Failure of an in vitro lymphoproliferative assay specific for bovine herpes virus type 1 to detect immunised or latently infected animals G. H. Wentinkl, V. P. M. G. Rutten2, A. C. A. van ExseP, W. A. C. de Jong2, H. VleugeP, and E. J. Hensen2 SUMMARY. The in vitro lymphoproliferative assay specific for bovine herpes virus type I (BHV1) was tested for its ability to predict whether an animal was protected against challenge with virulent BHV I and for its ability to identify animals latently infected with the virus. Three animals that had been in contact with a field strain of the virus, three that had been vaccinated with a modified live-virus vaccine seven weeks previously, six that had been vaccinated in the same way five months previously, and seven control animals that had had no previous contact with the virus were challenged with virulent BHV1. The 12 animals that had had previous contact with BHV1 all resisted the challenge well or fairly well, but six of them did not react positively in the in vitro lymphoproliferative assay. It was concluded that the assay did not give consistent evidence of the immune status of the animals. Four animals that had had previous contact with a field strain of BHV1 were treated with dexamethasone; they excreted BHV I
irrespective of whether they showed a positive response in the in vitro lymphoproliferative assay. INTRODUCTION
It has been shown that latent bovine herpes virus type 1 (BHV1) can readily become reactivated (3, 11) and that as a result cattle in infected herds may become reinfected at irregular intervals. The mechanisms by which immune cattle resist reinfection with BHV1 include T-cell-mediated cytotoxicity, antibody-dependent
cellular cytotoxicity, antibody-dependent complement-mediated cytolysis and virus neutralisation by circulating antibodies (4, 7, 12, 13, 15). It is agreed that T-cell mediated defence mechanisms are very important in herpes virus infections (12, 13, 15). The BHV1-specific cellular immune response has been demonstrated
in vitro using cells from immunised animals (4, 14). However, the question of whether the lymphoblastic assay, which measures the response of lymphocytes to antigen in vitro, can predict whether an animal is protected in vivo has hardly been considered. A previous study demonstrated that the lymphocytes taken from some animals that had previously, been exposed to BHV1 (either through natural infection or through vaccination) failed to show any blastogenic response to BHV1 (14). It was suggested that those animals whose lymphocytes did respond might be animals that were either better protected against BHV1 or were latently infected with the virus. A similar claim was made by Brochier et al. (1) and Straub (17), who used delayed type hypersensitivity tests and/or lymphoproliferation assays. The present study was undertaken in order to test this hypothesis by investigating
whether in vitro blastogenic responses could either predict the outcome of a challenge infection with a pathogenic field strain of BHV1 or detect animals that I
2
Animal Health Service, Molenwijkseweg 48, P.O. Box 4, 5280 AA Boxtel, The Netherlands. Department of Immunology, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80165, 3508 TD Utrecht, The Netherlands.
THE VETERINARY QUARTERLY, VOL. 12, No 3, JULY 1990
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were latently infected with the virus. Latency was tested by treating animals that had been previously exposed to a field strain of BHV1 with corticosteroids (2, 11). MATERIALS AND METHODS
Experimental design
Protection against a challenge with a pathogenic field strain of BHVI was evaluated (a) in cows that were serologically positive from BHV1 infected herds without vaccination history, (b) in cows that had been infected with a pathogenic field strain previously, and (c) in cows at various times after vaccination. In all, 19 animals were submitted to a challenge infection. Three animals (the field virus group) were serologically positive without vaccination history; three cows were challenged seven weeks and six five months after vaccination. Seven animals (the control group) were serologically negative when they were challenged. Two control animals were reinfected three and a half months after the first challenge.
Latency for BHV1 was tested in four cows, serologically positive for BHVI without vaccination history, coming from the same BHV1-infected herds as the cows mentioned above under a.
These animals were treated with corticosteroids to induce reactivation; they were subsequently monitored for the excretion of BHV1. Experimental animals
Twenty-three female animals of Maas-Rijn-IJssel and Friesian breeds, ranging in age from 20 months to 7 years, were used. All animals were purchased from commercial dairy farms after previous investigations of their immune status towards BHV I.
