U.S. patent application number 09/876849 was filed with the patent office on 2002-04-25 for poultry virus isolates and method.
Invention is credited to Newberry, Lisa A., Skeeles, John K..
Application Number | 20020048806 09/876849 |
Document ID | / |
Family ID | 26757706 |
Filed Date | 2002-04-25 |
United States Patent
Application |
20020048806 |
Kind Code |
A1 |
Skeeles, John K. ; et
al. |
April 25, 2002 |
POULTRY VIRUS ISOLATES AND METHOD
Abstract
In accordance with the present invention, numerous experiments
were conducted to determine the role of infectious bursal disease
(IBD) virus in the induction of lesions associated with
proventriculitis syndrome in chickens. Parameters examined included
age of the chicken at IBD virus exposure, concentration of IBD
virus at exposure, the strain of IBD virus, dietary influence in
the presence of IBD virus, mixed IBD virus infection, auto-immune
mediated IBD reactions associated with lesion production, viral
induced apoptotic tissue injury and isolation and characterization
of the causative agent. The experiments were carried out in SPF
white leghorns, with the experimental birds being examined for the
presence of gross and microscopic lesion at 4 and 11 days post
challenge. Tissue homogenates were analyzed for the presence of
IBDV at 4 and 11 days post challenge with Antigen Capture ELISA
(AC-ELISA). Determinations of neutralizing antibody levels and IgG
antibody responses were monitored as well as depletion of serum
complement following IBDV infection. Physical parameters were also
considered utilizing body weights and organ to body weight ratios
to determine IBDV effects following experimental challenge.
Physical parameters indicated that the primary viral response is
predominantly in the bursa of Fabricius, but changes were also
noted in the proventriculus. Physical changes in the proventriculus
occurred primarily during the acute stage of the IBD virus
infection. Gross and microscopic lesions in the proventriculus were
exacerbated by the presence of copper sulfate supplementation in
the feed. There were also very evident negative effects on the
weight gain of SPF white leghorn chickens given copper sulfate feed
supplementation. Infectious bursal disease virus strains show
different affinities for producing lesions in the proventriculus
following challenge. Standard challenge IBD virus USDA/STC stain
produced the most consistent lesions in the proventriculus often
accompanied by hemorrhage. This correlates well with antibody
enhanced pathology as observed with antigen-antibody complex
activation of serum complement. Evidence of yet another mechanism
of IBDV induced pathology, was demonstrated through the use of
TUNEL apoptosis staining of infected tissues. The degree of
apoptotic involvement was also reflected in the pathogenicity of
the IBD virus strain used for challenge. Infectious bursal disease
viral inclusions were demonstrated by thin section electron
microscopy in the proventriculus at 4 days post challenge. Viral
inclusions resemble previously documented inclusions produced in
the bursa following IBD infection. In accordance with the present
invention, infectious bursal disease viruses were isolated from
broiler chickens experiencing proventriculitis in, for example,
Oklahoma, Texas, West Virginia, and California. Proventriculitis is
a major problem to the broiler industry. Chicken virus isolates
Texas RB 3, Texas RB 4, HBS, F57-7, W/L 39, GAR 1, and the like
have been isolated. These viruses may be one of the major causes of
this condition and if this proves to be true, could be utilized as
or in a vaccine to prevent the disease condition. The viruses could
be a significant finding in the search for a causative agent for
proventriculitis in broiler chickens and as such may be utilized in
the development of a vaccine or vaccines. The viruses can be
attenuated to be used as a modified live vaccine or utilized in an
inactivated form in a killed vaccine.
Inventors: |
Skeeles, John K.;
(Fayetteville, AR) ; Newberry, Lisa A.;
(Springdale, AR) |
Correspondence
Address: |
HEAD, JOHNSON & KACHIGIAN
Suite 230
112 West Center Street
Fayetteville
AR
72701
US
|
Family ID: |
26757706 |
Appl. No.: |
09/876849 |
Filed: |
June 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60076136 |
Feb 27, 1998 |
|
|
|
Current U.S.
Class: |
435/235.1 ;
424/204.1; 435/236; 435/239 |
Current CPC
Class: |
A61K 2039/525 20130101;
C12N 7/00 20130101; C12N 2720/10021 20130101 |
Class at
Publication: |
435/235.1 ;
424/204.1; 435/236; 435/239 |
International
Class: |
A61K 039/12; C12N
007/00; C12N 007/04 |
Claims
What is claimed is:
1. A poultry virus isolate having the identifying characteristics
of an isolate selected from the group of F57-7, Texas RB 3, and
Texas RB 4.
2. A poultry vaccine utilizing at least one of the poultry virus
isolates of claim 1.
3. A vaccine suitable for immunizing chickens against
proventriculitis syndrome comprising an IBD virus having the
identifying characteristics of an isolate selected from the group
of F57-7, Texas RB 3, and Texas RB 4, and combinations or variants
thereof.
4. The vaccine of claim 3 defined further as containing an
attenuated, modified, inactivated, or killed form of the
isolate.
5. A method of protecting a chicken from clinical signs of
proventriculitis syndrome or disease caused by a virus having all
of the identifying characteristics of an isolate selected from the
group of F57-7, Texas RB 3, and Texas RB 4, comprising
administering an effective amount of the vaccine of claim 3 to at
least one of an egg or a chicken.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S.
provisional application Ser. No. 60/076,136, filed Feb. 27,
1998.
[0002] The present invention is directed to chicken virus isolates
or strains (Texas RB 3, Texas RB 4, HBS, F57-7, W/L 39, GAR 1)
believed to be a new strain or strains of infectious bursal disease
virus (IBDV) which infects the proventriculus, a vaccine made from
these isolates, a method of treatment, vaccination scheme, and the
like.
[0003] In accordance with the present invention, numerous
experiments were conducted to determine the role of infectious
bursal disease (IBD) virus in the induction of lesions associated
with proventriculitis syndrome in chickens. Parameters examined
included age of the chicken at IBD virus exposure, concentration of
IBD virus at exposure the strain of IBD virus, dietary influence in
the presence of IBD virus, mixed IBD virus infection, auto-immune
mediated IBD reactions associated with lesion production, viral
induced apoptotic tissue injury and isolation and characterization
of the causative agent.
[0004] The experiments were carried out in SPF white leghorns, with
the experimental birds being examined for the presence of gross and
microscopic lesion at 4 and 11 days post challenge. Tissue
homogenates were analyzed for the presence of IBDV at 4 and 11 days
post challenge with Antigen Capture ELISA (AC-ELISA).
Determinations of neutralizing antibody levels and IgG antibody
responses were monitored as well as depletion of serum complement
following IBDV infection. Physical parameters were also considered
utilizing body weights and organ to body weight ratios to determine
IBDV effects following experimental challenge.
[0005] Physical parameters indicated that the primary viral
response is predominantly in the bursa of Fabricius, but changes
were also noted in the proventriculus. Physical changes in the
proventriculus occurred primarily during the acute stage of the IBD
virus infection.
[0006] Gross and microscopic lesions in the proventriculus were
exacerbated by the presence of copper sulfate supplementation in
the feed. There were also very evident negative effects on the
weight gain of SPF white leghorn chickens given copper sulfate feed
supplementation.
[0007] Infectious bursal disease virus strains show different
affinities for producing lesions in the proventriculus following
challenge. Standard challenge IBD virus USDA/STC stain produced the
most consistent lesions in the proventriculus often accompanied by
hemorrhage. This correlates well with antibody enhanced pathology
as observed with antigen-antibody complex activation of serum
complement. Evidence of yet another mechanism of IBDV induced
pathology, was demonstrated through the use of TUNEL apoptosis
staining of infected tissues. The degree of apoptotic involvement
was also reflected in the pathogenicity of the IBD virus strain
used for challenge.
[0008] Infectious bursal disease viral inclusions were demonstrated
by thin section electron microscopy in the proventriculus at 4 days
post challenge. Viral inclusions resemble previously documented
inclusions produced in the bursa following IBD infection.
[0009] In accordance with the present invention, infectious bursal
disease viruses were isolated from broiler chickens experiencing
proventriculitis in. for example, Oklahoma, Texas, West Virginia,
and California.
[0010] IBDV Antigen Capture (AC) Elisa tested positive for Texas RB
3, Texas RB 4, HBS, F57-7, W/L 39, and GAR 1. Virus isolation in
SPF embryonated eggs was completed and 3rd and final passage for
the isolates. SPF chickens were inoculated with egg harvest for the
isolates. Tissues from inoculated birds and histopathology
confirmed lesions. AC-ELISA of tissue and serological confirmation
of the isolates was performed.
[0011] Proventriculitis is a major problem to the broiler industry.
These viruses may be one of the major causes of this condition and
if this proves to be true, could be utilized as or in a vaccine to
prevent the disease condition. The viruses are believed to be a
significant finding in the search for a causative agent for
proventriculitis in broiler chickens and as such may be utilized in
the development of a vaccine or vaccines. The viruses can be
attenuated to be used as a modified live vaccine or utilized in an
inactivated form in a killed vaccine.
[0012] The primary impetus for this invention came as a result of a
request for help from a number of commercial poultry processors.
Proventriculitis has been described in broiler chickens from a
number of geographical locations. This syndrome is characterized by
the enlargement of the proventriculus, with feed impaction, and
structural weakness. Affected birds can be either normal or
underweight at processing, with either a high or normal feed
conversion efficiency. Problems generally manifest at the
processing plant with breakage of the proventriculus during
mechanical evisceration, resulting in an increased number of
washouts, slowed or stopped processing lines, excessive trims, and
higher than normal levels of condemnation. This proventriculitis
syndrome has been linked to a number of environmental, nutritional,
genetic, and infectious causes.
[0013] Preliminary studies indicate that infectious bursal disease
virus is capable of producing lesions in the proventricular mucosa.
While proventriculitis induced by IBDV alone does not exactly mimic
what is observed in field situations, it is contented that IBDV
plays the role of a facilitator, and thus presents an opportunity
for other viral, bacterial, fungal or chemical pathogens to exert
an effect.
[0014] Some objects of this invention are to: (1) attempt to
establish infectious bursal disease virus as a causative agent in
the induction of lesions associated with proventriculitis, (2)
determine the pathogenic mechanism by which IBDV exerts an effect
on the proventriculus, and (3) to isolate and characterize IBDV
virus from field cases of proventriculitis.
INFECTIOUS BURSAL DISEASE VIRUS
[0015] Infectious bursal disease (IBD) is an acute viral infection
which predominantly affects domestic chickens. This viral disease
has also been described in commercial turkeys (22, 60, 81, 82,
119), and represents an occasional problem in ducks (81).
Infectious bursal disease virus infection and replication occurs
primarily in lymphoid tissues throughout the body. This results in
induction of significant pathological lesions within the follicles
of the bursa of Fabricius, which contains the highest concentration
of lymphoid cells. When infection occurs at an early age (<3
weeks), the disease syndrome may be further complicated by an
accompanying immunosuppression (33, 76, 95).
[0016] In 1962, Cosgrove (20) first described this disease syndrome
and an associated nephritis in a chicken flock in Gumboro, Del.
Early studies gave conflicting information concerning this disease.
It was later determined that the nephritis syndrome described was
actually a variant strain of infectious bronchitis virus and
another, as yet, unknown infectious agent (12). Studies of tissues
harvested from infected birds revealed the presence of virus
particles that were structurally different from infectious
bronchitis virus. The name Gumboro disease was adopted to describe
this new infectious disease syndrome (10,95).
[0017] Hitchner (50) proposed the final name of infectious bursal
disease virus to identify the etiological agent responsible for
inducing pathognomonic lesions of the cloacal bursa. It was finally
characterized as a member of the Birnaviridae family, which
reflects that it has a genome that consists of two segments of
dsRNA (10,26,76).
[0018] Infectious bursal disease virus (IBDV) infection represents
a disease of significant economic importance within the poultry
industry. Economic impact of IBDV infection is manifested by the
mortality it induces during the initial acute infection, and
secondly from the induction of an immunosuppressive syndrome in
chicks infected immediately after hatching. The virus is considered
ubiquitous in nature, with a worldwide distribution in all major
poultry producing areas with the exception of a few isolated
islands in the south Pacific (76,95,119).
[0019] Vaccination procedures for this virus are practiced
extensively by commercial producers and are such that all chickens
become seropositive to IBDV during their growout (35). Successful
vaccination programs are evident in that clinical cases of IBDV are
extremely rare. However, IBDV infections do still occur, and
usually become apparent only after the introduction of a secondary
pathogen. Modifications of the IBDV infection can occur in the
presence of maternal antibodies (135) or as a result of variant
IBDV strains that induce immunosuppression with no obvious clinical
disease (95,119).
[0020] Two serotypes of IBDV are currently recognized, they are
designated as serotype 1 and serotype 2 (81). The serotype 1 IBDV
group can be further divided into two subtypes, the so called
"Classic" and "Variant" (76). There is additional evidence of IBDV
isolates that represent a viral group that is transitional between
classic and variant (6,7,128). Serotype 1 IBDV classic viruses were
the first to be described in chickens, and as such, there is a
great deal of information available concerning their
characteristics. Variant IBDV strains of serotype 1 represent a new
faction of this viral disease syndrome. While variant strains of
serotype 1 IBDV maintain structural similarities and replication
strategies, they are capable of breaking through maternal antibody
generated against classical subtypes (110, 112). The appearance of
variants within the viral population appears to be a reflection of
the intensity of the vaccination program, with most variants
representing a viral subpopulation selected by the use of live IBDV
vaccines. Documented variant strains found within serotype 1 IBDV
show distinct sequence differences in the VP2 gene from the classic
viruses, indicating that mutations of the viral genome also
influence the emergence of variant IBDV strains (128).
[0021] Serotype 2 IBDV antibodies are found in both chickens and
turkeys, suggesting that this IBDV serotype also has a widespread
geographical distribution. This particular subtype is noted in
particular for their inability to induce detectable clinical
symptoms. The so-called a pathogenesis of IBDV serotype 2 has been
attributed to the virus lacking a selective tropism for the bursa
of Fabricius (22,99). Recent studies have also linked this a
pathogenesis to the inability of serotype 2 IBDV to utilize
macrophages for distribution of virus particles throughout the
host. This observation was made utilizing in-vitro cell culture in
which serotype 2 virus infected cells underwent a rapid lytic cycle
(138). Presence of antibodies to serotype 2 IBDV does not confer
protection against serotype 1 IBDV, although it has been determined
that both serotypes share a high degree of genetic homology
(43,59,60).
ETIOLOGY
[0022] IBDV is a classified as a member of the Birnaviridae family,
which has only one genus Birnavirus. The recognized prototype virus
in this classification is infectious pancreatic necrosis virus
(IPNV) of fish (trout). This virus family also includes Tellina
virus (bivalve mollusks, oysters)(26), Drosophila X (fruit fly)
(13), and Eel virus (fresh/saltwater eels). Cross virus
neutralization tests indicate that all the aquatic viruses within
this family are related to each other, but are different from the
avian and insect isolates (26).
[0023] Infectious bursal disease virus particles range in size from
55-60 nm, possess an external single shell, are non enveloped, and
exhibit icosahedral symmetry (26,39,48,68,101,104,119). The capsid
consists of 32 capsomeres arranged in a skewed (dextro) 5:3:2
symmetry (47,101). The G+C content is 55.3%, with a purine to
pyrimidine ratio of approximately 1. IBDV is resistant to
inactivation by ether, chloroform, pH levels between 2.0 and 12.0,
and has a T.sub.m of 95.5.degree. C., (12).
[0024] The buoyant density of complete particles was determined by
the use of cesium chloride gradients and ranges from 1.31 to 1.34
g/ml (47). Viral particles on the lower density range contain
significantly lower levels of dsRNA or are completely empty (85).
Lower density particles exhibit identical morphology but lack the
proteins of the proper size and concentration to initiate an
effective infection. Incomplete particles, are unable to initiate
infection, and constitute approximately 10% of any given Birnavirus
preparation (7,47,85,89).
PHYSIOCHEMICAL COMPOSITION
[0025] The IBDV genome of serotype 1 consists of two segments of
dsRNA (12, 88) of approximately equal size, segment A has a
molecular weight of 3.4.times.10.sup.6 and segment B has a
molecular weight of 2.8.times.10.sup.6 (10, 12 ,26, 47, 86, 95,
104, 119). Serotype 2 IBDV exhibit a difference in the size of both
segment A and B which are smaller by approximately 70 bp and 20 bp
respectively (10, 60), although there appears to be no difference
in function(11, 98). Five viral proteins have been identified for
IBDV; they are as follows with their approximate molecular weights:
VP1(90,000), VP2 (44,000), VP3 (35,000), VP4 (25,000), and VP5
(17,000) (25,119). Segment A codes for a polyprotein within a
single open reading frame(ORF) in the following order
N-VP2-VP5-VP4-VP3-C (4, 63, 83, 117). Segment B codes for a single
protein VP1 (83, 87, 103).
[0026] VP2 and VP3 are the major structural proteins (99, 130) and
collectively constitute 91% of the total viral protein. VP1 and VP4
are considered minor proteins (enzymes) and VP5 of unknown function
collectively represent the remaining 9% of viral protein. Viral
protein one (VP1) codes for a viral RNA polymerase, which is a
required element for replication of dsRNA. Since all IBDV's encode
their own viral polymerase, transcription and replication are
initiated immediately after the virus penetrates the host cell
without need for uncoating or any type of degradation of the
nucleocapsid (131). Infectious bursal disease virus utilizes the
strand displacement model of replication, with VP1 also functioning
as a viral replicase (130). Viral protein four (VP4) codes for a
viral protease which processes the large segment A polyprotein
precursor into mature viral proteins (62.63.83.98). VP5 has no
known function at this time (119).
[0027] Viral protein two (VP2) represents the major host protective
immunogen (61) and functions in virus adsorption to the host cell
(68). Variability in the VP2 amino acid sequence is found
predominantly within the antigenic binding epitope. Significant
changes in this genomic region are often associated with emergence
of variant IBDV strains (3, 4, 11, 14, 31, 69, 98).
[0028] Neutralizing antibody from the host animal is generated
against the antigenic structure within the VP2 protein (30).
Development of neutralizing antibodies in the host animal against
viral VP2 is highly conformational dependent (11). Experimental
studies utilizing renatured VP2 protein demonstrated the crucial
importance of maintaining the native state of the VP2 antigen
binding epitope. The renatured VP2 was capable of binding
neutralizing antibodies, however, antibodies generated against this
protein were not neutralizing (98).
[0029] Viral protein three (VP3) contains two conformational
independent non-overlapping binding epitopes (61, 127); one of
these epitopes is conserved across serotypes and one is distinct
for each serotype (87, 99). Antibodies specific to VP3 are the
first to appear in detectable levels in chickens following
infection (61).
[0030] Viral protein one (VP1) forms a dsRNA protein complex, by
covalently binding knoblike proteinaceous structures to the ends of
the two genomic segments, forming a circularized ring structure.
Viral protein one (VP1) exhibits the characteristics of an RNA
dependent RNA polymerase and functions also as an assembly protein.
Genome linked proteins are common in Birnaviruses (87, 103, 131),
and their presence indicates that replication of nucleic acid is by
strand displacement (semi-conservative) mechanism (13, 131) The
physiological function of VP1 protein is still unknown, although it
has indications of involvement in RNA replication as well as in
virus assembly (83, 87).
CLINICAL DISEASE
[0031] Chickens infected with IBDV exhibit trembling and ruffled
feathers with additional signs of depression and anorexia. These
symptoms are accompanied by a profuse urate-containing diarrhea
with some indication of bile stasis. The diarrhea syndrome is
further complicated by the birds refusal to eat or drink, with most
infected birds exhibiting evidence of dehydration. During acute
IBDV infection there is an initial elevation of body temperature,
which falls below normal shortly before death. Blood calcium levels
are lowered (20) and there is evidence of the presence of
additional blood clotting deficiencies (122,124).
[0032] Clinical IBDV is most commonly recognized in susceptible 3
to 6 week-old chickens. Although an early age resistance to
infection, regardless of maternal antibody has been proposed (29,
50, 100, 141). Infectious bursal disease virus infection, within
the first week of hatch leads to severe defects of humoral immune
response (49). Mortality from IBDV infection was shown to be
associated with a severe depletion of complement (123, 124), and
clotting abnormalities without changes in complement (124).
[0033] Virus persistence was measured in a number of studies which
considered the amount, and the route of virus inoculation, as well
as the age of the bird when the virus challenge was given. Virus
could be visualized, using the electron microscope, within fours
hours PI in the cecum, and within 5 hours in the duodenum and
jejunum. It was determined that IBDV could be reisolated from
infected organs within 24 hours post infection (PI), regardless of
the age of the bird, the dose given, or the route in which it was
administered. Virus could not be recovered beyond day 11, with
birds challenged at 1, 7, and 14 days of age. Birds challenged with
IBDV at 21 days of age tested positive for only 8 days (79). In
general, older birds at challenge experience shorter periods of
viremia, but have a more rapid and higher intensity immune response
(141). Bayyari et al. described extended periods of viremia
associated with increased levels of defective IBDV viral particles
following attenuation of IBDV in cell culture (7).
LABORATORY HOST SYSTEMS
[0034] Infectious bursal disease virus can be propagated in
specific pathogen free (SPF) embryonated eggs on the
chorioallantoic membrane (CAM) or by the yolk sac (YS) route at day
9 of embryonation. Chorioallantoic membrane inoculation is
considered to be the most sensitive method of virus isolation and
propagation. Embryos that become infected with IBDV show petechial
hemorrhages on the head, trunk, and feet, as well as edema within
the abdominal cavity. Internally, the livers of affected embryos
may demonstrate necrosis, but more commonly are pale and take on a
parboiled or cooked texture. The spleen is usually normal in size
with the exception of variant IBDV where there is significant
enlargement of the spleen, in both cases the spleen is generally
pale and may occasionally demonstrate necrosis. Embryonic bursae
are extremely small with occasional hemorrhage. Mortality, if it
occurs, is usually between 3 to 5 days PI, and, as a general rule,
is absent in the presence of variant IBDV strains. The highest
concentrations of the IBDV virus are found in the CAM, body and
viscera of the infected embryo (109).
[0035] Cell culture has become an increasingly economical and
efficient tool for measuring virus effects outside (in-vitro) the
host animal. A number of years ago it was determined that IBDV
could be adapted to grow in cell culture. Infectious bursal disease
virus propagation in primary cells derived from SPF chickens and/or
embryos results in the highest yield of virus particles (70).
Growth of IBDV, both classic and variant strains, has been
accomplished in the following primary cells: chicken bursal
lymphoid (CBL), chicken embryo kidney(CEK) and chicken embryo
fibroblast (CEF)(18, 46, 75, 82, 96, 126). Viral proteins of most
IBDV are present at detectable levels by 90 minutes post
inoculation (PI) in CEF cells. Virus replication then enters a
latent stage at approximately 4 hours (56), with mature virus
particles evident as early as 6 hours PI (85), and mature virus
particle release between 10-16 hours PI (59, 75, 77, 90, 94). Virus
replication in CBL cells closely parallels that of CEF cells,
however, IBDV infection of CEK cells is of extended duration (59).
Cytopathic effects induced by IBDV infection are visible by 16
hours PI (75, 94) and virus titers generally peak at 48 hours PI
(18). In addition, it has been determined that IBDV infection
activates complement in cultured lymphoid cells (71), and that IBDV
progeny virus release is followed within 2-8 hours by an increase
in the synthesis of interferon (34).
[0036] Several mammalian cell lines RK-13, VERO, BGM-70, and MA-104
will also support IBDV growth (34,42,56,75,104). Virus particle
yields are lower, and the replication cycle is prolonged when
compared to IBDV propagation in chick primary cells (56,75).
Repeated passage in these cell lines leads to loss of pathogenicity
and represent a means by which highly pathogenic IBDV can be
attenuated for utilization as vaccines (6,40,49,143).
[0037] Cell culture propagation of IBDV under conditions where
there is a high concentration of virus particles (MOI) and limited
cellular substrate will result in the production of incomplete
virus particles. Upon further passage in cell culture virus stocks
containing these defective interfering (DI) particles show reduced
virus yields and reduced pathogenicity (6,7,84). The mechanism that
triggers some IBDV viruses to produce DI particles and other IBDV
not to, is not clear at this point in time(6,89).
[0038] Cytopathic effects (CPE) of IBDV infection in cell culture
are described as plaque formation. Extensive passage in cell
culture can induce IBDV to form plaques of various sizes. It has
been determined that IBDV virus stocks which produce large plaque
CPE are more virulent than those that produce small plaque CPE
(23,73,113).
[0039] Cell culture propagation of IBDV is not without controversy.
A number of researchers have reported conflicting results from cell
culture derived IBDV stocks. especially when they are introduced
back into the chicken. These differences have raised some concern
as to whether in-vitro observations of IBDV infection correspond in
any way to what happens in-vivo , although the use of in-vitro
methods continues to be a widely accepted method of analysis for
IBDV infection (54).
VIRUS DETECTION
[0040] The presence of IBDV within infected tissues and/or the
presence of antibodies following infection can be determined with a
number of different laboratory tests. The agar gel precipitin test
(AGPT) can be used to detect IBDV group specific antibody. The test
can be both quantitative and qualitative, but is of extremely low
sensitivity, and therefore misses detection of low virus
concentrations and is only useful during the first few days of IBDV
infection (58,109).
[0041] Virus neutralization assays are highly specific for the
identification and characterization of IBDV isolates. The test is
performed utilizing primary CEF cell monolayers. Infectious bursal
disease virus of a known serotype is reacted at a constant
concentration with varying amounts of immune serum. The
antigen-antibody mixture is then inoculated onto cell monolayers
and incubated. Following incubation the cells are fixed and
stained. Endpoints are determined visually and neutralization index
titers are expressed as the reciprocal of the highest dilution of
antiserum that prevents IBDV from inducing cytopathic effect in
cell culture monolayers. Infectious bursal disease viruses can be
pathotyped using cross neutralization assays, and also quantitated
(7,32,109).
[0042] Fluorescently labeled antibodies are now readily available,
and offer a unique, yet diverse diagnostic tool. Immunofluorescent
staining of enzyme treated formalin fixed tissues using IBDV
polyclonal antiserum was capable of detecting IBDV (58,65), and
also non-specific presence of IgG complexes in the glomeruli of
IBDV infected chickens (74). Utilization of two fluorogenic labels
was also demonstrated as a means of quantitation of IBDV
replication in cell culture, as a reflection of live/dead cell
ratios (5).
[0043] The Enzyme Linked Immunosorbent Assay (ELISA) is by far the
most widely used diagnostic test in the modern poultry industry,
and is also capable of determining the presence of antibodies to
many other pathogens. The popularity of commercial ELISA kits
results from the ease of performance, accommodation of a large
number of samples, high sensitivity, and economical price.
Commercial ELISA kits for IBDV quantitate the presence of virus
specific antibodies only (109). Variations of the standard ELISA
are utilized to determine the presence of virus in IBDV infected
tissues, but remain predominantly as a research tool. The Antigen
Capture-ELISA developed by Snyder et al. (128) utilizes monoclonal
antibodies specific to the surface markers of IBDV to capture virus
from tissue homogenates. Field virus populations of IBDV were
screened and determined to be relatively homogenous falling into
three major antigen types. Antigenic types identified could be
segregated into distinct geographical regions of the United States
(128). This technology has since been modified to use polyclonal
antiserum to capture virus from infected tissues. Early indications
are that polyclonal serum may actually bind more virus than the
monoclonal antibody, due to the presence of antibodies to multiple
antigenic sites (41).
[0044] The development of a kinetic based ELISA was utilized to
determine the rate of antibody decay. Results from these assays
indicated that progeny from parents vaccinated 4 times or more had
higher rates of antibody decay than those being vaccinated only
twice (129).
[0045] Molecular biology procedures have also been refined for the
diagnosis, characterization, and identification of classic and
variant IBDV. Detection of IBDV in tissue was accomplished
utilizing a biotin labeled cDNA derived from segment A at the
VP2/VP4 junction. Infected tissues demonstrated specific staining,
however, the intensity of the stain did not correlate with virus
titer determined by other means (45,57). Randomly derived cDNA
probes demonstrated similar results (24). Dot blot hybridization
was utilized to detect IBDV in tissue with a probe derived from the
cloned sequence of VP1. IBDV could be detected by this method from
day 1 to day 24, with the test also demonstrating a wide
specificity encompassing both serotypes(58).
[0046] The polymerase chain reaction (PCR) has been modified so
that it can be utilized for the direct amplification of IBDV from
infected tissues (55,142). Because infected samples must first be
copied into cDNA by reverse transcriptase, and then amplified with
PCR, this procedure is called RT-PCR. A secondary analysis is
performed in which restriction enzymes are used to cut the
amplified product resulting in the formation of a banding pattern
that will identify it as to IBDV serotype classic or variant
(55).
IMMUNITY
[0047] Infectious bursal disease virus represents a true paradox.
in that it replicates in immune cells and destroys them, yet is
capable of stimulating an extremely high immune response (36,52).
Because of this immunogenic ability, IBDV infection can be
effectively controlled with use of one of any number of vaccination
programs. Most IBDV vaccination programs for commercial chickens
utilize a combination of a live virus vaccine and inactivated oil
emulsion vaccine containing serotype 1 IBDV. Commercial vaccines
are available in a number of formulations and concentrations
(35,93). The vaccine type employed by the producer will reflect
which IBDV is currently causing disease on their premises.
Vaccination regimens also vary as to route of administration,
timing of administration, number of repetitions, and dose given.
Infectious bursal disease virus vaccinations are given to the
breeder flock to boost maternal antibody levels that are
transferred to the progeny.
[0048] Maternal antibody levels are capable of protecting the chick
for the first few weeks, but are generally insufficient to protect
for the entire growout (29,135). In addition, maternally derived
antibody is not uniformly distributed to the progeny, so it is
evident that production flocks will contain both susceptible and
protected birds at any given time (135).
[0049] Earliest vaccinations for IBDV are administered in-ovo at
day 18 of embryonation. Virus neutralization (VN) antibody titers
in chicks that received the vaccine in-ovo resulted in a resistance
to challenge from virulent IBDV up to 10 weeks of age (116).
