No.

23 / 2000, page 21

DETECTION OF INFECTIOUS BRONCHITIS VIRUS

Dr J.J. De Wit (Deventer, The Netherlands)

Introduction A complicating factor from a diagnostic point of view

is the question whether and if, in which organ(s), a
Infectious bronchitis virus (IBV) is a major cause of persistent carrier state for vaccines and/or field viruses
economic losses in poultry and can be involved in respi- can develop. The two candidate sites mentioned for
ratory disease, nephritis, and both poor egg production persistence are caecal tonsils and kidney. Although
and quality. However, these signs are not specific to IBV. further studies are needed to know the nature of long-
Therefore, diagnostic tools are needed to identify IBV term infections and re-excretion, the phenomenon compli-
infections in relation to a clinical problem in the field. This cates the interpretation of IBV detection: The virus that
may also include typing of the isolate involved in order to is detected from an IBV suspected flock having been
enable the choice of a vaccination programme with the vaccinated or infected before, does not necessarily infer
best chance of achieving sufficient protection against an a recent infection. Therefore, even when IBV is detected
IBV infection in the next flock. in an IBV suspected flock, it is important to exclude other
possible (infectious and non-infectious) causes of the
In general, acute IBV infections can be diagnosed by disease.
detection of IBV virus (antigen) itself or the specific anti-
body response. The most common assays for routine
use of virus detection are virus isolation (VI), immunofluo- Level of immunity in chicken at the moment of infection
rescence assay (IFA), immunoperoxidase assay (IPA), The level of acquired immunity (by vaccination of previous
polymerase chain reaction (PCR), and for antibody detec- infection) at the moment of infection has a major influ-
tion the haemagglutination inhibition (HI) test, agar gel ence on the time and amount of IBV that can be detected
precipitation test (AGPT), and enzyme linked immuno- (Figure 1 and 2). Experimental IBV infections in vacci-
sorbent assay (ELISA). The virus neutralisation test (VNT) nated and unvaccinated birds show that homologous
is rarely used for routine diagnosis because it is relatively challenge virus is detected during a much shorter period
expensive and laborious. and at much lower (factor 102 - 104) levels than in unvac-
cinated chickens. The period during which IBV can be
Choosing between tests and subsequent interpretation of isolated from the trachea after a homologous infection of
the results can be very difficult and confusing and is quite vaccinated birds can be limited to a few days instead of
often thwarted by the poor documentation of the perform- a few weeks after infection of unvaccinated birds.
ance of tests after IBV infections in the field. Depending
on the demands and local circumstances, a best choice These data show, that for attempting to detect IBV field
or best combination of techniques for that situation can virus from vaccinates, it is important to sample at an early
be made. This paper attempts to present the current stage of the infection and to use a very sensitive test.
possibilities for detecting IBV infections, including discus-
sion about the possible advantages and disadvantages Number, choice and quality of sampled organs
of the available tests.
In the acute phase of an IBV infection of unprotected
chickens, many birds will obtain high amounts of IBV in
Detection of IB virus the trachea, so only a few birds have to be sampled.
However, in case of chronic infections or infections in
Factors influencing the success of IBV detection vaccinated birds, like layers and breeders, only low
amounts of virus may be present in only a low percentage
The level of success in detection of IBV after a disease of the birds. Detection requires the sampling of respira-
outbreak is influenced by many factors of which the time tory tract, kidney, caecal tonsils and cloaca of many more
between onset of infection and sampling, the level of birds.
immunity in the chicken at the moment of infection, and
the number, choice and quality of sampled organs are the To minimise the risk of inactivation of IBV in a carcass or
most important. sample, because of its limited thermostability, chilling of
the material to 4C should be done as soon as possible.
If virus isolation (VI) is to be attempted within a day no
Time elapsed between onset of infection and sampling other storage precaution is necessary. For longer storage
The upper respiratory tract is the primary site of IBV repli- the samples should be frozen at below -20C as soon as
cation, leading to viraemia and dissemination of the virus possible. If freezing is not possible the tissues should be
to other tissues (McMartin, 1993; Dhinakar Raj & Jones, placed in special media and glycerol (Cumming, 1969).
1997). All IBV strains can be isolated from the respiratory Under these conditions IBV will remain viable for many
tract, with the highest concentration of IBV in the trachea days, even if refrigeration is not available (McMartin,
during the first 3 to 5 days after infection. After this period, 1993).
the virus titre drops rapidly in the second week post infec-
tion to below the detection level.
Virus isolation (multiplication and / or detection of
When chickens are sampled in the chronic stage of an infectious IBV)
IBV infection, one is more likely to isolate IBV from the
intestinal tract (caecal tonsils or cloaca swabs) than from Virus isolation (VI) can be laborious, time-consuming and
the trachea (Cook, 1968, 1984; Alexander & Gough, costly. Increasingly, the classical way of isolation, that
1977; Alexander et al., 1978; El-Houadfi et al., 1986; De is giving a number of embryo passages until dwarfing,
Wit et al., 1998). curling or embryo mortality occur, is more often replaced
No. 23 / 2000, page 22

