Dielectric characterization of Babesia bovis using the dielectrophoretic crossover frequency.

Coinfection with the tick-transmitted pathogen Babesia spp. is becoming a serious health problem because of the erythrocyte invasion through Ixodes scapularis tick. The transmission of this protozoan by blood transfusion often results in high morbidity and mortality in recipients. A novel way to detect parasitized erythrocytes is by utilizing dielectrophoresis, an electrokinetic technique on a microfluidic platform, to improve the diagnostics of Babesia spp. The differences in the dielectric properties of Babesia spp.-infected erythrocytes versus healthy erythrocytes were exploited to design a fast and cost-effective diagnostic tool. One crucial factor for a successful diagnostic platform via dielectrophoretic separation is the dielectric characterization of Babesia-infected erythrocytes, which is investigated in this paper. The influence of medium conductivity and erythrocytes phenotype and genotype over the first crossover frequency (fco1 ) are used to quantify the dielectric properties of the infected cells. A sigmoidal curve was plotted via curve fitting of the single-shell model, which has been proven appropriate for parasitized cell populations where considerable cell geometry variation occurs. The difference in these curves is relevant for the separation of cells population. Microliters of sample and reagent were used throughout this experiment; the scale, results obtained, and simplicity of the system often make it very suitable for point-of-care babesiosis disease diagnostics.

Developing Anti-Babesia bovis Blood Stage Vaccines: A New Perspective Regarding Synthetic Vaccines.

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.

Babesia bovis Rad51 ortholog influences switching of ves genes but is not essential for segmental gene conversion in antigenic variation.

The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.

Novel Babesia bovis exported proteins that modify properties of infected red blood cells.

Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.

Establishment and application of a qPCR diagnostic method for Theileria annulata.

Bovine theileriosis caused by several Theileria species including Theileria annulata, Theileria parva, Theileria orientalis, Theileria mutans, and Theileria sinensis is a significant hemoprotozoan tick-borne disease. Among these, Theileria species, T. annulata, which causes tropical theileriosis (TT), is regarded as one of the most pathogenic and is responsible for high mortality. At present, most conventional diagnostic methods for tropical theileriosis are time-consuming and laborious and cannot distinguish newfound T. sinensis in China. Therefore, a high sensitivity and specificity real-time quantitative PCR method based on the TA19140 target molecule was developed, and the method was found to be specific for T. annulata. No cross-reaction was observed with T. sinensis, T. orientalis, Babesia bovis, Babesia bigemina, or Hyalomma anatolicum which is negative for T. annulata. A total of 809 field samples from different regions of China were analyzed by using the developed qPCR and conventional PCR. The positive samples for T. annulata detected by real-time qPCR and conventional PCR were 66/809 (8.16%) and 20/809 (2.47%), respectively, and all positive amplicons by qPCR were confirmed by Sanger sequencing. The results showed that the developed qPCR for the T. annulata 19,140 gene was more sensitive than conventional PCR. In addition, we first discovered that TA19140 was mainly expressed at the schizont and merozoite stages of T. annulata by relative quantification. The protein encoded by the TA19140 gene may be used as a potential diagnostic antigen for tropical theileriosis. In conclusion, a real-time quantitative PCR diagnostic method targeting the TA19140 gene was successfully established and could be used for both the quantitative and qualitative analysis of T. annulata infection from cattle and vector ticks, which will greatly help to control and diagnosis of tropical theileriosis.

Molecular epidemiology and characterization of Babesia bovis in cattle of Khyber Pakhtunkhwa province, Pakistan.

Babesiosis is a tick-borne disease found globally but most prominent in tropical and subtropical regions. It is responsible for huge mortality and morbidity, especially in developing countries like Pakistan. The current study was designed to determine the molecular epidemiology and characterization of Babesia bovis (B. bovis) infection in cattle populations of districts Mardan, Kohat and Swat of Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 434 tick-infested animals were sampled. Blood samples were collected, processed and then examined initially by microscopy for the presence of Babesia and were later confirmed through PCR by targeting cytochrome b gene, and the PCR products were sequenced. Phylogenetic analysis of sequenced isolates of the current study showed close sequence similarity with the reported strain of China. A non-significant association (p > 0.05) was observed between the prevalence of infections and risk factors. The overall prevalence of infection in all three districts was 10.11%. In district Swat (12.61%), the prevalence was recorded as the highest for B. bovis infection followed by district Mardan (10.60%) and district Kohat (06.90%). The Friesian breed of cattle, females and adult animals were highly susceptible to B. bovis infection. The prevalence of infection was recorded highest during the summer season and lowest during the winter season. This study concludes that B. bovis infection is prevalent in three studied districts of KP province and the sequenced isolates of the current study showed close sequence similarity with the reported strain of China.