Licensed vaccines are now available for mechanical administration
into the yolk sac at 18 days of embryonation (2,38). In general,
hatchery vaccination is somewhat questionable because of
interference from high levels of maternal antibodies (135). In the
hatchery, IBDV vaccination can also be given by microaerosol spray,
or subcutaneous injection. Field vaccination for IBDV is
customarily delivered in the drinking water.
[0050] Infectious bursal disease virus live vaccines are usually
derived from attenuated IBDV field virus strains. Attenuation is
accomplished by the repeated passage of the virus through cell
culture and/or embryonated eggs (126). Live vaccines also spread
laterally from bird to bird and carry with them the potential to
revert to virulence and, as such, pose a threat to the very birds
they were designed to protect (35). There is no evidence at present
to indicate that IBDV infection is spread vertically.
[0051] Infectious bursal disease virus vaccines are classified by
the degree of pathology they induce in the bursa as being mild,
intermediate, or invasive. Invasive vaccines induce a high degree
of bursal damage which is accompanied by immunosuppression, but
produce high antibody titers. Live invasive IBDV vaccines are
usually administered in the presence of low levels of maternal
antibodies in production birds or following priming with a killed
oil emulsion vaccine in breeders (35,76).
[0052] Genetically engineered vaccines for IBDV have been described
in which various pieces, mainly VP2, are expressed in a number of
different vectors. Field testing indicates that most of these
products stimulate antibody production, and since they contain only
pieces of the IBDV genome, do not pose a threat to virulence.
However, antibody produced using these constructs, at best, offer
only limited protection and are not economically feasible for
widespread application (62,80).
IMMUNOSUPPRESSION
[0053] Immunosuppression represents a physiological state in which
an individual exhibits temporary or permanent dysfunction of the
immune response, resulting from some type of damage to the immune
system. Whether temporary or permanent immunosuppression ultimately
leads to an increased susceptibility to other disease pathogens.
Two basic categories of immunosuppression are considered; one that
is antigen specific, and the other is a generalized
unresponsiveness of the immune system (119,120).
[0054] Antigen specific immunosuppression results in the loss of a
specific type, or types, of cell following exposure to the pathogen
and can occur directly or indirectly. Direct interference by the
pathogen occurs when replication takes place in a specific immune
cell or in a cell that in some way plays a role in innate immunity
(49). Indirect effects occur when the pathogen interferes with the
regulatory cells of the immune system. Both direct and indirect
immunosuppression invoke negative consequences for the complete
development of antigen specific antibodies. Generalized
immunosuppression is most often associated with genetic and/or
physiological deficiencies in which the immune cells are absent or
the body fails to produce cells or substances necessary to sustain
an immune response (49,72.120,121).
[0055] Immunosuppression following IBDV infection is characterized
by a severe lymphoid cell depletion in the bursa. There is some
evidence of additional cellular depletion in the thymus and
hematopoietic cell loss in the bone marrow. Lesions induced by IBDV
reflect that the virus prefers to replicate in IgM bearing
lymphocytes, but can, and will utilize other lymphocytes as well as
macrophages throughout the body. Loss of most of the functional
B-cells results in the inability to produce antibodies to IBDV or,
for that matter, any other pathogen that comes along afterward.
Infectious bursal disease virus infection, therefore, has the
potential to invoke both direct and indirect effects on the immune
system. This, in turn, results in reduced response to vaccines,
increased early mortality, and a significantly higher incidence of
secondary opportunistic pathogens (105,106).
[0056] Immunosuppression induced by IBDV infection is
experimentally assessed by the ability or inability of the bird to
mount an immune response to IBDV as well as other types of antigens
following infection or vaccination. It has been repeatedly
demonstrated that vaccination or infection with IBDV at an early
age will result in the suppression of the humoral immune response
to a number of different antigens (1, 19, 33, 35, 53, 68, 72, 100,
111, 119, 120, 121). In addition, other studies have determined
that there is a reduction in natural killer cell activity (115,
117), and reduced immune response to other classes of antigens
(102,118). Challenge with IBDV which is administered to
immunologically mature chickens may produce only partial
immunosuppression or no immunosuppression at all (28).
[0057] Bursectomized birds are still capable of producing an immune
response, but because a majority of the immune cells are no longer
present, the response is extremely weak. In addition, bursectomized
birds do not manifest the physical symptoms of clinical IBDV. The
proposed mechanism is that there are insufficient target cells to
attain viremia levels to induce lesions. The ability to produce low
levels of antibody indicate that immunity in birds to infectious
agents can develop, in part, from other lymphoid organs including
the spleen, and thymus (36, 95).
[0058] Classic and variant serotype 1 IBDV induce different types
and intensities of immunosuppression. In-vitro lymphoblast
transformation assays demonstrated that the variant IBDV exerted
more of an effect on the lymphoid cells, whereas the classic IBDV
targeted the humoral immune response (102). Utilization of certain
vaccines can also induce immunosuppression, and reflect the need to
utilize IBDV vaccines judiciously (35).
PATHOGENICITY
[0059] Primary pathologic lesions associated with IBDV infection or
vaccination of susceptible chickens are found predominantly within
the bursa of Fabricius (BF). Rapid onset of lesions within the BF
reflect the high concentration of target B-lymphocytes present
(120). This is accompanied by the lesser involvement of other
lymphoid organs, such as spleen, thymus, cecal tonsils and Peyers
patches (64,118). Pathology observed for classic IBDV infection
varies from that described for variant IBDV infection. Classic IBDV
pathology is well characterized and will be discussed thoroughly in
this section with reference to differences demonstrated by variant
IBDV (21,107). To summarize, variant IBDV pathology exhibits
similar lesions to the classic virus which are less severe, and can
be described as highly cytolytic with rapid bursal atrophy and a
minimal inflammatory response. One of the proposed mechanisms for
this difference in pathologies that variant IBDV, as well as
serotype 2 IBDV, induce cytopathology through the process of
apoptosis (135,136).
[0060] Pathology within the chicken lymphoid system is presumed to
result directly from IBDV infection of suitable target cells.
Primary target cells have been identified as IgM bearing B
lymphocytes, but other cells have also been implicated; they
include macrophages. non-B lymphocytes, endothelial cells and
reticuloendothelial cells (15,16,48,84,91).
[0061] The BF represents a unique organ structure found exclusively
in avian species. The bursa is contained within a blind pouch that
is located dorsal to the cloaca. This blind pouch is connected by a
short duct to the cloaca through which it receives contact with
environmental antigens. Exposure to these antigens appears to be a
non-specific mechanism at the control of peristaltic movement
through the bursal duct due to currents created by transport of
waste through the cloaca (105). The BF is divided into distinct
follicular regions that can be further divided into a outer dense
cortical zone filled with lymphocytes, which surrounds a loosely
packed medullary zone of reticuloepithelial cells (97,106). The
primary function of the BF is the development, maturation and
transformation of the B-lymphocyte, and as a result the bursa
strongly influences the humoral immune response of the chicken as
well as other avian species (36,67).
[0062] There is a direct correlation between the degree of
pathological lesions and symptoms induced by IBDV challenge and the
number of susceptible cells available for infection (44,51,
66,134). This is perhaps best demonstrated by infection of birds
with IBDV following removal of the bursa. Bursectomized birds have
little or no antibody production, but show mild lymphocytic
necrosis in the spleen and thymus. Presence of the bursa of
Fabricius is not necessary to establish infection with IBDV, but it
appears to be required for manifestation of the clinical infection
(96).
[0063] Bursal lesions appear as early as one day following
infection and are generally characterized by extensive necrosis of
lymphocytes in the cortex of the bursal follicle. Phagocytosis of
the necrotic lymphoid cells by the reticular cells then progresses
to a severe reticuloepithelial hyperplasia with accompanied
inflammation. This is followed by infiltration of heterophils, and
accumulation of pyknotic debris within the follicles. As the
follicles fill with cellular necrotic debris, the intrafollicular
area becomes edematous (105,106). Plasma cells and pyronionphilic
blast cells develop in the areas of reticuloepithelial hyperplasia
and subsequently become necrotic. Initial atrophy of the bursa is
followed by the rapid proliferation of the cortico medullary
epithelium and the formation of mucus secreting gland (118).
Following IBDV infection, the total bursal tissue surface area can
be reduced in excess of 70% with little or no lymphocyte
repopulation before age onset involution at approximately 12 weeks
of age (108). Secondary lymphoid organs are scattered throughout
the avian body. Collectively, these lymphoid tissues have more
B-lymphocytes than the bursa, however, they lack the high
concentration and as such reactions following IBDV infection go
relatively unnoticed (27).
[0064] Surveys of tissues from IBDV infected birds utilizing the
electron microscope detected morphological changes in the bursa as
early as 48 hours. Epithelial microvilli of the bursa were
initially reduced in number and size. As the infection progressed.
there was gradual involution of the bursal follicles, followed by
the development of surface erosions from the loss of epithelial
cells (92).
[0065] Bursae from IBDV vaccinated birds show marked lymphocyte
depletion and infiltration of mononuclear cells similar to what is
described for virus challenge (28). However, controlled exposure to
live virus vaccination results in overall reduction of the amount
of tissue damage. Histological changes were most pronounced in
groups of birds vaccinated at 28 days of age. Lesions were
characterized by the loss of demarcation between the cortex and
medulla, and a significant increase in the number of cells packed
into follicles with additional infiltration of mononuclear cells.
Depletion of cells from the medulla of the bursa was most severe by
5-7 days post vaccination (35).
[0066] Infectious bursal disease virus could be recovered from the
intestine from 4-10 days PI for birds challenged at 1, 7, and 14
days. Older challenged birds (5 weeks+) highest intestinal levels
were day 3-8 PI. Complement depletion was determined to be
significantly lower only in the older birds (5 week+)(29). IBDV
antigen can be detected in macrophages and lymphoid cells of the
cecum as early as 4 hours PI (84). Other lymphoid organs are
adversely affected by the presence of IBDV virus, with pathology
attributed to virus specific replication in target cells.
[0067] Following IBDV infection the spleen often demonstrates
reticuloendothelial cell hyperplasia in the lymphocyte beds that
surround the adenoid sheath arteries, with marked splenic fibrinoid
degeneration (17, 44, 114). The thymus exhibits a reduction in the
cortical mass (19) while in the cecal tonsil there were loss of
follicles and a decrease in the number of lymphocytes (17, 44,
114). Kidneys of infected birds demonstrate formation of homogenous
casts with infiltration of heterophils and the presence of
immunoglobulins (74). Post mortem examination of infected birds
often demonstrates infiltration of heterophils and edema in livers
associated with hepatic coagulative necrosis. Levels of interferon
production increase in the kidney, lungs, thymus, spleen, and bursa
following IBDV infection. Attenuated strains of IBDV similar to
those utilized for vaccination only induced an increase in
interferon levels in the bursa (34).
[0068] Other physical manifestations of IBDV infection do not
involve the lymphoid system. Secondary pathology induced by IBDV
infection is characterized by edema, congestion, hemorrhage,
necrosis and heterophilic infiltration in the skeletal muscles of
the chest and thigh. In addition, similar lesions have been
described (74,95,96), in the intestine (114) (120) and mucosa of
the proventriculus (116). Hemorrhage formation. in conjunction with
IBDV infection, has also been associated with a disseminated
intravascular coagulation defect (122) and the destruction of
thrombocytes (138). It can be postulated that IBDV would interfere
with immune reactions dependent on multiple immune structures
including both the bursa and the thymus (102).
[0069] Ivanyi et al hypothesized that immune complexes may play a
role in the pathogenesis of IBDV infections, which could account
for the secondary pathologic chances observed (53). Combinations of
antigen, antibody and complement represent complexes that
contribute to IBDV infection pathology by their physical and
pharmacological properties. The lesions of acute immune complex
disease typically develop in the vasculature and renal glomeruli
resulting in arteritis and glomerulonephritis. Fluorescent antibody
staining of infected tissue indicated the presence of gamma
globulins in renal glomeruli following infection with IBDV. The
presence of these immune complexes within the tissue indicate that
they may influence pathogenesis associated with IBDV infection in
chickens (74).
[0070] Simultaneous multiplication of IBDV and production of
antibodies in the bursa may produce immune complexes with cellular
damage occurring following the activation of complement, with
additional involvement of increased interferon levels. Complement
mediated destruction of bursal cells would result in a type of
non-inflammatory lymphocyte depletion. Skeeles et al. demonstrated
that complement levels increase with age, and associated the
increase with an increased severity of lesions induced in older
birds following infection with IBDV. It has also been demonstrated
that during acute IBDV infection there is a depletion of complement
in infected birds on days 3 and 5 PI, with complement levels
returning to normal by day 8. Administration of hyperimmune serum
during IBDV infection did not appear to increase disease severity
(123,124,125).
[0071] Apoptosis has also been proposed as one of the mechanisms by
which a number of viruses induce pathology. Recent studies have
indicated that IBDV also utilizes this mechanism (136,137).
Apoptosis is a genetically ordered sequence of events following a
very specific cellular signaling stimulus in which irreparably
damaged cells are disposed of, with minimal damage to surrounding
cells or tissue (133).
[0072] Apoptosis should not be confused with necrosis, as each
represents a totally different set of events albeit with some
overlap. Cellular necrosis results from physical injury and is not
in any way genetically controlled. Whereas apoptosis is a
genetically predetermined deliberate cellular response to specific
developmental and environmental stimuli. Injury events that produce
necrosis can trigger apoptosis in response to the presence of
cellular debris and inflammation. Likewise, it may be possible that
induction of apoptosis could indirectly produce necrosis in some
instances where cellular degeneration results from a disease
process. Necrosis is typified by the destruction of cytoplasmic
organelles and loss of the integrity of the plasma membrane.
Apoptosis is associated with the boiling of the cytoplasm,
condensation of chromatin, fractures of nuclear DNA and can be
distinguished from necrosis by lack of an inflammatory response
(139).
[0073] Where viruses are concerned, the inhibition of apoptosis has
resulted in persistent infections, latency, or enhanced virus
production. On the other hand, promotion of apoptosis has been
demonstrated to facilitate virus spread and release
(133,136,137,139).
PROVENTRICULITIS SYNDROME
[0074] Enlargement of the proventriculus has been recognized as a
problem in broiler chickens in Northwest Arkansas, as well as in
other high intensity poultry producing areas throughout the United
States for a number of years. The problem manifests itself at the
greatest cost in the processing plant, but may also be associated
with poor feed conversion.
[0075] Weakened proventriculi that are engorged with feed and
digesta are subject to breakage at the point of mechanical
evisceration. The contents of the proventriculus contaminate the
chicken carcass, resulting in increased levels of washouts,
downgrades, and condemnations.
[0076] Proventriculitis syndrome has been linked to a number of
possible causes. Early considerations involved a longer fasting
period before processing. However, increased fasts did not
facilitate the emptying of the proventriculus. The search for a
plausible explanation of this condition resulted in the
determination of a number of factors that adversely affect the
function, and structure of the avian proventriculus. A number of
dietary components have been associated with enlarged
proventriculi. Among them are: biogenic amines, lack of dietary
fiber, high levels of mold toxins (37), and copper sulfate
supplementation (140). In addition, a number of infectious agents
have been identified as exerting an effect on the proventriculus.
The pathological infectious agents identified as potential causes
of proventriculitis are as follows: reovirus, Marek's virus, avian
influenza, adenovirus (37) and velogenic NewCastle (vNDV) virus.
However, there is no evidence, at present, that links any of the
preceding factors with the syndrome currently being described.
[0077] Bayyari et al. demonstrated that proventriculitis syndrome
could be reproduced utilizing an undefined homogenate of affected
proventriculi collected from field birds at processing (8). The
syndrome could also be reproduced utilizing a filtrate of the same
homogenate, and the condition could be exacerbated by the addition
of copper sulfate in the feed. Induction of the proventricular
syndrome with the filtrate indicated the possibility that the
causative agent involved was some type of viral agent. Additional
clues indicated that the virus could possibly be infectious bursal
disease virus, as challenged birds seroconverted to IBDV (8,
9).
[0078] Further evidence that linked IBDV to this syndrome was found
following IBDV challenge after vaccination against IBDV.
Unvaccinated challenged control birds exhibited lesions associated
with proventriculitis. The following studies were undertaken to
elucidate the role of IBDV in the induction of a viral
proventriculitis syndrome in chickens. In addition, a number of
host and environmental factors will be investigated for the
influence they exert on the incidence and severity of this
syndrome.
[0079] In accordance with the present invention, variant E/1084
infectious bursal disease virus (IBDV) challenge given to specific
pathogen free (SPF) chicks at 7, 14, 21, and 28 days post hatch was
capable of producing microscopic lesions in the proventriculus, but
was only detected in the proventriculus of birds challenged at 28
days post hatch using the AC-ELISA. Birds challenged with Variant
E/1084 IBDV on 7, 14 and 21 days post hatch had lower antibody
titers at 11 days post challenge than those challenged at 28 days
post hatch. Antibody response to Variant E/1084 IBDV on all
challenge dates was higher than antibody response to USDA/STC IBDV
given at 28 days post hatch. Bursa:body weight (B:BW) ratios were
significantly different for birds challenge with Variant E/1084
IBDV on days 7 and 28 post hatch at 4 days post challenge. There
was evidence of early onset of bursal atrophy for these two
challenge groups. On day 11 post challenge all IBDV infected groups
demonstrated significant atrophy of the bursa regardless of age at
challenge or challenge virus given. Proventriculus:body weight
(P:BW) ratios were significantly different at 4 and 11 days post
challenge in birds that received Variant E/1084 IBDV on day 7 post
hatch and on day 11 post challenge in birds that received challenge
on day 21 post hatch. Internal gross lesions involving the papillae
were most evident in birds challenged on days 7, 14, and 28 post
hatch with Variant E/1084 IBDV, and also in birds challenged on day
28 post hatch with USDA/STC IBDV.
[0080] Specific pathogen free (SPF) white leghorn chickens were
challenged at 32 days post hatch with 3 different concentrations of
USDA/STC IBDV and 3 different concentrations of Variant E/1084
IBDV. Birds were examined at 2, 3, 4, and 11 days post challenge
for the presence of virus and lesions in the proventriculus and
bursa. AC-ELISA analysis indicated the presence of infectious
bursal disease virus (IBDV) was a factor of both the concentration
of virus inoculum given and time post exposure. AC-ELISA analysis
of the bursa reflected that the percentage of birds testing
positive was a factor of time post virus exposure. Serological
analysis indicated that all birds challenged with either strain of
IBDV were seropositive at 11 days post challenge, and total
antibody titer did not reflect the varied amounts of virus inoculum
given. Microscopic lesions in the proventriculus were more
pronounced in the groups receiving USDA/STC and occurred as an
acute lesion and as a chronic lesion, however, there was no
indication of a virus concentration effect. Microscopic lesions in
the bursa were present in all IBDV challenge groups at all sampling
times.
[0081] Thin section electron micrographs revealed the presence of
virus like particles within the proventriculus of SPF white leghorn
chickens at four days post challenge with IBDV. SPF white leghorn
chickens exhibit lesions in the bursa and proventriculus following
IBDV challenge. Lesion assessment, both grossly and
microscopically, indicates that the USDA/STC IBDV is capable of
producing the most severe effect on the proventriculus. Pathology
from infectious bursal disease virus infection is exacerbated by
the presence of copper sulfate supplementation in the feed. The
presence of REO S-1133 virus indicated interference in the immune
response or IBDV replication, as well as the number of tissue
homogenates testing positive for IBDV at 4 days post challenge.
Mortality was significantly increased for birds challenged with
USDA/STC IBDV in the presence of copper sulfate and/or REO S-1133
virus. Birds which received dietary copper sulfate supplementation
had reduced body weights at 4 days post challenge and at 11 days
post challenge.
[0082] The presence of apoptotic cells in the proventriculus and
bursa at 4 days post challenge with USDA/STC IBDV and Variant
E/1084 IBDV was determined using a modified TUNEL fluorescent
staining procedure. Specific green fluorescence was found in
highest quantity in the bursa, no differences in the intensity of
staining could be determined for each IBDV strain. Fluorescence was
focused predominantly in the medulla and cortex of infected bursae.
Proventricular fluorescent staining was found predominantly in the
villi and submucosa. Fluorescent intensity of proventricular
sections appeared highest in tissue sections from SPF leghorn
chickens challenged with USDA/STC IBDV.
[0083] Hemolytic complement levels were decreased at 4 days post
challenge in all IBDV challenge groups. Highest level of depletion
was in the SPF white leghorn chickens challenged with USDA/STC
IBDV. Complement levels were recovered by 6 days post challenge
which corresponded with measurable levels of neutralizing
antibodies against USDA/STC IBDV. IBD virus was detectable by
AC-ELISA at this time also, indicating that all the components
needed for the stimulation of complement mediated pathology are
present and as such remain as one possible mechanism for IBDV
induced tissue damage. Virus neutralizing antibodies appeared first
in the SPF white leghorn chickens challenged with Variant E/DEL at
6 days post challenge. All IBDV challenged groups had measurable
IgG titers at day 8, but there was a subsequent decrease in titer
at day 11.
[0084] Broiler chickens challenged with USDA/STC IBDV at 35 days
post challenge exhibited gross lesions in the bursa and
proventriculus at 4 days post challenge in the presence and absence
of copper sulfate. Antigen capture ELISA (AC-ELISA) analysis of
tissue homogenates indicates that the USDA/STC IBDV was present at
4 days post challenge and was not influenced by the presence of
dietary copper sulfate. Broiler chickens did produce an immune
response to IBDV following challenge.
[0085] To date submissions for AC-ELISA screening of broiler flocks
affected with proventriculitis have encompassed at least 46
facilities from 6 different states. Broiler chicken tissue
submissions come from flocks ranging in age from 14 to 35 days post
hatch. Virus immunoprecipitation following AC-ELISA screening has
resulted in the isolation of at least 5 infectious bursal disease
viruses of proventricular origin. Virus isolations are primarily
from broiler flocks ranging in age from 22-29 days post hatch.
[0086] Inoculation of proventricular origin IBDV isolates into 28
day post hatch SPF white leghorn chickens, revealed the production
of lesions associated with infectious bursal disease virus
infection. At 11 days post challenge all SPF white leghorn chickens
demonstrate IgG antibody titers to IBDV following screening of
serum with a commercial IBDV ELISA kit.
[0087] RT/PCR-RFLP analysis of new proventricular IBDV isolates
indicate that the viruses have a unique restriction enzyme pattern
within a 700 bp fragment of the VP2 genomic region. The new
isolates appear to share characteristics of both the Delaware
variant and a more recent California IBDV isolate from the
bursa.
BRIEF DESCRIPTION OF THE DRAWINGS
[0088] FIG. 1 is a diagram showing geometric meant titers (GMT) of
SPF white leghorn chickens at 4 and 11 days post challenge with
Variant E/1084 IBDV given at 7, 14, 21 and 28 days post hatch, and
with USDA/STC IBDV given at 28 days post hatch only. Negative
Control (NC), Variant E/1084 IBDV (E), USDA/STC IBDV (U).
[0089] FIG. 2 shows the serological response in white leghorn
chickens challenged at 32 days post hatch with USDA/STC IBDV and
Variant E/1084 IBDV at 2, 3, and 11 days post challenge.
[0090] FIG. 3 shows proventricular sections from SPF white leghorn
chickens at 32 days post hatch from negative control (A) and
negative control+copper sulfate (B) experimental groups, with
normal cellular structure. Mag.30,000.times.. Note the presence of
multiple vesicular bodies (P), dilated rough endoplasmic reticulum
(d), mitochondria (m) and golgi bodies (g).
[0091] FIG. 4 shows 2 inclusion bodies containing virus-like
particles associated with electron dense matrix and membranous
vesicles in the cytoplasm of a proventricular cell 4 days post
challenge with USDA/STC IBDV+copper sulfate in SPF white leghorn
chickens. (N)=Nucleus. Mag. 26,000.times..
[0092] FIG. 5 shows a membrane bound inclusion body containing an
aggregate of virus-like articles (v), adjacent to the cell nucleus
within the submucosa of the proventriculus at 4 days post challenge
with USDA/STC IBDV+copper sulfate in SPF white leghorn chickens.
Mag. 80,000.times.. (N)=Nucleus, (d)=dilated rough endoplasmic
reticulum, (r)=Ribosome. Compare the size of ribosome to the size
of virus-like particles.
[0093] FIG. 6 shows a large membrane bound inclusion body
containing densely packed virus-like particles and membranous
vesicles adjacent to the nucleus (N) of cell within the submucosa
of the proventriculus at 4 days post challenge with USDA/STC
IBDV+copper sulfate in SPF white leghorn chickens. Virus-like
particles are considerably larger than ribosomal granules.
Mitochondria (m), golgi bodies (g) and dilated rough endoplasmic
reticulum (d). Mag. 32,000.times..
[0094] FIG. 7 shows numerous membrane bound inclusion bodies
containing virus-like particles (VLP's) and vesicles occurring
closely together in the cytoplasm near the nucleus (N) of a cell in
the submucosa of the proventriculus at 4 days post challenge with
USDA/STC IBDV+copper sulfate in SPF white leghorn chickens. For
size comparison see ribosome (r) compare to virus like particles
(v).
[0095] FIG. 8 shows large membrane bound and a non-membrane bound
inclusion bodies containing virus-like particles in the cytoplasm
of a cell in the submucosa of the proventriculus at 4 days post
challenge with USDA/STC IBDV+copper sulfate in SPF white leghorn
chickens. Note characteristic mucus containing bodies occurring
also in the cytoplasm.
[0096] FIG. 9 is a diagram showing serum antibody titers in SPF
white leghorn chickens at 4 and 11 days post challenge with Variant
E/1084 IBDV in the presence or absence of REO virus and/or copper
sulfate.
[0097] FIG. 10 is a diagram showing serum antibody titers in SPF
white leghorn chickens at 4 and 11 days post-challenge with
USDA/STC IBDV in the presence or absence of REO virus and/or copper
sulfate.
[0098] FIG. 11 shows hemorrhage within the mucosa of proventriculus
of SPF white leghorn chickens at 28 days post hatch 4 days post
challenge with USDA/STC IBDV.
[0099] FIG. 12 shows pleomorphic cells within the proventriculus of
SPF white leghorn chickens challenged at 28 days post hatch with
USDA/STC IBDV only.
[0100] FIG. 13 shows vacuolated cells within the glands of the
proventriculus of SPF white leghorn chickens challenged at 28 days
post hatch with USDA/STC IBDV+Copper Sulfate at 4 days post
challenge (40.times.).
[0101] FIG. 14 is a diagram showing total mortality in SPF white
leghorn chickens following challenge with Variant E/1084 IBDV and
USDA/STC IBDV in the presence or absence of REO virus and/or copper
sulfate.
[0102] FIG. 15 is a diagram showing body weight means for SPF white
leghorn chickens at 4 and 11 days post challenge with Variant
E/1084 IBDV in the presence or absence of REO virus and/or copper
sulfate.
[0103] FIG. 16 is a diagram showing body weight means for SPF white
leghorn chickens at 4 and 11 days post challenge with USDA/STC IBDV
in the presence or absence of REO virus and/or copper sulfate.
[0104] FIG. 17 shows a TUNEL staining for the presence of
apoptosis. Bursa section from 32 day post hatch SPF white leghorn
chickens at 4 days post challenge with Variant E/1084 IBDV.
Positive fluorescence in the medulla and cortex of the bursal
follicle (20.times.).
[0105] FIG. 18 shows a TUNEL staining for the presence of
apoptosis. Bursa section from 32 day post hatch SPF white leghorn
chickens at 4 days post challenge with USDA/STC IBDV. Positive
fluorescence in the medulla and cortex of the bursal follicle
(20.times.).
[0106] FIG. 19 shows a TUNEL staining for the presence of apoptotic
cells. Proventriculus section from 32 day post hatch SPF white
leghorn chickens at 4 days post challenge with USDA/STC IBDV.
Positive fluorescence within the cells of the proventricular villus
(40.times.).
[0107] FIG. 20 shows a TUNEL staining for the presence of apoptotic
cells. Proventriculus section from 32 day post hatch SPF white
leghorn chickens at 4 days post challenge with Variant E/1084 IBDV.
Positive fluorescence within the cells in the tips of the
proventricular villi (40.times.).
[0108] FIG. 21 shows a TUNEL staining for the presence of apoptotic
cells. Proventriculus section from 32 day post hatch SPF white
leghorn chickens at 4 days post challenge with Variant E/1084 IBDV.
Positive fluorescence within the cells of the proventricular glands
and surrounding connective tissue (20.times.).
[0109] FIG. 22 shows a TUNEL staining for the presence of apoptotic
cells. Proventriculus section from 32 day post hatch SPF white
leghorn chickens negative control. Negative for green fluorescence,
DAPI staining indication of normal DNA structure (20.times.).
[0110] FIG. 23 is a diagram showing serum antibody levels from SPF
white leghorn chickens challenged at 28 days post hatch with
Variant E/DEL, Variant E/1084, and USDA/STC IBDV at 4, 6, 8 and 11
days post challenge.
[0111] FIG. 24 is a diagram showing hemolytic complement levels in
SPF white leghorn chickens challenged at 28 days post hatch with
Variant E/DEL, Variant E/1084, and USDA/STC IBDV at 4, 6, 8, and 11
days post challenge.
[0112] FIG. 25 is a diagram showing virus neutralization (VN)
levels from SPF white leghorn chickens challenged at 28 days post
hatch with Variant E/DEL, Variant E/1084, and USDA/STC IBDV at 4,
6, 8 and 11 days post challenge.
[0113] FIG. 26 shows the proventriculus, spleen and bursa from 43
day post hatch broiler without USDA/STC IBDV challenge and in the
absence of copper sulfate supplementation.
[0114] FIG. 27 shows the Proventriculus, spleen and bursa from a 43
day post hatch broiler at 4 days post challenge with USDA/STC IBDV
in the presence of copper sulfate dietary supplement.
[0115] FIG. 28 is a diagram showing serum antibody levels of
commercial broilers at day of hatch, 35 days post hatch and 4 and
11 days post challenge with USDA/STC IBDV in the presence or
absence of copper sulfate.
