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

Rizk, Mohamed Abdo; El‒Sayed, Shimaa Abd El‒Salam; AbouLaila, Mahmoud; Eltaysh, Rasha; Yokoyama, Naoki; Igarashi, Ikuo

doi:10.1038/s41598-017-13052-5

Cited by 33 publications

(49 citation statements)

References 31 publications

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“…In vitro B. divergens suppression of development, on the other hand, in blend with DA, luteolin, and PYR were increased by treatment with a fluoroquinolone (enoxacin, enrofloxacin, and trovafloxacin) [33]. Such non-improvement in the inhibitory effects of myrrh oil on B. divergens, when used in combination with the selected antibabesial drugs, might be attributed to the fact that parasite species, strain, and size affect the in vitro inhibitory activity of the drug combination on the Babesia growth [30,[36][37][38].…”

Section: Discussionmentioning

confidence: 99%

“…In vivo inhibition of myrrh oil test for B. microti (Munich strain) [32] was performed twice in BALB/c mice aged eight weeks old (purchased from CLEA Japan, Tokyo, Japan) using the previously described method [38]. Every mouse was housed under strict pathogen-free conditions.…”

Section: Chemotherapeutic Efficacy Of Myrrh Oil On the Growth Of B Mmentioning

confidence: 99%

“…Myrrh oil was administered intraperitoneally either only at a dose rate of 200 mg kg −1 or in conjunction with a subcutaneous dose of DA at a dosage level of 100 mg kg −1 myrrh oil and 12.5 mg kg −1 DA for mice in the fourth and fifth groups, respectively. The inhibitory effects of the drugs administered on the growth of B. microti were tracked each 48 h until 28 days after infection or the ending of parasitemia using fluorescence spectrophotometer [38]. After completion of the experiment, all the mice were humanely euthanized using inhalational agent, chloroform as a primary method of euthanasia, and cervical dislocation (physical euthanasia) was performed to all mice.…”

Section: Chemotherapeutic Efficacy Of Myrrh Oil On the Growth Of B Mmentioning

confidence: 99%

“…A NucleoSpin tissue kit (Macherey-Nagel, Düren, Germany) and a QIAamp DNA Blood Mini Kit (Qiagen, Tokyo, Japan) were used for tissues and blood DNA extraction. PCR cycling was accomplished, as mentioned earlier by Rizk et al [33,38].…”

Section: Pcr Detection Of B Microti In Micementioning

confidence: 99%

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

et al. 2020

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Chemotherapeutic Efficacy Of Myrrh Oil On the Growth Of B Mmentioning

confidence: 99%

Section: Chemotherapeutic Efficacy Of Myrrh Oil On the Growth Of B Mmentioning

confidence: 99%

Section: Pcr Detection Of B Microti In Micementioning

confidence: 99%

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

et al. 2020

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“…Additionally, removing blood from mice tail veins for in vitro cultivation allows environmental factors to in uence the results. Therefore, the use of BFA is inadequate to determine drug e cacy [20].…”

Section: Introductionmentioning

confidence: 99%

Establishment and Optimisation of Anti-Babesia microti Drug Efficacy Evaluation Method

Yin¹,

Zhang²,

Yi³

et al. 2020

Preprint

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BackgroundBabesiosis, an infectious zoonotic parasitic disease that occurs globally, is most commonly caused by Babesia microti. For a severe infection, a combination of exchange transfusion and support-therapy is used and, for a mild infection, single antibiotics, or a combination of antibiotics and antiprotozoal drugs, are used for 7–10 days for the treatment of babesiosis; however, as these treatments have problems, a new therapy is needed. Although aetiological and molecular biology methods are often used in drug screening, these methods have disadvantages and there is no standard anti-B. microti drug screening method.MethodThis study was consisted of basal test, drug-suppressive test and drug-therapeutic test. BALB/c mice were used as the animal model and robenidine hydrochloride (ROBH) was used as a positive drug. By modifying Peter’s 4-day suppression method, immunosuppressive and subpassage tests were conducted, in combination with microscopy and real-time fluorescent quantitative PCR (qPCR), the current anti-B. microti drug screening method was optimised. SPSS was used for data analysis.Result1) In the basal test, there were significant differences in the erythrocyte infection rate (EIR) and relative value of the target gene (P = 0.011 and 0.012, respectively) among days. The blood of infected mice 28, 35, and 37 days post-infection (dpi) could infect healthy second-generation mice, even if no parasites could be observed under a light microscope. Resurgence occurred when the immunity of an infected mouse decreased to a certain extent after using an immunosuppressant.2) In the drug-suppressive test, no B. microti was observed under the light microscope in any ROBH group after drug administration. Significant differences were observed in the EIR and relative value of the target gene (both P < 0.001) between the control group and ROBH groups. No B. microti was observed after the administration of an immunosuppressant or in second-generation mice in ROBH-treated groups; however, B. microti was observed in control and proguanil hydrochloride-treated groups.3) In the drug-therapeutic test, there were significant differences in the EIR and relative value of the target gene between the 100 mg/kg ROBH groups and the control group (P = 0.049 and < 0.001, respectively) but not between ROBH groups and atovaquone-azithromycin group (P = 0.587 and 0.418, respectively). None of the second-generation mice in any drug-treated group were infected, whereas all the second-generation mice in the control group were infected; parasites could be observed in the control group from the 2nd day after the administration of an immunosuppressant, several parasites were observed in the atovaquone-azithromycin group from the 10th day after the administration of an immunosuppressant, and no parasites were observed in ROBH groups. ConclusionROBH can be used as a positive drug in anti-B. microti drug screening trials. qPCR results can be used as a judgment standard in preliminary screening tests and the combination of immunosuppressive and subpassage experiments can be used to validate drug efficacy.

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Pyronaridine: An update of its pharmacological activities and mechanisms of action

Bailly

2020

Biopolymers

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Pyronaridine (PYR) is an erythrocytic schizonticide with a potent antimalarial activity against multidrug-resistant Plasmodium. The drug is used in combination with artesunate for the treatment of uncomplicated P. falciparum malaria, in adults and children. The present review briefly retraces the discovery of PYR and recent antimalarial studies which has led to the approval of PYR/artesunate combination (Pyramax) by the European Medicines Agency to treat uncomplicated malaria worldwide. PYR Pyronaridine (PYR, Figure 1), initially known as "antimalaria drug 7351", is a benzo-naphthyridine derivative originally synthesized in 1979 [1] and developed in China from the early 1980s. [2][3][4] Rapidly, experimental studies demonstrated that this compound was more active than chloroquine against different isolates of Plasmodium falciparum, the malaria pathogen.

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

Cited by 33 publications

References 31 publications

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

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

Establishment and Optimisation of Anti-Babesia microti Drug Efficacy Evaluation Method

Pyronaridine: An update of its pharmacological activities and mechanisms of action

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