Replication and Distribution of Toxoplasma gondii in the Small Intestine after Oral Infection with Tissue Cysts

Abstract

Natural infection by Toxoplasma gondii occurs via oral ingestion of tissue cysts that rupture in the small intestine, releasing zoites that infect locally before disseminating throughout the host. The studies presented here used fluorescent parasites combined with flow cytometry and multiphoton microscopy techniques to understand the events associated with parasite replication in the mucosa. At 3 days postinfection with tissue cysts, parasites were localized in small foci and flow cytometry revealed parasites present in macrophages, neutrophils, and monocytes in the lamina propria. By day 6 postinfection, there were large foci of replicating parasites; however, foci unexpectedly varied in the number of villi involved and were associated with the presence of viable tachyzoites within the intestinal lumen. Consistent with the flow cytometry data, neutrophils and monocytes in the lamina propria were preferentially associated with parasite plaques. In contrast, dendritic cells comprised a small fraction of the infected immune cell population and were localized at the periphery of parasite plaques. Together, these findings reveal the formation of localized sites of parasite replication and inflammation early during infection and suggest that sustained replication of T. gondii in the gut may be a function of pathogen luminal spread.

INTRODUCTION

While pathogens such as Escherichia coli, Giardia lamblia, and certain helminths survive in the hostile environment of the intestinal lumen, other orally acquired pathogens cross the intestinal epithelial cell barrier and evade host antimicrobial responses to establish infection. For Toxoplasma gondii, initial ingestion of oocysts or tissue cysts leads to infection of intestinal epithelial and lamina propria cells (1–3). The ability of T. gondii to disseminate from the intestine depends on its ability to evade an array of antimicrobial responses that are initiated rapidly after infection (4). Nevertheless, how parasites disseminate from the initial site of infection remains unclear and may involve the migration of extracellular tachyzoites released from lysed cells or the ability of T. gondii to take advantage of the motility of monocytes or dendritic cells to carry parasites throughout the body (5–9). Attempts to understand these early events are limited by what is known about cellular infection and control of parasites at the initial site of infection in the small intestine.

The current understanding of oral T. gondii infection maintains that initial cyst rupture results in the release of dormant zoites that infect the lamina propria of the small intestine by either infecting, invading through, or passing between intestinal epithelial cells (1–3, 10, 11). Various studies indicate that the major site of parasite replication is in the small intestinal ileum (1–3, 11, 12); however, recent quantitative PCR analysis studies by Courret and colleagues (9) found that the highest burdens are present within the jejunum. Regardless, once within the lamina propria, parasites invade and replicate within a variety of cell types, including neutrophils, macrophages, lymphocytes, and intestinal epithelial cells (2, 3, 9, 11). The consequences of oral infection with T. gondii cysts can vary depending on host and parasite genotype, ranging from asymptomatic infection to the development of severe pathology, including ulceration and lethal ileitis exacerbated by the breakdown of the intestinal epithelial cell barrier and bacterial translocation from the intestinal lumen into the lamina propria (2, 3, 12–16). The implication of this body of work is that following initial infection, there is a process of parasite dissemination throughout and away from the intestine; however, how this occurs is unclear.

In order to better understand the early events in the small intestine following oral challenge with T. gondii, transgenic parasites expressing fluorescent tdTomato protein were examined using flow cytometry to identify and quantify the infected cell types. Unexpectedly, multiphoton imaging of intestinal whole mounts revealed that infection leads to initial foci of replication that enlarge to encompass multiple villi, and these foci are surrounded by CD11c⁺ cells, while LysM⁺ cells are closely associated with areas of parasite replication. These studies provide new insights into events at the initial site of infection and suggest that luminal spread of parasites propagates new foci that may act as an additional source of parasites that continue to disseminate throughout the host.

MATERIALS AND METHODS

Mice.

C57BL/6, Swiss Webster, and CBA female mice were obtained from The Jackson Laboratories at 6 to 8 weeks of age. CD11c yellow fluorescent protein (YFP), major histocompatibility complex class II (MHC-II) green fluorescent protein (GFP), and LysM enhanced green fluorescent protein (eGFP) mice (a gift from David Sacks at NIAID, NIH), all on a C57BL/6 background, were bred in the specific-pathogen-free facility at the University of Pennsylvania. All experiments were conducted in accordance with United States and international guidelines and approved by the University of Pennsylvania Institutional Animal Care and Use Committee.

