Download Infectious Bronchitis Immunity: Its Study in Chickens Experimentally

1427
J. gen. Virol. (1986), 67, 1427-1434. Printed in Great Britain
Key words: IBV immunity/Escherichia coli/mixed inJbctions
Infectious Bronchitis Immunity: Its Study in Chickens Experimentally
Infected with Mixtures of Infectious Bronchitis Virus and Escherichia coli
By J A N E
K. A. COOK,*
H. WILLIAMS
SMITH
AND M. B, H U G G I N S
Houghton Poultry Research Station, Houghton, Huntingdon, Cambridgeshire, PE17 2DA, U.K.
(Accepted 20 March 1986)
SUMMARY
The live infectious bronchitis (IB) vaccine, H120, protected chickens against
intranasal challenge with a mixture of Escherichia coli strains (E. coli Pool) and IB virus
(IBV) strains of the same (Massachusetts) serotype as H120; it usually also protected
against challenge with the E. coli Pool and IBV strains of other serological types. W h e n
these challenge strains were themselves used as vaccines they usually protected against
challenge with a mixture of the E. coli Pool and an IBV strain of the Massachusetts
serotype (VF69-149) or an IBV strain not of the Massachusetts serotype (HVI-116).
Poor protection, when observed, was most c o m m o n in those experiments involving a
minority of the IBV strains that had been incriminated in recent outbreaks of disease in
vaccinated flocks of chickens. Much lower concentrations of IBV strain VF69-149 and
E. coli O18 were found in the nose, trachea and spleen o f H120-vaccinated chickens
killed at different times after they were given a mixture of these organisms than were
found in these sites in similarly challenged unvaccinated chickens. Some protection
against challenge with IBV and the E. coli Pool was also observed in chickens
vaccinated with an inactivated IBV strain; it was much less effective than that
obtained following vaccination with the corresponding live IBV strain.
INTRODUCTION
Research into immunity against infectious bronchitis (IB) has been seriously h a m p e r e d by
failure to produce consistently a quantifiable disease in chickens inoculated with IB viruses
(IBV). The need for a suitable experimental model has never been greater than at present
because of the recent incrimination of new IBV serotypes as a cause of disease in vaccinated
chicken flocks in The Netherlands (Davelaar et al., 1983) and in Britain (Cook, 1984). Smith et
al. (1985) attempted to satisfy this need by studying the effect of inoculating chickens
intranasally with pools of strains of IBV and Escherichia coli that are commonly involved in
natural outbreaks of the disease. A n experimental model was finally developed which closely
resembled the natural disease and in which a high mortality rate consistently occurred. This
p a p e r describes the application of the model to study vaccinal immunity against IB.
METHODS
Bacterial and viral strains. The E. coli Pool comprised an 078, an 02 : K 1 and an untypable strain, selected by
Smith et al. (1985) as being the most virulent representative of many strains of these three categories isolated from
natural outbreaks of IB/E. coli disease. Serogroups 078 and 02 :K 1 are the two most commonly incriminated in
the natural disease. An O18ac : K 1 : H7 strain, used as a nalidixic acid-resistantmutant and referred to hereafter as
O18, was included in the Pool because this serotype is sometimes associated with outbreaks of natural disease and
because the pathogenic characteristics of the particular strain used have been studied closely (Smith & Huggins,
1980).
The origin and relevant information concerning the IBV strains used is summarized in Table 1. The 10
Massachusetts-type strains which composed the IBV Pool (Smith et al., 1985) included M41, VF69-149 and the
commercial vaccine strain, H120. The method of propagating and maintaining the bacterial and viral strains
(except IBV-Beaudette which was propagated in embryonated chicken eggs) and the method of performing
bacterial and viral assays on cultures and on tissues of experimental animals have all been described previously
(Smith et al., 1985).
