2005 Poultry Science Association, Inc.
Infectious Bronchitis Virus
Surveillance in Ontario
Commercial Layer Flocks
B. Stachowiak,* D. W. Key,† P. Hunton,‡ S. Gillingham,§ and É. Nagy*,1
*Department of Pathobiology, Ontario Veterinary College, University of Guelph,
Guelph, Ontario, Canada, N1G 2W1; †Pennsylvania State University,
Animal Diagnostic Laboratory, Orchard Road, University Park, Pennsylvania 16802;
‡Ontario Egg Producers, 7195 Millcreek Drive, Mississauga, Ontario, L5N 4H1,
Canada; §Aviagen North America, Cummings Research Park,
5015 Bradford Drive, Huntsville, Alabama 35805
Primary Audience: Egg Producers, Veterinarians, Researchers
SUMMARY
Infectious bronchitis (IB) is one of the most important viral diseases of poultry and causes
major economic losses to the industry. IB status in commercial layers in southern Ontario was
assessed through a questionnaire. According to the data gathered from egg producers of Ontario,
repeated outbreaks of IB occurred even though flocks had been routinely vaccinated. Based on
this information, 13 farms throughout the region were selected for IB virus (IBV) surveillance.
IBV vaccinated and unvaccinated sentinel birds were placed in barns with different IB histories,
and after a 1-wk exposure tissues and pharyngeal swabs were collected and tested for the presence
of IBV. An initial study indicated that tracheal tissues were more often positive than samples from
cecal tonsils, kidneys, lungs, and pharyngeal swabs. Therefore, all subsequent IBV detection was
directly on tracheal tissues with nucleocapsid (N) gene specific reverse transcription (RT)
polymerase chain reaction (PCR). From the sentinel bird placements and N-gene RT-PCR analysis,
IBV was detected on 11 farms with differing IB histories. The study indicated that farms with
previous IB outbreaks could harbor the virus without clinical signs in the flocks. In addition, some
farms, although they did not report having IB associated problems, were IBV positive by N-gene
RT-PCR.
Key words: infectious bronchitis virus, nucleocapsid gene, sentinel bird, commercial laying hen
2005 J. Appl. Poult. Res. 14:141–146
DESCRIPTION OF PROBLEM
Infectious bronchitis (IB) is one of the most
important diseases of chickens and continues to
cause substantial economic losses to the industry. IB is caused by IB virus (IBV), which is
one of the primary agents of respiratory disease
in chickens worldwide [1].
All chickens are susceptible to IBV infection, and the respiratory signs include gasping,
coughing, rales, and nasal discharge. Sick chicks
usually huddle together and appear depressed.
The severity of the symptoms in chickens is
related to their age and vaccine status. The disease causes some mortality in chicks less than
3 wk of age; in contrast in older birds the clinical
1
To whom correspondence should be addressed: enagy@ovc.uoguelph.ca.
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signs may subside quickly. In layers there is a
noticeable decline in egg production, egg quality
is lowered, and eggs may be deformed with watery albumen. Other signs of IB, such as wet
droppings, are due to increased water consumption [2].
The type of virus strain infecting a flock
determines the pathogenesis of the disease, in
other words the degree and duration of lesions
in different organs. The upper respiratory tract
is the primary site of infection, but the virus
can also replicate in the reproductive, renal, and
digestive systems. The outcome of the infection
is very much dependent on the immune status
of the flock. Chickens that have recovered from
IB may have long-term damage in certain organs. For example atrophy of the oviduct contributes to the decline of egg production, which
may be up to 50% [3]; poor quality eggshell;
and watery albumen. Some IBV strains, such as
Aust T that are known for nephropathogenicity,
have high tropism to the oviduct epithelium. In
addition to commercial layers, eggs from IBVinfected breeder flocks have decreased hatchabilities [4]. After the acute phase of the infection, a persistent infection can be established in
specific organs, and the virus can be excreted
continuously [5]. Virus shedding can also be
stimulated by environmental and physical
stresses, leading to a new cycle of infection [6].
