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Short-term in vitro cultures of B. microti. (A) Giemsa-stained blood smears, indicating the developmental stages of B. microti 0, 6, and 24 h postinoculation. Percentages of stages were calculated as the number of B. microti-infected cells in at least 5,000 RBCs. (B) Detection of BmGPI12 in in vitro B. microti cultures collected 6 and 24 h following inoculation. At each time point, supernatant (S), hemolysate (H), and pellet (P) fractions from uninfected (U) or B. microti-infected (I) cultures were analyzed by immunoblotting. No signal could be detected with preimmune rabbit serum.
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Human babesiosis is an emerging zoonotic infectious disease caused by intraerythrocytic protozoan parasites of the genus Babesia . Most cases of human babesiosis are caused by Babesia microti and often manifest in individuals over the age of 50 or in patients with a compromised immune system. Those who develop symptomatic B. microti infection usual...
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The zoonotic babesiosis is a growing public health concern. This parasite mainly occurs in U.S. (B. microti) and Europe (B. divergens & B. venatorum). Emerging cases have been increasingly reported worldwide especially in immunosuppressed individuals. Other than tick bite, possible modes of infection include perinatal transmission and blood transfu...
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... Recent investigations have demonstrated that Babesia microti exports into the mammalian host a highly immunogenic protein named BmGPI12 (also known as BmSA1) (13)(14)(15)(16)(17)(18)(19). BmGPI12 is a glycosylphosphatidylinositol (GPI)-anchored protein that is a member of the B. microti BMN multigene family (13,16). ...
... BmGPI12 is a glycosylphosphatidylinositol (GPI)-anchored protein that is a member of the B. microti BMN multigene family (13,16). Recent studies have shown that an antigen-capture assay (BmGPAC) that relies on polyclonal antibodies against the full-length BmGPI12 protein can detect this antigen in the sera and plasma of infected animals and humans with high sensitivity and specificity (18). Furthermore, using Swiss-Webster (SW) mice, which naturally clear infection over time, or BALB/c mice treated with a combination of atovaquone and ELQ-334 to clear infection (20), it was found that the BmGPAC assay displays better correlation between active B. microti infection and BmGPI12 levels than serological and DNA amplification methods (18). ...
... Recent studies have shown that an antigen-capture assay (BmGPAC) that relies on polyclonal antibodies against the full-length BmGPI12 protein can detect this antigen in the sera and plasma of infected animals and humans with high sensitivity and specificity (18). Furthermore, using Swiss-Webster (SW) mice, which naturally clear infection over time, or BALB/c mice treated with a combination of atovaquone and ELQ-334 to clear infection (20), it was found that the BmGPAC assay displays better correlation between active B. microti infection and BmGPI12 levels than serological and DNA amplification methods (18). Therefore, the BmGPAC assay has the potential to differentiate between active infection and past exposure and could be used to document successful antibiotic therapy in babesiosis patients. ...
The apicomplexan pathogen Babesia microti is responsible for most cases of human babesiosis worldwide. The disease, which presents as a malaria-like illness, is potentially fatal in immunocompromised or elderly patients, making the need for its accurate and early diagnosis an urgent public health concern. B. microti is transmitted primarily by Ixodes ticks but can also be transmitted via blood transfusion. The parasite completes its asexual reproduction in the host red blood cell, where each invading merozoite develops and multiplies to produce four daughter parasites. While various techniques, such as microscopy, PCR, and indirect fluorescence, have been used over the years for babesiosis diagnosis, detection of the secreted B. microti immunodominant antigen BmGPI12 using specific polyclonal antibodies was found to be the most effective method for the diagnosis of active infection and for evaluation of clearance following drug treatment. Here, we report the development of a panel of 16 monoclonal antibodies against BmGPI12. These antibodies detected secreted BmGPI12 in the plasma of infected humans. Antigen capture assays identified a combination of two monoclonal antibodies, 4C8 and 1E11, as a basis for a monoclonal antibody-based BmGPI12 capture assay (mGPAC) to detect active B. microti infection. Using a collection of 105 previously characterized human plasma samples, the mGPAC assay showed 97.1% correlation with RNA-based PCR (transcription-mediated amplification [TMA]) for positive and negative samples. The mGPAC assay also detected BmGPI12 in the plasma of six babesiosis patients at the time of diagnosis but not in three matched posttreatment samples. The mGPAC assay could thus be used alone or in combination with other assays for accurate detection of active B. microti infection.