Establishment of a transient transfection system for Babesia sp. Xinjiang using homologous promoters.

Babesia species, the agentic pathogens of human and animal babesiosis, are spread worldwide. Over the last decade, genetic manipulation approaches have been applied with many protozoan parasites, including Plasmodium falciparum, Trypanosoma cruzi, Cryptosporidium parvum, Theileria annulata, Theileria parva, Babesia bovis, Babesia bigemina, Babesia ovata, Babesia gibsoni, and Babesia ovis. For Babesia sp. Xinjiang (BspXJ), which is the causative pathogen of ovine babesiosis mainly in China, the efficiency of these techniques remains unclear. Firstly, a plasmid bearing the elongation factor-1 alpha promoter and the firefly luciferase reporter gene and rap stop region were transfected into BspXJ by electroporation and nucleoporation to determine the most suitable transfection solution. Then, six program settings were evaluated to confirm the best for BspXJ transient transfection, and a series of different amounts of plasmid DNA were transfected to generate relatively high luminescence values. Finally, the activities of four promoters derived from BspXJ were evaluated using the developed transient transfection system. After evaluating of various transfection parameters, the human T cell nucleofector solution, program V-024 and 20 µg of plasmid DNA were selected as the most favorable conditions for BspXJ transient transfection. These findings provide critical information for BspXJ genetic manipulation, an essential tool to identify virulence factors and to further elucidate the basic biology of this parasite.

Vibrational Spectroscopic Based Approach for Diagnosing Babesia bovis Infection.

Babesia bovis parasites present a serious and significant health concern for the beef and dairy industries in many parts of the world. Difficulties associated with the current diagnostic techniques include the following: they are prone to human error (microscopy) or expensive and time-consuming (polymerase chain reaction) to perform. Little is known about the biochemical changes in blood that are associated with Babesia infections. The discovery of new biomarkers will lead to improved diagnostic outcomes for the cattle industry. Vibrational spectroscopic technologies can record a chemical snapshot of the entire organism and the surrounding cell thereby providing a phenotype of the organism and the host infected cell. Here, we demonstrate the applicability of vibrational spectroscopic imaging techniques including Atomic Force Microscopy Infrared (AFM-IR) and confocal Raman microscopy to discover new biomarkers for B. bovis infections. Furthermore, we applied Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) to detect B. bovis in red blood cells (RBCs). Based on changes in the IR spectral bands, with ATR-FTIR in combination with Partial Least Squares-Discriminant Analysis we were able to discriminate infected samples from controls with a sensitivity and specificity of 92.0% and 91.7%, respectively, in less than 2 min, excluding sample extraction and preparation. The proposed method utilized a lysis approach to remove hemoglobin from the suspension of infected and uninfected cells, which significantly increased the sensitivity and specificity compared to measurements performed on intact infected red blood cells (intact infected RBC, 77.3% and 79.2%). This work represents a holistic spectroscopic study from the level of the single infected RBC using AFM-IR and confocal Raman to the detection of the parasite in a cell population using ATR-FTIR for a babesiosis diagnostic.

Drug screening of food and drug administration-approved compounds against Babesia bovis in vitro.

Babesia (B.) bovis is one of the main etiological agents of bovine babesiosis, causes serious economic losses to the cattle industry. Control of bovine babesiosis has been hindered by the limited treatment selection for B. bovis, thus, new options are urgently needed. We explored the drug library and unbiasedly screened 640 food and drug administration (FDA) approved drug compounds for their inhibitory activities against B. bovis in vitro. The initial screening identified 13 potentially effective compounds. Four potent compounds, namely mycophenolic acid (MPA), pentamidine (PTD), doxorubicin hydrochloride (DBH) and vorinostat (SAHA) exhibited the lowest IC50 and then selected for further evaluation of their in vitro efficacies using viability, combination inhibitory and cytotoxicity assays. The half-maximal inhibitory concentration (IC50) values of MPA, PTD, DBH, SAHA were 11.38 ± 1.66, 13.12 ± 4.29, 1.79 ± 0.15 and 45.18 ± 7.37 µM, respectively. Of note, DBH exhibited IC50 lower than that calculated for the commonly used antibabesial drug, diminazene aceturate (DA). The viability result revealed the ability of MPA, PTD, DBH, SAHA to prevent the regrowth of treated parasite at 4 × and 2 × of IC50. Antagonistic interactions against B. bovis were observed after treatment with either MPA, PTD, DBH or SAHA in combination with DA. Our findings indicate the richness of FDA approved compounds by novel potent antibabesial candidates and the identified potent compounds especially DBH might be used for the treatment of animal babesiosis caused by B. bovis.

