Bovis Merozoites Research Articles

Nilgai antelope display no signs of infection upon experimental challenge with a virulent Babesia bovis strain

BackgroundBovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection.MethodsNilgai were needle inoculated with ≈108B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite.ResultsNilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer.ConclusionsNilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances.Graphical

Genetic diversity in Babesia bovis from southern Africa and estimation of B. bovis infection levels in cattle using an optimised quantitative PCR assay

Babesia bovis is a causal agent of bovine babesiosis, a disease which leads to mortality and morbidity and impacts the cattle industry worldwide. We amplified, cloned and sequenced the B. bovis merozoite surface antigen-2b (msa-2b) gene (∼940 bp) and the near full-length 18S rRNA gene (∼1600 bp) from cattle samples from South Africa and Mozambique to determine sequence variation between B. bovis parasites in the region. A TaqMan quantitative real-time PCR (qPCR) assay (18S rRNA gene) was optimised for the detection of B. bovis and estimation of parasitaemia in field samples from cattle from southern Africa. Phylogenetic analysis grouped the Msa-2b sequences in six clades and these were 59.7 to 99.6% identical to reference sequences. Sequence variation amongst B. bovis 18S rRNA sequences was found at 2 to 36 positions, and the sequences were 97 to 99% identical to published sequences. Mismatches between the B. bovis 18S rRNA sequences and a previously published qPCR forward primer (BoF) were observed; therefore, we developed a new forward primer (BoF2), and optimised the qPCR assay. Six 10-fold dilution series of B. bovis infected erythrocytes (2 × 108 to 2 × 103 infected red blood cells [iRBC]/ml) were analysed in triplicate in each of six separate qPCR runs, to determine the efficiency of the assay. The qPCR assay amplified the B. bovis 18S rRNA gene with 92.0 to 94.9% efficiency. The detection limit of the qPCR assay was approximately 6 iRBCs/μl. The performance of the optimised assay to diagnose B. bovis in field samples was assessed by testing DNA from 222 field samples of cattle from South Africa and Mozambique using three methods: the optimised qPCR assay, the reverse line blot (RLB) hybridisation assay, and the previously published qPCR assay. The detection rate of B. bovis using the optimised qPCR assay (31.1%, 69/222) was significantly higher (p<0.001) than both that using RLB (20.7%, 46/222) and the previously published qPCR assay (5.4%; 12/222). The B. bovis parasitaemia in samples from infected cattle ranged from 6 iRBCs/μl to 101,852 iRBCs/μl of blood. Our study revealed marked sequence variation between B. bovis parasites from southern Africa. The optimised qPCR assay will be useful in epidemiological studies and clinical diagnosis of B. bovis in southern Africa, and can be used to determine parasitaemia and potential carrier status in cattle populations, which is essential in the control of babesiosis.

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.

Isolation of viable Babesia bovis merozoites to study parasite invasion

Babesia parasite invades exclusively red blood cell (RBC) in mammalian host and induces alterations to host cell for survival. Despite the importance of Babesia in livestock industry and emerging cases in humans, their basic biology is hampered by lack of suitable biological tools. In this study, we aimed to develop a synchronization method for Babesia bovis which causes the most pathogenic form of bovine babesiosis. Initially, we used compound 2 (C2), a specific inhibitor of cyclic GMP-dependent protein kinase (PKG), and a derivative of C2, ML10. While both inhibitors were able to prevent B. bovis egress from RBC and increased percentage of binary forms, removal of inhibitors from culture did not result in a synchronized egress of parasites. Because using PKG inhibitors alone was not efficient to induce a synchronized culture, we isolated viable and invasive B. bovis merozoites and showed dynamics of merozoite invasion and development in RBCs. Using isolated merozoites we showed that BbVEAP, VESA1-export associated protein, is essential for parasite development in the RBC while has no significant role in invasion. Given the importance of invasion for the establishment of infection, this study paves the way for finding novel antigens to be used in control strategies against bovine babesiosis.

Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development.

