Accepted Manuscript

Broad spectrum protection of broiler chickens against inclusion

body hepatitis by immunizing their broiler breeder parents with a
bivalent live fowl adenovirus vaccine

Shelly Popowich, Ashish Gupta, Betty Chow-Lockerbie,

Lisanework Ayalew, Neil Ambrose, Davor Ojkic, Thushari
Gunawardana, Shanika Kurukulasuriya, Philip Willson, Suresh K.
Tikoo, Susantha Gomis

PII: S0034-5288(17)30968-2
DOI: doi:10.1016/j.rvsc.2018.03.003
Reference: YRVSC 3537
To appear in: Research in Veterinary Science
Received date: 18 September 2017
Revised date: 25 January 2018
Accepted date: 3 March 2018

Please cite this article as: Shelly Popowich, Ashish Gupta, Betty Chow-Lockerbie,
Lisanework Ayalew, Neil Ambrose, Davor Ojkic, Thushari Gunawardana, Shanika
Kurukulasuriya, Philip Willson, Suresh K. Tikoo, Susantha Gomis , Broad spectrum
protection of broiler chickens against inclusion body hepatitis by immunizing their broiler
breeder parents with a bivalent live fowl adenovirus vaccine. The address for the
corresponding author was captured as affiliation for all authors. Please check if
appropriate. Yrvsc(2018), doi:10.1016/j.rvsc.2018.03.003

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 ACCEPTED MANUSCRIPT

Broad spectrum protection of broiler chickens against inclusion body hepatitis by

immunizing their broiler breeder parents with a bivalent live fowl adenovirus vaccine

Shelly Popowich1 , Ashish Gupta1 , Betty Chow-Lockerbie1 , Lisanework Ayalew1 , Neil

Ambrose2 , Davor Ojkic3 , Thushari Gunawardana1 , Shanika Kurukulasuriya1 , Philip Willson4 ,

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Suresh K. Tikoo5 , and Susantha Gomis1*

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Department of Veterinary Pathology, Western College of Veterinary Medicine, University of

Saskatchewan, Saskatoon, SK, Canada, S7N 5B4.

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Sunrise Farms, Surrey, BC, Canada, V3W 1C9
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Animal Health Laboratory, University of Guelph, Guelph, ON, Canada, N1H 6R8.
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Canadian Centre for Health and Safety in Agriculture, University of Saskatchewan, Saskatoon,
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SK, Canada, S7N 5E5

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Vaccinology & Immunotherapeutics Program, School of Public Health, University of
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Saskatchewan, Saskatoon, SK Canada.

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* Corresponding author mailing address: Department of Veterinary Pathology, Western College

of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK Canada,

S7N 5B4. Phone: 306-966-7299, Email: susantha.gomis@usask.ca

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ABSTRACT

Historically, fowl adenovirus (FAdV) associated inclusion body hepatitis (IBH) was

considered a secondary disease in broiler chickens associated with immunosuppression.

However, we previously reported the occurrence of IBH as a primary disease in the broiler

chicken industry in Canada as a result of infections with various FAdV serotypes. Therefore, the

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objectives of this study were to develop an immunization strategy in broiler breeders using live

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fowl adenovirus (FAdV) 11-1047 and FAdV8a-TR59 to confer homologous and heterologous

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protection in broiler progeny against IBH and to study the efficacy of natural exposure of naïve

broiler breeders to a vaccine virus from live FAdV vaccinated birds as an immunization

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technique. Broiler breeders vaccinated orally with FAdV8a-TR59 (1x104 TCID50 /bird) and
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FAdV11-1047 (1x104 TCID50 /bird), FAdV8a-TR59 (1x106 TCID50 /bird) and FAdV11-1047

(1x106 TCID50 /bird) or FAdV8b (1x106 TCID50 /bird) transferred substantial levels of
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neutralizing antibodies to their progeny. The efficacy of the maternal antibodies was studied by
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challenging 14-day old broiler chickens with 1x107 TCID50 of FAdV2-685, FAdV7-x11a like,

