Misdiagnosis of Babesiosis as Malaria, Equatorial Guinea, 2014.

We report a case of babesiosis, caused by Babesia microti, in a missionary who worked in Equatorial Guinea but also visited rural Spain. The initial diagnosis, based on clinical features and microscopy, was malaria. The patient's recovery was delayed until she received appropriate treatment for babesiosis.

The efficacy of the ultraviolet C pathogen inactivation system in the reduction of Babesia divergens in pooled buffy coat platelets.

BACKGROUND: Babesia spp. is an intraerythrocytic parasite that causes human babesiosis and its transmission by transfusion has been extensively demonstrated. The aim of this study was to ascertain the efficacy of an ultraviolet C (UVC)-based pathogen inactivation system in the reduction of Babesia divergens-infected platelet (PLT) concentrates and to determine the parasite's ability to survive in PLT concentrates stored under blood bank conditions. STUDY DESIGN AND METHODS: This study was conducted using in vitro cultures of B. divergens. The detection limit of the culture assay was established and, subsequently, 15 buffy coat-derived PLT concentrates (BC-PCs) were inoculated with 10(7) B. divergens-infected red blood cells. Infected BC-PCs were irradiated with 0.2 J/cm(2) UVC light using the THERAFLEX UV-Platelets method (Macopharma). Viability and parasite growth were evaluated before and after inactivation. Culture growth kinetics were monitored by DNA incorporation of [(3) H]thymidine. The ability of B. divergens to survive in PLT concentrates was also analyzed. RESULTS: The limit of detection in cultures was established at 0.1 × 10(-6) % parasites. The THERAFLEX UV-Platelets system inactivated B. divergens to below the limit of detection in 12 of 15 BC-PCs (log reduction, >6.0) and to the limit of detection (log reduction, 5.0) in three of 15. It was also demonstrated that B. divergens remains viable in BC-PCs stored up to 7 days. CONCLUSION: Since B. divergens can survive in PLT concentrates and given the performance of UVC, this system could be considered as an alternative to prevent B. divergens and other Babesia species from being transmitted through PLT transfusions.

Multiomics analysis reveals B. MO1 as a distinct Babesia species and provides insights into its evolution and virulence.

Babesiosis, caused by protozoan parasites of the genus Babesia , is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of various Babesia species underscores the ongoing risk of new zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental shifts impacting the distribution and transmission dynamics of parasites, their vectors, and reservoir hosts. One such species, Babesia MO1, previously implicated in severe cases of human babesiosis in the midwestern United States, was initially considered closely related to B. divergens , the predominant agent of human babesiosis in Europe. Yet, uncertainties persist regarding whether these pathogens represent distinct variants of the same species or are entirely separate species. We show that although both B. MO1 and B. divergens share similar genome sizes, comprising three nuclear chromosomes, one linear mitochondrial chromosome, and one circular apicoplast chromosome, major differences exist in terms of genomic sequence divergence, gene functions, transcription profiles, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens , and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.

Characteristics of Human Babesiosis in Europe.

One of the Editor's choice articles in 2021 published in Pathogens was a review of human babesiosis in Europe [...].

Retrospective study of the epidemiological risk and serological diagnosis of human babesiosis in Asturias, Northwestern Spain.

BACKGROUND: Babesiosis is a globally growing tick-borne disease in humans. Severe babesiosis caused by Babesia divergens has been reported in two patients from Asturias (Northwestern Spain), suggesting an undetected risk for the disease. To analyze this risk, we retrospectively evaluated the seroprevalence of babesiosis in the Asturian population from 2015 through 2017, a period covering the intermediate years in which these two severe cases occurred. METHODS: Indirect fluorescent assay (IFA) and Western blot (WB) were performed to detect B. divergens IgG antibodies in 120 serum samples from Asturian patients infected with the tick-transmitted spirochete Borrelia burgdorferi sensu lato, a condition that indicates exposure to tick bites. RESULTS: This retrospective study confirmed a B. divergens seroprevalence rate of 39.2% according to IFA results. B. divergens incidence was 7.14 cases/100,000 population, exceeding previously reported seroprevalence rates. No differences in epidemiology and risk factors were found between patients infected solely with B. burgdorferi s.l. and those infected with B. burgdorferi s.l. and with IgG antibodies against B. divergens. This last group of patients lived in Central Asturias, had a milder clinical course and, according to WB results, developed different humoral responses against B. divergens. CONCLUSIONS: Babesia divergens parasites have circulated for several years in Asturias. Epidemiological evidence of babesiosis makes Asturias an emerging risk area for this zoonosis. Human babesiosis could also be relevant in other Spanish and European regions affected by borreliosis. Hence, the potential risk of babesiosis on human health in Asturias and other European forest regions needs to be addressed by the health authorities.

