|
SUMMARY
Avian infectious bronchitis (IB) is usually defined as an acute, contagious disease of chickens characterised primarily by respiratory signs. However, infections with the causative virus may also lead to nephritis (acute or chronic) and egg production problems in laying hens. The severity of respiratory infections with infectious bronchitis virus (IBV) can be greatly enhanced by the presence of other pathogens of the respiratory tract. Clinical signs are indicative, but not diagnostic, of IB, and confirmation requires the isolation or direct demonstration of the presence of IBV, although serology may also be useful in some circumstances. The widespread use of both live and inactivated vaccines may complicate both virus isolation and serology in the diagnosis of IB. The natural occurrence of antigenic variant strains may overcome any immunity induced by conventional vaccine. Recently coronaviruses have been isolated from turkeys and pheasants that are genetically similar to IBV.
Laboratory diagnosis is made by virus isolation in chicken embryos or in tracheal organ cultures. This can be supplemented by immunofluorescence, electron microscopy, polymerase chain reaction (PCR) techniques, haemagglutination inhibition (HI) tests or enzyme-linked immunosorbent assays (ELISA).
Identification of the agent: IBV may be isolated from tracheal mucosa and lung during the acute phase of the respiratory form of the disease. Faeces, kidneys or caecal tonsillar tissue are better sources of virus at other times.
Chicken embryos originating from specific pathogen free flocks or tracheal organ cultures (TOCs) from 20-day-old embryos are used for virus isolation. The inoculation of the allantoic cavity of chicken embryos of 9-11 days' incubation with IBV results in embryo stunting or death, usually within three serial passages. TOCs have the advantage that IBV produces stasis of the tracheal cilia on initial inoculation. The virus can be identified by neutralisation tests using specific antiserum. The antigen may be visualised in infected allantoic cells by immunofluorescence, or by electron microscopy after concentration by ultracentrifugation.
Antigenic typing of IBV is difficult and controversial. Within the same laboratory, a limited range of strains can be shown to be antigenically related or different by using various serological methods. The use of monoclonal antibodies may prove to be a useful method of distinguishing vaccine strains from field strains, and for defining serotypes. Genotyping of IB isolates using reverse-transcription PCR is becoming more widely available.
Serological tests: Regular monitoring of sera from flocks for IB antibody titres may help to indicate the level of vaccine response. Because many chicken sera, especially from older birds, contain antibodies that are highly cross-reactive against antigenically unrelated strains, sero-diagnosis of suspected disease outbreaks of IB cannot be used with a high degree of confidence. The HI test is rapid, inexpensive, practicable and under some circumstances may allow identification of infection with antigenic variant virus. Commercially produced kits that are very sensitive and suitable for monitoring immune response to vaccine are available for ELISAs. They appear to lack type specificity.
A positive diagnosis of IB is made by virus isolation together with tests to demonstrate a significant rise in specific antibody. Following preparation of monospecific antiserum to a virus isolate, a serological comparison can be made with existing strains to allow antigenic serotype identification.
Requirements for vaccines and diagnostic biologicals: Both live attenuated and oil emulsion inactivated vaccines are available. Live vaccines have been attenuated by serial passage in chicken embryos and confer a better local immunity of the respiratory tract. The use of some live vaccines carries the risk of residual pathogenicity associated with vaccine back-passage in flocks. However, proper mass application will generally result in safe application of live vaccines. The concerns about the use of live vaccines can be avoided by using inactivated vaccines.
Inactivated vaccines have to be given to birds individually, and a single inoculation does not confer protection unless preceded by the administration of a live vaccine. Both types of vaccine are available in combination with Newcastle disease vaccine; in some countries inactivated multivalent vaccines are available that include Newcastle disease, infectious bursal disease, reovirus and egg-drop syndrome 76 viral antigens.
A. INTRODUCTION
Avian infectious bronchitis (IB) was first described in the United States of America (USA) in the 1930s as an acute respiratory disease mainly of young chickens. A viral aetiology was established, and the agent was termed avian infectious bronchitis virus. Infectious bronchitis virus (IBV) is a member of the genus Coronavirus, family Coronaviridae, in the order Nidovirales. The virus has a nonsegmented, positive-sense, single-stranded RNA genome.
IBV affects chickens of all ages, which, apart from pheasants and guinea-fowl are the only species reported to be naturally affected. IB occurs world-wide and assumes a variety of clinical forms, the principal one being a classical respiratory syndrome. Infection of the oviduct can lead to permanent damage in young birds lacking maternal antibodies, and, in older birds, can lead to cessation of egg-laying or production of thin-walled and misshapen shells with loss of shell colour. IB can be nephrotropic causing acute nephritis, urolithiasis and mortality (10). After apparent recovery, chronic nephritis can produce sudden death some time later, especially in brown birds. The virus may persist in the intestinal tract and is excreted in the faeces for long periods. This occurs with vaccine strains as well as natural field strains (2).
There have been no reports of human infection with IBV.
