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The development of delayed cutaneous hypersensitivity in rabbits infected with the herpesvirus of malignant catarrhal fever
Comp. Immun. Mi~robiol. infect. Dis.. Vol. 4, No. 1, pp. 29 34, 1981. Printed in Great Britain.
0147 9571/81/0100294)6 $02.00/0 i', 1981 Pergamon Press Ltd.
THE D E V E L O P M E N T OF D E L A Y E D C U T A N E O U S H Y P E R S E N S I T I V I T Y IN RABBITS I N F E C T E D WITH THE H E R P E S V I R U S OF MALIGNANT CATARRHAL FEVER E. Z. MUSH! a n d F. R. RURANGIRWA Veterinary Research Department, Kenya Agricultural Research Institute, P.O. Box 32, Kikuyu, Kenya
Abstract--Delayed-type hypersensitivity (DTH) was demonstrated in malignant catarrhal fever (MCF) virus-infected rabbits intradermally injected with homologous viral antigens. The response was first detected four days post-infection (pi) and was maximal on day 7 pi. DTH could not be demonstrated alter the onset of pyrexia. The abrogation of DTH in MCF virusinfected rabbits is discussed in relation to the pathogenesis of the disease. K~v words." Malignant catarrhal fever, delayed cutaneous hypersensitivity
LE D E V E L O P P E M E N T D E S R E A C T I O N S D ' H Y P E R S E N S I B I L I T E R E T A R D E E C U T A N E E C H E Z LES L A P I N S I N F E C T E S A V E C L ' H E R P E S V I R U S D E LA F I E V R E C A T A R R H A L E M A L I G N E Rosum~Une hypersensibiliteretardee (DTH) a ete demontree chez les lapins infectes par le virus de la Fi6vre catarrhale maligne (MCF) par injection intradermique d'antig+nes viraux homologue. La reponse a d'abord ere detectee quatre jours apr~s l'infection eta ere maximale au 7+mejour de post-infection (pi). La DTH n'a pu 6tre dsmontree apr6s le pic de temperature. La disparition de la DTH chez les lapins infectes par le virus MCF est discutee en fonction de la pathog+nie de la maladie. Mot.s-cle/~s." Fi6vre catarrhale maligne, hypersensibilite retardee
INTRODUCTION The herpesvirus of bovine m a l i g n a n t catarrhal fever ( M C F ) induces a highly fatal lymphoproliferative disease in cattle a n d rabbits [1 4]. D u r i n g M C F virus infection, antibodies a p p e a r a n d have been detected by virus neutralizing a n d i m m u n o f l u o r e s c e n t tests [5]. The role of cellular i m m u n e response to M C F virus antigens in infected a n i m a l s has not been fully studied. Delayed-type hypersensitivity ( D T H ) has however been d e m o n s t r a t e d in most viral infections including M a r e k ' s disease ( M D ) virus [6]. W e report on the d e v e l o p m e n t of D T H in M C F virus-infected rabbits i n t r a d e r m a l l y inoculated with h o m o l o g o u s viral antigens. 29
30
E . Z . Musnl and F. R. RURANGIRWA
MATERIALS AND METHODS Rabbits
Adult New Zealand White rabbits weighing 2 kg and of either sex were used. Each rabbit was intraperitoneally inoculated with 2 ml of 10~',~, MCF virus-infected rabbit spleen suspension. The inoculum which had an infectivity of 102.8 TCIDs0/ml was obtained from a rabbit inoculated with MCF virus isolated from wildebeest calf secretions. Skin test antigen
The viral skin test antigen was prepared in primary calf kidney monolayers infected with the cell-free strain of MCF virus [7]. When the infected monolayers had about 75°/,, cytopathic effects (cpe), the medium was decanted and the cells were scraped and sonicated in an ultrasonic transducer* at a frequency of 41.23 kc/s for 30 s. The sonicated infected cell debris had an infectivity of 106'° TCID/ml and was heat-inactivated at 56'C for 30 min and stored at - 20 C. The cell control antigen was prepared in the same manner from noninfected calf kidney cell cultures. Skin test procedure
The skin test was performed by injecting 0.1 ml of the antigen intradermally into clipped skin on thoracic wall of rabbits. The thickness of the skin was measured with tuberculin testing callipers before injection and at 15 min 6, 24, and 48 hr after injection. This skin test was performed on days 0, 4, 7, 11, 14, 16 and 18 post-infection (pi). The increase in skin thickness was the skin thickness at the site injected with viral antigen minus the thickness of normal skin. Serum was collected twice a week and assayed for virus neutralizing antibodies in microtitre plates as previously described [8]. Pyrexia
x
1.5"
o
.1.5
/
E
/
E
~
/ l.0-
X
X'
X
/
I
8
I
-1.0~_
/
z
,0-
--'5-
-
-
-
tl
-.5
== s" / o
x 2O
DAYS POST INFECTION
Fig. 2. Development of DTH and virus-neutralizing antibodies in MCF virus infected rabbits, x Increase in skin thickness; • • virus-neutralizing antibodies. * Dawe Instruments, London, U.K.
