Intraerythrocytic Parasites Research Articles

Differential paired stage-specific expression of Babesia bovis cysteine-rich GCC2/GCC3 domain family proteins (BboGDP) during development within Rhipicephalus microplus

BackgroundBabesia bovis, an intra-erythrocytic apicomplexan parasite, is one of the causative agents of bovine babesiosis, the most important tick-borne disease of cattle in tropical and subtropical regions. Babesia bovis has a complex life-cycle that includes sexual development within the tick vector. The development of a transmission blocking vaccine to control bovine babesiosis requires the identification of antigens displayed on the surface of the parasite during its development within tick vectors. Four B. bovis cysteine-rich GCC2/GCC3 domain protein (BboGDP) family members were previously identified and are differentially expressed as discrete pairs by either blood stages or kinetes. In this study we focused on two family members, BboGDP1 and -3, that are expressed by Babesia parasites during tick infection.Methods and resultsTranscription analysis using quantitative PCR demonstrated that BboGDP1 and -3 were upregulated in in vitro-induced sexual stage parasites and during parasite development in the tick midgut. Moreover, protein expression analysis of BboGDP1 and -3 during the development of sexual stages in in vitro culture was consistent with their transcription profile. Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of BboGDP1 and -3 on in vitro-induced sexual stage parasites. In addition, fixed immunofluorescence analysis showed reactivity of anti-BboGDP1 and -3 polyclonal antibodies to kinetes.ConclusionsThe collective data indicate that BboGDP1 and -3 are expressed by kinetes and on the surface of sexual stages of the parasites. The identified parasite surface membrane proteins BboGDP1 and -3 are potential candidates for the development of a B. bovis transmission blocking vaccine.Graphical

2,3-Diphosphoglycerate and the Protective Effect of Pyruvate Kinase Deficiency against Malaria Infection-Exploring the Role of the Red Blood Cell Membrane.

Malaria remains a major world public health problem, contributing to poverty and inequality. It is urgent to find new efficacious tools with few adverse effects. Malaria has selected red blood cell (RBC) alterations linked to resistance against infection, and understanding the protective mechanisms involved may be useful for developing host-directed tools to control Plasmodium infection. Pyruvate kinase deficiency has been associated with resistance to malaria. Pyruvate kinase-deficient RBCs display an increased concentration of 2,3-diphosphoglycerate (2,3-DPG). We recently showed that 2,3-DPG impacts in vitro intraerythrocytic parasite growth, induces a shift of the metabolic profile of infected cells (iRBCs), making it closer to that of noninfected ones (niRBCs), and decreases the number of parasite progenies that invade new RBCs. As an increase of 2,3-DPG content may also have an adverse effect on RBC membrane and, consequently, on the parasite invasion, in this study, we explored modifications of the RBC morphology, biomechanical properties, and RBC membrane on Plasmodium falciparum in vitro cultures treated with 2,3-DPG, using atomic force microscopy (AFM)-based force spectroscopy and other experimental approaches. The presence of infection by P. falciparum significantly increased the rigidity of parasitized cells and influenced the morphology of RBCs, as parasitized cells showed a decrease of the area-to-volume ratio. The extracellular addition of 2,3-DPG also slightly affected the stiffness of niRBCs, making it more similar to that of infected cells. It also changed the niRBC height, making the cells appear more elongated. Moreover, 2,3-DPG treatment influenced the cell surface charge, becoming more negative in treated RBCs than in untreated ones. The results indicate that treatment with 2,3-DPG has only a mild effect on RBCs in comparison with the effect of the presence of the parasite on the host cell. 2,3-DPG is an endogenous host metabolite, which may, in the future, originate a new antimalarial tool with few adverse effects on noninfected cells.

