Genetic Characterization, Pathogenicity, and Protection Studies with an Avian

Adenovirus Isolate Associated with Inclusion Body Hepatitis

Author(s): I. R. Alvarado, P. Villegas, J. El-Attrache, E. Jensen, G. Rosales, F. Perozo, and L. B. Purvis
Source: Avian Diseases, 51(1):27-32.
Published By: American Association of Avian Pathologists
DOI: http://dx.doi.org/10.1637/0005-2086(2007)051[0027:GCPAPS]2.0.CO;2
URL: http://www.bioone.org/doi/full/10.1637/0005-2086%282007%29051%5B0027%3AGCPAPS
%5D2.0.CO%3B2

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AVIAN DISEASES 51:27–32, 2007

Genetic Characterization, Pathogenicity, and Protection

Studies with an Avian Adenovirus Isolate Associated
with Inclusion Body Hepatitis
I. R. Alvarado,A P. Villegas,AB J. El-Attrache,C E. Jensen,D
G. Rosales,D F. Perozo,A and L. B. PurvisA
A
Department of Population Health, University of Georgia, Athens, GA 30602
C
Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843
D
Aviagen Inc., 5015 Bradford Drive NW, Huntsville, AL 35805
Received 25 April 2006; Accepted 2 September 2006

SUMMARY. An avian adenovirus (AAV) was isolated from liver samples of two 2-wk-old broiler-breeder flocks obtained from
grandparents vaccinated at 10 and 17 wks of age with an autogenous inactivated vaccine containing the European AAV 8 (8565
strain) and 11 (1047 strain) serotypes (AAV8/11 vaccine). Affected broiler-breeders exhibited clinical signs and macroscopic and
microscopic lesions associated with inclusion body hepatitis (IBH). The isolated adenovirus, identified as Stanford, was molecularly
characterized as European serotype 9. The pathogenicity of the Stanford strain was confirmed after inoculation of specific-
pathogen-free (SPF) chickens at 1–7 days of age, causing 100% and 20% mortality, respectively. The level of protection against
IBH was evaluated in two broiler-breeder progenies from AAV 8/11–vaccinated grandparent flocks and a commercial broiler flock
by challenge at 1 or 7 days of age with the AAV 8 and 11 serotypes and/or the Stanford strain. The broiler-breeder progenies and
the commercial broiler flock exhibited protection against IBH after challenge. No significant differences in mean body weights were
observed at 3 wk of age in any of the evaluated groups. We conclude that broiler-breeder progenies from 30- to 50-wk-old
grandparents vaccinated with the AAV 8/11 vaccine were adequately protected against challenge with the AAV 8 and 11 serotypes
and the Stanford strain.

