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.


Introduction
In the worldwide livestock sector and animal trade, Babesia and Teileria, which are blood parasites transmitted by ticks and infect erythrocytes, result in considerable economic losses [1].Te disease's symptoms include sickness, lethargy, hemoglobinuria, jaundice, and mortality [2].Te most prevalent parasitic infestations in cattle are Babesia bovis (B.bovis) and B. bigemina [2].Te main causative agents of the disease in horses are B. caballi and Teileria equi (T.equi) [3].Unfortunately, no laboratory animals are accessible for cattle or equine Babesia infections.However, a rodent Babesia model infected with B. microti is used for evaluation new drugs [4].
Currently used anti-piroplasm drugs; imidocarb dipropionate and diminazene aceturate (DA) did not exhibit complete clearance of the infection from the infected animals [1].As a result, the search for alternative anti-piroplasm medicines becomes critical.In this regard, the antipiroplasm efcacy of Moringa oleifera leaves (MOL) methanolic extract is evaluated in the present study.Moringa oleifera Lam is the most extensively distributed Moringaceae family plant, with a wide range of medicinal and dietary benefts all over the world [5].Indeed, MOL is rich in natural cancer-preventive compounds and is considered a good source of macro-and micronutrients [4].MOL also has antibacterial [6,7], antimalarial [7], and anti-trypanosome [8] properties.However, there has been no research work on the efcacy of MOL extracts as an anti-piroplasm therapy.As a result, we investigated MOL's potency as an anti-piroplasm candidate against the in vitro growth of bovine cattle and equine piroplasm parasites, in addition to B. microti infection in mice.

Ethical Approval.
Te Animal Care and Use Committee at Obihiro University of Agriculture and Veterinary Medicine gave its approval to each study's experimental protocols (Approval No. 27-65).Te Fundamental Guidelines for the Proper Conduct of Animal Experiments and Related Activities at Academic Research Institutions, published by Japan's Ministry of Education's Culture, Sports, Science, and Technology, were followed in all the experiments.Te pathogen experiment's IDs were as follows: (bovine Babesia: 201708-4; equine piroplasma parasites: 201910-2; Babesia microti: 201709-05).
MOL was evaluated against the growth of B. bovis (Texas strain), B. bigemina (Argentina strain), B. caballi [10], and T. equi (United States Department of Agriculture) [10] parasites using a fuorescence spectrophotometer [3,12].MOL concentrations of 0.0005, 0.001, 0.005, 0.01, 0.025, 0.50, 1, 5, 10, and 25 mg/ml were utilized.For the in vitro investigation, the commonly used antibabesial DA drug was used as a control [12].As previously described in our study [13], the viability assay was utilized to follow up the in vitro regrowth of all screened parasites after MOL treatment was stopped.Te delayed-death efect of IC 99 MOL on B. bigemina in vitro growth was also assessed [9].
Te inhibitory efect of combination therapy consisting of MOL/DA was investigated against the in vitro growth of equine piroplasm and bovine Babesia parasites.Furthermore, MOL and potent MMV compounds (MMV665941, MMV396693, MMV006787, MMV665810, MMV007092, MMV085203, MMV666093, and MMV665875) from malaria box [2,14] were assessed against the in vitro growth of B. bovis (Babesia spp.exhibited the highest IC 50 among screened bovine Babesia parasites).Each experiment was performed in triplicates.

MOL In Vivo Inhibitory Assay and B. microti PCR Detection in Mice.
A fuorescence assay was used to evaluate the in vivo inhibitory efcacy of a MOL in mice infected with B. microti (Munich strain) [15].Twenty-fve female BALB/c mice (CLEA Japan, Tokyo, Japan) were used and divided equally into fve groups.Nontoxic doses from MOL either as monotherapy or combined therapy with DA were selected depending on previous study evaluated the antimalarial Activities of MOL extract against Plasmodium berghei ANKA infection in mice [8]. 10 µL of venous blood was collected from the tail of each mouse every four days and used for determination the selected hematological variables using a Celltac MEK-6450 automatic hematological analyzer (Nihon Kohden Corporation, Tokyo, Japan).Te potential of the MOL/DA combination to clear the infection by B. microti from mice's bodies was tested on day 44 post infection using nested PCR assay targeting the B. microti small isoform rRNA (ss-rRNA) gene [4,9].Each experiment was repeated two times.

Statistical Analysis.
A one-way ANOVA test was performed using GraphPad Prism (version 5.0 for Windows; GraphPad Software, Inc., San Diego, CA, USA) to detect the signifcant diferences between the analyzed groups.Statistics were signifcant at P values <0.05.

