Rhoptry-associated Protein Research Articles

First Molecular Evidence of Babesia caballi and Theileria equi in Imported Donkeys from Kyrgyzstan.

Equine piroplasmosis (EP) is an important tick-borne disease of equids, caused by Theileria equi, Theileria haneyi, and Babesia caballi. Nonetheless, there has been a scarcity of systematic reports on EP parasites in donkeys in Kyrgyzstan, Central Asia. In this study, piroplasms were screened in 1900 blood samples from imported donkeys from the Osh Oblast (southwestern Kyrgyzstan) by targeting partial 18S ribosomal RNA using the polymerase chain reaction (PCR). Through molecular and phylogenetic analyses, all positive samples were sequenced to identify the species and genotypes. The results indicated the presence of both B. caballi and T. equi, with prevalence rates of 8.4% (160/1900) and 12.2% (232/1900), respectively. By amplifying part of the Erythrocyte Merozoite Antigen 1 (EMA-1) and Rhoptry-Associated Protein (RAP-1) genes, B. caballi genotype B and T. equi genotype A were identified. To the best of our knowledge, this is the first report on piroplasm infection among donkeys from Kyrgyzstan.

Molecular genotyping of Babesia caballi

Babesia caballi is an intra-erythrocytic parasite causing equine piroplasmosis. Three B. caballi genotypes (A, B, and C) have been identified based on the 18 S rRNA and rhoptry-associated protein (rap-1) gene sequences. These variant parasite genotypes compromise the diagnostic utility of the WOAH-recommended serological assays in declaring horses free of equine piroplasmosis. Although a gene encoding a spherical body protein 4 (sbp4) has recently been identified as a potential antigen for the serological detection of B. caballi, the ability of this antigen to detect the different geographical strains has not been determined. The molecular distinction between variant B. caballi genotypes is limited and therefore we developed molecular typing assays for the rapid detection and quantification of distinct parasite genotypes. Field samples were screened for the presence of B. caballi using an established multiplex equine piroplasmosis qPCR assay. In this study, B. caballi genotype A was not detected in any field samples screened. However, phylogenetic analysis of the amplified sbp4 and 18 S rRNA genes confirmed the phylogenetic groupings of the South African isolates into either B. caballi genotypes B or C. A multiple sequence alignment of the sbp4 gene sequences obtained in this study together with the published sbp4 sequences representing B. caballi genotype A, were used to identify conserved regions within the gene to design three primer pairs and three genotype-specific TaqMan minor-groove binder (MGB™) probes. The qPCR assays were shown to be specific and efficient in the detection and differentiation between B. caballi genotypes A, B, and C and could be used as a diagnostic assay to prevent the unintentional spread of variant B. caballi genotypes globally.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

Detection of Plasmodium knowlesi in whole blood samples with sandwich enzyme-linked immunosorbent assay (ELISA) using rhoptry-associated protein 1 specific polyclonal antibodies.

Plasmodium knowlesi, a simian malaria species, is now known to infect humans. Due to disadvantages in the current diagnosis methods, many efforts have been placed into developing new methods to diagnose the disease. This study assessed the ability of the PkRAP-1 sandwich enzyme-linked immunosorbent (ELISA) to detect P knowlesi antigens in whole blood specimens. Western blot assay was conducted to evaluate the ability of raised mouse and rabbit anti-PkRAP-1 polyclonal antibodies to bind to the native proteins in P. knowlesi lysate. The polyclonal antibodies were then used in sandwich ELISA to detect P. knowlesi. In the sandwich ELISA, mouse and rabbit polyclonal antibodies were used as the capture and detection antibodies, respectively. The limit of detection (LOD) of the assay was determined using P. knowlesi A1H1 culture and purified recombinant PkRAP-1. Western blot results showed positive reactions towards the proteins in P. knowlesi lysate. The LOD of the assay from three technical replicates was 0.068% parasitaemia. The assay performance in detecting P. knowlesi was 83% sensitivity and 70% specificity with positive and negative predictive values of 74% and 80%, respectively. The anti-PkRAP-1 polyclonal antibodies did not cross-react with P. falciparum and healthy samples, but P. vivax by detecting all 12 samples. PkRAP-1 has the potential as a biomarker for the development of a new diagnostic tool for P. knowlesi detection. Further studies need to be conducted to establish the full potential of the usage of anti-PkRAP-1 antibodies for P. knowlesi detection.

