Microti In Mice Research Articles

Antiparasitic activity of FLLL-32 against four Babesia species, B. bovis, B. bigemina, B. divergens and B. caballi, and one Theileria species, Theileria equi in vitro, and Babesia microti in mice.

Introduction: FLLL-32, a synthetic analog of curcumin, is a potent inhibitor of STAT3's constitutive activation in a variety of cancer cells, and its anticancer properties have been demonstrated both in vitro and in vivo. It is also suggested that it might have other pharmacological activities including activity against different parasites. Aim: This study therefore investigated the in vitro antiparasitic activity of FLLL-32 against four pathogenic Babesia species, B. bovis, B. bigemina, B. divergens, and B. caballi, and one Theileria species, Theileria equi. In vivo anti-Babesia microti activity of FLLL-32 was also evaluated in mice. Methods: The FLLL-32, in the growth inhibition assay with a concentration range (0.005-50μM), was tested for it's activity against these pathogens. The reverse transcription PCR (RT-PCR) assay was used to evaluate the possible effects of FLLL-32 treatment on the mRNA transcription of the target B. bovis genes including S-adenosylhomocysteine hydrolase and histone deacetylase. Results: The in vitro growth of B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi was significantly inhibited in a dose-dependent manner (in all cases, p < 0.05). FLLL-32 exhibits the highest inhibitory effects on B. bovis growth in vitro, and it's IC50 value against this species was 9.57μM. The RT-PCR results showed that FLLL-32 inhibited the transcription of the B. bovis S-adenosylhomocysteine hydrolase gene. In vivo, the FLLL-32 showed significant inhibition (p < 0.05) of B. microti parasitemia in infected mice with results comparable to that of diminazene aceturate. Parasitemia level in B. microti-infected mice treated with FLLL-32 from day 12 post infection (pi) was reduced to reach zero level at day 16 pi when compared to the infected non-treated mice. Conclusion: The present study demonstrated the antibabesial properties of FLLL-32 and suggested it's usage in the treatment of babesiosis especially when utilized in combination therapy with other antibabesial drugs.

Evaluation of the Inhibitory Effect of Moringa oleifera Leaves Methanolic Extract against In Vitro Growth of Several Babesia Species and Theileria equi and the In Vivo Growth of Babesia microti.

The current study evaluated the inhibitory effect of Moringa oleifera leaves methanolic extract (MOL) against the in vitro growth of Babesia bovis (B. bovis), B. caballi, B. bigemina, and Theileria equi (T. equi), as well as in vivo growth of B. microti in mice. Active principles of MOL extract were determined using liquid chromatography mass spectrometry (LC-MS). MOL's anti-piroplasm efficacy was assessed both in vitro and in vivo using the SYBR Green I fluorescence assay. Every 96 hours, the hematological parameters, including red blood cell count (RBCs; 104/UL), hemoglobin content (HGB; g/dl), and hematocrit percent (HCT; %), in the treated mice were monitored using a Celltac MEK6450 automated hematological analyzer. LC-MS of MOL revealed that the most abundant polyphenolic catechism found in the MOL extract was isoquercetin and rutin. MOL inhibited B. bovis, B. caballi, B. bigemina, and T. equi in vitro growth in a dose-dependent way, with IC50 values of 45.29 ± 6.14, 19.16 ± 0.45, 137.49 ± 16.07, and 9.29 ± 0.014 μg/ml, respectively. MOL's in vitro antibabesial activity was enhanced when administrated simultaneously with either diminazene aceturate (DA) or MMV665875 compound from malaria box. In mice infected by B. microti, a combination of MOL and a low dose of DA (12.5 mg·kg-1) resulted in a significant (P < 0.05) reduction in B. microti growth. These findings suggest that MOL is an effective herbal anti-piroplasm therapy, especially when combined with a low dosage of either DA or MMV665875.

Evaluating the inhibitory effect of resveratrol on the multiplication of several Babesia species and Theileria equi on in vitro cultures, and Babesia microti in mice.

