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The antibody response in sheep infected with a Kenyan sheep and goat pox virus
J
COMP.
PATH,
1978. VOL. 88.
205
THE ANTIBODY WITH A KENYAN
RESPONSE IN SHEEP INFECTED SHEEP AND GOAT POX VIRUS
F.G. Veterinary
DAVIES Research
and C.OTEMA
Laborators,
P.O.
Kabete,
h”eq
INTRODUCTION
The problems associated with serum neutralization tests with sheep or goat pox viruses were outlined by Plowright and Ferris (1958). They used a lambtestis tissue-culture system for assay, in which the neutralization was rarel) complete and the indices obtained were low. Similar observations have been made by Martin (1976) and Davies (unpublished observations). Further, the serum neutralization test has been considered to be unreliable in determining the immune status of an animal (Ramyar and Hessami, 1970). An indirect fluorescent antibody test (FAT) has been used in this laboratory for the latter purpose and to carry out epidemiological investigations; some details of thtb test have already been reported (Davies, 1976). In this study, a serum neutralization test (SNT) has been developed with a bovine-foetal-muscle cell (BFMC) culture for assay and compared with the F.4T. This work has been carried out to study the specific antibody response following experimental infection with the Kenya sheep and goat pox virus (KSGPV) (Davies, 1976). MATERIALS
AND
METHODS
LLxperimental animals. Corriedale-cross wethers of 1 to 2 years of age were shown b) the FAT to be free from antibody to KSGPV. Approximately 10 000 TCID,, of the O--l80 strain of virus were given by subcutaneous injection to a group of 20 sheep. Nine developed generalized infections and were retained for antibody studies for 6 months. Four of these were kept for a further 12 months, a total period of 18 months (5 of the sheep were required for other purposes). Sera were also collected from these 4 sheep, 2 years after inoculation. Sera were collected before inoculation and then at monthly intervals and were stored at -20 “C. A group of 40 yearling Corriedale sheep brought to the laboratory from an area where no KSGPV infection had occurred, were bled on arrival and the sera titrated for antibody by FAT and micro-SNT. Virus strains. A strain of virus O-180, was isolated from a sheep in a mixed flock of sheep and goats, where both species were equally affected. It was passed once in BFMC and freeze dried. This was used for the sheep inoculations. The same strain was used to infect tissue cultures for the FAT and SNT, together with another strain O-240 isolated later in the same epizootic. The stock virus was used at the third to fifth tissue culture passages. Tissue culture. A continuous cell culture was prepared from foetal muscle taken from a bovine foetus of 4 to 5 months gestational age. This had been suggested by Professor E. K. Munz of the Institute for Tropical Medicine, Munich. The cultures remained
206
F. G. DAVIES
AND
C. OTEMA
unaltered in their sensitivity to the KSGPV for nearly 40 subcultures, when some morphological changes were observed. An earlier passage level, which had been stored in liquid nitrogen was thawed and used. The BFMC were grown in Eagles’ minimal essential medium with 5 per cent foetal calf serum; sodium bicarbonate was added to adjust the pH to 7.2. Penicillin and streptomycin sulphate were added at 100 iu and 100 pg per ml respectively. Mycostatin was also used at 100 units per ml. Maintenance media contained 2.5 per cent foetal calf serum. Cells were seeded in bottles and tubes at 300 000 cells per ml of growth medium; approximately 3000 to 4000 cells were added to each well of the microtitre plates. Monolayers were generally confluent within 2 days. Tube cultures were maintained on a roller drum and the microtitre plates were sealed with a transparent tape and kept in a 5 per cent CO, incubator at 35 to 37 “C. The BFMC were used for the SNT. The BHK 21 C 13 cell line (MacPherson and Stoker, 1962) was used to prepare KSGPV antigen for the FAT. This was grown and maintained as described by Davies