0000-7040 © 1986 SGM
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1428
J. K. A. COOK, H. WILLIAMS SMITH AND M. B. HUGGINS
Table 1. I B V strains used
IBV strain
Beaudette
H120
M41
VF69-149
Iowa 97
Connecticut
Holte
T
918
HVI-II6
D43
D41
Doorn 207
Doorn 3896
Doom 3128
2
3
4
7
8
9
10
12
Year of
isolation
1937
1960
1941
1969
1947
1951
1954
1962
1965
1967
1976
1976
1980
1980
1980
1982
1982 t
1982
1982
1982
1983
1983
1984
Country of
origin
Source
U.S.A.
Laboratory strain
Netherlands
Vaccine strain
U.S.A.
-~
U.K. (N. Ireland)[ Respiratory
disease
U.S.A.
Enteritis
Australia
Nephrosis
~ Respiratory
U.K. (G.B.)
[
disease
Serotype*
)
Reference to
serological typing
M
~ Darbyshire et aL (1979)
Nephrosis
Netherlands
} Aberrant
egg production
}Respiratory
t
disease
NM
.Cook (1984)
U.K. (G.B.)
Aberrant
egg production
) Cook & Huggins (1986)
*M, Massachusetts-type; NM, non-Massachusetts-type.
Experimental chickens and inoculationprocedures. Unsexed chickens from the Institute's specified pathogen-free
Rhode Island Red flock were used throughout. The pathogens of which they are known to be free include IBV and
Mycoplasma gallisepticum and M. synoviae. They were housed in strict isolation and fed ad libitum on a crumbed
commercial diet. Unless otherwise stated, live virus vaccination was carried out when chickens were 4 days old by
instilling into their anterior nares (i.n.) 0.1 ml of tracheal organ culture fluid containing, in the case of the H 120
strain approximately, log10, 4.0 median ciliostatic doses (CDs0) of virus, in the case of Beaudette approximately,
log10, 7.4 median egg infectious doses of virus and in the case of all other viral strains approximately, log~o, 5.0
CDs0 of virus. The same virus dose was given when vaccination was by the subcutaneous (s.c.) route. Unless
otherwise stated, all chickens were challenged at 25 days of age by i.n. inoculation of the same dose of virus as had
been used for vaccination, but to which had been added approximately, logto, 8.3 viable organisms of the four
strains that composed the E. coil Pool. Chicl~ens were observed daily for 14 days after challenge when the
experiments were terminated.
Preparation oJinactivated IBV vaccine. Nine-day-old chicken embryos were inoculated via the allantoic cavity
with approximately, logt 0, 3.0 CDs0 of the virus strain VF69-149. After 36 h incubation at 37 °C the embryos were
chilled, the allantoic fluid harvested and clarified by centrifugation at 8000 g for 30 rain. If required in
concentrated form, the clarified allantoic fluid was re-centrifuged at 30000 g for 40 min and the resultant pellet
resuspended in citrate buffer pH 6.4. The virus titres of the VF69-149 allantoic fluid and of the resuspended pellet
were, loglo, 6.5 and, loglo, 7.7 CDso/ml respectively, a concentration of approximately tenfold. Before use as a
vaccine each virus sample was inactivated by the addition of ~-propiolactone (BPL) to a final concentration of
0.05% and emulsified in an equal volume of Freund's complete adjuvant. The inactivated vaccine was
administered (0.5 ml) into the left gastrocnemius muscle (i.m.).
RESULTS
Protection provided by live I B V H I 2 0 against challenge with viruses o f the same serological type
and the E. coli Pool
I n t r a n a s a l v a c c i n a t i o n with live IBV H120 p r o v i d e d good protection against challenge 21
days later with a m i x t u r e of the E. eoli Pool a n d the IBV Pool, two of its c o m p o n e n t strains or
H120 itself, all of the Massachusetts serotype (Table 2). N o t only did the v a c c i n a t e d c h i c k e n s
survive the infection, b u t clinical respiratory infection was n o t observed, n o r were lesions o f
pericarditis or peritonitis seen in a n y of t h e m at the t e r m i n a t i o n of the e x p e r i m e n t (Fig. 1).