B. DIAGNOSTIC TECHNIQUES
Confirmation of diagnosis is based on demonstration of the virus, sometimes assisted by serology. Extensive use is made of live and inactivated vaccines, which may complicate diagnosis by serological methods as antibodies to vaccination and field infections cannot always be distinguished. Persistence of live vaccine virus may also confuse attempts at recovering the causative agent.
| 1. | Identification of the agent
|
| | a) | Sampling
|
| | | Samples taken from birds should relate to the disease under investigation. For acute respiratory disease, swabs from the upper respiratory tract of live birds or tracheal and lung tissues from recently killed birds should be kept on ice in transport medium containing penicillin (10,000 International Units [IU]/ml) and streptomycin (10 mg/ml). For birds showing nephritis or egg-production problems, samples may also be selected from the kidneys or oviduct, but the highest success rate of virus recovery has been reported from samples of large intestine, particularly the caecal tonsillar tissue, or faeces (2). Isolates from the intestinal tract, however, may have no relevance to the latest infection or clinical disease, and samples from the respiratory tract must be taken in all investigations.
|
| | | Tissue samples from the trachea, kidney, oviduct and caecel tonsils in sterile transport media with antibiotics and dry swabs from the respiratory tract or cloaca can also be submitted to specialist laboratories for reverse-transcription polymerase chain reaction (RT-PCR) analysis (7). For specimens requiring despatch to a diagnostic laboratory it is essential that samples in transport media be kept chilled or frozen throughout transport. Where delays of more than 3 days are expected, the samples should be frozen prior to dispatch and sent on dry ice. Samples should also be selected from fresh carcases for histopathological examination. Blood samples from acutely affected birds should also be submitted for serological analysis.
|
| | b) | Culture
|
| | | Suspensions of tissues (10-20% w/v) are prepared in sterile phosphate buffered saline (PBS) or nutrient broth for egg inoculation, or in tissue culture medium for tracheal organ culture (TOC) inoculation (10, 16). The suspensions are clarified by low-speed centrifugation and filtration through bacteriological filters (0.2 µ) before inoculation of eggs or TOCs.
|
| | | Embryonated hens' eggs and TOCs are widely used to titrate the virus or to make primary isolations of virus. Cell cultures are not used for primary isolation as it is usually necessary to adapt IBV isolates to growth in chicken embryos before cytopathic effects (CPE) of virus infection are seen.
|
| | | Eggs used in all culture work with IBV must originate from birds that have been neither infected nor vaccinated. Such eggs should preferably be from specific pathogen free (SPF) hens. Most commonly, 0.1-0.2 ml of sample supernatant is inoculated into the allantoic cavity of 9-11-day-old embryos. Embryos should be examined daily thereafter. Any deaths that occur within 24 hours are assumed to be nonspecific and the eggs are discarded. The initial inoculum usually has no effect on the embryo unless the strain is derived from a vaccine and already egg adapted. Normally, the allantoic fluids of all eggs are pooled after harvesting 3-7 days after infection; this pool is diluted 1/5 or 1/10 in antibiotic broth and further passaged into another set of eggs. This is repeated as desired. Typically, a field strain will induce teratological changes in the embryo at the second or third passage consisting of stunted and curled embryos with feather dystrophy and urate deposits in the embryonic mesonephros. Some mortality in later passages may occur. Other viruses, most notably adenoviruses, may also produce embryo lesions indistinguishable from IBV. The allantoic fluid should not agglutinate red blood cells and isolation of IBV must be confirmed by immunological or genotypic testing. Infective allantoic fluids are kept at -60°C or below for long-term storage, or at 4°C after lyophilisation.
|
| | | TOCs prepared from 20-day-old embryos can be used to isolate IBV directly from field material (16). An automatic tissue-chopper is desirable for the large-scale production of suitable transverse sections or rings of the trachea for this technique (20). The rings are about 0.5-1.0 mm thick, and are maintained in a medium consisting of Eagle's N-2-hydroxyethylpiperazine N'-2-ethanesulphonic acid (HEPES) in roller drums (15 rev/hour) at 37°C. Infection of tracheal organ cultures usually produces ciliostasis within 24-48 hours. Ciliostasis may be produced by other viruses and suspect IBV cases must be confirmed by immunological or genotypic methods.
|
| | c) | Methods for identification
|
| | | The virus neutralisation (VN) test in embryonated eggs and the immunodiffusion technique are useful for identification purposes (see below). Fluorescent antibody tests on cells present in the allantoic fluid of infected eggs may also demonstrate the presence of IBV (11) and direct negative-contrast electron microscopy will reveal particles with typical coronavirus morphology in allantoic fluid or TOC fluid concentrates. The specific presence of IBV in infective allantoic fluid may be detected by RT-PCR amplification and use of a DNA probe in a dot-hybridisation assay (27). Direct immunofluorescence staining of infected TOCs for the rapid detection of the presence of IBV has been described (3). Immuno-histochemistry, with a group-specific monoclonal antibody, can be used to identify IBV in infected chorioallantoic membranes (35).
|
| | d) | Serotype identification
|
| | | Antigenic and biological variation among IBV strains is well reported (10, 15, 22, 23, 26), but at present there is no agreed definitive classification system. Nevertheless, antigenic relationships and differences among strains are important, as vaccines based on one particular subtype may show little or no protection against viruses of a different antigenic group. As a result of the regular emergence of antigenic variants, the viruses, and hence the disease situation and vaccines used, may be quite different in different geographical locations. Constant assessment of the viruses present in the field is necessary to produce vaccines that will be efficacious in the face of antigenic variants that may arise. Serotyping of IBV isolates and strains has been done using VN tests in embryonated eggs (22), TOCs (21) and cell cultures (24). Neutralisation of fluorescent foci has also been applied to strain differentiation (18). The haemagglutination inhibition (HI) test has been employed for serotyping IBV (1, 29) and has proved useful providing early response sera are used.
