Rosario J Sosa1*, Jeevan B Sherchand1**, Nitaya Thammapalerd1, Daroon Kotimanusvanij2, Somchai Awakairt1*** Mario Riganti3, Savanat Tharavanij1
1Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand; 2Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; 3Department of Pathology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand

Abstract 
It is generally accepted that originally virulent strains of Entamoeba histolytica on prolonged cultivation in vitro, become avirulent or noninvasive when inoculated into the ceca of rats and guinea pigs or into the livers of hamsters. Certain manipulations could restore the virulence of an attenuated strain in culture. Among these are cholesterol treatment and hamster liver passage. The present study aimed at restoring virulence after 5 years in axenic culture of E. histolytica strain HM-1:IMSS, 4+ years in axenic culture of E. histolytica strain HTH-56:MUTM and 1 month in monoxenic culture of strain HTH-68:MUTM by giving prednisolone orally to the animals and then followed by liver passage of amebae, by liver passage of amebae together with Crithidia luciliae, and by liver passage of monoxenic amebae to the hamsters, respectively. Results of our experiments clearly indicated that virulence could be revived by so doing with success rates of abscess formation of 52%, 100% and 100%, respectively. In contrast, the failure to restore the virulence of amebae cultured in vitro for > 6 years in substrain HM-1:IMSS and in unmanipulated HTH-56:MUTM strain cultured in vitro for > 4 years occurred even by cholesterol treatment, liver passage in baby hamsters or splenectomy in the former and liver passage in the latter. The P15 line of E. histolytica strain HM-1:IMSS which had been periodically passed through the liver of hamsters to maintain its virulence served as positive control for abscess formation. To our knowledge, we suggest that periodic hamster liver passage offers the best method of restoring and maintaining the virulence of E. histolytica grown in axenic culture. Keywords: Entamoeba histolytica, virulence, in vitro cultivation, restoration, loss of virulence, prolonged culture