VACCINATION
OF CATTLE WITH DEXTRAN SULPHATE-BINDING BABESIA BIGEMINA ANTIGENS
M. W. KIJNG'U,* B. V. GOODGER,*~
G. R. BUSHELL,$ I. G. WRIGHT*
and D. J. WALTISBUHL*
* CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Private Bag No. 3, PO, Indooroopilly, Queensland 4068, Australia $ Griffith University, Division of Science and Technology, Nathan, Queensland 4111, Australia (Received 3 October 1991; accepted 10 February 1992)
Abstract-&.vmu M. W., GOODGER B. V., BUSHELL G. R., WRIGHT1. G. and WALTISBUHL D. J. 1992. Vaccination of cattle with dextran sulphate-binding Babes&z bigemina antigens. ~nter~utiQnu1~o~rn~~f~r Parasitology 22: 621-625. Dextran sulphate-bound Bubesiu bigemina antigens were used in a preliminary vaccination study and were shown to elicit a protective immune response in cattle. A dextran sulphatebinding fraction of B. bigemina was further subfractionated on a Phenyl Sepharose column to give two fractions-one that strongly bound to the column (bound fraction) and one that did not (unbound fraction). Two groups of cattle were each vaccinated with either the bound or the unbound fraction. These two groups of animals along with a control group were then challenged with B. bigemimz-infected erythrocytes. Both groups of vaccinated animals showed considerably lower mean daily parasitaemias as compared to the control group. INDEX KEY WORDS: Babesiu bigemina; dextran sulphate-bound antigens; vaccination.
BOVINEbabesiosis,
INTRODUCTION caused by various Babesia species,
is one of the most important diseases of cattle in tropical and subtropical countries of the world (McCosker, 1981; Kuttler, 1988; Wright & Riddles, 1989). Accepted and conventional vaccination against the disease uses attenuated living organisms, but this approach has a number of shortcomings. Various attempts have therefore been made to elicit immunity using dead vaccines consisting either of antigenic components of Babe& parasites or exoantigens derived from culture (Ristic & Kakoma, 1988; Wright, Goodger, Schuntner, Waltisbuhl & Duzgun, 1988; Goodger, 1989; Montenegro-James, Kakoma & Ristic, 1989; Wright & Riddles, 1989). Although considerable progress has been made in developing an experimental dead vaccine against B. his by workers at the CSIRO Laboratories, similar studies with Babesia bige~i~a have been hindered by the fact that B. bi~e~ina-infested erythrocytes were not as easy to concentrate as those infected with B. bovis (Mahoney, 1967). In a recent publication from this laboratory, it was reported that B. bigeemina-infected bovine erythrocytes
7 To whom all correspondence should be addressed.
could be concentrated in vitro by their selective binding to dextran sulphate columns (Goodger, Bushel1 & Wright, 1989). It was also noted that a soluble extract from erythrocytes infected with B. bigemina contained a subfraction that bound to dextran sulphate. Upon elution this fraction was shown to contain serologically detectable B. bigemina antigens. It was considered important to determine if the subfraction contained antigen(s) capable of eliciting a protective immune response; this manuscript describes preliminary vaccination studies with this fraction. MATERIALS AND METHODS Preparation of Babesia bigemina antigens for vaccination.
Blood containing approximately 20% B. bigemina-infected from serial passage in erythrocytes was obtained splenectomized calves and washed three times with phosphate-buffered saline (PBS), pH 7.2 by centrifugation at 2000 g for 15 min at 4°C. The washed, packed erythrocytes were sonically disrupted at 50-100 W for 2 min (BRAUN LABSONIC) and ultracentrifuged at 100,000 g for 60 min at 4°C. Two volumes of the resultant supernatant were applied to a 1 vol column of dextran sulphate previously equilibrated with PBS (Goodger et al., 1989). The column was washed with 5 vol of PBS and bound material then eluted with PBS containing 5% NaCl. Eluate fractions with absorbances greater than 0.01 at 280 nm were pooled and applied to a 1 vol column of Phenyl Sepharose CL-4B (Pharmacia) previously 621
