I thank her very much for taking care of my son
Ahsen and daughter Mishall and sparing my time to devote it for my studies.
vii
VITA
B.Sc. (Premedical Group)……………………… 1997 The University of Punjab
Lahore, Pakistan
B.Ed (Biology and Chemistry)………………..
1999 AIOU Islamabad, Pakistan
M.Sc. (Zoology/Microbiology)……….……….
2000 University of Arid Agriculture,
Rawalpindi, Pakistan
Research Assistant (Microbiology)…………….
1999-2001, Animal Health institute,
National Agriculture Research
Center, Islamabad Pakistan
Microbiology Research
2001-2006 University of Arid
Associate)………………………………………. Agriculture
Rawalpindi Pakistan
Fulbright Fellow ………………………………
February 2006 to December 2006
FAHRP/OARDC/The Ohio State
University, USA
Graduate Research Associate (Ph.D. Candidate)
2007-2011 FAHRP/OARDC
The Ohio State University, USA
Publications
1: Rauf, A., Khatri, M., Murgia, M.V. and Saif, Y.M. (2011) Expression of perforingranzyme pathway genes in the bursa of infectious bursal disease virus-infected chickens.
Dev Comp Immunol. In press.
viii
2: Functional Invariant NKT Cells in Pig Lungs Regulate the Airway Hyperreactivity: A
Potential Animal Model. Renukaradhya, G.J., Manickam, C., Khatri, M., Rauf, A., Li,
X., Tsuji, M., Rajashekara, G. and Dwivedi, V. J Clin Immunol. (2010).
Published Abstracts
1: Distribution and persistence of infectious bursal disease virus in chickens. Rauf
Abdul, Maria V. Murgia, M. Khatri, A. Rodriguez-Palacios, C-W. Lee and Y.M. Saif.
North central avian disease conference (61 Annual meeting March 15 & 16 2010) at St.
Paul River center St. Paul Minnesota
2: Prevalence of parvoviruses in commercial turkey flocks. Maria V. Murgia, Rauf
Abdul, Tang, Y; A. Gingerich, E; C-W. Lee and Y.M. Saif. North central avian disease
conference (61 Annual meeting March 15 & 16 2010) at St. Paul River center St. Paul
Minnesota
3: Persistence and distribution of infectious bursal disease virus in SPF and commercial
broiler chickens. Rauf Abdul, Maria V. Murgia, C-W. Lee, M. Khatri and Y.M Saif.
AVMA/AAAP Meeting. Georgia World Congress Center, Atlanta, GA. July31- August
4, 2010: Development of a Real-time PCR test for the detection of Turkey Parvoviruses
in fecal samples. Maria V. Murgia, Rauf Abdul and Y.M Saif. AVMA/AAAP Meeting.
Georgia World Congress Center, Atlanta, GA. July31- August 4, 2010
viii
5: Viral induced inflammatory cytokine, toll like receptors and cytotoxic T cells
components in infectious bursal disease infected chickens. Rauf Abdul, M. Khatri, Maria
V. Murgia and Y.M. Saif. Conference for Research workers in animal science at Chicago,
December 5 – 7, 2010.
Fields of Study
Major Field: Veterinary Preventive Medicine
Studies in Molecular Virology and Immunology
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TABLE OF CONTENTS
PERSISTENCE, DISTRIBUTION AND IMMUNOPATHOGENESIS OF
INFECTIOUS BURSAL DISEASE VIRUS IN CHICKENS ............................................ 1
DISSERTATION ................................................................................................................ 1
ABSTRACT ........................................................................................................................ ii
TABLE OF CONTENTS ................................................................................................. viii
LIST OF TABLES ............................................................................................................. xi
CHAPTER 1 ....................................................................................................................... 1
LITERATURE REVIEW ................................................................................................... 1
PERSISTENCE, PATHOGENESIS AND IMMUNOLOGY OF INFECTIOUS BURSAL
DISEASE VIRUS ............................................................................................................... 1
CHAPTER 2 ..................................................................................................................... 30
PERSISTENCE AND TISSUE DISTRIBUTION OF INFECTIOUS BURSAL DISEASE
VIRUS IN SPF AND COMMERCIAL BROILER CHICKENS ..................................... 30
2.1 SUMMARY ............................................................................................................ 30
2.2 INTRODUCTION ................................................................................................... 32
2.3 MATERIALS AND METHODS ............................................................................ 34
2.4 RESULTS................................................................................................................ 37
viii
2.5 DISCUSSION ......................................................................................................... 40
2.7 REFERENCES ........................................................................................................ 45
CHAPTER 3 ..................................................................................................................... 66
DIFFERENTIAL MODULATION OF CYTOKINES AND TOLL LIKE RECEPTORS
EXPRESSION IN THE BURSA OF CHICKENS INFECTED WITH CLASSICAL AND
VARIANT STRAINS OF INFECTIOUS BURSAL DISEASE VIRUS. ........................ 66
3.1 SUMMARY..………………………………………………………………………66
3.2 INTRODUCTION ................................................................................................... 67
3.3 MATERIALS AND METHODS ............................................................................ 70
3.4 RESULTS................................................................................................................ 74
3.5 DISCUSSION ......................................................................................................... 76
3.6 ACKNOWLEDGEMENTS .................................................................................... 80
3.7 REFERENCES ........................................................................................................ 81
CHAPTER 4 ................................................................................................................... 101
EXPRESSION OF PERFORIN-GRANZYME PATHWAY GENES IN THE BURSA
OF INFECTIOUS BURSAL DISEASE VIRUS-INFECTED CHICKENS .................. 101
4.1 SUMMARY...……………………………………………………………………100
4.2 INTRODUCTION ................................................................................................. 102
4.3 MATERIALS AND METHODS .......................................................................... 104
4.4 RESULTS.............................................................................................................. 108
ix
4.5 DISCUSSION ....................................................................................................... 110
4.6 ACKNOWLEDGEMENTS .................................................................................. 114
4.7 REFERENCES ...................................................................................................... 114
BIBLIOGRAPHY ........................................................................................................... 130
x
LIST OF TABLES
Table 2.1: Persistence and distribution of IBDV (STC & IN) strains in lymphoid tissues
of two-weeks old SPF chickens ........................................................................................ 47
Table 2.2: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid
tissues of two-weeks old SPF chickens ............................................................................ 48
Table 2.3: Persistence and distribution of IBDV (STC&IN) in non-lymphoid tissues of
two-weeks old SPF chickens ............................................................................................ 49
Table 2.4: Persistence and distribution of IBDV (STC & IN) strains in lymphoid tissues
of four-weeks old SPF chickens ....................................................................................... 50
Table 2.5: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid
tissues of four-weeks old SPF chickens ............................................................................ 51
Table 2.6: Persistence and distribution of IBDV (STC&IN) in non-lymphoid tissues of
four-weeks old SPF chickens ............................................................................................ 52
Table 2.7: Persistence and distribution of IBDV strains in lymphoid tissues of two-weeks
old in ovo vaccinated broilers chickens ............................................................................ 53
Table 2.8: Persistence and distribution of IBDV strains in non-lymphoid tissues of twoweeks old in ovo vaccinated broilers ................................................................................ 54
Table 2.9: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid
tissues of two-weeks old in ovo vaccinated broilers chickens ......................................... 55
xi
Table 2.10: Persistence and distribution of IBDV (STC & IN) strains in lymphoid tissues
of four-weeks old in ovo vaccinated broilers chickens ..................................................... 56
Table 2.11: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid
tissues of four-weeks old in ovo vaccinated broilers chickens ......................................... 57
Table 2.12: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid
tissues of four-weeks old in ovo vaccinated broilers ........................................................ 58
Table 2.13: Persistence and distribution of IBDV strains in lymphoid tissues of 2-weeks
old broilers having maternal antibodies ............................................................................ 59
Table 2.14: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid
tissues of 2-weeks old broilers having maternal antibodies.............................................. 60
Table 2.15: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid
tissues of 2-weeks old broilers having maternal antibodies.............................................. 61
Table 3.1: Sequence of primers for quantitative real-time RT-PCR analysis…………..85
xii
LIST OF FIGURES
Figure 2.1. Virus titter decay in tissue samples from chickens singly inoculated with
reference strains of infectious bursal disease virus……………………………………. 62
Figure 2.2. Persistence of two strains of infectious bursal disease virus after experimental
inoculation in SPF and commercial chickens. .................................................................. 63
Figure 2.3. Log10 viral titers in experimentally inoculated SPF and commercial chickens.
........................................................................................................................................... 64
Figure 2.4. Antibody titer in SPF chickens after inoculation with STC and IN strains…55
Figure 3.1. Histopathological lesions in bursa following IBDV-infection. ..................... 88
Figure 3.2. IBDV antigen detection by immunohistochemical staining in virus-infected
bursa. ................................................................................................................................. 90
Figure 3.3. T cells infiltration in the IBDV-infected bursa. ............................................. 92
Figure 3.4. Infiltration of macrophages in the IBDV-infected bursa. .............................. 94
Figure 3.5. Relative gene expression at mRNA level, of innate cytokines mRNA in
IBDV-infected bursa. ........................................................................................................ 96
Figure 3.6. Relative gene expression at mRNA level, of proinflamatory cytokines, iNOS
and chemokines, mRNA in IBDV-infected bursa. ........................................................... 98
xiii
Figure 3.7. Relative gene expression at mRNA level, of Toll like receptors and adaptor
molecules mRNA in IBDV-Infected bursa. .................................................................... 100
Figure 4.1. Detection of virus load and IBDV genome in bursa tissue. ........................ 119
Figure 4.2. Gene expression of PFN, Gzm-A and molecules involved in DNA repair and
apoptosis in the bursa of IBDV-infected chickens. ........................................................ 121
Figure 4.3. Accumulation of CD8+ T cells in the bursa. ................................................ 123
Figure 4.4. Infiltration of CD4+ T cells in the bursa. ..................................................... 125
Figure 4.5. Detection of PFN producing CD4+ and CD8+ T cells in BF. ...................... 127
Figure 4.6. Expression of Th-1 cytokines in the bursa of IBDV-infected chickens at PIDs
3, 5 and 7. ........................................................................................................................ 129
xiv
CHAPTER 1
LITERATURE REVIEW
PERSISTENCE, PATHOGENESIS AND IMMUNOLOGY OF
INFECTIOUS BURSAL DISEASE VIRUS
1.1 INTRODUCTION
Infectious bursal disease (IBD) is a globally distributed immunosuppressive viral disease
of young chickens, controlled by vaccination. The disease, also known as Gumboro
disease, is endemic in poultry-producing regions, inflicting significant economic losses to
the poultry industry worldwide (Flensburg et al., 2002; Saif, 1998; Sanchez et al., 2005).
Infectious bursal disease virus (IBDV), the causative agent of IBD, is comprised of two
serotypes, designated as serotypes 1 and 2 (McFerran, 1980). Only serotype 1 is
pathogenic for the domesticated chicken (Jackwood et al., 1985). Numerous serotype 1
strains, differing in antigenicity have been identified and classified as classic and variant.
Chickens are more susceptible to disease when they are 2–6 weeks of age and symptoms
are nonspecific, including depression, whitish diarrhea, anorexia, prostration, and death
(Chettle et al., 1989). Younger or older chickens may show milder disease symptoms, but
all age groups subsequently experience a transient immunosuppression (Dohms and
Jaeger, 1988; Kim et al., 1999; Sharma et al., 2000).
1
Classical IBDV has traditionally affected poultry worldwide since the first reported
incident from Gumboro, Delaware, USA. Classical strains cause bursal inflammation and
severe lymphoid necrosis in infected chicken, resulting in immunodeficiency and
moderate mortality from 20-30% in specific pathogen free (SPF) chicken (Lim et al.,
1999). Very virulent infectious bursal disease virus (vvIBDV) was isolated in the United
States in 2008 and the virus had 80 % sequence homology with classical IBDV strains
(Jackwood et al., 2009; Stoute et al., 2009). Variant strains were detected in the US in
1983. These strains were antigenically different from classic strains and caused a rapid
and severe bursal atrophy (Vakharia et al., 1994), and in contrast to classical strains
produced no clinical signs of illness. Antigenic variants have been recognized by their
ability to escape cross-neutralization by antiserum against the classical strains. The
Indiana (IN) strain is an antigenic variant strain of IBDV and was first isolated in our
laboratory (Ismail et al., 1990). This strain causes severe bursal atrophy at 5 and 10 days
post-inoculation in 3-weeks old SPF birds.
The IBDV being a non-zoonotic pathogen is ―not regarded as a human food safety issue,
nevertheless movement of birds with IBDV infections is a cause for concern because of
the possible introduction of new antigenic and pathogenic strains into a geographic area
can have a negative economic impact on the chickens grown in that region‖(Jackwood
and Sommer-Wagner, 2010). The relative heat resistance of IBDV and its ability to
survive 60 or more days in poultry litter (Mandeville et al., 2000), creates some important
epidemiological considerations. There have been questions’ regarding the persistence of
infectious IBDV in processed meat destined for export. Most of the studies conducted on
2
the persistence of IBDV in tissues are on SPF chickens. Limited data available on the
persistence of IBDV in SPF chickens cannot be superimposed on commercial broiler
chickens.
At present not much is known about the immune responses involved in the pathogenesis
of IBDV. The exact cause of clinical disease and death are still unclear and strains of
IBDV differ in their virulence. The IBDV infects and destroys actively dividing IgM
bearing B cells in the bursa of fabricius (Hirai et al., 1981; Rodenberg et al., 1994).
Infection with IBDV compromises both humoral and cellular immunity (Sharma et al.,
1989). Immunosuppression induced by IBDV is the primary cause of economic loss
associated with the virus (Khatri et al., 2005). Exposure to IBDV results in an acute, selflimiting disease. Under experimental conditions, the acute disease lasts for about a
weeks. During this acute phase, replicating virus causes extensive destruction of bursal
follicles (Tanimura and Sharma, 1997), resulting in reduction of circulating IgM+ B cells
(Hirai et al., 1981; Rodenberg et al., 1994).
Macrophages produce proinflamatory cytokines such as IL-6, IL-8, and IL-1β which are
important in initiating an inflammatory response at the site of infection. The local
inflammatory response recruits other phagocytic and non-phagocytic lymphoid cells
important for immunity. Thus, proinflamatory cytokines are a natural response to
infection and may be beneficial to the host defense. However, when produced in excess,
proinflammatory cytokines may enhance tissue destruction, impede recovery and harm
the host (Khatri et al., 2005).
3
Host immune cells use various receptors to perceive viral infections by recognizing
pathogen-associated molecular patterns (PAMPs) and subsequently induce an antiviral
response. Prominent among these are toll-like receptors (TLRs) (Kawai and Akira, 2006;
Sang et al., 2008; Werts et al., 2006). Several TLRs recognize viral PAMPs, of which toll
like receptor-3 (TLR3), detects double-stranded RNA (dsRNA) derived from viral
replication, whereas single-stranded RNA (ssRNA) are perceived by TLR7 and TLR8
(Sang et al., 2008). The TLR signaling proceeds via two pathways: the myeloid
differentiation factor 88 (MyD-88)-mediated pathway and the Toll-interleukin-1 receptor
(TIR)-domain-containing adaptor inducing IFN-β (TRIF)-mediated pathway (Kawai and
Akira, 2009; Takeuchi and Akira, 2009). TLR3 only activates the TRIF-mediated
pathway (Yokota et al., 2010).
The three main objectives envisioned for my research are:

Persistence and tissue distribution of infectious bursal disease virus in SPF and
commercial chickens.

Immunopathogenesis and modulation of Toll-like receptor 3 pathway in chickens
infected with a classical and a variant strain of infectious bursal disease virus.

Expression of perforin-granzyme pathway genes in the bursa of infectious bursal
disease virus-infected chickens.
1.2 PERSISTENCE OF IBDV IN CHICKEN TISSUES
The bursa and spleen were reported to have substantially higher concentration of the
virus as compared to any other tissue. The bursa harvested from chickens at 72 hours PI
4
yielded high virus titers followed by spleen and kidneys. No virus was detected beyond
10 days PI. IBDV was reported to persist in the chicken for a few days but the lesions
could be seen for at least 10 weeks, the longest interval evaluated in that study
(Winterfield et al., 1972). Chickens were inoculated with an attenuated cell culture
adapted virus at one day of age, the virus could be detected in cell culture inoculated with
homogenate of BF, spleen, thymus, liver, kidney and the lungs for up to 14 days PI in one
experiment and 10 days PI in another. No virus was detected after the levels of
neutralizing antibodies became significantly high. Also, the virus was not detected in
tissues from birds given the virus at 3 weeks and low virus neutralizing antibody titers
were detected, indicating an age resistance to chicken embryo fibroblast cells (CEF)
propagated virus or insufficient dose (Skeeles et al., 1979a; Skeeles et al., 1979b).
The IBDV was re-isolated most consistently from the bursa and less frequently from
thymus, liver, kidneys, lungs and spleen. No virus was isolated from the pancreas
(Mackenzie and Spradbrow, 1981). The virus was not detected beyond 11 days PI in
commercial chickens when inoculated at 1, 7 or 14 days of age. In birds inoculated at 21
days of age, IBDV was re-isolated up to days only. The precipitating antigen was
detected only in the bursa and only at 3rd, 4th and 5th day PI but was not detected in any
organ of chicken infected at 21 days of age (Mackenzie and Spradbrow, 1981).
When SPF chickens were inoculated with IBDV at 3 weeks of age, the viral RNA was
detected by RT/PCR up to 21 days PI, but attempts to isolate infectious virus from bursal
homogenates failed (Abdel-Alim and Saif, 2001a). Infectious virus was detected by
embryo inoculation up to 7 days PI in the bursa of SPF chickens inoculated at 2 or 3
5
weeks of age, whereas the viral RNA was detected by RT/PCR for up to 28 days PI. In
SPF chickens inoculated at 1 day of age, the bursa-derived virus or its RNA was detected
4
2.5
at 7 and 14 days PI when inoculated at a high dose, 10 EID /bird, or at a low dose, 10
50
EID /bird. In commercial 1-day-old broiler chickens, the bursa-derived virus was
50
4
detected at 7 and 14 days PI when inoculated at a high dose, 10 EID50/bird, whereas the
2.5
virus was detected only at 14 days PI when inoculated at a low dose, 10
EID50/bird. In
SPF and commercial chickens, vaccinated with a modified live IBDV vaccine, the virus
is known to persist in the bursa of SPF chickens up to 3 weeks but maternal antibodies in
the commercial chickens rapidly eliminate it from the bursa since no live vaccine virus
nor its RNA was detected in commercial broilers vaccinated at 1 day or 2 weeks of age
(Abdel-Alim and Saif, 2001a). In an another study it was documented that variant IBDV
was detected in virus-inoculated commercial broilers for up to 6 weeks and infectious
virus was recovered from all organs at 4 weeks PI (Elankumaran et al., 2002). This was
the first report that mentions that IBDV can be detected up to six weeks in the bursa.
Recently, the presence of infectious IBDV in broilers brought to processing plants was
reported. In that study bursa samples from 26 processing plants in the Eastern U.S. were
examined. The IBDV specific RT-PCR detected 25.5 % of the bursal samples from 11
different processing plants. Nucleotide sequence analysis conducted on 12 RT-PCR
positive samples indicated that the IBDV detected was not the commercially available
attenuated vaccine strains. Five RT-PCR positive samples were selected at random for
testing in specific-pathogen-free chickens. All five samples were positive for infectious
6
IBDV as confirmed by macroscopic lesions and bursa/body weight ratios. The five
viruses were re-isolated and identified in bursa tissue from chickens using RT-PCR and
nucleotide sequencing (Jackwood and Sommer-Wagner, 2010).
