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 (0.005-50 µM), 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 9.57 µM. 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 ± 2.46 µM. 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 5 mg/kg RVT and 8.5 mg/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.

Diminazene aceturate and imidocarb dipropionate-based combination therapy for babesiosis - A new paradigm.

In the present study, the effect of a combination therapy consisting of diminazene aceturate (DA) and imidocarb dipropionate (ID) on the in vitro growth of several parasitic piroplasmids, and on Babesia microti in BALB/c mice was evaluated using a fluorescence-based SYBR Green I test. We evaluated the structural similarities between the regularly used antibabesial medications, DA and ID, and the recently found antibabesial drugs, pyronaridine tetraphosphate, atovaquone, and clofazimine, using atom pair fingerprints (APfp). The Chou-Talalay approach was used to determine the interactions between the two drugs. A Celltac MEK-6450 computerized hematology analyzer was used to detect hemolytic anemia every 96 hours in mice infected with B. microti and in those treated with either mono- or combination therapy. According to the APfp results, DA and ID have the most structural similarities (MSS). DA and ID had synergistic and additive interactions against the in vitro growth of Babesia bigemina and Babesia bovis, respectively. Low dosages of DA (6.25 mg kg-1) and ID (8.5 mg kg-1) in conjunction with each other inhibited B. microti growth by 16.5 %, 32 %, and 4.5 % more than 25 mg kg-1 DA, 6.25 mg kg-1 DA, and 8.5 mg kg-1 ID monotherapies, respectively. In the blood, kidney, heart, and lung tissues of mice treated with DA/ID, the B. microti small subunit rRNA gene was not detected. The obtained findings suggest that DA/ID could be a promising combination therapy for treating bovine babesiosis. Also, such combination may overcome the potential problems of Babesia resistance and host toxicity induced by utilizing full doses of DA and ID.

Development of a promising antigenic cocktail for the global detection of Babesia caballi in horse by ELISA.

In this study, we designed novel truncated Babesia caballi (B. caballi) recombinant proteins from the previously used B. caballi proteins; 134-Kilodalton Protein (rBC134) and Merozoite Rhoptry 48 Protein (rBC48). Then, we evaluated the diagnostic performance of the newly designed proteins when used as a single antigen or when used as cocktail antigen consists of rBC134 full length (rBC134f) + newly designed rBC48 (rBC48t) or newly designed rBC134 (rBC134t) + rBC48t for the detection of B. caballi infection in horse using indirect enzyme-linked immunosorbent assay (iELISA). We used one dose and a half of each antigen in the cocktail formulas. The serum samples were collected from different endemic areas in addition to the sera collected from horses experimentally infected with B. caballi were used in the present study. Cocktail antigen in full dose of (rBC134f + rBC48t) exhibited the highest optical density (OD) values with B. caballi-infected sera and showed the lowest OD values with normal equine sera or B. caballi, and Theileria equi mixed infected sera in comparison with the single antigen. Interestingly, the same cocktail antigen exhibited the highest concordance rate (76.74%) and kappa value (0.79) in the screening of 200 field serum samples collected from five B. caballi endemic countries, including South Africa (n = 40), Ghana (n = 40), Mongolia (n = 40), Thailand (n = 40), and China (n = 40) using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. Moreover, the identified promising cocktail full dose antigen (rBC134f + rBC48t) showed that it can detect the infection as early as the 4th day post-infection in sera collected from experimentally infected horses. The obtained results revealed the reliability of the rBC134f + rBC48t cocktail antigen when used in full dose for the detection of specific antibodies to B. caballi in horses which will be useful for epidemiological surveys and control of equine babesiosis.

Spherical Body Protein 4 from Babesia bigemina: A Novel Gene That Contains Conserved B-Cell Epitopes and Induces Cross-Reactive Neutralizing Antibodies in Babesia ovata.