622
M. W.
KUNG’U
equilibrated with PBS-5% NaCl. The column was then eluted sequentially with PBS-5% NaCl, PBS and aqueous 0.1% sodium dodecyl sulphate (SDS). Absorbances of eluate fractions were obtained at 280 and 413 nm while antigenic activity of doubly diluted fractions was assessed by dot enzyme-linked immunosorbent assay (ELISA) (Hawkes, Niday & Gordon, 1982). Fractions with antigenic activity were concentrated to their original volume, their protein concentration estimated (Bradford, 1976) and then subsequently tested for vaccination efficacy. Experimental cattle and vaccination regime. Fifteen Bos zaurussteers 15-18 months old were obtained from a tick-free area and were confirmed free of babesial infections by thick film analysis (Mahoney 8z Saal, 1961), indirect fluorescent antibody test (IFAT; Goodger, 1973) and ELISA (Waltisbuhl, Goodger, Wright, Commins & Mahoney, 1987). Three groups of five cattle, designated Groups I, II, III, were used in the vaccination. Animals in Groups I and II were injected respectively with two different antigenic fractions (bound or unbound) obtained from Phenyl Sepharose chromatography. Each animal received 2 ml of antigen emulsified with 2 ml of Freund’s complete adjuvant (FCA). The injections were repeated 4 weeks later but without the FCA. Group III served as a control and were not injected with either FCA or normal erythrocyte fractions as neither induces non-specific immunity (Goodger, Commins, Wright & Mirre, 1984). All three groups were challenged 4 weeks after the second injection by intravenous injection of 1 x 10’ B. bigemina-infected erythrocytes of the virulent Townsville strain that had been stored in the vapour phase of liquid nitrogen. Vaccination parameters. Rectal temperatures and blood samples were taken daily. EDTA blood was analysed for packed cell volume (PCV) and parasitaemias estimated by thick blood film analysis. Serum samples prior to challenge were tested quantitatively for antibodies to B. bigemina by IFAT and qualitatively by immunoblotting following SDS electrophoresis of B. bigemina antigen. Sfatistical analysis. Results were recorded as mean and standard error of the mean. Differences between the means of all the vaccination parameters were tested for significance by Student’s ‘t’ test. RESULTS Analysis of antigen and antisera Figure 1 shows a typical elution pattern from Phenyl Sepharose with absorbances at 280 and 413 nm. Both the majority of the protein and practically all the 413 nm absorbing material (haemoglobin) bound avidly to the column and were eluted only with aqueous SDS. Serial dilution dot ELISA showed the unbound fraction had a titre of l/4, the PBS fraction had no titre and the SDS fraction had a titre of l/32 when reacted with bovine anti-B. bigemina serum. No reactions were noted with normal serum. After concentration, the unbound and bound fractions contained 102 and 224 pg protein per ml, respectively. The sera from both vaccination groups had mode titres of l/800 prior to challenge as assayed by IFAT.
et al.
?i
2g
0.2
B 4.
0
10
5
15
ElUatR
1. Elution pattern from a Phenyl Sepharose column of the dextran sulphate-bound B. bigemina antigens with absorbances at 280 () and 413 nm (-------). The first peak represents the unbound fraction while the last peak represents the bound fraction. The times when the PBS and the SDS were applied to the column are as indicated by the arrows. FIG.
