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Electron microscopical study to assess the in vitro effects of the synthetic trioxolane OZ78 against the liver fluke, Fasciola hepatica

Published online by Cambridge University Press:  07 August 2009

L. HALFERTY
Affiliation:
Parasite Therapeutics Research Group, School of Biological Sciences, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland
J. F. O'NEILL
Affiliation:
Parasite Therapeutics Research Group, School of Biological Sciences, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland
G. P. BRENNAN
Affiliation:
Parasite Therapeutics Research Group, School of Biological Sciences, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland
J. KEISER
Affiliation:
Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, P.O. Box, CH-4002 Basel, Switzerland
I. FAIRWEATHER*
Affiliation:
Parasite Therapeutics Research Group, School of Biological Sciences, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland
*
*Corresponding author: School of Biological Sciences, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland. Tel: +44 28 90972298. Fax: +44 28 90975877. E-mail: i.fairweather@qub.ac.uk

Summary

Adult Fasciola hepatica were incubated for 48 h in vitro in the synthetic peroxide, OZ78 at a concentration of 100 μg/ml and then prepared for scanning and transmission electron microscopy. There was limited disruption to the external fluke surface, with only slight swelling and blebbing of the interspinal tegument in the midbody and ventral tail regions. By contrast, significant disruption was observed to the ultrastructure of the tegument and subtegumental tissues. There was severe swelling of the basal infolds in the tegumental syncytium and the flooding spread internally to affect the subtegumental tissues. In the tegumental system, there was swelling of the cisternae of granular endoplasmic reticulum and of the mitochondria, with the latter showing signs of breaking down. Autophagic vacuoles and lipid droplets were present and the synthesis of tegumental secretory bodies was much reduced. The gastrodermal cells were severely affected, with swelling and degeneration of the mitochondria and the presence of autophagic vacuoles and lipid droplets. The granular endoplasmic reticulum was swollen and vesiculated and the cells contained few secretory bodies. Both the vitelline and testis follicles showed evidence of extensive cellular disruption and degeneration. This study confirms previous data indicating the potential flukicidal activity of OZ78.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2009

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References

REFERENCES

Anderson, H. R. and Fairweather, I. (1995). Fasciola hepatica: ultrastructural changes to the tegument of juvenile flukes following incubation in vitro with the deacetylated (amine) metabolite of diamphenethide. International Journal for Parasitology 25, 319333.CrossRefGoogle Scholar
Anderson, H. R., Fairweather, I., Bamford, D. R. and Montgomery, W. I. (1993). The effect of diamphenethide on protein synthesis by the liver fluke, Fasciola hepatica. International Journal for Parasitology 23, 10531062.CrossRefGoogle ScholarPubMed
Bennett, C. E. (1975). Scanning electron microscopy of Fasciola hepatica during migration in the mouse. Journal of Parasitology 61, 892898.CrossRefGoogle Scholar
Berman, P. A. and Adams, P. A. (1997). Artemisinin enhances heme-catalysed oxidation of lipid membranes. Free Radical Biology and Medicine 22, 12831288.CrossRefGoogle ScholarPubMed
