U.S. patent application number 10/156964 was filed with the patent office on 2003-04-10 for ibdv strain for in ovo administration.
Invention is credited to Loon Van, Adriaan Antonius Wilhelmus Maria, Mundt, Egbert.
Application Number | 20030068334 10/156964 |
Document ID | / |
Family ID | 8171620 |
Filed Date | 2003-04-10 |
United States Patent
Application |
20030068334 |
Kind Code |
A1 |
Mundt, Egbert ; et
al. |
April 10, 2003 |
IBDV strain for in ovo administration
Abstract
The present invention provides a novel infectious bursal disease
virus, strain 689 which is suited as vaccine candidate for in ovo
vaccination and is deposited at the European Collection of Cell
Cultures, Salisbury, Wiltshine, United Kingdom under No.
V00012609.
Inventors: |
Mundt, Egbert;
(Millienhagen, DE) ; Loon Van, Adriaan Antonius Wilhelmus
Maria; (Sambeek, NL) |
Correspondence
Address: |
INTERVET INC
405 STATE STREET
PO BOX 318
MILLSBORO
DE
19966
US
|
Family ID: |
8171620 |
Appl. No.: |
10/156964 |
Filed: |
May 29, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10156964 |
May 29, 2002 |
|
|
|
09878083 |
Jun 8, 2001 |
|
|
|
Current U.S.
Class: |
424/204.1 ;
424/199.1; 435/5; 435/7.1 |
Current CPC
Class: |
A61K 2039/552 20130101;
A61K 2039/55 20130101; A61P 31/12 20180101; A61P 37/04 20180101;
C12N 7/00 20130101; C12N 2720/10034 20130101; C12N 2720/10021
20130101; A61K 39/12 20130101 |
Class at
Publication: |
424/204.1 ;
424/199.1; 435/5; 435/7.1 |
International
Class: |
C12Q 001/70; G01N
033/53; A61K 039/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 9, 2000 |
EP |
00202042.8 |
Claims
1. A virus of an infectious bursal disease virus strain, a sample
of which is deposited at the European Collection of Cell Cultures,
Salisbury, Wiltshine, SP4 OJG, United Kingdom under accession No.
V00012609.
2. A virus according to claim 1, characterised in that the virus is
in a live form.
3. A vaccine against infectious busal disease, characterised in
that it comprises a virus according to claims 1 or 2 and a
pharmaceutically acceptable carrier or a diluent.
4. A vaccine according to claim 3, characterised in that the
vaccine comprises an effective dose per egg of 10.sup.1-10.sup.6
TCID.sub.50, (Tissue Culture Infectious Dose 50) preferably
10.sup.2-10.sup.5 TCID.sub.50.
5. A vaccine according to claim 3 or 4, characterised in that the
vaccine additionally comprises an embryo-safe vaccine strain of
another avian pathogen.
6. A method for the preparation of the virus according to claim 1
or 2, characterised in that the virus is cultured on a suitable
substrate and harvested from the substrate.
7. A method for preparation of the vaccine according to claim 3-5,
characterised in that a virus according to claim 1 or 2 is mixed
with a pharmaceutical suitable carrier or a diluent.
8. Use of the virus according to claim 1 or 2 for the manufacture
of a vaccine for the protection of birds against infectious bursal
disease for in ovo administration.
Description
[0001] The present invention is concerned with a virus of an
infectious bursal disease virus (IBDV) strain, a vaccine against
infectious bursal disease (IBD), a method for the preparation of
the virus, a method for the preparation of a vaccine and the use of
the virus for the manufacture of a vaccine.
[0002] Infectious bursal disease virus (IBDV), a member of the
birnaviridae family, is endemic in many poultry producing areas.
For IBDV, two serotypes exist, serotype 1 and 2. The two serotypes
may be differentiated by virus neutralisation (VN) test.