and Lund (1974). Indirect jluorescent antibody tests (FAT). These were carried out by methods described for African horse sickness by Davies and Lund (1974) and for Nairobi sheep disease (Davies, Jesset and Otieno, 1976). The virus was inoculated on to cultures of BHK cells grown on flying coverslips and harvested when direct staining showed that 15 to 30 per cent of the cells contained antigen. Fixation was in acetone at -20 “C for 10 min and storage at 4 “C. Two-fold dilutions of the sera to be tested were made in phosphate-buffered saline, starting at a dilution of 1 in 5. Two coverslips were tested at each dilution. A 1 in 10 000 dilution of Congo red was applied as a counterstain following the application of the anti-sheep conjugate. An anti-sheep gamma globulin preparation conjugated with fluorescein isothiocyanate was obtained from Nordic Pharmaceuticals, Tilburg, Netherlands. The slides were read on a Zeiss microscope with a halogen iodide bulb and a KP 500 interference filter. The intensity of the specific fluorescence was assessed subjectively on a scale 1 + to 4+ and the end points were where specific intracytoplasmic fluorescence of 3+ and 4+ intensity was readily seen. Serum neutralization tests (S.hfT). A group of 6 sera, 2 pre-inoculation and 4 immune, were used for the standardization of this test. Amethod with constant virus and varying serum was used. Initial experiments compared the use of cultures in tubes, 15-mm wells and microtitre (5 mm) wells. The volumes used in the 3 systems were the same namely, 0.05 ml of serum and 0.05 ml of the virus dilution. The amount of virus for standardization purposes was 100 TCID,,. Virus and serum dilutions were made in the maintenance medium without the serum and doubling dilutions of the sera were made, starting at 1 in 2. Inactivation was carried out at 56 “C for 30 min. The mixtures of virus and serum were held at 37 “C for 2 h. Absorption of virus was for 1 h in tube cultures, but there was no absorption period in the well cultures. Uninfected control cultures were maintained with all tests and the challenge virus was titrated for each series. The serum titre was that dilution completely suppressing a viral cytopathic effect. When the microtitre method was found to be suitable, all further work was carried out with this method alone.
RESULTS
Standardization
of the &VT
Viirus titrations. A cytopathic effect (CPE) appeared late on the second day of culture. After the fifth day there were no further changes in titre; the cultures were kept for 12 days. Similar virus titres were obtained when the dilutions were titrated in tube cultures, in 15-mm wells and in 5-mm wells. Plaque
POX
ANTIBODY
IN
207
SHEEP
counts could be made in the well cultures on the fourth day of culture and secondary plaques appeared by the sixth day when the fluid overlays were used. SAT in tubes, in 15mm wells and in S-mm wells. This was carried out to study the incomplete neutralization which was a characteristic of tests assayed in the iamb testis cultures (Plowright and Ferris, 1958). Cultures in both types 01 well gave similar results, with no evidence of incomplete neutralization. The tube cultures gave lower serum neutralizing indices (1 to 2 log, dilutions) and virus-specific CPE appeared in 2 tubes at the eighth day of culture.. In the microtitre system this was not found with any of the 6 sera tested, nor V,Y~S it seen in subsequent work carried out with many sera. Qtopathic effects in the micro-WT. CPE appeared on the third day of culture and the end-points changed up to the sixth day; the cultures were kept fbr 12 days. Ji’nts challenge. Challenges of 10 TCID,, and of 100 TCID,, were compared. The titres obtained with 100 TCID,, were lower than with 10 TCID,,,, Ijut results were more reproducible. This variability may have been due to difficult? in correctly administering 10 TCID,, to each well. The results of rcp~ated tests with 4 sera and 100 TCID,, are shown in Table 1. The rcproclucibilit\. 01’ rcasults was thought to be within the limits required for such ;I test.