FAdV8a-TR59, FAdV8b-SK or FAdV11-1047 which are the dominant serotypes causing IBH
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outbreaks in Canada. Broiler chickens from the low and high dose vaccinated breeders were
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significantly protected against all serotypes of FAdV (P<0.05). Comingling of unvaccinated

broiler breeders with FAdV-vaccinated broiler breeders was an effective immunization technique
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for in-contact naïve birds. This study confirms that IBH can be effectively controlled in Canada

by vaccination of broiler breeder parents with a bivalent vaccine containing live FAdV8a-TR59

and FAdV11-1047.
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Keywords: inclusion body hepatitis, fowl adenovirus, immunization, broiler breeder, broiler

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Abbreviations: AGID = agar gel immunodiffusion; DMEM/F-12 = Dulbecco’s Modified Eagle

Medium/Nutrient Mixture F-12; FAdV = fowl adenovirus; IBH = inclusion body hepatitis; LMH

= leghorn male hepatoma; TCID50 = tissue culture infective dose50 ; VN = virus neutralization

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INTRODUCTION

Inclusion body hepatitis (IBH) is an acute, viral disease in young, 2-7 week old broiler

chickens (Fitzgerald and Hess, 2013). Outbreaks of IBH are characterized by a sudden onset of

mortality that can exceed 30% (Fitzgerald and Hess, 2013) with a short clinical course of 4-5

days (Christensen, 1989; Fitzgerald and Hess, 2013). Clinical signs include severe depression,

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ruffled feathers and a crouching position. On necropsy, the affected birds have pale, swollen,

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friable, hemorrhagic livers with focal to extensive necrosis, and basophilic intranuclear inclusion

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bodies in hepatocytes (Fitzgerald and Hess, 2013).

Five fowl adenovirus (FAdV) species, designated with the letters A-E, are recognized in

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the genus Aviadenovirus within the family Adenoviridae based largely on molecular criteria
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(Harrach et al., 2012), in particular hexon gene sequence data (Ojkic et al., 2008; Niczyporuk,

2016, Schachner et al., 2016). FAdVs are further subdivided into twelve serotypes based on cross
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neutralization tests (Hess, 2000; Steer et al., 2011). Most FAdVs are considered non-pathogenic
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and only certain serotypes have been associated with IBH outbreaks. In IBH outbreaks, two or

three serotypes of FAdV may be isolated from a single bird (Meulemans et al., 2001; Mittal et
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al., 2014), suggesting that there is little cross protection between different serotypes (McFerran,
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2000).

Different serotypes of FAdV have been reported as primary causes of IBH in several
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countries (Zadravec et al., 2013; Gawel et al., 2016; Matos et al., 2016; Du et al., 2017; Morales

et al., 2017; Morshed et al., 2017; Sellers, 2017, Stoute, 2017). Prevalence of IBH as a primary

disease has increased in Canada, and outbreaks associated with FAdV8a-TR59, FAdV8b-SK,

FAdV11-1047 and occasionally with FAdV2-685 and FAdV7-x11a like have resulted in

mortality of up to 30% (Gomis et al., 2006; Ojkic et al., 2008; Dar et al., 2012). Currently, there
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is no commercial FAdV vaccine available in Canada. Hence, the objectives of this study were to

develop and evaluate an immunization strategy for broiler breeders using a combination of live

FAdV8a-TR59 and FAdV11-1047 for broad spectrum protection of their broiler progeny against

IBH caused by FAdV serotypes that are dominant in Canada, determine the effective dose of live

FAdV for broiler breeder vaccination and evaluate the sero-conversion of unvaccinated in-

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contact broiler breeders with vaccinated birds.

