avian infectious bronchitis

The domestic fowl is considered to be the natural host for IBV. However, IBV-like viruses infect other avian species. Coronaviruses with and without serological identity to IBV have been isolated from pheasants, and are associated with respiratory disease and nephritis (Lister et al., 1985; Gough et al., 1996). The gene sequences of the pheasant viruses are distinct from but clearly related to those of IBV from chickens. A pheasant isolate has been propagated in the allantoic cavity of domestic fowl embryos but it did not cause clinical disease when inoculated intranasally into three-week-old chicks (Lister et al., 1985). Some isolates of turkey coronavirus (TCoV) are very closely related to IBV (Guy, 2000) but have an enteric tropism. Therefore, TCoV is considered to be a species distinct from IBV. However, domestic fowl chicks have been experimentally infected with TCoV, the virus growing in the same tissues as in turkey. It is highly likely that other avian species have coronaviral infections.

Probably all breeds of domestic fowl are susceptible to IBV although the severity of the disease may vary. The disease is most severe in chicks, not only in terms of respiratory disease but also with respect to damage of kidneys and oviducts. In intensively reared birds, mortality is highest in broilers, secondary bacterial infection with Escherichia coli, other bacteria and mycoplasmas being the primary cause of mortality. The extent of losses is made worse by poor litter quality and poor ventilation, and nephritis may be exacerbated by food composition and temperature changes (Cumming, 1969; Meulemans and Berg, 1998).

IBV is distributed worldwide (de Wit et al., 2011a).

For current information on disease incidence, see OIE's WAHID Interface.

Clinical Diagnosis

The observations described in 'Disease Course' and 'Pathology' are suggestive of IB but are not diagnostic. Laboratory tests are required for definative diagnosis.

Differential Diagnosis

Several respiratory pathogens, including combinations thereof, may produce clinical signs similar to those described for IB. These include mild strains of Newcastle disease virus (Alexander, 1997), infectious laryngotracheitis virus (Bagust and Guy, 1997) and Haemophilus paragallinarum, causing infectious coryza (Blackall et al., 1997). The latter produces facial swelling that is rare in IB, although it is does occur as a consequence of secondary infection with coliform bacteria. Mycoplasma infections can also cause facial swelling. Egg drop syndrome (EDS) adenovirus (McFerran, 1997) produces a drop in egg production and quality similar to that caused by IB, but EDS virus does not affect internal egg quality and does not produce misshapen or ridged eggs. Kidney changes following infection by nephropathogenic strains of IBV may also resemble those caused by infectious bursal disease, mycotoxicosis and drug toxicities (Gelb, 1989).

Laboratory Diagnosis

Laboratory diagnosis is essential for confirmation of IBV infections. Ideally virus isolation (VI) should be performed, followed by tests, firstly to confirm that IBV is present, and secondly to identify the type of IBV. There are numerous ways of achieving all of these stages, each with their own advantages and disadvantages, and it is important to understand the strengths and weaknesses of the various techniques. De Wit (2000) has written a detailed, thoughtful and fully referenced review of all the procedures that have been tried, which have also been reviewed by Cavanagh and Gelb (2008). A brief discussion of the tests, supplemented with recommended protocols (the haemagglutination (HA) and HA inhibition (HI) test) has been produced by the Office Internationale Epizooties; this can also be accessed through the Internet (OIE, 1996).

Fowl can become persistently infected with IBV, which may be detected weeks or months later by virus isolation (Jones and Ambali, 1987). Isolation of IBV from older birds is not in itself, therefore, necessarily proof of the virus being responsible for any clinical disease or production losses that may be occurring at the time of detection. It is necessary to exclude other infections or non-infectious causes (De Wit, 2000).

