I.G. Wright

The development of a recombinant Babesia vaccine.

Crude extracts of Babesia bovis parasites were shown to induce levels of protection in susceptible cattle equivalent to that resulting from natural infection. The crude material was systematically fractionated and tested in numerous sequential vaccination/challenge experiments in adult cattle. Antigens in protective fractions were then purified by affinity chromatography with monoclonal antibodies. Three highly protective (more than 95% reduction in parasitaemias) antigens were thus identified. None of these antigens was immunodominant; a number of immunodominant antigens were identified and all were immunosuppressive and/or non-protective. The three protective antigens were cloned and expressed as either beta-galactosidase or glutathione-S-transferase (GST) fusion proteins. Two of these, GST-12D3 and GST-11C5, when used in combination were almost as protective as has been previously shown for the commercially available live attenuated vaccine. A short fragment of a third antigen (21B4) has also been shown to be protective. In two of the antigens, repetitive segments have been shown to be non-protective while the third antigen (12D3) does not contain repetitive domains. Homologues of these antigens exist in other Babesia species and it is anticipated that these may be candidate antigens for protective vaccines against those species.

An enzyme linked immunosorbent assay to diagnose Babesia bovis infection in cattle.

The optimum gel filtration fraction from lysate of Babesia bovis infected erythrocytes was determined for use as an antigen in an ELISA to diagnose B. bovis infection in cattle. Of four enzyme labels tested, horseradish peroxidase was the most suitable. The assay is both sensitive and specific in detecting antibody for 2–4 years after a single infection. False positive reactions were obtained only with some sera from some Anaplasma marginale infected cattle.

Characterisation of a family of multi-copy genes encoding rhoptry protein homologues in Babesia bovis, Babesia ovis and Babesia canis

A monoclonal antibody that had been raised against a protease-containing fraction of Babesia bovis, and shown to bind to a protein located in the rhoptries, was used to screen a B. bovis cDNA expression library. The sequence of the protein encoded by a positive clone was almost identical to the equivalent region of a previously described B. bovis 60-kDa rhoptry protein (Bv60). A tandem repeat of the gene encoding Bv60 was identified in all Australian isolates of B. bovis examined. Genes encoding homologous of Bv60 were cloned from Babesia ovis and Babesia canis. In B. ovis, 5 closely linked genes were identified. Four of these genes appeared to encode very similar proteins (Bo60.1-4). The protein (Bo60.5) encoded by the fifth B. ovis gene had 72% amino acid identity to Bo60.1-4 in the amino-terminal 306 amino acids, but no significant similarities in the carboxy-terminal region. In B. canis one gene (Bc60.2) was sequenced and a second closely linked gene was identified. A further member of the family, p58, has also been described previously from Babesia bigemina. Tandemly repeated genes subject to extensive gene conversion appear to be a feature of this family of babesial rhoptry protein homologous. No proteins significantly related to any members of the gene family were identified in a search of translated DNA and protein sequence databases. Thus the function of this family of proteins remains a matter for speculation.

The immune response of cattle to Babesia bovis (syn. B. argentina). Studies on the nature and specificity of protection

Abstract Studies of the immune response to Babesia bovis (syn. B. argentina ) in Bos taurus cattle, using the passive transfer of serum from immune animals, indicated that an effector mechanism was mediated by antibodies which reacted with the parasitized erythrocytes. During removal from the peripheral blood, the parasites did not show reduced viability on subinoculation into other non-infected animals, and thus were not dead or irreversibly damaged at this time. It was concluded that opsonization of infected erythrocytes was probably the basis of protection by the system. There was some evidence that minor variation of the protective antigen(s) occurred within strains of the parasite but this had little effect on the efficiency of the hosts immune response. However, there was no cross-protection between the antibodies against different strains. These interstrain differences in antibody specificity were reconciled with earlier observations that cross-immunity commonly occurs between different strains in infected animals. It was concluded that the mechanism of cross-immunity relied on priming of the hosts immune system by the protective antigen(s) of the strain so that a secondary response against the heterologous strain occurred soon after challenge.

An improved ELISA for the detection of antibodies against Babesia bovis using either a native or a recombinant B. bovis antigen.

Two new enzyme-linked immunosorbent assayes (ELISA) for the diagnosis ofBabesia bovis in cattle are described. The ELISA using a native antigen is more sensitive and less laborious than the assays described previously, because it does not require adsorption of sera with bovine erythrocytes. The second ELISA, using a recombinantB. bovis antigen expressed inEscherichia coli, was both sensitive and specific. It is suitable to replace the native antigen, thus avoiding large batch-to-batch variations in antigen preparations and the need to sacrifice experimental cattle.

