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Induction of protective immunity to Babesia divergens in Mongolian gerbils, Meriones unguiculatus, using culture-derived immunogens
Veterinary Parasitology, 26 (1987) 43-53 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
43
Induction of Protective Immunity to Babesia divergens in Mongolian Gerbils, Meriones unguiculatus, using Culture-Derived I m m u n o g e n s CAROLINE M.WINGER, ELIZABETH U. CANNING and J.D. CULVERHOUSE
Department o[ Biology, Imperial College, London SW7 2AZ ( Gt. Britain) ( Accepted for publication 16 March 1987)
ABSTRACT Winger, C.M., Canning, E.U. and Culverhouse, J.D., 1987. Induction of protective immunity to Babesta dtvergens in Mongolian gerbils, Mermnes unguiculatus, using culture-derived immunogens. Vet Parasttol, 26: 43-53. Immunogensderived from microaerophilous stationary phase (MASP) cultures of Babesta dtvergens grown in bovine erythrocytes were used to inoculate the laboratory host of B dwergens, the Mongolian gerbil, Mertones ungutculatus Ammals inoculated subcutaneously twice with preparations of freeze-thawed merozoites in complete Freund's adjuvant were fully protected against homologous challenge, as were gerbils immunised with a non-viable preparation of parasite-enriched lysed infected bovine erythrocytes. Animals which had been infected with small numbers of parasitised erythrocytes from cultures cooled to 4 ° C, allowed to recover, then challenged, also survived. All three groups had high antibody titres which dropped immediately after challenge and then rose again. Gerbils given culture supernatants containing soluble merozoite protein coat antigens were partmlly protected only after receiving a third inoculation. Non-lmmunisedanimals all died 4 days after challenge.
INTRODUCTION
In attempts to protect cattle from clinical disease due to species of Babesia, both live parasites and products from killed parasites have been investigated. The most widely used vaccines are those consisting of live parasites which have been attenuated by passage through splenectomised calves. These have been used in Australia for several years to protect against B.bovis and B. bigemina ( Callow, 1977). Protection can also be achieved by inoculation of irradiated parasites. Thus, Mahoney et al. (1973) obtained good protection against challenge with B. argentina ( --B. bovis). However, the irradiating dose is important: strong protection was conferred when low doses of irradiation, which inactivated the majority but not all of the parasites, were used but there was only partial pro0304-4017/87/$03.50
© 1987 Elsevier Scmnce Publishers B.V.
44 tection when the inoculum was treated with high doses of irradiation. Similar results have been achieved with B. major (Purnell et al. 1978) and with B. dwergens ( Lewis et al., 1979; Taylor et al., 1980 ). Inoculation of the attenuated and irradiated organisms results in mild parasitaemia and the animals become premune and are resistant to challenge. A disadvantage of the premunisation is that carrier animals may serve as sources of infection to non-vaccinated animals. Sterile immunity in cattle lasting at least 6 months has been induced by inoculation of B.bigemina or B boris, followed by chemosterilization ( Callow et al., 1974 ). Even with this system there remains the disadvantage that some vaccinated animals might develop severe infections. This disadvantage would be overcome if effective non-living vaccines were available. Mahoney and Wright (1976) found that a crude antigen, consisting of disrupted infected erythrocytes, conferred a high level of protection against challenge with a heterologous strain of B. boris. Also, a soluble antigen extracted from B. boris-infected erythrocytes protected cattle against homologous challenge (Mahoney et al., 1981 ) and some protection against B. divergens was given by fractions of parasitised erythrocytes (particularly the acidic fraction ) separated by isoelectric focussing ( Taylor et al., 1984 ). Wright et al., (1983, 1985) have used a monoclonal-derived antigen of B. boris and obtained complete protection against clinical disease when the vaccinated animals were challenged with a virulent homologous strain. Methods for in vitro culture of Babesia spp., in particular the microaerophilous stationary phase system (MASP), devised for B. bovis by Levy and Ristic (1980) have facilitated the collection and preparation of antigens for immunisation trials. Studies of the antigens from culture-derived merozoites have indicated that the major determinants associated with protective immunity may be those of the surface coat (Montenegro-James et al., 1983). The merozoite protein coat is sloughed off during the process of entry of merozoites into erythrocytes and accumulates as soluble exoantigen in the culture supernatant (James et al., 1981). The immunogenic properties of some culture-derived antigens have been studied in recent years. Smith and Ristic (1981) used non-viable merozoites, obtained from cultures of B. boris, and induced resistance in cattle to tickborne challenge 4 months after vaccination. Merozoite protein coat antigens obtained from the supernatant of MASP cultures have been used in vaccination trials in cattle with some success against homologous challenge ( Smith et al., 1981; Kuttler et al., 1982; Timms et al., 1983) and heterologous challenge (Montenegro-James et al., 1985). The susceptibility of the Mongolian gerbil, Meriones unguiculatus to the cattle parasite, B. divergens was discovered during investigation of a case of human babesiosis caused by B. divergens (Entrican et al., 1979; Lewis and Williams, 1979; Liddell et al., 1980). In this study we have used antigens prepared
45 from MASP cultures of B. divergens in an immunication trial in gerbils, which serve as convenient laboratory models for B. divergens infections in cattle. MATERIALS AND METHODS
Animals Gerbils were obtained from Intersimian Ltd. Animals aged between 12 and 18 weeks and of both sexes were used. All animals were healthy at the commencement of the trial. Babesia divergens strain A strain ofB. divergens, which had been obtained from a splenectomised calf at the Central Veterinary Laboratory, Weybridge, Surrey, had been adapted to MASP culture ( K o n r a d et al., 1984; Canning and Winger, 1987) and maintained through 100 passages. Cultures were grown in 25-cm 2 flasks in M199 (Gibco) and 40% bovine serum (Seralab) at 38°C in an atmosphere of 5% CO2 in air. Parasitaemias were monitored in Giemsa-stained blood smears every 24 h. Culture medium was replaced 24 h after subculture and subsequently twice daily. Subcultures were made after 72 h, or when a parasitaemia of 10-12% had been achieved, by making a 1 in 10 dilution of the infected cells into fresh normal bovine erythrocytes.
Antigen preparation Culture supernatants Culture supernatants were harvested when the cultures were at peak parasitaemia (10-12%). These were centrifuged at 10 000 g for 20 min and passed through a 0.22 z m filter to remove any particulate matter, especially merozoites.