|
| | | Monoclonal antibodies (MAbs), usually employed in enzyme-linked immunosorbent assays (ELISA), have proven useful in grouping and differentiating strains of IBV (25, 31). The limitations of MAb analysis for IB serotype definition are the lack of availability of MAbs or hybridomas and the need to produce new MAbs with appropriate specificity to keep pace with the ever-growing number of emerging IB-variant serotypes (28).
|
| | e) | Genotype identification
|
| | | The molecular basis of antigenic variation has been investigated, usually by nucleotide sequencing of the gene coding for the spike (S) protein or, more specifically, nucleotide sequencing of the gene coding for the S1 subunit of the S protein (5, 33) where most of the epitopes to which neutralising antibodies bind are found (32). An exact correlation with VN results has not been seen, in that while different serotypes generally have large differences (20-50%) in the deduced amino acid sequences of the S1 subunit (33), other viruses that are clearly distinguishable in neutralisation tests show only 2-3% differences in amino acid sequences (5). However, there is in general good agreement between data represented by the S1 sequence and the VN serotype, and it may eventually be possible to select vaccine strains on the basis of sequence data. It has been suggested that the nucleoprotein may play an important role in inducing protection against IB viruses. Recently, it has been shown that coronaviruses isolated from turkeys and pheasants are genetically similar to IBV, having approximately 90% nucleotide identity in the highly conserved region II of the 3' untranslated region (UTR) of the IBV genome (8, 9).
|
| | | Nucleotide sequencing (of particularly the S gene region) using appropriate primers is the most useful technique for the differentiation of IBV strains and has superseded restriction fragment length polymorphism analysis (RFLP) and the use of DNA probes. Nucleotide sequencing has also produced evidence that recombination between IB strains occurs often (6, 40). RT-PCR is now being used in a number of diagnostic laboratories for sequencing and characterising a wide range of variant serotypes of IBV from many countries (30).
|
| 2. | Serological tests
|
| | A number of tests have been described. Those considered here include VN (22), agar gel immunodiffusion (AGID) (39), HI (1) and ELISA (34). Each test has advantages and disadvantages in terms of practicality, specificity, sensitivity and cost. In general, for routine serological testing, the VN tests are too expensive and impractical, and AGID tests lack sensitivity. HI tests and ELISAs are suitable for routine serology although they differ in their specificity. As ELISAs are available in kit form with detailed instructions for their use, the HI test is described in detail below. Regular monitoring of sera from flocks for IB antibody titres may help to indicate the level of vaccine response. Because many chicken sera, especially from older birds, contain antibodies that are highly cross-reactive against antigenically unrelated strains, serodiagnosis of suspected disease outbreaks of IB cannot be used with a high degree of confidence.
|
| | a) | Virus neutralisation
|
| | | In VN tests, all sera should first be heated to 56°C for 30 minutes. Virus is mixed with serum and incubated for 30-60 minutes at 37°C or room temperature. Chicken embryos are most often employed, but antibodies can be measured using TOC or cell culture systems. Two methods have been used to estimate neutralising antibodies. One employs a constant serum concentration reacted with varying dilutions of virus (the alpha method) and the other employs a constant amount of virus and varying dilutions of serum (the beta method).
|
| | | In the alpha method, tenfold dilutions of egg-adapted virus are reacted with a fixed dilution (usually 1/5) of antiserum, and the mixtures are inoculated into groups of from five to ten eggs. The virus alone is titrated in parallel. End-points are calculated by the Kärber or the Reed and Muench methods. The results are expressed as a neutralisation index (NI) that represents the log10 difference in the titres of the virus alone and that of the virus/antiserum mixtures. The NI values may reach 4.5-7.0 in the case of homologous virus/serum mixtures; values of <1.5 are not specific, but a heterologous virus will give a value as low as 1.5.
|
| | | The beta method is the more widely used neutralisation test for antibody assay with chicken embryos. Two- or four-fold dilutions of antiserum are reacted in equal volumes with a dilution of virus, usually fixed at 100 or 200 EID50 (median embryo-infective doses) per 0.05 ml and 0.1 ml of each mixture inoculated into the allantoic cavity of each of from five to ten embryonated eggs. A control titration of the virus is performed simultaneously to confirm that the fixed virus dilution in the virus/serum mixtures was between 101.5 and 102.5 EID50. End-points of the serum titres are determined by the Kärber or Reed and Muench method as before, but here are expressed as reciprocals of log2 dilutions. This fixed-virus/varying-serum method is also employed for neutralisation tests in tracheal organ cultures using five tubes per serum dilution, as is conventional with other viruses (21). The results are calculated according to Reed and Muench, and the virus titres are expressed as median ciliostatic doses per unit volume (log10 CD50). Serum titres are again expressed as log2 dilution reciprocals. This test is more sensitive than others, but technical logistics hamper its more widespread adoption.
|
| | b) | Haemagglutination inhibition
|
| | | A standard protocol for a HI test for IBV has been described (1), and the test procedure detailed below is based on that standard. Many strains and isolates of IBV have been shown to agglutinate chicken red blood cells (RBCs) after enzyme treatment. The virus selected to produce antigen may be varied, depending on the requirements of diagnosis.
|
| | | . | Preparation of antigen
|
| | |
IBV antigen requires enzyme treatment to acquire haemagglutination (HA) activity. Originally this was shown to be facilitated by commercial phospholipase C type 1 enzyme, and it was recommended that the virus suspension be mixed with an equal volume of this enzyme to a final concentration of 1 unit/ml in the same buffer. However, it was found to be more efficient to use crude filtrate from Clostridium perfringens culture, and it seemed most likely that a contaminating enzyme rather than phospholipase was the constituent responsible for the activity. Subsequent work has indicated that this enzyme is most probably neuraminidase (36). Infective allantoic fluid is centrifuged at 30,000 g for 3 hours and the pellet is resuspended at 100-fold concentration in Clostridium perfringens type A filtrate and incubated at 37°C for 2 hours.
