In vitro anthelmintic effects of cysteine proteinases from plants against intestinal helminths of rodents Gillian Stepek 1† , Ann E. Lowe 1 , David J. Buttle 2 , Ian R. Duce 1 and Jerzy M. Behnke 1 * 1 School of Biology, University of Nottingham, NG7 2RD, UK: 2 Academic Unit of Molecular Medicine, University of Sheffield Medical School, S10 2RX, UK Abstract Infections with gastrointestinal (GI) nematodes are amongst the most prevalent worldwide, especially in tropical climates. Control of these infections is primarily through treatment with anthelmintic drugs, but the rapid development of resistance to all the currently available classes of anthelmintic means that alternative treatments are urgently required. Cysteine proteinases from plants such as papaya, pineapple and fig are known to be substantially effective against three rodent GI nematodes, Heligmosomoides polygyrus, Trichuris muris and Protospirura muricola, both in vitro and in vivo. Here, based on in vitro motility assays and scanning electron microscopy, we extend these earlier reports, demonstrating the potency of this anthelmintic effect of plant cysteine proteinases against two GI helminths from different taxonomic groups – the canine hookworm, Ancylostoma ceylanicum, and the rodent cestode, Rodentolepis microstoma. In the case of hookworms, a mechanism of action targeting the surface layers of the cuticle indistinguishable from that reported earlier appears to be involved, and in the case of cestodes, the surface of the tegumental layers was also the principal location of damage. Hence, plant cysteine proteinases have a broad spectrum of activity against intestinal helminths (both nematodes and cestodes), a quality that reinforces their suitability for development as a much- needed novel treatment against GI helminths of humans and livestock. Introduction Plant extracts have been used for centuries throughout the world, but particularly in developing countries, as traditional treatments for many illnesses, including parasitic infections (Waller et al., 2001; Giday et al., 2003). The introduction of synthetic chemotherapeutic drugs resulted in less interest being shown in these natural therapies until very recently, when there has been a resurgence in the interest of using natural products as treatments against many parasitic diseases, including those caused by worms (Hammond et al., 1997; Githiori et al., 2003; Anthony et al., 2005; Hounzangbe-Adote et al., 2005). This renewed interest is largely due to the need for alternative treatments with novel mechanisms of action as a result of the rapid and widespread development of resistance to all three current classes of anthelmintics in many species of parasitic helminths, but particularly those species found throughout the gastrointestinal (GI) tract of livestock (Waller, 1986; Gill & Lacey, 1998; Geerts & Gryseels, 2000; Kaminsky, 2003). The crude latex of papaya (Carica papaya) (Berger & Asenjo, 1940) and fig (Ficus species) (Robbins, 1930), and fruit of pineapple (Ananas comosus) (Berger & Asenjo, 1939) were shown to digest the cuticle of Ascaris suum, and hence kill the nematode, in vitro. However, it was not until much later that this mechanism was described in *Fax þ 44 115 951 3251 E-mail: jerzy.behnke@nottingham.ac.uk † Current address: Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, G61 1QH, UK Journal of Helminthology (2007) 81, 353–360 doi: 10.1017/S0022149X0786408X