Furthermore, subtypes of serotype 1 have been isolated. These so
called "variant" viruses of serotype 1 can be identified by
cross-neutralisation test (P. D. Lukert and Y. M. Saif, Diseases of
Poultry, 9.sup.th edition, Wolfe Publishing Ltd., Chapter 28,
648-663, 1991) a panel of monoclonal antibodies (Snyder, D. B. et
al, Arch. Virol.127, 89-101, 1992) or RT-PCT (Jackwood, D. J.
Proceedings of the International Symposium on Infectious Bursal
Disease and Chicken Anaemia, Rauischholzhausen, Germany, 155-161,
1994). Some of these subtypes of serotype 1 of IBDV have been
described in literature for example: Classical, Variant-E, GLS,
RS593 and DS326 strains (Van Loon et al, Proceedings of the
International Symposium on Infectious Bursal Disease and Chicken
Infectious Anaemia, Rauischholzhausen, Germany, 179-187, 1994).
[0003] IBDV causes infectious bursal disease (IBD) also called
Gumboro disease, an acute, highly contagious disease in chickens
that has lymphoid tissue as its primary target with a selective
tropism for cells of the bursa of Fabricius. The morbidity rate in
susceptible flocks is high with rapid weight loss and moderate
mortality. Chickens that recover from the disease may have immune
deficiencies because of the destruction of the bursa of Fabricius,
which is essential to the defence mechanism of the chicken. The
IBDV causes severe immunosuppression in chickens younger than 3
weeks of age and induces bursal lesions in chicken up to 3 months
old.
[0004] For many years the disease could be prevented by inducing
high levels of antibodies in breeder flocks by the application of
an inactivated vaccine, to chickens that had been primed with
attenuated live IBDV vaccine. This has kept economic losses caused
by IBD to a minimum. Maternal antibodies in chickens derived from
vaccinated breeders prevent early infection with IBDV and diminish
problems associated with immunosuppression. In addition, attenuated
live vaccines have also been used successfully in commercial
chicken flocks after maternal antibodies had declined.
[0005] Recently, very virulent strains of IBDV have caused
outbreaks of disease with high mortality in Europe. The current
vaccination program failed to protect chickens sufficiently.
[0006] Live classical IBDV vaccines are usually administered to
hatched chickens through drinking water, aerosol (coarse spray) or
intra-ocular (eye drops). These methods of post-hatch
administration have some disadvantages, most importantly they are
expensive because of the labour needed for their administration in
particular in large broiler flocks.
[0007] The use of vaccines as embryo vaccines (so called in ovo
vaccines) has been suggested previously (Sharma et al; Avian
diseases 29, 1155-1169, 1985).
[0008] Embryo vaccination (in ovo vaccination) in principle could
be advantageous due to the early age of resistance to the specific
disease and the administration of a uniform dose of vaccine into
each egg using semiautomatic machines with multiple injection
heads.
[0009] Usually conventional vaccines for post-hatch vaccination of
birds can not be used for in ovo vaccination. Late stage embryos
are highly susceptible to infection with most vaccine viruses
examined including those vaccine viruses, which can be safely used
in hatched chicken. As it is shown in Example 1 the commercially
available IBDV vaccines for intra-ocular, coarse spray and drinking
water administration in hatched chicken are not suitable for
administration in ovo.
[0010] An IBDV vaccine, for in ovo administration was made
available as, so called "complex vaccine". This vaccine is composed
of a mixture of an live IBD virus and chicken serum containing a
viral neutralising factor (VNF) in order to decrease the
pathogenicity of the live virus. (Whifill et al: Proceedings of XIX
World's Poultry Congress, Amsterdam, The Netherlands, 453-455,
1992). Other IBDV complex vaccines are commercially available under
the trademark Bursaplex.RTM. (Embrex Inc.) and Bursamune.RTM.(Fort
Dodge).
[0011] The commercially available complex vaccines have the
disadvantage of using a high virulent pathogenic IBDV strain (so
called hot strain) which reduces the hatchability of the vaccinated
eggs, if used alone and not complexed to a serum containing a virus
neutralising factor. The addition of such chicken serum to the live
IBD virus strain however, results in higher manufacturing costs for
the IBDV vaccine compared to those containing live IBDV strains
alone.
