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Rev. sci. tech. Off. int. Epiz., 2000,19 (2), 493-508

Avian infectious bronchitis virus

J. Ignjatovic & S. Sapats

Commonwealth Scientific and Industrial Research Organisation (CSIRO), Division of Animal Health, Australian Animal Health Laboratory, P.O. Box 24, Geelong, Victoria 3220, Australia

Summary In fect ious bronchi t is v i rus (IBV) is prevalent in all countr ies w i t h an intensive poul t ry industry, w i t h the inc idence of in fect ion approach ing 100% in most locat ions. Vacc inat ion is only part ial ly successfu l due to the cont inual emergence of ant igenic var iants . A t many si tes, mult ip le ant igenic types are s imul taneously present , requir ing the appl icat ion of mult ip le vacc ines . A l though many count r ies share some common ant igenic types, IBV strains w i th in a geographic region are unique and dist inct , examples are Europe, the United States of Amer ica and Aust ra l ia . Measures to res t r ic t the in t roduct ion of exotic IBV strains should there fore be cons idered. Infect ious bronchi t is has a s ign i f icant economic impact ; in bro i lers, product ion losses are due to poor we igh t gains, condemnat ion at processing and mortal i ty, wh i l s t in laying birds, losses are due to subopt imal egg product ion and downgrad ing of eggs. Chickens and commerc ia l l y reared pheasants are the only natural hosts for IBV. Other spec ies are not cons idered as reservoirs of IBV. The major i ty of IBV strains cause t rachea l lesions and respi ratory disease w i t h low mortal i ty due to secondary bacter ia l in fect ions, pr imar i ly in broi lers. Nephropathogen ic stra ins, in addi t ion to t rachea l lesions, also induce prominent kidney lesions w i t h mortal i ty of up to 25% in broi lers. Strains of both pathotypes in fect adul t birds and af fect egg product ion and egg qual i ty to a var iable degree. In fected ch icks are the major source of v i rus in the envi ronment. Contaminated equ ipment and mater ia l are a potent ial source for ind i rect t ransmiss ion over large d is tances. Virus is present in cons iderable t i t res in t rachea l mucus and in faeces in the acute and recovery phases of d isease, respect ively. Virus spreads horizontal ly by aerosol ( inhalat ion) or ingest ion of faeces or contaminated feed or water . The virus is highly in fect ious. Clinical signs w i l l develop in con tac t ch icks w i th in 36 h and in nearby sheds w i th in one to t w o days. Infect ion is resolved w i th in four teen days w i t h a rise in ant ibody t i t res. In a small number of ch icks, latent in fect ion is establ ished w i t h subsequent errat ic shedding of v i rus for a pro longed period of t ime via both faeces and aerosol . Movemen t of live birds should be considered as a potent ial source for the in t roduct ion of IBV. Isolat ion and ident i f icat ion of IBV is needed for posit ive diagnosis. The prefer red method of isolat ion is to passage a sample in embryonat ing spec i f ied-pa thogen-f ree ch icken eggs. Ident i f icat ion is ei ther by monoc lona l ant ibody based enzyme-l inked immunosorbent assay (ELISA) or polymerase chain react ion. Virus neutra l isat ion test in t rachea l organ cul ture is the best method for ant igenic typ ing. Continual use of l ive vacc ines compl ica tes diagnosis s ince no simple d iagnost ic tool can di f ferent iate a f ie ld f rom a vacc ine stra in. Nuc leot ide sequenc ing of the S1 g lycoprote in is the only method to d iscr iminate be tween all IBV strains. Serology is also compl ica ted by cont inual use of live vacc ines . For surve i l lance purposes, ELISA is the method of cho ice , regardless of the ant igenic type of IBV involved. The assay is used to moni tor the response to vacc ina t ion , but f ie ld

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chal lenge can only be detected if f lock ant ibody status is moni tored continually. The ant igenic type of a chal lenge strain involved cannot be ascer ta ined by ELISA.

Keywords Avian diseases - Avian infectious bronchitis - Avian infectious bronchitis virus -Epidemiology - International trade - Source of infection - Transmission.

Description of the disease Main presenting signs Avian infectious bronchitis (IB) is primarily a respiratory infection of chickens. Nevertheless, three clinical manifestations of infectious bronchitis virus (IBV) infection are generally observed in the field, namely: respiratory disease, reproductive disorders and nephritis. Clinical signs will vary based on the age of chicks, pathogenicity of the virus strain and existing level of immunity. For detailed reviews of clinical signs associated with IB and pathology induced by IBV, see Cavanagh and Naqui, and McMartin (14, 79) .

Respiratory disease Respiratory disease is the most frequently observed syndrome caused by IBV (14, 48 , 79) . In broiler chicks of between two and six weeks of age, the main clinical signs seen are difficulty in breathing, tracheal rales, coughing and sneezing with or without nasal discharge. A generalised weakness is observed, accompanied by depression. Feed consumption and body weight are markedly reduced. Clinical signs in uncomplicated infections can be of short duration, commonly lasting less than seven days. Secondary infections due to Escherichia coli often follow, thereby accentuating the respiratory signs (19, 37). The chronic respiratory disease that develops may last for several weeks, with mortality between 5% and 25%. On necropsy, the trachea is congested with excessive amounts of mucus, and where infection has been complicated with E. coli, airsacculitis, pericarditis and perihepatitis may be observed (37). Similarly, the presence of Mycoplasma results in more severe clinical signs, with depressed growth and airsacculitis (17, 65).

Reproductive disorders Infectious bronchitis virus infection at a young age and after maturity can both lead to reproductive problems in hens. In adult chickens clinical signs may not be present or may take the form of a mild respiratory disease with coughing, sneezing and rales which can go unnoticed unless the flock is examined carefully. A decline in egg production usually follows within seven to twelve days (78). The severity of the decline in egg production varies according to the stage of lay at infection and the strain of virus involved. Typically, these declines are between 3% and 10%, but reductions of up to 5 0 % have also been observed. In some flocks, a decline in egg production will be the only feature. However, in many flocks the decline in egg production is also associated with eggs of smaller size

and inferior shell and internal egg quality, seen as soft-, pale-shelled and misshapen eggs, and eggs with thin albumen (97). Flocks usually return to near normal production within one week, but occasionally six to eight weeks might elapse before a return to normal production. In many instances, production levels remain subnormal, usually 6% to 12% below pre-infection levels (7). On post-mortem examination, the oviduct length may be reduced and ovarian regression is noticed in some birds (97). If IBV infection occurs when chicks are less than two weeks of age, permanent damage of the oviduct may result, leading to poor laying capacity (26, 56) .