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MATERIALS AND METHODS

FAdV serotypes and virus propagation

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FAdV2-685, FAdV7-x11a like, FAdV8a-TR59 and FAdV11-1047 (Ojkic et al., 2008)
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and FAdV8b-SK (Gupta et al., 2018) were previously described. Virus propagation was

conducted in a Leghorn male hepatoma (LMH) cell line obtained from the American Type
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Culture Collection (ATCC#CRL-2117, VA) and maintained as described previously (Kawaguchi

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1987). Viruses used to immunize broiler breeders were prepared at the Animal Health

Laboratory, University of Guelph, Ontario, Canada. Plaque purified FAdV8a-TR59 or FAdV11-

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1047 was grown in LMH cells and a combination of FAdV8a-TR59 (1x104 TCID50 ) + FAdV11-
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1047 (1x104 TCID50 )/bird or FAdV8a-TR59 (1x106 TCID50 ) + FAdV11-1047 (1x106

TCID50 )/bird was used to vaccinate broiler breeders. Additionally, plaque purified FAdV8b-SK
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was grown in LMH cells and was used to vaccinate broiler breeders at 1x106 TCID50 /bird.

Plaque purification was performed as described before (Yates et al., 1976) using LMH cells.

Maintenance and vaccination of broiler breeders

Day-old broiler breeder parents were obtained from Aviagen Inc., Huntsville, AL. At

hatch, all birds were vaccinated against Marek’s Disease (Intervet Rismavac, Summit, NJ; Select
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HVT, West Perth, WA). Birds were housed at the Animal Care Unit, Western College of

Veterinary Medicine, University of Saskatchewan and feeding and lighting programs were

conducted according to Aviagen guidelines. Males and females were reared separately until 16

weeks of age. Thereafter, birds were randomly separated into 4 groups, three of those groups

consisting 15 females and 4 males and the fourth consisting of 20 females and 4 males. At 16

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weeks of age, 0.5 mL of FAdV live vaccine was administered to each bird by oral gavage.

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[group 1 = saline; group 2 = FAdV8a-TR59 (1x104 TCID50 ) + FAdV11-1047 (1x104

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TCID50 )/bird, group 3 = FAdV8a-TR59 (1x106 TCID50 ) + FAdV11-1047 (1x106 TCID50 )/bird

and group 4 = FAdV8b-SK (1x106 TCID50 /bird)]. Group 4 had 50% (n=12) of broiler breeders

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vaccinated with FAdV8b-SK (1x106 TCID50 /bird) and the remaining 50% (n=12) of naïve broiler
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breeders were not exposed to FAdVs. Prior to broiler breeder vaccination, agar gel

immunodiffusion (AGID) of sera samples was performed at the Animal Health Laboratory,
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University of Guelph to ensure broiler breeders were free of antibodies against FAdV. Following
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vaccination, sera samples were collected at 20, 35 and 50 weeks of age from groups 1 to 3 and at

23 weeks of age from group 4 to test neutralizing antibody levels against FAdV. Eggs from
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broiler breeders were collected between 30 and 45 weeks of age to hatch their broiler progeny.
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Virus neutralization (VN) test

VN testing was performed in LMH cells as described previously (Gupta et al., 2018) with
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a few modifications. Plaque purified FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-

SK or FAdV11-1047 serotypes were used to detect neutralizing antibodies against FAdVs in

broiler breeders and maternal antibodies against FAdVs in their broiler progeny. Serum samples

were heat inactivated at 56 C for 30 min. The samples were serially diluted 2-fold in 96 well

plates in triplicates with diluent (DMEM/F-12 containing gentamicin at10 µg/mL). A total of
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200 TCID50 of each FAdV serotype was added into each well of the respective 96 well plate and

the serum-virus mixture was incubated for 1 hr at 37 C with 5% CO 2 . The mixture was then

transferred onto freshly sub-cultured cells in 96 well plates (5x104 cells/well) starting from the

highest dilution. Subsequently, the plates were incubated at 37 o C with 5% CO 2 and results were

read on day 7 post-infection. The cut-off value for VN test was 200.

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Propagation and validation of FAdVs for broiler virus challenge

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Purification of FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK, or FAdV11-

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1047 from infected liver tissues from field cases of IBH in broiler chickens from Saskatchewan

and Ontario was carried out as described previously (Schat and Purchase, 1998). Briefly, livers

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infected with FAdVs from clinical cases of IBH were homogenized (Polytron PT 3000,
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Kinematica, AG, Littau, Switzerland) and centrifuged at 6000 rpm for 30 min as 40%

suspensions in Waymouth’s medium (Fisher Scientific, Waltham, MA). The prepared

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suspensions were freeze-thawed for 6 cycles then pelleted at 6000 rpm at 10 C for 30 min. The
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supernatant was filtered through sterile 0.22 μm filters. Eighty percent confluent LMH cells were

inoculated with 10% liver homogenates in phosphate buffered saline. The virus positive filtrate
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was titrated and stored at -80 C.

