Table 4.

Treatment of artemisinin and its derivatives against Neospora caninum and Eimeria tenella

Experiments	Species (strain)	Host/cell line	Drug	Treatment (dose, route, and time)	Effect	Reference
In vitro	N. caninum (KB-2)	Tachyzoites in mouse peritoneal macrophages (mouse strain was not stated)	Artemisinin	0.1, 1, and 10 μg/mL, started from 12 h p.i. to 30 h	The total number of tachyzoites per 100 infected macrophages reduced with a clear dose-dependent pattern, which were about 700, 500, and 340 at 0.1, 1, and 10 μg/mL, respectively	Kim et al. [71]
	N. caninum (KB-2)	Tachyzoites, Vero cells	Artemisinin	0.01, 0.1, 1, and 10 μg/mL, started from 12 h p.i. to 30 h	The total number of tachyzoites per 100 infected Vero cells was reduced with a clear dose-dependent pattern, which were about 980, 570, 300, and 260 at 0.01, 0.1, 1, and 10 μg/mL, respectively	Kim et al. [71]
	N. caninum (KB-2)	Tachyzoites, Vero cells	Artemisinin	0.1, 1, 10, and 20 μg/mL, started from 12 h p.i. to 12 days	10 or 20 μg/mL eliminated all microscopic foci of N. caninum in Vero cells by 11 days (third subculture) and 1 μg/ml eliminated all microscopic foci of N.caninum by 14 days (fourth subculture)	Kim et al. [71]
	N. caninum (NcIS491)	Tachyzoites, Vero cells	Artemisone	0.1, 0.5, 5.0, 10.0, or 15.0 g/mL, 5 h prior to infection 25.0 or 50.0 g/mL for	The parasite inhibitory effect was dose-dependent. Percentages of infected cells at 96 h of cultivation were 8.5%, 5.7%, 3.5%, 7.8%, 4.5%, and 1.0%, respectively. Percentages of parasite inhibition at 96 h of cultivation were 90.2%, 93.4%, 95.9%, 91.0%, 94.8%, 98.8%, and 87.3%, respectively	Mazuz et al. [72]
	N. caninum (NcIS491)	Tachyzoites, Vero cells	Artemisone	24 h, added at initial 15.3% of cells infected	The parasite inhibitory effects were 50.1% and 81.9%, respectively, compared to control	Mazuz et al. [72]
	N. caninum-β-galactosidase expression	Tachyzoites, HFF and Vero cells	Artemisone	Pre-treatment with 5 μM for 1 or 3 h. After 2 h, either 500 nM or 5 μM of the drug was added. The one with initial 500 nM is then increased stepwise for a period of 40 days	IC50 = 3 nM. Only after 21 days of continuous treatment with 5 μM of artemisone, all tachyzoites were eliminated	Müller et al. [74]
	N. caninum-β-galactosidase expression	Tachyzoites, HFF and Vero cells	GC007 (artemisinin derivative)	Pre-treatment with 5 μM for 1 or 3 h. After 2 h, either 500 nM or 5 μM of the drug was added. The one with initial 500 nM is then increased stepwise for a period of 40 days	IC50 = 29 nM. Only after 21 days of continuous treatment with 5 μM of GC007, all tachyzoites were eliminated	Müller et al. [74]
	N. caninum-β-galactosidase expression	Tachyzoites, HFF and Vero cells	GC012 (artemisinin derivative)	Pre-treatment with 5 μM for 1 or 3 h. After 2 h, either 500 nM or 5 μM of the drug was added. The one with initial 500 nM is then increased stepwise for a period of 40 days	IC50 = 54 nM. Tachyzoites readily adapted to a gradual increase in the concentration (0.5–10 μM) within 20 days, indicating the changes in tachyzoites were less dramatic	Müller et al. [74]
	N. caninum-GFP expression	Tachyzoites, HFF cells	Artemether	1, 10, 30, 40, 50, 60, 80, and 100 μg/mL for 2 h	IC50 = (1.0 ± 0.05) μg/mL, parasite growth concentration was 100 μg/mL	Qian et al. [75]
In vivo studies	N. caninum (NcIS491)	Gerbils (Meriones tristrami)	Artemisone	20 mg/kg, i.p., b.i.d. for 4 days	Among 9 treated gerbils, only 1 animal exhibited clinical signs and died 10 days p.i. vs. controls: 8 out of 9 untreated gerbils died with characteristic cerebral signs	Mazuz et al. [72]
	N. caninum (Nc-Spain7)	BALB/c male mice	Artemisone	50 mg/kg/day suspended in 100μL corn oil, i.g., q.d. for 6 days	Parasite burden in brains was 1–5 × 103 tachyzoites per μg DNA after treatment, lower than those of placebo (0.5–14 × 103), mefloquine (5–7.5 × 103), and artemiside (1–15 × 103) treatments; however parasite burden in lungs (1.4–2.2 × 103) after treatment, higher than those of placebo, mefloquine, and artemiside treatments	Müller et al. [73]
In vivo	E. tenella (S98N3 strain)	White leghorn chicken	Pure artemisinin	10 ppm, fed independently in food, p.o., 7 days p.i.	Significantly reduced oocyst output (0.7 × 106) vs. control (4.2 × 106) and reduced sporulation rate of oocyte (73.2% vs. 97.4%); chicken mortality rate reduced to 2.3% and lesion score reduced to 1.2 after treatment. SERCA expression in macrogamates reduced to 77.6% vs. 98.7% in control, indicating artemisinin was effective on the parasite	del Cacho et al. [78]
	E. tenella (S98N3 strain)	White leghorn chicken	Pure artemisinin	17 ppm in food, fed independently in food, p.o., 7 days p.i.	Significantly reduced oocyst output (0.2 × 106) vs. control (4.2 × 106), and reduced sporulation rate of oocyte (62% vs. 97.4%); the mortality rate of chickens reduced to 1.1% and lesion score reduced to 1. SERCA expression in macrogamates reduced to 66.4% vs. 98.7% in control	del Cacho et al. [78]

b.i.d.: two times per day

IC₅₀: concentration that causes 50% inhibition of growth

HFF: human foreskin fibroblast

i.g.: intragastric administration

i.p.: intraperitoneal injection

p.i.: post-infection

p.o.: oral administration

q.d.: one time per day

SERCA: sarco/endoplasmic reticulum Ca²⁺-ATPase

^*The IC50 is either in μg/mL or nM as in the original papers.