Introduction: Histone post-translational modification is one of the most studied factors influencing epigenetic regulation of protozoan parasite gene expression, which is mediated by histone deacetylases (KDACs) and acetyltransferases (KATs). Objective and methods: The present study investigated the role of resveratrol (RVT) as an activator of histone deacetylases in the control of various pathogenic Babesia sp. and Theileria equi in vitro, as well as B. microti infected mice in vivo using fluorescence assay. Its role in mitigating the side effects associated with the widely used antibabesial drugs diminazene aceturate (DA) and azithromycin (AZM) has also been investigated. Results: The in vitro growth of B. bovis, B. bigemina, B. divergens, B. caballi and Theileria equi (T. equi) was significantly inhibited (P < 0.05) by RVT treatments. The estimated IC50 values revealed that RVT has the greatest inhibitory effects on B. bovis growth in vitro, with an IC50 value of 29.51 ± 2.46µM. Reverse transcription PCR assay showed that such inhibitory activity might be attributed to resveratrol's stimulatory effect on B. bovis KDAC3 (BbKADC3) as well as its inhibitory effect on BbKATS. RVT causes a significant decrease (P < 0.05) in cardiac troponin T (cTnT) levels in heart tissue of B. microti- infected mice, thereby indicating that RVT may play a part in reducing the cardiotoxic effects of AZM. Resveratrol showed an additive effect with imidocarb dipropionate in vivo. Treatment of B. microti-infected mice with a combined 5mg/kg RVT and 8.5mg/kg ID resulted in an 81.55% inhibition at day 10 postinoculation (peak of parasitemia). Conclusion: Our data show that RVT is a promising antibabesial pharmacological candidate with therapeutic activities that could overcome the side effects of the currently used anti-Babesia medications.

Ascorbic acid co-administration with a low dose of diminazene aceturate inhibits the in vitro growth of Theileria equi, and the in vivo growth of Babesia microti.

In the current investigation, the effect of ascorbic acid on the in vitro growth of several piroplasm including Babesia bovis (Bartonella bovis), Baconia bigemina, B. caballi, and Theileria equi (T. equi), as well as against Brucella microti in mice was assessed. The antipiroplasm efficacy of ascorbic acid in vitro and in vivo was assessed using a fluorescence-based SYBR Green I test. Using atom pair fingerprint (APfp), we investigated the structural similarity between ascorbic acid and the commonly used antibabesial medicines, diminazene aceturate (DA) and imidocarb dipropionate (ID). In vitro cultures of B. bovis and T. equi were utilized to determine the ascorbic acid and DA interaction using the Chou-Talalay method. Ascorbic acid inhibited B. bovis, B. bigemina, T. equi, and B. caballi growth in vitro in a dose-dependent manner. The APfp results revealed that ascorbic acid and DA have a maximum structural similarity (MSS). On a T. equi culture in vitro, ascorbic acid showed a synergistic interaction with DA, with a combination index of 0.28. B. microti growth was decreased by 41% in vivo using ascorbic acid combined with a very low dosage of DA (6.25mgkg-1). The results imply that ascorbic acid /DA could be a viable combination therapy for the treatment of T. equi and that it could be utilized to overcome the resistance of Babesia parasites to full doses of the regularly used antibabesial medication, DA.

Repurposing of the Malaria Box for Babesia microti in mice identifies novel active scaffolds against piroplasmosis

BackgroundAn innovative approach has been introduced for identifying and developing novel potent and safe anti-Babesia and anti-Theileria agents for the control of animal piroplasmosis. In the present study, we evaluated the inhibitory effects of Malaria Box (MBox) compounds (n = 8) against the growth of Babesia microti in mice and conducted bioinformatics analysis between the selected hits and the currently used antibabesial drugs, with far-reaching implications for potent combinations.MethodsA fluorescence assay was used to evaluate the in vivo inhibitory effects of the selected compounds. Bioinformatics analysis was conducted using hierarchical clustering, distance matrix and molecular weight correlation, and PubChem fingerprint. The compounds with in vivo potential efficacy were selected to search for their target in the piroplasm parasites using quantitative PCR (qPCR).ResultsScreening the MBox against the in vivo growth of the B. microti parasite enabled the discovery of potent new antipiroplasm drugs, including MMV396693 and MMV665875. Interestingly, statistically significant (P < 0.05) downregulation of cysteine protease mRNA levels was observed in MMV665875-treated Theileria equi in vitro culture in comparison with untreated cultures. MMV396693/clofazimine and MMV665875/atovaquone (AV) showed maximum structural similarity (MSS) with each other. The distance matrix results indicate promising antibabesial efficacy of combination therapies consisting of either MMV665875 and AV or MMV396693 and imidocarb dipropionate (ID).ConclusionsInhibitory and hematology assay results suggest that MMV396693 and MMV665875 are potent antipiroplasm monotherapies. The structural similarity results indicate that MMV665875 and MMV396693 have a similar mode of action as AV and ID, respectively. Our findings demonstrated that MBox compounds provide a promising lead for the development of new antibabesial therapeutic alternatives.Graphical

