Dynamic methylation of histone H3K18 in differentiating Theileria parasites.

Lysine methylation on histone tails impacts genome regulation and cell fate determination in many developmental processes. Apicomplexa intracellular parasites cause major diseases and they have developed complex life cycles with fine-tuned differentiation events. Yet, apicomplexa genomes have few transcription factors and little is known about their epigenetic control systems. Tick-borne Theileria apicomplexa species have relatively small, compact genomes and a remarkable ability to transform leucocytes in their bovine hosts. Here we report enriched H3 lysine 18 monomethylation (H3K18me1) on the gene bodies of repressed genes in Theileria macroschizonts. Differentiation to merozoites (merogony) leads to decreased H3K18me1 in parasite nuclei. Pharmacological manipulation of H3K18 acetylation or methylation impacted parasite differentiation and expression of stage-specific genes. Finally, we identify a parasite SET-domain methyltransferase (TaSETup1) that can methylate H3K18 and represses gene expression. Thus, H3K18me1 emerges as an important epigenetic mark which controls gene expression and stage differentiation in Theileria parasites.

Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton.

Theileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and ß- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

Anti-piroplasmic activity of novobiocin as heat shock protein 90 inhibitor against in vitro cultured Theileria equi and Babesia caballi parasites.

Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP). Currently, imidocarb dipropionate (ID) is the only available drug for treating the clinical form of EP. Serious side effects and incomplete clearance of infection is a major drawback of ID. Heat-shock proteins (Hsp) play a vital role in the life cycle of these haemoprotozoans by preventing alteration in protein conformation. These Hsp are activated during transmission of EP sporozoites from the tick vector (poikilotherm) to the natural host (homeotherm) and facilitate parasite survival. In the present study, we targeted the heat shock protein 90 (Hsp-90) pathway of T. equi and B. caballi by using its inhibitor drug - novobiocin. Dose-dependent efficacy of novobiocin on the growth of T. equi and B. caballi was observed in in vitro culture. Additionally, we examined dose-dependent cell cytotoxicity on host peripheral mononuclear cells (PBMCs) and haemolytic activity on equine red blood cells (RBC). In vivo organ toxicity of novobiocin was also assessed in a mouse model. The IC50 (50 % inhibitory concentration) value of novobiocin against T. equi and B. caballi was 165 µM and 84.85 µM, respectively. Novobiocin significantly arrested the in vitro growth of T. equi and B. caballi parasites at 100 µM and 200 µM drug concentration, respectively. In vitro treated parasites had distorted nuclear material and showed no further viability. Based on the equine PBMCs and RBC, the drug was found to be safe even at 1000 µM concentration and the CC50 (50 % cytotoxicity concentration) values were 11.63 mM and 261.97 mM. Very high specific selective index (SSI) values (70.47 and 1587) were observed for equine PBMCs and RBC, respectively. Organ-specific biochemical markers and histopathological examination indicated no adverse effect of the drug at a dose rate of 50 mg kg body weight in the mouse model. The results demonstrate the growth inhibitory effect of novobiocin against T. equi and B. caballi parasites and its safety for host cell lines with very high SSI. Hence, it can be inferred that the Theileria/Babesia Hsp-90 family are potential drug targets worthy of further investigation.

Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi.

BACKGROUND: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. The endochin-like quinolones (ELQ)-300 and ELQ-316 have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp., with ELQ-316 also being effective against Babesia microti, without showing toxicity in mammals. METHODS: The inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. The percentage of parasitized erythrocytes was measured by flow cytometry, and the effect of the ELQ compounds on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay. RESULTS: We calculated the half maximal inhibitory concentration (IC50) at 72 h, which ranged from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 among all cultured parasites tested at 72 h. None of the parasites tested were able to replicate in cultures in the presence of ELQ-300 and ELQ-316 at the maximal inhibitory concentration (IC100), which ranged from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. CONCLUSIONS: The compounds ELQ-300 and ELQ-316 showed significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

Pathogenesis of liver lesions in Theileria orientalis-inoculated cattle and severe combined immunodeficiency mice with bovine erythrocyte transfusion.