Both antisera avidly stained parasites but in contrast there was enhanced staining of the infected erythrocyte with antisera to the unbound fraction (Fig. 2). Immunoblotting analysis following SDS electrophoresis showed that bovine antiserum to the unbound material gave moderate reactions at 5 1, 54, 62, 68 and 75 KDa (most of these were not photographically reproducible) whereas bovine antiserum to the bound material gave a strong doublet at 35 KDa and a weak band at 68 KDa (Fig. 4). No reactions were obtained with normal serum or with bovine antisera to B. bovis. Analogous fractions from normal erythrocytes gave no immunoblotting reactions with either of the B. bigemina antisera (data not shown). Vaccination results Both Group I (vaccinated with the bound fraction) and Group II (vaccinated with the unbound fraction) had lower mean daily parasitaemias than the control group throughout the experimental period, with statistically significant differences (P < 0.05) being detected on day 3 (Group I) and days 3 and 4 (Group II) (Fig. 3). There was also a significant difference (P < 0.05) in the mean daily temperature of vaccinates in Group I and the control group for days 2 and 5 while the PCV was significantly lower (P < 0.05) in control animals comparative to Group I animals on days 3 and 4 (results not shown). DISCUSSION Since it was previously shown that dextran sulphate
Dextran sulphate-binding B. bigemina antigens
623
FIG. 2a.
FIG. Zb. FIG.2. IFAT staining of B. higemina- infected erythrocytes after reaction with (a) bovine antisera to the Phenyl Slepharose-
bound fraction and (b) bovine antisera to the Phenyl Sepharose unbound fraction. Scale bars, 20.0 pm. B.
bigemina-infected
(Golodger et al., 1989) we set out to test whether
Sepharose column was based on the premise that any antigens that bound would be membrane deri ved and/
vau :ination with the babesial antigens implicated in the binding mechanism might play some role in of dextran elici sting immunity. The subfractionation lhate-binding antigen by passage through a Phenyl SUl&T
or associated and hence would be separable Iby virtue of their hydrophobic properties. The serolof $caf and vaccination results confirm this premise and indicate that the dextran sulphate-bound antigens can be
Id
bind
erythrocytes
624 16 Q o ,o m
16
‘ii
14
i
6-
3
4
5
6
7
Days after Infection FIG. 3. Mean daily parasitaemias in Group I (+-),
Group II (----) and the control group (.....) after challenge with B. ~~ge~~~a-infect~ erythrocytes.
divided into weakly and highly hydrophobic groups which contain unique antigens capable of eliciting
(a) (b) FIG. 4. Immunoblots of the (a) bound and (b) unbound antigens after reaction with bovine antisera to the Phenyl Sepharose bound and unbound fractions. respectively. The sizes of the major bands are indicated on the left (KDa).
some degree of immunity. Given the difficulty of obtaining single antigens by one stage chromatographi~ fractionation, it would have been sanguine to expect either fraction from Phenyl Sepharose to contain only a single antigen and this was confined by immunoblotting studies. The unbound fraction was more heterogeneous and further fractionation studies are needed to determine the protective moieties. In contrast, the bound fraction contained a major doublet antigen of approximately 35 KDa which is an obvious candidate for vaccine production and is presumably strongly involved in in vitro binding to dextran sulphate because of its strong hydrophobicity. As no reactions occurred with B. bovis antisera, this antigen could have a diagnostic serological role and warrants further investigation. The bound fraction also contained haemoglobin as seen by 413 nm absorbance and indicates that B. bigemina infection induces erythrocyte membran~haemoglobin binding as does B. bovis (Commins, Goodger, Waltisbuhl & Wright, 1988). This indicates probable lipid peroxidation and predisposal towards microcirculatory dysfunction and intravascular haemolysis. The results obtained in this experiment are encouraging in that the cost of cattle precluded dosage trials with either fraction and hence required us to make an estimate as to the amount of a protective vaccination dose. Our estimation was probably too low but the statistically significant differences between vaccinates and controls indicate that both antisera would be suitable for screening a B. bigemina cDNA library. Reactive antigens wili be cloned and then re-
Dextran tested
for
their
ability
to
protect
cattle
sulphate-binding against
B.
