Download Avian infectious bronchitis virus

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
Infectious bronchitis virus (IBV) is prevalent in all countries w i t h an intensive
poultry industry, w i t h the incidence of infection approaching 100% in most
locations. Vaccination is only partially successful due to the continual e m e r g e n c e
of antigenic variants. A t many sites, multiple antigenic types are simultaneously
present, requiring the application of multiple vaccines. A l t h o u g h many countries
share some c o m m o n antigenic types, IBV strains w i t h i n a geographic region are
unique and distinct, examples are Europe, the United States of A m e r i c a and
Australia. M e a s u r e s to restrict the introduction of exotic IBV strains should
therefore be considered.
Infectious bronchitis has a significant economic impact; in broilers, production
losses are due to poor w e i g h t gains, condemnation at processing and mortality,
w h i l s t in laying birds, losses are due to suboptimal egg production and
d o w n g r a d i n g of eggs.
Chickens and commercially reared pheasants are the only natural hosts for IBV.
Other species are not considered as reservoirs of IBV. The majority of IBV strains
cause t r a c h e a l lesions and respiratory disease w i t h low mortality due to
secondary bacterial infections, primarily in broilers. Nephropathogenic strains, in
addition to t r a c h e a l lesions, also induce prominent kidney lesions w i t h mortality of
up to 25% in broilers. Strains of both pathotypes infect adult birds and affect egg
production and egg quality to a variable degree.
Infected chicks are the major s o u r c e of virus in the environment. Contaminated
equipment and material are a potential source for indirect transmission over large
distances. Virus is present in considerable titres in tracheal mucus and in f a e c e s
in the acute and recovery phases of disease, respectively. Virus spreads
horizontally by aerosol (inhalation) or ingestion of faeces or contaminated feed or
water. The virus is highly infectious. Clinical signs w i l l develop in contact chicks
w i t h i n 36 h and in nearby sheds within one to t w o days. Infection is resolved
w i t h i n fourteen days w i t h a rise in antibody titres. In a small number of chicks,
latent infection is established w i t h subsequent erratic shedding of virus for a
prolonged period of time via both faeces and aerosol. M o v e m e n t of live birds
should be considered as a potential source for the introduction of IBV.
Isolation and identification of IBV is needed for positive diagnosis. The preferred
method of isolation is to passage a sample in embryonating specified-pathogenfree chicken eggs. Identification is either by monoclonal antibody based
enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction. Virus
neutralisation test in tracheal organ culture is the best method for antigenic
typing. Continual use of live vaccines complicates diagnosis since no simple
diagnostic tool can differentiate a field from a v a c c i n e strain. Nucleotide
sequencing of the S1 glycoprotein is the only method to discriminate b e t w e e n all
IBV strains.
Serology is also complicated by continual use of live vaccines. For surveillance
purposes, ELISA is the method of choice, regardless of the antigenic type of IBV
involved. The assay is used to monitor the response to v a c c i n a t i o n , but field
Rev. sci. tech. Off. int. Epiz., 19
494
challenge can only be detected if flock antibody status is monitored continually.
The antigenic type of a challenge strain involved cannot be ascertained 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, 7 9 ) .
Respiratory disease
Respiratory disease is the most frequently observed syndrome
caused by IBV (14, 4 8 , 7 9 ) . 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 ( 1 9 ,
37). The chronic respiratory disease that develops may last for
several weeks, with mortality between 5% and 2 5 % . 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).
Infectious bronchitis virus nephritis
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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Rev. sci. tech. Off. int. Epiz., 19 (2)
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, 9 4 ) . No correlation
has been observed between the degree of kidney damage and
the percentage of mortality observed in affected flocks ( 9 8 ) .
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 0 7 2
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 5 0 % )
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 1 0 % 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 ( 7 9 ) . The United States of
America (USA) Animal Health Association Report ( 1 0 5 ) 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 1 0 0 % . Within a region where vaccination has
not been practised, incidence of IBV infection was reported to
be between 8 0 % and 9 0 % ( 4 8 , 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 1 0 0 % , 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, 8 0 ) .
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 and 1 0 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.
3
4
Strain variation
Pathotypic variation
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 ( 1 4 ) . 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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Rev. sci. tech. Off. int. Epiz., 191
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 ( 2 8 ) , 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 ( 8 0 ) . The nephro­
pathogenic IBV strains are able to induce mortality,
principally in chicks under the age of ten weeks ( 2 8 ) , and
differ markedly in virulence. The nephropathogenic strains of
IBV isolated in Australia cause mortalities of between 5% and
80%
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, 8 1 ) .