Merozoites Merozoites were obtained by CO2 deprivation of MASP cultures of B. divergens with a parasitaemia of 10-12%. Cultures were placed in a desiccator over soda lime ( BDH Chemicals ) for 6-8 h at 38 ° C ( J. Konrad, personal communication, 1985). Merozoites which had accumulated in the culture supern a t a n t were partially purified, using a modification of the method described by Levy and Ristic (1980), to remove intact red cells and m a n y of the red cell membrane contaminants. Thus, 50 ml of culture were harvested and centrifuged at 2 0 0 x g for 10 min at 4°C. The upper 35 ml of supernatant were retained and centrifuged at 400 X g for 10 min at 4 ° C. Then, 30 ml of supernatant were retained and centrifuged at 1000×g for 20 min at 4 ° C. The pellet was resuspended in 15 ml of M199 and centrifuged at 1000 × g for 20 min at 4°C to
46
remove any remaining bovine serum. The pellet, which consisted of merozoites partially contaminated with erythrocyte ghosts, was again resuspended in two volumes of M199 and the merozoites were stored in aliquots at - 2 0 ° C.
Parasite-enriched lysis preparation ( PELP ) MASP cultures orB. divergens, at 10-12% parasitaemia, were centrifuged at 750 Xg for 5 min. After removal of the supernatant, the cells were resuspended in five volumes of phosphate buffered saline ( P B S ) , which had been diluted 1:1 with deionised distilled water, in order to effect hypotonic lysis of the more fragile infected cells. The preparation was incubated at 37" C for 5 min to complete the lysis before 10 volumes of isotonic PBS were added. The intact cells were centrifuged at 200 Xg for 10 min at 4 ~C and the supernatant was removed down to the last 2 ml. The supernatant was centrifuged at 400 x g for 10 min at 4 c C. After removal of all but the last 2 ml, the supernatant was then centrifuged at 1000 Xg for 20 min at 4 oC. The pellet, which consisted of extracellular parasites and erythrocyte membranes from infected erythrocytes, was resuspended in two volumes of M199 and stored in aliquots at - 20 ° C.
Cooled cultures MASP cultures at 1.0% parasitaemia were kept at 4 oC for I week before use.
Inoculation regimen Gerbils were divided into six groups of four. Serum was obtained from all groups before inoculation, in order to establish baseline antibody titres. Rectal temperatures were also taken. All animals were anaesthetised by an intramusuclar inoculation of 0.1 ml Rompun (Beyer) and 0.05 ml Vetalar (ParkeDavis) prior to immunisation. Group I was injected subcutaneously in the flank with an oil in water emulsion of 0.1 ml MASP culture supernatant and 0.1 ml of complete Freund's adjuvant (CFA) on Days 1, 21 and 42. Group 2 was injected subcutaneously in the flank with an oil in water emulsion of 0.1 ml MASP culture supernatant and 0.1 ml CFA on Days 21 and 42. Group 3 was injected subcutaneously in the flank with an oil in water emulsion of 0.1 ml of the merozoite preparation (containing 6 × 107 merozoites) and 0.1 ml CFA on Days 21 and 42. Group 4 was injected subcutaneously with an oil in water emulsion of 0.1 ml of P E L P and 0.1 ml CFA on Days 21 and 42. Group 5 was injected intraperitoneally with 1.8 × 106 B. divergens-infected erythrocytes from a cooled MASP culture on Day 1. Blood smears were taken daily until the animals had completely recovered from the infection. Group 6 (control group) was injected subcutaneously in the flank with an oil in water emulsion of 0.1 ml M199 and 0.1 ml CFA on Days 21 and 42.
47
All groups were challenged on Day 63 with 5.0 × 10 s B. divergens-infected erythrocytes of the homologous strain from M A S P culture. The erythrocytes were washed three times and injected intraperitoneally in 0.5 ml M199.
Experimental procedures Before challenge, gerbils were bled from the tail at weekly intervals. The blood was allowed to coagulate, and the serum was collected, diluted I in 20 in P B S and stored at - 2 0 ° C. After challenge, gerbils were bled daily for 10 days to obtain serum. Rectal temperatures were measured and Giemsa-stained blood smears were made. Percentage parasitaemias were obtained from the number of parasites in 1000 cells in stained smears. Attempts to estimate packed cell volumes were abandoned because removal of the volume required for the haematocrit tubes altered the cell concentration in the animals. Surviving animals in Group 5, the group which had been inoculated with live parasites from cooled cultures, were tested 9 months after challenge for possible persistence of parasites. Heparinised blood from the gerbils was pooled and 200 pl was injected into each of two normal gerbils. The Group 5 animals were then injected subcutaneously with 450 #g Dexafort (InterVet) in 0.15 ml. Blood smears were taken on Days 0, 1, 2, 3, 10 and 14, and examined for parasites. All animals were challenged with an intraperitoneal injection of 5 × 108 infected bovine erythrocytes from culture 1 month later, i.e. 10 months after initial infection, the same dose being given to two control gerbils which had not previously been exposed to infection.
Enzyme linked immunosorbent assay (ELISA ) Rigid polystyrene E L I S A plates (Flow) comprised of 96 wells were coated with 100 #l of a sonicated merozoite preparation diluted 1 in 10 in PBS. The assay was performed as described by Voller et al. (1976) using alkaline phosphatase-conjugated sheep anti-mouse immunoglobulin G ( S a M I g G ) (Sigma) and nitrophenyl phosphate (Sigma) as the substrate. S a M I g G was used as it was found to cross-react strongly with gerbil IgG, thus eliminating the necessity to raise an antiserum to gerbil Ig. Sera from each animal were titrated out to 1:81 920. RESULTS
Three animals died as a result of anaesthetic shock during the trial, one from each of Groups 1, 2 and 3.
48 Culture sup X 2
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Fig. 1. Antibody titres and parasitaemias m immumsed and control gerbils after challenge with 5 X l0 s B dwergens-infected erythrocytes of the homologous ( Weybridge ) strain.
Febrile reactions The baseline rectal temperatures taken immediately before challenge ranged from 35.4-38.7 ° C. There was a tendency towards increase in temperature up to the 3rd day post challenge in all groups but there was a marked drop to temperatures well below normal (26.9-33.3 °C) immediately before death. In the survivors the temperatures had reverted to normal by the 4th day.