|
| | | For HA and HI tests, procedures are best carried out at 4°C.
|
| | | . | Haemagglutination test
|
| | | i) | Dispense 0.025 ml of isotonic PBS, pH 7.0-7.4, into each well of a plastic microtitre plate.
|
| | | ii) | Place 0.025 ml of virus antigen in the first well. For accurate determination of the HA content, this should be done from a close range of an initial series of dilutions, i.e. 1/3, 1/4, 1/5, 1/6, etc.
|
| | | iii) | Make twofold dilutions of 0.025 ml volumes of the virus antigen across the plate.
|
| | | iv) | Dispense a further 0.025 ml of PBS into each well.
|
| | | v) | Dispense 0.025 ml of 1% (v/v) chicken RBCs to each well.
|
| | | vi) | Mix by tapping the plate gently and allow the RBCs to settle for about 40 minutes at 4°C, when control RBCs should be settled to a distinct button.
|
| | | vii) | HA is determined by tilting the plate and observing the presence or absence of tear-shaped streaming of the RBCs. The titration should be read to the highest dilution giving complete HA in which there is no streaming; this is 100% HA and represents 1 HA unit (HAU) and can be calculated accurately from the initial range of dilutions.
|
| | | . | Haemagglutination inhibition test
|
| | | The HI test is used in the diagnosis and routine flock monitoring of vaccine responses.
|
| | | i) | Dispense 0.025 ml of PBS into each well of a plastic microtitre plate.
|
| | | ii) | Place 0.025 ml of serum into the first well of the plate.
|
| | | iii) | Make twofold dilutions of 0.025 ml volumes of the serum across the plate.
|
| | | iv) | Add 4 HAU of virus antigen in 0.025 ml to each well and leave for 30 minutes.
|
| | | v) | Add 0.025 ml of 1% (v/v) chicken RBCs to each well and, after gentle mixing, allow the RBCs to settle for about 40 minutes when control RBCs should be settled to a distinct button.
|
| | | vi) | The HI titre is the highest dilution of serum causing complete inhibition of 4 HAU of antigen. The agglutination is assessed more exactly by tilting the plates. Only those wells in which the RBCs 'stream' at the same rate as the control wells (containing 0.025 ml RBC and 0.05 ml PBS only) should be considered to show inhibition.
|
| | | vii) | The validity of results should be assessed against a negative control serum, which should not give a titre >22, and a positive control serum, for which the titre should be within one dilution of the known titre.
|
| | | viii) | Sera are usually regarded as positive if they have a titre of 24 or more. However, it should be noted that even in SPF flocks, a very small proportion of birds may show a nonspecific titre of 24, but usually in birds over 1 year of age.
|
| | c) | Enzyme-linked immunosorbent assay
|
| | | The ELISA technique is the most sensitive serological method and gives earlier reactions and higher antibody titres than other tests (34). It lacks type or strain specificity, but is suitable for monitoring vaccination responses under field conditions. Commercial kits for ELISAs are available - these are based on several different strategies for the detection of IBV antibodies. Usually, such tests have been evaluated and validated by the manufacturer, and it is therefore important that the instructions specified for their use be followed carefully. The ELISA is widely used to identify IBV-infected flocks (broilers) based on high antibody titres. If IB reoccurs in the next flock on the farm, virus isolation attempts are performed and the virus is genotyped by RFLP or S1 sequencing.
|
| | d) | Agar gel immunodiffusion
|
| | | AGID can be used in diagnosis (39). The antigen is prepared from a homogenate of the chorioallantoic membranes of infected chicken embryos. The Beaudette embryo-lethal strain is often employed to produce antigen. The test lacks sensitivity and is liable to yield inconsistent results as the presence and duration of precipitating antibodies may vary with individual birds.
|
C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
All viruses in live virus vaccines must be attenuated or naturally apathogenic. At present, many countries only permit live vaccines based on the Massachusetts type. A single inoculation of an inactivated IB vaccine is unlikely to confer full protection to the end of lay unless preceded by a primary response, usually to a live vaccine. Inactivated vaccines have to be administered to birds individually, using intramuscular or subcutaneous injection, whereas live vaccines can be given as aerosols or in the drinking water, or by eyedrop. Live vaccines confer a better local immunity on the respiratory tract and may protect against a wider antigenic spectrum of field strains (16, 17). Live vaccine may not protect for the life of the layer flock as variant serotype challenge is very high on farms with flocks of multiple ages and production drops as early as 40 weeks of age are not uncommon. The use of some live vaccines carries the risk of residual pathogenicity associated with vaccine back-passage in flocks. However, proper mass application techniques (e.g. spray or drinking water) to achieve even coverage/ distribution of the vaccine in the flock and avoiding the use of suboptimal fractional doses of the vaccine will generally result in safe application of live vaccines. Concerns about the use of live vaccines can be avoided by using inactivated vaccines.
The more recent oil emulsion inactivated vaccines are now more efficacious, especially when preceded by a live virus vaccine. They also stimulate a more persistent antibody response. There are prospects for genetically engineered vaccines (4), and in-ovo vaccination is under development (38).