... Neither soluble nor vesicle-bound BmSA1 could be involved in erythrocyte invasion without physical connection to the merozoite. These forms of the protein are produced at a high rate and therefore could be exploited as a diagnostic marker of active infection [50]. ...
Human babesiosis results from a combination of tick tropism for humans, susceptibility of a host to sustain Babesia development, and contact with infected ticks. Climate modifications and increasing diagnostics have led to an expanded number of Babesia species responsible for human babesiosis, although, to date, most cases have been attributed to B. microti and B. divergens. These two species have been extensively studied, and in this review, we mostly focus on the antigens involved in host–parasite interactions. We present features of the major antigens, so-called Bd37 in B. divergens and BmSA1/GPI12 in B. microti, and highlight the roles of these antigens in both host cell invasion and immune response. A comparison of these antigens with the major antigens found in some other Apicomplexa species emphasizes the importance of glycosylphosphatidylinositol-anchored proteins in host–parasite relationships. GPI-anchor cleavage, which is a property of such antigens, leads to soluble and membrane-bound forms of these proteins, with potentially differential recognition by the host immune system. This mechanism is discussed as the structural basis for the protein-embedded immune escape mechanism. In conclusion, the potential consequences of such a mechanism on the management of both human and animal babesiosis is examined.
... Thekkiniath et al. developed an antigen capture assay for the detection of BmBAHCS1 that had a limit of detection of 20 pg/µL in in vitro samples [54]. However, it only identified six of seven clinical samples, failing to detect a sample with a parasitemia of 0.3% [54]. ...
... Thekkiniath et al. developed an antigen capture assay for the detection of BmBAHCS1 that had a limit of detection of 20 pg/µL in in vitro samples [54]. However, it only identified six of seven clinical samples, failing to detect a sample with a parasitemia of 0.3% [54]. Therefore, further improvements to the assay are required before its widespread adoption. ...
The biology of intraerythrocytic Babesia parasites presents unique challenges for the diagnosis of human babesiosis. Antibody-based assays are highly sensitive but fail to detect early stage Babesia infections prior to seroconversion (window period) and cannot distinguish between an active infection and a previously resolved infection. On the other hand, nucleic acid-based tests (NAT) may lack the sensitivity to detect window cases when parasite burden is below detection limits and asymptomatic low-grade infections. Recent technological advances have improved the sensitivity, specificity and high throughput of NAT and the antibody-based detection of Babesia. Some of these advances include genomics approaches for the identification of novel high-copy-number targets for NAT and immunodominant antigens for superior antigen and antibody-based assays for Babesia. Future advances would also rely on next generation sequencing and CRISPR technology to improve Babesia detection. This review article will discuss the historical perspective and current status of technologies for the detection of Babesia microti, the most common Babesia species causing human babesiosis in the United States, and their implications for early diagnosis of acute babesiosis, blood safety and surveillance studies to monitor areas of expansion and emergence and spread of Babesia species and their genetic variants in the United States and globally.
... Using a SYBR Green-I-based assay, qPCR analyses were conducted to quantify the expression of the BmCytb and BmCox1 genes in both ELQ-502 S and ELQ-502 R parasites from nymphs ( Figure 6A) and blood from infected mice ( Figure 6B). The expression level of BmGPI12 [24][25][26][27] was used as a control in each sample and results are presented as ratios of the quantification cycle (Cq) value of the target mRNA (BmCytb or BmCox1) versus the Cq value of BmGPI12 mRNA. For BmCytb, the ratios were mean 0.91 (SD 0.07) and mean 0.92 (SD 0.05) for ELQ-502 S and ELQ-502 R nymphs, respectively, whereas for BmCox1, these ratios were mean 0.95 (SD 0.09) and mean 0.96 (SD 0.03) for wild-type and ELQ-502 R nymphs, respectively. ...