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.

MMV020275 and MMV020490, promising compounds from malaria box for the treatment of equine piroplasmosis

Equine piroplasmosis is a tick-transmitted disease that is considered one of the most serious infectious diseases affecting equines. Searching for novel antipiroplasm drugs remains indispensable due to the emergence and spreading of resistant piroplasm parasites against the limited currently used drugs, diminazene aceturate and imidocarb dipropionate. Therefore, novel drugs with specified targets need to be identified and exploited. The inhibitory effects of Medicines for Malaria Venture (MMV) Malaria Box compounds with potent in vitro anti-equine piroplasmosis activity were evaluated against the growth of B. microti in mice in this study. Using a nested PCR assay targeting the B. microti ss-rRNA gene, we investigated the far-reaching impacts of effective combinations to inhibit parasite recrudescence. Using real-time PCR, this study revealed potential targets for the found potent compounds. When used as monotherapy, screening the Malaria Box against the in vivo growth of the B. microti parasite resulted in the discovery of new, potent antipiroplasm medicines, such as MMV020275 and MMV020490. In MMV020275-treated Theileria equi in vitro culture, a statistically significant difference (P<0.05) in the cGMP-dependent protein kinase (PKG) mRNA level was identified as a down-regulation in contrast to non-treated cultures. In conclusion, new potent antipiroplasm drugs, including MMV020275 and MMV020490 are identified. MMV020275 significantly down-regulate the mRNA levels of the PKG gene. Clofazimine enhanced the inhibitory efficacy of MMV compounds which is suggested to use in treatment of animal or human babesiosis in the future.

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.

In vivo antibabesial activity and bioinformatic analysis of compounds derived from the Medicines for Malaria Venture box against Babesia microti

Here, we have evaluated the inhibitory effects of Medicines for Malaria Venture (MMV) Malaria Box compounds that exhibited potent in vitro anti-bovine Babesia efficacy against the growth of B. microti in mice and conducted follow-up investigations of the structural similarity between the identified potent MMV compounds and the commonly used antibabesial drugs was performed using atom Pair fingerprints (APfp). Screening the Malaria Box against the in vivo growth of the B. microti parasite helped with the discovery of new, effective anti-bovine Babesia drugs, including MMV667488, MMV007285, and MMV019881. Of note, MMV019881 exhibited the highest anti-B. microti efficacy in vivo among the screened MMV compounds. The APfp results revealed that the maximum structural similarity (MSS) was observed between MMV007285, diminazene aceturate, and imidocarb dipropionate (ID). In the same way, clofazimine (CF) and MMV667488 showed the MSS with either each other based on the analysis. The distance matrix and molecular weight correlation findings highlight the possible potential antibabesial efficacy of MMV667488, ID, and CF when administrated as a combination therapy. In conclusion, in the current study new potent antibabesial drug, MMV019881 was identified. CF and MMV667488 showed the MSS with either each other based on the hierarchical clustering analysis (HCA) and such relation is confirmed by the distance matrix and molecular weight correlation findings. Such combination therapy might have a potential as a novel regime for treating animal or human babesiosis.

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. 12.5mg/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 25mg/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.

Discovering the Potent Inhibitors Against Babesia bovis in vitro and Babesia microti in vivo by Repurposing the Natural Product Compounds.

