Transfusion-transmitted Babesia spp.: bull's-eye on Babesia microti

Babesia spp. are intraerythrocytic protozoan parasites of animals and humans that cause babesiosis, a zoonotic disease transmitted primarily by tick vectors. Although a variety of species or types of Babesia have been described in the literature as causing infection in humans, the rodent parasite Babesia microti has emerged as the focal point of human disease, especially in the United States. Not only has B. microti become established as a public health concern, this agent is increasingly being transmitted by blood transfusion: estimates suggest that between 70 and 100 cases of transfusion-transmitted Babesia (TTB) have occurred over the last 30 years. A recent upsurge in TTB cases attributable to B. microti, coupled with at least 12 fatalities in transfusion recipients diagnosed with babesiosis, has elevated TTB to a key policy issue in transfusion medicine. Despite clarity on a need to mitigate transmission risk, few options are currently available to prevent the transmission of B. microti by blood transfusion. Future mitigation efforts may stress serological screening of blood donors in regionalized areas of endemicity, with adjunct nucleic acid testing during the summer months, when acute infections are prevalent. However, several hurdles remain, including the absence of a licensed blood screening assay and a thorough cost-benefit analysis of proposed interventions. Despite current obstacles, continued discussion of TTB without proactive intervention is no longer a viable alternative.     

Babesia microti primarily invades mature erythrocytes in mice

Babesia microti is a tick-borne red blood cell parasite that causes babesiosis in people. Its most common vertebrate reservoir is the white-footed mouse. To determine whether B. microti invades reticulocytes, as does the canine pathogen B. gibsoni, we infected the susceptible inbred mouse strains C.B-17.scid and DBA/2 with a clinical isolate of B. microti. Staining of fixed permeabilized red blood cells with 4',6'-diamidino-2-phenylindole or YOYO-1, a sensitive nucleic acid stain, revealed parasite nuclei as large bright dots. Flow cytometric analysis indicated that parasite DNA is primarily found in mature erythrocytes that expressed Babesia antigens but not the transferrin receptor CD71. In contrast, CD71-positive reticulocytes rarely contained Babesia nuclei and failed to express Babesia antigens. Accordingly, the frequency of YOYO-1-positive, CD71-negative cells strongly correlated with parasitemia, defined as the frequency of infected red blood cells assessed on Giemsa-stained blood smears. Importantly, the absolute numbers generated by the two techniques were similar. Parasitemia was modest and transient in DBA/2 mice but intense and sustained in C.B-17.scid mice. In both strains, parasitemia preceded reticulocytosis, but reticulocytes remained refractory to B. microti. In immunocompetent C.B-17 mice, reticulocytosis developed early, despite a marginal and short-lived parasitemia. Likewise, an early reticulocytosis developed in resistant BALB/cBy and B10.D2 mice. These studies establish that B. microti has a tropism for mature erythrocytes. Although reticulocytes are rarely infected, the delayed reticulocytosis in susceptible strains may result from parasite or host activities to limit renewal of the mature erythrocyte pool, thereby preventing an overwhelming parasitemia.     

Detection of Babesia microti by polymerase chain reaction.

Human babesiosis, which is caused by infection with the intraerythrocytic malarialike protozoan Babesia microti, has recently been diagnosed with increasing frequency in residents of New England. Diagnosis is difficult because of the small size of the parasite and the sparse parasitemia that is characteristic of most infections with this pathogen. We generated B. microti-specific DNA sequence information by universal primer amplification of a portion of the eukaryotic 16S-like gene; this was followed by direct DNA sequence analysis. Specific primers were synthesized on the basis of this sequence information for use in the polymerase chain reaction (PCR). The PCR-based system demonstrates a strong bias for detection of B. microti as opposed to Babesia gibsoni and does not amplify vertebrate DNA. The analytical sensitivity of the system is approximately three merozoites. Blood specimens from 12 patients with clinically diagnosed and parasitologically confirmed babesiosis from Nantucket Island, Mass., were PCR positive in a blinded test of this procedure. Thus, DNA amplification may provide an adjunct to conventional methods for the diagnosis of human babesiosis and may provide a new means of monitoring therapy or enhancing epidemiological surveillance for this emerging pathogen.     