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After propagation of each FAdV serotype, the hexon gene loop 1 region was amplified by

polymerase chain reaction, sequenced and analyzed by phylogenetic tree as described before
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(Ojkic et al., 2008). The phylogenetic tree was constructed by multiple alignment of reference

sequences with vaccine and challenge virus sequences using the Clustal W method. The tree was

built by neighbor-joining method at 1000 bootstrap values using Geneious software version

9.0.5. In addition, each FAdV serotype used for challenge was tested by cross neutralization

assay with sera raised against the different viruses used in the study.
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Broiler lethal FAdV challenge: Broiler chicken model

Broiler progeny from broiler breeder parents vaccinated with live FAdVs were randomly

allocated into groups, each containing 30 birds. At 14 d post-hatch, each group of broiler

progeny were inoculated intramuscularly in the thigh with 1x107 TCID50 of FAdV2-685,

FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK or FAdV11-1047. On day 10 post FAdV

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challenge, the remaining birds were euthanatized by cervical dislocation. Tissue sections from

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livers were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm

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thickness and stained with hematoxylin and eosin for histopathological studies. Liver samples

from birds that developed IBH were fixed in 5% glutaldehyde in sodium cocodylate (Canemco

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Inc., Quebec, Canada; pH 7.2) for electron microscopic studies.
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Statistical analysis

Survival and serum neutralizing antibody data were analyzed with the use of Prism
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(Prism 5.0, GraphPad Software Inc., San Diego, CA) with a significance level of P<0.05. The
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survival patterns and median survival times were compared using the Mantel-Cox (log-rank) test.

Ethics statement
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The animal experiments were approved by the University Committee on Animal Care
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and Supply Animal Research Ethics Board at the University of Saskatchewan and conducted

following the guidelines of the Canadian Council on Animal Care.

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RESULTS

Confirmation of FAdV serotypes used in the vaccine and for challenge-protection studies

To verify the identity of the FAdV serotypes included in the study (Ojkic et al., 2008),

the hexon gene loop 1 region was sequenced and compared with the reference strains. As
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expected, the sequences of the five FAdV serotypes had 100% amino acid sequence identity to

FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK and FAdV11-1047, respectively

(Figure 1). Furthermore, FAdV7-x11a like and FAdV8b-SK were neutralized with FAdV8a-

TR59 specific antisera, but not with FAdV11-1047 or FAdV2-685 specific anti-sera. FAdV8a-

TR59 and FAdV7-x11a like were neutralized with sera raised against FAdV8b-SK. In contrast,

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cross neutralization was observed between FAdV2-685 and FAdV11-1047, but both serotypes

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were not neutralized with FAdV8a-TR59 or FAdV8b-SK specific anti-sera (Table 1).

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Virus neutralizing and cross-protective antibody levels in broiler breeders

AGID was performed to test for the FAdV status of broiler breeders prior to vaccination.

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No antibodies were detected against FAdV in all broiler breeders prior to vaccination. Also,
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broiler breeders receiving saline had no detectable neutralizing antibodies against any of the

FAdV serotypes at 20, 23, 35 or 50 weeks of age.