Parasitaemias (Fig. 1) Parasitaemias of 27.5-46.3% were recorded in animals immunised with two doses of culture supernatant and in the control animals on the day of death. Two of three animals which received three immunising doses of supernatant survived, one of which had negligible parasitaemia and the other had 12% parasitaemia before recovery. The 3rd animal died with 35.2% parasitaemia. In the groups immunised with merozoites, P E L P and cooled cultures, parasitaemias were mainly negligible but two animals in the P E L P group attained 4% and 8% parasitaemia. When blood from two animals surviving 9 months after receiving blood from cooled cultures (Group 5 ) was transferred into normal gerbils, no infections were etablished. The original animals were solidly immune when challenged 1
49 113-
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Fig. 2. Pre-chaUenge antibody titres in gerbils immumsed with culture derived preparations of
Babesia dwergens
month later but the passage animals and controls, when challenged with the same dose, all died on the 5th day with parasitaemias of up to 51%. In addition, no infections arose when the original animals were treated with Dexafort.
Antibody titres (Figs. 1 and 2) In all groups, antibody titres reached their peaks at Weeks 7 and 8, then fell prior to challenge. The groups immunised with merozoites and P E L P showed a decrease in antibody titre on the 1st day after challenge. This was followed by a rise in titre from the 2nd day. There was little immediate change in antibody titres in the remaining test groups from the pre-challenge level, b u t all except the group immunised with two doses of culture supernatant showed a rise after the 2nd day.
Survival rates All control animals and the group immunised with two doses of culture supernatant were dead on the 5th day after challenge infection. One animal in
50 TABLE I Survival rates of immunised and control gerbils after challenge with Babesla dwergens Group
No. of animals to survive challenge
1 2 3 4 5 6
2/3 0/4 3/3 4/4 4/4 0/4
Culture supernatant ( × 3 ) Culture supernatant ( × 2 ) Merozoites PELP Cooled culture Control
the group immunised with three doses of culture supernatant died 5 days after the challenge. All animals in the groups receiving merozoites, P E L P and cooled culture survived the challenge (Table I). DISCUSSION
In this paper we have demonstrated the immunogenic properties of several
B. divergens immunogens derived from M A S P cultures. Soluble exoantigens of B. divergens from culture supernatants, unlike those of B. boris, ( Smith et a1.,1981; Kuttler et al., 1982; T i m m s et al., 1983) do not appear to provide adequate protection even to homologous challenge infection. Even three inoculations of culture supernatant protected only two out of three animals. The parasitaemias achieved by B. divergens in M A S P cultures are far lower than those achieved in cultures of B. bovis and a lower concentration of antigens in the supernatant could account for the failure of B. divergens supernatants to provide adequate protection. However, in a later immunisation trial, 10 times concentrated culture supernatant failed to provide any protection after two immunisations (C.M. Winger, E.U. Canning and J.D. Culverhouse, unpublished results, 1986), so the difference in antigen concentration may not be the reason for the difference in efficacy. Protein coat antigens of B. divergens appear to be very labile and will only retain their immunogenicity in an ELISA when stored at 4 °C for up to 3 days. Since protein coat antigens were collected in the culture supernatant over a period of 72 h at 38°C and were processed for a further 3 h before use, it is possible that a large proportion of the soluble antigens had been degraded. BSA (1%) was used to block E L I S A plates after the addition of antigen, and antibodies reacting to this were present in the sera from the groups immunised with culture supernatant. Blocking with goat serum failed to reduce this background. An E L I S A carried out using BSA as the antigen demonstrated the presence of antibodies to BSA with titres as high as 1/1280 in these groups. No
51
antibody to BSA was observed in any other groups. The high antibody titres to BSA observed in animals immunised with culture supernatants would be due mainly to heavy contamination of the immunogen with bovine albumin in the serum component of the culture medium. Effective immunity was induced in the animals immunised with the freeze-thawed merozoite preparation. These results are in agreement with those of Smith and Ristic (1981) who used non-viable merozoites of B. boris to immunise cattle. Further evidence for the immunogenicity of merozoite-derived antigens was obtained by Winger et al. (1986 } who induced partial protection of gerbils to homologous challenge by immunisation with an affinity purified 50-60 KD B. divergens merozoite antigen. The parasite-enriched cell lysis preparation also afforded protection against homologous challenge, as would be expected from an immunogen rich in parasite material and infected erythrocyte membranes, on which parasite antigens are possibly deposited (Schroeder and Ristic, 1968). Immunisation with a small number of intra-erythrocytic parasites from cooled cultures induced parasitaemias of up to 2% on the 3rd day after infection, but no parasites were evident 5 days later. Nine months after the challenge, no parasites were seen in blood smears taken both before and after the animals were immunosuppressed by treatment with dexamethasone, so the immunity was judged to be a true sterile immunity. This was confirmed by failure to induce a parasitaemia in two gerbils which received an inoculation of blood from the immunised animals. When all gerbils were given a subsequent challenge infection, the animals which had been immunised with cooled cultures survived the challenge. Passive transfer of whole blood from this group did not protect the two recipients and they succumbed to challenge infection. CONCLUSIONS
Vaccines currently in use againt B.divergens are based on live infected cattle blood and carry the risk that undetected viral or bacterial infections in the donor will be transmitted to the recipients. Immunogens obtained from cultures can be grown in bovine blood from a pathogen-free animal, thus eliminating this risk. The high proportion of host cell products in blood-derived vaccines can be decreased by using a merozoite preparation purified on a Percoll gradient (Rodriguez et al., 1986; X.-S. Jiang, C.M. Winger and E.U. Canning, unpublished data, 1986) in which intact erythrocytes are absent and erythrocyte membranes are reduced to a minimun. Vaccines of this type carry a lower risk of inducing an autoantibody response. With these advantages, culture-derived immunogens may have potential as vaccines as long as the cultures can be scaled up to commercial levels and they may be able to replace animal-derived
52
vaccines before non-living vaccines can be developed by recombinant DNA technology. ACKNOWLEDGEMENTS
We are pleased to acknowledge the generous financial support of the Wellcome Trust and are grateful to the Central Veterinary Laboratory, Weybridge, for regular supplies of fresh cattle blood, and to Mr. J. Donelly of the Central Veterinary Laboratory for provision of the Weybridge strain of B. divergens.