The effects and mechanisms of modified Xiaoyaosan on chronic unpredictable mild stress (CUMS)-induced depressive mice based on network pharmacology

Ethnopharmacological relevanceClinical research and basic scientific experiments have shown that modified Xiaoyaosan (MXYS) has antidepressant effects, whose system mechanism however has not been thoroughly characterized. Aim of the studyThis research was aimed at evaluating the treatment effects of MXYS on chronic unpredictable mild stress (CUMS)-induced depressive mice and exploring underlying mechanisms. Materials and methodsWhether MXYS has effects on depression was investigated via the depressive behaviors of mice, electron microscopy, real-time quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, immunofluorescence (IF) staining and the stereotaxic injection of adeno-associated viruses (AAVs). In addition, network pharmacology was applied to predict relevant molecular targets and possible mechanisms and perform further in vivo validation. ResultsMXYS is effective in ameliorating the depression-like symptoms of CUMS mice. It can stimulate autophagosome formation, activate the expression of microtubule-associated protein 1 light chain 3 (LC3B), autophagy-related gene 5 (Atg5), Atg7 and neuron-specific nuclear protein (NeuN), and decrease the protein expression sequestosome 1 (SQSTM1/p62). The autophagy-upregulating effect of MXYS was weakened by silencing. The network pharmacology analysis revealed that mitogen-activated protein kinase 1 (MAPK1), MAPK3, serine/threonine-protein kinase (AKT1), proto-oncogene tyrosine-protein kinase (SRC), PI 3 kinase p85 alpha (PIK3R1), catenin (cadherin-associated protein) beta 1 (CTNNB1) and human thrombin activator 1 (HRAS) may be of importance to treat depression by MXYS. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that metabolic and autophagy pathways, pathways in cancer and MAPK, phosphoinositide 3-kinase (PI3K)-Akt and rhoptry-associated protein 1 (Rap1) signaling pathways are involved in the antidepressant effects of MXYS. As suggested by Western blot, the anti-depression mechanism of MXYS is possibly associated with the extracellular signal-regulated protein kinase (ERK)/P38 MAPK signaling pathway. ConclusionThe findings indicate the possible antidepressant effects of MXYS on CUMS mice via triggering autophagy to alleviate neuronal apoptosis and prompting autophagy, which may involve the ERK/P38 MAPK signaling pathway.

Detection of Plasmodium knowlesi in whole blood samples with sandwich enzyme-linked immunosorbent assay (ELISA) using rhoptry-associated protein 1 specific polyclonal antibodies

ABSTRACT Background & objectives: Plasmodium knowlesi, a simian malaria species, is now known to infect humans. Due to disadvantages in the current diagnosis methods, many efforts have been placed into new method developments to diagnose the disease. This study assessed the ability of the PkRAP-1 sandwich enzyme-linked immunosorbent (ELISA) to detect P. knowlesi antigens in whole blood specimens. Methods: Western blot assay was conducted to evaluate the ability of raised mouse and rabbit anti-PkRAP-1 polyclonal antibodies to bind to the native proteins in P. knowlesi lysate. The polyclonal antibodies were then used in sandwich ELISA to detect P. knowlesi. In the sandwich ELISA, mouse and rabbit polyclonal antibodies were used as the capture and detection antibodies, respectively. The limit of detection (LOD) of the assay was determined using P. knowlesi A1H1 culture and purified recombinant PkRAP-1. Results: Western blot results showed positive reactions towards the proteins in P. knowlesi lysate. The LOD of the assay from three technical replicates was 0.068% parasitaemia. The assay performance in detecting P. knowlesi was 83% sensitivity and 70% specificity with positive and negative predictive values of 74% and 80%, respectively. The anti-PkRAP-1 polyclonal antibodies did not cross-react with Plasmodium falciparum and healthy samples, but Plasmodium vivax by detecting all 12 samples. Interpretation & conclusion: PkRAP-1 has the potential as a biomarker for the development of a new diagnostic tool for P. knowlesi detection. Further studies need to be conducted to establish the full potential of the usage of anti-PkRAP-1 antibodies for P. knowlesi detection.

