Evaluation of the in vitro and in vivo inhibitory effects of Artemisia herba-alba against the growth of piroplasm parasites.

Objective: The effect of Artemisia herba-alba methanolic extract monotherapy and combination therapies on the in vitro growth of several Babesia and Theileria parasites in vitro and mice was investigated in this study. Materials and Methods: Fluorescence assay using SYBR Green I stain was used to evaluate the antibabesial efficacy inhibitory of A. herba-alba either in vitro or in vivo. Hematological parameters in the treated mice were analyzed using a Celltac MEK-6450 computerized hematology analyzer. Results: Artemisia herba-alba reduced the growth of Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi, and Babesia caballi in vitro in a dose-dependent manner. The in vitro inhibitory impact of A. herba-alba on B. divergens and B. caballi cultures was amplified when combined with either diminazene aceturate (DA). In B. microti-infected mice, a combination therapy consisting of A. herba-alba and a low DA dose inhibited B. microti growth significantly (p < 0.05) better than treatment with 25 mg kg-1 DA. Conclusions: These data show that A. herba-alba, when paired with a modest DA dose, could be a promising medicinal plant for babesiosis treatment.

Pomegranate (Punica granatum) Peel Inhibits the In Vitro and In Vivo Growth of Piroplasm Parasites.

Pomegranate (Punica granatum) peel has seen a rapid surge in attention as a medical and nutritional product over the last decade. The impact of pomegranate peel methanolic extract monotherapy and combination therapy on the in vitro growth of Babesia (B.) bovis, B. bigemina, B. divergens, B. caballi, and Theileria (T.) equi, as well as B. microti in mice, was investigated in this work. Fluorescence-based SYBR green I assay was used for evaluating the inhibitory antibabesial efficacy of pomegranate (Punica granatum) peel against the growth of several piroplasm parasites in vitro and in vivo. Celltac α MEK-6450 computerized haematology analyzer was used for monitoring the haematological parameters of treated mice every 4 days. Pomegranate peel inhibited the in vitro growth of B. bovis, B. bigemina, B. divergens, T. equi, and B. caballi in a dose-dependent manner, with IC50 values of 154.45 ± 23.11, 40.90 ± 9.35, 72.71 ± 14.77, 100 ± 16.20, and 77.27 ± 16.94 µg/ml, respectively. On a B. bovis culture, the in vitro inhibitory effect of pomegranate peel was amplified when it was combined with diminazene aceturate (DA). Combination therapy of pomegranate peel and a low dose of DA (15 mg kg-1) inhibited B. microti growth significantly (P < 0.05) higher than the treatment with the full dose of DA (25 mg kg-1) in B. microti-infected mice. These findings suggest that pomegranate peel might be a potential medicinal plant for babesiosis treatment, especially when combined with a low dosage of DA.

Effect of methanolic extract from Capsicum annuum against the multiplication of several Babesia species and Theileria equi on in vitro cultures, and Babesia microti in mice.

Background and Aim: Piroplasmosis is a serious disease that infects animals, inflicting significant economic losses in the livestock industry and animal trade worldwide. Anti-piroplasm drugs now on the market have demonstrated host toxicity and parasite resistance. As a result, developing more effective and safer anti-piroplasm drugs becomes an urgent issue. This study aimed to evaluate the inhibitory effect of Capsicum annuum methanolic extract (CA) against the growth of Babesia bovis, Babesia divergens, Babesia caballi, and Theileria equi in vitro and against B. microti in mice. Materials and Methods: Fluorescence-based SYBR Green I assay was used to evaluate CA's inhibitory effect in vitro and in vivo when used either as a monotherapy or combined with diminazene aceturate (DA). The hematological parameters (HCT, hemoglobin, and red blood cells counts) were determined in the blood of mice every 96 h using Celltac a MEK-6450 electronic hematology analyzer. Results: The in vitro growth of B. bovis, B. divergens, T. equi, and B. caballi was inhibited by CA in a dose-dependent manner with IC50 values of 4.87±1.23, 44.11±8.03, 8.23±2.54, and 1.26±0.50 mg/mL, respectively. In B. microti-infected mice, a combination therapy consisting of CA and a low dose of DA showed a significant (p<0.05) inhibition of B. microti growth nearly similar to those obtained by treatment with the full dose of DA. Conclusion: The obtained results indicate that CA might be a promising medicinal plant for treating babesiosis, especially when used with a low dose of DA.