A cysteine protease of Babesia microti and its interaction with tick cystatins.

Babesiosis is a tick-borne protozoonosis caused by Babesia, which can cause fever, hemolytic anemia, hemoglobinuria, and even death. Babesia microti is a parasite found in rodents and can be pathogenic to humans. In this study, the full-length cDNA of a B. microti cysteine protease (BmCYP) was expressed and the recombinant rBmCYP protein analyzed and characterized. BmCYP is encoded by an ORF of 1.3 kb, with a predicted molecular weight of 50 kDa and a theoretical pI of 8.5. The amino acid sequence of BmCYP exhibits an identity of 32.9 to 35.2% with cysteine proteases of Babesia ovis, Babesia bovis, and Theileria, respectively. The results of the proteinase assays show that rBmCYP has cysteine protease enzymatic activity. In addition, we demonstrate that tick cystatins rRhcyst-1 and rRhcyst-2 were able to effectively inhibit the activity of rBmCYP; the inhibition rates were 57.2% and 30.9%, respectively. Tick cystatins Rhcyst-1 and Rhcyst-2 were differentially expressed in ticks that fed on Babesia-infected mice relative to non-infected control ticks. Our results suggest that BmCYP is a functional enzyme with cysteine protease enzymatic activity and may be involved in tick-B. microti interactions.

Correlations and repeatability between Babesia spp. infection levels using two dairy cattle breeding systems.

Babesia bovis and Babesia bigemina are tick-transmitted piroplasms that cause severe damage to the livestock industry in tropical regions of the world. Recent studies demonstrated differences in infection levels of these haemoparasites among bovine breeds and variation between individual cows regarding resistance to these diseases. This study aimed to estimate the repeatability and correlations between B. bovis and B. bigemina using two cattle breeding systems, an individual system (IS) and a collective paddock system (CPS). All animals were Holstein breed, and the levels of B. bovis and B. bigemina in blood samples were estimated by quantitative polymerase chain reaction (qPCR). The estimated correlations for the B. bigemina and B. bovis DNA copy number for IS and CPS were moderate and high, respectively, whereas repeatability estimates for both systems and both Babesia species were moderate. Although we cannot infer that the type of rearing system directly influenced the correlation and repeatability coefficients, it appears that the bovine parasitemia burden may be dependent on (or determine) the parasitemia burden on ticks because the bovines remained in the same place for a longer time in both systems. Thus, the babesiosis infection levels of the ticks may have been uniform, a phenomenon that also ensures greater uniformity in cattle infection. This factor may have favored the occurrence of infected ticks leading to higher repeatability estimates and correlations. Our study confirms high variability in resistance/susceptibility between breeds, and the high correlations found may be linked to this characteristic and the most intensive breeding type of dairy cattle. Besides, under the present study conditions, the estimated correlations suggest that measuring an infection level of one Babesia species can predict the level of infection of the other.

Phytochemical Characterization and Chemotherapeutic Potential of Cinnamomum verum Extracts on the Multiplication of Protozoan Parasites In Vitro and In Vivo.

Cinnamomum verum is a commonly used herbal plant that has several documented properties against various diseases. The existing study evaluated the inhibitory effect of acetonic extract of C. verum (AECV) and ethyl acetate extract of C. verum (EAECV) against piroplasm parasites in vitro and in vivo. The drug-exposure viability assay was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3) and human foreskin fibroblast (HFF) cells. Qualitative phytochemical estimation revealed that AECV and EAECV containing multiple bioactive constituents namely alkaloids, tannins, saponins, terpenoids and remarkable amounts of polyphenols and flavonoids. AECV and EAECV inhibited B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi multiplication at half-maximal inhibitory concentrations (IC50) of 23.1 ± 1.4, 56.6 ± 9.1, 33.4 ± 2.1, 40.3 ± 7.5, 18.8 ± 1.6 µg/mL, and 40.1 ± 8.5, 55.6 ± 1.1, 45.7 ± 1.9, 50.2 ± 6.2, and 61.5 ± 5.2 µg/mL, respectively. In the cytotoxicity assay, AECV and EAECV affected the viability of MDBK, NIH/3T3 and HFF cells with half-maximum effective concentrations (EC50) of 440 ± 10.6, 816 ± 12.7 and 914 ± 12.2 µg/mL and 376 ± 11.2, 610 ± 7.7 and 790 ± 12.4 µg/mL, respectively. The in vivo experiment showed that AECV and EAECV were effective against B. microti in mice at 150 mg/kg. These results showed that C. verum extracts are potential antipiroplasm drugs after further studies in some clinical cases.