MOL Methanolic Extract Inhibits the In Vitro Growth of
Babesia and Teileria.MOL exhibited the greatest inhibitory efect on the growth of B. bigemina and B. caballi, followed by B. bovis, according to the computed IC 50 s (Table 1).0.025 mg/mL MOL signifcantly inhibited (P < 0.05) the in vitro growth of B. bovis and B. bigemina (Figure 1).Furthermore, MOL treatments of 0.25 and 0.0005 mg/mL signifcantly inhibited (P < 0.05) T. equi and B. caballi growth, respectively (Figure 1).B. bovis and T. equi in vitro regrowth was inhibited at a dosage of 1 mg/mL in the subsequent viability test (Figures 1(a) and 1(c)).Notably, 0.5 mg/mL MOL inhibited B. bigemina in vitro regrowth (Figure 1(b)).At 5 mg/mL MOL, B. caballi regrowth was reduced (Figure 1(d)).Te nonsignifcant diference (P > 0.05) between the DMSO-containing positive control well and the untreated wells demonstrates that the diluent did not afect the efectiveness of the MOL methanolic extract.Furthermore, erythrocytes pretreated with a high dose of MOL methanolic extract 25 mg/mL showed no infuence on erythrocyte shape as compared to non-treated erythrocytes (Figure S1).
To study the delayed-death efect, the test was performed on the most susceptible Babesia parasite to the inhibitory efect of MOL, B. bigemina.Te parasite was treated with IC 99 MOL for 24 hours.Ten, the emitted fuorescence signals were determined.Peak fuorescence levels for untreated culture were reported at 72 and 96 hours (Figure 2(a)).At 24 hours, the IC 99 MOL methanolic extract resulted in 75.22% growth of B. bigemina (Figure 2(b)).Te inhibitory impact of MOL extract was then reduced to 68.16% after 48 hours.

DA and MMV Hits Impact the MOL Methanolic Extract's
In Vitro Efectiveness.MOL combined with either DA or MMV was investigated against various Babesia species.MOL/DA combination exhibited a higher inhibitory efect on the growth of B. bovis and equine Babesia/Teileria infections versus DA monotherapy at M6 (1/4 MOL: 12 DA) and M8 (1/8 MOL: 12 DA), respectively (Table 2).Such fndings confrmed MOL's potential anti-Babesia impact, particularly when prescribed in low dosage concurrently with DA.

MOL Methanolic Extract Clears B. microti Infection in
Mice.MOL's in vivo inhibitory activity against B. microti was tested in a mouse model.When compared to the positive control group, MOL treated mice demonstrated a signifcant suppression (P < 0.05) in the generated fuorescence signals after days 8 to 20 p.i (Figure 3(a)).Peak fuorescence values in 100 mg•kg −1 MOL therapy and 80 mg•kg −1 MOL plus 12.5 mg•kg −1 DA were 900.43 and 578.08 at 12 days p.i, respectively (Figure 3(a)).Whereas, peak fuorescence levels in the positive control group and 25 mg•kg −1 DA at 10 days p.i. were 2623 and 733.14, respectively (Figure 3(a)).Treatment with 100 mg•kg −1 MOL monotherapy for fve days resulted in 74.06% inhibition, compared to 72.04% inhibition with 25 mg•kg −1 DA (Figure 3(a)).At 10 and 12 days p.i, the inhibition in the fuorescence values was higher in mice treated with MOL/DA combination than in animals treated with 25 mg•kg −1 DA (Figure 3(a)).Oral injections of 80 mg•kg −1 MOL combined with a subcutaneous dosage of 12.5 mg•kg −1 DA inhibited the parasite growth at 10 and 12 days p.i by 78.57% and 75.47%, respectively, compared to 72.04% and 72.66% inhibitions in the presence of 25 mg•kg −1 DA at 10 and 12 days p.i. (Figure 3(a)).
MOL combined with a low dose of DA normalized the evaluated hematological variables almost identically to those treated with 25 mg•kg −1 DA (Figure 4).Such data demonstrated the antibabesial efcacy of MOL/DA combination therapy.
On day 44 post-infection, a nested PCR targeting the B. microti ss-rRNA gene was performed to detect parasite remains in the blood of treated mice.It should be noted that the parasite gene was not identifed in the blood of mice given the MOL/DA combination therapy (Figure 3

Discussion
In the present study, the in vitro and in vivoanti-piroplasm inhibitory efect of MOL was examined.Compared to the recently evaluated herbal antibabesial drugs, pomegranate (Punica granatum) peel [11], turmeric (Curcuma longa), and Zingiber ofcinale rhizome [9,10], MOL exhibits lower IC 50 values for the screened Babesia species (Table 1).Furthermore, the obtained data revealed that bovine or horse RBCs were unafected by extremely high MOL methanolic extract concentrations.