Infect ious bronchit is virus nephri t is The nephritic form of IB is characterised by mild and transient respiratory signs followed by depression, ruffled feathers, hunched stance, reluctance to move, excessive water intake, rapid weight loss and diarrhoea. Characteristically, wet litter is present. Death occurs four to five days after infection and ceases by day twelve after infection (27). On necropsy, carcasses are dehydrated and dark in colour, kidneys are enlarged and may be pale or marbled, and deposits of urates may be present in the ureters of some chicks.

Principal lesions In chickens with respiratory disease, the main histological lesions are found in the trachea. However, these lesions are not pathognomonic for IBV. The virus replicates in ciliated epithelial cells causing deciliation, oedema, desquamation, hyperplasia and mononuclear cell infiltration of the submucosa (93). These changes are observed between three and nine days after infection. Regeneration of tracheal epithelium resumes approximately ten to fourteen days after infection.

In the oviduct, the height of the epithelial cells is reduced, and this is accompanied by a reduction or complete loss of cilia. The epithelial cells, especially the goblet cells, become cuboidal (97).

Following infection with nephropathogenic strains, virus replication first occurs in the trachea, causing histological lesions identical to those induced by respiratory strains. The route of spread to the kidney tissues is unknown. In the kidney, cytopathic changes become apparent, initially in the tubular epithelium. As a result, an interstitial inflammatory response is generated and polymorphonuclear leukocytes can

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be seen, first in the medullary region and then in the cortex. Extensive tubular degradation and necrosis follow and these are prominent at between five and ten days after infection. Focal areas of uric acid precipitation appear in the kidney and may also accumulate in the ureters (92, 94) . No correlation has been observed between the degree of kidney damage and the percentage of mortality observed in affected flocks (98) . The reparative process commences in the kidneys approximately eleven days after infection.

Certain variant strains of IBV induce additional or differing pathology to that described above. Strain 793/B was associated with bilateral myopathy of the deep and superficial pectoral muscle in broiler breeders, previously unreported lesions in IBV infection (43). Strains of the Delaware 072 serotype were found to cause severe airsacculitis in the absence of secondary bacterial infection (S. Naqi, personal communication). Some strains of IBV are also reported to show particular affinity for, and persistence in, the proventriculus.

Economic losses associated with the disease Flock management and the strain of virus involved play a major role in the impact of IBV infection. The principal losses are from production inefficiencies. The respiratory disease is debilitating, resulting in poor utilisation of feed by young chicks, and hence poor weight gains. Condemnation at processing due to airsacculitis also contributes to production losses. Following an outbreak of IB, an estimated 3% to 8% of the broilers can be condemned at the processing plant, in comparison to flocks in which IBV is controlled, where condemnation can be below 1%.

In layers and breeders, the main production losses are from non-realisation of full egg-laying potential. This may be a result of one of the following:

a) delayed maturity

b) decline in production during infection (estimated at between 3% and 50%)

c) sub-optimal production after recovery.

Additionally, losses are incurred due to downgrading of eggs. In breeders, the fertility rate could be reduced during and after an outbreak (7). In cases of IB nephritis, in addition to losses from poor weight gains and downgrading of carcasses, losses from mortality may be in the order of 10% to 2 5 % .

McMartin estimates that an IBV infection in a commercial flock with the best possible management practice reduces income by approximately 3%, in comparison with a hypothetical flock free from IBV (79). The United States of America (USA) Animal Health Association Report (105) lists respiratory diseases as the most significant source of economic loss to the broiler industry, IBV being the major causative agent of respiratory infection in broilers (S. Naqi, personal communication).

Incidence and distribution The disease was first reported in the early 1930s, and since then has been documented in all countries with an intensive poultry industry. Infectious bronchitis virus is thought to be endemic at the majority of poultry sites, with an incidence approaching 100%. Within a region where vaccination has not been practised, incidence of IBV infection was reported to be between 8 0 % and 9 0 % (48, 1 0 2 , 1 0 3 ) .

The importance of IB varies; in some areas the disease is considered as an on-going problem, whereas in others, periodic outbreaks occur which in some cases become epidemics. In colder areas, the incidence is usually higher in the winter months due to closing of the bird houses which compromises ventilation. Many episodes are caused by poor or inadequate vaccination. Where IB persists on a site, or in the case of an epidemic, the emergence or introduction of antigenic variants may be responsible.

Morbidity and mortality In broiler flocks, morbidity is virtually 100%, whereas mortality is usually low. Early reports describe mortalities of 2 0 % to 3 0 % , and these were almost certainly due to mixed infections with other infectious agents such as E. coli or Mycoplasma. In younger chicks affected with IB nephritis, mortalities of up to 2 5 % are common (28, 80) .

Description of the aetiological agent Principal sites of replication The initial and principal site of viral replication is the epithelium of the trachea, where IBV can be detected at day one after infection. From this site, the virus spreads to other internal organs, such as the lung, spleen, liver, kidney, oviduct, ovaries, testes, digestive and intestinal tracts ( 3 1 , 5 1 , 74) . The highest titre of virus, between 1 0 3 and 1 0 4 median egg infective doses, is found in the trachea and kidney tissues between three and five days post infection (51), and these tissues are the usual source for virus isolation in the acute phase of the disease.

Strain variation P a t h o t y p i c v a r i a t i o n Strains of IBV differ in virulence or pathogenicity for the respiratory tract, kidney or oviduct. Although the virulence of many IBV strains has not been clearly defined, examples illustrate the predominant feature of each pathotype. The majority of IBV strains, including those of the Massachusetts (Mass) serotype, of which the M41 is the representative strain, produce prominent respiratory disease (14) . Most of these strains do not induce mortality when acting alone. However, in experimental and field infections with E. coli, variable mortality rates are obtained, indicating the differing

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pathogenic potential of strains to predispose chicks to the development of airsacculitis, pericarditis and perihepatitis (99).