[0012] The safety and efficacy of a live IBDV complex vaccine and
of a live IBDV vaccine was tested after in ovo administration by
Jungbck et at; (Abstracts of XI International Congress of the World
Veterinary Poultry Association, Budapest Hungary, 191ff, 1997). The
results show, that the efficacy requirements of the European
Pharmacopoea were not fulfilled by the tested vaccines after in ovo
administration, because more than 10% of the in ovo vaccinated
chicken showed strain severe lesions of the bursa of Fabricius
after challenge with a pathogenic IBDV strain.
[0013] It is therefore the aim of the current invention to provide
a safe and efficacious mild strain of IBDV that can be administered
in ovo, in the absence of a serum containing virus neutralising
factor, without having a negative impact on hatchability of the
vaccinated egg and furthermore, to provide a cost effective
manufacturing process for a vaccine for protection against IBD.
[0014] The present invention provides a virus of an IBDV strain
designated as strain 689, a sample of which is deposited at the
European Collection of Cell Culture, (ECACC) of Salisbury,
Wiltshire, SP4 OJG, United Kingdom under accession No.V00012609.
The IBD virus strain 689 according to the invention is distinct
from the existing IBDV (vaccine) strains, is safe for in ovo
administration, and is able to induce a solid immune response in
chickens.
[0015] A virus of the IBDV strain 689 refers to viruses derived
from a virus as deposited, as well as those progeny viruses derived
from a virus as deposited, and obtainable for example, by serial
passaging e.g. in embryonated eggs or in cell culture.
[0016] IBDV strain 689 was isolated from a chicken bursa of
Fabricius from the field showing mild signs of IBDV. The bursa of
Fabricius was homogenised and put on chicken embryo fibroblast
cells (CEF). After 5 days of incubation a cythopathogen effect
(CPE) was visible and characterised as IBDV with poly- and
monoclonal antibodies. A second passage was conducted on CEF and
this material (passage level 2) was used in Example 3. The IBDV
strain 689 was further plaque purified 3 times and a pre seed was
prepared followed by a master seed (passage level 7). The master
seed virus was used in the Example 2.
[0017] The IBDV strain 689 was identified by means of ELISA using
monoclonal antibodies (Moab) according to Van Loon et al (Van Loon,
A. A. W. M., D. Lutticken and D. B. Snyder. Rapid quantification of
infectious bursal disease (IBD) challenge, field or vaccine virus
strains. International symposium on infectious bursal disease and
chicken infectious anaemia, Rauischholzhausen, Germany, 179-187,
1994).
[0018] As can be seen from Example 4, the IBDV strain 689 belongs
together with the commercially available IBDV vaccine strain
Nobilis Gumboro strain D78 (Intervet International, Boxmeer, The
Netherlands) to the classical type of IBDV, because both strains
have an identical reaction pattern with the different monoclonal
antibodies and are both able to replicate on primary CEF inducing a
CPE.
[0019] The Examples 2 and 3 show that IBDV strain 689 is able to
induce a protective immune response to a challenge with virulent
classical IBDV and variant-E IBDV without causing severe lesions in
the bursa of Fabricius.
[0020] The virus according to the invention is provided in a live
and inactivated form, preferably in a live form.
[0021] Another aspect of the invention is a vaccine against IBD,
characterised in that it comprises a virus as defined above and a
pharmaceutically acceptable carrier or diluent and a method for the
preparation of the vaccine.
[0022] The vaccine containing the live virus can be prepared and
marketed in a form of a suspension or in a lyophilised form and
additionally contains a pharmaceutically acceptable carrier or
diluent customary for such compositions. Carriers include
stabilisers, preservatives and buffers. Suitable stabilisers are,
for example SPGA carbohydrates (such as sorbitol, mannitol, starch,
sucrose, dextran, glutamate or glucose), proteins (such as dried
milk serum, albumin, or casein) or degradation products thereof.
