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.     

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.     

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

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.     

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.     

Elevated anti-parasitic activity in peripheral blood monocytes and neutrophils of cattle infected with Babesia bovis

The innate immune response to bovine Babesia bovis infection in vivo has not previously been established. We used assays measuring phagocytosis and oxidative burst to investigate the immune response because they are indicative of the innate antimicrobial capacity of monocytes and neutrophils. Monocyte and neutrophil phagocytosis is thought to be non-specific in nature and so the phagocytosis of either opsonised Zymosan or Escherichia coli was used to indicate the non-specific phagocytic capacity of monocytes and neutrophils ex vivo. The kinetics of both phagocytic and oxidative burst activity in monocytes and neutrophils were followed twice weekly from pre-inoculation (day 0) through to 31 days after inoculation. Peripheral blood monocytes were found to display a pronounced oxidative burst, but a suppressed capacity to phagocytose during a primary infection. On the other hand, neutrophils exhibited an increased phagocytic capacity and reduced oxidative activity during a primary infection. These findings identified considerable antimicrobial activity evident in peripheral blood monocytes and neutrophils from cattle exposed to B. bovis as a primary exposure. This elevated antimicrobial activity was coincident with the time that parasite numbers peaked in the circulation and occurred prior to parasite clearance. These results suggest that peripheral blood monocytes and neutrophils are active mediators in the innate immune response to a primary B. bovis.     

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.     

Babesia bovis: immunity induced by vaccination with a lipid enriched fraction

A chloroform extract from Babesia bovis-infected erythrocytes was used to vaccinate a group of five naive cattle. Following vaccination, the vaccinates, along with a group of control cattle, were challenged with a virulent heterologous strain of B. bovis. The vaccinates, comparative to the controls, showed delayed as well as decreased parasitaemias. The serological and initial biochemical studies suggested that the immune response was elicited by lipid of babesial origin.     

Anaplasma marginale: Effect of challenge of cattle with varying doses of infected erythrocytes

Three groups, each of 6 Hereford cattle, were infected by the i.v. inoculation of 10¹⁰, 10⁸ or 10⁶ Anaplasma marginale-infected erythrocytes. The mean time taken to reach a 1% parasitaemia was 7.3, 13.9 and 19.9 days in the 10¹⁰, 10⁸ and 10⁶ infection dose groups, respectively. The rates of increase in parasitaemias during the exponential phase of parasite multiplication were similar for the 3 groups (doubling time 0.9 days). The exponential increase of the parasitaemia in the 10¹⁰ dose group extended to a higher level than in the 10⁸ or 10⁶ dose groups (to approximately 10% compared with 3%). The mean maximum parasitaemia attained in the 10¹⁰, 10⁸ and 10⁶ infection dose groups was 23.7, 14.7 and 8.7%, respectively. The time taken to reach the treatment criterion (packed cell volume decrease to 15% or lower) from a 1% parasitaemia was similar for the 3 groups. These results showed that the pathological outcome (anaemia) of anaplasmosis were similar over the 10 000-fold infective dose range tested.     

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

The dominant immunodiffusion antigen of Babesia 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.     

In vitro adherence of erythrocytes infected with Babesia bigemina and Babesia rodhaini to thrombospondin

The adherence of erythrocytes infected with Babesia bigemina and Babesia rodhaini to thrombospondin (TSP) in vitro is demonstrated. Blood with a range of parasitaemias was used and counts of cells which bound to TSP on plastic were significantly different from the controls with both Babesia species. These studies indicated that TSP receptors are present on the surface of red blood cells infected with the two Babesia species, although these parasites do not alter the membranes of infected erythrocytes obviously and do not cause cerebral symptoms in their hosts. Erythrocytes infected with either B. bigemina or B. rodhaini do not adhere to other erythrocytes in vivo, probably because these parasites induce mild infections in their hosts, but they can adhere to TSP in vitro.     