Distinctively different serotypes of IBV exist
throughout the world. The Massachusetts (Mass)
serotype, which was originally identified in the
United States, can be found in Europe and Asia,
and serotypes different from those in North
America have been isolated in other continents
[1]. For example, the Australian IB virus strains
do not have a close phylogenetic relationship
with well-known North American serotypes
such as Mass or Connecticut (Conn). However,
Australia was the first country to report problems
with nephropathogenic IB viruses, which are
now recognized as being present in most areas of
the world. Differing strains have been identified
based on their antigenic variation. Sequence
analyses of IBV pathotypes reveal that they are
distinct in each geographic region. In general,
the incidence of IB outbreaks is sporadic and
predominantly depends on the vaccination practices specific to each country, regardless of the
virus pathotype [6, 7].
Sentinel chickens raised for viral disease surveillance have been employed successfully to
investigate a variety of viruses such as West
Nile virus, infectious bursal disease virus and
IBV [8, 9, 10, 11]. Because sentinels are highly
susceptible to infection, the likelihood that they
will contract the infectious disease agent when
placed on suspect farms is increased. The use
of sentinel birds exposed to flocks with persistent
viral infections could increase the chances of
virus recovery and reduce false negative diagnosis [12]. Broiler and layer flocks have been monitored for the presence of IBV by sentinel birds
[10, 12]. Sentinel birds have also been used to
investigate persistent IB problems on farms for
the purpose of virus reisolation [10, 11] and to
demonstrate that IBV-vaccinated sentinel birds
could be used to evaluate the efficacy of vaccines [12].
A prestudy survey was conducted among
egg producers in Ontario to assess the number
of farms affected by IB. A few producers (13%)
reported continuous problems associated with
IB, whereas 87% of the responders did not indicate any IB problems. Based on the responses
to the questionnaire, the prevalence of IB was
14.2%.
The aims of this study were to survey the
status of IBV through sentinel birds in Ontario
layer operations and to adapt and evaluate a
method to detect IBV rapidly in tissues.
MATERIALS AND METHODS
Sentinel Birds
One-day-old Shaver Leghorn chickens [13]
were obtained and housed in the isolation unit
of the University of Guelph as outlined in the
Animal Utilization Protocol. The birds were vaccinated against Newcastle disease virus, and half
of them were vaccinated against IB [14] by an
eye drop method at 3 wk of age. At 6 wk of
age, 6 vaccinated and 6 unvaccinated chickens
were transported to selected farms. In the barns
the caged chickens were placed in close proximity to the flock and in direct airflow of ventilation
units. After 1 wk of exposure, the sentinel birds
were transported back to the laboratory where
they were euthanized, and samples of organs
(lung, kidney, and cecal tonsils), swabs (pharyngeal and cloacal), and blood were collected. The
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STACHOWIAK ET AL.: INFECTIOUS BRONCHITIS VIRUS SURVEILLANCE
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TABLE 1. Description of facilities and infectious bronchitis (IB) history of selected farms
N-gene specific RT-PCR results1
Farm
Flock
size
Flock
age
A
B-13
B-23
B-33
C
7,000
16,100
49,698
48,385
18,600
Multiple age
32 wk
32 wk
32 wk
38 wk
D
E
F
G
H
M
Q
R
S
X
12,000
18,200
3,400
2,672
7,400
34,000
19,250
11,178
13,121
59,600
59 wk
44 wk
NA5
20 wk
35 wk
40 wk
34 wk
32 wk
35 wk
60 wk
Clinical IB
Status2
Unvaccinated
sentinels
Vaccinated
sentinels
Total
Respiratory signs
Egg color discoloration
None4
None
<1 yr
6/6
3/6
4/6
6/6
5/6
4/6
2/6
3/6
3/6
4/5
10/12
5/12
7/12
9/12
9/11
2 mo
3 mo
None
8 mo
<1 yr
None
None
<1 yr
<2 yr
None
4/6
2/6
0/6
6/6
4/6
4/6
1/6
0/2
0/6
4/6
49/86
0/5
0/6
0/6
4/6
4/6
4/6
0/6
0/6
1/5
4/6
33/87
4/11
2/12
0/12
10/12
8/12
8/12
1/12
0/8
1/11
8/12
82/173
Breed
White Leghorns
White Leghorns
White Leghorns
Bovans
Bovans
Browns
Dekalb Delta
Babcock
White Leghorns
Dekalb Delta
White Leghorns
White Leghorns
ISA brown
Babcock
White Bovans
Hyline 98
1
Results of IB virus detection with N-gene specific reverse transcription-polymerase chain reaction (RT-PCR) in tracheal
tissues from sentinel birds.