RESUMEN. Caracterización genética, patogenicidad y estudios de protección con un adenovirus aviar asociado con hepatitis con
cuerpos de inclusión.
Se aisló un adenovirus aviar a partir de muestras de hı́gado obtenidas de dos lotes de reproductoras de dos semanas de edad
procedentes de lotes de abuelas que fueron vacunadas a las 10 y 17 semanas de edad con una vacuna autógena inactivada que
contenı́a adenovirus pertenecientes a los serotipos Europeos 8 (cepa 8565) y 11 (cepa 1047). Las reproductoras mostraron signos
clı́nicos y lesiones macro y microscópicas asociadas con la hepatitis con cuerpos de inclusión. El adenovirus aislado fue identificado
como la cepa Stanford y fue caracterizado molecularmente como serotipo Europeo 9. La patogenicidad de la cepa Stanford fue
confirmada después de la inoculación de aves libres de patógenos especı́ficos, donde se obtuvo una mortalidad del 100% en las aves
inoculadas al dı́a de edad, y de 20% en las inoculadas a los 7 dı́as. El nivel de protección contra la hepatitis fue evaluado en dos
progenies de reproductoras de engorde procedentes de lotes de abuelas vacunadas con la vacuna autógena con los serotipos 8 y 11,
lo mismo que en un lote de pollos de engorde seleccionado al azar. Estos grupos fueron desafiados al dı́a de edad y a los siete dı́as,
con las cepas de los serotipos 8 y 11, lo mismo que con la cepa Stanford. No se encontraron diferencias significantes en los
promedios de pesos corporales obtenidos a las tres semanas de edad en ninguno de los grupos evaluados. Se concluye que progenies
de engorde procedentes de abuelas vacunadas con los serotipos 8 y 11 de adenovirus aviar, fueron protegidas adecuadamente contra
los serotipos 8, 9 y 11.
Key words: avian adenovirus, molecular characterization, avian adenovirus pathogenicity, adenovirus inactivated vaccines,
hexon gene, inclusion body hepatitis
Abbreviations: AAV ¼ avian adenovirus; CAV ¼ chicken anemia virus; CELiC ¼ chicken embryo liver cells; CELO ¼ chicken
embryo lethal orphan; CV ¼ coefficient of variation; GMT ¼ geometric mean titer; HPS ¼ hydropericardium syndrome; IBDV ¼
infectious bursal disease virus; IBH ¼ inclusion body hepatitis; ELISA ¼ enzyme-linked immunosorbent assay; MAb ¼ maternal
antibody; PCR ¼ polymerase chain reaction; PDRC ¼ Poultry Diagnostic and Research Center; RFLP ¼ restriction fragment length
polymorphism; SPF ¼ specific-pathogen-free; TCID50 ¼ tissue culture infective dose producing a cytopathic effect in 50% of
cultures inoculated

The International Committee on Taxonomy of Viruses has
divided the Adenoviridae family into the Mastadenovirus, Siadeno-
virus, Atadenovirus, and Aviadenovirus genera (2). The Mastadeno-
virus genus contains the mammalian adenoviruses, including the
human, simian, bovine, porcine, and equine adenoviruses. The
Siadenovirus genus, formerly designated as group II avian adenovi-
ruses, includes the hemorrhagic enteritis virus, marble spleen disease
virus, and the avian adenovirus group 2 splenomegaly of chickens.
The Atadenovirus genus, formerly designated as group III avian
B
Corresponding author.
adenoviruses, includes the egg drop syndrome virus. The Aviadeno-
virus genus, formerly designated as group I avian adenoviruses
(AAV), contains 11 of the 12 recognized European adenovirus
serotypes classified in five (A to E) molecular groups. According to
the recent classification, the adenovirus strain identified as 380,
previously classified as European serotype 12, has been included in
molecular group D as European serotype 11, whereas the former
serotype 8 has been further divided into serotypes 8a and 8b (2).
AAV have been associated with inclusion body hepatitis (IBH) and
hydropericardium syndrome (HPS) (3,5,6,8,9), respiratory disease,
necrotizing pancreatitis, and gizzard erosions (13,14,16). Chickens
affected by IBH/HPS exhibit high mortality rates, liver damage, and

