Discovery of a recombinant Babesia canis supernatant antigen that protects dogs against virulent challenge infection.

Soluble parasite antigens (SPA) in supernatants of in vitro cultures of Babesia canis can be used to vaccinate dogs against virulent B. canis infection. The moment that immunity becomes apparent coincides with the appearance of antibodies against SPA in the serum of the vaccinated animals. This so-called vaccination-challenge serum (VC-serum) was used to precipitate antigens from B. canis culture supernatants in agarose gels. This antigen preparation was then used to analyse the reactivity of sera from vaccinated dogs on western blots. RESULTS: showed that the first appearance of antibody reactivity against a protein that migrated at the 39kDa position in SDS-PAGE gels was associated with the moment vaccinated dogs started to recover from a virulent challenge infection. In addition, pulse-chase experiments revealed that a 39-40kDa doublet was released into the supernatant of B. canis cultures starting 15min after the chase. This doublet was specifically precipitated by VC-serum, thus corroborating that the 39-40kDa doublet in SPA preparations was of parasite origin. Partial amino acid sequencing allowed the discovery of the gene that encoded the 39-40kDa doublet (canine Babesia antigen; CBA). The full-length gene was cloned and expressed in E. coli. The recombinant CBA protein (rCBA) was recognized by VC-serum, and antibodies against rCBA precipitated the 39kDa antigen of SPA preparations and of merozoites of B. canis. In addition, anti-rCBA serum reacted with the surface of B. canis merozoites (but not with B. rossi merozoites) in immunofluorescence. Vaccination of dogs with rCBA induced antibodies against rCBA, which recognized B. canis merozoites. Vaccinated dogs were protected against virulent challenge infection by limiting parasite proliferation. As a result, the development of clinical signs was prevented and the animals self-cured. In contrast, six out of seven non-vaccinated control dogs developed relatively high parasitaemia and serious clinical signs associated with poor tissue perfusion. This antigen can be used to replace the SPA antigen in the conventional B. canis vaccines, which eliminates the need for dog blood and serum for vaccine production.

Recombinant protein Bd37 protected gerbils against heterologous challenges with isolates of Babesia divergens polymorphic for the bd37 gene.

The Bd37gene encoding for a glycosyl-phosphatidyl-inositol anchored protein of Babesia divergens displays genetic polymorphisms among isolates. Five major polymorphic groups (clades) were shown by PCR-RFLP among different B. divergens isolates. Each group has been characterized according to a reference Bd37 gene (Rouen87, W8843, Y5, 6303E and 1705B). Recombinant (GST fusion) protein (Bd37r) expressed from the Bd37 gene, was used as antigen in a saponin-based formulation and was able to protect gerbils, after 2 injections at low dose vaccine concentration (1 mug per dose), against a virulent challenge with the B. divergens Rouen87 isolate. In spite of polymorphism of Bd37 gene, Bd37r induced complete immunoprotection against challenges with each of the 5 reference isolate groups defined by PCR-RFLP.

Association between sequence polymorphism in an epitope of Babesia divergens Bd37 exoantigen and protection induced by passive transfer.

In Europe, Babesia divergens is the major agent responsible for babesiosis in cattle and can occasionally infect splenectomised humans. Recently, we reported the characterisation of a 37 kDa exoantigen (Bd37) anchored in the merozoite membrane of B. divergens by a glycosylphosphatidyl-inositol. After phospholipase hydrolyse of the glycosylphosphatidyl-inositol anchor, the Bd37 antigen could be isolated in the plasma of the infected host and from the in vitro culture supernatants. Immunisation of mice with a gel-filtration protective fraction of B. divergens exoantigens, produced a monoclonal antibody (MAb), called F4.2F8-INT, directed against Bd37. In the present study, we report data on passive protection using MAb F4.2F8-INT. This MAb was able to completely protect against virulent challenges with B. divergens isolates Rouen 1987 (Rouen87) and Weybridge 8843 (W8843) but had no protective effect against another French isolate from Massif Central (6303E). Physical characterisation of the epitope recognised by F4.2F8-INT allowed us to explain the differences observed between these isolates by western blotting and passive protection. These results suggest that the antigen carrying this epitope could be used as a target in the development of a recombinant vaccine against B. divergens babesiosis.

Babesia divergens: cloning and biochemical characterization of Bd37.