Theileria orientalis (T. orientalis) is a bovine protozoal disease similar to malaria in humans. Although the common outcome of malaria in humans and T. orientalis infection in cattle is hepatic disorder, the mechanisms of its development remain unknown. In this study, we investigated hepatocyte injury characterized by accumulation of macrophages with ingested erythrocytes in sinusoid and extramedullary hematopoiesis in cattle and mice experimentally infected with T. orientalis (T. orientalis-infected cattle and T. orientalis-infected mice). Vacuolization of hepatic cells was frequently observed in the vicinity of the aggregated macrophages in the liver sinusoids of T. orientalis-infected mice. A significant percentage of the macrophages accumulated in the liver sinusoids of the severely infected cattle and mice (14.6% and 24.2 to 53.2%, respectively) reacted positively with interleukin-1, interleukin-6 and TNF-α antibodies. Increase in the production of these cytokines was confirmed in T. orientalis-infected cattle and mice by real-time RT-PCR. These findings strongly suggest that increased cytokine production by the macrophages that have phagocytosed T. orientalis-infected erythrocytes causes hepatic disorder in T. orientalis-infected animals.

Inhibitory effects of novel ciprofloxacin derivatives on the growth of four Babesia species and Theileria equi.

The problems of parasite resistance, as well as the toxic residues to most of the commercially available antipiroplasmic drugs severely weaken their effective, curative, and environmental safe employment. Therefore, it is clear that the development of treatment options for piroplasmosis is vital for improving disease treatment and control. Ciprofloxacin is a broad-spectrum antibiotic that targets mainly the DNA replication machinery by inhibiting DNA gyrase and topoisomerase enzymes. As a result, ciprofloxacin is used for treating several bacterial and parasitic infections. In this study, the efficacy of 15 novel ciprofloxacin derivatives (NCD) that had been developed against drug-resistant Mycobacterium tuberculosis was evaluated against piroplasm parasite multiplication in vitro. The half-maximal inhibitory concentration (IC50) values of the most effective five compounds of NCD (No. 3, 5, 10, 14, 15) on Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi were 32.9, 13.7, 14.9, and 30.9; 14.9, 25.8, 13.6, and 27.5; 34.9, 33.9, 21.1, and 22.3; 26.7, 28.3, 34.5, and 29.1; and 4.7, 26.6, 33.9, and 29.1 µM, respectively. Possible detrimental effects of tested NCD on host cells were assessed using mouse embryonic fibroblast (NIH/3T3) and Madin-Darby bovine kidney (MDBK) cell lines. Tested NCD did not suppress NIH/3T3 and MDBK cell viability, even at the highest concentration used (500 µM). Combination treatments of the identified most effective compounds of NCD/diminazene aceturate (DA), /atovaquone (AQ), and /clofazimine (CF) showed mainly synergistic and additive effects. The IC50 values of NCD showed that they are promising future candidates against piroplasmosis. Further in vivo trials are required to evaluate the therapeutic potential of NCD.

Protein Kinase Inhibitors Arrested the In-Vitro Growth of Theileria equi.

INTRODUCTION: Theileria equi is an intra-erythrocytic apicomplexean protozoa that infect equines. Protein kinases (PK), key molecules of the apicomplexean life cycle, have been implicated as significant drug targets. The growth inhibitory efficacy of PK inhibitors against Theileria/Babesia animal parasites have not been documented so far. METHODS: The present study aimed to carry out in-vitro growth inhibitory efficacy studies of four novel drug molecules-SB239063, PD0332991 isethionate, FR180204 and apigenin, targeting different protein kinases of T. equi. A continuous microaerophilic stationary-phase culture (MASP) system was established for propagation of T. equi parasites. This in-vitro culture technique was used to assess the growth inhibitory effect of protein kinase targeted drug molecules, whereas diminazene aceturate was taken as control drug against T. equi. The inhibitory concentration (IC50) was determined for comparative analysis. The potential cytotoxicity of the drug molecule was also assessed on horse's peripheral blood mononuclear cells (PBMCs) cell line. RESULTS: SB239063 and diminazene aceturate drugs significantly inhibited (p < 0.05) the in-vitro growth of T. equi parasite at 0.1 µM, 1 µM, 10 µM, 50 µM and 100 µM concentration at ≥ 48 h of incubation period and respective IC50 values were 4.25 µM and 1.23 µM. Furthermore, SB239063 was not cytotoxic to the horse PBMCs and found safer than diminazine aceturate drug. PD0332991 isethionate and FR180204 are extracellular signal-regulated kinase (ERK) inhibitors and significantly (p < 0.05) inhibited T. equi in-vitro growth at higher concentrations (≥ 48 h of incubation period) with respective IC50 value of 10.41 µM and 21.0 µM. Lower concentrations of these two drugs were not effective (p > 0.05) even after 96 h of treatment period. Apigenin (protein kinase-C inhibitor) drug molecule was unsuccessful in inhibiting the T. equi parasite growth completely. After 96 h of in-vitro treatment period, a parasite viability study was performed on drug-treated T. equi parasitized RBCs. These drugs-treated parasitized RBCs were collected and transferred to wells containing fresh culture media (without drug) and naïve host RBCs. Drug-treated RBCs collected from SB239063, PD0332991, diminazene aceturate treatment (1 µM to 100 µM concentration) were unsuccessful in growing/multiplying further. Apigenin drug-treated T. equi parasites were live after 96 h of treatment. CONCLUSION: It may be concluded that SB239063 was the most effective drug molecule (being lowest in IC50 value) out of the four different protein kinase inhibitors tested in this study. This drug molecule has insignificant cytotoxic activity against horse's PBMCs.