Pathogenicity of strains of IBV for the oviduct varies greatly. Many, but not all strains of IBV are able to replicate in the epithelial cells of the oviduct, causing pathological changes (26). The majority of those that are able to replicate will induce a decline in egg production, while others will not (78). Some of the respiratory strains cause only a slight reduction in egg production, but markedly affect the colour of the egg shell (23).

Nephropathogenic strains have been a predominant IBV pathotype only in Australia (28), with sporadic isolation in other countries. However, during the last decade, nephropathogenic strains have emerged in many countries including Italy, the USA, Belgium, France, the People's Republic of China j a p a n and Taipei China ( 1 3 , 7 0 , 1 0 6 , 1 1 1 ) . In some countries of Europe, these strains have become the predominant pathotype of concern (80) . The nephropathogenic IBV strains are able to induce mortality, principally in chicks under the age of ten weeks (28) , and differ markedly in virulence. The nephropathogenic strains of IBV isolated in Australia cause mortalities of between 5% and 8 0 % in experimental infections (66; J . Ignjatovic, F.D. Ashton, R. Reece, P. Scott and P. Hooper, unpublished observation), whereas those found in other countries result in mortality between 5% and 5 0 % (2, 81) .

Antigenic variation A multitude of IBV serotypes exist and this has become the major problem in diagnosis and control of IBV. Virus

Table I Distribution of serotypes of infectious bronchitis virus in different regions 122,32,34,41,43,54,55,64.71, 73,91, SB, 100,106)

neutralisation (VN) or haemagglutination-inhibition (HI) tests are used to determine the IBV serotype. The VN test is the preferred method, as the HI test lacks some specificity (24). Strains of the Mass serotype have been the predominant antigenic type in many countries, the exceptions being Australia and New Zealand (Table I). Although strains of Mass serotype are still frequently isolated, many unique and new serotypes have emerged world-wide (Table I). Comparisons of nucleotide sequences of IBV isolates from different parts of the world indicate that strains from a particular geographic area are more closely related to each other than to strains from other distant regions (Fig. 1). With the exception of strains of the Mass serotype, IBV strains from Europe differ from those found in the USA or Australia, and each geographic group can be distinguished by the uniqueness of the genetic sequences.

Host range Until recently, the chicken was considered as the only natural host of IBV. Chickens of all ages are susceptible, assuming that they have not had prior exposure to the virus and are not immune. Pheasants are the only other avian species that is now considered as a second natural host for IBV. Two reports from the United Kingdom (UK) have indicated recently that farm-reared pheasants are equally susceptible to IBV infection (44, 101). Clinically, the disease in pheasants resembled interstitial nephritis, with reduced egg production and high mortality. However, some species of pheasant are apparently not susceptible to IBV (5), or perhaps only certain strains of IBV are able to infect pheasants. Other avian species are not considered susceptible to infection with IBV. Antibodies to IBV were detected in turkeys, quails and in free-ranging rockhopper penguins (Eudyptes chrysocomes [chrysocome])

Serotypes identified Americas Europe Asia Australia New Zealand

Mass Mass Mass A-Vac1 A Conn D2C7 Conn B-Vic S B Florida 0212 Gray C-N1/62 C Clark 333 D3128 Ark S3 D-N9/74 D Ark 99 D389S N1/62-T E-Q1/73 SE17 D1466 Kb 8523-Japan F-V2/71 JMK PL-84084-France 793/B-llndia G-V1/71 Iowa 97 AZ23.74/ltaly NRZ-China H-N1/75 Iowa 609 81648-Belgium HV-Chtoa I-N2/75 Holte UK/918/67 SAIB3-China J-N3/63 Gray ÜK6/82 KMei-Korea K-T1/82 Main 209 UK/142/88 EJ95-Korea L-N1/88 DE/072/92 UK793/B A1121-Taiwan M-Q3/88 Chile 14 624/ltaly 0-NT2/89 50/96-Brazil P-N1/B1 22/97-Honduras Q-V18/91


Fig. 1 Phylogenetic relationship between strains of infectious bronchitis virus based on the nucleotide sequence of S1 glycoprotein available in GenBank Sequences were aligned using the program ClustalW (104) and used to calculate a distance matrix from which the most likely phylogenetic tree was inferred using neighbour-joining method

(62, 107), however, it is unclear whether IBV can be isolated from these species.

In vitro propagation systems The most effective in vitro propagation system is embryonating chicken eggs that originate from a specified-pathogen-free (SPF) flock (87). Eggs of between nine and eleven days of incubation are inoculated by the allantoic route. Allantoic fluid collected 36 h to 48 h after inoculation contains a significant amount of virus, typically between 1 0 5 and 1 0 7 median egg infective doses. However, for IBV to grow in eggs to an appreciable titre, adaptation of the virus is necessary, usually requiring three to five passages. Egg-adapted IBV induces morphological changes in embryos, such as dwarfing and curling, enabling positive IBV diagnosis (14). If adaptation of IBV is not desired, the field samples should be propagated in tracheal organ cultures (21).

Epidemiology Sources and routes of infection Contact with infected chickens is the most likely source of infection, tracheo-bronchial exudate and faeces of these birds being the major sources of virus. The virus spreads

horizontally by aerosol or ingestion. The rate of spread will depend on the virulence of virus and the immune status of the flock. The virus is highly infectious, and under natural conditions respiratory signs will develop in contact birds within 36 h. On a site, house-to-house spread will occur within one to two days, and between farms within three to four days. Faeces, and feed and drinking water that have been contaminated by faeces, are also sources of infection. The virus can survive for a considerable time in faeces and is suspected to represent a continuing source of re-infection in the recovery phase of the disease. Contaminated litter, footwear, clothing, utensils, equipment and personnel are all potential sources of virus for indirect transmission and have been implicated in IBV spread over large distances (36, 79 , 95) .