Suitable buffers are for example alkali metal phosphates. Suitable
preservatives are thimerosal, merthiolate and gentamicin. Diluents
include water, aqueous buffer (such as buffered saline) and polyols
(such as glycerol). If desired the live vaccine according to the
invention may contain an adjuvant. Examples of suitable compounds
and compositions with adjuvant activity are the same as mentioned
below.
[0023] In another aspect of the present invention a vaccine is
provided comprising the IBDV strain 689 in an inactivated form. The
major advantage of an inactivated vaccine is the high level of
protective antibodies of long duration that can be achieved. A
vaccine containing the inactivated IBDV virus can, for example
comprise one or more of the, above mentioned pharmaceutically
acceptable carriers or diluents suited for this purpose.
Preferably, an inactivated vaccine according to the invention
comprises one or more compounds with adjuvant activity. Suitable
compounds or compositions for this purpose include aluminium
hydroxide, -phosphate or -oxide, oil-in-water or water-in-oil
emulsion based on, for example a mineral oil, such as Bayol.RTM. or
Marcol.RTM. or a vegetable oil such as vitamin E acetate and
saponins.
[0024] The vaccine according to the present invention can be
administered to birds by any suitable means. Exemplary means for
post hatch administration are the oral administration (e.g. in feed
or drinking water), administration via parenteral routes e.g. by
intramuscular injection or via eye drop forms or spray vaccination
(aerosol). Inactivated vaccines are usually administered
parenterally, e.g. intramuscularly or subcutaneously.
[0025] The vaccine according to the invention can also be
administered in ovo, as described in U.S. Pat. No. 4,458,630. The
in ovo administration of the vaccine is most preferred.
[0026] The in ovo administration of the vaccine involves the
administration of the vaccine to an avian embryo while contained in
the egg. The vaccine may be administered to any suitable
compartment of the egg (e.g. allantois fluid, yolk sac, amnion, air
cell or into the embryo) as described in the art (Sharma; Am. J.
Vet. Res. 45, 1619-1623, 1984). Preferably the vaccine is
administered below the shell (aircell) membrane and chorioallantoic
membrane. Usually the vaccine is injected into embryonated eggs
during late stages of the embryonation, generally during the final
quarter of the incubation period (day 15-21), more preferably the
eggs are treated between the 15.-19. day of incubation and most
preferably at day 18 of the incubation period.
[0027] The mechanism of injection of the incubated eggs is not
particularly critical provided that it does not unduly damage
tissue and organs of the embryo or the extra-embryonic membranes
surrounding it so that the treatment will not decrease hatchability
or causes infection. The vaccine may be administered to the egg by
any means, which transports the compound through the shell. The
preferred method of administration is, however, by injection.
Depending on the precise stage of development and position of the
embryo the needle with a defined length will terminate in different
parts of the egg. For example, a small hole is pierced with a
needle (1-1 1/2 inch, about 22 gauge) attached to a syringe in the
shell of the large end of the egg and the vaccine is injected below
the inner shell membrane and the chorioallantoic membrane. A pilot
hole may be punched or drilled through the shell prior to insertion
of the needle to prevent damaging or dulling of the needle. In
addition if desired, the egg can be sealed e.g. by a sealing
apparatus with substantially bacteria-impermeable sealing material
such as wax or the like to prevent subsequent entry of undesirable
bacteria.
[0028] Subsequently, the vaccinated embryonated eggs are
transferred to an incubator to hatch (U.S. Pat. No. 4,458,630, WO
98/56413 and WO 95/35121). Preferably, the whole embryo vaccination
process is carried out using high-speed automated vaccination
systems, such as the commercially available INOVOJECT.RTM.. Such
devices are also disclosed in U.S. Pat. Nos. 4,681,063 and
4,903,635, 4,040,388, 4,469,047 and 4,593,646.
