Sporogonic development of Leucocytozoon smithi.

The sporogonic development of Leucocytozoon smithi in its black fly vector was studied by light and electron microscopy and was compared with that of other haemosporidians. Within 18 to 24 h after ingestion of gametocytes by black flies, ookinetes passing through the midgut epithelium were observed. Intracellular migration of ookinetes resulted in the apparent disruption and degeneration of host cells. Intercellular migration also occurred as was evidenced by the presence of ookinetes between midgut cells. Transformation of ookinete to spherical oocyst occurred extracellularly in three different sites. Although most oocysts were found between the host cell basal membrane and the basal lamina, large numbers also were found attached to the external surface of the basal lamina, projecting into the hemocoel. Ectopic development of oocysts in the midgut epithelium between cells was observed much less frequently than development on the basal side of the midgut. The oocyst wall of dense granules, believed to be of parasite origin, was distinguishable from the basal lamina of the host's midgut epithelium. As in other Leucocytozoidae, the cytoplasm of the oocyst differentiated into a single sporoblastoid from which 30-50 sporozoites were formed. Beginning on the third day post infection, elongation of segregated dense sporoblastoid material associated with pellicle thickening led to the formation of the finger-like sporozoite buds which projected into the oocyst cavity. Sporozoites within mature oocysts and salivary glands were structurally similar to sporozoites as described for other haemosporidians.     

PbGCbeta is essential for Plasmodium ookinete motility to invade midgut cell and for successful completion of parasite life cycle in mosquitoes

When malaria parasites enter to mosquitoes, they fertilize and differentiate to zygotes and ookinetes. The motile ookinetes cross the midgut cells and arrive to the basement membranes where they differentiate into oocysts. The midgut epithelium is thus a barrier for ookinetes to complete their life cycle in the mosquitoes. The ookinetes develop gliding motility to invade midgut cells successfully, but the molecular mechanisms behind are poorly understood. Here, we identified a single molecule with guanylate cyclase domain and N-terminal P-type ATPase like domain in the rodent malaria parasite Plasmodium berghei and named it PbGCbeta. We demonstrated that transgenic parasites in which the PbGCbeta gene was disrupted formed normal ookinetes but failed to produce oocyst. Confocal microscopic analysis showed that the disruptant ookinetes remained on the surface of the microvilli. The disruptant ookinetes showed severe defect in motility, resulting in failure of parasite invasion of the midgut epithelium. When the disruptant ookinetes were cultured in vitro, they transformed into oocysts and sporozoites. These results demonstrate that PbGCbeta is essential for ookinete motility when passing through the midgut cells, but not for further development of the parasites.     

SOAP,a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development

An essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite-producing oocysts on the mosquito midgut wall. For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion. After invasion ookinete-to-oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components. Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP). The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus. It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine-rich domains. Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds. Moreover, SOAP interacts strongly with mosquito laminin in yeast-two-hybrid assays. Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts. These results identify SOAP as a key molecule for ookinete-to-oocyst differentiation in mosquitoes.     

Penetration of the Mosquito (Aedes aegypti) Midgut Wall by the Ookinetes of Plasmodium gallinaceum

ABSTRACT We observed Plasmodium gallinaceum ookinetes in both intracellular and intercellular positions in the midgut epithelium of the mosquito Aedes aegypti. After epithelial cell invasion intracellular ookinetes lacked a parasitophorous vacuolar membrane and were surrounded solely by their own pellicle. Thus, the ookinete in the midgut epithelium of the mosquito differs from erythrocytic and hepatic stages in that the parasite in the vertebrate host is surrounded by a vacuole. The midgut epithelial cytoplasm around the apical end of invading ookinetes was replaced by fine granular material deprived of normal organelles. Membranous structure was observed within the fine granular area. Most ookinetes were seen intracellularly on the luminal side and intercellularly on the haemocoel side of the midgut epithelial cells. These observations suggest that the ookinete first enters into the midgut epithelial cell, then exits to the space between the epithelial cells and moves to the basal lamina where the ookinete develops to the oocyst.     

