- Email: [email protected]
A study of two avian adenovirus serotypes alone or in combination with avian infectious bronchitis in day-old chicks
J. COMP.
PATH.
1977.
VOL.
135
87.
A STUDY OF TWO AVIAN ADENOVIRUS ALONE OR IN COMBINATION WITH INFECTIOUS BRONCHITIS IN DAY-OLD
JANE Houghton
Poultry
K. A.
Research Station, Houghton,
SEROTYPES AVIAN CHICKS
COOK
Huntingdon,
Cambs. PE17
ZD.1,
England
INTRODUCTION
The pathogenicity of two avian adenovirus serotypes for day-old chicks has already been studied (Cook, 1974) and it has been shown that the Gallus adeno-like serotype (GAL virus) can cause mortality following intraperitoneal (i.p.) inoculation and depression in body weight gain following oral inoculation. Adenoviruses have been recovered from embryonated chicken eggs (Yates and Fry, 1957 ; Cook, 1968a) and antibody against them is widespread (Cook, 1970). The role of the chick embryo lethal orphan (CELO) serotype in mixed infections has been investigated. Ablashi, Chang and Yates (1965) have shown that a latent infection with this virus in embryonated eggs interferes with the propagation of Newcastle Disease (ND) and influenza A viruses. It has also been shown that infection of laying hens with CELO virus before inoculation with avian infectious bronchitis (AIB) virus significantly reduced the effect of the AIB virus on egg production and egg shell quality (Cook, 1972) and furthermore that the prior inoculation of embryonated chicken eggs with CELO virus caused a retardation of the growth rate of AIB virus inoculated subsequently (Cook, 1973). J or d an and Nassar (1974) have shown that when 17-day embryonated eggs, inoculated with the Phelps strain of CELO virus, were challenged with AIB virus at day-old, clinical signs of infectious bronchitis were not observed in the group which received both viruses; AIB virus could not be recovered from their tissues, nor did they produce AIB neutralizing antibody. Since vaccination with live AIB virus at a very young age is a common practice, it was decided to investigate the effect of two different serotypes of avian adenoviruses on the course of an avian infectious bronchitis infection; CELO virus was included since its role in mixed infections has already been studied and GAL virus because it is known to cause mortality in day-old chicks. MATERIALS
AND
METHODS
Embryonated eggs and chickens. Houghton Poultry Research Station Rhode Island Red embryonated eggs and chicks, obtained from specified pathogen-free (SPF) hens held in isolators (Cooper and Timms, 1972), were used throughout. Hatching eggs were transferred to an isolated building as 17-day embryos and chicks were hatched in the room in which they were to be inoculated.
136
JANE
Housing. Chicks were housed in floor as previously described (Cook, 196813).
K. A. COOK
pens on litter
under
conditions
of isolation
Viruses AIB virus. The H120 vaccine strain (Massachusetts serotype) was used diluted as recommended by the manufacturers and administered by spray.* The diluted virus had a mean titre of 6.5 log,,EIDj, (50 per cent egg infectious dose)/ml. Avian adenoviruses. The GAL serotype and the Phelps strain of the CELO serotype were used. Their origin and passage history in this laboratory have been described previously (Cook, 1974). Each virus was passaged once in SPF chickens before use in these experiments, and then GAL virus was grown in chick embryo kidney (CEK) cultures [mean titre 6.7 log,,TCID,,, (50 per cent tissue culture infective dose)/ml] and CELO virus was grown following inoculation into the allantoic cavity of g-day-old embryonated eggs (mean titre 8.5 log,,,TCID,,/ml). Experimental procedure. Two different inoculation schedules were used involving either inoculation of adenovirus at day-old and AIB virus 6 days later, or inoculation of adenovirus in ovo followed by AIB virus on hatching. The inoculation programmes used are indicated in Table 1. In the first experiment, a group of one-day-old chicks was inoculated i.p. with 0.2 ml of GAL virus in undiluted tissue culture fluid. Six days later, half this group, together with a previously uninoculated group of 34 chicks, was inoculated with AIB virus by spray. For each group of chicks, the recommended number of chick infectious doses of virus was contained in 2 ml of diluent which was then administered by spray. The chicks were allowed to dry for 15 min before removal to floor pens. Chicks were observed daily for clinical signs of infection and blood samples were taken from the wing vein of 10 chicks per group at weekly intervals for 4 weeks after AIB virus inoculation. At 1, 3, 7, 10 and 14 days after inoculation groups of 3 chicks were killed and recovery of each virus attempted from tissues. In the remaining experiments, groups of 17-day embryos were inoculated via the allantoic cavity with 0.1 ml of either CELO virus as a 1: 10 dilution of allantoic fluid, GAL virus as undiluted tissue culture fluid or Eagle’s minimum essential medium (MEM) as control. Groups were hatched in Curfew7 incubators in separate rooms. Immediately after hatching, chicks were weighed and one zdenovirusinoculated group and one previously uninoculated group were sprayed with AIB virus as described above, whilst in separate Curfew incubators. Chicks were observed daily for clinical signs of ill-health, weighed at 7 days of age and blood samples were collected and virus recovery attempted as above. Two similar experiments were carried out using each serotype of avian adenovirus. Virus recovery. At intervals after inoculation, lung and trachea, liver, spleen, kidney, caecal tonsil and, where possible, ovary and oviduct were removed and examined for the presence of virus. Tissues were homogenized and prepared for examination as previously described (Cook, 1974). Attempts to recover AIB virus were made by passage in g-day-old embryonatcd eggs (Cook, 196813) and to recover adenoviruses by passage in CEK cultures (Cook, 1972). Tissue homogenates obtained from groups inoculated with both viruses were divided into 2 portions, one portion being treated with the appropriate chicken anti-adenovirus serum to neutralize any adenovirus present. This portion was used in attempts to recover AIB virus. No attempt was made to remove AIB virus from specimens obtained from chicks inoculated with both viruses as it was assumed that AIB virus would not hinder adenovirus recovery in CEK cultures. Antibody determinations. Sera were examined for neutralizing antibodies to AIB virus by the u method, with g-day-old embryonated eggs for assay (Anon, 1971) and for adenovirus-neutralizing antibodies by the 8 method in CEK cultures (Cook, 1970). * Humbrol Ltc!, Hull, Yorks. t Curfew Applmces Ltd, Ottersbaw,
Chcrtsey,
Surrey.
