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Immunisation of sheep against heartwater with inactivated Cowdria ruminantium
Research in Veterinary Science 1995, 58, 46-49
Immunisation of sheep against heartwater with inactivated Cowdria ruminantium S. M. MAHAN, H. R. ANDREW, N. TEBELE*, University ofFIorida/USAID/SADC Heartwater Research Project, Veterinary Laboratory Diagnostic and Research Branch, PO Box CY 551, Causeway, Zimbabwe, M. J. BURRIDGE, A. F. BARBET, Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, PO Box 11080, Gainesville, Florida 32611-0880, USA
SUMMARY The immunisation of sheep with inactivated Cowdria ruminantium organisms (the causative agent of heartwater) emulsified in Freund's adjuvant induced protective immunity against a homologous challenge with virulent cell culture-derived C ruminantium organisms. This protective immunity was associated with the development of high titres of C ruminant&m-specific antibodies, a low level of rickettsiosis in brain endothelium and no mortalities. In contrast, when unimmunised sheep were challenged, they developed higher levels of rickettsiosis in brain endothelium, and some of them died. This method of immunisation against heartwater has the potential to replace the infection and treatment method that is in current use. HEARTWATER, caused by the rickettsia Cowdria ruminantium and transmitted by ticks of the genus Amblyomma is a disease of major economic importance which affects livestock production in sub-Saharan Africa and the Caribbean (PelTeau et al 1980, Uilenberg 1983, Burridge 1985). The disease is controlled with acaricides, by the chemotherapy of sick animals or by vaccination, using an infection and treatment method (Uilenberg 1990). The infection and treatment method of vaccination is used because animals that recover from the clinical disease are usually resistant to homologous and in many cases to heterologous challenge (Van Winkelhoff and Uilenberg 1981, Uilenberg et al 1983, Jongejan et al 1988). Recently, the possibility of using attenuated organisms had been reported, although attenuation appears to be strain specific (Jongejan 1991) and hence of limited value. The development of an improved vaccine against heartwater remains a feasible and desirable goal, and a method of immunisation, using cell culture-derived organisms, inactivated and mixed with Freund's adjuvant was therefore investigated. This method of immunisation protected sheep against a challenge with virulent homologous C ruminantium organisms.
dient of 0-10-20-30-40 per cent Percoll prepared in PBS. The gradients were centrifuged at 400 g for 30 minutes and the purified organisms were harvested from the 0 per cent layer, washed twice in PBS at 30,000 g for 30 minutes, resuspended in PBS and counted by using acridine orange (Knight et al 1989), to obtain a total count and fluorescein diacetate to obtain a viable count (Mishell and Shiigi 1980). Before they were used for immunisation, the C ruminantium organisms were inactivated with [3-propiolactone (0.4 per cent at 4°C for 30 minutes). The inactivation or loss of viability of the organisms was shown by their lack of infectivity for cultures of bovine endothelial cells and by their lack of staining with fluorescein diacetate.
