D.J. Waltisbuhl

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.

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.

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.

Babesia bovis: the effect of acute inflammation and isoantibody production in the detection of babesial antigens.

Immunoblots ofBabesia bovis antigen contain dominant antigens which react not only with antisera toB. bovis but with sera from naive calves recovering from an acute inflammatory reaction. It seems likely these antigens are from the host rather than the parasite.

Babesia bovis: Vaccination trial with a dominant immunodiffusion antigen in splenectomised calves

The dominant immunodiffusion antigen ofBabesia bovis was prepared from the lysate of infected erythrocytes by cation exchange chromatography, gel filtration and preparative native acrylamide electrophoresis. It was seemingly free of other babesial antigens and tested as a vaccine. In vaccinated calves, compared to controls, there was a delay in parasitaemia and at times a statistically significant difference in parasite numbers. However, the vaccinates showed little difference in pathophysiological parameters or survival rates from the controls. It was concluded that serodominance cannot necessarily be correlated with protection.

The lysate from bovine erythrocytes infected withBabesia bovis

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.A group ofBabesia 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 toB. bovis but did not produce erythrocytic isoantibodies. The vaccinated calves and a control group of four splenectomized calves were challenged with virulentB. 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.

Babesia bovis: Dextran sulphate as an adjuvant for and precipitant of protective immunogens

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.

Babesia bovis: Successful vaccination against homologous challenge in splenectomised calves using a fraction of haemagglutinating antigen

Abstract A soluble extract from lysed bovine erythrocytes infected with Babesia bovis, was separated by preparative agarose electrophoresis. A fraction with a mobility of plasma β globulins was shown to contain babesial antigen confined mainly to the infected erythrocyte and was used to vaccinate a group of four calves. Following challenge with homologous B. bovis all four calves vaccinated with the antigen survived the infection whereas all the calves in a control group of four died from infection.

Babesia bovis: vaccination studies with three groups of high molecular weight antigens from lysate of infected erythrocytes.

A void volume fraction and fractions of mean sizes 800 kdalton and 300 kdalton were isolated by gel filtration from lysate of bovine erythrocytes infected with Babesia bovis. All fractions had good serological activity, as assayed by ELISA and IFA. Groups of four splenectomized calves were vaccinated with each fraction and then challenged, together with groups of four control calves, with a homologous strain of B. bovis. The group vaccinated with the 300 kdalton fraction showed some protection, as indicated by delayed and significantly lower parasitaemias and by a 75% survival in the group. In contrast, all animals in the relevant control group died from infection. No evident protection was obtained with the other two fractions.

The characterisation of an esterase derived fromBabesia bovis and its use as a vaccine

An esterase was isolated from a crude extract ofBabesia bovis by affinity chromatography, using soy bean trypsin inhibitor as a ligand. In native form this enzyme had a molecular weight greater than 200000, but on denaturing gels major bands were observed with molecular weights of 20000, 10000 and 7000. Western transfer analysis revealed a major band with a molecular weight of 19000–20000. Both bovine and rabbit antisera avidly stained infected red cells, using indirect immunofluorescence. Weak parasite staining was also observed using this test.Two groups of five animals were vaccinated twice 4 weeks apart with esterase derived from 5×109 parasites as water-in-oil emulsions with Freunds complete adjuvant. Two control groups, each of five animals were also included. One group of vaccinates and a control group were challenged with virulent homologousB. bovis, whilst the other vacinated and the remaining control group were challenged with virulent heterologous organisms. In the homologous groups two controls but no vaccinates died, whereas in the heterologous groups four animals in each group died. Significant differences in parasitaemia, temperature rise and total haemolytic complement were observed in the homologous vaccinated group compared to their controls but no differences were observed between heterologous groups.