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The isolation of infectious bursal disease virus from turkeys in England
Br. vet.
1.
(1985) . 141, 141
THE ISOLATION OF INFECTIOUS BURSAL DISEASE VIRUS FROM TURKEYS IN ENGLAND
N . J . CHETTLE, R . K. EDDY and P . J . WYETH Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw, Weybridge, Surrey
SUMMARY The isolation of infectious bursal disease (IBD) virus from turkeys is described . The virus closely resembles TY89 which is the prototype for IBD serotype 2 . Using the tissue culture virus neutralization test, sera from five grandparent, 10 parent and 27 fattening flocks were all shown to contain antibodies to TY89 . Four • of the fattening flocks were monitored weekly from hatch . Each had maternally-derived antibody which persisted to between eight and 22 days of age . Every flock was naturally infected giving rise to antibodies which were first detectable from 36 to 57 days of age . This is believed to be the first report of natural infection of turkeys with IBD virus in England .
INTRODUCTION Infectious bursal disease (IBD) was first described in chickens in 1962 (Cosgrove, 1962) . The first reported natural outbreak of IBD in turkeys occurred in Northern Ireland (McNulty, Allan & McFerran, 1979) . McFerran et al. (1980), using a virus neutralization (VN) test, demonstrated that there were two serotypes of IBD and proposed G13 to be the prototype for serotype 1 and the Northern Ireland isolate, designated TY89, to be the prototype for serotype 2 . Experimental inoculation of turkeys using chicken isolates of IBD produced antibodies, but no symptoms (Giambrone et al., 1978 ; Weisman & Hitchner, 1978 ; Perelman & Heller, 1981 ; Jackwood, Saif & Hughes, 1982) . Turkeys inoculated experimentally with a strain of IBD isolated from turkeys were not clinically affected but a rise in antibodies was demonstrated (Jackwood et al, 1982) . Barnes, Wheeler & Reed (1982), reported naturally-occurring IBD antibodies in turkeys in Iowa ; they demonstrated that these were not serotype 1, but did not determine their relationship with TY89 . It was of interest to know whether IBD virus was infecting turkeys in England . A study was therefore undertaken on flocks from two major turkey-producing companies, widely separated geographically . This paper reports the isolation of a strain of IBD, designated 23/82 from fattening turkeys in one of the flocks. This strain was neutralized by TY89 antiserum using a VN test . The detection of type 2 antibodies in grandparent, parent and fattening flocks in England is also reported .
142
BRITISH VETERINARY JOURNAL . 141, 2
MATERIALS AND METHODS Experiment 1 Twenty turkeys from each of four fattening flocks (A, B, C and D) were blood sampled at weekly intervals from day old, when they were placed on the farm, until they were 64 days old . The sera were tested for the presence of antibodies to IBD serotypes 1 and 2 . At each sampling time ten bursae of Fabricius were pooled and attempts were made to isolate IBD virus . Experiment 2 Twenty turkeys from each of five grandparent and 10 parent flocks were blood sampled at 25 weeks of age, and 27 fattening flocks were blood sampled at slaughter (approximately 12 weeks of age) . The sera from all these were tested for the presence of antibodies to IBD serotypes 1 and 2 . Virus neutralization (VN) tests These were carried out using a constant-virus diluting-serum microtitre method . The TY89 strain of IBD was kindly supplied by Dr J . B . McFerran, Veterinary Research Laboratory, Stormont, N . Ireland . The G13 strain was a tissue culture adapted chicken strain of IBD . 0 . 05 ml virus dilution containing 100 TCID 50 was placed in each well of a microtitre plate . Serial dilutions of test sera were made in the diluted virus and the plates left for 30 min at room temperature . 0 . 2 ml of chick embryo fibroblast (CEF) cell suspension was then dispensed into each well . 'Pests were incubated at 37 °C for 96 h, after which the monolayers were observed microscopically for typical cytopathic effect (CPE) . End-points were determined by reading the reciprocal (log,) of the final dilution which did not show CPE . Virus isolations Bursae of Fabricius were homogenized as a 10% (w/v) suspension in Earle's balanced salt solution (EBSS) containing 2% M-HEPES and 100 units/ ml penicillin and 100 tg/ml streptomycin, then clarified at 3000 g for 20 min . The supernatant fluid was inoculated onto confluent CEF monolayers and incubated for 1 h at 37 °C . The monolayers were then washed twice with EBSS, covered with maintenance medium and incubated at 37 °C . The monolayers were examined daily for evidence of CPE . If no CPE was observed after six days the cells were disrupted by freezing and thawing . The resulting lysate was inoculated onto fresh monolayers . This was repeated three times . Cell cultures CEF cells were prepared by trypsinizing 10-day-old specific antibody negative embryos . Growth medium was Eagle's MEM containing 10% foetal calf serum (FCS), 0 . 5 g/1 sodium bicarbonate, 100 units/ml penicillin and 100 .tg/ml streptomycin . Maintenance medium was the same except FCS was reduced to 1% and sodium bicarbonate increased to 2 g/l . For the VN test a cell suspension was made from a 24-h CEF cell culture . The cell concentration was adjusted to 1 X 10'/ml in the same growth medium except that 2% M-HEPES was also incorporated .