Animals, immunisations and challenge inoculations Three groups of five merino sheep, six months old, were purchased from an area of Zimbabwe (Ruwa in the high veld) free of heartwater and AmbIyomma ticks. All the groups of sheep were inoculated five times subcutaneously at intervals of three to four weeks. Group 1 sheep each received 1 to 2 x 107 inactivated C ruminantium organisms; group 2 sheep each received the same number of C ruminantium organisms but mixed with Freund's complete adjuvant (FCA) for the first inoculation and with Freund's incomplete adjuvant (FIA) for the remaining inoculations; group 3 sheep were inoculated with PBS mixed with FCA for the first inoculation and thereafter with FIA. Samples of serum were obtained from the sheep before each injection to monitor the development of specific antiC ruminantium antibody responses by immunoblotting. In group 1, one sheep died of haemonchosis a condition not related to its immunisation with inactivated Cowdria organisms. All the surviving sheep received a homologous intravenous challenge of 106 viable C ruminantium organisms (Crystal Springs strain), after the final immunisation. The challenge inoculation dose containing the desired number of viable organisms was drawn into a syringe from a pool of pure C ruminantium, and all the inoculations were
MATERIALS AND M E T H O D S
Preparation of C ruminantium C ruminantium organisms (Crystal Springs strain, Zimbabwe isolate; Byrom and Yunker 1990) were harvested from terminally infected cultures of bovine endothelial cells and purified on discontinuous Percoll density gradients as follows: the debris of host cells were removed from the culture supernatant by centrifugation at 1500 g for 30 minutes at 4°C. The supernatants were then centrifuged at 30,000 g for 30 minutes at 4°C to pellet the organisms. The organisms were resuspended in 1 ml of phosphate buffered saline ([PBS] NaH2PO4.2H20 0.0028M, Na2HPO 4 0.0072M, NaC1 0.15M) pH 7.3 and loaded on to a step gra* Present address: ILRAD, PO Box 30709, Nairobi, Kenya
46
Protection of sheep against heartwater
administered intravenously. This challenge dose was chosen on the basis of the results of a challenge dose titration experiment in which a mortality rate of up to 80 per cent had been observed (data not shown). On the fourth day of the febrile reaction, brain biopsies (Synge 1978) were conducted on all the sheep and the percentage rickettsiosis was determined after staining the brain crush smears with Giemsa stain (Purchase 1945). The colonies were identified and the number of infected cells in 500 brain capillary endothelial cells were counted by three laboratory staff and an average of the three counts was taken. The results were transformed to an arcsin percentage and analysed by analysis of variance and Student's t test.
Immunoblotting Samples of C ruminantium antigen (20 gg) were electrophoresed on 12 per cent sodium dodecyl sulphatepolyacrylamide gels (SDS-PAGE), transferred electrophoretically to nitrocellulose and probed with titrated sheep sera (from 1/100 to 1/106 in PBS) as described by Mahan et al (1993). The end titre was determined as that which gave a reaction with any of the Cowdria antigens. Usually the reaction of the sera with the 32 kD and a 40 kD protein was the last to wane.
RESULTS
Antibody responses and responses of sheep to homologous challenge Before they were challenged the sheep of group 2, which were immunised with inactivated C ruminantium organisms mixed with FCA, had specific reciprocal antibody titres ranging from 5 × 10 5 to 10 6 (Fig 1, Table 1), whereas the sheep of group 1 which were immunised with the inactivated organisms alone, developed antibody titres of between 103 and 3 × 103 (Table 1). The sheep in Group 3 which
47
were inoculated only with Freund's adjuvant developed background antibody titres of less than 102 to 103 (Table 1). The sheep in groups 1 and 2 recognised immunogenic proteins of C ruminantium which have been described previously (Jongejan and Thielemans 1989, Rossouw et al 1990, Mahan et al 1993), but those in group 1 recognised them at lower titres (1/102 to 1/103) than those in group 2. All but two of the sheep exhibited febrile reactions (a rectal temperature of 40.5°C or higher) five to 10 days after the challenge. The lack of a febrile response in the two sheep was not due to the maladministration of the challenge inoculure. All the four sheep in group 1 and four of the five sheep in group 3 were positive for infection with C ruminantium by brain biopsy, whereas only one of the five sheep in group 2 was positive (Table 1). The degree ofrickettsiosis was significantly different (P=0-0238) in the three experimental groups. The sheep in groups 1 and 3 developed a greater degree of rickettsiosis, ranging from 0"9 + 0-5 to 14.7 _+2-0 per cent, than the one affected sheep in group 2, which had a rickettsiosis of only 0-2 per cent (Table 1). The rickettsiosis in the group 2 sheep was significantly different from that in the sheep &group 1 (P=0-0009) and group 3 (P=0.0233). The mortality rates also differed; two of the four sheep in group 1 died, three of the five sheep in group 3 died, but none of the the sheep in group 2 died (Table 1). In general, the sheep that died were those that exhibited high levels of rickettsiosis in brain capillary endothelium. One exception was sheep 083 in group 3 which survived although it had a similar degree of rickettsiosis to sheep 052 in group 1, which died.