[0029] The vaccine according to the invention comprises an
effective dosage of the IBDV 689 virus as the active component,
i.e. an amount of immunising IBDV material that will induce
immunity in the vaccinated birds against challenge by a virulent
IBDV virus. Immunity is defined herein, as the induction of a
significant higher level of protection in a population of birds,
after vaccination, compared to an unvaccinated group.
[0030] Depending on the inoculum administered, the site and manner
of administration, the species, age and condition of the subject,
the virus dose will range from 10.sup.1-10.sup.6 TCID.sub.50 (i.e.
one Tissue Culture Infectious Dose 50 is the dose at which 50% of
the infected tissue cultures show CPE), more preferably in a dose
of 10.sup.2-10.sup.5 TCID.sub.50.
[0031] Another aspect of the invention is a vaccine as described
above, characterised in that the vaccine additionally comprises an
embryo-safe vaccine strain of another avian pathogen.
[0032] The combined administration of more than one vaccine strain
is advantageous for economical reasons, because it requires fewer
vaccine inoculations in the egg. Moreover, the fewer an inoculum is
introduced into an egg, the less the risk of contaminating the eggs
exist.
[0033] With an embryo safe vaccine strain is meant a live vaccine
strain which, if inoculated into SPF eggs at incubation day 18,
results in the hatchability of the eggs of at least 70%, preferably
at least 80%.
[0034] Preferably, the combination vaccine additionally comprises
one or more embryo-safe vaccine strains of Mareks disease virus
(MDV) infectious bronchitis virus (IBV), Newcastle disease (NDV),
egg drop syndrome (EDS) virus, turkey rhinotracheitis virus (TRTV)
or reovirus.
[0035] Although, the IBDV vaccine according to the present
invention may be used effectively in chickens, also other poultry
as turkeys, guinea fowl and partridges may be successfully
vaccinated with the vaccine. Chickens include broilers,
reproduction stock and laying stock.
[0036] Another aspect of the invention is a method for the
preparation of the as disclosed above, characterised in that the
virus is cultured on a suitable substrate and harvested from the
substrate. IBD virus of the 689 strain according to the invention
can be obtained by conventional methods known in the art.
[0037] Briefly, a susceptible substrate is inoculated with IBDV
strain 689 and propagated until the virus replicated to a desired
titre after which IBDV containing material is harvested. Every
substrate which is able to support the replication of IBDV virus
can be used in the present invention, including embryonated chicken
eggs, primary (avian) cell cultures, such as embryo fibroblast
cells (CEF) or chicken kidney cells (CK), or mammalian cell lines
such as VERO cell line or baby hamster kidney (BHK) cell lines.
[0038] Preferably the IBDV strain 689 is propagated in primary
chicken embryo fibroblasts (CEF).
[0039] More in particular, primary chicken embryo fibroblasts (CEF)
cells can be prepared at a final concentration of
2.times.10.sup.6/ml. The cells are cultured in Eagles minimum
essential medium containing 5% foetal calf serum. To 15 ml of this
cell suspension 0.1 ml IBDV strain 689, obtained from a
plaque--purified clone, which was dissolved in 1 ml, can be added.
After incubation for 3-6 days in a high humidity incubator at
37.degree. C., the virus can be harvested.
[0040] Another aspect of the invention is the use of the virus
according to the invention, for the manufacture of a vaccine for
the protection of birds against IBD for in ovo administration.
EXAMPLE 1
Efficacy and Safety of Commercially Available IBDV Vaccine Strains
for in ovo Administration
[0041] In these studies safety and efficacy of the commercially
available IBDV vaccine strains Nobilis Gumboro strain D78 (Nobilis
D78) and Nobilis Gumboro strain PBG98 (Nobilis PBG98) of Intervet
International BV (Boxmeer, The Netherlands) for in ovo
administration were investigated.
[0042] Material and Methods
[0043] The commercially available vaccines were administered at day
18 of incubation at different dosages per egg. Parameters
investigated were hatchability, bursa of Fabricius lesions score
and the survival after challenge with the virulent classical IBDV
strain F52/70.