INFECTIOUS BURSAL DISEASE IN ENGLISH TURKEYS
143
Preparation of 23/82 antiserum Six five-week-old turkeys were inoculated intraocularly with 0 . 1 ml of the 23/82 isolate containing 1032 ' TCID 50 . They were blood sampled after 28 days . A pool of the sera was used as 23/82 standard antibody .
RESULTS
Experiment 1 Table I shows the mean VN titres for each of the four fattening flocks which were blood sampled weekly . Each flock had maternally-derived IBD type 2 antibody at day o ld . V N titres had fallen to less than 22 by days 8, 22, 22 and 29 in flocks A, B, C and D respectively, and an antibody response to TY89 was detected by days 57, 43, 50 and 36 respectively . The fall in titre of maternally-derived antibody was not strictly linear, but the values were means of 10 birds which were randomly selected and different at each sampling time . Antibodies to IBD type 1 were not detected in any of the sera .
Table I Mean virus neutralization titres* to IBD strain TY89 of 10 different birds sampled on each occasion from four fattening flocks
Days of age 1
8
15
22
29
36
43
50
57
64
Flock A
5 .9
2.0
2.0
2 .0
2 .0
2 .0
2.0
2 .0
5.6
8.7
Flock B
7.1
4.3
2. 6
2 .0
2 .0
2 .9
5.7
8 .3
9. 9
NT
Flock C
6.8
2.2
2. 7
2 .0
2.0
2 .0
2. 1
4. 2
7.8
NE
Flock D
7. 1
6.7
4. 5
3 .0
2.0
4. 5
6.3
10 . 3
10 . 4
NT
*k1ean reciprocal (log,) titre of 10 antisera . NT not tested .
Virus was isolated from flock D at 36 days of age and was designated 23/82 . Virus could not be isolated from any of the other flocks . Table II shows the antigenic relationships between TY89, G13 and the 23/82 isolate . TY89 and 23/82 cross-neutralized whereas G13 showed no significant cross with either .
Experiment 2 All the sera from the five grandparent flocks had titres of 2 10 or greater against 'hY89, as did those from the 10 parent flocks . The titres of all the sera from the two fattening flocks were greater than 2' when the turkeys were slaughtered at approximately 12 weeks of age . Antibodies to IBD type 1 were not detected in any of the sera . No symptoms of clinical disease associated with IBD were seen in any of the flocks from either experiment .
144
BRITISH VETERINARY JOURNAL, 141, 2 Table II The antigenic relationships* between TY89, G13 and the 23/82 isolate using the virus neutralization test Visus Antiserum
T289
23/82
G13
TY89 23/82 G13
13 10 5
13 10 5
4 4 11
* Reciprocal (loge ) of the final dilution which did not show cytopathic effect .
DISCUSSION These results demonstrate the presence of IBD serotype 2 in England . The presence of antibodies to serotype 2 in all the grandparent, parent and fattening flocks tested indicates that the infection is probably widespread . The VN titres, using the method described, correlate well with those of Skeeles et al. (1978) who used a similar method . McFerran et al. (1980) obtained higher titres ; they tested hyperimmune serum, however, and used a roller tube method of VN which is known to give higher titres than the microtitre method (T . J . Connor, personal communication) . Most infections of IBD serotype 1 in chickens in the UK are subclinical, but during the course of infection, destruction of the lymphocytes in the bursa of Fabricius in young chicks leads to immunosuppression leaving the chicks more susceptible to otherwise innocuous infectious agents . In addition, chicks which have had the disease give poor food conversion ratios and average weight gains may be reduced by as much as 7 . 9% at 56 days (Wyeth, O'Brien & Cullen, 1981) . Although no symptoms have been observed in association with serotype 2 infection in turkeys, it is possible that turkeys which are not exposed to IBD may grow more economically . Studies are now in progress to determine whether any specific tissue damage or immunosuppression may be caused by infection of young turkeys with IBD . If there is tissue damage it may be beneficial to develop a vaccine . If, however, it is shown that one serotype will protect against the other, then the currently available IBD vaccines may prove useful when applied to turkeys . If TY89 is shown to be widespread among chickens and there is no cross protection between the two serotypes, this may explain the apparent lack of protection afforded to some chickens after the application of the live vaccine currently available in Britain . The development of an IBD serotype 2 vaccine would be beneficial for these cases . Further work is required to investigate these aspects .
