Search Results (23)

Background: Bovine babesiosis, caused by the tick-borne apicomplexan parasite Babesia spp., is an economically significant disease that threatens the cattle industry worldwide. Babesia bovis is the most pathogenic species, leading to high morbidity and mortality in infected animals. One promising approach to vaccination against bovine babesiosis involves the use of multiple protective antigens, offering advantages over traditional live-attenuated vaccines. Tools such as immunobioinformatics and reverse vaccinology have facilitated the identification of novel antigens. Enolase, a “moonlighting” enzyme of the glycolytic pathway with demonstrated vaccine potential in other pathogens, has not yet been studied in B. bovis. Methods: In this study, the enolase gene from two B. bovis isolates was successfully identified and sequenced. The gene, consisting of 1366 base pairs, encodes a predicted protein of 438 amino acids. Its expression in intraerythrocytic parasites was confirmed by RT-PCR. Two peptides containing predicted B-cell epitopes were synthesized and used to immunize rabbits. Hyperimmune sera were then analyzed by ELISA, confocal microscopy, Western blot, and an in vitro neutralization assay. Results: The hyperimmune sera showed high antibody titers, reaching up to 1:256,000. Specific antibodies recognized intraerythrocytic merozoites by confocal microscopy and bound to a ~47 kDa protein in erythrocytic cultures of B. bovis as detected by Western blot. In the neutralization assay, antibodies raised against peptide 1 had no observable effect, whereas those targeting peptide 2 significantly reduced parasitemia by 71.99%. Conclusions: These results suggest that B. bovis enolase contains B-cell epitopes capable of inducing neutralizing antibodies and may play a role in parasite–host interactions. Enolase is therefore a promising candidate for further exploration as a vaccine antigen. Nonetheless, additional experimental studies are needed to fully elucidate its biological function and validate its vaccine potential. Full article

Babesia bovis is a protozoan parasite that causes babesiosis in cattle. It has been hypothesized that in apicomplexan parasites, translationally controlled tumor protein (TCTP) interferes with the host immune response by inhibiting B cell proliferation. The aim of this study was the characterization of B. bovis TCTP (BboTCTP) and the evaluation of its expression, immunogenicity and role in infection. The tctp gene was identified and sequenced from B. bovis isolates and revealed a high conservation. Expression was confirmed in intraerythrocytic stages by Western blot and confocal microscopy. Synthetic peptides containing predicted B cell epitopes were used to immunize cattle, followed by a challenge with a virulent B. bovis strain. Immunized animals showed milder clinical signs and faster recovery compared to controls. Sera from non-immunized animals exhibited lower total IgG levels after challenge (p < 0.05), while sera from immunized animals induced significant in vitro invasion inhibition (32–33%). These results suggest that BboTCTP is immunogenic and may play a role in modulating the host immune response. The results provide novel insights into B. bovis biology and support BboTCTP as a promising candidate for further evaluation as a vaccine antigen. Future studies should explore its immunomodulatory mechanisms and potential use in combined vaccine formulations. Full article

This study evaluated the effects of various concentrations of PVP-40 on the in vitro cryopreservation and recovery of Babesia bovis and Babesia bigemina. We also assessed a reduced dose of attenuated Babesia strains to determine its efficacy in preventing clinical disease. A microaerophilic stationary phase blood culture system was used to recover Babesia parasites that were cryopreserved in solutions with various PVP-40 concentrations and Babesia parasites in 20% PVP-40 were used to vaccinate naïve cattle. The animals were vaccinated intramuscularly with frozen parasites cryopreserved in 20% PVP-40, with a dose of either 1 × 10⁸ or 1 × 10⁷ erythrocytes infected with both attenuated B. bigemina and B. bovis produced from blood cultures. The control group received uninfected erythrocytes. During the vaccination, clinical parameters such as rectal temperature and hematocrit levels were unaffected. The animals were relocated to a farm in a Babesia hyperendemic area to test the efficacy of these live vaccines in controlling disease onset. Some vaccinated animals showed mild disease. In the vaccinated groups, parasites were detected in blood smears for only one day during the challenge. In contrast, the control group experienced fever for three consecutive days, a decline in hematocrit levels, and significant health deterioration. In this group, parasites were detected in smears for four consecutive days. All the animals in the control group required treatment to manage their high parasitemia and prevent mortality. In this study, we demonstrated that increasing the concentration of PVP-40 to cryopreserve parasites improved the recovery and proliferation of Babesia spp. in blood culture, and we also showed that when animals were vaccinated with cryopreserved, in vitro cultured, attenuated Babesia parasites in 20% PVP-40, they were effectively protected from severe clinical babesiosis. Full article

