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.     

Babesia bovis (Argentina): Studies on the composition and location of antigen associated with infected erythrocytes

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.     

New insights into the altered adhesive and mechanical properties of red blood cells parasitized by Babesia bovis

Sequestration of parasite-infected red blood cells (RBCs) in the microvasculature is an important pathological feature of both bovine babesiosis caused by Babesia bovis and human malaria caused by Plasmodium falciparum. Surprisingly, when compared with malaria, the cellular and molecular mechanisms that underlie this abnormal circulatory behaviour for RBCs infected with B. bovis have been relatively ignored. Here, we present some novel insights into the adhesive and mechanical changes that occur in B. bovis-infected bovine RBCs and compare them with the alterations that occur in human RBCs infected with P. falciparum. After infection with B. bovis, bovine RBCs become rigid and adhere to vascular endothelial cells under conditions of physiologically relevant flow. These alterations are accompanied by the appearance of ridge-like structures on the RBC surface that are analogous, but morphologically and biochemically different, to the knob-like structures on the surface of human RBCs infected with P. falciparum. Importantly, albeit for a limited number of parasite lines examined here, the extent of these cellular and rheological changes appear to be related to parasite virulence. Future investigations to identify the precise molecular composition of ridges and the proteins that mediate adhesion will provide important insight into the pathogenesis of both babesiosis and malaria.     

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.     

Serological and immunological studies with a hexane extract of Babesia bovis-infected erythrocytes.

Antigenic and immunogenic activities of a hexane extract from Babesia bovis-infected erythrocytes were investigated. Positive ELISA and IFAT reactions were obtained with bovine antisera to B. bovis and B. bigemina produced by natural infection and rabbit antisera to the hexane extract, respectively. In contrast, negative ELISA reactions were obtained with Anaplasma marginale antisera indicating that the antigen(s) is specific for the genus Babesia. The IFAT clearly demonstrated that the antigen was associated with the parasite and the infected erythrocyte and not present in uninfected erythrocytes. Furthermore, cross-reactions with Babesia bigemina antisera suggested that serological cross-reactivity in bovine Babesia species is at least due in part to lipid or lipid-associated antigens.     

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.     

Increased glucose permeability in Babesia bovis-infected erythrocytes

Glucose influx into bovine erythrocytes was found to be significantly increased upon infection with the parasite, Babesia bovis. The influx of glucose into the infected cells over 4 min was not saturable at high concentrations of glucose (240 mM), nor was it affected by established inhibitors of mammalian glucose transport, such as cytochalasin B and phloretin (0.1-100 microM). Glucose uptake into the parasitized cells was, however, inhibited by phloridzin (phloretin-2-beta-glucoside) at concentrations over the range of 10-500 microM. Further inhibition of glucose uptake by adenosine (2.5-15 mM) was found to occur in B. bovis-infected bovine erythrocytes, suggesting an interaction of adenosine with the new or altered component of glucose transport in the parasitized cells.     

Virulent and Avirulent Strains of Babesia bovis: The Relationship Between Parasite Protease Content and Pathophysiological Effect of the Strain

A virulent strain of Babesia bovis (“L” strain) was rendered avirulent by irradiation with 35 krads with a γ 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 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.     

α-Enolase, an adhesion-related factor of Mycoplasma bovis

Mycoplasma bovis is the causative agent of Mycoplasma bovis-associated disease (MbAD). Although the mechanisms underlying M. bovis adherence to host cells is not clear, recent studies have shown that the cell surface protein α-enolase facilitates bacterial invasion and dissemination in the infected host. In this study, we cloned, expressed and purified recombinant M. bovis α-enolase and induced polyclonal anti-α-enolase antibodies in rabbits. M. bovis α-enolase was detected in the cytoplasmic and membrane protein fractions by these antibodies. Triple immunofluorescence labeling combined with confocal laser scanning microscopy (CLSM) revealed that the plasminogen (Plg) enhanced the adherence of M. bovis to embryonic bovine lung (EBL) cells; the values obtained for adherence and inhibition are consistent with this finding. Interestingly, we found that trace amounts of trypsin acted as a more effective enhancer of cell adherence than Plg. Hence, our data indicate that surface-associated M. bovis α-enolase is an adhesion-related factor of M. bovis that contributes to adherence by binding Plg.     

