# In vitro antiprotozoal action of 7 spices, medicinal plants and essential oils against 3 stages of Eimeria tenella

Claire Girard, Anne Silvestre, Thibaut Chabrillat, Sylvain Kerros

► To cite this version:

Claire Girard, Anne Silvestre, Thibaut Chabrillat, Sylvain Kerros. In vitro antiprotozoal action of 7 spices, medicinal plants and essential oils against 3 stages of Eimeria tenella. World's Poultry Congress (WPC), 2021, Online, France. **hal-04126107**

HAL Id: hal-04126107

https://hal.inrae.fr/hal-04126107

Submitted on 13 Jun 2023

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# In vitro antiprotozoal action of 7 spices, medicinal plants and essential oils against 3 stages of *Eimeria tenella*

C. GIRARD(1), A. SILVESTRE(1), T. CHABRILLAT(1), S. KERROS(1)

(1)PHYTOSYNTHESE, F-63200 MOZAC, FRANCE

(2)INRAE, UNIVERSITÉ DE TOURS, ISP, F-37380 NOUZILLY, FRANCE

Growing societal concerns to replace coccidiostats in poultry farms have led to the use of alternatives such as plant bio-actives. To investigate the mode of action of natural molecules, we designed this *in vitro* study to screen anti-protozoal activity of 4 spices or medicinal plants described by non-volatile active molecules (*MedP*) and 3 essential oils (EO) against 3 different stages of *E. tenella*.

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## MATERIAL AND METHODS

* Natural products tested:

* MedP: Fabacea powder, Urticaceae extract, Punicaceae extract, Acanthaceae extract

* EO: Lilaceae EO, Lamiaceae EO, Lauraceae EO

* Negative control: H₂O₂, soybean hemicellulose, DMSO

* Protozoal strain: *Eimeria tenella* recombined strain

* 3 experimental steps after cytotoxicity assay (colorimetric MTS test, 1.5×10⁴ MDBK seed, incubation 5h at 37°C):

1. Direct anti-protozoal activity test: incubation at 41°C for 1h. Killed sporozoites were stained with Evans Blue Dye. Each sample tested in triplicate. Viability rate with fluorescence microscopy as % viability = number of live parasite in tested sample/ number of live parasite in negative control*100

2. Invasion inhibition test: 96 microplates: 1.5×10⁴ MDBK cells, 1.5×10⁴ *E. tenella* sporozoites expressed enzymatic activity; substrate added for 2h at 37°C, 4 replicates per tested sample. % invasion = absorbance (550 nm) tested sample/ absorbance (550 nm) negative control*100

3. Sporulation rate test: 5.10⁵ non-sporulated oocysts (in 2% potassium dichromate solution), incubation for 72h at 26°C; each sample tested in triplicate. % sporulation = number of sporulated oocysts in tested sample/number of sporulated oocyst in negative control*100

* Statistical test: Mann-Whitney test compared to negative control for each test (p<0.05).

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## RESULTS AND DISCUSSION

The 4 MedP samples (Fabacea powder, Urticaceae extract, Punicaceae extract, Acanthaceae extract) were less cytotoxic (lethal dose 10% (LD10) than the 3 EO samples (Lilaceae EO, Lamiaceae EO, Lauraceae EO) (MedP diluted at 10⁻², EO diluted at 10⁻⁴~10⁻⁶).

<figure><img src="image_4.png" /><figcaption>Fig. 1 DIRECT ANTI-PROTOZOAL ACTIVITY AGAINST *E. TENELLA* SPOROZOITES (**: p<0.05; **:p<0.01; ***: p<0.001)</figcaption></figure>

MedP samples showed direct anti-protozoal activity against *E. tenella* stages: sporozoite viability rates were between 76.2% and 27% (p=0.05). Lilaceae EO and Lauraceae EO exhibited 81% and 77% (p=0.05) sporozoite viability rates, respectively (Fig. 1).

<figure><img src="image_5.png" /><figcaption>Fig. 2 MDBK CELL INVASION BY *E. TENELLA* (**: p<0.05; **: p<0.01; ***: p<0.001)</figcaption></figure>

Punicaceae extract showed a reduction of 32% of cell invasion.

<figure><img src="image_6.png" /><figcaption>Fig. 3 SPORULATION INHIBITION OF *E. TENELLA* (**: p<0.05; **: p<0.01; ***: p<0.001)</figcaption></figure>

Acanthaceae extract and Urticaceae extract showed a sporulation inhibition of *E. tenella* oocysts (p=0.05) (Fig. 3). These medicinal plant extracts exhibited anti-protozoal activity against the different *E. tenella* stages contrary to the tested EOs which target only sporozoites. These results were in accordance with studies reporting several modes of action of medicinal plant extracts, both direct anti-protozoal activity and protective cell activity [1].

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## CONCLUSION

Medicinal plant extracts exhibited an anti-protozoal activity against the different *E. tenella* stages. EOs only target sporozoites in this study. These findings help to understand how plant bio-actives could act to reduce *E. tenella* pathogenicity. Further studies could be carried out to test *in vitro* and/or *in vivo* potential synergies between different natural compounds.

## REFERENCE

<footnote n="1">¹WUNDERLICH et al. (2014). *Towards Identifying Novel Anti-Eimeria Agents: Trace Elements, Vitamins, and Plant-Based Natural Products*. Parasitology Research 113, no 10: 3547-56</footnote>