Note: Descriptions are shown in the official language in which they were submitted.
~sz~
- 1 - 23199-6
This invention relates to the identification and char-
acterisation of the major structural protein of infectious bursal
~ disease (IBD) virus of chickens (host-protective immunogen) which
stimulates the production of antibody that neutralises the in-
fectivity of IBD virus in vitro and which protects susceptible
chickens against inEection with virulent IBD virus. The invention
further relates to the production of an effective sub-unit vaccine
against the virus utilising this major host-protective immunogen,
as ~ell as to the use of this immunogen in diagnostic tests,
assays and the like.
Infectious bursal disease virus is a pathogen of major
economic importance throughout the world poultry industry and is a
ubiquitous contaminant of commercial poultry environments. The
virus causes a highly contagious immunodepressive disease of young
chickens, and selectively proliferates in the bursa of Fabricius
(one of the two major avian immunological organs) thereby des
troying the precursors of the antibody producing plasma cells. In
young chickens (day old to 4 weeks) it directly causes morbidity
and mortality, while the capacity to produce antibody responses is
inhibited or depressed in chickens which survive infection. Such
1~19-jms
~~
:LZ'jZ~
chickens respond poorly to vaccination programs aimed at other
avian infections, remain highly susceptible to a variety of
bac-terial, mycoplasmal and viral pathogens and exhibit very poor
weight gain and food conversion ratios.
Protection against IBD virus infection is mediated by
humoral antibody alone and does not require the presence oE cell-
mediated immune effector systems. Thus chickens that receive an
adequate amount of maternal antibody from an imMune breeder hen
via the yolk sac are protected through the critical first ~ to 5
weeks after hatching.
Current vaccination strategies are aimed a-t achieving
the deposition of high levels of maternal antibody in fertilized
eggs to protect the chickens throughout these critical weeks after
hatching. The presently used vaccination regimens to control IBDV
involve injecting breeder hens (previously exposed to live IBDV)
with an inactivated oil-emulsion whole virus vaccine prior to the
onset of their period of egg production around 22 weeks of age.
This inactivated vaccine provokes a major secondary antibody res-
ponse -that is several orders of magnitude (> 100 fold) greater and
persists for longer than the responses obtained by repeated vac-
cination with live virus. This results in the transmission of
high levels of protec-tive antibody to each egg throughout the next
~0 weeks or so of the egg production cycle. If the antibody
levels can be boosted sufficiently it should be possible Eor
dG)4~1
broiler chickens, that are slaughtered at 6 weeks of age, to go
through their complete rearing period totally protected Erom IBD
virus by maternally derived antibody.
The presently available inactivated IBD virus vaccine is
expensive and difficult to produce since the virus cannot be grown
to sufficiently high titres in simple culture systems such as
embryonated eggs or -tissue culture. Currently the viral material
required for vaccine production is obtained by infecting six-week-
old specified-pathogen-free (SPF) chickens and then harvesting the
virus from the infected bursae 3 or 4 days after infection. This
procedure for producing an IBD virus vaccine from infected SPF
chickens is both laborious and expensive. The identification and
isolation of the major host-protective immunogen in accordance
with the present invention opens the way for development of a safe
and inexpensive sub-unit vaccine which is effective in stimulating
prolonged high-titre antibody responses in hens to allow the
transfer of sufficient maternal antibody to protect young
chickens, at least during the critical first few weeks after
hatching.
One object of the work leading to the present invention
has been to identify the IBD virus-encoded protein(s~ that induce
antibody in chickens following natural infection or the injection
of a commercial inactivated oil-emulsion whole virus vaccine.
~ 2S ~
Tn this work, the purification of the
Australian strain of IBD virus from chicken hursae by
successive rate-zonal and density-equilibrium
centrifugation using sucrose and caesium chloride
respectively has been studied. The virus so purified
has been analysed and found to be composed of two
major structural protein or polypeptide components of
molecular weights (MW~ approximately 37 kilodal-ton
(Kd) and 32 Kd, and three other components of ~Ws
approximately 91.5 Kd, 41.5 Kd and 29 Rd. Only the
major polypeptides reacted strongly with serum from
chickens naturally infected or hyperimmunized with IBD
virus, and of these the reaction of the 32 Kd
polypeptide with antibody was iypically the most
intense. The 3~ Kd polypeptide was a major component
of all preparations (as revealed by Coomassie-blue
staining of gels) of purified bursal grown ~ustralian
IBD virus, with a buoyant density in CsCl of 1.33
g/ml. The relative amounts of the other polypeptides
varied between preparations. It should be noted that
from preliminary work in this re~ard, the 32 Kd
polypeptide was estimated to have a molecular w~ight
of approximately 31 Kd when compared with standard
molecular wei~ht markers. As a result of further
work, howeverj the molecular weight of approximately
32 Kd is considered to be more accurate.
The major 32Kd polypeptide of the Australian
isolate is comparable in size to the VP-3 protein (M~l
of 32 to 35 Kd) detected in studi~s overseas on the
Cu-l isolate of IBD virus grown in vitro or in vivo
- 5 - ~ ~5 Z~ ~
(Nick et al, 1976; Dobos 1979; Todd & McNulty, 1979; Muller &
Becht, 1982). The major 37 Kd polypeptide is, however, smaller
than the VP-2 (MW of 40 to 41 Kd) of overseas isolates, while the
VP-X protein (MW of 47 to 4~ Kd - Dobos, 1979; Muller & Becht
1982) was not detectecl in preparations of the intact Australian
virus. ~he 41.5 Kd polypeptide of the Australian isolate is,
however, the precursor of the 37 Kd polypeptide, and it is
suggested that the 41.5 Kd polypep-tide is analagous to VP-X of
cverseas isolates.
It has no~7 been demonstrated that serum from naturally
infected or hyperimmunised chickens contains antibodies to all
polypeptides of the Australian isolate other than the minor
91.5 Kd polypeptide. It has also been shown that antibodies
specific for the 32 Kd polypeptide of IBD virus appear first
during the primary immune response of SPF chickens to the
infectious Virus, and these are also the predominant antibodies
detected in the primary response of chickens to an inacti~ated
oil-emulsion whole virus vaccine prepared from IBD virus
propogated in the bursa. Only later in the response to live virus
or following revaccination with an inactivated vaccine could
antibodies to other structural polypeptides be readily detected.
The 32 Kd polypeptide thus appears to be a major, if not the
major, immunogen of IBD virus.
The protective ability of chicken sera which contain
only antibodies specific for the 32 Kd polypeptide, as assessed by
Western blotting analysis on viral
~,S~ Z
polypeptides separated by SDS-PAGE, is attested to by
the capacity oE such sera to neutralise the
infectivity of IBD virus in vitro and to passively
protect highly~susceptible 2-day-old SPF chickens.
