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Ovine babesiosis: induction of a protective immune response with crude extracts of either Babesia bovis or B ovis
SHORT COMMUNICATIONS Research in Veterinary Science 1987, 43, 401-402
Ovine babesiosis: induction of a protective immune response with crude extracts of either Babesia bovis or B ovis M. ALABAY, A. DUZGUN, H. CERCI, Lalahan Nuclear Research Institute of Animal Health, Lalahan-Ankara, Turkey, I. G. WRIGHT*, D. J. WAL T1SBUHL, B. V. GOODGER, CSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag Number 3, PO, Indooroopilly, Queensland 4068, Australia
Adult sheep were vaccinated twice with crude extracts of either Babesia bovis or B avis parasites in Freund's complete adjuvant, four weeks apart. Three weeks later these two groups and a third susceptible group were challenged with B avis organisms. Both vaccinated groups had significantly lower parasitaemias than control animals.
peroxidase and 5-amino salicylic acid as a substrate. Sera were absorbed with either normal bovine or normal ovine red cell stroma before use (Waltisbuhl et al 1987). Before vaccination all sheep were screened to confirm their negative status. Antigen for use as an immunogen was frozen and thawed three times and cells or lysate emulsified with equal volumes THE induction of immunity to Babesia bovis infections by of Freund's complete adjuvant. Each vaccine dose consisted both crude and defined extracts of B bovis organisms has of 4 ml of emulsion. been recorded (Mahoney and Wright 1976, Kuttler et al Three groups of five Akkaraman sheep, nine to 12 months 1982, Wright et al 1983, Commins et a11985), although only old and of both sexes, were used for vaccination and a one comparable study with the small sheep parasite B avis further two splenectomised sheep were used as donor (Kyurtov 1979) has been published. In that study, plasma animals. One group of sheep (group I) was injected subfrom infected animals induced protection against subcutaneously twice, four weeks apart, with B avis antigen. A sequent virulent challenge. However, erythrocyte cytosecond group (group 2) was similarly vaccinated with B plasmic extracts from parasitised erythrocytes failed to bovis antigen. Vaccinated groups and a third untreated induce protective immunity. Because of the successful group used as controls were then challenged intravenously protective immunity produced against B bovis using extracts with I· 5 X 106 B avis infected erythrocytes three weeks after of that organism, the current study was undertake~ to the second injection. This group was not sham vaccinated determine whether cross-protective immunity could. be with Freund's complete adjuvant alone as this has been induced against subsequent B avis challenge using crude shown to have no effect on the outcome of babesiosis (I. G. extracts of either B bovis or B avis. Wright, unpublished data). Serum samples were collected weekly during the vaccinaB avis was isolated from a fatal clinical case in the Ankara region of Turkey. This isolate was used to make crude tion period and tested for the presence of either B avis or antigen preparations and was also stored as a stabilate in B bovis antibodies. Upon challenge, jugular blood was liquid nitrogen. B avis antigen was made from the Australian collected daily in trisodium citrate and haematocrits and Samford strain (Mahoney and Wright 1976). Approxi- thick blood films for parasitaemia estimates (Mahoney and mately 100per cent infected red blood cell preparations were prepared from both B avis and B bovis-infected blood by differential lysis in hypotonic saline solutions (Mahoney 1967). With B bovis preparations the optimal hypotonic 3 - - Untreated •••••• B bovis solution was 0·425 per cent, whereas with B avis it was O' 50 _.- Bovis to O' 55 per cent. The infected cells were diluted 1:2 in phosphate buffered saline and stored at - 20°C until required. Each millilitre of infected cell suspension contained approximately 2·5 X 109 parasites. For serodiagnostic antigen, 5 volumes of distilled water were added to I volume of infected erythrocyte suspension and the supernatant lysate obtained by centrifugation at 10,000 g for 30 minutes at 5°C. An ELISA procedure was developed similar to that described for B bovis (Waltisbuhl et al 1987). Both B avis and B bovis antigens were used in conjunction with a rabbit anti-ovine secondary antibody conjugated to horseradish Days
FIG 1: Mean 1091O parasitaemias for untreated animals and for animals immunised with either B ovis or B bovis extracts upon subsequent B ovis challenge
'Reprint requests to Dr I. G. Wright
401
402
M. Alabay, A. Duzgun, H. Cerci, I. G. Wright, D. J. Waltisbuhl, B. V. Goodger