Boray, J. C., Crowfoot, P. D., Strong, M. B., Allison, J. R., Schellenbaum, M., Von Orelli, M. and Sarasin, G. (1983). Treatment of immature and mature Fasciola hepatica infections in sheep with triclabendazole. Veterinary Record 113, 315317.CrossRefGoogle ScholarPubMed
Borstnik, K., Paik, I.-H., Shapiro, T. A. and Posner, G. H. (2002). Antimalarial chemotherapeutic peroxides: artemisinin, yinghaosu A and related compounds. International Journal for Parasitology 32, 16611667.CrossRefGoogle ScholarPubMed
Colhoun, L. M., Fairweather, I. and Brennan, G. P. (1998). Observations on the mechanism of eggshell formation in the liver fluke, Fasciola hepatica. Parasitology 116, 555567.CrossRefGoogle ScholarPubMed
Dawes, B. (1968). Further evidence on the effect of bithionol (“Actamer”) on Fasciola hepatica. Wiadomosci Parazytologiczne 14, 575577.Google ScholarPubMed
Devine, C., Brennan, G. P., Lanusse, C. E., Alvarez, L. I., Trudgett, A., Hoey, E. and Fairweather, I. (2009). Effect of the metabolic inhibitor, methimazole on the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica. Parasitology 136, 183192.CrossRefGoogle ScholarPubMed
Dubin, M. and Stoppani, A. O. M. (2000). Programmed cell death and apoptosis. The role of mitochondria. Medicina 60, 375386.Google ScholarPubMed
Eckstein-Ludwig, U., Webb, R., van Goethem, I., East, J., Lee, A., Kimura, M., O'Neill, P., Bray, P., Ward, S. and Krishna, S. (2003). Artemisinins target the SERCA of Plasmodium falciparum. Nature, London 424, 957961.CrossRefGoogle ScholarPubMed
Fairweather, I. (2005). Triclabendazole: new skills to unravel an old(ish) enigma. Journal of Helminthology 79, 227234.CrossRefGoogle ScholarPubMed
Fairweather, I. (2009). Triclabendazole progress report, 2005–2009: an advancement of learning? Journal of Helminthology 83, 139150.CrossRefGoogle ScholarPubMed
Fairweather, I., Anderson, H. R. and Baldwin, T. M. A. (1987). Fasciola hepatica: tegumental surface alterations following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide. Parasitology Research 73, 99–106.CrossRefGoogle ScholarPubMed
Fairweather, I., Anderson, H. R. and Threadgold, L. T. (1986). Fasciola hepatica: tegumental changes induced in vitro by the deacetylated (amine) metabolite of diamphenethide. Experimental Parasitology 62, 336348.CrossRefGoogle ScholarPubMed
Fairweather, I., Anderson, H. R. and Threadgold, L. T. (1988). Fasciola hepatica: morphological changes in vitelline cells following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide (DAMD). International Journal for Parasitology 18, 10611069.CrossRefGoogle ScholarPubMed
Fairweather, I., Threadgold, L. T. and Hanna, R. E. B. (1999). Development of Fasciola hepatica in the mammalian host. In Fasciolosis (ed. Dalton, J. P.), pp. 47–111. CAB International, Wallingford, Oxon., UK.Google Scholar
Fletcher, H. F., Hoey, E. M., Orr, N., Trudgett, A., Fairweather, I. and Robinson, M. W. (2004). The occurrence and significance of triploidy in the liver fluke, Fasciola hepatica. Parasitology 128, 6972.CrossRefGoogle ScholarPubMed
Gu, H. M., Warhurst, D. C. and Peters, W. (1983). Rapid action of qinghaosu and related drugs on incorporation of (3H) isoleucine by Plasmodium falciparum in vitro. Biochemical Pharmacology 32, 24632466.CrossRefGoogle ScholarPubMed
Halferty, L., Brennan, G. P., Hanna, R. E., Edgar, H. W., Meaney, M. M., McConville, M., Trudgett, A., Hoey, L. and Fairweather, I. (2008). Tegumental surface changes in juvenile Fasciola hepatica in response to treatment in vivo with triclabendazole. Veterinary Parasitology 155, 4958.CrossRefGoogle ScholarPubMed
Halferty, L., Brennan, M., Trudgett, A., Hoey, L. and Fairweather, I. (2009). Relative activity of triclabendazole metabolites against the liver fluke, Fasciola hepatica. Veterinary Parasitology 159, 126138.CrossRefGoogle ScholarPubMed