DISCUSSION Heartwater is an important disease which causes economic losses as a result of high mortalities in susceptible animals (Uilenberg 1983). However, animals that recover from the clinical disease are usually protected against
97.4
69
46
30
21.5
14.3 -+
054
068
4752
4764
082
FIG 1: Immunoblots demonstrating the recognition of C ruminantium antigens by titrated pre-challenge sera (1/102, 1/104, 1/105, 1/5 x 105 and 1/106) from sheep 054, 068, 4752, 4764, and 082. The first three blots from the left represent molecular weight markers and negative (-) and positive (+) control sera reactions with Cowdria antigens
48
S. M. Mahan, H. R. Andrew, N. Tebele, M. J. Burridge, A. F. Barbet
TABLE 1: Results of the challenge of sheep vaccinated with inactivated cell culture-derived C ruminantium organisms Group and treatment
Animal
Pre-challenge antibody titre*
Interval to febriiet Reaction (days)
Brain biopsy
Inactivated organisms
052 092 079 4762
3 x 103 3 x 103 3 x 103 103
7 10 9 9
+ + + +
2 Inactivated organisms and Freund's adjuvant
054 068 4752 4764 082
106 106 >5 x 105 106 106
7 5 7 7
-
3
4754 083 4765 055 069
103 102 <102 <102 <102
7 9 9 9
Per cent Rickettsiosis
Death
1
Freund's adjuvant
0.9 ± 2.5 ± 3.9± 6.1 ±
0.5 0.4 1-1 4.8
+ +
0 0
-
--
0
--
+
0 0-2 _+ 0"0
--
+ + + +
0
--
0-9 _+0'8 8"2 ± 3-9 12-3 + 3"2 14"7 ± 2.0
+ + +
* The antibody titre is the reciprocal of the last serum dilution which gave a positive reaction on immunoblots t Rectal temperature of 40.5°C or more Sheep 4752 and 4754 did not respond to the challenge inoculum
homologous and in most cases against heterologous challenge (Van Winkelhoff and Uilenberg 1981, Uilenberg et al 1983, Jongejan et al 1988). This finding makes the development of a vaccine a feasible proposition. Improved methods of immunisation against heartwater need to be developed to replace the currently available cumbersome method of infection and treatment (Uilenberg 1983, 1990). The recently reported method of vaccination of goats and sheep against heartwater by using culture-attenuated Senegalese C ruminant±urn organisms (Jongejan 1991) shows promise, but it has the drawback that cross-protection in heartwater is not complete and it would therefore be necessary to attenuate several strains of C ruminant±urn to cover the antigenic repertoire of the organisms. In addition, in the same study it was reported that it was not possible to attenuate the Welgevonden strain of C ruminant±urn (Jongejan 1991). In the present authors' experience it has not been possible to attenuate the Zimbabwean Crystal Springs strain of C ruminant±urn and after 60 and 192 passages it was still fatally infective for sheep. The attenuation of C ruminant±urn thus appears to be strain specific and its application is currently limited to the Senegalese strain. The data presented here indicate that it was possible to immunise sheep against a homologous challenge by using inactivated culture-derived C ruminant±urn organisms. Although a febrile reaction was observed in all three experimental groups, the sheep that received inactivated C ruminant±urn organisms mixed with Freund's adjuvant (group 2) were significantly protected against a virulent challenge compared with sheep that received the organisms or Freund's adjuvant alone. The protection of the sheep in group 2 was associated with the development of high titres of specific anti-C ruminant±urn antibodies, a low level of brain endothelial cell rickettsiosis and zero mortality. The stimulation of the protective responses appeared to be adjuvant dependant, because the sheep in group 1 recognised the same immunogenic proteins of C ruminant±urn but at lower antibody titres. This study demonstrates that protective homologous immunity against heartwater can be induced with inactivated organisms mixed with Freund's adjuvant. However, Freund's adjuvant is not suitable for commercial use because it can cause abscesses and inflammation at the injection site, and severe pain and fever (Edelman 1974, Hilleman and Beale 1983, Steiner et al 1960); it may also induce permanent injury to organs as a result of generalised
granulomatous proliferation (Behar and Tal 1959, Heymann et al 1959, Steiner et al 1960), autoimmune disease (Stewart-Tull 1980), amyloidosis (Tal and Laufer 1960), adjuvant arthritis (Pearson and Wood 1963, 1964, StewartTull 1988) and hypersensitisation (Pearson and Wood 1963, Edelman 1974, Stewart-Tull 1988). The discovery and testing of a commercially viable and effective adjuvant for use in combination with cultured inactivated C ruminant±urn organisms is now an important objective, as is the determination of the duration of the immunity induced by the method, and the extent of the protection it provides against challenge from infected ticks and heterologous isolates of the organism. The inactivated vaccine could incorporate any strain of C ruminant±urn, to cover the differences in antigen±city between the strains which are responsible for incomplete protection or lack of protection.