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Serum was collected from broiler breeders at 20, 35 and 50 weeks of age to examine the
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presence of neutralizing antibodies after vaccination at 16 weeks of age with a low dose (104

TCID50 /bird) or high dose (106 TCID50 /bird) of FAdV8a-TR59 and FAdV11-1047. Broiler
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breeder neutralizing antibodies against FAdV8a-TR59 at 20 weeks of age were 3.75 log10 ± 0.13
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(low dose group) and 3.75 log10 ± 0.25 (high dose group) (Figure 2A); at 35 weeks of age, 4.26

log10 ± 0.30 (low dose group) and 4.26 log10 ± 0.17 (high dose group) (Figure 2B) and at 50
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weeks of age 4.11 log10 ± 0.21 (low dose group) and 4.43 log10 ± 0.41 (high dose group) (Figure

2C). Similarly, mean neutralizing antibodies against FAdV11-1047 at 20 weeks of age were

2.84 log10 ± 0.39 (low dose group) and 3.38 log10 ± 0.27 (high dose group) (Figure 2A); at 35

weeks of age 3.43 log10 ± 0.38 (low dose group) and 3.66 log10 ± 0.17(high dose group) (Figure

2B); and at 50 weeks were 3.44 log10 ± 0.33 (low dose group) and 3.69 log10 ± 0.27 (high dose
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group) (Figure 2C). In addition, body weight, fertility and hatchability between vaccinated and

unvaccinated breeders were similar.

Serology of in-contact vaccinated birds

To assess the possibility of live virus shedding by vaccinated birds and indirect

immunization of sentinel birds, serum was collected at 23 weeks of age from broiler breeders

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that received FAdV8b-SK (1x106 TCID50 /bird) and naïve broiler breeders in contact with

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FAdV8b-SK vaccinated broiler breeders. The mean neutralizing antibody titers against

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FAdV8b-SK in the FAdV8b-SK vaccinated group were 3.44 log10 ± 0.13 while mean

neutralizing antibody titers in birds in contact with FAdV8b-SK vaccinated birds was 3.20 log10

± 0.37 (Figure 3).

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Neutralizing maternal antibody levels in broiler progeny

Sera from broiler progeny were collected at the time of hatch to measure the level of
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neutralizing maternal antibody transfer. Mean neutralizing antibodies at 0 d of age in broiler

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progeny against FAdV8a-TR59 were 3.54 log10 ± 0.37 and (low dose group) and 3.57 log10 ±

0.39 (high dose group) while for FAdV11-1047, mean neutralizing antibodies were 2.96 log10 ±
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0.25 (low dose group) and 3.08 log10 ± 0.27 (high dose group) (Figure 4).
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Protection of broiler progeny against FAdV challenge

Hatching eggs were collected between 30 to 45 weeks of age from vaccinated and
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unvaccinated broiler breeders and incubated. The progeny were challenged with FAdV2-685,

FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK or FAdV11-1047 14 d post hatch to study the

degree of protection from mortality. Mortality associated with IBH in broiler chickens from non-

vaccinated broiler breeders, peaked at 3 to 4 d after FAdV challenge and no further mortality

occurred after 6 d post challenge (Figure 5). Broiler progeny from broiler breeders vaccinated
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against FAdV8a-TR59 and FAdV11-1047 with either the low (1x104 TCID50 /bird) or high(1x106

TCID50 /bird) dose did not show significant clinical signs, lesions or mortality against any of the

FAdV serotypes (i.e. FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK or FAdV11-

1047) following challenge when compared to the broiler progeny from non-vaccinated broiler

breeders. In the saline control group, the highest (90%) and lowest (20%) mortality rates were

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observed in progeny challenged with FAdV8b-SK (Figure 5D) and FAdV7-x11a like (Figure

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5B), respectively. In the vaccinated groups, the highest (15%) and lowest (0%) mortality rates

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were observed in FAdV8b-SK (Figure 5D) and FAdV11 (Figure 5E) challenged groups,

respectively. Generally, broiler progeny from both the low and high dose vaccine groups were

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significantly protected against challenge with different serotypes of FAdVs as compared to the
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saline control group (P<0.05) (Figure 5). Statistically different protection was not observed

between progeny from the low and high dose group.

