Chagas disease is a zoonosis caused by the protozoan parasite Trypanosoma cruzi. Transmission cycles are maintained by haematophagous triatomine bug vectors that carry infective T. cruzi in their faeces. Most human infections are acquired by contamination of mucosal membranes with triatomine faeces after being bitten, however, T. cruzi can be transmitted by several other routes. Oral transmission is an increasingly important aspect of Chagas disease epidemiology, typically involving food or drink products contaminated with triatomines. This has recently caused numerous outbreaks and been linked to unusually severe acute infections. The long-term impact of oral transmission on infection dynamics and disease pathogenesis is unclear. We used highly sensitive bioluminescence imaging and quantitative histopathology to study orally transmitted T. cruzi infections in mice. Both metacyclic and bloodform trypomastigotes were infectious via the oral cavity, but only metacyclics led to established infections by intra-gastric gavage. Mice displayed only mild acute symptoms but later developed significantly increased myocardial collagen content (p = 0.017), indicative of fibrosis. Gastrointestinal tissues and skin were the principal chronic infection reservoirs. Chronic phase parasite load profiles, tissue distribution and myocardial fibrosis severity were comparable to needle-injected controls. Thus, the oral route neither exacerbates nor ameliorates experimental Chagas disease.

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. The mechanism of pathogenesis is poorly understood and the lack of a robust, predictive animal model has held back research. We screened a series of mouse models using gastrointestinal tracer assays and in vivo infection imaging systems to discover a subset exhibiting chronic digestive transit dysfunction and significant retention of faeces in both sated and fasted conditions. The colon was a specific site of both tissue parasite persistence, delayed transit and dramatic loss of myenteric neurons as revealed by whole-mount immunofluorescence analysis. DCD mice therefore recapitulated key clinical manifestations of human disease. We also exploited dual reporter transgenic parasites to home in on locations of rare chronic infection foci in the colon by ex vivo bioluminescence imaging and then used fluorescence imaging in tissue microdomains to reveal co-localisation of infection and enteric nervous system lesions. This indicates that long-term T. cruzi-host interactions in the colon drive DCD pathogenesis, suggesting that the efficacy of anti-parasitic chemotherapy against chronic disease progression warrants further pre-clinical investigation.

Leishmania donovani causes visceral leishmaniasis (VL), which is typically fatal without treatment. There is substantial variation between individuals in rates of disease progression, response to treatment and incidence of post-treatment sequelae, specifically post-kala-azar dermal leishmaniasis (PKDL). Nevertheless, the majority of infected people are asymptomatic carriers. Hamsters and mice are commonly used as models of fatal and non-fatal VL, respectively. Host and parasite genetics are likely to be important factors, but in general the reasons for heterogeneous disease presentation in humans and animal models are poorly understood. Host microbiota has become established as a factor in cutaneous forms of leishmaniasis but this has not been studied in VL. We induced intestinal dysbiosis in mice and hamsters by long-term treatment with broad-spectrum antibiotics in their drinking water. There were no significant differences in disease presentation in dysbiotic mice. In contrast, dysbiotic hamsters infected with L. donovani had delayed onset and progression of weight loss. Half of control hamsters had a rapid progression phenotype compared with none of the ABX-treated animals and the nine-month survival rate was significantly improved compared to untreated controls (40% vs. 10%). Antibiotic-treated hamsters also had significantly less severe hepatosplenomegaly, which was accompanied by a distinct cytokine gene expression profile. The protective effect was not explained by differences in parasite loads or haematological profiles. We further found evidence that the gut-liver axis is a key aspect of fatal VL progression in hamsters, including intestinal parasitism, bacterial translocation to the liver, malakoplakia and iron sequestration, none of which occurred in non-progressing murine VL. Diverse bacterial genera were cultured from VL affected livers, of which

Gastrointestinal (GI) disease affects a substantial subset of chronic Chagas disease (CD) patients, but the mechanism of pathogenesis is poorly understood. The lack of a robust, predictive animal model of chronic T. cruzi infection that exhibits functional digestive disease has held back research. To address this, we combined GI tracer assays and bioluminescence in vivo infection imaging systems for diverse parasite strains to discover models exhibiting chronic digestive transit dysfunction. We identified the colon as a specific site of both tissue parasite persistence and delayed transit. Digestive CD mice exhibited significant retention of faeces in both sated and fasted conditions. Histological and immunofluorescence analysis of the enteric nervous system (ENS) revealed a dramatic reduction in the number of neurons and a loss of immunoreactivity of the enteric neural network in the colon. This model therefore recapitulates key clinical manifestations of human digestive CD. We also exploited dual bioluminescent-fluorescent parasites to analyse rare chronic infection foci in the colon at the single cell level, revealing co-localisation with ENS lesions. This indicates that long-term T. cruzi-host interactions in the colon drive pathogenesis and thus chronic disease may be preventable using anti-parasitic chemotherapy.

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. There is a lack of evidence on the mechanism of pathogenesis and rationales for treatment. We used a mouse model that recapitulates key clinical manifestations to study how infection dynamics shape DCD pathology, and the impact of treatment with the front-line drug benznidazole. Curative treatment at 6 weeks post-infection resulted in sustained recovery of GI transit function, whereas sub-curative treatment led to infection relapse and gradual return of DCD symptoms. Neuro-immune gene expression profiles shifted from chronic inflammation to a tissue repair signature after cure, accompanied by increased glial cell activity and regenerative neurogenesis in the myenteric neuronal plexus. Delaying treatment until 24 weeks post-infection led to a partial reversal of the DCD phenotype, suggesting the accumulation of permanent tissue damage over the course of chronic infection. Our study shows that murine DCD pathogenesis is sustained by chronic T. cruzi infection and is not an inevitable consequence of acute stage denervation. The risk that irreversible enteric neuromuscular tissue damage and dysfunction will develop highlights the importance of prompt diagnosis and treatment. Finally, these findings support the concept of treating asymptomatic T. cruzi infected individuals with benznidazole to prevent DCD development.