- Email: [email protected]
Relative efficiency of techniques for detecting avian reticuloendotheliosis virus as a vaccine contaminant
Paramyxc, v i m Yucaipa in pig I:
data p resented here suggest that the to7wn pigeon is fully suscepltible to and excretes a Yucaipa-like:virus. This bird could thus be a link in the transmission o~fPMY from tjird to bird and perhaps from bird to man and a survey of town pigeons for paramyxovlruses woula oe worthvvhile.
R. D. J. (1980) Velerinow Bulletin 50.737-752 I, R. A., CORSTVET, R. E. &CLARK, G. T. (1%0)
!
.. .
E
LIC1CrLLS1a-~292-293
DINTER. Z., HERMODS;SON, S. & :HERMODSS;ON, L. (1%A) Virologv 22,297-304 FLEURY, H . J. A. & A LEXANDEI;1, D. J. (19'78) Bulletin I'lnrtilut Parteur 76,175-- ..--- - - .. . . . -. :LEURY, H. J. A. & A L ~ X A N U ~ U. K , J. (1YIY) Avran Ursea
Ackno
--A
?
This work was a 33867 t o H.F.).
23,742-744
y a grant fr.om the CN b*,hI;,."t;,,"
Receivedforp~,,,,,,.,..December20, Accepted ' Februory I ,
ALETA, E. F. & HEEFELS, Wochenschriff 89,31 VINDEVOGEL, H., TIU I D V MEULEMANS, G . (198
u.
....., ,.,
1982
C
(1
he tieriintlic PASTORE'.,T
,.
D
.. D
R e s e u ~ hin Veterinary Science 1983.3.4.377-379
Rela virus
ciency c)f techniques fa,r detecting avian reticuloendotheliosis ccine ca~ntamin ant
R. A. J. NICHOLAS, D. H. 'I nunn I U N
Centro
1
Loborator
ge, Surrey
70). Therefore contamination wi ducks (Witt ial hazard in the use of new isolat REV is inde :ys for the production of Marek of herpesvir the use of turkey, duck or chickc disease vacc embryos or cells tor the propagation of vaccine viruse Since REV is believed to have entered the avian class from mammalian source (Waite and Allen 1975) the hazard fro the substrate applies not only to production of avii vaccines but to mammalian and human vaccines also. TI cletection of REV is hampered because the virus does n c,awe gross effects in either embryos or cell cultures. TI c,omparison of various in vitro test procedures for detectil x
Three tecnn~queswere comparea tor sens~riv~ry In aerecung reticuloendotheliosis virus (REV) in a deliberately contaminated Marek's disease vaccine. The most sensitive and rapid test was the indirect fluorescent antibody test (FAT). The in direct immunoperoxidase test, a lthough sinnple to perforrn and only marginally less sensit]ive than thce FAT, was di fficult to 1interpret at low level!s of REV. Using immun oelectron nnicroscopy, virions we*reseen on1ly after .. and then not to the same level as that three suoculrures detected by the FAT or im munoperox idase test. Serum raised against the HPRS-1 strain of RIEV detecteci other Strains of this virus in the FA1r
.
. .
.
BEFOI
A
..
.
marenam ano mernoos ated and pl aque purifi~
The HPR S-l strain o f REV, is01 . .. on Poultrq Research x a t ~ o n , was used t>y Hought~
.