ADENOVIRUSES
AND
AIB
VIRUS
IN DAY-OLD
IS7
CHICKS
RESULTS
Embryos. In the control groups, inoculated only with Eagle’s MEM, embryo mortality was at the normally acceptable level of approximately 20 per cent (Table 2). Following inoculation of 1 ‘I-day embryos with CELO virus, 61 per cent of embryos died, showing a highly significant difference from the mortality in the control group (P < 0.001). In the groups of 17-day embryos inoculated with GAL virus, embryo mortality was similar to that in the control groups. TABLE 1 SCHEDULE
USED
Ii-day
TO
INOCULATE
AVIAN
ADENOVIRUSES
AND
,4ge when inoculated with each oirus &y-old chick
embryo
GAL GAL GAL GAL
r\IB ;\IB
AFTER
IN
OVO
OF OF
AIB
261159:
AIB
t AIB not administered Number dead/number : x.s. 7 not significant;
22 25 N.S.
until immediately inoculated. * P < 0.05; ***
ADENOVIRUS AT
61***
261120 )- 711287 chicks
AVIAN VIRUS
16
]- 196/321
I>iluent+-iiIB GAL GAL+ .-\I)3 Uninoculated
2
INOCULATION
ADMINISTRATION
+,\IBt
chick
MB MB
TABLE
Diluent CELO CELOf
G-dq-old
VIRUS
=\IB AIR
GEL0 GEL0
MORTALITY
.4IB
after P i
hatching. 0.001.
FOLLOWED
BY
DAY-OLD
3173 17/e 36163
1 44 57 N.S.
2/66 55/106 39/106
3 5’2 37*
l/38
3
138
JANE
K.
A.
COOK
Chicks. Following inoculation with AIB virus only, mortality was similar to that in the uninoculated control group (Table 2). In groups inoculated with GAL virus when 17-day embryos the mortality rate following hatching was 55/106 (52 per cent). However, when GAL virus inoculation was followed by administration of AIB virus, mortality was only 39/106 (37 per cent) which was significantly lower (P < O-05) than the mortality in the group inoculated only with GAL virus. Challenge with AIB virus after inoculation of 17-day embryos with CELO virus resulted in a higher chick mortality 36/73 (57 per cent) than did inoculation of CELO virus alone, when chick mortality was 27/62 (44 per cent), a difference in mortality which was not statistically significant. When chicks were inoculated with GAL virus at day-old and AIB virus 6 days later there was little difference in subsequent mortality between the 2 groups, it being 17/45 (38 per cent) in the groups inoculated only with GAL virus and 15137 (41 per cent) in the dually inoculated group. In all adenovirus-inoculated groups chick mortality occurred within 7 days of hatching. Respiratory
Infection
In all groups of chicks inoculated with AIB virus alone, whether at day-old or 6 days of age, coughing and riles were first observed 2 to 3 days after inoculation and continued in the majority of chicks for approximately 7 days, thereafter 1 or 2 chicks in some groups remained affected until up to 14 days after inoculation, When chicks already inoculated with GAL virus at day-old were inoculated with AIB virus 6 days later, the duration and severity of respiratory signs were very similar to that recorded in the groups inoculated with only AIB virus despite the higher mortality in the former group. When embryos inoculated with GAL virus were challenged with AIB virus on hatching, in the first experiment there was no difference in duration and severity of respiratory signs from that recorded in the group inoculated only with AIB virus, but when the experiment was repeated, onset of respiratory signs was delayed until 6 days after inoculation and then continued until the 16th day. No respiratory signs were recorded in any chicks inoculated with CELO virus prior to AIB virus, nor in any of the chicks inoculated only with adenovirus. General Condition Chicks inoculated with AIB virus alone appeared lively throughout, even when showing respiratory signs. In addition to the deaths, chicks inoculated with either of the adenovirus strains as embryos were generally depressed, with ruffled or drooping feathers and showed inappetance from hatching until approximately 7 to 8 days of age whether or not they were inoculated with AIB virus. Bodyweight at 7 days of age was depressed following inoculation of embryos with adenovirus alone and following inoculation with CELO and AIB viruses but there was little depression in weight gain in groups inoculated with GAL and AIB viruses.