Clinical IB status prior to sentinel bird placement.
3
The number indicates different barns on the same farm.
4
None = the farm did not indicate any problems associated with IB.
5
Not available.
2
flow chart of the experimental design is shown
in Figure 1.
practices and IB history. Some of the data gathered are summarized in Table 1.
Farm Selection for Sentinel Bird Placement
N-Gene Specific RT-PCR
to Detect IBV in Tissues
Thirteen farms participated in the study (Table 1). Some of the farms (A, C, D, E, M, Q)
were identified based on information obtained
from veterinary clinicians, who were aware of
the IB problems. Other farms (B, F, G, H, R,
S, and X) were selected based on the survey
described in the introduction. These farms were
located in one county of Ontario, where higher
incidences of IB-related problems were identified. The potential farms chosen for sentinel
placement had to have permission from the Ontario Egg Producers to be included in the study.
If a given producer did not wish to participate
in the study, the producer at the farm with the
next most recent IB outbreak was contacted. At
the time of the bird delivery, a detailed survey
was given to the producer to be filled out and
returned when birds were collected. The survey
asked for information regarding flock size,
breed, vaccination programs, farm management
Total RNA was extracted from approximately 100 mg of tissue and homogenized in 1
mL of TRIzol [15] and kept at room temperature
for 5 min. After the addition of 240 µL of chloroform it was shaken vigorously and incubated for
10 min. The sample was centrifuged and the
upper colorless aqueous phase was transferred
to a clean tube and the RNA was precipitated
by addition of an equal volume of isopropyl
alcohol. The RNA pellet was dissolved in 10
µL of Rnase-free water and stored at −70°C.
To detect all serotypes of IBV a conserved
region of the genome such as the nucleocapsid
(N) gene was targeted by reverse transcription
(RT) polymerase chain reaction (PCR). The
primer pair used in this study was published by
Handberg et al. [16], and the 2-step method was
followed as described [17]. Briefly, the random
hexamer primed cDNA synthesis was carried
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FIGURE 1. Flow chart of the experimental design. Farm
selection was based on a questionnaire directed to
all commercial layer operations in Ontario. Of the 12
sentinel birds, half of birds were vaccinated against
infectious bronchitis virus (IBV), and the other half was
not vaccinated.
out with Superscript II RNase H reverse transcriptase. The PCR reactions were conducted in
a GeneAmp Applied Biosystems 9600 thermocycler using Invitrogen reagents [15, 18].
The PCR products were analyzed in 0.8%
agarose gels prepared in 1× TAE (40 mM Tris,
5 mM sodium acetate, and 1 mM EDTA, pH 7.8).
The DNA bands were visualized by staining with
0.2 mg/mL of ethidium bromide and viewing on
an ultraviolet transilluminator at 366 nm wavelength using a BioRad Gel Doc 1000 system
[19].
RESULTS AND DISCUSSION
Three hundred sentinel chickens were raised,
and 1,800 samples were collected during this
study. Tissues and pharyngeal swabs were collected from each sentinel bird for testing to determine whether the sentinel birds had become infected with IBV during the farm placement.