Parasites and cell cultures.

Prugniaud (Pru) strain T. gondii parasites were maintained by serial passage in human foreskin fibroblast (HFF) monolayers cultivated in Eagle's minimal essential medium (Gibco) containing 1% fetal bovine serum (FBS), as previously described (17). Extracellular tachyzoites were purified by filtration through 3.0-μm-pore-size filters (Nuclepore) and washed in phosphate-buffered saline (PBS). Transgenic Pru strain parasites were engineered to express tandem dimers of tdTomato (abbreviated below as Tomato) (19) or tdTomato-ovalbumin (OVA) (18), and tissue cyst (bradyzoite) stages were maintained through serial passage in Swiss Webster and CBA mice (20). For infections, brains of chronically infected mice were mechanically separated by passage through a series of 18-gauge, 20-gauge, and 22-gauge needles; cysts in the brain homogenate were then counted, and approximately 50 cysts were delivered orally with a 20-gauge gavage needle.

The presence and viability of extracellular parasites from the lumen of infected mice were assessed using fluorescence microscopy and transfer of lumen contents to naive mice. C57BL/6 mice were infected orally with 50 Pru Tomato cysts and sacrificed 3 or 6 days postinfection. The small intestines of naive and infected mice were removed and rinsed in PBS prior to gentle PBS perfusion of the lumen. Large particulate matter in the lumen contents was removed from the supernatants after gentle vortexing and a rest period, while the remaining debris and host cells were removed by passing supernatants through a 5.0-μm-pore-size filter membrane (Whatman). The remaining material was collected by centrifugation at 400 × g for 5 min, resuspended in 500 μl of PBS, and aliquoted for use: 25-μl aliquots were stained with 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Invitrogen) for fluorescence microscopy, and the remaining suspension was used to intraperitoneally infect naive Swiss Webster mice. Peritoneal lavage was performed on day 7 postinfection, and Pru Tomato-infected cells were detected using flow cytometry.

Intestinal whole mounts and plaque counting.

To visualize plaques in the entire small intestine, a whole-mount technique developed by McDonald and Newberry was modified (21). Briefly, small intestines from naive and infected mice were separated into four equal segments and cleared of luminal contents by gentle perfusion with PBS. Segments were then cut open longitudinally and embedded lumen side up in 1.5% agarose. Paraformaldehyde (4%) was used to fix tissues overnight at 4°C; tissues were stored at 4°C in PBS for up to 1 week prior to counting. Parasite plaques were counted on a Nikon E600 epifluorescence scope using either a ×4 or ×10 objective. Plaques were measured as the number of infected villi and counted as distinct when separated by one or more uninfected villi.

Tile scan confocal imaging.

Small intestines from naive and infected animals were imaged with a Leica SP5 microscope using a ×10 air objective lens and LGK 7872 ML05 argon and 543-nm diode lasers. Preparation of the intestine for imaging consisted of washing the lumen and opening the intestine to expose villi, as described for whole mounting. Prepared segments, luminal surface up, were adhered to glass slides by a thin layer of Vetbond tissue adhesive (Fisher Scientific) and lightly moistened with Dulbecco's modified Eagle's medium (DMEM) containing no phenol red (Gibco). A cover glass was placed on the surface of the segments for imaging. Confocal z-plane images were acquired every 10 μm while fluorescence was detected; multiple z stacks were taken with a 10% overlapping area using Leica's LAS AF software. Merging of tiled images was completed using LAS AF software.

Multiphoton microscopy.

Small intestines, including Peyer's patches, from naive mice and mice that had been infected for 3 and 6 days were removed and maintained ex vivo in DMEM at room temperature. Intestinal segments clear of luminal contents were removed, gently patted dry, and opened longitudinally. Peyer's patches were mechanically removed for imaging, and both lumen and muscle surfaces were blotted dry for mounting. Segments, either intestinal or Peyer's patches, were placed onto a Vetbond-coated cover glass, lumen or muscle side up, and the cover glass was secured into the imaging chamber (Warner Instruments) with agarose (22). During imaging, segments were perfused with warm phenol-free DMEM, oxygenated (95% O₂, 5% CO₂), and kept at 37°C. Imaging was completed using a Leica SP5 two-photon microscope equipped with a ×20 water immersion lens, a picosecond laser (720 nm to 980 nm; Coherent Chameleon), and tunable internal detectors for detection of emissions of different wavelengths and secondary harmonic signals (SHGs). For optimal imaging depth, 495-nm and 560-nm filters in a nondescanned detector (NDD) system were used to image fluorescence and secondary harmonic resonance signals (SHG). Excitation of eGFP, YFP, and Tomato was achieved using a laser wavelength of 928 nm. Using the Leica LAS AF software (Leica Microsystems), z stacks with a step size of 2 μm were taken, while image movies were created from 24-μm-thick z stacks taken every 30 to 35 s.