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Immunity against IBV/E. coli infection
1429
Fig. 1. Chicken (left) vaccinated intranasally with IBV strain H 120 21 days before intranasal challenge
with a mixture of the E. eoliPool and IBV strain VF69-149.The other chicken had not been vaccinated;
note the pericarditis. Magnification x 1.
Table 2. Effect of vaccinating groups of chickens with live IBV strain H120 and challenging them
with a mixture of l B V strains of the same serotype as H120 and the E. coli Pool*
IBV
challenge
strain
H120
IBV Pool
M41
VF69-!49
of 22 chicks that died
following challenge
c
z
Vaccinated
Not vaccinated
0
14
0
77
0
73
5
55
* The chickens were vaccinated when they were 4 days old and challenged 21 days later.
Time taken for the live IBV H120 vaccination to produce immunity
Two experiments were performed in which groups of chickens were vaccinated with live
H120 virus either i.n. or s.c. and challenged at various times afterwards with the E. coli Pool and
the Massachusetts-type strain VF69-149. I n one experiment all groups were vaccinated when
4 days old and in the second all groups were challenged when 25 days old (Table 3). Good
protection was demonstrated in all groups in which the interval between vaccination and
challenge was 11 days or more. In the groups challenged when 25 days old, i.n. vaccination was
superior to s.c. vaccination.
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J. K. A. COOK, H. WILLIAMS SMITH AND M. B. H U G G I N S
1430
T a b l e 3. Effect of varying the interval between vaccination with live IBV strain H120 and
challenge with IBV strain VF69-149 and the E. coli Pool
Interval
between
vaccination
and challenge
(days)
0
2
4
7
11
14
18
of 30 chicks that died after they had been
~
,
Vaccinated at 4 days old and challenged
Vaccinated at different times before
at different times afterwards
challenge at 25 days old
•
~
~•
A
,
i.n.
s.c.
Not vaccinated
i.n.
s.c.
Not vaccinated
77
57
97
83
93
"]
40
50
73
87
97
57
-*
90
70
90
43
40
90
27
60
80
7
0
70
17
30
7
0
53
7
20
•
0
7
53
0
7
* , No observation.
T a b l e 4. Effect of vaccinating groups of chickens with live IBV strain H120 and challenging
them with IB V strains of other serological types and the E. coli Pool
IBV
challenge
strain*
% of 22 chicks that died
following challenge
r
z
•
Vaccinated
Not vaccinated
HVI-116
D41
918
T
D43
Iowa 97
Holte
Doorn 207
2
0
0
0
0
9
0
0
0
0
8
0
Doorn 3128
Doorn 3896
7
4
10
3
9
0
5
5
9
14
14
23
82
64
55
36
36
18
14
23
18
18
18
14
14
23
41
14
23
* The last 10 strains were isolated from flocks vaccinated against IB, those with the prefix Doom were isolated
in Holland, the others in the U.K. For other details, see Table 2.
Protection provided by live IBV H120 against challenge with viruses of other serological types and
the E. coli Pool
V a c c i n a t i o n w i t h live H 1 2 0 v i r u s p r o t e c t e d efficiently a g a i n s t c h a l l e n g e w i t h t h e E. coli P o o l
a n d I B V s t r a i n s H o l t e or I o w a 97 o f A m e r i c a n o r i g i n , t h e A u s t r a l i a n T s t r a i n , t h e B r i t i s h isolates
H V I - 116, D41 or 918 or t h e t h r e e D u t c h isolates r e c e n t l y i n c r i m i n a t e d as c a u s i n g i n f e c t i o n i n IBv a c c i n a t e d flocks ( T a b l e 4). It p r o t e c t e d less well a g a i n s t c h a l l e n g e w i t h t h e E. coli P o o l a n d
s o m e o f t h e I B V s t r a i n s r e c e n t l y i s o l a t e d in B r i t a i n f r o m d i s e a s e o u t b r e a k s i n I B - v a c c i n a t e d
flocks. T h i s a p p e a r e d to b e p a r t i c u l a r l y so w i t h isolates 3 a n d 9, a l t h o u g h , as i n t h e c a s e o f s o m e
o f t h e o t h e r viruses, t h e low m o r t a l i t y in t h e c o n t r o l g r o u p s m a d e c o m p a r i s o n o f a s s e s s m e n t o f
p r o t e c t i o n difficult.