Bovine babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia. Its main causative agents in the Americas are Babesia bigemina and Babesia bovis, while Babesia ovata affects cattle in Asia. All Babesia species secrete proteins stored in organelles of the apical complex, which are involved in all steps of the invasion process of vertebrate host cells. Unlike other apicomplexans, which have dense granules, babesia parasites instead have large, round intracellular organelles called spherical bodies. Evidence suggests that proteins from these organelles are released during the process of invading red blood cells, where spherical body proteins (SBPs) play an important role in cytoskeleton reorganization. In this study, we characterized the gene that encodes SBP4 in B. bigemina. This gene is transcribed and expressed in the erythrocytic stages of B. bigemina. The sbp4 gene consists of 834 nucleotides without introns that encode a protein of 277 amino acids. In silico analysis predicted a signal peptide that is cleaved at residue 20, producing a 28.88-kDa protein. The presence of a signal peptide and the absence of transmembrane domains suggest that this protein is secreted. Importantly, when cattle were immunized with recombinant B. bigemina SBP4, antibodies identified B. bigemina and B. ovata merozoites according to confocal microscopy observations and were able to neutralize parasite multiplication in vitro for both species. Four peptides with predicted B-cell epitopes were identified to be conserved in 17 different isolates from six countries. Compared with the pre-immunization sera, antibodies against these conserved peptides reduced parasite invasion in vitro by 57%, 44%, 42%, and 38% for peptides 1, 2, 3, and 4, respectively (p < 0.05). Moreover, sera from cattle infected with B. bigemina cattle contained antibodies that recognized the individual peptides. All these results support the concept of spb4 as a new gene in B. bigemina that should be considered a candidate for a vaccine to control bovine babesiosis.

Diminazene aceturate and imidocarb dipropionate-based combination therapy for babesiosis - a new paradigm.

In the present study, the effect of a combination therapy consisting of diminazene aceturate (DA) and imidocarb dipropionate (ID) on the in vitro growth of several parasitic piroplasmids, and on Babesia microti in BALB/c mice was evaluated using a fluorescence-based SYBR Green I test. We evaluated the structural similarities between the regularly used antibabesial medications, DA and ID, and the recently found antibabesial drugs, pyronaridine tetraphosphate, atovaquone, and clofazimine, using atom pair fingerprints (APfp). The Chou-Talalay approach was used to determine the interactions between the two drugs. A Celltac MEK-6450 computerized hematology analyzer was used to detect hemolytic anemia every 96 h in mice infected with B. microti and in those treated with either mono- or combination therapy. According to the APfp results, DA and ID have the most structural similarities (MSS). DA and ID had synergistic and additive interactions against the in vitro growth of Babesia bigemina and Babesia bovis, respectively. Low dosages of DA (6.25 mg kg-1) and ID (8.5 mg kg-1) in conjunction with each other inhibited B. microti growth by 16.5, 32, and 4.5% more than 25 mg kg-1 DA, 6.25 mg kg-1 DA, and 8.5 mg kg-1 ID monotherapies, respectively. In the blood, kidney, heart, and lung tissues of mice treated with DA/ID, the B. microti small subunit rRNA gene was not detected. The obtained findings suggest that DA/ID could be a promising combination therapy for treating bovine babesiosis. Also, such combination may overcome the potential problems of Babesia resistance and host toxicity induced by utilizing full doses of DA and ID.

Imidazo[1,2-a]pyridine: a Highly Potent Therapeutic Agent Clears Piroplasm Infection In Vitro.

BACKGROUND AND PURPOSE: The imidazo[1,2-a] pyridines have huge applications in medicinal chemistry with potent activity against wide spectrum of infectious agents. The efficacy of imidazo[1,2-a]pyridine on the in vitro growth of different piroplasms, including Babesia bovis, B. bigemina, B. divergens, B. caballi, and Theileria equi, was investigated in this study. METHODS: The anti-piroplasm efficacy of imidazo[1,2-a] pyridines was assessed using a fluorescence-based SYBR Green I assay. Furthermore, efficacy of imidazo[1,2-a]pyridine against piroplasms following discontinuation of treatment was also assessed using a viability assay. In vitro cultures of B. bovis and T. equi were used to assess the imidazo[1,2-a]pyridine and diminazene aceturate (DA) interaction. RESULTS: In vitro, imidazo[1,2-a]pyridine inhibited the growth of B. bovis, B. bigemina, B. caballi, and T. equi in a dose-dependent manner. The highest inhibitory effects of imidazo[1,2-a]pyridine were detected on the growth of B. caballi with IC50 value of 0.47 ± 0.07. Interestingly, the efficacy of imidazo[1,2-a]pyridine was higher against B. bigemina (IC50: 1.37 ± 0.15) compared to the positive-control DA (IC50: 2.29 ± 0.06). The viability test findings indicate that imidazo[1,2-a]pyridine had a long-lasting inhibitory effect on bovine Babesia parasites in vitro growth up to 4 days after treatment. Notably, when coupled with DA at 0.75 or 0.50 IC50, a high concentration (0.75 IC50) of imidazo[1,2-a]pyridine produced additive suppression of B. bovis growth which suggest that imidazo[1,2-a]pyridine/DA could be a promising combination therapy for the treatment of B. bovis. CONCLUSION: The obtained encouraging findings pave the way for in vitro and in vivo efficacy trials of imidazo[1,2-a]pyridine derivatives against several piroplasmids.