REFERENCES Callow, L.L.,1977. Vaccination against bovine babesiosls Adv. Exp. Med. and Biol., 93: 121-149. Callow, L.L., McGregor, W , Parker, R.J. and Dalghesh, R.J,. 1974. Immunity of cattle to Babes~a b~gemma following its elimination from the host, with observations on antibody levels detected by indirect fluorescent antibody test. Aust. Vet. J., 50: 12-15. Canning, E.U. and Winger, C.M., 1987. Babesiidae In: A.E.R. Taylor and J.R. Baker (Editors), Methods of cultivating parasites m vitro. Academic Press, New York, London, pp. 199-229. Entrican, J.H., Williams, H., Cook, I.A., Lancaster, W.M., Clark, J.C., Joyner, L.P. and Lewis, D., 1979. Babesiosis in man: report of a case from Scotland with observations on the infecting strain. J. Infect., 1: 227-234. James, M.A., Levy, M.G. and Ristic, M., 1981. Isolation and partial charactemsation of culture derived soluble Babesta bovis antigens. Infect. Immun., 31: 385-391. Konrad, J., Phlpps, L.P., Canning, E.U. and Donelly, J., 1984. Long term in vitro maintenance of Babesia dwergens m a stationary phase culture. Parasitology, 89: xxvi (abstract). Kuttler, K L., Levy, M.G., James, M.A. and Ristic, M., 1982. Efficacy of a non-viable culture demved Babes~a bovls vaccine. Am. J. Vet. Res., 43: 281-284. Levy, M.G. and Ristlc, M., 1980. Babesla bovts, continuous cultivation m microaerophilus stationary phase culture. Scmnce, 207:1218-1220. Lewis, D. and Williams, H., 1979. Infection of the Mongohan gerbil with the cattle plroplasm Babesm dwergens. Nature {London), 278: 170-171. Lewis, D., Purnell, R.E. and Brocklesby, D.W., 1979. Babesm divergens: the immunisatlon of splenectomised calves using irradiated piroplasms. Res. Vet Sci., 26: 220-222. Liddell, K.G., Lucas, S.B. and Williams, H., 1980. Babes~a dwergens infections in the Mongolian gerbil: characteristics of the human strain. Parasitology, 82: 205-224. Mahoney, D.F. and Wright, I.G., 1976. Babes~a argentina Immumsation of cattle with a killed antigen against infection with a heterologous strain. Vet. Parasltol., 2: 273-282. Mahoney, D.F., Wright, I.G. and Ketterer, P.J., 1973. Babes~a argentina: the infectivity and immunogenicity of irradiated blood parasites. Int. J Parasltol., 3:209-217 Mahoney, D.F., Wright, I.G. and Goodger, B.V., 1981. Bovine babesmsis. The immunisation of cattle with fractions of erythrocytes infected with Babes~a bows (Syn. B argentina). Vet. Immun. ImmunopathoL, 2: 145-146. Montenegro-James, S., James, M.A. and Ristlc, M., 1983. Localisation of culture-derived soluble Babesia bows antigens in the infected erythrocytes. Vet. Parasitol., 13: 311-316. Montenegro-James, S., Ristic, M., Benitez, M.T., Leon, E. and Lopez, R., 1985. Heterologous strain immunity in bovine babesiosis using a culture-derived soluble Babes~a boy,s immunogen. Vet Pars~tol., 18: 321-337.
53 Purnell, R.E., Brocklesby, D.W. and Stark, A.J., 1978. Protection of cattle against Babesm major by the inoculation of irradiated piroplasms. Res. Vet. Sci, 25: 388-390. Rodriguez, S.D., Buening, G.M., Vega, C.A. and Carson, C.A., 1986. Babesla bovts: purification and concentration of merozoites and infected bovine erythrocytes. Exp. Parasitol., 61: 236-243. Schroeder, W.F. and Ristic, M., 1968. Autoimmune response and pathogenesis of blood parasite disease. In: D. Weinman and M. Ristic (Editors), Infectious Blood Disease of Man and Animals. Academic Press, New York/London, pp. 63-77. Smith, R.D. and Ristic, M., 1981. Immunisation against bovine babesiosis with culture derived antigens. In: M. Ristic and J.P. Kreier, (Editors), Babesiosis. Academic Press, New York/ London, pp. 485-507. Smith, R.D., James, M.A., Ristic, M., Aikawa,M., Vega, Y. and Murguia, C.A., 1981. Bovine babesiosis: protection of cattle with culture-derived soluble Babesm bows antigen. Science, 212: 335-338. Taylor, S.M., Kenny, J., PurneU, R.E. and Lewis, D., 1980. Exposure of cattle immunised against redwater to tick challenge in the field: challenge by a homologous strain of B dwergens Vet. Rec., 106: 167-170. Taylor, S.M., Kenny, J., Mallon, T. and Elliott, C.T, 1984. The immunisation of cattle against Babesm dwergens with fractions of parasitised erythrocytes. Vet. Parasltol., 16: 235-242. Timms, P., Dalgleish, R.J., Barry, D.N., Dimmock, C.K. and Rodwell, B.J., 1983. Babesm bows: comparison of culture derived parasites, non-living antigen and conventional vaccine in the protection of cattle against heterologous challenge. Aust. Vet. J., 60: 75-77. Voller, A,. Bldwell, D.E. and Bartlett, A,. 1976. Enzyme ~mmunoassays in diagnostic medicine. Theory and Practice. Bull. W.H.O., 53: 3418-3421. Winger, C.M., Canning, E.U. and Culverhouse, J.D, 1987. A monoclonal antibody to Babesla dwergens which inhibits merozoite invasion. Parasitology, 94: 17-27. Wright, I.G,. White, M., Tracey-Patte, P.D., Donaldson, R.A, Goodger, B.V, WalUsbuhl, D.J. and Mahoney, D.F., 1983. Babes~a bows: isolation of a protective antigen by using monoclonal antibodies. Infect. Immun., 41: 244-250. Wright, I.G., Mirre, G.B., Rode-Bramanis, K., Chamberlain, M., Goodger, B.V. and Waltisbuhl, D.J., 1985. Protective vaccination against virulent Babesta boy,s with a low molecular weight antigen. Infect. Immun., 48: 109-113.