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Immunity against IB V/E. coli infection
1431
Table 5. Effect of vaccinating chickens with live IB V strains and challenging them with the
E. coli Pool and either IBV strains VF69-149 or HVI-116"
of 30 chicks that died following
challenge with the E. coli
Pool and IBV strain
IB vaccine
c
"
strain
VF69-149
HVI-116
None
70
67
VF69-149
0
3
HVI-116
0
3
Iowa 97
7
0
Beaudette
20
10
Connecticut
30
10
2
t7
7
3
17
7
9
13
7
12
7
27
* For other details, see Tables 2 and 4.
Table 6. Effect of vaccinating chickens intramuscularly once or twice with inactivated I B V
strain VF69-149 and challenging them with strain VF69-149 and the E. coli Pool
VF69-149 vaccine
One dose
Ten doses
One dose
Ten doses
One dose
Ten doses
One dose
Ten doses
One dose
Ten doses
Freund's complete
adjuvant
None
Interval
between vaccination
and challenge (days)
r
21 and 14
21 and 14
3
0
0
10
0
0
3
3
0
0
0
0
-
0
0
7
14
21
of 30 chicks that had died by the
following days after challenge
x
5
6
7
8
10
10
17
27
33
37
0
7
10
30
33
13
27
33
37
37
23
27
27
43
53
0
0
0
3
10
3
3
3
3
7
7
7
7
10
13
10
13
13
17
20
0
7
7
10
10
0
3
3
7
7
0
7
20
27
30
14
43
53
40
60
10
10
20
23
17
13
37
10
40
17
20
33
33
Protection produced by vaccination with live IB viruses of different serotypes against challenge
with the E. coli Pool and I B V strains VF69-149 and HVI-116
All of nine IBV strains gave some protection against subsequent challenge with the E. coli
Pool and IBV strain VF69-149 of the Massachusetts serotype or HVI-116, a non-Massachusettstype strain (Table 5). Of the four British strains which had been isolated from vaccinated flocks,
the two (3 and 9), against which H120 did not protect in the previous experiment (see Table 4),
provided only incomplete protection against challenge in this experiment. In a similar
experiment in which groups of 30 chickens were challenged with the E. eoli Pool and IB VF69149, the mortality rate was 5 7 ~ in the control group. I n the groups vaccinated with six nonMassachusetts strains not referred to in Table 5 and which had recently been isolated in Britain,
France, Israel and Morocco, the mortality rates following challenge were 3 ~ , 13 ~ , 13 ~ , 17 ~ ,
20 ~ and 33 ~ .
Protection produced by inactivated I B V
A reduced mortality rate was observed in groups of chickens vaccinated i.m. with inactivated
IBV strain VF69-149 at 21, 14 or 21 and 14 days before challenge with that strain and the E. coli
Pool. No reduction in mortality was noted in groups of chickens vaccinated 7 days before
challenge or at the time of challenge (Table 6). Increasing the vaccine dose tenfold had little
additional protective effect.
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J.K.A.