Image analysis.

Confocal and two-photon images with identical bit data were analyzed using Volocity software measurement and tracking tools (PerkinElmer). Raw data from Volocity were exported to the Microsoft Excel program, and intensity, distance, and volume values were used to determine the presence of fluorescence per unit area. Densitometry data were collected from images as fluorescence intensity along a transect line bisecting a single plaque or fluorescence intensity within successive regions of interest measured radially from a central infected villus by a single villus unit (see Fig. S1 in the supplemental material).

Characterization of infected cells.

C57BL/6 mice were infected orally with whole-brain homogenate containing approximately 50 Pru Tomato OVA or Pru Tomato cysts either 3 or 6 days prior to sacrifice. Naive mice and mice that had been infected for 3 and 6 days were sacrificed, and the small intestine, Peyer's patches, mesenteric lymph nodes, spleen, and blood were collected for processing. Small intestinal lamina propria was isolated through EDTA-dithiothreitol treatment, followed by collagenase treatment (0.5 mg/ml) and a Percoll gradient (23). Peyer's patches and spleen were digested for 30 min at 37°C in collagenase and mechanically disrupted. Mesenteric lymph nodes were mechanically disrupted for a single-cell suspension, and blood was treated with 0.1 M sodium citrate prior to layering on Histopaque (Sigma-Aldrich) and centrifugation. Cell suspensions were stained for dead cells with Invitrogen's Aqua Blue Live/Dead dye (24) and with fluorescently labeled antibodies for cell markers Ly6C, F4/80, CD3, CD19, NK1.1, CD11c, CD11b, CD8, CD4, MHC-II, and B220 (see Table S1 in the supplemental material). Data from cells were collected on a digital BD LSRII flow cytometer equipped with DIVA software and analyzed using FlowJo software.

RESULTS

T. gondii infection in the intestinal mucosa.

In order to better understand the early events that occur during toxoplasmosis, C57BL/6 mice were infected by oral gavage with Pru Tomato tissue cysts and cell populations from the lamina propria, Peyer's patches, mesenteric lymph nodes, blood, and spleen of infected mice were analyzed using flow cytometry to determine which cell types were infected with T. gondii. On the basis of the presence of Tomato protein fluorescence, infected cells within each organ were easily distinguished as a population of cells absent in naive controls, as shown for the lamina propria on days 3 and 6 postinfection (Fig. 1A). By day 3 postinfection, a small population of infected cells averaging approximately 0.01% of the population was readily detected within the lamina propria, Peyer's patches, and mesenteric lymph nodes (Fig. 1B). By day 6, the infected population was at least 10-fold higher at these sites than other areas, such as the blood and spleen (Fig. 1C).

Fig 1.

Flow cytometry analysis of immune cells from Pru Tomato-infected mice indicates the parasite burden in tissues on days 3 and 6 postinfection. Tissues from C57BL/6 mice infected by oral gavage with 50 Pru Tomato tissue cysts were processed and analyzed for cellular infection, as determined by the presence of Tomato fluorescence. (A) As seen in representative grayscale pseudocolored plots from single-cell suspensions of lamina propria cells, parasitized cells were gated on the basis of the absence of a population in naive mice. Values displayed in the gates indicate the percentage of total live cells for that sample. (B) Analysis of flow cytometry data accumulated for the lamina propria, Peyer's patches, mesenteric lymph nodes, blood, and spleen was used to determine the parasite burden on days 3 and 6 postinfection as a percentage of all live cells (n = 9). The limit of detection (dashed line) was determined as an average of the background detected in naive mice. Error bars represent the standard errors of the means.