[0116] FIG. 29 shows an enlarged proventriculus in a 22 day post
hatch production broiler chicken.
[0117] FIG. 30 shows the internal characteristics of acute
proventriculitis in 27 day post hatch production broiler. Note the
hemorrhages in the papillar ducts, loss of papillar definition, and
discoloration of the mucosal surface.
[0118] FIG. 31 is a diagram showing serum antibody response of SPF
white leghorn chickens challenged at 28 days post hatch with
USDA/STC IBDV and proventricular origin field IBDV isolates.
[0119] FIG. 32 shows a demonstration of lesions produced following
immunoprecipitation of AC-ELISA positive proventriculi after three
consecutive passages in SPF eggs.
[0120] FIG. 33 shows the RT/PCR-RFLP analysis of proventricular and
bursal homogenates from the two original proventricular IBDV
isolates at 4 days post challenge in 28 day post hatch SPF white
leghorn chickens.
[0121] FIG. 34A is a schematic cross-section representation of
normal papillae.
[0122] FIG. 34B is a schematic cross-section representation of
abnormal papillae affected by acute IBDV infection.
SECTION 1. DETERMINATION OF THE ROLE OF INFECTIOUS BURSAL DISEASE
VIRUS STRAIN VARIANT E/1084 IN THE INDUCTION OF PROVENTRICULITIS IN
SPF CHICKENS: AGE RELATED EFFECTS
SUMMARY
[0123] Variant E/1084 infectious bursal disease virus (IBDV)
challenge given to specific pathogen free (SPF) chicks at 7, 14,
21, and 28 days post hatch was capable of producing microscopic
lesions in the proventriculus, but was only detected in the
proventriculus of birds challenged at 28 days post hatch using the
AC-ELISA. Birds challenged with Variant E/1084 IBDV on 7,14 and 21
days post hatch had lower antibody titers at 11 days post challenge
than those challenged at 28 days post hatch. Antibody response to
Variant E/1084 IBDV on all challenge dates was higher than antibody
response to USDA/STC IBDV given at 28 days post hatch. Bursa:body
weight (B:BW) ratios were significantly different for birds
challenge with Variant E/1084 IBDV on days 7 and 28 post hatch at 4
days post challenge. There was evidence of early onset of bursal
atrophy for these two challenge groups. On day 11 post challenge
all IBDV infected groups demonstrated significant atrophy of the
bursa regardless of age at challenge or challenge virus given.
Proventriculus:body weight (P:BW) ratios were significantly
different at 4 and 11 days post challenge in birds that received
Variant E/1084 IBDV on day 7 post hatch and on day 11 post
challenge in birds that received challenge on day 21 post hatch.
Internal gross lesions involving the papillae were most evident in
birds challenged on days 7, 14, and 28 post hatch with Variant
E/1084 IBDV, and also in birds challenged on day 28 post hatch with
USDA/STC IBDV.
INTRODUCTION
[0124] Proventiculitis syndrome is a problem described in broiler
field production birds in which the proventriculus is swollen and
engorged with feed. The problem is described most often in
conjunction with birds being processed at an early age
(approximately 28-35 days). The syndrome is present in older birds
at processing but appears to be of a much lower incidence.
Withdrawal of feed prior to processing does not facilitate the
emptying of the proventriculus, or alleviate the problem. At
processing the enlarged proventriculus tears and the contents
contaminate the carcass, requiring further washing, excessive
trims, or high condemnation. It had been determined earlier that
IBDV/STC was capable of inducing lesions consistent with those
described for proventriculitis syndrome. It was also evident that
the intensity of lesions produced following IBDV challenge was
increased as the age of the birds increased (data not shown). The
purpose of this experiment was to determine if Variant E/1084 IBDV
was also capable of inducing the same type of lesion observed with
the USDA/STC classic IBDV, and whether or not the presence and
intensity of that lesion was increased as age of the birds
increased.
MATERIALS AND METHODS
[0125] One hundred and fifty SPF chickens derived from fertile SPF
eggs (HYVAC Inc., Adel, Iowa) were hatched at the University of
Arkansas Poultry Health Laboratory Isolation Facility. Chicks were
placed in negative pressure isolation units on day of hatch. Each
isolator contained 20 birds, with the exception of the negative
control group which contained 50 birds in order to accommodate all
posting dates with age matched non-infected controls. All birds
received water and the standard University of Arkansas diet
formulation as specified by age ad libitum. Lighting was maintained
continuously for brooding purposes throughout the entire study
utilizing a 250 watt light bulb in each isolator as well as diffuse
fluorescent lighting in the animal suite.
IBDV VIRUS CHALLENGE INOCULUM PREPARATION
[0126] Variant E/1084 challenge virus (Select Laboratories,
Gainsville, Ga.) was prepared from stock virus (1084-E). The
challenge virus aliquot was removed from the ultralow freezer
(-70.degree. C.), placed in a laminar flow hood, and thawed at room
temperature. The outside of the vial was wiped with 70% ethanol to
remove any surface contamination. The virus was then diluted 1:1000
in sterile deionized water. The Variant E/1084 challenge stock
titer was 10.sup.5 6 TCID.sub.50/ml . Virus challenge inoculum
titer was 10.sup.2.6TCID.sub.50/ml.
[0127] USDA/STC IBDV challenge inoculum was prepared from lot #92-1
(NVSL, Ames, Iowa) as follows: The virus was removed from the
ultralow freezer (-70.degree. C.), and thawed at room temperature
in a laminar flow hood. The outside of the virus containing glass
ampule was wiped with 70% ETOH, to remove any surface
contamination. The ampule of virus was snapped open by wrapping the
neck with a paper towel and breaking along the prescored line. The
virus was then diluted 1:10 by mixing 1.5 ml of USDA/STC IBDV virus
into 13.5 ml of Dulbecco's Phosphate Buffered Saline (D-PBS)
(Appendix). The USDA/STC IBDV challenge virus stock titer was
10.sup.4 1EID.sub.50/ml. Virus challenge inoculum titer was
10.sup.3 1 EID.sub.50/ml.
SPF BIRD CHALLENGE
[0128] Viral challenges with Variant E/1084 IBDV were staggered by
age and administered to individual isolators containing 20 SPF
white leghorn chickens. Challenges were administered on the
following days 7, 14, 21, and 28 post hatch (PH) with the prepared
inoculum as described previously. Challenge was administered to
each individual bird bilaterally to the eye (30 .mu.l per eye)
using an Eppendorf micropipettor (Brinkmann Inc., Westbury, N.Y.)
and a sterile pipette tip (Costar Corp., Cambridge, Mass.). In
addition, on day 28 PH one isolator of 20 birds was challenged with
USDA/STC IBDV virus inoculum as described previously. Challenge was
administered to each individual bird bilaterally to the eye (30
.mu.l per eye) using an Eppendorf micropipettor (Brinkmann Inc.,
Westbury, N.Y.) and a sterile pipette tip (Costar Corp., Cambridge,
Mass.)
SAMPLE COLLECTION
[0129] On days 4 and 11 post challenge 10 birds were necropsied
from each virus challenge group as well as an additional 10 birds
from the negative control. Each bird was weighed, bled, and
euthanized with CO.sub.2 gas. All bursae and proventriculi were
scored for the presence of gross lesions during necropsy. The
bursa, spleen, and proventriculus were individually weighed and
weights recorded. Tissues from each of the first five birds were
cut in half, with one half being placed into 10% buffered formalin,
and the other half being placed into a sterile sampling bag (Fisher
Scientific, Pittsburgh, Pa.). All tissues were pooled by organ and
experimental treatment group into the buffered formalin and into
the sampling bags. Tissues from the remaining five birds were
placed only in the sterile sampling bags. Tissues in sterile
sampling bags were frozen at -20.degree. C. for later analysis
utilizing Antigen Capture ELISA (AC-ELISA), as described by Snyder
et al (11).
ANTIGEN CAPTURE ELISA
[0130] AC-ELISA Plate Preparation: Monoclonal antibody B-29
(University of Maryland, College Park, Md., Intervet Inc.,
Millsboro, Del.) was diluted 1:10 in ice cold sterile distilled
water. The solution was then acidified by the addition of 0.4 N
sulfuric acid. The acidified monoclonal solution was incubated in
an ice bath for one hour at -20.degree. C. Following incubation the
acidified monoclonal antibody was diluted in 200 ml of coating
buffer bringing the final dilution of monoclonal antibody to
1:10,000. ELISA optimized 96 well microtiter plates Corning #
25805-96 (Corning Glass Works, Corning N.Y. 14831) were coated with
100 .mu.l per well of a 1:10,000 dilution of monoclonal antibody
B-29 (University of Maryland, College Park, Md.; Intervet, Inc.,
Millsboro Del.) ascitic fluid in coating buffer (Appendix). Plates
were covered with individual plate sealers (Dynatech, Chantilly
Va.), and incubated for 2 hours at 37.degree. C. and then overnight
at 4.degree. C. Coated plates were washed three times with washing
buffer (Appendix), with residual fluid being tapped out. Plates
were allowed to air dry at room temperature and were then covered
with individual plate sealers and stored at -20.degree. C. until
needed.
[0131] AC-ELISA Sample Preparation: Infectious bursal disease virus
suspect tissues were homogenized in AC-ELISA dilution buffer
(Appendix) at a ratio of 1:5 or approximately 20% tissue weight to
volume. Homogenization was accomplished by use of a rubber mallet,
followed by the addition of the dilution buffer. One hundred fifty
.mu.l of each bursal homogenate and 300 .mu.l of each
proventricular homogenate were placed in duplicate in a 96 well
round bottom plate. The concentration of the bursa tissue was
adjusted by addition of 150 .mu.l of AC-ELISA dilution buffer.
There was no additional dilution to the proventriculus tissue
homogenates.
[0132] AC-ELISA Analysis of Tissue Homogenates: Monoclonal antibody
coated plates were removed from -20.degree. C. storage and allowed
to come to room temperature (approximately 30 minutes). Monoclonal
coated plates then received 75 .mu.l of dilution buffer to each
well. Twenty-five .mu.l of suspect IBDV bursal tissue homogenates
and 75 .mu.l of suspect IBDV proventriculus tissue homogenates were
then transferred to the AC-ELISA plate using a costar 12 well
adjustable micro pipettor (Costar Corp., Cambridge, Mass.). The
final concentration of bursal homogenate on the AC-ELISA plates was
1:40 and the final concentration of the proventriculus tissue
homogenates was 1:10. AC-ELISA plates were incubated at 37.degree.
C. for 2 hours. Tissue samples were removed by inverting the plate,
and directing the contents into a container of disinfectant. Plates
were washed 3 times with washing buffer. The secondary antibody
chicken anti-IBDV (SPAFAS, Storrs, Conn.) was diluted 1:150 in
dilution buffer. Each well on the AC-ELISA plate received 100 .mu.l
of the secondary antibody. The AC-ELISA plate was incubated at
37.degree. C. for 1 hour. Secondary antibody was removed and the
AC-ELISA plate was washed three times with washing buffer. AC-ELISA
conjugate was prepared with #14-24-06 Peroxidase labeled Affinity
Purified Goat anti Chicken IgG (Kirkegaard and Perry Laboratories,
Inc., Gaithersburg, Md.) to a final concentration of 0.1 .mu.g/ml
or approximately 1:200 in dilution buffer. Each well on the
AC-ELISA plate received 100 .mu.l of conjugate. AC-ELISA plates
were incubated at 37.degree. C. for 1 hour. The conjugate was
removed as described earlier and plates were washed 3 times with
washing buffer. The substrate was prepared by using a # 50-62-00
ABTS Peroxidase Substrate System (2,2'-azino-di[3-ethyl-benzthia-
zoline sulfonate(6)]) (Kirkegaard and Perry Laboratories, Inc.,
Gaithersburg, Md.). The substrate was prepared by mixing equal
volumes of the 2 liquid components; ABTS and H.sub.2O.sub.2.
Substrate volume added was 100 .mu.l per well to the entire plate.
The reaction was allowed to develop until there was sufficient
color difference detectable by eye between the negative and
positive controls. The reaction was arrested by adding 10 .mu.l of
1% sodium dodecyl sulfate (Sigma Inc., St. Louis, Mo.). Plates were
read on an EL312e Microplate Biokinetics Reader (Biotek
Instruments, Winooski, Vt.) utilizing the Flock Profile.TM.
(Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.)
software at an absorbance of 405-450. Means were calculated for the
paired samples within each group and the homogenates were
determined to be positive or negative based on the mean absorbances
as compared to the negative and positive controls on each
plate.
BLOOD SAMPLE COLLECTION AND PROCESSING
[0133] Blood samples were collected individually using 4.5 ml
Sarstedt monovette.TM. syringes (Sarstedt, Inc., Arlington, Tex.)
containing clot activating beads and 22 gauge 11/2 inch needles
(Becton Dickinson and Company, Rutherford, N.J. 07070). Serum was
separated from the clot by centrifugation at 1000 rpm for 10
minutes using an IEC tabletop centrifuge. Serum was collected and
placed in a 96 well round bottom plate (Corning Glass Works,
Corning, N.Y.) format and also into Nalgene.TM. cryovials (Nalge
Company, Rochester, N.Y.) and frozen at -20.degree. C. until they
were analyzed using the commercial KPL Infectious Bursal Disease
Virus ELISA kit and the Flock Profile.TM. software (Kirkegaard and
Perry Laboratories, Inc, Gaithersburg Md.).
HISTOPATHOLOGY
[0134] Tissues were placed in 10% buffered formalin at necropsy.
Twenty-four hours later tissues were removed and manually trimmed
to a thickness of 1/8 inch and placed in tissue cassettes. Tissues
were dehydrated through graded alcohols, and cleared in two changes
of toluene. Tissues were then infiltrated with paraffin over a
16-hour period using an autoprocessor. Following paraffin
infiltration, tissues were positioned in paraffin blocks and
trimmed to expose a uniform tissue face. Prepared tissue blocks
were stored at -20.degree. C. until ready to be sectioned. Sections
were cut at a thickness of 5 .mu.m and floated on a water bath
containing 5% gelatin before they were placed on standard 1.times.3
inch slides. Tissue sections were then stained using the standard
method of Harris (41), for hematoxylin and eosin.
[0135] Tissues were examined microscopically for the presence of
histopathological lesions induced by IBDV infection. Lesions in the
proventriculus exhibited pleomorphism, lymphoid depletion and
heterophil infiltration. Lesions in the bursa were predominantly
lymphoid depletion with accompanied follicle degeneration Tissues
were given a numerical index score based on the presence or absence
of lesions as well as the intensity of the lesions. The scores
ranged from a low of "0" which indicates no lesions or pathology
observed to a high of "4" which indicates extensive tissue
damage.
STATISTICS
[0136] All data was entered into Microsoft.TM. Excel (Microsoft
Corp., Redmond Wash.), and were analyzed using SAS (SAS Institute
Inc., Cary, N.C.) (124). Statistical significance was determined at
the P<0.05 level using General Linear Models Procedure and least
squared means for body weight, bursa:body weight ratios,
spleen:body weight ratios, and proventriculus:body weight ratios.
In addition, statistical differences were determined for means of
the numerical index scores for gross lesions in the bursa and
proventriculus.
RESULTS
[0137] AC-ELISA screens of tissue pools indicated that Variant
E/1084 IBDV could be detected in the proventriculus (Table 1) on
day 4 post challenge, only in the groups that were challenged on
days 14 and 28 post hatch. Birds challenged with USDA/STC at day 28
post hatch were also positive for IBDV at 4 days post challenge in
the proventriculus. AC-ELISA screens of bursal tissue pools
indicated that Variant E/1084 was present in the bursa (Table 2) on
day 4 post challenge, in all groups with the exception of those
challenged on day 28. The bursae of birds challenged with USDA/STC
were also positive for IBDV at 4 days post challenge in the bursa.
On day 11 post challenge all birds tested negative for IBDV in both
the proventriculus and the bursa.
[0138] Serological analysis utilizing the commercial IBDV ELISA
test (FIG. 1.) indicate that all birds were negative for the
presence of IBDV IgG at 4 days post challenge. However, at 11 days
post challenge all IBDV challenged groups had seroconverted to
IBDV. Birds challenged with Variant E/1084 IBDV on day 7 and day 14
have a lower antibody titer than birds challenged at day 21 and day
28. The antibody titer difference between the younger and older
birds is not significantly different. It should also be noted that
the antibody response to USDA/STC IBDV challenge given at day 28 is
lower than all antibody titers of the Variant E/1084 IBDV challenge
groups regardless of age at challenge.
[0139] The presence of histopathological lesions in the
proventriculus from SPF chickens challenged with Variant E/1084
IBDV at 7, 14, 21 and 28 days post hatch are presented in Table 3.
Data is presented as a ratio of the number of tissues that exhibit
lesions of IBDV infection over the number of tissues screened. At 4
days post challenge 100% of the birds challenged with Variant
E/1084 IBDV on days 14, 21, and 28 were positive for IBDV lesions.
Birds challenged with Variant E/1084 IBDV at day 7 post hatch had
lesions present in 85% of the birds screened at 4 days post
challenge. Birds challenged with USDA/STC IBDV at day 28 post hatch
had lesions in 80% of the tissues screened at 4 days post
challenge. At 11 days post challenge birds who received Variant
E/1084 IBDV on days 7, 14, and 28 had no lesions in the
proventriculus. Birds challenged on day 21 with Variant E/1084 IBDV
and on day 28 with USDA/STC IBDV had a 20% and 40% incidence of
proventricular lesions respectively at 11 days post challenge.
There was a 40% incidence of proventricular lesions in the negative
control birds at 11 days post challenge. This may be an indication
that the virus had spread from the challenged birds to the
controls, however, there was no indication of the virus being
present by AC-ELISA (Table 1.).
[0140] The presence of microscopic bursal lesions in birds
challenged with Variant E/1084 IBDV on 7, 14, 21 and 28 days post
hatch are presented in Table 4. All groups, regardless of age at
challenge with Variant E/1084 IBDV, demonstrated 100% incidence of
lesions in the bursa on day 4 and day 11 post challenge. SPF white
leghorn chickens challenged with USDA/STC at 28 days post hatch
also demonstrated 100% incidence of lesions in the bursa on day 4
and day 11 post challenge. Please note again that the negative
control group from day 28 indicate the presence of lesions in 40%
of the birds sampled. However, there was no evidence of IBDV virus
when tissues were screened by the AC-ELISA test (Table 2).
[0141] Bursa to body weight ratios are a standard measure for IBDV
infection. Table 5 shows the mean bursa:body weight ratios of SPF
white leghorn chickens sampled at day 4 and day 11 following
challenge on days 7, 14, 21 and 28 post hatch. Values that are
placed in shaded boxes are significantly different at the P<0.05
confidence interval. On day 4 post challenge bursa:body weight
ratios were significantly lower in the two groups challenged on day
7 and 28 with Variant E/1084 IBDV. On day 11 post challenge all
groups regardless of age at challenge or the challenge inoculum are
significantly smaller than the negative control.
[0142] The proventriculus: body weight ratios were also calculated
for all challenge groups on 4 and 11 days post challenge and are
presented in Table 6. Numerical values which are significantly
different at P<0.05 confidence level are indicated by the shaded
boxes. The experimental group challenged at 7 days post hatch
exhibits significant enlargement of the proventriculus on day 4 and
day 11 post challenge. SPF white leghorn chickens challenged at 21
days post hatch demonstrate a significant difference only at day 11
post challenge. All other challenge groups were not significantly
different from the negative control.
[0143] Spleen: body weight ratios were also considered because
variant IBDV viruses consistently produce a splenomegaly during
infection. All challenge groups regardless of age at challenge,
demonstrated splenomegaly with spleen:body weight ratios that were
significantly higher than the negative control (Table 7). SPF white
leghorn chickens challenged on days 7, 14 and 21 post hatch with
Variant E/1084 IBDV also demonstrate spleen: body weight ratios
that are significantly higher on dav 11 post challenge than the
negative control. Birds challenged with Variant E/1084 IBDV and
USDA/STC at 28 days post hatch are not significantly different from
the negative control on day 11 post challenge.
[0144] Gross lesion scores were determined for the proventriculus
during necropsy. External (serosal) parameters evaluated were gland
prominence, length of the isthmus, and hypertrophy. Internal
(mucosal) parameters evaluated were impaction, papillae, and
hemorrhage. All of the parameters scored with the exception of the
papillae were not significantly different (data not shown).
Papillae scores from day 4 and day 11 post challenge with Variant
E/1084 IBDV on days 7, 14, 21 and 28 post hatch are presented in
Table 8. Mean papillae scores were only significantly different on
day 4 post challenge for the groups challenged on days 7, 14, and
28 post hatch with Variant E/1084 IBDV and also for the group
challenged with USDA/STC IBDV at 28 days post hatch.
DISCUSSION
[0145] Variant E/1084 IBDV was determined to be present by AC-ELISA
on day 4 post challenge in the bursa of all challenge ages with the
exception of day 28 post hatch. In contrast, Variant E/1084 IBDV
could only be found in the proventriculus on day 4 post challenge
and only in birds that were challenged at day 28 post hatch. This
indicates that in the younger SPF white leghorn chickens the IBDV
infection was focused predominantly in the bursa. Other lymphoid
areas in the chicken are not well defined at day of hatch and as
birds get older the peripheral lymphoid areas become better
established, thus providing the IRD virus other lymphoid targets in
which to replicate. The classic USDA/STC IBDV virus challenge given
at day 28 post hatch is detected in both the bursa and the
proventriculus. This may indicate that the classic virus particles
are more plentiful and can therefore establish a more extensive
viremia. Another consideration is that tissues that test negative
by AC-ELISA may reflect that these tissues were pooled and as such
tissues with low levels or no levels of virus could effectively
dilute tissues with high concentrations of virus to below test
detectable levels.
[0146] Serological measurements indicated that all birds were still
essentially seronegative 4 days post challenge and all challenged
birds were seropositve by 11 days post challenge. Geometric mean
titers reflect a lower antibody response to Variant E/1084 IBDV in
the younger birds. This syndrome is well documented by a number of
different authors, in which infection with IBDV shortly after hatch
can lead to immunosuppression (1,29,34,37,536,9). However,
regardless of the age of challenge, all titers to Variant E/1084
IBDV were higher than the antibody response to USDA/STC given at 28
days post hatch. The lower immune response to USDA/STC could
reflect the increased levels of damage induced in the bursa by this
strain as compared to the damage from Variant E/1084 IBDV infection
(37, 69).
[0147] Microscopic lesions were induced in the proventriculus by
both Variant E/1084 IBDV and USDA/STC IBDV on day 4 post challenge
but were less severe on day 11 post challenge. Therefore, it
appears that proventricular damage from IBDV infection occurs
predominantly during the acute stage of the virus infection.
Microscopic lesions of the bursa were found both on day 4 and day
11 in all challenged groups and were as expected for typical IBDV
infection (30, 37).
[0148] Ratios which consider the bursa, proventriculus, and spleen
weights in comparison to the body weight of each individual bird
represent excellent indicators of the acute and chronic stage of
the IBDV infection. The bursa to body weight ratio is considered a
standard measure of the infection, but in this case, gave some
indications that age may play a role in the intensity of the IBDV
infection. Birds challenged on day 7 and day 28 post hatch with
Variant E/1084 IBDV show evidence of bursal atrophy as early as
four days post challenge. Whereas, birds challenged on day 14 and
21 are not significantly different from the control values obtained
at 4 days post challenge, and the response is remarkably similar to
what is seen with birds challenged at 28 days post hatch with
USDA/STC IBDV. The possibility exists that birds challenged at two
and three weeks of age have the ability to modulate the IBDV virus
infection, or that this represents the period of time when
lymphocyte maturation and function are at their peak performance.
Modulation of the IBDV infection could also be facilitated by the
presence of antibodies, but antibody titers are below detectable
levels at this point.
[0149] Proventriculus to body weight ratios were significantly
larger at day 4 and day 11 post challenge only in birds challenged
with Variant E/1084 IBDV at day 7 post hatch. This ratio was also
significantly larger in birds challenged on day 21 post hatch at
day 11 post challenge. This does not correspond to the AC-ELISA
results which indicate that the Variant E/1084 IBDV was present in
the proventriculus only in the day 28 challenged birds. This again
may be a reflection of the dilution caused by pooling of tissues.
It also could reflect that proventricular enlargement is such a
transient change and that it occurs so quickly that the samples
were taken to late. In addition, it is important to consider that
microscopic lesions are present in the proventriculus which
indicate that some damage has occurred. This strongly suggests that
the virus was at one time present within the tissue, but apparently
at the time the sample was taken, the level of infection was below
detection capabilities of the AC-ELISA test.
[0150] The spleen to body weight ratios were good indicators of the
day 4 and day 11 challenge effects of the Variant E/1084 IBDV and
USDA/STC IBDV viruses. Chickens challenged at day 28 post hatch
with the Variant E/1084 IBDV or USDA/STC IBDV did not have spleens
that were significantly larger than the negative control at 11 days
post challenge. The most likely explanation for this is that at
this point in the experiment the negative controls were infected,
as demonstrated by the presence of lesions in 40% of the birds
screened by histopathology, and since their spleens were enlarged
the difference at 11 days post challenge is not detectable.
[0151] Papillae scores indicate that IBDV challenge does cause
changes in the tissue of the proventriculus, and can be
differentiated both grossly and microscopically. However, chickens
challenged on day 21 post hatch show adverse effects only during
the acute stage of the virus infection. Interestingly, lesions are
present in the tissue without the detection of virus within the
tissue, demonstrating that even low levels of IBDV can cause
damage.
SECTION 2 DETERMINATION OF THE ROLE OF INFECTIOUS BURSAL DISEASE
VIRUS STRAINS USDA/STC AND VARIANT E/1084 ON THE INDUCTION OF
LESIONS ASSOCIATED WITH PROVENTRICULITIS: INFLUENCE OF VIRUS
CONCENTRATION AND TIME
SUMMARY
[0152] Specific pathogen free (SPF) white leghorn chickens were
challenged at 32 days post hatch with 3 different concentrations of
USDA/STC IBDV and 3 different concentrations of Variant E/1084
IBDV. Birds were examined at 2, 3, 4, and 11 days post challenge
for the presence of virus and lesions in the proventriculus and
bursa. AC-ELISA analysis indicated the presence of infectious
bursal disease virus (IBDV) was a factor of both the concentration
of virus inoculum given and time post exposure. AC-ELISA analysis
of the bursa reflected that the percentage of birds testing
positive was a factor of time post virus exposure. Serological
analysis indicated that all birds challenged with either strain of
IBDV were seropositive at 11 days post challenge, and total
antibody titer did not reflect the varied amounts of virus inoculum
given. Microscopic lesions in the proventriculus were more
pronounced in the groups receiving USDA/STC and occurred as an
acute lesion and as a chronic lesion, however, there was no
indication of a virus concentration effect. Microscopic lesions in
the bursa were present in all IBDV challenge groups at all sampling
times.
INTRODUCTION
[0153] Proventriculitis syndrome is a problem within the broiler
industry that results in the enlargement and an associated
weakening of the walls of the glandular stomach. Affected
proventriculi fail to empty following feed withdrawal prior to
processing and at processing the proventriculus tears contaminating
the chicken carcass with digesta and feed. The end result is an
increase in the number of washouts, downgrades and
condemnations.
[0154] Previous experiments at the University of Arkansas have
indicated that infectious bursal disease virus (IBDV) plays a role
in the induction of proventriculitis syndrome. The purpose of this
experimental procedure was to determine if the amount and strain of
IBD virus influences the incidence or the severity of the
proventriculitis syndrome. In addition, an IBDV infection time
course will be examined to determine critical periods with respect
to the process of infection and the induction of lesions in the
proventriculus.
MATERIALS AND METHODS
[0155] Two hundred fifty specific pathogen free (SPF) white leghorn
chicks were hatched at the University of Arkansas Poultry Health
Laboratory Isolation Facility. Chicks were derived from fertile SPF
eggs obtained from HYVAC Inc., Adel, Iowa. Thirty-five chicks were
placed in each of 7 negative pressure isolation cages on day of
hatch. All birds received the standard University of Arkansas diet
formulation as specified by age and breed and along with water was
provided ad libitum. Lighting was maintained continuously for
brooding purposes throughout the entire study utilizing a 250 watt
incandescent light bulb. Birds were maintained under isolation
until they reached the age of 32 days post hatch.
IBDV CHALLENGE INOCULUM PREPARATION
[0156] USDA/STC IBDV lot# 92-1 was obtained from the National
Veterinary Services Laboratory (NVSL) in Ames, Iowa. The virus was
maintained at the University of Arkansas Poultry Health Laboratory
Isolation Facility at -70.degree. C. until needed. Two ampules of
virus were removed from the ultralow freezer and thawed at room
temperature in a laminar flow hood. The outside of the virus ampule
was wiped with 70% ETOH, to remove any surface contamination. The
ampule of virus was snapped open by wrapping the neck with a paper
towel and breaking along the prescored line. The contents of the
two separate IBDV ampules were combined and mixed thoroughly to
make the IBDV stock. Two virus inoculums were prepared from the
IBDV stock. The first virus inoculum preparation was undilute, and
1.5 ml of USDA/STC IBDV were transferred to a sterile 15 ml conical
tube. The second IBDV preparation was as per NVSL instructions in
which 1.5 ml of lot # 92-1 USDA/STC IBDV were diluted in 13.5 ml of
Dulbecco's Phosphate Buffered Saline (D-PBS). The third USDA/STC
IBDV virus challenge was achieved by utilizing the second IBDV
preparation and administering less volume per bird. The titer of
the USDA/STC IBDV virus was previously determined to be 10.sup.4.1
EID.sub.50/ml.
[0157] The Variant E/1084 strain IBDV challenge virus (Select
Laboratories, Gainsville, Ga.) was prepared from stock virus. The
challenge virus aliquot was removed from the ultralow freezer
(-70.degree. C.), placed in a laminar flow hood, and thawed at room
temperature. The outside of the cryovial tube was wiped with 70%
ETOH prior to opening to remove any surface contamination. The
virus was then diluted 1:100 and 1:1000 in sterile deionized water.