43
Induction of Protective Immunity to Babesia divergens in Mongolian Gerbils, Meriones unguiculatus, using Culture-Derived I m m u n o g e n s CAROLINE M.WINGER, ELIZABETH U. CANNING and J.D. CULVERHOUSE
Department o[ Biology, Imperial College, London SW7 2AZ ( Gt. Britain) ( Accepted for publication 16 March 1987)
ABSTRACT Winger, C.M., Canning, E.U. and Culverhouse, J.D., 1987. Induction of protective immunity to Babesta dtvergens in Mongolian gerbils, Mermnes unguiculatus, using culture-derived immunogens. Vet Parasttol, 26: 43-53. Immunogensderived from microaerophilous stationary phase (MASP) cultures of Babesta dtvergens grown in bovine erythrocytes were used to inoculate the laboratory host of B dwergens, the Mongolian gerbil, Mertones ungutculatus Ammals inoculated subcutaneously twice with preparations of freeze-thawed merozoites in complete Freund's adjuvant were fully protected against homologous challenge, as were gerbils immunised with a non-viable preparation of parasite-enriched lysed infected bovine erythrocytes. Animals which had been infected with small numbers of parasitised erythrocytes from cultures cooled to 4 ° C, allowed to recover, then challenged, also survived. All three groups had high antibody titres which dropped immediately after challenge and then rose again. Gerbils given culture supernatants containing soluble merozoite protein coat antigens were partmlly protected only after receiving a third inoculation. Non-lmmunisedanimals all died 4 days after challenge.
INTRODUCTION
In attempts to protect cattle from clinical disease due to species of Babesia, both live parasites and products from killed parasites have been investigated. The most widely used vaccines are those consisting of live parasites which have been attenuated by passage through splenectomised calves. These have been used in Australia for several years to protect against B.bovis and B. bigemina ( Callow, 1977). Protection can also be achieved by inoculation of irradiated parasites. Thus, Mahoney et al. (1973) obtained good protection against challenge with B. argentina ( --B. bovis). However, the irradiating dose is important: strong protection was conferred when low doses of irradiation, which inactivated the majority but not all of the parasites, were used but there was only partial pro0304-4017/87/$03.50
© 1987 Elsevier Scmnce Publishers B.V.
44 tection when the inoculum was treated with high doses of irradiation. Similar results have been achieved with B. major (Purnell et al. 1978) and with B. dwergens ( Lewis et al., 1979; Taylor et al., 1980 ). Inoculation of the attenuated and irradiated organisms results in mild parasitaemia and the animals become premune and are resistant to challenge. A disadvantage of the premunisation is that carrier animals may serve as sources of infection to non-vaccinated animals. Sterile immunity in cattle lasting at least 6 months has been induced by inoculation of B.bigemina or B boris, followed by chemosterilization ( Callow et al., 1974 ). Even with this system there remains the disadvantage that some vaccinated animals might develop severe infections. This disadvantage would be overcome if effective non-living vaccines were available. Mahoney and Wright (1976) found that a crude antigen, consisting of disrupted infected erythrocytes, conferred a high level of protection against challenge with a heterologous strain of B. boris. Also, a soluble antigen extracted from B. boris-infected erythrocytes protected cattle against homologous challenge (Mahoney et al., 1981 ) and some protection against B. divergens was given by fractions of parasitised erythrocytes (particularly the acidic fraction ) separated by isoelectric focussing ( Taylor et al., 1984 ). Wright et al., (1983, 1985) have used a monoclonal-derived antigen of B. boris and obtained complete protection against clinical disease when the vaccinated animals were challenged with a virulent homologous strain. Methods for in vitro culture of Babesia spp., in particular the microaerophilous stationary phase system (MASP), devised for B. bovis by Levy and Ristic (1980) have facilitated the collection and preparation of antigens for immunisation trials. Studies of the antigens from culture-derived merozoites have indicated that the major determinants associated with protective immunity may be those of the surface coat (Montenegro-James et al., 1983). The merozoite protein coat is sloughed off during the process of entry of merozoites into erythrocytes and accumulates as soluble exoantigen in the culture supernatant (James et al., 1981). The immunogenic properties of some culture-derived antigens have been studied in recent years. Smith and Ristic (1981) used non-viable merozoites, obtained from cultures of B. boris, and induced resistance in cattle to tickborne challenge 4 months after vaccination. Merozoite protein coat antigens obtained from the supernatant of MASP cultures have been used in vaccination trials in cattle with some success against homologous challenge ( Smith et al., 1981; Kuttler et al., 1982; Timms et al., 1983) and heterologous challenge (Montenegro-James et al., 1985). The susceptibility of the Mongolian gerbil, Meriones unguiculatus to the cattle parasite, B. divergens was discovered during investigation of a case of human babesiosis caused by B. divergens (Entrican et al., 1979; Lewis and Williams, 1979; Liddell et al., 1980). In this study we have used antigens prepared
45 from MASP cultures of B. divergens in an immunication trial in gerbils, which serve as convenient laboratory models for B. divergens infections in cattle. MATERIALS AND METHODS
Animals Gerbils were obtained from Intersimian Ltd. Animals aged between 12 and 18 weeks and of both sexes were used. All animals were healthy at the commencement of the trial. Babesia divergens strain A strain ofB. divergens, which had been obtained from a splenectomised calf at the Central Veterinary Laboratory, Weybridge, Surrey, had been adapted to MASP culture ( K o n r a d et al., 1984; Canning and Winger, 1987) and maintained through 100 passages. Cultures were grown in 25-cm 2 flasks in M199 (Gibco) and 40% bovine serum (Seralab) at 38°C in an atmosphere of 5% CO2 in air. Parasitaemias were monitored in Giemsa-stained blood smears every 24 h. Culture medium was replaced 24 h after subculture and subsequently twice daily. Subcultures were made after 72 h, or when a parasitaemia of 10-12% had been achieved, by making a 1 in 10 dilution of the infected cells into fresh normal bovine erythrocytes.
Antigen preparation Culture supernatants Culture supernatants were harvested when the cultures were at peak parasitaemia (10-12%). These were centrifuged at 10 000 g for 20 min and passed through a 0.22 z m filter to remove any particulate matter, especially merozoites.