Furthermore, chickens immunised with the purified
32 Kd polypeptide produced antibody detectable by
ElISA and the virus neutralisation assay, while
chickens ir~unised with the 37 Kd or ~1.5 Kd
polypeptides produced antibody detectable by ELISA,
but only low levels of virus neutralising ability.
Adsorption of the anti-32 Kd sera with the 37 Kd or
41.5 Kd polypeptides did not reduce the virus
neutralising titre of the anti-32 ~d sera. ~hese
results confirm that the 32 Kd polypeptide is a major
protective immunogen of IBD virus.
According to one aspect of the present
invention, there is provided a non-infectious sub-unit
vaccine for use against IBD virus which comprises the
structural polypeptide of approximate .~ 32 Kd
~ contained in the IBD virus, or an immunogenic peptide
derived therefrom, tog~ther with, if desired, an
adjuvant.
- In a related aspect, this invention provides
a method of increasing the level of protective
antibodies against IBD virus in poultry, particularly
breeding hens, which method comprises administering
the aforesaid vaccine to said poultry.
- 7 ~
In yet another aspect, this invention provides a method
of providing passive immunity to IBD virus in poultry, which
method comprises administering to said poultry an antiserum con-
taining antibodies specific for th~e 32 Kd structural polypeptide
or an immunogenic peptide derived therefrom.
The 32 Kd polypeptide may be isolated from IBD Virus,
for example IBD virus which has been grown in and purified from
infected bursae of Fabricius in chickens.
As mentioned above, the vaccine according to this inven-
tion may comprise an immunogenic peptide derived from the 3? Kdpolypeptide, for example~ by "genetic engineering" or chemical
synthesis. A suitable immunogenic peptide may be derived so that
it comprises all or at least the major immunogenic determinants of
the 32 Kd polypeptide contained in the IBD virus and thus exhibits
the same or similar immunogenicity to the 32 Kd polypeptide. If
required the 32 Kd polypeptide may also be coupled to a carrier
molecule to increase its immunogenicity and hence its efficacy as
a vaccine.
Preferably, the non-infectious sub-unit vaccine of this
inven~ion comprises an adjuvant. The vaccine may, for example, be
delivered in an aqueous-mineral oil emulsion, such as an emulsion
achieved by using an oil-phase emulsifier (e.g. Arlacel 80) and
:`~
an aqueous-phase emulsifier (e.g. Tween 80) as described by
-~D f' ~ ?~
23199-66
~:~S2~
Stone _ al., 1978. Additional adjuvants may also be included if
required, for example Al OH3 (Wells et al., 1979), saponin or a
derivative of muramyl dipeptide (Wells et al., 1982).
In other aspects of the present invention there are
provided methods for assaying both quantitatively and quali-ta-
tively the levels of protec-tive antibodies in poultry, including
breeding hens and their progeny, and methods for assaying the
relative concentrations of protective immunogen in preparations of
IBD virus produced for experimental and commercial inactivated
vaccines, which methods are characterised by the use as an immuno
gen of the polypeptide of approximate MW 32 Kd isolated from IBD
virus r or an immunogenic peptide derived therefrom. Further
details of the methods by which these immunoassays can be carried
out are well known in the art, and are accordingly not described
in detail here. ~hese methods include the well known ELISA and
radioimmunoassays.
In another aspect of the invention, there is provided
a process for preparing a non-infectious sub-unit vaccine for use
against IBD virus which comprises a structural polypeptide of
approximate M~1 32 Kd contained in the IBD virus, or an immuno-
genic peptide derived therefrom together with, if required, an
adjuvant which process comprises separating said 32 Kd polypeptide
or an immunogenic peptide derived therefrom from IBD virus and,
if required, mixing said separated 32 Kd polypeptide or said
immunogenic peptide with an adjuvant.
~5~ 23199-66
-8a-
The following detailed description relates to the isola-
tion and characterisation by elec-trophoresis and Western blotting
of an Australian isolate o:E IBD virus. In the accompanying dia-
grams:
Figure l shows the electrophoretic profile of the total
RNA isolated from IBD virus which had been fractionated on a 25
to 50% sucrose gradient (lO ml) at 28,000 rpm for 90 mins. The
white doublets towards the top of the gel in Eractions 3, 4 and
2~Z
9 from the gradient comprise the two seçments of
ds-RNA present in IBD virus. Arrow indicates
increasing concentration of sucrose.
Figure 2 shows (a) Polyacrylamide ge]
electrophoresis of purified IBD virus in a 12.5
gel using the discontinuous SDS-gel system
described by Laemmli (1970). The gel was stained
with Coomassie brilliant blue to reveal 2 major
bands of approximate ~ 37 Xd and 32 Kd and 3
others (arrowed) of approximate M~l 91.5 Kd,
41.5 Kd and 29 Kd.
(b) Autoradiograph of a Western blot of the virus
preparation from (a) after probing with serum
fro~ a chicken experimentally infected with live
IBD virus 2 months previously. ~olecular weight
standards are on the left side of each gel [(a)
Pharmacia standards and (b) Amersham
1 4c-standards]
~ Figure 3 shows the specificity of the serum antibody
response of a 6 week-old chicken to live IBD
virus, as assessed by reacting 1:500 dilutions of
serum collected 3,5,7,10 and 14 days after
infection with Western blots of the viral
polypeptides separated by SDS-PAGE. 14C-
~markers (Amersham, U.X.) are on the left-hand
side.
Figure 4 shows (a) the specificity of antibodies in
serum collected from six 6-week-old chickens, 14
~2~2~14~
days after they had been infected with IBD virus
(tracks 1-6) or in the serum from a chicken tha~
had been infected 28 days previously (track 7).
Only one (track 1) of the 6 chickens had antibody
with demonstrate specificity ~or other than the
32 Kd structures polypeptide by 14 days after
infection. Autoradiograph exposed for 7 da~s.
Amersham 14C-~bl markers on left-hand side.
Figure 5 shows the specificity of the primary
antibody response of 2 SPF chickens (l and 2)
injected at 5 weeks of age with a commercial
inactivated oil-emulsion vaccine. Serum obtained
from the chickens 4 and 8 weeks (a and b) a~ter
primary vaccination are compared with serum
obtained 4 weeks (c) after a second injection of
inactivated vaccine at 13 weeks of age. Amersham
14C-MW markers on left-hand side.
Figure 6 shows the specificity of antibodies in sera
- from a chicken infected at 5 weeks of a~e with
live virus when exam'ned 4 and 20 weeks post
infection ~a and b). The chicken was then
reimmunised with a commercial inactivated
whole-virus vaccine at 25 weeks of age and the
specificity of the response examined 4 and 8
weeks later (c) and d). Amersham 14C-~ markers
on left-hand side.
