Biomedical subjects
Publications and source records attributed to I G Wright.
A saline eluate from sucrose washed bovine erythrocytes infected with Babesia bovis contains at least 4 babesial antigens. The antigens are located not only in the parasite but also in the internal matrix of the infected erythrocyte.
An esterase was isolated from a crude extract of Babesia bovis by affinity chromatography, using soy bean trypsin inhibitor as a ligand. In native form this enzyme had a molecular weight greater than 200 000, but on denaturing gels major bands were observed with molecular weights of 20 000, 10 000 and 7 000. Western transfer analysis revealed a major band with a molecular weight of 19 000-20 000. 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 X 10(9) parasites as water-in-oil emulsions with Freund's complete adjuvant. Two control groups, each of five animals were also included. One group of vaccinates and a control group were challenged with virulent homologous B. bovis, whilst the other vaccinated 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.
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.
All six enzymes of de novo pyrimidine biosynthesis leading to the formation of UMP have been demonstrated in whole homogenates from two bovine Babesia species, B. bovis and B. bigemina. The specific activities of the respective enzymes were of the same order of magnitude as observed for the related parasite, Plasmodium berghei. The results indicate that both these parasites have the potential of obtaining their pyrimidine requirements by de novo synthesis. Subcellular fractionation established that dihydroorotate dehydrogenase, the fourth enzyme of the pathway, was of a particulate nature. Mammalian respiratory chain inhibitors and ubiquinone analogues caused inhibition of the Babesia dihydroorotate dehydrogenase. As observed for other eukaryotic systems, the dehydrogenase appears to be linked to a respiratory chain via ubiquinone.
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Three beagle dogs were immunised against the paralysing effects of holocyclotoxin, using toxic extracts derived from salivary glands of the Australian paralysis tick, Ixodes holocyclus. Upon subsequent challenge with four ED50's of toxin the animals were completely immune, whereas during the early stages 2 of 3 dogs became paralysed after injection with 0.5 ED50. Three immunised beagles, on which 15 I. holocyclus ticks had fed, showed no symptoms but a control beagle became paralysed after 4 I. holocyclus females fed on it. Treatment was instituted immediately upon the onset of clinical signs and the dog recovered uneventfully.
Three distinct monoclonal antibody-producing hybridomas have been produced against a partly purified protective fraction of Babesia bovis. All three stain the parasite or infected erythrocytes or both in precise and different manners when fluorescent-antibody techniques are used. The relevant antigens for each monoclonal antibody were isolated by immunoadsorption, their native molecular weights being 1.3 X 10(6), 180 X 10(3), and 44 X 10(3). Each antigen reacted in serological assays with homologous and heterologous bovine antisera to B. bovis. Susceptible splenectomized calves were immunized twice, 4 weeks apart, with the respective antigens and were challenged with virulent homologous organisms 2 weeks later. Strong protective immunity was induced by the antigen with a molecular weight of 44 X 10(3), but no significant protection was induced by either of the other two antigens.
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Intraerythrocytic forms of B. bovis were exposed to 350 Grays (Gy) gamma irradiation and were then injected intravenously into intact two and three year old Hereford steers. One of 15 steers died on initial infection and subsequently six steers were given a virulent heterologous challenge three weeks after recovery; all six animals were highly immune. The remaining eight animals were kept under quarantine conditions for 10 months and were then challenged with a different virulent heterologous strain of B. bovis. Seven of eight were highly immune, but one animal died. Subsequently a further 12 steers were injected intravenously with 1 X 10(8) irradiated organisms. All showed only mild transient clinical signs. After 12 months quarantine in a tick-free area these animals were then challenged with a virulent heterologous strain and all 12 were shown to be highly immune. Irradiation reduced the infective dose from 1 X 10(8) to 2.5 X 10(3) parasites. These parasites multiplied at the same rate, and achieved the same maximum parasitaemia as the parent non-irradiated strain, but the disease produced by them was not severe. A dose of 2.5 X 10(3) non-irradiated parasites was lethal to all of the four animals which received it. It was concluded that irradiation had produced a predominantly avirulent parasite population.