FIGURE 2. Agarose gel electrophoresis of reverse
transcription-polymerase chain reaction products using
infectious bronchitis virus (IBV) N-gene specific primers
and RNA extracted from tracheal tissues of sentinel
birds placed on farm B-1. Lanes 1–6: different sentinel
birds, lane 7: known positive sample, lane 8: IBV Mass
42, lane 9: negative control trachea, lane M: 100-bp
ladder.
Diagnostic laboratories usually first isolate
the virus in embryonated eggs and use the allantoic fluid to detect IBV specific RNA by RTPCR. To reduce the time and labor needed for
diagnosis, we first assessed the feasibility of the
N-gene specific RT-PCR to detect virus directly
in tissues without virus isolation. Trachea, lung,
kidney, and cecal tonsils from birds placed on
3 farms (A, Q, E) were analyzed. Representative
RT-PCR results are shown in Figure 2. A sample
was considered positive when the expected size
fragment (450 bp) was observed in the gel. As
expected, IB virus was detected more frequently
in tracheal tissues than in lungs, kidney, or cecal
tonsils. Overall 62.5% of the IBV-positive tissues were from farm A, and only 2 (8.3%) and
3 (12.5%) originated from farms E and Q, respectively (Table 2). In a study carried out by
Handberg et al. [16] IBV was detected in the
tracheas of experimentally inoculated chickens.
In this study we were able to detect the virus in
TABLE 2. N-gene specific reverse transcription-polymerase chain reaction results for RNA extracted from different
tissues for detection of infectious bronchitis virus (IBV)
Farm
A
Q
E
Total
1
Trachea
1
6/6
2/6
2/6
10/18
Lung
Kidney
Cecal tonsils
Total
IBV positive
3/6
1/6
0/6
4/18
2/6
0/6
0/6
2/18
4/6
0/6
0/6
4/18
15/24
3/24
2/24
20/72
62.5%
12.5%
8.3%
Number of positive per number of birds.
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STACHOWIAK ET AL.: INFECTIOUS BRONCHITIS VIRUS SURVEILLANCE
TABLE 3. Statistical analysis of virus detection by Ngene specific reverse transcription-polymerase chain
reaction (RT-PCR) done directly on tissues versus
infectious bronchitis virus (IBV) isolation followed by
RT-PCR evaluated by a 2 × 2 table1
Tissues +2
Tissues −3
Allantoic
fluid +2
Allantoic
fluid −3
Total
22
3
25
0
5
5
22
8
30
1
Sensitivity: 88% (confidence interval: 67.7 to 96.8%);
specificity: 100% (confidence interval: 46.3 to 100%).
2
+ = positive IBV detection.
3
− = negative IBV detection.
field-exposed birds. Although the trachea is the
preferred tissue for virus detection in the case of
acute infection, in subclinically affected flocks,
kidney and cecal tonsils might be a better choice
when testing for persistent infection [5, 20].
The sensitivity and specificity of direct virus
detection in tissues by RT-PCR were compared
to virus isolation followed by RT-PCR. The tracheal tissues were randomly selected from 30
sentinel birds representing farms A, D, E, M, and
Q. For virus isolation samples were passaged in
embryonated specific pathogen-free eggs 3 times
or until stunting or curling of the embryos was
observed, then RNA was extracted from the allantoic fluid and subjected to N-gene specific
RT-PCR. The results and statistical analysis of
the 2 approaches are summarized in Table 3.
Sensitivity and specificity percentages were determined using the EPI Info 6 program [21].
Direct virus detection in tissues was 100% specific and 88% sensitive when compared with
virus isolation followed by RT-PCR. These results were considered satisfactory to proceed
with the larger study.