27
28 I. R. Alvarado et al.
hydropericardium (10). IBH/HPS outbreaks have been reported as
a result of the association with immunosuppressive viral agents, such
as infectious bursal disease virus (IBDV) and chicken anemia virus
(CAV), and mycotoxins, such as aflatoxins (17,18,19). Also, some
AAV strains have been reported as primary pathogens of peracute
IBH/HPS episodes (1,3,10). Therefore, after isolation from clinical
cases, it has become necessary to determine the pathogenicity of
AAV strains for the host (7). Because AAV infections commonly
occur under commercial conditions, several strategies against immu-
nosuppressive diseases, such as effective control of IBDV and CAV,
which can predispose or increase the pathogenicity of AAV, have
been implemented (10,19). Also, inactivated vaccines have been used
in grandparent and broiler-breeder flocks to prevent vertical trans-
mission, inducing the production of maternal antibodies to protect
their progenies against AAV infection and clinical IBH/HPS (10).
The objectives of this study were 1) to isolate, characterize, and
evaluate the pathogenicity of an AAV (identified as Stanford) strain
isolated from a case of IBH in two sister broiler-breeder flocks; and
2) to evaluate the level of protection in two broiler-breeder progenies
from grandparent flocks vaccinated with the autogenous AAV 8/11
vaccine and a commercial broiler flock against challenge with the
homologous AAV 8 and 11 serotypes and the Stanford strain.
MATERIALS AND METHODS
Case history. Two sister broiler-breeder flocks, obtained from
grandparent flocks vaccinated at 10 and 17 wk of age with an
autogenous, inactivated vaccine containing the European AAV 8 (8565
strain) and 11 (1047 strain) serotypes (AAV 8/11 vaccine), were
diagnosed with inclusion body hepatitis at 2 wk of age. The flocks
experienced a slight increase in cumulative mortality (5.1%) at 2 wk,
with affected birds showing clinical signs of depression and ruffled
feathers. At macroscopic examination, birds showed normal develop-
ment, feed in the crop, swollen yellowish livers with petechial
hemorrhages, swollen kidneys, and some hemorrhages in skeletal
muscles. Histologic examination revealed the presence of multifocal
necrosis of hepatocytes with basophilic intranuclear inclusion bodies.
Liver sections were submitted to the Poultry Diagnostic and Research
Center (PDRC) of the University of Georgia for virus isolation.
Virus isolation. Liver tissues were processed for virus isolation as
described elsewhere (20). Briefly, liver samples were frozen and thawed
twice, macerated in tryptose phosphate broth (10% w/v), with 1%
penicillin (10,000 IU/ml) and streptomycin (10,000 lg/ml) (Sigma
Chemicals Co., St Louis, MO), and 1% amphotericin B (250 lg/ml)
(Sigma Chemicals Co.) and centrifuged at 7600 3 g for 5 min. The
supernatant was filtered through a sterile 0.22-lm syringe filter
(Whatman Inc., Clifton, NJ) and used to inoculate chicken embryo
liver cells (CELiC) from 14-day-old specific-pathogen-free (SPF)
chicken embryos (SPAFAS Inc., Norwich, CT).
Virus characterization. Viral DNA, extracted from inoculated
liver cells, was purified using the QIAamp DNA Mini Kit (Qiagen,
Valencia, CA), following the recommendations of the manufacturer.
Polymerase chain reaction (PCR) amplification of an 897-bp fragment
containing the L1 loop of the hexon gene (position 144 to 1041 based
on the hexon gene of the chicken embryo lethal orphan [CELO] strain,
accession number NC-001720 in GenBank) was performed with the
hexon A (59-CAARTTCAGRCAGACGGT-39) and hexon B (59-
TAGTGATGMCGSGACATCAT-39) primers following the procedure
described elsewhere (11). The amplified product was visualized in
a 1.5% agarose gel with ethidium bromide.
The 897-bp, amplified fragment was purified using the QIAquick
Spin kit (Qiagen) and used as a template for sequencing. Sequencing
reactions, using 30 ng of template per reaction, were performed with the
BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,
Inc., Foster City, CA), as described by the manufacturer. Sequencing
reactions were run in an ABI PRISM 310 Genetic Analyzer (Applied
Biosystems). Sequences were edited, saved, and analyzed with the
DNAStar software (DNAStar Inc., Madison, WI).