ACKNOWLEDGEMENTS This study was supported by the United States Agency for International Development Cooperative Agreement number AFR-0435-A-00-9084-00.
REFERENCES BEHAR, A. J. & TAL, C. (1959) Experimental liver necrosis produced by the injection of homologous whole liver with adjuvant. Journal of Pathological Bacteriology 77, 591-596 BURRIDGE, M. J. (i985) Heartwater invades the Caribbean. Parasitology Today 1, 1-3 BYROM, B. & YUNKER, C. E. (1990) Improved culture conditions for Cowdria ruminant±urn (Rickettsiales), the agent of heartwater disease of domestic ruminants. Cytotechnology 4, 285-290 EDELMAN, R. (1974) Vaccine adjuvants. Review of Infectious Diseases 2, 370-383 HEYMANN, W., HACKEL, D. B., HARWOOD, S., WILSON, S. G. F. & HUNTER, J. L. P. (1959) Production of nephrotic syndrome in rats by Freund's adjuvants and rat kidney suspensions. Proceedings of the Society of Experimental Biology, New York 100, 660-664 HILLEMAN, M. R. & BEALE, A. J. (1983) Adjuvants for human use. In New Approaches to Vaccine Development. Eds R. Bell and G. Torrigiani. Basel, Schwabe. pp i57-i66 JONGEJAN, F. (1991) Protective immunity to heartwater (Cowdria ruminant±urn infection) is acquired after vaccination with in vitro-attenuated rickettsiae. Infection and Immunity 59, 729-731 JONGEJAN, F. & THIELEMANS, M. J. C. (1989) Identification of an immunodominant antigenically conserved 32-Kilodalton protein from Cowdria ruminant±urn.Infection and Immunity 57, 3243-3246 JONGEJAN, F., UILENBERG, G., FRANSSEN, F. F. J., GUEYE, A. & MEUWEHUYS, J. (1988) Antigenic differences between stocks of Cowdria ruminant±urn. Research in Veterinary Science 44, 186-188
Protection o f sheep against heartwater
KNIGHT, S. T., NEECE, V. R. & WITT, D. J. (1989) Rapid culture-independent techniques for quantitation of Chlamydia trachomatis elementary bodies. Journal of Microbiological Methods 10, 255-263 MAHAN, S. M., TEBELE, N., MUKWEDEYA, D., SEMU, S., NYATHI, C. B., WASSINK, L. A., KELLY, P. J., PETER, T. & BARBET, A. F. (1993) An immunoblotting diagnostic assay for heartwater based on the immunodominant 32kD protein of Cowdria rumina~tium detects false positives in field sera. Journal of Clinical Microbiology 31, 2729-2737 MISHELL, B. B. & SHIIGI, S. M. (1980) Selected Methods in Cellular Immunology. New York, W. H. Freeman Company. p 19 PEARSON, C. M. & WOOD, F. D. (1963) Studies of arthritis and other lesions induced in rats by injection of mycobacterial adjuvant. VII. Pathologic details of the arthritis and spondylitis. American Journal of Pathology 42, 73-95 PEARSON, C. M. & WOOD, F. D, (1964) Passive transfer of adjuvant arthritis by lymph-nodes or spleen ceils. Journal of Experimental Medicine 120, 547-560 PERREAU, P., MOREL, P. C., BARRE, N. & DURRAND, P. (1980) Existence de la cowdriose (heartwater) a Cowdria ruminantium chez les ruminants des Antilles francaises (La Guadeloupe) at des Mascareignes (La Reunion et lie Maurice). Revue d' Elevage et de Medicine Veterinaire des Pays Tropicaux 33, 21-22 PURCHASE, H. S. (1945) A simple and rapid method for demonstrating Rickettsia ruminantium (Cowdry, 1925) in heartwater brain. Veterinary Record 57, 413-414 ROSSOUW, M., NEITZ, A. W. H., DE WAAL, D. T., DU PLESSIS, J. L., VAN GAS, L. & BRETT. S. (1990) Identification of the antigenic proteins of Cowdria ruminantium. Onderstepoort Journal of Veterinary Research 57, 215-221
49