Fig. 1. Cutaneous D T H response 48 hr after intradermal injection of heat-inactivated M C F virus antigens (seven days post-infection). WCII = Cell-free strain of M C F virus; N B K = normal bovine kidney cells.
Fig. 3. Histopathological changes in epidermis of M C F virus-infected rabbits 48 hr after intradermal injection of heat-inactivated M C F virus antigens. H. & E. stain x 100. 31
Delayed cutaneous hypersensitivityin rabbits
33
RESULTS Skin reactions
Circumscribed, indurated skin nodules appeared in infected rabbits at the sites inoculated with MCF virus antigens but not at the sites inoculated with uninfected cell culture antigens (Fig. 1). Two rabbits previously inoculated with 10°~i normal rabbit spleen suspension did not develop any skin nodules at the sites injected with MCF virus antigen. The DTH skin nodules were detected 24 hr post-injection and reached maximum size by 48 hr. This reaction was first detected in rabbits tested after four days of infection with MCFV. Maximum response was demonstrated in rabbits tested seven days post-MCFV infection but the nodules were smaller in animals tested there after (Fig. 2). This D T H reaction was not elicited in three rabbits tested after the onset of pyrexia. Histopathological examination of skin nodules taken 48 hr after injection showed mononuclear cell infiltration of the dermis and subcutis (Fig. 3). Immediate hypersensitivity [9] was not demonstrated at any stage of MCF virus infection. Virus-neutralizing antibodies
Virus-neutralizing antibodies to MCF virus were first detected on day 10 pi and the titres continued to rise until death (Fig. 2). DISCUSSION Delayed-type hypersensitivity is a classical correlate of CM1 and is important for recovery from herpesvirus infections [10]. The herpesvirus of MCF is strictly cell-associated in rabbits and cattle as the virus could not be detected cell-free in the tissues of these animals [4], MCF virus-infected cattle and rabbits die although the animals produce virusneutralizing antibodies [5]. All these are pointers to the importance of CMI in MCFinfected cattle and rabbits. We have demonstrated DTH in MCF virus-infected rabbits intradermally injected with homologous viral antigens. This response was completely suppressed in rabbits tested after the onset of clinical disease. Thus our results corroborate those of Wilks and Rossiter [1 1] who showed that in vitro lymphocyte response to phytohemagglutinin in MCF virusinfected rabbits dropped precipitously from the day of disease onset and remained low until death. The suppression of D T H during the clinical course of MCF is indicative of T-cell involvement. Indeed, histopathological examination has revealed focal necrosis within the paracortical areas of lymph nodes and destruction of the cortical areas in the thymus [12, 13]. It is possible, therefore, that the suppression/abrogation of D T H / C M I in MCFinfected rabbits could account for the very high mortality rate in this disease. In Marek's disease virus infection of chickens, another ceil-associated herpesvirus, T cells are involved leading to deficiency in cellular and humoral immune responses [14]. MCF, like MD, is also associated with proliferation of lymphoid cells [3, 12, 13] but whether they are T or B cells remains to be established. 2
34
E.Z. SUSHI and F. R. RURANGIRWA
Acknowledgement---This paper was published with the permission of the Director. Veterinary Research
Department, Kenya Agricultural Research Institute.
