0
aim-7Sl9j92 s5.w + o.al Pergamm Press Lid Societyfor Pamimiogy
1992Ausrmiian
RESEARCH NOTE _BABEsIA BOY12 ANALYSIS OF AND PRELIMINARY VACCINATION STUDIES WITH A DEFINED INFECTED ERYTHROCYTE MEMBRANE BINDING ANTIGEN B. V. GOODGER, D. J. WALTISBUHL, I. G. WRIGHT
and M. WHITE*
CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Private Bag No. 3 PO, Indooroopilly, Queensland 4068, Australia (Received 4 February 1992; accepted 6 March 1992)
B. V., WALTISBUHL D. J., WRIGHTI. G. and WHITEM. 1992. Babesia bovis: analysis of and preliminary vaccination studies with a defined infected erythrocyte membrane binding antigen. Internarionai Journalfar Parasirology 22: 533-535. Murine monoclonal antibodies (MAB) were produced against the ‘j3’ fraction of Bubesiu bovis. A MAB, WI ICS, selected on the criterion of its staining of erythrocytes infected with B. b&s, was purified. The antigen identified by MAB WI lC5 was extracted from B. bovis infected erythrocytes by afhnity chromatography and used in a vaccination trial to test its vaccine efficacy against homologous B. bovis infection in splenectomized calves. The vaccinated group showed significantly different parasitaemias from the control group and it was concluded that the B. bovis antigen 1lC5 induced a protective immune response when used as a vaccine. This antigen should be synthesized using recombinant DNA techniques to determine its efficacy and suitability as a commercial vaccine against B. bovis infection. AbStIRCG-GmDGER
INDEX KEY WORDS: Babesiu bovis; erythrocyte; vaccination; monoclonal antibody.
Bubesia b&s
produces
one of the most debilitating
acute infections of cattle. Vaccination is almost mandatory for naive cattle introduced into areas endemic for the tick vector Boophilus microplus. At present, vaccination is achieved by the inoculation of blood containing attenuated parasites. While this mode is efficacious it has some biological drawbacks including limited shelf-life and accidental dissemination of other pathogens such as bovine leucosis virus (Rogers, Dimmock, De Vos L Rodwell, 1988). The concept of vaccinating with killed Babesia parasites was pioneered by Mahoney (1967a) and shown to be feasible, even against heterologous challenge, by Mahoney 8~ Wright (1976). Since then a systematic fractionationvaccination regime has been pursued which has resulted in fractions of infected erythrocytes sufficiently enriched in protective antigens to elicit heterologous immunity. One such fraction was designated ‘p’ after a multifractionation procedure involving selective lysis, sonication, ultracentrifugation, gei filtration and preparative agarose gel electrophoresis (Goodger, Wright, Waltisbuhl & Mirre, 1985). It was decided
*Present address: DPI Plant Pathology Research, Meiers Road, Indooroopilly, Queensland 4068, Australia.
that further subfractionation of the /I fraction would be best achieved by production of murine monoclonal antibodies (MAB). This manuscript describes the production and selection of a P-specific MAB, the subsequent purification of the relevant antigen and an initial vaccination study using splenectomized calves. Monoclonal antibodies to the p fraction were produced by the procedures described in detail by Wright, White, Tracey-Patte, Donaldson, Goodger, Waltisbuhl & Mahoney (1983). One of the MABs, designated W 1lC5, was selected for vaccination studies because of two criteria: (i) by immunofluorescent antibody assay (IFA) it avidly stained acetone-fixed infected erythrocytes, and (ii) in immunoblots of the fi fraction it gave a distinctive ladder of reactive bands (Fig. 1). Statistical and biological studies strongly imply that the major immune mechanism against B. bovis infection is the removal of infected erythrocytes (Mahoney, Kerr, Goodger & Wright, 1979) and it thus seemed logical to select for an antigen strongly associated with the infected erythrocyte. It also seemed logical to select for novel immunogenicity and the ladder sequence in the immunoblots suggested the antigen was indeed unique and novel. The antibody class of Wl lC5 was shown to be IgG, by a commercial ELISA kit (CSL, Melbourne). The
533
B. V.
534
GOODGER.