Figure 1: Average virus excretion by M41 infected reproducible and specific product. A disadvantage of
unvaccinated broilers (Avian Pathology, 27, using a Mab to detect IBV antigen, or more accurately,
464-471) a certain epitope, is that a mutation of only one nucle-
otide, resulting in a different amino acid in the epitope
with which the Mab reacts, can prevent binding of the
Mab to that epitope. Depending on the Mab used, this
would suggest either that the strain is not IBV or is not of
that serotype. This problem can be reduced by choosing
Mabs against highly conserved or essential areas of the
virus, or by using a mixture of Mabs. The risk, that one
missing or changed epitope will cause a false negative
result in an antiserum-based test is much smaller, when
polyclonal antisera are directed against more epitopes.

The available tests for detecting IBV antigen will now be

considered in turn.

Agar-gel precipitation test (AGPT)

The group-specific AGPT can be used for the detection
of antigen. The test is very cheap, fast and requires few
Figure 2: Average virus excretion by M41 infected laboratory facilities. For best results, several antisera or
H 120 vaccinated broilers (Avian Pathology, an antiserum at different dilutions should be used, to
27, 464-471) prevent false negative results caused by imbalance of the
antigen: antiserum ratio (Lohr, 1981). Although the test
has an image of poor sensitivity, the published data using
the test directly on organs suggest that it is not lower than
that of recent techniques when used directly on organs.
Another application of the AGPT is for confirmation of
the presence of IBV in inoculated eggs (allantoic fluid or
CAM). In this situation, the sensitivity will be increased
due to the replication of the virus.

Immunofluorescent assay (IFA)

The IFA is a relatively cheap and fast technique for
detecting IBV antigen in chickens, eggs, TOCs and cell
culture. The IFA is group-specific when using polyclonal
anti-IBV serum or group-specific Mabs. When using type-
specific Mabs, the IFA can be a type-specific test (De Wit
et al, 1995).

The interpretation of the fluorescent reactions may be

by a combination of a shortened isolation procedure for complicated by non-(IBV)-specific reactions. The specifi-
multiplication of the virus and a second technique, such city of IFA can be improved by using Mabs instead of
as IFA, antigen ELISA or PCR for antigen detection. By hyperimmune anti-IBV serum (Yagyu & Ohta, 1990; De
using the combination of isolation and other techniques, Wit et al., 1995). Protection of the epithelium by reducing
the procedure can be shortened with maintenance of storage time (at low temperatures) between sampling and
sensitivity. fixation will also improve the specificity of the IFA.
Field strains of IBV can be isolated using embryonated
Because, unlike with VI, the antigen is not replicated
(SPF) eggs or trachea organ cultures (TOCs). In none of
before detection, the amount of antigen must be large
these systems, IBV causes specific lesions (McMartin,
enough to be detected by staining. Therefore, depending
1993). Therefore, the presence of IBV antigen has to be
on the test material, the sensitivity can vary from equal
confirmed by an IBV antigen detection method.
(first week of infection in unprotected birds) to or much
less than VI (chronic) infection in vaccinated birds.
Detection of IBV antigen
The IFA can also be used for detection of IBV antigen in
embryonated eggs, cell cultures, and TOCs.
The techniques that are suitable for detection of IBV-
specific antigen use IBV-specific antibodies. These anti-
bodies are either in the form of antisera or monoclonal
Immunoperoxidase assay (IPA)
antibodies (Mabs). Antisera are from an animal that was
infected with IBV or injected with certain parts of the virus. The IPA technique is, like the IFA, a staining technique
Consequently, antisera may contain antibodies against with a comparable sensitivity to that of IFA. After fixation
different parts of the virus. Standardisation and tech- and embedding of sections of organs or tissues, an
nical performance of antisera is hampered by the in anti-IBV specific peroxidase-labelled conjugate binds
vivo biological variation of the infected animal and virus. with and stains the antigen in the sample, after addition
Since a Mab only reacts with one or a small number of of the substrate and chromogen. Advantages of IPA
epitope(s) of the IBV antigen, it provides a well-defined, compared to IFA are, that IPA allows evaluation of antigen-
No. 23 / 2000, page 23