In the present study, we screened 502 natural product compounds against the in vitro growth of Babesia (B.) bovis. Then, the novel and potent identified compounds were further evaluated for their in vitro efficacies using viability and cytotoxicity assays. The in vivo inhibitory effects of the selected compounds were evaluated using B. microti “rodent strain” in mice model. Three potent compounds, namely, Rottlerin (RL), Narasin (NR), Lasalocid acid (LA), exhibited the lowest IC50 (half-maximal inhibitory concentration) as follows: 5.45 ± 1.20 μM for RL, 1.86 ± 0.66 μM for NR, and 3.56 ± 1.41 μM for LA. The viability result revealed the ability of RL and LA to prevent the regrowth of treated parasite at 4 × IC50 and 2 × IC50, respectively, while 4 × IC50 of NR was sufficient to stop the regrowth of parasite. The hematology parameters of B. microti in vivo were different in the NR-treated groups as compared to the infected/untreated group. Interestingly, intraperitoneal administration of NR exhibiting inhibition in the growth of B. microti in mice was similar to that observed after administration of the commonly used antibabesial drug, diminazene aceturate (DA) (76.57% for DA, 74.73% for NR). Our findings indicate the richness of natural product compounds by novel potent antibabesial candidates, and the identified potent compounds, especially NR, might be used for the treatment of animal babesiosis.

Artemisia annua and artemisinins are ineffective against human Babesia microti and six Candida sp.

Background:Artemisia annua L.is a well-established medicinal herb used for millennia to treat parasites and fever-related ailments caused by various microbes. Although effective against many infectious agents, the plant is not a miracle cure and there are infections where it has proved ineffective or limited. It is important to report those failures.Methods:Here artemisinin, artesunate and dried leaf slurries of A. annua were used daily for 6 days in vivo against Babesia microti in mice 2 days post infection at 100 µg artemisinin/kg body weight. Parasitemia was measure before and 15 days days post treatment. Artemisinin and extracts of A. annua also were tested in vitro against six Candida sp. at artemisinin concentrations up to 180 µM and growth measured after cultures were fed drugs once at different stages of growth and also after repeated dosing.Results:A. annua, artesunate, and artemisinin were ineffective in reducing or eliminating parasitemia in B. microti-infected mice treated at 100 µg artemisinin/kg body weight. Although the growth of exponential cultures of many of the tested Candida sp. was inhibited, the response was not sustained and both artemisinin and Artemisia were essentially ineffective at concentrations of artemisinin at up to 180 µM of artemisinin.Conclusions:Together these results show that artemisinin, its derivatives, and A. annua are ineffective against B. microti and at least six species of Candida.

Exploration of Serum Marker Proteins in Mice Induced by Babesia microti Infection Using a Quantitative Proteomic Approach.

Babesia microti is a protozoan that mainly parasitizes rodent and human erythrocytes. B. microti infection can result in changes in the expression levels of various proteins in the host serum. To explore the mechanism underlying the regulation of serum proteins by the host during B. microti infection, this study used a data-independent acquisition (DIA) quantitative proteomic approach to perform comprehensive quantitative proteomic analysis on the serum of B. microti-infected mice. We identified and analysed 333 serum proteins during the infectious stage and recovery stage within 30days of infection by B. microti in mice. Through quantitative analysis, we found 57 proteins differentially expressed in the infection stage and 69 proteins differentially expressed in the recovery stage. Bioinformatics analysis revealed that these differentially expressed proteins were mainly concentrated in organelles, cell parts, and extracellular regions that are mainly involved in immune system, metabolic, and cellular processes. Additionally, the differentially expressed proteins mainly had catalytic activity. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis showed that many of the differentially expressed proteins participate in the complement and coagulation cascade reaction, including complement C3, complement FP, and coagulation factor XII. The results of this study can provide more information for the selection of biomarkers for the early clinical monitoring of babesiosis and help in the treatment of babesiosis.

Impact of using pyronaridine tetraphosphate- based combination therapy in the treatment of babesiosis caused by Babesia bovis, B. caballi, and B. gibsoni in vitro and B. microti in mice.

