The in vitro uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice

Irvin A. D., Young E. R. and Purnell R. E. 1978. The in vitro uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice. International Journal for Parasitology8: 19–24. Blood and mice infected with Babesia microti and B. rodhaini, and from cattle infected with B. divergens and B. major, was incubated in Eagles medium for 24 h in the presence of tritiated purines and pyrimidines. Uptake of these compounds was assessed by liquid scintillation counting and by autoradiography. Hypoxanthine, adenosine and adenine were readily incorporated by all four species of parasites. Thymine, thymidine and uridine were generally not incorporated. Uptake of [³H]hypoxanthine by B. microti occurred within minutes of exposure to the precursor. The amount of [³H]hypoxanthine incorporated by B. rodhaini-infected erythrocytes was proportional to the percentage of parasitized cellsThe results suggest that structural analogues of hypoxanthine and other purines may be incorporated and act against intra-erythrocytic Babesia.     

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.     

Babesia rodhaini: the protective effect of pyruvate kinase deficiency in mice

Despite the evidence suggesting that mouse pyruvate kinase (PK) deficiency provides protection against malaria in rodents, there has been no investigation of a parallel protective effect against babesiosis caused by Babesia rodhaini. Here, we examined whether a PK-deficient co-isogenic mouse strain (CBA-Pk-1^slc) was protected against B. rodhaini infection. We demonstrated that deficiency in pyruvate kinase correlated with a significant protective effect, with survival rates of 50%, 58% and 56% in groups inoculated with 10, 10³ and 10⁵ parasitized erythrocytes, respectively. In contrast, control CBA (CBA-Pk-1⁺) mice exhibited 100% lethality, regardless of the infectious dose. In addition, CBA-Pk-1^slc mice showed decreased levels of parasitemia when compared to CBA-Pk-1⁺ mice, in groups given 10, 10³ or 10⁵ parasitized erythrocytes. These results indicate that similar to PK deficiency in rodents, PK deficiency in mice affects the in vivo growth of B. rodhaini and protects the mice from lethal babesiosis.     

Investigating disease severity in an animal model of concurrent babesiosis and Lyme disease

The incidence of babesiosis, Lyme disease and other tick-borne diseases has increased steadily in Europe and North America during the last five decades. Babesia microti is transmitted by species of Ixodes, the same ticks that transmit the Lyme disease-causing spirochete, Borrelia burgdorferi. B. microti can also be transmitted through transfusion of blood products and is the most common transfusion-transmitted infection in the U.S.A. Ixodes ticks are commonly infected with both B. microti and B. burgdorferi, and are competent vectors for transmitting them together into hosts. Few studies have examined the effects of coinfections on humans and they had somewhat contradictory results. One study linked coinfection with B. microti to a greater number of symptoms of overall disease in patients, while another report indicated that B. burgdorferi infection either did not affect babesiosis symptoms or decreased its severity. Mouse models of infection that manifest pathological effects similar to those observed in human babesiosis and Lyme disease offer a unique opportunity to thoroughly investigate the effects of coinfection on the host. Lyme disease has been studied using the susceptible C3H mouse infection model, which can also be used to examine B. microti infection to understand pathological mechanisms of human diseases, both during a single infection and during coinfections. We observed that high B. microti parasitaemia leads to low haemoglobin levels in infected mice, reflecting the anaemia observed in human babesiosis. Similar to humans, B. microti coinfection appears to enhance the severity of Lyme disease-like symptoms in mice. Coinfected mice have lower peak B. microti parasitaemia compared to mice infected with B. microti alone, which may reflect attenuation of babesiosis symptoms reported in some human coinfections. These findings suggest that B. burgdorferi coinfection attenuates parasite growth while B. microti presence exacerbates Lyme disease-like symptoms in mice.     

Babesia bigemina: immune response of cattle inoculated with irradiated parasites

Effects of various radiation dosages on the infectivity and immunogenicity of Babesia bigemina were studied. Calves infected with 1 × 10¹⁰, B. bigemina parasitized erythrocytes exposed to 24 krad developed progressive parasitemias. Some calves receiving 1 × 10¹⁰ parasitized erythrocytes irradiated at 36 krad did not develop progressive infections. Progressive infections were prevented by exposure to irradiation at 48 and 60 krad. A degree of acquired resistance to infection with B. bigemina developed in calves after inoculation with parasites irradiated at 48 and 60 krad. The resistance developed was sufficient to suppress multiplication of the Babesia and to permit calves to survive otherwise severe clinical infections due to challenge with nonirradiated parasites. Irradiated parasites were frozen without apparent loss of immunizing properties.     