'
I
" ",,,.,..iercially ,..rn ~ L
available ~ ~ herpesv~rus ~ ~ turkey vaccine. The other REV strains used were duc infectious anaemia, chick syncytial, spleen necrosis virus and REV C and REV F (Purchase et a1 1973). Antiserum was obtained three weeks aftel r r l r c c ~ U I rlightly intramuscular inoculations of specific 1 C:hicks with 10sTCID50 of HPRS-I virus. Primary chick embryo fibroblast s were prepared in plat iontaining glass coversiips and in tissue culturf:flasks. .-,t--:n~rr
c
r
~
~
a
~
1
~
~
~
,
~
H. Thorn
The lndlrect fluorescent antlboay test ( ~ A was I )c out on coverslip cu'ltures as paeviously de:scribed (Nit et al1983). For the indirect irnrnunoperc~xidaset a t coverslip cu --Llr"... G - d m;tI.nr of chick embryo fibruula3rs W G L G &LAr u chrrlrr accrvrlr five minutes at 4OC or in a mixture of periodate, lysine and paraformaldehyde (McLean and Nakane 1974) for 30 minutes at 4OC. The coverslips were incubated with a 1:20 . dilution of HPRS-1 antiserum for 20 minutes at -. room temperature, washed in running phosphate buffered saline and then incubated with a 1:1000 dilution of rabbi1t antichicken immunoglobulin conjugated with pero xidase (Nordic Laboratories) for the same period. After wiashing as before the coverslips were covered with diaminobenzidine solution (0.5 mg/ml activated wit h 0.01 pelr cent hydrogen peroxide) and left 2it room ternperature 1ror 20 , minutes. The coverslips were wasnea and air dried Ibefore mounting in DPX ( BDH). For immune electron micro:scopy, fornnvar coatedl grids were floated on H P RS-1 antiserum dilutec1 1:2 in tris buffer for 15 minutes andI washed fi1ve times in the same tbuffer. . .--.- ..L:-L ,*-A .c .~.on . rne sammes. wr~cclrw r l s l r Grids were then floatea I a minimi lyse-d wit1 ~ b l a s t cells chick embryo fibrl distilled water and stained wit11 2 per cent: phosphotc acid. Freezedried HPRS-1 virus was reconstituted In 5 rnl nf Eagle's minimal essential medium; this was termed ttle 10-1 dilution. Further dilutions of 10-3. 10-5. 10-7 and 10-9 were made and 0 . 2 ml of each dilution was added to 1.8 nnl of a suspension of herpesvirus of turkey containing ap~woximately 1W plaque forming units/ml. After neutrali sation with 2 ml of herpesvirus of turkey antiserum for two hours at 4°C. 0.1 ml of each sample was inoculated on to chick embryo fibroblast cultures. Three days later, co.verslip cultures were take]n for the FAT and innmunopero xidase test andI flask cultures for immune electron micro scopy. The re1naining chlick embryo fibroblast cells in flask furthe1r. flask tryps cultures were inised and dispensed Iinto .. . . .. . and coversl~pcultures, and after three days' lncu bation these were examined as above. This procedure was re peated every three to four days for a total of four passages. To determine whether the other strains of REV could equally be detected by the FAT using the HPRS-III am".r:r r serum, tissue culture fluid containing virus at a low passage level was inoculated on t o coverslip culture and thle FAT was carried out three days later. If the viruses could not be ed . detected, cells were passaged and the FAT was repeat1--
...---
:...
.."&A
S
W
~
. .
-
~
~-
..
~
3.-
.... ".
ncv
3rd passage LndpaIssage 1st passagc! 'AT IPT ItiM FAT IPT IEM FAT IPT IEM
1
~~-
3
.-.-W L I: ~ wrnmnson or recnncques ror me detection ur -
1"
-
ND
ND
+
10 10 10
lo +
Positive Dubious - Negative -. . N[J NO^ aone Fluorescent antibody test 4T FJ T lmmunoperoxidasetest IP' M lmmunoelectronmicrosc:oPY sults of the fourth subcultiIre have been omitted because they ?reidentical to those of the ttiird
+
-
F:
,,
The immu ne electronI microscopy detected REV afte'r thlree passagt!s but not t o the same level as th e FAT. Th e .nons were p~eomorpnicbut generally sphenca~ . witn a ameter of 90 t o 120 Inm. An ou ter layer bearing short )mpact fibre-like projtxtions was just visiblc: in some o~f thle particles. Results of rllG LlvJJ IGaLL.una ~ ~ p e r i m e nrevealed t that fcbur strains of REV, ie, HPRS-1, spleen necrosis, chick sy,ncytial and duck infectious anaemia, were detected by the F,AT using the HPRS-1 antiserum after one passage. REV c was detected after two passages and REV F after a f ulrther passa
. ..