INFECTIOUS BURSAL DISEASE IN ENGLISH TURKEYS
14$
ACKNOWLEDGEMENTS The authors are indebted to Mr W . Cox for his technical assistance in this work .
REFERENCES BARNES, H . J ., WHEELER, J . & REED, D . (1982) . Avian Diseases 26, 560 . COSCROVE, A . S . (1962) . Avian Diseases 6, 385 . GIAAIBRONE, J . J ., FLr. rsIER, D . J ., L(KERr, P . D ., PACE, R. K . & EIDSON, C . E . (1978) . Avian Diseases 22, 451 . JACKWOOD, D . J ., SATS, Y . M . & Hi CilEs, J . H . (1982) . Avian Diseases 26, 871 . MCFERRAN, J . B ., McNuLLY, M . S ., MCKILLOP, E . R., CONNOR, J . T ., MCCRACKEN, R . M ., D . S . & ALLAN, G . M . (1980) . Avian Pathology 9, 395 . MCNCLLY, M . S ., ALLAN, G . M . & MCFERRAN, J . B . (1979) . Avian Pathology 8,205 . PE.RELnlAN, B . & HELLER, E. D . (1981) . Refuah Velerinarilh 38, 12 . SKEELES, J . K ., LI KERL, P. D ., FLEICDER, D . J . & LEONARD, J . D . (1978) . Avian Diseases 23, 456 . WEISNIAN, J . & Hi LCFINER, S. B . (1978) . Avian Diseases22, 604 . WYEi ii, P . J ., O'BRIEN, J . D . P . & CCLLEN, G . A . (1981) . Avian Diseases 25, 228 . (Accepted for publication 23 February 1984)
1.
(1985) . 141, 141
THE ISOLATION OF INFECTIOUS BURSAL DISEASE VIRUS FROM TURKEYS IN ENGLAND
N . J . CHETTLE, R . K. EDDY and P . J . WYETH Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw, Weybridge, Surrey
SUMMARY The isolation of infectious bursal disease (IBD) virus from turkeys is described . The virus closely resembles TY89 which is the prototype for IBD serotype 2 . Using the tissue culture virus neutralization test, sera from five grandparent, 10 parent and 27 fattening flocks were all shown to contain antibodies to TY89 . Four • of the fattening flocks were monitored weekly from hatch . Each had maternally-derived antibody which persisted to between eight and 22 days of age . Every flock was naturally infected giving rise to antibodies which were first detectable from 36 to 57 days of age . This is believed to be the first report of natural infection of turkeys with IBD virus in England .
INTRODUCTION Infectious bursal disease (IBD) was first described in chickens in 1962 (Cosgrove, 1962) . The first reported natural outbreak of IBD in turkeys occurred in Northern Ireland (McNulty, Allan & McFerran, 1979) . McFerran et al. (1980), using a virus neutralization (VN) test, demonstrated that there were two serotypes of IBD and proposed G13 to be the prototype for serotype 1 and the Northern Ireland isolate, designated TY89, to be the prototype for serotype 2 . Experimental inoculation of turkeys using chicken isolates of IBD produced antibodies, but no symptoms (Giambrone et al., 1978 ; Weisman & Hitchner, 1978 ; Perelman & Heller, 1981 ; Jackwood, Saif & Hughes, 1982) . Turkeys inoculated experimentally with a strain of IBD isolated from turkeys were not clinically affected but a rise in antibodies was demonstrated (Jackwood et al, 1982) . Barnes, Wheeler & Reed (1982), reported naturally-occurring IBD antibodies in turkeys in Iowa ; they demonstrated that these were not serotype 1, but did not determine their relationship with TY89 . It was of interest to know whether IBD virus was infecting turkeys in England . A study was therefore undertaken on flocks from two major turkey-producing companies, widely separated geographically . This paper reports the isolation of a strain of IBD, designated 23/82 from fattening turkeys in one of the flocks. This strain was neutralized by TY89 antiserum using a VN test . The detection of type 2 antibodies in grandparent, parent and fattening flocks in England is also reported .