After vaccination, nine animals were kept in BHV1-free environments, monitored by serological control of negative contact animals, until challenges. Seven conventionally reared heifers, free of antibodies to BHV I, were purchased from farms free of BHVI as a control group for the challenge. Seven cows that were serologically positive for BHV1 without vaccination history were subdivided in accordance with their responses in the in vitro lymphoproliferative assay and allocated as follows: three of them, two of which had positive stimulation indices in the assays and one of which did not respond, were challenged with BHV1; the other four, two with positive stimulation indices and two which did not respond in the assay, were treated with corticosteroids to induce reactivation of latent infections of BHV1. Virus for the in vitro assays
The BHV1 strain from the Central Veterinary Institute (Lelystad, The Netherlands) was grown on a MDBK cell-line and used for all the serological tests and for the in vitro lymphocyte reactivity tests. Live virus was used to avoid any antigenic changes which might have been induced by the process of inactivation (6). Challenge virus
A pathogenic field strain of BHVI (19, 20) was used'. It was grown on bovine foetal tracheal cells, and the inoculum contained 106 TCID50/ml. Vaccine viruses
Three commercially available modified live-virus vaccines were used according to the manufacturers' instructions. A temperature-sensitive strain (RLB 106)2 (4, 8, 21) and avirulent strain3 (8) were administered intranasally. An avirulent Japanese strain of BHV14 (5) in an oil in water emulsion was administered intramuscularly. I
2 3
4
Made available by Dr. G. Wellemans of the INRV, Brussels, Belgium. Smith Kline, Zoetermeer, The Netherlands. Beecham. Amstelveen, The Netherlands. Duphar, Weesp, The Netherlands, experimental batch.
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BHV1 was isolated from a nasal swab taken from animal 17 two days after challenge, but not from animals 18 and 19. Only animal 17 showed a fourfold increase in antibody titre, and only animal 19 showed a significant increase in the SI. Challenge seven weeks after vaccination (animals 8, 9 and 10)
These animals showed only moderate clinical signs; the temperature of animal 10 increased above 40° C for one day. BHV1 was isolated for at least five days after the challenge from nasal swabs taken from all three animals, and they all showed clear serological responses and significant increases in lymphocyte reactivity (SI) to BHV1. Challenge five months after vaccination (animals 11, 12, 13, 14, 15 and 16) This group showed mild (11, 13, 14) or moderate (12, 15, 16) clinical signs of IBR; animal 12 had a temperature above 40° C for one day, and animals 15 and 16 severe necrotising foci in their nostrils. BHV1 was isolated from animals 11, 13 and 16 for at least five days, but it was
not isolated from 14. Animal 14 showed only a slight serological response and no increase in its SI for BHV1, whereas the other animals in this group showed clear increases in serological titre against BHV1 and significant increases in lymphocyte reactivity against BHV1. Reactivation of BHVI with corticosteroids (animals 20, 21, 22 and 23) The results are summarised in Table 2. Table 2. Results of repeated lymphocyte stimulation tests (cpm) and the calculated SIs during the period of two months before (individual tests at least 14 days apart) and the first day of BHV I excretion
from the nostrils and the clinical signs (see Table 1) after treatment with dexamethasone and prednisolone. Animal
cpm in BHV1 stimulated cells cpm in control cells SI
20 1544
22
21
435
2514
311
1506
189
3681
2372
982
2427
923
3294
613
1352
0,7
0,5
1,0
0,3
0,5
0,3
2,7
23
1234 785
1,6
22872
2416
23586
1190
1427
898
3660
1,8
16,0
2,7
6,4
2086
Isolation of BHV1
on day .... after start
5
5
4
4
0
0
0
0
+
+
+
+ +
of treatment
Days temperature > 40% Reddening of mucosa Necrotizing foci on nasal mucosa
+
+
.\
Remarks
Abortion: twins
BHV1 positive
none
none
Abortion BHV1 negative
In the period of two months before the immunosuppressive treatment began the lymphocyte reactivity of the animals with initial SIs > 1.5 (22 and 23) and SIs ...c. 1.5 (20 and 21) fluctuated, but they remained either positive or negative. The treatment with corticosteroids did not induce clinical signs of BHV1 infection in any of the animals, but BHV1 could be isolated from the nostrils of all of them four or five days after the treatment again. The two pregnant cows aborted, and BHV1 was isolated from one foetus delivered by cow 20. DISCUSSION
In a previous paper (14) the lymphoproliferative assay for BHV1 was discussed. In that study it was shown that 29 animals serologically negative for BHV1 from THE VETERINARY QUARTERLY, VOL. 12, No. 3, JULY 1990
177
.