Detection of IB virus/antigen/RNA

Collection of samples

At the acute stage of the infection (up to 5 days post-infection) live virus isolation is conventionally attempted by swabbing of the trachea or from trachea and lung tissue. Material should be conveyed to a laboratory on ice in transport medium containing penicillin (10,000 IU/ml) and streptomycin (10 mg/ml). Beyond one week after infection IBV is best sought from caecal tonsils or by cloacal swabs (Jones and Ambali, 1987). Kidney or oviduct material might be sampled in cases of nephritis and decreases in egg production, respectively.

If a live IBV isolate is not required and direct detection by reverse transcriptase polymerase chain reaction (RT-PCR) is to be attempted then swabs of the buccal cavity or oropharyngeal region may be made, allowed to dry, and then sent to the laboratory by mail at ambient temperature (Cavanagh et al., 1999). When broiler flocks are naturally infected with IBV in the field, the virus may be detectable in mouth swabs for several weeks by RT-PCR, the infection gradually working through the flock (Cavanagh et al., 1999).

Virus isolation/propagation

Virus isolation (VI) can be performed in embryonating eggs, tracheal organ cultures (TOCs, from 19-day-old embryos; Cook et al., 1976) or cell cultures (chick kidney or chick embryo kidney; Gillette, 1973). Cell cultures are not frequently used because field isolates would have to be adapted to grow successfully in them. Field strains will grow readily in eggs (9 to 11-day-old embryos, inoculation into the allantoic cavity; OIE, 1996) although several passages might be required to observe malformation of the embryo. TOCs have the advantage that field strains produce ciliostasis, easily seen by low power microscope, without adaptation.

Conventional isolation using embryos involves incubation with field material for 5-7 days, followed by several further passages with observation for malformation of the embryo (Cavanagh and Naqi, 1997). On initial infection a field strain would be expected to produce dwarfing of a few embryos, the percentage increasing with passage number, possibly with increasing mortality. Dwarfed embryos have deformed feet over the head with the thickened amnion adhered to it. Allantoic fluid is collected for further analysis. A shorter procedure involves the incubation of inoculated embryos for about 2 days (possibly repeating this once or twice), followed by testing for the presence of IBV by immunofluorescent antibody staining of cells sedimented from the allantoic fluid (Clarke et al., 1972), antigen capture ELISA (Naqi et al., 1993; Ignjatovic and Ashton, 1996) or RT-PCR (Andreasen et al., 1991; Adzhar et al., 1997).

Detection methods

Agar-gel precipitation test (AGPT)

This is simple, quick, inexpensive and more sensitive than is sometimes supposed. Lohr (1981) used the AGPT to detect IBV in tracheal exudates of birds from the field, sensitivity being half that of VI. AGPT is also used to detect IBV after growth in embryos, using allantoic fluid or the chorioallantoic membrane as the source of antigen (Alexander and Gough, 1977; Gelb et al., 1981; MAFF, 1984).

Immunofluorescent antibody (IFA)

This can be used to demonstrate the presence of IBV in whatever medium the virus has been grown. Direct detection by IFA of IBV in tissues from chickens can be complicated by non-specific reactions (De Wit, 2000). Bhattecharjee et al. (1994) used IFA in a very simple way to demonstrate the presence of IBV within 24 hours of inoculation of TOCs; the IF procedure was performed without fixation of the TOCs.

Immunoperoxidase antibody (IPA) staining can also be performed, with sensitivity similar to IFA.

Antigen capture ELISAs

These have not been very successful with tissue from infected birds but have been used successfully following propagation of virus in embryonated eggs (Ignjatovic and Ashton, 1996). These procedures generally require a non-type specific monoclonal antibody.

Haemagglutination (HA)

Treatment of IBV with neuraminidase to produce HA activity is normally done after concentration of the virus from allantoic fluid by ultracentrifugation. Ruano et al. (2000) have recently reported that treatment of unconcentrated infectious allantoic fluid with neuraminidase type V (from Clostridium perfringens, Sigma Chemical Company, Cat. No. N-2876) to produce IBV HA antigen. They mixed allantoic fluid with an equal volume of neuraminidase (at 1 unit/ml) for 30 min at 37°C. The HA test was performed on a ceramic plate by mixing 50 µl of neuraminidase-treated allantoic fluid with 50 µl of a 5% suspension of chicken red blood cells. HA occurred within a minute.