Structural definition of babesial RAP-1 proteins identifies a novel protein superfamily across Apicomplexa

Apicomplexan protozoa are intracellular parasites of medical and economic importance. These parasites contain specialized apical complex organelles, including rhoptries, that participate in the process of host cell invasion. Conserved antigens expressed in the rhoptries are rational vaccine targets, but whether conservation of protein structure is a functional requirement for invasion remains unknown. Novel protein structural modeling enables identification of structurally conserved protein families that are not evident by sequence analysis alone. Here we show by AlphaFold2 structural modeling that the rhoptry-associated protein 1 superfamily of the Piroplasmida hemoparasites Babesia and Theileria (pRAP-1) is structurally conserved, with the core conserved region being composed of a globin-like and a 4-helix bundle subdomain. Search for structurally related members of this protein family in other apicomplexan parasites revealed structural homologues of pRAP-1 in several species of Plasmodium, Toxoplasma gondii and other members of the Sarcocystidae family. Based on these structural findings, pRAP-1 is a conserved apical complex protein, but whether these proteins share functional features in different species remains unknown. Identification of widely conserved elements involved in infection in these parasites will enhance our knowledge of invasion mechanisms, and facilitate the design of methods for controlling diseases that affect humans and animals globally.

Recent increase in low complexity polygenomic infections and sialic acid-independent invasion pathways in Plasmodium falciparum from Western Gambia

BackgroundThe malaria parasite Plasmodium falciparum utilizes multiple alternative receptor-ligand interactions for the invasion of human erythrocytes. While some P. falciparum clones make use of sialic acid (SA) residues on the surface of the human glycophorin receptors to invade the erythrocyte, others use alternative receptors independent of sialic acid residues. We hypothesized that over the years, intensified malaria control interventions and declining prevalence in The Gambia have resulted in a selection of parasites with a dominant invasion pathways and ligand expression profiles.MethodsBlood samples were collected from 65 malaria-infected participants with uncomplicated malaria across 3 years (2015, 2016, and 2021). Genetic diversity was determined by genotyping the merozoite surface protein 2 (msp2) polymorphic gene of P. falciparum. Erythrocyte invasion phenotypes were determined using neuraminidase, trypsin, and chymotrypsin enzymes, known to cleave different receptors from the surface of the erythrocyte. Schizont-stage transcript levels were obtained for a panel of 6 P. falciparum invasion ligand genes (eba175, eba181, Rh2b, Rh4, Rh5, and clag2) using 48 successfully cultured isolates.ResultsThough the allelic heterozygosity of msp2 repeat region decreased as expected with reduced transmission, there was an increase in infections with more than a single msp2 allelotype from 2015 to 2021. The invasion phenotypes of these isolates were mostly SA independent with a continuous increase from 2015 to 2021. Isolates from 2021 were highly inhibited by chymotrypsin treatment compared to isolates from 2015 and 2016. Higher invasion inhibition for 2021 isolates was further obtained following erythrocyte treatment with a combination of chymotrypsin and trypsin. The transcript levels of invasion ligand genes varied across years. However, levels of clag2, a rhoptry-associated protein, were higher in 2015 and 2016 isolates than in 2021 isolates, while Rh5 levels were higher in 2021 compared to other years.ConclusionsOverall, these findings suggest increasing mixed infections with an increase in the use of sialic-acid independent invasion pathways by P. falciparum clinical isolates in the Western part of Gambia.Graphical

Molecular characterization and genetic diversity of Babesia bovis and Babesia bigemina of cattle in Thailand.

