DALRYMPLE, B. P., PETERS, J. M., BÖSE, R., and WRIGHT, I. G. 1996. A polymerase chain reaction method for the identification of genes encoding members of the Bv60/p58 family of rhoptry protein homologues in the genusBabesia. Experimental Parasitology84,96–100.

Studies were undertaken to determine whether treatments with low doses of natural human interferon alpha (HuIFN-alpha) administered by various routes could inhibit the development of the intra-erythrocytic protozoan Babesia microti in BALB/c mice. HuIFN-alpha treatment given intramuscularly significantly inhibited development of the parasitaemia of the parasite compared with infections in control mice.

Sera from cattle infected with Babesia bovis were found to contain antibodies to phosphatidyl-serine (PS), a negatively charged phospholipid normally found on the internal membrane of erythrocytes. In contrast, no autoantibodies were detected following Babesia bigemina infection indicating that the autoimmunity is not genus specific. During infection with Babesia bovis, PS translocates to the external membrane and it is suggested that this may result in PS behaving as an autoantigen owing to a transitional change. These autoantibodies may also play some role in the pathology of infection, especially the disturbed coagulation system associated with acute Babesia bovis infection.

The in vitro phagocytosis of bothBabesia bovis-infected red cells and of parasites exposed by lysis of infected red blood cells is demonstrated in a phagocytic mouse model. Twenty-fourB. bovis immune sera were tested alone or as a pool as were antibodies (DS antibodies) raised against aB. bovis protective fraction, prepared by dextran sulfate precipitation. All the immune sera failed to promote significant levels of phagocytosis, whereas the other antibodies (DS antibodies) consistently induced phagocytosis of infected cells in all the experiments carried out. This study shows that antibody specificity is critical to the opsonization of infected red cells and parasites during in vitro phagocytosis and suggests that phagocytosis is one of the mechanisms in the in vivo immune response againstBabesia species.

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.

Studies were undertaken to determine whether human interferon alpha (HuIFN-alpha) administered orally could inhibit the development of clinical disease caused by the intraerythrocytic protozoan Babesia bigemina and the intraerythrocytic rickettsia Anaplasma marginale in cattle. HuIFN-alpha did not inhibit intraerythrocytic multiplication of either of the two parasites, suggesting that there is no role for HuIFN-alpha administered orally in the control of these pathogens.

The immune response to Babesia bovis infection or vaccination was evaluated by measuring antibody and interferon gamma (IFN-gamma) production to protective recombinant and crude native B. bovis antigens. Cells from vaccinated or infected cattle failed to produce detectable IFN-gamma when stimulated with B. bovis antigens in vitro. In contrast, antibody was induced by protective recombinant B. bovis antigens. These findings are consistent with the argument that immunity to B. bovis infection is correlated most strongly with humoral rather than cell-mediated immune responses.

The three ribosomal DNA (rDNA) units have been cloned from an Australian isolate of Babesia bigemina. The organization of the units is very similar to that reported for a Mexican isolate of B. bigemina. In Babesia canis four rDNA units have been identified. Both Babesia rodhaini and Babesia microti contain two different rDNA units. A small number of different rDNA units appears to be a common feature of this group of protozoa. Restriction enzyme analysis of the rDNA units from these species and B. bovis suggests that the genus Babesia as currently defined does indeed include two distinct groups of organisms namely, B. bovis, B. bigemina and B. canis and B. rodhaini and B. microti.

Antigenic and immunogenic activities of a hexane extract from Babesia bovis-infected erythrocytes were investigated. Positive ELISA and IFAT reactions were obtained with bovine antisera to B. bovis and B. bigemina produced by natural infection and rabbit antisera to the hexane extract, respectively. In contrast, negative ELISA reactions were obtained with Anaplasma marginale antisera indicating that the antigen(s) is specific for the genus Babesia. The IFAT clearly demonstrated that the antigen was associated with the parasite and the infected erythrocyte and not present in uninfected erythrocytes. Furthermore, cross-reactions with Babesia bigemina antisera suggested that serological cross-reactivity in bovine Babesia species is at least due in part to lipid or lipid-associated antigens.

Analysis of the composition of samples of Babesia bovis and the influence of different environmental conditions on genetically distinct subpopulations. International Journal for Parasitology22: 731–737. 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.

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.

