01WApplied Poultry Science, Inc
J. J. GIAMBRONE2and T DORMITORIO
Depamnent of Poultry Science, Auburn University,AL 36849-5416
Phone: (334) 8442642
F M : (334) 844-2641
Degariment ofAvian Medicine, University of Georgia,Athens, GA 30601
IGI Vineland, Kneland, NJ 08360
Primary Audience: Breeder and Broiler Managers, Veterinarians, Poultry
Disease Experts and Vaccine Manufacturers
1 Alabama Agricultural Experiment Station Journal No. 12-965267
2 To whom correspondence should be addressed
GIAMBRONE et al. 363
Challenge results were determined using both
DESCRIPTION bursae size and microscopic pathology. How-
Infectious bursal disease virus (1BDV)- ever, we did not have access to the broiler
induced immunosuppression continues to be performance data as did the prior study. In
a problem in commercial broilers, resulting order to avoid promoting one product over
in increased condemnations, above average another, we did not intend to determine which
mortality, and suboptimal feed conversion, vaccine or program is the best, but rather to
and weight gains [ ] Antigenic variants of
l. determined whether current vaccines and pro-
IBDV and/or poor broiler breeder pullet vac- grams commonly used in the industry would
cination techniques plague the U.S. industry, protect against newly isolated variants of
resulting in increased susceptibility to sub- IBDV and if the new ELISA system containing
clinical IBDV-induced immunosuppression both STD and bursal-derived variant IBDV
. Recent studies [3, 41 have shown that antigens would correlate with resistance to
IBDV variants predominate in U.S. broiler- challenge at 2 wk of age with the variant IBDV
producing areas. Variant IBDV have been re-
cently isolated from Georgia [q, Mississippi AEI S
MTRL A AND METHODS
[l], and Arkansas . Maternal immunity
derived from breeder vaccination represents Chickens were challenged by eye and
the first line of defense against early IBDV nasal instillation with ldID50 of the serologic
infections. Pullet vaccination success is standard challenge virus from the Animal
based on serologicprofiling of the hens during Plant and Health Inspection Service (APHIS)
egg production. However, the two most in Ames, IA or the Delaware antigenic
commonly used commercial enzyme-linked variant E virus .
immunosorbent assay (ELISA) kits use a Broilers were from commercial breeder
serologic standard antigen as the indicator. flocks from Arkansas, Alabama, Georgia,
Antibody titers using this kit do not correlate Mississippi, and North Carolina. Breeder
well with broiler flock performance [q. An- flocks ranged in age from 30 to 50 wk when the
eggs were taken and hatched. Al breeders
other means of determining the susceptibility were vaccinated as pullets with two live and
of progeny to IBDV infections is to measure two inactivated IBDV vaccines. The vaccines
the size of the bursae during the grow out. were from various commercial U.S. manufac-
However, there are numerous infectious and turers and the inactivatedvaccines contained
noninfectious agents which can alter the sized both serologically standard and variant E
of the bursae [11. IBDV The first live vaccine contained an in-
One researcher  has developed an termediate STD IBDV and was given during
IBDV progeny challenge model for determin- the first 2 wk of age. The second live vaccine
ing the success of breeder hen vaccination contained an intermediate plus vaccine (less
programs. Using broiler progeny from the attenuated and more virulent than the first
Delmarva peninsula, he has determined that vaccine) and was given during 6 to 10 wk of
there is a direct correlation between resistance age. The first inactivated vaccine was given
to the Delaware variant E virus challenge at between 10 and 14 wk of age and the second
2 wk of age and performance in the field. The l
between 18 and 20 wk of age. Al inactivated
bursa weight:body weight ratio was correlated vaccines contained at least 50% bursal-de-
with resistance to IBDV infection. The greater rived antigen.