bearing cells as well as general tissue morphology. Also, tions, making a clear classification not always possible.
evaluation of the slides can be done in day-light using
a normal microscope, and storage of the slides is easy Classification systems are divided into two major groups:
because of the stability of the staining, in contrast with functional tests, that regard the biological function of
IFA, where the staining fades when not stored dark and a virus, and non-functional tests that look at the viral
frozen. Disadvantages of IPA compared to IFA are that genome. Typing by functional tests results in immu-
the technique is more laborious, takes a few days and is notypes or protectotypes, and antigenic (serotype or
sensitive to non-specific background staining by endog- epitope) types. Tests that look at the genome result in
enous peroxidase that is naturally present in the sample. genotypes. The preferred typing system depends on the
This endogenous peroxidase has to be removed during goal (e.g. selection of vaccination programmes, or epide-
the process of performing the IPA. miology), available techniques, experience and costs.

Antigen ELISA
Immunotypes or protectotypes
Because of the high amounts of virus that are needed for
detection by antigen ELISA, the sensitivity for detecting
Grouping of IBV strains into immunotypes (Cunningham,
IBV antigen directly in chicken organs is low. Elisa is more
1975) or protectotypes (Lohr, 1988; Hinze, et al., 1991) is
suited as confirmation test for detecting IBV antigen in
the most important system from a practical point of view,
allantoic fluid of inoculated eggs, especially when a large
because it provides direct information about the efficacy
number of samples has to be tested.
of a vaccine. Strains that induce protection against each
other, belong to the same immuno- or protectotype. The
number of protectotypes that exists is unknown, but
Detection of the IBV genome
cross-challenge experiments in chicken tend to reduce
the number of protectotypes types compared to sero-
Techniques that detect all or part of the IBV genome may
types, presumably because they are measuring the
be used for IBV detection. Although one- and two-step
complete immune response and not just a part of it
procedures are reported, the detection of genomic RNA
(McMartin, 1993).
is usually a three step procedure. The first step is repli-
cation of the virus from the (field) sample using embryo-
To determine the protectotype of a strain, a cross-immu-
nated eggs or TOCs. Subsequently, the genomic RNA is
nisation study (CIS) has to be performed. A CIS is labour-
translated by reverse transcriptase (RT) into copy-DNA
intensive, expensive and requires many animals and
(cDNA) and multiplied many times by polymerase chain
isolation facilities. A more economical in vitro alternative
reaction (PCR). The last step is classifying the strain into
for the CIS may be the cross-immunisation test (CIT). In a
a genotype using a third technique (see later).
CIS, vaccinated birds are challenged in different groups
with different strains. When performing a CIT, the chal-
Reverse transcriptase polymerase chain reaction lenge of vaccinated chickens is performed on TOCs of
(RT-PCR) those birds, testing the tracheal cross-immunity. Because
one bird provides many tracheal rings, each bird can be
A technique increasingly used is the reverse transcriptase
challenged with a number of different isolates. Although
polymerase chain reaction (RT-PCR). The sensitivity of
TOCs simulate the chicken better than most in vitro
the RT-PCR is usually low when performed directly on
methods, there might be differences. When the challenge
organs. Therefore, IBV is often first multiplied in embryo-
is done in chickens, the whole immune system is avail-
nated eggs or TOCs before the RT-PCR is performed.
able, whereas in TOCs, this is probably not the case.
The RT-PCR product is to be identified as originating from
Whether CIT is really a reliable alternative for the CIS
IBV by another technique such as sequencing, restriction
needs further work, preferably in comparative studies
enzyme fragment length polymorphism (RFLP), or hybridi-
using different strains of different tropism.
sation. Further, use of appropriate controls to check the
performance of the test and possible cross-contamina-
tions are required.
Antigenic types