Optimization of Babesia bovis transfection methods

The tick borne Babesia parasites remain an important limitation for development of cattle industries worldwide. A stable transfection of Babesia bovis will be useful for functional analysis of the recently sequenced B. bovis genome and to design improved methods to control Babesia infections. In this study, we describe a novel system for nucleofection of B. bovis infected erythrocytes and we optimize methods to introduce plasmids encoding the luciferase reporter gene into Babesia infected erythrocytes or free merozoites using either a BioRad GenePulser II electroporation system or nucleofection technology (Amaxa) A comparative study among four different transfection methods: transfection of infected erythrocytes and purified merozoites with 2 or 100 microg of plasmid, using electroporation (BioRad GenePulser II) or nucleofection (Amaxa) indicates that electroporation of infected erythrocytes with 100 microg of plasmid or nucleofection with 2 microg of plasmid are the most efficient ways to transfect B. bovis parasites. The data also indicate that nucleofection is more efficient than electroporation for transfecting small quantities of plasmids (2 microg range), whereas the inverse is true for transfection of larger quantities (100 microg range). This information will facilitate further development of efficient stable transfection systems.     

A study of the candidate virulence factors of Bacteroides fragilis

Bacteroides fragilis strains were classified as virulent or avirulent on the basis of their clearance from the subcutaneous tissues of mice. To determine the factors which may contribute to the virulence of B. fragilis strains, we studied encapsulation, hydrophobicity, growth rate, serum sensitivity, agglutination with erythrocytes of different origin, and neuraminidase production. The strains of the virulent group displayed a higher growth rate in broth and a lower sensitivity to the bactericidal activity of serum than the strains of the avirulent group. They also agglutinated different types of erythrocytes more strongly than did the avirulent strains. No significant differences were found between the two groups of strains as regards encapsulation, hydrophobicity and neuraminidase activity.     

An efficient microtest to study adherence of bacteria to mammalian cells

A simple, fast and highly reproducible microtest was developed for in vitro adherence studies. A rat epithelial cell line was investigated for the adherence of clinical and subclinical ovine and bovine Staphylococcus aureus strains isolated from mastitis. Staphylococcus aureus strains differed in their ability to adhere to epithelial cells, the degree of adherence being dependent on the concentration of bacteria used in the test.     

Increased adherence of oxidant-treated human and bovine erythrocytes to cultured endothelial cells

Bovine erythrocytes, which normally lack phosphatidyl choline in their membranes, when treated with either H2O2 or diamide (1-3 mM), showed a partial appearance of phosphatidyl ethanolamine (PE 40%) and phosphatidyl serine (PS, 30-33%) in the external leaflet of the bilayer and a concomitant increased (four- to five-fold) propensity to adhere to cultured bovine aortic endothelial cells. Similar treatment of normal human erythrocytes caused an alteration in the organization of the phospholipid bilayer and also resulted in their increased adherence to endothelial cells derived either from human umbilical vein or bovine aorta. Treatment of RBCs with H2O2 at low concentration (0.5 mM) resulted in cross-linking of spectrin without significant changes in the orientation of aminophospholipids but the RBCs exhibited 15-20% increase in adherence to endothelial cells. Pretreatment of either human or bovine erythrocytes with antioxidants such as vitamin E (2 mM) prevented both oxidant-induced reorganization of phospholipids in the bilayer and enhancement of adherence to endothelial cells. Introduction of either phosphatidyl serine or phosphatidyl ethanolamine but not phosphatidyl choline into erythrocyte membranes increased their adherence to endothelial cells threefold. Oxidant-treated RBCs exhibited enhanced binding and fluorescence of Merocyanine 540 dye (MC-540), which is sensitive to the packing of lipids in the lipid bilayer. On flow cytometric analysis, 78% of H2O2 (0.5 mM)-treated erythrocytes compared to 30% of untreated RBCs exhibited MC-540 binding and fluorescence, indicating differences in the lipid packing in the outer leaflet of the bilayer. Oxidant-treated erythrocytes adhere preferentially to endothelial cells rather than to bovine aortic smooth muscle cells and skin fibroblasts. It is suggested that the alterations in the erythrocyte membrane surface due to spectrin cross-linking and the organization of the phospholipids concomitant with less ordered packing in the external leaflet of the bilayer, either induced by oxidative manipulation in normal RBC or in pathological erythrocytes, play a role in erythrocyte-endothelial cell interaction.     

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.     

