M C Taylor2; A Ward2; A F Francisco1; S Jayawardhana2; M Lewis1; J M Kelly1;
1 Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT., UK; 2 London School of Hygiene and Tropical Medicine, UK
DiscussionTrypanosoma cruzi causes a chronic life-long infection. Parasites are rarely observed in the blood and infected patients may routinely be PCR-negative. Highly sensitive bioluminescence imaging (BLI) has allowed us to establish the GI tract as the major reservoir site in murine models of chronic T. cruzi infections. However, BLI is limited in its ability to identify parasites at a cellular level. To circumvent this, we generated trypanosomes expressing a luciferase-mNeonGreen fusion protein, such that parasites were both highly bioluminescent and fluorescent. In acutely infected mice many organs are infected and nests of amastigotes are readily identified in the heart, the adipose tissue and in other organs. In chronically infected animals, where parasites are mainly restricted to the GI tract, there were very few nests, with infections generally restricted to single or low numbers of amastigotes per cell. However, some of the nests detected in the chronic phase contained extremely large numbers of parasites. Using a combination of TUNEL assays for kDNA replication, and EdU labelling, we have been able to examine the replication of the parasites in the chronic phase. This has demonstrated that in all phases of the infection parasite replication within a single infected cell is asynchronous, regardless of the number of parasites within the nest. Differentiation from amastigote to trypomastigote also appears to be asynchronous and multiple morphological forms can exist within a single nest, including epimastigote-like cells and amastigotes with protruding flagella.