Phylogenetic analysis of the L1 amino acid sequences (correspond-
ing to residues 49 to 346 in the hexon protein of the reference CELO
strain) was performed with the Clustal W method of DNAStar, and
final phylogeny (dendogram) was produced by the neighbor-joining
method. The L1 amino acid sequence of the isolated adenovirus strain,
identified as Stanford, was compared with the sequences of the AAV 8
(8565 strain) and 11 (1047 strain) serotypes and also with reference
AAV L1 sequences available at GenBank.
Reference L1 sequences obtained from GenBank and used in the
phylogenetic analysis of the Stanford strain (DQ323986) included the
CELO (NC-001720), SR48 (AAN77072), 75 (N77075), 506
(AAN77076), 340 (AAN77078), CR119 (AAN77080), B-3A
(AAL13225), TR59 (AAN77082), 764 (AAN77084), X11A
(AAL13227), C2B (AAN77085), and 380 (AAL13228) sequences,
which correspond to each of the 12 European AAV serotypes,
respectively, and the AAV 8 (8565 strain, accession number
DQ323985) and 11 (1047 strain, accession number DQ323984)
serotypes, previously isolated and characterized in our laboratory.
Virus pathogenicity. The pathogenicity of the Stanford strain
was evaluated in SPF leghorns (SPAFAS Inc.). The 31-day-old SPF
birds were divided into three groups of 10 birds each and were
housed in filtered, positive air pressure, isolation units. Two groups
were subcutaneously inoculated with 104.5 tissue culture infective dose
producing a cytopathic effect in 50% of cultures inoculated (TCID50) of
the Stanford strain at 1 and 7 days of age, respectively. The third group
was not inoculated and was used as a negative control group. The SPF
birds were observed daily until 3 wk of age. Several parameters,
including mortality, clinical signs, and macroscopic and microscopic
lesions, were used to determine the pathogenicity of the Stanford strain.
CHALLENGE STUDIES
Antibody levels. The level of AAV maternal antibodies in challenged
birds was determined at 1 and 21 days of age by an indirect enzyme-linked
immunosorbent assay (ELISA) test (Roveko Ltd., Gaithersburg, MD).
Briefly, 50 ll of diluted (1:50) serum was added to each well on a test plate
covered with the AAV 8 (8565 strain) serotype. After 1 hr of incubation at
room temperature, the wells were washed twice with 300 ll of wash
solution, and 100 ll of conjugate-labeled secondary antibody was added
per well. After 30 min of incubation at room temperature, the wells were
washed, and 100 ll of substrate was added per well. The enzymatic reaction
was stopped after 20 min of incubation at room temperature by adding 100
ll of stop solution. The ELISA plates were read at 600–630 nm with a 492-
nm differential filter. Antibody titers were calculated using a software
program manufactured by IDEXX Laboratories Inc. (Westbrook, ME).
Challenge viruses. The AAV 8 and 11 serotypes, previously
isolated at PDRC from IBH outbreaks in broiler-breeder flocks
and included in the AAV 8/11 vaccine (Lohmann Animal Health
International, Waterville, ME), and the Stanford strain were propagated
and titrated in CELiC (20). Viral titers were standardized at 105.5
TCID50/ml and used as inoculum.
Chickens. Different types of birds were used in the challenge
studies. The broiler-breeder progenies were obtained from one of the
major broiler-breeder companies in the United States. The SPF broilers
were provided by the Southeastern Poultry Research Laboratory (Athens,
GA) and the commercial broilers were obtained from a local hatchery.
Experimental design. The broiler-breeder progenies (identified as
A and B from a 50- and 30-wk-old grandparent flocks, respectively) were
obtained from grandparent flocks vaccinated at 10 and 17 wk of age with
the AAV 8/11 vaccine. These broiler-breeder progenies were selected to
evaluate the level of protection afforded by maternal antibodies against
challenge with the AAV 8 and 11 serotypes and the Stanford strain. The
levels of protection in progenies A and B were also compared with the SPF
broilers and commercial broilers. For each of the two evaluated progenies,
350 1-day-old birds were used. The birds from each progeny were divided
into seven groups with 50 chickens each. Six groups were challenged
 Protection against inclusion body hepatitis 29