Molecular Screening of Blood Donors for Babesia in Tyrol, Austria

Introduction: Babesia is a tick-borne intraerythrocytic parasite that is globally ubiquitous, yet understudied. Several species of Babesia have been shown to be transfusion-transmissible. Babesia has been reported in blood donors, animals, and ticks in the Tyrol (Western Austria), and regional cases of human babesiosis have been described. We sought to characterize the risk of Babesia to the local blood supply. Methods: Prospective molecular testing was performed on blood donors who presented to regional, mobile blood collection drives in the Tyrol, Austria (27 May to October 4, 2021). Testing was conducted using the cobas^® Babesia assay (Roche Molecular Systems, Inc.), a commercial PCR assay approved for blood donor screening that is capable of detecting the 4 primary species causing human babesiosis (i.e., B. microti, B. divergens, B. duncani, and B. venatorum). A confirmatory algorithm to manage initial PCR-reactive samples was developed, as were procedures for donor and product management. Results: A total of 7,972 donors were enrolled and screened; 4,311 (54.1%) were male, with a median age of 47 years (IQR = 34–55). No positive cases of Babesia were detected, corresponding with an overall prevalence of 0.00% (95% CI: 0.00%, 0.05%). Discussion: The findings suggest that the prevalence of Babesia is low in Austrian blood donors residing in the Tyrol, even during months of peak tick exposure. Although one cannot conclude the absence of Babesia in this population given the limited sample size, the findings suggest that the regional risk of transfusion-transmitted babesiosis is low.

402: FATAL TICK BITE

Introduction: As of 2022, there has been no fatality reported in NJ from severe Babesiosis. We present a case of Babesiosis with rapid clinical deterioration to shock and death in an immunocompetent patient despite early recognition and proper recommended treatment through multidisciplinary approach. Description: 48-year-old obese male with Diabetes Mellitus and Hypertension presented to emergency department two weeks after hiking, with epigastric, non-radiating pain associated with fevers, headache, worsening fatigue, poor intake, and watery diarrhea for a week. Patient was febrile, tachypneic, hypoxic, and jaundiced. Initial bloodwork reflected leukocytosis, anemia, elevated reticulocyte count, thrombocytopenia, elevated indirect bilirubinemia, acute kidney Injury and lactic acidosis. Chest radiograph with bilateral lower lobe infiltrates. Babesia microti IgG and IgM were detected with intra and extra cellular paracitemia of 9.9%. Patient was admitted to intensive care unit for Acute respiratory, renal and liver failure. Initially started on Intravenous Azithromycin, Atovaquone and later Doxycycline and Zosyn were added given continued deterioration. Patient required mechanical ventilation and hemodialysis, steroids, Intravenous immunoglobulin. Red blood exchange therapy was completed within 12 hours of admission given high parasitemia. Despite aggressive measures during ICU stay, patient declined and expired 72 hours into hospital course. Discussion: Babesia are intraerythrocytic protozoan parasites of animals and humans, a disease transmitted by infected tick, Ixodes scapularis, which also transmits other causative agents of Lyme Disease. Babesiosis can range from mild to severe disease and death. Severity depends on babesia species, age, host immune status, route of transmission, and parasitemia level. Incidence is highest in summer months, middle-aged patients. Fatality rate reported at 3-9% in hospitalized patients, 20% among immunosuppressed or disease acquisition via blood transfusion. Clinicians need to be aware of the risk of fulminant illness when practicing in known endemic regions. It is important to keep zoonotic infections as part of differential in patient presenting with multi organ failure.

Ring stage classification of Babesia microti and Plasmodium falciparum using optical diffraction 3D tomographic technique

BackgroundBabesia is an intraerythrocytic parasite often misdiagnosed as a malaria parasite, leading to inappropriate treatment of the disease especially in co-endemic areas. In recent years, optical diffraction tomography (ODT) has shown great potential in the field of pathogen detection by quantification of three-dimensional (3D) imaging tomograms. The 3D imaging of biological cells is crucial to investigate and provide valuable information about the mechanisms behind the pathophysiology of cells and tissues.MethodsThe early ring stage of P. falciparum were obtained from stored stock of infected RBCs and of B. microti were obtained from infected patients during diagnosis. The ODT technique was applied to analyze and characterize detailed differences between P. falciparum and B. microti ring stage at the single cell level. Based on 3D quantitative information, accurate measurement was performed of morphological, biochemical, and biophysical parameters.ResultsAccurate measurements of morphological parameters indicated that the host cell surface area at the ring stage in B. microti was significantly smaller (140.2 ± 17.1 µm2) than that in P. falciparum (159.0 ± 15.2 µm2), and sphericities showed higher levels in B. microti-parasitized cells (0.66 ± 0.05) than in P. falciparum (0.60 ± 0.04). Based on biochemical parameters, host cell hemoglobin level was significantly higher and membrane fluctuations were respectively more active in P. falciparum-infected cells (30.25 ± 2.96 pg; 141.3 ± 24.68 nm) than in B. microti (27.28 ± 3.52 pg; 110.1 ± 38.83 nm). The result indicates that P. falciparum more actively altered host RBCs than B. microti.ConclusionAlthough P. falciparum and B. microti often show confusable characteristics under the microscope, and the actual three-dimensional properties are different. These differences could be used in differential clinical diagnosis of erythrocytes infected with B. microti and P. falciparum.Graphical