The immunoprotective potential of Babesia divergens antigens released in supernatants of in vitro cultures of the parasite is generally known. Among a number of parasite molecules, a 37 kDa protein has been found in the supernatants of Babesia divergens cultures. In this report the cloning and biochemical characterization of this protein, called Bd37, are described. In addition, the processing of the protein was studied in vitro. Results suggest that Bd37 is encoded by a single copy gene. Bd37 appears to be a merozoite-associated molecule attached to the surface by a glycosylphosphatidylinositol moiety containing a palmitate residue attached to the inositol ring. In addition, it is demonstrated that both extremities of the protein are linked by a disulphide bond. Results further indicate that a soluble, hydrophilic form of Bd37 can be released from the merozoite surface by GPI-specific phospholipase D. The potential role the Bd37 protein and the GPI anchor are discussed.

Chromosome number, genome size and polymorphism of European and South African isolates of large Babesia parasites that infect dogs.

Pulsed-field gel electrophoresis of intact chromosomes from 2 isolates of each of the 2 most pathogenic species of large Babesia parasites that infect dogs, i.e. Babesia canis (European species) and B. rossi (South African species), revealed 5 chromosomes in their haploid genome. The size of chromosomes 1-5 was found to be different in the 2 species, ranging from 0.8 to 6.0 Mbp. The genome size was estimated to be approximately 14.5 Mbp for B. canis and 16 Mbp for B. rossi, respectively. Within each species, the size of chromosomes 1-3 of B. canis and 1-2 of B. rossi was conserved between the 2 isolates, whereas the size of chromosomes 4-5 of B. canis and 3-5 of B. rossi was variable. Chromosomes 1-5 hybridized with a 28-mer telomeric oligonucleotide probe derived from Plasmodium berghei. When NotI-digested chromosomes of the 4 isolates were hybridized with the telomeric probe a maximum of 10 fragments was revealed. Moreover, hybridization of this telomeric probe to a Southern blot of genomic DNA from the 4 isolates, digested with a series of restriction enzymes, revealed a species-specific restriction map. Hybridization of intact or NotI-digested chromosomes of both species with 2 sets of 3 cDNA-antigen probes derived from each species, revealed no cross-hybridization between these B. canis and B. rossi genes.

Identification and characterization of corpuscular, soluble and secreted antigens of a Venezuelan isolate of Anaplasma marginale.

Anaplasma marginale is the etiological agent of anaplasmosis, a tick-transmitted disease with an important economic impact that affects cattle throughout the world. Although, North American isolates of A. marginale and their antigens have been extensively studied, relatively little information is available on the antigenic composition of South American isolates. The characterization of diverse geographical isolates of A. marginale will result in a thorough antigenic profile and may lead to the identification of additional diagnostic and immunoprophylactic tools. Short-term cultures of a Venezuelan isolate (Ta) of A. marginale were maintained for up to 13 days in vitro. During that period, the A. marginale remained viable and were propagated in the bovine erythrocyte culture system. During the initial days of culture, cell division and reinvasion were evidenced by a significant rise in parasitemia up to a 50%. A. marginale antigens were identified by metabolic labeling with (35S) methionine, followed by fractionation and immunoprecipitation with homologous and heterologous bovine sera. This yielded a complete antigenic set for the Ta isolate of A. marginale, including soluble, secreted and corpuscular polypeptide antigens. Fifteen immunodominant polypeptides were recognized by the bovine sera in the soluble and corpuscular fractions with relative molecular weights of 200, 150, 100-110, 86, 60, 50, 47, 40, 37, 33, 31, 25, 23, 19 and 16kDa. Seven polypeptides were present in the exoantigen fraction. The 31 and 19kDa antigens were recognized by the ANAR76A1 and ANAF16C1 monoclonal antibodies, respectively which are specific for MSP-4 and MSP-5 from North American isolates of A. marginale. Metabolic labeling with (14C) glucosamine prior to immunoprecipitation with bovine sera allowed the identification of glycoprotein antigens of 200, 100-150, 60, 55, 50, 45-43, 37, 33, 31, 22, 19 and 16kDa in the soluble fraction.

Characterization and molecular cloning of an adenosine kinase from Babesia canis rossi.