Compounds from the Medicines for Malaria Venture Box Inhibit In Vitro Growth of Babesia divergens, a Blood-Borne Parasite of Veterinary and Zoonotic Importance.

Babesiosis is an infectious disease with an empty drug pipeline. A search inside chemical libraries for novel potent antibabesial candidates may help fill such an empty drug pipeline. A total of 400 compounds (200 drug-like and 200 probe-like) from the Malaria Box were evaluated in the current study against the in vitro growth of Babesia divergens (B. divergens), a parasite of veterinary and zoonotic importance. Novel and more effective anti-B. divergens drugs than the traditionally used ones were identified. Seven compounds (four drug-like and three probe-like) revealed a highly inhibitory effect against the in vitro growth of B. divergens, with IC50s ≤ 10 nanomolar. Among these hits, MMV006913 exhibited an IC50 value of 1 nM IC50 and the highest selectivity index of 32,000. The atom pair fingerprint (APfp) analysis revealed that MMV006913 and MMV019124 showed maximum structural similarity (MSS) with atovaquone and diminazene aceturate (DA), and with DA and imidocarb dipropionate (ID), respectively. MMV665807 and MMV665850 showed MMS with each other and with ID. Of note, a high concentration (0.75 IC50) of MMV006913 caused additive inhibition of B. divergens growth when combined with DA at 0.75 or 0.50 IC50. The Medicines for Malaria Venture box is a treasure trove of anti-B. divergens candidates according to the obtained results.

Evaluation of the inhibitory effect of Zingiber officinale rhizome on Babesia and Theileria parasites.

The effect of Zingiber officinale rhizome methanolic extract (ZOR) on the in vitro growth of bovine Babesia (B. bovis, B. bigemina, and B. divergens) and equine piroplasm (B. caballi, and Theileria equi) parasites and on the growth of B. microti in mice was evaluated in this study. The possible in vitro synergistic interaction between ZOR and either diminazene aceturate (DA) or potent Medicines for Malaria Venture (MMV) hits from the malaria box was also investigated. In vitro, ZOR reduced the growth of B. bovis, B. bigemina, T. equi, and B. caballi in a dose-dependent manner. B. divergens was the most susceptible parasite to the in vitro inhibitory effect of ZOR. DA and MMV compounds enhanced the in vitro inhibitory antibabesial activity of ZOR. 12.5 mg/kg DA when administrated in combination with ZOR in mice exhibited a significant inhibition (P < 0.05) in B. microti growth better than those observed after treatment with 25 mg/kg DA monotherapy. These findings suggest that ZOR could be a viable medicinal plant for babesiosis treatment, particularly when combined with a modest dose of either DA or powerful anti-B. bigemina MMV hits.

In vitro screening of novel anti-Babesia gibsoni drugs from natural products.