Merozoites Merozoites were obtained by CO2 deprivation of MASP cultures of B. divergens with a parasitaemia of 10-12%. Cultures were placed in a desiccator over soda lime ( BDH Chemicals ) for 6-8 h at 38 ° C ( J. Konrad, personal communication, 1985). Merozoites which had accumulated in the culture supern a t a n t were partially purified, using a modification of the method described by Levy and Ristic (1980), to remove intact red cells and m a n y of the red cell membrane contaminants. Thus, 50 ml of culture were harvested and centrifuged at 2 0 0 x g for 10 min at 4°C. The upper 35 ml of supernatant were retained and centrifuged at 400 X g for 10 min at 4 ° C. Then, 30 ml of supernatant were retained and centrifuged at 1000×g for 20 min at 4 ° C. The pellet was resuspended in 15 ml of M199 and centrifuged at 1000 × g for 20 min at 4°C to
46
remove any remaining bovine serum. The pellet, which consisted of merozoites partially contaminated with erythrocyte ghosts, was again resuspended in two volumes of M199 and the merozoites were stored in aliquots at - 2 0 ° C.
Parasite-enriched lysis preparation ( PELP ) MASP cultures orB. divergens, at 10-12% parasitaemia, were centrifuged at 750 Xg for 5 min. After removal of the supernatant, the cells were resuspended in five volumes of phosphate buffered saline ( P B S ) , which had been diluted 1:1 with deionised distilled water, in order to effect hypotonic lysis of the more fragile infected cells. The preparation was incubated at 37" C for 5 min to complete the lysis before 10 volumes of isotonic PBS were added. The intact cells were centrifuged at 200 Xg for 10 min at 4 ~C and the supernatant was removed down to the last 2 ml. The supernatant was centrifuged at 400 x g for 10 min at 4 c C. After removal of all but the last 2 ml, the supernatant was then centrifuged at 1000 Xg for 20 min at 4 oC. The pellet, which consisted of extracellular parasites and erythrocyte membranes from infected erythrocytes, was resuspended in two volumes of M199 and stored in aliquots at - 20 ° C.
Cooled cultures MASP cultures at 1.0% parasitaemia were kept at 4 oC for I week before use.
Inoculation regimen Gerbils were divided into six groups of four. Serum was obtained from all groups before inoculation, in order to establish baseline antibody titres. Rectal temperatures were also taken. All animals were anaesthetised by an intramusuclar inoculation of 0.1 ml Rompun (Beyer) and 0.05 ml Vetalar (ParkeDavis) prior to immunisation. Group I was injected subcutaneously in the flank with an oil in water emulsion of 0.1 ml MASP culture supernatant and 0.1 ml of complete Freund's adjuvant (CFA) on Days 1, 21 and 42. Group 2 was injected subcutaneously in the flank with an oil in water emulsion of 0.1 ml MASP culture supernatant and 0.1 ml CFA on Days 21 and 42. Group 3 was injected subcutaneously in the flank with an oil in water emulsion of 0.1 ml of the merozoite preparation (containing 6 × 107 merozoites) and 0.1 ml CFA on Days 21 and 42. Group 4 was injected subcutaneously with an oil in water emulsion of 0.1 ml of P E L P and 0.1 ml CFA on Days 21 and 42. Group 5 was injected intraperitoneally with 1.8 × 106 B. divergens-infected erythrocytes from a cooled MASP culture on Day 1. Blood smears were taken daily until the animals had completely recovered from the infection. Group 6 (control group) was injected subcutaneously in the flank with an oil in water emulsion of 0.1 ml M199 and 0.1 ml CFA on Days 21 and 42.
47
All groups were challenged on Day 63 with 5.0 × 10 s B. divergens-infected erythrocytes of the homologous strain from M A S P culture. The erythrocytes were washed three times and injected intraperitoneally in 0.5 ml M199.
Experimental procedures Before challenge, gerbils were bled from the tail at weekly intervals. The blood was allowed to coagulate, and the serum was collected, diluted I in 20 in P B S and stored at - 2 0 ° C. After challenge, gerbils were bled daily for 10 days to obtain serum. Rectal temperatures were measured and Giemsa-stained blood smears were made. Percentage parasitaemias were obtained from the number of parasites in 1000 cells in stained smears. Attempts to estimate packed cell volumes were abandoned because removal of the volume required for the haematocrit tubes altered the cell concentration in the animals. Surviving animals in Group 5, the group which had been inoculated with live parasites from cooled cultures, were tested 9 months after challenge for possible persistence of parasites. Heparinised blood from the gerbils was pooled and 200 pl was injected into each of two normal gerbils. The Group 5 animals were then injected subcutaneously with 450 #g Dexafort (InterVet) in 0.15 ml. Blood smears were taken on Days 0, 1, 2, 3, 10 and 14, and examined for parasites. All animals were challenged with an intraperitoneal injection of 5 × 108 infected bovine erythrocytes from culture 1 month later, i.e. 10 months after initial infection, the same dose being given to two control gerbils which had not previously been exposed to infection.
Enzyme linked immunosorbent assay (ELISA ) Rigid polystyrene E L I S A plates (Flow) comprised of 96 wells were coated with 100 #l of a sonicated merozoite preparation diluted 1 in 10 in PBS. The assay was performed as described by Voller et al. (1976) using alkaline phosphatase-conjugated sheep anti-mouse immunoglobulin G ( S a M I g G ) (Sigma) and nitrophenyl phosphate (Sigma) as the substrate. S a M I g G was used as it was found to cross-react strongly with gerbil IgG, thus eliminating the necessity to raise an antiserum to gerbil Ig. Sera from each animal were titrated out to 1:81 920. RESULTS
Three animals died as a result of anaesthetic shock during the trial, one from each of Groups 1, 2 and 3.
48 Culture sup X 2
113
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--1
MerozoJtes
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i 20
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Control
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~
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"
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Days post Challenge Infect,on
Fig. 1. Antibody titres and parasitaemias m immumsed and control gerbils after challenge with 5 X l0 s B dwergens-infected erythrocytes of the homologous ( Weybridge ) strain.
Febrile reactions The baseline rectal temperatures taken immediately before challenge ranged from 35.4-38.7 ° C. There was a tendency towards increase in temperature up to the 3rd day post challenge in all groups but there was a marked drop to temperatures well below normal (26.9-33.3 °C) immediately before death. In the survivors the temperatures had reverted to normal by the 4th day.