ADENOVIRUSES
AND
AIB
VIRUS
IN DAY-OLD
139
CHICKS
Virus Recovery CELO or GAL viruses were recovered at all times and from all tissues examined, whether or not adenovirus inoculation was followed by AIB administration. The results of attempts to recover AIB virus from tissues are shown in Table 3. In each experiment AIB virus was recovered consistently from all TABLE DURATION
OF RECOVERY OF AIB VIRUS PRECEDED BY AVIAN
Days Tissne
Lung/trachea Liver Spleen Kidney Ovary/oviduct Caecal tonsil
for AIB
which AIB onb
14 14 14 14 14 14
3 AFTER INOCULATION ADENOVIRUS
cirus wm recowred CELO+.4IB
ALONE
after inoctrlatiort
miih
GAL+-MB
0
10
,”
il 7 10 0
0” 0
OK
tissues of chicks inoculated with that virus alone. The virus was never recovered from any tissue of chicks inoculated with CELO virus prior to AIB virus. However, AIB virus could be recovered for 10 days from the respiratory tract of chicks sprayed with AIB virus after GAL virus inoculation as 17-day embryos. Initially the virus was also recovered from each of the other tissues examined with the exception of the caecal tonsil. However, AIB virus could not be recovered from tissues of the groups inoculated with both viruses as consistently or with such ease as from groups inoculated only with AIB virus. Following inoculation with GAL virus at day-old and AIB virus 6 days later, AIB virus was recovered from the respiratory tract (the only tissue examined) with equal facility in both groups. Neutralizing
Antibody Determinations
In all groups inoculated with AIB virus alone, mean neutralizing antibody indices of 300 or greater were recorded by 28 days after inoculation (Fig. 1). Following inoculation with both CELO and AIB virus, no AIB neutralizing antibody was detectable by 28 days after inoculation. When AIB virus administration at either day-old or 6 days followed inoculation with GAL virus, very similar mean neutralizing antibody indices of 32 and 16 respectively were recorded and, although very low, these antibody levels were rising by 28 days. Mean adenovirus neutralizing antibody titres are shown in Fig. 2. In groups inoculated with adenovirus alone, neutralizing antibody titres had in each case reached 4.5 log,, or over by 28 days after inoculation regardless of when
140
K.
JANE
A.
COOK
adenovirus was administered. When GAL virus inoculation was followed by challenge with AIB virus, neutralizing antibody titres did not differ from those in groups which received only GAL virus. Following inoculation of AIB virus after CELO virus, mean neutralizing antibody titres to CELO virus were initially lower than those in the chicks inoculated only with CELO virus but were similar by the end of the experiments. 1 I I I I I I I I I (a)
Lb)
(c)
I 7
14
21
28
7
14
21
1
28
7
14
21
28
Days
1. AIB serum neutralization (a) CELO virus (I’i-day and AIB virus (day-old). oculated with AIB virus; 6 5-
I
b
I
I
indices following inoculation of AIB virus alone or after adenovirus, embryos) and AIB virus (day-old). (b) GAL virus (17-day embryos) (c) GAL virus (day-old) and AIB virus (6 days). (O---O) In(O--O) inoculated with adenovirus+AIB virus. I
I
I
I
I
I
I
I
I
(cl
(b)
(ai
I
,
A
-
-
4-
7
14
21
28
7
I4
21
28
7
14
21
28
Days
Fig. 2. Adenovirus serum neutralizing antibody titres after inoculation of adenovirus alone or followed by AIB virus. (a) CELO virus (17-day embryos) and AIB virus (day-old). (b) GAL virus (17-day embryos) and AIB virus (day-old). (c) GAL virus (day-old) and AIB virus (6 days). (A-A) Inoculated with adenovirus; (/J--A) inoculated with adenovirus+AIB virus. DISCUSSION
Inoculation of 17-day embryos with CELO but not GAL virus resulted in considerable embryo mortality. In contrast, mortality after hatching was higher following inoculation with GAL virus. No embryo or chick mortality was recorded by Jordan and Nassar (1974) following inoculation of 17-day
ADENOVIRUSES
AND
AIB
VIRUS
IN
DAY-OLD
141
CHICKS
embryos with the same strain of CELO virus. Since the experimental design was the same, the different findings may possibly be a result of variations in the breed of chicken used. The difference in mortality following in ovo inoculation of the two adenoviruses indicates that embryos are more susceptible to CELO virus whereas young chicks are more susceptible to GAL virus. CELO virus causes high embryo mortality following inoculation of 9 or 10 day-old embryos via the allantoic cavity but similar inoculation with GAL virus rarely causes mortalit) although the virus replicates to a titre of at least 5.5 log,,TCID,,/ml (unpublished observation). It has been shown (Cook, 1974) that inoculation 01 day-old chicks with GAL virus resulted in considerable mortality, but that such inoculation with CELO virus did not, and furthermore that recovery of CELO virus from tissues was more difficult than was recovery of GAL virus. Apart from the different mortality patterns the prior inoculation of the two serotypes of avian adenovirus produced very difFerent cfFects on the course of an AIB infection. In ovo inoculation with CELO virus prior to challenge kvitlr AIB virus resulted in complete absence of respiratory signs, failure to recover 1ZIB virus from any tissues examined even as early as one day after AIB virus inoculation and absence of an AIB neutralizing antibody response. These findings are in agreement with the observations of Jordan and Nassar (1974). ‘l~hq~ are in contrast, however, to the findings when inoculation with GAL \-irus preceded exposure to AIB virus. Respiratory signs in these groups \vere similar to those following exposure to AIB alone. Although virus recovery was more difficult, AIB virus could be recovered throughout from the respirator!. tract and initially from most other tissues examined. The greater ease of recovcry Of AIB virus from the respiratory tract may have been a result of the \,irus replicating to a higher titre in th.at site than elsewhere. Adenoviruses are known to produce latent infections (Pereira and Kelly, 1957) and it seems likely that these viruses persisted in the chicks throughout the course of these experiments. The inhibitory effect of avian adenoviruses on other viruses is already known and Ablashi et al. (1965), who demonstrated intcrlcrence between CELO virus and both ND virus and influenza A virus in chicken embryos, suggested two possible explanations-interferon production or competition for available receptor sites or enzymes. Although intrrliAron production by human strains of adenovirus (Ho and Kohler, 1967), b> animal strains (Guenov, 1970) and by the GAL serotype of avian adenovirus (Bakay, 1969) has been demonstrated, Pereira (1960), investigating a viral inhibitor produced by several adenoviruses, concluded that, whilst similar to interferon in many respects, it was distinct from it. The results described here demonstrate that two distinct serotypes of avian adenovirus are capable of apparently different degrees of interference due, possibly, to different rates of replication in chicken embryos and young chicks. SUMMARY
Administration of GEL0 but not GAL considerable embryo mortality.