Serological expression cloning of novel immunoreactive antigens of Babesia microti

Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent, Babesia microti. To develop more sensitive and specific assays for B. microti, we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique B. microti antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to Plasmodium sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during B. microti infection in humans.     

Identification and characterization of putative secreted antigens from Babesia microti

The need for improved diagnostic reagents to identify human long-term carriers of the zoonotic parasite Babesia microti is evidenced by numerous reported cases of transfusion-acquired infections. This report describes the identification and initial characterization of 27 clones representing seven genes or gene families that were isolated through serological expression cloning by using a technique that we specifically designed to screen for shed antigens. In this screen, sera from B. microti-infected SCID mice, putatively containing secreted or shed antigens from the parasites, were harvested and used to immunize syngeneic immunocompetent mice (BALB/c). After boosting, the sera from the BALB/c mice, containing antibodies against the immunodominant secreted antigens, were used to screen a B. microti genomic expression library. Analyses of the putative peptides encoded by the novel DNA sequences revealed characteristics indicating that these peptides might be secreted. Initial serological data obtained with recombinant proteins and a patient serum panel demonstrated that several of the proteins could be useful in developing diagnostic tests for detection of B. microti antibodies and antigens in serum.     

Macrophages are critical for cross-protective immunity conferred by Babesia microti against Babesia rodhaini infection in mice

Although primary infection of mice with Babesia microti has been shown to protect mice against subsequent lethal infection by Babesia rodhaini, the mechanism behind the cross-protection is unknown. To unravel this mechanism, we investigated the influence of primary infection of mice with nonlethal B. microti using different time courses on the outcome of subsequent lethal B. rodhaini infection. Simultaneous infections of mice with these parasites resulted in rapid increases in parasitemia, with 100% mortality in BALB/c mice, as observed with control mice infected with B. rodhaini alone. In contrast, mice with acute, resolving, and chronic-phase B. microti infections were completely protected against B. rodhaini, resulting in low parasitemia and no mortalities. Mice immunized with dead B. microti were not protected from B. rodhaini infection, although high antibody responses were induced. Interestingly, the protected mice had significantly decreased levels of antibody response, cytokines (including gamma interferon [IFN-γ], interleukin-2 [IL-2], IL-8, IL-10, and IL-12), and nitric oxide levels after infection with B. rodhaini. SCID mice and IFN-γ-deficient mice with chronic B. microti infections demonstrated protective responses comparable to those of immunocompetent mice. Likewise, in vivo NK cell depletion did not significantly impair the protective responses. Conversely, macrophage depletion resulted in increased susceptibility to B. rodhaini infection associated with changes in their antibody and cytokines profiles, indicating that macrophages contribute to the protection against this challenge infection. We conclude that future development of vaccines against Babesia should include a strategy that enhances the appropriate activation of macrophages.     

Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils

In order to investigate the possible role of Ixodes ricinus as a vector of zoonotic Babesia microti infection in Europe, a European rodent isolate (HK) and a zoonotic American isolate (GI) were studied in transmission experiments. PCR detected B. microti in the blood and spleens of infected gerbils (Meriones unguiculatus) and also in laboratory-induced infections of I. ricinus ticks. B. microti DNA was detected by PCR in all pooled samples of nymphs and the majority of adults that had fed as larvae and nymphs, respectively, on gerbils with acute infection of the European isolate, confirming that I. ricinus could serve as a vector in Europe. The American isolate, GI, proved to be equally infective for larval and nymphal I. ricinus as the HK strain, despite a very different appearance in gerbil erythrocytes. Nymphs infected with the HK and GI strains readily infected gerbils. In contrast to the finding in acute infections, ticks that fed on gerbils with chronic infections of HK and GI did not become infected. It was also found that the HK strain was not transmitted transovarially. The finding that a B. microti strain (GI) from a distant geographical region (United States) can infect and be transmitted by I. ricinus suggests that other European B. microti strains, in addition to the HK strain used here, are probably infective for I. ricinus, supporting the view that infection of humans with European B. microti may be a regular occurrence.     