Induction of IL-10-Producing CD1dhighCD5+ Regulatory B Cells following Babesia microti-Infection

Background

Understanding the induction of immune regulatory cells upon helminth infection is important for understanding the control of autoimmunity and allergic inflammation in helminth infection. Babesia microti, an intraerythrocytic protozoan of the genus Babesia, is a major cause of the emerging human disease babesiosis, an asymptomatic malaria-like disease. We examined the influence of acute B. microti infection on the development of regulatory B cells together with regulatory T cells.

Principal Findings

Our data demonstrate that B cells stimulated in vitro with B. microti produce interleukin (IL)-10. This cytokine is also secreted by B cells isolated from B. microti-infected mice in response to lipopolysaccharide stimulation. In addition, high levels of IL-10 were detected in the serum of mice after infection with B. microti. The frequency of IL-10-producing CD1d^highCD5⁺ regulatory B cells (Bregs) and CD4⁺CD25⁺FoxP3⁺ T cells increased during the course of B. microti infection. Furthermore, adoptive transfer of IL-10-producing B cells induced by B. microti infection led to increased susceptibility of recipient mice to infection with B. microti. In contrast, experiments with B cell-deficient (µMT) mice demonstrated that lack of B cells enhances susceptibility to B. microti infection.

Conclusions

This study is the first demonstration of the expansion of Bregs following infection by an intraerythrocytic protozoan parasite. These data suggest that B. microti infection in mice provides an excellent model for studying Breg-mediated immune responses and begins to elucidate the mechanism by which helminth infection regulates autoimmunity and allergic inflammation.     

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice. International journal for Parasitology3: 773–781. Babesia rodhaini infection was compared in irradiated, splenectomized and control mice. Although irradiation reduced the weight of the spleen by as much as 95 per cent, this reduction in size did not result in parasitaemia levels comparable to those seen in splenectomized mice, which were consistently higher. Parasitaemias were similar in irradiated and control mice, but the mean survival time in control mice was longer than that of irradiated or splenectomized mice, which were comparable. Splenectomy generally resulted in higher parasitaemias than those seen in non-splenectomized mice.Since B. rodhaini has a predeliction for invading reticulocytes, the apparent failure of irradiated mice to develop parasitaemias comparable to those of splenectomized mice, may have been due to the selective destruction of these immature red cells by irradiation.     

Quantitative proteomics and phosphoproteomic analyses of mouse livers after tick-borne Babesia microti infection

Babesia microti is a tick-borne protozoan parasite that infects the red blood cells of mice, humans, and other mammals. The liver tissues of BALB/c mice infected with B. microti exhibit severe injury. To further investigate the molecular mechanisms underlying liver injury and liver self-repair after B. microti infection, data-independent acquisition (DIA) quantitative proteomics was used to analyse changes in the expression and phosphorylation of proteins in liver tissues of BALB/c mice during a B. microti infection period and a recovery period. The expression of FABP1 and ACBP, which are related to fatty acid transport in the liver, was downregulated after infection with B. microti, as was the expression of Acox1, Ehhadh and Acaa1a, which are crucial rate-limiting enzymes in the process of fatty acid β oxidation. The phosphorylation levels of AMP-activated protein kinase (AMPK) and Hormone-sensitive lipase (HSL) were also downregulated. In addition, the expression of PSMB9, CTSC, and other immune-related proteins was increased, reflecting an active immune regulation mechanism in the mice. The weights of mice infected with B. microti were significantly reduced, and the phosphorylation levels of IRS-1, c-Raf, mTOR, and other proteins related to growth and development were downregulated.     

Impact of the large-scale deployment of artemether/lumefantrine on the malaria disease burden in Africa: case studies of South Africa,Zambia and Ethiopia

Background

Azathioprine triggers suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Eryptosis may accelerate the clearance of Plasmodium -infected erythrocytes. The present study thus explored whether azathioprine influences eryptosis of Plasmodium -infected erythrocytes, development of parasitaemia and thus the course of malaria.