virus
to 17-day embryos
resulted
jn
142
JANE
K.
A.
COOK
When in ovo inoculation of CELO virus was followed by administration of AIB virus on hatching, there was little difference in mortality between the group inoculated with CELO virus alone and the dually inoculated group. In contrast, in ovo inoculation of GAL virus prior to AIB virus resulted in lower chick mortality than did inoculation of GAL virus alone. Prior inoculation with CELO and GAL viruses caused very different effects on the course of an AIB infection. When CELO virus was inoculated prior to AIB virus, no respiratory signs were detected, AIB virus could not be recovered from tissues and no AIB neutralizing antibody was produced. When GAL virus was inoculated prior to AIB virus challenge, respiratory signs were as marked as in chicks inoculated with AIB virus alone, AIB virus could be recovered from most tissues but less easily and for a shorter time than after inoculation with AIB virus alone and there was only a very slight AIB neutralizing antibody response by 28 days after inoculation. Adenoviruses were readily recovered from all chicks, whether inoculated alone or with AIB virus. Neutralizing antibody titres to GAL virus were similar whether or not AIB virus was present. However, CELO virus neutralizing antibody reactions in the dually infected groups were initially delayed, but by 28 days after inoculation were similar to those in the group inoculated with CELO virus alone. ACKNOWLEDGMENTS
The author wishes to acknowledge the technical assistance of Miss F. R. Pickworth, Mrs A. P. Parker and Miss D. M. Schietzel and to thank Mr J. G. Rowe11 for assistance with the statistical analysis. REFERENCES
Ablashi, D. V., Chang, P. W., and Yates, V. J. (1965). The effect of a latent CELO virus infection in the chicken embryo on the propagation of Newcastle Disease and influenza viruses. Avian Diseases, 9, 407-417. Anon. (197 1). Methods for Examining Poultry Biologics and for Identifying and Quant;fving Avian Pathogens. National Academy of Science, National Research Council, Washington. Bakay, M. (1969). Interferon induction by GAL virus in chick embryo cells. Acta virologica,
13, 340-342.
Cook, J. K. A. (1968a). Isolation of a CELO virus from fertile chicken eggs. Veterinary Record, 82, 294.
Cook, J. K. A. (1968b). D uration of experimental infectious bronchitis in chickens. Research in Veterinary Science, 9, 506-514. Cook, J. K. A. (1970). I ncidence of chick embryo lethal orphan virus antibody in the fowl (Callus domesticus) in Britain. Research in Veterinary Science, 11, 343-348. Cook, J. K. A. (1972). A vian adenoviruses alone or followed by infectious bronchitis virus in laying hens. Journal of Comparative Pathology, 82, 119-128. Cook, J. K. A. (1973). Th e inhibition of infectious bronchitis virus multiplication by CELO virus in the fowl embryo. Research in Veterinary Science, 15, 375-378. Cook, J. K. A. (1974). Pathogenicity of avian adenoviruses for day-old chicks. Journal of Comparative Pathology, 84, 505-515. Cooper, D. M., and Timms, J. R. (1972). The rearing and maintenance of breeding chickens in isolators. I. Glass fibre isolators. Avian Pathology, 1, 47-57. Cuenov, I. (1970). Interference activity of animal adenoviruses in chick embryo cell cultures. Acta virologica, 14, 337-342.
ADENOVIRUSES
AND
AIB
VIRUS
IN
DAY-OLD
IF
CHICKS
143
M., and Kohler, K. (1967). Studies on human adenoviruses as inducers of interferon in chick cells. Archiv ftir die gesamte Virusforschung, 22, 69-78. Jordan, F. T. W., and Nassar, T. J. (1974). The effect of infectious bronchitis (IB) virus on day-old chicks previously infected as 17-day-old embryos with an avian adenovirus. Research in Veterinary Science, 16, 47-53. Pcreira, H. G. (1960). A virus inhibitor produced in HeLa cells infected with adenovirus. Virology, 11, 590-602. Pereira, H. G., and Kelly, B. (1957). Latent infection of rabbits by adenovirus type 3. Nature, 180, 615-616. \-ates, V. J., and Fry, D. E. (1957). Ob servations on a chicken embryo lethal orphan (GEL01 virus. American Journal of Veterinary Research, 18, 657-660. [Received for publication,
May 17th, 19761
PATH.