MATERIALS AND METHODS
~L252(~
Growth and purification of IBD virus.
The isolate 002/73 of IBD virus was originally
obtained in Australia by Firth (197~) from commercial
poultry with varying degrees of bursitis and
identified serologically as IBD virus at the Central
Yeterinary Laboratory, ~eybridge, UK. Following
propagation at a limiting a:ilution of infectivity, the
virus was routinely propagated by intraocular
inoculation of 4 to 6 week old specified pathogen free
(SPF) white leghorn chickens (CSIRO SPF Poultry Unit -
Maribyrnong, Victoria, Aust.). ~omogenates of
infected bursae of Fabricius were prepared as 10~
(w/v) suspensions in phosphate buffered saline (PBS)
and stored at -80C. The IBD virus stocks appeared to
be free of contamination by other poultry viruses on
electromicroscopic examination and did not cross react
in the agar-gel precipitation test with antisera to
avian reovirus.
The virus was purified by a modification of the method
of Todd ~ McNulty ~1979). An equal weight of chilled
PBS was added to the freshly harvested bursae which
were homogenised in an ice bath by 3 x 20s bursts of a
Polytron (PT-10-OD, Kinematica, GM~H, Luzern, Schweiz)
on setting 5. The homogenate was frozen to -80C and
thawed rapidly before an equal volume of the
fluorocarbon Arklone (Wertheim Labs., Melb. Australia)
was added and the mixture rehomogenised. After
centrifugation at 10,000 g for 30 min at 5C, the
aqueous phase (ca 7ml) was prepared in 0.1 M NaCl,
~25~C3aL~
0.01 M Tris-HCl buffer, pH 7.6. Afte~ centrifugation
at 28,000 rpm for 1.5 h in a Beckmann SW28 rotor at
5C, the gradients were harvested from the bottom in
1 ml fractions. These were then examined by gel
electrophoresis, Western blGtting with hyperimmune
sera, ELISA and by an assay for ds-RNA in order to
determine the position of complete or incomplete
particles of virus and soluble proteins of the virus
(in later preparations the virus was collected from
the interface of a stepwise gradient prepared by
overlaying l0 ml of 40~ sucrose on 5 ml of 60%
sucrose). Fractions containing complete (intact)
virus were pooled and layered onto chilled preformed
20 to ~0% (w/v) CsCl gradients (10 ml) which were
centrifuged at 30,000 rpm for 18 h at 5C in a
Beckmann ~0 Ti rotor and the band of IBD virus
harvested through the side of the tube. The virus was
dialysed against NaCl-Tris buffer to remove CsCl and
then against MaCl-Tris buffer containing 0.05~ (w/v)
sodium azide before being stored at 4C or else made
50% (v/vJ in glycerol and stored at -20C.
Chicken antisera to IBD virus
SPF chickens were infected intraocularly with IBD
virus and bled 0,3,5,7,10 and 14 days later, then
every two weeks. Chickens injected intramuscularly
with 0.5 ml of commercial inactivated oil-emulsion
vaccine (Arthur Webster P-ty.Ltd., Morthmead, NSW,
Aust.) were bled fortnightly, as were chickens given a
second intraocular infection with IBD virus or
D f~ R ~
3~ 2
previously-infected chickens yiven an intramuscular
injection of commercial inactivated vaccine. The sera
were collected by centrifugation at 400 g for 15 min
and stored at -20C.
Enzyme linked immunosorbent assav (ELLSA)
The ELISA method used to assess the presence of I~D
viral antigen in various gradient fractions or chicken
antibody to IBD virus was essentially that described
by York et al., (1983), except that the microtiter
trays (Nunc Immunoplate I) were coated with rabbit
anti-IBD virus IgG prepared by hy~erimmunising rabbits
with 002/73. To detect viral antigens in the
gradient, serial dilutions of the fractions were added
to the wells, which were then treated with a dilution
of chicken anti-serum to IBD virus which produced a
maximum OD450nm of 1Ø To titrate antibodies to IBD
virus, dilutions of chicken sera were added to the
wells that had first been coated with rabbit ant~body
followed by a standardised concentration of an extract
of infected bursae. The amount of chicXen antibody
binding to the viral antigen in each assay was then
quantitated by adding sheep IgG-anti-chicken
IgG-horseradish peroxidase conjugate, followed by
5-aminosalicylic acid. The trays were shaken for 30
min and the OD450nm was then read immediately on a
Titertek Multiscan (Flow Lahoratories, Australia).
Polyacrylamide ~el electrophoresis ~PAGE) and Western
blotting.
~.ZS2(~
la
Aliquots (40~1) of each sucrose-gradient fraction were
dried down under vacuum, resuspended in 20~1 of the
sample buffer described by Laemmli (1970) which
contained SDS and a trace of solid bromophenol-blue
dye, then heated for 3 min in a boiling water bath.
These samples were examined by discontinuous P~GE
(Laemmi, 1970~ with Coomassie blue staining. The
structural proteins of the virus, separated by
SDS-PAGE, were also examined by the Western blotting
procedure described by Burnette (1981). The viral
proteins were transferred to nitrocellulose
membrane-filter (Schleicher and Sch~ll BA83 0.2~m) and
probed with chicken antisera diluted 1: 500 in 1%
(w/v) gelatin in NaC1-Tris buffer. The chicken
antibodies binding to viral polypeptides were
identified with rabbit IgG anti-chicken IgG (Cappel
Labs, Cochranville, USA) diluted 1:1000 -n
NaC1-Tris-gelatin buffer followed by I~Ci of
125I-Protein A (Amersham, U.K.) in the same buffer.
An autoradiograph of the nitrocellulose membrane was
prepared using Fuji Rx Medical X-ray film and Ilford
Fast Tungstate intensif~ing screens usually for 16-24 h
at -70C.
Detection of_IBD virus by the RMA content of various
fractions.
Sucrose-gradient fractions were diluted to 1:4 with
10 mM Tris-HCl, 50 mM NaCl, 0.2~ SDS buffer, pH 7.05
and treated with 0.5 mg/ml ribonuclease-free Pronase
(Worthington, USA) for 1 h at 37C. The solutions
~25~ Z
were made 0.3 M with respect to NaCl and the nucleic
acids extracted with 1 volume of phenol at 56C for
5 min. One volume of chloroform was added to the
mixture which was then shaken at room temperature ror
10 min before being centrifuged at 12,0Q0 rpm for
2 min in an micro-centrifuge (Eppendorf, ~J.Germany~.
The nucleic acids in the upper aqueous phase were
recovered by precipitation with 2 volumes of ethanol
at -20C for 30 min followed by centrifugation. The
R-I~A pellets were washed thoroughly with 67% tv/v)
ethanol, dried and then dissolved in ~0~1 water.