BHV1-free herds always had SIs 1.5 in an in vitro lymphoproliferative assay specific for BHV1. Serologically positive animals either had SIs 1.5 (19 animals) or > 1.5 (17 animals). The results were reproducible for animals tested two or more times. Live virus was used as antigen to circumvent problems with alteration of virus antigen composition by inactivation procedures (6). The principal aim of the present investigation was to determine whether animals
that were serologically positive for BHV1, but nonresponding in in vitro lymphoproliferative assays (negative SIs) were less well protected against a
challenge with virulent virus than animals with a positive response in the assay (positive SIs). Before challenge, five of nine previously vaccinated animals (animals 8, 9, 11, 15 and 16) one of the three animals previously exposed to a field strain of the virus (animal 18), and both the control animals that were challenged for a second time (animals 4 and 5) had SIs 1.5. -
irrespective of whether they showed a positive response in the in vitro lymphoproliferative assay. INTRODUCTION
It has been shown that latent bovine herpes virus type 1 (BHV1) can readily become reactivated (3, 11) and that as a result cattle in infected herds may become reinfected at irregular intervals. The mechanisms by which immune cattle resist reinfection with BHV1 include T-cell-mediated cytotoxicity, antibody-dependent
cellular cytotoxicity, antibody-dependent complement-mediated cytolysis and virus neutralisation by circulating antibodies (4, 7, 12, 13, 15). It is agreed that T-cell mediated defence mechanisms are very important in herpes virus infections (12, 13, 15). The BHV1-specific cellular immune response has been demonstrated
in vitro using cells from immunised animals (4, 14). However, the question of whether the lymphoblastic assay, which measures the response of lymphocytes to antigen in vitro, can predict whether an animal is protected in vivo has hardly been considered. A previous study demonstrated that the lymphocytes taken from some animals that had previously, been exposed to BHV1 (either through natural infection or through vaccination) failed to show any blastogenic response to BHV1 (14). It was suggested that those animals whose lymphocytes did respond might be animals that were either better protected against BHV1 or were latently infected with the virus. A similar claim was made by Brochier et al. (1) and Straub (17), who used delayed type hypersensitivity tests and/or lymphoproliferation assays. The present study was undertaken in order to test this hypothesis by investigating
whether in vitro blastogenic responses could either predict the outcome of a challenge infection with a pathogenic field strain of BHV1 or detect animals that I
2
Animal Health Service, Molenwijkseweg 48, P.O. Box 4, 5280 AA Boxtel, The Netherlands. Department of Immunology, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80165, 3508 TD Utrecht, The Netherlands.
THE VETERINARY QUARTERLY, VOL. 12, No 3, JULY 1990
175
.
were latently infected with the virus. Latency was tested by treating animals that had been previously exposed to a field strain of BHV1 with corticosteroids (2, 11). MATERIALS AND METHODS
Experimental design
Protection against a challenge with a pathogenic field strain of BHVI was evaluated (a) in cows that were serologically positive from BHV1 infected herds without vaccination history, (b) in cows that had been infected with a pathogenic field strain previously, and (c) in cows at various times after vaccination. In all, 19 animals were submitted to a challenge infection. Three animals (the field virus group) were serologically positive without vaccination history; three cows were challenged seven weeks and six five months after vaccination. Seven animals (the control group) were serologically negative when they were challenged. Two control animals were reinfected three and a half months after the first challenge.
Latency for BHV1 was tested in four cows, serologically positive for BHVI without vaccination history, coming from the same BHV1-infected herds as the cows mentioned above under a.
These animals were treated with corticosteroids to induce reactivation; they were subsequently monitored for the excretion of BHV1. Experimental animals
Twenty-three female animals of Maas-Rijn-IJssel and Friesian breeds, ranging in age from 20 months to 7 years, were used. All animals were purchased from commercial dairy farms after previous investigations of their immune status towards BHV I.
After vaccination, nine animals were kept in BHV1-free environments, monitored by serological control of negative contact animals, until challenges. Seven conventionally reared heifers, free of antibodies to BHV I, were purchased from farms free of BHVI as a control group for the challenge. Seven cows that were serologically positive for BHV1 without vaccination history were subdivided in accordance with their responses in the in vitro lymphoproliferative assay and allocated as follows: three of them, two of which had positive stimulation indices in the assays and one of which did not respond, were challenged with BHV1; the other four, two with positive stimulation indices and two which did not respond in the assay, were treated with corticosteroids to induce reactivation of latent infections of BHV1. Virus for the in vitro assays
The BHV1 strain from the Central Veterinary Institute (Lelystad, The Netherlands) was grown on a MDBK cell-line and used for all the serological tests and for the in vitro lymphocyte reactivity tests. Live virus was used to avoid any antigenic changes which might have been induced by the process of inactivation (6). Challenge virus
A pathogenic field strain of BHVI (19, 20) was used'. It was grown on bovine foetal tracheal cells, and the inoculum contained 106 TCID50/ml. Vaccine viruses
Three commercially available modified live-virus vaccines were used according to the manufacturers' instructions. A temperature-sensitive strain (RLB 106)2 (4, 8, 21) and avirulent strain3 (8) were administered intranasally. An avirulent Japanese strain of BHV14 (5) in an oil in water emulsion was administered intramuscularly. I
2 3
4
Made available by Dr. G. Wellemans of the INRV, Brussels, Belgium. Smith Kline, Zoetermeer, The Netherlands. Beecham. Amstelveen, The Netherlands. Duphar, Weesp, The Netherlands, experimental batch.