1977.
VOL.
135
87.
A STUDY OF TWO AVIAN ADENOVIRUS ALONE OR IN COMBINATION WITH INFECTIOUS BRONCHITIS IN DAY-OLD
JANE Houghton
Poultry
K. A.
Research Station, Houghton,
SEROTYPES AVIAN CHICKS
COOK
Huntingdon,
Cambs. PE17
ZD.1,
England
INTRODUCTION
The pathogenicity of two avian adenovirus serotypes for day-old chicks has already been studied (Cook, 1974) and it has been shown that the Gallus adeno-like serotype (GAL virus) can cause mortality following intraperitoneal (i.p.) inoculation and depression in body weight gain following oral inoculation. Adenoviruses have been recovered from embryonated chicken eggs (Yates and Fry, 1957 ; Cook, 1968a) and antibody against them is widespread (Cook, 1970). The role of the chick embryo lethal orphan (CELO) serotype in mixed infections has been investigated. Ablashi, Chang and Yates (1965) have shown that a latent infection with this virus in embryonated eggs interferes with the propagation of Newcastle Disease (ND) and influenza A viruses. It has also been shown that infection of laying hens with CELO virus before inoculation with avian infectious bronchitis (AIB) virus significantly reduced the effect of the AIB virus on egg production and egg shell quality (Cook, 1972) and furthermore that the prior inoculation of embryonated chicken eggs with CELO virus caused a retardation of the growth rate of AIB virus inoculated subsequently (Cook, 1973). J or d an and Nassar (1974) have shown that when 17-day embryonated eggs, inoculated with the Phelps strain of CELO virus, were challenged with AIB virus at day-old, clinical signs of infectious bronchitis were not observed in the group which received both viruses; AIB virus could not be recovered from their tissues, nor did they produce AIB neutralizing antibody. Since vaccination with live AIB virus at a very young age is a common practice, it was decided to investigate the effect of two different serotypes of avian adenoviruses on the course of an avian infectious bronchitis infection; CELO virus was included since its role in mixed infections has already been studied and GAL virus because it is known to cause mortality in day-old chicks. MATERIALS
AND
METHODS
Embryonated eggs and chickens. Houghton Poultry Research Station Rhode Island Red embryonated eggs and chicks, obtained from specified pathogen-free (SPF) hens held in isolators (Cooper and Timms, 1972), were used throughout. Hatching eggs were transferred to an isolated building as 17-day embryos and chicks were hatched in the room in which they were to be inoculated.
136
JANE
Housing. Chicks were housed in floor as previously described (Cook, 196813).
K. A. COOK
pens on litter
under
conditions
of isolation
Viruses AIB virus. The H120 vaccine strain (Massachusetts serotype) was used diluted as recommended by the manufacturers and administered by spray.* The diluted virus had a mean titre of 6.5 log,,EIDj, (50 per cent egg infectious dose)/ml. Avian adenoviruses. The GAL serotype and the Phelps strain of the CELO serotype were used. Their origin and passage history in this laboratory have been described previously (Cook, 1974). Each virus was passaged once in SPF chickens before use in these experiments, and then GAL virus was grown in chick embryo kidney (CEK) cultures [mean titre 6.7 log,,TCID,,, (50 per cent tissue culture infective dose)/ml] and CELO virus was grown following inoculation into the allantoic cavity of g-day-old embryonated eggs (mean titre 8.5 log,,,TCID,,/ml). Experimental procedure. Two different inoculation schedules were used involving either inoculation of adenovirus at day-old and AIB virus 6 days later, or inoculation of adenovirus in ovo followed by AIB virus on hatching. The inoculation programmes used are indicated in Table 1. In the first experiment, a group of one-day-old chicks was inoculated i.p. with 0.2 ml of GAL virus in undiluted tissue culture fluid. Six days later, half this group, together with a previously uninoculated group of 34 chicks, was inoculated with AIB virus by spray. For each group of chicks, the recommended number of chick infectious doses of virus was contained in 2 ml of diluent which was then administered by spray. The chicks were allowed to dry for 15 min before removal to floor pens. Chicks were observed daily for clinical signs of infection and blood samples were taken from the wing vein of 10 chicks per group at weekly intervals for 4 weeks after AIB virus inoculation. At 1, 3, 7, 10 and 14 days after inoculation groups of 3 chicks were killed and recovery of each virus attempted from tissues. In the remaining experiments, groups of 17-day embryos were inoculated via the allantoic cavity with 0.1 ml of either CELO virus as a 1: 10 dilution of allantoic fluid, GAL virus as undiluted tissue culture fluid or Eagle’s minimum essential medium (MEM) as control. Groups were hatched in Curfew7 incubators in separate rooms. Immediately after hatching, chicks were weighed and one zdenovirusinoculated group and one previously uninoculated group were sprayed with AIB virus as described above, whilst in separate Curfew incubators. Chicks were observed daily for clinical signs of ill-health, weighed at 7 days of age and blood samples were collected and virus recovery attempted as above. Two similar experiments were carried out using each serotype of avian adenovirus. Virus recovery. At intervals after inoculation, lung and trachea, liver, spleen, kidney, caecal tonsil and, where possible, ovary and oviduct were removed and examined for the presence of virus. Tissues were homogenized and prepared for examination as previously described (Cook, 1974). Attempts to recover AIB virus were made by passage in g-day-old embryonatcd eggs (Cook, 196813) and to recover adenoviruses by passage in CEK cultures (Cook, 1972). Tissue homogenates obtained from groups inoculated with both viruses were divided into 2 portions, one portion being treated with the appropriate chicken anti-adenovirus serum to neutralize any adenovirus present. This portion was used in attempts to recover AIB virus. No attempt was made to remove AIB virus from specimens obtained from chicks inoculated with both viruses as it was assumed that AIB virus would not hinder adenovirus recovery in CEK cultures. Antibody determinations. Sera were examined for neutralizing antibodies to AIB virus by the u method, with g-day-old embryonated eggs for assay (Anon, 1971) and for adenovirus-neutralizing antibodies by the 8 method in CEK cultures (Cook, 1970). * Humbrol Ltc!, Hull, Yorks. t Curfew Applmces Ltd, Ottersbaw,
Chcrtsey,
Surrey.