Samples of RNA were electrophoresed under
non-denaturing conditions in 1% (w/v) agarose slab
gels in 20 mM phosphate buffer, pH 6.8, together with
~DNA standards (Boehringer, W.Germany). When the gels
were stained with acridine orange and illuminated with
W light, the ds-DNA or ~IA appeared as green bands
while single-stranded (ss) R~A appeared as red bands
(McMaster & Carmichael, 1977).
RESULTS
Purification of I~DV from infected bursae
Following centrifugation of the clari~ied bursal
homogenates in 25 to 50% continuous sucrose gradients,
a major band of material was visible approximately 3/5
the way down the gradient. SDS-PAG~ analysis of the
sucrose fractions indicated that the highest
concent~ation of ~iral proteins was in the visible
band, while fractions immediately above and below the
2,S2~2
16
major band contained lesser amounts of the viral
proteins. Similarly, the ~LISA for viral antigen
revealed peaks of IBD viral antigen throughout the
gradient, although the visible band again contained
the highest relative concentration.
The electrophoretic profiles of total RNA from
different sucrose-gradient ~Eractions located 2
segments of viral RNA in fractions 3 and 4,
corresponding to the major visible band and in
fraction 9 near the top of the gradient (Fig.l).
Colour reaction with acridine orange showed that the
two viral ~A segments were double-stranded. Fraction
10 contained only low ~^l RNAs (e.g. tRNA) and
fractions 6-9 contained mainly ribosomal RNA (18S and
28S) and low ~l RNAs. The viral ds-RMA bands were
much more resistant to ribonuclease digestion than the
ribosomal ss-RNAs. When electrophoresed under
non-denaturing conditions with ds-~DNA as standsrds,
the two viral RNA segments appeared to have molecular
weights of 2.5 x 106 and 2.2 x 106 respectively.
CsCl density-e~uilibrium centrifugation of complete
virus from the continuous sucrose gradients revealed
one major band which was visible under reflected light
and had a mean buoyant density of 1.33 g/ml. When the
crude virus from the interface of a 40-60% stepwise
sucrose gradient was further purified on CsCl, a
second, less dense, band was frequently seen which
appeared by electron microscopy to contain a high
proportion of "core" particles.
~ zs~2o~æ
The effect of the duration of infection on the yield
of virus
No band of IBD virus was visible in CsC1 gradients of
virus from bursae harvested from chickens 2 days after
infection. A distinct band of virus was visible in
CsCl gradients of bursae from chickens infected for 3
daysy but was more diffuse when the virus was purified
from bursae harvested 4 days after infection. An
ELI5A for IBD viral antigen also found maximum titres
of antigen in bursal homogenates obtained 3 or 4 days
after infection. Consequently, virus was routinely
prepared from bursae that were collected 3 days after
infection.
Amino acid analysis of an acid hydrolysate, assuming a
mean amino acid-residue weight of 110, showed that up
to 250~g of viral protein could be obtained from a
single bursa.
Coomassie-blue staining of SDS-PAGE of purified virus
As shown in Fig.2, purified preparations of intact
virus contained 2 major polypeptides with approximate
MW of 37 ~d and 32 Kd, and 3 other components (arrows
in Fig.2) with approximate M~ of 91.5 Kd, 41.5 Kd and
2q Kd. Although the polypeptide of ~ 32 Kd was a
major component of all preparations of virus,
densitometer tracings from polyacrylamide gels of
different preparations of virus revealed that the
~L'2,5~2
18
relative amounts of the polypeptides varied between
preparations.
The kinetics and specificity of the primary antibody
res~onse of chickens infectled with IBD virus.
L
The appearance of serum antibody -to IBD virus, as
determined by ELISA, was followed in 6 SPF chickens
infected intraocularly at 6 weeks of age with isolate
002/73. Antibody was first detected on day 5 (mean
titre 250), rising quickly to a mean titre of 10,000
on day 10 and 17,~00 on day 14. The analyses by
Western blotting of the sera ob-tained from one of
these chickens is shown in Fig.3. Antikody binding to
the 32 Kd polypeptide of IBD virus was detected on day
5, with the intensity of binding increasing with time
after infection. The antibodies present in the
circulation of this chicken remained relatively
specific ~or the 32 Kd polypeptide, at least until day
14 of the response. ~estern blots of the antibody
present in sera obtained 14 days after infection from
all 6 chickens are shown in Fig.~ (tracks 1-6). The
sera from 5 of these 6 chickens demonstrated
specificity for the 32 Kd polypeptide, even when the
autoradiographs were exposed for 7 days. By 28 days
after a primary infection the chickens had produced
antibodies to all I~D virus polypeptides, except the
91.5 Kd polypeptide ~Fig.4, track 7~.
Response of SPF chickens to vaccination with an
inactivated oil-emulsion IBD vaccine
-- 19 --
Six chickens, when 5 ~eeks of age, were injected intramuscularly
with 0.5 ml of a commercial inactivated whole virus vaccine. The
sera from the 2 chickens with the highest ELISA titres at 8 weeks
post-vaccination (titres of 25, 600 and 51, 200 respectively) were
analysed by Western blotting. ~t 4 and 8 weeks after vaccination
the primary antibody response of both chickens to the inactivated
vaccine was relatively specific for the 32 Kd polypeptide of IBD
virus (Fig.5, tracks a and b). By 4 weeks after a second intra-
muscular injection of inactivated vaccine at 13 weeks of age,
however, both chickens had produced serum antibodies which reacted
with the 3 most abundant structural proteins (Fig.5, tracks c)~
Response of sensitised chickens to an inactivated
oil-emulsion IBD vaccine
Chickens that had been given live IBD virus at 5 weeks of age were
injected at 25 weeks of age with a commercial inactivated vaccine~
Sera were obtained 4 weeks and 20 weeks after the primary infec-
tion and then 4 and ~ weeks following revaccination. Analysis by
Western blotting showed that initially there had been a response
to 4 of the structural proteins, which had waned by 20 weeks post-
infection (Fig.6). An injection of inactivated vaccine at thattime resulted in a heightened response to all the IBD viral poly-
peptides, including the 91.5 Kd polypeptide (Fig.6, track d).
~L~5~
- 20 -
The following detailed description describes experiments
detected towards defining the major immunogen of IBD virus and
assessing the protective efficacy o antibodies to that immunogen
in vitro and in vivo. In the accompanying drawings:
Figure 7 shows Western-blots of immune serum collected from three
10-week-old chickens (tracks 1, 2 and 3), 1~ days after in-
fection with IBD virus (002/73). Nitrocellulose strips
reacted with 25~1 of serum diluted 1:100. Hyperimmune serum
(track 4) included as a positive control.
