D. F. Mahoney

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.

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.

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.

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.

AcuteBabesia bovis infection: A study of the vascular lesions in kidney and lung

SummaryA histological study of kidney and lung from cattle acutely infected with the intra-erythrocytic protozoan parasiteBabesia bovis was undertaken. Samples of the same tissue were examined using haematoxylin-eosin, Lendrums acid picro-Mallory, and phosphotungstic acid-haematoxylin fibrin stains, direct fluorescent antibody techniques using rabbit anti-B. bovis cryofibrinogen and rabbit anti-bovine fibrinogen conjugates, and electron microscopy. Although positive fibrin reactions were observed with histochemical stains, examination of the same material by the electron microscope and fluorescent reagents failed to confirm its presence. It was concluded that the fibrin reactions were given by sludged erythrocytes packed tightly together in capillaries and coated with cryofibrinogen complexes.

Babesia bovis (argentina): studies on the composition and location of antigen associated with infected erythrocytes.

Abstract Specific antisera against Babesia bovis (= argentina) antigens were prepared in rabbits by inoculation of precipitates from an extract of infected erythrocytes and absorption of the antisera with normal bovine components. Of three babesial antigens detected, one appeared to contain a modified stromal component. The antisera, when conjugated with fluoroscein isothiocyanate, stained aggregated infected erythrocytes in the microcirculation and located antigen in glomeruli and blood vessel endothelium. It was suggested that a babesial enzyme-fibrinogen complex contributes to the pathological changes of infection such as sludging and adherence of erythrocytes to blood vessel walls.

Changes in the haemolytic activity of serum complement during acute Babesia bovis infection in cattle.

The haemolytic activity of serum complement was measured in cattle during acuteBabesia bovis infection. The level fell significantly on day 5 post infection (p.i.) and was not detectable by day 10 p.i. After clinical recovery between days 15 and 20 p.i. haemolytic activity was again detected in serum and gradually returned to the pre-infection level. The components of both classical and alternative pathways were involved in complement depletion and factors such as protease secretion by the parasites, antigenantibody reactions, and the release of haemoglobin were implicated as causes of the decline. The absence of complement activity during the acute phase of the disease may interfere with immune mechanisms at a critical time for the host.

Babesia bovis (argentina): Components of the cryofibrinogen complex and their contribution to pathophysiology of infection in splenectomized calves

SummaryA cryofibrinogen complex, found in the plasma of cattle acutely infected withBabesia bovis (argentina), was characterized. The fibrinogenlike proteins of the complex were isolated and the structure of their polypeptide chains analysed. In general, the chain structure was similar to that of soluble non-crosslinked fibrin (fibrinogen) although chains indicating some degree of fibrin crosslinking were often detected. Only rarely did the chain structure suggest that fibrinolysis occurred. It was concluded that the complex was produced by activation of the coagulation system but that fibrinolysis did not occur to any marked degree. The complex was implicated in assistance to the sludging of erythrocytes in the internal organs which is a feature of the pathogenesis of the disease.

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.

Immunization against babesiosis: Current studies and future outlook

Experiments were conducted on the induction of immunity to Babesia bovis in cattle with antigen extracted from erythrocytes infected with the parasite. Protection after inoculation with crude parasite-erythrocyte stroma mixtures was as strong as that induced by natural infection. B. bovis appears to contain several different protective antigens, and one of these was partially purified by immunoabsorption using a monoclonal antibody. It is a protein consisting of a single peptide of low molecular weight and appears to be located in or on the surface of the parasite.