Serotypes
Strain classification
The classical functional typing system is serotyping by
Typing of IBV strains is useful for implementation of control VN-test, which is based on the reaction between an IBV
measures, for research purposes and for understanding strain and chicken-induced IBV serotype-specific anti-
the epidemiology and evolution of IBVs. Classification of bodies.
strains is hampered by the lack of standardisation of tests
A disadvantage of serotyping is the lack of standardisa-
used world wide, use of different names for the same type
tion between the different systems and users. The results
of virus, the number of different test systems available
of different laboratories are therefore not always compa-
and, maybe most important, the nature of this virus. A
rable.
typing system suggests that a strain clearly belongs to
only one type. However, the IBV genome consists of
Epitope types (monoclonal antibody)
single stranded RNA with a high mutation frequency.
Molecular studies with IBV have shown that new IBV By incorporating a Mab into the test, it is possible to
serotypes and genotypes can emerge as a result of check whether a certain epitope or a number of epitopes
only a very few changes or mutations in the amino acid is present on a strain. When this (these) epitope(s)
sequence of the spike gene, while the major part of the is (are) related to a certain serotype, it can be used
virus genome remains unaltered. Also, several workers to produce a serotype-specific test such as antigen-
demonstrated or provided circumstantial evidence that ELISAs or IFAs. Use of serotype-specific Mabs, which are
IBV can undergo recombination during mixed infections. directed against hypervariable parts of the S1 protein,
As a result, IBV strains may show multiple cross-reac- has a certain risk of false-negative results. A mutation in
No. 23 / 2000, page 24