1.3 TARGET ORGAN
The target organ for pathogenic serotype 1 is the bursa of fabricius (BF). The BF reaches
the maximum development between 3-6 weeks of age and at this time chickens are most
susceptible to the disease. The IBDV infection results in high mortality during the acute
stage of the disease or in B cell deficiency after recovery from infection (Becht, 1980;
Kaufer and Weiss, 1980) .
Chickens infected with IBDV immediately after hatching develop a chronic infection
with atrophy of BF and B cell depletion (Hudson et al., 1975; Winterfield et al., 1972).
Chickens infected with IBDV when older than 12 weeks do not show clinical signs
(Becht, 1980). The bursectomized chickens survive the IBDV infections which is lethal
for normal chicken (Kaufer and Weiss, 1980).
High concentrations of antigens and high infectivity titers were found in BF of infected
chickens, whereas only traces of antigen and low virus titers were detected in the thymus,
spleen (Kaufer and Weiss, 1980) and peripheral blood (Burkhardt and Muller, 1987;
Mundt et al., 2003). In vitro infection studies have shown that IBDV replicates in the
population of proliferating B cells (Muller, 1986; Skeeles et al., 1979b) but not in very
immature lymphobalsts or competent B cells (Becht, 1980).
Apathogenic serotype 2 strains do not replicate in bursal lymphoid cells or in other
lymphoid cells (Nieper and Muller, 1996). Treatment of chicken with cyclophosphamide
7
(CY) or surgical bursectomy at 4 weeks of age is known to prevent IBDV infection
(Kaufer and Weiss, 1980). Bursectomized chickens did not show the disease and had
transient lesions in the lymphatic tissues. However, these chickens had virus and
produced antibodies against it. The virus concentration in the bursectomized chickens
was about 1000 times lower than the non-bursectomized chickens (Kaufer and Weiss,
1980). It appeared that the availability of a large number of bursal cells is an essential
factor in the development of IBD (Ismail et al., 1987).
1.4 PATHOGENESIS
Pathogenesis is defined as the method used by the virus to cause injury to the host with
mortality, disease or immuno-suppression as a consequence (van den Berg et al., 2000).
The injuries can be evaluated at the level of whole animal, the organ and the cell. IBDV
usually infects young chickens between 3-6 weeks of age and causes a clinical disease,
while sub-clinically infecting older birds. The outcome of IBDV infection is dependent
on the strain and amount of the infecting virus, the age and breed of the birds, route of
inoculation and presence or absence of neutralizing antibodies (Muller et al., 2003).
Sequential studies of tissues from orally infected chickens using immuno-fluorescence
detected the viral antigen in macrophages and lymphoid cells in the cecum at 4 hr PI and
in the lymphoid cells of duodenum and jejunum at 5 hr PI (Muller et al., 1979). The virus
reaches the liver at 5 hrs PI and enters the bloodstream from where it is distributed to
other organs; the bursal infection is followed by viremia. The virus persists in the bursa
of experimentally inoculated SPF chickens up to 3 weeks of age but the presence of
8
maternal antibodies in the commercial chicken decreases the duration of its existence in
bursa (Abdel-Alim and Saif, 2001a).
Various studies have shown that the variant and classic viruses exhibit similar pathology
but differ from each other with respect to their pathogenicity and immunogenicity
(Hassan et al., 1996). Variant viruses (Var A) were reported to induce bursal atrophy with
minimal or no immune response in contrast to the classic viruses (IM) which induce a
severe inflammatory response (Sharma et al., 1989). However, it was noticed
subsequently that variant viruses are not homogenous as a group as thought previously
(Hassan et al., 1996).
Host systems used to propagate the virus have a profound effect on the pathogenicity of
the virus isolates. Significant differences occurred in the pathogenicity and
immunogenicity of the virus propagated in BF or in the blue grates monkey-70 (BGM70) cells. However, the antigenicity of the viruses propagated in BF or the BGM-70 cells
were not significantly different (Hassan et al., 1996; Hassan and Saif, 1996). Some
strains of IBDV can adapt to CEF while others are refractory to grow in it. The SAL
strain was adapted and passaged successfully in CEF cells while IN strain was unable to
grow in CEF (Hassan et al., 1996; Hassan and Saif, 1996). The back passage of either IN
or SAL in SPF chickens maintained or increased the virulence of both viruses (Hassan et
al., 1996; Hassan and Saif, 1996). Wild type viruses from B lymphocytes of BF were
reported to be different than those grown in chicken embryo fibroblast (CEF).
Differentiating B lymphocytes in the BF provide the optimal micro-environment for
9
highly efficient virus replication; CEF and other cells seem to lack that environment
(Lange et al., 1987).
1.5 IMMUNOLOGY
The IBDV is ubiquitous in commercial chickens environment and chickens acquire the
infection orally or by inhalation. The virus is transferred from the gut to other tissues by
phagocytic cells like macrophages. In macrophages of the gut associated tissues it could
be detected as early as 4 hours after oral inoculation using immunofluorescence (Muller
et al., 1979). The virus then reaches the bursa via the blood where the most extensive
virus replication occurs. By 13 hours post-inoculation (PI) most follicles are positive for
virus and by 16 hours PI a second and pronounced viremia occurs accompanied by
secondary replication in other organs resulting in disease and death (van den Berg et al.,
2000).
The target organs for the virus are the IgM+ bearing B cells. During the acute phase of
the disease the bursa undergoes atrophy as the bursal follicles get depleted of B cells.
Virus replication causes extensive damage to lymphoid cells in medullary and cortical
regions of the follicle. Apoptosis of the neighboring B cells augments the destruction of
the bursal morphology. By this time an ample amount of viral antigen can be detected in
other organs (Granzow et al., 1997; Kim et al., 1999). Maternally derived antibodies
(MDA) protect chickens against subclinical disease and immunosuppression (Giambrone
and Clay, 1986). The MDA is known to protect the chickens for 3 weeks of age (Lasher
and Davis, 1997).
10
T cells are resistant to infection by IBDV(Hirai et al., 1979). During the acute phase of
the disease lesions appear in the thymus which are quickly overcome within a few days
(Sharma et al., 2000). A profound influx of T cells is reported in and around the site of
virus replication. The infiltrated T cells could be detected from one to twelfth weeks postinoculation, although the viral antigen disappears by the third weeks. The IBDV induced
cytotoxic T cell limit the spread of the virus by destroying the cells expressing the viral
antigen and thus can initiate the recovery process. At the same time IBDV-induced T
cells might enhance the viral lesions by producing inflammatory cytokines. T helper cells
produce inflammatory cytokines like IFN-γ which activates the macrophages to produce
nitric oxide (NO) (Sharma et al., 2000). Both humoral and cellular arms of the immune
system are compromised during the IBDV infection due to lysis of the B cells and altered
antigen-presenting cells.
The IBDV causes a transient inhibition of in vitro proliferative activity of T cells to
mitogens. The virus stimulates the macrophages to produce T cell cytokine like IFN-γ to
produce nitric oxide (NO) and other cytokines with anti-proliferative activity. IBD did
not affect natural killer cells levels in chickens (Sharma et al., 2000).The NO production
after IBD virus infection exerts antiviral effect since the immune-suppressed chickens
that failed to induce NO had more severe disease and higher degree of virus replication.
But it does not seem to correlate with the hemorrhagic lesions which result from the
reaction of host-factors and the determinants responsible for virus virulence and virus
clearance (Poonia and Charan, 2005).
11
The IBDV induced damage to humoral immunity is reversible. Antibody production
correlates with the morphologic restoration of the bursal follicles. Mitogenic response of
T cells returned to the normal levels. During the course of mitogenic inhibition, T cells of
infected chicken also failed to secrete IL-2 upon in vitro stimulation (Sharma and
Fredericksen, 1987).
The T cells play a significant role in the pathogenesis of IBDV. Intra bursal T cells and
T-cell-mediated responses play significant role in viral clearance and promoting recovery
from infection. They defend the host cell by reducing the viral burden but at the same
time produce inflammatory cytokines and nitric oxide inducing factor that enhance
tissues destruction and also delay the recovery process (Rautenschlein et al., 2002b).
Intrabursal T cells were activated by in vitro stimulation with IBDV. The activated cells
had increased surface expression of chicken MHC class II molecule, Ia and IL-2 receptor
CD25. In addition, these cells have an up regulated IFN-γ gene expression (Kim et al.,
2000). Splenocytes exposed to IBDV produced nitric oxide inducing factor (IFN-γ)
(Rautenschlein et al., 2002b). Intrabursal T cells inhibited the mitogenic response of
normal splenocytes by 90%. This bursal T cell-induced mitogen inhibition was found to
be dose-dependent and not MHC-restricted (Kim and Sharma, 2000). In contrast to the
bursal T cells, the splenocytes from IBDV exposed chickens did not have suppressive
activity. Mitogenic inhibition by bursal T cells is mediated by soluble factors, the nature
of which is still unknown (Rautenschlein et al., 2002b). Chickens that survive the disease,
clear the virus and recover from its pathologic effects (Sharma et al., 2000). It has been
shown that the more virulent the virus the stronger is the suppression of the humoral and
12
cell mediated immunity. Virulent virus also produced a detectable NO production in
serum.
Humoral immunity is the primary mechanism of the protective immune response.
Infection with IBDV results in the formation of antibodies to the group and serotype
specific antigens (Jackwood et al., 1985). Field exposure or vaccination results in VN
titers higher than 1:1000. But weak responses are obtained in chickens immunized with
purified viral polypeptides (Fahey et al., 1985), since viral protein conformation is
important in eliciting a high VN antibody response (Azad et al., 1987).
Antibody production is stimulated at the primary site of viral replication in gut associated
tissue and they can be detected as soon as 3 days PI. These antibodies prevent the spread
of the virus to other tissues. Due to the rapid onset of antibodies, the necrotic foci that
form in the bursa of fabricius stop expanding and are completely eliminated (Becht,
1980).
1.6 ROLE OF T CELLS IN THE IMMUNOPATHOGENESIS OF IBDV
Appearance of viral antigen in bursa is accompanied by an infiltration of T cells while
IgM+ cells undergo a precipitous decrease and the immunoglobulin level remains the
same (158). Infiltrating T cells were first detected at one day post-inoculation through
flowcytometry and were shown to persist up to 12 weeks (Sharma et al., 2000). The ratio
of CD4 and CD8 cells were the same during the seven days PI, but CD8 cells became
predominant afterwards (Kim et al., 2000).
The IBDV induced bursal T cells have increased surface expression of MHC-II and IL-2
receptors, with elevated expression of cytokine genes like IFN-γ and IL-6 (Sharma et al.,
13
2000). T cells from the bursa of the recovered bird proliferate when exposed in vitro to
purified IBDV. While the spleen cells from IBDV exposed-chicken produced nitric oxide
stimulating factor when stimulated in vitro with purified IBDV. Bursal T cells also
suppressed the mitogenic proliferation of the spleen from normal, virus free chicken
(Rautenschlein et al., 2002b).
T cell immunodeficiency can modulate pathogenicity of the virus since it has been shown
that the thymectomized birds have a higher viral burden in the bursa, with lower
inflammatory lesions in bursa. In addition, downregulated IFN-γ and IL-2 genes in bursal
cells, have a lower incidence of apoptotic bursal cells (Sharma et al., 2000) and undergo a
quick follicular recovery than T cell intact birds (Rautenschlein et al., 2002b).
1.7 EFFECT OF IBDV ON INNATE IMMUNITY
IBDV have been shown to modulate the macrophage function by altering the in vitro
phagocytic activity (162). Macrophages from the infected chicken have upregulated
cytokine gene expression and produce increased levels of NO (Kim et al., 1998). There
are reports that macrophages and monocytes may be susceptible to infection with the
virus (Burkhardt and Muller, 1987; Inoue et al., 1997; Kaufer and Weiss, 1976; Kaufer
and Weiss, 1980; Khatri et al., 2005; Komine et al., 1989; Palmquist et al., 2006).
1.8 IMMUNOSUPPRESSION
Immunosuppression caused by IBDV has a significant economic impact due to
widespread nature of the disease in commercial chickens. IBDV infection at an early age
compromises the humoral and local immune responses of chickens. Allan made the
earliest observation about the immunosuppressive potential of IBDV (Allan et al., 1972).
14
The extent of the immunosuppressive effect is related to the age at infection. The most
pronounced damage results if the infection occurs within the first 2-3 weeks of hatch
(Allan et al., 1972). Chickens less than three weeks of age do not exhibit clinical signs
but are immunosuppressed (Saif, 1991). Following the ingestion of the virus, lymphoid
cells and macrophages in the intestine are infected and carry the virus to the bursa of
fabricius (BF) (Muller et al., 1979). Clinical signs and lesions of IBDV appear shortly
thereafter. Chickens infected with IBDV are more susceptible to various other infections.
Chickens exposed to IBDV at 1 day of age had lower antibody responses and were more
prone to infection with Newcastle disease virus (NDV) (Faragher et al., 1972). The
infected chicken had a decreased humoral antibody response to vaccines as well (Hirai et
al., 1974). Immunosuppression resulted in lower flock performance, more secondary
infections, poor feed conversion, less protective response to vaccines and higher rate of
carcass condemnation at the processing level (Sharma et al., 2000).
The immunosuppressive effects of IBDV are dependent on the strain of the virus (Craft
et al., 1990; Higashihara et al., 1991; Mazariegos et al., 1990; Sharma et al., 1989).
Chicken infected with IBDV at an earlier age succumbed to other infections like
inclusion body hepatitis (Bacon et al., 1986), reovirus (Montgomery and Maslin, 1991),
coccidiosis (Anderson et al., 1977), Marek’s disease (Sharma, 1984), hemorrhagicaplastic anemia and gangrenous dermatitis (Rosenberger et al., 1975), infectious
laryngotracheitis (Rosenberger and Gelb, 1978), infectious bronchitis (Pejkovski et al.,
1979), chicken anemia agent, salmonellosis, Escherichia coli, colibacillosis Mycoplasma
synoviae (Giambrone et al., 1977b) and Eimeria tenella (Anderson et al., 1977;
15
Giambrone et al., 1977a). Peripheral blood lymphocytes (PBL) from chickens infected
with IBDV have depressed proliferative responses to stimulation with the mitogens
concanavalin A or phorbol myristate acetate (Rautenschlein et al., 2002b; Sharma et al.,
2000) .
1.9 EFFECTS OF VIRUS ON HUMORAL IMMUNITY
The IBDV has a predilection for the immature (Sivanandan and Maheswaran, 1980a)
actively dividing B lymphocytes and causes lytic infection of IgM bearing B cells
+
resulting in the decrease in circulating IgM cells. Infected chicken produce less level of
antibodies against the antigen (Kim et al., 1999). Only primary antibody responses are
affected and secondary responses remain unaltered (Rodenberg et al., 1994; Sharma et
al., 1989). IBDV induced humoral deficiency is reversible and overlaps with the
restoration of bursal morphology (Sharma et al., 1989; Sharma et al., 2000).
Chickens infected with IBDV at 1 day of age were found to be completely deficient in
serum immunoglobulin G and produced only a monomeric immunoglobulin M (IgM)
(Ivanyi, 1975; Ivanyi and Morris, 1976). The IgG levels varied depending on the age at
the time of infection (Hirai et al., 1979). The number of B cells in peripheral blood was
reduced after infection with IBDV, but T cells were not appreciably affected (Hirai et al.,
1979; Sivanandan and Maheswaran, 1980b). The adverse effect on antibody responses is
due to the damage to B cells in the bursa and in blood. Since the virus has a predilection
for actively dividing B cells as compared to the mature B cells (Sivanandan and
Maheswaran, 1980a).
16
1.10 EFFECT OF VIRUS ON CELLULAR IMMUNITY
The extent of which cellular immune response is affected is not well understood.
However, it is known that the effect of IBDV on CMI is transient and less pronounced
than the effect on humoral response. Infected chickens show a poor cellular response to
certain antigens and show increased susceptibility of disease that are under the control of
cellular immune defense (Anderson et al., 1977). The thymic lesions were transient and
appeared within the first weeks of infection, peaked at 3-4 days PI and then subsided. The
presence of thymic lesions were not associated with active viral replication of the virus in
the thymic cells as shown by the immunoflourescence (IF) and antigen capture ELISA. In
addition, T cells from infected chickens during the early stages of virus infection fail to
respond optimally to mitogens in vitro (Confer et al., 1981).
Maximum depression in the cellular immunity was shown to occur at 6 weeks postinfection by using the lymphoblast transformation assay. The reason for the delay in this
response is not clear considering that the virus persists in the host for approximately 3
weeks. It was speculated that this depression is the overall depression of T-cell function
during the virus infection (Sivanandan and Maheswaran, 1981). The effect of IBDV on
two CMI functions i.e. natural killer cell cytotoxicity and mitogenic response had been
studied. It was reported that IBDV had an inconsistent effect on the natural killer cell
cytotoxicity but caused a transient early depression of the blastogenic response of spleen
cells to phytohemaglutinin (Sharma and Lee, 1983). In vitro mitogen hyporesponsiveness of T cells is mediated by the suppressor cells in the spleens of the infected
17
chicken; the mechanisms of reduced in vivo cellular immunocompetence are not known
(Lam, 1991).
The IBDV infected chickens were shown to have a normal natural killer cell population,
mononuclear phagocytic activity and delayed-type hyper sensitivity reaction (Granzow et
al., 1997; Hudson et al., 1975). Neither did virus infection alter the normal proportions of
CD4 and CD8 subsets of T cells in the circulation and spleen (Sharma et al., 1993).
It was reported that variant A strain of IBD had a significantly higher effect on CMI as
compared to the standard Edgar strain when given to 1-day-old chicken which lingered
on up to 5 weeks. A similar effect was reported in chicken infected at 3 weeks of age
(Craft et al., 1990).
Harderian gland an, another component of immune system associated with the local
immune system of the respiratory tract. IBDV infection of 1-5 day-old chickens produced
a dramatic decrease in plasma cell content of the harderian gland that lasted up to 7
weeks (Dohms et al., 1981). Broilers infected with IBDV at 3 weeks of age had reduced
antibody titers to Brucella abortus (T cell independent antigen) and sheep red blood cells
(SRBC, a T cell dependent antigen) in extracts from harderian gland and serum.
Decreasing antibody responses to B. abortus were evident at a later time as compared to
SRBC antibody response (Dohms et al., 1981).
1.11 MECHANISMS OF IMMUNOSUPPRESSION
Reduction in the number of B cells in the BF due to viral infection is the major cause of
immunosuppression. Suppression of B cell function might be caused by damage to
helper T cells or other cells involved in generating the immune responses (Sharma et al.,
18
1989). Chickens infected with IBDV have suppressor cells in the spleen, which cause in
vitro mitogenic hypo responsiveness to concavalin A. These cells prevent normal spleen
cells from responding to the mitogen (Sharma and Fredericksen, 1987). The impairment
of T cells and development of suppressor cells (Sharma and Fredericksen, 1987) was
demonstrated in vitro by using proliferation tests (Confer and MacWilliams, 1982;
Confer et al., 1981; Sharma and Lee, 1983).
1.12 APOPTOSIS
Apoptosis or the programmed cell death plays a major role in the immuno-pathology of
IBDV characterized by destruction of cells of BF (Becht and Muller, 1991; Burkhardt
and Muller, 1987; Kaufer and Weiss, 1980). Only 20% of the lymphoid cells in the BF
contain replicating IBDV. The severe damage to the bursa can be ascribed to apoptosis
(Burkhardt and Muller, 1987). In addition to necrosis, marked atrophy of the BF occurs
without eliciting an inflammatory response that is a characteristic sign of the apoptotic
process. Replication of the virus in BF results in secondary viremia thus spreading the
virus to other tissues.