STEINER, J. W., LANGER, B. & SCHATZ, D. L. (1960) The local and systemic effects of Freund's adjuvant and its fractions. Archives of Pathology 70, 424-434 STEWART-TULL, D. E. S. (1980) The immunological activities of bacterial peptidoglycans. Annual Reviews of Microbiology 34, 311-340 STEWART-TULL, D. E. S. (1988) Immunostimulation with peptidoglycan and its synthetic derivatives. Progress in Drug Research 32, 305-328 SYNGE, B. A. (1978) Brain biopsy for the diagnosis of heartwater. Tropical Animal Health and Production 10, 605-609 TAL, C. & LAUFER, A. (1960) Amyloidosis in mice following infection with Freund's Adjuvant, its components given separately or in homologous liveradjuvant mixture. British Journal of Experimental Pathology 41, 605-609 UILENBERG, G. (1983) Heartwater (Cowdria ruminantium infection): Current status. Advances in Veterinary Science and Comparative Medicine 27, 427-480 UILENBERG, G, (1990) Present and future possibilities for the control of cowdriosis and anaplasmosis. Veterinary Quarterly 12, 39-45 UILENBERG, G., ZIVKOVIC, D., DWINGER, R. H., TER HUURNE, A. A. H. M. & PERLE, N. M. (1983) Cross immunity between strains of Cowdria ruminantium. Research in Veterinary Science 35, 200-205 VAN WINKELHOFF, A, J. & UILENBERG, G. (1981) Heartwater: Cross immunity studies with strain of Cowdria ruminantium isolated in West and South Africa. Tropical Animal Health and Production 13, 160-164
Received November 17, 1993 Accepted July 7, 1994
Immunisation of sheep against heartwater with inactivated Cowdria ruminantium S. M. MAHAN, H. R. ANDREW, N. TEBELE*, University ofFIorida/USAID/SADC Heartwater Research Project, Veterinary Laboratory Diagnostic and Research Branch, PO Box CY 551, Causeway, Zimbabwe, M. J. BURRIDGE, A. F. BARBET, Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, PO Box 11080, Gainesville, Florida 32611-0880, USA
SUMMARY The immunisation of sheep with inactivated Cowdria ruminantium organisms (the causative agent of heartwater) emulsified in Freund's adjuvant induced protective immunity against a homologous challenge with virulent cell culture-derived C ruminantium organisms. This protective immunity was associated with the development of high titres of C ruminant&m-specific antibodies, a low level of rickettsiosis in brain endothelium and no mortalities. In contrast, when unimmunised sheep were challenged, they developed higher levels of rickettsiosis in brain endothelium, and some of them died. This method of immunisation against heartwater has the potential to replace the infection and treatment method that is in current use. HEARTWATER, caused by the rickettsia Cowdria ruminantium and transmitted by ticks of the genus Amblyomma is a disease of major economic importance which affects livestock production in sub-Saharan Africa and the Caribbean (PelTeau et al 1980, Uilenberg 1983, Burridge 1985). The disease is controlled with acaricides, by the chemotherapy of sick animals or by vaccination, using an infection and treatment method (Uilenberg 1990). The infection and treatment method of vaccination is used because animals that recover from the clinical disease are usually resistant to homologous and in many cases to heterologous challenge (Van Winkelhoff and Uilenberg 1981, Uilenberg et al 1983, Jongejan et al 1988). Recently, the possibility of using attenuated organisms had been reported, although attenuation appears to be strain specific (Jongejan 1991) and hence of limited value. The development of an improved vaccine against heartwater remains a feasible and desirable goal, and a method of immunisation, using cell culture-derived organisms, inactivated and mixed with Freund's adjuvant was therefore investigated. This method of immunisation protected sheep against a challenge with virulent homologous C ruminantium organisms.