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.

Inhibitory effects of Syzygium aromaticum and Camellia sinensis methanolic extracts on the growth of Babesia and Theileria parasites.

Currently, chemotherapeutics against piroplasmosis are also associated with toxicity and the emergence of drug-resistant parasites. Therefore, the discovery of new drug compounds is necessary for the effective control of bovine and equine piroplasms. Syzygium aromaticum (clove) and Camellia sinensis (green tea) have several documented medicinal properties. In the present study, the growth-inhibiting effects of S. aromaticum and C. sinensis methanolic extracts were evaluated in vitro and in vivo. The half-maximal inhibitory concentration (IC50) values for methanolic S. aromaticum against Babesia bovis, B. bigemina, B. divergens, B. caballi, and Theileria equi were 109.8 ± 3.8, 8.7 ± 0.09, 76.4 ± 4.5, 19.6 ± 2.2, and 60 ± 7.3 µg/ml, respectively. Methanolic C. sinensis exhibited IC50 values of 114 ± 6.1, 71.3 ± 3.7, 35.9 ± 6.8, 32.7 ± 20.3, and 60.8 ± 7.9 µg/ml against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, respectively. The toxicity assay on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines showed that methanolic S. aromaticum and methanolic C. sinensis affected only the viability of the MDBK cell line with half-maximal effective concentrations (EC50) of 894.7 ± 4.9 and 473.7 ± 7.4 µg/ml, respectively, while the viability of NIH/3T3 and HFF cell lines was not affected even at 1000 µg/ml. In the in vivo experiment, methanolic S. aromaticum and methanolic C. sinensis oral treatments at 150 mg/kg inhibited the growth of Babesia microti in mice by 69.2% and 42.4%, respectively. These findings suggest that methanolic S. aromaticum and methanolic C. sinensis extracts have the potential as alternative remedies for treating piroplasmosis.

Ellagic acid microspheres restrict the growth of Babesia and Theileria in vitro and Babesia microti in vivo.