Babesia bigemina is one of the most important etiological agents of bovine babesiosis, a tick-borne disease posing a major threat in the livestock industry globally, including South Africa. Despite the huge economic impact of cattle babesiosis in South Africa, antigenic variation observed among B. bigemina strains worldwide has impeded the successful development of a single vaccine with the potential to eliminate the disease. As such, there is still a dearth of information regarding the conservation of B. bigemina genes encoding functionally important proteins that play a crucial role during the invasion of bovine erythrocytes by merozoites. Fifty blood samples previously collected from cattle in eight provinces of South Africa were genetically tested for the presence of B. bigemina DNA fragments using four nested PCR-based assays. The genes targeted coded for SpeI-AvaI restriction fragment, rhoptry-associated protein 1 (BgRAP-1), apical membrane antigen 1 (BgAMA-1) and β-tubulin (BgβTUB). PCR-generated fragments of randomly selected samples were sequenced. BLAST searches in GenBank were performed with newly determined sequences to search for homologous sequences. Neighbor-joining phylogenies were inferred from aligned, contiguous sequences of BgRAP-1, BgAMA-1 and BgβTUB genes. Nested PCR assays generated single fragments of 170 bp, 472 bp, 765 bp and 302 bp for SpeI-AvaI, BgRAP-1, BgAMA-1 and BgβTUB fragments, respectively. Of the 50 bovine samples tested by nested PCR, 82% (42/50; 95% CI = 69.2–90.2%), 68% (34/50; 95% CI = 54.2–79.2%), 50% (25/50; 95% CI = 36.6–63.4%) and 46% (23/50; 95% CI = 33.0–59.6%) possessed B. bigemina-specific SpeI-AvaI, BgRAP-1, BgAMA-1 and BgβTUB DNA fragments, respectively. The BgRAP-1, BgAMA-1 and BgβTUB sequences of South African B. bigemina isolates shared 98–100% similarity with previously reported sequences of strains originating from cattle in countries other than South Africa. The high genetic conservation observed among geographical isolates of B. bigemina suggests the conserved functional role of BgRAP-1 and BgAMA-1 proteins as potential candidates that could be incorporated in recombinant subunit vaccines. Full article

Bovine babesiosis caused by the tick-borne apicomplexan parasite Babesia bovis remains a threat for cattle worldwide, and new vaccines are needed. We propose using immune-subdominant (ISD) antigens as alternative vaccine candidates. We first determined that RAP-1 NT and RRA are subdominant antigens using recombinant antigens in ELISAs against sera from B. bovis-protected cattle. Protected animals demonstrated high antibody responses against the known immunodominant rRAP-1 CT antigen, but significantly lower levels against the rRAP-1 NT and rRRA antigens. Next, a group of cattle (n = 6) was vaccinated with rRRA and rRAP-1 NT using a FliC–Emulsigen mix as the adjuvant, and there was a control group (n = 6) with the adjuvant mix alone. All but one immunized animal demonstrated elicitation of strong humoral immune responses against the two ISD antigens. Acute babesiosis occurred in both groups of cattle upon a challenge with the virulent B. bovis, but a significant delay in the average rate of decrease in hematocrit in the vaccinated group, and an early monocyte response, was found in half of the vaccinated animals. In conclusion, we confirmed the immune subdominance of rRRA and rRAP-1 NT and the ability of FliC to increase immunogenicity of ISD antigens and generate useful information toward developing future subunit vaccines against B. bovis. Full article