dient of 0-10-20-30-40 per cent Percoll prepared in PBS. The gradients were centrifuged at 400 g for 30 minutes and the purified organisms were harvested from the 0 per cent layer, washed twice in PBS at 30,000 g for 30 minutes, resuspended in PBS and counted by using acridine orange (Knight et al 1989), to obtain a total count and fluorescein diacetate to obtain a viable count (Mishell and Shiigi 1980). Before they were used for immunisation, the C ruminantium organisms were inactivated with [3-propiolactone (0.4 per cent at 4°C for 30 minutes). The inactivation or loss of viability of the organisms was shown by their lack of infectivity for cultures of bovine endothelial cells and by their lack of staining with fluorescein diacetate.
Animals, immunisations and challenge inoculations Three groups of five merino sheep, six months old, were purchased from an area of Zimbabwe (Ruwa in the high veld) free of heartwater and AmbIyomma ticks. All the groups of sheep were inoculated five times subcutaneously at intervals of three to four weeks. Group 1 sheep each received 1 to 2 x 107 inactivated C ruminantium organisms; group 2 sheep each received the same number of C ruminantium organisms but mixed with Freund's complete adjuvant (FCA) for the first inoculation and with Freund's incomplete adjuvant (FIA) for the remaining inoculations; group 3 sheep were inoculated with PBS mixed with FCA for the first inoculation and thereafter with FIA. Samples of serum were obtained from the sheep before each injection to monitor the development of specific antiC ruminantium antibody responses by immunoblotting. In group 1, one sheep died of haemonchosis a condition not related to its immunisation with inactivated Cowdria organisms. All the surviving sheep received a homologous intravenous challenge of 106 viable C ruminantium organisms (Crystal Springs strain), after the final immunisation. The challenge inoculation dose containing the desired number of viable organisms was drawn into a syringe from a pool of pure C ruminantium, and all the inoculations were
MATERIALS AND M E T H O D S
Preparation of C ruminantium C ruminantium organisms (Crystal Springs strain, Zimbabwe isolate; Byrom and Yunker 1990) were harvested from terminally infected cultures of bovine endothelial cells and purified on discontinuous Percoll density gradients as follows: the debris of host cells were removed from the culture supernatant by centrifugation at 1500 g for 30 minutes at 4°C. The supernatants were then centrifuged at 30,000 g for 30 minutes at 4°C to pellet the organisms. The organisms were resuspended in 1 ml of phosphate buffered saline ([PBS] NaH2PO4.2H20 0.0028M, Na2HPO 4 0.0072M, NaC1 0.15M) pH 7.3 and loaded on to a step gra* Present address: ILRAD, PO Box 30709, Nairobi, Kenya
46
Protection of sheep against heartwater
administered intravenously. This challenge dose was chosen on the basis of the results of a challenge dose titration experiment in which a mortality rate of up to 80 per cent had been observed (data not shown). On the fourth day of the febrile reaction, brain biopsies (Synge 1978) were conducted on all the sheep and the percentage rickettsiosis was determined after staining the brain crush smears with Giemsa stain (Purchase 1945). The colonies were identified and the number of infected cells in 500 brain capillary endothelial cells were counted by three laboratory staff and an average of the three counts was taken. The results were transformed to an arcsin percentage and analysed by analysis of variance and Student's t test.