Failure of antibiotics gentamycin, tylosin, lincomycin and spectinomycin to eliminate Mycoplasma bovis in artificially infected frozen bovine semen

To study the effect of antibiotics upon Mycoplasma bovis in fresh bovine semen just before freezing, specimens of bovine semen were artificially infected with 1 of 9 different strains of M. bovis. Inocula of each strain were prepared to contain 10(5) to 10(6)/mL colony-forming units of M. bovis at 3 different stages of the growth phase. The infected semen was diluted with a Tris extender by a 3-step procedure using an antibiotic mixture of gentamicin, tylosin, lincomycin and spectinomycin (GTLS). This semen-antibiotic mixture was placed into French straws that were stored at -196 degrees C. The control semen specimens contained no antibiotics Mycoplasmas were counted after 8 d of storage in 3 decimal dilutions of the frozen semen. No evident effect was noticed upon the 9 tested strains of mycoplasmas in the semen frozen with the antibiotics, compared with that of the untreated control samples. It was further shown that this lack of effect was irrespective of the stage of the growth phase of the mycoplasmas. It was concluded that the antibiotic mixture (GTLS) in semen specimens is not capable of total elimination of mycoplasmas in frozen bovine semen.     

Babesia bovis: evidence for selection of subpopulations during attenuation

DNA probes were used to detect variation in subpopulations of virulent and serially passaged Babesia bovis. Two distinct patterns were evident after hybridization to genomic DNA; the first was a basic profile typical of virulent B. bovis and the second, a more variable array, was characteristic of B. bovis after various stages of attenuation. Tick transmission of avirulent B. bovis causes reversion to the virulent genomic pattern, suggesting that selective enrichment of a small residual subpopulation caused reversion to a virulent profile of subpopulations. Certain genomic fragments, predominant in either virulent or avirulent parasite forms, are putative "markers" or actual elements responsible for these biological characteristics.     

Haemagglutination of Bacteroides fragilis

Abstract The relationship between the ability to cause haemagglutination (HA) and the presence of capsules and/or pili was examined for 50 clinical isolates of Bacteroides fragilis . HA was tested using a slide technique, and bovine, porcine, guinea pig, rat, rabbit, horse, human, chicken and pigeon erythrocytes. Chicken and pigeon erythrocytes were the best indicators for HA with 43 (86%) of the strains tested causing HA and 39 (78%) with strong reactions. Capsule staining showed that the same 43 strains causing HA also produced a demonstrable capsule. No pili were found on either encapsulated or non-encapsulated strains using transmission electron microscopy. These results suggest that adherence of B. fragilis is related to the presence of capsular material, not pili.     

Modification of host erythrocyte membranes by trypsin and chymotrypsin treatments and effects on the in vitro growth of bovine and equine Babesia parasites

In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC showed that the trypsin-treated bovine RBC, but not the chymotrypsin-treated ones, significantly reduced the growth of B. bovis and B. bigemina as compared to the control. In contrast, the growth of B. equi and B. caballi was not affected by any of these proteases. Thus, the bovine, but not the equine, Babesia parasites require the trypsin-sensitive membrane (sialoglyco) proteins to infect the RBC.     

Evaluation of susceptibility of Mycobacterium bovis to antituberculous drugs by radiometric BACTEC 460TB system

Susceptibility of Mycobacterium bovis strains to antituberculous drugs (isoniazid and rifampin) was detected by radiometric BACTEC 460TB system. M.bovis strains were isolated from tissue samples showing tuberculous lesions collected at an abbattoir from cattle belonging to 47 tuberculosis outbreaks occurring in Northern Italy in 1995-1999. Forty-six out of 61 strains (75.4%) resulted susceptible to both isoniazid and rifampin. Thirteen strains (21.3%) were resistant to isoniazid only. No strains showed resistance to rifampin only. Two strains (3.3%) resulted resistant to both drugs, showing antituberculous multidrug-resistance. Given the compulsory eradication program of bovine tuberculosis by elimination of infected animals and the ban on antituberculous drug treatments in animals, detection of resistant M. bovis strains appears of great interest.     

Babesia bovis--effects of phospholipid translocation and adenosine tri-phosphate consumption

The adenosine tri-phosphate concentration of Babesia bovis-infected erythrocytes was significantly (P less than 0.001) less than that of pre-infection erythrocytes. In addition, phosphatidyl serine was detected on the plasmatic surface of the infected erythrocyte. These two related findings could play important roles in the microvascular stasis characteristic of acute B. bovis infection.