BACKGROUND: There are no effective vaccines against Babesia and Theileria parasites; therefore, therapy depends heavily on antiprotozoal drugs. Treatment options for piroplasmosis are limited; thus, the need for new antiprotozoal agents is becoming increasingly urgent. Ellagic acid (EA) is a polyphenol found in various plant products and has antioxidant, antibacterial and effective antimalarial activity in vitro and in vivo without toxicity. The present study documents the efficacy of EA and EA-loaded nanoparticles (EA-NPs) on the growth of Babesia and Theileria. METHODS: In this study, the inhibitory effect of EA, ß-cyclodextrin ellagic acid (ß-CD EA) and antisolvent precipitation with a syringe pump prepared ellagic acid (APSP EA) was evaluated on four Babesia species and Theileria equi in vitro, and on the multiplication of B. microti in mice. The cytotoxicity assay was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3) and human foreskin fibroblast (HFF) cell lines. RESULTS: The half-maximal inhibitory concentration (IC50) values of EA and ß-CD EA on B. bovis, B. bigemina, B. divergens, B. caballi and T. equi were 9.58 ± 1.47, 7.87 ± 5.8, 5.41 ± 2.8, 3.29 ± 0.42 and 7.46 ± 0.6 µM and 8.8 ± 0.53, 18.9 ± 0.025, 11 ± 0.37, 4.4 ± 0.6 and 9.1 ± 1.72 µM, respectively. The IC50 values of APSP EA on B. bovis, B. bigemina, B. divergens, B. caballi and T. equi were 4.2 ± 0.42, 9.6 ± 0.6, 2.6 ± 1.47, 0.92 ± 5.8 and 7.3 ± 0.54 µM, respectively. A toxicity assay showed that EA, ß-CD EA and APSP EA affected the viability of cells with a half-maximal effective concentration (EC50) higher than 800 µM. In the experiments on mice, APSP EA at a concentration of 70 mg/kg reduced the peak parasitemia of B. microti by 68.1%. Furthermore, the APSP EA-atovaquone (AQ) combination showed a higher chemotherapeutic effect than that of APSP EA monotherapy. CONCLUSIONS: To our knowledge, this is the first study to demonstrate the in vitro and in vivo antibabesial action of EA-NPs and thus supports the use of nanoparticles as an alternative antiparasitic agent.

The effects of trans-chalcone and chalcone 4 hydrate on the growth of Babesia and Theileria.

BACKGROUND: Chemotherapy is a principle tool for the control and prevention of piroplasmosis. The search for a new chemotherapy against Babesia and Theileria parasites has become increasingly urgent due to the toxic side effects of and developed resistance to the current drugs. Chalcones have attracted much attention due to their diverse biological activities. With the aim to discover new drugs and drug targets, in vitro and in vivo antibabesial activity of trans-chalcone (TC) and chalcone 4 hydrate (CH) alone and combined with diminazene aceturate (DA), clofazimine (CF) and atovaquone (AQ) were investigated. METHODOLOGY/PRINCIPAL FINDINGS: The fluorescence-based assay was used for evaluating the inhibitory effect of TC and CH on four Babesia species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, the combination with DA, CF, and AQ on in vitro cultures, and on the multiplication of a B. microti-infected mouse model. The cytotoxicity of compounds was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half maximal inhibitory concentration (IC50) values of TC and CH against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 69.6 ± 2.3, 33.3 ± 1.2, 64.8 ± 2.5, 18.9 ± 1.7, and 14.3 ± 1.6 µM and 138.4 ± 4.4, 60.9 ± 1.1, 82.3 ± 2.3, 27.9 ± 1.2, and 19.2 ± 1.5 µM, respectively. In toxicity assays, TC and CH affected the viability of MDBK, NIH/3T3, and HFF cell lines the with half maximum effective concentration (EC50) values of 293.9 ± 2.9, 434.4 ± 2.7, and 498 ± 3.1 µM and 252.7 ± 1.7, 406.3 ± 9.7, and 466 ± 5.7 µM, respectively. In the mouse experiment, TC reduced the peak parasitemia of B. microti by 71.8% when administered intraperitoneally at 25 mg/kg. Combination therapies of TC-DA and TC-CF were more potent against B. microti infection in mice than their monotherapies. CONCLUSIONS/SIGNIFICANCE: In conclusion, both TC and CH inhibited the growth of Babesia and Theileria in vitro, and TC inhibited the growth of B. microti in vivo. Therefore, TC and CH could be candidates for the treatment of piroplasmosis after further studies.

Secreted parasite Pin1 isomerase stabilizes host PKM2 to reprogram host cell metabolism.

Metabolic reprogramming is an important feature of host-pathogen interactions and a hallmark of tumorigenesis. The intracellular apicomplexa parasite Theileria induces a Warburg-like effect in host leukocytes by hijacking signaling machineries, epigenetic regulators and transcriptional programs to create a transformed cell state. The molecular mechanisms underlying host cell transformation are unclear. Here we show that a parasite-encoded prolyl-isomerase, TaPin1, stabilizes host pyruvate kinase isoform M2 (PKM2) leading to HIF-1α-dependent regulation of metabolic enzymes, glucose uptake and transformed phenotypes in parasite-infected cells. Our results provide a direct molecular link between the secreted parasite TaPin1 protein and host gene expression programs. This study demonstrates the importance of prolyl isomerization in the parasite manipulation of host metabolism.