the epitope to which the Mab is directed, does not neces- tion of the virus. The correlation between RFLP pattern
sarily mean that the strain has changed to another sero- and serotype can be high, but as reported by Hein et
type. So, an IBV virus that does not react with a panel al. (1998), different isolates, typed by RFLP as belonging
of serotype-specific Mabs is preferably re-tested by a to the same genotype, can be of different serotypes or
different technique to check whether another serotype is protectotypes. Therefore, it is recommended that where
involved, or whether possibly one of the epitopes was there is suspicion in the field that the RFLP-genotype
changed by mutation and therefore missed by the panel of recent isolates does not provide accurate information
of Mabs that was used. about the true antigenic nature of IBV isolates, conven-
tional testing and especially in vivo studies are also
Genotypes used.
Grouping of strains based on genetic characterisation of
c: RNase T1 fingerprinting analysis
(a part of) the genome (or its c-DNA) results in genotypes.
Methods include sequencing part(s) of the genome or After multiplication of IBV, extracted and purified viral
determining the position of enzyme cleavage sites (RFLP, RNA is digested with ribonuclease (RNase) T1, resulting
RNase T1 fingerprinting). Genomic information is objec- in oligonucleotides. After staining, the oligonucleotides
tive and provides essential information for epidemiolog- are resolved on two-dimensional gel electrophoresis,
ical studies. A disadvantage of genotyping for use in the resulting in a fingerprint of the genome. The fingerprint
field, is that direct translation of information about a part can be compared with fingerprints of known strains.
of the genome of an IBV strain to biological function or This complex and labour-intensive technique is hardly
antigenicity of the virus is not possible or not without used anymore for IBV because it does not work well
risk. Isolates of the same serotype or protectotype can for genomes with less than 95 % homology (what is not
differ substantially in some genes (Cavanagh et al., unusual for IBV) and shows poor correlation between
1992a), while different serotypes or protectotypes can genotype and serotype.
have remarkably high similarity between their genomes
(Clewley et al., 1981, Kusters et al., 1987; Williams et
al., 1992; Cavanagh et al., 1992a, 1992b). Although Detection of antibodies
high correlations between the genotype and serotype
of strains have been reported, other papers present IBV infections can be diagnosed by detecting the appear-
conflicting data about strains genotype and serotype or ance of, or rise in, antibody titre of IBV specific anti-
even protectotype. bodies. Generally, in order to be able to correlate a
clinical problem with an IBV infection, paired sampling is
Therefore, for practical use in the field, exclusive use
required. The first sample is taken at onset of disease,
of genotyping methods is not recommended. Especially
and the second sampling several weeks later. The paired
when there is suspicion in the field that the genotype
sera should be tested in the same test run, to prevent
of recent isolates does not provide accurate information
wrong conclusions caused by day-to-day variation of the
about the true antigenic nature of IBV isolates, conven-
tests. At least a four-fold rise in titre is required for a posi-
tional testing (serotyping) and especially in vivo studies
tive diagnosis.
are required.
Factors influencing the success of IBV antibody detec-
a: sequencing
tion
Sequencing and subsequent comparison of the amino
acid sequences of viral proteins is a very useful instru- Interpretation of serological results can be complicated
ment to help locating conserved domains in proteins that by a number of factors including presence of immunity
might be essential for their structure and function and at time of vaccination/infection, cross-reactions between
for epidemiological studies. Based on sequence data, serotypes, and occurrence of new or unexpected IBV
a phylogenic tree can be made, revealing the genomic strains. Furthermore, lack of attempts to standardise tests
relatedness between different strains. However, it must be between different laboratories adds to the difficulty in
remembered that the place of a certain strain in a phyl- interpreting results.
ogenic tree can differ depending on the genotyping
techniques used, or on which part of the genome is Presence of immunity at time of vaccination/infection
analysed. Sequence data only provide information about
A very important factor that influences the degree of
the primary structure (sequence of amino acids) of the
humoral response after an infection or vaccination is the
protein. Detected differences in sequence of two strains
immunity derived from previous vaccination or infection
can not be translated to differences in antigenity or
(Gazdzinski et al., 1977; Macdonald et al., 1981; Darby-
biological function. Also the occurrence of recombina-
shire & Peters, 1984, 1985; De Wit et al., 1997). The
tions between different IBV strains hampers the transla-
degree of humoral response after infection in vaccinated
tion of data of a genotype to a serotype or protectotype.
chickens can be decreased and slowed down compared
to the humoral response of unprotected birds (Figure 3
b: restriction enzyme fragment length polymorphism
and 4). As a result, the sensitivity of antibody detecting
(RFLP)
tests can be much lower in vaccinated chickens as
After amplification of cDNA of the S1 gene by PCR compared to unvaccinated chickens.
and purification by electrophoresis, the PCR product is
digested with restriction enzymes that cut cDNA into frag- Cross-reactions between serotypes
ments at certain highly specific cleavage sites. The
A complicating factor in serotyping an IBV infection by
RFLP patterns are then compared with patterns of repre-
serology is the cross reaction which may occur after an
sentatives of known serotypes. This test can provide a
infection. These cross reactions especially occur in birds
fast diagnosis, including typing of the virus. However,
that had multiple contacts (e.g. vaccinations) with IBV
cleavage sites of enzymes used in RFLP are not related
(Table 1), particularly when more than one serotype is
and cannot be translated to biological or antigenic func-
involved (Gelb & Killian, 1987; Karaca & Naqi, 1993; De
No. 23 / 2000, page 25

Figure 3: Average HI and ELISA titre after D8880 Wit et al., 1997).
infection of unvaccinated broilers
For interpreting serological results, these heterologous
cross reactions in serotype-specific tests have to be
differentiated from the homologous response. When the
cross reactions are high, as may be expected in older
birds that have been vaccinated (and probably infected)
several times with different serotypes, interpretation of the
serological data can be very difficult or even impossible.
In those conditions, subjectivity of the interpreting person
is a real danger for the quality of the diagnosis. Obvi-
ously, a particular IB-serotype can only be identified if that
serotype is included in the panel of IBVs tested. If not,
the highest cross reaction may be misinterpreted as the
serotype involved.