The role of vertical transmission in the epidemiology of IBV has not been clearly established. Vertical transmission of IBV has been suspected in one field case (76) and was demonstrated in another study in which experimentally infected hens layed infected eggs between one and six weeks after infection (20) . Virus was also isolated from the day-old chicks of these hens. This suggests that IBV can be transmitted vertically. Infectious bronchitis virus was also recovered from the semen of cockerels for up to two weeks after inoculation (20), indicating the possibility that the oviduct of a susceptible hen, and also an egg in the oviduct, could be infected with such semen.

Principal routes and duration of excretion During the acute phase of the disease, virus is copiously shed into the respiratory tract, and coughing birds shed virus into the environment. Infectious bronchitis virus can be recovered routinely from the trachea and lungs, between days one and seven post infection, in considerable titres. The virus can also be isolated from the cloacal contents from days one to twenty-four post infection. Faeces contain a significant amount of virus for a prolonged period of time - during an active infection, a recovery phase and during the course of chronic infections ( 3 , 1 9 ) .

Evidence of the carrier state or latency A true latency, such as that observed in herpesvirus infections, is not believed to occur in IBV infections. However, in some circumstances, IBV is thought to persist for a considerable time in infected birds at some privileged sites such as the kidney and the alimentary tract, particularly the caecal tonsils (4, 16, 35) . Persistence and prolonged excretion of IBV in faeces by a small number of chicks between 4 9 and 227 days after infection has been demonstrated ( 4 , 1 9 ) , R e - e x c r e t i o n is suspected to be triggered by adverse environmental factors or changed physiological conditions. Chicks infected at one-day-old with enterotropic G -Strain, re-excreted virus at onset of lay into both the trachea and cloaca and continued to excrete the virus for a prolonged period (57) . These chicks also re-excreted virus at twelve weeks following treatment with the immunosuppressive agent cyclosporin (6).

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Persistently infected birds are considered to be the likely source of re-infection in layers which occurs at regular intervals (19) . Persistence may be characteristic of some IBV strains only (3).

Methods of spread Method of spread is airborne or mechanical transmission between birds, houses and farms. Airborne transmission is via aerosol and occurs readily between birds kept at a distance of over 1.5 m. Prevailing winds might also contribute to spread between farms that are separated by a distance of as much as 1,200 m (29). Mechanical transmission of virus is via personnel, material and equipment, and plays a role in transmission of IBV between flocks or farms. Movement of live birds, either as one-day-old chicks or as adult birds, should be considered as a potential source for the introduction of IBV. Although this has not been directly demonstrated, the strong experimental evidence of IBV persistence and re-excretion are an indication of the potential risk (3). Vectors do not appear to be a factor in the spread of IBV.

Susceptibility of other species to infection Other species are not considered susceptible to natural infection with IBV. Infectious bronchitis virus can be propagated in suckling mice if inoculated by the intra-cerebral route (30) . However, such virus passage appears to result in selection of a virus population which is not pathogenic for chicks (110). Suckling rabbits and guinea-pigs are also susceptible to intra-cerebral inoculation with IBV.

Stability to environmental inactivation and disinfectants Infectious bronchitis virus is temperature sensitive; some strain differences may exist in terms of thermal sensitivity, but in general, most strains are inactivated at 56°C for 15 min to 45 min or at 45°C for 90 min (89). At room temperature, the virus will survive for only a few days. Viral infectivity is affected by storage at 4°C, and for long term storage, a temperature of - 7 0 ° C is recommended. Lyophilised virus stored at 4°C will retain infectivity for at least twenty-one months (50). Infectious bronchitis virus is easily inactivated by many common disinfectants including 7 0 % ethanol, chloroform, 1% phenol, 1% formalin and iodine (58). The virus is more stable at a low pH than at a high pH. At pH 3.0, the virus is stable for fourteen days. During vaccine administration, IBV longevity is affected by water quality and contents or additives (58). The virus appears to be able to survive for a considerable period of time in litter containing faeces.

Risks of importing the disease through poultry and poultry products Infectious bronchitis is a List B notifiable disease, and countries importing poultry genetic material should require testing to be performed (88). Hatching eggs do not represent a risk if the flock of origin is antibody-free. Day-old chicks and

older birds that are antibody-negative and have not been vaccinated prior to despatch are also low risk. However, these birds must be kept in quarantine conditions for at least four weeks, and for up to fifty days after arrival at a new location (88). Live birds of all ages that have been vaccinated and are antibody-positive, represent a risk since a small percentage of such birds may excrete virus until at least twenty weeks after infection. Processed poultry meat which has undergone treatment at temperatures above 56°C for 30 min to 45 min and is destined for human consumption is also considered a low risk. Infectious bronchitis virus has not been implicated as an accidental vaccine contaminant.

Measures to reduce the risk of infect ious bronchi t is virus spread through t rade

Controlled studies regarding the possibility of IBV transmission through trade in poultry products have not been performed. There is circumstantial evidence however, which indicates that unrestricted trade might have contributed to the spread of some IBV types. In the most rigorous analysis, and if the precautionary principle is adopted, eggs and live birds should be considered as potential sources and carriers of IBV. However, current trade practices have accepted that the risk of spread of IBV through trade in eggs and live chicks is low. Eggs are disinfected after collection as is required for the control of other pathogens. Chicks that are destined for export are hatched on premises with an acceptable or approved level of biosecurity. Whether vaccination of chicks against IBV will be practised at the hatchery will be determined between trading partners or may be regulated by the importing country. Countries importing eggs for hatching or live chicks might wish to quarantine the flock for 3 to 4 weeks in the presence of local antibody-free sentinel chicks.

Consequences of introduction into a susceptible population Although IBV is distributed world-wide, strong consideration should be given to measures to restrict the introduction of exotic IBV variants. As outlined, many countries share common IBV serotypes (Table I). However, in many of these countries or regions, new economically important strains have arisen that require different vaccines for control. An incursion of an unrelated IBV will necessitate the introduction of new vaccines, which will ultimately be reflected in higher costs, particularly in management, since a greater number of vaccines will need to be administered. In addition to the immediate economic significance, introduction of an exotic strain increases the pool of genetically different viruses that circulate on a site. This increases the likelihood of generating new and more variable strains through processes such as recombination (68). In both the USA and Australia, although variants continue to emerge, IBV strains have remained specific for each region (Fig. 1), illustrating the benefits of quarantine.