Human babesiosis is an emerging tick-borne malaria-like illness caused by Babesia parasites following their development in erythrocytes. Here, we show that a mutation in the Babesia microti mitochondrial cytochrome b (Cytb) that confers resistance to the antibabesial drug ELQ-502 decreases parasite fitness in the arthropod vector. Interestingly, whereas the mutant allele does not affect B. microti fitness during the mammalian blood phase of the parasite life cycle and is genetically stable as parasite burden increases, ELQ-502 R mutant parasites developing in the tick vector are genetically unstable with a high rate of the wild type allele emerging during the nymphal stage. Furthermore, we show that B. microti parasites with this mutation are transmitted from the tick to the host, raising the possibility that the frequency of Cytb resistance mutations may be decreased by passage through the tick vector, but could persist in the environment if present when ticks feed.
... Our results agree with previous study by Skariah and coworkers, who showed that MyD88 deficiency did not affect Bm parasitemia in mice (Skariah et al., 2017). Furthermore, the lack of stimulation of TLR2 and TLR4 in transfected HEK293 cells to the whole blood from Bm-infected C3H mice, which contained iRBCs as well as egressed parasites that should display GPI-anchored proteins on their surface such as BmGPI12 (Thekkiniath et al., 2019;Thekkiniath et al., 2018), in our in vitro experiment confirmed the lack of response of these PRRs to Bm (Figure 6). These results also confirmed our in vivo data. ...
Toll-like receptors (TLRs) are a class of membrane-spanning proteins of host cells. TLR2 and TLR4 are displayed on the surface of macrophages, neutrophils and dendritic cells and recognize structurally conserved microbial signatures defined as Pathogen associated molecular patterns (PAMPs). C3H mice are susceptible to tick-borne pathogens; Lyme disease causing Borrelia burgdorferi that manifests arthritis and carditis and Apicomplexan protozoan, Babesia microti (Bm) that causes significant parasitemia associated with erythrocytopenia and haemoglobinuria. B. burgdorferi lacks typical TLR4 ligand lipopolysaccharides (LPS) and Bm TLR ligand(s) remain unknown. Only Borrelia lipoproteins that signal through TLR2 are established as PAMPs of these pathogens for TLR2/TLR4. Infection of C3H mice with each pathogen individually resulted in increase in the percentage of splenic B, T, and FcR+ cells while their co-infection significantly diminished levels of these cells and caused increased B. burgdorferi burden in the specific organs. The most pronounced inflammatory arthritis was observed in co-infected C3H/HeJ mice. Parasitemia levels and kinetics of resolution of Bm in both mice strains were not significantly different. Transfected HEK293 cells showed pronounced signaling by B. burgdorferi through TLR2 and to some extent by TLR4 while Bm and infected erythrocytes did not show any response confirming our results in mice.
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... Additional studies also are needed to determine whether blood smear or PCR should be the initial test used to diagnose acute babesiosis and monitor patients during therapy. More rapid, sensitive, specific, and cost-effective diagnostic tests are being developed but require clinical validation [53,54,56,[74][75][76][77][78]. ...
The purpose of this guideline is to provide evidence-based guidance for the most effective strategies for the diagnosis and management of babesiosis. The diagnosis and treatment of co-infection with babesiosis and Lyme disease will be addressed in a separate Infectious Diseases Society of America (IDSA), American Academy of Neurology (AAN), and American College of Rheumatology (ACR) guideline [1]. Recommendations for the diagnosis and treatment of human granulocytic anaplasmosis can be found in the recent rickettsial disease guideline developed by the Centers for Disease Control and Prevention [2]. The target audience for the babesiosis guideline includes primary care physicians and specialists caring for this condition, such as infectious diseases specialists, emergency physicians, intensivists, internists, pediatricians, hematologists, and transfusion medicine specialists.