Rabbits have been immunized against the effects of the paralyzing toxin of the Australian paralysis tick Ixodes holocyclus by injecting them with preparations extracted from tick salivary glands. Immunized rabbits were able to withstand doses of toxin known to kill unimmunized rabbits. Neutralizing antibodies were detectable in serum after 2-4 doses of the crude extract or of the relatively pure antigenic fraction. When injections were continued at intervals of from 2-7 weeks, hyperimmunity was retained for at least 68 weeks. Hyperimmune serum, reaching a very high titre of neutralizing antibodies, was obtained after 3-6 injections. Titres tended to decline when boosting ceased, but after a "rest period' high titres were restored by further boosting with normally lethal doses of toxin. No symptoms of tick paralysis developed despite low titres prior to boosting. Thus, once hyperimmunity had been established, high titres of circulating antibodies were not immediately essential for immunity to tick paralysis. An IgG fraction was obtained from rabbit serum using a Protein A-Sepharose method; 33.4 micrograms of IgG protein fully neutralized, and 19.5 micrograms IgG half neutralized, 1 micrograms of crude toxin protein. This procedure with rabbits may permit the production of a purified tick-paralysis antitoxin more suitable for human use than the existing antitoxin based on canine hyperimmune serum.
Preliminary studies have demonstrated changes to the lipid metabolism of cattle acutely infected with Babesia bovis. Total lipid, total cholesterol, and phospholipids decreased significantly during infection. Associated with this was a decrease in concentration of serum lipoproteins and a loss of their electrophoretic heterogeneity. These changes are discussed in relation to the pathophysiological changes during infection.
Two groups of five two-year-old Hereford steers were infected with two virulent strains of B. bovis. The plasma concentrations of creatine kinase (CK), lactate dehydrogenase (LDH), and creatinine rose early in infection and showed highly significant increases terminally. Extensive skeletal muscle degeneration was observed terminally as was degeneration of myocardium, although this was much less extensive. Terminal clinical signs included posterior muscle wasting, weakness, and recumbency. It was concluded that the extensive changes to muscle and to CK and LDH were caused by anoxia due primarily to shock and stasis of the circulation induced by the parasites.
A group of five splenectomised calves was immunised with a soluble extract prepared from erythrocytes infected with Babesia bovis and then challenged by the intravenous inoculation of virulent B. bovis along with appropriate control animals. All controls died but four of the five immunised animals survived. All survivors showed significantly lower activation of the coagulation and kinin systems and did not develop the hypotensive shock syndrome characteristic of acute B. bovis infection. It was concluded that the concentrations of fibrinogen, kininogen, and bradykinin, and the production of soluble fibrin, cryofibrinogen, and high molecular weight fibrinogen complexes were useful criteria on which to assess the efficacy of the immune response.
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 plasma of cattle recovering from severe babesia bovis infection contained cold precipitable protein which consisted of immune complexes formed in antibody excess. The major immunoglobulin in the complexes was IgM although IgG1 and IgG2 were also present at lower concentrations. In addition, fibrinogen, alpha 2 macroglobulin and C3 component of complement were detected. As the complexes were produced after parasiteamia fell below the detectable level and the inflammatory response to B. bovis was waning, the complexes did not appear to have much pathological significance.
Further pathophysiological studies of the hypotensive shock syndrome associated with acute Babesia bovis infection in cattle have demonstrated changes in the plasma concentrations of conglutinin, complement C3, fibronectin and immunoconglutinin. Decreases in the first three components are presumably due to their contribution towards intravascular sludging of infected erythrocytes within the microvascular system during the acute phase of infection. In contrast, the increase in fibronectin and immunoconglutinin in the recovery phase suggests the involvement of these proteins in the reticulo-endothelial clearance of infected or damaged erythrocytes, immune complexes and cryofibrinogen.
A virulent strain of Babesia bovis ("L" strain) was rendered avirulent by irradiation with 35 krads with a gamma source. Another virulent strain of B. bovis ("C" strain) was made avirulent by rapid blood passage through 12 splenectomised calves. Both the parent virulent and their respective avirulent strains were injected into susceptible cattle. A nonfatal disease was observed in those intact cattle that had received avirulent parasites; however, a fatal disease was produced in those animals that had received virulent parasites and in splenectomised calves that had received avirulent parasites. Blood kinin levels rose and plasma kininogen levels fell significantly in those animals infected with both virulent strains. Nonsignificant changes occurred with these parameters in animals infected with avirulent parasites. Preparations of disrupted parasites were obtained from the four parasite populations. Both virulent strains contained high levels of protease. The avirulent forms contained insignificant amounts. As parasite doubling times and maximum parasitaemias were the same for all four parasite populations, we conclude that these enzymes are not obligatory for parasite multiplication in the vertebrate host. Their role in producing pathological changes in the host is discussed.