bigemina. Acknowledgements-The authors are grateful to the Australian International Development Assistance Bureau for financial support, Dr B. P. Dalrymple for reading the manuscript and Mr Kurts Rode-Bramanis for technical assistance. REFERENCES BRADFORDM. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing of protein dye-binding. Analytical the principle Biochemistry 72: 248-254. COMMINSM. A., GOODGER B. V., WALTISBUHLD. J. &WRIGHT I. G. 1988. Babesia bovis: studies of parameters influencing microvascular stasis of infected erythrocytes. Research in Veterinary Science 44: 226-228. GRUDGER B. V. 1973. Babesia argentina: intraerythrocytic location of babesial antigen extracted from parasite suspensions. International Journalfor Parasitology 3: 387391. GCODCER B. V., COMMINSM. A., WRIGHT I. G. & MIRRE G. B. 1984. Vaccination of cattle against heterologous challenge with fractions of lysate from infected erythrocytes. Zeitschrift ftir Parasitenkunde 70: 321-329. GOODGER B. V. 1989. Babesial vaccination with dead antigen. In: Veterinary Protozoan and Hemoparasite Vaccines (Edited by WRIGHT I.G.), pp. 939-941. CRC Press, Boca Raton, FL. G~ODGER B. V., BUSHELL G. R. & WRIGHT I. G. 1989. Concentration of Babesia bigemina-infected erythrocytes using a dextran sulphate affinity column. International Journal for Parasitology 19: 939-941. HAWKES R., NIDAY E. & GORDON J. 1982. A dot-immunobinding assay for monoclonal and other antibodies.
B. bigemina antigens
625
Analytical Biochemistry 119: 142-147. KUTTLER K. L. 1988. World-wide impact of babesiosis. In: Babesiosis of Domestic Animals and Man (Edited by RISTIC M.), pp. l-22. CRC Press, Boca Raton, FL. MAHONEY D. F. & SAAL J. R. 1961. Bovine babesiosis: thick blood films for the detection of parasitaemia. Australian Veterinary Journal 37: 4447. MAHONEY D. F. 1967. Bovine babesiosis: preparation and assessment of complement fixing antigens. Experimental Parasitology 20: 232-241. MCCOSKER P. J. 198 1. The global importance of babesiosis. In: Babesiosis (Edited by &TIC M. & KREIER J. P.), pp. l24. Academic Press, New York. MONTENEGRO-JAMES S., KAKOMA I. & RISTIC M. 1989. Culture-derived Babesia exoantigens as immunogens. In: Veterinary Protozoan and Hemoparasite Vaccines (Edited by WR1oH.r I. G.), pp. 61-97. CRC Press, Boca Raton, FL. I&TIC M. & KAKOMA I. 1988. Exoantigens of Babesia. In: Babesiosis of Domestic Animals and Man (Edited by RISTIC M.), pp. 131-141. CRC Press, Boca Raton, FL. WAL~ISBUHLD. J., GOODGER B. V., WRIGHT I. G., COMMINS M. A. & MAHONEY D. F. 1987. An enzyme-linked immunosorbent assay to diagnose Babesia bovis infection in cattle. Parasitology Research 73: 126-l 3 I. WRIGHT I. G., GOODGER B. V., SCHUNTNERC. A., WALTISBUHL D. J. & DUZGUN A. 1988. Use of nuclear techniques in the study of some tick-borne haemoparasitic diseases. In: Nuclear Techniques in the Study and Control qf Parasitic Diseases of Livestock. Proceedings of the Final Research Co-ordination Meeting on the Use of Nuclear Techniques in the Study and Control of Parasitic Diseases of Farm Animals, Vienna, 1987, pp. 157-172. International Atomic Energy Agency, Austria. WRIGHT I. G. & RIDDLESP. 1989. Biotechnology in tick-borne diseases: present status, future perspectives. In: Biotechnology for Livestock Production (an FAO/Plenum publication), pp. 325-340. Plenum Publishing Corporation.