Occurrence of new or unexpected IBV strains

When serology is used for serotyping the IBV infection,
a selection of serotype-specific tests will be requested,
based on experience, tradition or availability. Not-
requested serotypes, like new or unexpected serotypes,
will not be included in the requested panel of serotype-
Figure 4: Average HI and ELISA titre after D8880
specific tests. Under these circumstances, there is a high
infection of H120-vaccinated broilers
risk of wrongly appointing the serotype with the highest
cross reactions as being the serotype involved in the
infection. Therefore, to minimise this risk of missing the
correct serotype, serotype-specific tests for all known
serotypes (based on typing of isolates) circulating in
the region should be used. It is important always to
remember this limitation of serotype specific diagnostic
tests.

Tests for antibody detection can be grouped into group-

specific and serotype-specific tests.

Group-specific tests

Group-specific tests for detection of antibodies against

IBV do not differentiate between antibodies induced by
strains of different serotypes. Although the antigen that
is used in the test is of one serotype (usually Massachu-
setts), it also detects antibodies against other serotypes.
The AGPT and ELISA are such tests.

Table 1: Mean VN titres after an IBV infection in non-vaccinated and vaccinated broilers (Avian Pathology,

Group Vaccination Challange Mean VN titre a

with H120 at day 1 with IBV at day 28 M41 D274 D1466 D8880
day day day day
28 49 28 49 28 49 28 49

A1 - - -b - - - - -
B1 - - - - - - - - - -
A2 - M41 - 7.6 - - - - - -
B2 - M41 - 8.3 - - - - -

B5 - D274 - - - 7.1 - - - -
B6 - D1466 - - - - - 9.5 - -
B7 - D8880 - - - - - - - 9.9

A3 yes - 4.1 4.9 - - - - - -

A3 yes - 4.0 4.8 - - - - - -

A4 yes M41 4.4 7.6 - - - - - 4.0

B4 yes M41 4.3 6.9 - - - - - -

A5 yes D274 4.4 6.7 - 6.7 - - - 5.5

A6 yes D1466 - 5.1 - - - 5.3 - 4.1
A7 yes D8880 4.6 6.6 - 5.8 - - - 8.1
a VN titres expressed as log2 of the reciprocal of the highest serum dilution that showed complete inhibition of CPE.
b VN titre <4
No. 23 / 2000, page 26