REFERENCES 1. Daubney, R. and Hudson, J. R., Transmission experiments with bovine malignant catarrhal, J. comp. Path. Ther. 49, 63 89 (1936). 2. Mettam, R. W. M., Snotsiekte in cattle, 9th and lOth Reps Dir. Vet. Educ. Res. Union ~[ South A[rica, pp. 395 432 (1923). 3. Piercy. S. E., Studies on bovine malignant catarrhal fever. VI. Adaptation to rabbits, Br. vet. J. |II, 484- 491 (1955). 4. Plowright, W., Studies of malignant catarrhal lever of cattle. D.V.Sc. Thesis University of Pretoria, South Africa. (1964). 5. Rossiter, P. B., Mushi. E. Z. and Plowright, W., The development of antibodies in rabbits and cattle infected experimentally with an African strain of malignant catarrhal fever virus, Vet. Microbiol. 2, 57 66 (1977). 6. Byerly, J. k. and Dawe, D. L., Delayed hypersensitivity reactions in Marek's disease virus-infected chickens, Am. J. vet. Res, 33, 2267 2273 (1972). 7. Plowright, W., Macadam, R. F. and Armstrong, J. S., Growth and characterisation of the virus of bovine malignant catarrhal fever in East Africa, J. gen. Microbiol. 39, 252 266 (1965). 8. Mushi, E. Z. and Plowright, W., Microtitre technique for the assay of malignant catarrhal fever virus and neutralising antibody, Res. vet. Sci. 27, 230 232 (1979). 9. Coombs, R. R. A. and Gell, P. G. H., Classification of allergic reactions responsible for clinical hypersensitivity and disease. In Clinical A.~pects ¢~l'Immunology, P. G. H. Gell, R. R. A. Coombs and P. J. Lachmann (Eds), pp. 761 778. Blackwell Scientific Publications, Oxtbrd (1975). 10. Allison, A. C., Interactions of antibody, complement components and various cell types in immunity against virus diseases and pyogenic bacteria, Tramplanm. Rev. 19, 3 55 (1974). I 1. Wilks, C. R. and Rossiter, P. B,, An immunosuppressive factor in serum of rabbits lethally infected with the herpesvirus of bovine malignant catarrhal fever, J. inlect. Dis. 137, 403 409 (1978). 12. Edington, N., Patel, J., Russell, P. H. and Plowright, W., The nature of the acute lymphoid proliferation in rabbits infected with the herpes virus of bovine malignant catarrhal fever, Eur. J. Cancer 15, 151 .%1522 (1980). 13. Selman, 1. E., Wiseman, A., Murray, M. and Wright, N. G., A clinico-pathological study of bovine malignant catarrhal fever in Great Britain, Vet. Rec. 94, 483 490 (1974). 14. Purchase, H. G., Marek's disease virus and the herpesvirus of turkeys, Prog. med. Virol. 18, 178 197 (1974).
0147 9571/81/0100294)6 $02.00/0 i', 1981 Pergamon Press Ltd.
THE D E V E L O P M E N T OF D E L A Y E D C U T A N E O U S H Y P E R S E N S I T I V I T Y IN RABBITS I N F E C T E D WITH THE H E R P E S V I R U S OF MALIGNANT CATARRHAL FEVER E. Z. MUSH! a n d F. R. RURANGIRWA Veterinary Research Department, Kenya Agricultural Research Institute, P.O. Box 32, Kikuyu, Kenya
Abstract--Delayed-type hypersensitivity (DTH) was demonstrated in malignant catarrhal fever (MCF) virus-infected rabbits intradermally injected with homologous viral antigens. The response was first detected four days post-infection (pi) and was maximal on day 7 pi. DTH could not be demonstrated alter the onset of pyrexia. The abrogation of DTH in MCF virusinfected rabbits is discussed in relation to the pathogenesis of the disease. K~v words." Malignant catarrhal fever, delayed cutaneous hypersensitivity
LE D E V E L O P P E M E N T D E S R E A C T I O N S D ' H Y P E R S E N S I B I L I T E R E T A R D E E C U T A N E E C H E Z LES L A P I N S I N F E C T E S A V E C L ' H E R P E S V I R U S D E LA F I E V R E C A T A R R H A L E M A L I G N E Rosum~Une hypersensibiliteretardee (DTH) a ete demontree chez les lapins infectes par le virus de la Fi6vre catarrhale maligne (MCF) par injection intradermique d'antig+nes viraux homologue. La reponse a d'abord ere detectee quatre jours apr~s l'infection eta ere maximale au 7+mejour de post-infection (pi). La DTH n'a pu 6tre dsmontree apr6s le pic de temperature. La disparition de la DTH chez les lapins infectes par le virus MCF est discutee en fonction de la pathog+nie de la maladie. Mot.s-cle/~s." Fi6vre catarrhale maligne, hypersensibilite retardee