Journal of Tropical Medicine
(1) Nested PCR experiment was used to test the ability of MOL/DA combined therapy to further remove parasite nucleic acid from the animal's blood.Combination therapy was efective in removing parasite nucleic acid from the blood of treated mice.Such fnding is similar with the efcacy of enoxacin/DA [4], PYR/DA [9].(2) Bovis in vitro growth suppression was increased after treatment with MOL/MMV compounds, particularly MOL/MMV665875.Rizk et al. [13] revealed that the cysteine protease (CP) gene could be one of the targets of MMV665875 (Probe similar chemical) for in vitro growth inhibition of several protozoan parasites.CPs are ubiquitous in all living species and are required for many protozoan parasites to enter the cells of their hosts [11,22].For Plasmodium, CPs play an important role in parasite egression by degrading both hemoglobin and erythrocyte cytoskeletal proteins, causing the infected erythrocyte to rupture [10].As a result, CPs provide interesting new therapeutic targets for a variety of protozoan illnesses, including trypanosomiasis [10], malaria [11], schistosomiasis [23], and leishmaniasis [24].A prior in vitro investigation [20] for piroplasm parasites showed the of CPs in B. bovis.Following that, the genome project [25] established the existence of CPs in Babesia and described CP in parasites of B. bovis.Furthermore, Okubo et al. [20] proposed that CPs play an important role in B. bovis invasion of host RBCs.Furthermore, Ascencio et al. [26] revealed that T. equi had an unusually large number of C1A-CP paralogs, which could be associated with the evolution of the schizont stage.However, the precise role of babesial CPs is unknown.Te fndings suggest that MOL may be targeted to the CP gene in the Babesia parasite.Further investigation is required to determine the mechanism of improvement in the inhibitory impact of MOL when combined with the screened MMV hits, particularly MMV665875.Although the current study demonstrated an improvement in the in vitro inhibitory efect of MOL when combined with diferent MMV    Journal of Tropical Medicine hits from the malaria box on B. bovis growth, the synergetic or antagonistic relationships between these combination therapies against the growth of B. microti in mice have not yet been evaluated.As a result, additional research is required to assess the potential inhibitory efects of these combinations on the growth of B. microti in a mouse model.

Conclusions
Babesia bigemina and B. caballi were the most sensitive parasites to the in vitro inhibitory efect of MOL, followed by B. bovis.MOL in combination with MMV665875 exhibited the highest inhibitory efcacy against B. bovis growth.In vitro, the combination of MOL and DA suppressed piroplasm growth, particularly in equine piroplasm parasites.Emitted fuorescence signals were signifcantly reduced in mice treated with a combination treatment consists of lower doses of MOL and DA.Furthermore, the MOL/DA combination therapy was efective in eliminating the B. microti ss-rRNA gene from treated mice's blood.Te obtained fndings revealed that MOL may be efective in the treatment of animal piroplasmosis, particularly when combined with a low dose of either MMV665875 or DA.

Data Availability
Necessary data are available in the manuscript.Additional information can be received from the corresponding authors upon reasonable request.

Journal of Tropical Medicine
(b)).Te B. microti residual gene, on the other hand, was found in the blood of mice administered DA alone (Figure 3(b)).Tese data suggest MOL/DA combo therapy's advantages over the routinely recommended antibabesial medication DA.

Figure 1 :
Figure 1: Inhibitory efect of Moringa oleifera leaves on B. bovis, B. bigemina, T. equi, and B. caballi on the fourth day of treatment.(a) B. bovis.(b) B. bigemina.(c) T. equi.(d) B. caballi.Each value represents the mean ± standard deviation of triplicate trials after subtraction of the background fuorescence for non-parasitized RBCs.Asterisks indicate a signifcant diference (P < 0.05) between the control-and MOLtreated cultures.MOL viability test fndings for screened piroplasm parasites are indicated by +"live" and -"dead."

Figure 2 :
Figure 2: Delayed-death efect on B. bigemina.(a) Inhibitory efect of Moringa oleifera leaves (MOL) in comparison with control culture at diferent incubation periods after 24 h exposure to IC 99 MOL.Each value represents the mean ± standard deviation of triplicate trials.P < 0.05 indicates a signifcant diference between the treated and the non-treated cultures.(b) Percent of inhibition in B. bigemina growth in comparison with non-treated culture at diferent incubation times after exposure to IC 99 MOL.

Table 3 :Figure 3 :
Figure 3: In vivo chemotherapeutic efcacy of moringa oleifera leaves (MOL), diminazene aceturate (DA), and the combination of both drugs on the growth of Babesia microti.(a) Drug inhibitory efects.(b) PCR of the ss-rRNA gene in the blood of B. microti-infected mice.All mice were intraperitoneally injected with 1 × 10 7 B. microti-RBCs.Te treatment started when parasitemia reached approximately 1% in the infected mice and continued for 5 successive days.In the control group, mice were injected with I/P doses of DMSO in phosphate bufer saline (PBS) (0.02%).DA and MOL were administrated in subcutaneous and oral dosages, respectively.Drugs in the combination therapy were administrated during the same inoculation time.Each value represents the mean ± standard deviation of fve mice per experimental group.Asterisks indicate signifcant diferences (ANOVA; * P < 0.05) between the MOH-treated and control groups.PC, positive control; NC, negative control.M indicates a 100 bp DNA ladder.Treatment time is indicated by an arrow (4-8 days post inoculation).

Table 1 :
IC 50 values of Moringa oleifera leaves evaluated for bovine Babesia and equine Babesia and Teileria parasites.