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DISCUSSION

IBH has been described in many countries including Canada as a primary disease causing
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significant economic losses in the broiler chicken industry (Christensen and Saifuddin, 1989;
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Gomis et al., 2006; Ojkic et al., 2008; Zadravec et al., 2013; Gawel et al., 2016; Matos et al.,

2016; Du et al., 2017; Morales et al., 2017; Morshed et al., 2017; Sellers, 2017, Stoute, 2017). In
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Canada, IBH is primarily caused by FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-

SK or FAdV11-1047 (Philippe et al., 2007; Ojkic et al., 2008; Dar et al., 2012). Although

commercial live vaccines are available to protect chickens against various viral diseases (Muller

et al., 2012; Ou and Giambrone, 2012; Dimitrove et al., 2017, Jordan 2017), live FAdV vaccines
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are in the preliminary stages of development (Schonewille et al., 2010; Mansoor et al., 2011;

Steer et al., 2011; Gupta et al., 2018).

FAdV vaccine protection is mainly evaluated based on induction of virus neutralizing

antibodies (Alvarado et al., 2007; Gupta et al., 2018). Multiple serotypes of FAdV are prevalent

in Canada (Philippe et al., 2007; Ojkic et al., 2008; Dar et al., 2012) and cross neutralizing

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antibodies induced to heterologous serotypes is variable (Gupta et al., 2018). Hence, a proper

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mix of antigens in a FAdV vaccine is required for broad-spectrum protection of chickens against

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IBH. In this study, the potential use of a combination of live FAdV8a-TR59 and FAdV11-1047

as a bivalent vaccine in broiler breeders for broad spectrum protection of their progeny against

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IBH was evaluated. In addition, the effect of varying vaccine doses on neutralizing antibody
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titers and the level of flock immunity after vaccinating only 50% of broiler breeders was

examined.
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Administration of live vaccines through drinking water or aerosolized particles is

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advantageous. Since it requires less labor and no handling of birds, it is cost effective (Collett,

2013). Gupta et al., 2018 reported that vaccinating broiler breeders with a live wild type FAdV is
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safe at 16 weeks of age without producing noticeable gross or histopathological lesions. Vertical
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transmission was also not observed as vaccination was completed and viral shedding ceased

prior to the onset of egg production. In this study, we demonstrated that a bivalent broiler breeder
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FAdV vaccine (i.e. a combination of FAdV8a-TR59 and FAdV11-104) given orally at 16 weeks

of age significantly protected their progeny against FAdV2-685, FAdV7-x11a like, FAdV8a-

TR59, FAdV8b-SK or FAdV11-1047 challenge. Previously, Alvarado et al., 2007 demonstrated

maternal antibody derived cross protection against IBH in broilers by vaccinating broiler breeder

parents with an autogenous FAdV8A/FAdV11 bivalent vaccine. Similarly, Kim et al., 2014
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reported cross protection against FAdVs (FAdV4, 5, 8a 8b, 11) by vaccinating chickens with an

oil emulsion inactivated FAdV4 vaccine; however, interspecies cross protection is rarely

observed among FAdVs (Steer et al., 2011). We were also able to demonstrate that a single oral

vaccination at 16 weeks of age with a vaccine dose as low as 1x104 TCID50 /bird was able to

produce an adequate quantity of neutralizing antibodies against FAdVs in broiler breeders.

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Nonetheless, increasing the dose of the vaccine to 1x106 TCID50 /bird did not significantly

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increase the level of neutralizing antibodies or degree of protection in the progeny. It is evident

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from this that using a low dose has an advantage of economizing on the antigen while

maintaining an optimal antibody response. Although FAdV7-x11a like, FAdV8a-TR59 and

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FAdV8b-SK belong to separate genotyping clusters, there was strong two-way cross
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neutralization, except for FAdV7-x11a like which remains to be determined. Similarly, two-way

cross neutralization was observed between FAdV11-1047 and FAdV2-685 which cluster
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separately in a phylogenetic tree. One way cross neutralization between FAdV8a-TR59 and
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FAdV8b-SK (Gupta et al., 2018) and two-way cross neutralization between FAdV2 and FAdV11

(Steer et al., 2011) were previously reported suggesting the presence of common neutralizing
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epitopes between these FAdVs in the different genotyping clusters. Contrary to our results, Steer
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et al., 2011 did not demonstrate cross neutralization between FAdV7, FAdV8a and FAdV8b.

This might be due to the difference in the cut-off of value of the VN test between the studies.
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Our in-vitro cross neutralization results between FAdV7-x11a like, FAdV8a-TR59 and FAdV8b-

SK are supported by the challenge-protection results in the broiler progeny.