The inhibitory efficacies of pyronaridine tetraphosphate (PYR), when used in combination with two novel and potent antibabesial drugs; clofazimine (CF), and MMV396693 were evaluated in the current study against the growth of Babesia bovis, B. caballi, and B. gibsoni in vitro and B. microti in mice. The in vitro study against the selected parasites was performed using combination of PYR with either CF or MMV396693 in ratios ranged from 0.75:0.75 to 0.25:0.25. Combined application of PYR/MMV396693 revealed additive and indifferent interactions against the in vitro growth of all screened Babesia parasites. PYR in combination with CF, achieved indifferent and antagonistic interactions with all used concentration ratios against the in vitro growth of B. bovis and B. caballi. Treatment with PYR-CF combination therapy caused significant inhibition (P<0.05) of the fluorescence values at days 12, 14, 16, 18, and 22 p.i. in comparison with control mice. Of note, treatment with combination therapy exhibited inhibition in the growth of B. microti (23.16%) greater than those caused by PYR alone. In summary, the obtained results highlight the improvement in the in vivo antibabesial efficacy of PYR when used in combination with CF rather than using PYR alone but such inhibition is still lower than those caused by either DA or CF monotherapies.

In vitro and in vivo growth inhibitory activities of cryptolepine hydrate against several Babesia species and Theileria equi.

Piroplasmosis treatment has been based on the use of imidocarb dipropionate or diminazene aceturate (DA), however, their toxic effects. Therefore, the discovery of new drug molecules and targets is urgently needed. Cryptolepine (CRY) is a pharmacologically active plant alkaloid; it has significant potential as an antiprotozoal and antibacterial under different in vitro and in vivo conditions. The fluorescence assay was used for evaluating the inhibitory effect of CRY on four Babesia species and Theileria equi in vitro, and on the multiplication of B. microti in mice. The toxicity assay was evaluated 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 CRY on Babesia bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 1740 ± 0.377, 1400 ± 0.6, 790 ± 0.32, 600 ± 0.53, and 730 ± 0.025 nM, respectively. The toxicity assay on MDBK, NIH/3T3, and HFF cell lines showed that CRY affected the viability of cells with a half-maximum effective concentration (EC50) of 86.67 ± 4.43, 95.29 ± 2.7, and higher than 100 μM, respectively. In mice experiments, CRY at a concentration of 5 mg/kg effectively inhibited the growth of B. microti, while CRY–atovaquone (AQ) and CRY–DA combinations showed higher chemotherapeutic effects than CRY alone. Our results showed that CRY has the potential to be an alternative remedy for treating piroplasmosis.

Myrrh Oil in Vitro Inhibitory Growth on Bovine and Equine Piroplasm Parasites and Babesia microti of Mice.

The present experimental study was conducted for the assessment of the efficacy of in vitro inhibition of myrrh oil on the propagation of Babesia bovis, B. divergens, B. bigemina, Theileria equi, and B. caballi and in vivo efficacy on B. microti in mice through fluorescence assay based on SYBR green I. The culture of B. divergens B. bovis and was used to evaluate the in vitro possible interaction between myrrh oil and other commercial compound, such as pyronaridine tetraphosphate (PYR), diminazene aceturate (DA), or luteolin. Nested-polymerase chain reaction protocol using primers of the small-subunit rRNA of B. microti was employed to detect any remnants of DNA for studied parasitic species either in blood or tissues. Results elucidated that; Myrrh oil significantly inhibit the growth at 1% of parasitic blood level for all bovine and equine piroplasm under the study. Parasitic regrowth was inhibited subsequently by viability test at 2 µg/mL for B. bigemina and B. bovis, and there was a significant improvement in the in vitro growth inhibition by myrrh oil when combined with DA, PYR, and luteolin. At the same time; mice treated with a combination of myrrh oil/DA showed a higher inhibition in emitted fluorescence signals than the group that challenged with 25 mg/kg of diminazene aceturate at 10 and 12 days post-infection. In conclusion, this study has recommended the myrrh oil to treat animal piroplasmosis, especially in combination with low doses of DA.

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.