In the search for immunoprotective antigens of the intraerythrocytic Babesia canis rossi parasite, a new cDNA was cloned and sequenced. Protein sequence database searches suggested that the 41-kDa protein belongs to the phosphofructokinase B type family (PFK-B). However, because of the low level sequence identity (< 20%) of the protein both with adenosine and sugar kinases from this family, its structural and functional features were further investigated using molecular modelling and enzymatic assays. The sequence/structure comparison of the protein with the crystal structure of a member of the PFK-B family, Escherichia coli ribokinase (EcRK), suggested that it might also form a stable and active dimer and revealed conservation of the ATP-binding site. However, residues specifically involved in the ribose-binding sites in the EcRK sequence (S and N) were substituted in its sequence (by H and M, respectively), and were suspected of binding adenosine compounds rather than sugar ones. Enzymatic assays using a purified glutathione S-transferase fusion protein revealed that this protein exhibits rapid catalysis of the phosphorylation of adenosine with an apparent Km value of 70 nM, whereas it was inactive on ribose or other carbohydrates. As enzymatic assays confirmed the results of the structure/function analysis indicating a preferential specificity towards adenosine compounds, this new protein of the PFK-B family corresponds to an adenosine kinase from B. canis rossi. It was named BcrAK.

Secreted antigens of the amastigote and promastigote forms of Leishmania infantum inducing a humoral response in humans and dogs.

To study the antigens secreted by promastigote and amastigote forms of Leishmania infantum which are able to induce a humoral response in human patients and dogs, we have carried out immunoprecipitation assays with different supernatants of in vitro cultured parasites, metabolically labelled with [35S]methionine, using serum samples from human patients and dogs. In addition, some metabolic labelling experiments were performed daily during the in vitro culture parasite's life cycle to follow the time course excretion-secretion of parasitic antigens. The results demonstrated that the two different hosts developed an antibody response against secreted antigens of both stages of Leishmania infantum. Nevertheless, the humoral response directed against the excreted-secreted antigens of the promastigote forms was qualitatively and quantitatively different when we compare the human and the dog immune responses. On the other hand, when the excreted-secreted antigens of the amastigote forms are immunoprecipitated with either human or canine immune serum, the humoral response is similar. In addition, the time course study showed that excretion-secretion of antigens was qualitatively and quantitatively modulated during the parasitic in vitro life cycle.

Babesia canis canis, Babesia canis vogeli, Babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal RNA genes.

The parasites Babesia canis and Babesia gibsoni (phylum Apicomplexa) are responsible for canine babesiosis throughout the world. Babesia canis was previously described as a group of three biologically different subspecies, namely B. canis canis, B. canis vogeli, and B. canis rossi. We report partial sequences of small subunit ribosomal RNA gene (ssu-rDNA) of each subspecies amplified in vitro with primers derived from a semi-conserved region of the ssu-rDNA genes in other Babesia species. The polymerase chain reaction combined with a restriction fragment length polymorphism analysis, using HinfI and TaqI restriction enzymes, confirmed the separation of B. canis into three subspecies. These sequences were compared with previously published sequences of other Babesia species. A phylogenetic approach showed that the three subspecies of B. canis belong to the clade of Babesia species sensu stricto where B. canis canis clusters with B. canis rossi whereas B. canis vogeli might form a monophyletic group with the cluster B. divergens and B. odocoilei. Our results show that the three subspecies of B. canis can readily be differentiated at the molecular level and suggest that they might be considered as true species.

Human babesiosis.

The first demonstrated case of human babesiosis in the world was reported in Europe, in 1957. Since then, a further 28 babesial infections in man have been reported in Europe. Most (83%) of the infections were in asplenic individuals and most (76%) were with Babesia divergens, a cattle parasite. Parasitaemias varied from 1%-80% of red blood cells. The usual clinical manifestations of severe B. divergens infection were severe intravascular haemolysis with haemoglobinuria. The most efficient treatment consisted of a massive blood-exchange transfusion, followed immediately by chemotherapy with clindamycin. Hundreds of cases of human infection with Babesia spp. have been reported in the U.S.A. Most cases were infected by ticks carrying the rodent parasite B. microti, but other emerging. Babesia spp. (currently known as WA1, CA1, and MO1) are increasingly involved. Several cases were the result of blood transfusion. In terms of clinical manifestations, human infections with B. microti varied widely, from asymptomatic infection to a severe, rapidly fatal disease. Parasitaemia ranged between <1% and 85%. The splenectomized, the elderly, the immunocompromised and HIV-infected patients were predisposed to severe infection. Infection with B. microti often remained subclinical or asymptomatic and were only detected through serological surveys. The currently recommended treatment of symptomatic cases is quinine plus clindamycin. A few other cases of human babesial infection have been described in China, Egypt, Mexico, South Africa and Taiwan.

