In Vitro Screening of the Open-Source Medicines for Malaria Venture Malaria Box Reveals Novel Compounds with Profound Activities against Theileria annulata Schizonts.

Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leukocytes and thereby cause fatal diseases. The hydroxynaphthoquinone buparvaquone is currently the only option for the treatment of theileriosis, and resistance development has been reported. It is therefore tempting to investigate the repurposing of compounds effective against related apicomplexan parasites, such as Plasmodium Here, we present the results of a screen of 400 compounds included in the open-access Medicines for Malaria Venture (MMV) malaria box on TaC12 cells, a macrophage-derived cell line immortalized by T. annulata schizonts. Using a combination of the classical alamarBlue vitality assay and a recently developed quantitative reverse transcriptase real-time PCR method based on the Theileria TaSP gene, we have identified 5 compounds, characterized their effects on the ultrastructure of TaC12 cells, and investigated whether they easily induce resistance formation. Two compounds, the quinolinols MMV666022 and MMV666054, have 50% inhibitory concentrations (IC50s) of 0.5 and 0.2 µM on TaC12 cells and 5.3 and 5.2 µM on BoMac cells, respectively. Thus, with therapeutic indexes of 11 and 18, they represent promising leads for further development of antitheilerial chemotherapeutics.

Comparisons among two serological tests and microscopic examination for the detection of Theileria annulata in cattle in northern Sudan.

We tested the agreement between microscopic examination (ME), a surface protein-detecting enzyme-linked immunosorbent assay (TaSP ELISA) and an indirect fluorescent assay (IFA) for detection of Theileria annulata in 2,661 naturally infected cattle from northern Sudan (samples collected between June 2001 and July 2002). In the ME, we detected piroplasms in 364/2,661 cattle (14%), and the kappas between the ME and the serological tests were poor (TaSP ELISA 10%; IFA 8%). The TaSP ELISA detected 885/2,661 cattle as positive, and the Rogan-and-Gladen corrected true prevalence of this sample was estimated to be 30%. The relative sensitivity and specificity of the IFA (compared to the previously validated TaSP ELISA) were 70.7% and 81.8%, respectively.

Molecular genetic characterization and subcellular localization of Theileria annulata mitochondrial heat-shock protein 70.

A Theileria annulata mitochondrial heat-shock protein of the 70-kDa family (Tamthsp70) was isolated by screening of the cDNA library of a T. annulata-infected bovine lymphoblastoid cell line with an antibody raised against T. annulata schizonts. The Tamthsp70 coding sequence was found to be most closely related to a previously reported mitochondrial hsp70 gene of Eimeria tenella exhibiting a similarity of 67% with mitochondrial hsp70 genes of eukaryotic plants (Pisum sativum, Phaseolus vulgaris) and with dnaK proteins of prokaryotes (Rhizobium meliloti, Agrobacterium tumefaciens). The Tamthsp70 mRNA is expressed within the sporozoite, schizont, and merozoite stages of the parasite, which suggests that it is constitutively transcribed throughout the life cycle. The gene encodes a polypeptide of 681 amino acids and exhibits a mitochondrial targeting sequence and several sequence motifs common to mitochondrial hsp70 and prokaryotic dnaK proteins. The protein level of the Tamthsp70 protein after heat shock decreased slightly during the exposure of infected cells to a temperature of 42 degrees C in comparison with cells cultured at 37 degrees C. By immunofluorescence the protein was located in the area in which the schizonts reside within infected cells. Immunoelectron microscopy showed that the hsp70 protein was predominantly localized in the mitochondria of the parasites. However, it was also found in small amounts in the cytoplasm of the parasite and host cell. This indicates (1) that Tamthsp70 is very probably translated in the parasite cytoplasm and then transported across the mitochondrial membrane into the mitochondrial matrix and (2) that it is transported across the parasite membrane into the host-cell cytoplasm.