The titer of the IBDV Variant E/1084 challenge stock virus was
10.sup.5.6TCID.sub.50/ml. Virus challenge inoculum titers were as
follows: 1:100 1084-E=10.sup.3.6 TCID.sub.50/ml and for the 1:1000
1084-E=10.sup.2.6 TCID.sub.50/ml.
SPF BIRD CHALLENGE
[0158] All SPF white leghorn chickens were maintained under
isolation until they reached 32 days post hatch. Three isolation
cages of birds were challenged with one of three different amounts
of each IBDV virus strain. Infectious bursal disease virus
challenges were administered bilaterally to each eye using an
Eppendorf pipettor (Brinkmann, Inc., Westbury, N.Y.) and a sterile
pipette tip (Costar Corp., Cambridge, Mass.). Virus inoculum
volumes were adjusted to reduce or increase the number of virus
particles given to each bird as outlined as in Table 9. Viral
inoculums were diluted and administered in different volumes in
order to standardize the amount of virus given per bird. Volumes
administered and dilutions prepared were based solely on the
predetermined infectivity titers provided with each challenge
virus. Comparison of the two IBDV strains should be relegated to
paired groups that are identified by a letter designation in the
final column of Table 9.
SAMPLE COLLECTION
[0159] On days 2, 3, 4 and 11 post challenge 8 SPF white leghorn
chickens were necropsied from each virus strain challenge group and
also from the negative control. Each bird was weighed, bled, and
euthanized by CO.sub.2 asphyxiation. All birds were necropsied at
which time the bursae and proventriculi were scored for the
presence of gross lesions. The bursa, spleen and proventriculus
were weighed individually and results recorded. Individual tissues
from each bird were cut in half, with one half being placed into
10% buffered formalin, and the other half being placed into an
individual sterile sampling bag (Fisher Scientific, Pittsburgh,
Pa.). Tissues in sterile sampling bags were frozen at -20.degree.
C. for later analysis utilizing IBDV Antigen Capture ELISA
(AC-ELISA) as described by Snyder et al (138).
ANTIGEN CAPTURE ELISA PROCEDURE
[0160] AC-ELISA test plates were prepared as described earlier in
Section 1. Tissues were homogenized in AC-ELISA dilution buffer at
a ratio of 1:5 tissue weight to volume. Homogenates were analyzed
in duplicate against monoclonal B-29 (University of Maryland,
College Park, Md.; Intervet, Inc., Millsboro Del.) AC-ELISA plates
as described earlier in Section 1. Plates were read and means were
calculated for the paired samples within each group and the
homogenates were determined to be positive or negative based on the
mean absorbance values as compared to the negative and positive
controls on each plate.
BLOOD SAMPLE COLLECTION AND PROCESSING
[0161] Blood samples were collected individually using 4.5 ml
Sarstedt monovette.TM. syringes (Sarstedt, Inc., Arlington, Tex.)
that contain clot activating beads and 22 gauge 11/2 inch needles
(Becton Dickinson and Company, Rutherford, N.J.). Serum was
separated from the clot by centrifugation at 1000 rpm for 10
minutes using an IEC tabletop centrifuge and placed in both a 96
well round bottom plate (Corning Glass Works, Corning N.Y.) and
into Nalgene.TM. cryovials (Nalge Company, Rochester, N.Y.). All
serum samples were frozen at -20.degree. C. until they were
analyzed using the commercial KPL Infectious Bursal Disease Virus
ELISA Kit and the Flock Profile.TM. software (Kirkegaard and Perry
Laboratories, Inc., Gaithersburg, Md.).
HISTOPATHOLOGY
[0162] Tissues were placed in buffered formalin at necropsy, and
processed twenty-four hours later by the method described in
Section 1 (55). Tissues were examined for the presence of
histopathological lesions induced by IBDV infection. Lesions in the
lamina propria of the proventriculus were predominantly
pleomorphism of the lymphoid cells in the mucosa along with
lymphocyte depletion. Lesions in the bursa were predominantly
lymphoid depletion with accompanied follicle degeneration.
[0163] Tissues were given a numerical index score based on the
presence or absence as well as the intensity of the lesions
present. The score ranged from a low of "0" which indicates no
lesions or pathology observed to a high of "4" which indicates
extensive tissue damage.
STATISTICS
[0164] All data was entered into Microsoft.TM. Excel (Microsoft
Corp., Redmond, Wash.) and was analyzed using SAS (124) (SAS
Institute Inc., Cary N.C.) Statistical significance was determined
at the P<0.05 level using General Linear Models Procedure and
least squared means for body weight, bursa: body weight ratios,
spleen: body weight ratios, and proventriculus: body weight ratios.
In addition, statistical differences were determined for means of
the numerical index scores for gross lesions in the bursa and
proventriculus.
RESULTS
[0165] AC-ELISA analysis of individual proventricular tissue
homogenates harvested at 2, 3, 4, and 11 days post challenge with
USDA/STC IBDV revealed that IBDV detection was variable and
dependent on the day post challenge in which samples were obtained.
SPF white leghorn chickens challenged with USDA/STC IBDV 0.5.times.
concentration were 12.5% positive at 2 days, 75% positive at 3
days, and 33% positive at 4 days post challenge (Table 10). SPF
white leghorn chickens challenged with USDA/STC IBDV 1.times.
concentration were 50% positive at 2 days, 88% positive at 3 days,
and 80% positive at 4 days post challenge (Table 10). SPF white
leghorn chickens challenged with USDA/STC IBDV 10.times.
concentration were negative at 2 and 4 days, and 100% positive at 3
days post challenge (Table 10). All SPF white leghorn chickens
challenged with USDA/STC IBDV tested negative by AC-ELISA for
proventricular tissue homogenates harvested at 11 days post
challenge regardless of the concentration of IBDV virus given
(Table 10).
[0166] Individual proventricular homogenates from SPF white leghorn
chickens challenged with Variant E IBDV were analyzed by AC-ELISA
at 2, 3, 4 and 11 days post challenge at three different viral
challenge concentrations. Levels of IBDV detection varied with the
concentration given and the day on which samples were obtained. SPF
white leghorn chickens challenged with Variant E IBDV 1.times.
concentration were 88% positive at 2 days, 63% positive at 3 days,
and 38% positive at 4 days post challenge (Table 10). SPF white
leghorn chickens challenged with Variant E IBDV 2X concentration
were 25% positive at 2 days, 38% positive at 3 days, and 13%
positive at 4 days post challenge (Table 10). SPF white leghorn
chickens challenged with Variant E IBDV 20X concentration were 88%
positive at 2 and 3 days, and negative at 4 days post challenge
(Table 10). All SPF white leghorn chickens challenged with Variant
E IBDV tested negative by AC-ELISA for proventricular tissue
homogenates at 11 days post challenge regardless of the level of
IBDV challenge given (Table 10).
[0167] Individual bursal tissue homogenates from SPF white leghorn
chickens challenged with USDA/STC IBDV were analyzed with AC-ELISA
at 2. 3, 4, and 11 days post challenge at three different
concentrations (Table 11). SPF white leghorn chickens challenged
with USDA/STC IBDV 0.5.times. concentration were 100% positive at 2
days, 86% positive at 3 days, and 100% positive at 4 days post
challenge. SPF white leghorn chickens challenged with USDA/STC IBDV
1.times. concentration were 100% positive at 2, 3 and 4 days post
challenge. SPF white leghorn chickens challenged with USDA/STC IBDV
10.times. concentration were 38% positive at 2 days, and 100%
positive at 3 and 4 days post challenge. All SPF white leghorn
chickens challenged with USDA/STC IBDV tested negative by AC-ELISA
for bursal homogenates collected at 11 days post challenge
regardless of IBDV concentration given.
[0168] Individual bursal tissue homogenates from SPF white leghorn
chickens challenged with Variant E IBDV were analyzed with AC-ELISA
at 2, 3, 4 and 11 days post challenge at three different virus
concentrations (Table 11). SPF white leghorn chickens challenged
with Variant E IBDV 1.times. concentration were 100% positive at 2
and 3 days, and 13% positive at 4 days post challenge. SPF white
leghorn chickens challenged with Variant E IBDV 2X concentration
were 13% positive at 2 days, 50% positive at 3 days, and 88%
positive at 4 days post challenge. SPF white leghorn chickens
challenged with Variant E IBDV 20.times. were 75% positive at 2
days, 88% positive at 3 days, and 100% positive at 4 days post
challenge. All SPF white leghorn chickens challenged with Variant E
IBDV tested negative for bursal homogenates collected at 11 days
post challenge regardless of the IBDV concentration given.
[0169] Serological analysis of all IBDV challenge groups are found
in FIG. 2. Antibody titers were negative on days 2, 3, and 4 post
challenge with all SPF white leghorn chickens challenged with
USDA/STC IBDV. However, all SPF white leghorn chickens challenged
with USDA/STC IBDV were seropositive at 11 days post challenge.
Antibody titers to USDA/STC IBDV increased slightly with respect to
the original concentration of virus given at challenge but were not
significantly different. Antibody titers were negative on day 2
post challenge with all SPF white leghorn chickens challenged with
Variant E/1084 IBDV. On day 3 post challenge, there is a small
increase in titer with birds that received the 1.times. Variant
E/1084 IBDV challenge concentration. On day 4 post challenge, there
is a significant increase in the antibody titer with birds
challenged with the 1.times. Variant E/1084 IBDV. All SPF white
leghorn chickens challenged with Variant E/1084 IBDV were
seropositive at 11 days post challenge. with no significant
differences in final immune titer between challenge groups.
[0170] Microscopic lesions in the proventriculus were found at 2,
3, and 4 days post challenge with both the USDA/STC IBDV, and
Variant E/1084 IBDV (Table 12). The incidence level of lesions
varied with the challenge concentration given and the time at which
the sample was taken. Incidence levels of proventricular lesions
following challenge with USDA/STC IBDV at 2 days post challenge
were 80%, 20%, and 20% with respect to the increasing virus
inoculum. Incidence levels of proventricular lesions at 3 days post
challenge of 100% for all challenge groups. Incidence levels of
proventricular lesions were 100%, 40%, and 40% with respect to
increasing USDA/STC IBDV inoculum. All SPF white leghorn chickens
at 11 days post challenge with USDA/STC IBDV regardless of the
concentration given were negative for the presence of microscopic
lesions in the proventriculus (Table 12).
[0171] Microscopic lesions present in the proventriculus of SPF
white leghorn chickens challenged with Variant E IBDV at 2 days
post challenge were present at incidence levels of 60%, 0%, and 20%
with respect to increasing viral inoculum. SPF white leghorn
chickens at 3 days post challenge with Variant E IBDV demonstrated
proventricular lesion levels of 50%, 20%, and 100% , with respect
to increasing viral inoculum. At 4 days post challenge,
proventricular lesion levels following challenge with Variant
E/1084 IBDV were 40%, 80%, and 60% with respect to increasing virus
inoculum. By day 11 post challenge with Variant E/1084 IBDV all
challenged groups regardless of the virus inoculum given showed no
evidence of lesions in the proventriculus (Table 12).
[0172] Microscopic lesions in the bursa at 2 days post challenge
with USDA/STC IBDV were 80%, 40%, and 20% with respect to
increasing virus inoculum. SPF white leghorn chickens at 3, 4, and
11 days post challenge with USDA/STC IBDV indicate that all
challenge groups demonstrated 100% incidence of lesions in the
bursa (Table 13).
[0173] Microscopic lesions in the bursa at 2 days post challenge
with Variant E/1084 IBDV were 100%, 40%, and 40% with respect to
increasing virus inoculum. At 3 days post challenge with Variant
E/1084 IBDV, bursal lesions were found in 100% of the birds in the
1.times. and 20.times. challenge groups, whereas the 2.times.
challenge group had an incidence of only 40%. At 4 and 11 days post
challenge 100% of the SPF white leghorn chickens challenged with
Variant E/1084 IBDV demonstrated lesions in the bursa regardless of
the virus inoculum given (Table 13).
[0174] Proventiculus to body weight ratios (P:BW) were only
significantly different for birds challenged with the USDA/STC IBDV
virus at 2 days post challenge with the 0.5.times. and 10.times.
virus inoculums, and for all USDA/STC IBDV challenge groups at 11
days post challenge. At 2 days post challenge, statistical
differences were significantly smaller, and at day 11 post
challenge differences were significantly larger than the negative
control. There were no differences noted for the SPF white leghorn
chickens challenged with Variant E/1084 IBDV at any challenge level
or at any day post challenge (Table 14).
[0175] Bursa to body weight ratios (B:BW) demonstrated a
significant reduction in size at 4 days post challenge for SPF
white leghorn chickens challenged with Variant E/1084 IBDV given at
the 1.times. and 20.times. concentration. All challenge groups,
regardless of virus inoculum or IBDV strain type, were
significantly smaller at 11 days post challenge (Table 15).
[0176] Spleen to body weight ratios (S:BW) were significantly
different at 2 days post challenge with the Variant E/1084 IBDV
challenge given at 1.times. concentration. All challenge groups
were significantly larger at 3 and 4 days post challenge regardless
of virus inoculum or strain of IBDV given. On day 11 post challenge
no significant differences were noted for any of the IBDV challenge
groups with respect to strain or amount of inoculum given (Table
16).
DISCUSSION
[0177] AC-ELISA demonstrated that challenge with different levels
of USDA/STC IBDV influences the presence of virus and the intensity
of lesions in the proventriculus at different time intervals post
challenge. However, this appears to be a function of the acute
infection only as all challenge groups test negative by day 11 post
challenge. SPF white leghorn chickens that received the lower
concentration of USDA/STC IBDV inoculum had a gradual increase in
the number of birds that test positive on each successive day,
whereas SPF white leghorn chickens that received the 10.times.
USDA/STC IBDV tested positive in the proventriculus only on day 3
post challenge. This may be related to a phenomenon previously
described by a number of authors in which high virus dosage
interferes with the normal replication of the virus (7, 19).
Similar analysis of bursal homogenates from the same SPF white
leghorn chickens indicated that groups which received the lower
challenge inoculums show a gradual increase in the number of
positive samples. SPF white leghorn chickens that received the
10.times. challenge show low levels of virus at 2 days post
challenge and progress to 100% level at day 3 and 4 post
challenge.
[0178] Microscopic lesions in the proventriculi were of higher
incidence in SPF white leghorn chickens that received the 1.times.
concentration of USDA/STC IBDV at 2 days post challenge. However,
tissue samples screened on days 3 and 4 post challenge indicated no
influence from the amount of virus inoculum given. This effect is
also seen in the incidence of bursal lesions in SPF white leghorn
chickens that received the 1.times. concentration of USDA/STC at 2
days post challenge; the effect disappears on day 3 and day 4 post
challenge.
[0179] AC-ELISA analysis of proventricular homogenates at 2, 3, 4,
and 11 days post challenge with Variant E/1084 IBDV indicates both
a time and concentration effect. SPF white leghorn chickens that
received the 1.times. concentration of Variant E/1084 IBDV have an
incidence level of 88% at 2 days post challenge and then show a
gradual decline in the number of positive samples. SPF white
leghorn chickens challenged with the 2.times. Variant E/1084 show a
slight increase in the number of positives from day 2 to day 3, but
by day 4 the incidence was lower. Interestingly, these birds
demonstrate fewer positive tissue homogenates than the 1.times.
challenge group, even though receiving twice as much virus. SPF
white leghorn chickens challenged with the 20.times. concentration
of Variant E/1084 IBDV were initially equivalent with the 1.times.
dosage at 2 days post challenge, but virus presence does not
decline until day 4 post challenge. Analysis of bursal homogenates
taken from the same birds followed along similar trends as far as
virus challenge concentration were concerned. The 1.times.
challenge group appears to be declining in the number of tissue
positives while the 2.times. and 20.times. groups appear to be
increasing.
[0180] Microscopic lesions of the proventriculus following
challenge with Variant E/1084 IBDV did not correlate well with any
of the challenge groups when compared to AC-ELISA results. At 2
days post challenge the relationship between virus concentration
and the level of lesions present appears to be inverse in that the
lowest challenge concentration has the highest incidence of lesions
present in the proventriculus. No discernible pattern was
determined for the different virus concentration groups on day 3
post challenge with Variant E/1084 IBDV. Microscopic lesions of the
bursa following challenge with Variant E!1084 IBDV do not appear to
be a function of virus concentration, however, the lesion incidence
level increased over time with all challenge groups.
[0181] Significant differences in the proventriculus to body weight
ratios were only noted in groups which received USDA/STC IBDV. The
effect appears to have two separate manifestations, because during
the acute infection, proventriculi are significantly smaller than
the negative control but during the chronic infection, the
proventriculus enlarges.
[0182] Bursa to body weight ratios indicate that atrophy begins as
early as day 4 for one group of SPF white leghorn chickens
challenged with Variant E/1084 IBDV. However, there appears to be
no effect based on challenge concentration or IBDV strain
utilized.
[0183] Spleen to body weight ratios indicate that splenomegally is
present in all groups on days 3 and 4 post challenge. Again the
1.times. concentration Variant E/1084 IBDV shows an effect by 2
days post challenge. This challenge group is highly suspicious in
that it consistently does things earlier than the other challenge
groups. It is suspected that this group may have already been
incubating the virus when the actual challenge was given.
[0184] Serological measurements on days 2, 3, 4, and 11 indicate
that all SPF white leghorn challenge groups seroconverted by day 11
post challenge. The chickens challenged with Variant E/1084 IBDV
show a slight response on day 3 post challenge, and in the case of
the 1.times. challenge group a most noticeable spike in antibody
titer at day 4 post challenge. This is a clear indication that
these birds were possibly incubating the IBDV virus earlier than
when challenged. Attempts were made to try to determine if these
chickens were incubating another serotype as well as Variant IBDV.
Incorporation of specific monoclonals to differentiate standard
from variant viruses using the AC-ELISA gave mixed results, in that
some of the tissues harvested tested as standard IBDV and others
tested as Variant IBDV, while still others tested completely
negative (data not shown).
[0185] In the assessment of virus effects on the proventriculus
based on different IBDV strains, it would appear that the USDA/STC
IBDV strain exerts more of an effect on the proventriculus than
Variant E/1084 IBDV. The effects seen do not appear to be in any
way linked to the concentration of virus present but is more a
factor of time elapsed post exposure. Each IBD virus strain could
also be demonstrating different host cell preferences based on
their passage histories in cell culture and eggs. Also, it should
be noted that two observations were noticed with the USDA/STC IBDV;
during the acute phase there is a significant reduction in the size
of the proventriculus, and in the chronic phase there is a
significant enlargement of the proventriculus.
SECTION 3 THE INTERACTION OF INFECTIOUS BURSAL DISEASE VIRUS, REO
VIRUS, AND COPPER SULFATE IN THE PRODUCTION OF LESIONS ASSOCIATED
WITH PROVENTRICULITIS IN SPF WHITE LEGHORN CHICKENS
SUMMARY
[0186] Thin section electron micrographs revealed the presence of
virus like particles within the proventriculus of SPF white leghorn
chickens at four days post challenge with IBDV. SPF white leghorn
chickens exhibit lesions in the bursa and proventriculus following
IBDV challenge. Lesion assessment, both grossly and
microscopically, indicates that the USDA/STC IBDV is capable of
producing the most severe effect on the proventriculus. Pathology
from infectious bursal disease virus infection is exacerbated by
the presence of copper sulfate supplementation in the feed. The
presence of REO S-1133 virus indicated interference in the immune
response or IBDV replication, as well as the number of tissue
homogenates testing positive for IBDV at 4 days post challenge.
Mortality was significantly increased for birds challenged with
USDA/STC IBDV in the presence of copper sulfate and/or REO S-1133
virus. Birds which received dietary copper sulfate supplementation
had reduced body weights at 4 days post challenge and at 11 days
post challenge.
Introduction
[0187] Proventriculitis syndrome has been described in broiler
chickens from a number of geographical regions. This syndrome is
characterized by an enlargement of the proventriculus, accompanied
by feed impaction, and structural weakness of the organ. Affected
birds can be either normal or underweight at processing, with
either a high or normal feed conversion efficiency. Problems can
occur at the processing plant with breakage of the proventriculus
during mechanical evisceration and contamination of the carcass
with digesta and feed. This results in an increased number of
washouts, slowed or stopped processing lines, excessive trims, and
higher than normal levels of condemnation. This proventriculitis
syndrome has been linked to a number of environmental, nutritional,
genetic, and infectious causes (38).
[0188] Recent studies completed at the University of Arkansas
indicate that acute infectious bursal disease virus (IBDV)
infection that occurs between days 21-35 can produce lesions in the
proventricular mucosa. The proventriculitis induced by IBDV alone
does not exactly mimic what is observed in field situations, but
studies indicate that IBDV may play the role of a facilitator, and
thus presents an opportunity for other agents present in the
chicken house environment (and in the chicken) to exert an effect,
and as such predisposes affected birds to the possibility of
secondary infections.
[0189] This study was undertaken to determine the effect on the
proventriculus of two different IBD (USDA/STC) (Variant E/1084)
virus challenges, in the presence of respiratory enteric orphan
(REO) virus strain S-1133, when administered at 28 days post hatch.
In addition, the mixed virus challenge was further complicated by
the presence or absence of a common feed additive, copper sulfate
given at the rate of 1 lb/ton.
MATERIALS AND METHODS
[0190] Four hundred SPF white leghorn chickens derived from fertile
SPF eggs (HYVAC inc. Adel, Iowa) were hatched at the University of
Arkansas Poultry Health Laboratory Isolation Facility. Thirty
chicks were placed in each negative pressure isolation cage on day
of hatch. All birds received water and the standard University of
Arkansas diet formulation as specified by age and breed ad libitum.
Chicks given the 1 lb/ton copper sulfate dietary supplement
received it beginning on day 1 and throughout the entire study.
Lighting was maintained continuously for brooding purposes
throughout the entire study utilizing a 250 watt incandescent light
bulb in each isolator.
IBDV VIRUS CHALLENGE INOCULUM PREPARATION
[0191] The Variant E/1084 Strain IBDV challenge virus (Select
Laboratories, Gainsville, Ga.) was prepared from stock virus. The
challenge virus aliquot was removed from the ultralow freezer
(-70.degree. C.), placed in a laminar flow hood, and thawed at room
temperature. The outside of the vial was wiped with 70% ethanol to
remove any surface contamination. The virus was then diluted 1:1000
in sterile deionized water. The Variant E/1084 challenge stock
virus titer was 10.sup.5 6 TCID.sub.50/ml. Virus challenge inoculum
titer was 10.sup.2.6 TCID.sub.50/ml.
[0192] USDA/STC IBDV challenge inoculum was prepared from lot #92-1
(NVSL, Ames Iowa) as follows: The virus was removed from the
ultralow freezer (-70.degree. C.), and thawed at room temperature
in a laminar flow hood. The outside of the virus containing glass
ampule was wiped with 70% ETOH, to remove any surface
contamination. The ampule of virus was snapped open by wrapping the
neck with a paper towel and breaking along the prescored line. The
virus was then diluted 1:10 by mixing 1.5 ml of USDA/STC IBDV virus
into 13.5 ml of Dulbecco's Phosphate Buffered Saline (D-PBS)
(Appendix). The USDA/STC IBDV challenge stock virus titer was
10.sup.4 1 EID.sub.50/ml. USDA/STC IBDV virus challenge inoculum
titer was 10.sup.3.1 EID.sub.50/ml.
[0193] REO virus strain S-1133 was prepared from a viral stock
maintained at the Poultry Health Laboratory Isolation Facility. The
cryovial containing a yolk suspension of the 8th egg passage of REO
virus S-1133 was removed from the ultralow freezer storage
(-70.degree. C.) and allowed to thaw at room temperature in a
laminar flow hood. The outside of the vial was wiped with 70%
ethanol to remove any surface contamination. The cryovial was then
opened and 0.1 ml of the virus containing yolk suspension was
diluted to 1:1000 in 99.9 ml of sterile D-PBS . The REO virus
strain S-1133 stock virus titer was 10.sup.6.8 EID.sub.50/ml. REO
virus challenge inoculum titer was 10.sup.4 8 EID.sub.50/ml.
SPF BIRD CHALLENGE
[0194] SPF white leghorn chickens were challenged on day 28 post
hatch (PH) with Variant E/1084 IBDV, USDA/STC IBDV, and/or REO
virus S-1133 with prepared inoculums as described previously.
Infectious bursal disease virus challenge was administered to each
individual bird bilaterally to the eye (30 .mu.l per eye) using an
Eppendorf micropipettor (Brinkmann Inc., Westbury, N.Y.) and a
sterile pipette tip (Costar Corp., Cambridge, Mass.). REO virus S-1
133 challenge inoculum was given at a rate of 0.1 ml per individual
bird and was administered orally using an Eppendorf micropipettor
(Brinkmann Inc., Westbury, N.Y.) and a sterile pipette tip (Costar
Corp., Cambridge, Mass.). Experimental groups and challenge
inoculums are given in Table 17.
SAMPLE COLLECTION
[0195] On days 4 and 11 post challenge 10 birds were necropsied
from each virus challenge group and an additional 10 birds from the
negative control. Each bird was weighed, bled, and euthanized by
CO.sub.2 asphyxiation. All birds were necropsied at which time the
bursa, and proventriculus were scored for the presence of gross
lesions. The bursa, spleen, and proventriculus were individually
weighed and results recorded. Tissues from each of the birds were
cut in half, with one half being placed into 10% buffered formalin,
and the other half being placed into a sterile sampling bag (Fisher
Scientific, Pittsburgh, Pa.). Tissues in sterile sampling bags were
frozen at -20.degree. C. for later analysis utilizing IBDV Antigen
Capture ELISA(AC-ELISA), as described by Snyder et al (138).
[0196] In addition, proventriculus samples were collected for
electron microscopic evaluation for the presence of USDA/STC IBDV
and Variant E/1084 IBDV at 4 days post challenge. Tissue samples
from the papillae region of the proventriculus were collected at
necropsy and manually trimmed to approximately 2 mm.sup.2. Tissue
specimens were fixed in a modified Karnovsky's fixative (74)
containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.05 M
cacodylate buffer, pH 7.2. Tissues were then post-fixed in 1%
osmium tetroxide in 0.05 M cacodylate buffer, pH 7.2. Tissues were
stained overnight in 0.5% aqueous uranyl acetate, dehydrated
through a graded ethanol series, and embedded in Spurr's medium
(143).
[0197] Thin sections were cut with a glass knife, captured on a 300
mesh copper grid and stained with 2% aqueous uranyl acetate
followed by lead citrate. Grids were examined for the presence of
IBDV using a JEOL 100 CX electron microscope.
ANTIGEN CAPTURE ELISA PROCEDURE
[0198] AC-ELISA test plates were prepared as described earlier in
Section 1. Tissues were homogenized in AC-ELISA dilution buffer at
a ratio of 1:5 tissue weight to volume. Homogenates were analyzed
in duplicate against monoclonal B-29 (University of Maryland,
College Park, Md.; Intervet Inc., Millsboro Del.) AC-ELISA plates
as described earlier in Section 1. Plates were read and means were
calculated for the paired samples within each group and the
homogenates were determined to be positive or negative based on the
mean absorbance values as compared to the negative and positive
controls on each plate.
BLOOD SAMPLE COLLECTION AND PROCESSING
[0199] Blood samples were collected individually using 4.5 ml
Sarstedt monovette.TM. syringes (Sarstedt, Inc., Arlington, Tex.)
containing clot activating beads and 22 gauge, 11/2 inch needles
(Becton Dickinson and Company, Rutherford, N.J.). Serum was
separated from the clot by centrifugation at 1000 rpm for 10
minutes using an IEC tabletop centrifuge. Serum was collected and
placed in both a 96 well round bottom plate (Corning Glass Works,
Corning N.Y.) and into Nalgene.TM. cryovials (Nalge Company,
Rochester, N.Y.). All serum samples were frozen at -20.degree. C.
until they were analyzed using the commercial KPL Infectious Bursal
Disease Virus ELISA Kit and the Flock Profile.TM. software
(Kirkegaard and Perry Laboratories, Inc., Gaithersburg, Md.).
HISTOPATHOLOGY
[0200] Tissues were placed in buffered formalin at necropsy, and
processed twenty-four hours later by the method described in
Section 1 (55). Tissues were examined for the presence of
histopathological lesions induced by IBDV infection. Tissues were
given a numerical index score based on the presence or absence as
well as the intensity of the lesions present. The score ranged from
a low of "0" which indicates no lesions or pathology observed to a
high of "4" which indicates extensive tissue damage.
FEED ANALYSIS
[0201] Feed samples were pulled at the end of the study and sent
for analysis to establish that the two diets varied only in the
different levels of copper sulfate present. Feed samples were
analyzed at the Center of Excellence for Poultry Science, Central
Analytical Laboratory. The diet without copper sulfate
supplementation contained 22.3% protein, 89.0% dry matter, 5.6%
ash, and 17.44 ppm copper. The diet with copper sulfate
supplementation contained 22.6% protein, 89.5% dry matter, 5.6%
ash, and 95.87 ppm copper.
STATISTICS
[0202] All data was entered into Microsoft.TM. Excel (Microsoft
Corp., Redmond, Wash.). and were analyzed using SAS (10) (SAS
Institute Inc., Cary, N.C.). Statistical significance was
determined at the P<0.05 level using General Linear Models
Procedure and least squared means for body weight, bursa: body
weight ratios, spleen: body weight ratios. and proventriculus: body
weight ratios. In addition, statistical differences were determined
for means of the numerical index scores for gross lesions in the
bursa and proventriculus.
RESULTS
[0203] Tissue sections studied with the electron microscope
revealed the presence of virus like particles (VLP's), of
approximately the same size (60 nm) and shape as described for IBD
virus (FIGS. 4-8). These particles were observed in the cytoplasm
of the mucosal epithelium surrounding the proventricular papillae.
The particles were found individually or in surrounded by a
cytoplasmic membrane, appearing as virus containing inclusion
bodies. The inclusion bodies also contained fibrillar and electron
lucent material, and membranous vesicles of various sizes, which
are similar to those interpreted as phagocytic vacuoles or
lysosomes. Structure similar to those virus containing inclusion
bodies but without the virus like particles were also present in
the negative control tissues (FIG. 3). Virus containing inclusion
bodies were found predominantly in SPF white leghorn chickens
challenged with USDA/STC IBDV+copper sulfate.