INTRODUCTION The herpesvirus of bovine m a l i g n a n t catarrhal fever ( M C F ) induces a highly fatal lymphoproliferative disease in cattle a n d rabbits [1 4]. D u r i n g M C F virus infection, antibodies a p p e a r a n d have been detected by virus neutralizing a n d i m m u n o f l u o r e s c e n t tests [5]. The role of cellular i m m u n e response to M C F virus antigens in infected a n i m a l s has not been fully studied. Delayed-type hypersensitivity ( D T H ) has however been d e m o n s t r a t e d in most viral infections including M a r e k ' s disease ( M D ) virus [6]. W e report on the d e v e l o p m e n t of D T H in M C F virus-infected rabbits i n t r a d e r m a l l y inoculated with h o m o l o g o u s viral antigens. 29
30
E . Z . Musnl and F. R. RURANGIRWA
MATERIALS AND METHODS Rabbits
Adult New Zealand White rabbits weighing 2 kg and of either sex were used. Each rabbit was intraperitoneally inoculated with 2 ml of 10~',~, MCF virus-infected rabbit spleen suspension. The inoculum which had an infectivity of 102.8 TCIDs0/ml was obtained from a rabbit inoculated with MCF virus isolated from wildebeest calf secretions. Skin test antigen
The viral skin test antigen was prepared in primary calf kidney monolayers infected with the cell-free strain of MCF virus [7]. When the infected monolayers had about 75°/,, cytopathic effects (cpe), the medium was decanted and the cells were scraped and sonicated in an ultrasonic transducer* at a frequency of 41.23 kc/s for 30 s. The sonicated infected cell debris had an infectivity of 106'° TCID/ml and was heat-inactivated at 56'C for 30 min and stored at - 20 C. The cell control antigen was prepared in the same manner from noninfected calf kidney cell cultures. Skin test procedure
The skin test was performed by injecting 0.1 ml of the antigen intradermally into clipped skin on thoracic wall of rabbits. The thickness of the skin was measured with tuberculin testing callipers before injection and at 15 min 6, 24, and 48 hr after injection. This skin test was performed on days 0, 4, 7, 11, 14, 16 and 18 post-infection (pi). The increase in skin thickness was the skin thickness at the site injected with viral antigen minus the thickness of normal skin. Serum was collected twice a week and assayed for virus neutralizing antibodies in microtitre plates as previously described [8]. Pyrexia
x
1.5"
o
.1.5
/
E
/
E
~
/ l.0-
X
X'
X
/
I
8
I
-1.0~_
/
z
,0-
--'5-
-
-
-
tl
-.5
== s" / o
x 2O
DAYS POST INFECTION
Fig. 2. Development of DTH and virus-neutralizing antibodies in MCF virus infected rabbits, x Increase in skin thickness; • • virus-neutralizing antibodies. * Dawe Instruments, London, U.K.
Fig. 1. Cutaneous D T H response 48 hr after intradermal injection of heat-inactivated M C F virus antigens (seven days post-infection). WCII = Cell-free strain of M C F virus; N B K = normal bovine kidney cells.
Fig. 3. Histopathological changes in epidermis of M C F virus-infected rabbits 48 hr after intradermal injection of heat-inactivated M C F virus antigens. H. & E. stain x 100. 31
Delayed cutaneous hypersensitivityin rabbits
33
RESULTS Skin reactions
Circumscribed, indurated skin nodules appeared in infected rabbits at the sites inoculated with MCF virus antigens but not at the sites inoculated with uninfected cell culture antigens (Fig. 1). Two rabbits previously inoculated with 10°~i normal rabbit spleen suspension did not develop any skin nodules at the sites injected with MCF virus antigen. The DTH skin nodules were detected 24 hr post-injection and reached maximum size by 48 hr. This reaction was first detected in rabbits tested after four days of infection with MCFV. Maximum response was demonstrated in rabbits tested seven days post-MCFV infection but the nodules were smaller in animals tested there after (Fig. 2). This D T H reaction was not elicited in three rabbits tested after the onset of pyrexia. Histopathological examination of skin nodules taken 48 hr after injection showed mononuclear cell infiltration of the dermis and subcutis (Fig. 3). Immediate hypersensitivity [9] was not demonstrated at any stage of MCF virus infection. Virus-neutralizing antibodies