Figure 8 shows (a) Protein profile (OD 280nm) of an S200 column
fractionation of day 10 immune serum from a 10-week-old
chicken infected with IBD virus (002/73).
(b) Antibody activity de~ectable by ELISA (OD 450nm) in a
1:10 dilution of each fraction.
Figure 9 shows Western-blots of whole virus with pools of the
(a) IgM, and
(b) IgG fractions of 10 day immune sera obtained from two
10-week-old chickens (1 and 2) infected with IBD virus
(002/73),
0 Figure 10 shows ~estern-blots of sera from adult chickens obtained
3 weeks after immunisation with approximately 50~g of puri-
fied structural polypeptides oE IBD virus (002/73). Chickens
A, B and C immunised with 32 Kd polypeptide: D, E and F
~L2~2C3~2
- 21 -
immunised with 37 Kd polypeptide; G, H and I immunised with
41.5 Kd polypeptide. Track K reacted with hyperimmwne serum.
Amersham C14 MW markers on left-hand side.
Figure 11 shows Western-blots of 2 anti-32 Kd polypeptide sera (A
and B) before (a) and after adsorption with (b) the 37 Kd
polypeptide or (c) the 41.5 Kd polypeptide and of 2 anti-37
Kd polypeptide sera (E and F) before (a) and after adsorption
with (d) the 32 Kd polypepticle. The extraneous antibody
activity removed by adsorption is arrowed on each original
serum (a). Refer to Table 5.
Figure 12 shows Western-blots of day 10 immune serum from a
chicken infected with 002/73 (L) and day 28 immune serum from
a chicken immunised with inactivated vaccine (K) before (a)
and after adsorption with the (b) 32 Xd, (c) 37 Kd or (d)
41.5 Xd polypeptides. Refer to Table 5.
MATERIALS AND METHODS
Animals
White Leghorn chickens of the "CSIRO-W" line were supplied by the
CSIRO Specified Pathogen Free (SPF) Poultry Unit, Maribyrnong,
Victoria and transferred into flexible-film plastic poultry iso-
lators, Dennett and Bagust (1979) at one-day-old.
A~L
Virus
The Australian isolate of IBD virus (002/73) used in these studies
was originally described by Firth (1974) and identified sero-
logically as IBD virus at the Central Veterinary Laboratory,
Weybridge, U.K. Following one passage at limiting dilution of
infectivity, the virus was routinely passaged by intraocular
(i~o.) infection of ~ to 6 week old SPF chickens. Virus infect-
ivity was titrated by inoculating 3-day-old SPF chickens i.o. with
25~1 of log1o dilutions of a 10% (w/v) homogenate of infected
bursae. The bursae were harvested from 'hese chickens 72h later,
homogenised and IBD viral antigen detected by an enzyme-linked
immunosorbent assay (ELISA). The titre of virus was expressed as
the reciprocal of the dilution of virus that infected 50~ of the
chickens inoculated (CID50).
IBD virus adapted to propagate in chick embryo fihroblast (CEF)
culture was initially made available by A. Webster Pty. Ltd.,
Sydney, Australia. The virus has been designated TC-IBD virus
(GT101) and was routinely passaged in CEF cultures. GT101 virus
is neutralised in vitro by chicken antisera to type-1 IBD, virus,
but not type-2 IBD virus (unpublished data - Central Veterinary
Laboratory, Weybridge, UK).
Purification of virus
IBD virus (002/73 was grown in bursae then purified as described
above. Briefly, a 50~ homogenate of the infected bursae in O.OlM
Tris-HCl, 0.15M NaCl, pH 7.6 (TBS) was frozen/thawed and
homogenised with an equal
IL25j2~
volume of the fluorocarbon Arklone (Wertheim Labs.,
Melbourne, Australla). The clarified aclueous phase
was centrifuged on s-tepwise gradients of ~0% and 60
(w/v) sucrose. The sucrose interface was collected
and centrifuged on preformed 25~ to 50gO (w/v) CsCl
gradients. The purified intact virus banded at a
density on CsCl of 1.33g/ml.
Polyacryl mide gel electrophoresis ~PAGE~ and
immunoblotting of viral proteins.
Viral polypeptides were analysed in 12.5% (w/v)
polyacrylamide slab gels using the discontinuous SDS
gel system of Lael~mli ~1970), then transferred from
the gel onto nitrocellulose paper by the Western
blotting technique described by Burnette (1981~ and
reacted with chicken antibodies as described above.
Brieflyr the nitrocellulose membrane filter was
blocked with a 5% ~w/v) solution of dried skim milk
powder (blotto) in TBS (Johnson, et al, 1984), cut
into 5mm strips then reacted with a 1:100 dilution of
chick antisera in blotto, followed by a 1:1000
dilution of rabbit anti-chicken IgG (Cappel Labs.,
Cochranville, USA) in blotto and finally 1 ~Ci of
125I-Protein A (Amersham, U.K.). Autoradiographs were
prepared using Fugi Medical X-ray film and Ilford Fast
Tungstate intensifying screens.
Purification of the structural pol~peptides OL IBD
virus.
~L~52~
24
Purified virus was boiled with SDS for 2 min in the
absence of reducing agents and the peptiAes separated
by SDS-PAG~. The gels were lightly stained with
Coomassie-blue, destained and the 29 Kd, 32 Kd, 37 Kd,
41.5 Kd and 91.5 Kd bands of protein cut from the
gels. The polypeptides were eluted from the gel
strips into 0.05M Tris-acetic acid buffer (pH8.0)
containing 0.1% (w/v) SDS at 50 volts for 40h. The
eluted polypeptides were d:ialysed against multiple
changes of distilled H2O for 48h and the approximate
concentration of the polypeptides assessed by SDS-PAGE
against known concentrations of MW markers
(Pharmacia, Sweden). The purity of the polypeptides
was assessed by Western-blotting with hyperimmune
chicken serum.
Chicken antisera to whole virus or the structural
polypeptides of IBD vir
Six to ten-week-old SPF chickenc were inoculated i.o.
with infectious virus (002/73) and bled 10 and 14 days
later. The serum was collected by centrifugation and
stored at -20C. SPF chickens were also injected
intramuscularly (i.m.) with 0.5ml of a commercial
inactivated IBD vaccine (Arthur Webster, Pty.Ltd.),
and sera collected 28 days later, a-t the peak of the
primary antibody response.
Adult SPF chickens were injected i.m. with
approximately ~O~g of either the 32 Kd, 37 Kd or
41.5 Kd polypeptides of IBD virus emulsified in 2
ii20~
volumes of Freund's complete adjuvant (FCA) (Difco,
U.S.A.). Three weeks later the chickens were
reinjected i.m. with the same amount of the respective
polypeptides in Freund's incomplete adjuvant (FIA)
(Difco). Approximately 40ml bleeds were collected at
approximately weekly intervals into preservative free
heparin (a final concentration of lOU/ml~ and the
plasma collected and fro2en at -20C. When the plasma
was thawed, the fibrin clot was removed by
centrifugation. Other chickens were immunised with
the 29 Kd and 91.5 Kd polypeptides, except that the
amounts of polypeptide injected were very much less
and the antibody responses were correspondingly weak.