The United States of America (US) has the highest annual number of human babesiosis cases caused by Babesia microti (Bm). Babesia, like malaria-causing Plasmodium, are protozoan parasites that live within red blood cells (RBCs). Both infectious diseases can be associated with hemolysis and organ damage, which can be fatal. Since babesiosis was made a nationally notifiable condition by the Centers for Disease Control and Prevention (CDC) in January 2011, human cases have increased, and drug-resistant strains have been identified. Both the Bm ligand(s) and RBC receptor(s) needed for invasion are unknown, partly because of the difficulty of developing a continuous in vitro culture system. Invasion pathways are relevant for therapies (e.g., RBC exchange) and vaccines. We hypothesize that there is at least one RBC surface antigen that is essential for Bm invasion and that all Bm hosts express this. Because most RBC surface antigens that impact Plasmodium invasion are in human blood group (hBG) systems, which are generated by 51 genes, they were the focus of this study. More than 600 animals with at least one hBG system gene ortholog were identified using the National Center for Biotechnology Information (NCBI) command-line tools. Google Scholar searches were performed to determine which of these animals are susceptible to Bm infection. The literature review revealed 28 Bm non-human hosts (NHH). For 5/51 (9.8%) hBG system genes (e.g., RhD), no NHH had orthologs. This means that RhD is unlikely to be an essential receptor for invasion. For 24/51 (47.1%) hBG system genes, NHH had 4–27 orthologs. For the ABO gene, 15/28 NHH had an ortholog, meaning that this gene is also unlikely to generate an RBC antigen, which is essential for Bm invasion. Our prior research showed that persons with blood type A, B, AB, O, RhD+, and RhD- can all be infected with Bm, supporting our current study’s predictions. For 22/51 (43.1%) hBG system genes, orthologs were found in all 28 NHH. Nineteen (37.3%) of these genes encode RBC surface proteins, meaning they are good candidates for generating a receptor needed for Bm invasion. In vitro cultures of Bm, experimental Bm infection of transgenic mice (e.g., a CD44 KO strain), and analyses of Bm patients can reveal further clues as to which RBC antigens may be essential for invasion. Full article

Bovine babesiosis, caused by the protozoan Babesia bigemina, is one of the most important hemoparasite diseases of cattle in Mexico and the world. An attenuated B. bigemina strain maintained under in vitro culture conditions has been used as a live attenuated vaccine; however, the biological mechanisms involved in attenuation are unknown. The objective of this study was to identify, through a comparative transcriptomics approach, the components of the B. bigemina virulent parasites that are differentially expressed in vivo, as opposed to those expressed by B. bigemina attenuated vaccine parasites when inoculated into naïve cattle. The biological material under study was obtained by inoculating spleen-intact cattle with infected erythrocytes containing either the attenuated strain or a virulent field strain. After RNA extraction, transcriptomic analysis (RNA-seq) was performed, followed by bioinformatic Differential Expression (DE) analysis and Gene Ontology (GO) term enrichment. The high-throughput sequencing results obtained by analyzing three biological replicates for each parasite strain ranged from 9,504,000 to 9,656,000, and 13,400,000 to 15,750,000 reads for the B. bigemina attenuated and virulent strains, respectively. At least 519 differentially expressed genes were identified in the analyzed strains. In addition, GO analysis revealed both similarities and differences across the three categories: cellular components, biological processes, and molecular functions. The attenuated strain of B. bigemina derived from in vitro culture presents global transcriptomic changes when compared to the virulent strain. Moreover, the obtained data provide insights into the potential molecular mechanisms associated with the attenuation or pathogenicity of each analyzed strain, offering molecular markers that might be associated with virulence or potential vaccine candidates. Full article

Bovine babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia. Its main causative agents in the Americas are Babesia bigemina and Babesia bovis, while Babesia ovata affects cattle in Asia. All Babesia species secrete proteins stored in organelles of the apical complex, which are involved in all steps of the invasion process of vertebrate host cells. Unlike other apicomplexans, which have dense granules, babesia parasites instead have large, round intracellular organelles called spherical bodies. Evidence suggests that proteins from these organelles are released during the process of invading red blood cells, where spherical body proteins (SBPs) play an important role in cytoskeleton reorganization. In this study, we characterized the gene that encodes SBP4 in B. bigemina. This gene is transcribed and expressed in the erythrocytic stages of B. bigemina. The sbp4 gene consists of 834 nucleotides without introns that encode a protein of 277 amino acids. In silico analysis predicted a signal peptide that is cleaved at residue 20, producing a 28.88-kDa protein. The presence of a signal peptide and the absence of transmembrane domains suggest that this protein is secreted. Importantly, when cattle were immunized with recombinant B. bigemina SBP4, antibodies identified B. bigemina and B. ovata merozoites according to confocal microscopy observations and were able to neutralize parasite multiplication in vitro for both species. Four peptides with predicted B-cell epitopes were identified to be conserved in 17 different isolates from six countries. Compared with the pre-immunization sera, antibodies against these conserved peptides reduced parasite invasion in vitro by 57%, 44%, 42%, and 38% for peptides 1, 2, 3, and 4, respectively (p < 0.05). Moreover, sera from cattle infected with B. bigemina cattle contained antibodies that recognized the individual peptides. All these results support the concept of spb4 as a new gene in B. bigemina that should be considered a candidate for a vaccine to control bovine babesiosis. Full article