ADENOVIRUSES
AND
AIB
VIRUS
IN DAY-OLD
IS7
CHICKS
RESULTS
Embryos. In the control groups, inoculated only with Eagle’s MEM, embryo mortality was at the normally acceptable level of approximately 20 per cent (Table 2). Following inoculation of 1 ‘I-day embryos with CELO virus, 61 per cent of embryos died, showing a highly significant difference from the mortality in the control group (P < 0.001). In the groups of 17-day embryos inoculated with GAL virus, embryo mortality was similar to that in the control groups. TABLE 1 SCHEDULE
USED
Ii-day
TO
INOCULATE
AVIAN
ADENOVIRUSES
AND
,4ge when inoculated with each oirus &y-old chick
embryo
GAL GAL GAL GAL
r\IB ;\IB
AFTER
IN
OVO
OF OF
AIB
261159:
AIB
t AIB not administered Number dead/number : x.s. 7 not significant;
22 25 N.S.
until immediately inoculated. * P < 0.05; ***
ADENOVIRUS AT
61***
261120 )- 711287 chicks
AVIAN VIRUS
16
]- 196/321
I>iluent+-iiIB GAL GAL+ .-\I)3 Uninoculated
2
INOCULATION
ADMINISTRATION
+,\IBt
chick
MB MB
TABLE
Diluent CELO CELOf
G-dq-old
VIRUS
=\IB AIR
GEL0 GEL0
MORTALITY
.4IB
after P i
hatching. 0.001.
FOLLOWED
BY
DAY-OLD
3173 17/e 36163
1 44 57 N.S.
2/66 55/106 39/106
3 5’2 37*
l/38
3
138
JANE
K.
A.
COOK
Chicks. Following inoculation with AIB virus only, mortality was similar to that in the uninoculated control group (Table 2). In groups inoculated with GAL virus when 17-day embryos the mortality rate following hatching was 55/106 (52 per cent). However, when GAL virus inoculation was followed by administration of AIB virus, mortality was only 39/106 (37 per cent) which was significantly lower (P < O-05) than the mortality in the group inoculated only with GAL virus. Challenge with AIB virus after inoculation of 17-day embryos with CELO virus resulted in a higher chick mortality 36/73 (57 per cent) than did inoculation of CELO virus alone, when chick mortality was 27/62 (44 per cent), a difference in mortality which was not statistically significant. When chicks were inoculated with GAL virus at day-old and AIB virus 6 days later there was little difference in subsequent mortality between the 2 groups, it being 17/45 (38 per cent) in the groups inoculated only with GAL virus and 15137 (41 per cent) in the dually inoculated group. In all adenovirus-inoculated groups chick mortality occurred within 7 days of hatching. Respiratory
Infection
In all groups of chicks inoculated with AIB virus alone, whether at day-old or 6 days of age, coughing and riles were first observed 2 to 3 days after inoculation and continued in the majority of chicks for approximately 7 days, thereafter 1 or 2 chicks in some groups remained affected until up to 14 days after inoculation, When chicks already inoculated with GAL virus at day-old were inoculated with AIB virus 6 days later, the duration and severity of respiratory signs were very similar to that recorded in the groups inoculated with only AIB virus despite the higher mortality in the former group. When embryos inoculated with GAL virus were challenged with AIB virus on hatching, in the first experiment there was no difference in duration and severity of respiratory signs from that recorded in the group inoculated only with AIB virus, but when the experiment was repeated, onset of respiratory signs was delayed until 6 days after inoculation and then continued until the 16th day. No respiratory signs were recorded in any chicks inoculated with CELO virus prior to AIB virus, nor in any of the chicks inoculated only with adenovirus. General Condition Chicks inoculated with AIB virus alone appeared lively throughout, even when showing respiratory signs. In addition to the deaths, chicks inoculated with either of the adenovirus strains as embryos were generally depressed, with ruffled or drooping feathers and showed inappetance from hatching until approximately 7 to 8 days of age whether or not they were inoculated with AIB virus. Bodyweight at 7 days of age was depressed following inoculation of embryos with adenovirus alone and following inoculation with CELO and AIB viruses but there was little depression in weight gain in groups inoculated with GAL and AIB viruses.