Hanna, R. E. B., Edgar, H., Moffett, D., McConnell, S., Fairweather, I., Brennan, G. P., Trudgett, A., Hoey, E. M., Cromie, L., Taylor, S. M. and Daniel, R. (2008). Fasciola hepatica: histology of the testis in egg-producing adults of several laboratory-maintained isolates of flukes grown to maturity in cattle and sheep and in flukes from naturally infected hosts. Veterinary Parasitology 157, 222234.CrossRefGoogle ScholarPubMed
Happich, F. A. and Boray, J. C. (1969). Quantitative diagnosis of chronic fasciolosis. 2. The estimation of daily total egg production of Fasciola hepatica and the number of adult flukes in sheep by faecal egg counts. Australian Veterinary Journal 45, 329331.CrossRefGoogle ScholarPubMed
Irwin, S. W. B. and Threadgold, L. T. (1970). Electron microscope studies on Fasciola hepatica.VIII. The development of the vitelline cells. Experimental Parasitology 28, 399411.CrossRefGoogle ScholarPubMed
Jefford, C. W. (2007). New developments in synthetic peroxide drugs as artemisinin mimics. Drug Discovery Today 12, 487495.CrossRefGoogle ScholarPubMed
Jung, J., Kim, H., Nam, K. Y. and No, K. T. (2005). Three-dimensional structure of Plasmodium falciparum Ca2+-ATPase (PfATP6) and docking of artemisinin derivatives to PfATP6. Bioorganic and Medicinal Chemistry Letters 15, 29942997.CrossRefGoogle ScholarPubMed
Keiser, J. and Morson, G. (2008 a). Fasciola hepatica: tegumental alterations in adult flukes following in vitro and in vivo administration of artesunate and artemether. Experimental Parasitology 118, 228237.CrossRefGoogle ScholarPubMed
Keiser, J. and Morson, G. (2008 b). Fasciola hepatica: surface tegumental responses to in vitro and in vivo treatment with the experimental fasciolicide OZ78. Experimental Parasitology 119, 8793.CrossRefGoogle ScholarPubMed
Keiser, J., Utzinger, J., Tanner, M., Dong, Y. and Vennerstrom, J. L. (2006 b). The synthetic peroxide OZ78 is effective against Echinostoma caproni and Fasciola hepatica. Journal of Antimicrobial Chemotherapy 58, 11931197.CrossRefGoogle ScholarPubMed
Keiser, J., Utzinger, J., Vennerstrom, J. L., Dong, Y., Brennan, G. P. and Fairweather, I. (2007 a). Activity of artemether and OZ78 against triclabendazole-resistant Fasciola hepatica. Transactions of the Royal Society of Tropical Medicine and Hygiene 101, 12191222.CrossRefGoogle ScholarPubMed
Keiser, J., Xiao, S., Tanner, M. and Utzinger, J. (2006 a). Artemether and artesunate are effective fasciolicides in the rat model and in vitro. Journal of Antimicrobial Chemotherapy 57, 11391145.CrossRefGoogle ScholarPubMed
Keiser, J., Xiao, S.-H., Dong, Y., Utzinger, J. and Vennerstrom, J. L. (2007 b). Clonorchicidal properties of the synthetic trioxolane OZ78. Journal of Parasitology 93, 12081213.CrossRefGoogle ScholarPubMed
Li, W., Mo, W., Shen, D., Sun, L., Wang, J., Lu, S., Gitschier, J. M. and Zhou, B. (2005). Yeast model uncovers dual roles of mitochondria in the action of artemisinin. PloS Genetics 1, 329334.CrossRefGoogle ScholarPubMed
Mas-Coma, S., Bargues, M. D. and Valero, M. A. (2005). Fascioliasis and other plant-borne trematode zoonoses. International Journal for Parasitology 35, 12551278.CrossRefGoogle ScholarPubMed
Mason, R. P., Walter, M. F. and Mason, P. E. (1997). Effect of oxidative stress on membrane structure: small-angle X-ray diffraction analysis. Free Radical Biology and Medicine 23, 419425.CrossRefGoogle ScholarPubMed
McConville, M., Brennan, G. P., Flanagan, A., Edgar, H. W. J., Hanna, R. E. B., McCoy, M., Gordon, A. W., Castillo, R., Hernández-Campos, A. and Fairweather, I. (2009 a). An evaluation of the efficacy of compound alpha and triclabendazole against two isolates of Fasciola hepatica. Veterinary Parasitology 162, 7588.CrossRefGoogle ScholarPubMed