Evaluation of 4-Amino 2-Anilinoquinazolines against Plasmodium and Other Apicomplexan Parasites In Vitro and in a P. falciparum Humanized NOD-scid IL2Rγnull Mouse Model of Malaria.

A series of 4-amino 2-anilinoquinazolines optimized for activity against the most lethal malaria parasite of humans, Plasmodium falciparum, was evaluated for activity against other human Plasmodium parasites and related apicomplexans that infect humans and animals. Four of the most promising compounds from the 4-amino 2-anilinoquinazoline series were equally as effective against the asexual blood stages of the zoonotic P. knowlesi, suggesting that they could also be effective against the closely related P. vivax, another important human pathogen. The 2-anilinoquinazoline compounds were also potent against an array of P. falciparum parasites resistant to clinically available antimalarial compounds, although slightly less so than against the drug-sensitive 3D7 parasite line. The apicomplexan parasites Toxoplasma gondii, Babesia bovis, and Cryptosporidium parvum were less sensitive to the 2-anilinoquinazoline series with a 50% effective concentration generally in the low micromolar range, suggesting that the yet to be discovered target of these compounds is absent or highly divergent in non-Plasmodium parasites. The 2-anilinoquinazoline compounds act as rapidly as chloroquine in vitro and when tested in rodents displayed a half-life that contributed to the compound's capacity to clear P. falciparum blood stages in a humanized mouse model. At a dose of 50 mg/kg of body weight, adverse effects to the humanized mice were noted, and evaluation against a panel of experimental high-risk off targets indicated some potential off-target activity. Further optimization of the 2-anilinoquinazoline antimalarial class will concentrate on improving in vivo efficacy and addressing adverse risk.

Cocktail Babesia bovis antigens for global detection of Babesia bovis infection in cattle.

The diagnostic performance of a cocktail formula consisting of two Babesia (B.) bovis recombinant proteins, including spherical body protein 1 (BbSBP-1) and spherical body protein 4 (BbSBP-4), was evaluated in the present study for the global detection of B. bovis infection in cattle and for the differentiation between B. bovis and B. bigemina infections. The efficacy and the practicality of the rBbSBP-1 and rBbSBP-4 cocktail formula for differentiation between the infection caused by both parasites were assessed using indirect enzyme-linked immunosorbent assay (iELISA) with serum samples collected from cattle experimentally infected by B. bovis (n = 33) or B. bigemina (n = 30). Cocktail antigen exhibited the highest optical density (OD) values with B. bovis-infected sera and the lowest OD values with normal bovine sera or B. bigemina-infected sera in comparison with the single antigen. A total of 581 field serum samples collected from four countries with known B. bovis endemicity: Ghana (n = 154), Egypt (n = 162), Thailand (n = 96), and South Africa (n = 169) were screened also in the current study using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. A cocktail formula (rBbSBP-1 and rBbSBP-4) exhibited the highest concordance rate (89.90%) and kappa value (0.73). The obtained results revealed the reliability of the rBbSBP-1 and rBbSBP-4 cocktail antigen for the detection of specific antibodies to B. bovis in cattle and demonstrated the usefulness of cocktail antigen for differentiation between B. bovis and B. bigemina infections compared with the single antigen in cattle, which will be useful for epidemiological surveys and control of bovine babesiosis.

Genetic diversity of Babesia bovis in beef cattle in a large wetland in Brazil.