Overwhelming Intravascular Hemolysis and Acute Renal Failure in Babesiosis Requiring Erythrocytapheresis

Babesia is an obligate intra-erythrocytic parasite transmitted by a tick called Ixodes scapularis . They invade erythrocytes and can present in various hematologic manifestations that can range from mild anemia to severe pancytopenia, splenic rupture, disseminated intravascular coagulation(DIC) or even hemophagocytic lymphohistocytosis1. They are typically self limiting in immunocompetent hosts, however management in splenectomized patients is complex and requires multidisciplinary care. We present the case of a 60 year old homeless male with a history of splenectomy secondary to a motor vehicle accident 6 years ago who presented to us with severe sepsis. On arrival his systolic blood pressure was in low 90s, tachycardic, tachypneic and saturating 93% on room air. Initial labs were significant for hyponatremia (125), hyperkalemia (5.6), acute renal failure (creatinine 2.18, baseline 0.8-1 and GFR 32, baseline 96-98), hyperuricemia(12.5), hyperphosphatemia(5) with congruent hypocalcemia(7.3).On blood counts hemoglobin(hb) was noted at 9.2 with baseline of 15 with appropriate reticulocytosis, leukocytosis(17.4) with monocytosis(26, normal is less than 11). Other labs include lactate dehydrogenase(LDH) which was elevated five times the normal limit(852) and serum haptoglobin was less than 8 (normal:11-44). He was appropriately fluid resuscitated and started on vancomycin and cefepime by emergency department. Overnight, a critical lab result was received, peripheral smear(pic 1) was showing intraerythrocytic parasites. By next morning, hematology confirmed the presence of Maltese cross on peripheral smear and parasite burden was determined at 20%. We subsequently transitioned the patient to atovaquone 750mg twice daily and IV azithromycin 500 mg daily. Doxycycline 100mg BID was later added by Infectious diseases(ID)to the regimen to empirically cover Lyme disease as both babesia and borrelia share the same tick vector. By day 2 of hospitalization patient was producing less than 0.5ml/kg/hour of urine output with decrease in GFR to 16 and concomitant rise in creatinine to 3.86, hb dropped to 7.8 with with notable rise in LDH(1080). Hematology opined for an urgent exchange transfusion specifically erythrocytapheresis and IR was consulted for a peripherally inserted central catheter insertion for the same. Post exchange transfusion, patient was noted to have improving kidney functions with decrease in parasite burden on serial peripheral smears. His hb remained stable around 10 and GFR improved to 63, at which point he was deemed stable for discharge. ID stepped down therapy to oral azithromycin 500mg daily and atovaquone 750 mg BID and doxycycline for total duration of two weeks at discharge. The indications of exchange transfusion in severe babesiosis are not well established. Erythrocytapheresis is typically considered in patients with severe hemolysis (hb less than 10), parasitemia greater than 10% or organ failure. However we did not find any prospective randomized studies to support this approach. The data to support erythrocytapheresis versus whole blood or plasma exchange is limited at best in immunocompetent patients2,3,4,5 .Though it should be noted that a prompt reduction in parasitemia markedly improves mortality in immunocompromised patient such as ours especially when used adjunctively to antimicrobial therapy 6.

Phosphatidylinositol 4-kinase is a viable target for the radical cure of Babesia microti infection in immunocompromised hosts.