Babesia hylomysci and B. divergens: presence of antioxidant enzymes destroying hydrogen peroxide.

Catalase and glutathione peroxidase (Gpx), two enzymes destroying hydrogen peroxide, were reported in two Babesia species: B. divergens cultivated in vitro and B. hylomysci obtained in vivo. On the use of specific substrate and inhibitor, we confirmed that the Gpx activity detected was selenium-dependent. Moreover, the two Babesia species contain glutamate dehydrogenase activity. This enzyme is capable of providing to the cell the reduced nicotinamide adenine dinucleotide phosphate (NADPH) necessary for regeneration of the reduced glutathione. Gpx activity is weaker in B. divergens than in B. hylomysci and seems to be compensated by higher levels of catalase activity. Such a balance between the two enzymes may depend on the selenium concentration available for the parasite.

Different Babesia canis isolates, different diseases.

Using surface immunofluorescence isolate-specific antigens were detected on the membrane of erythrocytes infected with Babesia parasites. In addition, the strains reacted differently with Plasmagel in that the European isolate (B.c. canis) could be purified on Plasmagel effectively, whereas infected erythrocytes of the South-African isolate (B.c. rossi) could not. Experimental infection of dogs with Babesia canis isolates from geographically different areas revealed different pathology. The European isolate obtained from France exhibited transient parasitaemia, usually below 1%, associated with low PCV values and congestion of internal organs. Clinical disease was correlated with an effect on the coagulation system, and not with peripheral parasitaemia. Infection of dogs with South-African-derived isolate induced high parasitaemia usually much higher than 1%, which required chemotherapeutic treatment. In these animals clinical disease was correlated with peripheral parasitaemia and not with parameters of the coagulation system. The results show that the etiology of disease caused by these isolates of B.c. canis and B.c. rossi is different. This might have implications for the development of vaccines against these infections.

Survey of Babesia divergens antibody kinetics in cattle in western France.

Data collected from a longitudinal survey carried out over a 2-year period, in four dairy herds in western France, were used to assess Babesia divergens antibody kinetics. Farms were visited once a month. The total number of animals monitored was 236 including calves, heifers and cows of the Holstein and Normande breeds. An ELISA was used to follow the humoral response levels against Babesia divergens. When the study began, in the autumn of 1991 (200 animals), half of the animals were already seropositive (57.5%). In all four herds, the percentage of positive animals decreased during winter, and increased during spring. Antibody levels remained stable in 49 animals, high for some, very low or negative for others. Most seropositive animals showed at least one antibody peak at some time during the 2-year period, but some presented two to five. Among the calves, 61.3% showed seroconversion during the first pasture season. Similarly, 60% of the newly purchased cows showed increases in antibody levels during the 3 months after their introduction into a new herd, regardless of the initial antibody level. Only three dairy cows had expressed a clinical babesiosis, these cows were already seropositive. Clinical incidence is low in the four farms, nevertheless serological prevalence and incidence are high.

Continuous in vitro culture of Babesia divergens in a serum-free medium.

Babesia divergens was cultivated in RPMI 1640 (25 mM HEPES) supplemented with 10% human serum (RPMI-10% HS) with a high percentage of parasitized erythrocytes (PPE) (> or = 40%). Standardization of in vitro tests, purification of exoantigens, biochemical studies and the safety of the culture handler motivated the development of a serum-free defined medium. Removal of serum greatly reduced the PPE but, after a period of adaptation, the culture was continuous and the parasite was able to develop a 3% routine PPE. Addition of vitamins or reduced glutathione in basal medium (RPMI) did not improve the PPE. The supplementation of basal medium with lipidic carrier (Albumax I or bovine serum albumin-Cohn's fraction V) promoted the growth of B. divergens with high PPE (> 30%) close to those obtained in RPMI-10% HS. Neither protein nor lipid fractions alone were able to restore the growth of B. divergens. Nevertheless, the whole lipid fraction from serum or Albumax I added to delipidated albumin partially restored the growth (7% PPE), indicating that the presentation of specific lipids by a carrier is crucial for the parasite. All the data indicate that Albumax I can replace human serum offering the advantages of safety, standardization for chemosensitivity tests, and exoantigen purification.