Bovine babesiosis: The immunization of cattle with fractions of erythrocytes infected with Babesia bovis (syn B. argentina)

Soluble antigen which protected susceptible cattle against challenge with Babesia bovis was extracted from B. bovis-infected erythrocytes by sonic disintegration and separation of the soluble from the insoluble matter by ultracentrifugation. The material was then fractionated by the precipitation of fibrinogen-like proteins. The precipitate contained the babesial antigens that were located on the stroma of the infected erythrocytes. Antigen originally located on the parasite remained in solution. Both fractions conferred protection on splenectomized calves against challenge with B. bovis. However, the fraction containing the parasite antigens appeared to have more potential for development as a killed vaccine because it was not heavily contaminated with antigenic material from bovine erythrocytes.

Immunopathophysiology of babesial infections

Humans infected with Plasmodium falciparum and bovines infected with Babesia bovis display severe haemolysis, alterations in red cell deformability and rigidity, and endothelial cell damage leading to pulmonary oedema and cerebral dysfunction. Much of the pathology associated with these infections is not easily attributable to the relatively small numbers of parasites present. This paper considers the possible roles of soluble mediators released from macrophages, and chemical and physical changes in the infected red cell membrane, in the pathogenesis of babesial infections, and also discusses the effects of prior immunization with various immunogens.

Cloning and characterisation of cDNA clones encoding two Babesia bovis proteins with homologous amino- and carboxy-terminal domains

A dextran sulphate protein (DSP) fraction derived from Babesia bovis has previously been shown to induce a protective immune response in cattle. A B. bovis cDNA library was screened with both the complete anti-DSP serum and a subfraction of the anti-DSP serum affinity purified on a native B. bovis protein of approx. 80 kDa. cDNA clones encoding two different B. bovis proteins were identified. The product of one gene, Bv80, has a single divergent copy of a sequence of 149 amino acids (approx. 30% amino acid identity) in both the amino- and carboxy-terminal domains. These domains are separated by an array of short variant repeat sequences rich in proline and glutamic acid. The product of the other gene, BvVAl (homologous to the previously described 225-kDa B. bovis protein)[19], is predicted to have a single divergent copy of a sequence of 170-171 amino acids (approx. 35% amino acid identity) in both the amino- and carboxy-terminal domains. These domains are also separated by an array of repeats. The 73-amino acid repeat unit of this array is composed of a number of variant derivatives of shorter repeat units. Detailed analysis of genomic clones flanking two alleles of the gene encoding BvVAl/225 kDa identified further members of a multi-gene family. This region of the genome of B. bovis has been subject to a large number of amplification processes.

Analysis of the composition of samples of Babesia bovis and the influence of different environmental conditions on genetically distinct subpopulations

A recombinant DNA probe specific for a tandemly repeated sequence located within the BoVA1 gene of Babesia bovis was used to analyse 10 independent samples of B. bovis. Twelve different alleles of the BoVA1 gene and flanking regions were identified in the 18 different subpopulations analysed. Most samples of B. bovis originally derived from single animals contained more than one genetically distinct subpopulation. However, only one population of parasites was identified in samples of the Ka line used in Australia from 1979 until 1990 as the live attenuated vaccine strain. In contrast, the replacement attenuated vaccine line, Ta, contained two genetically distinct subpopulations of parasites. Changes in the ratios of subpopulations of parasites were identified during attenuation and under different culture conditions. Batch-to-batch variation in the composition of doses of the live attenuated vaccine may lead to differences in efficacy and in severity of the infection associated with vaccination.

The irradiation ofBabesia bovis

Babesia bovis parasites attenuated by 35 krads γ irradiation and parasites not exposed to irradiation, were injected into intact 2-year-old Hereford steers. All five animals receiving non-irradiated blood died but the five animals which received irradiated blood were only mildly affected.Highly significant differences were observed in changes to plasma fibrinogen, serum fibrinogen-like proteins, packed cell volume, partial thromboplastin time, prothrombin time, blood kinins, and plasma kininogen levels in the control animals but non-significant changes in these parameters occurred in the group receiving irradiated blood. Significant changes in the antiplasmin, α2M, and the antithrombin levels occurred in control cattle but not in the group receiving irradiated blood. Parasite multiplication rates and maximum parasitaemias were similar in both groups. Irradiation reduced the dose of living parasites from 1×108 to 2.5×103, but this was not the reason for the mild reactions. It was concluded that irradiation had selected an avirulent parasite population.Babesia bovis parasites attenuated by 35 krads gamma irradiation and parasites not exposed to irradiation, were injected into intact 2-year-old Hereford steers. All five animals receiving non-irradiated blood died but the five animals which received irradiated blood were only mildly affected. Highly significant differences were observed in changes to plasma fibrinogen, serum fibrinogen-like proteins, packed cell volume, partial thromboplastin time, prothrombin time, blood kinins, and plasma kininogen levels in the control animals but non-significant changes in these parameters occurred in the group receiving iradiated blood. Significant changes in the antiplasmin alpha 2M, and the antithrombin levels occurred in control cattle but not in the group receiving irradiated blood. Parasite multiplications rates and maximum parasitaemias were similar in both groups. Irradiation reduced the dose of living parasites from 1 x 10(8) to 2.5 x 10(3), but this was not the reason for the mild reactions. It was concluded that irradiation had selected an avirulent parasite population.