[0204] AC-ELISA analysis of bursal and proventricular tissue
homogenates from SPF white leghorn chickens challenged with Variant
E/1084 IBDV tested positive only on day 4 post challenge. The
presence of REO virus and/or copper sulfate did not significantly
reduce the number of bursal tissues that tested positive. However,
the combination of Variant E/1084, REO virus S-1133, and copper
sulfate did reduce the number of virus infected proventriculi
(Table 18).
[0205] AC-ELISA analysis of bursal and proventricular tissue
homogenates from SPF white leghorn chickens challenged with
USDA/STC IBDV tested positive only on day 4 post challenge (Table
19). Experimental treatments with USDA/STC IBDV+REO virus S-1133 or
USDA/STC IBDV+copper sulfate showed a reduction in the number of
bursal samples which tested positive. However, the combination of
USDA/STC IBDV, REO virus S-1133, and copper sulfate showed no
significant reduction in the number of samples which tested
positive. The number of positive test samples from the
proventriculus in IBDV challenged groups appears to only be
adversely affected by the presence of copper sulfate (Table
19).
[0206] Serological testing utilizing the KPL infectious bursal
disease virus ELISA kit, of serum samples obtained on day 4 post
challenge with Variant E/1084 IBDV indicate that all experimental
SPF white leghorn chickens had no measurable levels of serum
antibody to IBDV (FIG. 9). On day 11 post challenge with Variant
E/1084 IBDV the highest antibody response was to the virus alone.
Antibody titers to the Variant E/1084+REO+copper sulfate were not
significantly different from the virus alone. However, the antibody
response to Variant E/1084 IBDV+copper sulfate was significantly
suppressed (FIG. 9).
[0207] Serological testing of serum samples obtained on day 4 post
challenge with USDA/STC IBDV indicates that all experimental SPF
white leghorn chickens had no measurable levels of serum antibody
to IBDV (FIG. 10). On day 11 post challenge with USDA/STC IBDV, the
highest antibody response was USDA/STC IBDV+copper sulfate. The
USDA/STC IBDV alone, USDA/STC IBDV+REO+copper sulfate, and the
1/2.times. USDA/STC IBDV+copper sulfate had antibody titers that
were significantly lower than the USDA/STC IBDV+copper sulfate. The
lowest antibody response was with birds that received USDA/STC
IBDV+REO (FIG. 10). Gross lesion scores were only significantly
different on day 4 post challenge with both strains of IBDV (Table
20). Challenge with Variant E/1084 IBDV resulted in an increase in
the gross lesions involving the papillae only and was most
pronounced in the challenge group that received Variant E/1084
IBDV+copper sulfate. It should be noted that the addition of REO
virus S-1133 in the presence of dietary copper sulfate and Variant
E/1084 IBDV resulted in the reduction of the number of lesions
present. While the gross lesion incidence in this group was still
significantly higher than the negative control, it was
approximately 50% less than the value of Variant E/1084 alone and
25% less than value of Variant E/1084 IBDV+copper sulfate (Table
20).
[0208] Gross lesions in the proventriculus of SPF white leghorn at
4 days post challenge with USDA/STC IBDV were more extensive than
those with Variant E/1084 IBDV (Tables 20 and 21). Hemorrhage
values are significantly increased in the presence of USDA/STC
IBDV+copper sulfate, and with USDA/STC IBDV+REO. However, the
combination of USDA/STC IBDV+REO+copper sulfate is not
significantly different from the negative control values (Table
21). Hemorrhage associated with IBDV infection was contained within
the mucosa of the proventriculus and were visible grossly as well
(FIG. 11).
[0209] Papillae gross scores for birds challenged with Variant
E/1084 in the presence or absence of copper sulfate and REO virus
were significantly different from the negative control (Table 20).
Papillae gross scores were significantly different from the
negative control in all USDA/STC IBDV challenge groups with the
exception of the USDA/STC IBDV+copper sulfate. Papillae scores were
highest for the challenge group which received USDA/STC IBDV+REO,
similar for the challenge groups that received the USDA/STC IBDV
only and the USDA/STC IBDV+REO+copper sulfate and lower in the
1/2.times. USDA/STC IBDV+copper sulfate (Table 21).
[0210] Gross lesions in the gizzard were assessed as to the degree
of organ rigidity at necropsy. Numerical value assignments were
assessed according to the softness of the tissue, and a higher
score value reflects a tissue that is soft and pliable. Lower value
scores reflect a tissue that is extremely rigid. Gizzard rigidity
in birds challenged with Variant E/1084 in the presence or absence
of copper sulfate and/or REO virus was not significantly different
from the negative control values at 4 days post challenge (Table
20). Gizzard rigidity was highest in the USDA/STC IBDV challenge
group. Gizzards from the groups which received USDA/STC IBDV+copper
sulfate and USDA/STC IBDV+REO S-1133 were also significantly harder
than the negative control values (Table 21).
[0211] Microscopic tissue lesions in the proventriculus following
IBDV challenge were found predominantly in the lamina propria of
the mucosa and were characterized- by pleomorphism of the lymphoid
cells in the mucosa (FIG. 12). This cellular pleomorphism was
sometimes accompanied by lymphocyte depletion, similar to what is
described in the bursa following IBDV challenge. The addition of
copper sulfate in the infection scheme introduced a new type of
lesion within the secretory glands of the proventriculus. The cells
which line the glandular ducts appeared to be swollen and contain
large vacuoles (FIG. 13). Lesions in the bursae were predominantly
lymphoid depletion with an accompanied follicle degeneration.
[0212] Microscopic tissue lesions in the bursa following challenge
with Variant E/1084 IBDV were present in all the SPF white leghorn
chickens sampled on day 4 and day 11. Microscopic tissue lesions in
the proventriculus following challenge with Variant E/1084 IBDV
were present only on day 4 post challenge. There was a reduction in
the number of SPF white leghorn chickens exhibiting lesions in the
Variant E/1084+copper sulfate treatment (Table 22).
[0213] Microscopic tissue lesions in the bursa following challenge
with USDA/STC IBDV were present in all the SPF white leghorn
chickens sampled on day 4 and day 11 (Table 23). Microscopic tissue
lesions in the proventriculus following challenge with USDA/STC
IBDV were present only on day 4 post challenge. There was no
significant reduction in the presence of lesions by any of the
treatments (Table 23).
[0214] Bursa to body weight and spleen to body weight ratios of SPF
white leghorn chickens at 4 days post challenge with Variant E/1084
IBDV were significantly different in all IBDV challenged groups
(Table 24). There were no significant differences in the
proventriculus to body weight ratios in any of the experimental
groups at day 4 post challenge. At day 11 post challenge all IBDV
challenged groups had significantly lower bursa to body weight
ratios. The experimental group that received Variant E/1084 IBDV
only was significantly different for the spleen to body weight
ratios. The proventriculus to body weight ratios at day 11 post
challenge were significantly different only for the experimental
group that received Variant E/1084 IBDV+REO+copper sulfate (Table
24).
[0215] Bursa to body weight ratios of SPF white leghorn chickens at
4 days post challenge with USDA/STC IBDV were significantly
different for groups that received USDA/STC IBDV+REO+copper
sulfate, and USDA/STC IBDV+REO (Table 25). At 4 days post challenge
all USDA/STC IBDV challenged groups had significantly different
spleen to body weight ratios. At 4 days post challenge the
proventriculus to body weight ratios were significantly different
only in the group that received USDA/STC IBDV only. On day 11 post
challenge all experimental groups that received USDA/STC IBDV were
significantly atrophied. On day 11 post challenge only the USDA/STC
IBDV+copper sulfate group was significantly different from the
negative control. On day 11 post challenge USDA/STC IBDV only,
USDA/STC IBDV+copper sulfate, and USDA/STC IBDV+REO+copper sulfate
were significantly different from the negative control (Table
25).
[0216] Mortality for the study was 44% for SPF white leghorn
chickens challenged with USDA/STC IBDV+REO+copper sulfate, 40% for
1/2.times. USDA/STC IBDV+copper sulfate, 21% for USDA/STC+copper
sulfate, 15% for USDA/STC IBDV+REO, and 11% for USDA/STC IBDV only
(FIG. 14). Mortality began at approximately 3 days post challenge,
peaked at 4 days post challenge, and continued until 5 days post
challenge. Variant E/1084 did not produce any mortality throughout
the entire study (data not shown).
[0217] Mean body weight differences were compared for 4 and 11 days
post challenge with Variant E/1084 IBDV (FIG. 15). All SPF white
leghorn chickens gained weight between day 4 and day 11 post
challenge, however, all experimental groups had lower weight gains
than the negative control group. REO virus infection in the
presence and absence of copper sulfate, and Variant E/1084 did not
significantly decrease weight gain. However, copper sulfate only,
Variant E/1084 IBDV only, and Variant E/1084 IBDV+copper sulfate
body weight gain was significantly lower than the negative control
(FIG. 15).
[0218] Mean body weight differences were compared for 4 and 11 days
post challenge with USDA/STC IBDV (FIG. 16). All SPF white leghorn
chickens challenged with USDA/STC IBDV gained weight between day 4
and day 11 post challenge, however, all experimental groups had
lower weight gains than the negative control group. Day 11 body
weight means were only significantly different in the USDA/STC
IBDV+REO+copper sulfate, and the copper sulfate only groups.
DISCUSSION
[0219] Electron micrographs reveal the presence of viral inclusion
bodies at 4 days post challenge. These inclusions are similar in
size and structural characteristics to those described by a number
of other authors for bursal tissues infected with IBDV (17, 40, 52,
73, 86). While micrographs presented are from only one experimental
group, USDA/STC IBDV+copper sulfate, they in no way indicate that
other experimental IBDV groups were lacking in viral inclusions.
Rather that pictures were selected based on the quality of the
sections cut and the reproducibility of the photos.
[0220] AC-ELISA indicated the presence of USDA/STC and Variant
E/1084 IBDV in the bursa and proventriculus at 4 days post
challenge. The presence of REO virus S-1133 and copper sulfate did
not interfere with the number of bursal tissues that tested
positive by AC-ELISA. However, in IBDV challenged groups in the
presence of REO S-1133 virus or copper sulfate there was a
reduction in the number of tissues that tested positive by
AC-ELISA. Both, the copper sulfate and REO virus S-1133 exert their
effects on the chicken primarily in the proventriculus and the
possibility exists that there is competition for limited substrate
in the proventriculus and that the presence of both mediates the
reaction. In contrast, the combination of IBDV+REO+copper sulfate
reduced the number of samples that tested positive in the bursa but
not in the proventriculus indicating the possibility of other
influencing factors.
[0221] Serological evidence showed a reduced response in the
presence of copper sulfate for Variant E/1084 and an increased
response to USDA/STC IBDV. USDA/STC IBDV antibody response was
significantly reduced in the presence of REO S-1133 virus. Antibody
response to USDA/STC IBDV has consistently been lower than antibody
response to Variant E/1084 IBDV. This has been generally attributed
to the higher degree of damage induced by USDA/STC IBDV and a lack
of cells capable of producing antibodies. It would appear that the
addition of REO S-1133 virus further reduces the production of
antibodies against USDA/STC IBDV, possibly as it exerts its own
immunosuppressive mechanisms or through interference.
[0222] Gross lesions in the proventriculus involving the papillae
were noticeably increased in the presence of copper sulfate+Variant
E/1084. However, papillar lesions were reduced in the presence of
REO S-1133. Gross lesions associated with USDA/STC IBDV included
hemorrhage, and involvement of papillae in all but the USDA/STC
IBDV+copper sulfate challenge group. Papillar lesions were of
highest incidence in the presence of USDA/STC IBDV+REO S-1133 and
with the addition of copper sulfate there appears to be a mediation
of the reaction. Gizzard rigidity was also noticeably higher in SPF
white leghorn chickens which received USDA/STC IBDV alone, or
+copper sulfate, or +REO S-1133. The combination of all three did
not appear to exert any effect.
[0223] Microscopic lesions are present in the bursa at day 4 and 11
post challenge with both IBDV strains as expected. Microscopic
lesions in the proventriculus were however, only present on day 4
post challenge. There appears to be no treatment influence with
USDA/STC IBDV, but the presence of copper sulfate reduced the
number of birds showing lesions with Variant E/1084 IBDV.
[0224] Organ to body weight ratios were significantly different in
the bursa and spleen on day 4 post challenge, but effects were seen
in the proventriculus only on day 11 post challenge. The
proventriculus was most affected by the combination of Variant
E/1084+REO+copper sulfate and copper sulfate+REO challenge groups.
Reduced overall body weights were noticed for all IBDV challenged
groups as well as those receiving copper sulfate with no virus.
Bayyari et al (5) described similar effects in broiler chickens
given dietary copper sulfate in the presence of an undefined
pathogen and Wideman et al (152) demonstrated similar findings with
diets containing excessive levels of copper sulfate.
[0225] Mortality was significantly increased in the USDA/STC IBDV
in the presence of REO S-1133 and copper sulfate, however, there
was no increase in mortality with Variant E/1084. All mortality
occurred prior to necropsy in the USDA/STC IBDV groups. SPF white
leghorn chickens that died prior to necropsy were not tested for
virus in the bursa and proventriculus and represent potential
positive samples had they been tested.
SECTION 4 INFECTIOUS BURSAL DISEASE VIRUS INDUCED APOPTOSIS
ASSOCIATED WITH PRODUCTION OF LESIONS IN THE PROVENTRICULUS AND
BURSA
SUMMARY
[0226] The presence of apoptotic cells in the proventriculus and
bursa at 4 days post challenge with USDA/STC IBDV and Variant
E/1084 IBDV was determined using a modified TUNEL fluorescent
staining procedure. Specific green fluorescence was found in
highest quantity in the bursa, no differences in the intensity of
staining could be determined for each IBDV strain. Fluorescence was
focused predominantly in the medulla and cortex of infected bursae.
Proventricular fluorescent staining was found predominantly in the
villi and submucosa. Fluorescent intensity of proventricular
sections appeared highest in tissue sections from SPF leghorn
chickens challenged with USDA/STC IBDV.
INTRODUCTION
[0227] Apoptosis has been proposed as one of the mechanisms by
which a number of viruses induce pathology (49, 70), recent studies
have indicated that IBDV also utilizes this mechanism (6, 13, 145,
148, 149). Apoptosis is a genetically ordered sequence of events
following a very specific cellular signaling stimulus in which
irreparably damaged cells are disposed of with minimal damage to
surrounding cells or tissue.
[0228] Apoptosis should not be confused with necrosis, as each
represents a totally different set of events albeit with some
overlap. Cellular necrosis results from physical injury and is not
in any way genetically controlled, whereas apoptosis is a
genetically predetermined deliberate cellular response to specific
developmental and environmental stimuli. Injury events that produce
necrosis can trigger apoptosis in response to the presence of
cellular debris and inflammation. Likewise, it may be possible that
induction of apoptosis could indirectly produce necrosis in some
instances where cellular degeneration results from a disease
process. Necrosis is typified by the destruction of cytoplasmic
organelles and loss of the integrity of the plasma membrane (12,
92, 151). Apoptosis is associated with the boiling of the
cytoplasm, chromatin condensation (12, 49, 70, 92, 151), fractured
nuclear DNA, and can be distinguished from necrosis by lack of an
inflammatory response (12, 92, 151).
[0229] Where viruses are concerned, the inhibition of apoptosis has
resulted in persistent infections, latency, or enhanced virus
production (49, 70, 145, 148, 149). On the other hand, promotion of
apoptosis has been demonstrated to facilitate virus spread and
release (12, 92, 151).
[0230] Infectious bursal disease virus infection occurs sometimes
without overt clinical disease symptoms. Previous experiments
utilizing IBDV challenge in SPF white leghorn chickens presented a
number of tissues in which the cellular damage present could not be
directly linked to necrosis following a viral inflammatory
response. Experimental data also indicated that virus persistence
in tissues (AC-ELISA) could be associated with more severe
pathology during the acute stage of the infection. The purpose of
this study was to determine the presence of apoptotic cells in the
bursa and proventriculus following challenge with USDA/STC IBDV and
Variant E/1084 IBDV.
MATERIALS AND METHODS
[0231] Formalin fixed tissues from experiments described in Section
3 were utilized in this staining procedure. All tissues utilized
were harvested at 4 days post challenge with a 1.times.
concentration of USDA/STC IBDV or Variant E/1084 IBDV. Tissues from
specific birds were selected following an initial screening with
hematoxylin and eosin stained sections. Tissue preparation and
sectioning were as follows. Tissues were placed in 10% buffered
formalin at necropsy. Twenty-four hours later tissues were removed,
manually trimmed to a thickness of 1/8 inch and placed in tissue
cassettes. Tissues were dehydrated through graded alcohols and
cleared in two changes of toluene. Tissues were then infiltrated
with paraffin over a 16 hour period using an autoprocessor.
Following paraffin infiltration, tissues were positioned in
paraffin blocks and trimmed to expose a uniform tissue face.
Prepared tissue blocks were stored at -20.degree. C. until ready to
be sectioned. Sections were cut at a thickness of 5 .mu.m and
floated on a water bath containing 5% gelatin to facilitate
attachment (55). Sections were captured onto standard 1.times.3
inch glass microscope slides that had been coated with poly
L-lysine solution (Appendix) (Sigma, Inc., St. Louis, Mo.). Tissue
blocks were sectioned and multiple slides were prepared for each
specimen. Slides with tissue sections were stored at 4.degree. C.
until they were deparaffinized for staining.
TUNEL TISSUE STAINING PROCEDURE
[0232] The TUNEL procedure detects the presence of apoptotic cells
by the in situ labeling of breaks in the nuclear DNA within
individual cells. TUNEL stands for TdT-mediated dUTP-biotin nick
end labeling and is based on the specific binding of terminal
deoxynucleotidyl transferase(TdT) to exposed 3'-OH ends of DNA
fragments generated through apoptotic mechanisms (12). The
following procedure outlines a modified TUNEL procedure utilized
for apoptotic assessment of bursal and proventricular tissues
following challenge with IBDV.
[0233] Tissue sections were prepared as described earlier and
removed from storage at 4.degree. C. Tissue sections had been
previously assigned numbers so that the individual screening of
them would be blinded to the treatments. Each experimental group
was represented by tissues from 3 SPF white leghorn chickens, and
all sections were cut and stained in triplicate. Prepared slides
with suspect tissue sections were deparaffinized and hydrated
through the following solutions: twice in 100% Xylene for 5 minutes
each, twice in 96% ethanol for 3 minutes each, 90% ethanol for 3
minutes, 80% ethanol for 3 minutes, and double distilled water
(ddH.sub.2O) for 10-20 minutes. Tissue sections were then treated
with a proteinase K (Sigma, Inc., St. Louis, Mo.) solution (5 ug/ml
with 0.5% Triton X-100) in Dulbecco's phosphate buffered saline
(D-PBS) (Appendix) for 5-6 minutes. Slides were then washed 2 times
with D-PBS for 5 minutes each. The slides were then placed on a
flat slide tray and tissue sections were covered with equilibration
buffer (Appendix) and incubated at room temperature for 10 minutes.
Equilibration buffer was removed by wicking onto a Kimwipe.TM.
(Kimberly Clark Corp., Roswell, Ga.). Tissue sections were then
covered with TdT 12-dUTP reaction mixture (Appendix). Sections were
incubated at 37.degree. C. with high humidity for 1 hour. Reaction
buffer was removed and slides were placed in stop buffer (330 mM
NaCl, 30 mM NaCitrate, pH 8.0) at room temperature for 15 minutes.
Slides were then counter stained using 0.1 ug/ml DAPI
(4',6-Diamidine-2'-phenylindole dihydrochloride) (Boehringer
Mannheim Biochemica, Indianapolis, Ind.) in D-PBS for 15 minutes.
Slides were washed 3 times with D-PBS, coverslips were mounted and
sealed with nail polish. Slides were stored at 4.degree. C.
overnight before viewing for the presence of fluorescence. Slides
were viewed using an Olympus BX50 microscope equipped with a
Olympus PM-C35X camera and Omega dye specific fluorescence filters
(DAPI 385-395) (FITC 475-497). An Image Pro computer analysis
system equipped with an Optronic cd video camera under the control
of Image Pro Plus software (Media Cybernetics, Silver Spring, Md.)
was utilized to capture images from stained slides at the same
time.
RESULTS
[0234] Tissue sections were viewed for the presence of specific
green fluorescence in the cellular nuclei and scored as positive
when present and negative when absent (Table 26). No quantitation
of the amount of fluorescence present was attempted, but visual
differences were noticeable as to intensity of the fluorescence and
the localization of the fluorescence within tissue sections. The
degree of staining intensity as a reflection of the total
involvement of the tissue section is reflected in the +or - score
in Table 25. Fluorescence was present in all bursal and
proventricular tissues from SPF white leghorn chickens that were
challenged with USDA/STC IBDV or Variant E/1084 IBDV 4 days prior
to sample collection. Staining intensity of the bursal sections was
higher within the medulla and cortex of the bursal follicles (FIG.
17), with low levels of fluorescence in the connective tissue
surrounding the follicles (FIG. 18). Staining of the proventricular
tissues was found predominantly within the villi (FIGS. 19 and 20)
with occasional staining in the glands (FIG. 21). The specific
green fluorescence was noticeably absent from negative control SPF
white leghorn chickens (FIG. 22). Tissues which demonstrated more
severe lesions with H and E staining also reflected the presence of
apoptotic cells deeper in the area of the glands (FIG. 21).
DISCUSSION
[0235] Formalin fixed paraffin embedded proventricular tissues
harvested from SPF white leghorn chickens at 4 days post challenge
with USDA/STC IBDV and Variant E/1084 IBDV demonstrated evidence of
the presence of apoptolic cells. Although the intensity and the
incidence of this particular type of staining were not quantitated,
visual observations were that higher levels of apoptotic cells were
present in the proventriculus of birds challenged with USDA/STC
IBDV. This is not to say that apoptotic cells were totally absent
from proventriculi that were exposed to Variant E/1084 but just
that there number appears to be less. This may in fact be a
reflection of where the section of tissue is actually cut, and not
a reflection of viral pathogenicity. Also, please note that these
sections were taken at only one time point following IBDV
challenge, in SPF white leghorn chickens that were given what is
considered a standard challenge of each IBDV strain. Based on the
known infectivity titers of these two virus preparations, the
USDA/STC IBDV had an advantage over Variant E/1084 IBDV. Previous
studies have indicated that lesions are more severe in the
proventriculus of SPF white leghorn chickens following challenge
with the USDA/STC IBDV.
[0236] Bursal tissues also demonstrated the presence of apoptotic
cells following challenge with both the USDA/STC IBDV and Variant
E/1084 IBDV. No visual differences could be determined for
fluorescent intensity and quantity of stained bursal cells between
the two challenge strains of IBDV. This is more or less a
reflection of the large number of cells that fluoresce in infected
bursae following staining.
SECTION 5 MEASUREMENT OF HEMOLYTIC COMPLEMENT LEVELS AND VIRUS
NEUTRALIZING ANTIBODY TITERS IN SPF WHITE LEGHORN CHICKENS 4, 6, 8
AND 11 DAYS POST CHALLENGE WITH INFECTIOUS BURSAL DISEASE VIRUS
STRAINS USDA/STC, VARIANT E/DEL, AND VARIANT E/1084.
SUMMARY
[0237] Hemolytic complement levels were decreased at 4 days post
challenge in all IBDV challenge groups. Highest level of depletion
was in the SPF white leghorn chickens challenged with USDA/STC
IBDV. Complement levels were recovered by 6 days post challenge
which corresponded with measurable levels of neutralizing
antibodies against USDA/STC IBDV. IBD virus was detectable by
AC-ELISA at this time also, indicating that all the components
needed for the stimulation of complement mediated pathology are
present and as such remain as one possible mechanism for IBDV
induced tissue damage. Virus neutralizing antibodies appeared first
in the SPF white leghorn chickens challenged with Variant E/DEL at
6 days post challenge. All IBDV challenged groups had measurable
IgG titers at day 8, but there was a subsequent decrease in titer
at day 11.
INTRODUCTION
[0238] Infectious bursal disease virus (IBDV) infection induces a
higher level of morbidity and mortality in older chickens than in
younger chickens. Neutralizing antibodies are at detectable levels
within 3 days post challenge with IBDV. This corresponds well with
the 3 and 5 day time period in which the clinical signs and
mortality are most severe with IBDV infection (79, 134, 135, 136).
Bursal lesions induced in older chickens during IBDV infection have
been described as an Arthus-type reaction (134). An arthus reaction
results from the interaction of antigen-antibody complexes and
serum complement (C') and is usually localized in an area where the
antigen-antibody complexes are plentiful. Some of the pathology
observed with IBDV infection can at least in part be attributed to
this type of hyperimmune reaction (135). Lesions in the bursa,
following IBDV infection, are well characterized, but evidence
suggests that this virus affects other lymphoid areas in the
chicken, and few if any lesions have been thoroughly
characterized.
[0239] Previous experiments at the University of Arkansas have
demonstrated that IBDV infection and related pathology also occurs
in the proventriculus. Acute infection often results in the
presence of hemorrhages in the papillae of the proventriculus.
Pathology observed following IBDV infection in the proventriculus
could also be a result of an Arthus type reaction. The purpose of
this study was two fold: (1) to determine if there were different
levels of circulating serum complement during IBDV infection with
different strains of IBDV; (2) to measure neutralizing antibody
levels, and time of appearance following infection with IBDV. Our
goal at this point was to link complement levels and neutralizing
antibody titers with the appearance and intensity of lesions in the
proventriculus following IBDV infection.
MATERIALS AND METHODS
[0240] One hundred specific pathogen free (SPF) chicks were hatched
at the University of Arkansas Poultry Health Laboratory Isolation
Facility. Chicks were derived from fertile SPF eggs obtained from
HYVAC Inc., Adel, Iowa. Chicks were placed in negative pressure
isolation cages on day of hatch. Twenty-five chicks were placed in
each of 4 isolation cages. All birds received water and the
standard University of Arkansas diet formulation as specified by
age and breed ad libitum. Lighting was maintained continuously for
brooding purposes throughout the entire study utilizing a 250 watt
incandescent light bulb. Birds were maintained under isolation
until they reached the age of 28 days post hatch.
IBDV CHALLENGE INOCULUM PREPARATION
[0241] USDA/STC IBDV lot# 92-1 was obtained from the National
Veterinary Services Laboratory (NVSL) in Ames, Iowa. The virus was
maintained at the University of Arkansas Poultry Health Laboratory
Isolation Facility at -70.degree. C. until needed. One ampule of
virus was removed from the ultralow freezer, and thawed at room
temperature in a laminar flow hood. The outside of the virus ampule
was wiped with 70% ETOH to remove any surface contamination. The
ampule of virus was snapped open by wrapping the neck with a paper
towel and breaking along the prescored line. The USDA/STC IBDV
inoculum was prepared as per NVSL instructions in which 1.5 ml of
lot # 92-1 USDA/STC IBDV were diluted in 13.5 ml of Dulbecco's
Phosphate Buffered Saline (D-PBS) (Appendix). The titer of the
USDA/STC IBDV virus was previously determined to be 10.sup.4 1
EID.sub.50/ml.
[0242] The Variant E (E/DEL) challenge virus (Intervet America,
Millsboro Del.) was prepared from stock virus lot # 060490. The
lyophilized challenge virus aliquot was removed from the ultralow
freezer (-70.degree. C.) and placed in a laminar flow hood. The
outside of the vacuum sealed lyophilization container was wiped
with 70% ETOH prior to removal of the aluminum seal to remove any
surface contamination. The butyl rubber septum was also disinfected
with 70% ETOH before insertion of needle with fluid for
resuspension. A three cc syringe (Becton Dickinson and Company,
Rutherford, N.J.) was filled with two ml of sterile D-PBS through a
16 gauge 11/2-inch needle (Becton Dickinson and Company,
Rutherford, N.J.). The needle was then inserted through the butyl
rubber seal, and the contents of the syringe was drawn into the
vacuum of the lyophilization vial. The lyophilization vial was
gently agitated to facilitate the rehydration of the viral pellet.
The vial was then inverted and 0.5 ml of the Variant E/DEL IBDV
suspension were withdrawn from the vial into the syringe. The
contents of the syringe was then diluted into 74.5 ml of sterile
D-PBS. The syringe was rinsed twice with the diluent solution to
remove any residual virus and was then discarded into the hazardous
medical waste container. The remainder of the challenge virus stock
was aliquoted to cryovials, labeled and frozen at -70.degree. C.
Challenge virus stock titer was 10.sup.5 7 EID.sub.50ml following
dilution at 1:150 the challenge Var E/DEL concentration was
10.sup.3.0 EID.sub.50/ml.
[0243] The Variant E/1084 strain IBDV challenge virus (Select
Laboratories, Gainsville, Ga.) was prepared from stock virus. The
challenge virus aliquot was removed from the ultralow freezer
(-70.degree. C.), placed in a laminar flow hood, and thawed at room
temperature. The outside of the cryovial tube was wiped with 70%
ETOH prior to opening to remove any surface contamination. The
virus was then diluted 1:100 and 1:1000 in sterile deionized water.
The titer of the IBDV Variant E/1084 challenge stock virus was 1056
TCID.sub.50/ml. Virus challenge inoculum titers were as follows:
1:100 1084-E=10.sup.3 6 TCID.sub.50/ml and for the 1:1000 1084-E=10
2.6 TCID.sub.50/ml.
SPF BIRD CHALLENGE
[0244] All SPF white leghorn chickens were maintained under
isolation until they reached 28 days post hatch. Each of three
isolation cages of SPF white leghorn chickens were challenged with
one of three different IBDV virus strains, with the fourth
isolation cage remaining as the negative control. All IBDV
challenges were administered bilaterally to each eye using an
Eppendorf pipettor (Brinkmann, Inc., Westbury, N.Y.) and a sterile
pipette tip (Costar Corp., Cambridge, Mass.). All IBDV challenge
inoculum's were given at the standard 1.times. challenge dose.