Subsequently, we assessed vaccine virus exposure of naïve broiler breeders in contact

with vaccinated breeders. Interestingly, non-vaccinated in-contact broiler breeders had

comparable levels of serum neutralizing antibodies as in oral gavage vaccinated broiler breeders
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7 weeks post contact. This suggests infection of naïve in-contact birds by the vaccine virus

following virus shedding of vaccinated breeders. This is consistent with our previous study that

demonstrated virus shedding by broiler breeders up to 3 weeks post FAdV8b-SK or FAdV11-

1047 infection (Gupta et al., 2018). Similar virus shedding patterns were also observed by

chickens infected with different FAdVs (Clemmer, 1972; Deng et al., 2013). However, fecal

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shedding only continues until effective levels of neutralizing antibodies are produced (Clemmer,

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1972; Gupta et al., 2018). The vaccinated and non vaccinated in-contact broiler breeders

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maintained high level of antibodies against FAdV throughout their life. This finding indicates

that flock immunity against FAdV can be maintained by vaccinating only half of the broiler

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breeders with a live FAdV vaccine ensuring the transfer of adequate neutralizing maternal
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antibodies to their progeny.

Maternally derived neutralizing antibodies are important in inactivation of FAdVs and

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protection of chicks from infection and development of clinical disease (Toro et al., 2002;
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Alvarado et al., 2007; Gupta et al., 2017; Gupta et al., 2018). In this study, a live FAdV8a-TR59

and FAdV11-1047 vaccine protected progeny of vaccinated broiler breeders from FAdV2-685,
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FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK or FAdV11-1047 challenge at a significant level

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compared to progeny from unvaccinated broiler breeder parents. Since virus shedding stops after

neutralizing antibodies are produced and no vertical transmission occurs during the onset of egg
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production (Gupta et al., 2018), a combination of live FAdV8a-TR59 and FAdV11-1047 oral

breeder vaccine administered at 16 weeks of age is a practical and effective method of

controlling IBH in broilers in Canada.

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In summary, cost effective and successful broad-spectrum protection of broilers against

IBH can be achieved in Canada by immunizing broiler breeder parents with a bivalent live

breeder vaccine composed of FAdV8a-TR59 and FAdV11-1047.

ACKNOWLEDGEMENTS

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The authors are grateful to the animal health technicians at the Animal Care Unit,

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Western College of Veterinary Medicine, University of Saskatchewan and Elizabeth Hillyer and

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the late Keith Harron, Animal Health Laboratory, University of Guelph. Special thanks to Drs.

Gregorio Rosales and Eric Jensen, Aviagen North America, Huntsville, Alabama for donation of

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broiler breeders for animal studies. Financial support was provided by grants from Saskatchewan
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Chicken Industry Development Fund [413862], Saskatchewan Agriculture Development Fund

[412921], Natural Sciences and Engineering Research Council of Canada and Agriculture [CRD
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PJ428582-11] and Agri-Food Canada (AAFC) Growing Forward 2 [AIP-P107].

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Table 1. Summary of cross neutralization between FAdV- serotypes included in the study.

Chicken antisera
FAdV strain
8a-TR59 8b-SK 7-x11a 2-685 11-1047

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8a-TR59 + + NT - -

8b-SK + + NT - -

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7-x11a + + NT - -

11-1047 - - NT + +

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2-685 - - NT + +
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“+” Neutralizing; “-” Non-neutralizing; “NT”- Not tested. Cut off value for the neutralization assay was
200.
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Figure 1: Phylogenetic tree analysis of vaccine and challenge viruses with other reference FAdV
hexon gene sequences from Genbank (accession numbers are indicated on the left side of each
virus strain). FAdVs used as vaccine; FAdVs included for challenge protection study.
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Figure 2: Neutralizing antibodies against FAdVs in broiler breeders at 20 (A), 35 (B) and 50 (C)
weeks of age for serotypes FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK, FAdV11-1047 and
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FAdV2-685.
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1 0 0 ,0 0 0 S a lin e