ELISA for anti-IBD virus antibody and IBD vlral
anti~en.
Anti-IBD virus antibody in chicken sera and IBD viral
antigen in bursal homogenates were both quantitated
using the ELISA as described above.
-
Plaque-reduction serum neutralising (SN) assay
Serial dilutions of heat inactivated 56C/30 min)
serum in biocarbona~e buffered medium l99 (Gibco, USA)
were added to an equal volume of TC-IBD virus, which
had been diluted to approximately ~00 plaque-forming
units (pfu)/ml. Virus-serum mi~tures were incubated
at room temperature for 60 min with occasional shaking
and then 0.lml of each mixture inoculated into 3
secondary CEF cultures (35mm diam.plastic petri
~L25;2(~2
26
dishes, Kayline, Sth.Australia) to test for residual
viral infectivity. The virus was allowed to adsorb to
monolayers for 60 min at 37C before each dish was
overlayed with 2ml of 0.7~O (w/v~ agar (Baco-Difco,
USA) in HEPES (0.015 M) buffered medium 199 containing
5% (w/v) calf serum~ The cultures were incubated at
37C for a further 6 days and stained by the addition
of 0.15~o (w/v) neutral red -in a lgo agar overlay. The
end-point of the neutralisation assay was the dilution
of serum which caused a 50~ reduction in the number of
IBD virus plaques.
The Micro-SN assay
Serial dilutions of inactivated serum were prepared in
a volume of 25~1 of 199 containing 10% (w/v) TPB
(Difco) and 2~o heat inactivated fetal calf serum in
flat-bottomed microtiter trays (Linbro, USA) before an
equal volume of TC-IBD virus (500 pfu/ml) was added
and incubated at 37~ for lh. Following incuba-tion,
50~1 of a 7.5 105 cell/ml suspension of secondary CEF
cells were added and the trays incubated at 37 DC for 5
days. The trays were then stained with 1~ (w/v)
crystal violet in methanol and the end point read as
the last dilution to completely inhibit virus
replication.
assive protection of chickens with specific antibody
r
Chickens were injectcd intraperitoneally (i.p.) at 2
days of age with various immune serum or control serum
2s~
- 27 -
free of antibody to IBD virus. One day later the chickens were
challenged i.o. with 25~l of bursal homogenate, usually containing
a minimum of 1000 CID50 of IBD virus 002/73. Three days after
infection, the chickens were exsanguinated and their bursae re-
moved, weighed and made into a 5% homogenate in saline. Both the
levels of antibody in the sera and the presence of viral antigen
in the bursae were quantitated by ELISA.
Column chromatography
Five ml of immune serum was separated on a 2.5cm x 100cm S200
(Pharmacia) column by elution with 0.01M phosphate, 0.15M NaCl, pH
7.6 (PBS). The fractions were assayed for antibody activity by
ELISA before the IgM region and IgG regions were pooled, deleting
one 6ml fraction overlapping the two regions. The pools were
concentrated to 4ml using an ~M100A membrane (Amicon, USA) and
sterile filtered.
Affinity chromatography
Only the 32 Kd, 37 Kd and 41.5 Kd polypep~ides were obtained in
sufficient quantity to prepare absorption columns. The polypep-
tides were obtained from unstained gels, guided by Coomassie
stained strips taken from each margin of the gel. The purity of
the eluted polypeptides was assessed by Western blotting with
hyperimmune serum. The polypeptides were quantitated by a Lowry
protein assay (Hartree, 1972) and between 150 and 300 ~g of the
respective polypeptides were reacted with lml of
~25~
28
Affigel ~0 (Biorad, USA) according to the
manufacturers instructions.
One ml of each antipolypeptide sera was run slowly
through the adsorption columns and eluted with 3
volumes of PBS. The columns were reactivated by
passing 5ml of lM proprionic acid through the columns
followed by 20ml of PBS. sera from chickens immunised
with whole virus or inactivated vaccine were adsorbed
by running 0O25ml of serum onto the columns and
eluting with 1.75ml of PBS. The effectiveness of the
adsorption was assessed by immunoblotting.
P~ESULTS
Passive protection with monospecific anti-32 Kd sera
When antisera obtained from 6-week-old chickens, 14
days after the had been infected with IBD virus, were
analysed by ~estern-blotting~ 5 were found to contain
only antibodies specific for the 32 Kd structural
polypeptide. The sera had E~ISA titers between 6,620
and 51,200 and virus neutralisation titers of 6,250 or
greater (~able 1~. The ability of these sera to
passively protect chickens was assessed by injecting
lml of each serum into each o four 2-day-old
chickens, which were challenged 1 day later with 1000
CID50 of 002/73. All the chickens that received
immune serum resisted infection and had circulating
antibody when exsanguinated 3 days after challenge,
while those that received antibody negative serum had
~f~Pkfi~ /S
~ 29 -
no antibody and were susceptible to infection (Table 1).
The experiment was repeated with day 10 and day 14 immune serum
from three 10-week-old chickens infected with IBD virus. Again
these sera were monospecific by Western-blotting (Fig.7) and had
ELISA titers between 57,000 and 77,000 at day 10 and 70,000 and
166,000 at day 14. Two ml of each serum was injected into each of
3 chickens. In addition to the groups of chickens that were
challenged with 1000 CIDso of 002/~3, small groups oE chickens
that received either pooled immune serum or control serum remained
unchallenged and served as in-contact controls. Neither the
chickens that received immune serum and virus (Table 2) nor the
in-contact chickens were infected. The chickens that were given
normal chicken serum and virus were all susceptible to infection.
Characterisation of the protective antibodies in
anti-32 Kd serum
When sera from two 10-week-old chickens, obtained 10 days after
infection with IBD virus, were fractionated b~ S200 column chroma-
tography and assayed for antibody detectable by the ELISA, all
activity was confined to the IgG region of the column profile
(Fig.8). The IgM region and ~he IgE regions were pooled and
titrated by the micro-SN assay. Both the IgM and IgG pools of
these sera were found to have virus neutralising activity; The
- 30 -
IgG pool having t~ice the SN-titer of the IgM pool (~able 3,.
Immunoblotting whole virus with these antibody pools showed that
this assay also primarily detected IgG antibodies which were
specific for the 32 Kd polypeptide (Fig.9).