Titres
log,
geometric
Serum Antibody Responses of Sheep to KSGPV
means.
ilssa_yed b_vL\licro-SA‘T
aud l:A T
The peak antibody titres occurred 1 month after infection? as sho~~n in Fig. 1. There was then a gradual fall up to the sixth month, aftrr \
Reciprocal
P
6, I
of serum
dilution
m I
(5x
log21
0 I
I
5. 2 m
Reciprocal
of serum
dilution
(logz)
POX
ANTIBODY
IN
209
SHEEP
No fluorescence was seen in virus-infected coverslips stained with the preinoculation sera: nor in uninfected preparations stained with high titre postinoculation sera. DISCUSSION
The results show that both serological tests may he used to assay antibod) to KSGPV. They are relatively simple. The FAT has been used for man) years and has proved to be reliable; non-specific fluorescence was not seen in the systems used. It should be appreciated that the actual titrcs obtained will \.ary from laboratory to laboratory depending on conjugates, light sources and counterstains. The incomplete neutralization which has been observed in SNT carried out in lamb testis or kidney cells, and the low titres obtained in these tests, ha\~e reduced the value of SNT in work with this virus. In the BFMC microsystem, these difficulties did not occur and the titres obtained with a constant virus method were comparable with those obtained on F.4T. Gispen, Huisman, Brand-Saathof and Hekker (1974) compared the SNT and FAT in sera from people many years after vaccination with vaccinia and found good correlation between the 2 tests. The KSGPV is very closely related to the virus of lumpy skin disease Capstick, 1959) and shows complete cross-reactions on the FAT, and this is whether the KSGPV or lumpy skin virus antigen is used with antisera prepared against either virus. There are also low titre relationships with certain strains of cowpox virus-one strain of cowpox had no relationship with KSGPV or lumpy skin disease (Davies, 1975), while another had. This may he due to the diEercnces in cowpox strains, some of which may be more closely related IO vaccinia than others (Baxby, 1975; Gibbs, 1976), or it may be that different methods of preparation of the rabbit sera had affected the results. The extent of thcsc relationships is currently being studied by these serological tests. SUMMARY
The antibody responses to a Kenya sheep and goat pox virus were studied over an l&month-period by means of an indirect fluorescent antibody test and a micro-serum virus neutralization test. The latter assay was carried out in a continuous cell line prepared from bovine foetal muscle. Both tests proved suitable for work with this disease and avoided the difficulty of incomplete neutralization that has been observed in previously described systems with this \-irus. ACKNOWLEDGMENT
This paper is published Kenya.
by kind permission of the Director
of Veterinary
Services,
REFERENCES
Baxby, D. (1975). Laboratory characteristics of British and Dutch strains of cowpox virus.
210
F. G.
DAVIES
AND
C.
OTEMA
Davies, F. G. (1975). Characteristics of a Kenyan camel pox virus. Journal of Hygiene, 75, 381-385. Davies, F. G. (1976). Characteristics of a virus causing a pox disease in sheep and goats in Kenya, with observations on the epidemiology and control. Journal of Hygiene, 76, 163- 17 1. Davies, F. G., and Lund, L. J. (1974). The application of fluorescent antibody techniques to the virus of African horse sickness. Research in Veterinary Science, 17, 128-130. Davies, F. G., Jesset, D. M., and Otieno, S. (1976). The antibody response of sheep following infection with Nairobi sheep disease virus. Journal of Comparative Pathology, 86, 497-503. Gibbs, E. P. J. (1976). Personal communication. Gispen, R., Huisman, J., Brand-Saathof, B., and Hekker, A. C. (1974). Immunofluorescence test for persistent pox virus antibodies. Archiv fiir die gesamte Virusforschung, 44, 391-395. Macpherson, I. A., and Stoker, M. G. P. (1962). Polyoma transformation of hamster cell clones, an investigation of genetic factors affecting cell competence. Virology, 16, 147-151. Martin, W. (1976). Personal communication. Plowright, W., and Ferris, R. D. (1958). The growth and cytopathogenicity of sheep pox virus in tissue cultures. British Journal of Experimental Pathology, 39, 426-435. Ramyar, H., and Hessami, M. (1970). Studies on the duration of immunity conferred by a live modified sheep pox tissue culture virus vaccine. zentralblatt fiir Veteriniirmedirin, B 17, 869-874. [Received for publication,
May 5th, 19771
COMP.
