Camellia sinensis Aqueous Extract: A Promising Candidate for Hepatic Eimeriosis Treatment in Rabbits.

Eimeria stiedae (E. stiedae) is a common coccidian species that infects the liver and causes economic losses for the rabbit industry. This study aimed to determine the efficiency of green tea aqueous extract (GTE) as a natural treatment for eimeriosis caused by E. stiedae. Male rabbits Cuniculus L. (Oryctolagus) of the New Zealand White rabbit strain (4-4.5 months) were used, as they are suitable for research and conducting experiments. Thirty rabbits were allocated into six groups, with five rabbits in each group; the G1 group (non-infected untreated) served as a negative control group; the G2 group was not infected and treated with 250 mg GTE; the G3 group was not infected and treated with 500 mg GTE; the G4 group was untreated and was infected with 3 × 104 Sporulated E. stiedae oocysts, which served as a positive control group; the G5 group was infected and treated with 250 mg GTE; and the G6 group was infected and treated with 500 mg GTE. The hematological and biochemical analyses of each group of rabbit sera were carried out. Phytochemical analysis was performed to evaluate the active components in GTE leaves using the following methods: IR spectroscopy, liquid chromatography-mass spectrometry (LC-MS), energy-dispersive X-ray spectroscopy (EDX), and scanning electron microscopy. The infected rabbit groups treated with GTE at both doses of 250 and 500 mg/kg exhibited a significant decrease in the extent of E. stiedae oocyst shedding compared with the infected untreated group at 14, 21, and 28 days post-infection. Also, treatment with green tea showed improvement in liver weight compared with the enlarged livers of infected, untreated rabbits. The disturbance in serum liver enzymes' gamma-glutamyl transferase (GGT) and aspartate aminotransferase (AST/GOT) levels, as well as serum glucose, potassium, uric acid, cholesterol, and urea levels, were improved after the treatment of infected rabbit groups with green tea compared with the infected untreated group. Moreover, in this study, the images of the egg stages of the parasite were taken using a fluorescence microscope at 25 µm and 26 µm magnifications. This study provides promising results for the effective cell absorption of the aqueous extract of green tea, which was confirmed in the analyzed images using a scanning electron microscope at 5 µm and 20 µm magnifications.

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.

Pycnogenol a promising remedy for diabetic keratopathy in experimentally induced corneal alkali burns in diabetic rats.

AIM: This study aimed to investigate the efficiency of topically applied pycnogenol (PYC) in healing the standardized alkaline corneal ulcer in diabetic and normal rats. MATERIALS AND METHODS: The corneal alkali-burn injury (CA-I) model was unilaterally developed in Wistar rats by filter paper saturated with 0.01 M of NaOH and touching the eyes for 45 s. Rats were divided into four groups: Normal control (NC), normal PYC (NPYC), diabetic control (DC), and diabetic PYC (DPYC). Both NPYC and DPYC groups were daily treated with PY eye drops three times, whereas NC and DC ones were treated with ordinary saline for six successive days. RESULTS: The wound healing of corneal epithelial was improved in the NPYC group compared to the NC group. Meanwhile, it was significantly improved (P < 0.05) in the DPYC group than in the DC group. Histological examination revealed that corneal re-epithelialization was more accomplished in the DPYC group than in the DC group. In addition, the inflammatory cells were augmented in the DC group more than those in the DPYC one. CONCLUSION: The findings obtained revealed the efficiency of PYC for enhancing the corneal re-epithelialization and reducing the inflammatory reaction post alkali burn in rats, and thus it could be beneficially valuable as a treatment for the diabetic keratopathy.

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.

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.

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.

Comparative therapeutic effect of steroidal and non-steroidal anti-inflammatory drugs on pro-inflammatory cytokine production in water buffalo calves (Bubalus bubalis) naturally infected with bronchopneumonia: a randomized clinical trial.

In the current study, we compared the therapeutic effects of a non-steroidal and a steroidal anti-inflammatory drug on the production of pro-inflammatory cytokines, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-12p40 (IL-12p40), interferon gamma (IFNγ), and tumor necrosis factor alpha (TNF-α) in the blood of water buffalo (Bubalus bubalis) calves naturally infected by bronchopneumonia. Twenty-seven buffalo calves (7 ± 2-month-old, 163 ± 12 kg) reared in smallholder farms in El-Dakahlia province in Egypt were identified to have bronchopneumonia and randomly allocated into three equal groups. Ten clinically healthy buffalo calves with negative bronchoalveolar lavage results were served as negative control. Diseased calves were treated with tulathromycin alone, a combination of tulathromycin with dexamethasone (steroidal anti-inflammatory drug) or tulathromycin with flunixin meglumine (non-steroidal anti-inflammatory drug). The results revealed significant elevations (P < 0.05) in the production of selected cytokines in all diseased calves in comparison with healthy animals. Six days post-treatment, a significant inhibition (P < 0.05) in the production of all assessed cytokines was observed in the blood of all treated calves. Interestingly, the serum concentrations of IL-1ß and IL-12p40 were returned to the normal levels in pneumonic calves treated with the combination therapy of tulathromycin and flunixin meglumine. A strong significant positive correlation (P < 0.05) was detected between clinical sum scoring and IL-12p40 and TNF-α concentrations. The obtained results indicate the selectively potent anti-inflammatory effect of flunixin meglumine on the production of pro-inflammatory cytokines in pneumonic buffalo calves and highlight the efficacy of flunixin meglumine in the treatment of bronchopneumonia in buffalo calves when used in combination with tulathromycin.

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.