Babesia gibsoni is a tick-transmitted intraerythrocytic apicomplexan parasite that causes babesiosis in dogs. Due to the strong side effects and lack of efficacy of current drugs, novel drugs against B. gibsoni are urgently needed. Natural products as a source for new drugs is a good choice for screening drugs against B. gibsoni. The current study focuses on identifying novel potential drugs from natural products against B. gibsoniin vitro. Parasite inhibition was verified using a SYBR green I-based fluorescence assay. A total of 502 natural product compounds were screened for anti-B. gibsoni activity in vitro. Twenty-four compounds showed high growth inhibition (>80%) on B. gibsoni and 5 plant-derived compounds were selected for further study. The half-maximal inhibitory concentration (IC50) values of lycorine (LY), vincristine sulfate (VS), emetine·2HCl (EME), harringtonine (HT) and cephaeline·HBr (CEP) were 784.4 ± 3.3, 643.0 ± 2.8, 253.1 ± 1.4, 23.4 ± 1.2, and 108.1 ± 4.3 nM, respectively. The Madin-Darby canine kidney (MDCK) cell line was used to assess cytotoxicity of hit compounds. All compounds showed minimal toxicity to the MDCK cells. The effects of hit compounds combined with diminazene aceturate (DA) on B. gibsoni were further evaluated in vitro. VS, EME, HT or CEP combined with DA showed synergistic effects against B. gibsoni, whereas LY combined with DA showed an antagonistic effect against B. gibsoni. The results obtained in this study indicate that LY, VS, EME, HT and CEP are promising compounds for B. gibsoni treatment.

Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

BACKGROUND: Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. METHODS: Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. RESULTS: Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. CONCLUSIONS: Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

Gas chromatography-mass spectrometry analysis, phytochemical screening and antiprotozoal effects of the methanolic Viola tricolor and acetonic Laurus nobilis extracts.

BACKGROUND: The antiprotozoal and antioxidant activities of Viola tricolor and Laurus nobilis have been reported recently. Thus, the existing study pursued to assess the growth inhibition effect of methanolic extract of V. tricolor (MEVT) and acetonic extract of L. nobilis (AELN) against five Babesia parasites and Theileria equi in vitro and in vivo. RESULTS: MEVT and AELN suppressed Babesia bovis, B. bigemina, B. divergens, B. caballi, and T. equi growth at half-maximal inhibitory concentration (IC50) values of 75.7 ± 2.6, 43.3 ± 1.8, 67.6 ± 2.8, 48 ± 3.8, 54 ± 2.1 µg/mL, and 86.6 ± 8.2, 33.3 ± 5.1, 62.2 ± 3.3, 34.5 ± 7.5 and 82.2 ± 9.3 µg/mL, respectively. Qualitative phytochemical estimation revealed that both extracts containing multiple bioactive constituents and significant amounts of flavonoids and phenols. The toxicity assay revealed that MEVT and AELN affected the mouse embryonic fibroblast (NIH/3 T3) and Madin-Darby bovine kidney (MDBK) cell viability with half-maximum effective concentrations (EC50) of 930 ± 29.9, 1260 ± 18.9 µg/mL, and 573.7 ± 12.4, 831 ± 19.9 µg/mL, respectively, while human foreskin fibroblasts (HFF) cell viability was not influenced even at 1500 µg/mL. The in vivo experiment revealed that the oral administration of MEVT and AELN prohibited B. microti multiplication in mice by 35.1 and 56.1%, respectively. CONCLUSIONS: These analyses indicate the prospects of MEVT and AELN as good candidates for isolating new anti-protozoal compounds which could assist in the development of new drug molecules with new drug targets.

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.

Phytochemical Screening and Antiprotozoal Effects of the Methanolic Berberis vulgaris and Acetonic Rhus coriaria Extracts.