Lumefantrine and o-choline - Parasite metabolism specific drug molecules inhibited in vitro growth of Theileria equi and Babesia caballi in MASP culture system.

Theileria equi and Babesia caballi are tick-borne apicomplexan haemoprotozoan parasites of equines and are responsible for considerable economic losses to stakeholders. Chemotherapeutic drugs that are available not only require multiple dosages but also prompt multiple organ toxicity in treated host though incapable of clearing parasitaemia completely. In this study, we have screened the in vitro inhibitory efficacy of four different drug molecules (o-choline, DABCO®, lumefantrine and eugenol) against T. equi and B. caballi, targeting different parasite metabolism pathways. Imidocarb dipropionate and diminazene aceturate were used as reference control drugs. The 50% in vitro growth inhibitory concentration (IC50) of lumefantrine, o-choline, DABCO® and eugenol for T. equi were: 30.90 µM; 84.38 µM; 443 µM; 120 µM and for B. caballi growth inhibition were: 5.58 µM; 135.29 µM; 150 µM; 197.05 µM, respectively. Imidocarb dipropionate inhibited the in vitro growth of T. equi at IC50 of 257.5 nM, while diminazene aceturate inhibited the in vitro growth of B. caballi at IC50 of 22 nM. DABCO® and eugenol were not so effective in inhibiting the in vitro growth of T. equi and B. caballi, while lumefantrine and o-choline significantly (p ≤ 0.05) inhibited the in vitro growth of these piroplasms targeting haem digestion and parasite membrane phospholipid synthesis.

Establishment of a mouse-tick infection model for Theileria orientalis and analysis of its transcriptome.

Oriental theileriosis caused by Theileria orientalis is an economically significant disease in cattle farming. The lack of laboratory animal models and in vitro culture systems is a major obstacle in the drive to better understand the biology of this parasite. Notably, research on the sporozoite stage of T. orientalis has rarely been undertaken, although such investigations are of paramount importance for vaccine development based on blocking sporozoite invasion of its host animals. In the present study, we established a mouse-tick infection model for propagating T. orientalis in mice and for producing the sporozoite stage in tick salivary glands. Splenectomized severe combined immunodeficient mice transfused with bovine erythrocytes were infected with T. orientalis. The larval ticks of Haemaphysalis longicornis were then fed on the T. orientalis-infected mice. The piroplasm and sporozoite stages were microscopically observed in the mouse blood and nymphal salivary glands, respectively. The transcriptomics data generated from the piroplasm and sporozoite stages revealed a stage-specific expression pattern for the parasite genes. The mouse-tick infection model and the transcriptomics data it has provided will contribute to a better understanding of T. orientalis biology and will also provide much needed information for the design of effective control measures targeting oriental theileriosis.

The effects of nitidine chloride and camptothecin on the growth of Babesia and Theileria parasites.

The treatment of bovine and equine piroplasmosis is limited to diminazene aceturate (DA) and imidocarb dipropionate. To address this challenge, we need to explore novel drug compounds and targets. Topoisomerases are potential drug targets because they play a vital role in solving topological errors of DNA strands during replication. This study documented the effectiveness of topoisomerase inhibitors, nitidine chloride (NC) and camptothecin (Cpt), on the growth of Babesia and Theileria parasites. The half maximal inhibitory concentrations (IC50s) against B. bovis, B. bigemina, B. caballi, and T. equi were 1.01 ±â€¯0.2, 5.34 ±â€¯1.0, 0.11 ±â€¯0.03, and 2.05 ±â€¯0.4 µM for NC and 11.67 ±â€¯1.6, 4.00 ±â€¯1.0, 2.07 ±â€¯0.6, and 0.33 ±â€¯0.02 µM for Cpt, respectively. The viability experiment revealed that 4, 10, and 4 µM treatments of NC or 48, 8, and 8 µM treatments of Cpt were sufficient to stop the in vitro regrowth of B. bovis, B. bigemina, and B. caballi, respectively. However, T. equi regrew in all of the concentrations used. Moreover, increasing the concentration of NC and Cpt to 16 µM and 1.2 µM (8 × IC50) did not eliminate T. equi. The micrographs of B. bigemina and B. caballi taken at 24 h and 72 h showed deformed merozoites and remnants of parasites within the red blood cell (RBC), respectively. The treatments of 25 mg/kg DA and 20 mg/kg NC administered intraperitoneally and 20 mg/kg NC given orally showed 93.7, 90.7, and 83.6% inhibition against Babesia microti (B. microti), respectively, compared to the untreated group on day 8. In summary, NC and Cpt were effective against Babesia and Theileria parasites in vitro. Moreover, 20 mg/kg NC administered intraperitoneally was as effective as 25 mg/kg DA against B. microti in mice and showed no toxic symptoms in mice. The results indicate that NC may, after further evaluations, prove to be an alternative drug against bovine and equine piroplasmoses.