Babesia bovis and B. bigemina are the most common tick-borne parasites that cause bovine babesiosis which effects livestock production, leading to economic losses in tropical and subtropical areas of the world. The aims of this study were to determine the molecular detection, genetic diversity and antigenicity prediction of B. bovis based on spherical body protein 2 (sbp-2) gene and B. bigemina based on rhoptry-associated protein 1a (rap-1a) gene in cattle in Thailand. By PCR assay, the molecular detection of B. bovis and B. bigemina infection revealed levels of 2.58% (4/155) and 5.80% (9/155), respectively. The phylograms showed that B. bovis sbp-2 and B. bigemina rap-1a sequences displayed 5 and 3 clades with similarity ranging between 85.53 to 100% and 98.28 to 100%, respectively, when compared within Thailand strain. Diversity analysis of sbp-2 and rap-1a sequences showed 18 and 4 haplotypes, respectively. The entropy analysis illustrated 104 and 7 polymorphic sites of sbp-2 and rap-1a nucleic acid sequences, respectively, while those of sbp-2 and rap-1a amino acid sequences showed 46 and 4 high entropy peaks, respectively. Motifs analysis exhibited the distribution and conservation among sbp-2 and rap-1a sequences. The continuous and discontinuous B-cell epitopes have also been evaluated in this work. Therefore, our findings may be used to ameliorate the understanding inputs of molecular phylogeny, genetic diversity and antigenicity of B. bovis and B. bigemina Thailand stains.

Molecular detection of Babesia and Theileria from crossbred cattle in Sirajganj and Rangpur districts of Bangladesh.

Babesia and Theileria are potential threats to the livestock industry, causing considerable economic losses. These tick-borne blood parasites are more prevalent in crossbred cattle than local cattle in Bangladesh. To confirm the species of Babesia and Theileria in crossbred cattle from the northern part of Bangladesh using conventional and molecular tools. A total of 385 crossbred cattle blood samples were subjected to DNA extraction and PCR. For molecular detection, B. bigemina rhoptry-associated protein 1a, B. bovis spherical body protein-4, and Theileria spp. 18S rRNA were used as the marker genes. Using PCR, only 72 (18.7%) samples were found piroplasm positive, of which 12.2% Theileria, 4.7% Babesia, and 1.8% mixed infections. Both Babesia (7.3%), Theileria (7.7%) and mixed (2.8%) infections were detected in Sirajganj, and only Theileria (20.4%) was detected in Rangpur district. By PCR and nPCR we detected B. bigemina and T. annulata in Sirajganj district, and Theileria sp. in Rangpur district. The target gene sequences of isolated pathogens confirmed B. bigemina and T. annulata, and Theileria sp from these samples. Blood smears of all samples were also examined microscopically for Babesia and/or Theileria spp. and 14.3% of samples were found positive, of which 5.9% Babesia and 8.3% Theileria. Generally, the pathogens detected in Sirajgang and Rangpur were genetically related to South Asia, particularly South East Asian isolates. These findings provide information for a better understanding of the epidemiology of Babesia and Theileria as well as to improve the approaches for diagnosis and control of tick-borne diseases in Bangladesh.

Molecular Identification of Babesia spp. and Anaplasma marginale in Water Buffaloes in Veracruz and Tabasco, Mexico: A Retrospective Study.

Two hundred and thirty-three blood samples of water buffalo were collected on four farms in Veracruz state and Tabasco state, Mexico, to detect and confirm the identities of Babesia and Anaplasma spp. sequences. Nested PCR assays were used for the amplification of specific genes encoding B. bovis rhoptry-associated protein (RAP-1), B. bigemina SpeI-AvaI restriction fragment, and Anaplasma marginale major surface protein 5 (MSP5). Using DNA sequencing and BLASTn analysis for DNA homology hemoparasite identification, the identities of the hemoparasites were established by comparing the nucleotide sequences obtained in this study with those available in the GenBank database at the National Center for Biotechnology Information (NCBI). Water buffalo infection with at least one of the hemoparasites under study was detected in 45% (105/233) of the blood samples, while a mixed infection with B. bovis and B. bigemina was detected in 6.4% (15/233) of samples. For this cross-sectional study, mixed infections with the three hemoparasites were not detected. BLASTn analysis revealed that the nucleotide sequences of the water buffalo isolates shared sequence identity values ranging from 88 to 100% with previously published gene sequences of B. bovis, B. bigemina, and A. marginale. The current results confirm that water buffalo, as cattle, are also carriers of hemoparasite infections that are tick-transmitted, and suggest that they probably have an important role in the epidemiology of bovine babesiosis in Mexico.