It has been suggested that early post-infection, the cells containing the viral antigen are
protected from apoptosis to ensure viral replication. Anti- viral mechanisms kick in and
destroy the neighboring cells to prevent the spread of the virus. During the late infection,
the infected cells undergo apoptosis thus seeding the virus to other cells. The IBDV
infection of a susceptible chicken has been shown to induce apoptosis in the bursa as well
as thymus (Inoue et al., 1994; Lam, 1997; Ojeda et al., 1997; Tanimura and Sharma,
1997; Tanimura and Sharma, 1998; Vasconcelos and Lam, 1995).
19
Morphological and biochemical features of apoptosis were also observed after in vitro
infection of IBDV in chicken peripheral blood lymphocytes (Vasconcelos and Lam,
1994) and chicken embryo fibroblasts (Tham and Moon, 1996). Apoptosis occurs in
lymphocytes of various organs like thymus (Inoue et al., 1994), bursa and spleen (Lam,
1997). Some researchers believed that apoptosis induced by IBDV in cell cultures
following in vitro infection was an early genetic response of the host cells and was
independent of virus replication while others showed that appearance of CPE coincided
with virus replication (Jungmann et al., 2001; Tham and Moon, 1996). (Jungmann et al.,
2001), however, showed that the proportion of apoptotic cells increased from 5.8 % at 4
hrs PI to 64.5% at 48 hrs PI in chicken embryo cells after infection with IBDV strain Cu1. However, treatment of CE cell cultures with UV inactivated IBDV did not induce
apoptosis (Jungmann et al., 2001).
Whether apoptosis is triggered via virus receptor activation is not yet known. Double
labeling technique revealed that during the course of early infection maximum number of
antigen- expressing cells were not apoptotic. It was only later in the infection that the
double-labeled cells appeared. Double labeling technique determined the distribution of
both apoptotic cells and cells containing viral antigen in the same section of BF.
Double labeling studies for apoptotic or antigen positive cells revealed that apoptosis in
bursa occurs both in IBDV positive and IBDV negative cells (Tanimura and Sharma,
1998) whereas apoptosis in the thymus occurs in the antigen negative cells only
(Tanimura and Sharma, 1998). It was concluded that IBDV induced apoptosis indirectly
in non-bursal organs. It has been postulated that IBDV impairs the withdrawal of
20
apoptotic cells and therefore results in the increased number of the apoptotic cells (Ojeda
et al., 1997). Apoptotic cells were located mostly in an area between the cortex and
medulla whereas majority of cells positive for viral antigens were found in medulla.
Indirect mechanisms might also be involved in the induction of apoptosis and could have
induced apoptosis in vivo resulting in rapid depletion of cells in BF. In the infected
follicles large numbers of cells were apoptotic but very few contained the viral antigen
(Jungmann et al., 2001; Tanimura and Sharma, 1998). Interferon production occurs after
IBDV infection and is thought to be the major apoptosis-inducing factor in the
neighboring cells along with TNF-α (Jungmann et al., 2001). Viral proteins VP2 and VP5
have been implicated to play a role in apoptosis. The VP2 induced apoptosis in
mammalian cells but not in CE cells (Fernandez-Arias et al., 1997). The VP5 deletion
mutant of IBDV induced lesser degree of apoptosis in infected CE cells and replicated
slower than the parental strain (Yao et al., 1998).
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31
CHAPTER 2
PERSISTENCE AND TISSUE DISTRIBUTION OF INFECTIOUS
BURSAL DISEASE VIRUS IN SPF AND COMMERCIAL BROILER
CHICKENS
2.1 SUMMARY
Infectious bursal disease virus (IBDV) is an important poultry pathogen that primarily
infects young chickens and leads to severe immunosuppression. There have been
questions regarding the persistence of IBDV in processed meat destined for export. This
study was initiated to determine the persistence, distribution and quantification of IBDV
strains in lymphoid and non-lymphoid tissues of specific pathogen free chickens (SPF)
and commercial broiler chickens. Five separate experiments were conducted using 2 and
4 weeks old SPF chickens and 2 weeks old commercial broilers having maternally
derived antibodies. Four different types of lymphoid and seven different non-lymphoid
tissues were sampled from individual chickens in each experiment. Viral RNA (vRNA)
30
was extracted from tissues and subjected to real time RT-PCR. In lymphoid tissues the
virus strains were detected for the longest duration in bursal tissues followed by spleen,
thymus and bone marrow. In non-lymphoid tissues both of the strains were detected the
longest in caecum followed by liver, kidney, pancreas, lungs, thigh muscles and breast
muscles. The STC strain, a serotype 1 classic virus, vRNA was detected in bursa of SPF
chickens up to 8 weeks post-inoculation (PI) and in commercial broiler chickens it was
detected up to 6 weeks PI. In addition, we were able to isolate the STC strain up to 4 and
3 weeks PI from the bursal homogenates of SPF and commercial chickens respectively.
The IN strain, vRNA was detected up to 5 weeks PI in SPF and up to 3 weeks PI from
commercial chickens in bursal tissue. The IN strain was isolated up to 10 days postinoculation (DPI) from bursal tissues collected from both SPF and commercial chickens.
This is the first detailed report on the persistence and distribution of classic and variant
strains of IBDV in different tissues of SPF and commercial chickens. The study indicated
that the persistence and distribution of IBDV in non-lymphoid tissues, specifically, thigh
muscles and breast muscles is significantly different between SPF and commercial
chickens. In commercial broilers the STC was detected at DPI 3 in thigh muscles and
only one breast muscle sample was RT-PCR positive at DPI 14. IN strain was not
detected in thigh muscles while it was detected in breast muscles up to two weeks PI in
commercial chickens. Whereas in SPF chickens STC and IN strains, vRNA were detected
in thigh muscles up to 4 and 2 weeks PI and in breast muscles up to 3 and 2 weeks PI,
respectively. Although the virus can persist in SPF chicken bursa for 4 weeks, it is very
31
unlikely that the infectious virus will be present in the processed meat. In addition, the
RT-PCR results are not sufficient to indicate the presence of the infectious virus.
2.2 INTRODUCTION
Infectious bursal disease (IBD) is one the most important chicken diseases that affects
young chickens around the world. IBD virus is an icosahedral, bisegmented and
polyploid virus. Its characteristics of being polyploid and having bisegmented genome
continue to present us with new challenges as it can genetically mutate to avoid vaccine
immunity and can also reassort its genome to become more virulent virus (Luque et al.,
2009).
The IBD is considered ―communicable disease‖ and is of socio-economic importance
within countries and important in the international trade of poultry products (Australia,
2001 ). Presence of IBDV in chicken meat is questioned in international trade of poultry
products. IBDV being a non zoonotic pathogen is ―not a human food safety issue,
nevertheless movement of birds with IBDV infections is cause for concern because the
introduction of new antigenic and pathogenic strains into a geographic area can have a
negative economic impact on the chickens grown in that region. Contaminating trucks,
processing plant personnel and equipment servicing multiple poultry farms can lead to an
increased risk of spreading IBDV‖ (Jackwood and Sommer-Wagner, 2010).
Following an infection, the persistence of IBDV in chickens has been reported and the
results were dependent on the type of test used. Using molecular assays, the RNA of
IBDV was detected in bursa tissue at 24 days post-inoculation (Henderson and Jackwood,
1990). In a later study, viral RNA was detected by RT-PCR up to 28 DPI in bursa tissue
32
but using embryonated eggs and specific-pathogen-free chickens, infectious virus was
only detected for 7–14 days post-inoculation in the bursa tissue (Abdel-Alim and Saif,
2001a). In that study, the persistence of the virus was dependent on the titer of the
inoculums and quantity of maternal antibodies. In another study, it was documented that
variant IBDV was detected in virus-challenged commercial broilers for up to 6 weeks and
infectious virus capable of replication could be isolated from all organs at 4 weeks PI
(Elankumaran et al., 2002).
Control strategies for IBD include vaccination of broiler breeder flocks and the resulting
transfer of maternal immunity that protect their progeny. When this strategy is successful,
maternal immunity should prevent IBDV infection for several weeks following hatch
(Al-Natour et al., 2004; Wyeth and Cullen, 1978). This is an effective control strategy
because it protects the young chick’s immune system from being permanently
compromised (Saif, 2004). In regions where IBDV is endemic, broiler chicks will
become infected with the virus as maternal immunity wanes (Wyeth et al., 1981). During
a study of the distribution of IBDV in the United States it was typically observed that
IBDV infects the broilers at 3–6 weeks of age (Jackwood and Sommer-Wagner, 2005).
Most of the studies conducted on the persistence of IBDV in tissues were on specific
pathogen free (SPF) chickens. Such data does not reflect the situation in young
commercial broiler chickens which usually have maternal antibodies. Hence to
understand the pathogenesis and epidemiology of IBDV it was necessary to undertake
this comprehensive study. The goal of this study was to determine persistence and
distribution of IBDV in chicken tissues.
33
2.3 MATERIALS AND METHODS
Chicken and embryonated eggs: One day old commercial broiler chicks (in ovo
vaccinated used in experiment 3 and 4 and commercial broiler chicks having maternally
derived antibodies used in experiment 5) were purchased from a local hatchery. The
specific pathogen free (SPF) chicken eggs (Charles River Laboratories Inc; Wilmington,
MA, USA) were incubated in our facility at Ohio Agriculture Research and Development
Center, The Ohio State University. The hatched chickens were kept at a disease
containment building that had rooms supplied with HEPA filtered intake and exhaust air.
At two weeks of age (experiment 1, 3, and 5) and 4 -weeks of age (experiment 2 and 4)
the birds were transferred to hard sided isolators supplied with HEPA filtered intake and
exhaust air.
Viruses: A serotype 1 classical IBDV (cIBDV), standard challenge (STC) strain
(Jackwood and Saif, 1987) and a serotype 1 variant IBDV (vIBDV) strain IN (Ismail et
al., 1990) isolated in our laboratory were used for challenge studies. Each virus strain was
propagated separately in two weeks old SPF chickens. Bursas were harvested from
inoculated birds at 5 DPI, homogenized aseptically, and 10 % W/V suspension was made
in minimal essential medium (MEM). The bursal homogenates were titrated in 10 day old
SPF chicken embryonated eggs via chorioallantioc membrane (CAM) route as described
earlier (Abdel-Alim and Saif, 2001a). The titer was expressed as mean embryo infectious
dose50 (EID50) per milliliter (ml) on the basis of lesions and mortality and was calculated
by the method of Reed and Muench (Reed and Muench, 1983). The bursal homogenates
were filtered and stored at -70 OC for later use.
34
Experimental Design: Five separate experiments were conducted. Each experiment
included three groups. Group A was challenged, intraocularly with STC strain, group B
was challenged intraocularly with IN strain of IBDV and groups C was kept as negative
control. SPF chickens were used in experiments 1 and 2. In ovo vaccinated commercial
broiler chickens were used in experiment 3 and 4. In experiment 5 commercial chickens
having maternally derived antibodies were used.
In experiment 1, 81 two weeks old SPF chickens were allocated equally into three
groups. Group A, chickens were challenged each with 104EID50 of STC strain. Groups B,
chickens were challenged each with 104EID50 of IN strain and group C 27 chickens
served as virus free control. The lymphoid tissues (thymus, bursa, spleen and bone
marrow) and non-lymphoid tissues (thigh muscles, breast muscles, pancreas, kidney,
caecum, liver and lungs) were collected at the following time point: 3, 5, 7, 10, 14, 21,
28, 35 and 42 DPI. Three chickens per group were euthanized at each designated time
points. Each tissue (approximately 2 grams in weight) was collected aseptically using
separate sterile surgical scissors and forceps. The same procedure as mentioned for
experiment 1 was used for experiment 2, 3, 4, and 5 with the following modifications. In
the second experiment the SPF chickens were inoculated at 4 weeks of age and the
samples were collected at DPI 7, 14, 21, 28, 35, 42, 49, and 56.
Experiments 3 and 4 were conducted on in ovo vaccinated commercial broilers whereas
in experiment 5 commercial broiler having maternally derived antibodies were used. The
sampling procedure for experiment 3, 4 and 5 was similar to that of experiment 2.
35
Tissues processing: Each tissue was suspended in sterile phosphate buffered saline
(PBS) in 14 ml sterile tube (Fisher scientific, USA), properly labeled for every tissue.
The tissues were homogenized separately using Labgen homogenizer (Cole-Parmer,
USA) supplied with sterile disposable plastic probes to rule out any sample to sample
carry over cross contamination. After homogenization the tissues were frozen and thawed
three times and stored at -70
O
C. The homogenized tissues were subjected to
centrifugation at 4000 RPM (Allegra X- 15 R centrifuge Beckman Coulter) for 20
minutes at 4 OC. The clarified supernatant from each sample was collected and used for
virus isolation and viral RNA extraction.
Viral RNA extraction and Real-time RT PCR: The processed tissue samples were
subjected to viral RNA extraction by using the Mag MAXTM magnetic beads processor
and Mag MAXTM viral RNA extraction kit as per manufacturer’s instructions (Applied
Biosystems). Real- time RT-PCR was performed on all the collected tissues, using
published strain specific primers and probe sets (Peters et al., 2005). Real-Time RT-PCR
was performed using 7500 ABI RT-PCR system on 96 well plates. One step RT-PCR
AgPath-IDTM kit (Applied Biosystems) was used for the RT-PCR reaction following the
manufacturer’s instructions. The reaction profile used was: 45 oC for 10 minutes, 95 oC
for 10 minutes for the RT step and 40 cycles of 95 oC for 15 seconds, 60 oC for 45
seconds for the PCR step (Rauf et al., 2011).
Standard Curve generation: Serial 10 fold dilutions of RNA of known viral titer for
STC and IN strains were made and standard curves were generated. Viral RNA load in
36
each tissue (approximately 2 grams in weight) was then estimated based on the standard
curves developed (Rauf et al., 2011).
Virus Isolation: All RT-PCR positive bursal tissues were subjected to virus isolation
using ten day old SPF embryonated chicken eggs. Additionally RT-PCR positive bursal
homogenates were inoculated into 2 weeks old SPF chickens to further confirm the
presence of infectious virus.
ELISA procedure: In each experiment blood samples were collected from chickens
before inoculation and at each sampling point after inoculation with virus. Commercially
available IBDV ELISA kit (IDEXX laboratories) was used. The sera were diluted,
ELISA conducted and titers calculated in accordance with the manufacturer’s
recommendations. Geometric mean titer (GMT) was calculated as previously published
(Ashraf et al., 2006).
Statistical analysis: Statistical analysis: Data was analyzed by STATA using
multivariate analysis and generalized linear model.
2.4 RESULTS
Persistence and tissue distribution of STC and IN strains in SPF chickens:
In experiment 1, the STC vRNA was detected in bursal tissues up to 42 DPI and in
spleen, thymus and bone marrow up to 35 DPI. The IN strain vRNA was detected up to
35 DPI in bursal tissues, up to 28 DPI in spleen and up to 14 DPI in thymus and bone
marrow (Table; 2.1). In non-lymphoid tissues the STC vRNA was detected in thigh
muscles and lungs up to 28 DPI and in breast muscles, pancreas, kidneys, caecum and
37
liver up to 35 DPI. The IN strain vRNA was detected in caecum up to 28 DPI, in thigh
muscles, breast muscle, pancreas, kidneys, and liver up to 14 DPI. The IN strain vRNA
was detected in lungs up to 7 DPI (Table; 2.2-3).
In experiment 2, the STC strain vRNA was detected in bursas and spleens up to 56 DPI,
in bone marrow up to 49 DPI and in thymus up to 35 DPI. The IN strain vRNA was
detected up to 28 DPI in bursal tissues, up to 21 DPI in spleens, up to 7 DPI in bone
marrow and was not detected in thymus (Table; 2.4). In non-lymphoid tissues the STC
strain vRNA was detected in thigh muscles and breast muscles up to 28 DPI, in pancreas
up to 35 DPI, in kidneys, caecum and liver up to 42 DPI. The IN strain vRNA was
detected in caecum up to 21 DPI, in pancreas and liver up to 7 DPI. The IN strain vRNA
was not detected in breast muscle, lungs and kidneys. The IN strain vRNA was detected
in thigh muscles samples at DPI 28 and 35 (Table; 2.5-6).
Persistence and tissue distribution of STC and IN strains in ovo vaccinated
commercial broilers:
In experiment 3, the STC vRNA was detected up to 35 DPI in bursa and spleen, in
thymus up to 5 DPI and in bone marrow up to 28 DPI. The IN strain vRNA was detected
up to 28 DPI in bursa, 21 DPI in spleen and 7 DPI in thymus, while it was not detected in
bone marrow (Table; 2.7). In non-lymphoid tissues STC strain vRNA was detected in
caecum and liver up to 35 DPI, in thigh muscles and lungs up to 14 DPI, pancreas and
kidneys up to 10 DPI and in breast muscles was not detected. The IN strain vRNA was
not detected in breast muscles pancreas and lungs at any DPI. The IN strain vRNA was
38
detected in caecum up to 21 DPI, and in thigh muscle and liver it was detectable up to 3
DPI (Table; 2.8-9).
In experiment 4 the STC strain vRNA was detected up to 42 DPI in bursa and thymus,
and up to 28 DPI in spleen and bone marrow (Table; 2.10). The IN strain vRNA was
detected up to 42 DPI in bursa, 35 DPI in thymus and 21 DPI in spleen, while it was not
detected in bone marrow. In non-lymphoid tissues STC strain vRNA was detected in
thigh muscles, caecum and pancreas up to 35 DPI, in kidney, lungs and liver up to 28 DPI
and in breast muscles up to DPI 7. The IN strain, vRNA, was not detected in thigh and
breast muscles. The IN strain, vRNA was detected in pancreas, kidney and caecum up to
DPI 28, and in liver it was detected only at DPI 3 (Table; 2.11-12).
Persistence and tissue distribution of STC and IN strains in commercial broilers:
In experiment 5 commercial broilers having maternally derived antibodies were
inoculated with the two strains (STC and IN). The STC strain vRNA was detected up to
42 DPI in bursa; in spleen it was detected up to 14 DPI and in thymus and bone marrow
up to 7 DPI. The IN strain vRNA was detected up to 21 DPI in bursa, 7 DPI in spleen and
3 DPI in thymus and bone marrow (Table; 2.13). In non-lymphoid tissues STC strain
vRNA was detected in caecum up to 21DPI, pancreas, kidneys, lungs and liver up to 7
DPI. In thigh muscles it was detected up to DPI 3 and in breast muscles only one sample
was detected positive at DPI 14. The IN strain vRNA was not detected in thigh muscles
and was detected in breast muscles, pancreas, kidneys, caecum, lungs, liver up to DPI 7
(Table; 2.14).
39
Virus Isolation: Persistence of infectious virus was confirmed by inoculating the all RTPCR positive, bursal homogenate from SPF and commercial chickens (having maternally
derived antibodies) in to chickens embryos. The STC virus was isolated up to 4 and 3
weeks PI from SPF and commercial chickens respectively. The IN strain was isolated up
to 10 DPI from both SPF and commercial chickens (Figures; 2.2 A-D).
ELISA:
The SPF chickens were negative for antibodies before inoculation and IBDV specific
antibodies were detected post-inoculation in STC and IN inoculated groups (Fig; 3.4AB). No antibodies were detected in virus free control SPF chickens during the
experimental period. The antibodies titers were measured, in commercial broiler (having
maternally derived antibodies and in ovo vaccinated chickens), data not shown. It was
observed that at one day of age the antibody titer were high and at 2 weeks of age (at the
time of inoculation) the antibodies titers declined to approximately 1/2 of the titers
recorded at one day of age, data not shown.
In brief, the overall persistence, distribution and vRNA load (approximately 2 grams/
tissue) was significantly different between SPF and commercial broiler chickens (Fig;
2.3). While comparing the persistence, distribution and vRNA load of the two strain used,
the STC strain was significantly different than the IN strain (Fig; 2.1).