BackgroundBovine babesiosis caused by Babesia bovis (B. bovis) has had a significant effect on the mobility and mortality rates of the cattle industry worldwide. Live-attenuated vaccines are currently being used in many endemic countries, but their wide use has been limited for a number of reasons. Although recombinant vaccines have been proposed as an alternative to live vaccines, such vaccines are not commercially available to date. Apical membrane antigen-1 (AMA-1) is one of the leading candidates in the development of a vaccine against diseases caused by apicomplexan parasite species. In Plasmodium falciparum (P. falciparum) AMA-1 (PfAMA-1), several antibodies against epitopes in the plasminogen, apple, and nematode (PAN) motif of PfAMA-1 domain I significantly inhibited parasite growth. Therefore, the purpose of this study was to predict an epitope from the PAN motif of domain I in the B. bovis AMA-1 (BbAMA-1) using a combination of linear and conformational B-cell epitope prediction software. The selected epitope was then bioinformatically analyzed, synthesized as a peptide (sBbAMA-1), and then used to immunize a rabbit. Subsequently, in vitro growth- and the invasion-inhibitory effects of the rabbit antiserum were immunologically characterized.ResultsOur results demonstrated that the predicted BbAMA-1 epitope was located on the surface-exposed α-helix of the PAN motif in domain I at the apex area between residues 181 and 230 with six polymorphic sites. Subsequently, sBbAMA-1 elicited antibodies capable of recognizing the native BbAMA-1 in immunoassays. Furthermore, anti-serum against sBbAMA-1 was immunologically evaluated for its growth- and invasion-inhibitory effects on B. bovis merozoites in vitro. Our results demonstrated that the rabbit anti-sBbAMA-1 serum at a dilution of 1:5 significantly inhibited (p < 0.05) the growth of B. bovis merozoites by approximately 50–70% on days 3 and 4 of cultivation, along with the invasion of merozoites by approximately 60% within 4 h of incubation when compared to the control groups.ConclusionOur results indicate that the epitope predicted from the PAN motif of BbAMA-1 domain I is neutralization-sensitive and may serve as a target antigen for vaccine development against bovine babesiosis caused by B. bovis.

N-Glycosylation in Piroplasmids: Diversity within Simplicity.

N-glycosylation has remained mostly unexplored in Piroplasmida, an order of tick-transmitted pathogens of veterinary and medical relevance. Analysis of 11 piroplasmid genomes revealed three distinct scenarios regarding N-glycosylation: Babesia sensu stricto (s.s.) species add one or two N-acetylglucosamine (NAcGlc) molecules to proteins; Theileria equi and Cytauxzoon felis add (NAcGlc)2-mannose, while B. microti and Theileria s.s. synthesize dolichol-P-P-NAcGlc and dolichol-P-P-(NAcGlc)2 without subsequent transfer to proteins. All piroplasmids possess the gene complement needed for the synthesis of the N-glycosylation substrates, dolichol-P and sugar nucleotides. The oligosaccharyl transferase of Babesia species, T. equi and C. felis, is predicted to be composed of only two subunits, STT3 and Ost1. Occurrence of short N-glycans in B. bovis merozoites was experimentally demonstrated by fluorescence microscopy using a NAcGlc-specific lectin. In vitro growth of B. bovis was significantly impaired by tunicamycin, an inhibitor of N-glycosylation, indicating a relevant role for N-glycosylation in this pathogen. Finally, genes coding for N-glycosylation enzymes and substrate biosynthesis are transcribed in B. bovis blood and tick stages, suggesting that this pathway is biologically relevant throughout the parasite life cycle. Elucidation of the role/s exerted by N-glycans will increase our understanding of these successful parasites, for which improved control measures are needed.