A new real-time PCR assay for improved detection of the parasite Babesia microti

Babesiosis is an emerging zoonosis with important public health implications, as the incidence of the disease has risen dramatically over the past decade. Because the current gold standard for detection of Babesia is microscopic examination of blood smears, accurate identification requires trained personnel. Species in the genus cannot be distinguished microscopically, and Babesia can also be confused with the early trophozoite stage (ring forms) of Plasmodium parasites. To allow more accurate diagnosis in a format that is accessible to a wider variety of laboratories, we developed a real-time PCR assay targeting the 18S rRNA gene of Babesia microti, the dominant babesiosis pathogen in the United States. The real-time PCR is performed on DNA extracted from whole-blood specimens and detects Babesia microti with a limit of detection of ～100 gene copies in 5 μl of blood. The real-time PCR assay was shown to be 100% specific when tested against a panel of 24 organisms consisting of Babesia microti, other Babesia species, Plasmodium species, tick-borne and other pathogenic bacteria, and other blood-borne parasites. The results using clinical specimens show that the assay can detect infections of lower parasitemia than can be detected by microscopic examination. This method is therefore a rapid, sensitive, and accurate method for detection of Babesia microti in patient specimens.     

鼠感染Babesia microti和 Babesia rodhaini后早期 IL-12、IL-10诱生的研究

目的 研究Babesia microti 和Babesia rodhaini感染后早期IL-12和IL-10诱生情况,以探讨B. microti和 B. rodhaini不同种属感染的发病机制与免疫应答效应.方法 使用ELISA方法分别检测Babesia microti 和 Babesia rodhaini 感染鼠后的0、3、6、9、12、18、24、36、48、72、96 h血清中IL-12及IL-10的浓度.结果 B. microti感染后3 h,小鼠血液中IL-12产生达一个高峰,至感染后24 h达最高峰值,与对照组比较差异显著,同组鼠在感染的早期(96 h前)血液中IL-10水平无明显变化.而B. rodhaini感染鼠,在感染后3～72 h,血清中IL-10和IL-12水平与对照组比较均无明显差异.在感染后96 h,血清中IL-10和IL-12水平开始下降,与对照组比较差异显著.结论 B. microti 感染鼠早期诱生的细胞因子主要为IL-12,而B. rodhaini感染后早期IL-12和IL-10水平无明显变化.     

A polymorphic multigene family encoding an immunodominant protein from Babesia microti

Human babesiosis in the United States is caused predominantly by Babesia microti, a tick-transmitted blood parasite. Improved testing methods for the detection of infection with this parasite are needed, since asymptomatic B. microti infection represents a potential threat to the blood supply in areas where B. microti is endemic. We performed immunoscreening of an expression library of genomic DNA from a human isolate of B. microti (strain MN1). Among 17 unique immunoreactive clones, we identified 9 which represent a related family of genes with little sequence homology to other known sequences but with an architecture resembling that of several surface proteins of Plasmodium. Within this family, a tandem array of a degenerate six-amino-acid repeat (SEAGGP, SEAGWP, SGTGWP, SGTVGP) was found in various lengths between relatively well conserved segments at the N and C termini. In order to examine within-clone variation, we developed a PCR protocol for direct recovery of a specific bmn1-6 homologue directly from 30 human blood isolates, 4 corresponding hamster isolates, and 5 geographically corresponding Peromyscus leucopus (white-footed mouse) isolates. Isolates from the hamsters had the same sequences as those found in the corresponding human blood, suggesting that genetic variation of bmn1-6 does not occur during passage. However, clones from different patients were often substantially different from each other with regard to the number and location of the degenerate repeats within the bmn1-6 homologue. Moreover, we found that strains that were closely related geographically were also closely related at the sequence level; nine patients, all from Nantucket Island, Mass., harbored clones that were indistinguishable from each other but that were distinct from those found in other northeastern or upper midwestern strains. We conclude that considerable genetic and antigenic diversity exists among isolates of B. microti from the United States and that geographic clustering of subtypes may exist. The nature of the bmn1-6 gene family suggests a mechanism of antigenic variation in B. microti that may occur by recombination, differential expression, or a combination of both mechanisms.     