2.5 DISCUSSION
This is the first comprehensive report on the persistence, distribution and quantification
of viral RNA concentration of IBDV in SPF and commercial broiler chickens. We found
that the classical strain STC, vRNA was detected longer than previously documented for
40
SPF and commercial broiler chickens. For both viral strains the highest vRNA
concentration was found in the bursa followed by the caecum spleen, liver, kidney,
pancreas, thymus, lungs, bone marrow, thigh muscles and breast muscles. This finding is
particularly important in understanding IBDV pathogenesis. The persistence and
distribution of IBDV strains was significantly different between SPF and commercial
broiler chickens. Attempts were made to verify whether the RT-PCR positive results
were indicative of the presence of infectious virus or viral RNA. The RT-PCR positive
bursal homogenates (from SPF and commercial broilers having maternally derived
antibodies) were inoculated to chickens embryos. The STC strain was isolated up to 28,
DPI and 21 DPI from SPF and commercial chickens respectively. Whereas IN strain was
isolated up to DPI 10, either from SPF or commercial chickens.
When we inoculated 2 groups of commercial chickens (one group having maternal
derived antibodies and the other was in ovo vaccinated) and compared them with SPF
chickens (experiment 1 and 2), there was no significant difference in the persistence of
virus in bursal tissues between the SPF and commercial chickens. Significant differences
were determined in the persistence of virus/viral RNA in non-lymphoid tissues of SPF
and commercial chickens. Previously in our laboratory(Abdel-Alim and Saif, 2001a),
SPF chickens were inoculated with IBDV at 3 weeks of age, the viral RNA was detected
by RT/PCR until 21 DPI, but attempts to isolate infectious virus from bursal
homogenates failed. Infectious virus was detected by embryo inoculation up to 7 DPI in
the bursa of SPF chickens inoculated at 2 or 3 weeks of age, whereas the viral RNA was
detected by RT/PCR for up to DPI 28. In SPF chickens inoculated at 1 day of age, the
41
bursa-derived virus or its RNA was detected at DPI 7 and 14, when inoculated at a high
4
2.5
dose, 10 EID50/bird or at a low dose, 10 EID50/bird. In commercial 1-day-old broiler
chickens, the bursa-derived virus was detected at 7 and 14 days PI when inoculated at a
4
high dose, 10 EID50/bird, whereas the virus was detected only at 14 days PI when
inoculated at a low dose, 102.5 EID50/bird. In SPF and commercial chickens, vaccinated
with a modified live IBDV vaccine, the virus is known to persist in the bursa of SPF
chickens for 3 weeks but maternal antibodies in the commercial chickens readily
eliminate it from the bursa since no live vaccine virus nor its RNA were detected in
commercial broilers vaccinated at 1 day or 2 weeks of age (Abdel-Alim and Saif, 2001a).
Previously Elankumaran et al., 2002, reported that a variant strain of IBDV was detected
in virus-challenged commercial broilers for up to 6 weeks and infectious virus was reisolated from all tissues up to 4 weeks PI (Elankumaran et al., 2002).
It is surprising to detect the viral RNA in SPF chickens up to DPI 56 and virus capable of
replication could only be isolated from the bursal homogenates of the same experiment
only up to DPI 28. It is possible that some of the RT-PCR positive samples only
contained viral RNA and no infectious virus (Jackwood and Sommer-Wagner, 2010). It
might be possible that virus persist in very low amount in the bursal cells that can be
detected by RT-PCR but is not enough to establish successful infection when inoculated
into chicken embryos or chickens.
In a study it was shown that IBDV persistently infect the chicken B-lymphoid DT40
cells, a tumor cell line derived from the bursa of fabricius of a chicken infected with
avian leukosis virus. Establishment of the persistent infection is associated with an
42
extensive remodeling of the hypervariable region of the VP2 capsid polypeptide,
accumulating 14 amino acid changes during the first 60 days of the persistent infection.
The amino acid sequence of the non-structural VP5 polypeptide, involved in virus
dissemination, is not altered during the persistent infection (Delgui et al., 2009). There
are reports that macrophages and monocytes can be infected with the virus (Kaufer and
Weiss, 1976, 1980; Burkhardt and Muller, 1987; Komine et al., 1989; Inoue et al., 1992;
Lam, 1998; Khatri et al., 2005; Palmquist et al., 2006). Chicken surviving IBDV
infections are immunosuppressed despite repopulation of the bursa with B cells. It was
shown that infection of neonatal chicks with a classical virulent IBDV strain (F52/70)
causes severe bursal B cell depletion with recovery after about one week (Withers et al.,
2005). In that study it was observed that two types of bursal follicles developed: normal
large reconstituted follicles and small poorly developed follicles lacking a discernible
cortex and medulla. It was demonstrated that the presence of large numbers of
undifferentiated follicles was associated with inability to mount antibody responses to
IBDV itself and after immunization with Salmonella enteritidis bacterin, indicating that B
cells in these follicles are unable to produce peripheral B-cells with an effective
immunoglobulin repertoire (Withers et al., 2005). Additionally, a number of
inflammatory foci were observed in the recovering bursa. These foci contained few B
cells at the margins, but large numbers of CD4+ and CD8+ cells, scattered T-cells and
macrophages, and small central aggregates of dendritic like cells expressing the CD40
antigen (Withers et al., 2005). One might speculate the possibility that some of these cells
may contain small amount of virus or viral antigen that can be detected by RT-PCR. It
43
will be interesting to further explore the possibility of the persistent infection of IBDV in
chickens.
Based on the findings in our persistence experiments we conclude that, thigh muscles and
breast muscle were the least positive organs for virus/vRNA persistence. Hence we
speculate that the persistence of IBDV in commercial chicken meat (breast and thigh
muscles) is unlikely to be a major concern for international trade. The persistence and
distribution of IBDV RNA in chickens depends on the presence of maternally derived
antibodies. The presence of vRNA is not indicative of the presence of the infectious
virus as indicated by virus isolation; hence virus isolation has to be performed to prove
the presence of infectious virus. In addition, one has to be cautious in interpreting the
results from experimental infection. We have shown earlier (Abdel-Alim and Saif, 2001a;
Al-Natour et al., 2004), that inoculums and antibody titers are important determinants of
virus persistence. Hence, results from experimental infections are at best a guide but not
necessarily represents the field conditions.
2.6 ACKNOWLEDGEMENTS
We acknowledge the salaries and research support provided by state and federal funds
appropriated to the Ohio Agriculture Research and Development Center, The Ohio State
University. We are grateful to Dr. Juliette Hanson, Gregory Myers and Kingsly Berlin for
their help in animal work. We acknowledge Dr. Alexander Rodriguez-Palaci for
statistical analysis of the data.
44
2.7 REFERENCES
Abdel-Alim, G.A. and Saif, Y.M. (2001) Detection and persistence of infectious bursal
disease virus in specific-pathogen-free and commercial broiler chickens. Avian
Dis 45(3), 646-54.
Al-Natour, M.Q., Ward, L.A., Saif, Y.M., Stewart-Brown, B. and Keck, L.D. (2004)
Effect of diffferent levels of maternally derived antibodies on protection against
infectious bursal disease virus. Avian Dis 48(1), 177-82.
Anon. (1999) International Animal Health Code: mammals, birds and bees. Office
International des Epizooties; 8th ed. Office International des Epizooties 8th ed.
Ashraf, S., Abdel-Alim, G. and Saif, Y.M. (2006) Detection of antibodies against
serotypes 1 and 2 infectious bursal disease virus by commercial ELISA kits.
Avian Dis 50(1), 104-9.
Australia. (2001. ) Generic import risk analysis (IRA) for uncooked chicken meat. IRA
Report, 35.
Delgui, L., Gonzalez, D. and Rodriguez, J.F. (2009) Infectious bursal disease virus
persistently infects bursal B-lymphoid DT40 cells. J Gen Virol 90(Pt 5), 1148-52.
Elankumaran, S., Heckert, R.A. and Moura, L. (2002) Pathogenesis and tissue
distribution of a variant strain of infectious bursal disease virus in commercial
broiler chickens. Avian Dis 46(1), 169-76.
Henderson, K.S. and Jackwood, D.J. (1990) Comparison of the dot blot hybridization
assay with antigen detection assays for the diagnosis of infectious bursal disease
virus infections. Avian Dis 34(3), 744-8.
Jackwood, D.J. and Sommer-Wagner, S.E. (2005) Molecular epidemiology of infectious
bursal disease viruses: distribution and genetic analysis of newly emerging viruses
in the United States. Avian Dis 49(2), 220-6.
Jackwood, D.J. and Sommer-Wagner, S.E. (2010) Detection and characterization of
infectious bursal disease viruses in broilers at processing. Prev Vet Med 97(1),
45-50.
Luque, D., Rivas, G., Alfonso, C., Carrascosa, J.L., Rodriguez, J.F. and Caston, J.R.
(2009) Infectious bursal disease virus is an icosahedral polyploid dsRNA virus.
Proc Natl Acad Sci U S A 106(7), 2148-52.
45
Peters, M.A., Lin, T.L. and Wu, C.C. (2005) Real-time RT-PCR differentiation and
quantitation of infectious bursal disease virus strains using dual-labeled
fluorescent probes. J Virol Methods 127(1), 87-95.
Rauf, A., Khatri, M., Murgia, M.V. and Saif, Y.M. (2011) Expression of perforingranzyme pathway genes in the bursa of infectious bursal disease virus-infected
chickens. Dev Comp Immunol.
Reed, L.J. and Muench, H.A. (1983) A simple method of estimation fifty per cent endpoint. . Am. J. Hyg 27, 493-497.
Saif, Y.M. (2004) Control of infectious bursal disease virus by vaccination. Dev Biol
(Basel) 119, 143-6.
Wyeth, P.J. and Cullen, G.A. (1978) Susceptibility of chicks to infectious bursal disease
(IBD) following vaccination of their parents with live IBD vaccine. Vet Rec
103(13), 281-2.
Wyeth, P.J., O'Brien, J.D. and Cullen, G.A. (1981) Improved performance of progeny of
broiler parent chickens vaccinated with infectious bursal disease oil-emulsion
vaccine. Avian Dis 25(1), 228-41.
46
Table 2.1: Persistence and distribution of IBDV (STC & IN) strains in lymphoid tissues of two-weeks old SPF chickens
IBDV genome detection by real time RT-PCR
Thymus
DPI*
STC
Bursa
IN
STC
Spleen
IN
STC
Bone marrow
IN
STC
IN
47
3
3/3 (4.54±0.69)**
3/3 (5.08±0.48)
3/3 (7.12±0.86)
3/3 (9.13±0.12)
3/3 (4.97±0.91)
3/3 (5.54±0.57)
3/3 (4.46±0.47)
3/3 (4.98±0.48)
5
3/3 (4.38±0.12)
3/3 (4.45±0.47)
3/3 (5.75±0.95)
3/3 (6.33±0.84)
3/3 (4.22±0.72)
3/3 (4.26±0.51)
3/3 (1.96±0.69)
2/3 (3.46±0.47)
7
3/3 (4.57±0.73)
2/3 (3.78±0.02)
3/3 (5.91±0.32)
3/3 (5.4±00.15)
3/3 (4.35±0.46)
3/3 (3.16±0.19)
3/3 (1.98±0.50)
0/3
10
2/3 (3.3±0.43)
3/3 (3.57±0.76)
3/3 (5.60±0.25)
3/3 (5.71±1.04)
3/3 (3.19±0.12)
3/3 (2.66±0.74)
1/3 (1.26±00)
3/3 (6.98±0.76)
14
3/3 (2.81±0.13)
3/3 (2.75±0.49)
3/3 (5.38±0.30)
3/3 (4.68±0.44)
3/3 (3.57±0.41)
3/3 (2.97±0.2)
3/3 (0.79±0.14)
1/3 (6.78±00)
21
0/3
0/3
3/3 (4.08±0.25)
3/3 (4.25±0.22)
3/3 (0.78±0.17)
0/3
1/3 (0.68±00)
28
2/3 (1.21±0.57)
0/3
3/3 (3.54±0.36)
3/3 (3.41±0.61)
2/3 (2.2±0.04)
1/3 (2.43±00)
1/3 (1.39±00)
0/3
35
1/3 (1.87±00)
0/3
3/3 (3.53±0.63)
1/3 (3.86±00)
1/3 (1.31±00)
0/3
1/3 (0.65±00)
0/3
42
0/3
0/3
3/3 (3.17±0.61)
0/3
0/3
0/3
0/3
0/3
Dayinoculation
post-inoculation
**: positive/total
Number positive/total
inoculated
(average
log± 10
EID50/1ml
± Standard
* DPI: DPI:
Day post
**: Number
inoculated (average
log 10
EID 50/50ul
Standard
deviation).
47
0/3
deviation).
Table 2.2: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid tissues of two-weeks old SPF chickens
IBDV genome detection by real time RT-PCR
Thigh muscles
DPI*
STC
Breast muscles
IN
STC
Pancreas
IN
STC
Kidney
IN
STC
IN
48
3
3/3(3.92±0.90**
3/3 (3.22±0.78)
2/3 (2.69±0.014)
3/3 (3.02±0.32)
3/3 (3.85±0.17)
3/3 (3.45±1.14)
3/3 (4.09±0.89)
3/3 (3.71±0.47)
5
2/3 (2.01±0.50)
3/3 (1.84±0.15)
1/3 (2.61±00)
0/3
3/3 (3.26±0.47)
3/3 (2.42±0.67)
2/3 (3.17±0.84)
3/3 (2.84±0.82)
7
3/3 (1.85±0.54)
1/3 (1.69±00)
1/3 (1.94±00)
0/3
3/3 (3.09±0.73)
2/3 (2.63±0.65)
3/3 (3.42±0.38)
1/3(1.9±00)
10
1/3 (0.54±00)
1/3 (3.02±00)
1/3 (0.57±00)
3/3 (2.83±0.61)
3/3 (2.45±0.31)
0/3
2/3 (2.71±0.50)
2/3 (2.70±0.44)
14
2/3 (1.33±0.16)
1/3 (2.81±00)
2/3 (0.55±0.49)
2/3 (2.85±0.44)
3/3 (2.22±0.19)
1/3 (1.85±00)
3/3 (2.24±0.40)
3/3 (2.52±0.56)
21
1/3 (0.33±00)
0/3
1/3 (0.57±00)
0/3
2/3 (2.05±0.53)
0/3
2/3 (0.94±0.48)
0/3
28
2/3 (0.55±0.34)
0/3
0/3
0/3
1/3 (0.68±00)
0/3
3/3 (1.23±0.80)
0/3
35
0/3
0/3
1/3 (0.14±00)
0/3
2/3 (0.81±0.5)
0/3
1/3 (0.43.±00)
0/3
42
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
* DPI: DPI:
Day post
**: Number
positive/total
inoculated inoculated
(average log(average
10 EID 50/50ul
± Standard
deviation).
Dayinoculation
post-inoculation
**: Number
positive/total
log 10
EID50/1ml
± Standard
48
deviation).
Table 2.3 : Persistence and distribution of IBDV (STC&IN) in non-lymphoid tissues of two-weeks old SPF chickens
IBDV genome detection by real time RT-PCR
Caecum
DPI*
STC
Lungs
IN
STC
Liver
IN
STC
IN
49
3
3/3 (4.63±0.73) **
3/3 (5.2±0.53)
3/3 (3.46±1.93)
3/3 (3.37±0.93)
3/3 (4.31±1.21)
3/3 (5.69±0.25)
5
3/3 (4.68±0.40)
3/3 (4.47±0.51)
3/3 (3.55±0.71)
3/3 (2.92±0.78)
3/3 (4.43±0.32)
3/3 (4.5±0.95)
7
3/3 (3.21±1.13)
3/3 (3.95±0.02)
3/3 (3.11±0.08)
1/3 (1.67±00)
2/3 (3.71±0.26)
3/3 (2.99±0.33)
10
3/3 (2.08±1.25)
3/3 (2.7±0.12)
3/3 (1.95±0.73)
0/3
3/3 (2.05±0.80)
2/3 (2.97±0.33)
14
3/3 (1.75±0.62)
3/3 (2.59±0.37)
3/3 (1.29±0.29)
0/3
3/3 (2.39±0.65)
2/3 (1.56±0.87)
21
3/3 (0.83±0.49)
3/3 (2.81±1.5)
3/3 (0.69±0.14)
0/3
2/3 (1.92±0.40)
0/3
28
2/3 (1.86±0.12)
1/3 (4.6±00)
1/3 (0.51±00)
0/3
3/3 (1.42±0.10)
0/3
35
1/3 (1.66±0)
0/3
0/3
0/3
1/3 (2.23±00)
0/3
0/3
0/3
0/3
0/3
0/3
0/3
42
* DPI: Day post inoculation **: Number positive/total inoculated (average log 10 EID 50/50ul ± Standard deviation).
DPI: Day post-inoculation **: Number positive/total inoculated (average log 10 EID50/1ml ± Standard deviation).
49
Table 2.4: Persistence and distribution of IBDV (STC & IN) strains in lymphoid tissues of four-weeks old SPF chickens
IBDV genome detection by real time RT-PCR
Thymus
Bursa
STC
IN
STC
7
2/3 (2.33±0.48)**
0/3
3/3 (4.38±0.59)
14
3/3 (1.45±0.24)
0/3
21
1/3 (1.44±00)
28
IN
Bone marrow
STC
IN
STC
IN
2/3 (5.38±0.03)
3/3 (3.51±0.58)
3/3 (3.27±0.39)
3/3 (1.67±0.07)
1/3 (1.7±00)
3/3 (3.56±0.34)
3/3 (3.59±0.3)
3/3 (1.98±0.66)
2/3 (1.19±1.04)
0/3
0/3
0/3
3/3 (3.31±1.00)
3/3 (3.68±0.1)
3/3 (1.54±1.15)
1/3 (0.75±00)
1/3 (0.19±00)
0/3
2/3 (1.75±0.11)
0/3
3/3 (3.71±0.6)
3/3 (3.36±1.14)
2/3 (2.15±0.02)
0/3
1/3 (0.62±00)
0/3
35
3/3 (0.83±0.35)
0/3
3/3 (3.24±0.24)
0/3
3/3 (1.49±0.11)
0/3
0/3
0/3
42
0/3
0/3
2/3 (3.01±0.24)
0/3
2/3 (1.35±0.12)
0/3
0/3
0/3
49
0/3
0/3
2/3 (2.23±0.88)
0/3
1/3 (0.42±00)
0/3
1/3 (0.51±00)
0/3
56
0/3
0/3
2/3 (1.56±0.70)
0/3
1/3 (0.41±00)
0/3
0/3
0/3
50
DPI*
Spleen
DPI:
DayDay
post-inoculation
Numberpositive/total
positive/total
inoculated
(average
EID /1ml ± Standard deviation).
* DPI:
post inoculation **:
**: Number
inoculated
(average
log 10 log
EID10
50/50ul ±50Standard deviation).
50
Table 2.5: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid tissues of four-weeks old SPF chickens
IBDV genome detection by real time RT-PCR
Thigh muscles
Breast muscles
Pancreas
Kidney
51
DPI*
STC
IN
STC
IN
STC
IN
STC
IN
7
1/3 (2.35±00)**
0/3
2/3 (1.5±0.42)
0/3
3/3 (2.24±0.15)
1/3 (2.25±00)
3/3 (2.4±0.38)
0/3
14
0/3 (00±00)
0/3
0/3
0/3
2/3 (1.19±0.28)
0/3
3/3 (2.15±0.53)
0/3
21
1/3 (0.47±00)
0/3
0/3
0/3
3/3 (1.48±0.79)
0/3
3/3 (0.95±0.46)
0/3
28
1/3 (0.092±00)
0/3
2/3 (0.83±0.47)
0/3
3/3 (1.62±0.44)
0/3
3/3 (1.85±0.32)
0/3
35
0/3
0/3
0/3
0/3
3/3 (1.03±0.11)
0/3
3/3 (1.18±0.19)
0/3
42
0/3
0/3
0/3
0/3
0/3
0/3
1/3 (1.6±00)
0/3
49
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
56
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
DPI:
Day
post-inoculation
**:Number
Number
positive/total
inoculated
(average
log50/50ul
10 EID
Standard deviation).