Integrating network pharmacology, transcriptomics, and molecular simulation to reveal the mechanism of tert-butylhydroquinone for treating diabetic retinopathy

Diabetic retinopathy (DR), a common microvascular complication of diabetes mellitus, is a significant cause of blindness. As one of the crucial factors in the pathogenesis of DR, oxidative stress provides new insights for the treatment of DR. Tert-butylhydroquinone (TBHQ), an efficient phenolic antioxidant, has been proved to inhibit diabetic retina injury. However, the mechanism of TBHQ for DR treatment is still unclear. The present study was designed to investigate the potential mechanism of TBHQ for treating DR. Firstly, the potential targets of TBHQ and DR were selected to construct protein-protein interaction network, which was applied to illustrate the potential mechanism of TBHQ against DR. Combined with transcriptomics and molecular simulation, the potential mechanisms were systematically verified. The results showed that TBHQ inhibited retinal microvascular injury by regulating oxidative stress, inflammation, cell proliferation-death regulation, and vascular system development. The mechanisms of these activities were associated with hypoxia-inducible factor-1 (HIF-1), nuclear factor-erythroid 2 related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), forkhead box O (FoxO), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), and rhoptry-associated protein1 (Rap1) signaling pathways and their related targets nitric oxide synthase 3 (NOS3), mitogen-activated protein kinase 8 (MAPK8), prostaglandin-endoperoxide synthase 2 (PTGS2), and heme oxygenase 1 (HMOX1). In conclusion, a systematic perspective for the mechanism of TBHQ against DR was revealed by present study which lays a foundation for the application of TBHQ in treating DR.

Reduning Attenuates LPS-Induced Human Unmilical Vein Endothelial Cells (HUVECs) Apoptosis Through PI3K-AKT Signaling Pathway.

The molecular mechanism of Reduning (RDN) in the treatment of sepsis was analyzed based on network pharmacology. The system pharmacology method was administered to search the active ingredients and targets of RDN, identify the sepsis-related genes, and determine the targets of RDN in the treatment of sepsis. Cytoscape was used to build a “drug component-target” network to screen key compounds. A protein-protein interaction (PPI) network was constructed using STRING, and core targets were revealed through topological analysis. 404 shared targets of RDN and sepsis were introduced into DAVID Bioinformatics Resources 6.8 for GO and KEGG enrichment analysis to predict their possible signaling pathways and explore their molecular mechanisms. GO enrichment analysis highlighted that they were largely related to protein phosphorylation, inflammatory reaction, and positive regulation of mitogen-activated protein kinase (MAPK) cascade. KEGG enrichment analysis outlined that they were enriched in PI3K-AKT signaling pathway, calcium signaling pathway, rhoptry-associated protein 1 (Rap1) signaling pathway, and advanced glycation end products and receptors for advanced glycation end products (AGE-RAGE) signaling pathway. Molecular biological validation results exposed that RDN could significantly improve the protein expression of p-AKT and p-PI3K, alleviate apoptosis-related proteins expression level and decrease apoptosis rate in LPS-induced HUVECs. In conclusion, it was illustrated that RDN could considerably constrain LPS-induced apoptosis by activating the PI3K-AKT signaling pathway, which advocated a basis for fundamental mechanism research and clinical application of RDN in the treatment of sepsis.

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.

Molecular investigation of Babesia caballi in horses from the state of Rio de Janeiro, Brazil: Epidemiological aspects associated with the infection.