Genetic control of Propionibacterium acnes-induced protection of mice against Babesia microti.

Using various strains of inbred mice, we found that Propionibacterium acnes-induced protection against the hemoprotozoan parasite Babesia microti was controlled by a dominant gene(s) not linked to the major histocompatibility gene (H2) complex of mice. P. acnes-induced resistance to infection was not merely an amplification of the normal immune response to B. microti, since innate resistance to infection was controlled by different genes. Expression of the nonspecific protection induced by P. acnes was found to be transferable with cells of bone marrow origin, and it did not necessarily accompany the induction of hepatosplenomegaly. The implication of these results for the understanding of the mechanism of P. acnes-induced protection is discussed.     

Multiplex Assay Detection of Immunoglobulin G Antibodies That Recognize Babesia microti Antigens

Human babesiosis, a blood-borne infection caused by several species of Babesia, including B. microti, is an emerging disease that is endemic in the Northeast, upper Midwest, and Pacific Northwest regions of the United States. Risk factors for babesiosis include exposure to the infected tick vector and blood transfusions from infected donors. In this work, we cloned and expressed two of the immunodominant antigens from B. microti and used them in a multiplex bead format assay (MBA) to detect parasite-specific IgG responses in human sera. The MBA using recombinant B. microti secreted antigen 1 (BmSA1) protein was more specific (100%) and slightly more sensitive (98.7%) than the assay using a truncated recombinant BMN1-17 construct (97.6% and 97.4%, respectively). Although some antibody reactivity was observed among sera from confirmed-malaria patients, only one Plasmodium falciparum sample was simultaneously positive for IgG antibodies to both antigens. Neither antigen reacted with sera from babesiosis patients who were infected with Babesia species other than B. microti. Both positive and negative MBA results were reproducible between assays and between instruments. Additional studies of these recombinant antigens and of the multiplex bead assay using blood samples from clinically defined babesiosis patients and from blood donors are needed to more clearly define their usefulness as a blood screening assay.     

The effect of age on the infection and immunoresponsiveness of mice to Babesia microti

5he effect of age on the immunological responses to Babesia microti infection in the mouse was investigated. Aged mice experienced reduced and delayed peak parasitemias compared to younger animals; however, the old mice failed to clear the parasites from the blood and experienced fluctuating parasitemias until death. Babesiosis produced suppression of responses to nonspecific B and T cell mitogens concomitant with rising autoantibody plaque forming cells reactive with untreated and bromelain modified mouse erythrocytes. Similar observations of increased susceptibility to babesiosis with age and immunosuppression have been made in human babesiosis. Thus, the murine model for this hemoprotozoan infection is faithful to the human immunological responses.     

Case report: spontaneous splenic rupture during acute parasitemia of Babesia microti

Babesia is a malaria-like protozoan parasite spread by Ixodes ticks primarily from the white-footed deer mouse to humans. Typically it causes subclinical disease, but occasionally causes acute febrile disease with hepatosplenomegaly. We report a case of spontaneous splenic rupture of a 56-yr-old man with acute Babesia microti infection.