Babesia duncani multi-omics identifies virulence factors and drug targets.

Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.

Babesia and Human Babesiosis.

Babesia is a genus of intraerythrocytic protozoan parasites belonging to the exclusively parasitic phylum Apicomplexa [...].

Babesia and Theileria Identification in Adult Ixodid Ticks from Tapada Nature Reserve, Portugal.

This study, conducted in a nature reserve in southern Portugal, investigated the frequency and diversity of tick-borne piroplasms in six species of adult ixodid ticks removed from 71 fallow deer (Dama dama) and 12 red deer (Cervus elaphus), collected over the period 2012-2019. The majority of 520 ticks were Ixodes ricinus (78.5%), followed by Rhipicephalus sanguineus sensu lato, Hyalomma lusitanicum, Haemaphysalis punctata, Dermacentor marginatus, and Ixodes hexagonus. The R. sanguineus ticks collected from the deer were clearly exophilic, in contrast to the endophilic species usually associated with dogs. Four tick-borne piroplasms, including Theileria spp., and the zoonotic species, Babesia divergens and Babesia microti, were detected. B. divergens 18S rDNA, identical to that of the bovine reference strain U16370 and to certain strains from red deer, was detected in I. ricinus ticks removed from fallow deer. The sporadic detection of infections in ticks removed from the same individual hosts suggests that the piroplasms were present in the ticks rather than the hosts. Theileria sp. OT3 was found in I. ricinus and, along with T. capreoli, was also detected in some of the other tick species. The natural vector and pathogenic significance of this piroplasm are unknown.

Human Babesiosis in Europe.

Babesiosis is attracting increasing attention as a worldwide emerging zoonosis. The first case of human babesiosis in Europe was described in the late 1950s and since then more than 60 cases have been reported in Europe. While the disease is relatively rare in Europe, it is significant because the majority of cases present as life-threatening fulminant infections, mainly in immunocompromised patients. Although appearing clinically similar to human babesiosis elsewhere, particularly in the USA, most European forms of the disease are distinct entities, especially concerning epidemiology, human susceptibility to infection and clinical management. This paper describes the history of the disease and reviews all published cases that have occurred in Europe with regard to the identity and genetic characteristics of the etiological agents, pathogenesis, aspects of epidemiology including the eco-epidemiology of the vectors, the clinical courses of infection, diagnostic tools and clinical management and treatment.

Integration of Genomic and Transcriptomic Data to Elucidate Molecular Processes in Babesia divergens.

Emerging pathogens have developed ingenious life cycles to facilitate their growth and survival in the host organism. Detailed knowledge of the life cycle of these pathogens is increasingly necessary if we are to design new strategies to prevent infection and transmission. Multi-omics platforms provide useful data at different biological levels, and integration of these data into current approaches can facilitate holistic assessment of emerging pathogens. In this chapter, we bring together various methods and apply an integrative approach for analysis of genomic and transcriptomic data in Babesia divergens, an Apicomplexa emerging parasite that invades red blood cells and causes redwater fever in cattle and the most severe form of babesiosis in humans in Europe. The integrative methodology described herein can be helpful to identify genes active at specific points during life cycle of Apicomplexa parasites.

Integration of Functional Genomic, Transcriptomic, and Metabolomic Data to Identify Key Features in Genomic Expression, Metabolites, and Metabolic Pathways of Babesia divergens.