Current methods for the control of the cattle tick Boophilus microplus and the agent of bovine babesiosis, Babesia bovis are unsatisfactory. Effective immunological control of both parasites would have great advantages. However, naturally acquired immunity to the lick is generally unable to prevent serious production losses. A vaccine against the tick, based on a novel form of immunization, is being developed. A protective antigen has been isolated from the tick, characterized and produced as an effective, recombinant protein. A vaccine incorporating this antigen is currently undergoing field trials. In the Australian situation, improved tick control will probably increase endemic instability with respect to B. bovis. Fortunately, a trivalent, recombinant B. bovis vaccine has also been developed This too is now undergoing pre-registration field trials.

Murine monoclonal antibodies (MAB) were produced against the 'beta' fraction of Babesia bovis. A MAB, W11C5, selected on the criterion of its staining of erythrocytes infected with B. bovis,was purified. The antigen identified by MAB W11C5 was extracted from B. bovis infected erythrocytes by affinity chromatography and used in a vaccination trial to test its vaccine efficacy against homologous B. bovis infection in splenectomized calves. The vaccinated group showed significantly different parasitaemias from the control group and it was concluded that the B. bovis antigen 11C5 induced a protective immune response when used as a vaccine. This antigen should be synthesized using recombinant DNA techniques to determine its efficacy and suitability as a commercial vaccine against B. bovis infection.

A series of hydroxyacetophenones was prepared for evaluation as leukotriene B4 (LTB4) receptor antagonists, culminating in 1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5- yl)heptyl]oxy]phenyl]ethanone (compound 35, LY255283). Using an assay for inhibition of specific [3H]LTB4 binding to human PMN, we found that substitution of a nonpolar substituent in the 5-position was required for activity. Best activity was realized with hydrogen in the 3-position, hydroxyl in the 2-position, short chain alkyl ketone in the 1-position, and a six- or eight-carbon chain linking the oxygen in the 4-position with an unsaturated terminal function. Compound 35, having an IC50 of 87 nM in the binding assay, was chosen for further preclinical evaluation.

Dextran sulphate, a chemical with some specificity for lipoproteins, was used to precipitate a fraction from a soluble extract of Babesia bovis-infected erythrocytes. The precipitate, in combination with dextran sulphate as an adjuvant, was used to vaccinate naive calves. The vaccinates and a group of control calves were challenged with virulent homologous B. bovis. The vaccinates showed delayed and decreased parasitaemias comparative to the controls. The antibody response to vaccination was primarily against the infected erythrocyte being of both IgG1 and IgG2 classes. We believe this is the first report of B. bovis antibody being detected in the IgG2 class. Lipase inhibition and chemical analysis suggested babesial lipid or lipoprotein was sufficiently immunogenic to produce serologically detectable antibody and presumably to elicit immunity.

Dextran sulphate-bound Babesia bigemina antigens were used in a preliminary vaccination study and were shown to elicit a protective immune response in cattle. A dextran sulphate-binding fraction of B. bigemina was further subfractionated on a Phenyl Sepharose column to give two fractions--one that strongly bound to the column (bound fraction) and one that did not (unbound fraction). Two groups of cattle were each vaccinated with either the bound or the unbound fraction. These two groups of animals along with a control group were then challenged with B. bigemina-infected erythrocytes. Both groups of vaccinated animals showed considerably lower mean daily parasitaemias as compared to the control group.

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of Babesia ovis antibodies is described. In an initial study, a crude Babesia bovis antigen and a synthetic B. bovis-derived antigen (designated 11C5) were used to screen 46 B. ovis-positive and 55 negative sheep sera. A 95% correlation between the two antigenic preparations was found with the positive sera; no negative sera gave positive reactions. The synthetic antigen was then used in the screening of 1466 sera collected from sheep from 18 regions of Turkey. A high incidence of B. ovis-positive reactions was found from all regions (60-80%) in sheep over 1 year old, while from two smaller samples the incidence in young sheep was much less (28 and 52%). This test is superior to existing ones because the synthetic antigen can be produced in a highly reproducible state, is specific and is stable over extended periods of time.

Summary The effect of immune serum and complement on the in vitro phagocytosis of Babesia rodhaini was investigated. Infected erythrocytes and parasites released from erythrocytes by lysis were phagocylosed by mouse peritoneal macrophages in vitro when the infected erythrocytes or parasites were exposed to hyperimmune B. rodhaini serum. Complement, in the presence of immune serum, did not reproducibly enhance phagocytosis of infected erythrocytes or parasites alone. When adjusted for its effect on normal erythrocytes and normal serum, complement generally inhibited rather than enhanced phagocytosis. When coupled with other published data, our data suggest that the activation of the immune system in vivo against this species of Babesia involves a series of mechanisms of which phagocytosis is but one.