McConville, M., Brennan, G. P., Flanagan, A., Hanna, R. E. B., Edgar, H. W. J., Castillo, R., Hernández-Campos, A. and Fairweather, I. (2009 b). Surface changes in adult Fasciola hepatica following treatment in vivo with the experimental fasciolicide, compound alpha. Parasitology Research (in the Press).Google ScholarPubMed
McConville, M., Brennan, G. P., McCoy, M., Castillo, R., Hernández-Campos, A., Ibarra, F. and Fairweather, I. (2006). Adult triclabendazole-resistant Fasciola hepatica: surface and subsurface tegumental responses to in vitro treatment with the sulphoxide metabolite of the experimental fasciolicide compound alpha. Parasitology 133, 195208.CrossRefGoogle ScholarPubMed
McCoy, M., Fairweather, I., Brennan, G. P., Kenny, J. M., Ellison, S. and Forbes, A. B. (2005). The efficacy of nitroxynil and triclabendazole administered synchronously against juvenile triclabendazole-resistant Fasciola hepatica in sheep. Research in Veterinary Science 78 (Suppl. A), 33.Google Scholar
McKinstry, B., Brennan, G. P., Halferty, L., Forbes, A. B. and Fairweather, I. (2007). Ultrastructural changes induced in the tegument and gut of Fasciola hepatica following in vivo and in vitro drug treatment with nitroxynil (Trodax). Parasitology Research 101, 929941.CrossRefGoogle ScholarPubMed
McKinstry, B., Fairweather, I., Brennan, G. P. and Forbes, A. B. (2003). Fasciola hepatica: tegumental surface alterations following treatment in vivo and in vitro with nitroxynil (Trodax). Parasitology Research 91, 251263.CrossRefGoogle ScholarPubMed
McKinstry, B., Halferty, L., Brennan, G. P. and Fairweather, I. (2009). Morphological response of triclabendazole-susceptible and triclabendazole-resistant isolates of Fasciola hepatica to treatment in vitro with nitroxynil (Trodax). Parasitology Research 104, 645655.CrossRefGoogle ScholarPubMed
Meaney, M., Allister, J., McKinstry, B., McLaughlin, K., Brennan, G. P., Forbes, A. B. and Fairweather, I. (2006). Fasciola hepatica: morphological effects of a combination of triclabendazole and clorsulon against mature fluke. Parasitology Research 99, 609621.CrossRefGoogle ScholarPubMed
Meaney, M., Allister, J., McKinstry, B., McLaughlin, K., Brennan, G. P., Forbes, A. B. and Fairweather, I. (2007). Fasciola hepatica: ultrastructural effects of a combination of triclabendazole and clorsulon against mature fluke. Parasitology Research 100, 10911104.CrossRefGoogle ScholarPubMed
Meaney, M., Fairweather, I., Brennan, G. P. and Forbes, A. B. (2004). Transmission electron microscope study of the ultrastructural changes induced in the tegument and gut of Fasciola hepatica following in vivo drug treatment with clorsulon. Parasitology Research 92, 232241.CrossRefGoogle ScholarPubMed
Meaney, M., Fairweather, I., Brennan, G. P., McDowell, L. S. L. and Forbes, A. B. (2003). Fasciola hepatica: effects of the fasciolicide clorsulon in vitro and in vivo on the tegumental surface, and a comparison of the effects on young- and old-mature flukes. Parasitology Research 91, 238250.CrossRefGoogle Scholar
Meaney, M., Haughey, S., Brennan, G. P. and Fairweather, I. (2005). Ultrastructural observations on oral ingestion and trans-tegumental uptake of clorsulon by the liver fluke, Fasciola hepatica. Parasitology Research 95, 201212.CrossRefGoogle ScholarPubMed
Mitchell, M. (2002). Update on fascioliasis in cattle and sheep. In Practice 24, 378385.CrossRefGoogle Scholar
O'Neill, P. (2004). A worthy adversary for malaria. Nature, London 430, 838839.CrossRefGoogle ScholarPubMed