Table 1. Experimental design to evaluate the level of protection provided by maternal antibodies against challenge with the AAV 8 (8565 strain)
and 11 (1047 strain) serotypes or the Stanford strain in broiler-breeder progenies A and B from a 50- and 30-wk-old grandparent flocks, respectively.
Protection against challenge with the Stanford strain in progenies A and B was compared with the protection level present in a commercial broiler
flock and SPF broilers. The birds in the different groups were challenged at 1 or 7 days of age with 104.5 TCID50 of the AAV 8 and 11 serotypes or
the Stanford strain.A

Progeny A Progeny B Commercial broiler SPF broilers

(AAV 8/11 MAb þ) (AAV 8/11 MAb þ) flock (MAb þ) (MAb
)
Challenge age (days) 1 7 1 7 1 7 1 7
Serotype (strain)
8 (8565) 50 50 50 50
11 (1047) 50 50 50 50
9 (Stanford) 50 50 50 50 50 50 10 10
Unchall control 50 50 50 10
A
MAb ¼ maternal antibody; unchall ¼ unchallenged.

subcutaneously at 1 or 7 days of age with 104.5 TCID50 of the AAV 8 and
11 serotypes and the Stanford strain, respectively (Table 1). The
remaining progeny group was not challenged and was used as a negative
control. The birds from the commercial broiler flock and the SPF broilers
were divided in three groups of 50 and 10 birds each, respectively.
The commercial and SPF broilers were challenged only with the Stanford
strain at 1 or 7 days of age (Table 1). All the birds were kept in floor pens
with feed and water at libitum until the end of the study.
Evaluation of protection. The level of protection afforded by
maternal antibodies against challenge with the AAV 8 and 11 serotypes
and the Stanford strain was evaluated using daily mortality, macroscopic
and microscopic lesions, reisolation of the challenge virus from livers in
CELiC and performance. The performance of progenies A and B and the
commercial broiler flock was evaluated by comparing 3-wk-old body
weights of the challenged and nonchallenged groups.
Statistical analysis. Statistical analysis was performed in the SAS
system for Windows V8 (SAS institute Inc., Cary, NC) using the
maternal antibody titers and body weights as units. Means for each were
analyzed separately using the Student–Newman–Keuls test. The
challenged groups were compared with the noninoculated control group
using the Dunnett’s test. Significant differences in the scores were
established at P-values less than 0.05 (P , 0.05).
RESULTS
Virus isolation and characterization. An adenovirus
strain, identified as Stanford, was isolated from liver samples of
two sister broiler-breeder flocks experiencing IBH. Cytopathic effect,
characterized by the presence of round and refractile cells, was
observed after two consecutive passages in CELiC when compared
with normal, noninoculated cells. Positive PCR amplification of the
897-bp fragment containing the L1 loop of the hexon gene was
observed in DNA isolated from infected cells. By phylogenetic
analysis of the amino acid L1 sequences, the Stanford strain was
closely related (99.7%) with the 764 strain, classified as European
AAV 9 serotype (Fig. 1). By phylogenetic analysis, the 8565 and
1047 adenovirus strains were more closely related (100% and
97.7%, respectively) with the TR-59 and C2B reference strains,
which correspond to the European AAV 8 and 11 serotypes,
respectively (Fig. 1). The Stanford strain exhibited similarities of
81.7% and 75.3% when compared with the AAV 8 and 11
serotypes, respectively.
Virus pathogenicity. All the SPF leghorns inoculated with the
Stanford strain at one day of age died between 3 and 5 days post-
inoculation. One (10%), eight (80%), and one (10%) birds died at 3,
4, and 5 days of age, respectively. In the group inoculated at 7 days of
age, two birds died, one (10%) at 11 days and one (10%) at 14 days
of age, while no clinical signs were observed in the remaining birds.
No mortality was observed in the control group, as expected. Clinical
signs included depression, ruffled feathers, and crouching position.
Macroscopic examination of the dead birds showed the presence of