Guidelines for the production of veterinary vaccines are given in Chapter I.1.7. Principles of veterinary vaccine production. The guidelines given here and in Chapter I.1.7 are intended to be general in nature. National and international standards that apply in the country in which IB vaccines are manufactured must be complied with. The licensing authority should provide information and guidance on requirements. These are now often presented in general terms, as applying to all vaccines - avian and mammalian, live and inactivated, or viral and bacterial vaccines. There may also be specific requirements applying to IB vaccines, live and inactivated. As examples, references are given to the European and USA regulations (12-14, 37).
The list of extraneous agents that must be shown to be absent continues to grow. Manufacturers must be familiar with those that currently apply in their country. Recent additions are avian nephritis virus and avian pneumovirus.
For IB vaccines, important differences among countries may arise regarding the challenge virus to be used for potency tests, and its validation. Traditionally, a virulent M-41 strain of the Massachusetts type has been used for challenge tests of both live and inactivated vaccines. Although this type is still common, it is often not the only or the dominant type in many countries and it may be advisable to prepare vaccines from other types. It is logical for challenges to be made by the same type as present in the vaccine. Establishing criteria for validating the challenge virus may be more difficult for non-Massachusetts types, because of their lower virulence in general. Inactivated vaccines are usually expected to protect against drops in egg production. The traditional M-41 challenge, as described in this chapter, should cause a drop of at least 67% in the unvaccinated controls, but when using other types much lower drops may be regarded as satisfactory, depending on published evidence of the effects of these strains in the field. There is also a tendency to relax the criteria for Massachusetts type challenges, and the European Pharmacopoeia now defines a satisfactory drop for Massachusetts types to be at least 35%, and for non-Massachusetts types to be at least 15%, provided that the drop is 'commensurate with the documented evidence' (14).
| 1. | Seed management
|
| | a) | Characteristics of the seed
|
| | | The seed-lot system should be employed for whatever type of vaccine is produced, and for challenge strains. Each virus must be designated as to strain and origin and must be free from contamination with other strains of IBV. Separate storage facilities should be provided between the strains of virus intended for vaccines or for challenge.
|
| | | For live virus vaccines, many countries permit only strains of the Massachusetts type. Some countries allow other strains, usually on the basis that those strains are already present in their national flocks. The antigenic type incorporated in both live and inactivated vaccines requires justification if there is doubt as to its existence in a country.
|
| | b) | Method of culture
|
| | | All seed viruses are grown in the allantoic sac of developing chicken embryos or in suitable cell cultures. The eggs should be from an SPF flock.
|
| | c) | Validation as a vaccine
|
| | | . | Purity
|
| | | Every seed lot must be free from bacterial, fungal, mycoplasmal and viral contamination.
|
| | | For the detection of extraneous viruses, the seed is first treated with a high-titred monospecific antiserum prepared against the strain under examination or against one of identical type. This mixture is cultured in a variety of ways, designed to confirm the absence of any viruses considered from past experience to be potential contaminants. The antiserum must not contain antibodies to adenovirus, avian encephalomyelitis virus, avian rotavirus, chicken anaemia virus, fowlpox virus, infectious laryngotracheitis virus, influenza A virus, Newcastle disease virus, infectious bursal disease virus, leukosis virus, reovirus, Marek's disease virus, turkey herpesvirus, adeno-associated virus, egg-drop syndrome 76 (EDS76) virus, avian nephritis virus, avian pneumovirus or reticulo-endotheliosis virus. The inoculum given to each unit of the culture system used should contain a quantity of the neutralised IBV component under test that had an initial infectivity of at least ten times the minimum field dose. These systems include:
|
| | | 1. | SPF chicken embryos, incubated for 9-11 days, inoculated via both allantoic sac and chorioallantoic membrane (two passages);
|
| | | 2. | Chicken embryo fibroblast cultures, for leukosis virus subgroups A and B. The COFAL test (test for avian leukosis using complement fixation) or double-antibody sandwich ELISA for group-specific leukosis antigen is performed on cell extracts harvested at 14 days. An immunofluorescence test for reticulo-endotheliosis virus is done on cover-slip cultures after two passages.
|
| | | 3. | SPF chicken kidney cultures that are examined for CPEs, cell inclusions and haemadsorbing agents passaged at intervals of no fewer than 5 days for up to 20 days' total incubation.
|
| | | 4. | SPF chickens of minimum vaccination age inoculated intramuscularly with 100 field doses, and on to the conjunctiva with ten field doses; this is repeated 3 weeks later when the chickens are also inoculated both into the foot pad and intranasally with ten field doses. Observations are made for 6 weeks overall, and serum is collected for tests for avian encephalomyelitis, infectious bursal disease, Marek's disease, Newcastle disease and Salmonella pullorum infection.
|
| | | . | Potency
|
| | | Vaccines intended to protect against loss of egg production should be tested for duration of antibody response. Mean HI titres should be >6 log2 up to at least 60 weeks of age. Serological tests should be done at intervals frequent enough to show that titres have not been boosted by extraneous IBV infection.
|
| | | Vaccines intended for protection of broiler chickens or rearing chickens against the respiratory form of the disease should be similarly tested for duration of antibody responses; in the case of broilers this would be up to the normal age for slaughtering, and in the case of rearers up to the age when a booster vaccination would be administered (often at 16-18 weeks of age).
|
| | | . | Safety
|
| | | Tests on seed virus should include a test for any potential ability to revert to virulence. Live and inactivated vaccine seed must be tested for safety as in Section C.4.b.