Berberis vulgaris (B. vulgaris) and Rhus coriaria (R. coriaria) have been documented to have various pharmacologic activities. The current study assessed the in vitro as well as in vivo inhibitory efficacy of a methanolic extract of B. vulgaris (MEBV) and an acetone extract of R. coriaria (AERC) on six species of piroplasm parasites. The drug-exposure viability assay was tested on three different cell lines, namely mouse embryonic fibroblast (NIH/3T3), Madin-Darby bovine kidney (MDBK) and human foreskin fibroblast (HFF) cells. Qualitative phytochemical estimation revealed that both extracts containing alkaloid, tannin, saponins and terpenoids and significant amounts of flavonoids and polyphenols. The GC-MS analysis of MEBV and AERC revealed the existence of 27 and 20 phytochemical compounds, respectively. MEBV and AERC restricted the multiplication of Babesia (B.) bovis, B. bigemina, B. divergens, B. caballi, and Theileria (T.) equi at the half-maximal inhibitory concentration (IC50) of 0.84 ± 0.2, 0.81 ± 0.3, 4.1 ± 0.9, 0.35 ± 0.1 and 0.68 ± 0.1 µg/mL and 85.7 ± 3.1, 60 ± 8.5, 90 ± 3.7, 85.7 ± 2.1 and 78 ± 2.1 µg/mL, respectively. In the cytotoxicity assay, MEBV and AERC inhibited MDBK, NIH/3T3 and HFF cells with half-maximal effective concentrations (EC50) of 695.7 ± 24.9, 931 ± 44.9, ˃1500 µg/mL and 737.7 ± 17.4, ˃1500 and ˃1500 µg/mL, respectively. The experiments in mice showed that MEBV and AERC prohibited B. microti multiplication at 150 mg/kg by 66.7% and 70%, respectively. These results indicate the prospects of these extracts as drug candidates for piroplasmosis treatment following additional studies in some clinical cases.

Assay methods for in vitro and in vivo anti-Babesia drug efficacy testing: Current progress, outlook, and challenges.

Absence of an effective high-throughput drug-screening system for Babesia parasites is considered one of the main causes for the presence of a wide gap in the treatment of animal babesiosis when compared with other hemoprotozoan diseases, such as malaria. Recently, a simple, accurate, and automatic fluorescence assay was established for large-scale anti-Babesia (B. bovis, B. bigemina, B. divergens, B. caballi and T. equi) drug screening. Such development will facilitate anti-Babesia drug discovery, especially in the post-genomic era, which will bring new chemotherapy targets with the completion of the Babesia genome sequencing project currently in progress. In this review, we present the current progress in the various assays for in vitro and in vivo anti-Babesia drug testing, as well as the challenges, highlighting new insights into the future of anti-Babesia drug screening.

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.

Screening the Medicines for Malaria Venture Pathogen Box against piroplasm parasites.

Diminazene aceturate (DA) and imidocarb dipropionate are commonly used in livestock as antipiroplasm agents. However, toxic side effects are common in animals treated with these two drugs. Therefore, evaluations of novel therapeutic agents with high efficacy against piroplasm parasites and low toxicity to host animals are of paramount importance. In this study, the 400 compounds in the Pathogen Box provided by the Medicines for Malaria Venture foundation were screened against Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi. A fluorescence-based method using SYBR Green 1 stain was used for initial in vitro screening and determination of the half maximal inhibitory concentration (IC50). The initial in vitro screening performed using a 1 µM concentration as baseline revealed nine effective compounds against four tested parasites. Two "hit" compounds, namely MMV021057 and MMV675968, that showed IC50 < 0.3 µM and a selectivity index (SI)> 100 were selected. The IC50s of MMV021057 and MMV675968 against B. bovis, B. bigemina, T. equi and B. caballi were 23, 39, 229, and 146 nM, and 2.9, 3, 25.7, and 2.9 nM, respectively. In addition, a combination of MMV021057 and DA showed additive or synergistic effects against four tested parasites, while combinations of MMV021057 with MMV675968 and of MMV675968 with DA showed antagonistic effects. In mice, treated with 50 mg/kg MMV021057 and 25 mg/kg MMV675968 inhibited the growth of Babesia microti by 54 and 64%, respectively, as compared to the untreated group on day 8. Interestingly, a combination treatment with 6.25 mg/kg DA and 25 mg/kg MMV021057 inhibited B. microti by 91.6%, which was a stronger inhibition than that by single treatments with 50 mg/kg MMV021057 and 25 mg/kg DA, which showed 54 and 83% inhibition, respectively. Our findings indicated that MMV021057, MMV675968, and the combination treatment with MMV021057 and DA are prospects for further development of antipiroplasm drugs.

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.

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.

Flavonoids isolated from the flowers of Pulsatilla flavescens and their anti-piroplasm activity.