50/1ml ±deviation).
* DPI:
Day
post inoculation **:
positive/total
inoculated
(average
log 10 EID
± Standard
51
Table 2.6: Persistence and distribution of IBDV (STC&IN) in non-lymphoid tissues of four-weeks old SPF chickens
IBDV genome detection by real time RT-PCR
Caecum
Lungs
Liver
52
DPI*
STC
IN
STC
IN
STC
IN
7
3/3 (3.45±1.77)**
3/3 (3.03±0.29)
3/3 (1.7±0.76)
0/3
3/3 (3.87±3.01)
2/3 (2.78±0.014)
14
3/3 (2.76±0.85)
0/3
3/3 (3.5±3)
0/3
3/3 (3.1±2.54)
0/3
21
3/3 (1.7±0.37)
1/3 (2.00±00)
3/3 (3.7±0.78)
0/3
3/3 (3.2±0.12)
0/3
28
3/3 (2.3±0.16)
0/3
2/3 (3.4±1.66)
0/3
3/3 (3.1±0.47)
0/3
35
2/3 (0.95±0.08)
0/3
0/3
0/3
3/3 (3.44±2.15)
0/3
42
1/3 (1.04±00)
0/3
1/3 (38.20±0.48)
0/3
2/3 (3.6±1.39)
0/3
49
0/3
0/3
0/3
0/3
0/3
0/3
56
0/3
0/3
0/3
0/3
0/3
0/3
* DPI: Day post-inoculation **: Number positive/total inoculated (average log 10 EID50/1ml ± Standard deviation).
52
Table 2.7: Persistence and distribution of IBDV strains in lymphoid tissues of two-weeks old in ovo vaccinated broilers chickens
IBDV genome detection by real time RT-PCR
Thymus
DPI*
STC
Bursa
IN
STC
Spleen
IN
STC
Bone marrow
IN
STC
IN
53
3
0/3 (00±00)**
1/3 (2.95±00)
2/3 (4.90±1.24)
3/3 (5.64±1.71)
3/3 (1.87±0.37)
2/3 (3.57±0.57)
2/3 (0.71±0.42)
0/3
5
2/3 (1.94±0.12)
0/3 (00±00)
3/3 (4.28±1.02)
1/3 (4.23±00)
2/3 (1.30±0.23)
0/3
0/3
0/3
7
0/3
1/3 (4.23±00)
2/3 (5.44±0.57)
2/3 (4.89±0.92)
2/3 (1.24±0.51)
0/3
0/3
0/3
10
0/3
0/3
2/3 (4.75±0.12)
2/3 (4.05±0.55)
1/3 (1.91±00)
1/3 (2.31±00)
2/3 (0.98±0.05)
0/3
14
0/3
0/3
2/3 (0.71±0.88)
3/3 (3.23±0.83)
3/3 (3.61±0.76)
1/3 (1.60±00)
3/3 (1.46±0.68)
0/3
21
0/3
0/3
3/3 (3.47±0.76)
3/3 (3.35±0.86)
1/3 (1.80±00)
1/3 (6.95±00)
0/3
1/3 (5.67±00)
28
0/3
0/3
2/3 (0.93±0.15)
1/3 (3.26±00)
1/3 (0.78±00)
0/3
1/3 (1.99±00)
0/3
35
0/3
0/3
2/3 (1.39±0.60)
0/3
1/3 (0.59±00)
0/3
0/3
0/3
42
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
* DPI: Day post-inoculation **: Number positive/total inoculated (average log 10 EID50/1ml ± Standard deviation).
53
Table 2.8 : Persistence and distribution of IBDV strains in non-lymphoid tissues of two-weeks old in ovo vaccinated broilers
IBDV genome detection by real time RT-PCR
Thigh muscles
DPI*
STC
Breast muscles
IN
STC
Pancreas
IN
STC
Kidney
IN
STC
IN
54
3
1/3 (1.02±00)**
1/3 (3.21±00)
0/3
0/3
1/3 (0.51±00)
0/3
0/3
2/3 (3.29±0.79)
5
3/3 (1.78±0.17)
0/3
0/3
0/3
1/3 (0.54±00)
0/3
2/3 (0.49±0.62)
0/3
7
1/3 (1.47±00)
0/3
0/3
0/3
0/3
0/3
0/3
1/3 (2.64±00)
10
1/3 (0.56±00)
0/3
0/3
0/3
1/3 (0.56±00)
0/3
1/3 (1.61±00)
0/3
14
2/3 (1.33±0.16)
0/3
0/3
0/3
0/3
0/3
0/3
0/3
21
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
28
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
35
0/3
0/3
0/3
0/3
2/3 (1.31±0.42)
0/3
0/3
0/3
42
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
* DPI: Day post inoculation **: Number positive/total inoculated (average log 10 EID 50/1ml ± Standard deviation).
54
Table 2.9: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid tissues of two-weeks in ovo vaccinated broilers
chickens
IBDV genome detection by real time RT-PCR
Caecum
DPI*
STC
Lungs
IN
STC
Liver
IN
STC
IN
55
3
3/3 (0.92±0.14)**
3/3 (3.16±0.51)
0/3
0/3
1/3 (4.31±1.21)
3/3 (2.93±1.06)
5
2/3 (2.35±0.80)
1/3 (2.28±00)
1/3 (1.06±00)
0/3
2/3 (4.43±0.32)
0/3
7
2/3 (1.13±0..09)
1/3 (3.28±00)
1/3 (1.11±00)
0/3
0/3
0/3
10
3/3 (1.99±0.40)
0/3
1/3 (0.65±00)
0/3
2/3 (1.66±0.20)
0/3
14
1/3 (0.79±00)
0/3
2/3 (1.16±0.20)
0/3
0/3
0/3
21
1/3 (2.23±00)
2/3 (4.45±0.63)
0/3
0/3
1/3 (0.28±00)
0/3
28
1/3 (1.27±00)
0/3
0/3
0/3
1/3 (1.54±00)
0/3
35
1/3 (0.67±00)
0/3
0/3
0/3
1/3 (0.57±00)
0/3
42
0/3 (00±00)
0/3
0/3
0/3
0/3
0/3
* DPI: Day post-inoculation **: Number positive/total inoculated (average log 10 EID50/1ml ± Standard deviation).
55
Table 2.10: Persistence and distribution of IBDV (STC & IN) strains in lymphoid tissues of four-weeks in ovo vaccinated broilers
chickens
IBDV genome detection by real time RT-PCR
Thymus
DPI*
STC
Bursa
IN
STC
Spleen
IN
STC
Bone marrow
IN
STC
IN
56
3
2/3 (2.61±1.40)**
1/3 (3.26±00)
3/3 (7.40±0.20)
2/3 (5.54±0.85)
3/3 (3.47±1.37)
1/3 (3.03±00)
1/3 (0.71±0.42)
0/3
7
1/3 (1.57±00)
2/3 (2.94±0.27)
1/3 (5.15±00)
3/3 (6.94±0.18)
2/3 (2.40±0.70)
3/3 (3.22±0.60)
1/3 (0.65±00)
0/3
14
0/3 (00±00)
0/3
3/3 (2.01±0.44)
2/3 (3.97±0.56)
2/3 (1.41±1.83)
0/3
1/3 (3.85±00)
0/3
21
2/3 (1.19±0.52)
0/3
3/3 (4.51±0.19)
3/3 (4.10±0.34)
3/3 (3.07±0.91)
1/3 (2.63±00)
1/3 (5.92±00)
0/3
28
2/3 (2.52±1.94)
1/3 (3.25±00)
3/3 (4.53±0.39)
3/3 (3.29±0.28)
2/3 (3.22±0.05)
0/3
1/3 (7.36±00)
0/3
35
1/3 (4.69±00)
1/3 (3.94±00)
3/3 (3.51±0.43)
3/3 (3.22±0.05)
0/3
0/3
0/3
0/3
42
1/3 (3.23±00)
0/3
2/3 (3.23±0.014)
2/3 (2.60±0.30)
0/3
0/3
0/3
0/3
* DPI: Day post-inoculation **: Number positive/total inoculated (average log 10 EID50/1ml ± Standard deviation).
* DPI: Day post inoculation **: Number positive/total inoculated (average log 10 EID 50/50ul ± Standard deviation).
56
Table 2.11: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid tissues of four-weeks in ovo vaccinated
broilers chickens
IBDV genome detection by real time RT-PCR
Thigh muscles
DPI*
STC
Breast muscles
IN
STC
Pancreas
IN
STC
Kidney
IN
STC
IN
1/3 (1.19±00)**
0/3
1/3 (1.33±00)
0/3
3/3 (2.1±1.06)
0/3
2/3 (2.57±1.70)
0/3
7
0/3
0/3
0/3
0/3
0/3
1/3 (1.60±00)
1/3 (0.36±00)
1/3 (1.53±00)
14
0/3
0/3
1/3 (1.41±00)
0/3
1/3 (1.99±00)
0/3
0/3
0/3
21
1/3 (6.63±00)
0/3
0/3
0/3
2/3 (1.27±0.42)
1/3 (7.25±00)
1/3 (1.94±00)
1/3 (7.98±00)
28
1/3 (4.54±00)
0/3
0/3
0/3
2/3 (4.14±1.14)
1/3 (7.04±00)
2/3 (4.47±0.49)
2/3 (8.67±2.78)
35
1/3 (7.09±00)
0/3
0/3
0/3
1/3 (4.69±00)
0/3
0/3
0/3
42
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
57
3
** DPI:
**:Number
Numberpositive/total
positive/total
inoculated
(average
10 EID
50/1ml ± Standard deviation).
DPI: Day
Day post-inoculation
post inoculation **:
inoculated
(average
log log
10 EID
50/50ul ± Standard deviation).
57
Table 2.12: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid tissues of four-weeks in ovo vaccinated
broilers
IBDV genome detection by real time RT-PCR
Caecum
DPI*
STC
Lungs
IN
STC
Liver
IN
STC
IN
58
3
3/3 (4.72±0.14)**
0/3
1/3 (1.95±0.5)
1/3 (2.±00)
2/3 (2.20±0.91)
3/3 (2.93±1.06)
7
2/3 (2.54±0.80)
3/3 (4.66±0.38)
1/3 (1.51±00)
2/3 (2.38±0.56)
2/3 (1.27±0.38)
0/3
14
1/3 (0.51±00)
1/3 (2.45±00)
0/3 (3.21±00)
0/3
0/3
0/3
21
3/3 (2.94±0.43)
1/3 (3.03±00)
2/3 (3.25±2.2)
0/3
0/3
0/3
28
3/3 (1.86±0.11)
1/3 (2.67±00)
3/3 (4.50±2.7)
0/3
2/3 (3.41±0.75)
0/3
35
3/3 (1.14±0.60)
0/3
0/3
0/3
0/3
0/3
42
0/3
0/3
0/3
0/3
0/3
0/3
* DPI: Day post-inoculation **: Number positive/total inoculated (average log 10 EID50/1ml ± Standard deviation).
58
Table 2.13: Persistence and distribution of IBDV strains in lymphoid tissues of 2-weeks old broilers having maternal antibodies
IBDV genome detection by real time RT-PCR
Thymus
DPI*
STC
Bursa
IN
STC
Spleen
IN
STC
Bone marrow
IN
STC
IN
2/3 (2.01±1.11)**
3/3 (4.18±0.5)
3/3 (6.16±0.58)
3/3 (7.19±0.40)
3/3 (2.88±0.88)
1/3 (3.72±00)
1/3 (0.88±00)
0/3
7
1/3 (1.23±00)
0/3
3/3 (5.15±1.17)
3/3 (5.2±0.56)
3/3 (3.54±0.80)
2/3 (3.66±0.1)
2/3 (1.03±0.22)
0/3
14
0/3
0/3
3/3 (4.81±0.39)
2/3 (4.43±0.56)
3/3 (1.21±0.76)
0/3
0/3
0/3
21
0/3
0/3
3/3 (4.16±0.21)
3/3 (2.87±0.6)
0/3
0/3
0/3
0/3
28
0/3
0/3
3/3 (2.59±1.15)
0/3
0/3
0/3
0/3
0/3
35
0/3
0/3
3/3 (2.80±0.66)
0/3
0/3
0/3
0/3
0/3
42
0/3
0/3
3/3 (2.28±2.8)
0/3
0/3
0/3
0/3
0/3
59
3
* DPI: Day post-inoculation **: Number positive/total inoculated (average log 10 EID50/1ml ± Standard deviation).
59
Table 2.14: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid tissues of 2-weeks broilers having maternal
antibodies
IBDV genome detection by real time RT-PCR
Thigh muscles
DPI*
STC
Breast muscles
IN
STC
Pancreas
IN
STC
Kidney
IN
STC
IN
1/3 (1.26±00)**
0/3
0/3
0/3
1/3 (1.72±1.06)
0/3
1/3 (1.72±00)
1/3 (7.45±00)
7
0/3
0/3
0/3
2/3 (1.79±0.11)
2/3 (2.21±0.88)
1/3 (1.60±00)
2/3 (1.54±0.47)
1/3 (6.8±00)
14
0/3
0/3
1/3 (1.41±00)
0/3
0/3
0/3
0/3
0/3
21
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
28
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
35
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
42
0/3
0/3
0/3
0/3
0/3
0/3
0/3
0/3
60
3
* DPI:
Day post
**: Number
positive/total
inoculatedinoculated
(average log
10 EID50log
/50ul
Standard
* DPI:
Dayinoculation
post-inoculation
**: Number
positive/total
(average
10±EID
± Standard deviation).
50/1ml deviation).
60
Table 2.15: Persistence and distribution of IBDV (STC & IN) strains in non-lymphoid tissues of 2-weeks broilers having maternal
antibodies
IBDV genome detection by real time RT-PCR
Caecum
DPI*
STC
Lungs
IN
STC
Liver
IN
STC
IN
61
3
3/3 (3.47±0.92)**
2/3 (3.62±0.38)
1/3 (1.95±0.5)
2/3 (2.69.±00)
3/3 (2.63±1.00)
2/3 (2.77±1.06)
7
3/3 (4.21±0.57)
2/3 (4.18±0.54)
1/3 (1.51±00)
2/3 (1.56±0.02)
3/3 (2.70±1.19)
1/3 (3.66±00)
14
1/3 (1.29±00)
0/3
0/3
0/3
0/3
0/3
21
3/3 (1.13±0.15)
0/3
0/3
0/3
0/3
0/3
28
0/3
0/3
0/3
0/3
0/3
0/3
35
0/3
0/3
0/3
0/3
0/3
0/3
42
0/3
0/3
0/3
0/3
0/3
0/3
* DPI: Day post-inoculation **: Number positive/total inoculated (average log 10 EID50/1ml ± Standard deviation).
61
Viral titers over time (all tissues)
3
5
7
10
14
21
28
35
42
49
56
42
49
56
42
49
56
IN
2
4
6
8
Indiana strain, n=792
0
CT–value
value
CTof
LogLog
titter
10 ofEID
10
Log
/ 1ml
10
50
3
5
7
10
14
21
28
35
STC
2
4
6
8
STC strain, n=810
0
log_with40
0
2
4
6
8
Non-inoculatedC controls, n=72
3
5
7
10
14
21
28
35
Days post inoculation
Figure 2.1. Virus titer decay in tissue samples from chickens singly inoculated with
reference strains of infectious bursal disease virus. STC virus was significantly more
persistent overtime (GLM, p<0.001). The number of tissue samples tested is presented in
italics (n=).
62
SPF chickens
Log10 EID50/ 1ml
(A)
Commercial chickens
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
0
Log10 EID50/ 1mll
(C)
8
7
14
21
28
42
49
56
8
7
6
6
5
5
4
4
3
3
2
2
1
8
1
0
7
0
7
14
21
28
35
42
days post-inoculation
6 bursa
2-week-old
spf 2 IN
Cecum:
spf 4 IN
4-week-old
5 bursa
6
35
7
7
7
Virus isolated
0
0
80
8
(B)
caecum spf 2 IN
49
56
0
7
14
(D)
21
28
35
42
49
56
bursa spf 2 IN
0
7
14
21
28
bursa
spf 4 IN
42 49 56
35
bursa
spf 2 IN days post-inoculation
6
caecum spf 2 IN
bursa
spf 4 IN
5
caecum spf 4 IN
caecum
spf 2 INT. muscle:
42-week-old
thigh
spf 2 IN
2-week-old
4-week-old
caecum
spf 4 IN
thigh
spf 4 IN 7
4-week-old
3
thigh spf 2 IN
4 Persistence of two strains of infectious
2
Figure 2.2.
bursal disease virus after experimental
caecum spf 4 IN
thigh spf 4 IN 7
inoculation
in SPF
and commercial chickens.
(A) Persistence of STC strain in SPF
3 thigh
1
spf 2 IN
chickens. Virus isolation was confirmed in bursal homogenates which is represented by
4 IN 7 of STC strain0 in commercial chickens. Virus isolation was
2 thigh
shaded line.
(B) spf
Persistence
21 28 of 35
42 49
56
confirmed1 up 21 DPI as shown by shaded0 line.7 (C) 14
Persistence
IN strain
in SPF
chickens. Virus isolation was confirmed in bursal homogenates which is represented by
0 up to 10 DPI. (D) Persistence of IN strain in commercial chickens. Virus
shaded line
isolation was
10 DPI
by shaded
0 confirmed
7
14up to21
28 as shown
35 42
49 line.
56
5
4
3
2
1
0
0
7
14
21
28
35
42
49
56
63
2
0
3
5
7
10
14
21
28
35
42
49
56
35
42
49
56
6
8
SPF Chickens
0
2
4
50 ul
Log
EID 50/ /1ml
Log
EID
10 10
50
log_with40
4
6
8
Commercial
Commercialbroilers
broilers
3
5
7
10
14
21
28
Figure 2.3. Log10 viral titers in experimentally inoculated SPF and commercial
chickens. Data represents all tissues tested from animals infected with IN and STC strains
of IBDV between 2 and 4 weeks of age (n=1,654 samples). Commercial broilers (2weeks-old) were studied until day 42 PI; SPF experiments were conducted until day 56
PI.
64
Antibody response aganist STC
S/P
(A)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
DPI
Antibody response aganist IN
S/P
(B)
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
D.P.I
Figure 2.4. Antibody titer in SPF chickens after inoculation with STC and IN strains.
Antibodies titers were measured by IDDEX kit and observed O.D values were converted
in to S/P ratio as per manufacturer instructions. (A) Antibody titer in STC challenged
chickens and (B) Antibody titer in IN strain challenged chickens.
65
CHAPTER 3
DIFFERENTIAL MODULATION OF CYTOKINES AND TOLL LIKE
RECEPTORS EXPRESSION IN THE BURSA OF CHICKENS
INFECTED WITH CLASSICAL AND VARIANT STRAINS OF
INFECTIOUS BURSAL DISEASE VIRUS.
3.1 SUMMARY
Infectious bursal disease (IBD) is an important immunosuppressive disease of chickens.