Virus-neutralizing antibodies to MCF virus were first detected on day 10 pi and the titres continued to rise until death (Fig. 2). DISCUSSION Delayed-type hypersensitivity is a classical correlate of CM1 and is important for recovery from herpesvirus infections [10]. The herpesvirus of MCF is strictly cell-associated in rabbits and cattle as the virus could not be detected cell-free in the tissues of these animals [4], MCF virus-infected cattle and rabbits die although the animals produce virusneutralizing antibodies [5]. All these are pointers to the importance of CMI in MCFinfected cattle and rabbits. We have demonstrated DTH in MCF virus-infected rabbits intradermally injected with homologous viral antigens. This response was completely suppressed in rabbits tested after the onset of clinical disease. Thus our results corroborate those of Wilks and Rossiter [1 1] who showed that in vitro lymphocyte response to phytohemagglutinin in MCF virusinfected rabbits dropped precipitously from the day of disease onset and remained low until death. The suppression of D T H during the clinical course of MCF is indicative of T-cell involvement. Indeed, histopathological examination has revealed focal necrosis within the paracortical areas of lymph nodes and destruction of the cortical areas in the thymus [12, 13]. It is possible, therefore, that the suppression/abrogation of D T H / C M I in MCFinfected rabbits could account for the very high mortality rate in this disease. In Marek's disease virus infection of chickens, another ceil-associated herpesvirus, T cells are involved leading to deficiency in cellular and humoral immune responses [14]. MCF, like MD, is also associated with proliferation of lymphoid cells [3, 12, 13] but whether they are T or B cells remains to be established. 2
34
E.Z. SUSHI and F. R. RURANGIRWA
Acknowledgement---This paper was published with the permission of the Director. Veterinary Research
Department, Kenya Agricultural Research Institute.
REFERENCES 1. Daubney, R. and Hudson, J. R., Transmission experiments with bovine malignant catarrhal, J. comp. Path. Ther. 49, 63 89 (1936). 2. Mettam, R. W. M., Snotsiekte in cattle, 9th and lOth Reps Dir. Vet. Educ. Res. Union ~[ South A[rica, pp. 395 432 (1923). 3. Piercy. S. E., Studies on bovine malignant catarrhal fever. VI. Adaptation to rabbits, Br. vet. J. |II, 484- 491 (1955). 4. Plowright, W., Studies of malignant catarrhal lever of cattle. D.V.Sc. Thesis University of Pretoria, South Africa. (1964). 5. Rossiter, P. B., Mushi. E. Z. and Plowright, W., The development of antibodies in rabbits and cattle infected experimentally with an African strain of malignant catarrhal fever virus, Vet. Microbiol. 2, 57 66 (1977). 6. Byerly, J. k. and Dawe, D. L., Delayed hypersensitivity reactions in Marek's disease virus-infected chickens, Am. J. vet. Res, 33, 2267 2273 (1972). 7. Plowright, W., Macadam, R. F. and Armstrong, J. S., Growth and characterisation of the virus of bovine malignant catarrhal fever in East Africa, J. gen. Microbiol. 39, 252 266 (1965). 8. Mushi, E. Z. and Plowright, W., Microtitre technique for the assay of malignant catarrhal fever virus and neutralising antibody, Res. vet. Sci. 27, 230 232 (1979). 9. Coombs, R. R. A. and Gell, P. G. H., Classification of allergic reactions responsible for clinical hypersensitivity and disease. In Clinical A.~pects ¢~l'Immunology, P. G. H. Gell, R. R. A. Coombs and P. J. Lachmann (Eds), pp. 761 778. Blackwell Scientific Publications, Oxtbrd (1975). 10. Allison, A. C., Interactions of antibody, complement components and various cell types in immunity against virus diseases and pyogenic bacteria, Tramplanm. Rev. 19, 3 55 (1974). I 1. Wilks, C. R. and Rossiter, P. B,, An immunosuppressive factor in serum of rabbits lethally infected with the herpesvirus of bovine malignant catarrhal fever, J. inlect. Dis. 137, 403 409 (1978). 12. Edington, N., Patel, J., Russell, P. H. and Plowright, W., The nature of the acute lymphoid proliferation in rabbits infected with the herpes virus of bovine malignant catarrhal fever, Eur. J. Cancer 15, 151 .%1522 (1980). 13. Selman, 1. E., Wiseman, A., Murray, M. and Wright, N. G., A clinico-pathological study of bovine malignant catarrhal fever in Great Britain, Vet. Rec. 94, 483 490 (1974). 14. Purchase, H. G., Marek's disease virus and the herpesvirus of turkeys, Prog. med. Virol. 18, 178 197 (1974).