-
1
Results The results of the comparative assessment of techniques for the detection of REV are shown in Table 1. The FAT proved to be the most sensitive and rapid technique, detecting virus from the lo-' dilution after three passages. Preliminary work with the immunoperoxidase test sbowed that periodate, lysine and paraformaldehyde fixatiorI led to a better differentiation between infected and unirifected cultures than did acetone fixation. However, it tjecame increasingly diffic ult t o disti~nguish inf eaed areasi from uninfected areas WIith diminishling levels o f virus. I n c:easing ~ the period of cell culture inclubation dicI not enharice the differentiation.
Discussion The direct and indirect FAT was used to show that REV ill multiply in cell culture by Cook (1969) although Smith et al (1977) reported that low intensity and non-specific st aining frecluently led to inconc:lusive resillts. In thiis :esent stud)r the FAT vvas not onl:y the most sensitive te!it blut results wc:re unequiv~ ocal. The immulnoperoxidase test has Irecently bee:n applied t o . .. . . . . . le aerecr~onof another retrovirus, avlan ~eukosisvlrus, in cc:I1 cultures by Mizuno and Arai (1981). Good differentiati,on was obtained in staining between infected and unin~ f e a e dcultures at the lower dilutions of REV but at the I.: dilutions detection became much less definite and ,,igher more time consuming. Negative contrast electron micros;copy is wid ely used as a c.~f its relati! re speed and means of detecting viru s because . simplicity particularly where the virus has a characteristic r?orphology. REV particles, however, are spherical and not tlistinctive when negatively stained and unless they are grouped together by the use of specific antiserum, as in our study, they are very difficult to detect. Purchase and Witter (1975) stated that viruses in the re~iculoendotheliosisgroup cross react in the FAT. All the vi ruses used in this study were detectable by the FAT using H'PRS-1 antiserum. However, REV C required two subCIlltures and REV F three passages before being detected; ttlis reflected either their inferior growth rate in cell culture 01 r the lower virus content of the initial inoculum or some rn ino or antigenic differences detected by the FAT.
..
.
.
'ios~v i m
Dete
PURCHASE, H. G., LUDFORD, C., NAZERIAN, K. & CO. H. W. (1973) Journal of the National Cancer Institute 5 489-499 PURCHASE, G. WmR, R. L. (1975) Topics
References
DENNETT, D. P- (1979) BAGUST* T. J., GRIMES, T. M. AustmIian VetmmnatyJoumal55.153-157 Microbiology and Immunologv71,103-123 BAINS. B. S. & NIELSEN, 1. I:)..(1979) Promedings of the 28th iARMA, P. S., JAIN, D. K., MISHRA, N. K., VE RNON, M. 1 Western Poultry Disease Con1fncnce and 13th Poultry Health PAUL, P. S. & POMEROY, B. S. (1975) Journal of the Nation Symposium. pp 82-85 Cancer Institute 54,1255-1359 COOK, M. K. (1%9) Journal of the National1 Concer Ins?itute 43. IMITH, E. J., SOLOMON. J. J. & WIT'ITR. R. L. ( I ~ I I Jn v n 203-212 GRIMES, T. M. & PURCHASEi, H. G. (1973) Australian VeterZN, P. T. (IS175) Journal Igf Virology 1 WAITE, Dire- M.21, inary Journal 49,4 66-471 HANSON. J. & HO WELL. J. (11979) Cbnadian Veterinary Journal 872-879 ,-** WITTER, R. L., PUKLHASb, H. CJ.&BUKWYN 20,161-164 Journalof the National Concer Institute 45,567-57 JACKSON. C. A. w.. UUNN. S. E. & SMITH. D. I WASA, N.. YOSHIDA. I. & TANIGUCHI, T. (1976) Nation Australian VeterinaryJournal 53.457-478 Institute of Animal Health Quarterly 16,141-151. McDOUGALL. J. S., BIGGS, P. M. &SHILL EIGEL. R. F., THEILEN. G. H. & TWIEHAUSI, M. J. (1% Avian Pathology 7.557-568 Journal of the National Concerlnstitute37,709-72 9 McLEAN, I. W. & NAKANE, P. K. (1974, , chemirtry and Qtochemirrry 22.1077-1083 MIZUNO, Y. & ARAI, K. (1981) National Institute of A n ~ m a ~ Health Quarterly 21,6367 a ber 23,1982 NICHOLAS. R. A. J.. WOOD. Receivec 'HORNTON , D. H. rpeprea reuruary 17, 1983 (1983)Journal o f Biological Stanaamuatron (In ,-.., nnxd . __-, r
-
,
data p resented here suggest that the to7wn pigeon is fully suscepltible to and excretes a Yucaipa-like:virus. This bird could thus be a link in the transmission o~fPMY from tjird to bird and perhaps from bird to man and a survey of town pigeons for paramyxovlruses woula oe worthvvhile.