Methods

Human erythrocytes were infected in vitro with Plasmodium falciparum (P. falciparum) (strain BinH) in the absence and presence of azathioprine (0.001 – 10 μM), parasitaemia determined utilizing Syto16, phosphatidylserine exposure estimated from annexin V-binding and cell volume from forward scatter in FACS analysis. Mice were infected with Plasmodium berghei (P. berghei) ANKA by injecting parasitized murine erythrocytes (1 × 10⁶) intraperitoneally. Where indicated azathioprine (5 mg/kg b.w.) was administered subcutaneously from the eighth day of infection.

Results

In vitro infection of human erythrocytes with P. falciparum increased annexin V-binding and initially decreased forward scatter, effects significantly augmented by azathioprine. At higher concentrations azathioprine significantly decreased intraerythrocytic DNA/RNA content (≥ 1 μM) and in vitro parasitaemia (≥ 1 μM). Administration of azathioprine significantly decreased the parasitaemia of circulating erythrocytes and increased the survival of P. berghei -infected mice (from 0% to 77% 22 days after infection).

Conclusion

Azathioprine inhibits intraerythrocytic growth of P. falciparum, enhances suicidal death of infected erythrocytes, decreases parasitaemia and fosters host survival during malaria.     

Nippostrongylus brasiliensis: mast cells and histamine levels in tissues of infected and normal rats.

The role of the spleen during Babesia microti and B. hylomysci infection was investigated by examining the course of infection in both intact and splenectomized mice. The presence of the spleen was critical during the early stages of infection to control excessive multiplication of either parasite, a role taken over by other lymphoid sites as the infection progressed. Mice splenectomized prior to or within 1 week of B. microti inoculation developed extended infections with some deaths, and others were unable to check their parasitemias. All intact mice, and those splenectomized 1 week after infection with B. microti, recovered completely with subsequent development of sterile immunity. Mice splenectomized prior to or within 1 week of B. hylomysci inoculation succumbed to hyperacute infections: Some of the intact mice, and those splenectomized 12 days after infection, recovered but continued to harbor a low-grade infection with periodical recrudescences. Erythrophagocytosis of infected and uninfected erythrocytes was detected in saline preparations and impression smears of spleen and bone marrow and rarely in blood smears of infected mice. This coincided with anemia, splenomegaly, and relatively high levels of opsonizing antibodies, especially during B. microti infection. The colloidal carbon clearance method was used to investigate the phagocytic activity of the reticuloendothelial system. Carbon clearance rates increased rapidly during both infections, but peak B. hylomysci parasitemia coincided with reticuloendothelial phagocytic depression and death of the host. Babesia microti stimulated a consistently higher reticuloendothelial phagocytic activity with higher erythrophagocytosis both in the spleen and bone marrow than did B. hylomysci.     

Studies on cell fusion between Babesia rodhaini-infected mouse erythrocytes and baby hamster kidney cells.

Heterokaryons were formed by fusion of B. rodhaini-infected mouse erythrocytes and baby hamster kidney (BHK) cells, using Sendai virus. The erythrocyte membrane rapidly lysed inside the BHK cell cytoplasm releasing free parasites. There was no evidence that parasite multiplication occurred inside the BHK cells, nor that parasitized BHK cells were infective for mice.Transient erythrocyte homokaryons were observed in some preparations.The approach indicates a possible method for the in vitro cultivation of Babesia.     

Vertical Transmission of Babesia microti in BALB/c Mice: Preliminary Report

Babesia spp. (Apicomplexa, Piroplasmida) are obligate parasites of many species of mammals, causing a malaria-like infection- babesiosis. Three routes of Babesia infection have been recognized to date. The main route is by a tick bite, the second is via blood transfusion. The third, vertical route of infection is poorly recognized and understood. Our study focused on vertical transmission of B. microti in a well-established mouse model. We assessed the success of this route of infection in BALB/c mice with acute and chronic infections of B. microti. In experimental groups, females were mated on the 1^st day of Babesia infection (Group G0); on the 28^th day post infection (dpi) in the post- acute phase of the parasite infection (G28); and on the 90^th and 150^th dpi (G90 and G150 group, respectively), in the chronic phase of the parasite infection. Pups were obtained from 58% of females mated in the post-acute phase (G28) and from 33% of females in groups G90 and G150. Mice mated in the pre-acute phase of infection (G0) did not deliver pups. Congenital B. microti infections were detected by PCR amplification of Babesia 18S rDNA in almost all pups (96%) from the experimental groups G28, G90 and G150. Parasitaemia in the F1 generation was low and varied between 0.01–0.001%. Vertical transmission of B. microti was demonstrated for the first time in BALB/c mice.     