OF CATTLE WITH DEXTRAN SULPHATE-BINDING BABESIA BIGEMINA ANTIGENS
M. W. KIJNG'U,* B. V. GOODGER,*~
G. R. BUSHELL,$ I. G. WRIGHT*
and D. J. WALTISBUHL*
* CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Private Bag No. 3, PO, Indooroopilly, Queensland 4068, Australia $ Griffith University, Division of Science and Technology, Nathan, Queensland 4111, Australia (Received 3 October 1991; accepted 10 February 1992)
Abstract-&.vmu M. W., GOODGER B. V., BUSHELL G. R., WRIGHT1. G. and WALTISBUHL D. J. 1992. Vaccination of cattle with dextran sulphate-binding Babes&z bigemina antigens. ~nter~utiQnu1~o~rn~~f~r Parasitology 22: 621-625. Dextran sulphate-bound Bubesiu bigemina antigens were used in a preliminary vaccination study and were shown to elicit a protective immune response in cattle. A dextran sulphatebinding fraction of B. bigemina was further subfractionated on a Phenyl Sepharose column to give two fractions-one that strongly bound to the column (bound fraction) and one that did not (unbound fraction). Two groups of cattle were each vaccinated with either the bound or the unbound fraction. These two groups of animals along with a control group were then challenged with B. bigemimz-infected erythrocytes. Both groups of vaccinated animals showed considerably lower mean daily parasitaemias as compared to the control group. INDEX KEY WORDS: Babesiu bigemina; dextran sulphate-bound antigens; vaccination.
BOVINEbabesiosis,
INTRODUCTION caused by various Babesia species,
is one of the most important diseases of cattle in tropical and subtropical countries of the world (McCosker, 1981; Kuttler, 1988; Wright & Riddles, 1989). Accepted and conventional vaccination against the disease uses attenuated living organisms, but this approach has a number of shortcomings. Various attempts have therefore been made to elicit immunity using dead vaccines consisting either of antigenic components of Babe& parasites or exoantigens derived from culture (Ristic & Kakoma, 1988; Wright, Goodger, Schuntner, Waltisbuhl & Duzgun, 1988; Goodger, 1989; Montenegro-James, Kakoma & Ristic, 1989; Wright & Riddles, 1989). Although considerable progress has been made in developing an experimental dead vaccine against B. his by workers at the CSIRO Laboratories, similar studies with Babesia bige~i~a have been hindered by the fact that B. bi~e~ina-infested erythrocytes were not as easy to concentrate as those infected with B. bovis (Mahoney, 1967). In a recent publication from this laboratory, it was reported that B. bigeemina-infected bovine erythrocytes
7 To whom all correspondence should be addressed.
could be concentrated in vitro by their selective binding to dextran sulphate columns (Goodger, Bushel1 & Wright, 1989). It was also noted that a soluble extract from erythrocytes infected with B. bigemina contained a subfraction that bound to dextran sulphate. Upon elution this fraction was shown to contain serologically detectable B. bigemina antigens. It was considered important to determine if the subfraction contained antigen(s) capable of eliciting a protective immune response; this manuscript describes preliminary vaccination studies with this fraction. MATERIALS AND METHODS Preparation of Babesia bigemina antigens for vaccination.
Blood containing approximately 20% B. bigemina-infected from serial passage in erythrocytes was obtained splenectomized calves and washed three times with phosphate-buffered saline (PBS), pH 7.2 by centrifugation at 2000 g for 15 min at 4°C. The washed, packed erythrocytes were sonically disrupted at 50-100 W for 2 min (BRAUN LABSONIC) and ultracentrifuged at 100,000 g for 60 min at 4°C. Two volumes of the resultant supernatant were applied to a 1 vol column of dextran sulphate previously equilibrated with PBS (Goodger et al., 1989). The column was washed with 5 vol of PBS and bound material then eluted with PBS containing 5% NaCl. Eluate fractions with absorbances greater than 0.01 at 280 nm were pooled and applied to a 1 vol column of Phenyl Sepharose CL-4B (Pharmacia) previously 621