Diagnostic methods Positive diagnosis of IB cannot be made on clinical signs alone, since other avian pathogens such as Newcastle disease, infectious laryngotracheitis and avian pneumoviruses can produce similar clinical signs. Involvement of IBV should be confirmed by virus isolation and, if desired, the antigenic type can be determined by VN. Serological tests to document a recent IBV infection are only valid if no prior flock exposure to IBV has occurred, or if the antibody status of the flock is monitored and enables unambiguous detection of a specific rise in IBV antibody titres.

Laboratory tests to confirm diagnosis A number of tests are available (87); those considered most useful are discussed below. In adoption of any of the tests, consideration should be given to the diagnostic capability of laboratories involved and to the purpose of the positive IBV identification. None of the existing in vitro diagnostic tests will provide all of the necessary information. Only in vivo cross-protection experiments will demonstrate if the vaccine used on the premises is effective against the isolated field strain.

Virus isolat ion The most common diagnostic method is to passage samples in embryonating SPF chicken eggs. The test is lengthy since three to five passages are required for a field isolate to become adapted to the egg and cause gross embryo pathology typical of IBV. A sample is classified as negative if no embryo changes are induced at the sixth egg passage. Allantoic fluid of the third to sixth passages usually contains enough virus to be confirmed by an antigen-detecting ELISA or electron microscopy, if such a facility is available. Fluorescent staining of amniotic cells using reference IBV sera may be used to confirm virus isolation (18) . Tracheal organ culture is the most sensitive method for virus isolation and serotyping, particularly if adaptation of virus is not desired, however a specialised laboratory is required (21) . Propagation of virus can also be performed in chicken embryo kidney cells, although this method has been used only on material already propagated in eggs, and some selection of a particular virus population is likely to occur during this process (75).

Ant igen-detect ing enzyme-l inked immunosorbent assay Antigen-detecting ELISA is the most rapid test to confirm the presence of IBV, whether in a field sample or in allantoic fluid (53, 6 1 , 67 , 8 5 , 86 ) . All antigenic types can be detected, however commercial kits are not widely available and diagnostic laboratories may need to source the reagents from the laboratories in which the monoclonal antibodies have been produced. An antigen-detecting ELISA kit and monoclonal antibodies developed in Australia are available commercially.

R e v e r s e t r a n s c r i p t a s e p o l y m e r a s e c h a i n r e a c t i o n Reverse transcriptase polymerase chain reaction (RT-PCR) is a reliable and sensitive method which can be used to test field samples. The test can be tailored to detect all IBV strains by using primers for the conserved N protein or S2 glycoprotein of IBV ( 1 , 1 1 2 ) . However, RT-PCR is complex to perform and requires a level of expertise that may not be available in a diagnostic laboratory.

Strain di f ferent iat ion No easy method exists to differentiate between various IBV strains, due to the existence of multiple serotypes. Any of the following methods can be used, but each has some disadvantages. The most accurate strain differentiation is achieved by nucleotide sequencing of the S1 glycoprotein, however this cannot be done on a routine basis. Serotyping using VN in tracheal organ cultures or embryonating eggs is the recommended method (47, 55) . The VN test is lengthy and expensive, since multiple sera and IBV strains must be used in a two-way neutralisation assay (87) . Strains of Mass, Connecticut (Conn), Arkansas (Ark) or D207 serotypes, can be differentiated by monoclonal antibodies in ELISA (60 , 67, 86) . However, isolation of an IBV strain that reacts with a serotype-specific monoclonal antibody does not always guarantee that the isolate is identical to the expected serotype. Some of the recently developed methods based on RT-PCR aim to differentiate certain serotypes (63, 69 , 72) . Further developments in this area, particularly RT-PCR followed by restriction fragment analysis may provide more user-friendly, comprehensive and rapid differentiation methods.

Diagnosis is complicated by the simultaneous presence of vaccine and field strains, which is suspected to occur in many field samples (15). No simple diagnostic tools are available to distinguish between strains of IBV in the same sample. Some molecular probes enable differentiation of strains simultaneously present in the sample (15, 45) . However, these probes will only detect IBV genotypes that have been recognised previously.

Specificity of tests for surveillance and imports For surveillance purposes, an antibody-detecting ELISA is the most appropriate test. A number of commercial kits are available which will all detect seroconversion to IBV regardless of the antigenic type or origin of the virus. In an antibody negative flock that has been exposed to IBV, ELISA antibodies will appear approximately ten days after infection, and will continue to rise until approximately four weeks after infection, at which time the level of antibodies will stabilise (46). An ELISA will detect an antibody response in broilers following vaccination at one day of age, and also in layers and breeders vaccinated at three weeks of age and re-vaccinated at twelve to eighteen weeks of age ( 8 3 , 1 0 9 ) .

Imported poultry that are serologically negative should be quarantined for at least twenty-one days in the presence of sentinel SPF chicks. Imported chicks should be tested for


antibody levels at the time of import and twenty-one days later. Chicks which have been recently infected will give a negative result at the first testing but will seroconvert during the observation period and become antibody positive twenty-one days later. Sentinels will also seroconvert, confirming in-contact transmission. Chicks which are immune at the time of import should be quarantined for a period of at least seven to eight weeks, preferably longer, in the presence of sentinel SPF chicks. If imported poultry is persistently infected, the antibody level will remain unchanged during the observation period. However, if the birds are excreting IBV, sentinel chicks will seroconvert.

If chicks are immune at the time of import and are persistently infected, a quarantine period of seven to eight weeks may not be sufficient, since some of the persistently infected birds may re-excrete virus only under certain conditions, for example at commencement of lay.

Public health implications Risk to public health No risks to human health are suspected or have been demonstrated to arise from IBV. Neutralising antibodies have been detected in people working with commercial chicken flocks (82), but the significance of this remains unknown.

Possibility of transmission from human to human or human to animal No evidence exists to suggest that humans act as a reservoir for active replication of IBV and no evidence has been found of transmission from human to human, or human to animal. Humans can only transmit IBV to chicks by mechanical means. Reported isolation of an 'avian IBV-like* virus from humans was later demonstrated to be an isolate of human Coronavirus and not an IBV Coronavirus (59) .