One ml oE the IgM and IgG pools from each serum were injected into
groups of four 2-day-old chickens, which were then challenged 1
day later with 10 CIDso f virus. The IgG antibody pools were
found to confer protection while chickens injected with the IgM
pools of antibody were all susceptible to infection (Table 3).
Antibody responses to purified viral polypeptides
Adult SPF chickens did not produce substantial titers of antibody
that could be detected by either the ELISA or micro-SN assay
during the early stages of the response to approximately 50~9 of
either the 32 Kd, 37 Kd or 41.5 Kd purified viral polypeptides in
FCA. Western-blotting with serum obtained 3 weeks after immuni-
sation, however, showed that the chickens had synthesised anti-
bodies to their respective po~ypeptides (Fig.10). Chicken B,
which produced the strongest response to the 32 Kd polypeptide,
also produced antibodies to the 37 Kd and 41.5 Kd polypeptides
(Fig.10, track B) and the sera from the 3 chickens (~, E and F)
injected with the 37 Kd polypeptide were almost indistinguishable
from sera from the 3 chickens (G, H and I) injected with the 41.5
Kd polypeptide
~2S2~
(Fig.10, tracks D to I). Tt was noted that all of the
chickens (D to I~ injected with either the 37 Kd or
41.5 Kd polypeptides also produced antibodies that
reacted with low MW material on the blots,
material that was not recognised by hyperimmune serum
from vaccinated chickens ~Fig.10, track K).
One week after a second injection of viral
polypeptides in FIA, 1 of the 3 chickens injected with
the 32 Kd polypeptide had a micro-SM titre of 256
(Table 4). Further bleedings at 3, 4 and 6 weeks
after the second immunisation showed that 2 of the
chickens injected with the 32 Kd polypeptide had
neutralising titers for IBD virus of between 160 and
1280, while those injected with the 37 Kd or 41.5 Kd
polypeptides had neutralising titers of 40 or less.
One o:F the chickens injected with the 41.5 Kd
polypeptides had the highest ELISA titers followed the
second injection of polypeptides (Table 4).
Affinity chromatography of an-ti-pol~peptide sera
Probing ~estern blots of the virus with sera o~tained
3 to 6 weeks after the second injection of the
purified polypeptides showed that they all contained
antibodies to the other polypeptides. Monospecific
antisera were prepared by passing antipolypeptide sera
through adsorption columns prepared by binding the
various viral polypeptides to Affigel-10.
~,~52~2
32
Passing the anti-32 Kd sera through a 37 Kd column
removed all antibody acti~ity to the 37 Kd and 41~5 ~d
polypeptides (Fig.ll, A and B), but did not reduce the
micro-SN titre (Table 5). The 41.5 Kd column was less
efficient, but gave similar results in that it did not
reduce the SN-titer of the anti-32 Kd sera. Passing
the 2 anti-37 Kd sera with the highest S~ activity,
through the 32 Kd adsorpticn column removed all
anti-32 Kd antibody (Fig.Ll, E and F) r but did not
reduce the low levels of SN antibodies present in
these sera (Table 5). The mcre discriminating
plaque-reduction assay confirmed that passing the
anti-3~ Kd sera through the 37 Kd column, did not
reduce the activity in the sera ~Table 6). Passing
the antipolypeptide sera through the homologous
adsorption column either had no detectable effect on
the micro-SN titre or reduced it by no more than 50~0
(Table 5).
Day ln serum from an infected chicken and day 28 serum
froma chicken injected with an inactivated I~D
vaccine, were also passed through the absorption
columns. In neither case did the 37 Kd and 41 5 Kd
columns affect the Western-blotting patterns (Fig.12) or
the micro-SN titers of the serum (Table 5). Passing
the serum through the 32 Rd col~mn, however, mar~edly
reduced the intensity of the Western-blotting pattern
(Fig.12) and reduced the micro-SN titre of the sera by
half (Table 5~. In an attempt to improve the efficacy
of absorption, day 10 serum was diluted 1:100 prior to
being passed through the 32 ~d column. In this case,
;20~2
- 33 -
the plaque-reduction assay tTable 6) showed that the virus neu-
tralising activity in a 1:20,000 dilution of serum was reduced by
almost 50%.
Synergism between anti-polypeptide sera
Mixing equal volumes of an anti-32 Rd serum, which did not have a
detectable micro-SN titre, with 3 different anti-37 Kd sera, did
not enhance the SN titre of the mixtures more than could be
accounted for by the activity of the anti-37 Kd sera alone
(Table 7). Similarly, mixing an anti-32 Kd serum which had a
micro-SN titre of 320 with the 3 anti-37 Kd sera, resulted in
mixtures with SN titers which could be completely accounted for
by the activity of the anti-32 Kd serum (Table 7).
Passive protection with anti-32 Kd polypePtide serum
The concentration of antibody in 1 to 2ml of anti-32 Kd poly
peptide serum was found to be too low to produce detectable levels
of circulating antibody when injected i.p. into young chickens.
An (NH~)2SO4 precipitate of 20ml of anti-32 Kd polypeptide serum
(Chicken B) was prepared, redissolved in ~ml of PBS, sterile
filtered and lml injected each of 3 chickens of 2 days of age.
These chickens, together with 3 control chickens, were challenged
one date later with 10 CIDso f 002~73, exsanguinated 3 days after
challenge and their bursae and serum assessed by the ELISA. The 3
chickens that received the precipitated antibody had residual
- 33a -
ELISA titres between 160 and 320 and 2 of them had no detectable
viral antigen in their bursae. The 3 control chickens had no
detectable antibody and all had ELISA titres of viral antigen
~128.
~eutralisation of ty~e-1 IBD viruses by chicken
anti-serum specific for the 32 Kd polypeptide
Five S~F chickens of 7 weeks of age were infected with IBD virus
(002/73) and bled on days 10, 11 and 12 post-infection. The 15
individual sera were assessed by Western-blotting, and all con-
tained antibodies specific for the 32 Kd polypeptide of IBD virus.
The sera were pooled and sent to the Central Veterinary Labora-
tory, Weybridge, UK. When assessed by the serum neutralisation
assay, the pooled serum was found to neutralise the type-1 strains
PGB-98, Cu-1 and G-13 in addition to GT-101, but not the type-2
IBD virus strain Ty-89.
- 34 -
Table 1. Passive pro~ection of chickens wiLh serum conLaining anLibodies
specific for Lhe 32k polypep~ide of IBD v;rus (002/73)
Group Antibody titers of Mean ELI~SA titers of t
No. donor serum*
__ _ _ .
viral antigen in residual antibody
_ ELISA S~ bursae in circulation
1 <20 <?0 >256 <20
2 6,620 6,250 <1~477
317,000 >6,250 <11,830
422,300 >6,250 <11,740
525,600 >6,250 <11,930
6>25,600>6,250 <13,340
* Each group of 3 chickens of 2 days of age was injected i.p. ~i~h 1 ml
of serum from 5 different donor chickens obtained 14 days after they
had been infected with IBD virus (Groups 2-6) or antibody negative
serum from an SPF chicken (Group 1). Western blots of immune serum
- shown in Fig.4.
t The chickens were challenged i.o. with 1000 CIDso of IBD virus 1 day
after receiving the serum and then exsanguinated 3 days later. The
bursae were examined by ELISA for the presence of IBD viral antigen
and the corresponding sera assayed for residual specific antibody.