D. J. WALTISBUHL, I. G. WRIGHTand M. WHITE
200 B
97’ 67’ 431
ladder. No reactions were obtained with normal mouse serum or normal bovine serum. Four calves, judged free of Babesia by IFA and Giemsa staining, were injected subcutaneously (SC) with an emulsion containing 2 ml of Freund’s Complete Adjuvant and 2 ml of llC5 eluate. The vaccination was repeated at 4 weeks. At 7 weeks the vaccinates, along with five control calves, were splenectomized and at 8 weeks both groups were challenged with 1 x lo6 infected erythrocytes of B. bovis Samford strain (Mahoney & Wright, 1976). Prior to challenge, serum samples were obtained and, thereafter, serum and whole blood samples were obtained daily. The mean parasitaemias of each group are depicted in Table 1. Both groups had detectable parasites on day 7 post-challenge with the difference between the means being statistically significant on days 7 (PC O.OOS), 9 (PcO.05) and 10 (P
aim-7Sl9j92 s5.w + o.al Pergamm Press Lid Societyfor Pamimiogy
1992Ausrmiian
RESEARCH NOTE _BABEsIA BOY12 ANALYSIS OF AND PRELIMINARY VACCINATION STUDIES WITH A DEFINED INFECTED ERYTHROCYTE MEMBRANE BINDING ANTIGEN B. V. GOODGER, D. J. WALTISBUHL, I. G. WRIGHT
and M. WHITE*
CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Private Bag No. 3 PO, Indooroopilly, Queensland 4068, Australia (Received 4 February 1992; accepted 6 March 1992)
B. V., WALTISBUHL D. J., WRIGHTI. G. and WHITEM. 1992. Babesia bovis: analysis of and preliminary vaccination studies with a defined infected erythrocyte membrane binding antigen. Internarionai Journalfar Parasirology 22: 533-535. Murine monoclonal antibodies (MAB) were produced against the ‘j3’ fraction of Bubesiu bovis. A MAB, WI ICS, selected on the criterion of its staining of erythrocytes infected with B. b&s, was purified. The antigen identified by MAB WI lC5 was extracted from B. bovis infected erythrocytes by afhnity chromatography and used in a vaccination trial to test its vaccine efficacy against homologous B. bovis infection in splenectomized calves. The vaccinated group showed significantly different parasitaemias from the control group and it was concluded that the B. bovis antigen 1lC5 induced a protective immune response when used as a vaccine. This antigen should be synthesized using recombinant DNA techniques to determine its efficacy and suitability as a commercial vaccine against B. bovis infection. AbStIRCG-GmDGER
INDEX KEY WORDS: Babesiu bovis; erythrocyte; vaccination; monoclonal antibody.
Bubesia b&s
produces
one of the most debilitating
acute infections of cattle. Vaccination is almost mandatory for naive cattle introduced into areas endemic for the tick vector Boophilus microplus. At present, vaccination is achieved by the inoculation of blood containing attenuated parasites. While this mode is efficacious it has some biological drawbacks including limited shelf-life and accidental dissemination of other pathogens such as bovine leucosis virus (Rogers, Dimmock, De Vos L Rodwell, 1988). The concept of vaccinating with killed Babesia parasites was pioneered by Mahoney (1967a) and shown to be feasible, even against heterologous challenge, by Mahoney 8~ Wright (1976). Since then a systematic fractionationvaccination regime has been pursued which has resulted in fractions of infected erythrocytes sufficiently enriched in protective antigens to elicit heterologous immunity. One such fraction was designated ‘p’ after a multifractionation procedure involving selective lysis, sonication, ultracentrifugation, gei filtration and preparative agarose gel electrophoresis (Goodger, Wright, Waltisbuhl & Mirre, 1985). It was decided
*Present address: DPI Plant Pathology Research, Meiers Road, Indooroopilly, Queensland 4068, Australia.
that further subfractionation of the /I fraction would be best achieved by production of murine monoclonal antibodies (MAB). This manuscript describes the production and selection of a P-specific MAB, the subsequent purification of the relevant antigen and an initial vaccination study using splenectomized calves. Monoclonal antibodies to the p fraction were produced by the procedures described in detail by Wright, White, Tracey-Patte, Donaldson, Goodger, Waltisbuhl & Mahoney (1983). One of the MABs, designated W 1lC5, was selected for vaccination studies because of two criteria: (i) by immunofluorescent antibody assay (IFA) it avidly stained acetone-fixed infected erythrocytes, and (ii) in immunoblots of the fi fraction it gave a distinctive ladder of reactive bands (Fig. 1). Statistical and biological studies strongly imply that the major immune mechanism against B. bovis infection is the removal of infected erythrocytes (Mahoney, Kerr, Goodger & Wright, 1979) and it thus seemed logical to select for an antigen strongly associated with the infected erythrocyte. It also seemed logical to select for novel immunogenicity and the ladder sequence in the immunoblots suggested the antigen was indeed unique and novel. The antibody class of Wl lC5 was shown to be IgG, by a commercial ELISA kit (CSL, Melbourne). The
533
B. V.
534
GOODGER.
D. J. WALTISBUHL, I. G. WRIGHTand M. WHITE
200 B
97’ 67’ 431
ladder. No reactions were obtained with normal mouse serum or normal bovine serum. Four calves, judged free of Babesia by IFA and Giemsa staining, were injected subcutaneously (SC) with an emulsion containing 2 ml of Freund’s Complete Adjuvant and 2 ml of llC5 eluate. The vaccination was repeated at 4 weeks. At 7 weeks the vaccinates, along with five control calves, were splenectomized and at 8 weeks both groups were challenged with 1 x lo6 infected erythrocytes of B. bovis Samford strain (Mahoney & Wright, 1976). Prior to challenge, serum samples were obtained and, thereafter, serum and whole blood samples were obtained daily. The mean parasitaemias of each group are depicted in Table 1. Both groups had detectable parasites on day 7 post-challenge with the difference between the means being statistically significant on days 7 (PC O.OOS), 9 (PcO.05) and 10 (P