R. D. J. (1980) Velerinow Bulletin 50.737-752 I, R. A., CORSTVET, R. E. &CLARK, G. T. (1%0)
!
.. .
E
LIC1CrLLS1a-~292-293
DINTER. Z., HERMODS;SON, S. & :HERMODSS;ON, L. (1%A) Virologv 22,297-304 FLEURY, H . J. A. & A LEXANDEI;1, D. J. (19'78) Bulletin I'lnrtilut Parteur 76,175-- ..--- - - .. . . . -. :LEURY, H. J. A. & A L ~ X A N U ~ U. K , J. (1YIY) Avran Ursea
Ackno
--A
?
This work was a 33867 t o H.F.).
23,742-744
y a grant fr.om the CN b*,hI;,."t;,,"
Receivedforp~,,,,,,.,..December20, Accepted ' Februory I ,
ALETA, E. F. & HEEFELS, Wochenschriff 89,31 VINDEVOGEL, H., TIU I D V MEULEMANS, G . (198
u.
....., ,.,
1982
C
(1
he tieriintlic PASTORE'.,T
,.
D
.. D
R e s e u ~ hin Veterinary Science 1983.3.4.377-379
Rela virus
ciency c)f techniques fa,r detecting avian reticuloendotheliosis ccine ca~ntamin ant
R. A. J. NICHOLAS, D. H. 'I nunn I U N
Centro
1
Loborator
ge, Surrey
70). Therefore contamination wi ducks (Witt ial hazard in the use of new isolat REV is inde :ys for the production of Marek of herpesvir the use of turkey, duck or chickc disease vacc embryos or cells tor the propagation of vaccine viruse Since REV is believed to have entered the avian class from mammalian source (Waite and Allen 1975) the hazard fro the substrate applies not only to production of avii vaccines but to mammalian and human vaccines also. TI cletection of REV is hampered because the virus does n c,awe gross effects in either embryos or cell cultures. TI c,omparison of various in vitro test procedures for detectil x
Three tecnn~queswere comparea tor sens~riv~ry In aerecung reticuloendotheliosis virus (REV) in a deliberately contaminated Marek's disease vaccine. The most sensitive and rapid test was the indirect fluorescent antibody test (FAT). The in direct immunoperoxidase test, a lthough sinnple to perforrn and only marginally less sensit]ive than thce FAT, was di fficult to 1interpret at low level!s of REV. Using immun oelectron nnicroscopy, virions we*reseen on1ly after .. and then not to the same level as that three suoculrures detected by the FAT or im munoperox idase test. Serum raised against the HPRS-1 strain of RIEV detecteci other Strains of this virus in the FA1r
.
. .
.
BEFOI
A
..
.
marenam ano mernoos ated and pl aque purifi~
The HPR S-l strain o f REV, is01 . .. on Poultrq Research x a t ~ o n , was used t>y Hought~
.