V a c c in a te d (F A d V 8 b -S K )
L o g 1 0 N e u tr a liz in g A n t ib o d y L e v e l

In -c o n ta c t (w ith F A d V 8 b -S K v a c c in a te d )
1 0 ,0 0 0

1 ,0 0 0

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F A d V 7 -x 1 1 a lik e

F A d V 7 -x 1 1 a lik e

F A d V 7 -x 1 1 a lik e
F A d V 2 -6 8 5

F A d V 2 -6 8 5

F A d V 2 -6 8 5
F A d V 8 b -S K

F A d V 8 b -S K

F A d V 8 b -S K
F A d V 1 1 -1 0 4 7

F A d V 1 1 -1 0 4 7

F A d V 1 1 -1 0 4 7
F A d V 8 a -T R 5 9

F A d V 8 a -T R 5 9

F A d V 8 a -T R 5 9
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Figure 3: Neutralizing antibodies against FAdVs in broiler breeders vaccinated with FAdV8b-
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SK (106 ) and unvaccinated broiler breeders in-contact with FAdV8b-SK vaccinated birds at 23
weeks of age for serotypes FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK, FAdV11-1047 and
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FAdV2-685.
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1
10
100
1,000
10,000
100,000

F A d V 7 -x 1 1 a lik e

F A d V 8 a -T R 5 9

F A d V 8 b -S K
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F A d V 7 -x 1 1 a lik e

F A d V 8 a -T R 5 9

F A d V 8 b -S K
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F A d V 1 1 -1 0 4 7

F A d V 2 -6 8 5
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F A d V 7 -x 1 1 a lik e

F A d V 8 a -T R 5 9
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F A d V 1 1 -1 0 4 7

F A d V 2 -6 8 5
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F A d V 8 a -T R 5 9 (1 0 ) + F A d V 1 1 - 1 0 4 7 ( 1 0 )
F A d V 8 a -T R 5 9 (1 0 ) + F A d V 1 1 - 1 0 4 7 ( 1 0 )

Figure 4: Neutralizing antibodies against FAdVs of broiler progenies at 0 d of age for serotypes FAdV7-
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100 100

90 90

80 80

70 70
S u r v iv a l (% )

S u r v iv a l (% )
60 60

50 50

40 40

30 30

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20 20

10 10

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0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10

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D a y s P o s t C h a lle n g e D a y s P o s t C h a lle n g e

A B
100 100

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90 90
80 80
70 70
S u r v iv a l ( % )

S u r v iv a l (% )

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50 50

40 40

30 30

20 20
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0 0

0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
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D a y s P o s t C h a lle n g e D a y s P o s t C h a lle n g e

C D
S a lin e
100 4 4
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F A d V 8 a -T R 5 9 (1 0 ) + F A d V 1 1 -1 0 4 7 (1 0 )
90 6 6
F A d V 8 a -T R 5 9 (1 0 ) + F A d V 1 1 -1 0 4 7 (1 0 )
80
S u r v iv a l (% )

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30
20
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0 1 2 3 4 5 6 7 8 9 10
D a y s P o s t C h a lle n g e

Figure 5: Survival of broilers following challenge with FAdV2-685 (A), FAdV7-x11a like (B),
FAdV8a-TR59 (C), FAdV8b-SK (D) and FAdV11-1047 (E). Broiler progenies are from broiler
breeder parents vaccinated against FAdV8a-TR59 (104 TCID50 /bird) + FAdV11-1047 (104
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TCID50 /bird) or FAdV8a-TR59 (106 TCID50 /bird) + FAdV11-1047 (106 TCID50 /bird) were
protected against any of the FAdV serotypes following challenge (P<0.05).

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Highlights

 Broiler breeder parents immunized with a bivalent vaccine composed of FAdV8a-TR59 and

FAdV11-1047 transfers adequate neutralizing antibodies to their progeny.

 The maternal antibodies confer broad spectrum protection against IBH in broilers challenged with

FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK or FAdV11-1047.

 Flock immunity against IBH can be acquired by vaccinating only 50% of broiler breeders.

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