Abstract
There is no effective natural alternative control for gastrointestinal nematodes (GIN) of small ruminants, with Haemonchus contortus being the most economically important GIN. Despite frequent reports of multidrug-resistant GIN, there is no new commercial anthelmintic to substitute failing ones. Although trematocidal activity of artemisinin analogs has been reported in sheep, neither artemisinin nor its plant source (Artemisia annua) has been evaluated for anthelmintic activity in ruminants. This study evaluated the anthelmintic activity of A. annua crude extracts in vitro and compared the most effective extract with artemisinin in sheep naturally infected with H. contortus. A. annua leaves extracted with water, aqueous 0.1 % sodium bicarbonate, dichloromethane, and ethanol were evaluated in vitro by the egg hatch test (EHT) and with the bicarbonate extract only for the larval development test (LDT) using H. contortus. The A. annua water, sodium bicarbonate (SBE), ethanol, and dichloromethane extracts tested in vitro contained 0.3, 0.6, 4.4, and 9.8 % of artemisinin, respectively. The sodium bicarbonate extract resulted in the lowest LC99 in the EHT (1.27 μg/mL) and in a LC99 of 23.8 μg/mL in the LDT. Following in vitro results, the SBE (2 g/kg body weight (BW)) and artemisinin (100 mg/kg BW) were evaluated as a single oral dose in naturally infected Santa Inês sheep. Speciation from stool cultures established that 84–91 % of GIN were H. contortus, 8.4–15.6 % were Trichostrongylus sp., and 0.3–0.7 % were Oesophagostomum sp. Packed-cell volume and eggs per gram (EPG) of feces were used to test treatment efficacy. The SBE tested in vivo contained no artemisinin, but had a high antioxidant capacity of 2,295 μmol of Trolox equivalents/g. Sheep dosed with artemisinin had maximum feces concentrations 24 h after treatment (126.5 μg/g artemisinin), which sharply decreased at 36 h. By day 15, only levamisole-treated sheep had a significant decrease of 97 % in EPG. Artemisinin-treated and SBE-treated sheep had nonsignificant EPG reductions of 28 and 19 %, respectively, while sheep in infected/untreated group had an average EPG increase of 95 %. Sheep treated with artemisinin and A. annua SBE maintained blood hematocrits throughout the experiment, while untreated/infected controls had a significant reduction in hematocrit. This is the first time oral dose of artemisinin and an aqueous extract of A. annua are evaluated as anthelmintic in sheep. Although oral dose of artemisinin and SBE, at single doses, were ineffective natural anthelmintics, artemisinin analogs with better bioavailability than artemisinin should be tested in vivo, through different routes and in multiple doses. The maintenance of hematocrit provided by artemisinin and A. annua extract and the high antioxidant capacity of the latter suggest that they could be combined with commercial anthelmintics to improve the well-being of infected animals and to evaluate potential synergism.
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Acknowledgments
This work received financial support from the Brazilian Agricultural Research Corporation (Embrapa) and scholarships and work grants from FAPESP and CNPq. The authors are highly thankful to MSc. Rodrigo Giglioti from Unesp, Dr. Glyn M. Figueira from the University of Campinas, and Dr. Moacir R. Forim from the University of São Carlos for their assistance in this study. Acknowledgements are due to Dr. John Phillips (USDA-ARS) and Dr. Aida Jimenez (University of Charleston, Beckley-WV) for the advice with the statistical analysis and to Mr. Barry Harter, Robert Arnold, and Mrs. J. Diane Snuffer (USDA-ARS) for their help with the chromatographic analyses and figures.
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The authors declare no conflict of interest. The mention of proprietary brands and names is solely for the convenience of the reader and does not imply endorsement of the authors or of the USDA over similar products.
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Cala, A.C., Ferreira, J.F.S., Chagas, A.C.S. et al. Anthelmintic activity of Artemisia annua L. extracts in vitro and the effect of an aqueous extract and artemisinin in sheep naturally infected with gastrointestinal nematodes. Parasitol Res 113, 2345–2353 (2014). https://doi.org/10.1007/s00436-014-3891-z
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DOI: https://doi.org/10.1007/s00436-014-3891-z