622
M. W.
KUNG’U
equilibrated with PBS-5% NaCl. The column was then eluted sequentially with PBS-5% NaCl, PBS and aqueous 0.1% sodium dodecyl sulphate (SDS). Absorbances of eluate fractions were obtained at 280 and 413 nm while antigenic activity of doubly diluted fractions was assessed by dot enzyme-linked immunosorbent assay (ELISA) (Hawkes, Niday & Gordon, 1982). Fractions with antigenic activity were concentrated to their original volume, their protein concentration estimated (Bradford, 1976) and then subsequently tested for vaccination efficacy. Experimental cattle and vaccination regime. Fifteen Bos zaurussteers 15-18 months old were obtained from a tick-free area and were confirmed free of babesial infections by thick film analysis (Mahoney 8z Saal, 1961), indirect fluorescent antibody test (IFAT; Goodger, 1973) and ELISA (Waltisbuhl, Goodger, Wright, Commins & Mahoney, 1987). Three groups of five cattle, designated Groups I, II, III, were used in the vaccination. Animals in Groups I and II were injected respectively with two different antigenic fractions (bound or unbound) obtained from Phenyl Sepharose chromatography. Each animal received 2 ml of antigen emulsified with 2 ml of Freund’s complete adjuvant (FCA). The injections were repeated 4 weeks later but without the FCA. Group III served as a control and were not injected with either FCA or normal erythrocyte fractions as neither induces non-specific immunity (Goodger, Commins, Wright & Mirre, 1984). All three groups were challenged 4 weeks after the second injection by intravenous injection of 1 x 10’ B. bigemina-infected erythrocytes of the virulent Townsville strain that had been stored in the vapour phase of liquid nitrogen. Vaccination parameters. Rectal temperatures and blood samples were taken daily. EDTA blood was analysed for packed cell volume (PCV) and parasitaemias estimated by thick blood film analysis. Serum samples prior to challenge were tested quantitatively for antibodies to B. bigemina by IFAT and qualitatively by immunoblotting following SDS electrophoresis of B. bigemina antigen. Sfatistical analysis. Results were recorded as mean and standard error of the mean. Differences between the means of all the vaccination parameters were tested for significance by Student’s ‘t’ test. RESULTS Analysis of antigen and antisera Figure 1 shows a typical elution pattern from Phenyl Sepharose with absorbances at 280 and 413 nm. Both the majority of the protein and practically all the 413 nm absorbing material (haemoglobin) bound avidly to the column and were eluted only with aqueous SDS. Serial dilution dot ELISA showed the unbound fraction had a titre of l/4, the PBS fraction had no titre and the SDS fraction had a titre of l/32 when reacted with bovine anti-B. bigemina serum. No reactions were noted with normal serum. After concentration, the unbound and bound fractions contained 102 and 224 pg protein per ml, respectively. The sera from both vaccination groups had mode titres of l/800 prior to challenge as assayed by IFAT.
et al.
?i
2g
0.2
B 4.
0
10
5
15
ElUatR
1. Elution pattern from a Phenyl Sepharose column of the dextran sulphate-bound B. bigemina antigens with absorbances at 280 () and 413 nm (-------). The first peak represents the unbound fraction while the last peak represents the bound fraction. The times when the PBS and the SDS were applied to the column are as indicated by the arrows. FIG.