Antigenic variation
A multitude of IBV serotypes exist and this has become the
major problem in diagnosis and control of IBV. Virus
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])
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)
Americas
Europe
Mass
Conn
Florida
Clark 333
Ark 99
SE17
JMK
Iowa 97
Iowa 609
Holte
Gray
Main 209
DE/072/92
Chile 14
50/96-Brazil
22/97-Honduras
Mass
D2C7
0212
D3128
D389S
D1466
PL-84084-France
AZ23.74/ltaly
81648-Belgium
UK/918/67
ÜK6/82
UK/142/88
UK793/B
624/ltaly
Serotypes identified
Asia
Mass
Conn
Gray
Ark S3
N1/62-T
Kb 8523-Japan
793/B-llndia
NRZ-China
HV-Chtoa
SAIB3-China
KMei-Korea
EJ95-Korea
A1121-Taiwan
Australia
New Zealand
A-Vac1
B-Vic S
C-N1/62
D-N9/74
E-Q1/73
F-V2/71
G-V1/71
H-N1/75
A
B
C
D
I-N2/75
J-N3/63
K-T1/82
L-N1/88
M-Q3/88
0-NT2/89
P-N1/B1
Q-V18/91
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Rev. sci. tech. Off. int. Epiz., 19 (2)
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 3 6 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 ( 3 6 , 7 9 ,
95).
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 3 6 h to 4 8 h after
inoculation contains a significant amount of virus, typically
between 1 0 and 1 0 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).
5
7
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
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 ( 7 6 ) 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, 3 5 ) . Persistence and prolonged excretion o f IBV i n
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 nis
suspected to be triggered by adverse environmental factors o r
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 ( 5 7 ) . These chicks
also re-excreted virus at twelve weeks following treatment
with the immunosuppressive agent cyclosporin (6).
498
Persistently infected birds are considered to be the likely
source of re-infection in layers which occurs at regular
intervals (19). Persistence may b e 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 ( 2 9 ) . 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 9 0 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 ( 5 8 ) . 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 ( 5 8 ) . 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
Rev. sci. tech. Off. int. Epiz., 1
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 3 0 min to 4 5 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 infectious bronchitis
virus spread through trade
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.
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Rev. sci. tech. Off. int. Epiz., 19 (2)
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 isolation
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 ( 1 8 ) . 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 ( 2 1 ) . 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).
Antigen-detecting enzyme-linked 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 , 6 7 , 8 5 , 8 6 ) . 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.
Reverse transcriptase polymerase chain reaction
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 differentiation
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, 5 5 ) . 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 D 2 0 7 serotypes, can
be differentiated by monoclonal antibodies in ELISA (60, 6 7 ,
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 ( 6 3 , 6 9 , 7 2 ) . 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 ( 1 5 , 4 5 ) . 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
500
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 b e 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 b e
Rev. sci. tech. Off. int. Epiz., 19
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 ( 1 1 , 12, 9 9 ) . 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 ( 9 0 ) . 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
501
Rev. sci. tech. Off. int. Epiz., 19 (2|
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 ( 4 9 ) . 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 M 4 1 , Ma5, H52
and H 1 2 0 . Other monovalent vaccines for regional use are
also available, such as Conn 4 6 , Ark 9 9 , Florida, JMK, 4 / 7 2 ,
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 vaccines
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, 4 2 ) . 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 M 4 1 ; 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 vaccine failures
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 D 2 7 4 can be differentiated by
serotype-specific monoclonal antibodies ( 6 7 , 86). Nucleotide
sequencing of the S I gene can differentiate all vaccine strains
and is the method of choice. No simple serological methods
502
Rev. sci. tech. Off. int. Epiz., 19 (2)
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
multiple field strains of IBV can mask the detection of
serotype-specific antibodies (10).
Virus de la bronchite infectieuse aviaire
J. Ignjatovic & S. Sapats
Résumé
Le virus de la bronchite infectieuse est présent dans tous les pays pratiquant une
aviculture industrielle intensive, l'incidence étant souvent proche de 1 0 0 % . La
vaccination ne donne pas des résultats pleinement satisfaisants en raison de
l'apparition permanente de variants antigéniques. Dans de nombreux élevages,
des types antigéniques multiples sont présents simultanément, d'où la nécessité
de recourir à des v a c c i n s multiples. Nombre de pays o n t en c o m m u n certains
types antigéniques, mais les souches du virus de la bronchite infectieuse au sein
d'une même zone géographique sont uniques et distinctes ; c'est n o t a m m e n t le
cas de l'Europe, des États-Unis d'Amérique et de l'Australie. Il convient donc
d'envisager des mesures visant à restreindre l'introduction de s o u c h e s exotiques
de ce virus.