... Babesia microti surface antigen 1 (BmSA1)has been reported as a diagnostic marker with high reactivity , and the ELISA detection method has also been established (Luo et al., 2011;Thekkiniath et al., 2018). However, there is no relevant report on its function or its specific role in parasite invasion. ...
... Both LC-MS/MS and secretion assays showed the presence of BmSA1 in the in vitro supernatant, indicating that BmSA1 exists in B. microti in two forms. This characteristic was consistent with that of most merozoite surface coat proteins in apicomplexan parasites (Man et al., 2017;Thekkiniath et al., 2018). One of the two existing forms is focused on the GPI-attachment site, where the proprotein cleavage occurs and which localizes a few amino acids before the C-terminal hydrophobic domain. ...
Babesia microti, a tick-borne intraerythrocytic zoonotic protozoan, causes most of human babesiosis in the world, and patients usually experience intermittent fever, fatigue, and chills, followed by a combination of additional symptoms and even death in severe cases. Unfortunately, there is no curable drug or effective vaccine available, and the mechanism of related virulence factors in invasion to host cells during the merozoite stage is unclear. Here, we evaluated a secreted protein annotated as B. microti surface antigen 1 (BmSA1) and identified from in vitro culture supernatant by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). BmSA1 fragment was expressed in Escherichia coli to prepare polyclonal antiserum. Western blot analysis revealed the existence of BmSA1 in the lysate of the parasites and the hemolysate of infected red blood cells (iRBCs). Laser confocal microscopy confirmed BmSA1 as a secreted protein with diffuse distribution around the parasites in red blood cells (RBCs). The adhesion capacity of BmSA1 against the host RBCs was tested by RBC binding assays using the recombinant BmSA1 protein (rBmSA1), which was shown to specifically bind to host RBCs. Further in vitro antiserum-neutralization test demonstrated that the growth of parasites could be significantly inhibited by the anti-BmSA1 antiserum. These results indicate that BmSA1 is a crucial factor for B. microti invasion into host RBCs with an important role in host-parasite interactions during the merozoite stage and has the potential use as a vaccine candidate due to its high secretion amount.
... Publication of the full B. microti genome in 2012 has enabled identification of higher copy number detection targets, allowing the routine detection of less than 10 B. microti parasites per ml of blood [18,20]. Additional technological advances such as sample concentration, high copy detection targets such as BMN multigene family members, and bead-based target capture may further enhance the sensitivity of NAT assays [21,22]. Detection of Babesia antigen(s) offers additional markers of active infection. ...
Persistent infection is a characteristic feature of babesiosis, a worldwide, emerging tick-borne disease caused by members of the genus Babesia. Persistence of Babesia infection in reservoir hosts increases the probability of survival and transmission of these pathogens. Laboratory tools to detect Babesia in red blood cells include microscopic detection using peripheral blood smears, nucleic acid detection (polymerase chain reaction and transcription mediated amplification), antigen detection, and antibody detection. Babesia microti, the major cause of human babesiosis, can asymptomatically infect immunocompetent individuals for up to two years. Chronically infected blood donors may transmit the pathogen to another person through blood transfusion. Transfusion-transmitted babesiosis causes severe complications and death in about a fifth of cases. Immunocompromised patients, including those with asplenia, HIV/AIDS, malignancy, or on immunosuppressive drugs, often experience severe disease that may relapse up to two years later despite anti-Babesia therapy. Persistent Babesia infection is promoted by Babesia immune evasive strategies and impaired host immune mechanisms. The health burden of persistent and recrudescent babesiosis can be minimized by development of novel therapeutic measures, such as new anti-parasitic drugs or drug combinations, improved anti-parasitic drug duration strategies, or immunoglobulin preparations; and novel preventive approaches, including early detection methods, tick-avoidance, and blood donor screening.