Comparison of cattle responses to mono-specific or combined inoculations with Babesia bigemina and Babesia bovis vaccine strains

Combined inoculation of cattle with vaccine strains of Babesia bigemina and Babesia bovis induced lower antibody titers to B. bigemina than to B. bovis (previous study). Three groups of heifers were used to detect if the low antibody level was due to competition between Babesia species: individuals of G1 and G2 were inoculated with 10 million B. bigemina and B. bovis, respectively, and those of G3 with 10 million of each parasite. The prepatent periods, maximum parasitaemias and antibody titers (indirect immunofluorescent antibody test) were evaluated. The mean prepatent periods (days) for B. bigemina was of 5.6 (G1) and 5.2 (G3) and 7.0 (G2) and 6.7 (G3) for B. bovis (P > 0.05, "t" test). No differences were found in the parasitaemias. The only difference was found in the antibody titers to B. bovis, that were lower (P < 0.05 "t" test) from week 7 onwards when B. bovis was used in combination. The biological significance of this difference is unclear.     

Transcriptional response of peripheral blood mononuclear cells from cattle infected with Mycobacterium bovis

Mycobacterium bovis is the causative agent of most cases of bovine tuberculosis. The identification of bTB biomarkers in specific stages of the disease will contribute to a better understanding of the immunopathology associated with tuberculosis and will enable their use in disease diagnosis and prognosis. The aim of this study was to evaluate the gene expression profile induced after specific stimulation of bovine peripheral blood mononuclear cells from cattle infected with M. bovis using the Affymetrix® GeneChip® Bovine Genome Array. A total of 172 genes showed differential expression profile that was statistically significant with log2-fold change >2.5 and <−2.5. Twenty-four out of these genes were upregulated and 148 were downregulated in bovine peripheral blood mononuclear cells of M. bovis-infected cattle. The highest differentially-expressed genes were related to immune and inflammatory responses, apoptosis, endocytosis, cellular trafficking and genes encoding proteins involved in cellular matrix degradation. Microarray results were confirmed in another group of infected cattle by RT-qPCR for the CD14, IL-1R, THBS1, MMP9 and FYVE genes. This study confirms previous findings that have shown that M. bovis infection in cattle results in the downregulation of immune response-related genes. Moreover, it validates the use of microarray platforms in combination with RT-qPCR to identify biomarkers of bovine tuberculosis. In addition, we propose CD14, IL-1R, THBS1, MMP9 and FYVE as potential biomarkers of bovine tuberculosis.     

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P>0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P<0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P>0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P<0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P<0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar.     

The effect of mitomycin C treatment of infected erythrocytes on infection with Babesia microti and Babesia rodhaini in mice

In vitro treatment of Babesia microti infected erythrocytes with mitomycin C before their injection into mice prolonged the prepatent period of infection, reduced the levels of the infection in the ‘breakthrough’ parasitaemia and induced protection against reinfection. Treatment of B. microti with mitomycin C at a concentration of 25 μg ml^?1 resulted in a mean peak parasitaemia of 6.2% in the infected mice compared with 46.5% in control mice injected with untreated B. microti parasites. In addition, mice survived a normally fatal B. rodhaini infection if injected with 6.2 × 10⁷ infected erythrocytes treated with 25 μg ml^?1 mitomycin C and four of five mice survived infection with 6.2 × 10⁵ similarly treated infected erythrocytes. However, the degree of protection against B. rodhaini was dependent on the concentration of mitomycin C used to treat the parasites and treatment of 5 × 10⁷ infected erythrocytes with 50 μg ml^?1 resulted in survival of only four of the five infected mice. In addition, when 100 μg ml^?1 of mitomycin C was used to treat B. rodhaini parasites, the course of infection, although delayed, was indistinguishable from that seen in the control mice and all the mice died. The latter results and the lack of efficacy of comparable numbers of heat killed parasites suggested the necessity for sufficient, non-replicating, mitomycin C treated parasites to metabolize and produce and/or present protective antigens to the host.     

The toxicity of adenosine analogues against Babesia bovis in vitro.