ADENOVIRUSES
AND
AIB
VIRUS
IN DAY-OLD
139
CHICKS
Virus Recovery CELO or GAL viruses were recovered at all times and from all tissues examined, whether or not adenovirus inoculation was followed by AIB administration. The results of attempts to recover AIB virus from tissues are shown in Table 3. In each experiment AIB virus was recovered consistently from all TABLE DURATION
OF RECOVERY OF AIB VIRUS PRECEDED BY AVIAN
Days Tissne
Lung/trachea Liver Spleen Kidney Ovary/oviduct Caecal tonsil
for AIB
which AIB onb
14 14 14 14 14 14
3 AFTER INOCULATION ADENOVIRUS
cirus wm recowred CELO+.4IB
ALONE
after inoctrlatiort
miih
GAL+-MB
0
10
,”
il 7 10 0
0” 0
OK
tissues of chicks inoculated with that virus alone. The virus was never recovered from any tissue of chicks inoculated with CELO virus prior to AIB virus. However, AIB virus could be recovered for 10 days from the respiratory tract of chicks sprayed with AIB virus after GAL virus inoculation as 17-day embryos. Initially the virus was also recovered from each of the other tissues examined with the exception of the caecal tonsil. However, AIB virus could not be recovered from tissues of the groups inoculated with both viruses as consistently or with such ease as from groups inoculated only with AIB virus. Following inoculation with GAL virus at day-old and AIB virus 6 days later, AIB virus was recovered from the respiratory tract (the only tissue examined) with equal facility in both groups. Neutralizing
Antibody Determinations
In all groups inoculated with AIB virus alone, mean neutralizing antibody indices of 300 or greater were recorded by 28 days after inoculation (Fig. 1). Following inoculation with both CELO and AIB virus, no AIB neutralizing antibody was detectable by 28 days after inoculation. When AIB virus administration at either day-old or 6 days followed inoculation with GAL virus, very similar mean neutralizing antibody indices of 32 and 16 respectively were recorded and, although very low, these antibody levels were rising by 28 days. Mean adenovirus neutralizing antibody titres are shown in Fig. 2. In groups inoculated with adenovirus alone, neutralizing antibody titres had in each case reached 4.5 log,, or over by 28 days after inoculation regardless of when
140
K.
JANE
A.
COOK
adenovirus was administered. When GAL virus inoculation was followed by challenge with AIB virus, neutralizing antibody titres did not differ from those in groups which received only GAL virus. Following inoculation of AIB virus after CELO virus, mean neutralizing antibody titres to CELO virus were initially lower than those in the chicks inoculated only with CELO virus but were similar by the end of the experiments. 1 I I I I I I I I I (a)
Lb)
(c)
I 7
14
21
28
7
14
21
1
28
7
14
21
28
Days
1. AIB serum neutralization (a) CELO virus (I’i-day and AIB virus (day-old). oculated with AIB virus; 6 5-
I
b
I
I
indices following inoculation of AIB virus alone or after adenovirus, embryos) and AIB virus (day-old). (b) GAL virus (17-day embryos) (c) GAL virus (day-old) and AIB virus (6 days). (O---O) In(O--O) inoculated with adenovirus+AIB virus. I
I
I
I
I
I
I
I
I
(cl
(b)
(ai
I
,
A
-
-
4-
7
14
21
28
7
I4
21
28
7
14
21
28
Days
Fig. 2. Adenovirus serum neutralizing antibody titres after inoculation of adenovirus alone or followed by AIB virus. (a) CELO virus (17-day embryos) and AIB virus (day-old). (b) GAL virus (17-day embryos) and AIB virus (day-old). (c) GAL virus (day-old) and AIB virus (6 days). (A-A) Inoculated with adenovirus; (/J--A) inoculated with adenovirus+AIB virus. DISCUSSION
Inoculation of 17-day embryos with CELO but not GAL virus resulted in considerable embryo mortality. In contrast, mortality after hatching was higher following inoculation with GAL virus. No embryo or chick mortality was recorded by Jordan and Nassar (1974) following inoculation of 17-day
ADENOVIRUSES
AND
AIB
VIRUS
IN
DAY-OLD
141
CHICKS
embryos with the same strain of CELO virus. Since the experimental design was the same, the different findings may possibly be a result of variations in the breed of chicken used. The difference in mortality following in ovo inoculation of the two adenoviruses indicates that embryos are more susceptible to CELO virus whereas young chicks are more susceptible to GAL virus. CELO virus causes high embryo mortality following inoculation of 9 or 10 day-old embryos via the allantoic cavity but similar inoculation with GAL virus rarely causes mortalit) although the virus replicates to a titre of at least 5.5 log,,TCID,,/ml (unpublished observation). It has been shown (Cook, 1974) that inoculation 01 day-old chicks with GAL virus resulted in considerable mortality, but that such inoculation with CELO virus did not, and furthermore that recovery of CELO virus from tissues was more difficult than was recovery of GAL virus. Apart from the different mortality patterns the prior inoculation of the two serotypes of avian adenovirus produced very difFerent cfFects on the course of an AIB infection. In ovo inoculation with CELO virus prior to challenge kvitlr AIB virus resulted in complete absence of respiratory signs, failure to recover 1ZIB virus from any tissues examined even as early as one day after AIB virus inoculation and absence of an AIB neutralizing antibody response. These findings are in agreement with the observations of Jordan and Nassar (1974). ‘l~hq~ are in contrast, however, to the findings when inoculation with GAL \-irus preceded exposure to AIB virus. Respiratory signs in these groups \vere similar to those following exposure to AIB alone. Although virus recovery was more difficult, AIB virus could be recovered throughout from the respirator!. tract and initially from most other tissues examined. The greater ease of recovcry Of AIB virus from the respiratory tract may have been a result of the \,irus replicating to a higher titre in th.at site than elsewhere. Adenoviruses are known to produce latent infections (Pereira and Kelly, 1957) and it seems likely that these viruses persisted in the chicks throughout the course of these experiments. The inhibitory effect of avian adenoviruses on other viruses is already known and Ablashi et al. (1965), who demonstrated intcrlcrence between CELO virus and both ND virus and influenza A virus in chicken embryos, suggested two possible explanations-interferon production or competition for available receptor sites or enzymes. Although intrrliAron production by human strains of adenovirus (Ho and Kohler, 1967), b> animal strains (Guenov, 1970) and by the GAL serotype of avian adenovirus (Bakay, 1969) has been demonstrated, Pereira (1960), investigating a viral inhibitor produced by several adenoviruses, concluded that, whilst similar to interferon in many respects, it was distinct from it. The results described here demonstrate that two distinct serotypes of avian adenovirus are capable of apparently different degrees of interference due, possibly, to different rates of replication in chicken embryos and young chicks. SUMMARY
Administration of GEL0 but not GAL considerable embryo mortality.