Immunoblotting Samples of C ruminantium antigen (20 gg) were electrophoresed on 12 per cent sodium dodecyl sulphatepolyacrylamide gels (SDS-PAGE), transferred electrophoretically to nitrocellulose and probed with titrated sheep sera (from 1/100 to 1/106 in PBS) as described by Mahan et al (1993). The end titre was determined as that which gave a reaction with any of the Cowdria antigens. Usually the reaction of the sera with the 32 kD and a 40 kD protein was the last to wane.
RESULTS
Antibody responses and responses of sheep to homologous challenge Before they were challenged the sheep of group 2, which were immunised with inactivated C ruminantium organisms mixed with FCA, had specific reciprocal antibody titres ranging from 5 × 10 5 to 10 6 (Fig 1, Table 1), whereas the sheep of group 1 which were immunised with the inactivated organisms alone, developed antibody titres of between 103 and 3 × 103 (Table 1). The sheep in Group 3 which
47
were inoculated only with Freund's adjuvant developed background antibody titres of less than 102 to 103 (Table 1). The sheep in groups 1 and 2 recognised immunogenic proteins of C ruminantium which have been described previously (Jongejan and Thielemans 1989, Rossouw et al 1990, Mahan et al 1993), but those in group 1 recognised them at lower titres (1/102 to 1/103) than those in group 2. All but two of the sheep exhibited febrile reactions (a rectal temperature of 40.5°C or higher) five to 10 days after the challenge. The lack of a febrile response in the two sheep was not due to the maladministration of the challenge inoculure. All the four sheep in group 1 and four of the five sheep in group 3 were positive for infection with C ruminantium by brain biopsy, whereas only one of the five sheep in group 2 was positive (Table 1). The degree ofrickettsiosis was significantly different (P=0-0238) in the three experimental groups. The sheep in groups 1 and 3 developed a greater degree of rickettsiosis, ranging from 0"9 + 0-5 to 14.7 _+2-0 per cent, than the one affected sheep in group 2, which had a rickettsiosis of only 0-2 per cent (Table 1). The rickettsiosis in the group 2 sheep was significantly different from that in the sheep &group 1 (P=0-0009) and group 3 (P=0.0233). The mortality rates also differed; two of the four sheep in group 1 died, three of the five sheep in group 3 died, but none of the the sheep in group 2 died (Table 1). In general, the sheep that died were those that exhibited high levels of rickettsiosis in brain capillary endothelium. One exception was sheep 083 in group 3 which survived although it had a similar degree of rickettsiosis to sheep 052 in group 1, which died.