Human babesiosis is a global emerging tick-borne disease caused by infection with intra-erythrocytic parasites of the genus Babesia. With the rise in human babesiosis cases, the discovery and development of new anti-Babesia drugs are essential. Phosphatidylinositol 4-kinase (PI4K) is a widely present eukaryotic enzyme that phosphorylates lipids to regulate intracellular signaling and trafficking. Previously, we have shown that MMV390048, an inhibitor of PI4K, showed potent inhibition against Babesia species, revealing PI4K as a druggable target for babesiosis. However, twice-administered, 7-day regimens failed to clear Babesia microti parasites from the immunocompromised host. Hence, in this study, we wanted to clarify whether targeting PI4K has the potential for the radical cure of babesiosis. In a B. microti-infected SCID mouse model, a 64-day-consecutive treatment with MMV390048 resulted in the clearance of parasites. Meanwhile, an atovaquone (ATO) resistant parasite line was isolated from the group treated with ATO plus azithromycin. A nonsynonymous variant in the Y272C of the cytochrome b gene was confirmed by sequencing. Likewise, MMV390048 showed potent inhibition against ATO-resistant parasites. These results provide evidence of PI4K as a viable drug target for the radical cure of babesiosis, which will contribute to designing new compounds that can eradicate parasites.

Evaluation of the inhibitory efficacy of quaternary ammonium compounds on in vitro growth of Theileria equi parasite in MASP culture

Equine piroplasmosis has become a global problem of the equine husbandry sector. Haemoprotozoans evolved very quickly and developed resistance against most of the current available drugs. Phospholipid membrane synthesis by choline kinase enzyme is vital for propagation of intra-erythrocytic protozoa parasites. This pathway was targeted in the present study. Quaternary ammonium salts (QAS) and their analogues act against choline and hamper the biosynthesis process for phosphatidylcholine. We analysed anti-T. equi activity of three QAS - decamethonium bromide (DMB), decyl trimethyl ammonium bromide (DTAB) and dodecyl trimethyl ammonium bromide (DDTAB). Theileria equi parasites in vitro treated with different concentrations of DMB, DDTAB and DTAB. Drug treated T. equi failed to multiply further in the viability test. The IC50 value of DMB, DDTAB and DTAB for growth inhibition of T. equi was 14.0 μM, 469.51 nM and 558.40 nM, respectively. DMB, DDTAB and DTAB treated T. equi parasites were observed to be devoid of internal structures, showing pyknotic and degenerative appearances. Various concentration of DMB, DDTAB and DTAB were analysed for their cytotoxicity and haemolytic activity on horse's PBMCs and RBCs. DMB was less than 10% cytotoxic to PBMCs, while DDTAB and DTAB were 40%–50% cytotoxic at 1000 μM concentrations. The respective CC50 values were 7202.96 μM, 1026.26 μM and 1263.95 μM. DMB and DTAB showed least haemolytic activity (<3%); whereas DDTAB was more haemolytic to RBCs at highest concentration of 2000 μM. The respective CC50 values of these drugs were 224495.3 μM, and 39101.35 μM; 713.54 μM. Specific selective index for DMB, DDTAB and DTAB values with respect to host's PBMC and RBC cells, were 514.50, 2185.81, 2263.52 and 16035.38, 1519.75, 70023.91, respectively. These data indicated its non-toxicity to host's cells and selective potential of anti-T. equi in vitro activity.

Isolation and molecular characterization of Polychromophilus spp. (Haemosporida: Plasmodiidae) from the Asian long-fingered bat (Miniopterus fuliginosus) and Japanese large-footed bat (Myotis macrodactylus) in Japan.

Bats (order, Chiroptera) account for more than one-fifth of all mammalian species in the world and are infected by various intra-erythrocytic parasites of the family Plasmodiidae (Apicomplexa: Haemosporida), including Polychromophilus Dionisi, 1899. Recent advance in the molecular characterization of haemosporidian isolates has enabled their accurate identification, particularly in the last decade. Studies are actively conducted in tropical regions, Europe, and Australia; however, data on haemosporidian infection in bats in Asian temperate areas, including Japan, remain limited. In this study, 75 bats of 4 species (Miniopterus fuliginosus, Myotis macrodactylus, Rhinolophus nippon, and Rhinolophus cornutus) were captured at three sites in western Japan (Yamaguchi Prefecture), and haemosporidian parasites were screened microscopically and molecularly via nested polymerase chain reaction (PCR) targeting the cytochrome b (cytb), cytochrome c oxidase subunit I (cox-1), apicoplast caseinolytic protease C (clpc), and nuclear elongation factor 2 (EF2) genes. The survey detected Polychromophilus melanipherus in 15 (40.5%) miniopterid bats (M. fuliginosus) and Polychromophilus murinus in 6 (46.2%) vespertilionid bats (M. macrodactylus), whereas none of the 25 rhinolophid bats (R. nippon and R. cornutus) was infected, indicating the robust host specificity for miniopterid (P. melanipherus) and vespertilionid (P. murinus) bats regardless of orthotopic nesting. The 15 Polychromophilus cytb sequences obtained from 11 miniopterid and 4 vespertilionid bats were classified into six cytb haplotypes (three for each species), showing no region-specific variation in a phylogenetic tree of Polychromophilus isolates in the Old World. Similarly, multiple haplotypes (seven for cox-1 and nine for clpc) and genotypes (three for EF2) were characterized for the Japanese isolates of Polychromophilus, and the results were consistent with those based on a haemosporidian cytb analysis. Bat flies (Nycteribia allotopa and another undetermined Nycteribia sp.) collected from the body surface of bats harbored Polychromophilus oocysts on the external surface of the midgut. This is the first study to report the isolation and molecular characterization of Polychromophilus spp. in miniopterid and vespertilionid bats in the temperate area of Asia (western Japan). Future studies should evaluate the global prevalence of haemosporidian infections in bats.