RT-PCR

This has been used either directly on RNA extracted from tracheal/mouth/oropharyngeal swabs (Li et al., 1993; Jackwood et al., 1997; Cavanagh et al., 1999; Worthington et al., 2008) or extracted from infectious allantoic fluid after propagation in the laboratory (Lin et al., 1991; Andreasen et al., 1991; Kwon et al., 1993; Adzhar et al., 1996, 1997). Flinders Technology Associates (FTA) cards have been used successfully for the collection, inactivation and transport of IBV samples (Moscoso et al., 2005).

As there are many types of IBV it is advisable, especially in the early stages of an investigation of infection in a region, to use RT-PCRs designed to detect many types of IBV, using so-called 'universal' oligonucleotides i.e. those corresponding to highly conserved sequences in the IBV genome (Lin et al., 1991; Kwon et al., 1993; Adzhar et al., 1996; Keeler et al., 1998; Handberg et al., 1999; Cavanagh et al., 1999). It is advisable to have more than one set of 'universal' oligonucleotides, corresponding to different genes, in case one set is not appropriate for a given type of virus (Adzhar et al., 1996).

The DNA product of the RT-PCR can be studied further to type an isolate (described below 'Typing IBV isolates').

Detection of antibodies to IBV

Detection of antibodies to IBV is frequently used as part of disease surveillance and for monitoring vaccination. Ideally one should have paired sera, collected before and after a suspected IB infection, especially as birds will probably carry IB antibodies following vaccination.

Agar-gel immunodiffusion test

Several dilutions of antibody may be required to prevent false positives caused by imbalance of the antigen-antiserum ratio (Lohr, 1981). The test is also liable to yield inconsistent results, as the presence and duration of precipitating antibodies may vary with individual birds (Gough and Alexander, 1977; OIE, 1996). This test is perhaps best used for detecting the presence of virus after laboratory propagation, rather than antibody.

ELISA

ELISA is much more sensitive than AGPT and does not suffer from the problems mentioned above. ELISAs can be purchased commercially or prepared (Garcia and Bankowski, 1981; Mockett and Darbyshire, 1981; Snyder and Marquardt, 1989), provided there is access to high-speed centrifugation to concentrate the virus. IB antibody can be detected within one week of infection, so the first set of paired sera must be taken immediately after infection is suspected. As with AGPT, ELISA is a group-specific test, i.e. it may be used for many types of IBV, as different types of IBV will have epitopes in common. Chen et al. (2011) have described a type-specific blocking ELISA for the detection of IBV antibodies.

Virus neutralisation (VN) and haemagglutination-inhibition (HI) tests

VN and HI tests are less suitable than ELISA for the detection of infection by IBV, because both tests are susceptible to serotype differences; antibodies from a fowl may not give VN or HI with a standard reference strain of IBV if they are of different serotypes. These tests are best used when IB has been confirmed or strongly suspected and the typing of the causative strain is required (see 'Typing of IBV isolates'). Notwithstanding, HI antibodies are usually more cross-reactive than VN antibodies, so the HI test, using a standard antigen preparation against one type of IBV, has a reasonable chance of detecting antibodies following a field infection. As most fowl are vaccinated against IB on one or more occasions, cross-reactive antibodies are likely to be encountered following an anamnestic response induced by a field infection.

The HI test with field sera is best used in conjunction with a panel of laboratory strains representing the IBV types most likely to be present in a region.