C O O K , H. W I L L I A M S SMITH AND M. B. H U G G I N S
T a b l e 7. Amount of I B V in the nose, trachea and spleen of H120-vaccinated chickens at different
times after they were challenged intranasally with I B V strain VF69-149 and E. coli 018
logt0 Amount of IBV strain VF69-149 per g tissue*
r"
Time
after
challenge
~s)
A
Nose
.~
Trachea
Spleen
~(
Unvaccinated
chickens
1/12
Vaccinated
chickens
<2"0t
<2.0
5.8
3.3
5.5
3.0
5.6
2.3
4.7
<2.0
4.6
<2.0
5.5
<2.0,<2.0,2.0
4.7
<2.0
4.2
<2.0,<2.0,2.3
4.5
<2.0
<2.0,<2.0,2.5
<2.0
2.2
<2-0
2.8
<2.0
1
2
3
4
5
6
7
8
9
10
12
14
"it"
`i
Unvaccinated
chickens
Vaccinated
chickens
Unvaccinated
chickens
Vaccinated
chickens
<2.0
7.2
5.7
7.5
5.2
4.4
5.3
5.0
3.4
4.4
3.8
2.4
2.4
<2.0
2-5
2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2.0
<2-0
<2.0
<2.0
2.4
<2.0,<2.0,2.0
3.2
<2.0
2.4
<2.0
2.5
<2.0
<2.0
<2,0
2.0
<2,0
<2.0,<2.0,2.2
<2.0
2.3
<2,0
2.2
<2.0
* Three vaccinated and three unvaccinated chickens were killed at each of the stated times after challenge and
the amount of IBV VF69-149 in the three organs determined; the medians for each set of three determinations are
shown, except where only one of the three components yielded a positive result, when all three are shown.
t Results are expressed as CDso; the lowest detectable concentration was, log10, 2.0 CDso per g.
T a b l e 8. Amount ofE. coli 018 in the nose, trachea and spleen of Hl20-vaccinated chickens at
different times after they were challenged intranasally with IBV strain VF69-149 and E . coli 018
log10 Amount of E. coli O18 per g tissue*
&
(
Time
after
challenge
(days)
1/12
1
2
3
4
5
6
7
8
9
10
12
14
~,
Nose
Trachea
Spleen
e
x
~r
~
`i r
x
Unvaccinated Vaccinated Unvaccinated
Vaccinated
Unvaccinated
Vaccinated
chickens
chickens
chickens
chickens
chickens
chickens
4.5
4.6
6.6
7.3
7.5
8.2
8.0
8.0
7.8
6.5
7-3
4-7
5-2
4.5
< 1.5, < 1.5, 1.5 <1.5, < 1.5, 3.9
<1.5
<1.5
4.2
< 1.5
< 1.5
< 1.5
< 1.5
4.9
3.5
< 1.5, < 1.5, 2.3 < 1.5, < 1.5, 2.2
< 1.5
3.7
3.5
< 1.5, < 1.5, 1.5
2.0
< 1.5, < 1-5, 2.0
3.0
6.5
< 1-5
2.4
< 1.5
3.0
6.8
< 1-5, < 1.5, 2.6 < 1-5, < 1.5, 2.7
< 1.5
< 1.5
7.0
< 1,5
2.4
< 1.5
< 1.5, < 1.5, 4.8
6.3
< 1-5, < 1-5, 4.8
3.3
< 1-5
2.3
6.6
< 1.5, < 1.5, 2.3
2.7
< 1.5, < 1.5, 1.5
3.7
6.6
< 1.5
3.6
< 1.5
< 1-5
4.3
< 1.5
2.4
< 1.5
< 1-5
3.9
< 1-5
< l-5
< 1-5
< 1-5
2.7
< 1-5
< 1-5
< 1-5
* Results are expressed as no. of viable organisms. The lowest detectable concentration was, loglo, 1.5 per g. For
other details see footnotes to Table 7.
Fate of l B V strain VF69-149 and E. coli 018 given to chickens that had been vaccinated with
live I B V H120
T h e c o n c e n t r a t i o n s o f I B V V F 6 9 - 1 4 9 a n d E. coli O 1 8 w e r e e s t i m a t e d in t h e t i s s u e s o f H 1 2 0 vaccinated or unvaccinated chickens killed at different times after challenge with a mixture of
these two organisms. High concentrations of both were found in the nose and trachea of the
u n v a c c i n a t e d c h i c k e n s f r o m a b o u t t h e 1st to t h e 9 t h d a y a f t e r c h a l l e n g e ( T a b l e s 7 a n d 8). B o t h
o r g a n i s m s w e r e f o u n d in t h e s p l e e n o f t h e m a j o r i t y o f t h e s e c h i c k e n s . N o n e , o r m u c h l o w e r
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Immunity against IBV/E. coli infection
1433
concentrations of both organisms, were found in the nose and trachea of the vaccinated
chickens. IBV was found in only one and E. coli in only two spleens examined from the
vaccinated chickens.