the ratio the more resistant the bird was to Vaccines containing bursal-derived anti-
challenge. This very important field study gen have been shown to be more protective
initiated our current experiments. than antigens from cell culture because IBDV
The purpose of these experimentswas to propagated in cell culture results in many non-
implement this IBDV challenge program [ I complete viral particles which may induce
Auburn University and to determine the efi- antibodies which are non-neutralizing. Since
cacy of broiler breeder vaccination programs the authors were not present during the pullet
in commercial flocks around the southeastern vaccinations, we can not comment as to the
U.S. using newly isolated antigenic variants effectiveness of vaccine administration. The
from the southeastern U.S. and a new ELISA commercial vaccine names for each breeder
kit containing both STD and variant IBDV flock were known, but were not revealed to
364 BROILER IMMUNE STATUS
avoid commercialism. The objective of these weighed. Bursae weight:body weight ratios
experiments was not to determine which vac- were determined for each of the challenge
cine or program was the best, but rather to groups with chicks from each of the sixbreeder
determine whether current vaccines and pro- flocks. All bursae were placed in formalin and
grams commonly used in the industry would processed for microscopic observations using
protect against newly isolated variant IBDV, Hematoxylin and Eosin staining. Bursae were
and if the new ELISA system containing both scored from 0 to 4 based on increasing severity
STD and variant IBDV antigens would corre- of lesions .
late with resistance to challenge at 2 wk of age Bursae weight:body weight ratios were
with the variant IBDV. analyzed using the Statistical Analysis System
Al broilers were fed a commercial broiler
l [lo]. Bursae weight:body weight ratios two
starter and given feed and water adlibiturn.All standard deviations below the mean of the
were held in modified Horsfall-Bauer isola- unchallenged control were considered not
tion units maintained with filtered air under protected. A percent protection score was de-
negative pressure. termined based on the percentage of birds
Ten buds per flock were bled at 2 wk of within a flock that were protected. Flocks with
age and sera analyzed for antibody against a a percent protection score above 30% were
standard IBDV antigen using a commercial considered adequately protected. This figure
ELISA kit. The kit was from Kirkagaard and was derived from a previously published work
Perry Lab, Inc. (KPL) of Gaithersburg, MD [q. that study, chicks from flocks with per-
and contained an STD (D78) and variant E cent protection scores below 30% had poor
antigen produced from bursal-derived field performance. In the present experiments,
material. This fact is important since most in- we did not have access to broiler flock perfor-
activated vaccines contain variant E produced mance.
in the bursa.
A total of four challenge studies were RESULTS DISCUSSION
done during an 8-month period. Each repre-
Table 1 presents IBDV serologic and
sented chickens from a separate integrator. challenge data, based on bursa:body weight
Progeny from six breeder flocks were tested data (% protection scores) for Experiment 1.
during each experiment. Fifty-five chicks were Data showed that all flocks, except Flock 6,
obtained from each broiler flock for a total of were well protected against the STD
330 birds per experiment. At 2 wk of age, 15 challenge. Results for the IBDV E and GA
chicks from each flock were challenged with variants were similar. All flocks, except
one of the three IBDV's (Groups 1-3) and 10 Flocks 5 and 6, were adequately protected. In
chicks (Group 4) were not challenged. In each Experiment 2, results for the STD and MISS
of the four experiments, Group 1chicks were l
isolates were similar (Table 2). Al flocks,
challenged with the antigenic STD IBDV except Flock 1, were adequately protected
APHIS isolate, and Group 2 with the antigenic against these two isolates. In contrast, only
Delaware E variant . In Experiment 1, Flock 3 was adequately protected against vari-
Group 3  was challenged with the antigenic ant E. In Experiment 3, challenge results for
variant GA isolate [SI. In Experiment 2, the STD and ARK-1 isolates were similar
Group 3 received the variant MISS [l]isolate. (Table 3). All flocks were adequately pro-
In Experiment 3, Group 3 received the variant tected against these isolates. In contrast, only
ARK-1 , and in Experiment 4, Group 3 re- Flock 2 was adequately protected against
ceived the variant ARK-2 IBDV. The ARK-2 variant E. In Table 4, data for Experiment 4
IBDV was isolated in 1997from the Northwest showed that only Flocks 1,2, and 3 were ade-
Arkansas and determined to be variant using quately protected against the STD isolate.
molecular techniques . The 10 control birds Results for the variant E and ARK-2 isolates
per flock were also bled at this time and sera were similar. Only Flock 2 was adequately
measured for antibodies against IBDV using protected against these isolates.
the ELISA. The four groups were kept i n In the interest of brevity, microscopic
separate isolation units. lesion scores were not listed in table form.