DISCUSSION Heartwater is an important disease which causes economic losses as a result of high mortalities in susceptible animals (Uilenberg 1983). However, animals that recover from the clinical disease are usually protected against
97.4
69
46
30
21.5
14.3 -+
054
068
4752
4764
082
FIG 1: Immunoblots demonstrating the recognition of C ruminantium antigens by titrated pre-challenge sera (1/102, 1/104, 1/105, 1/5 x 105 and 1/106) from sheep 054, 068, 4752, 4764, and 082. The first three blots from the left represent molecular weight markers and negative (-) and positive (+) control sera reactions with Cowdria antigens
48
S. M. Mahan, H. R. Andrew, N. Tebele, M. J. Burridge, A. F. Barbet
TABLE 1: Results of the challenge of sheep vaccinated with inactivated cell culture-derived C ruminantium organisms Group and treatment
Animal
Pre-challenge antibody titre*
Interval to febriiet Reaction (days)
Brain biopsy
Inactivated organisms
052 092 079 4762
3 x 103 3 x 103 3 x 103 103
7 10 9 9
+ + + +
2 Inactivated organisms and Freund's adjuvant
054 068 4752 4764 082
106 106 >5 x 105 106 106
7 5 7 7
-
3
4754 083 4765 055 069
103 102 <102 <102 <102
7 9 9 9
Per cent Rickettsiosis
Death
1
Freund's adjuvant
0.9 ± 2.5 ± 3.9± 6.1 ±
0.5 0.4 1-1 4.8
+ +
0 0
-
--
0
--
+
0 0-2 _+ 0"0
--
+ + + +
0
--
0-9 _+0'8 8"2 ± 3-9 12-3 + 3"2 14"7 ± 2.0
+ + +
* The antibody titre is the reciprocal of the last serum dilution which gave a positive reaction on immunoblots t Rectal temperature of 40.5°C or more Sheep 4752 and 4754 did not respond to the challenge inoculum
homologous and in most cases against heterologous challenge (Van Winkelhoff and Uilenberg 1981, Uilenberg et al 1983, Jongejan et al 1988). This finding makes the development of a vaccine a feasible proposition. Improved methods of immunisation against heartwater need to be developed to replace the currently available cumbersome method of infection and treatment (Uilenberg 1983, 1990). The recently reported method of vaccination of goats and sheep against heartwater by using culture-attenuated Senegalese C ruminant±urn organisms (Jongejan 1991) shows promise, but it has the drawback that cross-protection in heartwater is not complete and it would therefore be necessary to attenuate several strains of C ruminant±urn to cover the antigenic repertoire of the organisms. In addition, in the same study it was reported that it was not possible to attenuate the Welgevonden strain of C ruminant±urn (Jongejan 1991). In the present authors' experience it has not been possible to attenuate the Zimbabwean Crystal Springs strain of C ruminant±urn and after 60 and 192 passages it was still fatally infective for sheep. The attenuation of C ruminant±urn thus appears to be strain specific and its application is currently limited to the Senegalese strain. The data presented here indicate that it was possible to immunise sheep against a homologous challenge by using inactivated culture-derived C ruminant±urn organisms. Although a febrile reaction was observed in all three experimental groups, the sheep that received inactivated C ruminant±urn organisms mixed with Freund's adjuvant (group 2) were significantly protected against a virulent challenge compared with sheep that received the organisms or Freund's adjuvant alone. The protection of the sheep in group 2 was associated with the development of high titres of specific anti-C ruminant±urn antibodies, a low level of brain endothelial cell rickettsiosis and zero mortality. The stimulation of the protective responses appeared to be adjuvant dependant, because the sheep in group 1 recognised the same immunogenic proteins of C ruminant±urn but at lower antibody titres. This study demonstrates that protective homologous immunity against heartwater can be induced with inactivated organisms mixed with Freund's adjuvant. However, Freund's adjuvant is not suitable for commercial use because it can cause abscesses and inflammation at the injection site, and severe pain and fever (Edelman 1974, Hilleman and Beale 1983, Steiner et al 1960); it may also induce permanent injury to organs as a result of generalised
granulomatous proliferation (Behar and Tal 1959, Heymann et al 1959, Steiner et al 1960), autoimmune disease (Stewart-Tull 1980), amyloidosis (Tal and Laufer 1960), adjuvant arthritis (Pearson and Wood 1963, 1964, StewartTull 1988) and hypersensitisation (Pearson and Wood 1963, Edelman 1974, Stewart-Tull 1988). The discovery and testing of a commercially viable and effective adjuvant for use in combination with cultured inactivated C ruminant±urn organisms is now an important objective, as is the determination of the duration of the immunity induced by the method, and the extent of the protection it provides against challenge from infected ticks and heterologous isolates of the organism. The inactivated vaccine could incorporate any strain of C ruminant±urn, to cover the differences in antigen±city between the strains which are responsible for incomplete protection or lack of protection.
ACKNOWLEDGEMENTS This study was supported by the United States Agency for International Development Cooperative Agreement number AFR-0435-A-00-9084-00.
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Received November 17, 1993 Accepted July 7, 1994