Pulsatilla species are known as "Yargui", and their flowers are traditionally used in Mongolia as a tonic and for the treatment of inflammatory diseases. By chemical investigation of P. flavescens flowers, 21 flavonoids, including a new chalcone C-glucoside, chalconaringenin 2'-O-ß-D-glucopyranosyl-5'-ß-D-glucopyranoside, and two new flavanone C-glucosides, (2R)- and (2S)-naringenin 8-ß-D-glucopyranosyl-4'-O-ß-D-glucopyranoside, were isolated. The absolute configurations of the seven flavanone glucosides were elucidated by ECD spectra. For the isolated compounds, inhibitory activity against Babesia caballi and Theileria equi, which cause fatal diseases in horses, was estimated. Although most of the isolated chalcone and flavanone derivatives did not show any anti-piroplasm activity, all the isolated flavone and flavonol derivatives showed moderate effects against B. caballi and/or T. equi.

Performance and consistency of a fluorescence-based high-throughput screening assay for use in Babesia drug screening in mice.

In this study, we evaluated the validity of a fluorescence-based assay using SYBR Green I (SG I) stain for screening antibabesial compounds against B. microti in mice. Two different hematocrits (HCTs; 2.5% and 5%) were used. Correlating relative fluorescence units (RFUs) with parasitemia showed significant linear relationships with R2 values of 0.97 and 0.99 at HCTs of 2.5% and 5%, respectively. Meanwhile, the Z' factors in a high-throughput screening (HTS) assay were within the permissible limit (≥0.5) at 2.5% HCT and lower than this value at 5% HCT. Taken together, the highest signal-to-noise (S/N) ratios were obtained at 2.5% HCT; therefore, we concluded that 2.5% was the best HCT for applying fluorescence assay in antibabesial drug screening in mice. Additionally, positive control mice and those treated with diminazene aceturate, pyronaridine tetraphosphate, and an allicin/diminazene aceturate combination showed peak parasitemia and fluorescence values on the same day post-inoculation. Moreover, using different concentrations of SG I revealed that the optimal concentration was 2x. In summary, considering that all experiments were applied under optimal laboratory conditions, fluorescence assay at 2.5% HCT using 2x SG I for B. microti parasite offers a novel approach for drug screening in mice.

Molecular Characterization of Babesia bovis M17 Leucine Aminopeptidase and Inhibition of Babesia Growth by Bestatin.

The M17 leucine aminopeptidase (M17LAP) enzymes of the other apicomplexan parasites have been characterized and shown to be inhibited by bestatin. Though Babesia bovis also belongs to the apicomplexan group, it is not known whether its M17LAP could display similar biochemical properties as well as inhibition profile. To unravel this uncertainty, a B. bovis M17LAP (BbM17LAP) gene was expressed in Escherichia coli , and activity of the recombinant enzyme as well as its inhibition by bestatin were evaluated. The inhibitory effect of the compound on growths of B. bovis and Babesia gibsoni in vitro was also determined. The expression of the gene fused with glutathione S-transferase (GST) yielded approximately 81-kDa recombinant BbM17LAP (rBbM17LAP). On probing with mouse anti-rBbM17LAP serum, a green fluorescence was observed on the parasite cytosol on confocal laser microscopy, and a specific band greater than the predicted molecular mass was seen on Western blotting. The Km and Vmax values of the recombinant enzyme were 139.3 ± 30.25 and 64.83 ± 4.6 µM, respectively, while the Ki was 2210 ± 358 µM after the inhibition. Bestatin was a more potent inhibitor of the growth of B. bovis [IC50 (50% inhibition concentration) = 131.7 ± 51.43 µM] than B. gibsoni [IC50 = 460.8 ± 114.45 µM] in vitro. The modest inhibition of both the rBbM17LAP activity and Babesia parasites' growth in vitro suggests that this inhibition may involve the endogenous enzyme in live parasites. Therefore, BbM17LAP may be a target of bestatin, though more studies with other aminopeptidase inhibitors are required to confirm this.

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.