The epidemiological aspects of Babesia caballi infection were evaluated in 516 horse samples from Rio de Janeiro, Brazil. The presence and infestation level of ticks on horses, breed conditions, and animal management were evaluated on each farm through an epidemiological questionnaire. The gene that codes for rhoptry-associated protein-1 (RAP-1) of B. caballi was amplified by nested PCR (nPCR). Among the horses sampled, 17.2% (n=89/516) presented B. caballi DNA. The characterized samples showed 99-100% similarity with other isolates of B. caballi based on the RAP-1 gene, available in GenBank. In the final logistic regression model, the variables associated with B. caballi infection in horses were as follows: age below two years (OR=3.33; IC=1.7-6.5), farms located in low altitudes (OR=3.52; IC=1.7-7.3) and Dermacentor nitens infestation (OR=1.91; IC=1.1-3.4). Furthermore, a high level of D. nitens infestation in horses was also a factor associated with positivity for B. caballi (OR=2.11; IC=1.25-3.54). In summary, young horses bred in low altitude regions characterized with high temperatures, and infested by D. nitens, mainly with a higher level of infestation, are more likely to be infected by B. caballi. This epidemiological study provides statical evidence that the D. nitens tick play a role as the biological vector of B. caballi in the studied region.

Protection and Alleviated Inflammation Induced by Virus-like Particle Vaccines Containing Plasmodium berghei MSP-8, MSP-9 and RAP1.

Virus-like particles (VLP) are a highly efficient vaccine platform used to present multiple antigenic proteins. Merozoite surface protein 8 (MSP-8), 9 (MSP-9) and rhoptry-associated protein 1 (RAP1) of Plasmodium berghei are the important proteins in erythrocyte invasion and the replication of parasites. In this study, we generated three VLPs expressing MSP-8, MSP-9 or RAP1 together with influenza virus matrix protein M1 as a core protein, and the protection and alleviated inflammation induced by VLP immunization were investigated. Mice were immunized with a mixture of three VLPs, MSP-8, MSP-9 and RAP1, and challenge-infected with P. berghei. As a result, VLPs immunization elicited higher levels of P. berghei or VLPs-specific IgG antibody responses in the sera upon boost compared to that upon prime and naive. Upon challenge infection with P. berghei, higher levels of CD4+ T cell and memory B cell responses in the spleen were also found in VLPs-immunized mice compared to non-immunized control. Importantly, VLP immunization significantly alleviated inflammatory cytokine responses (TNF-α, IFN-γ) both in the sera and spleen. VLP vaccine immunization also assisted in diminishing the parasitic burden in the peripheral blood and prolonged the survival of immunized mice. These results indicated that a VLPs vaccine containing MSP-8, MSP-9 and RAP1 could be a vaccine candidate for P. berghei infection.

Babesia microti Immunoreactive Rhoptry-Associated Protein-1 Paralogs Are Ancestral Members of the Piroplasmid-Confined RAP-1 Family.

Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites of the order Piroplasmida, responsible for diseases in humans and animals. Members of the piroplasmid rhoptry-associated protein-1 (pRAP-1) family have a signature cysteine-rich domain and are important for parasite development. We propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode two paralogue pRAP-1-like proteins named BmIPA48 and Bm960. The two genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical ~225 bp sequence located in their 5′ putative regulatory regions. BmIPA48 and Bm960 proteins contain a N-terminal signal peptide, share very low sequence identity (<13%) with pRAP-1 from other species, and harbor one or more transmembrane domains. Diversification of the piroplasmid-confined prap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of pRAP-1 at the parasite-host interface, possibly in parasite adhesion, attachment, and/or evasion of the host immune defenses. Both BmIPA48 and Bm960 are recognized by antibodies in sera from humans infected with B. microti and might be promising candidates for developing novel serodiagnosis and vaccines.

