Rapid communication Apoptosis of Leishmania (Leishmania) chagasi amastigotes in hamsters infected with visceral leishmaniasis Jose ´ A.L. Lindoso a,b , Paulo C. Cotrim a,c , Hiro Goto a,d, * a Laborato ´rio de Soroepidemiologia e Imunobiologia, Instituto de Medicina Tropical de Sa ˜o Paulo, Universidade de Sa ˜o Paulo, Avenida Dr. Ene ´as de Carvalho Aguiar, 470, 05403-000 Sa ˜o Paulo, SP, Brazil b Instituto de Infectologia Emı ´lio Ribas, SES-SP, Sa ˜o Paulo, Brazil c Departamento de Mole ´stias Infecciosas, Faculdade de Medicina, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil d Departamento de Medicina Preventiva, Faculdade de Medicina, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil Received 30 July 2003; received in revised form 19 September 2003; accepted 19 September 2003 Abstract Apoptosis in amastigotes from hamsters infected with visceral leishmaniasis was absent 30-day post-infection but appeared 90-day post- infection in the liver and spleen, as analysed using the TUNEL method. Necrosis was not present in these tissues and the nuclei of macrophages harbouring apoptotic amastigotes were preserved. Amastigote DNA fragmentation was demonstrated using agarose gel electrophoresis. DNA fragmentation was evident 90-day post-infection, coinciding with the occurrence of apoptosis of amastigotes in the tissues. Apoptosis of Leishmania amastigotes in vivo may constitute a mechanism that regulates growth of the parasite population during infection. q 2004 on behalf of Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Leishmania (Leishmania) chagasi; Visceral leishmaniasis; DNA fragmentation; In vivo; TUNEL method Leishmania (Leishmania) chagasi is a protozoan that causes visceral leishmaniasis in Brazil, while in the Old World it is caused by Leishmania (L.) donovani, and in the southeast of Europe and the Mediterranean area by Leishmania (L.) infantum. The infectious agent is an intracellular organism that proliferates within the cells of the mononuclear phagocyte system, mainly in the spleen and liver. Regu- lation of the parasite population is necessary for its own survival, and one important mechanism known to regulate cell populations in tissues and organs is apoptosis (Cohen and Duke, 1992). This process was initially described in multicellular organisms; however, death by a process suggestive of apoptosis has been observed in vitro in some unicellular organisms such as Tetrahymena (Davis et al., 1992), Dictyostelium discoideum (Cornilon et al., 1994) and Trypanosoma brucei rhodesiense (Welburn et al., 1996). Recently, an apoptosis-like process has been obser- ved under various stimuli in Leishmania (L.) amazonensis promastigotes (Moreira et al., 1996), Leishmania (L.) donovani promastigotes (Das et al., 2001), L. infantum axenic amastigotes (Sereno et al., 2001), L. major promastigotes (Arnoult et al., 2002) and L. donovani promastigotes (Lee et al., 2002). In trypanosomatids, apoptosis is claimed to be a mechanism of parasite population control triggered by overcrowding and/or nutrient restriction (Welburn et al., 1997; Knight, 2002). In the present work, we evaluate the occurrence of apoptosis in Leishmania (L.) chagasi amastigotes in the spleen and liver of hamsters infected with visceral leishmaniasis. Out-bred, 45–60-day-old male hamsters (Mesocricetus auratus), supplied by the Animal Breeding Facility of the Medical School of the University of Sa ˜o Paulo, were maintained at the Animal Facility of the Tropical Medicine Institute of Sa ˜o Paulo of the University of Sa ˜o Paulo. Hamsters received water and food ad libitum during the experiments. All procedures in this study complied with the ethical guidelines set down by the Institute for Animal Experimentation. 0020-7519/$30.00 q 2004 on behalf of Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2003.09.010 International Journal for Parasitology 34 (2004) 1–4 www.parasitology-online.com * Corresponding author. Address: Laborato ´rio de Soroepidemiologia e Imunobiologia, Instituto de Medicina Tropical de Sa ˜o Paulo, Universidade de Sa ˜o Paulo, Avenida Dr Ene ´as de Carvalho Aguiar, 470, 05403-000 Sa ˜o Paulo, SP, Brazil. Tel.: þ55-11-3066-7023; fax: þ 55-11-3062-3622. E-mail address: hgoto@usp.br (H. Goto).