A fatal case of babesiosis in Missouri: identification of another piroplasm that infects humans.

OBJECTIVE: To characterize the etiologic agents (MO1) of the first reported case of babesiosis acquired in Missouri. DESIGN: Case report, serologic testing, animal inoculations, and molecular studies. SETTING: Southeastern Missouri. PATIENT: A 73-year-old man who had had a splenectomy and had a fatal case of babesiosis. MEASUREMENTS: Serum specimens from the patient were assayed by indirect immunofluorescent antibody testing and immunoprecipitation for reactivity with antigens from various Babesia species. Whole blood obtained from the patient before treatment was inoculated into hamsters and jirds and into calves and bighorn sheep that had had splenectomy and were immunosuppressed with dexamethasone. Piroplasm-specific nuclear small-subunit ribosomal DNA was recovered from the patient's blood by using broad-range amplification with the polymerase chain reaction; a 144 base-pair region of the amplification product was sequenced; and phylogenetic analysis was done to compare MO1 with various Babesia species. RESULTS: Indirect immunofluorescent antibody testing showed that the patient's serum had strong reactivity with Babesia divergens, which causes babesiosis in cattle and humans in Europe, but that it had minimal reactivity with B. microti and WA1, which are the piroplasms previously known to cause zoonotic babesiosis in the United States. Immunoprecipitations showed that MO1 is more closely related to B. divergens than to B. canis (a canine parasite). None of the experimentally inoculated animals became demonstrably parasitemic. Phylogenetic analyses, after DNA sequencing, showed that MO1 is most closely related to B. divergens (100% similarity). CONCLUSIONS: Although MO1 is probably distinct from B. divergens, the two share morphologic, antigenic, and genetic characteristics; MO1 probably represents a Babesia species not previously recognized to have infected humans. Medical personnel should be aware that patients in the United States can have life-threatening babesiosis even though they are seronegative to B. microti and WA1 antigen.

Babesia divergens: an ELISA with soluble parasite antigen for monitoring the epidemiology of bovine babesiosis.

An enzyme linked immunosorbent assay (ELISA) for bovine babesiosis caused by Babesia divergens was developed to analyse the evolution of the serological status of cattle living in an enzootic area. The antigen used was a soluble extract of B. divergens obtained from in vitro culture. Specificity, evaluated with negative sera, was 96.6%. The ELISA was compared to indirect immunofluorescence analysis (IFA) on naturally or experimentally infected animals. It appeared that IFA was positive for at least seven or eight weeks; on the contrary, B. divergens-specific antibodies were only transitorily detected by ELISA. Furthermore, the ELISA was more sensitive than the IFA for the detection of post-infection antibody increases, particularly when infection-pressure was low. These results suggest that IFA and ELISA could be complementary tools in epidemiological surveys; indeed, this ELISA could be the most efficient tool to detect new infections in cohort monitoring.

A 37-kilodalton glycoprotein of Babesia divergens is a major component of a protective fraction containing low-molecular-mass culture-derived exoantigens.

The supernatants of in vitro cultures of Babesia divergens Rouen 1987 in human erythrocytes, obtained by using a semidefined medium based on human high-density lipoproteins, were fractionated by gel filtration chromatography into four fractions, F1 to F4. The crude supernatant as well as each fraction adjuvanted with Quil-A protected gerbils from mortality due to a homologous infectious challenge. Analysis of the humoral response of the 10 protected gerbils with fraction F4, containing major proteins with molecular masses lower than 50 kDa, showed that a few antigens (from 50 to 17 kDa) could be important candidates for an improved vaccine against B. divergens babesiosis. As an immunodominant response was directed against the 37-kDa antigen (Bd37) in two different B. divergens strains tested, a polyclonal antibody directed against Bd37 was produced in a rabbit. In an immunofluorescence assay, the anti-Bd37 antiserum strongly labelled small internal vesicles of the merozoites and the cell surface was diffusely labelled after fixation, whereas on live merozoites, this labelling was not observed. [3H]glucosamine-radiolabelling experiments demonstrate that Bd37 is a glycoprotein. The Bd37 protein can also be labelled with [14C]palmitate but not with [3H]myristic acid. In Triton X-114 temperature phase partitioning of B. divergens-infected erythrocyte extracts, Bd37 was exclusively found into the detergent phase, indicating that the palmitoylated Bd37 protein was in the membrane fraction. In the in vitro supernatant, the glycoprotein Bd37 was found in a nonpalmitoylated form, indicating excretion and/or release of the glycoprotein from the merozoite.

