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.     

The proliferative response of mouse lymphoid tissues during infections with Babesia microti or Babesia rodhaini

Inchley C. J., Grieve E. M. and Preston P. M. 1987. The proliferative response of mouse lymphoid tissues during infections with Babesia microti or Babesia rodhaini. International Journal for Parasitology17: 945–950. Proliferative responses induced by the lethal protozoan parasite, Babesia rodhaini, or the non-lethal species, B. microti were measured in the lymphoid tissues of infected mice. Both stimulated equally rapid DNA synthesis in the spleen, but spleen enlargement during the first week was more pronounced after infection with B. rodhaini, suggesting an earlier influx of cirulating cells than during B. microti infections. Termination of B. rodhaini infections by chemotherapy revealed that the recruitment of cells, but not the proliferative response, was dependent on the presence of live parasites. The spleen enlargement typical of B. microti-infected mice developed during the second week, but up to half of this response could be attributed to compensatory erythropoiesis. Babesia microti, but not B. rodhaini, induced a proliferative response in peripheral lymph nodes, and transiently depressed cell division in the thymus.     

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.     

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.     

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.     

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.     

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.     

Artesunate,a potential drug for treatment of Babesia infection

The effects of artesunate, a water-soluble artemisinin derivative, against Babesia species, including Babesia bovis, Babesia gibsoni and Babesia microti were studied. Cultures of B. bovis and B. gibsoni were treated with 0.26, 2.6, 26 and 260 μM artesunate, showing inhibition of parasite growth at concentrations equal to and greater than 2.6 μM artesunate by days 3 post-treatment for B. gibsoni and B. bovis in a dose-dependent manner. Consistent with in vitro experiments, artesunate was effective in the treatment of mice infected with B. microti at doses equal to and greater than 10 mg/kg of body weight on days 8–10 post-infection. Taken together, these results suggest that artesunate could be a potential drug against Babesia infection.     

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.     

The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks

Irvin A.D., Boarer C.D.H., Kurtti T.J. and Ocama J.G.R. 1981. The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks. International Journal for Parasitology11:451–456. The uptake of radio-labelled nucleic acid precursors by blood and tick salivary gland forms of Theileria pana was studied. Piroplasms took up tritiated purines, particularly hypoxanthine, but not pyrimidines. Similar uptake was recorded by T. parva, both in tick saliva and in salivary glands maintained in vitro. Intermediate parasite stages were those most readily labelled in glands; this reflected active nucleic acid synthesis associated with rapid parasite division. Radio-labelling of T. parva in tick salivary glands could be of value in procedures used for concentrating and purifying theilerial sporozoites.     

The role of Ixodes trianguliceps tick larvae in circulation of Babesia microti in the Middle Urals

The nested PCR method with primers flanking a conserved fragment of the Babesia microti ss-rDNA gene was used to examine 834 larvae of Ixodes trianguliceps ticks engorged to a varying degree, taken off 237 hosts of 12 species (rodents and insectivores). The hosts were collected in southern taiga forests in the lowmountain area of the Middle Urals (Chusovoi District, Perm Province) in 2003–2010. Babesia DNA was detected in 89 (10.7%) larvae from 8 species of small mammals. According to the data obtained by PCR and microscopic methods, either B. microti DNA or the parasites themselves were found in the blood of 45.2% of the mammals. The nucleotide sequences of 15 amplicons of Babesia DNA obtained from larvae of I. trianguliceps ticks and their hosts were identical to those of B. microti available in GenBank. In 13 cases, they were similar to B. microti US-type (a human pathogen) and in two cases (those from I. trianguliceps and from the vole Clethrionomys rufocanus from which it was removed), to B. microti of the Munich strain which is not pathogenic to humans. The duration of feeding on small mammals seems to exert the main influence on the infection rate of I. trianguliceps larvae. The fully engorged larvae contained B. microti DNA more often and usually in greater amounts than those collected during the first days of blood-sucking. The latter usually revealed Babesia DNA in the minimum quantity (< 0.064 ng/μl). According to the data obtained, transovarial transmission of Babesia in I. trianguliceps is unlikely. The processes of horizontal and transstadial transmission appear to be of crucial importance for the functioning of the natural foci of babesiosis.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.     

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.     

A comparison of Babesia infections in intact,surgically splenectomised,and congenitally asplenic (Dh/+) mice

Infections with Babesia rodhaini and B. microti were studied in congenitally asplenic (Dh/+) mice, surgically splenectomised mice and intact mice. Mice without spleens were more susceptible to infections than intact mice, but Dh/+ mice were less susceptible than surgically splenectomised mice, indicating that some functional splenic activity had been taken over by other tissues in Dh/+ mice. It is suggested that this functional activity may be mediated by natural killer (NK) cells, and that Dh/+ mice could prove of value in the study of babesiosis in general and NK activity in particular.Male mice were more susceptible to infection than females.     

Babesia microti: Pathogenesis of parasite of human origin in the hamster

The pathogenesis of the disease in hamsters caused by the first human Babesia isolant, tentatively named Babesia microti, and the immunologic relationship of the organism to Babesia canis were studied. The patent phase of the disease was characterized by severe anemia and marked parasitemia which occurred between the 6th and 41st day following infection. An increase in total white cell count with a neutrophilia, eosinophilia, monocytosis, and lymphocytosis was observed during the patent phase. The patent phase was followed by development of a carrier state. This was demonstrated by relapse following splenectomy 113 days after infection. No statistically significant differences were observed between the serum profiles of infected and noninfected animals during the period monitored. A serologic relationship between B. microti and B. canis was revealed by the use of gel diffusion and indirect fluorescent antibody (IFA) tests. The IFA test was used to monitor serum antibody responses during the patent and carrier phases of the disease. Crossabsorption studies between B. canis and B. microti revealed that the two organisms possess common and specific antigens.     

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.     

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.     

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.     

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.     

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.