Fig. 1. Phylogenetic tree constructed on the basis of the amino acid sequences of the L1 loop of the hexon protein (corresponding to residues
49–346 in the hexon protein of reference CELO strain) with the Clustal W method of DNAStar showing the relationship of the AAV 8 (8565
strain) and 11 (1047 strain) serotypes and the Stanford strain (in boxes) and several reference strains representing each of the 12 AAV serotypes
(corresponding European serotype in parenthesis) available at GenBank.
30 I. R. Alvarado et al.
Fig 2. Histologic examination of livers from dead SPF leghorns exhibiting basophilic intranuclear inclusion bodies (arrows) after subcutaneous
inoculation at 1 or 7 days of age with 104.5 TCID50 of the Stanford strain.
yellowish and friable livers with ecchymotic hemorrhages, dehy-
dration, and kidney urates. The microscopic examination of livers
from dead birds showed the presence of basophilic intranuclear
inclusion bodies in association with multifocal vacuolar necrosis
(Fig. 2). The Stanford strain was successfully reisolated in CELiCs
previously inoculated with processed liver samples from dead birds
inoculated at 1 or 7 days of age. No macroscopic or histopathologic
lesions were observed in other tissues from the SPF birds, including
the bursa of Fabricius or the thymus.
Antibody levels. Maternal antibody levels in progeny A ranged
from 323 to 11,690, with a geometric mean titer (GMT) of 3393
and a coefficient of variation (CV) of 65%. Antibody levels in prog-
eny B ranged from 149 to 4700, with a GMT of 927 and a CV of
89.51%. In the commercial broilers, antibody levels ranged from
602 to 18,798, with a GMT of 4361 and a CV of 82%. No
maternal adenovirus antibodies were detected in the SPF broilers,
as expected. Progeny B exhibited significantly lower (P , 0.05)
antibody titers when compared with progeny A and the commercial
broilers. At 3 wk of age, no antibody titers were observed in any of
the groups, as measured by the ELISA test.
Evaluation of protection. Total mortality in the different
groups (broiler-breeder progenies A and B, commercial broilers, and
SPF broilers) from which the AAV 8 and 11 serotypes and the Stanford
strain were reisolated after challenge at 1 or 7 days are shown in Table 2.
In progeny A, one and four birds died in the groups challenged
at one day of age with the AAV 8 and 11 serotypes, respectively. The
challenge virus was reisolated from only one (challenged with the
AAV 11 serotype at one day of age) of the five dead birds.
In progeny B, one, two, and one birds died in the groups
challenged at one day of age with the AAV 8 and 11 serotypes
and the Stanford strain, respectively, whereas four and one birds died
in the groups challenged at 7 days with the AAV 11 serotype and the
Stanford strain, respectively. The challenge virus was reisolated from
only two dead birds challenged at one day of age, one inoculated
with the AAV 8 and one inoculated with the AAV 11 serotypes. In
the nonchallenged groups, in progenies A and B, two and one birds
died, respectively; however, no challenge virus was reisolated from
those birds after three consecutive passages in CELiC.
In the commercial broiler flock, the Stanford strain was reisolated
from the only dead bird in the group, challenged at 7 days of age. In
the SPF broiler groups, all (100%) and four out of 10 (40%) birds
died after challenge with the Stanford strain at 1 and 7 days of age,
respectively. In these groups, the challenge virus was reisolated from
all the dead birds.
Mean body weights at 21 days of age and statistical analysis using
individual body weights in the different treatment groups are
presented in Table 3.
In progeny A, mean body weights of 670, 786, and 765 g, and
808, 723, and 745 g were observed in the birds challenged with the
AAV 8 and 11 serotypes and the Stanford strain, respectively. A
mean body weight of 763 g was observed in the unchallenged group.
Only the body weights in the birds challenged at one day of age with
the AAV 8 serotype were significantly lower (P , 0.05) when
compared with the other groups.
In progeny B, mean body weights of 745, 675, and 706 g, and 731,
732, and 698 g were observed in the birds challenged with the AAV 8
and 11 serotypes and the Stanford strain, respectively. A mean body
weight of 631 g was observed in the unchallenged control group. In the
groups challenged at one day of age, significantly higher body weights
were observed in the AAV 8 challenged group, whereas in the groups
challenged at 7 days of age, significantly lower body weights were
observed in the unchallenged control group.
The commercial broilers exhibited mean body weights of 705
and 720 g in the birds challenged with the Stanford strain at 1 and
7 days, respectively. A mean body weight of 700 g was observed
in the unchallenged control group. No significant differences in
body weights were observed between the Stanford-challenged and
control groups.
DISCUSSION
An AAV strain was isolated from two sister broiler-breeder flocks,
with few birds showing clinical signs or macroscopic and
 Protection against inclusion body hepatitis 31