Methods of prevention and control Exclusion and eradication None of the countries which have an Intensive poultry industry are free from IBV. Although attempts have been made, at the regional level, to keep flocks free from IBV, none have been successful. Given the highly infectious nature of the virus, even the strictest preventative measures are sometimes not sufficient. In one case, flocks well separated from other commercial farms became infected despite strict attention to quarantine (95). In another case, all flocks in a region became infected by a variant IBV strain from another region although import regulations prohibited the entry of live poultry and eggs (79). Only the strictest of quarantine measures, such as those used to maintain SPF poultry, and which cannot be

justified commercially, appear to be able to exclude IBV. Under normal flock management with 'all-in/all-out' operations, cleaning and disinfection between batches will limit the level of infection to a minimum; however, exclusion of IBV has not been achieved through such measures. Under the present circumstances, eradication is not a consideration, but may become a long-term prospect with the introduction of recombinant IBV vaccines, which would replace live vaccines, and thus reduce the levels of infectious virus in the environment.

Breeding to increase resistance Breeding to increase resistance to IBV is not practised. As evident from both field and experimental studies, all chicks are susceptible to infection with IBV. However, the outcome of the infection may vary in different lines. In experimental studies that included inbred laboratory lines, varying mortality rates were observed in lines inoculated with a pool of respiratory IBV strains and E. coli (11 , 12, 99) . Chicks of one 'susceptible' line showed more severe respiratory signs and had a prolonged recovery phase in comparison to chicks of a 'resistant' line. However, no differences were found between these 'susceptible' and 'resistant' lines in the rate of virus replication (90). Similar genetic differences between inbred lines have also been observed following infection with a nephropathogenic strain; one line exhibited high mortality from nephritis whereas no mortality occurred in another line J. Ignjatovic, R. Reece and F.D. Ashton, unpublished observation). Another study noted that White Leghorns were severely affected by IB nephritis in comparison to a layer line from Australia which exhibited only mild clinical disease (27).

Vaccination The only practical means of controlling IB is vaccination, which is routinely used throughout the intensive poultry industry. The following factors are a feature of IB vaccination:

a) vaccinal immunity is not long-lasting and re-vaccination is necessary

b) the selection of an appropriate antigenic type for the region is important, given the existence of wide antigenic variation

c) timing and method of vaccine application will vary for different flocks and may require adjustment according to practical experiences.

Type of vaccine used and effectiveness in protecting against infection and disease Live and inactivated vaccines are used. Vaccination programmes and procedures may differ from one country to another, or even from one farm to another within the same country, depending on local conditions.

Live vaccines Live vaccines are in widespread use. These vaccines represent IBV strains that have been passaged in embryonated chicken eggs to achieve a reduction in virulence for the respiratory

Rev. sci. tech. Off. int. Epiz., 19 (2| 501

tract. Consequently, depending on the level of attenuation, IBV vaccines can be either mild or virulent. Live IBV vaccines are administered by either coarse spray, aerosol or drinking water, depending upon the vaccine used (49). Embryo vaccination is not practised but vaccines suitable for such a purpose are in development. Vaccination of day-old chicks at the hatchery, with vaccines of low virulence, is practised in most countries since this is a simple way of handling the birds. More virulent vaccines are used for booster vaccination at approximately seven to ten days, usually in the drinking water. Vaccines of low virulence are suitable for chicks with a lower level of maternal immunity and the advantage of these vaccines is that they do not cause the respiratory reactions or lead to a decline in growth rate, which can occur with vaccines of greater virulence (39) . The disadvantage of milder vaccines is that the level of immunity provided is low, and is only sufficient to protect the respiratory tract. Often these vaccines will not protect tissues such as the kidney and oviduct against challenge with strains which are nephropathogenic or pathogenic for the reproductive tract (33). Broilers in sparsely populated areas may be adequately protected with a single IB vaccination, whereas those in areas with a high density of farms and those on multi-age sites require early vaccination with follow-up vaccinations. The vaccination procedure used must immunise all chicks concurrently, whether this is in day-old or in follow-up vaccinations, so as to minimise 'back passaging' of vaccine virus. Some live vaccines have a significant degree of residual virulence and a tendency to increase airsacculitis, particularly in adverse environmental conditions (52) .

Many antigenic types of IB vaccines are available and this is a major problem for many poultry growers. The choice of vaccines will decide the success of vaccination and should be based on prior information as to which antigenic types are prevalent in the region. The vaccines used most frequently are based on Mass strains, examples of these are M41, Ma5, H52 and H120. Other monovalent vaccines for regional use are also available, such as Conn 46 , Ark 99 , Florida, JMK, 4/72, D247, etc. Often two different antigenic types of vaccines are required (108) . Indiscriminate use of live vaccines is strongly discouraged. Live vaccine strains from other parts of the world should not be used or introduced if prevailing endemic strains are of a different serotype or genetic lineage. In the USA, application of variant vaccines requires approval by the regulatory authority and this is issued only on a regional basis. Such practice has restricted the distribution of some unique antigenic types into other regions.

Inactivated vacc ines

Inactivated vaccines are intended for use in layers and breeders. The vaccines are administered by subcutaneous inoculation at thirteen to eighteen weeks of age, to pullets which have been previously primed with live attenuated vaccines. Inactivated vaccines provide high and uniform levels of antibodies that persist for longer periods than those

induced by live vaccination (8, 9, 42) . These high levels of antibodies are particularly useful in providing protection for the internal organs by preventing spread of the virus. In layers and breeders, inactivated vaccines provide protection against reductions in egg production, which might not always be afforded by live vaccination (8). In addition, in breeders, progeny chicks will be protected by maternally transferred antibodies. Progeny chicks that originate from breeders vaccinated with inactivated vaccines have high and uniform maternal antibody levels in comparison to broilers from dams vaccinated with live vaccines only. Most of the inactivated vaccines are of one type, Mass M41; however, bivalent vaccines that incorporate additional variant antigens may also be necessary ( 3 8 , 8 4 ) . Inactivated vaccines are produced from IBV-infected allantoic fluid, which is inactivated and usually formulated as oil emulsion vaccine. The disadvantage of such vaccines is the expense.