Characterization of GASA-1, a new vaccine candidate antigen of Babesia bovis

Surface proteins bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors are considered essential for the survival of pathogenic protozoans. In the case of the tick-transmitted hemoparasite Babesia bovis, the most virulent causative agent of bovine babesiosis, the GPI-anchored proteome was recently unraveled by an in silico approach. In this work, one of the identified proteins, GASA-1 (GPI-Anchored Surface Antigen-1), was thoroughly characterized. GASA-1 is 179 aa long and has the characteristic features of a GPI-anchored protein, including a signal peptide, a hydrophilic core and a hydrophobic tail that harbors a GPI anchor signal. Transcriptomic analysis shows that it is expressed in pathogenic and attenuated B. bovis strains. Notably, the gasa-1 gene has syntenic counterparts in B. bigemina and B. ovata, which also encode GPI-anchored proteins. This is highly unusual since all piroplasmid GPI-anchored proteins described so far have been found to be species-specific. Sequencing of gasa-1 alleles from B. bovis geographical isolates originating from Argentina, USA, Brazil, Mexico and Australia showed over 98 % identity in both nucleotide and amino acid sequences. A recombinant form of GASA-1 (rGASA-1) was generated in E. coli and anti-rGASA-1 antibodies were raised in mice. Fixed and live immunofluorescence assays showed that GASA-1 is expressed in in vitro cultured B. bovis merozoites and surface-exposed. Moreover, incubation of B. bovis in vitro cultures with anti-GASA-1 antibodies partially, but significantly, reduced erythrocyte invasion, indicating that this protein bears neutralization-sensitive antibody epitopes. Splenocytes of rGASA-1-inoculated mice showed a specific proliferative response when exposed to the recombinant protein, indicating that GASA-1 bears T-cell epitopes. Finally, sera from a group of B. bovis-infected cattle reacted with the recombinant protein, demonstrating that GASA-1 is expressed during natural infection of bovines with B. bovis, and suggesting that it is immunodominant. The high degree of conservation among B. bovis isolates and the presence of syntenic genes in other Babesia species suggest a relevant role of GASA-1 and GASA-1-like proteins for parasite survival, especially considering that, due to their surface location, they are exposed to the selection pressure of the host immune system. The highlighted features of GASA-1 make it an interesting candidate for the development of vaccines against bovine babesiosis.

Genetic Diversity of Babesia bovis MSA-1, MSA-2b and MSA-2c in China.

The apicomplexan parasite Babesia bovis is a tick-borne intracellular hemoprotozoan parasite that is widespread across China. Genetic diversity is an important strategy used by parasites to escape the immune responses of their hosts. In our present study, 575 blood samples, collected from cattle in 10 provinces, were initially screened using a nested PCR (polymerase chain reaction) for detection of B. bovis infection. To perform genetic diversity analyses, positive samples were further amplified to obtain sequences of three B. bovis merozoite surface antigen genes (MSA-1, MSA-2b, MSA-2c). The results of the nested PCR approach showed that an average of 8.9% (51/575) of cattle were positive for B. bovis infection. Phylogenetic analyses of the predicted amino acid sequences revealed that unique antigen variants were formed only by Chinese isolates. Our findings provide vital information for understanding the genetic diversity of B. bovis in China.

The application of the HyPer fluorescent sensor in the real-time detection of H2O2 in Babesia bovis merozoites in vitro

In recent years, genetically encoded fluorescent probes have allowed a dramatic advancement in time-lapse imaging, enabling this imaging modality to be used to investigate intracellular events in several apicomplexan parasite species. In this study, we constructed a plasmid vector to stably express a genetically encoded H2O2 sensor probe called HyPer in Babesia bovis. The HyPer-transfected parasite population was successfully developed and subjected to a time-lapse imaging analysis under in vitro culture conditions. HyPer was capable of sensing an increasing H2O2 concentration in the parasite cells which was induced by the administration of paraquat as a superoxide donor. HyPer fluorescence co-staining with MitoTracker Red indicated the mitochondria as the major source of reactive oxygen species (ROS) in parasite cells. The fluctuating ROS dynamics in the parasite gliding toward, attaching to, and invading the target red blood cell was visualized and monitored in real time with the HyPer expressing parasite population. This is the first report to describe the application of the HyPer probe in an imaging analysis involving Babesia parasites. Hyper-expressing parasites can be widely utilized in studies to investigate the mechanisms of emergence and the reduction of oxidative stress, as well as the signal transduction in the parasite cells during host invasion and intercellular development.