Table 2. Total mortality observed in broiler-breeder progenies A and B, the commercial broilers, and SPF broilers after subcutaneous
inoculation with 104.5 TCID50 of the AAV 8 (8565 strain) and 11 (1047 strain) serotypes and the Stanford strain at 1 or 7 days of age, and number
of birds from which the challenged virus was reisolated.AB

Progeny A (AAV Progeny B (AAV Commercial SPF broilers

8/11 MAb þ) 8/11 MAb þ) broilers (MAb þ) (MAb
)
Challenge age (days) 1 7 1 7 1 7 1 7
Serotype (strain)
8 (8565) 1 0 1 (1) 0 ND ND ND ND
11 (1047) 4 (1) 0 2 (1) 4 ND ND ND ND
9 (Stanford) 0 0 1 1 0 1 (1) 10 (10) 4 (4)
Unchall control 2 1 0 0
A
MAb ¼ maternal antibody; unchall ¼ unchallenged; ND ¼ not done.
B
Number of dead birds from which the challenge strains were reisolated are shown in parenthesis.

microscopic lesions associated with IBH. The isolated strain,
identified as Stanford, exhibited typical cytopathic effect in CELiCs,
and its presence was confirmed by PCR amplification of the 897-bp
segment of the L1 loop of the hexon gene. In previous studies, PCR
amplification with the hexon A and B primers followed by restric-
tion fragment length polymorphism (RFLP) with the BsiWI, MluI,
StyI, and BstXI restriction enzymes has been successfully used to
differentiate the 12 AAV European serotypes (11,12). Because phy-
logenetic analysis of this 897-bp fragment has shown consistent
results when compared with the PCR/RFLP assay (11,12), we com-
pared the amino acid sequence of the L1 loop of the hexon gene of
the Stanford strain with reference strains representing each of the 12
European serotypes. The phylogenetic analysis of the Stanford strain
showed a very high (99.7%) similarity with the 706 strain, which
belongs to the European serotype 9. Moreover, the 8565 and 1047
adenovirus strains, previously classified by virus neutralization as
European serotypes 8 and 11, were more closely related with the
TR59 (European serotype 8) and C2B (European serotype 11)
strains. The pathogenicity of the Stanford strain in birds without
maternal antibodies was confirmed by the elevated mortality
observed in the SPF leghorn and SPF broilers inoculated at 1 or 7
days of age. The absence of any histopathologic lesions in the bursa
of Fabricius or the thymus of the dead birds at different ages was
interpreted as lack of IBDV and CAV infection.
The level of protection provided by maternal antibodies was
evaluated in selected progenies from AAV 8/11–vaccinated
grandparent flocks against challenge with the AAV 8 and 11
serotypes and the Stanford strain. Higher maternal antibody levels
were observed in the commercial broilers when compared with
broiler-breeder progenies A and B, which might be because of the
constant exposure of the commercial broiler breeders to adenovirus
strains under typical field conditions, with the development of
immunity and transfer of high levels of maternal antibodies to their
broiler progeny. In contrast, maternal antibodies present in the
broiler-breeder progenies were derived from grandparent flocks
vaccinated at 10 and 17 wk of age with the AAV 8/11 vaccine with
no apparent previous exposure to live virus during the growth and
production periods because of strict biosecurity practices at the
grandparent level. The lack of antibodies at 21 days of age in the
broiler-breeder progenies and the commercial broilers challenged at
1 or 7 days of age might be because of a normal decline of maternal
antibodies after challenge with neutralization of the challenge strains
and the short time between challenge and testing. An increase in the
antibody titers because of the development of an active immune
response after challenge might have been observed if the serologic
evaluation had been performed at a later age.
Considering that the birds were challenged subcutaneously with
a considerably high dose of virus (104.5 TCID50/bird), which most
likely would not be the case of a natural challenge in the field, very
low mortality was observed in the evaluated progenies. Also, a similar
level of protection was observed in the broiler-breeder progenies and
the commercial broiler flock. From these results, it can be concluded
that under field conditions, progenies from 30- to 50-wk-old parents
vaccinated twice with the AAV 8/11 vaccine are adequately pro-
tected against challenge with the homologous serotypes and the
Stanford strain.
A slight increase in mortality associated with IBH at 2 wk of age
in the two sister broiler-breeder flocks was observed. Because the
grandparent hens are vaccinated only with inactivated vaccines
without previous exposure to live virus, a few hens might not
develop enough antibodies to transmit to their respective progenies.
Chickens derived from these grandparent hens would lack, or have
very low levels of, maternal antibodies, being susceptible to
adenovirus infection in the field at an early age when compared