PATH,
1978. VOL. 88.
205
THE ANTIBODY WITH A KENYAN
RESPONSE IN SHEEP INFECTED SHEEP AND GOAT POX VIRUS
F.G. Veterinary
DAVIES Research
and C.OTEMA
Laborators,
P.O.
Kabete,
h”eq
INTRODUCTION
The problems associated with serum neutralization tests with sheep or goat pox viruses were outlined by Plowright and Ferris (1958). They used a lambtestis tissue-culture system for assay, in which the neutralization was rarel) complete and the indices obtained were low. Similar observations have been made by Martin (1976) and Davies (unpublished observations). Further, the serum neutralization test has been considered to be unreliable in determining the immune status of an animal (Ramyar and Hessami, 1970). An indirect fluorescent antibody test (FAT) has been used in this laboratory for the latter purpose and to carry out epidemiological investigations; some details of thtb test have already been reported (Davies, 1976). In this study, a serum neutralization test (SNT) has been developed with a bovine-foetal-muscle cell (BFMC) culture for assay and compared with the F.4T. This work has been carried out to study the specific antibody response following experimental infection with the Kenya sheep and goat pox virus (KSGPV) (Davies, 1976). MATERIALS
AND
METHODS
LLxperimental animals. Corriedale-cross wethers of 1 to 2 years of age were shown b) the FAT to be free from antibody to KSGPV. Approximately 10 000 TCID,, of the O--l80 strain of virus were given by subcutaneous injection to a group of 20 sheep. Nine developed generalized infections and were retained for antibody studies for 6 months. Four of these were kept for a further 12 months, a total period of 18 months (5 of the sheep were required for other purposes). Sera were also collected from these 4 sheep, 2 years after inoculation. Sera were collected before inoculation and then at monthly intervals and were stored at -20 “C. A group of 40 yearling Corriedale sheep brought to the laboratory from an area where no KSGPV infection had occurred, were bled on arrival and the sera titrated for antibody by FAT and micro-SNT. Virus strains. A strain of virus O-180, was isolated from a sheep in a mixed flock of sheep and goats, where both species were equally affected. It was passed once in BFMC and freeze dried. This was used for the sheep inoculations. The same strain was used to infect tissue cultures for the FAT and SNT, together with another strain O-240 isolated later in the same epizootic. The stock virus was used at the third to fifth tissue culture passages. Tissue culture. A continuous cell culture was prepared from foetal muscle taken from a bovine foetus of 4 to 5 months gestational age. This had been suggested by Professor E. K. Munz of the Institute for Tropical Medicine, Munich. The cultures remained
206
F. G. DAVIES
AND
C. OTEMA
unaltered in their sensitivity to the KSGPV for nearly 40 subcultures, when some morphological changes were observed. An earlier passage level, which had been stored in liquid nitrogen was thawed and used. The BFMC were grown in Eagles’ minimal essential medium with 5 per cent foetal calf serum; sodium bicarbonate was added to adjust the pH to 7.2. Penicillin and streptomycin sulphate were added at 100 iu and 100 pg per ml respectively. Mycostatin was also used at 100 units per ml. Maintenance media contained 2.5 per cent foetal calf serum. Cells were seeded in bottles and tubes at 300 000 cells per ml of growth medium; approximately 3000 to 4000 cells were added to each well of the microtitre plates. Monolayers were generally confluent within 2 days. Tube cultures were maintained on a roller drum and the microtitre plates were sealed with a transparent tape and kept in a 5 per cent CO, incubator at 35 to 37 “C. The BFMC were used for the SNT. The BHK 21 C 13 cell line (MacPherson and Stoker, 1962) was used to prepare KSGPV antigen for the FAT. This was grown and maintained as described by Davies and Lund (1974). Indirect jluorescent antibody tests (FAT). These were