In Vitro Inhibitory Effects and Bioinformatic Analysis of Norfloxacin and Ofloxacin on Piroplasm.

PURPOSE: The in vitro inhibitory effect of two fluroquinolone antibiotics, norfloxacin and ofloxacin, was evaluated in this study on the growth of several Babesia and Theileria parasites with highlighting the bioinformatic analysis for both drugs with the commonly used antibabesial drug, diminazene aceturate (DA), and the recently identified antibabesial drugs, luteolin, and pyronaridine tetraphosphate (PYR). METHODS: The antipiroplasm efficacy of screened fluroquinolones in vitro and in vivo was assessed using a fluorescence-based SYBR Green I assay. Using atom Pair signatures, we investigated the structural similarity between fluroquinolones and the antibabesial drugs. RESULTS: Both fluroquinolones significantly inhibited (P < 0.05) the in vitro growths of Babesia bovis (B. bovis), B. bigemina, B. caballi, and Theileria equi (T. equi) in a dose-dependent manner. The best inhibitory effect for both drugs was observed on the growth of T. equi. Atom Pair fingerprints (APfp) results and AP Tanimoto values revealed that both fluroquinolones, norfloxacin with luteolin, and ofloxacin with PYR, showed the maximum structural similarity (MSS). Two drug interactions findings confirmed the synergetic interaction between these combination therapies against the in vitro growth of B. bovis and T. equi. CONCLUSION: This study helped in discovery novel potent antibabesial combination therapies consist of norfloxacin/ofloxacin, norfloxacin/luteolin, and ofloxacin/PYR.

Animal models of the immunology and pathogenesis of human babesiosis.

Animal models of human babesiosis have provided a basic understanding of the immunological mechanisms that clear, or occasionally exacerbate, Babesia infection and those pathological processes that cause disease complications. Human Babesia infection can cause asymptomatic infection, mild to moderate disease, or severe disease resulting in organ dysfunction and death. More than 100 Babesia species infect a wide array of wild and domestic animals, and many of the immunologic and pathologic responses to Babesia infection are similar in animals and humans. In this review, we summarize the knowledge gained from animal studies, their limitations, and how animal models or alternative approaches can be further leveraged to improve our understanding of human babesiosis.

In vivo activity and atom pair fingerprint analysis of MMV665941 against the apicomplexan parasite Babesia microti, the causative agent of babesiosis in humans and rodents.

The effect of MMV665941 on the growth of Babesia microti (B. microti) in mice, was investigated in this study using a fluorescence-based SYBR Green I test. Using atom Pair signatures, we investigated the structural similarity between MMV665941 and the commonly used antibabesial medicines diminazene aceturate (DA), imidocarb dipropionate (ID), or atovaquone (AV). In vitro cultures of Babesia bovis (B. bovis) and, Theileria equi (T. equi) were utilized to determine the MMV665941 and AV interaction using combination ratios ranged from 0.75 IC50 MMV665941:0.75 IC50 AV to 0.50 IC50 MMV665941:0.50 IC50 AV. The used combinations were prepared depending on the IC50 of each drug against the in vitro growth of the tested parasite. Every 96 h, the hemolytic anemia in the treated mice was monitored using a Celltac MEK-6450 computerized hematology analyzer. A single dose of 5 mg/kg MMV665941 exhibited inhibition in the B. microti growth from day 4 post-inoculation (p.i.) till day 12 p.i. MMV665941 caused 62.10%, 49.88%, and 74.23% inhibitions in parasite growth at days 4, 6 and 8 p.i., respectively. Of note, 5 mg/kg MMV665941 resulted in quick recovery of hemolytic anemia caused by babesiosis. The atom pair fingerprint (APfp) analysis revealed that MMV665941 and atovaquone (AV) showed maximum structural similarity. Of note, high concentrations (0.75 IC50) of MMV665941 and AV caused synergistic inhibition on B. bovis growth. These findings suggest that MMV665941 might be a promising drug for babesiosis treatment, particularly when combined with the commonly used antibabesial drug, AV.