Possible reasons for vacc ine fa i lures A number of factors influence the outcome of live vaccination (77). Vaccine preparations should be appropriately stored at all times and not used after the expiry date. The efficacy of vaccines will decline if stored for long periods at room temperature and if diluted in an inappropriate solution. Vaccine failure may also result if the chosen vaccine is not protecting against the prevalent antigenic type; if the vaccine is too mild or too virulent; or if the vaccinating dose per chick is too low due to excessive dilution of the vaccine. The route of vaccine application will affect the level and the duration of immunity as well as the vaccine reaction. The chosen route will depend upon previous experience or on the degree of clinical reaction in the flock. Manufacturers state the preferred method of vaccine application which can vary according to the strain.

Inactivated vaccines, if applied correctly, perform well in most situations. The lack of efficacy of inactivated vaccines may be due to inappropriate priming or because challenge strains are of a variant serotype which is not included in the common inactivated IB vaccines.

Ability to distinguish between vaccine and field strains and between immune responses to these strains No reagents or simple molecular markers are able to distinguish a vaccine from a field strain. The simple procedure is to determine whether a field sample causes prominent dwarfing and pathology in embryonating eggs in the first inoculation. If so, the sample is likely to be a vaccine strain. The result needs to be confirmed by other tests such as VN and nucleotide sequencing. Certain IBV vaccines, such as Mass, Conn, Ark or D274 can be differentiated by serotype-specific monoclonal antibodies (67, 86) . Nucleotide sequencing of the S I gene can differentiate all vaccine strains and is the method of choice. No simple serological methods


are able to differentiate between exposure of commercial flocks to different IBV serotypes. A VN test can be used (25), but considerable cross-reaction can occur (40) , and extensive testing is required involving IBV strains of many serotypes. Similarly, the use of an HI test is restricted, since exposure to

Virus de la bronchite infectieuse aviaire J. Ignjatovic & S. Sapats

multiple field strains of IBV can mask the detection of

serotype-specific antibodies (10).

Résumé Le virus de la bronchi te in fect ieuse est présent dans tous les pays prat iquant une avicul ture industr iel le intensive, l ' inc idence étant souvent proche de 100%. La vacc inat ion ne donne pas des résul tats p le inement sat isfaisants en raison de l 'appari t ion permanente de var iants ant igéniques. Dans de nombreux élevages, des types ant igéniques mult ip les sont présents s imul tanément , d'où la nécessi té de recour i r à des vacc ins mult iples. Nombre de pays ont en commun certains types ant igéniques, mais les souches du virus de la bronchi te in fect ieuse au sein d'une même zone géographique sont uniques et dist inctes ; c 'est no tamment le cas de l 'Europe, des États-Unis d 'Amér ique et de l 'Austral ie. Il conv ient donc d 'envisager des mesures v isant à restre indre l ' in t roduct ion de souches exot iques de ce vi rus.

Cette maladie a d ' importantes conséquences économiques : chez les poulets de chair, les pertes sont dues aux retards de cro issance, aux saisies à l 'abattoir et à la morta l i té, alors que chez les pondeuses, elles sont l iées à une per turbat ion de la ponte et à un déc lassement des œufs. Les poulets et fa isans d'élevage sont les seuls hôtes naturels du virus de la bronchi te in fect ieuse. Les autres espèces ne sont pas cons idérées comme des réservoirs de ce v i rus. La plupar t des souches du virus provoquent des lésions de la t rachée et des maladies respirato i res avec une légère mortal i té due à des sur in fect ions bactér iennes, essent ie l lement chez les poulets de chair. Certaines souches néphropathogéniques occas ionnent , outre des lésions de la t rachée, des lésions rénales, la mortal i té pouvant alors s 'élever à 25 % chez les poulets de chair. Ces deux types de souches in fectent les sujets adultes et af fectent à des degrés divers la ponte et la qual i té des œufs.

Les volai l les in fectées sont la pr incipale source du virus dans l 'environnement. Les instal lat ions et le matér ie l contaminés représentent une source potent iel le de t ransmiss ion indi recte sur de longues d is tances. Le virus est présent à des t i tres t rès élevés dans la muqueuse t rachéa le lors des phases aiguës de la maladie, ainsi que dans les déject ions en période de guér ison. Le virus se propage hor izonta lement par aérosols ( inhalat ion) ou ingest ion de déject ions, d'al iments ou d'eau contaminés. Son pouvoir in fect ieux est élevé. Les signes cl iniques appara issent chez les poussins 36 heures après leur con tac t avec des sujets atteints ; dans les bât iments vois ins, l ' infect ion apparaît dans un délai d'un à deux jours. La guér ison interv ient au bout de 14 jours, avec l 'élévation des t i t res d 'ant icorps. Chez un peti t nombre de poussins, on observe une in fect ion latente avec él iminat ion du virus pendant une longue pér iode, dans les déject ions et les aérosols. Les déplacements de volai l les v ivantes doivent être considérés comme une source potent iel le d ' in t roduct ion du virus de la bronchi te in fect ieuse. Les œufs et les poussins provenant d 'élevages reconnus indemnes sont les seuls à ne présenter aucun r isque.

Le diagnost ic doit être établ i par l ' isolement et l ' ident i f icat ion du v i rus. La méthode d ' iso lement pr iv i légiée est celle de l ' inoculat ion d'œufs de poule embryonnés


exempts d 'organismes pathogènes spéc i f iques. Le v i rus est ident i f ié soi t par une épreuve immuno-enzymat ique (ELISA) ut i l isant des ant icorps monoc lonaux, so i t par l 'ampl i f icat ion en chaîne par polymérase. La neutra l isat ion v i ra le dans une cul ture réal isée à part ir d 'écouvi l lons t rachéaux est la mei l leure méthode de typage ant igénique. L'uti l isation permanente de vacc ins à virus v ivant compl ique le diagnost ic car il n'existe pas d'outi l d iagnost ique simple permet tant de dist inguer les souches sauvages des souches vacc ina les . Le séquençage des nuc léot ides de la g lycoproté ine S1 est la seule techn ique permet tant de dist inguer les di f férentes souches du v i rus de la bronch i te in fect ieuse. L'uti l isation permanente de vacc ins à v i rus v ivant compl ique éga lement les examens séro logiques. Pour les besoins de l 'ép idémio-surve i l lance, l 'épreuve ELISA est la plus ind iquée, quel que soi t le type ant igénique du virus concerné . L'épreuve est ut i l isée pour suivre la réponse à la vacc ina t ion , mais l 'appari t ion d'une in fect ion naturel le ne peut être déce lée que si le s tatut immunolog ique de l 'élevage est contrô lé régul ièrement . Le type ant igénique de la souche responsable ne peut être ident i f ié par l 'épreuve ELISA.