carried out by methods described for African horse sickness by Davies and Lund (1974) and for Nairobi sheep disease (Davies, Jesset and Otieno, 1976). The virus was inoculated on to cultures of BHK cells grown on flying coverslips and harvested when direct staining showed that 15 to 30 per cent of the cells contained antigen. Fixation was in acetone at -20 “C for 10 min and storage at 4 “C. Two-fold dilutions of the sera to be tested were made in phosphate-buffered saline, starting at a dilution of 1 in 5. Two coverslips were tested at each dilution. A 1 in 10 000 dilution of Congo red was applied as a counterstain following the application of the anti-sheep conjugate. An anti-sheep gamma globulin preparation conjugated with fluorescein isothiocyanate was obtained from Nordic Pharmaceuticals, Tilburg, Netherlands. The slides were read on a Zeiss microscope with a halogen iodide bulb and a KP 500 interference filter. The intensity of the specific fluorescence was assessed subjectively on a scale 1 + to 4+ and the end points were where specific intracytoplasmic fluorescence of 3+ and 4+ intensity was readily seen. Serum neutralization tests (S.hfT). A group of 6 sera, 2 pre-inoculation and 4 immune, were used for the standardization of this test. Amethod with constant virus and varying serum was used. Initial experiments compared the use of cultures in tubes, 15-mm wells and microtitre (5 mm) wells. The volumes used in the 3 systems were the same namely, 0.05 ml of serum and 0.05 ml of the virus dilution. The amount of virus for standardization purposes was 100 TCID,,. Virus and serum dilutions were made in the maintenance medium without the serum and doubling dilutions of the sera were made, starting at 1 in 2. Inactivation was carried out at 56 “C for 30 min. The mixtures of virus and serum were held at 37 “C for 2 h. Absorption of virus was for 1 h in tube cultures, but there was no absorption period in the well cultures. Uninfected control cultures were maintained with all tests and the challenge virus was titrated for each series. The serum titre was that dilution completely suppressing a viral cytopathic effect. When the microtitre method was found to be suitable, all further work was carried out with this method alone.
RESULTS
Standardization
of the &VT
Viirus titrations. A cytopathic effect (CPE) appeared late on the second day of culture. After the fifth day there were no further changes in titre; the cultures were kept for 12 days. Similar virus titres were obtained when the dilutions were titrated in tube cultures, in 15-mm wells and in 5-mm wells. Plaque
POX
ANTIBODY
IN
207
SHEEP
counts could be made in the well cultures on the fourth day of culture and secondary plaques appeared by the sixth day when the fluid overlays were used. SAT in tubes, in 15mm wells and in S-mm wells. This was carried out to study the incomplete neutralization which was a characteristic of tests assayed in the iamb testis cultures (Plowright and Ferris, 1958). Cultures in both types 01 well gave similar results, with no evidence of incomplete neutralization. The tube cultures gave lower serum neutralizing indices (1 to 2 log, dilutions) and virus-specific CPE appeared in 2 tubes at the eighth day of culture.. In the microtitre system this was not found with any of the 6 sera tested, nor V,Y~S it seen in subsequent work carried out with many sera. Qtopathic effects in the micro-WT. CPE appeared on the third day of culture and the end-points changed up to the sixth day; the cultures were kept fbr 12 days. Ji’nts challenge. Challenges of 10 TCID,, and of 100 TCID,, were compared. The titres obtained with 100 TCID,, were lower than with 10 TCID,,,, Ijut results were more reproducible. This variability may have been due to difficult? in correctly administering 10 TCID,, to each well. The results of rcp~ated tests with 4 sera and 100 TCID,, are shown in Table 1. The rcproclucibilit\. 01’ rcasults was thought to be within the limits required for such ;I test.