<1 = undiluted bursal homogenate.
2~Z
- 35 -
Table 2. Passive protection of chickells wi~h serum containin~ antibodies
specific for the 32k polypep~ide of I~D virus*
. ._
Mean El ,ISA titers of
Donor Serum ELISA titer No of Virus viral anLigen residual antibody
Type-Number of Donor Serum Chickens Inoculated in bursae in circulation
. __ _ .
SPF <5 3 _ <1 <5
SPF <5 3 + >12$ <5
Day 10 Immuue
6357 77,000 3 + <1 3,900
6362 58,000 3 + <1 1,900
6-366 76,000 2 + <1 3,000
Pool N.T. 2 _ <1 2,200
Day 14 Immune
. ..__
6357 166,000 3 + <1 5~800
6362 78,000 3 + <1 6,600
6366 70,000 2 + <1 3,800
Pool N.T. 2 _ <1 8,000
* Experimental design as per Table 1, excepc that groups of 3 chickens received 2ml
of serum i.p., which was lethal for some chickens and some of the groups remained
unchallenged, but in contact with the challenged chickens. Western-blots of sera
shown in Fig. 7.
N.T. Not tested.
~2szl~4~ .
36
Table 3. Passive protcction of chickens* with IgG and IgM anLlbodies
specific for the 32k polypeptide of IBD virus
.
Donor Titer of antibody t Mean ELISA titer of
Antibody in donol serum viral antigen residual antibody
- Serum No.ELISA Micro-SN in bursal in circulation
.. . , .. __
IgM-1 <40 3,200 >128 N.A.
IgM-2 <40 12,800 >128 N.A.
IgG-1 12,800 6,400 <8 515
IgG-2 25,600 25,600 <8 800
. _ , `
* Groups of 3 chickens of 2 days of age were injected I.P. with IgM or IgG antibody
obtained from 2 donor chickens, 10 days after being infected with IBD virus. The
antibodies, particularly the IgG antibodies, were specific for the 32k polypeptide
of IBD virus (Fig.9 ).
t The chickens wer-e challenged at 3 days of age with 10 I~so of 002/73 and
exsanguinated 3 days later. The bursae were homogenised and assessed by ELISA
for the presence of antigen and the sera assayed for residual IgG antibody.
N.A. Not applicable.
-
~ 2 5~7
37
Table 4. Antibody responses of SPF chickens immunised wiLh Lhe
32k, 37k or 41.5k structural polypepLides of IBD virus
Tlme after ~InmunlsaLion* (weeks~
Antigen Antibody
Preparation Assay
0 3 4 6 7 9
. _
32k polypeptide
Chicken A ELISA <100 <100<100100 300 300
Micro-SN <4 4 4 320 160 320
Chicken B ELISA <100 <1003006001,200 800
Micro-SN <4 4 2561,2801,280 640
. _ .
37k polypeptide
Chicken E ELISA <100 <1008004001,200 1,200
Micro-SN <4 4 ~, 40 40 20
Chicken F ELISA <100 <1003001,2001,6001,600
Micro-SN <4 <4 8 20 40 40
. _ .
41.5k polypeptide
Chicken G ELISA <100 <100674006,40012,8009,600
_ Micro-SN <4 <4 4 40 10 20
Chicken H ELISA <100 <1004001,6001,600 800
Micro-SN <4 <4 <4 20 <10 <20
* Three adult SPF chickens injected i.m. with approximately 50~ of the respective
polypeptides in FCA. Three weeks after the first immunisation they were reinjected
with the polypeptides in FIA. Only 2 of each group of 3 chickens synthesised antibody
detectable by the micro-neutralisation assay.
~5j2C~Z
38 ~
Table 5. The effect of adsorption with the viral polypepLi _s on the
micro-SN titer of antisera from chickens immunised with
. _ _ _ _ _
IBD viral polypeptides, inactivaLed whole virlls vaccine or live virus.
_ . . _
SN-titer post-adsorption with viral polypeptides of mol wt
Antisera Original
SN-titer _ .
32k 37k 41.5k
Anti-32k _ _ _ _ __ .
1,280t 1,280 1,280t 1,280t
B 2,560t 1,280 2,560t 2,560t
- . . _
Anti-37k
_ 32t 32 64t N.T.
F 16t 16 32t N.T.
_ _ . __
Inactivated* 12,800 6,400 12,800 25,600
Vaccine
_ . . _
Live** 51,200 25,600 102,400 102,400
Vaccine _
* Sera obtained 28 days* or 10 days** post-vaccinaLion and were specific for the 32k
** polypeptide by Western-blotting as shown in Fig. 12.
t Western-blots of sera pre and post-adsorption shown in Fig.ll.
N.T. NoL tested.
~252(11~
39 ~
Table 6. The effect of adsorption wlth the viral po]ypeptldes on Lhe
percenta~e plaque-reduction of dilutions of antisera from chickens immunised
with IBD viral polypeptides, inactivated whole virus vaccine or live virus
% p1aque-reduction following
Dilution % plaque- adsorption with viral
Antisera of serum reduction polypeptides of mol w~
_ 32k37k 41.5k
Anti-32k
At 1:2G0 61.5 61.5 92.3 N.T,
Bt 1.200 92.3 88.5 80.8 N.T.
_
Inactivated 1:16,000 69.2 42.3 80.8 61.5
Vaccinet
Live 1:20,000 80.8 80.8 84.6 N.D.
Vaccinet 42.3
.
t Antisera as per Table 5.
* Antiserum diluted 1:100 prior to adsorption.
N.T. Not tested.
,~ZS;2~
--'10 --
Table 7. Micro-SN antibody tlL~rs of 1:1 mixtures of
anti-32k and anti-371c sera
-
Anti-37k sera
.__ . .
Anti-~2k Original SN-titers 40 40 20
. . - . .. _
C <20 20* 80* 20*
A 320 320* 160* 160*
.
* Micro-SN titer of 1:1 mixtures have been doubled for direct comparison with titer of
original sera.
~2~
- 41 -
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~5~1~42
42
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~25~2
43
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~ It will be appreciated that many
modifications and variations may be made to the
particular methods described above by way of
illustration of the present invention, and that the
present invention includes all such modifications
which fall within the scope of the invention as
broadly described above.