... The immunodominant BmGPI12 of B. microti is encoded by a member of the bmn multigene family and is one of the most highly expressed genes of the parasite during its development within red blood cells (Lodes et al, 2000;Cornillot et al, 2012Cornillot et al, , 2016Silva et al, 2016). Consistent with the secretion of BmGPI12 from the parasite into the red blood cytoplasm and subsequently into the host (Luo et al, 2011(Luo et al, , 2012Cornillot et al, 2016;Thekkiniath et al, 2018), immunoblot analyses using anti-BmGPI12 antibodies on blood collected from mice and fractionated to collect plasma (S), erythrocyte cytoplasm (H), and membrane (P) fractions showed the presence of BmGPI12 in all three fractions from animals infected with B. microti strains (LabS1 or PRA99) (Fig 1A), but not from uninfected animals ( Fig 1A). As a control, immunoblot analyses conducted using a monoclonal antibody against the mouse erythrocyte membrane protein, TER-119 (glycophorin A-associated protein [Ly-76]), identified this protein in the membrane (P) fractions of both uninfected and B. microti-infected erythrocytes (Kina et al, 2000), but not in the plasma or erythrocyte cytoplasm fractions ( Fig 1A). ...
The apicomplexan parasite Babesia microti is the primary agent of human babesiosis, a malaria-like illness and potentially fatal tick-borne disease. Unlike its close relatives, the agents of human malaria, B. microti develops within human and mouse red blood cells in the absence of a parasitophorous vacuole, and its secreted antigens lack trafficking motifs found in malarial secreted antigens. Here, we show that after invasion of erythrocytes, B. microti undergoes a major morphogenic change during which it produces an interlacement of vesicles (IOV); the IOV system extends from the plasma membrane of the parasite into the cytoplasm of the host erythrocyte. We developed antibodies against two immunodominant antigens of the parasite and used them in cell fractionation studies and fluorescence and immunoelectron microscopy analyses to monitor the mode of secretion of B. microti antigens. These analyses demonstrate that the IOV system serves as a major export mechanism for important antigens of B. microti and represents a novel mechanism for delivery of parasite effectors into the host by this apicomplexan parasite.
... Especially ELISA seems to be a distinctly time-saving immunological method, because large amounts of samples can be screened at once and they are not influenced by the personnel's subjectivity. In 2018, Thekkiniath et al. [140] described a novel antigen capture ELISA method with very high sensitivity, detecting BmGPI12 protein of B. microti. According to another 2018 study by Cai et al. [33], ELISA test using rBmSA5-1-1 protein demonstrates high specificity and sensitivity for the serologic detection of B. microti in mice and it also confirms suitability of ICT in rapid diagnostics of B. microti infection. ...
Babesiosis is a tick-borne disease of veterinary and medical concern. The disease is caused by parasitic protists of the genus Babesia that are transmitted by ticks of the family Ixodidae. In addition to transmission by ticks, several cases of infections through blood transfusion and congenital transmission have been reported in humans. The parasites invade and destroy the erythrocytes of their hosts. In humans, the disease manifestations are broad, from asymptomatic through mild flu-like infection to severe malaria-like disease with a potentially fatal outcome, mainly in immunedeficient and/or elderly individuals. Out of the approximately 100 known Babesia species, only a few (B. microti, B. duncani, B. divergens, B. venatorum) have been associated with human babesiosis. Babesiosis is endemic in temperate regions of the northern hemisphere. Autochthonous infections in the USA are caused mainly by B. microti and B. duncani. In Europe, B. divergens is the most common etiological agent. In China, several cases of infections with B. microti and B. venatorum have been reported. Human babesiosis is ranked among emerging diseases. The number of confirmed cases has increased during the last decade, the risk areas have spread and new foci have been discovered. As a consequence, babesiosis has become a growing public health challenge, although a considerably high proportion of infections remain asymptomatic or misdiagnosed. Changing climate and a number of other biotic and abiotic factors affect the distribution of babesiosis. The infectivity and pathogenicity of the individual Babesia species may vary, depending on the strain and geographic region. Accurate diagnosis of babesiosis is of particular importance as misdiagnosis can lead to confusion with malaria. Filling in the still existing gaps in the knowledge of the life cycle, ecology and distribution of Babesia species is essential for the diagnosis and prevention of the disease.