Both antisera avidly stained parasites but in contrast there was enhanced staining of the infected erythrocyte with antisera to the unbound fraction (Fig. 2). Immunoblotting analysis following SDS electrophoresis showed that bovine antiserum to the unbound material gave moderate reactions at 5 1, 54, 62, 68 and 75 KDa (most of these were not photographically reproducible) whereas bovine antiserum to the bound material gave a strong doublet at 35 KDa and a weak band at 68 KDa (Fig. 4). No reactions were obtained with normal serum or with bovine antisera to B. bovis. Analogous fractions from normal erythrocytes gave no immunoblotting reactions with either of the B. bigemina antisera (data not shown). Vaccination results Both Group I (vaccinated with the bound fraction) and Group II (vaccinated with the unbound fraction) had lower mean daily parasitaemias than the control group throughout the experimental period, with statistically significant differences (P < 0.05) being detected on day 3 (Group I) and days 3 and 4 (Group II) (Fig. 3). There was also a significant difference (P < 0.05) in the mean daily temperature of vaccinates in Group I and the control group for days 2 and 5 while the PCV was significantly lower (P < 0.05) in control animals comparative to Group I animals on days 3 and 4 (results not shown). DISCUSSION Since it was previously shown that dextran sulphate
Dextran sulphate-binding B. bigemina antigens
623
FIG. 2a.
FIG. Zb. FIG.2. IFAT staining of B. higemina- infected erythrocytes after reaction with (a) bovine antisera to the Phenyl Slepharose-
bound fraction and (b) bovine antisera to the Phenyl Sepharose unbound fraction. Scale bars, 20.0 pm. B.
bigemina-infected
(Golodger et al., 1989) we set out to test whether
Sepharose column was based on the premise that any antigens that bound would be membrane deri ved and/
vau :ination with the babesial antigens implicated in the binding mechanism might play some role in of dextran elici sting immunity. The subfractionation lhate-binding antigen by passage through a Phenyl SUl&T
or associated and hence would be separable Iby virtue of their hydrophobic properties. The serolof $caf and vaccination results confirm this premise and indicate that the dextran sulphate-bound antigens can be
Id
bind
erythrocytes
624 16 Q o ,o m
16
‘ii
14
i
6-
3
4
5
6
7
Days after Infection FIG. 3. Mean daily parasitaemias in Group I (+-),
Group II (----) and the control group (.....) after challenge with B. ~~ge~~~a-infect~ erythrocytes.
divided into weakly and highly hydrophobic groups which contain unique antigens capable of eliciting
(a) (b) FIG. 4. Immunoblots of the (a) bound and (b) unbound antigens after reaction with bovine antisera to the Phenyl Sepharose bound and unbound fractions. respectively. The sizes of the major bands are indicated on the left (KDa).
some degree of immunity. Given the difficulty of obtaining single antigens by one stage chromatographi~ fractionation, it would have been sanguine to expect either fraction from Phenyl Sepharose to contain only a single antigen and this was confined by immunoblotting studies. The unbound fraction was more heterogeneous and further fractionation studies are needed to determine the protective moieties. In contrast, the bound fraction contained a major doublet antigen of approximately 35 KDa which is an obvious candidate for vaccine production and is presumably strongly involved in in vitro binding to dextran sulphate because of its strong hydrophobicity. As no reactions occurred with B. bovis antisera, this antigen could have a diagnostic serological role and warrants further investigation. The bound fraction also contained haemoglobin as seen by 413 nm absorbance and indicates that B. bigemina infection induces erythrocyte membran~haemoglobin binding as does B. bovis (Commins, Goodger, Waltisbuhl & Wright, 1988). This indicates probable lipid peroxidation and predisposal towards microcirculatory dysfunction and intravascular haemolysis. The results obtained in this experiment are encouraging in that the cost of cattle precluded dosage trials with either fraction and hence required us to make an estimate as to the amount of a protective vaccination dose. Our estimation was probably too low but the statistically significant differences between vaccinates and controls indicate that both antisera would be suitable for screening a B. bigemina cDNA library. Reactive antigens wili be cloned and then re-
Dextran tested
for
their
ability
to
protect
cattle
sulphate-binding against
B.
