Babesia bovis: purification and concentration of merozoites and infected bovine erythrocytes

Babesia bovis merozoites, externalized by removal of infected erythrocytes from ordinary culture conditions, were completely separated from red blood cells and stroma by centrifugation in a Percoll gradient. A merozoite band formed at a point corresponding to about 1.087 g/ml specific density. Infected red blood cells were concentrated approximately fourfold to obtain greater than 49.0% parasitemia after centrifugation in Percoll. Most highly enriched fractions positioned between 1.121 and 1.123 g/ml specific density. Full parasite viability was retained.     

Use of helper T cells to identify potential vaccine antigens of Babesia bovis

Babesia bovis is an economically important hemoprotozoon parasite o f cattle that is prevalent in many, tropical and subtropical regions o f the world. Effective vaccines against this tick-transmitted parasite consist o f live organisms attenuated by passage through splenectomized calves. However, the nature o f acquired resistance to challenge infection with heterologous isolates of this parasite has not been clearly defined. Unsuccessful attempts to select protective antigens have relied upon the use of antibodies to identify immunodominant proteins. In this review, Wendy Brown and Allison Rice-Ficht discuss the limitations of this approach and the rationale behind using helper T cells to select potential vaccine antigens.     

Theileria parva: bovine helper T cell clones specific for both infected lymphocytes and schizont membrane antigens

Two Theileria parva-specific bovine helper T cell clones were used to identify T. parva-derived antigens expressed on the surface of schizont infected lymphoblastoid cells. Although the clones proliferated in response to both the immunizing (Muguga) and heterologous stocks of T. parva, the patterns of the responses differed, showing that the two clones recognized different antigenic epitopes. Both clones were stimulated by autologous infected cells, without an additional source of antigen-presenting cells, as well as by purified schizonts and by a subcellular membrane fraction prepared from infected lymphoblastoid cells, when antigen-presenting cells were present. The membrane fraction was shown to be enriched for schizont membranes as indicated by the presence of a schizont surface antigen detected by immunoblotting using a schizont-specific monoclonal antibody. Elimination of schizonts with the anti-theilerial drug, parvaquone, resulted in reduced antigenicity of the membrane fraction as detected by both the T cell clones and the schizont-specific monoclonal antibody. We conclude that the T. parva-infected cell surface antigens recognized by the T cell clones are of schizont membrane origin. Although the antigens have not yet been characterized biochemically, the monoclonal antibody-specific epitope appears to be distinguishable from the T cell epitopes.     

Characterisation of erythrocyte invasion by Babesia bovis merozoites efficiently released from their host cell after high-voltage pulsing

Apicomplexa are a phylum of obligate intracellular parasites critically dependent on invasion of a host cell. An in vitro assay for erythrocyte invasion by Babesia bovis was established, employing free merozoites obtained after the application of high-voltage to the parasitised erythrocytes. The invasion proceeds efficiently in phosphate-buffered saline solution without the requirement for any serum or medium components. The kinetics of invasion can be measured over a time span of 5-60 min after which invasion is completed at an average efficiency of 41%. The fast kinetics and high efficiency exceed those of most previously established apicomplexan invasion assays. The manipulation of intracellular calcium concentration inhibits invasion. Preincubation of merozoites at 37 degrees C also reduces invasion, possibly by the premature secretion of protein. Proteins that are shed into the environment during invasion were directly detectable by protein staining after 2-D gel electrophoresis. The limitations posed by the immunological detection of proteins released during in vitro invasion by other apicomplexan parasites can, therefore, be avoided by this method. A unique feature of the assay is the reversible uncoupling of invasion and intracellular development, the latter taking place only under serum-rich medium conditions. In addition, host cell attachment is uncoupled from invasion by cytochalasin B.     

Babesia bovis: molecular and biological characteristics of cloned parasite lines

An in vivo limiting dilution technique was used to produce several Babesia bovis cloned lines with which to study the basis of virulence and immunogenicity in this parasite. DNA hybridization using a cloned DNA fragment from the BabR locus demonstrated that the cloned lines were a more restricted genetic population than the parent strain. Biosynthetic labeling and immunoprecipitation studies indicated that the cloned lines differed from each other and from the parentals in the expression of a small number of polypeptides and antigens. Animal trials with three of the lines demonstrated that the parental line contains both virulent and avirulent parasite populations, at least three of which are not tick transmissible, and that while the lines do provide significant protection against heterologous challenge, they may not give as effective protection as the parental line. These experiments demonstrated the existence of subpopulations with distinctive molecular and biological properties, providing evidence that the attenuation process is based on the selection of preexisting parasite subpopulations combined with the ability of these parasites to vary genetically.     