To determine what specific cell types were infected with T. gondii, cell populations from the lamina propria, Peyer's patches, mesenteric lymph nodes, blood, and spleen of mice infected for 3 or 6 days with Pru Tomato tissue cysts were analyzed using flow cytometry to distinguish macrophages, monocytes, neutrophils, B cells, T cells, and dendritic cell subsets (for markers, see Table S1 in the supplemental material). At day 3 postinfection, the parasite burden in the blood and spleen was too low to accurately identify and phenotype the infected cells (Fig. 1B). Results of the analysis from infected cell populations that were above the limit of detection (Fig. 1B) are presented in Fig. 2 and indicate that cellular infection varied only slightly between lymphoid-associated tissues (Peyer's patches, mesenteric lymph nodes, spleen, and blood). In comparison, the profile of infected cells in the lamina propria was significantly different from what was observed in the lymphoid tissues on both days 3 and 6 postinfection (Fig. 2). Although the infected cell population in all tissues was reflective of the composition of the total cell population, greater numbers of phagocytic cells, most notably, monocytes, neutrophils, and, to a lesser extent, dendritic cells, contained parasites in the lamina propria (Fig. 2). Another notable difference between the infected population of the lamina propria and the lymphoid tissues was in the lymphocyte populations; however, this difference was largely reflective of the availability of these cells in each organ (Fig. 2 and data not shown). In other words, the percentage of the infected population composed of lymphocytes was high in lymphoid organs, where these cells are the most abundant, whereas the percentage of the infected population composed of lymphocytes in the lamina propria was lower, as the lamina propria harbors fewer lymphocytes (Fig. 2 and data not shown).

Fig 2.

Characterization of infected cell populations from the lamina propria, Peyer's patches, mesenteric lymph nodes, blood, and spleen on days 3 and 6 postinfection. Each graph represents the total parasitized cell population in a tissue on day 3 or day 6 postinfection; values indicate the percentage of the population for each cell type. Graphs representing the same tissues are highlighted as follows: lamina propria (blue), Peyer's patches (orange), mesenteric lymph nodes (green), blood (pink), and spleen (yellow). Due to the limited number of events collected, data from the spleen and blood are displayed only for day 6 postinfection (6dpi). The infected cell population in each tissue was phenotyped using flow cytometry analysis. Markers to identify macrophages, monocytes, neutrophils, B cells, CD4⁺ T cells, CD8⁺ T cells, CD11b⁺ dendritic cells (DC), CD8α⁺ dendritic cells, CD4/CD8α double-negative (DN) dendritic cells, and plasmacytoid dendritic cells (pDC) were used, and cells not falling into any of these categories were classified “others.” All cell types are presented in the key. Markers used to distinguish each cell type are described in Table S1 in the supplemental material.

T. gondii replication within the small intestine results in multiple distinct plaques.

To establish where parasite replication takes place in the intestine and to distinguish the lamina propria from the intestinal lumen, multiphoton imaging was performed using MHC-II GFP reporter mice on a C57BL/6 background (in which intestinal epithelial cells are marked due to their expression of MHC-II protein) infected with Pru Tomato by oral ingestion (25). In naive mice, the well-ordered epithelial cell layer clearly distinguished individual intestinal villi and could be imaged for several hours without observable destruction of the tissue (Fig. 3A and unpublished observations). Unlike images from naive tissue, multiphoton images of villi from MHC-II GFP infected mice showed varied structural architectures, ranging from areas similar to those in naive animals with a well-defined epithelial layer to areas with indiscernible epithelial cells and increased MHC-II GFP cells in the lamina propria (Fig. 3B and C and data not shown). These differences were observed in addition to decreased stability of infected tissue, including the sloughing of the epithelial cell layer after prolonged imaging.

Fig 3.

Visualization and quantification of parasites in the small intestine. Sections of the small intestine measuring 5 to 10 mm were taken from naive and infected MHC-II GFP mice and mounted for use in live multiphoton imaging. For images, z stacks up to 120 μm deep (5-μm steps) were merged into one maximum-focus plane, scale bars were added, and fine noise was filtered using Volocity software (resolution, 0.98 μm/pixel). In the images, MHC-II GFP is seen in green, Pru Tomato is seen in red, and secondary harmonic resonance signals are blue. (A) Image from a naive sample. (B and C) Representative images demonstrate the typical size of Pru Tomato plaques observed within villi. (B) Image from day 3 postinfection (3 dpi) where two plaques of replicating parasites are present (arrows); (left) with the GFP channel; (right) without the GFP channel. (C) On day 6 postinfection (6 dpi), replicating parasites in multivillus plaques are easily distinguished. (D and E) Quantification of the size (D) and number (E) of plaques on days 3 and 6 postinfection was determined by visual inspection for Pru Tomato in whole mounted small intestine using an epifluorescence scope equipped with a ×10 objective (n = 12 and 9, respectively). Intestinal sections from the stomach to the cecum roughly correspond to the duodenum (Duo), proximal jejunum (Jej 1), distal jejunum (Jej 2), and ileum. (D) Plaque size was determined by counting the number of adjacent infected villi; all plaques are represented. Error bars indicate standard errors of the means.