Parasitaemias (Fig. 1) Parasitaemias of 27.5-46.3% were recorded in animals immunised with two doses of culture supernatant and in the control animals on the day of death. Two of three animals which received three immunising doses of supernatant survived, one of which had negligible parasitaemia and the other had 12% parasitaemia before recovery. The 3rd animal died with 35.2% parasitaemia. In the groups immunised with merozoites, P E L P and cooled cultures, parasitaemias were mainly negligible but two animals in the P E L P group attained 4% and 8% parasitaemia. When blood from two animals surviving 9 months after receiving blood from cooled cultures (Group 5 ) was transferred into normal gerbils, no infections were etablished. The original animals were solidly immune when challenged 1
49 113-
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control • cooled culture LJ PELP cs
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Fig. 2. Pre-chaUenge antibody titres in gerbils immumsed with culture derived preparations of
Babesia dwergens
month later but the passage animals and controls, when challenged with the same dose, all died on the 5th day with parasitaemias of up to 51%. In addition, no infections arose when the original animals were treated with Dexafort.
Antibody titres (Figs. 1 and 2) In all groups, antibody titres reached their peaks at Weeks 7 and 8, then fell prior to challenge. The groups immunised with merozoites and P E L P showed a decrease in antibody titre on the 1st day after challenge. This was followed by a rise in titre from the 2nd day. There was little immediate change in antibody titres in the remaining test groups from the pre-challenge level, b u t all except the group immunised with two doses of culture supernatant showed a rise after the 2nd day.
Survival rates All control animals and the group immunised with two doses of culture supernatant were dead on the 5th day after challenge infection. One animal in
50 TABLE I Survival rates of immunised and control gerbils after challenge with Babesla dwergens Group
No. of animals to survive challenge
1 2 3 4 5 6
2/3 0/4 3/3 4/4 4/4 0/4
Culture supernatant ( × 3 ) Culture supernatant ( × 2 ) Merozoites PELP Cooled culture Control
the group immunised with three doses of culture supernatant died 5 days after the challenge. All animals in the groups receiving merozoites, P E L P and cooled culture survived the challenge (Table I). DISCUSSION
In this paper we have demonstrated the immunogenic properties of several
B. divergens immunogens derived from M A S P cultures. Soluble exoantigens of B. divergens from culture supernatants, unlike those of B. boris, ( Smith et a1.,1981; Kuttler et al., 1982; T i m m s et al., 1983) do not appear to provide adequate protection even to homologous challenge infection. Even three inoculations of culture supernatant protected only two out of three animals. The parasitaemias achieved by B. divergens in M A S P cultures are far lower than those achieved in cultures of B. bovis and a lower concentration of antigens in the supernatant could account for the failure of B. divergens supernatants to provide adequate protection. However, in a later immunisation trial, 10 times concentrated culture supernatant failed to provide any protection after two immunisations (C.M. Winger, E.U. Canning and J.D. Culverhouse, unpublished results, 1986), so the difference in antigen concentration may not be the reason for the difference in efficacy. Protein coat antigens of B. divergens appear to be very labile and will only retain their immunogenicity in an ELISA when stored at 4 °C for up to 3 days. Since protein coat antigens were collected in the culture supernatant over a period of 72 h at 38°C and were processed for a further 3 h before use, it is possible that a large proportion of the soluble antigens had been degraded. BSA (1%) was used to block E L I S A plates after the addition of antigen, and antibodies reacting to this were present in the sera from the groups immunised with culture supernatant. Blocking with goat serum failed to reduce this background. An E L I S A carried out using BSA as the antigen demonstrated the presence of antibodies to BSA with titres as high as 1/1280 in these groups. No
51
antibody to BSA was observed in any other groups. The high antibody titres to BSA observed in animals immunised with culture supernatants would be due mainly to heavy contamination of the immunogen with bovine albumin in the serum component of the culture medium. Effective immunity was induced in the animals immunised with the freeze-thawed merozoite preparation. These results are in agreement with those of Smith and Ristic (1981) who used non-viable merozoites of B. boris to immunise cattle. Further evidence for the immunogenicity of merozoite-derived antigens was obtained by Winger et al. (1986 } who induced partial protection of gerbils to homologous challenge by immunisation with an affinity purified 50-60 KD B. divergens merozoite antigen. The parasite-enriched cell lysis preparation also afforded protection against homologous challenge, as would be expected from an immunogen rich in parasite material and infected erythrocyte membranes, on which parasite antigens are possibly deposited (Schroeder and Ristic, 1968). Immunisation with a small number of intra-erythrocytic parasites from cooled cultures induced parasitaemias of up to 2% on the 3rd day after infection, but no parasites were evident 5 days later. Nine months after the challenge, no parasites were seen in blood smears taken both before and after the animals were immunosuppressed by treatment with dexamethasone, so the immunity was judged to be a true sterile immunity. This was confirmed by failure to induce a parasitaemia in two gerbils which received an inoculation of blood from the immunised animals. When all gerbils were given a subsequent challenge infection, the animals which had been immunised with cooled cultures survived the challenge. Passive transfer of whole blood from this group did not protect the two recipients and they succumbed to challenge infection. CONCLUSIONS
Vaccines currently in use againt B.divergens are based on live infected cattle blood and carry the risk that undetected viral or bacterial infections in the donor will be transmitted to the recipients. Immunogens obtained from cultures can be grown in bovine blood from a pathogen-free animal, thus eliminating this risk. The high proportion of host cell products in blood-derived vaccines can be decreased by using a merozoite preparation purified on a Percoll gradient (Rodriguez et al., 1986; X.-S. Jiang, C.M. Winger and E.U. Canning, unpublished data, 1986) in which intact erythrocytes are absent and erythrocyte membranes are reduced to a minimun. Vaccines of this type carry a lower risk of inducing an autoantibody response. With these advantages, culture-derived immunogens may have potential as vaccines as long as the cultures can be scaled up to commercial levels and they may be able to replace animal-derived
52
vaccines before non-living vaccines can be developed by recombinant DNA technology. ACKNOWLEDGEMENTS
We are pleased to acknowledge the generous financial support of the Wellcome Trust and are grateful to the Central Veterinary Laboratory, Weybridge, for regular supplies of fresh cattle blood, and to Mr. J. Donelly of the Central Veterinary Laboratory for provision of the Weybridge strain of B. divergens.