Neonatally induced tolerance to soluble protein antigens. I. Analysis of B-cell and T helper cell function in mice tolerant to bovine serum albumin or fowl gamma-globulin

High dose tolerance to either bovine serum albumin (BSA) or fowl γ-globulin (FGG) was induced in CBA mice by neonatal injection. Tolerance to BSA lasted about 9 weeks, and that to FGG, about 18 weeks. Splenic B-cell function was analyzed using quantitative in vivo assays and in vitro limiting dilution analysis. Tolerogen-specific IgM- and non-IgM-producing B cells are depleted at least threefold in the spleens of tolerant mice. Tolerogen-specific T-helper-cell function was examined by immunization with haptenated antigens. Analysis of the recovery from tolerance indicates that the return to normal function in the tolerogen-specific B-cell and T helper fractions coincides with the return to normal responsiveness by the whole animal.     

The lysate from bovine erythrocytes infected with Babesia bovis. Analysis of antigens and a report on their immunogenicity when polymerized with glutaraldehyde

A group of Babesia bovis antigens obtained by a lengthy biochemical procedure involving disruption of infected erythrocytes has previously been shown to be highly protective. This study shows that these antigens can be found in a simple lysate of infected erythrocytes. The antigens have been characterized by gel filtration and nitrocellulose transfer and consist of a wide spectrum of molecular sizes. Some of the antigens exist in complex form and are easily dissociated. The lysate was polymerized with glutaraldehyde and injected per se into four splenectomized calves. All the calves produced antibody to B. bovis but did not produce erythrocytic isoantibodies. The vaccinated calves and a control group of four splenectomized calves were challenged with virulent B. bovis. Statistically, the vaccinated group differed significantly in parasitaemia, temperature change and pathophysiological parameters from the control group. All of the control group died whereas two of the vaccinated group survived infection.     

Comparative functional analysis of helper T lymphocyte responses to soluble and particulate antigens

An adaptable and sensitive assay to analyze the roles of helper T lymphocytes (TH) which recognize soluble or cell-surface bound antigens in the induction of cytotoxic T lymphocyte precursors (CTLp) is described. Long-term T cell lines that recognize purified protein derivative, keyhole limpet hemocyanin, or Corynebacterium parvum were used in these studies. The ability of T cells from these lines to induce cytotoxic T lymphocyte or antibody responses were compared with their ability to proliferate or release interleukin 2 (IL 2). The results demonstrate that these T cell lines are able to react to soluble antigen by proliferation and IL 2 release. Moreover, the same cell lines are able to interact with CTLp or with the precursors of antibody-secreting B cells to induce a response. In the induction of CTLp we observed an inverse correlation between the number of TH cells required and the concentration of antigen used to pulse the antigen presenting cells. However the correlation between the ability of TH lines to proliferate specifically in response to antigen and to act as helpers for CTLp and B cells was not absolute as cells with compromised proliferative capacity were able to efficiently deliver inductive signals.     

Proteins and antigens of merozoites and sporozoites of Eimeria bovis (Apicomplexa)

Proteins and antigens of first-generation merozoites and sporozoites of Eimeria bovis were examined using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and lactoperoxidase iodination procedures. SDS-PAGE gels revealed both common and unique protein bands in merozoite and sporozoite extracts, ranging in molecular weight (Mr) from 15,000 to 215,000. Nitrocellulose immunoblots of separated proteins, when probed with sera obtained from immunized calves, revealed numerous IgG-binding antigens of Mr 18,000 to 180,000 in merozoites and Mr 28,000 to approximately 118,000 in sporozoites. Although merozoite and sporozoite preparations each contained antigens of different molecular weights, 4 antigens had the same migratory distance in both preparations (Mr 58,000, 70,000, 83,000, 98,000). Of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 wk later by subcutaneous injection with 2 X 10(7) live merozoites emulsified in Freund's complete adjuvant consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. Autoradiographic analysis of radioiodinated parasites revealed major surface proteins on merozoites of between 15,000 and 18,000 Mr and 3 surface proteins on sporozoites of Mr 28,000, 77,000, and 183,000. All but the 183,000 protein elicited an IgG antibody response in the host.     