Assessment of Draxxin® (tulathromycin) as an inhibitor of in vitro growth of Babesia bovis, Babesia bigemina and Theileria equi.

Babesia bovis, Babesia bigemina and Theileria equi are worldwide tick-borne hemoprotozoan that cause diseases characterized by fever, anemia, weight loss and abortion. A common feature of these diseases are transition from acute to chronic phases, in which parasites may persist in the host for life, and becoming a reservoir for tick transmission. The live-attenuated vaccines for B. bovis and B. bigemina are not available for worldwide use due to legal restrictions and other concerns such as potential erythrocyte antigen and pathogen contamination, and a vaccine for T. equi is not available. The use of chemotherapeutics is essential to treat and control these diseases, but several studies have shown the development of drug-resistance by these parasites, and safe and effective alternative drugs are needed. Tulathromycin, a macrolide antibiotic, has proven to be effective against a vast range of bacteria and Plasmodium yoelli, a Babesia and Theileria related intra-erythrocytic apicomplexan. Draxxin® (tulathromycin) is currently licensed to treat infections that cause respiratory diseases in cattle in several countries. In this study, the activity of Draxxin® was tested in vitro on cultured B. bovis, B. bigemina and T. equi. Addition of the drug to in vitro cultures resulted in cessation of parasite replication of the three species tested, B. bovis, B. bigemina and T. equi, with estimated IC50 of 16.7 ±â€¯0.6 nM; 6.2 ±â€¯0.2 nM and 2.4 ±â€¯0.1 nM, respectively, at 72 h. Furthermore, neither parasites nor parasite DNA were detectable in cultures treated with IC100, suggesting Draxxin® is a highly effective anti-Babesia/Theileria drug. Importantly, the IC50 calculated for Draxxin® for the Babesia/Theileria parasites tested is lower that the IC50 calculated for some drugs currently in use to control these parasites. Collectively, the data strongly support in vivo testing of Draxxin® for the treatment of bovine babesiosis and equine piroplasmosis.

In vitro growth inhibition of Theileria equi by bumped kinase inhibitors.

INTRODUCTION: Theileria equi, an etiologic agent of equine piroplasmosis, is a tick-transmitted hemoprotozoan of the phylum Apicomplexa. Recent outbreaks of piroplasmosis in the United States have renewed interest in safe and effective treatment options. Although imidocarb dipropionate (IMD) is the drug of choice for clearance of T. equi, adverse reactions and recently documented resistance support the need for alternative therapeutic strategies. The recently described bumped kinase inhibitors (BKIs) are a new class of compounds that could potentially be used as safe and effective alternatives to IMD. In an initial effort to evaluate this potential, herein we determined the T. equi growth inhibitory activity of 11 BKIs relative to that of IMD and the previously tested BKI 1294. Because some BKIs have known human ether-à-go-go related gene (hERG) channel activity, we also assessed the hERG activity of each compound with the goal to identify those with the highest potency against T. equi coupled with the lowest potential for cardiotoxicity. RESULTS: Six BKIs inhibited T. equi growth in vitro, including the previously evaluated BKI 1294 which was used as a positive control. All six compounds were significantly less potent (higher 50% effective concentration (EC50)) than IMD. Two of those compounds were more potent than BKI 1294 control but had similar hERG activity. Although the remaining three compounds had similar to lower potency than BKI 1294, hERG EC50 was higher for three of them (BKI 1735, BKI 1369 and BKI 1318). CONCLUSIONS: The BKI compounds evaluated in this study inhibited T. equi in vitro and had diverse hERG activity. Based on these considerations, three compounds would be suitable for further evaluation. While these results provide a foundation for future work, in vivo pharmacokinetic, pharmacodynamics, and safety studies are needed before BKI compounds can be recommended for clinical use in T. equi infected horses.