|
| | | . | Efficacy
|
| | | To demonstrate efficacy, a trial vaccine must be made from the master seed and the working seed at five passages from the master seed and subjected to tests that demonstrate their protective effect.
|
| | | For live vaccines, a minimum of ten SPF chickens aged 3-4 weeks are vaccinated intranasally or by eyedrop with the recommended dose. Ten unvaccinated control birds from the same age and source are retained separately. All birds of both groups are challenge inoculated intranasally or by eyedrop 3-4 weeks later, each with 103.0-103.5 EID50 of the virulent Massachusetts M-41 strain. A swab of the trachea is taken from each bird 4-5 days after challenge and placed in 3 ml of antibiotic broth. Each fluid is tested for IBV by the inoculation (0.2 ml) of five embryonated eggs after 9-11 days of incubation. An alternative test to that of taking swabs is to kill birds at 4-6 days after challenge and examine microscopically the tracheal rings for ciliary activity (19). Failure to resist challenge is indicated by an extensive loss of ciliary motility. The live vaccine is suitable for use if at least 90% of the challenge vaccinated birds show no evidence of IBV in their trachea, while 80% or more of the control birds should have evidence of the presence of the virus.
|
| | | To assess an inactivated vaccine intended to protect laying birds, 30 or more SPF chickens are vaccinated as recommended at the earliest permitted age. If a primary vaccination with live vaccine is first undertaken, an additional group of birds is given only the primary vaccination. In both cases, these primary vaccinations should be done at no later than 3 weeks of age. The inactivated vaccine is given 4-6 weeks after the live priming vaccination. A further group of 30 control birds are left unvaccinated. All groups are housed separately until 4 weeks before peak egg production, and then are housed together. Individual egg production is monitored and once it is regular, all birds are challenged, egg production being recorded for a further 4 weeks. The challenge should be sufficient to ensure loss of production during the 3 weeks after challenge. The loss in the control group should be at least 67%; the group that received primary live virus vaccine followed by inactivated vaccine should remain at the previous level, and the group given only a primary vaccination should show an intermediate drop in production. Sera are collected from all birds at vaccination, 4 weeks later, and at challenge; there should be no response in the control birds.
|
| | | To assess an inactivated vaccine intended to protect birds against respiratory disease, 20 SPF chickens aged 4 weeks are vaccinated as recommended. An additional 20 control birds of the same age and origin are housed with this first group. Antibody responses are determined 4 weeks later; there should be no response in the control birds. All birds are then challenged with 103 CID50 (50% chick infective dose) of virulent virus, killed 4-7 days later, and tracheal sections are examined for ciliary motility. At least 80% of the unvaccinated controls should display complete ciliostasis, whereas the tracheal cilia of a similar percentage of the vaccinated birds should remain unaffected.
|
| | | Both live and inactivated vaccines containing Newcastle disease, infectious bursal disease, reovirus and EDS76 viruses are available in some countries. The efficacy of the different components of these vaccines must each be established independently and then as a combination in case interference between different antigens exists.
|
| 2. | Method of manufacture
|
| | All virus strains destined for live vaccines are cultured in the allantoic sac of SPF chicken embryos or in suitable cell cultures. For inactivated vaccines, hens' eggs from healthy non-SPF flocks may be used. The pooled fluid is clarified and then titrated for infectivity. For live vaccines this fluid is lyophilised in vials, and for inactivated vaccines it is blended with high-grade mineral oil to form an emulsion to which a preservative is added.
|
| 3. | In-process control
|
| | The required antigen content is based on initial test batches of vaccine of proven efficacy in laboratory and field trials. Infectivity titrations are done in chicken embryos.
|
| | Live vaccine should contain not less than 103.5 EID50 per dose per bird until the expiry date indicated, and not less than 102.5 EID50 per dose per bird after incubation at 37°C for 7 days at the time of issue. For inactivated vaccine, the inactivating agent and inactivation procedure must be shown under manufacture to be effective on both IBV and potential contaminants. With the use of beta-propiolactone or formalin, any live leukosis viruses and Salmonella species must be eliminated; and with other inactivating agents, the complete range of potential contaminants must be rendered ineffective. Before inactivation procedures, it is important to ensure homogeneity of suspensions, and a test of inactivation should be conducted on each batch of both bulk harvest after inactivation and the final product.
|
| | Tests of inactivation should be appropriate to the vaccine concerned and should consist of two passages in cell cultures, embryos or chickens, using inoculations of 0.2 ml and ten replicates per passage.
|
| 4. | Batch control
|
| | a) | Sterility
|
| | | Every batch of live vaccine should be tested for the absence of extraneous agents as for the seed virus (see Chapter I.1.5.).
|
| | b) | Safety
|
| | | . | For live vaccines
|
| | | Use no fewer than ten chickens from an SPF flock that are of the minimum age stated on the label for vaccination. Administer by eyedrop to each chicken ten doses of the vaccine reconstituted so as to obtain a concentration suitable for the test. Observe the chickens for 21 days. For vaccines intended for chickens that are 2 weeks old or more, use the chickens inoculated in the 'test for extraneous agents using chickens' (see Section C.1.c.4.). If during the period of observation, more than two chickens die from causes not attributable to the vaccine, repeat the test. The vaccine complies with the test if no chicken shows serious clinical signs, in particular respiratory signs, and no chicken dies from causes attributable to the vaccine.
|
| | | . | For inactivated vaccines
|
| | | Inject a double dose of vaccine by the recommended route into each of ten 14-28-day-old chickens from an SPF flock. Observe the chickens for 21 days. Ascertain that no abnormal local or systemic reaction occurs.