Bovine babesiosis is caused by the Apicomplexa parasites from the genus Babesia. It is one of the most important tick-borne veterinary diseases worldwide; Babesia bovis being the species associated with the most severe clinical signs of the disease and causing the greatest economic losses. Many limitations related to chemoprophylaxis and the acaricides control of transmitting vectors have led to the adoption of live attenuated vaccine immunisation against B. bovis as an alternative control strategy. However, whilst this strategy has been effective, several drawbacks related to its production have prompted research into alternative methodologies for producing vaccines. Classical approaches for developing anti-B. bovis vaccines are thus discussed in this review and are compared to a recent functional approach to highlight the latter’s advantages when designing an effective synthetic vaccine targeting this parasite. Full article

Babesiosis is a tick-borne zoonotic disease, which is caused by various species of intracellular Babesia parasite. It is a problem not only for the livestock industry but also for global health. Significant global economic losses, in particular in cattle production, have been observed. Since the current preventive measures against babesiosis are insufficient, there is increasing pressure to develop a vaccine. In this review, we survey the achievements and recent advances in the creation of antibabesiosis vaccine. The scope of this review includes the development of a vaccine against B. microti, B. bovis, B. bigemina, B. orientalis and B. divergens. Here, we present different strategies in their progress and evaluation. Scientists worldwide are still trying to find new targets for a vaccine that would not only reduce symptoms among animals but also prevent the further spread of the disease. Molecular candidates for the production of a vaccine against various Babesia spp. are presented. Our study also describes the current prospects of vaccine evolution for successful Babesia parasites elimination. Full article

Human babesiosis is an emerging tick-borne disease, caused by haemoprotozoa genus of Babesia. Cases of transfusion-transmitted and naturally acquired Babesia infection have been reported worldwide in recent years and causing a serious public health problem. Babesia duncani is one of the important pathogens of human babesiosis, which seriously endangers human health. The in vitro culture systems of B. duncani have been previously established, and it requires fetal bovine serum (FBS) to support long-term proliferation. However, there are no studies on serum-free in vitro culture of B. duncani. In this study, we reported that B. duncani achieved long-term serum-free culture in VP-SFM AGT^TM (VP-SFM) supplemented with AlbuMax^TM I. The effect of adding different dilutions of AlbuMax^TM I to VP-SFM showed that 2 mg/mL AlbuMax^TM I had the best B. duncani growth curve with a maximum percentage of parasitized erythrocytes (PPE) of over 40%, and it can be used for long-term in vitro culture of B. duncani. However, the commonly used 20% serum-supplemented medium only achieves 20% PPE. Clearly, VP-SFM with 2 mg/mL AlbuMax^TM I (VP-SFMA) is more suitable for the in vitro proliferation of B. duncani. VP-SFM supplemented with CD lipid mixture was also tested, and the results showed it could support the parasite growth at 1:100 dilution with the highest PPE of 40%, which is similar to that of 2 mg/mL AlbuMax^TM I. However, the CD lipid mixture was only able to support the in vitro culture of B. duncani for 8 generations, while VP-SFMA could be used for long-term culture. To test the pathogenicity, the VP-SFMA cultured B. duncani was also subjected to hamster infection. Results showed that the hamster developed dyspnea and chills on day 7 with 30% PPE before treatment, which is similar to the symptoms with un-cultured B. duncani. This study develops a unique and reliable basis for further understanding of the physiological mechanisms, growth characteristics, and pathogenesis of babesiosis, and provides good laboratory material for the development of drugs or vaccines for human babesiosis and possibly other parasitic diseases. Full article