The HI-test is like the VNT a serotype-specific test when

Agar gel precipitation test (AGPT) used to detect antibodies after a single inoculation with
IBV. Although the correlation between both test systems
Performing the group-specific AGPT requires almost no
is high under these circumstances, the specificity of the
laboratory facilities. With this test, inclusion of a positive
HI is considered to be lower than that of the VNT (King &
control serum adjacent to the well with test serum is
Hopkins, 1983; Gelb & Killian, 1987; De Wit et al., 1997).
important to be able to differentiate non-specific precipi-
tation bands from IBV-specific bands (Woernle, 1966).
The serotype-specificity of the HI-test is much lower
Because of its ability to differentiate non-specific from
following re-infection with IBV, especially when the second
specific reactions, the AGPT has a high specificity,
or subsequent serotype is heterologous (King & Hopkins,
although the precipitation lines for IBV are more difficult
1983; Gelb & Killian, 1987; Brown & Bracewell, 1988;
to read then for other agents like Gumboro or Newcastle
De Wit et al., 1997). Sequential inoculation with IBV can
virus. Although the test is relatively simple, there is an
induce antibodies that react in HI test with serotypes to
absolute lack of standardisation of the test for IBV.
which the birds had not been exposed.
Precipitins are usually detected about a week post-infec-
Serotype-specific ELISA
tion or vaccination, usually followed by a fast decrease
of percentage of positive samples a few weeks later, A new approach of producing serotype-specific tests was
although extended periods are reported. Because of the tried by Karaca & Naqi (1993) by using serotype-
transient reactions of the AGPT, positive results indicate specific Mabs as blocking agent in two blocking ELISAs.
a recent contact with IBV. Whereas the results after primary inoculation seemed to
be serotype-specific, heterologous cross reactions were
The reported sensitivity of the AGPT varies from 0 to
detected by both the blocking ELISAs after a secondary
100 %, depending on the IBV strain inoculated, applica-
inoculation with the homologous virus.
tion route, age of infection, presence of MDA or vaccinal
immunity at the time of challenge, and test performance.
Relative sensitivities of AGPT, ELISA, HI-test and VNT
Therefore, use of a non-validated AGPT is not recom-
mended. Many studies have compared the performance of two
or more serological tests. Due to the many variations
Group-specific ELISA in experimental conditions, test performance and kind
of antibodies that are detected by the different tests,
Reports of indirect ELISAs show them to be group-
different results are reported. But generally, antibodies
specific. Antibodies can first be detected by ELISA within
can first be detected by ELISA, followed by AGPT and
a week after vaccination or infection. Because of the short
HI-test, and last by VNT.
period between infection and the detection of the first
antibodies by ELISA, the first of paired sampling must
be done at the first signs of IBV, which usually appear
Last recommendations for use of serology
between 18 and 36 h after infection. If the first sampling
is not done in time, seroconversion can be missed.
The data of all serotype-specific antibody tests show that
the presence of antibodies against a certain serotype
-IBV-IgM ELISA
in vaccinated chickens is not necessarily a proof of an
IBV-specific IgM is only present temporarily after an infec- infection caused by that serotype. Therefore, one should
tion or vaccination. Therefore, its detection is indicative be very cautious in concluding that a new serotype is
for a recent infection or vaccination. However, the reports present in a region, only on the basis of detecting anti-
regarding IgM detection after IBV vaccination and infec- bodies by a serotype-specific test in sera of vaccinated
tion are limited and partly conflicting. Therefore, more chickens. Detection of the virus itself is more reliable.
data collected under different conditions are needed When serology is used for screening a region for the
before IgM detection techniques are suitable for use in presence of a certain serotype of IBV, testing by VNT of
the field. sera of unvaccinated chickens is preferred.

Serology is better suited to monitor the presence of

Serotype-specific tests already known serotypes (clinical or subclinical infec-
tions), especially in younger birds. When group specific
Serotype-specific tests for detection of antibodies against tests like AGPT or ELISA detect IBV infections without
IBV differentiate between antibodies induced by strains clear titres in serotype-specific tests, possibly a new sero-
of different serotypes. type has arrived. For older birds that have been vacci-
nated (and maybe infected) several times using vaccines
Virus Neutralisation test (VNT) of different serotypes, serology is far better suited to
monitor the presence of IBV infections in general than
The VNT is the gold standard test for the detection of IBV
being able to serotype them. Again, detection and typing
serotype-specific antibodies. The specificity is very high
of the virus itself is preferred in these circumstances.
after a single IBV inoculation (Table 1). Cross-reactions
can occur after multiple contacts with different IBV
Use of serology to check the take of vaccines often
serotypes (Gelb & Killian, 1987; De Wit et al., 1997),
requires very sensitive tests that are properly validated
although some studies also report low cross reactions
for that purpose. Lack of detectable amounts of vaccine-
after repeated inoculations with the same serotypes
induced antibodies do not prove that their is no vaccine-
(Karaca & Naqi, 1993).
induced protection, as this can be local protection.
Haemagglutination inhibition (HI) test
The first haemagglutination inhibiting antibodies are
References
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No. 23 / 2000, page 27

De Wit, J.J., Jong, M.C.M. de, Pijpers, A., & Verheijden,

Alexander, D.J., & Gough, R.E. (1977). Isolation of J.H.M. (1998). Transmission of infectious bronchitis
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infected chickens. Research in Veterinary Science, chickens. Avian Pathology, 27, 464-471
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Dhinakar Raj & Jones (1997). Infections bronchitis virus:
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