At 7 days after challenge, all birds were Results correlated with data calculated from
killed, weighed, and their bursae extracted and the bursa weight:body weight ratios. Results
GIAMBRONE et al. 365
FLOCK # mA VARIANTE GA ELISA~
1 70 66 58 5,391
2 92 92 92 11,687
3 82 90 70 3,633
4 54 86 30 4,657
5 40 ga sa 4,406
6 Wa 2oa laa 6,513
FLOCK # mA VARIANTE IS
1 14a 27" xa 56
2 40 Oa 87 1,502
3 65 54 75 3,514
4 37 Oa 94 4,235
5 53 Wa 100 3,628
6 60 27a 60 6,152
TABLE 3. IBVD challenge results of progeny (Experiment 3)
BGeometricmean enzyme-linked immunosorbent assay.
'Statistically different f o other numbers within the same column.
were also variable for individual flocks and flock ranged from a low of 14 to a high of
IBDV isolates. They ranged from a low of 0 to 16,112. The correlation of variation (CV) was
a high Of 4 Flocks with an average lesion score
. low (less than 10%)for all flocks tested. Flocks
of 2 or less were generally adequately pro- with GMTs of 1,500 or more were always ade-
tected against IBDVas measured by the bursal quately protected against the STD challenge.
weight:body weight ratios. However, correlation between ELISA analysis
Individual geometric mean ELISA anti- and resistance to the variant E isolate was not
body titers (GMT) at 2 wk of age for a broiler consistent. Flocks with antibody titers as high
366 BROILER IMMUNE STATUS
=Serological standard APHIS IBDV.
’Geometric mean enzyme-linkedimmunosorbent assay.
aStatisticallydifferent from other numbers within the same column. I
as 16,112 were poorly protected against this progeny that had lower ELISA titers and were
isolate. Therefore, producers with high titers more susceptible to challenge against the
using the new ELISA system may mistakenly variants than were progeny from younger
believe they their broiler progeny are ade- breeder flocks.
quately protected against antigenic variants. In the present experiments, progeny were
Flocks with titers, below 1,500 were always taken from states which represent nearly 60%
poorly protected against these isolates. Inves- of the U.S. broiler production. Our data con-
tigators from a prior study [Idid not have firmed previous results [I, which showed that
access to this new ELISA system, so we cannot producers from Delmarva were doing a good
compare their ELISA results to ours. job of immunizingtheir flocks against the stan-
IBDV-induced immunosuppression con- dard virus, but significantly less so against the
tinues to cause significant economic losses in variant E. Our data also agreed with previous
the US. broiler industry despite improved work [I, which showed a lack of correlation
vaccine programs. All commercial broiler between ELISA titers and resistance to the
breeder pullets in the U.S. are now receiving variant E virus as well as the newly isolated
combinations of live and inactivated vaccines ARK-2 and GA variants at 2 wk of age. How-
containing both serologicstandard and variant ever, since we did not have access to the broiler
viruses. Recent studies have shown that the performance data, we cannot make assump-
incidence of variant IBDV is increasing tions as to the correlation of ELISA titer and
throughout the U.S. [3,4] and that resistance percent protection scores with broiler flock
to IBDV variant challenge at 2 wk of age cor-
relates with performance in the Delmarva area Viruses have many antigenic sites which
more so than does serologic profiling of breed-
are capable of eliciting an antibody response
ers using commercial ELISA kits [I.
in the host. Some sites elicit neutralizing and
results showed that resistance to the variant E
virus was significantly less than to the standard some non-neutralizing antibodies. The ELISA
virus. That study had access to broiler perfor- does not measure the neutralizing IBDV anti-
mance data not available in the present study. body, and therefore there is not always good
Researchers showed that flocks that had less correlation between ELISA and resistance to
than 30% protection agahst STD and variant IBDV infection. In addition, resistance to
IBDV at 2 wk of age performed poorly i the n IBDV infection may also rely on cell-mediated
field. immunity. However, this new ELISA from
Data presented in the tables in this study KPL was an improvement over previous kits
does not reveal the age of the breeders when since titers of 2-wk-old birds were higher and
the eggs were taken for the progeny challenge CV’s were lower than data generated with
studies. However, with hen age the protection older kits in similar studies. Also, there was
against the variant challenge waned, but was good correlation between the ELISA titer and
relatively constant for the STD challenge. protection against the STD, MISS, and ARK-1
Breeder flocks older than 40 wk produced viruses.