The tick-transmitted disease bovine babesiosis causes significant economic losses in many countries around the world. Current control methods include modified live-attenuated vaccines that have limited efficacy. Recombinant proteins could provide effective, safe, and low-cost alternative vaccines. We compared the expression of the Babesia bovis thrombospondin-related anonymous protein (TRAP) family from parasites in bovine blood, in vitro induced sexual stages, and kinetes from tick hemolymph. Quantitative PCR showed that in blood and sexual stages, TRAP3 was highly transcribed as compared to the other TRAPs. In contrast, the TRAP1 gene was highly transcribed in kinetes as compared to the other TRAPs. Fixed immunofluorescence assays showed that TRAP2, 3, and 4 proteins were expressed by both blood and sexual stages. Conversely, TRAP1 protein, undetected on blood and induced sexual stages, was the only family member expressed by kinetes. Live IFA revealed that TRAP2, 3, and 4 proteins were expressed on the surface of both B. bovis blood and sexual stages. Modeling of B. bovis TRAP1 and TRAP4 tertiary structure demonstrated both proteins folded the metal-ion-dependent adhesion site (MIDAS) domain structure of Plasmodium TRAP. In conclusion, TRAP proteins may serve as potential vaccine targets to prevent infection of bovine and ticks with B. bovis essential for controlling the spread of bovine babesiosis. Full article

Babesiosis is a disease caused by tickborne hemoprotozoan apicomplexan parasites of the genus Babesia that negatively impacts public health and food security worldwide. Development of effective and sustainable vaccines against babesiosis is currently hindered in part by the absence of definitive host correlates of protection. Despite that, studies in Babesia microti and Babesia bovis, major causative agents of human and bovine babesiosis, respectively, suggest that early activation of innate immune responses is crucial for vertebrates to survive acute infection. Trained immunity (TI) is defined as the development of memory in vertebrate innate immune cells, allowing more efficient responses to subsequent specific and non-specific challenges. Considering that Mycobacterium bovis bacillus Calmette-Guerin (BCG), a widely used anti-tuberculosis attenuated vaccine, induces strong TI pro-inflammatory responses, we hypothesize that BCG TI may protect vertebrates against acute babesiosis. This premise is supported by early investigations demonstrating that BCG inoculation protects mice against experimental B. microti infection and recent observations that BCG vaccination decreases the severity of malaria in children infected with Plasmodium falciparum, a Babesia-related parasite. We also discuss the potential use of TI in conjunction with recombinant BCG vaccines expressing Babesia immunogens. In conclusion, by concentrating on human and bovine babesiosis, herein we intend to raise awareness of BCG TI as a strategy to efficiently control Babesia infection. Full article

Development of in vitro culture and completion of genome sequencing of several Babesia parasites promoted the efforts to establish transfection systems for these parasites to dissect the gene functions. It has been more than a decade since the establishment of first transfection for Babesia bovis, the causative agent of bovine babesiosis. However, the number of genes that were targeted by genetic tools in Babesia parasites is limited. This is partially due to the low efficiencies of these methods. The recent adaptation of CRISPR/Cas9 for genome editing of Babesia bovis can accelerate the efforts for dissecting this parasite’s genome and extend the knowledge on biological aspects of erythrocytic and tick stages of Babesia. Additionally, glmS ribozyme as a conditional knockdown system is available that could be used for the characterization of essential genes. The development of high throughput genetic tools is needed to dissect the function of multigene families, targeting several genes in a specific pathway, and finally genome-wide identification of essential genes to find novel drug targets. In this review, we summarized the current tools that are available for Babesia and the genes that are being targeted by these tools. This may draw a perspective for the future development of genetic tools and pave the way for the identification of novel drugs or vaccine targets. Full article

Babesia bovis, an etiological agent of bovine babesiosis, causes a significant burden to the cattle industry worldwide. The most efficient method to mitigate bovine babesiosis is a live vaccine produced by serial passage in splenectomized cattle. However, there are several concerns regarding live vaccine production, including variation between batches and the use of many animals. In this study, we report a B. bovis-SF strain continuously cultured in a medium free of components of animal origin enriched with a chemically defined lipid mixture (CD lipid mixture) and the use of a perfusion bioreactor to harvest a large amount of B. bovis. Six culture media were compared, including VP-SFM, CD-CHO, CD-Hydrolyzed, CD-CHO, SFM, and ADMEM/F12. We found that the VP-SFM medium performed the best for B. bovis growth, with a maximum percentage of parasitized erythrocytes (PPE) of 8.6%. The effect of six dilutions of a commercial mixture of CD lipids added to VP-SFM showed that the CD lipid mixture at a dilution of 1:100 had the best B. bovis growth curve, with a maximum PPE of 13.9%. Propagation of the in vitro B. bovis culture was scaled up in a perfusion bioreactor using VP-SFM with a CD lipid mixture, and the PPE reached over 32%. The continuous in vitro B. bovis culture in a medium free of animal origin components could potentially reduce and replace the use of animals to produce a reagent for diagnostics and live vaccines to control bovine babesiosis. Full article