Upon invasion of red blood cells (RBCs), the Apicomplexa parasite Babesia divergens remains within the RBC for several hours and reproduces asexually, resulting in infective free merozoites that egress and destroy the host cell. Free merozoites rapidly seek and invade new uninfected RBCs. This repetitive cycle allows B. divergens to build a complex population of intraerythrocytic and extracellular stages in the bloodstream of humans and cattle, thus causing babesiosis. To compare biological aspects between B. divergens stages, including the different nature of their metabolism, could be key to our understanding of pathogenesis. Thus, we are currently assessing differences in the B. divergens metabolism of intra- and extracellular (free merozoites) life stages by the use of an integrative approach combining functional genomic, transcriptomic, differential expression, and metabolomic data acquired from sequencing and various analytical platforms. To our knowledge, this is the first effort to describe, in detail, the experimental procedures and integration of different omics to explore the regulation of the metabolism, invasion and proliferation mechanisms of B. divergens. This integrative approach can be used as a reference to study other Apicomplexa parasites.

Four-Dimensional Characterization of the Babesia divergens Asexual Life Cycle, from the Trophozoite to the Multiparasite Stage.

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle.IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis.

Babesia divergens apical membrane antigen 1 and its interaction with the human red blood cell.

Multiple parasite ligand-erythrocyte receptor interactions must occur for successful Babesia and Plasmodium invasion of the human red cell. One such parasite ligand is the apical membrane antigen 1 (AMA1) which is a conserved apicomplexan protein present in the micronemes and then secreted onto the surface of the merozoite. Much evidence exists for a vital role for AMA1 in host cell invasion; however, its interaction with the host erythrocyte has remained controversial. In this paper, we present a detailed characterization of a Babesia divergens homolog of AMA1 (BdAMA1), and taking advantage of the relatively high amounts of native BdAMA1 available from the parasite culture system, show that proteolytic products of native BdAMA1 bind to a trypsin- and chymotrypsin-sensitive receptor on the red blood cell. Moreover, immuno-electron microscopic images of the B. divergens merozoite captured during invasion offer additional evidence of the presence of BdAMA1 on the red cell membrane. Given the importance of AMA1 in invasion and the central role invasion plays in pathogenesis, these studies have implications both for novel drug design and for the development of new vaccine approaches aimed at interfering with AMA1 function.

Initial characterization of Pf62, a novel protein of Plasmodium falciparum identified by immunoscreening.

In order to find new antigens from Plasmodium falciparum, a complementary DNA (cDNA) library was constructed and screened. The study of expression library of P. falciparum was performed in an attempt to identify new antigens that could have potential relevance for the falciparum-malaria diagnosis and/or protection. Between the positive clones detected (ring erythrocyte surface antigen, merozoite erythrocyte surface antigen, RHOP H3, CSP, LSA), a new gene that correspond to a new protein (Pf62) was isolated and characterized. This antigen was useful for the diagnosis of malaria in enzyme-linked immunosorbent assay tests. The cDNA corresponding to this antigen and structure of the gene were characterized. Pf62 is a single copy gene that contains one exon. The Pf62 cDNA has an open reading frame of 1,599 nucleotides that code for a putative protein of 532 amino acids with a predicted molecular mass of 62 kDa. The polypeptide contains in the central section two regions of repeats of 21 and 19 amino acids, respectively. The localization of the Pf62 protein was performed by immunoblot, indirect immunofluorescence assay and immunoelectron microscopy. Pf62 is localized in the cytoplasm of the parasite and also on the surface of the infected erythrocyte. Serologic assays by using synthetic peptides designed from different antigenic regions of the Pf62 protein resulted in acceptable data of sensitivity and specificity in symptomatic malaria patients.

Comparative and functional genomics of the protozoan parasite Babesia divergens highlighting the invasion and egress processes.

Babesiosis is considered an emerging disease because its incidence has significantly increased in the last 30 years, providing evidence of the expanding range of this rare but potentially life-threatening zoonotic disease. Babesia divergens is a causative agent of babesiosis in humans and cattle in Europe. The recently sequenced genome of B. divergens revealed over 3,741 protein coding-genes and the 10.7-Mb high-quality draft become the first reference tool to study the genome structure of B. divergens. Now, by exploiting this sequence data and using new computational tools and assembly strategies, we have significantly improved the quality of the B. divergens genome. The new assembly shows better continuity and has a higher correspondence to B. bovis chromosomes. Moreover, we present a differential expression analysis using RNA sequencing of the two different stages of the asexual lifecycle of B. divergens: the free merozoite capable of invading erythrocytes and the intraerythrocytic parasite stage that remains within the erythrocyte until egress. Comparison of mRNA levels of both stages identified 1,441 differentially expressed genes. From these, around half were upregulated and the other half downregulated in the intraerythrocytic stage. Orthogonal validation by real-time quantitative reverse transcription PCR confirmed the differential expression. A moderately increased expression level of genes, putatively involved in the invasion and egress processes, were revealed in the intraerythrocytic stage compared with the free merozoite. On the basis of these results and in the absence of molecular models of invasion and egress for B. divergens, we have proposed the identified genes as putative molecular players in the invasion and egress processes. Our results contribute to an understanding of key parasitic strategies and pathogenesis and could be a valuable genomic resource to exploit for the design of diagnostic methods, drugs and vaccines to improve the control of babesiosis.