The causative agent, infectious bursal disease virus (IBDV), consists of two serotypes, 1
and 2. Serotype 1 consists of classic IBDV (cIBDV) and variant IBDV (vIBDV). Both of
these strains vary in antigenicity and pathogenicity. The goal of this study was to
compare the immunopathogenesis of cIBDV and vIBDV. Three-weeks-old specific
pathogen free chickens were inoculated intraocularly with standard challenge strain
(STC) (cIBDV) and a variant strain Indiana (IN) (vIBDV). The cIBDV produced more
pronounced bursal damage and inflammatory response as compared to vIBDV. There
were significant differences in the expression of innate (IFN-α and IFN-β),
proinflammatory cytokine and mediator (IL-6 and iNOS) in cIBDV- and vIBDV-infected
bursas. The expression of genes of chemokines, IL-8 and MIP-α was also higher in
66
cIBDV -infected chickens compared to control during the early phase of infection. The
expression of Toll like receptor 3 (TLR3) was downregulated at PIDs 3, 5, and 7 in the
bursas of vIBDV-infected chickens whereas TLR3 was upregulated at PIDs 3 and 5 in
cIBDV-infected bursas. In vIBDV-infected bursa, TLR7 expression was downregulated
at PIDs 3 and 5 and upregulated at PID 7. However, TLR7 was upregulated at PIDs 3 and
7 in cIBDV-infected bursas. The expression of MyD88 was downregulated whereas TRIF
gene expression was upregulated in cIBDV- and vIBDV-infected bursa. These findings
will be useful in understanding the differential immuno-pathogenesis of classical and
variant strains of IBDV.
3.2 INTRODUCTION
Infectious bursal disease (IBD) is one of the most important naturally occurring viral
diseases of commercial chickens worldwide (Lukert P.D., 2003). The causative agent,
IBD virus (IBDV) belongs to the family Birnaviridae. The virus causes an acute, highly
contagious and immunosuppressive disease in chickens (Lukert P.D., 2003). The virus
infects and destroys actively dividing IgM-bearing B cells in the bursa of fabricius (Hirai
et al., 1981; Rodenberg et al., 1994). IBDV exists in different antigenic and pathogenic
forms (Jackwood et al., 2006). Initial isolates designated as classical strains (cIBDV) of
the serotype 1 viruses were considered to be a single antigenic type. In the early 1980s,
antigenic variants (vIBDV) of the virus were identified in the United States (Lukert P.D.,
2003). These variant viruses were able to cause disease in the presence of immunity to
cIBDV viruses (Ismail and Saif, 1991). The antigenic variants typically do not cause
clinical signs of disease but can cause a marked immunosuppression (Jackwood et al.,
67
2006). The immunosuppression caused by variants and classical strains of IBDV is often
associated with secondary viral respiratory infections and bacterial infections (Bautista et
al., 2004; Li et al., 2010; Subler et al., 2006). The IBDV induced immunosuppression
also renders chicken flocks refractory to live attenuated vaccines against other viral
diseases such as avian influenza virus, infectious bronchitis and Newcastle disease virus
(Muller et al., 2003; Ramirez-Nieto et al., 2010).
Following infection and replication of IBDV, T cells infiltrate the bursa of infected
chickens (Kim et al., 2000; Sharma et al., 2000). Although B cells are considered the
major targets for IBDV, it has been shown that the virus can infect and possibly replicate
in macrophages (Khatri et al., 2005; Kim et al., 1998; Palmquist et al., 2006). Following
viral infections, including IBDV, activated macrophages produce various mediators, such
as proinflammatory cytokines, interleukin-1 (IL-1) and IL-6, chemokines, and nitric
oxide (NO) (Glass et al., 2003; Heitmeier et al., 1998; Khatri et al., 2005; Khatri and
Sharma, 2006).
Host cells use various receptors to detect viral infections by recognizing pathogenassociated molecular patterns (PAMPs) and subsequently induce an antiviral response.
Prominent among these are Toll-like receptors (TLRs) (Kawai and Akira, 2006; Sang et
al., 2008; Werts et al., 2006). Several TLRs recognize viral PAMPs: TLR3, detects
double-stranded RNA (dsRNA) derived from viral replication whereas single-stranded
RNA (ssRNA) are detected by TLR7 and TLR8 (Sang et al., 2008). The TLR signaling
proceeds via two pathways; the myeloid differentiation factor 88 (MyD88)-mediated
pathway and the Toll-interleukin-1 receptor (TIR)-domain-containing adaptor inducing
68
IFN-β (TRIF)-mediated pathway (Kawai and Akira, 2009; Takeuchi and Akira, 2009).
The TLR signaling pathways arise from intracytoplasmic TIR domains, which are
conserved among all TLRs. The TLR7 specifically involves MyD88-dependent pathway,
whereas TRIF is implicated in the TLR3-mediated MyD88-independent pathway (Takeda
and Akira, 2004).
The IBD is controlled by vaccination and the vaccines are very effective against classical
strains but with the emergence of variant and the very virulent strains of IBDV in the
United States (Stoute et al., 2009), there are several incidents of vaccine failure (Berg and
Meulemans, 1991; Ismail et al., 1990; Okoye and Shoyinka, 1983; Snyder, 1990; Stoute
et al., 2009). This highlights the need to examine the differential immuno-pathogenesis of
classical and variant strains of IBDV in order to devise better control strategies.
In this study, for the first time, we examined the differential immuno-pathogenesis of
classical and variant strains of IBDV. As compared to vIBDV, cIBDV induced early
bursal lesions, extensive infiltration of T cells in the bursa and induced higher expression
of proinflammatory cytokine and mediators; IL-6 and iNOS. Further, there were
differences in the expression of TLR3 and TLR7 and their adapter molecules, TRIF and
MyD88, in the bursa of cIBDV and vIBDV-infected chickens. Elucidation of the TLRs
signaling pathway and factors leading to activation of the innate immune response to
IBDV infection may provide new strategies for the development of cross-protective
vaccines against IBDV.
69
3.3 MATERIALS AND METHODS
Chickens and virus strains:
Specific pathogen free (SPF) chicken eggs (Charles River Laboratories Inc; Wilmington,
MA, USA) were incubated and hatched in our facility at The Ohio Agricultural Research
and Development Center, The Ohio State University. Chickens were kept in a disease
containment building. At 3-weeks of age, prior to inoculation with virus, the chickens
were transferred to an isolation unit. The standard challenge strain, (STC) (Abdel-Alim
and Saif, 2001a) representing cIBDV strain, and a variant Indiana (IN) representing
(vIBDV), were propagated in chickens and titrated in eggs as described earlier (Ismail et
al., 1990).
Experimental design:
Eighty-four SPF chickens were allocated to 3 groups; thirty-six chickens in both group 1
and group 2 were inoculated intraocularly with 104 EID50/200ul of either vIBDV or
cIBDV strains respectively and 12 chickens in group 3 were inoculated similarly with
PBS to serve as virus-free controls (Abdel-Alim and Saif, 2001b). At post-inoculation
days (PIDs) 3, 5 and 7, twelve chickens each from the virus-infected groups and 4
chickens from the virus-free group were euthanized and bursas were collected. Four
pools of 3 bursas each from virus-infected groups were prepared. The harvested bursal
tissues
were
examined
for
the
following:
1-
histopathological
lesions,
2-
immunohistochemical detection of virus antigen, T cells and macrophages, 3- isolation
of mononuclear cells and expression of virus-induced innate, proinflammatory cytokines,
70
chemokines, Toll like receptors (TLRs), and their adaptor molecules by quantitative real
time RT-PCR (qRT-PCR).
Microscopic lesions:
At PIDs 3, 5 and 7, four bursas each from virus-free control, cIBDV- and vIBDVinfected chickens were harvested, fixed in 10 % phosphate buffered formalin, sectioned
and stained with hematoxylin and eosin for the detection of histopathological lesions.
Bursal follicular lesions were observed microscopically and lesion scores were
determined. The bursal lesions were scored as follows: lesion score 1 represents 1-25% of
lymphoid follicles affected, 2 represents 26-50% of lymphoid follicles affected, 3
represents
51-75% of lymphoid follicles affected
and
4 represents
76-100% of
lymphoid follicles affected (Kim et al., 1999; Rautenschlein et al., 2007).
Detection of IBDV-antigen, macrophages and T cells in virus-infected bursas:
The IBDV antigens, T cells, and macrophages were detected in snap frozen sections of
bursas
of
vIBDV-,
cIBDV-infected
and
virus-free
control
chickens
by
immunohistochemistry (Khatri et al., 2005; Rauf et al., 2011). Briefly, small sections of
bursal tissue were embedded in Tissue-Tek® O.C.T compound (Sakura Finetek, CA,
USA), sectioned 5 μM thick with the help of cryostat microtome (Leica CM 1510S
Germany), spotted on double positive glass slides, and dehydrated at 37 oC overnight.
The dehydrated sections were fixed with acetone (75% acetone and 25% ethyl alcohol)
for 10 minutes followed by 3 washes with phosphate buffered saline (PBS). The tissue
sections were then blocked with 2% goat serum for 1 hour (hr). After blocking, the
sections were incubated with respective primary and secondary antibodies. A biotin71
streptavidin-peroxidase method using R.T.U vectastain(R) kit (Vector Laboratories,
Burlingame, CA) was adopted for the detection of viral antigen, T cells and macrophages
in frozen sections of vIBDV-, cIBDV-infected and virus-free chickens bursas. The
primary antibodies used for the detection of T cells and macrophages were: mouse antichicken CD3, (diluted 1:200) and mouse anti-chicken monocytes/macrophage KUL01,
(diluted 1:400) (Southern Biotech, Birmingham, AL, USA). The primary antibody used
for the detection of IBDV antigen was biotinylated mouse anti-IBDV polyclonal antibody
(diluted 1:100). The development of dark brown color indicated a positive reaction. The
group means ± SEM of vIBDV- and cIBDV-infected cells, macrophage or T cells per
field was determined at 20 X magnification after counting 5 fields /bursa/ chicken and
compared with virus-free control groups.
Isolation of bursal mononuclear cells:
Twelve bursas either from cIBDV- or vIBDV-infected groups at each PID were pooled
into 4 pools of three bursas each and bursas were also collected from 4 virus-free control
chickens at each PID. Mononuclear cells were isolated from bursas as previously
described (Khatri et al., 2005; Rauf et al., 2011). Briefly, mononuclear cells suspension
was prepared from bursas by density gradient centrifugation (gradient density 1.090) over
Ficoll-Hypaque (GE healthcare Bio-Sciences Uppsala Sweden) and washed twice in cold
RPMI 1640 (Gibco, Carlsbad, CA). The cell pellets were lysed with Trizol reagent and
stored at -70 for RNA extraction.
72
RNA extraction and qRT-PCR:
Total RNA from bursal mononuclear cells of virus-infected and virus-free control
chickens was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA) according to
the manufacturer’s instructions. qRT-PCR was used for the quantification of genes
specific for the expression of messenger RNAs (mRNAs) for innate (IFN-α, IFN-β) and
proinflammatory (IL-6 and iNOS) cytokines, chemokines (IL-8 and MIP-α), Toll like
receptors and their adaptor molecules (TLR3, TLR7, MyD88 and TRIF) (Khatri et al.,
2005). The primers for 28S, IFN-α, IFN-β, IL-6, iNOS, IL-8, MIP-α, TLRL3, TLR7,
TRIF and MyD88 were designed according to previously published sequences (Hghihghi
et al., 2010; Jarosinski et al., 2002; Sijben et al., 2003). RT-PCR was performed using
Power SYBER® Green RNA – to-CT TM 1 step RT-PCR kit (Applied Biosystems, Foster
City, CA). Amplification and detection were performed in an automated 7500 Real
time RT-PCR system (Applied Bio System, Foster City, CA). Fold increase of target
gene expression over uninfected controls was calculated with the 2-ΔΔCT method
(Giulietti et al., 2001; Khatri et al., 2005; Palmquist et al., 2006; Rauf et al., 2011).
Statistical analysis:
Graph Pad Prism version 5 for Windows was used for graphical presentation of data.
Student’s t-test was used to detect significant differences between vIBDV- and cIBDVinfected chickens. P < 0.05 was considered to be statistically significant.
73
3.4 RESULTS
Infection of chickens with cIBDV and vIBDV:
Inoculation of chickens with vIBDV and cIBDV resulted in a typical IBDV infection. All
of the chickens inoculated with cIBDV showed morbidity and 8% mortality whereas
mortality was not observed in chickens infected with vIBDV. Virus-free control chickens
had no clinical signs or macroscopic lesions during the course of the experiment.
Histologically, cIBDV induced early and more pronounced bursal damage as compared
to vIBDV (Fig. 1A). Severe follicular lesions were observed histologically in the bursa of
cIBDV-infected chickens at PID 3 with a mean bursal lesions score of 3.25 ± 0.50
whereas in vIBDV-infected group, the lesion score at PID 3 was 1.75 ±0.50. At PIDs 5
and 7, the lesion score increased to 3.75 ± 0.50 and 4 ± 00 respectively in cIBDVinfected bursa. In vIBDV-infected bursa, lesion score was 3.25 ± 0.50 and 3.75 ± 0.50 at
PIDs 5 and 7, respectively (Fig. 3.1B).
Detection of IBDV-antigen, T cells and macrophages in virus-infected bursa:
We detected viral antigen, T cells and macrophages in cIBDV- and vIBDV-infected
bursa at PID 3, 5 and 7 by immunohistochemistry. Number of IBDV antigen positive
cells was significantly higher (P<0.05) in cIBDV-infected bursa as compared to bursa of
vIBDV-infected chickens at all the PIDs tested (Fig. 3.2A and B). Similar to viral antigen
positive cells, we observed significantly higher (P<0.05) number of infiltrating T cells in
the bursa of cIBDV-infected chickens as compared to vIBDV-infected chickens at PID 3
and 5 (Fig. 3.3A and B). However, both viral strains induced infiltration of similar
number of macrophages in the bursa which was maximum at PID 5 (Fig. 3.4A and B).
74
These results indicate that cIBDV replicated extensively in the bursa and induced more
pronounced infiltration of T cells as compared to vIBDV.
Virus induced expression of innate cytokines:
Both vIBDV and cIBDV induced innate cytokine response in virus infected chicken
bursas. The innate cytokine IFN-α was upregulated at PID 3, 5 and 7 in the bursa of
vIBDV-infected chickens (Fig. 3.5A). In cIBDV-infected chickens, IFN-α was
downregulated at PID 3 and upregulated at PID 5 and 7 (Fig. 3.5A). IFN-β was
upregulated in cIBDV- and vIBDV-infected chickens at PIDs 3, 5 and 7 (Fig. 3.5B).
Expression of proinflammatory cytokine, mediator and chemokines in IBDVinfected bursa:
Classical strains of IBDV are known to induce a strong inflammatory response in the
bursa. In this study, we detected stronger upregulation in the gene expression of IL-6 in
the bursa of cIBDV-infected chickens than vIBDV-infected chickens during the early
stage of infection (Fig. 3.6A). Similarly, cIBDV induced significantly higher (P<0.05)
expression of iNOS as compared to vIBDV at PID 3 (Fig. 3.6B). Expression of the
chemokine IL-8 was also higher in cIBDV-infected bursa at PID 3 (Fig. 3.6C). MIP-α
expression was significantly elevated (P<0.05) in cIBDV-infected chickens at PID 3 and
substantially elevated at PID 7 as compared to vIBDV-infected chickens (Fig. 3.6D).
These data suggest that both viral strains activate proinflammatory cytokine and
chemokine responses which are more pronounced in cIBDV-infected chickens.
75
Expression of toll like receptors and adaptor molecules in virus-infected bursa:
The gene expression data of TLRs and their adaptor molecules are illustrated in Fig. 3.7.
We noted downregulation in the expression of TLR3 in vIBDV-infected bursa at all PIDs
tested (Fig. 3.7A). However, in cIBDV-infected bursa, TLR3 gene expression was
significantly upregulated at PID 3 and 5 as compared to vIBDV, and it was
downregulated at PID 7 (Fig. 3.7A). The expression level of TLR 7 showed a general
trend of downregulation during the early stage of infection in chickens infected with
either viral strain (Fig. 3.7B).
Expression of TRIF was upregulated in both cIBDV- and vIBDV-infected chickens at all
PIDs (Fig. 3.7C). The gene expression of MyD88 in vIBDV-infected bursas was
downregulated at all time points. MyD88 was upregulated at PID 3 and downregulated at
PID 5 and 7 in cIBDV-infected chickens (Fig. 3.7D).
3.5 DISCUSSION
Innate immune responses orchestrate the antiviral activities through the proliferation of
different effector cells such as macrophages, T-cells and effector molecules such as
cytokines, chemokines and Toll like receptors. In this study, while comparing the
pathogenesis of cIBDV and vIBDV, we noted that cIBDV produced more pronounced
bursal damage as compared to vIBDV during early stages of the infection. Both viral
strains induced pronounced infiltration of macrophages and T cells in the bursa of
infected chickens. The expression of the proinflammatory cytokines and chemokines in
cIBDV-infected bursa was significantly higher than vIBDV-infected bursa. Importantly,
76
we observed that cIBDV infection upregulated the expression of TLR3 whereas vIBDV
infection downregulated its expression.
Following infection, IBDV replicates extensively in B cells and causes bursal atrophy
(Sharma et al., 2000). This study revealed that there were distinct differences in the early
pathogenic events of the classical and variant strains of IBDV. Although both viruses
caused bursal atrophy and lymphoid cell depletion, cIBDV replicated extensively in the
bursa and induced more bursal lesions. Bursal lesions in cIBDV-infected bursa were
accompanied by infiltration of inflammatory cells and well pronounced plical edema. In
contrast, bursal lesions induced by vIBDV were accompanied by thickening of the intra
follicular septum and a less marked inflammatory response. We also noted the inhibition
of the expression of the antiviral cytokine IFN-α in cIBDV-infected chickens early in the
infection. The lack of IFN-α early in the infection would provide an opportunity for the
virus to establish an infection. In a previous study (Eldaghayes et al., 2006), inhibition of
IFN-α in virulent IBDV-infected chickens was also observed.
Although antibody mediated immunity plays an important role, it has been demonstrated
that cell mediated immunity is also crucial against IBDV infection. This study compared
for the first time the infiltration of T cells and macrophages in cIBDV- and vIBDVinfected bursa. The infiltration of T cells was significantly higher (P<0.05) in cIBDVinfected bursa during the early stages of the infection. Previously, it was shown that a
virulent strain of IBDV induced extensive infiltration of T cells as compared to an
avirulent strain (Rautenschlein et al., 2003). In our study, T cell infiltration was higher in
cIBDV-infected chickens and cIBDV appeared to be more virulent (based on bursal
77
lesions score). These findings indicate that infiltration of T cells may be related to the
virulence of the virus strain. The T cells are hypothesized to mediate the virus clearance
and may also be responsible for the exacerbated bursal lesions (Rautenschlein et al.,
2002a). Higher infiltration of T cells in cIBDV-infected chickens may have contributed
to the enhanced bursal damage observed during the early stage of the infection. Although
not tested in this study, both classical and variant strains caused functional impairment of
T cells and T cells responded poorly to mitogens in vitro (Sharma et al., 1989).
Macrophages are the central effector cells of the innate immune system. Cytokines
produced by innate immune cells influence the nature of the adaptive immune response
(Mogensen et al., 2004). We observed the recruitment of macrophages in cIBDV- and
vIBDV-infected bursa. Previously, we have shown that macrophages from IBDVinfected chickens produce proinflammatory cytokine (Khatri et al., 2005). In the present
study, expression of the proinflammatory cytokine IL-6 and mediator iNOS was
significantly upregulated in cIBDV-infected chickens as compared to vIBDV-infected
chickens. The increased expression of IL-6 and iNOS correlated well with the previous
reports where based on histological observations, it was shown that variant IBDV
induced mild inflammatory response as compared to classical IBDV (Ismail and Saif,
1991; Sharma et al., 1989).
The mechanisms responsible for the recruitment of
macrophages to the bursa of IBDV-infected chickens are not known. However, it is likely
that the chemokines, IL-8 and MIP-α, may have a role in this regard. Chicken IL-8 acts as
a chemoattractant for heterophils and monocytes (Barker et al., 1993). In this study, we
found that the expression of these chemokines was significantly upregulated in cIBDV78
infected birds and slightly upregulated in vIBDV-infected birds. Although the number of
macrophages in cIBDV- and vIBDV-infected bursa was the same, cytokine and
chemokine data suggest that macrophages were likely to be more activated in cIBDVinfected chickens. Activated macrophages in chickens are known to be a source of proinflammatory cytokines (Khatri et al., 2005; Palmquist et al., 2006).