The procedures for performing the IBV HI test are described in detail by Alexander et al. (1983), King and Hopkins (1984) and OIE (1996). A complicating factor is that IBV does not haemagglutinate spontaneously; pre-treatment with an enzyme preparation is necessary. This can be done with a commercially available phosholipase C type 1 enzyme, from Clostridium welchii. However, results can be erratic, because the active agent for producing HA activity is a contaminant (probably a neuraminidase), whose presence is variable, rather than phosholipase (Schultze et al., 1992). It has been found to be efficient to use crude filtrate from a Clostridium welchii culture as a source of the active enzyme (OIE, 1996).

Typing IBV isolates

There are many types of IBV; dozens have been described and there are probably a greater number still to be discovered (de Wit et al., 2011a; Worthington et al., 2008). New genotypes have been reported in China (Liu and Kong, 2004; Liu et al., 2009; Ma et al., 2012) and in countries where previously data was lacking e.g. Egypt (Abdel-Moneim et al., 2012), and Iraq (Mahmood et al., 2011). The QX serotype, first described in China (Wang et al., 1998), spread to many countries, where it became economically important (Worthington et al., 2008; de Wit et al., 2011a, b; Abro et al., 2012).

Typing can be performed with antibodies (antisera or monoclonal antibodies) in VN or HI tests or, more commonly, by using nucleic acid technology.

Typing with antibodies

Virus neutralization tests

The VN test is expensive and impractical for routine use, as the tester needs to have access to a panel of many IBV types, given the considerable antigenic diversity exhibited by the virus. Furthermore, IBVs that are extremely similar at the sequence level may behave as different serotypes if the changes affect key VN epitopes (Cavanagh et al., 1992a; Cavanagh et al., 1992b). VN tests can be performed on embryonating eggs, tracheal organ cultures (TOCs) or cell cultures. The constant virus (for example, 100 infectious units)-varying serum method is most commonly used (OIE, 1996).

Haemagglutination-inhibition test

The HI test is widely used to type field isolates, and is a valuable tool. However, it is somewhat subject to interpretation by the diagnostician, because of cross-reactions. Conversely, serum raised against a single inoculation of IBV may be strain rather than type-specific (King and Hopkins, 1983; Brown and Bracewell, 1985, 1988). An isolate may be tested against a panel of antisera raised against types of IBV known or suspected to be in an area.

A serum might show HI activity with more than one virus of the panel but show a higher titre with one, tentatively indicating the identity of the field virus. However, it is possible to wrongly assign an isolate to a particular serotype in this way, the serum simply cross-reacting more strongly with one virus type in the panel than the others. If a new type of IBV is suspected, then the virus should be isolated and a type-specific antiserum raised in specific-pathogen free (SPF) birds using a single infection (Gelb, 1989); this should then be used in HI tests with future isolates.

Monoclonal antibodies (Mabs)

Panels of monoclonal antibodies have been used successfully to identify types of IBV but only on a limited scale (Koch et al., 1986; Karaca et al., 1992; Karaca and Naqi, 1993). This is because of the very large number of IBV types and the time and cost of making monoclonal antibodies to new types.

Typing with nucleic acid technology

One approach to the use of RT-PCR is to use universal oligonucleotides designed to bind to the RNA of many, if not all, types of IBV (Lin et al., 1991; Kwon et al., 1993; Adzhar et al., 1996; Keeler et al., 1998; Handberg et al., 1999; Cavanagh et al., 1999; Jones et al., 2005; Worthington et al., 2008). DNA products can be analyzed by nucleic acid sequencing to characterize isolates. Alternatively, when the presence of a particular type of IBV in a region has been established then type-specific RT-PCRs might be used. Sequencing can then be used to put IBVs into epidemiological and phylogenetic contexts.

Nucleic acid sequencing

This is the 'gold standard' amongst nucleic acid approaches to IBV characterization. The S1 gene is generally chosen for typing IBV as it is the S1 protein that is the inducer of protective immunity, and is the major protein in which mutations occur that in turn helps a variant to avoid immunity induced by other types.