|
| | c) | Potency
|
| | |
The potency test is developed from the results of efficacy tests on the master seed virus. Live vaccines are tested for potency by titration of infectivity, and inactivated vaccines by measuring antibody production. The potency test for a batch of inactivated vaccine consists of vaccinating 20 SPF chickens, 4 weeks of age, and showing that their mean HI titre 4 weeks later is not less than 6 log2.
|
| | d) | Duration of immunity
|
| | | Vaccine must be shown to have the required potency to achieve the claimed duration of immunity at the end of the claimed shelf life.
|
| | e) | Stability
|
| | | At least three batches should be tested for stability and must give satisfactory results for 3 months beyond the claimed shelf life.
|
| | | The stability of a live vaccine must be measured by maintenance of an adequate infectivity titre.
|
| | | The stability of an inactivated vaccine is measured at intervals by batch potency tests. The concentration of preservative and persistence through the shelf life should be assessed. There should be no physical change in the vaccine and it should regain its former emulsion state after one quick shake.
|
| | f) | Preservatives
|
| | | There are maximum level requirements for the use of antibiotics, preservatives and residual inactivating agents.
|
| | g) | Precautions (hazards)
|
| | | IBV itself is not known to present any danger to staff employed in vaccine manufacture or testing. Extraneous agents may be harmful, however, and the initial stages of handling a new seed virus should be carried out in a safety cabinet. It is a wise precaution with all vaccine production to take steps to minimise exposure of staff to aerosols of foreign proteins. Persons allergic to egg materials must never be employed in this work.
|
| 5. | Tests on the final product
|
| | a) | Safety
|
| | | A safety test must be carried out on each batch of final product, as in Section C.4.b.
|
| | b) | Potency
|
| | | A potency test must be carried out on each batch of final product, as in Section C.4.c., at manufacture and at the end of the stated shelf life.
|
REFERENCES
| 1. | Alexander D.J., Allan W.H., Biggs P.M., Bracewell C.D., Darbyshire J.H., Dawson P.S., Harris A.H., Jordan F.T., MacPherson I., McFerran J.B., Randall C.J., Stuart J.C., Swarbrick O. & Wilding G.P. (1983). A standard technique for haemagglutination inhibition tests for antibodies to avian infectious bronchitis virus. Vet. Rec., 113, 64.
|
| 2. | Alexander D.J., Gough R.E. & Pattison M. (1978). A long-term study of the pathogenesis of infection of fowls with three strains of avian infectious bronchitis virus. Res. Vet. Sci., 24, 228-233.
|
| 3. | Bhattacharjee P.S., Naylor C.J. & Jones R.C. (1994). A simple method for immunofluorescence staining of tracheal organ cultures for the rapid identification of infectious bronchitis virus. Avian Pathol., 23, 471-480.
|
| 4. | Binns M.M., Boursnell M.E.G., Tomley F.M., Mockett A.P.A., Cavanagh D. & Brown T.D.K. (1986). Prospects for a genetically engineered vaccine against infectious bronchitis. Israel J. Vet. Med., 42, 124-127.
|
| 5. | Cavanagh D. (1991). Sequencing approach to IBV antigenicity and epizootiology. In: Proceedings of the Second International Symposium on Infectious Bronchitis. Rauischholzhausen, Germany, June 1991, 147-160.
|
| 6. | Cavanagh D., Davis, P.J. & Cook J.K.A. (1992). Infectious bronchitis virus: evidence for recombination within the Massachusetts serotype. Avian Pathol., 21, 401-408.
|
| 7. | Cavanagh D., Mawditt K., Britton P. & Naylor C.J. (1999). Longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using type-specific polymerase chain reactions. Avian Pathol., 28, 593-605.
|
| 8. | Cavanagh. D., Mawditt K., Sharma. M., Drury S.E., Ainsworth H.L., Britton P. & Gough R.E. (2001). Detection of a coronavirus from turkey poults in Europe genetically related to infectious bronchitis virus of chickens. Avian Pathol., 30, 355-368.
|
| 9. | Cavanagh D., Mawditt K., Welchman D. de B., Britton P. & Gough R.E. (2002) Coronaviruses from pheasants (Phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys. Avian Pathol., 31, 81-93.
|
| 10. | Cavanagh D. & Naqi S.A. (1997). Infectious Bronchitis. In: Diseases of Poultry, Tenth Edition, Calnek B. W., Barnes H.J., Beard C.W., McDougald L.R. & Saif Y.M., eds. Iowa State Press, Iowa, USA, 511-526.
|
| 11. | Clarke J.K., McFerran J.B. & Gay F.W. (1972). Use of allantoic cells for the detection of avian infectious bronchitis virus. Arch. Gesamte Virusforsch., 36, 62-70.
|
| 12. | Commission of the European Communities (Interim Publication Nov. 1993). The Rules Governing Medicinal Products in the European Community. Volume VII: Guidelines for the Testing of Veterinary Medicinal Products.
|
| 13. | Council of Europe (1997). European Pharmacopoeia, Third Edition. Vaccinum bronchitidis infectivae aviariae vivum cryodesiccatum. Editions of the Council of Europe, Strasbourg, France, 1795-1796.
|
| 14. | Council of Europe (1997). European Pharmacopoeia, Third Edition. Vaccinum bronchitidis infectivae aviariae inactivatum. Council of Europe, Strasbourg, France, 1745-1746.