Babesia divergens: identification and characterization of BdHSP-20, a small heat shock protein.

This study describes the identification and characterization of the Babesia divergens alpha-crystallin/small heat shock protein 20 (BdHSP-20). BdHSP-20 was recognized by the DG7 monoclonal antibody (DG7 mAb) originally produced by Precigout et al. [Precigout, E., Valentin, A., Carcy, B., Gorenflot, A., Nakamura, K., Aikawa, M., Schrevel, J. 1993. Babesia divergens: characterization of a 17-kDa merozoite membrane protein. Experimental Parasitology 77, 425-434] against B. divergens merozoites. We used DG7 mAb to immunoscreen a B. divergens cDNA library to clone the gene encoding the small heat shock protein. Bdhsp-20 is a single copy gene interrupted by one intron. The deduced gene product (BdHSP-20) clearly belongs to the alpha-crystallin family and shows significant homology to Babesia bovis, Plasmodium falciparum and Toxoplasma gondii sHSPs, with the highest degree of sequence identity around the catalytic domain. Nutritient stress (serum depletion) treatment of the parasites induced the upregulation of BdHSP-20 gene expression observed by semi-quantitative PCR and immunoprecipitation. This regulation pattern suggests that BdHSP-20 could probably be of importance for parasite survival in the case of environmental stress. BdHSP-20 has previously been shown to be highly conserved among different strains and antibodies against the protein drastically reduce parasitemia in vitro.

Kinetics of the invasion and egress processes of Babesia divergens, observed by time-lapse video microscopy.

Based on confocal fluorescence and bright field video microscopy, we present detailed observations on the processes of invasion and egress of erythrocytes by the apicomplexan parasite Babesia divergens. Time-lapse images reveal numerous unexpected findings associated with the dynamics of B. divergens and its ability to manipulate the erythrocyte during both processes in its asexual cycle under in vitro conditions. Despite the speed at which these processes occur and the small size of the parasite, we capture infective merozoites moving vigorously and causing striking deformations in the erythrocyte's plasma membrane during an active invasion. We also observed intraerythrocytic dynamic stages as paired pyriforms, double paired pyriforms, tetrads, unattached pyriform sister cells and multiple parasite stages resulting in the release of large numbers of merozoites over a short period. Of considerable interest is that time-lapse images reveal a novel mechanism of egress used by B. divergens to exit the human erythrocyte. The release occurs when B. divergens parasites establish contacts with the plasma membrane of the erythrocyte from within, before exiting the cell. Visualization and analysis of the images enabled us to obtain useful information and broaden our knowledge of complex and crucial events involved with parasitisation of human erythrocytes by B. divergens.

A fatal case of Babesia divergens infection in Northwestern Spain.

We describe a fatal case caused by the intra-erythrocytic Babesia divergens parasite in an elderly woman. This is the third case of fatal babesiosis reported in the last 15 years in Europe, and the only one in a patient with an intact spleen.

Ultrastructure of the Babesia divergens free merozoite.

The invasive form of the apicomplexan parasite Babesia divergens, the free merozoite, invades the erythrocytes of host vertebrates, leading to significant pathology. Although invasion is an active process critical for parasite survival, it is not yet entirely understood. Using techniques to isolate the viable free merozoite, as well as electron microscopy, we undertook a detailed morphological study and explored the sub-cellular structure of the invasive B. divergens free merozoite after it had left the host cell. We examined characteristic apicomplexan features such as the apicoplast, the inner and discontinuous double membrane complex, and the apical complex; some aspects of erythrocyte entry by B. divergens were also defined by electron microscopy. This study adds to our understanding of B. divergens free merozoites and their invasion of human erythrocytes.