'
I
" ",,,.,..iercially ,..rn ~ L
available ~ ~ herpesv~rus ~ ~ turkey vaccine. The other REV strains used were duc infectious anaemia, chick syncytial, spleen necrosis virus and REV C and REV F (Purchase et a1 1973). Antiserum was obtained three weeks aftel r r l r c c ~ U I rlightly intramuscular inoculations of specific 1 C:hicks with 10sTCID50 of HPRS-I virus. Primary chick embryo fibroblast s were prepared in plat iontaining glass coversiips and in tissue culturf:flasks. .-,t--:n~rr
c
r
~
~
a
~
1
~
~
~
,
~
H. Thorn
The lndlrect fluorescent antlboay test ( ~ A was I )c out on coverslip cu'ltures as paeviously de:scribed (Nit et al1983). For the indirect irnrnunoperc~xidaset a t coverslip cu --Llr"... G - d m;tI.nr of chick embryo fibruula3rs W G L G &LAr u chrrlrr accrvrlr five minutes at 4OC or in a mixture of periodate, lysine and paraformaldehyde (McLean and Nakane 1974) for 30 minutes at 4OC. The coverslips were incubated with a 1:20 . dilution of HPRS-1 antiserum for 20 minutes at -. room temperature, washed in running phosphate buffered saline and then incubated with a 1:1000 dilution of rabbi1t antichicken immunoglobulin conjugated with pero xidase (Nordic Laboratories) for the same period. After wiashing as before the coverslips were covered with diaminobenzidine solution (0.5 mg/ml activated wit h 0.01 pelr cent hydrogen peroxide) and left 2it room ternperature 1ror 20 , minutes. The coverslips were wasnea and air dried Ibefore mounting in DPX ( BDH). For immune electron micro:scopy, fornnvar coatedl grids were floated on H P RS-1 antiserum dilutec1 1:2 in tris buffer for 15 minutes andI washed fi1ve times in the same tbuffer. . .--.- ..L:-L ,*-A .c .~.on . rne sammes. wr~cclrw r l s l r Grids were then floatea I a minimi lyse-d wit1 ~ b l a s t cells chick embryo fibrl distilled water and stained wit11 2 per cent: phosphotc acid. Freezedried HPRS-1 virus was reconstituted In 5 rnl nf Eagle's minimal essential medium; this was termed ttle 10-1 dilution. Further dilutions of 10-3. 10-5. 10-7 and 10-9 were made and 0 . 2 ml of each dilution was added to 1.8 nnl of a suspension of herpesvirus of turkey containing ap~woximately 1W plaque forming units/ml. After neutrali sation with 2 ml of herpesvirus of turkey antiserum for two hours at 4°C. 0.1 ml of each sample was inoculated on to chick embryo fibroblast cultures. Three days later, co.verslip cultures were take]n for the FAT and innmunopero xidase test andI flask cultures for immune electron micro scopy. The re1naining chlick embryo fibroblast cells in flask furthe1r. flask tryps cultures were inised and dispensed Iinto .. . . .. . and coversl~pcultures, and after three days' lncu bation these were examined as above. This procedure was re peated every three to four days for a total of four passages. To determine whether the other strains of REV could equally be detected by the FAT using the HPRS-III am".r:r r serum, tissue culture fluid containing virus at a low passage level was inoculated on t o coverslip culture and thle FAT was carried out three days later. If the viruses could not be ed . detected, cells were passaged and the FAT was repeat1--
...---
:...
.."&A
S
W
~
. .
-
~
~-
..
~
3.-
.... ".
ncv
3rd passage LndpaIssage 1st passagc! 'AT IPT ItiM FAT IPT IEM FAT IPT IEM
1
~~-
3
.-.-W L I: ~ wrnmnson or recnncques ror me detection ur -
1"
-
ND
ND
+
10 10 10
lo +
Positive Dubious - Negative -. . N[J NO^ aone Fluorescent antibody test 4T FJ T lmmunoperoxidasetest IP' M lmmunoelectronmicrosc:oPY sults of the fourth subcultiIre have been omitted because they ?reidentical to those of the ttiird
+
-
F:
,,
The immu ne electronI microscopy detected REV afte'r thlree passagt!s but not t o the same level as th e FAT. Th e .nons were p~eomorpnicbut generally sphenca~ . witn a ameter of 90 t o 120 Inm. An ou ter layer bearing short )mpact fibre-like projtxtions was just visiblc: in some o~f thle particles. Results of rllG LlvJJ IGaLL.una ~ ~ p e r i m e nrevealed t that fcbur strains of REV, ie, HPRS-1, spleen necrosis, chick sy,ncytial and duck infectious anaemia, were detected by the F,AT using the HPRS-1 antiserum after one passage. REV c was detected after two passages and REV F after a f ulrther passa
. ..
-
1
Results The results of the comparative assessment of techniques for the detection of REV are shown in Table 1. The FAT proved to be the most sensitive and rapid technique, detecting virus from the lo-' dilution after three passages. Preliminary work with the immunoperoxidase test sbowed that periodate, lysine and paraformaldehyde fixatiorI led to a better differentiation between infected and unirifected cultures than did acetone fixation. However, it tjecame increasingly diffic ult t o disti~nguish inf eaed areasi from uninfected areas WIith diminishling levels o f virus. I n c:easing ~ the period of cell culture inclubation dicI not enharice the differentiation.