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

BACKGROUND: An 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. METHODS: A 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). RESULTS: Screening 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). CONCLUSIONS: Inhibitory 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.

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.

The GP-45 Protein, a Highly Variable Antigen from Babesia bigemina, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates.

In B. bigemina, the 45 kilodaltons glycoprotein (GP-45) is the most studied. GP-45 is exposed on the surface of the B. bigemina merozoite, it is believed to play a role in the invasion of erythrocytes, and it is characterized by a high genetic and antigenic polymorphism. The objective of this study was to determine if GP-45 contains conserved B-cell epitopes, and if they would induce neutralizing antibodies. The comparative analysis of nucleotide and amino acids sequences revealed a high percentage of similarity between field isolates. Antibodies against peptides containing conserved B-cell epitopes of GP-45 were generated. Antibodies present in the sera of mice immunized with GP-45 peptides specifically recognize B. bigemina by the IFAT. More than 95% of cattle naturally infected with B. bigemina contained antibodies against conserved GP-45 peptides tested by ELISA. Finally, sera from rabbits immunized with GP-45 peptides were evaluated in vitro neutralization tests and it was shown that they reduced the percentage of parasitemia compared to sera from rabbits immunized with adjuvant. GP-45 from geographically distant isolates of B. bigemina contains conserved B-cell epitopes that induce neutralizing antibodies suggesting that this gene and its product play a critical role in the survival of the parasite under field conditions.

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 (6.25 mg kg-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.

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.

International interlaboratory validation of a nested PCR for molecular detection of Babesia bovis and Babesia bigemina, causative agents of bovine babesiosis.

Babesia bovis and B. bigemina are tick-transmitted parasites causing bovine babesiosis, characterized by significant morbidity and mortality leading to economic losses to the livestock industry in tropical and subtropical regions worldwide. Animals that recover from acute infection remain carriers with low parasitemia acting as a source of transmission, and often escape detection. An improved diagnosis of a B. bovis and/or B. bigemina infection of carrier animals is enabled by the availability of detection methods with high sensitivity. To this end, two nested PCR assays targeting the cytochrome b (cytb) genes of B. bovis and B. bigemina (cytb-nPCR), have been recently developed and an increased sensitivity with respect to reference protocols has been shown (Romero-Salas et al., 2016). In this study, the specificity against a panel of hemoparasites that potentially co-occur with B. bovis and B. bigemina was demonstrated to ensure applicability of the cytb-nPCR assays in a wide range of regions where bovine babesiosis is endemic. Furthermore, we compared both reported cytb-nPCR assays with reference nPCR and qPCR protocols for (i) their capability to detect carrier animals in the field, and (ii) their reproducibility when performed in different laboratories by independent operators. We show that, in a panel of bovine field samples (n = 100), the cytb-nPCR assays detected a considerably higher number of 25% B. bovis and 61% B. bigemina-positive animals compared to 7% and 20% B. bovis and 55% and 49% B. bigemina-positive animals when tested by reference nPCR and qPCR protocols, respectively. Cytb-nPCRs were also found superior in the detection of carrier animals when field samples from Africa were analyzed. In addition, both the B. bovis and B. bigemina cytb-nPCR assays were independently validated in a single blinded study in three laboratories. Importantly, no significant differences in the number/percentage of infected animals was observed using cytb-nPCR assays. In summary, the cytb-nPCR assays detected a considerably higher number of chronically infected B. bovis and B. bigemina carrier animals compared to reference nPCR and qPCR protocols, when applied in different epidemiological field situations. Furthermore, a high reproducibility between laboratories could be demonstrated.

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.