Fig 1.
Sequence of steps to acquire and analyze whole slide images.
(A) After loading the slides in the scanner, image acquisition starts with a “prescan” step in which the equipment takes a low resolution grayscale image followed by an automated setup process termed “profile” in which the tissue is detected and focused. (B) The equipment scans all regions of the slide defined by the profile and generates a virtual slide, which is saved on disk. These first steps take just few minutes and many slides can be sequentially scanned. (C, Ci) The operator can then review the virtual slide(s) and work on each one by selecting the area(s) of interest with the use of morphometric software. In the present work, the number and area of granulomas and area of inflammatory infiltrates were quantitated by using Pannoramic Viewer 1.15.2 SP2 RTM software. A representative digital slide shows a section from the small intestine of an S. mansoni-infected mouse in which the above parameters were manually delineated for subsequent automatic quantification.
Table 1.
Granuloma area analyzed in digital slides from target organs of S. mansoni-infected animals.
Fig 2.
Representative types of granulomas and their frequencies in target organs of rodents naturally or experimentally infected with S. mansoni.
Four types of granulomas were identified in virtual slides: Pre-granulomatous exudative (PE); necrotic-exudative (NE); exudative-productive (EP) and productive (P) as described in material and methods. Bar = 250 μm.
Fig 3.
Intensity of the granulomatous response in target organs of rodents naturally or experimentally infected with S. mansoni.
(A) Mean percentage of granulomatous response in livers and intestines (mean ± SEM). (B-J) Representative virtual slides of tissues with different levels of granuloma formation. In the liver (B, E, H), the lowest response is shown by the natural infection in N. squamipes (B) compared to the experimental infection in mice (E, H), while in the small intestines (C, F, I), the highest granuloma formation was observed in this wild reservoir (C). Different letters indicate significant differences between the means (P < 0.0001 for all comparisons between different letters in the respective groups). Bar = 1000 μm (B, D, F); 950 μm (H); 750 μm (C); 650 μm (E, G, I, J).
Fig 4.
Liver and intestine areas taken by inflammatory infiltrates in the natural and experimental infections with S. mansoni.
(A) Representative image of hepatic tissue from a mouse experimentally infected. After acquisition of whole slide images, areas with inflammatory infiltrates (marked in red) outside typical granulomas (marked in green) were measured. In (B), morphometric analyses reveal a very low incidence of infiltrates in the liver of infected wild rodent (natural infection) while mice experimentally infected show a very high proportion of infiltrates. In the small intestine, infiltrates are moderately higher in the natural compared to the acute experimental infection. Different letters indicate significant differences between the means (P < 0.0001 for all comparisons between different letters in the respective groups).
Fig 5.
Eosinophil numbers in the natural and experimental infections with S. mansoni.
(A) Representative image of a hepatic granuloma from a naturally infected wild reservoir (N. squamipes) showing accumulation of eosinophils (indicated in high magnification in Ai by arrowheads). (B) Quantitative analyses revealed a lower number of eosinophils per granuloma area (μm2) in all target organs in the natural infection compared to the experimental infections. Different letters indicate significant differences between the means (P < 0.0001 for all comparisons between different letters in the respective groups). (C) Proportion of eosinophils within hepatic granulomas in each model of the infection. (****) P < 0.0001. Eosinophil numbers were quantitated per granuloma area, considering all types of granuloma (B) or per most frequent type of hepatic granuloma (C). Data represent mean ± SEM. Morphometric evaluation was done with the use of Histoquant software. Bar = 400 μm (A), 100 μm (Ai).
Fig 6.
Confluent granulomas in livers and small and large intestines of Nectomys squamipes and Swiss mice infected with Schistosoma mansoni.
(A-H) General morphology of confluent granulomas in different organs. Graph I shows the proportions of confluent granulomas in livers and in intestines taken from naturally infected N. squamipes (at day one of capture after confirmation of the infection) and from experimentally infected Swiss mice at days 55 (acute phase) and 120 (chronic phase) of infection. Confluent granulomas were quantified using Pannoramic Viewer software. (***) indicates significant differences between the means (P < 0.001). Bar = 100 μm (A, D), 220 μm (B, C); 150 μm (E, F, G, H).
Fig 7.
Path of Schistosoma mansoni eggs in the small (A, B) and large intestines (C, D) in the rodents Nectomys squamipes (A, C) and Swiss mice (B, D). The granulomatous inflammatory reaction (IR) produces a path which leads the translocation of parasite eggs (*) from the inner layers to the lumen of the intestines, as shown by the arrows. In (Ai), a S. mansoni egg is going out of the villi into the lumen of the small intestine. Liver and intestine fragments were taken from naturally infected N. squamipes (at day one of capture after confirmation of the infection) and from experimentally infected Swiss mice at days 55 (acute phase) and 120 (chronic phase) of infection. Bar = 160 μm (A); 120 μm (B, C, D); 80 μm (Ai).