Babesia bovis is the etiological agent of bovine babesiosis, a disease transmitted by Rhipicephalus microplus, which affects cattle herds in tropical and subtropical regions of the world, causing significant economic losses due to decreasing meat and milk yield. This study used molecular techniques to determine the occurrence and genetic diversity of B. bovis, based on the genes encoding the spherical body protein (sbp-2) and the merozoite surface antigens (MSAs) genes, in a herd of 400 Nellore (Bos indicus) sampled from beef cattle farms in the Pantanal region, state of Mato Grosso do Sul, Midwestern Brazil. The results of the nested PCR assays based on the sbp-2 gene indicated that 18 (4.5%) calves were positive for B. bovis; out of them, while 77.7% (14/18) were positive for the B. bovis msa-2b fragment, 66.6% (12/18) were positive for the msa-2c fragment. The phylogenetic analysis based on the maximum likelihood method using 14 sequences from msa-2b clones and 13 sequences from msa-2c clones indicated that the sequences detected in this study are clearly distributed in different cladograms. These findings corroborated the diversity analysis of the same sequences, which revealed the presence of 14 and 11 haplotypes of the msa-2b and msa-2c genes, respectively. Furthermore, the entropy analyses of amino acid sequences revealed 78 and 44 high entropy peaks with values ranging from 0.25 to 1.53 and from 0.27 to 1.09 for MSA-2B and MSA-2C, respectively. Therefore, the results indicate a low molecular occurrence of B. bovis in beef cattle sampled in the Brazilian Pantanal. Despite this, a high degree of genetic diversity was found in the analyzed B. bovis population, with possibly different haplotypes coexisting in the same animal and/or in the same studied herd.

Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka.

Bovine babesiosis is a serious threat to the cattle industry. We prepared blood DNA samples from 13 cattle with clinical babesiosis from the Badulla (n = 8), Jaffna (n = 3), and Kilinochchi (n = 2) districts in Sri Lanka. These DNA samples tested positive in PCR assays specific for Babesiabovis (n = 9), Babesia bigemina (n = 9), and Babesiaovata (n = 1). Twelve cattle were positive for B. bovis and/or B. bigemina One cow was negative for the tested Babesia species but was positive for Babesia on microscopic examination; the phylogenetic positions of 18S rRNA and cytochrome oxidase subunit III gene sequences suggested that the cow was infected with Babesia sp. Mymensingh, which was recently reported from a healthy cow in Bangladesh. We then developed a novel Babesia sp. Mymensingh-specific PCR assay and obtained positive results for one other sample. Analysis of gene sequences from the cow with positive B. ovata-specific PCR results demonstrated that the animal was infected not with B. ovata but with Babesia sp. Hue-1, which was recently reported from asymptomatic cattle in Vietnam. The virulence of Babesia sp. Hue-1 is unclear, as the cow was coinfected with B. bovis and B. bigemina However, Babesia sp. Mymensingh probably causes severe clinical babesiosis, as it was the sole Babesia species detected in a clinical case. The present study revealed the presence of two bovine Babesia species not previously reported in Sri Lanka, plus the first case of severe bovine babesiosis caused by a Babesia species other than B. bovis, B. bigemina, and Babesiadivergens.

Comparisons of the topographic characteristics and electrical charge distributions among Babesia-infected erythrocytes and extraerythrocytic merozoites using AFM.

Tick-borne Babesia parasites are responsible for costly diseases worldwide. Improved control and prevention tools are urgently needed, but development of such tools is limited by numerous gaps in knowledge of the parasite-host relationships. We hereby used atomic force microscopy (AFM) and frequency-modulated Kelvin probe potential microscopy (FM-KPFM) techniques to compare size, texture, roughness and surface potential of normal and infected Babesia bovis, B. bigemina and B. caballi erythrocytes to better understand the physical properties of these parasites. In addition, AFM and FM-KPFM allowed a detailed view of extraerythrocytic merozoites revealing shape, topography and surface potential of paired and single parasites. B. bovis-infected erythrocytes display distinct surface texture and overall roughness compared to noninfected erythrocytes. Interestingly, B. caballi-infected erythrocytes do not display the surface ridges typical in B. bovis parasites. Observations of extraerythrocytic B. bovis, B. bigemina and B. caballi merozoites using AFM revealed differences in size and shape between these three parasites. Finally, similar to what was previously observed for Plasmodium-infected erythrocytes, FM-KPFM images reveal an unequal electric charge distribution, with higher surface potential above the erythrocyte regions that are likely associated with Babesia parasites than over its remainder regions. In addition, the surface potential of paired extraerythrocytic B. bovis Mo7 merozoites revealed an asymmetric potential distribution. These observations may be important to better understand the unique cytoadhesive properties of B. bovis-infected erythrocytes, and to speculate on the role of differences in the distribution of surface charges in the biology of the parasites.