Evaluation of the inhibitory effect of N-acetyl-L-cysteine on Babesia and Theileria parasites.

N-acetyl-L-cysteine is known to have antibacterial, antiviral, antimalarial, and antioxidant activities. Therefore, the in vitro inhibitory effect of this hit was evaluated in the present study on the growth of Babesia and Theileria parasites. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi, and Babesia caballi that were tested was significantly inhibited (P < 0.05) by micromolar concentrations of N-acetyl-L-cysteine. The inhibitory effect of N-acetyl-L-cysteine was synergistically potentiated when used in combination with diminazene aceturate on B. bovis and B. caballi cultures. These results indicate that N-acetyl-L-cysteine might be used as a drug for the treatment of babesiosis, especially when used in combination with diminazene aceturate.

Inhibition of the in vitro growth of Babesia bigemina, Babesia caballi and Theileria equi parasites by trifluralin analogues.

Bovine and equine babesiosis caused by Babesia bovis, Babesia bigemina and Babesia caballi, along with equine theileriosis caused by Theileria equi are global tick-borne hemoprotozoan diseases characterized by fever, anemia, weight losses and abortions. A common feature of these diseases are transition from acute to chronic phases, in which parasites may persist in the hosts for life. Antiprotozoal drugs are important for managing infection and disease. Previous research demonstrated that trifluralin analogues, designated (TFLAs) 1-15, which specifically bind to regions of alpha-tubulin protein in plants and protozoan parasites, have the ability to inhibit the in vitro growth of B. bovis. The inhibitory activity of TFLAs 1-15 minus TFLA 5 was tested in vitro against cultured B. bigemina, B. caballi and T. equi. The four TFLAs with greatest inhibitory activity were then analyzed for hemolytic activity and toxicity against erythrocytes. All TFLAs tested in the study showed inhibitory effects against the three parasite species. TFLA 2, TFLA 11, TFLA 13 and TFLA 14 were the most effective inhibitors for the three species tested, with estimated IC50 between 5.1 and 10.1µM at 72h. The drug's solvent (DMSO/ethanol) did not statistically affect the growth of the parasites nor cause hemolysis. Also, TFLA 2, 13 and 14 did not cause statistically significant hemolytic activity on bovine and equine erythrocytes at 15µM, and TFLA 2, 11 and 13 had no detectable toxic effects on bovine and equine erythrocytes at 15µM, suggesting that these drugs do not compromise erythrocyte viability. The demonstrated ability of the trifluralin analogues to inhibit in vitro growth of Babesia spp. and Theileria equi, and their lack of toxic effects on erythrocytes supports further in vivo testing and eventually their development as novel alternatives for the treatment of babesiosis and theileriosis.

Effects of dihydroorotate dehydrogenase (DHODH) inhibitors on the growth of Theileria equi and Babesia caballi in vitro.

Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), which affects equine production in various parts of the world. However, a safe and effective drug is not currently available for treatment of EP. Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine synthesis pathway and has been known as a novel drug target for several apicomplexan protozoan parasites. In this study, we evaluated four DHODH inhibitors; atovaquone (ATV), leflunomide (LFN), brequinar (Breq), and 7-hydroxy-5-[1,2,4] triazolo [1,5,a] pyrimidine (TAZ) on the growth of T. equi and B. caballi in vitro and compared them to diminacene aceturate (Di) as the control drug. The growth of T. equi and B. caballi was significantly hindered by all inhibitors except TAZ. The half maximal inhibitory concentration (IC50) of ATV, LFN, Breq and Di against T. equi was approximately 0.028, 109, 11 and 40 µM, respectively, whereas the IC50 of ATV, LFN, Breq and Di against B. caballi was approximately 0.128, 193, 5.2 and 16.2 µM, respectively. Using bioinformatics and Western blot analysis, we showed that TeDHODH was similar to other Babesia parasite DHODHs, and confirmed that targeting DHODHs could be useful for the development of novel chemotherapeutics for treatment of EP.