Saal 1961) were performed. Parasitaemias were calculated as the 10gIO number /JI-l blood ± SD. Rectal temperatures were also taken daily. Both vaccinated groups had significantly lower parasitaemias than .the untreated group from day I after infection to day 10 after infection, at which point the experiment was terminated. The parasitaemias were not significantly different between vaccinated groups. The mean maximum parasitaemias were O' 88 ± O' 50 (group I, day 8, P<0·005), 1·02 ± 0·30 (group 2, day 3, P
while immunodominant antigens are not protective (Goodger et al 1986). This experiment has demonstrated that common protective antigens exist in both B ovis and B bovis and it may be that a synthetic vaccine developed against one species will be effective against the other. Already some evidence exists for this with B bovisand B bigeminasharing common protective antigens (Wright et al 1987). Acknowledgements The authors would like to thank the Turkish Atomic Energy Authority, CSIRO and the Joint Food and Agriculture Organisation/International Atomic Energy Agency Division, Vienna for their support of these studies. References CALLOW, L. L., MELLORS, L. T. & McGREGOR, W. (1979) International Journal for Parasitology 9, 333-338 COMMINS, M. A., GOODGER, B. V. & WRIGHT, I. G. (1985) International Journal for Parasitology 15, 491-495 GOODGER, B. V., COMMINS, M. A., WRIGHT, I. G., MIRRE, G. B., WALTlSBUHL, D. J. & WHITE, M. (1986) Zeitschrift fur Parasitenkunde 72,715-722 KUTTLER, K. L., LEVY, M. G., JAMES, M. A. & RISTlC, M. (1982) American Journal of Veterinary Research 43, 281-284 KYURTOV, N. (1979) Veterinarnomeditsinki Nauki 16, 15-22 MAHONEY, D. F. (1967) Experimental Parasitology 20, 232-241 MAHONEY, D. F. & SAAL, J. A. (1961) Australian Veterinary Journal 37, 44-47 MAHONEY, D. F. & WRIGHT, I. G. (1976) Veterinary Parasitology 2, 273-282 WALTlSBUHL, D. J., GOODGER, B. V., WRIGHT, I. G., COMMINS, M. A. & MAHONEY, D. F. (1987) Parasitology Research 73, 126-131 WRIGHT, I. G., MAHONEY, D. F., MIRRE, G. B., GOODGER, B. V. & KERR, J. D. (1982) Veterinary Immunology and Immunopathology 3,591-601 WRIGHT, I. G., WHITE, M., TRACEY-PATTE, P. D., DONALDSON, R. A., GOODGER, B. V., WALTISBUHL, D. J. & MAHONEY, D. F. (1983) Infection and Immunity 41, 244-250 WRIGHT, I. G., GOODGER, B. V., LEJ\TCH, G., AYLWARD, J. H., RODE-BRAMAN IS, K. & WALflSBUHL, D. J. (1987) Infection and Immunity 155, 364-368
Received June 23, 1987 Accepted July 2, 1987
Ovine babesiosis: induction of a protective immune response with crude extracts of either Babesia bovis or B ovis M. ALABAY, A. DUZGUN, H. CERCI, Lalahan Nuclear Research Institute of Animal Health, Lalahan-Ankara, Turkey, I. G. WRIGHT*, D. J. WAL T1SBUHL, B. V. GOODGER, CSIRO, Division of Tropical Animal Science, Long Pocket Laboratories, Private Bag Number 3, PO, Indooroopilly, Queensland 4068, Australia
Adult sheep were vaccinated twice with crude extracts of either Babesia bovis or B avis parasites in Freund's complete adjuvant, four weeks apart. Three weeks later these two groups and a third susceptible group were challenged with B avis organisms. Both vaccinated groups had significantly lower parasitaemias than control animals.
peroxidase and 5-amino salicylic acid as a substrate. Sera were absorbed with either normal bovine or normal ovine red cell stroma before use (Waltisbuhl et al 1987). Before vaccination all sheep were screened to confirm their negative status. Antigen for use as an immunogen was frozen and thawed three times and cells or lysate emulsified with equal volumes THE induction of immunity to Babesia bovis infections by of Freund's complete adjuvant. Each vaccine dose consisted both crude and defined extracts of B bovis organisms has of 4 ml of emulsion. been recorded (Mahoney and Wright 1976, Kuttler et al Three groups of five Akkaraman sheep, nine to 12 months 1982, Wright et al 1983, Commins et a11985), although only old and of both sexes, were used for vaccination and a one comparable study with the small sheep parasite B avis further two splenectomised sheep were used as donor (Kyurtov 1979) has been published. In that study, plasma animals. One group of sheep (group I) was injected subfrom infected animals induced protection against subcutaneously twice, four weeks apart, with B avis antigen. A sequent virulent challenge. However, erythrocyte cytosecond group (group 2) was similarly vaccinated with B plasmic extracts from parasitised erythrocytes failed to bovis antigen. Vaccinated groups and a third untreated induce protective immunity. Because of the successful group used as controls were then challenged intravenously protective immunity produced against B bovis using extracts with I· 5 X 106 