DISCUSSION
Previous attempts to study IBV immunity in the chicken have depended on virus recovery
from the respiratory tract (Winterfield et al., 1972) or on assessing the presence or absence of
ciliary activity in the trachea (Darbyshire, 1980) following IBV challenge. Our experimental
model resembles the natural disease produced by IBV and E. coli. In it the degree of protection is
assessed by the fate of the intact animal following challenge. Consequently, it is a more realistic
approach to studying IBV immunity than the earlier methods. The fact that H120, whose
vaccinal efficiency in the field is well established, provided excellent protection against
challenge with IB viruses of the same serotype as H120 and the E. coli Pool supports this view.
The much poorer protection provided by inactivated IB vaccine is also in keeping with field
experience. It might, however, in part be a reflection of the reduced immunological competence
of young chickens. The use of young chickens in the present studies was considered necessary
because the mortality rate in the model infection is considerably reduced in chickens over
5 weeks of age (Smith et al., 1985).
The finding in H120-vaccinated chickens of no, or very low, concentrations of IBV strain
VF69-149 and E. coli strain O18 in the nose and trachea following challenge with a mixture of
these organisms, their apparent inability to invade the tissues and the contrasting observations
in the challenged non-vaccinated chickens is in keeping with the hypothesis formed after studies
on the pathogenesis of the experimental disease (Smith et al., 1985). This hypothesis is that the
development of generalized E. coli infection, a common consequence of IB infection in the field,
results from prior damage to the respiratory tract by IBV. This damage permits pathogenic E.
coli to proliferate there and then invade the tissues. The virtual absence of IBV in the nose,
trachea and spleen of the challenged, vaccinated chickens confirms the comprehensive nature of
the protection provided by live H120 vaccination.
Plaque reduction tests in chick kidney cell culture (Hopkins, 1974) and cross-neutralization
tests in tracheal organ cultures (Johnson & Marquardt, 1975; Darbyshire et al., 1979; Cook,
1984) have demonstrated the serological diversity among IBV strains. The present studies,
however, confirm reports (Hitchner et al., 1964; Raggi & Lee, 1965; Winterfield & Fadly, 1972)
that the results of serological tests do not correlate with those of immunological studies and that
protection against IBV infection in the chicken is more widely based than the results of in vitro
serological tests indicate. For example, they reveal that live H 120 vaccination protected against
challenge with a variety of IBV serotypes, specially chosen to assess the breadth of its
immunological ability. These serotypes had a worldwide geographical distribution and included
ones against which H 120 vaccine has been reported to give incomplete protection (Davelaar et
al., 1984). Because many of them did not cause high mortality when given alone to experimental
chickens, they could be used as live vaccines. They, too, usually gave good protection against
challenge with the E. coli Pool and IB viruses of both the Massachusetts serotype (VF69-149) and
a non-Massachusetts serotype (HVI-116), viruses selected because they had caused high
mortality when given to chickens with the E. coli Pool (Smith et al., 1985). As was observed
following live H120 vaccination, the protection in a few instances was poor. It was perhaps
significant that this was so in the experiments involving a minority of the IBV strains currently
believed to be causing outbreaks of disease in vaccinated flocks in the field (Cook, 1984; Cook &
Huggins, 1986). The results reported here, however, suggest that, in general, inadequacy of the
currently used vaccines cannot be held responsible for the majority of these outbreaks.
We are grateful to Mrs M. M. Ellis and Mrs T. Berrill for their capable technical assistance. We are also
indebted to Professor P. M. Biggs, Mrs S. Pilcher and Mr B. Wells and his staff for helping in a variety of ways.
REFERENCES
COOK, J. K. A. (1984). The classification of new serotypes of infectious bronchitis virus isolated from poultry flocks
in Britain between 1981 and 1983. Avian Pathology 13, 733-741.
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1434
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(Received 18 January 1986)
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