Stable transfection system for Babesia sp. Xinjiang

BackgroundStable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis and mainly prevails across China, the platform of those techniques remains absent. Genetic manipulation techniques are powerful tools to enhance our knowledge on parasite biology, which may provide potential drug targets and diagnostic markers.MethodsWe evaluated the inhibition efficiency of blasticidin (BSD) and WR99210 to Bxj. Then, a plasmid was constructed bearing selectable marker BSD, green fluorescent protein (GFP) gene, and rhoptry-associated protein-1 3′ terminator region (rap 3′ TR). The plasmid was integrated into the elongation factor-1 alpha (ef-1α) site of Bxj genome by cross-over homologous recombination technique. Twenty μg of plasmid was transfected into Bxj merozoites. Subsequently, drug selection was performed 24 h after transfection to generate transfected parasites.ResultsTransfected parasite lines, Bxj-c1, Bxj-c2, and Bxj-c3, were successfully obtained after transfection, drug selection, and colonization. Exogenous genes were integrated into the Bxj genome, which were confirmed by PCR amplification and sequencing. In addition, results of western blot (WB) and indirect immunofluorescence assay (IFA) revealed that GFP-BSD had expressed for 11 months.ConclusionsIn our present study, stable transfection system for Bxj was successfully developed. We anticipate that this platform will greatly facilitate basic research of Bxj.Graphical abstract

Molecular detection and identification of Babesia bovis and Trypanosoma spp. in one-humped camel (Camelus dromedarius) breeds in Egypt.

Background and Aim:Camels are a unique source of milk and meat, which helps recover from several diseases that affect humans worldwide. In Egypt, one of the great obstacles for this industry is tick-borne diseases. This study aimed to characterize blood parasite infections, such as Babesia (B.) bovis and Trypanosoma (T.) spp. in one-humped camel (Camelus dromedarius) (n=142) breeds in Halayeb and Shalateen, Egypt, through phylogenetic analysis.Materials and Methods:The prevalence of B. bovis and Trypanosoma spp. was identified in camels using polymerase chain reaction (PCR) assays targeting the Rhoptry-Associated Protein-1 and internal transcribed spacer 1 genes, respectively. A nested PCR technique was conducted to detect B. bovis. At the same time, KIN multispecies PCR assay was employed to diagnose and classify trypanosome DNA in camels.Results:B. bovis was detected in 4/142 camels with an infection rate of 2.81%. Sequencing and phylogenetic analyses revealed that the strain of B. bovis isolated from this population was closely related to strains isolated from Argentine, the United States, and Brazil. Moreover, Trypanosoma evansi was detected in 8/142 camels with an infection rate of 5.63%. Sequencing and phylogenetic analyses revealed that this isolated strain T. evansi was closely related to Trypanosoma theileri detected from cattle in Brazil.Conclusion:The obtained data indicated the existence of B. bovis and T. evansi in camels from two provinces of Egypt. The obtained findings have economic significance and reflect the importance of implementing effective prevention and control methods across Egypt to reduce the incidence of B. bovis and T. evansi in camels.

Assessing the Immunochromatographic Test Strip for Serological Detection of Bovine Babesiosis in Uganda.

In Uganda, bovine babesiosis continues to cause losses to the livestock industry because of shortages of cheap, quick, and reliable diagnostic tools to guide prescription measures. In this study, the presence of antibodies to Babesia bigemina and Babesia bovis in 401 bovine blood samples obtained from eastern and central areas of Uganda were detected using enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic test strips (ICTs). The ELISA and ICT test used targeted the B. bigemina C-terminal rhoptry-associated protein (RAP-1/CT17) and B. bovis spherical body protein-4 (SPB-4). Using ELISA, single-ICT and dual-ICT, positive samples for B. bovis were detected in 25 (6.2%), 17 (4.3%), and 14 (3.7%) samples respectively, and positive samples for B. bigemina were detected in 34 (8.4%), 27 (6.7%), and 25 (6.2%), respectively. Additionally, a total of 13 animals (3.2%) had a mixed infection. The correlation between ELISA and single-ICT strips results revealed slight agreement with kappa values ranging from 0.088 to 0.191 between both methods, while the comparison between dual-ICT and single-ICT results showed very good agreement with kappa values >0.80. This study documented the seroprevalence of bovine babesiosis in central and eastern Uganda, and showed that ICT could, after further optimization, be a useful rapid diagnostic test for the diagnosis of bovine babesiosis in field settings.