SAMPLE COLLECTION
[0245] On days 4 and 11 post challenge 10 SPF white leghorn
chickens were necropsied from each virus strain challenge group and
also from the negative control. Each bird was weighed, bled, and
euthanized by CO.sub.2 asphyxiation. All birds were necropsied at
which time the bursa and proventriculus were scored for the
presence of gross lesions. The bursa, spleen and proventriculus
were weighed individually and results recorded. Individual tissues
from each bird were cut in half, with one half being placed into
10% buffered formalin, and the other half being placed into an
individual sterile sampling bag (Fisher Scientific, Pittsburgh,
Pa.). Tissues in sterile sampling bags were frozen at -20.degree.
C. for later analysis utilizing IBDV Antigen Capture ELISA
(AC-ELISA) as described by Snyder et al (138).
ANTIGEN CAPTURE ELISA PROCEDURE
[0246] AC-ELISA test plates were prepared as described earlier in
Section 1. Tissues were homogenized in AC-ELISA dilution buffer at
a ratio of 1:5 tissue weight to volume. Homogenates were analyzed
in duplicate against IBDV monoclonal B-29 (University of Maryland,
College Park, Md.; Intervet Inc., Millsboro Del.) AC-ELISA plates
as described earlier in Section 1. Plates were read and means were
calculated for the paired samples within each group and the
homogenates were determined to be positive or negative based on the
mean absorbance values as compared to the negative and positive
controls on each plate.
BLOOD SAMPLE COLLECTION AND PROCESSING
[0247] Blood samples were collected individually on 4, 6, 8 and 11
days post challenge, using 4.5 ml Sarstedt monovette.TM. syringes
(Sarstedt, Inc. Arlington, Tex.) that contain clot activating beads
and 22 gauge 1 inch needles (Becton Dickinson and Company,
Rutherford, N.J.). All blood was collected by cardiac puncture on
days 4 and 11 and from the brachial vein on days 6 and 8 post
challenge. In order to reduce stress and insure a sufficient
recovery period birds bled on days 6 and 8 post challenge were
rotated based-on wing band numbers, with each successive bleed
utilizing only 5 birds from the previous bleed. Serum was separated
from the clot by centrifugation at 1000 rpm for 10 minutes using an
IEC tabletop centrifuge and placed in both a 96 well round bottom
plate (Corning Glass Works, Corning N.Y.) and into two Nalgene.TM.
cryovials (Nalge Company, Rochester, N.Y.). All serum samples were
processed and frozen at -20.degree. C. or -70.degree. C. within 2
hours of collection. Serum remained frozen until analyzed using the
commercial KPL Infectious Bursal Disease Virus ELISA Kit and the
Flock Profile.TM. software (Kirkegaard and Perry Laboratories,
Inc., Gaithersburg, Md.), as well as virus neutralization, and
hemolytic complement assay.
VIRUS NEUTRALIZATION
[0248] Virus neutralization (VN) assay of serum from experimental
SPF leghorn chickens challenged at 28 days post hatch with three
different IBDV strains was carried out utilizing the 13 procedure.
In this assay the known virus concentration remains constant while
the serum concentration is varied which allows for a quantitative
measurement of antibody present to the known virus. This procedure
is carried out in two distinct parts. In the first part, the
diluted virus is mixed with diluted serum and allowed to incubate
for a given period of time in order for neutralization to take
place. The second part requires a method of analysis for any
residual virus that might be present and can be any viable
indicator assay system (45, 119).
[0249] The virus utilized in this VN analysis was the Edgar strain
of IBDV virus stock at the 18th passage in primary cell culture.
The virus concentration was adjusted to 100 TCID.sub.50/0.025 ml by
diluting the stock virus 1:150 in sterile tissue culture medium.
Then 0.025 ml of diluted IBDV was placed in each well of 11 columns
by 8 rows of a 96 well flat bottom sterile microtiter tissue
culture plate (Corning Glass Works, Corning N.Y.). Column 12
received neither virus or serum and served as a cell culture
viability control. Each serum sample had been previously heat
inactivated at 56.degree. C. for 30 minutes to remove non-specific
viral inhibitory components and diluted 1:5 in sterile tissue
culture medium (GIBCO-BRL, Grand Island, N.Y.). Then 0.025 ml of
each diluted serum was placed in duplicate wells on row A of each
microtiter plate using an Eppendorf micropippetor (Brinkmann, Inc.,
Westbury, N.Y.) and a sterile pippette tip (Costar Corp.,
Cambridge, Mass.). The sera were then serially diluted by using a
12-pette micropipettor (Costar Corp., Cambridge, Mass.) to transfer
and mix each consecutive 0.025 ml dilution step. Pipette tips were
changed after each successive dilution step to eliminate any
residual carryover from the previous dilution. Infectious bursal
disease virus and suspect sera were allowed to incubate at room
temperature for 1 hour.
[0250] Primary chicken embryo fibroblast (CEF) cells were prepared
from 9 day old SPF embryonated eggs (HYVAC, Inc., Adel Iowa). All
eggs were candled to determine viability, and only eggs with
healthy, active embryos were utilized. Eggs were placed in a
laminar flow hood and the outside shell was disinfected with 70%
ETOH. The alcohol was allowed to evaporate and the shell above the
air cell was aseptically removed with a pair of sterile forceps.
Forceps were then dipped in 95% ETOH and flame sterilized before
removal of the air cell membrane. Embryos were removed from the
eggs and placed in sterile, warm D-PBS in a 150 mm petri dish
(Fisher Scientific, Pittsburgh, Pa.). Each embryo was processed by
which the head, feet, wings and internal viscera were removed,
leaving tissue of predominantly epithelial type cells. Tissues were
washed two times in warm sterile D-PBS to remove nucleated red
blood cells prior to enzymatic digestion. Tissue was manually
homogenized by pushing through a 20 cc syringe (Becton Dickinson
and Company, Rutherford, N.J.) and then subjected to a 15 minute
digestion at room temperature with 0.25% trypsin solution
(GIBCO-BRL, Grand Island, N.Y.) at a ratio of 4 ml of 0.25% trypsin
per embryo utilized. Trypsinization was arrested by the addition of
fetal bovine serum (FBS) (GIBCO-BRL, Grand Island, N.Y.), and
liberated cells were separated from the trypsin digestion solution
by centrifugation at 1000 rpm for 10 minutes in an IEC tabletop
centrifuge. The supernatant was removed and the cellular pellet was
washed one additional time in tissue culture medium, and pelleted
by repeating the centrifugation step. The supernatant was again
discarded and the cellular pellet was resuspended in a known volume
of fresh sterile tissue culture medium. Cells were counted using a
hemacytometer and assessed for viability by the trypan blue
(GIBCO-BRL, Grand Island, N.Y.) dye exclusion method. The final
concentration of CEF cells was adjusted to 1.times.10.sup.6 cells
per ml in 1X Minimum Essential Medium (GIBDCO-BRL, Grand Island,
N.Y.) supplemented with 5% FBS.
[0251] Following the one hour incubation at room temperature, 100
.mu.l of CEF cells were added to each well of the 96 well
microtiter plate. Plates were then incubated at 37.degree. C. with
high humidity and an atmosphere of 5% CO.sub.2 (Precision
Scientific, Chicago, Ill.) for four days. Plates were examined
visually for the presence of viral induced cytopathic effects (CPE)
using a Nikon-diaphot phase contrast inverted microscope (Nikon
Inc., Garden City, N.Y.). Lids were removed from each plate and
then the contents of each well were emptied into a container of
disinfectant. Remaining cellular monolayers were fixed by the
addition of 95% ETOH. Following fixation, the alcohol was removed,
and the plates were stained with crystal violet for approximately
30 seconds and then rinsed thoroughly in cold tap water. Virus
neutralization endpoints were determined by the visual presence of
intact cellular monolayers and are expressed as a mean
neutralization antibody titer in FIG. 25.
HEMOLYTIC COMPLEMENT ASSAY
[0252] Hemolytic complement assay was utilized to determine the
effect each of the different strains of IBDV exerted on levels of
serum complement in SPF white leghorn chickens following challenge.
Serum complement was analyzed for 10 individual birds on days 4, 6,
8, and 11 post challenge with IBDV. Blood was collected as
described earlier, and serum aliquots were stored at -70.degree. C.
until they could be analyzed for hemolytic complement activity.
[0253] Sheep red blood cells (SRBC's) (Remel, Inc., Lenexa Kans.)
were washed 3 times with 5 to 10 volumes of GBB-EDTA (Appendix).
Following each wash cells were pelleted at 900 RPM for 20 minutes.
Following the final wash SRBC's were resuspended in GBB-EDTA buffer
to a final concentration of 5%. SRBC's were filtered through cotton
to remove any clumps or clots. Then 0.5 ml of the filtered blood
suspension was removed and lysed with 7.0 ml of 0.1% sodium
carbonate solution. The optical density of the SRBC lysate was
determined using a JENWAY 6105 UV/VIS spectrophotometer (Jenway
Ltd., Essex, England) at a wavelength of 541 mu. An optical density
reading of 0.700 corresponds to 1.times.10.sup.9 SRBC's per ml of
suspension. The final volume of the SRBC suspension was adjusted
using the following formula, V.sub.f=V.sub.i.multidot.O.D./0.700- .
Rabbit anti-sheep rbc hemolysin (Sigma Inc., St. Louis, Mo.) was
diluted 1:100 in GBB-EDTA, mixed with an equal volume of 5% SRBC's,
and incubated at room temperature for 45 minutes. Sensitized SRBC's
were washed once in GBB-EDTA buffer and twice in BB.sup.2++ buffer
(Appendix), pelleting cells after each wash at 900 RPM in an IEC
table top centrifuge for 20 minutes. SRBC's were brought to their
original 5% concentration in GVB+buffer (Appendix) (134). Serum
samples were removed from the -70.degree. C. freezer, and thawed at
room temperature. Fifty .mu.l of each serum sample was diluted 1:2
in NaCl-Veronal Buffer with Ca and Mg (Appendix), and placed in
duplicate in row A of a 96 well round bottom microtiter plate
(Corning Glass Works, Corning N.Y.). Each row was then diluted in
NaCl-Veronal Buffer with Ca and Mg in two fold steps using a
12-pette micropipettor and sterile pipette tips (Costar Corp.,
Cambridge, Mass.). Sensitized sheep red blood cells (sSRBC) were
then added (0.050 ml) to each well and plates were covered and
allowed to incubate at 37.degree. C. for 1 hour. Plates were then
viewed on a light box. The presence of a bright red color dispersed
throughout the contents of the well was an indication that the
sSRBC's had been lysed, the presence of compact buttons of sSRBC's
was an indication that lysis had not occurred. Serum hemolytic
complement titers were recorded as the reciprocal of the highest
dilution at which lysis occurred.
HISTOPATHOLOGY
[0254] Tissues were placed in buffered formalin at necropsy, and
processed twenty-four hours later by the method described in
Section 1 (55). Tissues were examined for the presence of
histopathological lesions induced by IBDV infection.
[0255] Tissues were given a numerical index score based on the
presence or absence as well as the intensity of the lesions
present. The score ranged from a low of "0" Which indicates no
lesions or pathology obseried to a high of "4" which indicates
extensive tissue damage.
STATISTICS tatistics
[0256] All data was entered into Microsoft.TM. Excel (Microsoft
Corp., Redmond, Wash.) and was analyzed using SAS (124) (SAS
Institute Inc., Cary N.C.). Statistical significance was determined
at the P<0.05 level using the General Linear Models Procedure
and least squared means for body weight. bursa:body weight ratios,
spleen: body weight ratios, and proventriculus:body weight ratios.
In addition, statistical differences were determined for means of
the numerical index scores for gross lesions in the bursa and
proventriculus.
RESULTS
[0257] Serum antibody levels from SPF white leghorn chickens
challenged at 28 days post hatch with Variant E/DEL, Variant E/1084
and USDA/STC IBDV were not at detectable levels at 4 days post
challenge (FIG. 23). Variant E/DEL initiated a small antibody
response at 6 days post challenge. All IBDV challenged groups had
antibody titers by day 8 post challenge. Interestingly, antibody
titers at 8 days post challenge were higher than antibody titers at
day 11 for all IBDV groups. The highest antibody titers were
produced against Variant E/DEL at 8 days post challenge (FIG.
23).
[0258] Hemolytic serum complement levels were reduced for all IBDV
challenged groups at 4 days post challenge, with USDA/STC IBDV the
most severely affected, followed by Variant E/DEL, and Variant
E/1084 (FIG. 24). Complement levels at 6 days post challenge were
essentially normal when compared to the negative control for all
IBDV challenged groups. Complement levels at 8 days post challenge
were elevated for USDA/STC IBDV, lower for Variant E/DEL IBDV, and
significantly lower for Variant E/1084 IBDV. Complement levels at
11 days post challenge were essentially normal with a slight
elevation in Variant E/DEL IBDV (FIG. 24).
[0259] Neutralizing antibody was present in all IBDV challenge
groups at 4 days post challenge (FIG. 25). Neutralizing antibody
levels were higher in the USDA/STC IBDV challenge group at 4 days
post challenge when compared to all other challenge groups.
Neutralizing antibody titers at 6 days post challenge were highest
with USDA/STC IBDV, followed by Variant E/DEL and Variant E/1084.
Neutralizing antibody titers to IBDV at 8 days post challenge were
highest against USDA/STC IBDV, followed by Variant E/1084, and
Variant E/DEL. Neutralization antibody titers at day 11 post
challenge are all at the maximum dilution value tested regardless
of the challenge virus strain (FIG. 25).
[0260] Tissues harvested at day 4 and 11 post challenge were
analyzed for the presence of IBDV antigen by AC-ELISA. The bursae
of all IBDV challenge groups were positive at 4 days post challenge
(Table 27). The spleens of fifty percent of the Variant E/1084 IBDV
challenged SPF leghorn chickens were positive at 4 days post
challenge, all other groups tested negative in the spleen.
Proventricular homogenates tested positive for all IBDV challenge
groups at 4 days post challenge. However, Variant E/DEL had a much
lower incidence of virus detection as compared to Variant E/1084
and USDA/STC IBDV (Table 27). All tissues for all IBDV challenge
groups tested negative by AC-ELISA at 11 days post challenge (Table
28).
[0261] Microscopic lesions in the bursa and proventriculus at 4
days post challenge were detected in all IBDV challenge groups
(Table 29). However, both the Variant E/DEL IBDV, and Variant
E/1084 IBDV seem to have less of a trophism for the proventriculus
than USDA/STC IBDV (Table 29). Microscopic lesions were detected in
the bursa at 11 days post challenge in all IBDV challenge groups,
but were absent from the proventriculus (Table 30).
DISCUSSION
[0262] Serum antibody appears at day 6 post challenge for only one
experimental group Variant E/DEL. On day 8 post challenge serum
antibody levels are measurable for all IBDV experimental challenge
groups. but again the highest titers are against both of the
Variant E IBDV strains. Antibody levels at 11 days post challenge
are still highest for the Variant E/IBDV groups as the USDA/STC
IBDV group continues to be the poorest antibody producer. This may
reflect the more extensive damage present in the bursa following
challenge with USDA/STC IBDV and because of the inability to mount
an effective immune response because of damage to immune cells.
[0263] Complement levels in all IBDV challenged groups were
decreased below the negative controls at day 4 post challenge as
described in a number of papers (79, 134, 135, 136). It would
appear that hemolytic complement reaches normal levels at 6 days
post challenge. However, at day 8 post challenge birds challenged
with USDA/STC IBDV continue to increase complement levels seeming
to overcompensate for the deficiency at 4 days post challenge. By
day 11, complement levels appear normal for all but the Variant
E/DEL which appear to be increasing. The levels of complement
following IBDV challenge appear to be cyclic at their lowest levels
when virus populations are highest (detectable by AC-ELISA), and
increasing with the corresponding increased levels of neutralizing
antibodies, and serum IgG titers. The induction of pathology as a
result of the presence of IBDV antibody-antigen complexes and
complement interaction would seem to fall on day 6 post challenge.
Interestingly, lesions observed in the tissues examined at 4 and 11
days following IBDV challenge were the most severely affected in
the USDA/STC IBDV challenge group. This corresponds well with the
presence of a high neutralizing antibody titer on day 6 post
challenge, at a time when complement levels are returning to
normal. Ideally, complement levels, neutralizing antibody levels,
IgG antibody titers and virus AC-ELISA should be measured at much
closer intervals to fully elucidate this viral induced pathogenic
mechanism.
SECTION 6 THE INTERACTION OF INFECTIOUS BURSAL DISEASE VIRUS
(USDA/STC) AND COPPER SULFATE IN THE PRODUCTION OF LESIONS
ASSOCIATED WITH PROVENTRICULITIS IN BROILER CHICKENS
SUMMARY
[0264] Broiler chickens challenged with USDA/STC IBDV at 35 days
post challenge exhibited gross lesions in the bursa and
proventriculus at 4 days post challenge in the presence and absence
of copper sulfate. Antigen capture ELISA (AC-ELISA) analysis of
tissue homogenates indicates that the USDA/STC IBDV was present at
4 days post challenge and was not influenced by the presence of
dietary copper sulfate. Broiler chickens did produce an immune
response to IBDV following challenge.
INRODUCTION
[0265] Previous experiments at the University of Arkansas indicated
that challenge with infectious bursal disease virus (IBDV) was
exacerbated by the presence of dietary copper sulfate
supplementation. Bayyari et al (5) reported a similar finding with
broiler chickens given supplemental copper sulfate in the presence
of challenge with a proventricular tissue homogenate containing an
undefined pathogen. SPF white leghorn chickens showed decreased
weights, increased lesions in the proventriculus and higher
mortality following challenge with USDA/STC IBDV in the presence of
copper sulfate. The purpose of this experiment was to attempt to
reproduce the syndrome seen in SPF white leghorn chickens in
commercial broiler chickens.
MATERIALS AND METHODS
[0266] One hundred twenty commercial broiler chickens were procured
from a local hatchery. Chicks received Marek's vaccine at the
hatchery by subcutaneous injection but were given no other
vaccines. Chicks were dispersed into battery cages at day of hatch,
and sequestered by treatment group. All birds received the standard
University of Arkansas diet formulation as specified by age and
water ad libitum. Chicks given the diet supplemented with copper
sulfate received it beginning on day 1 and throughout the entire
study at the rate of 1 lb/ton.
VIRUS CHALLENGE INOCULUM PREPARATION
[0267] USDA/STC IBDV challenge inoculum was prepared from lot #92-1
(NVSL, Ames, Iowa) as follows: The virus was removed from the
ultralow freezer (-70.degree. C.), and thawed at room temperature
in a laminar flow hood. The outside of the virus containing glass
ampule was wiped with 70% ETOH, to remove any surface
contamination. The ampule of virus was snapped open by wrapping the
neck with a paper towel and breaking along the prescored line. The
virus was then diluted 1:10 by mixing 1.5 ml of USDA/STC IBDV virus
into 13.5 ml of Dulbecco's Phosphate Buffered Saline (D-PBS)
(Appendix A).
BBROILER CHALLENGE
[0268] Broiler chickens were challenged on day 35 post hatch (PH)
with the USDA/STC IBDV prepared inoculum as described previously
Infectious bursal disease virus challenge was administered to each
individual bird bilaterally to the eye (30 .mu.l per eye) using an
Eppendorf micropipettor (Brinkmann Inc., Westbury, N.Y.) and a
sterile pipette tip (Costar Corp., Cambridge, Mass.).
SAMPLE COLLECTION
[0269] On days 4 and 11 post challenge 25 birds were necropsied
from each virus challenge group and an additional 10 birds from the
negative control. Each bird was weighed, bled, and euthanized by
CO.sub.2 asphyxiation. All birds were necropsied at which time the
bursa and proventriculus were scored for the presence of gross
lesions. The bursa, spleen, and proventriculus were individually
weighed and results recorded. Tissues from each of the birds were
cut in half, with one half being placed into 10% buffered formalin,
and the other half being placed into a sterile sampling bag (Fisher
Scientific, Pittsburgh, Pa.). Tissues in sterile sampling bags were
frozen at -20.degree. C. for later analysis utilizing IBDV Antigen
Capture ELISA(AC-ELISA), as described by Snyder et al (138).
ANTIGEN CAPTURE ELISA PROCEDURE
[0270] AC-ELISA test plates were prepared as described earlier in
Section 1. Tissues were homogenized in AC-ELISA dilution buffer at
a ratio of 1:5 tissue weight to volume. Homogenates were analyzed
in duplicate against IBDV monoclonal B-29 AC-ELISA plates as
described earlier in Section 1. Plates were read and means were
calculated for the paired samples within each group and the
homogenates were determined to be positive or negative based on the
mean absorbance values as compared to the negative and positive
controls on each plate.
BLOOD SAMPLE COLLECTION AND PROCESSING
[0271] Blood samples were collected on days 1 and 35 post hatch and
days 4 and 11 post challenge from individual chickens using 4.5 ml
Sarstedt monovette.TM. syringes (Sarstedt, Inc., Arlington, Tex.)
containing clot activating beads and 22 gauge 11/2-inch needles
(Becton Dickinson and Company, Rutherford, N.J.). Serum was
separated from the clot by centrifugation at 1000 rpm for 10
minutes using an IEC tabletop centrifuge. Serum was collected and
placed in both a 96 well round bottom plate (Corning Glass Works,
Corning N.Y.) and into Nalgene.TM. cryovials (Nalge Company,
Rochester, N.Y.) All serum samples were frozen at -20.degree. C.
until they were analyzed using the commercial KPL Infectious Bursal
Disease Virus ELISA Kit and the Flock Profile.TM. software
(Kirkegaard and Perry, Laboratories, Inc., Gaithersburg, Md.).
HISTOPATHOLOGY
[0272] Tissues were placed in buffered formalin at necropsy, and
processed twenty-four hours later by the method described in
Section 1 (55). Tissues were examined for the presence of
histopathological lesions induced by IBDV infection. Lesions in the
lamina propria of the proventriculus were predominantly
pleomorphism of the lymphoid cells in the mucosa along with
lymphocyte depletion. Lesions in the bursa were predominantly
lymphoid depletion with accompanied follicle degeneration.
[0273] Tissues were given a numerical index score based on the
presence or absence as well as the intensity of the lesions
present. The score ranged from a low of "0" which indicates no
lesions or pathology observed to a high of "4" which indicates
extensive tissue damage.
FEED ANALYSIS
[0274] Feed samples were pulled at the end of the study and sent
for analysis to establish that the two diets varied only in the
different levels of copper sulfate present. Feed samples were
analyzed at the Center for Excellence in Poultry Science, Central
Analytical Laboratory. The diet without copper sulfate
supplementation contained 14.95 ppm copper. The diet with copper
sulfate supplementation contained 126.35 ppm copper.
STATISTICS
[0275] All data was entered into Microsoft.TM. Excel (Microsoft
Corp., Redmond, Wash.) and was analyzed using SAS (3) (SAS
Institute Inc., (Cary, N.C.). Statistical significance was
determined at the P<0.05 level using General Linear Models
Procedure and least squared means for body weight, bursa:body
weight ratios, spleen: body weight ratios, and proventriculus:body
weight ratios. In addition statistical differences were determined
for means of the numerical index scores for gross lesions in the
bursa and proventriculus.
RESULTS
[0276] The proventriculi from broilers challenged with USDA/STC
IBDV in the presence of copper sulfate at 35 days post hatch had
gross lesions involving the discoloration of the mucosal lining and
enlargement of the papillae (FIG. 27) as compared to an
unchallenged hatchmate without copper sulfate supplementation (FIG.
26). Virus induced effects are evident in the bursa (atrophy) and
spleen (splenomegaly) (FIG. 27) when compared to the negative
control bursa and spleen (FIG. 26).
[0277] Organ to body weight ratios were compared for each
experimental group at day 4 and day 11 post challenge with USDA/STC
IBDV. Significant differences were noted only in the spleen at 4
days post challenge for the USDA/STC IBDV and USDA/STC IBDV+copper
sulfate (Table 31). Bursa to body weights ratios at 11 days post
challenge were significantly higher for all challenge groups that
received USDA/STC IBDV. Spleen to body weight ratios were
significantly lower only in the USDA/STC IBDV challenged group
(Table 32).
[0278] Gross lesion scores for the proventriculus were significant
for only two criteria, which was different for each necropsy day.
Papillae were significantly affected in the USDA/STC IBDV
challenged group only at 4 days post challenge. Feed impaction was
of significantly higher incidence in the challenge group that
received USDA/STC IBDV only (Table 33).
[0279] AC-ELISA analysis of bursal and proventricular homogenates
at 4 and 11 days post challenge with USDA/STC IBDV indicated the
presence of virus in both the bursa and the proventriculus at 4
days post challenge only. The number of AC-ELISA detectable
positives was not significantly different between USDA/STC
challenged groups in the presence or absence of copper sulfate for
either the bursa or the proventriculus.
[0280] Microscopic lesions at 4 and 11 days post challenge were
assessed for the proventriculus and bursa (Table 35). Bursal
lesions at 4 days post challenge were present in the USDA/STC IBDV
challenged groups in the presence or absence of copper sulfate.
Proventricular lesions at 4 days post challenge are present in the
USDA/STC IBDV challenged groups in the presence or absence of
copper sulfate. Bursal lesions at 11 days post challenge were
present in all experimental groups. Proventricular lesions were not
present in any of the experimental groups at 11 days post
challenge.
[0281] Maternal serum antibody titers were in excess of 9000 at day
of hatch but had dropped below detectable levels by 35 days post
hatch. Immune response following challenge was detectable at 11
days post challenge. Post challenge titers did not approach
antibody levels present at hatch (FIG. 28).
DISCUSSION
[0282] The gross lesions visible following challenge with USDA/STC
IBDV in the proventriculus indicate that there is an effect on the
mucosal surface of the proventriculus (FIG. 27). The enlarged
papillae were also present in the unchallenged group that received
copper sulfate but the yellow discoloration was absent (data not
shown). This coloration is reminiscent of the pigment that is
absorbed from corn in the diet, however, birds exhibiting yellow
mucosal proventricular linings did not exhibit pale shanks or
bleaching. The enlarged papillae have been seen previously in SPF
white leghorn chickens following challenge with USDA/STC IBDV in
the presence and absence of copper sulfate but were much less
evident.
[0283] AC-ELISA indicates the presence of virus in the bursa and
proventriculus. Virus presence within the tissues does not appear
to be influenced by the presence of copper sulfate. AC-ELISA has
not been capable of picking up tissue homogenate positives in any
of the experimentally challenged birds beyond day 4 post challenge.
This appears to be a reflection of the sensitivity of the assay as
well as the rapid viral replication and clearance.
[0284] Organ to body weight ratios indicated that the virus was
capable of exerting an effect. There were no significant
differences in body weights following challenge with USDA/STC IBDV
in the presence or absence of dietary copper sulfate. Bayyari et al
reported a reduction in body weights and feed conversion following
challenge of day old broilers with an undefined pathogen in the
presence of copper sulfate. This phenomenon was also noted in SPF
white leghorn chickens following challenge with USDA/STC IBDV in
the presence of copper sulfate. The microscopic tissue lesions
should confirm whether or not the lesions are typical of IBDV
infection.
[0285] Microscopic lesions in the bursa were present in the
USDA/STC IBDV challenged group at a higher frequency than the
USDA/STC IBDV+copper sulfate group. It would appear that infection
of the bursa is delayed or there is some type of interference when
copper sulfate is present. At 11 days post challenge lesions are
present in all experimental groups indicating that the USDA/STC
IBDV virus had spread laterally within the experimental house.
Proventricular lesions at 4 days post challenge are of higher
incidence in the USDA/STC IBDV+copper sulfate experimental group.
At 11 days post challenge lesions are absent from the
proventriculus in all experimental groups, which is similar to what
is described in SPF white leghorn chickens.
[0286] Serum antibody titers demonstrate that immunity to
infectious bursal disease virus is not of long duration when
relying entirely on maternally derived antibody. It is necessary to
point out that IBDV challenge did not take place until 35 days post
hatch. The goal was to allow for the catabolism of maternally
derived antibody so that is was possible to infect these birds with
IBDV. Antibody levels were not checked on a regular interval and
may have waned well before challenge took place.
SECTION 7 ISOLATION AND CHARACTERIZATION OF INFECTIOUS BURSAL
DISEASE VIRUS FROM BROILER CHICKENS EXHIBITING SYMPTOMS OF
PROVENTRICULITIS
SUMMARY
[0287] To date submissions for AC-ELISA screening of broiler flocks
affected with proventriculitis have encompassed 46 facilities from
6 different states. Broiler chicken tissue submissions come from
flocks ranging in age from 14 to 35 days post hatch. Virus
immunoprecipitation following AC-ELISA screening has resulted in
the isolation of 5 infectious bursal disease viruses of
proventricular origin. Virus isolations are primarily from broiler
flocks ranging in age from 22-29 days post hatch.
[0288] Inoculation of proventricular origin IBDV isolates into 28
day post hatch SPF white leghorn chickens, revealed the production
of lesions associated with infectious bursal disease virus
infection. At 11 days post challenge all SPF white leghorn chickens
demonstrate IgG antibody titers to IBDV following screening of
serum with a commercial IBDV ELISA kit.
[0289] RT/PCR-RFLP analysis of new proventricular IBDV isolates
indicate that the viruses have a unique restriction enzyme pattern
within a 700 bp fragment of the VP2 genomic region. The new
isolates appear to share characteristics of both the Delaware
variant and a more recent California IBDV isolate from the
bursa.
INTRODUCTION
[0290] Poultry producers have continued to report increased
incidence of proventriculitis in broiler flocks as well as the
associated processing complications. Producers were given an option
to submit tissues to the University of Arkansas from affected
flocks for initial screening by Antigen capture ELISA (AC-ELISA)
assay. The AC-ELISA technology was obtained through a cooperative
agreement with University of Maryland, College Park, Md. and
Intervet Inc., Millsboro, Del. Proventricular tissues from field
submissions which tested positive by AC-ELISA were then
immunoprecipitated using a IBDV monoclonal antibody B29 (University
of Maryland, College Park, Md.; Intervet Inc., Millsboro, Del.) in
an attempt to isolate any IBDV present. The purpose of this
experiment was to isolate and characterize new IBDV isolates of
proventricular origin from broiler chickens experiencing
proventriculitis.