The TLRs have been established to play a pivotal role in the activation of innate
immunity by recognizing specific patterns of microbial components. In the present study,
we demonstrate for the first time, the induction of TLRs and their adaptor proteins, TRIF
and MyD88 in IBDV-infected chickens. TLR3 and TLR7 are the only TLRs implicated
in antiviral responses in chickens (Hghihghi et al., 2010).
Strikingly, TLR3 was
downregulated in vIBDV-infected bursa whereas it was upregulated in cIBDV-infected
bursa. In addition to TLR3, melanoma differentiation-associated gene 5 (MDA5) and
retinoic acid inducible gene I (RIG-I) are also receptors for dsRNA (Andrejeva et al.,
2004; Yoneyama et al., 2004). The MDA5 and RIG-1 activate IRF3 and NF-κB for the
induction of innate immunity. Previously, we have shown that NF-κB regulates IBDVinduced cytokine production (Khatri and Sharma, 2006). Therefore, it is possible that
vIBDV induced activation of the innate response may be MDA5/RIG1 mediated.
Previously, TLR3 upregulation was shown in chickens infected with very virulent MDV
and H5N1 avian influenza infection (Abdul-Careem et al., 2009; Karpala et al., 2008).
The expression of TLR3 adaptor protein TRIF was upregulated in cIBDV- and vIBDVinfected bursa. Further in vitro studies using a siRNA approach will be needed to
79
delineate the role of TRIF-dependent or independent TLR3/MDA5/RIG1-mediated innate
immune activation by cIBDV and vIBDV.
While examining the expression of TLR7 in infected bursas, we found that TLR7 gene
expression was downregulated in vIBDV-infected bursa. However, in cIBDV-infected
bursas TLR7 gene expression was upregulated. TLR7 primarily acts as a receptor for
ssRNA; however, dsRNA duplexes of 19–21 bp in length can activate mammalian TLR7
(Heil et al., 2004; Ward et al., 2005). It is possible that the dsRNA genome of IBDV may
activate avian TLR7. In our study, MyD88 was downregulated in cIBDV- and vIBDVinfected bursa. Previously, it was shown that dsRNA-triggered, TLR3-mediated signaling
is independent of MyD88 (Jiang et al., 2004).
In conclusion, this study reports striking differences in the pathogenesis and activation of
host responses by cIBDV and vIBDV. Compared to vIBDV, cIBDV produced more
pronounced bursal damage, accumulation of T cells, and inflammatory response (IL-6
and iNOS expression). Further studies are needed to identify the role of viral proteins
responsible for mediating the differential host responses against classical and variant
IBDV and the cellular source of cytokines and chemokines produced in the bursa. The
findings of this study provide new insights that could be useful for the design of effective
vaccines against classical and variant IBDV strains.
3.6 ACKNOWLEDGEMENTS
We acknowledge the salaries and research support provided by state and federal funds
appropriated to the Ohio Agriculture Research and Development Center, The Ohio State
University. We are grateful to Dr. Juliette Hanson, Gregory Myers and Kingsly Berlin for
80
their help in animal work. We acknowledge Dr. Michele Williams for critically reviewing
the manuscript.
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85
Table 3.1. Sequence of primers for quantitative real-time RT-PCR analysis
Target
sequence
Primers
Reference
28 S
IL-6
iNOS
IL-8
IFN-α
IFN-β
TLR3
TRIF
TLR7
MyD88
Forward 5-GGCGAAGCCAGAGGAAACT-3
Reverse 5-GACGACCGATTTGCACGTC-3
Forward 5-GCTCGCCGGCTTCGA-3
Reverse 5-GGTAGGTCTGAAAGGCGAACAG-3
Forward 5-GAGTGGTTTAAGGAGTTGGATCTGA3
Reverse 5-TTCCAGACCTCCCACCTCAA-3
Forward 5-GCCCTCCTCCTGGTTTCA G-3
Reverse 5-TGGCACCGCAGCTCATT-3
Forward 5-GACAGCCAACGCCAAAGC
Reverse 5-GTCGCTGCTGTCCAAGCATT
Forward 5-CCTCCAACACCTCTTCAACATG
Reverse 5-TGGCGTGCGGTCAAT
Forward 5-TCAGTACATTTGTAACACCCCGCC-3
Reverse 5-GGCGTCATAATCAAACACTCC-3
Forward 5-GCTGACCAAGAACTTCCTGTGC-3
Reverse 5-AGAGTTCTCATCCA AGGCCACC-3
Forward 5-TTCTGGCCACAGATGTGACC-3
Reverse 5-CCTTCAACTTGGCAGTGCAG-3
Forward 5-AGAAGGTGTCGGAGGATGGT-3
Reverse 5-GCTGGATGCTATTGCCTCGCTG-3
86
(Sijben et al.,
2003).
(Sijben et al.,
2003).
(Jarosinski et al.,
2002).
(Sijben et al.,
2003).
(Eldaghayes et al.,
2006)
(Eldaghayes et al.,
2006).
(Hghihghi et al.,
2010).
(Hghihghi et al.,
2010).
(Hghihghi et al.,
2010).
(Hghihghi et al.,
2010).
Fig.3.1:
Histopathological lesions in bursa following IBDV-infection. (A) bursa of virus free
control chickens at PIDs 3, 5 and 7
(a, b and c; 20 X); bursa of vIBDV-infected
chickens at PIDs 3, 5 and 7 (d, e and f; 20 X); bursa of cIBDV-infected chickens at 3, 5
and 7 PID (g, h and I; 20 X) (note mild to moderate follicular depletion in d, e, and f and
severe follicular depletion in g, h and i). (B) Bursal lesions score of follicular depletion in
virus free control, vIBDV and cIBDV-infected chickens. * indicates statistically
significant differences between vIBDV- and cIBDV-infected groups (p < 0.05).
87
(A)
vIBDV
Virus free
cIBDV
d
g
b
e
h
c
f
i
PID 7
PID 5
PID 3
a
(B)
5
PID 3
Lesion score
4
PID 5
*
PID 7
3
2
1
0
virus free
vIBDV-Infected cIBDV-Infected
Figure 3.1. Histopathological lesions in bursa following IBDV-infection.
88
Fig.3.2:
IBDV antigen detection by immunohistochemical staining in virus-infected bursa. SPF
chickens were inoculated with 104EID50 of cIBDV or vIBDV and bursal tissues were
collected at PIDs 3, 5 and 7. Bursal sections from virus free chickens (a, b and c; 20X),
vIBDV-infected chickens (d, e, and f; 20X) and cIBDV-infected chickens (g, h and I;
200X) were examined for the presence of IBDV antigen by immunohistochemistry using
mouse anti IBDV antibody. Brown color indicated by arrow represents the positive
staining. (B) IBDV positive cells were counted (40X) at PID 3, 5 and 7. The values
represent the mean ±SEM of 5 fields/bursa/chicken on designated PID. * indicates
statistically significant differences between vIBDV- and cIBDV-infected groups (p <
0.05).
89
(A)
Virus free
d
g
PID 3
a
cIBDV
vIBDV
e
c
f
h
PID 5
b
PID 7
i
Mean Antign+cells / microscopic field
(B)
200
*
PID 3
PID 5
150
*
PID 7
*
100
50
0
vIBDV-infected
cIBDV-infected
Figure 3.2. IBDV antigen detection by immunohistochemical staining in virus-infected
bursa.
90
Fig.3.3:
T cells infiltration in the IBDV-infected bursa. SPF chickens were inoculated with
104EID50 of cIBDV or vIBDV and bursal tissues were collected at PID 3, 5 and 7. Bursal
sections from virus free chickens (a, b and c; 20X), vIBDV-infected bursa (d, e, and f;
20X) and cIBDV-infected bursa (g, h, and i; 20X) were examined for the presence of T
cells by immunohistochemistry using anti-chicken CD3+ monoclonal antibody. Brown
color indicated by arrow represents the positive staining. (B) T cells were counted (40X)
at PID 3, 5 and 7. The values represent the mean ±SEM of 5 fields/bursa/chicken on
designated PID. * indicates statistically significant differences between vIBDV- and
cIBDV-infected groups (p < 0.05).
91
(A)
cIBDV
vIBDV
Virus-free
d
gg
b
e
h
PID 5
PID 3
a
c
i
PID 7
f
(B)
Mean Tcells / microscopic field
200
*
PID 3
150
PID 5
PID 7
*
100
50
0
Virus-free
vIBDV-infected
cIBDV-infected
Figure 3.3. T cells infiltration in the IBDV-infected bursa.
92
Fig. 3.4:
Infiltration of macrophages in the IBDV-infected bursa. SPF chickens were inoculated
with 104EID50 of cIBDV or vIBDV and bursal tissues were collected at PID 3, 5 and 7.
Bursal sections from virus free chickens (a, b and c; 20X), vIBDV-infected chickens (d,
e, and f; 20X) and cIBDV-infected chickens (g, h, and i; 20X) were examined for the
presence of macrophages by immunohistochemistry using anti-chicken macrophage
antibody. Brown color indicated by arrow represents the positive staining. (B)
Macrophage positive cells were counted (20X) at PIDs 3, 5 and 7. The values represent
the mean ±SEM of 5 fields/bursa/chicken on designated PID. * indicates statistically
significant differences between vIBDV- and cIBDV-infected groups (p < 0.05).
93
(A)
vIBDV
Virus free
cIBDV
d
g
b
e
h
PID 5
PID 3
a
(f)
c
f
PID 7
i
Mean Macs +cells / microscopic field
(B)
100
PID 3
75
PID 5
PID 7
50
25
0
control group
vIBDV-infected
cIBDV-infected
Figure 3.4. Infiltration of macrophages in the IBDV-infected bursa.
94
Fig.4.5: Relative gene expression at mRNA level, of innate cytokines mRNA in IBDVinfected bursa. At PIDs 3, 5 and 7, bursal mononuclear cells were isolated from cIBDVor vIBDV-infected and virus-free control chickens and examined for (A) IFN-α and (B)
IFN-β gene expression by qRT-PCR. Results are shown as transcription of the target
gene relative to housekeeping gene 28S. The data is expressed as fold change expression
in infected chickens over virus-free control. The values represent the mean ±SE of 4
pools of 3 bursa each at designated PID. * indicates statistically significant differences
between cIBDV- and vIBDV -infected groups (p < 0.05).
95
Fig. 5.
(A)
Relative fold change of IFN- 
10
-10
PID 3
-30
PID 5
PID 7
-50
-70
*
vIBDV-infected
cIBDV-infected
(B)
Relative fold change of IFN-
8
PID 3
PID 5
6
PID 7
4
*
2
0
vIBDV-infected
cIBDV-infected
Figure 3.5. Relative gene expression at mRNA level, of innate cytokines in IBDVinfected bursa.
96
Fig.3.6: Relative gene expression at mRNA level, of proinflamatory cytokines, iNOS and
chemokines, mRNA in IBDV-infected bursa. At PIDs 3, 5 and 7, bursal mononuclear
cells were isolated from cIBDV, vIBDV and virus-free control chickens and examined
for (A) IL-6 (B) iNOS, (C), IL-8 and (D) MIP-α gene expression by qRT-PCR. Results
are shown as transcription of the target gene relative to housekeeping gene 28S. The data
is expressed as fold change expression in infected chickens over virus-free control. The
values represent the mean ±SE of 4 pools of 3 bursa each at designated PID. * indicates
statistically significant differences between vIBDV and cIBDV virus infected groups (p <
0.05).
97
Figure 3.6. Relative gene expression at mRNA level, of proinflamatory cytokines, iNOS
and chemokines, in IBDV-infected bursa.
98
Fig. 3.7: Relative gene expression at mRNA level, of Toll like receptors and adaptor
molecules mRNA in IBDV-Infected bursa. At PIDs 3, 5 and 7, bursal mononuclear cells
were isolated from cIBDV, vIBDV and virus-free control chickens and examined for (A)
TLR3, (B) TLR7, (C) TRIF and (D) MyD88 gene expression by qRT-PCR. Results are
shown as transcription of the target gene relative to housekeeping gene 28S. The data is
expressed as fold change expression in infected chickens over virus-free control. The
values represent the mean ±SE of 4 pools of 3 bursa each at designated PID. * indicates
statistically significant differences between vIBDV- and cIBDV-infected groups (p <
0.05).
99
Figure 3.7. Relative gene expression at mRNA level, of Toll like receptors and adaptor
molecules in IBDV-Infected bursa.
100
CHAPTER 4
EXPRESSION OF PERFORIN-GRANZYME PATHWAY GENES IN
THE BURSA OF INFECTIOUS BURSAL DISEASE VIRUS-INFECTED
CHICKENS
4.1 SUMMARY
Infectious bursal disease (IBD) is an economically important immunosuppressive disease
of chickens. The IBD virus (IBDV) actively replicates in B cells and causes severe bursal
damage. Generally, T cells are refractory to infection with IBDV but are known to
promote virus clearance. However, the mechanisms of T cell mediated viral clearance are
not well understood. In this study, we evaluated the molecular mechanisms of cytotoxic T
cell responses in the pathogenesis of IBD in chickens. Infection of chickens with IBDV
was accompanied by the infiltration of CD4+ and CD8+ T cells in the bursa. There was an
upregulation in the gene expression of important cytolytic molecules; perforin
(PFN), granzyme-A (Gzm-A), DNA repair and apoptotic proteins; high mobility proteins
group (HMG) and poly (ADP-ribose) polymerase (PARP) in the bursa of Fabricius (BF)
whereas expression of NK (natural killer) lysin was downregulated. Importantly, PFN
101
producing CD4+ and CD8+ T cells were also detected in the bursa of IBDV-infected
chickens by immunohistochemistry. The Th1 cytokines, IL-2 and IFN-γ expression was
also strongly upregulated, suggesting the activation of T cells. The findings of this study
highlight the mechanisms of IBD pathogenesis and the role of cytotoxic T cells in the
clearance of virus-infected cells.
4.2 INTRODUCTION
Infectious bursal disease is an important immunosuppressive viral disease of chickens
which causes substantial economic losses to poultry producers around the world. The
IBD virus belongs to the family Birnaviridae and has a polyploid, bisegmented genome.
The virus preferentially replicates in IgM-bearing B cells in the bursa of Fabricius (BF).
The acute phase of the disease lasts for about 7 to 10 days. During this phase, bursal
follicles are depleted of B cells and the BF becomes atrophic (Sharma et al., 2000).
A strong antibody response against IBDV is considered important in defense against
virulent virus. However, it is well established that antibody alone is not sufficient to
provide complete protection. T cells rapidly infiltrate the BF starting at an early stage of
virus infection (Tanimura and Sharma, 1997). Colocalization of T cells with replicating
virus suggested that T cells may be involved in the host defense. Also, chickens rendered
deficient in antibody production by chemical bursectomy recover fully from infection and
develop an anamnestic cellular immune response (Rautenschlein et al., 2002b). Studies
with
surgical
and
chemical thymectomy
have
indicated
that
inactivated IBDV also requires T cell involvement (Yeh et al., 2002).
102
protection
by
Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells play central roles in the
immune response against virus-infected or transformed cells. Cytotoxic cells kill target
cells by releasing a membrane-disrupting protein known as perforin (PFN) and
granzymes (Gzms) secretion by exocytosis and induce apoptosis of the target cell (Smyth
and Trapani, 1995; Trapani and Smyth, 2002). The granule-exocytosis pathway activates
cell death through either caspase-dependent or caspase-independent pathways (Sarin et
al., 1997; Trapani et al., 1998; Trapani and Smyth, 2002). Another well recognized
mechanism of cell cytotoxicity which functions independent of PFN is the engagement of
Fas ligand triggering the caspase-dependent pathway of apoptosis (Nagata and Golstein,
1995; Van Parijs and Abbas, 1996). In the granule exocytosis pathway, Gzm-A causes
DNA damage through activation of the SET (endoplasmic reticulum-associated)
complex. The cleavage of the nucleosome assembly protein SET and the DNA-binding
protein, high mobility group-2 (HMG-2) by Gzm-A interferes with DNA replication,
repair, and transcription. Poly (ADP-ribose) polymerase (PARP) which primarily
regulates the caspase-dependent cell lysis pathway, also interacts with the SET complex
and depletes the cellular energy stores at the time of DNA fragmentation to further
facilitate apoptosis (Mullbacher et al., 1999; Trapani and Smyth, 2002) .
Not much is known about the mechanisms of cytotoxic T cell responses against IBDV in
chickens. Previously, gene expression of Gzm-A and PFN has been reported in Marek’s
disease virus (MDV)-infected chickens (Sarson et al., 2008). In this study we have
demonstrated the infiltration of T cell subsets in the BF, gene expression of PFN, Gzm-A
and molecules involved in DNA repair and apoptosis and the presence of PFN producing
103
CD4+ and CD8+ T cells in IBDV-infected bursa. Importantly, IL-2 and IFN-γ were also
detected in cells isolated from IBDV-infected bursa suggesting that activated T cells may
be involved in antiviral immunity and mediation of virus clearance from the BF. The
findings of this study will help in understanding the role of T cells in the pathogenesis of
IBD and may help in the design of better vaccines effective against both classical and
variant IBDV.
4.3 MATERIALS AND METHODS
Chickens and virus: Specific pathogen free (SPF) chicken eggs (Charles River
Laboratories Inc. Wilmington, MA, USA) were incubated and hatched at our facilities at
The Ohio Agriculture Research and Development Center, The Ohio State University.
The chickens were kept in a disease containment building that had rooms supplied with
HEPA filtered intake and exhaust air. At 3-weeks of age chickens were transferred to
hard sided isolators supplied with HEPA filters and exhaust air. The classical IBDV
strain STC (Abdel-Alim and Saif, 2001a) maintained in our laboratory was used to infect
chickens.
Experimental design: Forty-eight 3 weeks-old chickens were allotted into 2 groups; 12
chickens were used as virus free controls and 36 chickens were inoculated intraocularly
each with 104 embryo infectious dose50 (EID50)/200 µl of IBDV (Abdel-Alim and Saif,
2001b). At post-inoculation days (PIDs) 3, 5 and 7, twelve chickens from virus infected
group and 4 chickens from the virus free group were euthanized and BF was collected.
Bursas were examined for the following: (a) gross lesions, (b) quantification of viral load
by quantitative real time RT-PCR (qRT-PCR) and viral genome detection by RT-PCR,
104
(c) isolation of mononuclear cells and expression of PFN, Gzm-A, HMG, PAPR and, IL2 and
IFN-γ gene by qRT-PCR and (d) detection of T cell subsets and PFN by
immunohistochemistry.
Isolation of bursal mononuclear cells: Twelve bursa collected at each PID were divided
into 4 pools of three bursa each from infected chickens. BF was also collected from 4
virus-free control chickens at each PID. Mononuclear cells were isolated from the BF as
previously described (Khatri et al., 2005; Pertile et al., 1995). Briefly, a mononuclear
cells suspension was prepared from BF by density gradient centrifugation over FicollHypaque (GE healthcare Bio-Sciences Uppsala Sweden) (gradient density 1.090) and
washed twice in cold RPMI 1640 (Gibco, Carlsbad). The cell pellet was lysed by adding
Trizol reagent (Invitrogen, Carlsbad, CA) and stored at -70 OC for RNA extraction.
RNA extraction and qRT-PCR: Total RNA from bursal mononuclear cells of IBDVinfected and virus free control chickens was extracted using the Trizol reagent
(Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Quantitative RTPCR (qRT-PCR) was used to examine the gene expression of PFN, Gzm-A, HMG,
PARP, NK lysin, IL-2 and IFN-γ using previously published gene specific primers
(Eldaghayes et al., 2006; Sarson et al., 2008). Two-fold dilutions of total RNA were used
to standardize gene amplification. Fifty nanograms of total RNA was found to be optimal
and used throughout. qRT-PCR was performed using Power SYBER Green RNA – toCT 1 step RT-PCR kit (Applied Bio System, Foster City CA). Amplification and
detection were performed in an automated 7500 Real time RT-PCR system (Applied bio
system, Foster city CA) (Khatri et al., 2005).