At 3 days postinfection, individual or a few closely associated villi that contained multiple parasites were observed, as shown in maximum focus both with and without GFP channel data to reveal parasites (Fig. 3B, showing a split panel of a z stack with GFP data on the left and the same z stack without GFP data on the right). To determine the number of infected villi on day 3 postinfection, the entire small intestine was whole mounted and paraformaldehyde fixed for epifluorescence imaging. Through this process, it was determined that infected villi were primarily found within the proximal jejunum. Additionally, parasite replication was typically contained within a single villus, although as many as five adjacent infected villi were observed (Fig. 3D and E). The number of areas of parasite replication increased throughout the small intestine between days 3 and 6 postinfection (Fig. 3E). By day 6 postinfection, imaging showed that multiple adjacent villi were densely packed with replicating parasites in plaque structures present throughout the small intestine (Fig. 3D and E). The average number of infected villi per plaque was 11 but varied from as few as 1 infected villus to more than 100 infected villi (Fig. 3D). When areas of parasite replication were examined in a T. gondii-resistant mouse strain (BALB/c) on day 6 postinfection, fewer intestinal plaques were observed (duodenum = 8, proximal jejunum [Jej 1] = 10, distal jejunum [Jej 2] = 2, ileum = 1), but the size (x = 9 villus units) and distribution of individual plaques were similar to those in C57BL/6 mice (data not shown). Thus, the phenomenon of plaque formation was not unique to C57BL/6 mice.

Analysis of plaque composition.

As monocytes and neutrophils are major cell populations infected with T. gondii (Fig. 2), LysM eGFP mice (C57BL/6 background) were used to characterize the localization of these cells at the site of infection (26). In the lamina propria of naive mice, cells observed in z stacks were predominantly eGFP intermediate, with an occasional eGFP-high cell being observed; these findings are consistent with the flow cytometry analysis that indicated a predominantly macrophage population (Fig. 4A, B, and E and data not shown) (26, 27). In infected LysM eGFP mice, large areas of parasite replication were readily apparent and closely associated with an increased population of eGFP-high cells (neutrophils and inflammatory monocytes) in the lamina propria (Fig. 4C, D, and F). Maximum-focus images and three-dimensional (3D) projections of infected villi highlighted the close proximity between parasites and LysM eGFP cells, as well as differences in cell organization between naive and infected animals (Fig. 4A and B versus C and D). Tile scanning confocal microscopy revealed the distribution of cells throughout the entirety of infected areas and the surrounding tissue (Fig. 4E and F). Results from densitometry analysis of villi from naive and Pru Tomato-infected mice showed a general increase in eGFP fluorescence intensity after infection, as the fluorescence intensity for eGFP was greater where there was the highest intensity of Tomato protein (Fig. 4G). This analysis highlights that an increased population of LysM eGFP cells is associated with areas of parasite replication.

Fig 4.