Of the 402 egg producers throughout southern Ontario, 13 farms participated in the sentinel
bird study. The selected farms were at least 25
km apart from each other and were not centered
in one area. Of the surveyed farms, 11 were
positive by the N-gene specific RT-PCR when
tissues of unvaccinated and vaccinated sentinel
birds were tested after a 1-wk exposure. The
vaccinated chickens were used to evaluate the
protection afforded by the most commonly used
vaccination programs for Ontario broilers, lay-
145
ers, and breeders. Overall, IBV was more often
detected in tracheal tissues of unvaccinated than
vaccinated birds (Table 1). It seemed that the
mild IBV vaccine did not protect the sentinel
chickens against infection. If the unvaccinated
chickens became infected so did the vaccinated
birds, regardless of the IB history of the farm.
The antibody (Ab) level of vaccinated birds may
not have been high enough to protect the chickens from infection. Based on the data of de Herdt
et al. [22], the Ab titers of vaccinated sentinel
chickens in this study were considered relatively
low, partly because of the single vaccination.
Moreover, Gelb et al. [12] demonstrated that
ocular vaccination with the Mass serotype alone
does not prevent IBV infection. However, vaccination with the Mass serotype could reduce the
rate of mortality because this serotype provides
some heterologous protection against other IBV
strains [23]. Indeed, according to this study, the
clinical signs were less severe in vaccinated
birds than in unvaccinated birds placed on farm
A. Clinical signs of IB were observed at the time
of sentinel bird placement in this layer flock.
The IBV infection is highly contagious, and
the virus is easily transmitted [2]. Indeed the
sentinel birds exposed to the layer flocks (farms
A and B-1) with clinical signs contracted the
virus. Also, farms with outbreaks less than 3 mo
(farms D and E) prior to sentinel bird placement
were positive by N-gene specific RT-PCR. IBV
can establish a persistent infection, and the virus
can be isolated from flocks up to 39 d postinfection [5]. Moreover, IBV can survive in fecal
material [1], permitting infection of susceptible
birds. Farms with a history of IB outbreaks could
harbor the virus without exhibiting clinical signs
of the disease or presenting problems as was
shown by our study. Farms B-2, B-3, M, and X
did not report problems associated with IB, yet
IBV was detected on these farms by the N-gene
specific RT-PCR.
Detection of IBV with N-gene specific RTPCR cannot differentiate a vaccine strain from
field virus isolates. Therefore, the next step will
be to partially characterize these viruses by using
an S1-gene specific primer in the PCR reaction
followed by restriction fragment length polymorphism and sequence analyses [17].
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CONCLUSIONS AND APPLICATIONS
1. The main advantage of IBV detection by direct tissue extraction was increased speed of obtaining
results as opposed to virus isolation from eggs. The detection of IBV could be reduced from
weeks to 1 d.
2. The cDNA generated for N gene specific PCR could be used in the PCR with S1-gene specific
primers so the IBV isolate can be quickly characterized. Nevertheless, virus isolation from
specific pathogen-free eggs cannot be eliminated. Infectious virus may be necessary for additional
tests, for example, to evaluate the efficacy of vaccine protection or to assess the serotype of
the isolate. The combination of the 2 virus detection systems could be the optimal diagnostic tool.
3. Use of vaccinated and unvaccinated sentinel birds was shown to be highly effective as a means
of identifying IBV-positive barns regardless of whether they had a history of IB-related signs.
4. Knowledge of the persistence of the virus in these barns in the absence of detectable clinical
signs is of great importance to the development of effective long-term vaccination programs in
individual barns and in multiple-barn complexes.
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Acknowledgment
This work was supported by the Ontario Egg Producers, the
Ontario Ministry of Agriculture and Food, and the Poultry Industry
Council, Ontario, Canada. We thank Sophie St-Hilaire (Department
of Population Medicine, University of Guelph) for her advice on
statistical analysis and Helena Grgic (Department of Pathobiology,
University of Guelph), and David Bridle (Isolation Unit, University
of Guelph) for their assistance in the experimental work.
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