[0044] The hatchability was investigated by calculation of the
relative hatchability (hatchability of test IBDV strains compared
with the hatchability of none-treated eggs). The score of the bursa
of Fabricius lesions (lymphocytic depletion in the follicles) was
determined after microscopically examination of the HE stained
bursa: 0 (no lesions), 1 (0-20% depletion), 2 (20-40% depletion), 3
(40-60% depletion), 4 (60-80% depletion) and 5 (severe lesions,
80-100% depletion).
[0045] Results
[0046] The results are summarised in Table 1.
[0047] Nobilis PBG98 has a negative influence on hatchability at
the protective dose levels of >90%.
[0048] At 15 days of age, the average bursal lesion score for eggs
injected with a dose of 5, 6 or 7 log10 TCID.sub.50/egg of IBDV
strain PBG98, was 0, 0, 0, respectively.
[0049] Nobilis D78 has a negative influence on bursal lesions. At
15 days old, the average bursal lesion score of animals vaccinated
with a dose of 4.3, 5.3 or 6.3 log10 TCID50/egg of Nobilis D78
were, 3.4, 4.7 and 5.0, respectively. These results indicate that
IBDV strain Nobilis D78 can induce severe (complete lymphocytic
depletion) lesions in the bursa of Fabricius after in ovo
vaccination.
1TABLE 1 Overview of in ovo experiments with the commercial vaccine
Nobilis D78 and Nobilis PBG98. SPF birds MDA + birds Dose Hatch @ %
Survival after Dose Hatch Strain (TCID50/egg) (%) challenge
(TCID50/egg) (%) D78 4.3 96 100 Field 100 5.3 98 100 6.3 95 100
PBG98 2 98 50 Field 102 3 90 ND 4 90 100 5 77 100 6 82 100 7 51 100
PBS 98 50 100 -- 100 20 @ - relative hatchability PBS - phosphate
buffered saline
[0050] Conclusion
[0051] The commercially available IBDV vaccine strains vaccines
Nobilis Gumboro strain PBG98 and Nobilis Gumboro strain D78 are not
suitable for in ovo administration. Nobilis D78 induces severe
lesion in the bursa of Fabricius after in ovo administration and
Nobilis PGB98 has a negative influence on hatchablity at the
protective dose levels of >90%.
EXAMPLE 2
Efficacy and Safety of the IBDV 689 Strain After in ovo
Administration and Challenge with a Virulent IBDV Variant-E
Strain
[0052] The effect of the different vaccines is assessed by
measurement of the serological response and resistance to challenge
obtained from a Delaware Variant-E strain at the age of 14 days
old.
[0053] Material and Methods
[0054] The vaccines investigated were the IBDV strain 689 and the
commercial classical IBDV vaccine Nobilis Gumboro strain D78
(Nobilis D78). The IBDV strain 689, 10.sup.5.3 TCID.sub.50/egg, was
applied via the in ovo route. Nobilis D78, 10.sup.3.5
TCID.sub.50/egg was applied via the in ovo route.
[0055] The presence of IBDV in the bursa of Fabricius and
microscopic lesions in the bursa of Fabricius of 3 animals per
group were investigated and the bursa lesion score determined as
described before, 3 and 17 days after vaccination and 3 and 10 days
after challenge.
[0056] Efficacy: The protection against challenge obtained from a
virulent IBDV strain variant-E, at the age of 14 days old was
determined. The serological response against IBDV was determined
with the Virus Neutralisation (VN)-test, 14 days after vaccination.
A classical IBDV strain was used as VN-IBD-virus.
[0057] Results
[0058] Bursa of Fabricius lesions: As shown in Table 2, IBDV strain
689 does only induce mild lesions after vaccination. Nobilis D78
induces moderate to severe lesions after vaccination. In 4 out of
12 animals (33%) vaccinated with Nobilis D78, complete lymphocytic
depletion was observed 10 days after challenge. Individual data
showed that animals vaccinated with both vaccines yielded complete
protection against challenge.