Fluorescence imaging in naive and infected LysM eGFP small intestine. (A to D) Multiphoton z stacks (approximately 120 μm deep, 5-μm steps) from naive (A and B) and Pru Tomato tissue cyst-infected (C and D) mice were merged into a single plane (A and C) or 3D projected (B and D) to demonstrate the distribution of LysM eGFP cells and parasites in villi (resolution, 0.98 μm/pixel). The coloration in the images is as follows: LysM eGFP is in green, Pru Tomato is in red, and secondary harmonic resonance is in blue. Minimal processing was performed to remove noise. Scale bars were added to indicate size (Volocity). LysM eGFP cells appeared uniformly distributed within the lamina propria of villi in naive mice but were preferentially clustered among parasites on day 6 postinfection (A and B versus C and D). (E and F) Tile scanning confocal microscopy of whole mounted intestine samples, including plaques and surrounding tissue, from naive mice (E) and from mice at day 6 postinfection (F). Multiple adjacent z stacks (5 to 15 stacks, 10-μm steps, 3 μm/pixel) were stitched together to form one maximum-focus image using LAS software (Leica). (G) Tile scanned images were used to perform densitometry analysis for GFP and Tomato fluorescence intensity per villus (Volocity software). Fluorescence intensity (arbitrary units) was measured from a central villus within a plaque outward by villus units. Increased fluorescence intensity equated with the increased presence of cells in images. Panel G includes the average fluorescence intensity in villi from naive mice as well as villi containing no parasites from infected mice (Infected background) for purposes of comparison. Data in the graph are averaged from the analysis of multiple plaques or villi.

In plaques on day 6 postinfection, the population of LysM eGFP-intermediate cells per villus decreased compared with what was observed in naive mice (Fig. 4). As many of these cells were CD11b-intermediate (CD11b^int) dendritic cells (data not shown), CD11c YFP animals (C57BL/6 background) were imaged ex vivo to more specifically visualize dendritic cell localization during infection. z stacks of the jejunum and ileum revealed that within densely infected villi, fewer CD11c YFP cells were present compared with the number observed in villi from naive mice (Fig. 5A and E versus C and F). This observation was confirmed by densitometry analysis of the fluorescence intensity of YFP per villus (Fig. 5G). Further, 3D projection of the z stack shown in Fig. 5C indicated that unlike MHC-II GFP cells (Fig. 4D), CD11c YFP cells present within infected villi were predominantly separate from parasites (Fig. 5D). The overall intensity of CD11c YFP fluorescence in the villi of infected mice was elevated, but it was heterogeneous. In comparison, uniform fluorescence was observed in naive mice (Fig. 5E versus F). YFP intensity in villi at the periphery of plaques increased over that seen in that seen in areas of the intestine that did not contain detectable parasites or the villi of naive mice (Fig. 5G). By analyzing the fluorescence intensity across a line transecting a large portion of the intestine, it was clear that CD11c YFP cells were absent where Pru Tomato fluorescence was the greatest, increased as Pru Tomato fluorescence decreased, and decreased again outside the area of parasite replication before returning to background levels (Fig. 5H). Thus, despite the local recruitment of dendritic cells to sites of infection, these cells were most prominent around the periphery of parasite plaques.

Fig 5.

Fluorescence imaging of small intestine samples from naive and infected CD11c YFP mice. (A to D) Naive (A and B) and Pru Tomato tissue cyst-infected (C and D) CD11c YFP mice were imaged (multiphoton z stacks ∼120 μm deep, 5-μm steps), and the distribution of cells in the lamina propria was examined (resolution, 0.98 μm/pixel; a scale bar is included). Image colors are green for CD11c YFP, red for Pru Tomato, and blue for secondary harmonic resonance signals. When visualized as either a maximum-focus (A and C) or a 3D projection (B and D) image, CD11c YFP cells were uniformly distributed in naive mice but elevated in the villi at the periphery of plaques. Noise was removed from the final images using Volocity's fine filter tool. (E and F) Intestinal whole mounts from naive CD11c YFP mice (E) and CD11c YFP mice at day 6 postinfection (F) were imaged by tile scanning, as was done for LysM eGFP mice (see the legend to Fig. 4; 5 to 15 z stacks stitched by the LAS software, 10-μm steps, 3 μm/pixel). (G and H) Densitometry analysis on day 6 postinfection of multiple plaques (G) or across a line transecting the center of a single plaque (H) showed that YFP fluorescence intensity (arbitrary units) was decreased in villi where Tomato fluorescence was the most intense but increased as Tomato fluorescence decreased (Volocity software). Naive intestine and sections of infected intestine that were without parasites (Infected background) were also analyzed (G).

Late dissemination of T. gondii through the intestinal lumen.