Table 3. Mean body weights in grams at 3 wk of age and statistical analysis using individual body weights in the different treatment groups of
broiler-breeder progenies A and B and the commercial broiler flock as units.AB

Progeny A (AAV Progeny B (AAV Commercial

8/11 MAb þ) 8/11 MAb þ) broilers (MAb þ)
Challenge age (days) 1 7 1 7 1 7
Serotype (strain)
8 (8565) 670 B 808 A 745 A 731 A ND ND
11 (1047) 786 A 723 A 675 B 732 A ND ND
9 (Stanford) 765 A 745 A 706 B 698 A 705 A 720 A
Unchall control 763 A 631 B 700 A
A
MAb ¼ maternal antibody; unchall ¼ unchallenged; ND ¼ not done.
B
Mean body weights with different uppercase letter are significantly different (P , 0.05).
32 I. R. Alvarado et al.
with chickens with high maternal antibodies. The presence of high
maternal antibodies would allow the birds to be protected against the
development of IBH during the first weeks, with the development of
age-related resistance observed at a later age (4,15). Another possible
explanation might be a recent infection of the grandparent flocks
with the Stanford strain, without protective antibodies against that
particular strain present in the hens, and the consequent vertical
transmission of the virus to the progenies. However, if that had been
the case, a higher number of broiler-breeder progeny chickens should
have been affected by IBH. Moreover, considering the strict
biosecurity measures observed in grandparent facilities, this
hypothesis does not seem to be probable. The presence of AAV
strains of broad antigenicity showing partial cross-neutralization
with other strains has been observed (9), and might explain the
protection observed in our study of the broiler-breeder progenies
against challenge with the Stanford strain. Another explanation
might be a previous infection in the grandparent flocks with the
development and transmission of neutralizing antibodies to their
progenies. However, because the indirect ELISA test used in this
study cannot differentiate the antibody response against a particular
AAV serotype, the presence of specific antibodies against the
Stanford strain in the broiler-breeder progenies cannot be evaluated.
Maternal antibodies have been shown to protect chickens against
the development of IBH; however, growth retardation in the
presence of maternal antibodies has also been observed (15). In this
study, when statistical analyses of individual body weights were
performed, challenged groups did not exhibit significantly lower
body weights when compared with the negative control groups, with
the exception of the group inoculated at one day of age with the
AAV 8 serotype in progeny A. Moreover, some of the challenged
groups exhibited even higher mean body weights when compared
with the unchallenged control groups. Based on the mean body
weight results, broiler-breeder progenies from 30- to 50-wk-old
grandparent flocks vaccinated twice with the AAV 8/11 vaccine
appear to be protected against other potential effects of natural field
infections with the homologous AAV serotypes and the Stanford
strain.
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