Akram Salama

An epidemiological survey of bovine Babesia and Theileria parasites in cattle, buffaloes, and sheep in Egypt

Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivity is thought to be greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood-DNA samples sourced from cattle (n=439), buffaloes (n=50), and sheep (n=105) reared in Menoufia, Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were the only parasites detected in buffaloes and each of these parasites was only found in two individual DNA samples (both 2%), while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively. Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical Membrane Antigen-1 genes were highly conserved among the samples, with 99.3-100% and 95.3-100% sequence identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences were relatively diverse (87.8-100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major Piroplasm Surface Protein (MPSP) gene sequences were classified as types 1 and 2. This is the first report of T. orientalis in Egypt, and of type 2 MPSP in buffaloes. Detection of MPSP type 2, which is considered a relatively virulent genotype, suggests that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt.

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites

A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

Evaluation of in vitro and in vivo inhibitory effects of fusidic acid on Babesia and Theileria parasites

Fusidic acid known to has antibacterial, antifungal, and antimalarial activities. Fusidic acid blocks translation elongation factor G gene in Plasmodium falciparum. In the present study, the inhibitory effects of fusidic acid on the in vitro growth of bovine and equine Babesia parasites were evaluated. The inhibitory effect of fusidic acid on the in vivo growth of Babesia microti was also assessed. The in vitro growth of four Babesia species that were tested was significantly inhibited (P<0.05) by micromolar concentrations of fusidic acid (IC(50) values=144.8, 17.3, 33.3, and 56.25 μM for Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi, respectively). Combinations of fusidic acid with diminazene aceturate synergistically potentiated its inhibitory effects in vitro on B. bovis and B. caballi. In B. microti-infected mice, fusidic acid caused significant (P<0.05) inhibition of the growth of B. microti at the dose of 500 mg/kg BW relative to control group. These results indicate that fusidic acid might be incorporated in treatment of babesiosis.

Specific antibody to a conserved region of Babesia apical membrane antigen-1 inhibited the invasion of B. bovis into the erythrocyte

Apical membrane antigen-1 (AMA-1) is a microneme protein that exists in all apicomplexan parasites and plays an indispensable role in the invasion into host cell. Central region of ectodomains I and II of Babesia bovis apical membrane antigen-1 (BbAMA-1P) is highly conserved with these of Babesia species and may be beneficial for vaccine development against babesiosis. In the present study, recombinant protein encoding the central region of B. bovis AMA-1 (rBbAMA-1P) was produced in Escherichia coli and its antiserum was prepared in mice for further molecular characterization. Anti-rBbAMA-1P serum specifically reacted with corresponding authentic protein of B. bovis as determined by Western blotting and IFAT. Cultured B. bovis treated with anti-rBbAMA-1P serum showed significant reduction in the in vitro growth of the parasites. Moreover, preincubated free merozoites with 1mg/ml anti-rBbAMA-1P serum inhibited their efficiency in the invasion into erythrocytes (RBCs) by 61% and 70% at 3h and 6h, respectively. Our data suggest that the central region of domains I and II of BbAMA-1 may serve as a vaccine candidate against babesiosis.

Molecular Characterization of a New Babesia bovis Thrombospondin-Related Anonymous Protein (BbTRAP2)

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite’s growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

Evaluation of the inhibitory effects of miltefosine on the growth of Babesia and Theileria parasites

Miltefosine, a membrane-active synthetic ether-lipid analogue, has antiproliferative and antiparasitic effects. In this study, the inhibitory effects of miltefosine were evaluated against three Babesia species and Theileria equi in vitro and against Babesia microti in mice. The drug showed significant growth inhibition from an initial parasitemia of 1% for Babesia bovis, Babesia bigemina, Babesia caballi, and T. equi with IC50 values of 25, 10.2, 10.4, and 99 μM, respectively. Complete inhibition was observed at 200 μM of miltefosine on the third day of culture for the three Babesia species and 400 μM on the fourth day for T. equi. Reverse-transcription PCR (RT-PCR) showed that miltefosine inhibited the transcription of choline-phosphate cytidylyltransferase in B. bovis. Miltefosine at a dose rate of 30 mg/kg resulted in a 71.7% inhibition of B. microti growth in BALB/c mice. Miltefosine might be used for drug therapy in babesiosis.

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP).

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.

Animal level risk factors associated with Babesia and Theileria infections in cattle in Egypt

In present study, blood samples were collected randomly from 439 cows at three main regions of Egypt (northern, central and southern). Molecular diagnosis of Babesia and Theileria infections by PCR amplification of DNA (gene) fragments, then cloning and sequencing of the positive samples were conducted. A questionnaire was created to imply the assumed risk factors and logistic regression statistical analysis was carried out to appraise the potential factors on the animal level. The results revealed that 49 (11.16%) and 45 (10.25%) cattle were infected with Babesia and Theileria parasites, respectively. B. bigemina (7.97%) and T. annulata (9.56%) were the most prevalent parasites. For Babesia sp., final multivariate logistic regression analysis showed a significant association between the infection and irregular use of antiprotozoal drugs (P = 0.003; OR: 0.28; 95% CI: 0.12–0.65), management practice (P = 0.029; OR: 6.66; 95% CI: 1.21–36.59) and ecology area (P = 0.006; OR: 5.62; 95% CI: 1.63–19.31). However, for Theileria sp. infection, animal breed (P = 0.003; OR: 0.44; 95% CI:.45–1.00) and irregular use of antiprotozoal drugs (P<0.001; OR: 4.22; 95% CI: 2.62–5.60) were the potential risk factors. The results of the present study declare the prevalent bovine Babesia and Theileria sp. in Egypt based on molecular description. An impression on the potential risk factors associated with infections was obtained. Recognition of the potential risk factors associated with tick borne disease may be helpful to construct the best preventive measures.

Serum vitamin A and E, copper, zinc and selenium concentrations and their relationship with health outcomes in dromedary hospitalized camels ( Camelus dromedarius )

The goals of this study were to measure serum vitamin A (retinol) and E (α-tocopherol) and trace elements concentrations (copper, zinc and selenium) during diseases condition and to determine their association with hematological parameters and immune status of hospitalized camels. A total of 95 dromedary camels [healthy (n=65); hospitalized camels (n=30)] were included in this study. Vitamin A and E concentrations were significantly lower in hospitalized camels than apparently healthy ones (P<0.05). Hospitalized camels had lower concentrations of zinc and selenium compared to healthy camels (P<0.05). Vitamin E, copper, zinc and selenium concentrations were positively correlated with phagocytic activity in hospitalized camels (P<0.05). The likelihood of deficiency of vitamin A and E, zinc and selenium concentrations were significant in female hospitalized camels than males and in young age hospitalized camels < 6 years old compared to old ones (P<0.05). Decreased vitamin A and E and trace elements concentrations were associated with hospitalized camels’ phagocytic activity and index. The prevalence of low vitamin A and E, zinc and selenium concentrations were frequent in female hospitalized camels and hospitalized camels of age < 6 years old suggesting severe oxidative stress.

Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules

[This corrects the article DOI: 10.1371/journal.pone.0185372.].