GIAMBRONE et al. 367
Since IBDV infections continue to occur Further studies would also be warranted
in the U.S. and new antigenic variant viruses to correlate resistance to challenge infection
continueto evolve such as recently occurred in with the presence of virus-neutralizing anti-
Georgia [SI, Mississippi [l], and Arkansas , bodies and/or cell-mediated immune response
continual monitoring of IBDV pullet vaccina- against the homologous antigenic variant
virus. Other future studies could also examine
tion programs using a number of serologically whether the use of new live variant vaccines,
different IBDV's seems warranted. In addi- inactivated vaccines containing a higher per-
tion, the MISS and ARK1 variants are both centage of bursal derived material in pullet
antigenically and pathologically (cause in- flocks, and/or a mid lay boost with an inacti-
flammation of the bursae) distinct from the vated vaccine would provide better protection
Delaware E, GA, and ARK-2 variants. against variant IBDV's.
1. Producers continue to do a good job in vaccinating broiler breeder pullets to prevent
infection against serologic standard IBDV in the progeny, but results are variable against
the newly evolving variant IBDV's.
2. The vaccines and programs tested in t i study were effective in controlling the five
antigenicvariant IBDV's we examined.
3. The new ELISA test containing both STD- and bursal-derived variant E antigen correlates
well with resistance to protection against the STD IBDV and with some but not all antigenic
1. Giambrone, JJ., 1986. Isolation of variant infec- 7. Odor, E, 1995. U ate from the lab on IBDV
tious bursal disease viruses from commercial broiler monitoring. Pages 1 6 1 E n : Proc. IBD International
chickens. Poultry Sci. 6547. (Abs). Summit, Athens, GA.
2. Jackwood, D.H. and Y.M. S 4 1990. Antigenic
a 8. RosenFrger, J.K, S.S. cloud, and A. Metq 1987.
diversity of infectious bursal disease virus. Avian Dis. Use of infectious virus variant vaccines in broiler and
31:766-70. broiler breeders. Pages 105-106 in: Proc. 36th Western
Poultry Dis. Conf., Davis, CA.
3. Jackwood,DJ. and S.E Sommer, 1999. Restriction
fragment len h polymorphism in the W 2 ene of infec- 9. Skeeles, J.K., P.D. Lukert,EV. DeBuysscher, OJ.
tious bursal &ase viruses.Avian Dis. 41:d27437. .
Fletcher, and 1 Brown, 1979. Infectious bursal disease
4. Snyder, D.B., D.P. Lann,P.K. Savage, F.S. Yancey, virus infections. 1. Complement and virus-neutralizing
S.A. Mengel, andW.W. Marquardl, 1986. Differentiation antibody response following infection of susceptible
of infectious bursal disease viruses directly from in- chickens. Avian Dis. 2395-106.
fected tissues with neutralizing monoclonal antibodies: 10. Zar, J.H., 1984. The Analysis of Variance. Bio
Evidence of a ma'or antigenic shift in recent field isolates. Statistical Analyses. Prentice-Hall,Englewood Cliffs, NJ.
Avian Dis. 32:53!%539.
5. Rosales, A.G., P. Villegas, P.O. Lukert, OJ. ACKNOWLEDGEMENTS
Fletcher, M.A. Mohamed, and J. Brown, 1989. Isolation,
identification, and pathogenicity of two field strains of We thank the various broiler producers for sending
infectious bursal disease virus. Avian Dis. 333541. the day-old chicks as well as the Alabama Agricultural
Experiment Station, IGI Vineland, and the US. Poultry
6. Bayyari, G., J. Story, J. Beasley, and J.K. S k l e s , and Egg Association for helping fund the study.
1986.Pathogenicity studies of an Arkansas variant infec-
tious bursal disease vim. Avian Dis. 40:51&532.