Discussion The direct and indirect FAT was used to show that REV ill multiply in cell culture by Cook (1969) although Smith et al (1977) reported that low intensity and non-specific st aining frecluently led to inconc:lusive resillts. In thiis :esent stud)r the FAT vvas not onl:y the most sensitive te!it blut results wc:re unequiv~ ocal. The immulnoperoxidase test has Irecently bee:n applied t o . .. . . . . . le aerecr~onof another retrovirus, avlan ~eukosisvlrus, in cc:I1 cultures by Mizuno and Arai (1981). Good differentiati,on was obtained in staining between infected and unin~ f e a e dcultures at the lower dilutions of REV but at the I.: dilutions detection became much less definite and ,,igher more time consuming. Negative contrast electron micros;copy is wid ely used as a c.~f its relati! re speed and means of detecting viru s because . simplicity particularly where the virus has a characteristic r?orphology. REV particles, however, are spherical and not tlistinctive when negatively stained and unless they are grouped together by the use of specific antiserum, as in our study, they are very difficult to detect. Purchase and Witter (1975) stated that viruses in the re~iculoendotheliosisgroup cross react in the FAT. All the vi ruses used in this study were detectable by the FAT using H'PRS-1 antiserum. However, REV C required two subCIlltures and REV F three passages before being detected; ttlis reflected either their inferior growth rate in cell culture 01 r the lower virus content of the initial inoculum or some rn ino or antigenic differences detected by the FAT.
..
.
.
'ios~v i m
Dete
PURCHASE, H. G., LUDFORD, C., NAZERIAN, K. & CO. H. W. (1973) Journal of the National Cancer Institute 5 489-499 PURCHASE, G. WmR, R. L. (1975) Topics
References
DENNETT, D. P- (1979) BAGUST* T. J., GRIMES, T. M. AustmIian VetmmnatyJoumal55.153-157 Microbiology and Immunologv71,103-123 BAINS. B. S. & NIELSEN, 1. I:)..(1979) Promedings of the 28th iARMA, P. S., JAIN, D. K., MISHRA, N. K., VE RNON, M. 1 Western Poultry Disease Con1fncnce and 13th Poultry Health PAUL, P. S. & POMEROY, B. S. (1975) Journal of the Nation Symposium. pp 82-85 Cancer Institute 54,1255-1359 COOK, M. K. (1%9) Journal of the National1 Concer Ins?itute 43. IMITH, E. J., SOLOMON. J. J. & WIT'ITR. R. L. ( I ~ I I Jn v n 203-212 GRIMES, T. M. & PURCHASEi, H. G. (1973) Australian VeterZN, P. T. (IS175) Journal Igf Virology 1 WAITE, Dire- M.21, inary Journal 49,4 66-471 HANSON. J. & HO WELL. J. (11979) Cbnadian Veterinary Journal 872-879 ,-** WITTER, R. L., PUKLHASb, H. CJ.&BUKWYN 20,161-164 Journalof the National Concer Institute 45,567-57 JACKSON. C. A. w.. UUNN. S. E. & SMITH. D. I WASA, N.. YOSHIDA. I. & TANIGUCHI, T. (1976) Nation Australian VeterinaryJournal 53.457-478 Institute of Animal Health Quarterly 16,141-151. McDOUGALL. J. S., BIGGS, P. M. &SHILL EIGEL. R. F., THEILEN. G. H. & TWIEHAUSI, M. J. (1% Avian Pathology 7.557-568 Journal of the National Concerlnstitute37,709-72 9 McLEAN, I. W. & NAKANE, P. K. (1974, , chemirtry and Qtochemirrry 22.1077-1083 MIZUNO, Y. & ARAI, K. (1981) National Institute of A n ~ m a ~ Health Quarterly 21,6367 a ber 23,1982 NICHOLAS. R. A. J.. WOOD. Receivec 'HORNTON , D. H. rpeprea reuruary 17, 1983 (1983)Journal o f Biological Stanaamuatron (In ,-.., nnxd . __-, r
-
,