Immunization of Cattle With Recombinant Structural Ectodomains I and II of Babesia bovis Apical Membrane Antigen 1 [BbAMA-1(I/II)] Induces Strong Th1 Immune Response.

Both strong innate and adaptive immune responses are an important component of protection against intraerythrocytic protozoan parasites. Resistance to bovine babesiosis is associated with interferon (IFN)-γ mediated responses. CD4+ T cells and macrophages have been identified as major effector cells mediating the clearance of pathogens. Previously, the apical membrane antigen 1 (AMA-1) was found to significantly induce the immune response inhibiting B. bovis merozoite growth and invasion. However, a detailed characterization of both humoral and cellular immune responses against the structure of B. bovis AMA-1 (BbAMA-1) has not yet been established. Herein, the present study aimed to express the recombinant BbAMA-1 domain I+II protein [rBbAMA-1(I/II)], which is the most predominant immune response region, and to characterize its immune response. As a result, cattle vaccinated with BbAMA-1(I/II) significantly developed high titters of total immunoglobulin (Ig) G antibodies and a high ratio of IgG2/IgG1 when compared to control groups. Interestingly, the BbAMA-1(I/II)-based formulations produced in our study could elicit CD4+ T cells and CD8+ T cells producing IFN-γ and tumor necrosis factor (TNF)-α. Collectively, the results indicate that immunization of cattle with BbAMA-1(I/II) could induce strong Th1 cell responses. In support of this, we observed the up-regulation of Th1 cytokine mRNA transcripts, including IFN-γ, TNF-α, Interleukin (IL)-2 and IL-12, in contrast to down regulation of IL-4, IL-6 and IL-10, which would be indicative of a Th2 cytokine response. Moreover, the up-regulation of inducible nitric oxide synthase (iNOS) was observed. In conclusion, this is the first report on the in-depth immunological characterization of the response to BbAMA-1. According to our results, BbAMA-1 is recognized as a potential candidate vaccine against B. bovis infection. As evidenced by the Th1 cell response, it could potentially provide protective immunity. However, further challenge-exposure with virulent B. bovis strain in immunized cattle would be needed to determine its protective efficacy.

A prime-boost combination of a three-protein cocktail and multiepitopic MVA as a vaccine against Babesia bigemina elicits neutralizing antibodies and a Th1 cellular immune response in mice

In the intraerythrocytic protozoan parasites of the genus Babesia both innate and adaptive immune responses are necessary to confer protection against clinical disease. In particular, the adaptive immune response involves the production of neutralizing antibodies as well as the presentation of parasite antigens to CD4+ T lymphocytes by professional antigen-presenting cells. Therefore, the development of alternative vaccines that replace the use of live attenuated strains should include relevant epitopes targeting both B and T cell responses. The aim of this study was to design new Babesia bigemina immunogens and evaluate the humoral and cellular responses in mice. To achieve this, three B. bigemina recombinant antigens called Apical Membrane Antigen 1 (AMA-1), Rhoptry Associated Protein 1 (RAP-1) and the Thrombospondin Related Anonymous Protein 1 (TRAP-1) were obtained. Besides, two recombinant modified vaccinia virus Ankara vectors coding for chimeric constructs containing bioinformatically predicted B and T cell epitopes from the same three antigens were generated. These immunogens were evaluated in prime-boost heterologous schemes. Among the combinations tested, priming with a cocktail of the three proteins followed by a booster immunization with a mix of both viruses induced the highest activation of IFN-γ+ CD4+ and CD8+ antigen-specific T cell responses. Remarkably, all vaccine schemes containing antigen cocktails also induced antibodies that were capable of neutralizing merozoite invasion of bovine erythrocytes in vitro at a level comparable to an anti B. bigemina hyperimmune bovine serum. Our results offer a new perspective for vaccines against B. bigemina combining bioinformatics predictions and prime-boost immunization regimes for future control measures against bovine babesiosis.

Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant.

Aims:Genetically encoded green fluorescent protein (GFP)-based redox biosensors are widely used to monitor specific and dynamic redox processes in living cells. Over the last few years, various biosensors for a variety of applications were engineered and enhanced to match the organism and cellular environments, which should be investigated. In this context, the unicellular intraerythrocytic parasite Plasmodium, the causative agent of malaria, represents a challenge, as the small size of the organism results in weak fluorescence signals that complicate precise measurements, especially for cell compartment-specific observations. To address this, we have functionally and structurally characterized an enhanced redox biosensor superfolder roGFP2 (sfroGFP2).Results:SfroGFP2 retains roGFP2-like behavior, yet with improved fluorescence intensity (FI) in cellulo. SfroGFP2-based redox biosensors are pH insensitive in a physiological pH range and show midpoint potentials comparable with roGFP2-based redox biosensors. Using crystallography and rigidity theory, we identified the superfolding mutations as being responsible for improved structural stability of the biosensor in a redox-sensitive environment, thus explaining the improved FI in cellulo.Innovation:This work provides insight into the structure and function of GFP-based redox biosensors. It describes an improved redox biosensor (sfroGFP2) suitable for measuring oxidizing effects within small cells where applicability of other redox sensor variants is limited.Conclusion:Improved structural stability of sfroGFP2 gives rise to increased FI in cellulo. Fusion to hGrx1 (human glutaredoxin-1) provides the hitherto most suitable biosensor for measuring oxidizing effects in Plasmodium. This sensor is of major interest for studying glutathione redox changes in small cells, as well as subcellular compartments in general. Antioxid. Redox Signal. 37, 1–18.

A comprehensive review on classifying fast-acting and slow-acting antimalarial agents based on time of action and target organelle of Plasmodium sp.

The clinical resistance towards malarial parasites has rendered many antimalarials ineffective, likely due to a lack of understanding of time of action and stage specificity of all life stages. Therefore, to tackle this problem a more incisive comprehensive analysis of the fast- and slow-acting profile of antimalarial agents relating to parasite time-kill kinetics and the target organelle on the progression of blood-stage parasites was carried out. It is evident from numerous findings that drugs targeting food vacuole, nuclear components, and endoplasmic reticulum mainly exhibit a fast-killing phenotype within 24 haffecting first-cycle activity. Whereas drugs targeting mitochondria, apicoplast, microtubules, parasite invasion, and egress exhibit a largely slow-killing phenotype within 96-120 h, affecting second-cycle activity with few exemptions as moderately fast-killing. It is essential to understand the susceptibility of drugs on rings, trophozoites, schizonts, merozoites, and the appearance of organelle at each stage of the 48-hintraerythrocytic parasite cycle. Therefore, these parameters may facilitate the paradigm for understanding the timing of antimalarials action in deciphering its precise mechanism linked with time. Thus, classifying drugs based on the time of killing may promote designing new combination regimens against varied strains of Plasmodium falciparum and evaluating potential clinical resistance.

Climate predictors and climate change projections for avian haemosporidian prevalence in Mexico.