2TABLE 2 Average bursal lesion score Days after vaccination Days
after challenge Virus 3 17 3 10 IBDV Strain 689 1.7 1.7 1.0C 0.6
Nobilis D78 4.3 3.7 1.72C 2.8 None vaccinated 5.0A controls C =
chronic lesions; A = acute lesions
[0059] Serological response against IBDV: It can be seen in Table
3, that IBDV strain 689 and Nobilis D78 induced good levels of
antibodies against classical IBDV.
3TABLE 3 Serological response 17 days after vaccination Virus
Classical VN virus IBDV Strain 689 7.1 .+-. 2.2 Nobilis D78 0.8
.+-. 2.4 None vaccin. controls <1.0 .+-. 0.0 VN-titre is
expressed as log2 of the dilution.
[0060] Detection of IBDV antigen in the bursa of Fabricius with an
ELISA systems 3 and 17 days after vaccination and three days after
challenge: As shown in Table 4, the IBDV strain 689 could be
detected in 2 out of 3 chickens three days after vaccination.
Seventeen days after vaccination no viral antigen could be
detected. No virus could be detected three days after challenge
indicating that the animals were protected against challenge.
Nobilis D78 virus could be isolated from all animals, three days
after vaccination.
4TABLE 4 Presence of IBDV in the bursa of Fabricius. Days after
vaccination Days after challenge Virus 3 17 3 % protection* IBDV
strain 689 2/3 0/3 0/3 100 Nobilis D78 3/3 0/3 0/3 100 None vaccin.
controls 6/6 0 *Number of positive animals with viral antigen
present per total number investigated.
[0061] Conclusions
[0062] The IBDV strain 689 is very mild after in ovo
administration. In contrast, after in ovo administration of the
commercial Nobilis Gumboro D78 strain some birds showed complete
lymphocytic depletion even when a low dose (3.5 log10/egg) was
applied.
[0063] IBDV strain 689 is able to induce a serological response and
complete protection against challenge with a virulent IBDV
variant-E virus.
EXAMPLE 3
Efficacy and Safety of the IBDV 689 Strain After in ovo
Administration and Challenge with a Classical Virulent IBDV
Strain
[0064] Efficacy of the IBDV strain 689 is assessed by measurement
of the serological response and resistance to challenge obtained
from administering the classical virulent IBDV strain F52/70 at 14
days after hatching.
[0065] Material and Methods
[0066] Eggs were divided in 4 groups of 35 eggs each. On day 18 of
incubation, three groups were vaccinated with IBDV strain 689 in
different dosage per egg. The negative control was injected with
diluent only.
[0067] One day after hatching the bursa of Fabricius of 5 chickens
per group was examined for lesions and the presence of viral
antigen.
[0068] Fourteen days after hatching, serological response of all
animals was investigated and the bursa of Fabricius was examined
for lesions and the presence of viral antigen. For investigation of
the serological response a classical IBDV strain was used as Virus
Neutralisation-IBDV-virus.
[0069] All animals are challenged fourteen days after hatching with
the classical virulent IBDV strain F52/70 via eye drop
administration.
[0070] At three days after challenge the bursa of Fabricius of 5
chickens per group was examined for lesions and the presence of
viral antigen. Ten days after challenge the bursa of Fabricius of
all remaining animals was examined for microscopic lesions. The
relative hatchability was investigated.
[0071] Results:
[0072] Hatchability: The relative hatchabilty (hatchability
compared to none-infected eggs) for eggs vaccinated with <1.3,
2.4 or 4.7 log10 TCID50/egg were: 116, 93 and 89%, respectively.
The in ovo vaccination had no negative effect on hatchability.
[0073] Bursa of Fabricius lesions: As shown in Table 5, IBDV strain
689 does only induce mild lesions after vaccination. This confirms
the mild nature of the IBDV strain 689. Individual data showed that
all vaccinated animals were completely protected.