Recognition of soluble Theileria parva antigen by bovine helper T cell clones: characterization and partial purification of the antigen

Theileria parva-specific bovine BoT4+ Th cell clones were used to characterize Ag associated with T. parva schizont-infected lymphoblastoid cells. All of the clones tested responded to cells infected with the immunizing (Muguga) as well as heterologous stocks of T. parva, indicating that the T cells are specific for an Ag shared by several geographically diverse parasites. The response was apparently MHC-restricted, and induced by Ag expressed on the infected cell surface. In the presence of autologous APC, the clones were also stimulated by a soluble high speed supernatant (HSS), but not by a schizont membrane-enriched, subcellular fraction prepared from homogenates of infected cells. The clones produced IFN-gamma and T cell growth factor in response to HSS. The soluble Ag was absent in cells from which schizonts had been eliminated by treatment with the anti-theilerial drug, parvaquone. Fractionation of HSS by hydroxylapatite chromatography revealed two antigenic peaks that separated from the majority of the protein. Fractionation of HSS by gel filtration with the use of HPLC revealed several peaks of activity ranging in Mr from 270 kDa to less than 5 kDa. Further fractionation of HSS by both hydroxylapatite chromatography and gel filtration yielded three major peaks of activity (Mr 43, 12, 4.2 kDa). We conclude that a T cell-dependent schizont-associated soluble Ag is also expressed on the surface of T. parva-infected cells.     

Antigenic relationship between Plasmodium falciparum and Babesia bovis: reactivity with antibodies to culture-derived soluble exoantigens

Antigenic similarities between Plasmodium and Babesia parasites of the phylum Apicomplexa have been previously demonstrated primarily by the serological cross reactivity observed in the indirect fluorescent antibody (IFA) test. We have now studied the antigenic relationship between the human malaria parasite, Plasmodium falciparum, and the hemoparasitic agent of cattle, Babesia bovis, using rabbit monospecific antibodies produced against individual culture-derived P. falciparum polypeptides and bovine polyspecific antibodies to B. bovis exoantigens. These respective antibodies were found to be distinctly cross reactive in the IFA test using infected erythrocytes (squirrel monkey--P. falciparum; bovine--B. bovis) as antigen substrates. Immunofluorescence was shown to be highly specific for parasite surfaces. Additionally, the degree of reactivity with soluble exoantigens contained in Plasmodium and Babesia culture supernatants was monitored by a two-site enzyme immunoassay employing the cross-reactive antibodies. Further evidence for antigenic cross reactivity between P. falciparum and B. bovis parasites was shown with the in vitro inhibition assay. Antibodies to P. falciparum and B. bovis were found to be highly inhibitory for the in vitro growth of P. falciparum in human erythrocytes.     

Ability of T cell subsets and their soluble mediators to modulate the replication of Mycobacterium bovis in bovine macrophages

Peripheral blood mononuclear cells (PBMCs) from cattle vaccinated with Bacillus Calmette-Guerin (BCG) were obtained and expanded in vitro by incubation with purified protein derivative. The ability of these cells to modulate the replication of virulent Mycobacterium bovis in autologous-infected macrophages was compared to cells from non-vaccinated controls. Cells from non-vaccinated animals were shown to confer a significant degree of mycobacteriostatic activity to autologous-infected macrophages. This activity was not inhibited by including a neutralizing antibody versus interferon-gamma (IFN-gamma), and was dependent on direct contact between PBMCs and infected macrophages. Addition of autologous PBMCs from BCG-vaccinated cattle was shown to significantly enhance macrophage resistance to M. bovis, and this increased macrophage resistance was partly abrogated by including a neutralizing antibody to IFN-gamma. Addition of T cells from non-vaccinated animals to infected macrophages was associated with a modest increase in macrophage release of TNF-alpha and nitric oxide, whereas PBMCs from vaccinated animals increased very significantly the release of these factors. Neutralization of nitric oxide (NO), by inclusion of monomethyl-L-arginine, significantly diminished the ability of PBMCs from vaccinated animals to enhance macrophage resistance to M. bovis, but had no impact on the ability of T cells from naive animals to modulate macrophage function. The ability of naive cells to increase macrophage anti-M. bovis activity was largely mediated by CD4+ T cells, whereas both CD4+ T cells and CD8+ T cells conferred macrophage resistance to M. bovis in vaccinated animals. These data highlight the role of IFN-gamma and NO in the immune resistance of cattle to M. bovis.     