Cette maladie a d'importantes c o n s é q u e n c e s économiques : chez les poulets de
chair, les pertes sont dues aux retards de croissance, aux saisies à l'abattoir et à
la mortalité, alors que chez les pondeuses, elles sont liées à une perturbation de
la ponte et à un déclassement des œufs.
Les poulets et faisans d'élevage sont les seuls hôtes naturels du virus de la
bronchite infectieuse. Les autres espèces ne sont pas considérées c o m m e des
réservoirs de ce virus. La plupart des souches du virus provoquent des lésions de
la t r a c h é e et des maladies respiratoires avec une légère mortalité due à des
surinfections bactériennes, essentiellement chez les poulets de chair. Certaines
s o u c h e s néphropathogéniques occasionnent, outre des lésions de la t r a c h é e ,
des lésions rénales, la mortalité pouvant alors s'élever à 25 % chez les poulets de
chair. Ces deux types de souches infectent les sujets adultes et affectent à des
degrés divers la ponte et la qualité des œufs.
Les volailles infectées sont la principale source du virus dans l'environnement.
Les installations et le matériel contaminés représentent une source potentielle de
transmission indirecte sur de longues distances. Le virus est présent à des titres
très élevés dans la muqueuse trachéale lors des phases aiguës de la maladie,
ainsi que dans les déjections en période de guérison. Le virus se propage
horizontalement par aérosols (inhalation) ou ingestion de déjections, d'aliments
ou d'eau contaminés. Son pouvoir infectieux est élevé. Les signes cliniques
apparaissent chez les poussins 36 heures après leur c o n t a c t avec des sujets
atteints ; dans les bâtiments voisins, l'infection apparaît dans un délai d'un à deux
jours. La guérison intervient au bout de 14 jours, avec l'élévation des titres
d'anticorps. Chez un petit nombre de poussins, on observe une infection latente
avec élimination du virus pendant une longue période, dans les déjections et les
aérosols. Les déplacements de volailles vivantes doivent être considérés comme
une source potentielle d'introduction du virus de la bronchite infectieuse. Les
œufs et les poussins provenant d'élevages reconnus indemnes sont les seuls à ne
présenter aucun risque.
Le diagnostic doit être établi par l'isolement et l'identification du virus. La méthode
d'isolement privilégiée est celle de l'inoculation d'œufs de poule embryonnés
503
Rev. sci. tech. Off. int. Epiz., 19 (2)
exempts d'organismes pathogènes spécifiques. Le virus est identifié soit par une
épreuve immuno-enzymatique (ELISA) utilisant des anticorps monoclonaux, soit
par l'amplification en chaîne par polymérase. La neutralisation virale dans une
culture réalisée à partir d'écouvillons t r a c h é a u x est la meilleure méthode de
typage antigénique. L'utilisation permanente de v a c c i n s à virus vivant complique
le diagnostic car il n'existe pas d'outil diagnostique simple permettant de
distinguer les souches sauvages des s o u c h e s vaccinales. Le s é q u e n ç a g e des
nucléotides de la glycoprotéine S1 est la seule t e c h n i q u e permettant de
distinguer les différentes souches du virus de la bronchite infectieuse.
L'utilisation permanente de v a c c i n s à virus vivant complique également les
examens sérologiques. Pour les besoins de l'épidémio-surveillance, l'épreuve
ELISA est la plus indiquée, quel que soit le type antigénique du virus c o n c e r n é .
L'épreuve est utilisée pour suivre la réponse à la v a c c i n a t i o n , mais l'apparition
d'une infection naturelle ne peut être décelée que si le statut immunologique de
l'élevage est contrôlé régulièrement. Le type antigénique de la souche
responsable ne peut être identifié 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 virus de la bronquitis infecciosa aviar es prevalente en todos los países que
albergan explotaciones avícolas de tipo intensivo, hasta el punto de que en
muchas áreas la incidencia de la infección ronda el 100%. Debido a la continua
aparición de variantes antigénicas, las v a c u n a c i o n e s resultan eficaces sólo en
parte. En muchas explotaciones están presentes diversos tipos antigénicos a la
vez, lo que requiere la aplicación simultánea de varias vacunas. Aunque hay
algunos tipos antigénicos comunes a muchos países, las cepas víricas presentes
en cada región geográfica revisten un carácter único y distintivo (valgan, entre
otros ejemplos, los de Europa, Estados Unidos de A m é r i c a o Australia). Por ello
sería necesario estudiar la aplicación de medidas capaces de contener la
penetración de cepas exóticas.
