Trypanosoma cruzi : Where is it? Does it ever sleep? Why is a sterile outcome rarely achieved?

Trypanosoma cruzi infectionis responsible for Chagas disease, a condition that causes unacceptable morbidity and mortality throughout poorer populations of Latin America. Due to international migration, Chagas disease cases and asymptomatic T.cruzi infections are becoming a public health concern globally. At present there is no practical vaccine and the front-line drugs are rarely used due to low levels of diagnostic testing, long treatment regimens, unacceptable side effects and low efficacy. Here, we have employed state-of-the-art bioluminescent-fluorescent parasite reporter cell lines in experimental murine models to investigate parasite localisation in the chronic stage of infection, to assess if the recently reported dormant life-cycle stage can be demonstrated in vivo, and to better understand why infections are almost always life-long. In both the BALB/c and C3H/HeN murine models the gastrointestinal tract (GIT), specifically the colon and stomach, were confirmed to be primary sites of parasite persistence. Inthe colon, parasites were most commonly found in the circular smooth muscle layer, occupying the cytoplasm of the smooth muscle myocytes. The skeletal muscle in C3H/HeN, but not BALB/c mice, was identified as a key site of persistence, with the skeletal muscle fibres being the primary host cell type occupied. The skin in both models was shown to routinely harbour bioluminescent infection foci and therefore is likely to be a site of persistence, and critical to onward transmission. Infection in the acute stage is characterised by a large number of infected host cells, with rarely >50 amastigotes per cell. After transition to the chronic stage, even in reservoir sites such as the colon, the number of infected cells was extremely low. However, within these extremely rare infected myocytes, parasite numbers could be very high, up to 2000 in some cases. To monitor the rate of parasite replication in vivo, we injected infected mice with the thymidine analogue Ed U(5-Ethynyl-2-deoxyuridine). Incorporation of this analogue into parasite or host DNA identifies cells that are in S-phase during the period of exposure. This revealed that during the chronic stage of infection, there was a ~3-fold slow-down in the inferred rate of parasite replication, compared with the acute stage. Using a pulse-chase protocol we showed that long-term occupation of colonic wall myocytes is not a common feature of the chronic stage infection. We cannot exclude the possibility that parasites can enter a dormant state, but we found no definitive evidence for the existence of a truly dormant life-cycle stage in our T. cruzi infection model. Our interpretation of current data is that T. cruzi replication kinetics in vivo are more analogous to the 6general slow-down described in Leishmania, rather than the definitive long-term cell cycle arrest seen in the Plasmodium hypnozoites or Toxoplasma gondii bradyzoites. The technical approaches developed during this thesis allowed us, for the first time, to investigate the immunological context of large numbers of rare infected host cells in the colonic gut wall at late infection time-points. This revealed that a subset of infected host cells can act as a niche in which parasites are able to exist in large ‘mega-nests ’in the local absence of otherwise protective circulating T-cells. Investigating the molecular mechanisms responsible for this, and how they facilitate the long-term survival of the parasite, will be a focus of future work. We also found that serum antibody is incapable of maintaining the tight tissue load control characteristic of chronic infections, in the absence of circulating T-cells.