During the process of imaging plaques from infected MHC-II GFP mice (Fig. 3), unexpected single parasites were detected outside of villi within the intestinal lumen, as illustrated in Fig. 6A and B (22, 25). These single parasites were first observed on day 6 postinfection, immediately after dissection of the intestine from the mouse, and were considered to be outside of villi on the basis of their placement outside of the MHC-II GFP-positive epithelial layer, as seen in the 3D rotation of the image from Fig. 6B (Fig. 6C). To assess whether these parasites were viable, the lumen from infected mice was perfused with PBS and passed through a 5.0-μm-pore-size filter to eliminate most host cells and large debris prior to concentration of the remaining cellular material. A PBS wash was performed prior to perfusion to remove nonadherent cells and parasites found on the outside of the intestine and limit contamination of the luminal washes. Microscopic analysis of the filtrate from animals infected 3 and 6 days prior showed the presence of extracellular Tomato-positive tachyzoites (data not shown). Further, intraperitoneal injection of the concentrated material into Swiss Webster mice resulted in infection 35% and 80% of the time, respectively (Fig. 6D). While these results must be interpreted with care, they indicate that viable tachyzoites are present within the intestinal lumen days after initial infection and that these parasites may be involved in the dissemination of the parasites throughout the gut.

Fig 6.

High-resolution multiphoton images of infected MHC-II GFP mice. (A) Representative 3D reconstructions of villi from an infected MHC-II GFP (green) mouse showed single parasites (red, arrow) in the intestinal lumen adjacent to a villus (z stack with 5-μm steps, 0.98 μm/pixel, secondary harmonic structures are in blue; tissue was prepared as described in the legend to Fig. 3). On day 6 postinfection, multiple tachyzoites were observed on the lumen side of the intestinal epithelial barrier when tissue was imaged within the first hour after dissection. (B) Maximum focus of a representative z stack highlights a single luminal tachyzoite in the area of three uninfected villi (labeled 1, 2, and 3). (C) Image stills from various positions around the 3D reconstruction of panel B illustrate the proximity of a tachyzoite to the outside surface of villus number 2 (Volocity). (D) Parasites isolated from mouse luminal contents at 3 and 6 days postinfection transferred infection to naive Swiss Webster mice, as determined using flow cytometric analysis for Tomato-positive cells in peritoneal lavage fluid (n = 9 mice per time point). Precautions used to ensure that infection was transferred by extracellular parasites found in luminal contents included washing of the small intestine and removal of debris and host cells by filtration of luminal washes.

DISCUSSION

This study is the first to utilize multiphoton microscopy of live ex vivo intestinal tissue as well as fixed whole mounted tissue to assess the intestinal interactions and biology of oral T. gondii infection after tissue cyst ingestion. Typically, studies on T. gondii intestinal infection have used immunohistochemical analysis with antibodies that recognize either bradyzoite or tachyzoite antigens, and while parasite presence throughout the small intestine is accepted, much of the literature has focused on ileal infection, with few observations of parasite foci or lesions being made (1–3, 12, 14, 28). Although several studies have shown that parasites are present within the intestine beyond the initial stages of infection, much of the work has stressed the early dissemination of parasites from the mucosal tissues and not the parasite burden at this site (2, 29). Analysis of parasite DNA content and a recent immunohistochemical study showed that the parasite burden throughout the intestine increased exponentially over the first 6 days, indicating that parasite replication at this site is largely unchecked (9, 12).

The current study identified discrete areas of T. gondii infection among villi. These areas closely resembled the parasite plaques observed in undisturbed infected cell monolayers. The presence of few infected villi 3 days after ingestion of tissue cysts implies that each counted plaque is the result of an initial bradyzoite infection in a single villus and that multiple infected villi are indicative of plaque expansion. Preliminary experiments support plaque expansion in both susceptible and resistant mice, but evidence of fewer plaques in resistant mice remains consistent with the T. gondii oral infection literature that indicates differences in susceptibility and parasite density in inbred mouse strains (12, 15, 20). Also, oral infectivity studies comparing various T. gondii strains and cyst stages result in similar infections, albeit with differences in the density of infection, distribution of infection, and severity of intestinal pathology (3, 12). On the basis of these results, it is possible that plaque formation and expansion occur not only in various mouse strains but also after infection with any T. gondii strain or cyst type.