5TABLE 5 Average bursal lesion score in the bursa of Fabricius 3
and 14 days after vaccination and 3 and 10 days after challenge
IBDV strain 689 Days after vaccination Days after challenge Dose
(log 10 TCID50/egg) 3 17 3 10 <1.3 0 1.0 1.0C 0.8 2.4 0.8 1.6
0.8 1.0 47 2.2 1.4 1.8C 2.0 None vaccinated controls 0 0 5.0A 5.0 C
= chronic lesions; A = acute lesions
[0074] Serological response against IBDV: As shown in Table 6,
seventeen days after vaccination, all vaccinated animals averaged
between 7.3 and 8.7 log2/group, which are good serological
responses against classical IBDV.
6TABLE 6 Serological response 17 days after vaccination IBDV strain
689 Dose (log10 TCID50/egg) Classical VN virus <1.3 7.3 .+-. 2.1
2.4 8.7 .+-. 1.4 4.7 8.2 .+-. 1.7 None vaccinated controls <4.0
.+-. 0.0 VN-titre is expressed as log2 of the dilution.
[0075] Detection of IBDV antigen in the bursa of Fabricius with an
ELISA systems three and seventeen days after vaccination and three
days after challenge:
[0076] As shown in Table 7, three days after vaccination, vaccine
virus could be isolated from 2 out of 5 and 4 out of 5 birds
vaccinated with a dose of 2.4 or 4.7 log10 TCID.sub.50/egg,
respectively. No vaccine virus could be detected in animals
vaccinated with a dose of <1.3 log10 TCID.sub.50/egg. No vaccine
virus could be detected in any of the vaccinated animals just
before challenge.
[0077] No virus could be detected in any of the vaccinated animals,
three days after challenge indicating that all animals were
protected against challenge. In contrast all animals in the
none-vaccinated control group contained viral antigen three days
after challenge.
7TABLE 7 Presence of IBDV in the bursa of Fabricius. IBDV strain
689 Days after vaccination Days after challenge Dose (log10
TCID50/egg) 3 17 3 % protection* <1.3 0/5 0/5 0/5 100 2.4 2/5
0/5 0/5 100 4.7 4/5 0/5 0/5 100 None vaccinated controls 5/5 0
*Number of positive animals with viral antigen present per total
number investigated.
[0078] Conclusions
[0079] IBDV Strain 689 is very mild. The IBDV-strain 689 is able to
induce a serological response and complete protection against the
classical virulent IBDV strain F52/70.
EXAMPLE 4
Virus Identification
[0080] Identification of IBDV strains by means of monoclonal
antibody panel test
[0081] Material and Methods:
[0082] Chicken embryo fibroblasts in micro titre plates were
infected with different IBDV strains and are treated after
incubation for three to five days at 37.degree. C. with different
monoclonal antibodies. The binding of the monoclonal antibodies to
the different IBDV strains is visualised by fluorescence labelled
conjugate (goat-anti-mouse).
[0083] Results:
[0084] As can be seen in Table 8, IBDV strain 689 and the classical
commercial vaccine strain Nobilis D78 have an identical reaction
pattern with the different monoclonal antibodies (Moab).
8TABLE 8 Panel pattern of different IBDV viruses with different
Moab. Virus/Moab B29 8 R63 BK9 67 57 B69 IBDV Strain 689 + + + - -
- + Nobilis D78 + + + - - - + Control IBDV strains: Classical + + +
- - - + variant-E + + + + + - - GLS + + - - - + - + epitope present
on virus, - epitope not present on virus.
[0085] Growth on Chicken Embryo fibroblasts (CEF): As can be seen
in Table 9, IBDV strain 689 and Nobilis D78 are both able to
replicate on CEF inducing a CPE.
9TABLE 9 Ability to grow on CEF and inducing specific IBDV-CPE.
Virus CEF growth IBDV Strain 689 + Nobilis D78 + + = does induce
CPE on CEF; - = does not cause CPE on CE.
[0086] Conclusion:
[0087] IBDV strain 689 grows on CEF and belongs to the classical
type of IBDV.
* * * * *