Evaluation of the inhibitory effect of ivermectin on the growth of Babesia and Theileria parasites in vitro and in vivo

BackgroundTreatment is the principle way to control and eliminate piroplasmosis. The search for new chemotherapy against Babesia and Theileria has become increasingly urgent due to parasite resistance to current drugs. Ivermectin (IVM) was the world’s first endectocide, capable of killing a wide variety of parasites and vectors, both inside and outside the body. It is currently authorized to treat onchocerciasis, lymphatic filariasis, strongyloidiasis, and scabies. The current study documented the efficacy of IVM on the growth of Babesia and Theileria in vitro and in vivo.MethodsThe fluorescence-based assay was used for evaluating the inhibitory effect of IVM on four Babesia species, including B. bovis, B. bigemina, B. divergens, B. caballi, and Theileria equi, the combination with diminazene aceturate (DA), clofazimine (CF), and atovaquone (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/3 T3), and human foreskin fibroblast (HFF) cell lines.ResultsThe half-maximal inhibitory concentration (IC50) values determined for IVM against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 53.3 ± 4.8, 98.6 ± 5.7, 30.1 ± 2.2, 43.7 ± 3.7, and 90.1 ± 8.1 μM, respectively. Toxicity assays on MDBK, NIH/3 T3, and HFF cell lines showed that IVM affected the viability of cells with a half-maximal effective concentration (EC50) of 138.9 ± 4.9, 283.8 ± 3.6, and 287.5 ± 7.6 μM, respectively. In the in vivo experiment, IVM, when administered intraperitoneally at 4 mg/kg, significantly (p < 0.05) inhibited the growth of B. microti in mice by 63%. Furthermore, combination therapies of IVM–DA, IVM–AQ, and IVM–CF at a half dose reduced the peak parasitemia of B. microti by 83.7%, 76.5%, and 74.4%, respectively. Moreover, this study confirmed the absence of B. microti DNA in groups treated with combination chemotherapy of IVM + DA and IVM + AQ 49 days after infection.ConclusionsThese findings suggest that IVM has the potential to be an alternative remedy for treating piroplasmosis.

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

BackgroundThere 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.MethodsIn this study, the inhibitory effect of EA, β-cyclodextrin ellagic acid (β-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.ResultsThe half-maximal inhibitory concentration (IC50) values of EA and β-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, β-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.ConclusionsTo 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.

Inhibitory effects of methanolic Olea europaea and acetonic Acacia laeta on growth of Babesia and Theileria

Objective: To evaluate the antipiroplasmic activities of methanolic extract of Olea europaea (MOE) and acetonic extract of Acacia laeta (AAL) against Babesia and Theileria parasites in vitro and evaluate the chemotherapeutic effects of these extracts against Babesia (B.) microti in vivo. Methods: Fluorescence assay using SYBR Green 1 nucleic acid stain was used to detect inhibitory effects of the two extracts as well as the combination effects of the two extracts with diminazene aceturate and atovaquone on four Babesia species and Theileria equi in vitro while for in vivo experiments, 8-weekold female BALB/c mice were injected intraperitoneally with 1 × 107B. microti-iRBCs and treated orally at a dose of 150 mg/kg of both extracts. Results: The half maximal inhibitory concentration (IC50) values of AAL against B. bovis, B. bigemina, B. divergens, B. caballi, and Theileria equi were lower than those of MOE extracts. Toxicity assay on Madin–Darby bovine kidney, mouse embryonic fibroblast (MH/3T3), and human foreskin fibroblast cell lines showed that MOE and AAL affected only the viability of Madin–Darby bovine kidney cell line with half maximal effective concentrations (EC50) of (794.7±41.9) and (873.9±17.5) μg/mL, respectively. The oral treatments of MOE and AAL at 150 mg/kg inhibited the growth of B. microti in mice by 80.4% and 64.4%, respectively. The MOE and diminazene aceturate combination showed a higher chemotherapeutic effect than that of monotherapy. Conclusions: MOE and AAL have the potential to be an alternative remedy for treating piroplasmosis. Furthermore, the combination therapy of MOE + DA was more potent against B. microti infection in mice than their monotherapies.