Titres
log,
geometric
Serum Antibody Responses of Sheep to KSGPV
means.
ilssa_yed b_vL\licro-SA‘T
aud l:A T
The peak antibody titres occurred 1 month after infection? as sho~~n in Fig. 1. There was then a gradual fall up to the sixth month, aftrr \
Reciprocal
P
6, I
of serum
dilution
m I
(5x
log21
0 I
I
5. 2 m
Reciprocal
of serum
dilution
(logz)
POX
ANTIBODY
IN
209
SHEEP
No fluorescence was seen in virus-infected coverslips stained with the preinoculation sera: nor in uninfected preparations stained with high titre postinoculation sera. DISCUSSION
The results show that both serological tests may he used to assay antibod) to KSGPV. They are relatively simple. The FAT has been used for man) years and has proved to be reliable; non-specific fluorescence was not seen in the systems used. It should be appreciated that the actual titrcs obtained will \.ary from laboratory to laboratory depending on conjugates, light sources and counterstains. The incomplete neutralization which has been observed in SNT carried out in lamb testis or kidney cells, and the low titres obtained in these tests, ha\~e reduced the value of SNT in work with this virus. In the BFMC microsystem, these difficulties did not occur and the titres obtained with a constant virus method were comparable with those obtained on F.4T. Gispen, Huisman, Brand-Saathof and Hekker (1974) compared the SNT and FAT in sera from people many years after vaccination with vaccinia and found good correlation between the 2 tests. The KSGPV is very closely related to the virus of lumpy skin disease Capstick, 1959) and shows complete cross-reactions on the FAT, and this is whether the KSGPV or lumpy skin virus antigen is used with antisera prepared against either virus. There are also low titre relationships with certain strains of cowpox virus-one strain of cowpox had no relationship with KSGPV or lumpy skin disease (Davies, 1975), while another had. This may he due to the diEercnces in cowpox strains, some of which may be more closely related IO vaccinia than others (Baxby, 1975; Gibbs, 1976), or it may be that different methods of preparation of the rabbit sera had affected the results. The extent of thcsc relationships is currently being studied by these serological tests. SUMMARY
The antibody responses to a Kenya sheep and goat pox virus were studied over an l&month-period by means of an indirect fluorescent antibody test and a micro-serum virus neutralization test. The latter assay was carried out in a continuous cell line prepared from bovine foetal muscle. Both tests proved suitable for work with this disease and avoided the difficulty of incomplete neutralization that has been observed in previously described systems with this \-irus. ACKNOWLEDGMENT
This paper is published Kenya.
by kind permission of the Director
of Veterinary
Services,
REFERENCES
Baxby, D. (1975). Laboratory characteristics of British and Dutch strains of cowpox virus.
210
F. G.
DAVIES
AND
C.
OTEMA
Davies, F. G. (1975). Characteristics of a Kenyan camel pox virus. Journal of Hygiene, 75, 381-385. Davies, F. G. (1976). Characteristics of a virus causing a pox disease in sheep and goats in Kenya, with observations on the epidemiology and control. Journal of Hygiene, 76, 163- 17 1. Davies, F. G., and Lund, L. J. (1974). The application of fluorescent antibody techniques to the virus of African horse sickness. Research in Veterinary Science, 17, 128-130. Davies, F. G., Jesset, D. M., and Otieno, S. (1976). The antibody response of sheep following infection with Nairobi sheep disease virus. Journal of Comparative Pathology, 86, 497-503. Gibbs, E. P. J. (1976). Personal communication. Gispen, R., Huisman, J., Brand-Saathof, B., and Hekker, A. C. (1974). Immunofluorescence test for persistent pox virus antibodies. Archiv fiir die gesamte Virusforschung, 44, 391-395. Macpherson, I. A., and Stoker, M. G. P. (1962). Polyoma transformation of hamster cell clones, an investigation of genetic factors affecting cell competence. Virology, 16, 147-151. Martin, W. (1976). Personal communication. Plowright, W., and Ferris, R. D. (1958). The growth and cytopathogenicity of sheep pox virus in tissue cultures. British Journal of Experimental Pathology, 39, 426-435. Ramyar, H., and Hessami, M. (1970). Studies on the duration of immunity conferred by a live modified sheep pox tissue culture virus vaccine. zentralblatt fiir Veteriniirmedirin, B 17, 869-874. [Received for publication,
May 5th, 19771