Borrelia burgdorferi Promotes the Establishment of Babesia microti in the Northeastern United States

Babesia microti and Borrelia burgdorferi, the respective causative agents of human babesiosis and Lyme disease, are maintained in their enzootic cycles by the blacklegged tick (Ixodes scapularis) and use the white-footed mouse (Peromyscus leucopus) as primary reservoir host. The geographic range of both pathogens has expanded in the United States, but the spread of babesiosis has lagged behind that of Lyme disease. Several studies have estimated the basic reproduction number (R ₀) for B. microti to be below the threshold for persistence (<1), a finding that is inconsistent with the persistence and geographic expansion of this pathogen. We tested the hypothesis that host coinfection with B. burgdorferi increases the likelihood of B. microti transmission and establishment in new areas. We fed I. scapularis larva on P. leucopus mice that had been infected in the laboratory with B. microti and/or B. burgdorferi. We observed that coinfection in mice increases the frequency of B. microti infected ticks. To identify the ecological variables that would increase the probability of B. microti establishment in the field, we integrated our laboratory data with field data on tick burden and feeding activity in an R ₀ model. Our model predicts that high prevalence of B. burgdorferi infected mice lowers the ecological threshold for B. microti establishment, especially at sites where larval burden on P. leucopus is lower and where larvae feed simultaneously or soon after nymphs infect mice, when most of the transmission enhancement due to coinfection occurs. Our studies suggest that B. burgdorferi contributes to the emergence and expansion of B. microti and provides a model to predict the ecological factors that are sufficient for emergence of B. microti in the wild.     

Babesia hylomysci and Babesia microti: Dexamethasone treatment of infected mice

The effect of dexamethason on Babesia hylomysci and B. microti was investigated in LACA mice. The drug enhanced both infections by depressing the immune mechanisms of the host when treatment was initiated before parasite inoculation, but had no effects on established and subpatent infections. The degree of parasitemia in the treated mice seemed to depend on the tropism of either parasite toward mature erythrocytes or reticulocytes. B. hylomysci, which favors mature erythrocytes, produced fulminating infections in treated mice. B. microti, which prefers reticulocytes, produced similar parasitemia patterns in treated and untreated mice, but only the treated mice succumbed to the infection. The drug, which suppressed cellular proliferation in the spleens of infected animals, together with its direct lympholytic effects, drastically changed the architecture of the organ.     

Infectivity and Immunogenicity of Irradiated Babesia rodhaini*

SYNOPSIS. Babesia rodhaini-parasitized mouse blood exposed to varied doses of γ radiation up to 30 kRad was inoculated into mice. Mice inoculated with nonirradiated B. rodhaini developed progressive infections and died 7–11 days postinoculation. Mice infected with B. rodhaini-parasitized blood exposed to doses up to and including 22 kRad developed progressive parasitemias which were delayed in comparison to mice inoculated with non-irradiated B. rodhaini. Some mice receiving parasitized blood irradiated at 26 kRad did not develop progressive parasitemias. Progressive infections were prevented by exposure to irradiation at 30 kRad. The results of 2 separate experiments revealed that one inoculation of parasitized blood exposed to 30 kRad or higher apparently stimulated a resistance to a challenge infection with nonirradiated parasitized blood. While 20 of 20 control mice died as a result of challenging infections, 9 of 28 mice previously exposed to irradiated parasitized blood survived. The injection of irradiated nonparasitized blood did not produce a discernible acquired resistance to B. rodhaini. Presumably the irradiated parasitized blood was responsible for the development of acquired resistance to B. rodhaini.     

Trichuris muris: effect of concurrent infections with rodent piroplasms on immune expulsion from mice.