La bronquitis infecciosa da lugar a considerables perjuicios económicos: en
pollos asaderos, las pérdidas productivas están ligadas al escaso aumento de
peso, el descarte de ejemplares no aptos para el consumo, y a la tasa de
mortalidad; en aves ponedoras, las pérdidas derivan de la menor producción de
huevos y de su inferior calidad.
Los únicos huéspedes naturales de este virus son los pollos y los faisanes criados
a escala industrial. Las demás especies no están consideradas como reservorios
víricos. La mayoría de las cepas del virus provocan lesiones en la tráquea y la
subsiguiente enfermedad respiratoria, con cierta mortalidad, aunque escasa,
ligada a infecciones bacterianas secundarias, principalmente en pollos asaderos.
A d e m á s de las lesiones traqueales, las cepas nefropatogénicas provocan
importantes lesiones renales y dan lugar a una tasa de mortalidad de hasta el 25%
en pollos asaderos. Las aves adultas se ven afectadas por cepas de ambos tipos
patogénicos, con consecuencias variables en cuanto a la producción y la calidad
de los huevos.
504
Rev. sci. tech. Off. int. Epiz., 19 (
Los pollitos infectados constituyen la principal fuente excretora del virus al
entorno. El material o los productos contaminados s o n un posible f o c o de
transmisión indirecta a larga distancia. En las fases aguda y de c o n v a l e c e n c i a , el
virus está presente a títulos considerables en el m u c u s traqueal y las heces,
respectivamente. La infección se transmite horizontalmente, ya sea por aerosol
(inhalación) o por ingestión de heces o agua y alimentos contaminados. Se trata
de un virus extremadamente infeccioso: los pollitos expuestos a la fuente del
virus manifestarán signos clínicos en un plazo de 36 horas, y los ejemplares de
corrales adyacentes lo harán al cabo de uno a dos días. La infección remite
dentro de los 14 días siguientes, con un aumento del título de anticuerpos, aunque
unos pocos pollitos la conservarán en forma latente y seguirán excretando el
virus erráticamente durante largo tiempo, por vía tanto f e c a l como respiratoria.
Por ello debe considerarse que el movimiento de ejemplares vivos constituye un
posible mecanismo de introducción del virus de la bronquitis infecciosa aviar.
Sólo serán inocuos a ciencia cierta los huevos y pollitos procedentes de
bandadas seronegativas.
Para el diagnóstico de confirmación es necesario aislar e identificar el virus. El
mejor método de aislamiento consiste en inocular una muestra dentro de huevos
embrionarios de pollo libres del patógeno especificado. Para la identificación
puede usarse indistintamente un ensayo inmunoenzimático (ELISA) con
anticuerpos monoclonales o la amplificación en cadena por la polimerasa (PCR).
La prueba de neutralización viral sobre un cultivo de tejido t r a q u e a l , por su parte,
es el mejor método de tipificación antigénica. El empleo incesante de vacunas
vivas complica el diagnóstico, pues no hay ninguna t é c n i c a de diagnóstico
sencilla capaz de distinguir entre cepas salvajes y cepas de v a c u n a . El único
método que permite discriminar entre todas las cepas del virus es la
s e c u e n c i a c i ó n de la glicoproteina S 1 .
El uso continuo de v a c u n a s vivas dificulta también el uso de t é c n i c a s serológicas.
Para fines de vigilancia, y con independencia del tipo antigénico del que se trate,
el método más indicado es el ELISA. Esta t é c n i c a se utiliza para controlar la
respuesta a la v a c u n a c i ó n , pero para detectar una infección de campo es preciso
hacer un seguimiento continuo de los niveles de anticuerpos que presenta la
bandada. La t é c n i c a ELISA no permite determinar el tipo antigénico de la cepa
causante de una infección.
Palabras clave
Bronquitis infecciosa aviar - Comercio internacional - Enfermedades aviares Epidemiología - Fuente de infección - Transmisión - Virus de la bronquitis infecciosa
aviar.
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