142
BRITISH VETERINARY JOURNAL . 141, 2
MATERIALS AND METHODS Experiment 1 Twenty turkeys from each of four fattening flocks (A, B, C and D) were blood sampled at weekly intervals from day old, when they were placed on the farm, until they were 64 days old . The sera were tested for the presence of antibodies to IBD serotypes 1 and 2 . At each sampling time ten bursae of Fabricius were pooled and attempts were made to isolate IBD virus . Experiment 2 Twenty turkeys from each of five grandparent and 10 parent flocks were blood sampled at 25 weeks of age, and 27 fattening flocks were blood sampled at slaughter (approximately 12 weeks of age) . The sera from all these were tested for the presence of antibodies to IBD serotypes 1 and 2 . Virus neutralization (VN) tests These were carried out using a constant-virus diluting-serum microtitre method . The TY89 strain of IBD was kindly supplied by Dr J . B . McFerran, Veterinary Research Laboratory, Stormont, N . Ireland . The G13 strain was a tissue culture adapted chicken strain of IBD . 0 . 05 ml virus dilution containing 100 TCID 50 was placed in each well of a microtitre plate . Serial dilutions of test sera were made in the diluted virus and the plates left for 30 min at room temperature . 0 . 2 ml of chick embryo fibroblast (CEF) cell suspension was then dispensed into each well . 'Pests were incubated at 37 °C for 96 h, after which the monolayers were observed microscopically for typical cytopathic effect (CPE) . End-points were determined by reading the reciprocal (log,) of the final dilution which did not show CPE . Virus isolations Bursae of Fabricius were homogenized as a 10% (w/v) suspension in Earle's balanced salt solution (EBSS) containing 2% M-HEPES and 100 units/ ml penicillin and 100 tg/ml streptomycin, then clarified at 3000 g for 20 min . The supernatant fluid was inoculated onto confluent CEF monolayers and incubated for 1 h at 37 °C . The monolayers were then washed twice with EBSS, covered with maintenance medium and incubated at 37 °C . The monolayers were examined daily for evidence of CPE . If no CPE was observed after six days the cells were disrupted by freezing and thawing . The resulting lysate was inoculated onto fresh monolayers . This was repeated three times . Cell cultures CEF cells were prepared by trypsinizing 10-day-old specific antibody negative embryos . Growth medium was Eagle's MEM containing 10% foetal calf serum (FCS), 0 . 5 g/1 sodium bicarbonate, 100 units/ml penicillin and 100 .tg/ml streptomycin . Maintenance medium was the same except FCS was reduced to 1% and sodium bicarbonate increased to 2 g/l . For the VN test a cell suspension was made from a 24-h CEF cell culture . The cell concentration was adjusted to 1 X 10'/ml in the same growth medium except that 2% M-HEPES was also incorporated .
INFECTIOUS BURSAL DISEASE IN ENGLISH TURKEYS
143
Preparation of 23/82 antiserum Six five-week-old turkeys were inoculated intraocularly with 0 . 1 ml of the 23/82 isolate containing 1032 ' TCID 50 . They were blood sampled after 28 days . A pool of the sera was used as 23/82 standard antibody .
RESULTS
Experiment 1 Table I shows the mean VN titres for each of the four fattening flocks which were blood sampled weekly . Each flock had maternally-derived IBD type 2 antibody at day o ld . V N titres had fallen to less than 22 by days 8, 22, 22 and 29 in flocks A, B, C and D respectively, and an antibody response to TY89 was detected by days 57, 43, 50 and 36 respectively . The fall in titre of maternally-derived antibody was not strictly linear, but the values were means of 10 birds which were randomly selected and different at each sampling time . Antibodies to IBD type 1 were not detected in any of the sera .
Table I Mean virus neutralization titres* to IBD strain TY89 of 10 different birds sampled on each occasion from four fattening flocks
Days of age 1
8
15
22
29
36
43
50
57
64
Flock A
5 .9
2.0
2.0
2 .0
2 .0
2 .0
2.0
2 .0
5.6
8.7
Flock B
7.1
4.3
2. 6
2 .0
2 .0
2 .9
5.7
8 .3
9. 9
NT
Flock C
6.8
2.2
2. 7
2 .0
2.0
2 .0
2. 1
4. 2
7.8
NE
Flock D
7. 1
6.7
4. 5
3 .0
2.0
4. 5
6.3
10 . 3
10 . 4
NT
*k1ean reciprocal (log,) titre of 10 antisera . NT not tested .