Mice concurrently infected with the rodent piroplasms Babesia hylomysci or B. microti during a primary infection with the nematode Trichuris muris showed marked immunodepression, and the normal immune expulsion of the nematode was delayed. Immunodepression was most severe when the Babesia infections reached peak parasitaemia during the preexpulsion phase of the worm infection. Decline in parasitaemia to subpatent levels was associated with a reappearance of the immune response and expulsion of the worm. Babesia infections had little effect upon the expulsion of challenge infections of T. muris from mice previously immunized against the worm. Acute Babesia infections were found to exert a profound immunodepressive effect upon the agglutinating antibody response of mice to sheep red blood cells.     

Studies on immune responses to parasite antigens in mice: VII. Cells secreting antibodies to modified mouse erythrocytes in Babesia rodhaini-infected mice

Increased numbers of plaque-forming cells (PFC) secreting antibodies to bromelain-treated mouse erythrocytes were detected in the spleens of BALB/c·nu/+ mice heavily infected with Babesia rodhaini but not in the spleens of infected hypothymic BALB/ c·nu/nu mice. Whether the products of these cells, antierythrocyte autoantibodies, play any role in the destruction of intact erythrocytes in parasitized mice is unknown, but it is noteworthy that only low numbers of PFC were detected, and the modification of mouse erythrocytes was obligatory for detection of PFC.     

Tritrichomonas foetus: Stable anaerobic resistance to metronidazole in vitro

Stable anaerobic resistance of Tritrichomonas foetus to metronidazole was induced in vitro by cultivation of trichomonads in the Diamond's TYM medium with metronidazole in concentrations sublethal to the parasites. Nine metronidazole-resistant strains were derived from four drug-susceptible clones of the T. foetus strain KV-1. Subculturing the parasites at both increasing and constant pressure of the drug resulted in development of resistance if the medium contained at least 3 μg ml^?1 of metronidazole and the organisms were exposed to the drug for 3 to 8 months. The development of resistance was gradual and in all clones investigated proceeded through similar sequence of stages: (1) Survival without growth and subsequent reproduction at low metronidazole concentrations (1 to 5 μg ml^?1. (2) Survival and reproduction at moderate concentrations of the drug (10 to 15 μg ml^?1. (3) Resistance to 100 μg ml^?1 metronidazole, unstable in absence of selective pressure of the drug. (4) Resistance to high concentrations of metronidazole, stable when the organisms were maintained under nonselective conditions. The trichomonads with fully developed resistance were able to grow in anaerobic culture at 100 μg ml^?1 metronidazole and could be maintained indefinitely under these conditions. The minimal lethal concentrations for metronidazole obtained with these strains in an anaerobic in vitro assay were, at 48 h, 500 to 1000 μg ml^?1. This is 100 to 400 times higher than those obtained with the parent clones. The fully developed resistance was stable in organisms maintained in the absence of the drug over 2 years. The substrains with unstable resistance regained the susceptibility to high concentrations of metronidazole after 80 to 100 transfers in drug-free media. These strains, however, retained their resistance to moderate doses of metronidazole and full resistance could be restored by subculture in the presence of 10 μ ml^?1 metronidazole.     

Further studies on the uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice

Irvin A. D. and Young E. R. 1979. Further studies on the uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice. International Journal for Parasitology9: 109–114. An in vitro culture technique developed earlier was used to study the metabolism of nucleic acid precursors by Babesia microti and B. rodhaini of mice and by B. divergens and B. major of cattle. [³H]Hypoxanthine was readily incorporated by all species of parasite, and the presence of leucocytes did not affect this uptake. When parasites were maintained in culture their ability to incorporate [³H]hypoxanthine fell rapidly after 24 h, but when B. major was maintained at 4°C its subsequent ability to incorporate [³H]hypoxanthine persisted for at least 3 days. This finding could be of practical value in assessing infectivity of stored blood in vitro.On autoradiography, [³H]hypoxanthine appeared to be incorporated into both DNA and RNA of parasites. Salvage pathways for purine metabolism appeared to be important in all species of Babesia whereas for pyrimidine metabolism salvage pathways were more important for murine babesias and the de novo pathway more important for bovine species. This difference may relate to different permeabilities of bovine and murine erythrocyte membranes or may be a more fundamental species difference.     

Characterisation of the gene encoding a protective antigen from Babesia microti identified it as η subunit of chaperonin containing T-complex protein 1

Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the η subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT η subunit of B. microti and functions as a chaperonin.