virus
to 17-day embryos
resulted
jn
142
JANE
K.
A.
COOK
When in ovo inoculation of CELO virus was followed by administration of AIB virus on hatching, there was little difference in mortality between the group inoculated with CELO virus alone and the dually inoculated group. In contrast, in ovo inoculation of GAL virus prior to AIB virus resulted in lower chick mortality than did inoculation of GAL virus alone. Prior inoculation with CELO and GAL viruses caused very different effects on the course of an AIB infection. When CELO virus was inoculated prior to AIB virus, no respiratory signs were detected, AIB virus could not be recovered from tissues and no AIB neutralizing antibody was produced. When GAL virus was inoculated prior to AIB virus challenge, respiratory signs were as marked as in chicks inoculated with AIB virus alone, AIB virus could be recovered from most tissues but less easily and for a shorter time than after inoculation with AIB virus alone and there was only a very slight AIB neutralizing antibody response by 28 days after inoculation. Adenoviruses were readily recovered from all chicks, whether inoculated alone or with AIB virus. Neutralizing antibody titres to GAL virus were similar whether or not AIB virus was present. However, CELO virus neutralizing antibody reactions in the dually infected groups were initially delayed, but by 28 days after inoculation were similar to those in the group inoculated with CELO virus alone. ACKNOWLEDGMENTS
The author wishes to acknowledge the technical assistance of Miss F. R. Pickworth, Mrs A. P. Parker and Miss D. M. Schietzel and to thank Mr J. G. Rowe11 for assistance with the statistical analysis. REFERENCES
Ablashi, D. V., Chang, P. W., and Yates, V. J. (1965). The effect of a latent CELO virus infection in the chicken embryo on the propagation of Newcastle Disease and influenza viruses. Avian Diseases, 9, 407-417. Anon. (197 1). Methods for Examining Poultry Biologics and for Identifying and Quant;fving Avian Pathogens. National Academy of Science, National Research Council, Washington. Bakay, M. (1969). Interferon induction by GAL virus in chick embryo cells. Acta virologica,
13, 340-342.
Cook, J. K. A. (1968a). Isolation of a CELO virus from fertile chicken eggs. Veterinary Record, 82, 294.
Cook, J. K. A. (1968b). D uration of experimental infectious bronchitis in chickens. Research in Veterinary Science, 9, 506-514. Cook, J. K. A. (1970). I ncidence of chick embryo lethal orphan virus antibody in the fowl (Callus domesticus) in Britain. Research in Veterinary Science, 11, 343-348. Cook, J. K. A. (1972). A vian adenoviruses alone or followed by infectious bronchitis virus in laying hens. Journal of Comparative Pathology, 82, 119-128. Cook, J. K. A. (1973). Th e inhibition of infectious bronchitis virus multiplication by CELO virus in the fowl embryo. Research in Veterinary Science, 15, 375-378. Cook, J. K. A. (1974). Pathogenicity of avian adenoviruses for day-old chicks. Journal of Comparative Pathology, 84, 505-515. Cooper, D. M., and Timms, J. R. (1972). The rearing and maintenance of breeding chickens in isolators. I. Glass fibre isolators. Avian Pathology, 1, 47-57. Cuenov, I. (1970). Interference activity of animal adenoviruses in chick embryo cell cultures. Acta virologica, 14, 337-342.
ADENOVIRUSES
AND
AIB
VIRUS
IN
DAY-OLD
IF
CHICKS
143
M., and Kohler, K. (1967). Studies on human adenoviruses as inducers of interferon in chick cells. Archiv ftir die gesamte Virusforschung, 22, 69-78. Jordan, F. T. W., and Nassar, T. J. (1974). The effect of infectious bronchitis (IB) virus on day-old chicks previously infected as 17-day-old embryos with an avian adenovirus. Research in Veterinary Science, 16, 47-53. Pcreira, H. G. (1960). A virus inhibitor produced in HeLa cells infected with adenovirus. Virology, 11, 590-602. Pereira, H. G., and Kelly, B. (1957). Latent infection of rabbits by adenovirus type 3. Nature, 180, 615-616. \-ates, V. J., and Fry, D. E. (1957). Ob servations on a chicken embryo lethal orphan (GEL01 virus. American Journal of Veterinary Research, 18, 657-660. [Received for publication,
May 17th, 19761