The toxicities of 20 analogues of deoxyadenosine or adenosine were tested in vitro against the intraerythrocytic parasite Babesia bovis. IC37 values (the concentration of compound required to reduce cell survival to 37%) were determined for each compound. Tubercidin (7-deaza-adenosine), 2-bromo-adenosine, 8-bromo-3-ribosyl adenine and 6-phenylamino-deoxyadenosine were shown to be the most toxic towards B. bovis. Comparison of the toxicity results for these compounds in B. bovis with those in human melanoma cell lines indicated a differential toxicity, in that many of the compounds were toxic towards B. bovis but were relatively non-toxic towards human melanoma cell lines and vice versa. These results suggest that the mechanism of toxicity of the deoxyadenosine and adenosine analogues, whose normal metabolism involves transport, metabolism and incorporation into nucleic acids, may vary significantly between B. bovis and mammalian cells, allowing such drugs to be considered for parasite chemotherapy.     

Babesia rodhaini, Babesia bovis, and Babesia bigemina: analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst.

The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments.     

Reduction in virulence of Babesia bovis due to rapid passage in splenectomized cattle.

A marked loss of virulence of Babesia bovis for normal cattle was observed during its rapid serial, blood passage in splenectomized calves. In 2 strains studied closely, responses to infection in cattle inoculated with parasites collected from the 11th passages were minimal, although the splenectomized donors were severely affected. The change was reversed by passaging in intact hosts, and in one experiment the parasites had become very virulent at passage 5. The finding has proved useful in the preparation of safe, living vaccines to control babesiosis. Selection either for immunogenic antigens, against immunosuppressive ones, or a combination of these effects may cause the decrease in virulence.     

Early potent protection against heterologous SIVsmE660 challenge following live attenuated SIV vaccination in Mauritian cynomolgus macaques

Background

Live attenuated simian immunodeficiency virus (SIV) vaccines represent the most effective means of vaccinating macaques against pathogenic SIV challenge. However, thus far, protection has been demonstrated to be more effective against homologous than heterologous strains. Immune correlates of vaccine-induced protection have also been difficult to identify, particularly those measurable in the peripheral circulation.

Methodology/Principal Findings

Here we describe potent protection in 6 out of 8 Mauritian-derived cynomolgus macaques (MCM) against heterologous virus challenge with the pathogenic, uncloned SIVsmE660 viral stock following vaccination with live attenuated SIVmac251/C8. MCM provided a characterised host genetic background with limited Major Histocompatibility Complex (MHC) and TRIM5α allelic diversity. Early protection, observed as soon as 3 weeks post-vaccination, was comparable to that of 20 weeks vaccination. Recrudescence of vaccine virus was most pronounced in breakthrough cases where simultaneous identification of vaccine and challenge viruses by virus-specific PCR was indicative of active co-infection. Persistence of the vaccine virus in a range of lymphoid tissues was typified by a consistent level of SIV RNA positive cells in protected vaccinates. However, no association between MHC class I /II haplotype or TRIM5α polymorphism and study outcome was identified.

Conclusion/Significance

This SIV vaccine study, conducted in MHC-characterised MCM, demonstrated potent protection against the pathogenic, heterologous SIVsmE660 challenge stock after only 3 weeks vaccination. This level of protection against this viral stock by intravenous challenge has not been hitherto observed. The mechanism(s) of protection by vaccination with live attenuated SIV must account for the heterologous and early protection data described in this study, including those which relate to the innate immune system.     

Temperature and external electric field influence in membrane permeability of Babesia infected erythrocytes

1. Modification of erythrocyte membrane properties infected by Babesia canis was studied using the effect of electric pulses of short duration. 2. This process induces the formation of pores in the membrane and the releasing of hemoglobin and other cytoplasmic proteins into the external medium. 3. The rate of molecular permeation across the electrically perforated membranes depends on several factors: electric-field strength, pulse number, pulse duration, temperature and cellular concentration. 4. Even for low parasitemia, differences in the effect of these parameters were observed between infected and non-infected erythrocytes. 5. Here we describe an influence of electric field intensity and temperatures on the opening pores.