Eimeria tenella subsp. sporozoites

Haraguchi, Asako, Nagasawa, Jyunki, Kuramochi, Kouji, Tsuchida, Sayaka, Kobayashi, Atsushi, Hatabu, Toshimitsu, Sasai, Kazumi, Ikadai, Hiromi, Ushida, Kazunari & Matsubayashi, Makoto, 2024, Anticoccidial activity of the secondary metabolites in alpine plants frequently ingested by wild Japanese rock ptarmigans, International Journal for Parasitology: Parasites and Wildlife 25 (4), pp. 123153-123153 : 123153-

2.3. In vitro viability assay for E. tenella sporozoites

Sporozoites were suspended at 5 × 106 sporozoites/ml in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque), which was free of phenol red and contained antibiotics (Antibiotic-Antimycotic Mixed Stock Solution, Nacalai Tesque). The medium containing sporozoites was dispensed at 99 μl/well into a 96-well plate (AGC Techno Glass Co., Ltd., Shizuoka, Japan), and supplemented with 1 μl of the natural components extracted from alpine plants or the commercially available pure compounds. A sporozoite viability assay was performed using the natural components extracted from the alpine plants (components A-1 to I-1) at the concentration of 100 μM and lasalocid at the concentration of 1 μM. As the blank and solvent controls, we used RPMI medium containing sporozoites without any compounds or with DMSO (Nacalai Tesque), respectively. Using the natural and commercial compounds showing antiparasitic activities, IC 50 were analyzed in the ranging of 1–100 μM, and that of lasalocid was from 0.01 to 1 μM.

Subsequently, the samples were incubated for 24 h at 37 ◦ C under 5% CO 2. Then, 10 μl of Cell Count Reagent SF (Nacalai Tesque) was added, and the samples were incubated for 3 h under the same conditions. Finally, 10 μl of 0.1 M hydrochloric acid (Nacalai Tesque) was added and mixed to stop the reaction. A spectrophotometer (Nivo Multimode Microplate Reader, PerkinElmer, Boston, MA, USA) was used to measure the absorbance at 450 nm and 620 nm, and the sporozoite viability was calculated using the following formula:

Sporozoite viability (%) = {(Abs 450nm - Abs 620nm)compound – (Abs 450nm - Abs 620nm) blank} / {(Abs450 - Abs620)DMSO - (Abs 450nm - Abs 620nm)blank} × 100

Each experiment was repeated three to five times with a single well for each compound per experiment.

2.4. In vitro cytotoxicity assay of the natural components from alpine plants

Madin-Darby bovine kidney (MDBK) cells (NBL-1 strain, Japanese Collection of Research Bioresources (JCRB) Cell Bank, Osaka, Japan) were maintained in Minimum Essential Media (MEM; Nacalai Tesque) with 10% fetal bovine serum (FBS; Cosmo Bio, Tokyo, Japan) and 100 U/ml penicillin/100 μg/ml streptomycin (Nacalai Tesque) in an incubator at 37 ◦ C with 5% CO 2. When the cells reached approximately 70% confluence, they were detached using Accutase (Nacalai Tesque) and passaged.

The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to test the effects of the natural components obtained from the alpine plants (components A-1 to I-1) and lasalocid on MDBK cell viability. MDBK cells were seeded at 2 × 10 4 cells/well in 96-well plates (AGC Techno Glass). After cultivation at 37 ◦ C under 5% CO 2 for 24 h, each compound was added at the final concentrations of 0.1, 1, 10, 50, or 100 μM. DMSO (Nacalai Tesque) was used as a solvent control. After cultivation under the same conditions for 24 h, 10 μl of MTT (Nacalai Tesque) was added. Then, the cells were incubated for 2 h. Subsequently, 100 μl of 99% isopropyl alcohol (Nacalai Tesque) was added and vigorously mixed to completely dissolve the formazan. The absorbance at a wavelength of 562 nm was measured with a spectrophotometer (Nivo Multimode Microplate Reader, PerkinElmer). Cell viability was calculated according to the following formula:

Cell viability (%) = Abs 562nm compounds / Abs 562nm DMSO × 100

For each compound, the highest concentration at which no significant effect was seen on the cell viability (the maximum non-toxic concentration) was determined. Each experiment was repeated three times

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with three technical replicates per compound per experiment.