Fig 1.
(A) Schematic representation of prophylactic treatment, infection, and monitoring of mice. WT and IFNγ deficient mice were treated biweekly with 100mg/kg of BNZ over 24 weeks. After the second week of treatment, mice were infected (5–6 animals/group) intraperitoneally with 103 trypomastigotes of the Luciferase-expressing Colombiana strain of T. cruzi. Control groups of mice were infected and left untreated. (B) Representative images showing bioluminescent signal in mice (ventral view) at 14 days post-infection exposure. (C) Parasite bioluminescence quantification following D-luciferin injection was measured at the indicated times in the study period. Each data point represents the mean of bioluminescence from 3–6 mice expressed on a logarithmic scale as mean photons/second ±/- standard error of the mean. Grayed box marks the prophylaxis period. * p<0.03 relative to both WT groups; ** p<0.008 relative to both BNZ-treated groups; *** p<0.005 relative to BNZ-treated IFNγ KO group; n.s. bracketed data points are not statistically different. (D) Percentage of CD8+ blood lymphocytes specific for the T. cruzi TSKb20 epitope (top) and expressing the T cell central memory marker CD127 (bottom) during the study course (E) T. cruzi DNA determined by quantitative real time polymerase chain reaction in the skeletal muscle, heart, and intestine of untreated and BNZ-treated mice at week 33 of the study (~30 weeks post-infection). Dashed line indicates limit of quantification based on the range of the standard curve.
Fig 2.
Initial screening results with multiplex serology and PCR to identify T. cruzi-negative dogs to enroll in the prophylaxis study.
(A) Schematic representation of dog screening and prophylaxis trial. Heatmaps of antibody levels (mean fluorescence intensity; MFI) and qPCR measurement of T. cruzi DNA in blood were used to classify dogs as uninfected (B), T. cruzi-infected (C), or seronegative but with a blood PCR result that did not reach the cut-off for being considered positive (D). Recombinant T. cruzi proteins are defined in the Materials and Methods; lysate = a sonicate of T. cruzi trypomastigotes and amastigotes; GFP = recombinant green fluorescent protein (negative antigen control); Parvo = Parvovirus vaccine antigen (positive serological control).
Fig 3.
Multiplex serological and blood PCR changes over the transmission season in dogs receiving or not BNZ prophylaxis.
(A) Serological and PCR negative dogs (see Fig 2) were randomly assigned to the untreated or BNZ treatment groups and then rescreened at ~12 and ~24 weeks after the beginning of prophylaxis. One dog in each group (gray fill) was moved to BNZ treatment protocol after the 12 week sampling point due to high serum cardiac troponin I (cTnI) levels. One dog in each group was lost to follow-up before the 24 week sampling time due to death or sold by owner. Antigens used in the multiplex assay are as described in Fig 2 and the Materials and Methods. Blood PCR was considered positive if below the cut-off Ct value of 35. (B) Fraction of uninfected dogs in the initial screening group and their decrease in frequency as assessed in (A) at the midpoint (12 weeks) and end of treatment (24 weeks) sample timepoints (top) and comparison of the percentage of uninfected dogs in the untreated and prophylaxed groups over the study period (bottom).
Fig 4.
Multiplex serological and blood PCR changes over the transmission season in dogs that were seronegative but PCR undetermined in the pre-transmission season screening (see Fig 2).
Antigens used in the multiplex assay are as described in Fig 2 and the Materials and Methods. * moved to a high dose BNZ treatment protocol after 12 week sample point.