Babesia bovis BOV57, a Theileria parva P67 homolog, is an invasion-related, neutralization-sensitive antigen

Babesia bovis BOV57, which is a homolog of the Theileria parva vaccine candidate antigen P67, is expressed in both the tick and blood stages of the life cycle of this parasite. However, the vaccine potential of BOV57 remained to be investigated. In the present study, we generated recombinant BOV57 (rBOV57) and prepared polyclonal antibodies against rBOV57 in mice and rabbits. Indirect immunofluorescence assays conducted with the mouse anti-rBOV57 antibody demonstrated that BOV57 localized at the apical end of paired merozoites in infected bovine red blood cells, whereas the antigen was found in the parasite membrane around the apical end of intraerythrocytic single and extracellular merozoites. In an invasion-inhibition assay, the rabbit anti-rBOV57 antibody potentially inhibited RBC invasion of B. bovis merozoites in vitro. In addition, the invasion inhibition mediated by rabbit anti-rBOV57 antibody resulted in a reduced growth rate of B. bovis in the in vitro culture. These findings indicated that B. bovis BOV57 plays a critical role in the invasion of merozoites into red blood cells, suggesting its potential as a subunit vaccine candidate against B. bovis infection in cattle. Furthermore, we analyzed the genetic diversity of bov57 gene sequences isolated from Sri Lanka, Mongolia, the Philippines, and Vietnam. The bov57 gene sequences derived from Mongolia, the Philippines, and Vietnam were conserved, whereas insertion and/or deletion mutations resulted in sequence diversity among the Sri Lankan samples. In summary, BOV57 is an invasion-related, neutralization-sensitive antigen encoded by the bov57 gene, which displays higher sequence diversity than previously reported.

A recombinant multi-antigen vaccine formulation containing Babesia bovis merozoite surface antigens MSA-2a1, MSA-2b and MSA-2c elicits invasion-inhibitory antibodies and IFN-γ producing cells.

Background Babesia bovis is a tick-transmitted protozoan hemoparasite and the causative agent of bovine babesiosis, a potential risk to more than 500 million cattle worldwide. The vaccines currently available are based on attenuated parasites, which are difficult to produce, and are only recommended for use in bovines under one year of age. When used in older animals, these vaccines may cause life-threatening clinical symptoms and eventually death. The development of a multi-subunit recombinant vaccine against B. bovis would be attractive from an economic standpoint and, most importantly, could be recommended for animals of any age. In the present study, recombinant ectodomains of MSA-2a1, MSA-2b and MSA-2c antigens were expressed in Pichia pastoris yeast as secreted soluble peptides.ResultsThe antigens were purified to homogeneity, and biochemically and immunologically characterized. A vaccine formulation was obtained by emulsifying a mixture of the three peptides with the adjuvant Montanide ISA 720, which elicited high IgG antibody titers against each of the above antigens. IgG antibodies generated against each MSA-antigen recognized merozoites and significantly inhibited the invasion of bovine erythrocytes. Cellular immune responses were also detected, which were characterized by splenic and lymph node CD4+ T cells producing IFN-γ and TNF-α upon stimulation with the antigens MSA-2a1 or MSA-2c.ConclusionsThese data strongly suggest the high protective potential of the presented formulation, and we propose that it could be tested in vaccination trials of bovines challenged with B. bovis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1862-1) contains supplementary material, which is available to authorized users.

Identification and functional analysis of a novel mitochondria-localized 2-Cys peroxiredoxin, BbTPx-2, from Babesia bovis.