bigemina. Acknowledgements-The authors are grateful to the Australian International Development Assistance Bureau for financial support, Dr B. P. Dalrymple for reading the manuscript and Mr Kurts Rode-Bramanis for technical assistance. REFERENCES BRADFORDM. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing of protein dye-binding. Analytical the principle Biochemistry 72: 248-254. COMMINSM. A., GOODGER B. V., WALTISBUHLD. J. &WRIGHT I. G. 1988. Babesia bovis: studies of parameters influencing microvascular stasis of infected erythrocytes. Research in Veterinary Science 44: 226-228. GRUDGER B. V. 1973. Babesia argentina: intraerythrocytic location of babesial antigen extracted from parasite suspensions. International Journalfor Parasitology 3: 387391. GCODCER B. V., COMMINSM. A., WRIGHT I. G. & MIRRE G. B. 1984. Vaccination of cattle against heterologous challenge with fractions of lysate from infected erythrocytes. Zeitschrift ftir Parasitenkunde 70: 321-329. GOODGER B. V. 1989. Babesial vaccination with dead antigen. In: Veterinary Protozoan and Hemoparasite Vaccines (Edited by WRIGHT I.G.), pp. 939-941. CRC Press, Boca Raton, FL. G~ODGER B. V., BUSHELL G. R. & WRIGHT I. G. 1989. Concentration of Babesia bigemina-infected erythrocytes using a dextran sulphate affinity column. International Journal for Parasitology 19: 939-941. HAWKES R., NIDAY E. & GORDON J. 1982. A dot-immunobinding assay for monoclonal and other antibodies.
B. bigemina antigens
625
Analytical Biochemistry 119: 142-147. KUTTLER K. L. 1988. World-wide impact of babesiosis. In: Babesiosis of Domestic Animals and Man (Edited by RISTIC M.), pp. l-22. CRC Press, Boca Raton, FL. MAHONEY D. F. & SAAL J. R. 1961. Bovine babesiosis: thick blood films for the detection of parasitaemia. Australian Veterinary Journal 37: 4447. MAHONEY D. F. 1967. Bovine babesiosis: preparation and assessment of complement fixing antigens. Experimental Parasitology 20: 232-241. MCCOSKER P. J. 198 1. The global importance of babesiosis. In: Babesiosis (Edited by &TIC M. & KREIER J. P.), pp. l24. Academic Press, New York. MONTENEGRO-JAMES S., KAKOMA I. & RISTIC M. 1989. Culture-derived Babesia exoantigens as immunogens. In: Veterinary Protozoan and Hemoparasite Vaccines (Edited by WR1oH.r I. G.), pp. 61-97. CRC Press, Boca Raton, FL. I&TIC M. & KAKOMA I. 1988. Exoantigens of Babesia. In: Babesiosis of Domestic Animals and Man (Edited by RISTIC M.), pp. 131-141. CRC Press, Boca Raton, FL. WAL~ISBUHLD. J., GOODGER B. V., WRIGHT I. G., COMMINS M. A. & MAHONEY D. F. 1987. An enzyme-linked immunosorbent assay to diagnose Babesia bovis infection in cattle. Parasitology Research 73: 126-l 3 I. WRIGHT I. G., GOODGER B. V., SCHUNTNERC. A., WALTISBUHL D. J. & DUZGUN A. 1988. Use of nuclear techniques in the study of some tick-borne haemoparasitic diseases. In: Nuclear Techniques in the Study and Control qf Parasitic Diseases of Livestock. Proceedings of the Final Research Co-ordination Meeting on the Use of Nuclear Techniques in the Study and Control of Parasitic Diseases of Farm Animals, Vienna, 1987, pp. 157-172. International Atomic Energy Agency, Austria. WRIGHT I. G. & RIDDLESP. 1989. Biotechnology in tick-borne diseases: present status, future perspectives. In: Biotechnology for Livestock Production (an FAO/Plenum publication), pp. 325-340. Plenum Publishing Corporation.