During T. gondii infection, several hematopoietic cell types, including antigen-specific T cells, macrophages, neutrophils, monocytes, natural killer cells, and dendritic cells, have been identified to be important in controlling parasite replication and activating antimicrobial response mechanisms (4, 30–36). Previous studies have linked bacterial translocation from the gut during oral T. gondii infection as contributing to the development of acute ileitis and a population of bacterium-specific memory T cells that may play a role in the formation of inflammatory bowel disease at later time points (2, 14, 37). It seems likely that the defined areas of parasite replication and associated tissue damage described in this study would represent portals for bacteria. The organization of LysM⁺ and CD11c⁺ cells in and around parasite plaques, respectively, indicates that cells responding to infection accumulate specifically in the infected area. The distribution of neutrophils and monocytes among parasites is reminiscent of the intestinal lesion organization seen during infections with other protozoan pathogens, such as Neospora caninum (38). As plaques are areas where replicating T. gondii and host cells are in close association, these sites are the likely source of cellular infection. This idea is consistent with the observation of increased percentages of infected neutrophils and monocytes in the lamina propria, where LysM⁺ cells were visualized within plaques (3, 6, 8, 9, 39, 40).

Compared with naive animals, it is clear that dendritic cells are absent from densely infected villi. Hypotheses to explain this observation include the rapid infection and lysis of dendritic cells, the migration of infected dendritic cells out of plaques, and the downregulation of YFP in these cells. Loss of YFP in infected areas seems unlikely, as bone marrow-derived CD11c YFP dendritic cells infected in vitro did not downregulate YFP, as assessed using flow cytometry; further, dendritic cell absence within infected villi was also observed in mice expressing GFP through an alternative dendritic cell marker, CX3CR1 (unpublished results) (41). While there are few CD11c YFP dendritic cells within densely infected villi, there is an increased population of these cells in villi at the periphery of plaques. It is possible that dendritic cells surround infected areas to limit parasite dissemination by creating a granuloma-like structure, as seen in lymph nodes after Listeria monocytogenes infection, or that these cells guide responding cells to plaques and activate antimicrobial responses (42). Further analysis of plaque structures, cellular migration, and the expression of cytokines and chemokines by dendritic cells specifically surrounding plaques is necessary to assess these possibilities.

While activated immune cells near plaques may attempt to control parasite dissemination, it is clear that both the size and number of plaques increased over time. Plaque expansion may occur via retrograde transmission of parasites through the lamina propria to adjacent villi, but the observation that few parasites were detected throughout the tissue suggests that dissemination is controlled and implies that there are other mechanisms of villus-to-villus spread. One such mechanism could be parasite transfer through the intestinal lumen from lysed intestinal epithelial cells to adjacent villi mediated by their close proximity. This model is supported by the isolation of viable extracellular tachyzoites in the lumen of the intestine at both 3 and 6 days postinfection, observed both here and by Manwell et al. (43). Also, a model of luminal spread of tachyzoites may explain the continued presence of single villus plaques at 6 days postinfection as well as the lack of plaques observed after intraperitoneal infection with similar parasites (unpublished results). It is possible that viable parasites released into the intestinal lumen are blocked from infecting new epithelial cells by mucus production or antibody responses; however, this should not be the case, as parasites are capable of infecting host cell monolayers after moving through a mucus-like substance (Matrigel) and parasite-specific IgA responses are observed later during infection (5, 44). Indeed, luminal spread is utilized by related protozoan species, including Cryptosporidium parvum and Eimeria species (43, 45, 46). However, unlike these examples, there is currently no evidence for an alternative stage of T. gondii that may increase the survival of parasites while within the intestinal lumen.

Although autoinfection by T. gondii may be a secondary product of parasite release into the lumen after natural cell lysis, the benefits of such infection may include the evasion of antimicrobial responses mediated by dendritic cells and macrophages generated around plaques and continued dissemination of parasites throughout the host. Indeed, precedence for the importance of luminal spread and a pool of replicating pathogens in the intestinal mucosa has been established in models of L. monocytogenes, Salmonella enterica, and Yersinia pseudotuberculosis infection (47–49). Melton-Witt et al. proposed that luminal shedding acts as a mechanism for bacterial spread and further dissemination after showing that L. monocytogenes shed from villi into the lumen can infect Peyer's patches (47). Additionally, data generated using each of these oral infection models have shown that replicating bacteria in the intestine are important for early systemic colonization of the host, but bacteria that migrate to the mesenteric lymph nodes are delayed in their systemic dissemination and filtered to the spleen (47–49). Like these models, the continued replication of T. gondii in mucosal tissues as well as the formation of secondary infection sites could act as foci that seed systemic dissemination and may result in multiple waves of dissemination from the gut.