Comparative evaluation of DNA extraction kit, matrix sample and qPCR assays for bovine babesiosis monitoring.

Bovine babesiosis caused by protozoan parasites Babesia bovis and B. bigemina is one of the most important causes of losses for the livestock industry in tropical and subtropical regions of the world. Therefore, highly sensitive and specific tools for these hemoparasites detection and monitoring are required, especially in carrier animals, in which low parasite levels were usually present. In this context, qPCR assays have been successfully and fairly used in last years. Aiming to improve the performance of Babesia levels monitoring by qPCR, some of main aspects of this methodology that may influence results were tested: DNA extraction kits, whole blood EDTA pre-treatment, blood source (tip of tail or jugular vein), erythrocytes isolation, FTA card interference and qPCR system of detection. Under our experimental conditions, both EDTA pre-treatment and FTA card application have no influence on the sensitivity of detection, and two DNA extraction kits provided higher sensitivity compared to others. As expected, blood samples collected from the tip of tail vessels presented higher levels of B. bovis DNA compared to those obtained from the jugular vein, and erythrocytes processed isolated has also improved the sensitivity compared to whole blood. Moreover, both qPCR assays here developed using hydrolysis probes for B. bovis and B. bigemina detection, presented enhanced reproducibility compared to qPCR assays using intercalating dye system. Even, qPCR for B. bigemina using hydrolysis probe here developed presented higher sensitivity compared to intercalating dye system. This study has contributed to the improvement of molecular diagnosis of bovine babesiosis, which may improve epidemiological studies related to these pathogens.

Novel biotechnological platform based on baculovirus occlusion bodies carrying Babesia bovis small antigenic peptides for the design of a diagnostic enzyme-linked immunosorbent assay (ELISA).

Baculoviruses are large DNA virus of insects principally employed in recombinant protein expression. Its ability to form occlusion bodies (OBs), which are composed mainly of polyhedrin protein (POLH), makes them biotechnologically attractive, as these crystals (polyhedra) can incorporate foreign peptides and can be easily isolated. On the other hand, peptide microarrays allow rapid and inexpensive high-throughput serological screening of new candidates to be incorporated to OBs. To integrate these 2 biotechnological approaches, we worked on Babesia bovis, one of the causative agents of bovine babesiosis. Current molecular diagnosis of infection with B. bovis includes enzyme-linked immunosorbent assay (ELISA) techniques, which use merozoite lysate obtained from infected bovine erythrocytes. However, it is important to produce recombinant antigens that replace the use of crude antigens. Here, we describe a new biotechnological platform for the design of indirect ELISAs based on 5 antigenic peptides of 15 amino acid residues of B. bovis (ApBb), selected from a peptide microarray and expressed as a fusion to POLH. An Sf9POLHE44G packaging cell line infected with recombinant baculoviruses carrying POLH-ApBb fusions yielded higher levels of chimeric polyhedra, highlighting the advantage of a trans-contribution of a mutant copy of polyhedrin. Finally, the use of dissolved recombinant polyhedra as antigens was successful in an ELISA assay, as B. bovis-positive sera recognized the fusion POLH-ApBb. Thus, the use of this platform resulted in a promising alternative for molecular diagnosis of relevant infectious diseases.

qPCR estimates of Babesia bovis and Babesia bigemina infection levels in beef cattle and Rhipicephalus microplus larvae.

Babesia spp. are tick-transmitted intraerythrocytic apicomplexan parasites that infect wild and domestic animals. Babesia bovis and B. bigemina are endemic and responsible for enormous economic losses to the livestock industry in most of the Brazilian territory, wherein the tick Rhipicephalus microplus is the unique vector. Better understanding of epidemiology and parasite-host interactions may improve the tools for disease control and genetic management for selection of resistant animals. This study aimed to detect, quantify and measure the correlation between B. bigemina and B. bovis infection levels in bovine blood and into tick, by absolute quantification of hemoparasite DNA using qPCR. Blood bovine samples and larvae pools from 10 engorged R. microplus females were collected from each Canchim heifers (5/8 Charolais + 3/8 zebu, n = 36). All evaluated samples were positive for both Babesia species tested. Correlations of B. bovis and B. bigemina levels between cattle and tick host were 0.58 and 0.66, respectively. These high positive correlation coefficients indicate that parasitemia load in the bovine may be dependent on or may determine the parasitemia load in the ticks.