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.

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.

Allicin is an active ingredient of garlic that has antibacterial, antifungal, antiviral, and antiprotozoal activity. However, the inhibitory effects of allicin on Babesia parasites have not yet been examined. In the present study, allicin was tested as a potent inhibitor against the in vitro growth of bovine and equine Babesia parasites and the in vivo growth of Babesia microti in a mouse model. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia caballi, or Theileria equi was inhibited by allicin in a dose-dependent manner and had IC50 values of 818, 675, 470, and 742 μM, respectively. Moreover, allicin significantly inhibited (P < 0.001) invasion of B. bovis, B. bigemina, B. caballi, and T. equi into the host erythrocyte. Furthermore, mice treated with 30 mg/kg of allicin for 5 days significantly (P < 0.05) reduced the parasitemia of B. microti over the period of the study. To further examine the potential synergism of allicin with diminazene aceturate, growth inhibitory assays were performed in vitro and in vivo. Interestingly, combinations of diminazene aceturate with allicin synergistically potentiated its inhibitory effects in vitro and in vivo. These results indicate that allicin might be beneficial for the treatment of babesiosis, particularly when used in combination with diminazene aceturate.

Bovine piroplasmosis is caused by tick-borne hemoprotozoans of the genera Babesia and Theileria and is the most prevalent in tropical and subtropical countries, causing a major economic impact worldwide. In the current study, a total of 405 cattle of different ages, sexes, and breeds were randomly sampled for surveying and diagnosis of babesiosis and theileriosis using three methods: direct microscopy (blood smears), indirect fluorescent antibody test (IFAT) and polymerase chain reaction (PCR). Giemsa-stained blood smears revealed that, out of 405 examined cattle, 33 (8.15 %) were infected with Babesia sp. and 65 (16.05 %) with Theileria sp. (total number of infected cattle was 98). Mixed infection was seen in 11 (2.72 %) animals. Moreover, application of the three diagnostic assays on 158 randomly sampled cattle indicated that 17 (10.76 %) and 33 (20.89 %) were positive for Babesia and Theileria spp. by the direct smear technique, 25 (15.82 %) and 33 (20.89 %) by IFAT (fluorescence was greenish yellow for Babesia and yellowish for Theileria), and 20 (12.66 %) and 38 (24.05 %) by PCR. Using primers specific for Babesia and Theileria spp., we found that diagnostic bands appeared at ~350 and ~370 bp, respectively indicating the presence of these piroplasms. Statistically, there was a non-significant difference of the positivity in response to the three techniques; thus, any of these methods can be described as useful for diagnosing blood parasites in both domesticated animals and birds. On the basis of the obtained results, it could be concluded that direct microscopy can be used in acute infections, whereas IFAT and PCR are useful in chronicity.