O'Neill, J. F., Johnston, R. C., Halferty, L., Brennan, G. P., Keiser, J. and Fairweather, I. (2009). Adult triclabendazole-resistant Fasciola hepatica: morphological changes in the tegument and gut following in vivo treatment with artemether in the rat model. Journal of Helminthology 83, 151163.CrossRefGoogle ScholarPubMed
Overend, D. J. and Bowen, F. L. (1995). Resistance of Fasciola hepatica to triclabendazole. Australian Veterinary Journal 72, 275276.CrossRefGoogle ScholarPubMed
Robinson, G. and Threadgold, L. T. (1975). Electron microscope studies of Fasciola hepatica: XII. The fine structure of the gastrodermis. Experimental Parasitology 37, 2036.CrossRefGoogle ScholarPubMed
Robinson, M. W., Trudgett, A., Hoey, E. M. and Fairweather, I. (2002). Triclabendazole-resistant Fasciola hepatica: β-tubulin and response to in vitro treatment with triclabendazole. Parasitology 124, 325338.CrossRefGoogle ScholarPubMed
Rohn, T. R., Hinds, T. R. and Vincenzi, F. F. (1996). Inhibition of Ca2+-pump ATPase and the Na+/K+-pump ATPase by iron-generated free radicals. Protection by 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrol0[2,3-d]pyrimidine sulfate (U-89843D), a potent, novel, antioxidant/free radical scavenger. Biochemical Pharmacology 51, 471476.CrossRefGoogle Scholar
Skuce, P. J., Anderson, H. R. and Fairweather, I. (1987). The interaction between the deacetylated (amine) metabolite of diamphenethide (DAMD) and cytochemically demonstrable Na+/K+-ATPase activity in the tegument of Fasciola hepatica. Parasitology Research 74, 161167.CrossRefGoogle ScholarPubMed
Skuce, P. J. and Fairweather, I. (1990). The effect of the hydrogen ionophore closantel upon the pharmacology and ultrastructure of the adult liver fluke Fasciola hepatica. Parasitology Research 76, 241250.CrossRefGoogle ScholarPubMed
Stitt, A. W. and Fairweather, I. (1990). Spermatogenesis and the fine structure of the mature spermatozoon of the liver fluke, Fasciola hepatica. Parasitology 101, 395407.CrossRefGoogle ScholarPubMed
Stitt, A. W. and Fairweather, I. (1993). Fasciola hepatica: tegumental surface changes in adult and juvenile flukes following treatment in vitro with the sulphoxide metabolite of triclabendazole (Fasinex). Parasitology Research 79, 529536.CrossRefGoogle ScholarPubMed
Stitt, A. W. and Fairweather, I. (1994). The effect of the sulphoxide metabolite of triclabendazole (“Fasinex”) on the tegument of mature and immature stages of the liver fluke, Fasciola hepatica. Parasitology 108, 555567.CrossRefGoogle ScholarPubMed
Stitt, A. W. and Fairweather, I. (1996). Fasciola hepatica: disruption of the vitelline cells in vitro by the sulphoxide metabolite of triclabendazole. Parasitology Research 82, 333339.CrossRefGoogle ScholarPubMed
Stitt, A. W., Fairweather, I. and Mackender, R. O. (1995). The effect of triclabendazole (“Fasinex”) on protein synthesis by the liver fluke, Fasciola hepatica. International Journal for Parasitology 25, 421429.CrossRefGoogle ScholarPubMed
Sumegi, B., Kovacs, K., Veres, B., Radnai, B., Varbiro, G., Bognar, Z., Toth, A. and Gallyas, F. (2005). Oxidative stress and the endoplasmic reticulum. In Endoplasmic Reticulum: a Metabolic Compartment (NATO Science Series, Vol. 363) (ed. Benedetti, A., Banhegyi, G. and Burchell, A.), pp. 121130. I.O.S. Press, Amsterdam, The Netherlands.Google Scholar
Threadgold, L. T. (1963). The tegument and associated structures of Fasciola hepatica. Quarterly Journal of Microscopical Science 104, 505512.Google Scholar
Threadgold, L. T. (1967). Electron-microscope studies of Fasciola hepatica. III. Further observations on the tegument and associated structures. Parasitology 57, 633637.CrossRefGoogle Scholar