Virus was isolated from flock D at 36 days of age and was designated 23/82 . Virus could not be isolated from any of the other flocks . Table II shows the antigenic relationships between TY89, G13 and the 23/82 isolate . TY89 and 23/82 cross-neutralized whereas G13 showed no significant cross with either .
Experiment 2 All the sera from the five grandparent flocks had titres of 2 10 or greater against 'hY89, as did those from the 10 parent flocks . The titres of all the sera from the two fattening flocks were greater than 2' when the turkeys were slaughtered at approximately 12 weeks of age . Antibodies to IBD type 1 were not detected in any of the sera . No symptoms of clinical disease associated with IBD were seen in any of the flocks from either experiment .
144
BRITISH VETERINARY JOURNAL, 141, 2 Table II The antigenic relationships* between TY89, G13 and the 23/82 isolate using the virus neutralization test Visus Antiserum
T289
23/82
G13
TY89 23/82 G13
13 10 5
13 10 5
4 4 11
* Reciprocal (loge ) of the final dilution which did not show cytopathic effect .
DISCUSSION These results demonstrate the presence of IBD serotype 2 in England . The presence of antibodies to serotype 2 in all the grandparent, parent and fattening flocks tested indicates that the infection is probably widespread . The VN titres, using the method described, correlate well with those of Skeeles et al. (1978) who used a similar method . McFerran et al. (1980) obtained higher titres ; they tested hyperimmune serum, however, and used a roller tube method of VN which is known to give higher titres than the microtitre method (T . J . Connor, personal communication) . Most infections of IBD serotype 1 in chickens in the UK are subclinical, but during the course of infection, destruction of the lymphocytes in the bursa of Fabricius in young chicks leads to immunosuppression leaving the chicks more susceptible to otherwise innocuous infectious agents . In addition, chicks which have had the disease give poor food conversion ratios and average weight gains may be reduced by as much as 7 . 9% at 56 days (Wyeth, O'Brien & Cullen, 1981) . Although no symptoms have been observed in association with serotype 2 infection in turkeys, it is possible that turkeys which are not exposed to IBD may grow more economically . Studies are now in progress to determine whether any specific tissue damage or immunosuppression may be caused by infection of young turkeys with IBD . If there is tissue damage it may be beneficial to develop a vaccine . If, however, it is shown that one serotype will protect against the other, then the currently available IBD vaccines may prove useful when applied to turkeys . If TY89 is shown to be widespread among chickens and there is no cross protection between the two serotypes, this may explain the apparent lack of protection afforded to some chickens after the application of the live vaccine currently available in Britain . The development of an IBD serotype 2 vaccine would be beneficial for these cases . Further work is required to investigate these aspects .
INFECTIOUS BURSAL DISEASE IN ENGLISH TURKEYS
14$
ACKNOWLEDGEMENTS The authors are indebted to Mr W . Cox for his technical assistance in this work .
REFERENCES BARNES, H . J ., WHEELER, J . & REED, D . (1982) . Avian Diseases 26, 560 . COSCROVE, A . S . (1962) . Avian Diseases 6, 385 . GIAAIBRONE, J . J ., FLr. rsIER, D . J ., L(KERr, P . D ., PACE, R. K . & EIDSON, C . E . (1978) . Avian Diseases 22, 451 . JACKWOOD, D . J ., SATS, Y . M . & Hi CilEs, J . H . (1982) . Avian Diseases 26, 871 . MCFERRAN, J . B ., McNuLLY, M . S ., MCKILLOP, E . R., CONNOR, J . T ., MCCRACKEN, R . M ., D . S . & ALLAN, G . M . (1980) . Avian Pathology 9, 395 . MCNCLLY, M . S ., ALLAN, G . M . & MCFERRAN, J . B . (1979) . Avian Pathology 8,205 . PE.RELnlAN, B . & HELLER, E. D . (1981) . Refuah Velerinarilh 38, 12 . SKEELES, J . K ., LI KERL, P. D ., FLEICDER, D . J . & LEONARD, J . D . (1978) . Avian Diseases 23, 456 . WEISNIAN, J . & Hi LCFINER, S. B . (1978) . Avian Diseases22, 604 . WYEi ii, P . J ., O'BRIEN, J . D . P . & CCLLEN, G . A . (1981) . Avian Diseases 25, 228 . (Accepted for publication 23 February 1984)