B avis infected erythrocytes three weeks after of that organism, the current study was undertake~ to the second injection. This group was not sham vaccinated determine whether cross-protective immunity could. be with Freund's complete adjuvant alone as this has been induced against subsequent B avis challenge using crude shown to have no effect on the outcome of babesiosis (I. G. extracts of either B bovis or B avis. Wright, unpublished data). Serum samples were collected weekly during the vaccinaB avis was isolated from a fatal clinical case in the Ankara region of Turkey. This isolate was used to make crude tion period and tested for the presence of either B avis or antigen preparations and was also stored as a stabilate in B bovis antibodies. Upon challenge, jugular blood was liquid nitrogen. B avis antigen was made from the Australian collected daily in trisodium citrate and haematocrits and Samford strain (Mahoney and Wright 1976). Approxi- thick blood films for parasitaemia estimates (Mahoney and mately 100per cent infected red blood cell preparations were prepared from both B avis and B bovis-infected blood by differential lysis in hypotonic saline solutions (Mahoney 1967). With B bovis preparations the optimal hypotonic 3 - - Untreated •••••• B bovis solution was 0·425 per cent, whereas with B avis it was O' 50 _.- Bovis to O' 55 per cent. The infected cells were diluted 1:2 in phosphate buffered saline and stored at - 20°C until required. Each millilitre of infected cell suspension contained approximately 2·5 X 109 parasites. For serodiagnostic antigen, 5 volumes of distilled water were added to I volume of infected erythrocyte suspension and the supernatant lysate obtained by centrifugation at 10,000 g for 30 minutes at 5°C. An ELISA procedure was developed similar to that described for B bovis (Waltisbuhl et al 1987). Both B avis and B bovis antigens were used in conjunction with a rabbit anti-ovine secondary antibody conjugated to horseradish Days
FIG 1: Mean 1091O parasitaemias for untreated animals and for animals immunised with either B ovis or B bovis extracts upon subsequent B ovis challenge
'Reprint requests to Dr I. G. Wright
401
402
M. Alabay, A. Duzgun, H. Cerci, I. G. Wright, D. J. Waltisbuhl, B. V. Goodger
Saal 1961) were performed. Parasitaemias were calculated as the 10gIO number /JI-l blood ± SD. Rectal temperatures were also taken daily. Both vaccinated groups had significantly lower parasitaemias than .the untreated group from day I after infection to day 10 after infection, at which point the experiment was terminated. The parasitaemias were not significantly different between vaccinated groups. The mean maximum parasitaemias were O' 88 ± O' 50 (group I, day 8, P<0·005), 1·02 ± 0·30 (group 2, day 3, P
while immunodominant antigens are not protective (Goodger et al 1986). This experiment has demonstrated that common protective antigens exist in both B ovis and B bovis and it may be that a synthetic vaccine developed against one species will be effective against the other. Already some evidence exists for this with B bovisand B bigeminasharing common protective antigens (Wright et al 1987). Acknowledgements The authors would like to thank the Turkish Atomic Energy Authority, CSIRO and the Joint Food and Agriculture Organisation/International Atomic Energy Agency Division, Vienna for their support of these studies. References CALLOW, L. L., MELLORS, L. T. & McGREGOR, W. (1979) International Journal for Parasitology 9, 333-338 COMMINS, M. A., GOODGER, B. V. & WRIGHT, I. G. (1985) International Journal for Parasitology 15, 491-495 GOODGER, B. V., COMMINS, M. A., WRIGHT, I. G., MIRRE, G. B., WALTlSBUHL, D. J. & WHITE, M. (1986) Zeitschrift fur Parasitenkunde 72,715-722 KUTTLER, K. L., LEVY, M. G., JAMES, M. A. & RISTlC, M. (1982) American Journal of Veterinary Research 43, 281-284 KYURTOV, N. (1979) Veterinarnomeditsinki Nauki 16, 15-22 MAHONEY, D. F. (1967) Experimental Parasitology 20, 232-241 MAHONEY, D. F. & SAAL, J. A. (1961) Australian Veterinary Journal 37, 44-47 MAHONEY, D. F. & WRIGHT, I. G. (1976) Veterinary Parasitology 2, 273-282 WALTlSBUHL, D. J., GOODGER, B. V., WRIGHT, I. G., COMMINS, M. A. & MAHONEY, D. F. (1987) Parasitology Research 73, 126-131 WRIGHT, I. G., MAHONEY, D. F., MIRRE, G. B., GOODGER, B. V. & KERR, J. D. (1982) Veterinary Immunology and Immunopathology 3,591-601 WRIGHT, I. G., WHITE, M., TRACEY-PATTE, P. D., DONALDSON, R. A., GOODGER, B. V., WALTISBUHL, D. J. & MAHONEY, D. F. (1983) Infection and Immunity 41, 244-250 WRIGHT, I. G., GOODGER, B. V., LEJ\TCH, G., AYLWARD, J. H., RODE-BRAMAN IS, K. & WALflSBUHL, D. J. (1987) Infection and Immunity 155, 364-368
Received June 23, 1987 Accepted July 2, 1987