Toner, E., McConvery, F., Brennan, G. P., Meaney, M. and Fairweather, I. (2009). A scanning electron microscope study on the route of entry of triclabendazole into the liver fluke, Fasciola hepatica. Parasitology 136, 523535.CrossRefGoogle Scholar
Uhlemann, A.-C., Wittlin, S., Matile, H., Bustamante, L. Y. and Krishna, S. (2007). Mechanism of antimalarial action of the synthetic trioxolane RBX1160 (OZ277). Antimicrobial Agents and Chemotherapy 51, 667672.CrossRefGoogle ScholarPubMed
Utzinger, J., Xiao, S.-H., Tanner, M. and Keiser, J. (2007). Aremisinins for schistosomiasis and beyond. Current Opinion in Investigational Drugs 8, 105116.Google ScholarPubMed
Vennerstrom, J. L., Arbe-Barnes, S., Brun, R., Charman, S. A., Chiu, F. C., Chollet, J., Dong, Y., Dorn, A., Hunziker, D., Matile, H., McIntosh, K., Padmanilayam, M., Tomas, J. S., Scheurer, C., Scorneaux, B., Tang, Y., Urwyler, H., Wittlin, S. and Charman, W. N. (2004). Identification of an antimalarial synthetic trioxolane drug development candidate. Nature, London 430, 900904.CrossRefGoogle ScholarPubMed
Wakabayashi, T. and Karbowski, M. (2001). Structural changes of mitochondria related to apoptosis. Biological Signals and Receptors 10, 2656.CrossRefGoogle ScholarPubMed
World Health Organization (2007). Report of the WHO Informal Meeting on Use of Triclabendazole in Fascioliasis Control, held at WHO Headquarters, Geneva, Switzerland, October 2006. World Health Organization, Geneva, Switzerland.Google Scholar
Wolstenholme, A., Fairweather, I., Prichard, R., von Samson-Himmelstjerna, G. and Sangster, N. (2004). Drug resistance in veterinary helminths. Trends in Parasitology 20, 469476.CrossRefGoogle ScholarPubMed
Woodrow, C., Haynes, R. and Krishna, S. (2005). Artemisinins. Postgraduate Medical Journal 81, 7178.CrossRefGoogle ScholarPubMed
Xiao, S.-H. (2005). Development of antischistosomal drugs in China, with particular consideration to praziquantel and the artemisinins. Acta Tropica 96, 153167.Google Scholar
Xiao, S.-H., Binggui, S., Utzinger, J., Chollet, J. and Tanner, M. (2002 a). Transmission electron microscopic observations on ultrastructural damage in juvenile Schistosoma mansoni caused by artemether. Acta Tropica 81, 5361.Google ScholarPubMed
Xiao, S.-H., Binggui, S., Utzinger, J., Chollet, J. and Tanner, M. (2002 b). Ultrastructural alterations in adult Schistosoma mansoni caused by artemether. Memorias do Instituto Oswaldo Cruz 97, 717724.CrossRefGoogle ScholarPubMed
Xiao, S.-H. and Catto, B. A. (1989). In vitro and in vivo studies of the effect of artemether on Schistosoma mansoni. Antimicrobial Agents and Chemotherapy 33, 15571562.CrossRefGoogle ScholarPubMed
Xiao, S.-H., Guo, J., Chollet, J., Wu, J.-T., Tanner, M. and Utzinger, J. (2004). Effect of artemether on Schistosoma mansoni: dose-efficacy relationship, and changes in worm morphology and histopathology. Chinese Journal of Parasitology and Parasitic Diseases 22, 148153.Google ScholarPubMed
Xiao, S.-H., Keiser, J., Chollet, J., Utzinger, J., Dong, Y., Endriss, Y., Vennerstrom, J. L. and Tanner, M. (2007). In vitro and in vivo activities of synthetic trioxolanes against major human schistosome species. Antimicrobial Agents and Chemotherapy 51, 14401445.CrossRefGoogle ScholarPubMed
Xiao, S.-H., Wu, Y.-L., Tanner, M., Wu, W.-M., Utzinger, J., Mei, J.-Y., Scorneaux, B., Chollet, J. and Zhai, Z. (2003). Schistosoma japonicum: in vitro effects of artemether combined with haemin depend on cultivation media and appraisal of artemether products appearing in the media. Parasitology Research 89, 459466.CrossRefGoogle ScholarPubMed