Mots-clés Bronchite infectieuse aviaire - Commerce international - Épidemiologie - Maladies aviaires - Source de l'infection - Transmission - Virus de la bronchite infectieuse aviaire.

El virus de la bronquitis infecciosa aviar J. Ignjatovic & S. Sapats

Resumen El v i rus de la bronqui t is in fecc iosa aviar es prevalente en todos los países que albergan exp lo tac iones avícolas de t ipo intensivo, hasta el punto de que en muchas áreas la inc idencia de la in fecc ión ronda el 100%. Debido a la cont inua apar ic ión de var iantes ant igén icas, las vacunac iones resul tan e f icaces sólo en parte. En muchas explotac iones están presentes diversos t ipos ant igénicos a la vez, lo que requiere la ap l icac ión s imul tánea de var ias vacunas. Aunque hay algunos t ipos ant igénicos comunes a muchos países, las cepas vír icas presentes en cada región geográf ica revisten un carác ter único y dist int ivo (valgan, entre otros e jemplos, los de Europa, Estados Unidos de Amér ica o Austra l ia) . Por ello sería necesar io estudiar la ap l icac ión de medidas capaces de contener la penet rac ión de cepas exót icas. La bronqui t is in fecc iosa da lugar a cons iderables perjuic ios económicos : en pollos asaderos, las pérdidas product ivas están l igadas al escaso aumento de peso, el descar te de ejemplares no aptos para el consumo, y a la tasa de mor ta l idad; en aves ponedoras, las pérdidas der ivan de la menor p roducc ión de huevos y de su infer ior ca l idad. Los únicos huéspedes naturales de este v i rus son los pollos y los fa isanes cr iados a escala industr ia l . Las demás especies no están cons ideradas como reservor ios vír icos. La mayoría de las cepas del v i rus provocan lesiones en la t ráquea y la subsiguiente enfermedad respirator ia, con cierta mor ta l idad, aunque escasa, l igada a in fecc iones bacter ianas secundar ias , pr inc ipa lmente en pollos asaderos. Además de las lesiones t raquea les , las cepas nef ropatogénicas provocan importantes lesiones renales y dan lugar a una tasa de morta l idad de hasta el 25% en pollos asaderos. Las aves adultas se ven afectadas por cepas de ambos t ipos patogénicos, con consecuenc ias var iables en cuanto a la p roducc ión y la ca l idad de los huevos.


Los poll i tos in fectados const i tuyen la pr inc ipal fuente excretora del v i rus al entorno. El mater ia l o los productos contaminados son un posible foco de t ransmis ión indirecta a larga d is tancia. En las fases aguda y de conva lecenc ia , el v i rus está presente a t í tulos cons iderables en el mucus t raquea l y las heces, respect ivamente. La in fecc ión se t ransmi te hor izonta lmente, ya sea por aerosol ( inhalación) o por ingest ión de heces o agua y al imentos contaminados. Se trata de un v i rus ext remadamente in fecc ioso: los poll i tos expuestos a la fuente del v i rus mani festarán signos cl ínicos en un plazo de 36 horas, y los e jemplares de corra les adyacentes lo harán al cabo de uno a dos días. La in fecc ión remite dentro de los 14 días s iguientes, con un aumento del t í tulo de ant icuerpos, aunque unos pocos poll i tos la conservarán en forma latente y segui rán excre tando el virus er rá t icamente durante largo t iempo, por vía tanto feca l como respirator ia. Por ello debe considerarse que el movimiento de ejemplares vivos const i tuye un posible mecanismo de in t roducc ión del v i rus de la bronquit is in fecc iosa aviar. Sólo serán inocuos a c iencia c ierta los huevos y pol l i tos procedentes de bandadas seronegat ivas. Para el d iagnóst ico de conf i rmac ión es necesar io aislar e ident i f icar el v i rus. El mejor método de ais lamiento consiste en inocular una muestra dentro de huevos embr ionar ios de pollo l ibres del patógeno espec i f icado. Para la ident i f icac ión puede usarse indist in tamente un ensayo inmunoenzimát ico (ELISA) con ant icuerpos monoc lona les o la ampl i f icac ión en cadena por la pol imerasa (PCR). La prueba de neutra l ización viral sobre un cul t ivo de te j ido t raquea l , por su parte, es el mejor método de t ip i f i cac ión ant igénica. El empleo incesante de vacunas vivas compl ica el d iagnóst ico, pues no hay ninguna técn ica de diagnóst ico senci l la capaz de dist inguir entre cepas salvajes y cepas de vacuna . El único método que permite d iscr iminar entre todas las cepas del v i rus es la secuenc iac ión de la g l icoprote ina S1 . El uso cont inuo de vacunas vivas di f icul ta también el uso de técn i cas serológicas. Para f ines de v ig i lanc ia , y con independenc ia del t ipo ant igénico del que se t ra te , el método más indicado es el ELISA. Esta técn ica se utiliza para cont ro lar la respuesta a la vacunac ión , pero para detectar una in fecc ión de campo es preciso hacer un seguimiento cont inuo de los niveles de ant icuerpos que presenta la bandada. La técn ica ELISA no permite determinar el t ipo ant igénico de la cepa causante de una in fecc ión.

Palabras clave Bronquitis infecciosa aviar - Comercio internacional - Enfermedades aviares -Epidemiología - Fuente de infección - Transmisión - Virus de la bronquitis infecciosa aviar.

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