Type-specific RT-PCRs

When the existence of one or more types of IBV (including vaccinal strains) in a region has been established and they have been characterized by sequencing it should be possible to design oligonucleotide primers specific for each type. The oligonucleotides should be chosen such that the different types of IBV produce DNA products of different sizes, which can be easily differentiated by agarose gel electrophoresis. Subsequently field isolates (or simply RNA isolated from swabs) can be identified by RT-PCR without further routine analysis (Cavanagh et al., 1999). However, occasionally DNA should be analyzed by sequencing. The presence of two or more types of IBV in the same sample can be easily distinguished (Cavanagh et al., 1999).

Any RT-PCR strategy for detecting and differentiating IBV must be flexible. One cannot rely solely on type-specific RT-PCRs. By definition, if a type of IBV new to a region were to be present, it would not be detected. Other approaches should also be used, for example, RT-PCR with universal oligonucleotides followed by sequencing (Capua et al., 1999; Cavanagh et al., 1999; Worthington et al., 2008; de Wit et al., 2011a).

A type-specific RT-PCR might not be sufficiently specific; the oligonucleotides may 'cross-react' with other types of IBV (Capua et al., 1999).

Immunology of the disease

Dhinakar Raj and Jones (1997) have reviewed the immunology of IB. Infection with IB results in the production of antibodies in serum which are detected much earlier by ELISA than by a VN test (Mockett and Darbyshire, 1981). Immunoglobulin IgM reaches a peak concentration about 8 days post-infection and then declines (Mockett and Cook, 1986). IgG can be detected within 4 days of infection, peaks at three weeks and gradually declines. Inactivated vaccines are given to laying birds to induce and maintain a high level of serum antibody which is transmitted to progeny (maternally-derived antibody). Serum antibodies do not correlate with protection, i.e. after vaccination some chicks may have low IBV serum antibody but still be protected against homologous challenge. Local immunity is important (Hawkes et al., 1983) including at the oviduct mucosa (Dhinakar Raj and Jones, 1996). The Harderian gland is the source of antibodies in lachrymal fluid and is believed to play an important role in the development of immunity following vaccination by spray (Davelaar and Kouwenhoven, 1981). Different breeds of chicken may differ in the amounts of serum IgG and lachrymal IgA that they produce (Toro et al., 1996).

Immunization and Vaccines

Vaccination is a key element of control allied with management of the environment (Cavanagh and Gelb, 2008; de Wit et al., 2011a). Both live and killed vaccines are available. However, killed vaccines alone do not induce immunity; a live vaccine is required to prime immunity prior to application of killed vaccine. Broilers are commonly given live vaccine by coarse spray at the hatchery. Depending on the disease situation, they may be vaccinated again at 2-3 weeks of age with vaccine in drinking water. A vaccination programme for breeders and commercial layers might involve live IB vaccination by drinking water at 3 and 8-10 weeks of age, followed by killed vaccine at around 16-18 weeks of age (Pattison and Cook, 1996).

In many countries live vaccines are limited to the 'Massachusetts' type. In the USA, 'Massachusetts' and 'Connecticut' types are used widely, with 'Florida', 'Arkansas' and 'JMK' used regionally, as appropriate. In Europe Massachusetts strains are also widely used, plus vaccines against serotypes not found in North America e.g. D274, D1466, and the ‘4/91’ or ‘793/B’ serotypes. Elsewhere, too, vaccines have been developed against new serotypes e.g. in Korea (Lim et al., 2012). As there are many more serotypes of IBV than there are IB vaccines, it is common to apply combinations of the available IB vaccines, either simultaneously or in sequence (Terregino et al., 2008; de Wit et al., 2011a, b).

Farm-level Control

In addition to vaccination, good biosecurity should be practiced, including attention to temporary storage and disposal of carcasses. Multi-age sites are particularly at risk. As with other viral diseases, good air quality is important to reduce stress on the respiratory mucosa that together with IBV infection can predispose birds to secondary bacterial infections. Control of ventilation, heating, and misting if litter is very dry are important if disease incidence is to be reduced. Overcrowding will exacerbate the effects of infection.