Cysteine-based peroxidases, known as peroxiredoxins (Prx) or thioredoxin peroxidases (TPx), are important antioxidant enzymes that prevent oxidative damage caused by reactive oxygen species (ROS). In this study, we identified a novel mitochondrial 2-Cys Prx, BbTPx-2, from a bovine Babesia parasite, B. bovis. BbTPx-2 complementary DNA (cDNA) encodes a polypeptide of 254 amino acid residues. This protein has a mitochondrial targeting peptide at the N-terminus and two conserved cysteine residues of the typical 2-Cys Prx. By using a thiol mixed-function oxidation assay, the antioxidant activity of recombinant BbTPx-2 was revealed, and its antioxidant activity was comparable to that of a cytosolic 2-Cys Prx from B. bovis, BbTPx-1. Notably, we confirmed that BbTPx-2 was expressed in the mitochondrion of B. bovis merozoites. Taken together, the results suggest that the mitochondrial BbTPx-2 is an antioxidative enzyme for scavenging ROS in B. bovis.

Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand

Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5–100%, and the similarity values among the translated amino acid sequences were 42.8–100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9–68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0–39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand.

RBC invasion and invasion-inhibition assays using free merozoites isolated after cold treatment of Babesia bovis in vitro culture

Babesia bovis is an apicomplexan hemoprotozoan that can invade bovine red blood cells (RBCs), where it multiplies asexually. RBC invasion assays using free viable merozoites are now routinely used to understand the invasion mechanism of B. bovis, and to evaluate the efficacy of chemicals and antibodies that potentially inhibit RBC invasion by the parasite. The application of high-voltage pulses (high-voltage electroporation), a commonly used method to isolate free merozoites from infected RBCs, reduces the viability of the merozoites. Recently, a cold treatment of B. bovis in vitro culture was found to induce an effective release of merozoites from the infected RBCs. In the present study, we incubated in vitro cultures of B. bovis in an ice bath to liberate merozoites from infected RBCs and then evaluated the isolated merozoites in RBC invasion and invasion-inhibitions assays. The viability of the purified merozoites (72.4%) was significantly higher than that of merozoites isolated with high-voltage electroporation (48.5%). The viable merozoites prepared with the cold treatment also invaded uninfected bovine RBCs at a higher rate (0.572%) than did merozoites prepared with high-voltage electroporation (0.251%). The invasion-blocking capacities of heparin, a polyclonal rabbit antibody directed against recombinant B. bovis rhoptry associated protein 1, and B. bovis-infected bovine serum were successfully demonstrated in an RBC invasion assay with the live merozoites prepared with the cold treatment, suggesting that the targets of these inhibitors were intact in the merozoites. These findings indicate that the cold treatment technique is a useful tool for the isolation of free, viable, invasion-competent B. bovis merozoites, which can be effectively used for RBC invasion and invasion-inhibition assays in Babesia research.

Molecular and biochemical characterization of methionine aminopeptidase of Babesia bovis as a potent drug target

Aminopeptidases are increasingly being investigated as therapeutic targets in various diseases. In this study, we cloned, expressed, and biochemically characterized a member of the methionine aminopeptidase (MAP) family from Babesia bovis (B. bovis) to develop a potential molecular drug target. Recombinant B. bovis MAP (rBvMAP) was expressed in Escherichia coli (E. coli) as a glutathione S-transferase (GST)-fusion protein, and we found that it was antigenic. An antiserum against the rBvMAP protein was generated in mice, and then a native B. bovis MAP was identified in B. bovis by Western blot assay. Further, an immunolocalization assay showed that MAP is present in the cytoplasm of the B. bovis merozoite. Analysis of the biochemical properties of rBvMAP revealed that it was enzymatically active, with optimum activity at pH 7.5. Enhanced enzymatic activity was observed in the presence of divalent manganese cations and was effectively inhibited by a metal chelator, ethylenediaminetetraacetic acid (EDTA). Moreover, the enzymatic activity of BvMAP was inhibited by amastatin and bestatin as inhibitors of MAP (MAPi) in a dose-dependent manner. Importantly, MAPi was also found to significantly inhibit the growth of Babesia parasites both in vitro and in vivo; additionally, they induced high levels of cytokines and immunoglobulin (IgG) titers in the host. Therefore, our results suggest that BvMAP is a molecular target of amastatin and bestatin, and those inhibitors may be drug candidates for the treatment of babesiosis, though more studies are required to confirm this.