MATERIALS AND METHODS
[0291] Tissues from birds with proventriculitis were collected from
affected farms and placed in individual sterile sample bags (Fisher
Scientific, Pittsburgh, Pa.) and frozen until they could be shipped
to the University of Arkansas. Suspect tissues were received at the
University of Arkansas Poultry Health Laboratory and stored at
-20.degree. C. until they could be processed.
[0292] Each farm from which affected tissues were collected was
given a University of Arkansas case number so that they could be
identified. All tissues were prepared for Antigen Capture ELISA
(AC-ELISA) analysis by the following procedure. Tissues were
thawed, weighed, and homogenized. Homogenization was accomplished
as described earlier in Section 1. Following homogenization,
tissues were resuspended in a volume of AC-ELISA Sample Dilution
Buffer (Appendix), that was equivalent to 4 times the weight of the
tissue sample. The resulting end dilution being 1:5 or 20% tissue
by weight to volume. Duplicate sample templates were then prepared
for the AC-ELISA test.
[0293] All bursal sample homogenates were screened for the presence
of infectious bursal disease virus (IBDV) at a final tissue
concentration of 1:40. All proventricular tissue homogenates were
screened for the presence of IBDV at a final tissue concentration
of 1:10. Samples were screened only against monoclonal antibody B29
(University of Maryland, College Park, Md.; Intervet Inc.,
Millsboro, Del.). After completion of all the AC-ELISA test
requirements as described earlier in Section 1, plates were read
using BIO-TEK model EL 312e ELISA reader. Optical densities for
each well were determined, recorded by the reader, and printed.
Samples were determined to be positive or negative based on the
optical density reading they produced in comparison to the negative
and positive control.
VIRAL IMMUNOPRECIPITATION
[0294] Samples that were determined to be positive, were then
immunoprecipitated utilizing IBDV monoclonal antibody B29 and
Pansorbin Cells.TM. (Calbiochem, LaJolla, Calif.) using methods
described by Kessler (75) and Harris (41) Briefly Pansorbin cells
(10% w/v cell suspension in D-PBS) were washed three times in
NET+NP40 buffer (Appendix), and the pellet was resuspended in
NET+NP40 to the original volume to restore the 10% suspension.
Sixty .mu.l of proventricular tissue homogenate was added to 20
.mu.l of IBDV Mab B-29 ascitic fluid and incubated at room
temperature for 60 minutes. Twenty-five .mu.l of NET+NP40 and 25
.mu.l of washed pansorbin cells were added to the antigen antibody
complex and incubated at room temperature for 15 minutes. The
Pansorbin antigen:antibody complex was then pelleted for 2 minutes
in an Eppendorf microfuge (Brinkmann Inc., Westbury, N.Y.) at 6000
rpm. The pellet was then resuspended and washed three times in
NET+NP40 buffer. Following the final wash the pellet was retained.
The precipitated virus:antibody complex was inoculated into SPF
eggs.
SPF EGG INOCULATION
[0295] Eggs utilized were laboratory grade specific pathogen free
(SPF) (HYVAC Inc., Adel, Iowa). Eggs were incubated to the 9th day
of development, at which time they were removed from the incubator,
and candled to determine viability. All viable eggs were then
manually manipulated to expose the chorioallantoic membrane (CAM).
Each sample was then injected onto the CAM of 5 fertile SPF eggs.
Immunoprecipitated samples were introduced onto the CAM utilizing a
1 cc tuberculin syringe and 26G 1/2-inch needle (Becton Dickinson
and Company, Rutherford, N.J.). Egg shell openings were then closed
using melted wax. Inoculated eggs were incubated at 37.degree. C.
with high humidity. Eggs were candled daily to determine viability
of experimental embryos. As embryo deaths occurred, non-viable
embryos were removed, recorded and place at 4.degree. C. until all
embryos could be visually examined for the presence or absence of
viral lesions. After 7 days of incubation, all remaining viable
embryos were chilled at 4.degree. C. overnight.
[0296] Examination of embryos for viral lesions was accomplished by
the removal of the external side shell and shell membrane of each
egg. Chorioallantoic membranes were visually examined for the
presence of viral infection. The number of affected membranes was
recorded for each experimental isolate. The chick embryo was then
removed from the egg and any external lesions present were
recorded. The final examination was of the internal organs of each
embryo. Target organs examined were the liver, spleen,
proventriculus, and bursa of Fabricius. All affected tissues
including the chorioallantoic membrane were harvested into a
sterile container, labeled, homogenized and diluted in Dulbecco's
phosphate buffered saline (D-PBS) with 10 .mu.g/ml gentamycin
(Schering Plough Union, N.J.) and 250 .mu.g/ml of Amphotericin B
(GIBCO-BRL, Grand Island, N.Y.). The egg inoculation procedure was
then repeated up to three passages at which time samples not
exhibiting viral lesions were discarded.
SPF BIRD CHALLENGE
[0297] Tissues and CAMs harvested from affected embryos on the
third passage were then homogenized, and inoculated into 28 day
post hatch SPF white leghorn chickens (HYVAC, Inc., Adel, Iowa).
The inoculum was administered 30 .mu.l bilaterally to the eye using
an Eppendorf micropipettor (Brinkmann Inc., Westbury, N.Y.) and a
sterile pipette tip (Costar Corp., Cambridge, Mass.). Approximately
50% of the SPF white leghorn chickens were then necropsied at 4 and
11 days post exposure. Birds were weighed, bled, euthanized by
CO.sub.2 asphyxiation, and examined by necropsy for the presence of
viral lesions. The bursa and spleen were visually scored for the
presence of gross lesions. In addition, bursa, spleen, and
proventriculus were harvested, weighed, and cut into three pieces
with one section going into 10% buffered formalin, one section for
AC-ELISA (138) and the final section for reverse transcriptase
polymerase chain reaction with restriction fragment length
polymorphism analysis (RT/PCR-RFLP) (60). Serum antibody titers
were determined utilizing a commercial ELISA kit for IBDV
(Kirkegaard and Perry, Laboratories Inc., Gaithersburg, Md.).
HISTOPATHOLOGY
[0298] Tissues were placed in buffered formalin at necropsy, and
processed twenty-four hours later by the method described in
Section 1 (55). Tissues were examined for the presence of
histopathological lesions induced by IBDV infection.
[0299] Tissues were given a numerical index score based on the
presence or absence as well as the intensity of the lesions
present. The score ranged from a low of "0" which indicates no
lesions or pathology observed to a high of "4" which indicates
extensive tissue damage.
STATISTICS
[0300] All data was entered into Microsoft.TM. Excel (Microsoft
Corp., Redmond, Wash.). and were analyzed using SAS (5) (SAS
Institute Inc., Cary, N.C.). Statistical significance was
determined at the P<0.05 level using General Linear Models
Procedure and least squared means for body weight, bursa:body
weight ratios, spleen: body weight ratios, and proventriculus:body
weight ratios. In addition, statistical differences were determined
for means of the numerical index scores for gross lesions in the
bursa and proventriculus.
RESULTS
[0301] FIG. 29 demonstrates an abnormally enlarged proventriculus
in a 22 day post hatch broiler. FIG. 30 illustrates the internal
lesions in a 27 day post hatch broiler that are commonly associated
with proventriculitis syndrome in the field. Note the hemorrhages
in the papillae, the loss of papillae definition, and the abnormal
coloration of the mucosal surface.
[0302] Microscopic lesions in the bursa of SPF white leghorn
chickens challenged with field IBDV isolates were present in all
but two challenged groups at day 4 and 11 post challenge.
Microscopic lesions in the proventriculi of SPF white leghorn
chickens challenged with field IBDV isolates were present on day 4
in all but two of the challenge groups. There were no microscopic
lesions in the proventriculus at 11 days post challenge (Table 35).
Samples W/L #22 and #33 consistently tested negative in inoculated
birds although they appeared to produce lesions in embryonated
eggs. SPF leghorn chickens challenged with these two isolates did
not produce serum antibodies to IBDV. These isolates will be
screened for the presence of other pathogens but at this point do
not appear to be IBDV.
[0303] Gross lesion scores from the proventriculus were not
significantly different from the negative control at 4 or 11 days
post challenge for any of the suspect virus challenge groups (data
not shown).
[0304] AC-ELISA analysis of bursal and proventricular homogenates
from SPF white leghorn chickens challenged with USDA/STC IBDV and
proventricular origin field isolates at 28 days post hatch tested
positive in the bursa at 4 days post challenge with the exception
of the two isolates discussed earlier. All bursal homogenates
tested negative at 11 days post challenge. Proventricular
homogenates from field isolates had fewer positives when compared
to the USDA/STC IBDV.
[0305] Organ to body weight ratios of SPF white leghorn chickens
demonstrate the induction of significant effects on the bursa and
spleen at 4 days post challenge with field isolates RB/Texas 3 and
4, Farm 57-7, and W/L #39 (Table 37). There were no significant
differences in the proventriculus to body weight ratio on day 4
post challenge (Table 37). The bursa to body weight ratio was lower
than the negative control in all challenge groups with the
exception of W/L # 22 and #33 at 11 days post challenge (Table
38).
[0306] RT/PCR-RFLP analysis of new proventricular IBDV isolates
RB/Texas 3 and RB/Texas 4 indicate that the viruses have a unique
restriction enzyme pattern within a 700 bp fragment of the VP2
genomic region. The agarose gel electrophoretic separation of
digested and undigested PCR products is demonstrated in FIG. 33.
Samples #24 and #34 (not shown) are PCR amplifications of bursa and
proventriculus respectively from SPF white leghorn chickens
challenged with isolate RB/Texas 3. Sample # 34 did not amplify
during the RT/PCR reaction. Samples #46 and #56 are RT/PCR
amplification of bursa and proventriculus respectively from SPF
white leghorn chickens challenged with isolate RB/Texas 4. RT/PCR
products from both RB/Texas 3 and RB/Texas 4 are identical with
respect to their restriction profile in this 700 bp VP2 fragment.
This was expected as both isolates were obtained from the same
poultry facility.
[0307] As shown in FIG. 31, serum antibody response of 28 day post
hatch SPF white leghorn chickens following challenge with USDA/STC
IBDV and proventricular origin IBDV field isolates indicate that
all challenge groups with the exception of W/L #22 and #33 produced
measurable levels of antibodies at 11 days post challenge.
DISCUSSION
[0308] Immunoprecipitation of IBDV antigen from AC-ELISA positive
tissue homogenates is quite easy to perform. This procedure
represents a rapid method for separating and concentrating IBDV
from field samples that could potentially contain many virus types.
The uniqueness of the binding of the IBDV Mab B29 without
neutralization of virus infectivity makes this particular procedure
adaptable to a any number of assay systems, and as we have
demonstrated here works quite well for virus isolation in eggs
(FIG. 32).
[0309] Proventricular origin IBDV suspect isolates demonstrated
repeated characteristics of bursal lesion production, antibody
stimulation, and AC-ELISA analysis which indicate that they are
indeed IBDV. These particular isolates are capable of producing
lesions in the proventriculus but do not appear to be significantly
more pathogenic than the USDA/STC IBDV isolate for the
proventriculus. There are two considerations at this point: field
isolates may have a lower affinity for infecting the
proventriculus, or there may be differences in virus inoculum titer
(Table 36).
[0310] The new isolates appear to share characteristics of both the
Delaware variant and a more recent California IBDV isolate from the
bursa. RT/PCR characterization of two isolates indicate that they
may represent a unique group of IBD viruses.
[0311] The viral isolates Texas RB 3, Texas RB 4, HBS, F57-7, W/L
39, GAR 1, and/or others are deposited with the American Type
Culture Collection (ATCC) patent depository, 10801 University
Blvd., Manasses, Va. 20110-2209, Telephone 703 365-2700 on behalf
of the University of Arkansas, Arkansas, U.S.A. and have respective
deposit accession numbers ______.
FURTHER DISCUSSION
[0312] It was a goal of this invention that the data compiled
provide significant inroads toward solving what appears to be a
very widely distributed poultry industry problem involving
proventriculitis syndrome. It is also necessary at this point to
recognize that although this data establishes the ability of
infectious bursal disease virus infection to induce lesions in
other lymphoid areas in the chicken, it does not establish that all
proventriculitis occurs as a result of infectious bursal disease
virus infection. However, at this point it appears safe to say that
infectious bursal disease virus represents a significant portion of
the problem identified as proventriculitis.
[0313] In consideration of the information provided in this patent
application, the poultry industry might wish to review current
vaccination protocols concerning infectious bursal disease virus
prevention and control. An additional emphasis should also be
applied to the consideration of infectious bursal disease virus and
the role it plays as an enteric pathogen. If poultry industry dogma
continues to consider only the immunosuppressive nature of
infectious bursal disease virus infection, then it will continue to
do battle with a virus it does not understand.
[0314] Infectious bursal disease virus has historically
demonstrated the ability to adapt and escape whatever control
measures the industry has managed to put in place. Of course the
possibility exists that infectious bursal disease virus infection
of the proventriculus has been simply overlooked all this time. But
the sudden onset of widespread proventriculitis in multiple
geographical areas might also suggest that these new IBDV isolates
are simply the latest adaptation of an extremely hard to control
virus.
[0315] Future considerations in the control of infectious bursal
disease virus should encompass and consider infectious bursal
disease as an enteric pathogen. The eventual goal of this work and
the work of a number of individuals to follow is the reduction in
the incidence of proventriculitis and the possibility of an
improved feed conversion, with a new generation of vaccines derived
from infectious bursal disease virus isolates of enteric
origin.
[0316] The present invention is directed to at least poultry virus
isolates Texas RB 3, Texas RB 4, HBS, F57-7, W/I 39, and GAR 1 and
the use of these virus isolates as or in vaccines for
proventriculitis.
[0317] Numerous experiments were conducted to determine the role of
infectious bursal disease (IBD) virus in the induction of lesions
associated with proventriculitis syndrome in chickens. Parameters
examined included age of the chicken at IBD virus exposure,
concentration of IBD virus at exposure, the strain of IBD virus,
dietary influence in the presence of IBD virus, mixed IBD virus
infection, auto-immune mediated IBD reactions associated with
lesion production, viral induced apoptotic tissue injury and
isolation and characterization of the causative agent.
[0318] The experiments were carried out in SPF white leghorns, with
the experimental birds being examined for the presence of gross and
microscopic lesion at 4 and 11 days post challenge. Tissue
homogenates were analyzed for the presence of IBDV at 4 and 11 days
post challenge with Antigen Capture ELISA (AC-ELISA).
Determinations of neutralizing antibody levels and IgG antibody
responses were monitored as well as depletion of serum complement
following IBDV infection. Physical parameters were also considered
utilizing body weights and organ to body weight ratios to determine
IBDV effects following experimental challenge.
[0319] Physical parameters indicated that the primary viral
response is predominantly in the bursa of Fabricius, but changes
were also noted in the proventriculus. Physical changes in the
proventriculus occurred primarily during the acute stage of the IBD
virus infection.
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1TABLE 1 Antigen Capture ELISA analysis of proventriculi tissue
pools collected at 4 and 11 days post challenge with Variant E/1084
IBDV given at 7, 14, 21 and 28 days post hatch and after challenge
with USDA/STC IBDV at 28 days post hatch only. 4 Days Post 11 Days
Post Age at Challenge Challenge Challenge Day 7 Variant E/1084 - -
Day 7 Negative Control - - Day 14 Variant E/1084 +/- - Day 14
Negative Control - - Day 21 Variant E/1084 - - Day 21 Negative
Control - - Day 28 Variant E/1084 + - Day 28 USDA/STC + - Day 28
Negative Control - -
[0478]
2TABLE 2 Antigen Capture ELISA analysis of bursal tissue pools
collected at 4 and 11 days post challenge with Variant E/1084 IBDV
given at 7, 14, 21 and 28 days post hatch and after challenge with
USDA/STC IBDV at day 28 post hatch only. 11 Days Post Age at
Challenge 4 Days Post Challenge Challenge Day 7 Variant E/1084 + -
Day 7 Negative Control - - Day 14 Variant E/1084 + - Day 14
Negative - - Control Day 21 Variant E/1084 + - Day 21 Negative - -
Control Day 28 Variant E/1084 - - Day 28 USDA/STC + - Day 28
Negative - - Control
[0479]
3TABLE 3 Microscopic proventricular lesions at 4 and 11 days post
challenge with Variant E/1084 IBDV given at 7, 14, 21 and 28 days
post hatch and after challenge with USDA/STC IBDV at 28 days post
hatch only. Age at Challenge Day 4 Post* Day 11 Post Day 7 NC 1/10
0/5 Day 7 Var E 17/20 0/4 Day 14 NC 0/5 0/5 Day 14 Var E 5/5 0/5
Day 21 NC 0/5 0/5 Day 21 Var E 5/5 1/5 Day 28 NC 0/5 2/5 Day 28 Var
E 5/5 0/5 Day 28 USDA 4/5 2/5 *Ratios represent the number of
tissues exhibiting lesions over the total number of tissues
screened.
[0480]
4TABLE 4 Microscopic bursal lesions of SPF white leghorn chickens
at 4 and 11 days post challenge with Variant E/1084 IBDV given at
7, 14, 21 and 28 days post hatch and after challenge with USDA/STC
IBDV given at 28 days post hatch only. Age at Challenge Day 4 Post
* Day 11 Post Day 7 NC 0/10 0/5 Day 7 Var E 10/10 4/4 Day 14 NC 0/5
0/5 Day 14 Var E 5/5 5/5 Day 21 NC 0/5 0/5 Day 21 Var E 5/5 5/5 Day
28 NC 0/5 2/5 Day 28 Var E 5/5 5/5 Day 28 USDA 5/5 4/4 * Ratios
represent number of tissues exhibiting lesions over the total
number of tissues screened.
[0481]
5TABLE 5 Bursa: Body weight ratios of SPF white leghorn chickens 4
and 11 days post challenge with Variant E/1084 IBDV given at 7, 14,
21 and 28 days post hatch and after challenge with USDA/STC IBDV at
28 days post hatch only. CHALLENGE DATE Days Neg. 28 28 PH**
Control 7 14 21 Var E USDA 11 3.46 .+-. 0.33 1 18 5.06 .+-. 0.33 2
4.15 .+-. 0.33 25 5.19 .+-. 0.33 3 4.76 .+-. 0.33 32 5.50 .+-. 0.33
4 5 4.52 .+-. 0.37 39 5.97 .+-. 0.37 6 7 *Numerical values within
shaded boxes are significantly different from the negative control
values at the P < 0.05 confidence interval. **Days PH = Days
post hatch and refers to the chronological age of the bird at
necropsy.
[0482]
6TABLE 6 Proventriculus:Body weight ratios for SPF white leghorn
chickens at 4 and 11 days post challenge with Variant E/1084 IBDV
given at 7, 14, 21 and 28 days post hatch and after challenge with
USDA/STC IBDV given at 28 days post hatch only. Days PH* Neg. *
Control 7 14 21 28 28 USDA 11 7.84 .+-. 0.25 9.35 .+-. 0.17 18 7.44
.+-. 0.25 8.16 .+-. 0.25 7.35 .+-. 0.2 5 25 6.30 .+-. 0.25 6.76
.+-. 0.2 6.49 .+-. 0.25 5 32 5.76 .+-. 0.25 6.68 .+-. 0.25 6.08
.+-. 0.25 6.09 .+-. 0.28 39 5.48 .+-. 0.28 5.60 .+-. 0.25 6.08 .+-.
0.28 *Numerical values within shaded boxes are significantly
different from the negative control values at the P < 0.05
confidence interval. **Days PH = Days post hatch and refers to the
chronological age of the bird at necropsy.
[0483]
7TABLE 7 Spleen:Body weight ratios for SPF white leghorn chickens
at 4 and 11 days post challenge with Variant E/1084 IBDV at 7, 14,
21 and 28 days post hatch, and challenge with USDA/STC IBDV given
at day 28 post hatch only. CHALLENGE DATE Days PH* Neg. * Control 7
14 21 28 28 USDA 11 0.98 .+-. 0.16 1.86 .+-. 0.11 18 1.37 .+-. 0.16
1.84 .+-. 0.16 2.22 .+-. 0.16 25 1.5 .+-. 0.16 2.45 .+-. 2.81 .+-.
0.16 0.16 32 1.74 .+-. 0.16 2.50 .+-. 0.16 2.86 .+-. 0.16 2.77 .+-.
0.18 39 2.81 .+-. 0.18 2.56 .+-. 0.16 2.59 .+-. 0.18 *Numerical
values within shaded boxes are significantly different from the
negative control values at P < 0.05 confidence interval. **Days
PH = Days post hatch and refers to the chronological age of the
bird at necropsy.
[0484]
8TABLE 8 Papillae scores of infected proventriculi from SPF white
leghorn chickens at 4 and 11 days post challenge with Variant
E/1084 IBDV at 7, 14, 21 and 28 days post hatch, and challenged
with USDA/STC IBDV given at 28 days post hatch only. Challenge Date
Days Neg. 28 28 PH** Control 7 14 21 Var E USDA 11 0.00 .+-. 0.20
0.75 .+-. 0.14 18 0.00 .+-. 0.20 0.40 .+-. 0.20 0.70 .+-. 0.20 25
0.00 .+-. 0.20 0.00 .+-. 0.20 0.40 .+-. 0.20 32 0.00 .+-. 0.20 0.40
.+-. 0.20 1.60 .+-. 0.20 0.63 .+-. 0.23 39 0.13 .+-. 0.23 0.50 .+-.
0.20 0.13 .+-. 0.23 * Numerical values within shaded boxes are
significantly different from the negative control values at the P
< 0.05 confidence interval. **Days PH = Days post hatch and
refers to the chronological age of the bird at necropsy.
[0485]
9TABLE 9 Virus inoculum preparation, route and volume of
administration, and paired virus titer groups based on virus stock
titers of USDA/STC IBDV and Variant E/1084 IBDV. Virus Equivalent
Challenge Inoculum Volume Treatment Virus Titer Group Dilution
Inoculated Identification Groups Negative NA NA NC 0 Control
USDA/STC undilute 30 .mu.l/eye USDA 10X A USDA/STC 1:10 30
.mu.l/eye USDA 1X B USDA/STC 1:10 15 .mu.ul/eye USDA 0.5 X C
Variant E/1084 1:1000 15 .mu.l/eye Variant E 1X C Variant E/1084
1:1000 30 .mu.l/eye Variant E 2X B Variant E/1084 1:100 30
.mu.l/eye Variant E 20 X A
[0486]
10TABLE 10 AC-ELISA analysis of proventricular homogenates from SPF
white leghorn chickens challenged at 32 days post hatch with
different concentrations of USDA/STC IBDV and Variant E/1084 IBDV
at 2, 3, 4, and 11 days post challenge. Days Post Challenge
Treatment 2 3 4 11 Negative Control 0/8 0/8 0/8 0/8 (0%) (0%) (0%)
(0%) USDA - 0.5X 1/8 6/8 2/6 0/8 (13%) (75%) (33%) (0%) USDA - 1X
4/8 7/8 4/5 0/8 (50%) (88%) (80%) (0%) USDA - 10x 0/8 8/8 0/8 0/8
(0%) (100%) (0%) (0%) Variant E - 1X 7/8 5/8 3/8 0/8 (88%) (63%)
(38%) (0%) Variant E - 2X 2/8 3/8 1/8 0/8 (25%) (38%) (13%) (0%)
Variant E - 20X 7/8 7/8 0/8 0/8 (88%) (88%) (0%) (0%) * Ratios
represent the number of birds which tested positive over the number
of birds tested by AC-ELISA.
[0487]
11TABLE 11 AC-ELISA analysis of bursal homogenates from SPF white
leghorn chickens challenged at 32 days post hatch with different
concentrations of USDA/STC IBDV and Variant E/1084 IBDV at 2, 3, 4,
and 11 days post challenge. Days Post Infection Treatment 2 3 4 11
Negative 0/8 0/8 0/8 0/8 Control (0%) (0%) (0%) (0%) USDA-0.5X 8/8
6/7 6/6 0/8 (100%) (86%) (100%) (0%) USDA-1X 8/8 8/8 5/5 0/8 (100%)
(100%) (100%) (0%) USDA-10X 3/8 8/8 8/8 0/8 (38%) (100%) (100%)
(0%) Variant E-1X 8/8 8/8 1/8 0/8 (100%) (100%) (13%) (0%) Variant
E-2X 3/8 4/8 7/8 0/8 (38%) (50%) (88%) (0%) Variant E-20X 6/8 7/8
8/8 0/8 (75%) (88%) (100%) (0%) * Ratios represent the number of
birds which tested positive over the total number of birds tested
by AC-ELISA.
[0488]
12TABLE 12 Microscopic proventricular lesions in SPF white leghorn
chickens challenged at 32 days post hatch with different
concentrations of USDA/STC IBDV and Variant E (1084-E) IBDV at 2,
3, 4, and 11 days post challenge. Days Post Infection Treatment 2 3
4 11 Negative 0/5 0/5 0/5 0/5 Control (0%) (0%) (0%) (0%) USDA-0.5X
4/5 5/5 5/5 0/5 (80%) (100%) (100%) (0%) USDA-1X 1/5 5/5 2/5 0/5
(20%) (100%) (40%) (0%) USDA-10X 1/5 5/5 2/5 0/5 (20%) (100%) (40%)
(0%) Variant E-1X 3/5 2/4 2/5 0/5 (60%) (50%) (40%) (0%) Variant
E-2X 0/5 1/5 4/5 0/5 (0%) (20%) (80%) (0%) Variant E-20X 1/5 5/5
3/5 0/5 (20%) (100%) (60%) (0%) * Ratios represent the number of
birds exhibiting lesions over the number of birds screened.
[0489]
13TABLE 13 Microscopic bursal lesions in SPF white leghorn chickens
challenged at 32 days post hatch with different concentrations of
USDA/STC IBDV and Variant E (1084-E) IBDV at 2, 3, 4, and 11 days
post challenge. Days Post Infection Treatment 2 3 4 11 Negative 0/5
0/5 0/5 0/5 Control (0%) (0%) (0%) (0%) USDA - 0.5 4/5 5/5 5/5 5/5
(80%) (100%) (100%) (100%) USDA - 1X 2/5 5/5 5/5 5/5 (40%) (100%)
(100%) (100%) USDA - 10X 1/5 5/5 5/5 5/5 (20%) (100%) (100%) (100%)
Variant E - 1X 5/5 5/5 5/5 5/5 (100%) (100%) (100%) (100%) Variant
E - 2X 2/5 2/5 5/5 5/5 (40%) (40%) (100%) (100%) Variant E - 2/5
5/5 5/5 5/5 20X (40%) (100%) (100%) (100%) * Ratios represent the
number of birds exhibiting lesions over the number of birds
screened.
[0490]
14TABLE 14 Proventriculus: Body weight ratios for SPF white leghorn
chickens challenged at 32 days post hatch with different
concentrations of USDA/STC IBDV and Variant E/1084 IBDV at 2, 3, 4,
and 11 days post challenge. Days Post Challenge Challenge Groups 2
3 4 11 Negative Control 6.28 .+-. 0.22.sup.ab 5.52 .+-. 0.22.sup.a
6.14 .+-. 0.22.sup.a 5.6 .+-. 0.21.sup.d USDA 0.5X 8 5.92 .+-.
0.22.sup.a 5.68 .+-. 0.25.sup.a 9 USDA 1X 6.33 .+-. 0.22.sup.a 5.74
.+-. 0.22.sup.a 6.12 .+-. 0.28.sup.d 10 USDA 10X 11 5.49 .+-.
0.22.sup.a 6.16 .+-. 0.22.sup.a 12 Variant E 1X 5.99 .+-.
0.22.sup.abc 5.96 .+-. 0.22.sup.a 6.46 .+-. 0.22.sup.d 6.12 .+-.
0.23.sup.bcd Variant E 2X 6.32 .+-. 0.22.sup.ab 5.97 .+-.
0.22.sup.a 5.99 .+-. 0.22.sup.d 6.1 .+-. 0.21.sup.cd Variant E 20X
5.71 .+-. 0.22.sup.bc 5.84 .+-. 0.22.sup.a 6.17 .+-. 0.22.sup.a
5.72 .+-. 0.28.sup.d *Values in shaded boxes with superscripts with
different letters indicate significant differences at the P <
0.05 confidence interval.
[0491]
15TABLE 15 Bursa: Body weight ratios for SPF white leghorn chickens
challenged at 32 days post hatch with different concentrations of
USDA/STC IBDV and Variant E/1084 IBDV at 2, 3, 4, and 11 days post
challenge. Days Post Challenge Challenge Groups 2 3 4 11 Negative
Control 5.52 .+-. 0.44.sup.ab 5.63 .+-. 0.44.sup.a 5.13 .+-.
0.44.sup.a 5.95% .+-. 0.41.sup.a USDA 0.5X 5.97 .+-. 0.44.sup.a
4.42 .+-. 0.44.sup.a 4.63 .+-. 0.51.sup.ab 13 USDA 1X 5.46 .+-.
0.44.sup.ab 4.70 .+-. 0.44.sup.a 4.95 .+-. 0.56.sup.ab 14 USDA 10X
4.39 .+-. 0.44.sup.b 3.77 .+-. 0.44.sup.a 4.40 .+-. 0.44.sup.ab 15
Variant E 1X 6.06 .+-. 0.44.sup.a 4.53 .+-. 0.44.sup.a 16 17
Variant E 2X 4.54 .+-. 0.44.sup.b 4.78 .+-. 0.44.sup.a 4.73 .+-.
0.44.sup.ab 18 Variant E 20X 6.14 .+-. 0.44.sup.a 4.34 .+-.
0.45.sup.a 19 20 *Values in shaded boxes with superscripts with
different letters indicate significant differences at the P <
0.05 confidence interval.
[0492]
16TABLE 16 Spleen: Body weight ratios for SPF white leghorn
chickens challenged at 32 days post hatch with different
concentrations of USDA/STC IBDV and Variant E/1084 IBDV at 2, 3, 4,
and 11 days post challenge. Days Post Challenge Challenge Groups 2
3 4 11 Negative Control 2.20 .+-. 0.21.sup.bc 1.67 .+-. 0.21.sup.d
1.77 .+-. 0.21.sup.c 2.19 .+-. 0.20.sup.a USDA 0.5X 2.78 .+-.