105
Quantitation of the mRNA was determined by the comparative cycle threshold (CT)
method. Bursal mononuclear cells from virus-free control chickens were used as the
reference sample (calibrator) at each time point. The relative change in mRNA
concentration (∆CT) for each test sample was then determined from the difference
between the calibrator CT and the CT of each test sample. Each sample was run in
duplicate. Fold increase of 28S, PFN, Gzm-A, HMG, PARP and NK lysin transcript
expression over uninfected controls was calculated with the 2--∆∆CT method (Giulietti et
al., 2001; Khatri et al., 2005; Palmquist et al., 2006).
RT-PCR to detect IBDV genome: Total RNA from bursal mononuclear cells of IBDVinfected and virus free control chickens was extracted using the Trizol reagent
(Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. One step RTPCR kit AgPath-IDTM (Applied bio system, Foster city CA) was used for the
amplification of IBDV genome using previously published primers and probe (Peters et
al., 2005). Serial tenfold dilutions of known tittered IBDV-STC strain were used to
generate the standard curve and CT values of the test samples were used to estimate EID50
values.
Detection of T cell subsets and PFN in IBDV-infected BF: The T cells subsets, CD4+
and CD8+ T cells and PFN were detected in frozen sections of virus-free and IBDVinfected bursal tissues by immunohistochemistry (Brewer et al., 2004; Khatri et al.,
2005). Briefly small pieces of bursal tissue were embedded in tissues– Tek O.C.T
compound (Sakura Finetek, CA, USA) using cryomold standard disposable vinyl
specimen molds (25 mm x 20mm x 5mm) (Sakura Finetek, CA, USA) and stored at -70
106
o
C for 30 minutes to obtain optimal cutting temperature for frozen sections. The frozen
sections were cut to a thin layer of tissue (5 μm thick) with the help of cryostat
microtome (Leica CM 1510S) and spotted on double positive glass slides and dehydrated
at 37 oC overnight. The dehydrated sections were fixed with chilled acetone (75%
acetone and 25% ethyl alcohol) for 5 minutes at -20 OC followed by 3 washing steps with
PBS (phosphate buffered saline). The tissues sections were then blocked with 2 % goat
serum for 1 hour. The primary and secondary antibodies used for the detection of CD4+
and CD8+ T cells were: mouse anti-chicken CD4 and CD8α at 1:200 dilutions (Southern
Biotech, Birmingham, AL, USA) and biotinylated goat anti-mouse IgG (H+L) 1:500
dilution (Vector laboratories, Burlingame, CA). After washing, the sections were
incubated with ABC reagent (Vector laboratories, Burlingame, CA) for 30 min. The
primary and secondary antibodies used for the detection of PFN staining were: polyclonal
rabbit anti-human PFN antibody (1:50) (BioVision research products, CA, USA) and
goat polyclonal anti rabbit IgG (H+L)–HRP at 1:400 dilutions (Abcam Inc; Cambridge
MA, USA). The reaction was developed with 3-3′diaminobenzidine (DAB) substrate. The
dark brown chromogens detected on the tissues were considered as positive CD4+ and
CD8+ T cells and PFN cells respectively. The immunostained sections were evaluated
using Olympus 1X 70 microscope (Olympus Optical Co., Ltd., Tokyo, Japan). The
number of CD4+ and CD8+T cells and PFN positive cells were counted as previously
described (Rautenschlein and Haase, 2005). Briefly the group means of the numbers of T
cells and PFN-positive cells were determined per microscopic fields, at a magnification
of 20X after counting 5 fields/BF/bird.
107
PFN producing CD4+ or CD8+ T cells in the bursa were detected by double staining
(Brewer et al., 2004). In double staining, PFN staining was developed with DAB (brown
color) and CD4+ or CD8+ T cells staining was developed by incubating the sections with
commercial Vector-SG peroxidase substrate kit (grey blue color) (Vector laboratories,
Burlingame, CA).
Statistical analysis: Student’s t test was used to detect significant differences between
virus-free and IBDV-infected chickens. P<0.05 was considered to be statistically
significant.
4.4 RESULTS
Response of chickens to IBDV: Inoculation of chickens with IBDV resulted in a typical
IBDV infection. Two chickens out of 36 in IBDV-inoculated group died at PID 3. The
IBDV infection was confirmed by gross bursal lesions at necropsy and by virus
quantification and detection of viral genome in the BF (Fig. 1A and B). Based on a
standard curve developed for known titered IBDV, the CT values obtained from qRTPCR were converted into log10 EID50. The virus titer in BF at PID 3, 5 and 7 was 6.59 ±
0.16, 6.2 ± 0.51 and 6.02 ± 0.37 EID50/50μl (Fig. 4.1A).
Expression of PFN, Gzm-A and molecules involved in DNA repair and apoptosis in
bursal mononuclear cells: The gene expression of PFN, Gzm-A, HMG and PARP in
bursal mononuclear cells was detected by qRT-PCR. Expression of PFN was upregulated
early in the infection at PIDs 3 and 5 and was downregulated (3 fold) at PID 7 (Fig.
4.2A), whereas upregulated expression of Gzm-A was detected at all PIDs tested and was
at peak at PID 7 (Fig. 2B). Similarly, HMG gene expression was upregulated at PIDs 3, 5
108
and 7 with peak upregulation at PID 5 (75 fold) (Fig. 4.2 C). PARP gene expression was
downregulated early at PID 3 (3 fold) whereas it was 20- and 34- fold up regulated at PID
5 and 7, respectively (Fig. 4.2 D). The gene expression of NK lysin was 30-, 40- and 45fold downregulated at PIDs 3, 5, and 7 respectively (Fig. 4.2 E).
Detection of CD4+, CD8+ T cells and PFN in IBDV-infected BF: At PIDs 3, 5 and 7, T
cell subsets; CD8+ and CD4+ T cells and PFN were detected in the BF of IBDV-infected
chickens by immunohistochemistry (Figs. 3A, 4A and 5A). The highest numbers of CD8+
cells were found on PID 7 (44.8 ± 9.8) whereas at PIDs 3 and 5 the numbers of CD8+
positive cells were 39.7 ± 13.5 and 33.7 ± 11.8, respectively (Figure 3 B). The highest
numbers of CD4+ cells were found at PID 3 (37.2 ± 3.1) whereas at PIDs 5 and 7 the
numbers of CD4+ positive cells were 28 ± 3.1 and 22.8 ± 5.1, respectively (Fig. 4.4 B).
The highest numbers of PFN positive cells were found on PID 7 (24.9 ± 3.6) whereas at
PIDs 3 and 5 the numbers of PFN positive cells were 16.2 ± 5.5 and 24.6 ± 3.1,
respectively (Fig. 4.5 D).
We also detected the PFN producing CD4+ and CD8+ T cells by double staining in
IBDV-infected bursa (Fig. 4.5 B and 4.5 C).
Expression of Th-1 cytokines IBDV-infected BF: To examine whether the infiltrating
T cells in the bursa are activated, we detected the expression of Th-1 cytokines in bursal
cells by qRT-PCR. Infection with IBDV resulted in increased mRNA expression of IL-2
and IFN-γ in bursal cells (Fig 4.6). The IL-2 gene expression at PIDs 3 and 5 was
upregulated whereas at PID 7 it was downregulated (Fig. 4.6A). The accumulation of
IFN-γ mRNA was detectable at PID 3 which further increased at PID 5 and 7. At PID 7,
109
bursal cells from IBDV-inoculated chickens had approximately 200-fold higher levels of
IFN-γ mRNA than bursal cells from control birds (Fig. 4.6B).
4.5 DISCUSSION
Elimination of invading pathogens requires a coordinated effort between effector
lymphocytes for containment and clearance of virus-infected cells (Trapani and Smyth,
2002). In this study, we detected extensive infiltration of CD4+ and CD8+ T cells in the
bursa of IBDV-infected chickens. We demonstrated for the first time, the upregulated
gene expression of cytolytic molecules PFN, Gzm-A, DNA repair and apoptosis
molecules, HMG and PAPR in bursal cells of IBDV-infected chickens. Most importantly,
the infiltrating CD4+ and CD8+ T cells were activated as evidenced by PFN production
and IFN-γ expression in the bursal cells.
Following infection, IBDV replicates extensively in B cells and causes bursal atrophy.
Virus replication is accompanied with accumulation of T cells in the BF at the site of
virus replication. These T cells are hypothesized to mediate the virus clearance in 7-10
days after infection. As demonstrated previously (Eldaghayes et al., 2006; Kim et al.,
2000; Tanimura and Sharma, 1997), we also noted extensive virus replication and CD4+
and CD8+ T cell accumulation in the bursa during the acute phase of infection at PIDs 3,
5 and 7. Consistent with the previous findings (Kim et al., 2000), we also observed peak
CD8+ T cell infiltration at PID 7. Similar to our findings, acute HIV infection also
induced CD4+ and CD8+ T cells infiltration in the gastric mucosa of HIV-infected
humans (Epple et al., 2010).
110
To understand the mechanisms underlying T cell mediated virus clearance from the
bursa, we examined the expression of PFN and Gzm-A. Cytotoxic T cells kill virusinfected cells through the release of lytic proteins mainly PFN and Gzms that are secreted
via exocytosis of pre-formed granules following recognition of infected targets
(Hersperger et al., 2010; Kuerten et al., 2008). We detected higher expression of cytolytic
proteins Gzm-A and PFN in bursal cells and PFN producing CD4+ and CD8+ T cells
indicating the involvement of PFN and Gzms mediated cytotoxic mechanisms for the
clearance of IBDV. In several human viral infections, CTLs play a crucial role in
clearance of the virus during the acute phase of infection. For example, in poliovirus
infection, cytotoxic T cells are activated, secrete IFN-γ in response to poliovirus antigen
and are cytotoxic via PFN and Gzm pathway (Wahid et al., 2005). Similarly, CTLs clear
the herpes simplex virus infection via PFN mediated cytotoxic mechanisms (Dobbs et al.,
2005). Although we observed the gene expression of Gzm-A, we were not able to detect
Gzm-A in IBDV- infected bursal tissues. This is most likely due to the lack of cross
reactivity of polyclonal anti-human Gzm-A antibody with chicken.
The expression of PFN was detected at PID 3 and 5 but was downregulated at PID 7
whereas Gzm-A expression was upregulated at all the PIDs tested. This differential
expression of PFN and Gzm-A may be related to different stages of CTL activation. PFN
expression has been shown to precede functional cytotoxicity by 12 to 24 hours
(Lichtenheld and Podack, 1989). In addition, PFN may induce a cell redistribution of
Gzm-A preceding apoptosis. PFN is localized together with Gzm-A in cytolytic granules
of CD8+ T cells. In mammals, a study of PFN and Gzm promotor sequences has indicated
111
that these proteins may be differentially regulated at a transcriptional level (Lichtenheld
and Podack, 1989). Signal requirements for PFN and Gzm-A mRNA induction may be
different (Salcedo et al., 1993).
We detected strong expression of HMG in bursal cells at PID 3, 5 and 7. Expression of
HMG has been detected in lymphoid tissues of chickens (Pedrini et al., 1992). As HMG
is cleaved during activation of the SET complex by Gzm-A, higher expression of HMG
was expected. Higher expression of HMG was also detected during the cytolytic phases
of MDV infection in chickens (Sarson et al., 2008). Similarly, higher expression PARP
was also observed in the virus-infected bursa. In the exocytosis pathway mediated by
Gzm-A, PARP activation results in depletion of cellular energy reserves thereby
facilitating the cell apoptosis (Pinkoski and Green, 2003). PARP protein involvement in
virus-induced apoptosis has been demonstrated in several viral infections (Mok et al.,
2007; Nargi-Aizenman et al., 2002).
Typically, CD4+ T cells are better known to provide helper functions for antigen
presenting cells and can restrict viral replication by secreting cytokines (Christensen et
al., 1999). However, there is also data that virus-specific CD4+ T cells can directly kill
infected cells (Heller et al., 2006; Misko et al., 1984). In the present study, we also
detected PFN positive CD4+ T cells in IBDV-infected bursa at all PIDs tested. In humans,
CD4+ T cells positive for the cytotoxic effector molecules PFN and Gzm-A were detected
in peripheral blood
(Appay et al., 2002). These CD4+ cytotoxic cells were fully
differentiated effector cells and some were specific for the β-herpesvirus human
cytomegalovirus. Similarly, in HIV-infected patients PFN positive virus-specific CD4+
112
cytotoxic T cells were detected during the early stage of infection (Appay et al., 2002).
Whether infiltrating PFN expressing CD4+ T cells have IBDV specific cytolytic function
is not known.
Surprisingly, in the present study, we observed downregulation of gene expression of NK
lysin, which is produced by both CTLs and NK cells. The role of NK cells in the
pathogenesis of IBDV is not clearly defined. In earlier studies, Kumar et al., (Kumar et
al., 1998) observed severe functional impairment of NK cells isolated from IBDVinfected chickens whereas Sharma and Lee (Sharma and Lee, 1983) reported no adverse
effect of IBDV infection on the NK cell activity. As we detected the PFN producing T
cells in infected bursa confirming that functional T cells are present in the virus-infected
bursa, we speculate that downregulated NK lysin is attributed to NK cells which might
not be involved in mediating the cytotoxic response against IBDV.
IFN-γ, a type II interferon, is mainly produced by activated T cells and NK cells and IFNγ secretion is widely used for the assessment of antigen specific Th1 immune responses
(Meier et al., 2003; Yuan et al., 2008). IFN-γ secretion by activated T-helper cells and
CTLs is suggestive of effective antiviral immunity. We also detected higher levels of
IFN-γ and IL-2 in the bursa of IBDV-infected chickens, suggesting that the possible
source of these Th-1 cytokines could be from activated CD4+ and CD8+ T cells. IFN-γ
production enhanced the cytotoxic activities of viral antigen specific CD8+ T cells in
influenza virus-infected mice and vaccine induced host responses against very virulent
MDV infection in chickens (Haq et al., 2010; Ishikawa et al., 2010).
113
In this study, we demonstrated the gene expression of cytolytic molecules PFN and GzmA and IFN- γ in BF of IBDV-infected chickens. These results strongly suggest that
activated cytotoxic CD4+ and CD8+ T cells mediate the clearance of IBDV infection via
the PFN and Gzm-A pathway. The findings of this study will be useful for designing
effective control strategies for IBDV based on vaccines that can augment T cell responses
in addition to an antibody response. Further studies will be needed to specifically
delineate the role of specific T cell subsets in the mediation of anti-IBDV immunity.
4.6 ACKNOWLEDGEMENTS
We acknowledge the salaries and research support provided by state and federal funds
appropriated to the Ohio Agriculture Research and Development Center, The Ohio State
University. We are grateful to Dr. Juliette Hanson, Gregory Myers and Kingsly Berlin for
their help in animal work. We acknowledge Dr. D.J. Jackwood, Dr. Anastasia Vlasova
and Dr. Michele Williams for critically reviewing the manuscript.
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Fig.4.1: Detection of virus load and IBDV genome in bursa tissue. At PIDs 3, 5 and 7,
bursal tissues were collected from IBDV-infected and virus free control chickens for the
quantification of virus load by qRT-PCR (A) and viral genome detection by RT-PCR (B).
(A) Represents the log10 virus titer at PIDs 3, 5 and 7 in IBDV-infected chickens. Values
represent the mean virus titer ±SEM of 3 chickens at each time point. (B) IBDV genome
was detected in the bursa of virus-infected chickens at PIDs 3, 5 and 7 whereas viral
genome was absent in mock-inoculated controls. Lane 1, 100 base pair marker, lane 2
positive control, lanes 3, 4 and 5 bursa samples from IBDV-infected chickens at PIDs 3,
5 and 7; and lanes 6, 7 and 8, BF samples from virus free control at PIDs 3, 5 and 7
respectively.
118
Figure 4.1. Detection of virus load and IBDV genome in bursa tissue.
119
Fig. 4.2: Gene expression of PFN, Gzm-A and molecules involved in DNA repair and
apoptosis in the bursa of IBDV-infected chickens. At PID 3, 5 and 7, bursal mononuclear
cells were isolated from IBDV-infected and virus-free control chickens and examined for
the expression of PFN (A), Gzm-A (B), HMG (C), PARP (D) and NK lysin (E) by qRTPCR. The data is expressed as fold change in gene expression in infected chickens over
virus free control. The values represent the mean ± SEM of 4 pools of 3 BF at each PID.
120
Figure 4.2. Gene expression of PFN, Gzm-A and molecules involved in DNA repair and
apoptosis in the bursa of IBDV-infected chickens.
121
Fig. 4.3: Accumulation of CD8+ T cells in the bursa. Three-weeks-old chickens were
inoculated with IBDV and frozen sections of bursa were prepared. At PIDs 3, 5 and 7
bursal sections from virus free chickens (a, b, and c) and IBDV-infected chickens (d, e,
and f) were examined for the presence of CD8+ T cells by immunohistochemistry using
anti-chicken CD8 mAb. Brown color shown by arrow indicates the positive staining. (B)
Quantification of CD8+ T cells in the bursa. CD8+ T were counted at magnification (20X)
at PID 3, 5 and 7. The values represent the mean ±SEM of 5 fields/BF/bird at each PID.
*Significantly different from the control group P<0.05.
122
Figure 4.3. Accumulation of CD8+ T cells in the bursa.
123
Fig.4.4: Infiltration of CD4+ T cells in the bursa. Three-weeks-old chickens were
inoculated with IBDV and frozen sections of bursa were prepared. (A) At PIDs 3, 5 and 7
bursal sections from virus free chickens (a, b, and c) and IBDV-infected chickens (d, e,
and f) were examined for the presence of CD4+ T cells by immunohistochemistry using
anti-chicken CD4 mAb. Brown color shown by arrow indicates the positive staining. (B)
Quantification of CD4+ T cells in the bursa. CD4+ T cells were counted (20X) at PID 3, 5
and 7. The values represent the mean ± SEM of 5 fields/BF/bird at each PID.
*Significantly different from the control group P<0.05.
124
Figure 4.4. Infiltration of CD4+ T cells in the bursa.
125
Fig. 4.5: Detection of PFN producing CD4+ and CD8+ T cells in BF. (A) Chickens were
inoculated with IBDV and bursal tissues were collected. At 3, 5 and 7 PID bursal sections
from virus free chickens (a, b and c) and IBDV-infected chickens (d, e, and f) were
examined (40X) for the presence of PFN by immunohistochemistry using anti-human
PFN mAb. Development of brown color shown by arrow indicates positive staining for
PFN. (B) Double staining for CD4+ and (C) for CD8+cells, positive for PFN; blue
staining shown by arrow head represents CD4+ or CD8+ positive T cells and brown
staining shows PFN positive cells respectively (D) The numbers of PFN positive cells
were counted (20X) at PID 3, 5 and 7. The values represent mean ± SEM of 5
fields/BF/bird at each PID. *Significantly different from the control group P<0.05.
126
Figure 4.5. Detection of PFN producing CD4+ and CD8+ T cells in BF.
127
Fig. 4.6: Expression of Th-1 cytokines in the bursa of IBDV-infected chickens at PIDs 3,
5 and 7. (A) IL-2 and (B) IFN-γ gene expression was quantified in bursal mononuclear
cells. The data is expressed as fold change in gene expression in infected chickens over
virus free control. The values represent the mean ±SEM of 4 pools of 3 BF at each PID.
128
Figure 4.6. Expression of Th-1 cytokines in the bursa of IBDV-infected chickens at
PIDs 3, 5 and 7.
129
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