Type-specific PCR assays for Babesia bovis msa-1 genotypes in Asia: Revisiting the genetic diversity in Sri Lanka, Mongolia, and Vietnam

Babesia bovis is the most virulent Babesia organism, resulting in a high mortality rate in cattle. The genetic diversity of B. bovis merozoite surface antigens (MSAs), such as MSA-1, MSA-2b, and MSA-2c, might be linked to altered immune profiles in the host animals. The present study aimed to develop type-specific PCR assays for Asian msa-1 genotypes, thereby re-analyzing the genetic diversity of msa-1 in Sri Lanka, Mongolia, and Vietnam. Specific primers were designed for nine Asian msa-1 genotypes, which had been detected based on the phylogeny constructed using msa-1 gene sequences retrieved from the GenBank database. Specificity of the type-specific PCR assays was confirmed using plasmids containing the inserts of msa-1 gene fragments that represent Asian genotypes. Furthermore, no amplicons were observed by these PCR assays when DNA samples of Babesia bigemina, Babesia ovata, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, Anaplasma marginale, and Anaplasma bovis, and non-infected bovine blood were analyzed. In total, 109 B. bovis-positive blood DNA samples sourced from Sri Lanka (44 cattle), Mongolia (26 cattle), and Vietnam (23 cattle and 16 water buffaloes) were then screened by the type-specific PCR assays. The sequences derived from all of the PCR amplicons were phylogenetically analyzed. Out of 109 DNA samples, 23 (20 from cattle and 3 from water buffaloes) were positive for at least one genotype. In agreement with previous studies, five and four different genotypes were detected among the DNA samples from Sri Lanka and Vietnam, respectively. In contrast, four genotypes, including three novel genotypes, were detected from Mongolia. Five DNA samples were found to be co-infected with multiple genotypes. The sequences of the PCR amplicons clustered phylogenetically within the corresponding clades. These findings indicated that the type-specific PCR assays described herein are useful for the determination of genotypic diversity of the B. bovis msa-1 gene in Asia.

Eimeria bovis infection modulates endothelial host cell cholesterol metabolism for successful replication.

During first merogony Eimeria bovis forms large macromeronts in endothelial host cells containing >120 000 merozoites I. During multiplication, large amounts of cholesterol are indispensable for the enormous offspring membrane production. Cholesterol auxotrophy was proven for other apicomplexan parasites. Consequently they scavenge cholesterol from their host cell apparently in a parasite-specific manner. We here analyzed the influence of E. bovis infection on endothelial host cell cholesterol metabolism and found considerable differences to other coccidian parasites. Overall, free cholesterol significantly accumulated in E. bovis infected host cells. Furthermore, a striking increase of lipid droplet formation was observed within immature macromeronts. Artificial host cell lipid droplet enrichment significantly improved E. bovis merozoite I production confirming the key role of lipid droplet contents for optimal parasite proliferation. The transcription of several genes being involved in both, cholesterol de novo biosynthesis and low density lipoprotein-(LDL) mediated uptake, was significantly up-regulated at a time in infected cells suggesting a simultaneous exploitation of these two cholesterol acquisition pathways. E. bovis scavenges LDL-derived cholesterol apparently through significantly increased levels of surface LDL receptor abundance and LDL binding to infected cells. Consequently, LDL supplementation significantly improved parasite replication. The up-regulation of the oxidized LDL receptor 1 furthermore identified this scavenger receptor as a key molecule in parasite-triggered LDL uptake. Moreover, cellular cholesterol processing was altered in infected cells as indicated by up-regulation of cholesterol-25-hydroxylase and sterol O-acyltransferase. Overall, these results show that E. bovis considerably exploits the host cell cholesterol metabolism to guarantee its massive intracellular growth and replication.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0230-z) contains supplementary material, which is available to authorized users.

Suitable in vitro culture of Eimeria bovis meront II stages in bovine colonic epithelial cells and parasite-induced upregulation of CXCL10 and GM-CSF gene transcription.