0.21.sup.b 21 22 2.78 .+-. 0.19.sup.a USDA 1X 2.74 .+-. 0.21.sup.b
23 24 2.70 .+-. 0.19.sup.a USDA 10X 2.31 .+-. 0.21.sup.bc 25 26
2.76 .+-. 0.19.sup.a Variant E 1X 27 28 29 2.48 .+-. 0.22.sup.a
Variant E 2X 2.12 .+-. 0.21.sup.c 30 31 2.79 .+-. 0.20.sup.a
Variant E 20X 2.66 .+-. 0.21.sup.bc 32 33 2.76 .+-. 0.26.sup.a
*Values in shaded boxes with superscripts with different letters
indicate significant differences at the P < 0.05 confidence
interval.
[0493]
17TABLE 17 Challenge group identification and outline of viral
inoculums and volumes administered to SPF white leghorn chickens at
28 days post hatch. Challenge Groups Challenge Given Negative
Control None Negative Control + Copper Sulfate None USDA/STC
USDA/STC 30 .mu.l bilateral eyedrop USDA/STC + Copper Sulfate
USDA/STC 30 .mu.l bilateral eyedrop 0.5X USDA/STC + Copper Sulfate
USDA/STC 15 .mu.l bilateral eyedrop Variant 1084-E Variant E 30
.mu.l bilateral eyedrop Variant 1084-E + Copper Sulfate Variant E
30 .mu.l bilateral eyedrop REO S1133 REO S1133 100 .mu.l oral REO
S1133 + Copper Sulfate REO S1133 100 .mu.l oral USDA/STC + REO
S1133 USDA/STC 30 .mu.l bilateral eyedrop REO S1133 100 .mu.l oral
USDA/STC + REO S1133 + Copper USDA/STC 30 .mu.l bilateral Sulfate
eyedrop REO S1133 100 .mu.l oral Variant E/1084 + REO S1133 Variant
E 30 .mu.l bilateral eyedrop REO S1133 100 .mu.l oral Variant
E/1084 + REO + Copper Variant E 30 .mu.l bilateral Sulfate eyedrop
REO S1133 100 .mu.l oral
[0494]
18TABLE 18 AC-ELISA analysis of bursal and proventricular
homogenates from SPF white leghorn chickens at 4 and 11 days post
challenge with Variant E/1084 IBDV in the presence or absence of
REO virus and/or copper sulfate. Bursa Proventriculus Treatment Day
4 Day 11 Day 4 Day 11 NC 0/10 0/10 0/10 0/10 NC + CS 0/10 0/10 0/10
0/10 Reo Only 0/10 0/10 0/10 0/10 Reo + CS 0/10 0/10 0/10 0/10 Var
E Only 8/10 0/10 3/10 0/10 Var E + CS 7/10 0/10 3/10 0/10 Var E +
REO + CS 9/10 0/10 0/10 0/10 * Ratios represent the number of
individual tissue homogenates that test positive over the total
number of samples tested for that challenge group NC = Negative
Control CS = Copper Sulfate
[0495]
19TABLE 19 AC-ELISA analysis of bursal and proventricular
homogenates from SPF white leghorn chickens at 4 and 11 days post
challenge with USDA/STC IBDV in the presence or absence of REO
virus and/or copper sulfate. Bursa Proventriculus Treatment Day 4
Day 11 Day 4 Day 11 NC 0/10 0/10 0/10 0/10 NC + CS 0/10 0/10 0/10
0/10 REO Only 0/10 0/10 0/10 0/10 REO + CS 0/10 0/10 0/10 0/10
USDA/STC Only 8/10 0/10 5/10 0/10 USDA/STC + CS 6/10 0/10 2/10 0/10
1/2 USDA + CS 9/10 0/10 4/10 0/10 USDA/STC + REO + 9/10 0/8 4/10
0/8 CS USDA/STC + REO 6/10 0/10 4/10 0/10 * Ratios represent the
number of individual tissue homogenates that test positive over the
total number of samples tested for that challenge group. NC =
Negative Control CS = Copper Sulfate
[0496]
20TABLE 20 Gross lesion scores from the proventriculus of SPF white
leghorn chickens at 4 days post challenge with Variant E/1084 IBDV,
in the presence or absence of REO virus, and/or copper sulfate.
Treatment Hemorrhage Papillae Gizzard NC 0.00.sup.c 0.00.sup.d
1.00.sup.ab NC + CS 0.00.sup.c 0.00.sup.d 1.60.sup.a Reo Only
0.00.sup.c 0.21.sup.cd 0.57.sup.bc Reo + CS 0.00.sup.c 0.00.sup.d
0.43.sup.bc Var E. Only 0.08.sup.c 0.54.sup.c 1.00.sup.ab Var E +
CS 0.11.sup.c 1.00.sup.c 0.78.sup.bc Var E + Reo + 0.27.sup.c
0.27.sup.c 0.78.sup.bc CS * Values in shaded boxes with different
superscripts are significantly different from the negative control
at the P < 0.05 confidence interval. NC = Negative Control CS =
Copper Sulfate
[0497]
21TABLE 21 Gross Lesions in the proventriculus of SPF white leghorn
chickens at 4 days post challenge with USDA/STC IBDV in the
presence or absence of REO virus and/or copper sulfate. Treatment
Hemorrhage Papillae Gizzard NC 0.00.sup.c 0.00.sup.d 1.00.sup.ab NC
+ CS 0.00.sup.c 0.00.sup.d 1.60.sup.a Reo Only 0.00.sup.c
0.21.sup.cd 0.57.sup.bc Reo + CS 0.00.sup.c 0.00.sup.d 0.43.sup.bc
USDA/STC Only 0.42.sup.c 0.75.sup.a 0.75.sup.bc USDA/STC + CS
1.64.sup.a 0.27.sup.bcd 0.18.sup.c 1/2 USDA/STC + CS 0.89.sup.b
0.56.sup.abc 1.00.sup.ab USDA/STC + Reo + 0.14.sup.c 0.71.sup.ab
0.29.sup.c CS USDA/STC + Reo 1.00.sup.b 0.82.sup.b 0.18.sup.c *
Values in shaded boxes with different superscripts are
significantly different from the negative control at the P <
0.05 confidence interval. NC = Negative Control CS = Copper
Sulfate
[0498]
22TABLE 22 Miscroscopic lesions in the bursa and proventriculus of
SPF white leghorn chickens at 4 and 11 days post challenge with
Variant E/1084 IBDV in the presence or absence of REO virus and/or
copper sulfate. Bursa Proventriculus Treatment Day 4 Day 11 Day 4
Day 11 NC 0/10 0/10 0/10 0/10 NC + CS 0/10 0/10 0/10 0/10 Reo 0/10
0/10 0/10 0/10 Reo + CS 0/10 0/10 0/10 0/10 Var. E 10/10 10/10
10/10 0/10 Var. E + CS 10/10 10/10 7/10 0/10 Var. E + CS + Reo
10/10 10/10 10/10 0/10 * Ratios represent the number of samples
exhibiting lesions over the total number of samples screened for
that challenge group. NC = Negative Control CS = Copper Sulfate
[0499]
23TABLE 23 Microscopic lesions in the bursa and proventriculus of
SPF white leghorn chickens at 4 and 11 days post challenge with
USDA/STC IBDV in the presence or absence of REO virus and/or copper
sulfate. Bursa Proventriculus Treatment Day 4 Day 11 Day 4 Day 11
NC 0/10 0/10 0/10 0/10 NC + CS 0/10 0/10 0/10 0/10 Reo Only 0/10
0/10 0/10 0/10 Reo + CS 0/10 0/10 0/10 0/10 USDA STC Only 10/10
10/10 9/10 0/10 USDA/STC + CS 10/10 10/10 10/10 0/10 1/2 USDA/STC +
CS 10/10 8/8 9/10 0/8 USDA/STC + Reo + 9/9 8/8 9/9 0/8 CS USDA/STC
+ Reo 10/10 10/10 10/10 0/10 * Ratios represent the number of
samples exhibiting lesions over the total number of samples
screened for that challenge group. NC = Negative Control CS =
Copper Sulfate
[0500]
24TABLE 24 Organ to body weight ratios of SPF white leghorn
chickens at 4 and 11 days post challenge with Variant E/1084 IBDV
in the presence or absence of REO virus, and/or copper sulfate.
Spleen Bursa Day Proventriculus Treatment Day 4 Day 11 Day 4 11 Day
4 Day 11 NC 5.74.sup.a 6.59.sup.ab 1.63.sup.c 2.26.sup.de
6.17.sup.bcd 6.06.sup.d NC + CS 4.85.sup.ab 5.96.sup.bc 1.52.sup.c
1.85.sup.de 5.86.sup.cd 6.19.sup.cd Reo Only 5.51.sup.a 5.67.sup.c
1.75.sup.c 2.18.sup.e 5.95.sup.cd 5.92.sup.d Reo + CS 5.69.sup.a
7.10.sup.a 1.96.sup.c 2.29.sup.de 6.31.sup.bcd 6.54.sup.bcd Var E
Only 3.17.sup.a 1.31.sup.d 3.25.sup.a 3.47.sup.a 6.11.sup.bcd
6.63.sup.bcd Var E + CS 3.74.sup.cd 1.44.sup.d 3.07.sup.a
3.08.sup.ab 6.44.sup.abc 6.54.sup.bcd Var E + Reo + 3.49.sup.cd
1.40.sup.d 2.51.sup.b 2.48.sup.bcd 6.40.sup.abc 7.46.sup.a CS
*Values in shaded boxes with different superscripts are
significantly different from the negative control at the P <
0.05 confidence interval. NC = Negative Control CS = Copper
Sulfate
[0501]
25TABLE 25 Organ to body weight ratios of SPF white leghorn
chickens at 4 and 11 days post challenge with USDA/STC IBDV in the
presence or absence of REO virus and/or copper sulfate. Spleen
Bursa Day Proventriculus Treatment Day 4 Day 11 Day 4 11 Day 4 Day
11 NC 5.74.sup.a 6.59.sup.ab 1.63.sup.c 2.26.sup.de 6.17.sup.bcd
6.06.sup.d NC + CS 4.85.sup.ab 5.96.sup.bc 1.52.sup.c 1.85.sup.e
5.86.sup.cd 6.19.sup.cd Reo Only 5.51.sup.a 5.67.sup.bc 1.75.sup.c
2.18.sup.e 5.95.sup.cd 5.92.sup.d Reo + CS 5.69.sup.a 7.10.sup.a
1.96.sup.c 2.29.sup.de 6.31.sup.bcd 6.54.sup.bcd USDA/STC
4.33.sup.bc 1.44.sup.d 3.23.sup.a 2.86.sup.abcd 7.05.sup.a
7.44.sup.a Only USDA/STC + 4.78.sup.ab 1.51.sup.d 2.81.sup.ab
2.97.sup.abc 6.20.sup.bcd 6.83.sup.abc CS 1/2 USDA/ 4.76.sup.ab
1.29.sup.d 3.23.sup.a 2.35.sup.cde 6.05.sup.bcd 6.17.sup.cd STC +
CS USDA/STC + 3.06.sup.d 0.98.sup.d 3.09a 2.42.sup.bcde 6.65.sup.ab
7.10.sup.ab Reo + CS USDA/STC + 3.17.sup.d 1.12.sup.a 3.12.sup.a
2.91.sup.abcd 5.70.sup.a 6.59.sup.bcd Reo *Values in shaded boxes
with different superscripts are significantly different from the
negative control at the P < 0.05 confidence interval. NC =
Negative Control CS = Copper Sulfate
[0502]
26TABLE 26 TUNEL staining of tissue sections 4 days post challenge
with USDA/STC IBDV, Variant E/1084 IBDV, and REO (S-1133) in the
presence or absence of copper sulfate feed supplement. Experimental
Group Bursa Proventriculus Negative Control - - Negative Control +
Copper Sulfate - - USDA/STC +++ ++ Variant E ++ + REO +/- -
USDA/STC + REO +++ ++ Variant E + REO ++ + USDA/STC + Copper
Sulfate +++ +++ Variant E + Copper Sulfate ++ ++ REO + Copper
Sulfate - - USDA/STC + REO + Copper ++++ +++ Sulfate Variant E +
REO + Copper Sulfate +++ ++ 1/2 USDA/STC + Copper Sulfate +++ +++ *
+ indicates the presence of specific fluorescence following TUNEL
staining. multiple "+'s" indicate visual intensity assessment.
[0503]
27TABLE 27 AC-ELISA analysis of individual tissue homogenates from
SPF white leghorn chickens challenged at 28 days post hatch with
Variant E/DEL, Variant E-1084, and USDA/STC IBDV at 4 days post
challenge. Challenge Group Bursa Spleen Proventriculus Negative
Control 0/10 0/10 0/10 Variant E/DEL 10/10 0/10 3/10 Variant E/1084
7/10 5/10 7/10 USDA/STC 10/10 0/10 8/10 * Ratios represent the
number of samples testing positive over the total number of samples
tested for each experimental group.
[0504]
28TABLE 28 AC-ELISA analysis of individual tissue homogenates from
SPF white leghorn chickens challenged at 28 days post hatch with
Variant E/DEL, Variant E/1084, and USDA/STC IBDV at 11 days post
challenge Challenge Group Bursa Spleen Proventriculus Negative
Control 0/10 0/10 0/10 Variant E/DEL 0/10 0/10 0/10 Variant E/1084
0/10 0/10 0/10 USDA/STC 0/10 0/10 0/10 * Ratios represent the
number of samples testing positive over the total number of samples
tested for each experimental group.
[0505]
29TABLE 29 Histological lesions in the bursa and proventriculus of
SPF white leghorn chickens challenged at 28 days post hatch with
Variant E/DEL, Variant E/1084, and USDA/STC IBDV at 4 days post
challenge. Challenge Group Bursa Proventriculus Negative Control
0/10 0/10 Variant E/DEL 10/10 4/10 Variant E/1084 7/10 8/10
USDA/STC 9/10 10/10 * Ratios represent the number of tissues
positive for the presence of lesions over the total number of
tissues screened for each experimental group.
[0506]
30TABLE 30 Histological lesions in the bursa and proventriculus of
SPF white leghorn chickens challenged at 28 days post hatch with
Variant E/DEL, Variant E/1084, and USDA/STC IBDV at 11 days post
challenge. Challenge Group Bursa Proventriculus Negative Control
0/10 0/10 Variant E/DEL 10/10 0/10 Variant E/1084 10/10 0/10
USDA/STC 10/10 0/10 * Ratios represent the number of tissues
positive for the presence of lesions over the total number of
tissues screened for each experimental group.
[0507]
31TABLE 31 Organ to body weight ratios of broiler chickens
challenged at 35 days post hatch with USDA/STC IBDV in the presence
or absence of copper sulfate at 4 days post challenge. Treatment
Bursa Spleen Proventriculus NC 2.29.sup.a 1.81.sup.b 5.14.sup.a NC
+ CS 2.44.sup.ab 1.70.sup.b 5.15.sup.a USDA/STC 2.94a 2.78.sup.a
5.24.sup.a USDA + CS 2.37.sup.b 2.49.sup.a 5.11 * Values in shaded
boxes with different superscripts are significantly different from
the negative control at P <0.05 confidence interval.
[0508]
32TABLE 32 Organ to body weight ratios of broiler chickens
challenged at 35 days post hatch with USDA/STC IBDV in the presence
or absence of copper sulfate at 11 days post challenge. Treatment
Bursa Spleen Proventriculus NC 3.01.sup.a 2.64.sup.a 5.03.sup.a NC
+ CS 2.86.sup.a 2.36.sup.ab 4.95.sup.a USDA/STC 0.90.sup.a
1.89.sup.c 5.30.sup.a USDA + CS 0.89.sup.a 2.03.sup.bc 5.12.sup.a *
Values in shaded boxes with different superscripts are
significantly different from the negative control at P <0.05
confidence interval.
[0509]
33TABLE 33 Gross Lesion scores from broiler chickens challenged at
35 days post hatch with USDA/STC IBDV in the presence or absence of
copper sulfate at 4 and 11 days post hatch. Papillae Impaction
Treatment Day 4 Day 11 NC 0.25.sup.b 0.39.sup.b NC + CS 0.57.sup.b
0.23.sup.b USDA/STC 1.60.sup.a 0.84.sup.a USDA + CS 0.76.sup.b
0.08.sup.b * Values in shaded boxes with different superscripts are
significantly different from the negative control at P <0.05
confidence interval.
[0510]
34TABLE 34 AC-ELISA analysis of bursal and proventricular
homogenates from 35 post hatch broilers at 4 and 11 days post
challenge with USDA/STC IBDV in the presence or absence of copper
sulfate. Bursa Proventriculus Experimental Group Day 4 Day 11 Day 4
Day 11 Negative Control 0/10 0/10 0/10 0/10 Negative Control + CS
0/10 0/10 0/10 0/10 USDA/STC IBDV 7/10 0/10 4/10 0/10 USDA/STC IBDV
+ CS 6/10 0/10 5/10 0/10
[0511]
35TABLE 35 Microscopic lesions in the bursa and proventriculus of
broiler chickens challenged at 35 days post hatch with USDA/STC
IBDV in the presence or absence of copper sulfate at 4 and 11 days
post challenge. Bursa Proventriculus Challenge Group Day 4 Day 11
Day 4 Day 11 Negative Control 0/10 10/10 0/10 0/10 Negative Control
+ CS 0/10 9/10 0/10 0/10 USDA/STC 9/10 10/10 4/10 0/10 USDA/STC +
CS 6/10 10/10 5/10 0/10
[0512]
36TABLE 36 AC-ELISA analysis of bursal and proventricular
homogenates for SPF white leghorn chickens challenged at 28 days
post hatch with USDA/STC IBDV and suspect IBDV field isolates at 4
and 11 days post challenge. Bursa Proventriculus Challenge Group
Day 4 Day 11 Day 4 Day 11 Negative Control 0/10 0/10 0/10 0/10
USDA/STC 9/10 0/9 10/10 0/9 RB Texas 3 6/11 0/10 5/11 0/10 RB Texas
4 7/11 0/9 2/11 0/10 Farm 57-7 9/9 0/9 6/9 0/9 W/L #22 0/8 0/9 0/9
0/9 W/L #33 0/8 0/9 0/8 0/9 W/L #39 7/8 0/9 5/8 0/9 * Ratios
represent the number of tissue homogenates testing positive over
the total number of tissue homogenates tested for each challenge
group.
[0513]
37TABLE 37 Organ to body weight ratios of SPF white leghorn
chickens challenged at 28 days post hatch with USDA/STC IBDV and
suspect IBDV field isolates at 4 days post challenge. Challenge
Groups Bursa Spleen Proventriculus Negative Control 5.50.sup.b
1.86.sup.c 6.35.sup.a USDA/STC 4.48.sup.bc 2.09.sup.a 6.58.sup.a RB
Texas 3 3.63.sup.c 3.64.sup.a 6.86.sup.a RB Texas 4 3.68.sup.c
3.81.sup.a 7.15.sup.a Farm 57-7 3.84.sup.c 2.76.sup.a 6.27.sup.a
W/L #22 6.55.sup.a 1.74.sup.c 6.01.sup.a W/L #33 5.20.sup.b
1.21.sup.c 6.68.sup.a W/L #39 3.79.sup.c 2.57.sup.a 6.55.sup.a *
Values in shaded boxes with different superscripts are
significantly different from the negative control at P <0.05
confidence interval.
[0514]
38TABLE 38 Organ to body weight ratios of SPF white leghorn
chickens challenged at 28 days post hatch with USDA/STC IBDV and
suspect IBDV field isolates at 11 days post challenge. Challenge
Groups Bursa Spleen Proventriculus Negative Control 4.64.sup.a
2.14.sup.a 5.71.sup.a USDA/STC 1.38.sup.b 2.58.sup.a 6.41.sup.a RB
Texas 3 1.28.sup.b 3.42.sup.a 6.90.sup.a RB Texas 4 1.13.sup.b
2.98.sup.a 7.35.sup.a Farm 57-7 1.56.sup.b 2.08.sup.a 6.30.sup.a
W/L #22 6.19.sup.a 1.80.sup.a 6.21.sup.a W/L #33 6.67.sup.a
1.72.sup.a 6.26.sup.a W/L #39 1.46.sup.b 2.21.sup.a 6.03.sup.a *
Values in shaded boxes with different superscripts are
significantly different from the negative control at P <0.05
confidence interval.
[0515] The present invention is directed to proventricular origin
infectious bural disease viruses and any derivatives of these
viruses that might be used as vaccines. This would include any
viruses that are modified or attenuated by passage in cultured
cells, eggs or any other living system. This would also include any
of these viruses that are inactivated for use in a killed vaccine.
The present invention is also directed to new species of IBDV.
[0516] The present invention is directed to poultry virus isolates
or strains believed to be a new strain or strains of infectious
bursal disease virus (IBDV) which infects the proventriculus. More
particularly, the present invention is directed to one or more
newly isolated strains referred to as the Texas RB Strain., Texas
RB 3, Texas RB 4, F57-7, HBS, W/L 39, GAR 1, and the like of
infectious disease virus which was isolated from broiler chickens.
The isolated strains have been adapted to embryonated chicken eggs
and have been passed in SPF White Leghorn Chickens 1.times.. The
virus is a proventricular origin infectious bursal disease virus
and a candidate for a vaccine for chickens against an enteric form
of infectious bursal disease virus. It was noted that a number of
IBDV infected broiler chickens suffered from proventriculitis, each
having swollen, enlarged proventriculus, with the inner surface
having hemorrhage and flattened papillae. The virus strains
isolated from the infected proventriculi may be used to produce a
modified live vaccine or an inactivated vaccine form for
vaccinating for IBDV and/or proventriculitis. Also, it was
discovered that a combination of IBDV and the feed additive copper
sulfate (CS) increased the mortality rate and that a combination of
IBDV, Reovirus and CS further increased the mortality rate.
[0517] ARS-94015 is a proventricular tissue homogenate collected
from a pool of affected field broiler proventriculi. It will induce
a proventriculitis when used to experimentally infect broiler and
white leghorn chickens. This material has been subjected to
isolation procedures and the resulting product has been partially
characterized.
[0518] Other proventricular homogenates from affected field broiler
proventriculi have been subjected to virus isolation procedures and
isolates of infectious bursal virus successfully obtained. These
isolates are identified as Texas IB 3, Texas RB 4, F57-7, W/L 39,
HBS, and GAR 1.
[0519] Proventriculitis, proventricular enlargement and
proventricular tearing have been observed for many years in the
broiler industry. Research at the University of Arkansas has shown
that infectious bursal disease virus can cause proventriculitis and
may be a major cause of this condition. Unique infectious bursal
disease viruses have been isolated from the proventriculus of
affected broilers from the field. These viruses have been shown to
infect and induce pathology in the proventriculus of experimentally
infected chickens and this pathology has been shown to be
exacerbated by copper sulfate and reovirus.
[0520] Vaccines can be made from these proventricular orign IBDVs.
These vaccines may very well protect vaccinated broilers against
proventriculitis, proventricular enlargement, proventricular
tearing and other gastrointestinal pathology. The vaccines derived
from these unique proventricular origin IBDVs may have great
commercial value.
[0521] The present invention is directed to IBDVs which are
proventricular in origin. The present invention is also directed to
derivatives of the proventricular IBDVs which can be made to
include attenuated, inactivated, molecular altered or mutated that
can or possibly could be used as a vaccine against
proventriculitis, proventricular enlargement, proventricular
tearing or other abnormalities involving the gastrointestinal tract
excluding the bursa of Fabricius which is an organ of immunity that
communicates with the popsterior gut.
[0522] In regards to method of administration, a vaccine derived
from a proventricular origin IBDV of the present invention would be
given by standard acceptable methods that are commonly practiced in
the poultry industry including being given by aerosol, drinking
water, eyedrop, injection (SQ or inovo), feed, etc.
[0523] Our research has shown that proventricular origin IBDV and
certain challenge strains of IBDV do infect and cause pathology in
the proventriculus when given on days 7, 14, 21 and 28 post hatch.
IBDV is an RNA virus and as such does not cause intra-nuclear
inclusions.
[0524] Wideman et al. and Jensen et al. were able to induce
proventriculitis by feeding elevated levels of copper sulfate only.
Copper sulfate is a known irritant of the gastrointestinal tract
and of mucus membranes. Copper sulfate has been known to cause
proventricular enlargement for many years. Our work combined
IBDV+copper. The levels of copper used in our research induced no
lesions in the proventriculus either gross or microscopic. Our
research showed that when IBDV and copper was given together there
was elevated mortality from this combination. There is a
synergistic effect between copper and IBDV. Proventricular origin
IBDV is an irritant to the proventriculus and possibly other areas
of the gastrointestinal tract. The fact that this synergism exists
is an interesting observation. Since copper is widely used in the
broiler industry this is another reason for protecting chickens
against the gastrointestinal effects of IBDV along with the
possible lethal synergistic effect of IBDV and copper together.
[0525] The problem with trying to vaccinate against IBDV is vaccine
timing. All poultry houses are seeded with IBDVs. As soon as the
maternal immunity drops these viruses will induce infection in the
chicken and cause serious problems. When infection occurs these
viruses damage the intestinal tract and contribute to
proventriculitis and the so-called malabsorption/feed passage
syndrome.
[0526] If the above is true, then we should be able to vaccinate
for the condition. The proventricular origin viruses may be a
better alternative than what presently exists. By vaccinating the
birds at 2-3 weeks post hatch, and again at 4-5 weeks, and then
again at 6-7 weeks, we may be able to avoid the problem of the
timing of vaccination and the interference that occurs from
maternal immunity. It is conceivable that these viruses may also be
able to induce local immunity in the face of maternal antibody.
[0527] Since about 65-70% of the cost of raising poultry is the
poultry feed a vaccine or method of treatment which reduces or
eliminates the clinical manifestation of proventriculitis and/or
the so-called malabsorption/feed passage syndrome may be able to
reduce the overall cost of raising poultry by, for example, 5% or
more.
[0528] Conventional poultry vaccines are given to immunosuppression
or to boost maternal immunity. In accordance with the present
invention, we can vaccinate to protect against the clinical
manifestation of proventriculitis by vaccinating the birds one or
more times, preferably at least twice, two or more weeks post
hatch. Also, it may be necessary to use a relatively "hot" virus in
the vaccine to protect against the clinical manifestation of
proventriculitis.
[0529] Four-week old SPF white leghorn chickens were challenged by
eyedrop with ehter USDA/STC or Variant E IBDV. Four days post
challenge 10 birds from each group were weighed, bled, euthanized,
and necropsied. Tissues were scored for gross lesions, collected,
and individually weighed. Samples from the proventriculus, bursa
and spleen were placed in 10% buffered formalin, and processed for
standard hematoxylin eosin and apoptosis staining. All tissues were
paraffin embedded and cut into 5 urn sections. Tissue sections for
apoptosis staining were placed on poly-L-lysine coated slides,
deparaffinized, and dehydrated through graded alcohols. Sections
were then stained to determine the presence of apoptotic cells
using the Terminal dinucleotidyl d-UTP Nick End Labeling (TUNEL)
procedure and counterstained with 4',6-Diamidine-2'-phenylindole
dihydrochloride (DAPI). Slides were viewed and photographed with an
(Dlympus microscope to determine the presence/absence of cells with
specific green fluorescence.
[0530] Tissues harvested from negative control SPF leghorns
demonstrated occasional small areas with apoptotic staining in the
proventriculus and bursa. Birds challenged with the Variant E and
USDA/STC IBDV demonstrated extensive Apoptotic staining in the
proventriculus and bursa at 4 days post challenge. Apoptotic
staining in the bursa was evident within infected bursal follicles.
Apoptotic cells were present in the glands, lamina propria and
extensively in the villi of the proventriculus. No differences were
noted between the number of apoptotic cells present in tissues from
birds challenged with Variant E IBDV or USDA/STC IBDV.
[0531] 1. Overview of Research Findings
[0532] Infectious Bursal Disease (IBD) virus infection induces
gross and histopathologic lesions within the proventriculus at 4
days post challenge in SPF white leghorn chickens. The presence of
IBD virus has also been associated with proventriculitis syndrome
in commercial broiler chickens.
[0533] Pathology in the proventriculus associated with IBD virus
infection is transient as demonstrated by the absence of lesions at
11 days post challenge. Current studies involve the determination
of the mechanism(s) by which acute IBD virus infection induces
lesions in the proventriculus.
[0534] Formalin fixed tissue sections from affected proventriculi
and bursae were evaluated for the presence of fragmented cellular
DNA a common indicator of cellular apoptotic activity. Tissue
sections were stained by the terminal deoxynucleotidyl transferase
mediated dUTP-biotin nick end labeling (TUNEL) method to visually
detect the presence of nuclear fragmentation.
[0535] 2. Bottom Line
[0536] Tissues were harvested from SPF white leghorn chickens
challenged with USDA/STC or Variant E IBD viruses 4 days prior.
TUNEL staining revealed the presence of DNA fragmentation in bursal
and proventricular tissues from challenged birds only.
[0537] Staining intensity in bursal sections was highest in the
medulla and cortex of the bursal follicles with low levels in the
surrounding connective tissue. Proventricular staining was
localized within the villi with occasional involvement of the
glandular tissue. However, the amount or intensity of fluorescent
labeling was not quantitated.
[0538] TUNEL staining intensity in the proventriculus and bursa was
highly associated with confirmed IBDV histopathogy lesions,
suggesting that cellular mechanisms of apoptosis are involved in
the induction of lesions associated with IBD virus infection.
[0539] Thus, it will be appreciated that as a result of the present
invention, poultry viral isolates, vaccines, and method are
provided by which the principal objective, among others, is
completely fulfilled. It is contemplated and will be apparent to
those skilled in the art from the preceeding description and
accompanying drawings, that modifications and/or changes may be
made in the illustrated embodiments without departure from the
present invention. Accordingly, it is expressly intended that the
foregoing description and accompanying drawings are illustrative of
preferred embodiments only, not limiting, and that the true spirit
and scope of the present invention be determined by reference to
the appended claims.
* * * * *