Long-term, inter-annual and seasonal variation in temperature and precipitation influence the distribution and prevalence of intraerythrocytic haemosporidian parasites. We characterized the climatic niche behind the prevalence of the three main haemosporidian genera (Haemoproteus, Plasmodium and Leucocytozoon) in central-eastern Mexico, to understand their main climate drivers. Then, we projected the influence of climate change over prevalence distribution in the region. Using the MaxEnt modelling algorithm, we assessed the relative contribution of bioclimatic predictor variables to identify those most influential to haemosporidian prevalence in different avian communities within the region. Two contrasting climate change scenarios for 2070 were used to create distribution models to explain spatial turnover in prevalence caused by climate change. We assigned our study sites into polygonal operational climatic units (OCUs) and used the general haemosporidian prevalence for each OCU to indirectly measure environmental suitability for these parasites. A high statistical association between global prevalence and the bioclimatic variables ‘mean diurnal temperature range’ and ‘annual temperature range’ was found. Climate change projections for 2070 showed a significant modification of the current distribution of suitable climate areas for haemosporidians in the study region.

Babesia duncani as a Model Organism to Study the Development, Virulence, and Drug Susceptibility of Intraerythrocytic Parasites In Vitro and In Vivo.

Human babesiosis is a malaria-like illness caused by tick-borne intraerythrocytic Babesia parasites of the Apicomplexa phylum. Whereas several species of Babesia can cause severe disease in humans, the ability to propagate Babesia duncani both in vitro in human erythrocytes and in mice makes it a unique pathogen to study Babesia biology and pathogenesis. Here we report an optimized B. duncani in culture-in mouse (ICIM) model that combines continuous in vitro culture of the parasite with a precise model of lethal infection in mice. We demonstrate that B. duncani-infected erythrocytes as well as free merozoites can cause lethal infection in C3H/HeJ mice. Highly reproducible parasitemia and survival outcomes could be established using specific parasite loads in different mouse genetic backgrounds. Using the ICIM model, we discovered 2 new endochin-like quinolone prodrugs (ELQ-331 and ELQ-468) that alone or in combination with atovaquone are highly efficacious against B. duncani and Babesia microti.

Babesia gibsoni

Babesia gibsoni is an intraerythrocytic apicomplexan parasite that causes babesiosis in dogs. Since its first report in India in 1910, B. gibsoni has spread globally. It is transmitted mainly by tick bite but many reports have demonstrated transmission by dog fighting, and blood transfusion, and a few reports have mentioned transplacental transmission to the developing fetus. The parasite life cycle consists of a sexual developmental phase in the midgut lumen of tick vectors (gamogony) and two asexual developmental phases occurring in the tick salivary glands (sporogony) and host red blood cells (merogony).

Draft Genome Sequences of Mexican Babesia bovis Virulent and Attenuated Strains.

ABSTRACTBabesia bovis, a tick-borne intraerythrocytic protozoan parasite that belongs to the phylum Apicomplexa, is one of the etiological agents of bovine babesiosis, a highly prevalent disease in tropical and subtropical countries that causes significant morbidity and deaths in cattle. This report presents the draft genome sequences of attenuated and virulent B. bovis strains of Mexican origin.

Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein gene of Babesia gibsoni isolates in dogs in South India.

Babesia gibsoni, the causative agent of canine piroplasmosis, is a tick-borne intraerythrocytic protozoan parasite predominantly reported in Asian countries. The present study aimed at genotypic characterization of B. gibsoni isolates prevalent in dogs in Kerala, a southern state of India. Blood samples were collected from 272 dogs in Kerala and B. gibsoni infection was detected by microscopy and polymerase chain reaction (PCR). Molecular confirmation of B. gibsoni parasites was carried out by 18S rRNA nested-PCR, followed by sequencing. Nested-PCR detected a higher percentage of dogs (40.44%) positive for B. gibsoni infection than microscopy where 15.81% dogs were detected positive for infection. Genetic characterization of B. gibsoni isolates (n=11) prevalent in dogs in the state of Kerala was carried out by PCR amplification and sequencing of the 855bp thrombospondin-related adhesive protein (TRAP) gene fragment. Phylogenetic analysis of the B. gibsoni TRAP (BgTRAP) gene revealed that B. gibsoni isolates from Kerala formed a distinct cluster with the isolates from north India and Bangladesh, away from other East Asian isolates. Nucleotide analysis of the tandem repeats of BgTRAP gene showed considerable genetic variation among Indian isolates that was shared by B. gibsoni isolates of Bangladesh but not by the isolates of East Asian countries. The results of the present study further confirmed that B. gibsoni parasites in a distinct genetic clade are endemic in dogs in India and Bangladesh. However, elaborate studies are required for better understanding of the genetic diversity of B. gibsoni.