We here established a suitable in vitro cell culture system based on bovine colonic epithelial cells (BCEC) for the development of Eimeria bovis merozoites I and the characterization of early parasite-induced innate epithelial host cell reactions as gene transcription of proinflammatory molecules. Both primary and permanent BCEC (BCEC (rim) and BCEC(perm)) were suitable for E. bovis merozoite I invasion and subsequent development of meronts II leading to the release of viable merozoites II. E. bovis merozoite II failed to develop any further neither into gamont nor oocyst stages in BCEC in vitro. E. bovis merozoite I induced innate epithelial host cell reactions at the level of CXC/CCL chemokines (CXCL1, CXCL8, CXCL10, CCL2), IL-6, and GM-CSF gene transcription. Overall, both BCEC types were activated by merozoite I infections since they showed significantly enhanced gene transcript levels of the immunomodulatory molecules CXCL10 and GM-CSF. However, gene transcription profiles of BCEC(prim) and BCEC(perm) revealed different reaction patterns in response to merozoite I infection with regard to quality and kinetics of chemokine/cytokine gene transcription. Although both BCEC types equally showed most prominent responses for CXCL10 and GM-CSF, the induction of CXCL1, CXCL8, CCL2, and IL-6 gene transcripts varied qualitatively and quantitatively. Our results demonstrate that BCEC seem capable to respond to E. bovis merozoite I infection by the upregulation of CXCL10 and GM-CSF gene transcription and therefore probably contribute to host innate effector mechanisms against E. bovis.

Calcium ions are involved in egress of Babesia bovis merozoites from bovine erythrocytes.

Bovine babesiosis is a livestock disease known to cause economic losses in endemic areas. The apicomplexan parasite Babesia bovis is able to invade and destroy the host’s erythrocytes leading to the serious pathologies of the disease, such as anemia and hemoglobinuria. Understanding the egress mechanisms of this parasite is therefore a key step to develop new therapeutic strategies. In this study, the possible involvement of Ca2+ in the egress of B. bovis merozoites from infected erythrocytes was investigated. Egress was artificially induced in vitro using calcium ionophore A23187 and thapsigargin to increase Ca2+ concentration in the cytosol of the parasite cells. The increased intracellular Ca2+ concentration following these treatments was confirmed using live cell Ca2+ imaging with confocal laser scanning microscopy. Based on our findings, we suggest a Ca2+ signalling pathway in the egress of B. bovis merozoites.

Differential inhibition of host cell cholesterol de novo biosynthesis and processing abrogates Eimeria bovis intracellular development.

Eimeria bovis macromeront formation in bovine endothelial host cells is an energy- and nutrient-demanding process. Obligate intracellular replicating coccidians are generally considered as auxotrophic for cholesterol synthesis and scavenge cholesterol from the host cell by either enhancing the uptake of extracellular cholesterol sources or by upregulating the host cellular de novo biosynthesis. We here focused on the latter mechanism and analyzed the effects of several inhibitors targeting the host cellular mevalonate biosynthesis pathway and cholesterol processing. The following inhibitors were used: lovastatin, squalestatin, CI976 and C75 targeting HMG-CoA reductase, squalene synthase, acyl-CoA:cholesterol acyltransferase, and fatty acid synthase, respectively. In summary, all inhibitors significantly interfered with E. bovis meront formation and merozoite production in a dose-dependent manner. Dose effect responses identified lovastatin as the most effective compound, followed by CI976, C75, and squalestatin, respectively. Overall, merozoite production was inhibited by 99.6, 99.7, 84.6, and 70.2% via lovastatin (1 μM), CI976, C75, and squalestatin (all 5 μM) treatments, respectively. Concerning macromeront formation, both the rate and size of developing meronts were affected by inhibitor treatments. The effects were characterized by developmental arrest and meront degradation. In the case of CI976 treatment, we additionally observed detrimental effects on host cellular lipid droplet formation leading to meront developmental arrest irrespective of the time point of treatment onset. These analyses clearly indicate that successful E. bovis intracellular development strictly depends on the host cellular de novo biosynthesis of cholesterol and on the adequate subsequent processing thereof.