|
| 15. | Cook J.K.A. (1984). The classification of new serotypes of infectious bronchitis virus isolated from poultry flocks in Britain between 1981 and 1983. Avian Pathol., 13, 733-741.
|
| 16. | Cook J.K.A., Darbyshire J.H. & Peters R.W. (1976). The use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis virus. Arch. Virol., 50, 109-118.
|
| 17. | Cook J.K.A., Orbell S.J., Woods M.A. & Huggins M.B. (1999). Breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes. Avian Pathol., 28, 477-485.
|
| 18. | Csermelyi M., Thijssen R., Orthel F., Burger A.G., Kouwenhoven B. & Lutticken D. (1988). Serological classification of recent infectious bronchitis virus isolates by the neutralisation of immunofluorescent foci. Avian Pathol., 17, 139-148.
|
| 19. | Darbyshire J.H. (1985). A clearance test to assess protection in chickens vaccinated against avian infectious bronchitis virus. Avian Pathol., 14, 497-508.
|
| 20. | Darbyshire J.H., Cook J.K.A. & Peters R.W. (1978). Growth comparisons of avian infectious bronchitis virus strains in organ cultures of chicken tissues. Arch. Virol., 56, 317-325.
|
| 21. | Darbyshire J.H., Rowell R.G., Cook J.K.A. & Peters R.W. (1979). Taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures. Arch. Virol., 61, 227-238.
|
| 22. | Dawson P.S. & Gough R.E. (1971). Antigenic variation in strains of avian infectious bronchitis virus. Arch. Gesamte Virusforsch., 34, 32-39.
|
| 23. | Hofstad M.S. (1958). Antigenic differences among isolates of avian infectious bronchitis virus. Am. J. Vet. Res., 19, 740-743.
|
| 24. | Hopkins S.R. (1974). Serological comparisons of strains of infectious bronchitis virus using plaque purified isolates. Avian Dis., 18, 231-239.
|
| 25. | Ignjatovic J., McWaters P. & Galli L. (1991). Antigenic relationship of Australian infectious bronchitis viruses: analysis using polyclonal and monoclonal antibodies. In: Proceedings of the Second International Symposium on Infectious Bronchitis. Rauischholzhausen, Germany, June 1991, 161-167.
|
| 26. | Ignjatovic J. & Sapats S. (2000). Avian infectious bronchitis virus. Rev. sci. tech. Off. int. Epiz., 19 (2), 493-508.
|
| 27. | Jackwood M.W., Kwon H.M. & Hilt D.A. (1992). Infectious bronchitis virus detection in allantoic fluid using the polymerase chain reaction and a DNA probe. Avian Dis., 36, 403-409.
|
| 28. | Karaca K., Naqi S.A. & Gelb J. Jr (1992). Production and characterisation of monoclonal antibodies to three infectious bronchitis virus serotypes. Avian Dis., 36, 903-915.
|
| 29. | King D.J. & Hopkins S.R. (1984). Rapid serotyping of infectious bronchitis virus isolates with the haemagglutination inhibition test. Avian Dis., 28, 727-733.
|
| 30. | Kingham B.F., Keeler C.L. Jr, Nix W.A., Ladman B.S. & Gelb J. Jr (2000). Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S-1 gene. Avian Dis., 44, 325-335.
|
| 31. | Koch G., Hartog L., Kant A., Van Roozelaar D. & De Boer G.F. (1986). Antigenic differentiation of avian bronchitis virus variant strains employing monoclonal antibodies. Israel J. Vet. Med., 42, 80-97.
|
| 32. | Koch G., Kant A., Cook J.K.A. & Cavanagh D. (1992). Location of antigenic sites defined by neutralising monoclonal antibodies on the S1 avian infectious bronchitis virus glycopolypeptide. J. Gen. Virol., 73, 591-596.
|
| 33. | Kusters J.G., Niesters H.G.M., Lenstra J.A., Horzinek M.C. & Van Der Zeijst B.A.M. (1989). Phylogeny of antigenic variants of avian coronavirus IBV. Virology, 169, 217-221.
|
| 34. | Mockett A.P.A. & Darbyshire J.H. (1981). Comparative studies with an enzyme-linked immunosorbent assay (ELISA) for antibodies to avian infectious bronchitis virus. Avian Pathol., 10, 1-10.
|
| 35. | Naqi S.A. (1990) A monoclonal antibody-based immunoperoxidase procedure for rapid detection of infectious bronchitis virus in infected tissues. Avian Dis., 34. 893-898.
|
| 36. | Schultze B., Cavanagh D. & Herrler G. (1992). Neuraminidase treatment of avian infectious bronchitis coronavirus reveals a haemagglutinating activity that is dependent on sialic acid-containg receptors on erythrocytes. Virology, 189, 792-794.
|
| 37. | United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS) (1 January, 1994). Code of Federal Regulations. § 113. 327 Bronchitis Vaccine. US Government Printing Office, Washington D.C., USA.
|
| 38. | Wakenell P. (1991). Vaccination of chicken embryos against infectious bronchitis virus. In: Proceedings of the Second International Symposium on Infectious Bronchitis. Rauischholzhausen, Germany, June 1991, 239-240.
|
| 39. | Witter R.L. (1962). The diagnosis of infectious bronchitis of chickens by the agar gel precipitin test. Avian Dis., 6, 478-492.
|
| 40. | Zwaagstra K.A., Van Der Zeijst B.A.M. & Kusters J.G. (1992). Rapid detection and identification of avian bronchitis virus. J. Clin. Microbiol., 30, 79-84.
|
*
* *
|