Babesia bovis AMA-1, MSA-2c and RAP-1 contain conserved B and T-cell epitopes, which generate neutralizing antibodies and a long-lasting Th1 immune response in vaccinated cattle

Vaccines against bovine babesiosis must, ideally, induce a humoral immune response characterized by neutralizing antibodies against conserved epitopes and a cellular Th1 immune response. In Babesia bovis, proteins such as AMA-1, MSA-2c, and RAP-1 have been characterized and antibodies against these proteins have shown a neutralizing effect, demonstrating the implication of B and T-cell epitopes in the immune response. There is evidence of the existence of B and T-cell epitopes in these proteins, however, it remains to be defined, the presence of conserved peptides in strains from around the world containing B and T-cell epitopes, and their role in the generation of a long-lasting immunity. The aim in this paper was to identify peptides of Babesia bovis AMA-1, MSA-2c, and RAP-1 that elicit a neutralizing and long-lasting Th1 immune response. Peptides containing B-cell epitopes of AMA-1, MSA-2c and RAP-1, were identified. The immune response generated by each peptide was characterized in cattle. All peptides tested induced antibodies that recognized intraerythrocytic parasites, however, only 5 peptides generated neutralizing antibodies in vitro: P2AMA-1 (6.28%), P3MSA-2c (10.27%), P4MSA-2c (10.42%), P1RAP-1 (32.45%), and P4RAP-1 (36.98%). When these neutralizing antibodies were evaluated as a pool, the inhibition percentage of invasion increased to 52.37%. When the T cellular response was evaluated, two peptides: P3MSA2c and P2AMA1 induced a higher percentage (>70%) of activated CD4 +/CD45RO+ T cells than unstimulated cells. Additionally, both peptides induced the production of gamma interferon (IFN−) in PBMCs from vaccinated cattle after one year proving the implication of a long-lasting Th1 immune response. In conclusion, we identified conserved peptides containing B and T-cell epitopes in antigens of B. bovis that elicit a Th1 immune response and showed evidence that peptides from the same protein elicit different immune responses, which has implication for vaccine development in bovine babesiosis.

Uncomplicated Plasmodium vivax malaria: mapping the proteome from circulating platelets

BackgroundThrombocytopenia is frequent in Plasmodium vivax malaria but the role of platelets in pathogenesis is unknown. Our study explores the platelet (PLT) proteome from uncomplicated P. vivax patients, to fingerprint molecular pathways related to platelet function. Plasma levels of Platelet factor 4 (PF4/CXCL4) and Von Willebrand factor (VWf), as well as in vitro PLTs—P. vivax infected erythrocytes (Pv-IEs) interactions were also evaluated to explore the PLT response and effect on parasite development.MethodsA cohort of 48 patients and 25 healthy controls were enrolled. PLTs were purified from 5 patients and 5 healthy controls for Liquid Chromatography–Mass spectrometry (LC–MS/MS) analysis. Plasma levels of PF4/CXCL4 and VWf were measured in all participants. Additionally, P. vivax isolates (n = 10) were co-cultured with PLTs to measure PLT activation by PF4/CXCL4 and Pv-IE schizonts formation by light microscopy.ResultsThe proteome from uncomplicated P. vivax patients showed 26 out of 215 proteins significantly decreased. PF4/CXCL4 was significantly decreased followed by other proteins involved in platelet activation, cytoskeletal remodeling, and endothelial adhesion, including glycoprotein V that was significantly decreased in thrombocytopenic patients. In contrast, acute phase proteins, including SERPINs and Amyloid Serum A1 were increased. High levels of VWf in plasma from patients suggested endothelial activation while PF4/CXCL4 plasma levels were similar between patients and controls. Interestingly, high levels of PF4/CXCL4 were released from PLTs—Pv-IEs co-cultures while Pv-IEs schizont formation was inhibited.ConclusionsThe PLT proteome analyzed in this study suggests that PLTs actively respond to P. vivax infection. Altogether, our findings suggest important roles of PF4/CXCL4 during uncomplicated P. vivax infection through a possible intracellular localization. Our study shows that platelets are active responders to P. vivax infection, inhibiting intraerythrocytic parasite development. Future studies are needed to further investigate the molecular pathways of interaction between platelet proteins found in this study and host response, which could affect parasite control as well as disease progression.