Characterization of a new 60 kDa apical protein of Plasmodium falciparum merozoite expressed in late schizogony.

Immunological cross-reactivity studies between the Apicomplexa Babesia divergens and Plasmodium falciparum allowed us to identify a P falciparum 60 kDa protein (Pf60) using an antiserum directed against a B divergens 37 kDa culture-derived exoantigen. In immunofluorescence assays (IFA), Pf60 appears as a doublet of fluorescent spots associated to the apical pole of merozoites. The doublet co-locates with two rhoptry components: the protein RAP-1 and the 140/130/110 (105) kDa rhoptry protein complex suggesting the rhoptry location of Pf60. The biosynthesis of Pf60, established by labeling experiments with [35S]methionine on synchronized cultures, and by immunofluorescence detection, occurred during late schizogony. The physico-chemical properties of Pf60, the absence of identified precursor forms and the absence of co-precipitation with other proteins indicated a new class of rhoptry protein. Pf60 was detected in all the different geographic P falciparum strains so far tested, with a slight variability in molecular mass ranging from 58 to 60 kDa. During the invasion process of erythrocytes by merozoites, the IFA showed the presence of the Pf60 in the apex of free merozoites, but not in invading merozoite, as well as in new ring-infected erythrocytes. Furthermore, immunoprecipitation assays indicated the presence of Pf60 in the culture medium, and its absence in new ring-infected erythrocytes. All together these results suggest a possible involvement of the Pf60 protein in the invasion process.

Babesia divergens: characterization of a 17-kDa merozoite membrane protein.

Large amounts of viable merozoites were purified from in vitro cultures of Babesia divergens by a two-step sieving procedure. A monoclonal antibody produced against B. divergens merozoites, mAb DG7, stained the merozoite plasma membrane and an intra-parasitic linear organelle in indirect immunofluorescence. Immunogold labeling in electron microscopy demonstrated that the antigen recognized by mAb DG7 was localized just beneath the merozoite plasma membrane. Immunoprecipitations of metabolically labeled ([35S]methionine) B. divergens antigens showed that the epitope recognized by mAb DG7 was present on a 17-kDa polypeptide (Bd17) and was shared in all B. divergens geographical isolates tested so far. Bd17 was always present in the in vitro culture supernatants of all these isolates. Furthermore, Triton X-114 phase separation of babesial antigens demonstrated the hydrophilic character of Bd17 which suggests that it is an extrinsic protein present on the cytosol side of the parasite membrane. When added to the culture medium, mAb DG7, purified from ascite fluids, drastically altered the growth of the parasite with concentrations inhibiting 50% of development (IC50) ranging between 16.6 and 26.1 micrograms/ml).

Cellular and humoral immune responses induced in cattle by vaccination with Babesia divergens culture-derived exoantigens correlate with protection.

Previous results with the Babesia divergens gerbil vaccination model were extended in studies with cattle. Two calves were vaccinated with culture-derived B. divergens exoantigens, and two others were treated with control supernatant; both preparations were adjuvanted with Quil-A saponin. A parasite-specific humoral response was observed after the first vaccine injection and was boosted by two succeeding vaccine injections. Sera from the two vaccinated calves immunoprecipitated eight major parasitic proteins (with molecular masses ranging between 17 and 110 kDa) whose patterns were close to those observed in gerbil vaccine assays. The cellular immune response, monitored by lymphoproliferation assays, was slightly delayed in comparison with the humoral response; a significant proliferation occurred only after the second vaccine injection. Mononuclear cell proliferation was dose dependent in the presence of (i) lysates of B. divergens-parasitized erythrocytes, (ii) exoantigens of the whole supernatant, or (iii) protective exoantigens of two low-molecular-mass fractions obtained after supernatant gel filtration chromatography. An infectious challenge was administered 3 weeks after the third vaccine injection, with 3.6 x 10(10) B. divergens-parasitized erythrocytes. Erythrocyte count, rectal temperature, and parasitemia of the animals were monitored daily until they returned to initial values. All parameters indicated that the exoantigens induced protection from B. divergens infection for the two vaccinated calves.