REFERENCES Callow, L.L.,1977. Vaccination against bovine babesiosls Adv. Exp. Med. and Biol., 93: 121-149. Callow, L.L., McGregor, W , Parker, R.J. and Dalghesh, R.J,. 1974. Immunity of cattle to Babes~a b~gemma following its elimination from the host, with observations on antibody levels detected by indirect fluorescent antibody test. Aust. Vet. J., 50: 12-15. Canning, E.U. and Winger, C.M., 1987. Babesiidae In: A.E.R. Taylor and J.R. Baker (Editors), Methods of cultivating parasites m vitro. Academic Press, New York, London, pp. 199-229. Entrican, J.H., Williams, H., Cook, I.A., Lancaster, W.M., Clark, J.C., Joyner, L.P. and Lewis, D., 1979. Babesiosis in man: report of a case from Scotland with observations on the infecting strain. J. Infect., 1: 227-234. James, M.A., Levy, M.G. and Ristic, M., 1981. Isolation and partial charactemsation of culture derived soluble Babesta bovis antigens. Infect. Immun., 31: 385-391. Konrad, J., Phlpps, L.P., Canning, E.U. and Donelly, J., 1984. Long term in vitro maintenance of Babesia dwergens m a stationary phase culture. Parasitology, 89: xxvi (abstract). Kuttler, K L., Levy, M.G., James, M.A. and Ristic, M., 1982. Efficacy of a non-viable culture demved Babes~a bovls vaccine. Am. J. Vet. Res., 43: 281-284. Levy, M.G. and Ristlc, M., 1980. Babesla bovts, continuous cultivation m microaerophilus stationary phase culture. Scmnce, 207:1218-1220. Lewis, D. and Williams, H., 1979. Infection of the Mongohan gerbil with the cattle plroplasm Babesm dwergens. Nature {London), 278: 170-171. Lewis, D., Purnell, R.E. and Brocklesby, D.W., 1979. Babesm divergens: the immunisatlon of splenectomised calves using irradiated piroplasms. Res. Vet Sci., 26: 220-222. Liddell, K.G., Lucas, S.B. and Williams, H., 1980. Babes~a dwergens infections in the Mongolian gerbil: characteristics of the human strain. Parasitology, 82: 205-224. Mahoney, D.F. and Wright, I.G., 1976. Babes~a argentina Immumsation of cattle with a killed antigen against infection with a heterologous strain. Vet. Parasltol., 2: 273-282. Mahoney, D.F., Wright, I.G. and Ketterer, P.J., 1973. Babes~a argentina: the infectivity and immunogenicity of irradiated blood parasites. Int. J Parasltol., 3:209-217 Mahoney, D.F., Wright, I.G. and Goodger, B.V., 1981. Bovine babesmsis. The immunisation of cattle with fractions of erythrocytes infected with Babes~a bows (Syn. B argentina). Vet. Immun. ImmunopathoL, 2: 145-146. Montenegro-James, S., James, M.A. and Ristlc, M., 1983. Localisation of culture-derived soluble Babesia bows antigens in the infected erythrocytes. Vet. Parasitol., 13: 311-316. Montenegro-James, S., Ristic, M., Benitez, M.T., Leon, E. and Lopez, R., 1985. Heterologous strain immunity in bovine babesiosis using a culture-derived soluble Babes~a boy,s immunogen. Vet Pars~tol., 18: 321-337.
53 Purnell, R.E., Brocklesby, D.W. and Stark, A.J., 1978. Protection of cattle against Babesm major by the inoculation of irradiated piroplasms. Res. Vet. Sci, 25: 388-390. Rodriguez, S.D., Buening, G.M., Vega, C.A. and Carson, C.A., 1986. Babesla bovts: purification and concentration of merozoites and infected bovine erythrocytes. Exp. Parasitol., 61: 236-243. Schroeder, W.F. and Ristic, M., 1968. Autoimmune response and pathogenesis of blood parasite disease. In: D. Weinman and M. Ristic (Editors), Infectious Blood Disease of Man and Animals. Academic Press, New York/London, pp. 63-77. Smith, R.D. and Ristic, M., 1981. Immunisation against bovine babesiosis with culture derived antigens. In: M. Ristic and J.P. Kreier, (Editors), Babesiosis. Academic Press, New York/ London, pp. 485-507. Smith, R.D., James, M.A., Ristic, M., Aikawa,M., Vega, Y. and Murguia, C.A., 1981. Bovine babesiosis: protection of cattle with culture-derived soluble Babesm bows antigen. Science, 212: 335-338. Taylor, S.M., Kenny, J., PurneU, R.E. and Lewis, D., 1980. Exposure of cattle immunised against redwater to tick challenge in the field: challenge by a homologous strain of B dwergens Vet. Rec., 106: 167-170. Taylor, S.M., Kenny, J., Mallon, T. and Elliott, C.T, 1984. The immunisation of cattle against Babesm dwergens with fractions of parasitised erythrocytes. Vet. Parasltol., 16: 235-242. Timms, P., Dalgleish, R.J., Barry, D.N., Dimmock, C.K. and Rodwell, B.J., 1983. Babesm bows: comparison of culture derived parasites, non-living antigen and conventional vaccine in the protection of cattle against heterologous challenge. Aust. Vet. J., 60: 75-77. Voller, A,. Bldwell, D.E. and Bartlett, A,. 1976. Enzyme ~mmunoassays in diagnostic medicine. Theory and Practice. Bull. W.H.O., 53: 3418-3421. Winger, C.M., Canning, E.U. and Culverhouse, J.D, 1987. A monoclonal antibody to Babesla dwergens which inhibits merozoite invasion. Parasitology, 94: 17-27. Wright, I.G,. White, M., Tracey-Patte, P.D., Donaldson, R.A, Goodger, B.V, WalUsbuhl, D.J. and Mahoney, D.F., 1983. Babes~a bows: isolation of a protective antigen by using monoclonal antibodies. Infect. Immun., 41: 244-250. Wright, I.G., Mirre, G.B., Rode-Bramanis, K., Chamberlain, M., Goodger, B.V. and Waltisbuhl, D.J., 1985. Protective vaccination against virulent Babesta boy,s with a low molecular weight antigen. Infect. Immun., 48: 109-113.