In vitro adherence of bovine erythrocytes infected with Babesia bovis to thrombospondin and laminin

This study was undertaken to examine how eight putative adhesive agents bound to plastic surfaces affected the capacity of bovine erythrocytes, infected with either virulent or avirulent strains of Babesia bovis, to adhere in vitro. Thrombospondin (TSP) induced B. bovis-infected bovine erythrocytes to adhere and adherence was augmented when the infected blood was cultured for 24 h before the assay. Moreover, TSP also caused erythrocytes infected with avirulent strains of B. bovis to adhere to plastic in vitro. Laminin promoted the adherence of infected, and to a lesser extent, of uninfected erythrocytes.     

Proteinases in the lysate of bovine erythrocytes infected with Babesia bovis: initial vaccination studies

The lysate of erythrocytes infected with Babesia bovis was tested for proteinases using an electrophoretic method in which substrate was included in the acrylamide matrix. Two babesial proteinases, which seemingly exist in both free and complexed forms, were detected. One of the proteinases was prepared by chromatography and preparative electrophoresis and used to vaccinate four splenectomized calves. The latter, along with a group of control splenectromized calves, were challenged with a strain of B. Bovis from which the proteinase was obtained. All the control calves died whereas only one of the vaccinates died. The protection was evident as a suppression of parasitaemia.     

Babesia bovis: analysis of and preliminary vaccination studies with a defined infected erythrocyte membrane binding antigen.

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.     

Yersinia enterocolitica activates a T helper type 1-like T cell subset in reactive arthritis.

The profile of lymphokines secreted by 14 T cell clones and 24 T cell lines reactive with Yersinia Ag isolated from the synovial fluid cells of two HLA-B27+ patients with Yersinia-triggered reactive arthritis was characterized. In response to Ag-specific or -nonspecific stimulation, all of the Yersinia-reactive T cell clones and lines had a pattern of lymphokine secretion resembling that of murine (Th1) cells. A total of 50% of T cell lines and clones randomly isolated from a reactive arthritis patient, without prior in vitro stimulation with Yersinia Ag, also exhibited a Th1-like profile of cytokine secretion upon nonspecific activation. This indicates that the selective expansion of this subset of T cells had already occurred in vivo. The possibility that the predominance of Th1-like T cells was an artefact generated by the T cell cloning procedure was excluded; 50% of the randomly isolated T cell clones and lines produced IL-4, IL-5, or both cytokines upon nonspecific activation. These results indicate that Yersinia Ag selectively activate a Th1-like subset of T cells in patients with Yersinia-triggered reactive arthritis. Accumulation of such cells in the synovial tissue of patients with reactive arthritis may play a key role in the pathogenesis of this disease.     

Regulation of myelin basic protein-specific helper T cells in multiple sclerosis: generation of suppressor T cell lines.

Suppressor T cell (Ts) lines specific for myelin basic protein (MBP)-reactive helper T cell (Th) clones were generated from two patients with multiple sclerosis (MS) following a primary culture of peripheral blood mononuclear cells (PBMC) with MBP and cyclosporine A (CsA). These suppressor T cell lines were maintained in culture by alternate stimulation with MBP and antigen-presenting cells (APC). The Ts lines expressed preferentially the CD4 phenotype (5/6 Ts lines tested) and exhibited potent antigen-specific suppressor activity on the proliferation of MBP-specific Th clones and not on the T cell lines with other antigen specificity. For some Ts lines, a Ts-to-Th ratio of 1 was sufficient to inhibit the proliferation of MBP-specific T cells by 90%. The suppressor T cells obtained were weakly responsive to MBP and required the presence of the autologous PBMC for proliferation. Furthermore, proliferation of these suppressor T cell lines was restricted by HLA-DR molecules (for CD4+ Ts lines) and HLA class I (for a CD8+ Ts line). The suppressor T cell lines generated and the techniques described in this study may be helpful in our understanding of the events involved in the immune regulation in MS and other autoimmune diseases.     

Babesia bovis, Babesia bigemina, Babesia canis, Babesia microti and Babesia rodhaini: comparison of ribosomal RNA gene organization.

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 form 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.