176
THE VETERINARY QUARTERLY. VOL. 12, No. 3, JULY 1990
BHV1 was isolated from a nasal swab taken from animal 17 two days after challenge, but not from animals 18 and 19. Only animal 17 showed a fourfold increase in antibody titre, and only animal 19 showed a significant increase in the SI. Challenge seven weeks after vaccination (animals 8, 9 and 10)
These animals showed only moderate clinical signs; the temperature of animal 10 increased above 40° C for one day. BHV1 was isolated for at least five days after the challenge from nasal swabs taken from all three animals, and they all showed clear serological responses and significant increases in lymphocyte reactivity (SI) to BHV1. Challenge five months after vaccination (animals 11, 12, 13, 14, 15 and 16) This group showed mild (11, 13, 14) or moderate (12, 15, 16) clinical signs of IBR; animal 12 had a temperature above 40° C for one day, and animals 15 and 16 severe necrotising foci in their nostrils. BHV1 was isolated from animals 11, 13 and 16 for at least five days, but it was
not isolated from 14. Animal 14 showed only a slight serological response and no increase in its SI for BHV1, whereas the other animals in this group showed clear increases in serological titre against BHV1 and significant increases in lymphocyte reactivity against BHV1. Reactivation of BHVI with corticosteroids (animals 20, 21, 22 and 23) The results are summarised in Table 2. Table 2. Results of repeated lymphocyte stimulation tests (cpm) and the calculated SIs during the period of two months before (individual tests at least 14 days apart) and the first day of BHV I excretion
from the nostrils and the clinical signs (see Table 1) after treatment with dexamethasone and prednisolone. Animal
cpm in BHV1 stimulated cells cpm in control cells SI
20 1544
22
21
435
2514
311
1506
189
3681
2372
982
2427
923
3294
613
1352
0,7
0,5
1,0
0,3
0,5
0,3
2,7
23
1234 785
1,6
22872
2416
23586
1190
1427
898
3660
1,8
16,0
2,7
6,4
2086
Isolation of BHV1
on day .... after start
5
5
4
4
0
0
0
0
+
+
+
+ +
of treatment
Days temperature > 40% Reddening of mucosa Necrotizing foci on nasal mucosa
+
+
.\
Remarks
Abortion: twins
BHV1 positive
none
none
Abortion BHV1 negative
In the period of two months before the immunosuppressive treatment began the lymphocyte reactivity of the animals with initial SIs > 1.5 (22 and 23) and SIs ...c. 1.5 (20 and 21) fluctuated, but they remained either positive or negative. The treatment with corticosteroids did not induce clinical signs of BHV1 infection in any of the animals, but BHV1 could be isolated from the nostrils of all of them four or five days after the treatment again. The two pregnant cows aborted, and BHV1 was isolated from one foetus delivered by cow 20. DISCUSSION
In a previous paper (14) the lymphoproliferative assay for BHV1 was discussed. In that study it was shown that 29 animals serologically negative for BHV1 from THE VETERINARY QUARTERLY, VOL. 12, No. 3, JULY 1990
177
.
BHV1-free herds always had SIs 1.5 in an in vitro lymphoproliferative assay specific for BHV1. Serologically positive animals either had SIs 1.5 (19 animals) or > 1.5 (17 animals). The results were reproducible for animals tested two or more times. Live virus was used as antigen to circumvent problems with alteration of virus antigen composition by inactivation procedures (6). The principal aim of the present investigation was to determine whether animals
that were serologically positive for BHV1, but nonresponding in in vitro lymphoproliferative assays (negative SIs) were less well protected against a
challenge with virulent virus than animals with a positive response in the assay (positive SIs). Before challenge, five of nine previously vaccinated animals (animals 8, 9, 11, 15 and 16) one of the three animals previously exposed to a field strain of the virus (animal 18), and both the control animals that were challenged for a second time (animals 4 and 5) had SIs 1.5. -