Penetration of the mosquito (Aedes aegypti) midgut wall by the ookinetes of Plasmodium gallinaceum.

We observed Plasmodium gallinaceum ookinetes in both intracellular and intercellular positions in the midgut epithelium of the mosquito Aedes aegypti. After epithelial cell invasion intracellular ookinetes lacked a parasitophorous vacuolar membrane and were surrounded solely by their own pellicle. Thus, the ookinete in the midgut epithelium of the mosquito differs from erythrocytic and hepatic stages in that the parasite in the vertebrate host is surrounded by a vacuole. The midgut epithelial cytoplasm around the apical end of invading ookinetes was replaced by fine granular material deprived of normal organelles. Membranous structure was observed within the fine granular area. Most ookinetes were seen intracellularly on the luminal side and intercellularly on the haemocoel side of the midgut epithelial cells. These observations suggest that the ookinete first enters into the midgut epithelial cell, then exists to the space between the epithelial cells and moves to the basal lamina where the ookinete develops to the oocyst.     

Electron Microscopic and Histochemical Studies of Sporozoite Formation in Plasmodium berghei*

SYNOPSIS. Observations were made on the differentiation of fine structure during sporogonic development of Plasmodium berghei. The oocyst in the process of sporozoite formation is an encapsulated structure 30-40 μ in diameter. It typically develops while in an extracellular position, attached to the basement membrane of the mosquito midgut and projecting into the mosquito hemocoel. Occasionally, however, ookinetes passing thru the midgut epithelial cells may become impacted within a cell so that the resulting oocyst develops intracellularly. Each oocyst has a large differentiating region, the sporoblastoid body. This body contains large dividing nuclei which are Feulgenpositive, and a cytoplasm which includes mitochondria, dense rodlike structures, cytoplasmic membranes, cisternae and vacuolar structures, Golgi material, and ribosomes which are both free and membrane-associated. Sporozoite budding takes place along the surface of the sporoblastoid body. Bits of a new membrane condense under the plasma membrane which bounds the sporoblastoid body. These 2-membraned sites then bulge out, continue to elongate, and eventually become sporozoites. The various nuclear and cytoplasmic components of the sporoblastoid body are passed into the sporozoites during their elongation. In addition, the sporozoite develops a system of elogate, subpellicular microtubules, possibly contractile in function. The pellicle of the sporozoite is broken by an opening, the cytostome (micropyle). The anterior end is truncate.     

PSOP1, putative secreted ookinete protein 1, is localized to the micronemes of Plasmodium yoelii and P. berghei ookinetes

Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Activated gametocytes in the mosquito midgut egress from erythrocytes followed by fertilization and zygote formation. Zygotes differentiate into motile invasive ookinetes, which penetrate the midgut epithelium before forming oocysts beneath the basal lamina. Ookinete development and traversal across the mosquito midgut wall are major bottlenecks in the parasite life cycle. In ookinetes, surface proteins and proteins stored in apical organelles have been shown to be involved in parasite-host interactions. A group of ookinete proteins that are predicted to have such functions are named PSOPs (putative secreted ookinete protein). PSOP1 is possibly involved in migration through the midgut wall, and here its subcellular localization was examined in ookinetes by immunoelectron microscopy. PSOP1 localizes to the micronemes of Plasmodium yoelii and Plasmodium berghei ookinetes, indicating that it is stored and possibly apically secreted during ookinete penetration through the mosquito midgut wall.     

Ultrastructural studies on oocysts, sporulation and sporozoites of Schellackia cf. agamae from the intestine of the starred lizard Agama stellio.

Young intracellular oocysts of Schellackia cf. agamae in the gut epithelium of agama stellio were bound by several fine membranes. Later-stage oocysts and sporoblasts in the lamina propria were intercellular and were bound by a thin but firm tri-layered wall. Oocysts had a large central refractile body which, during sporulation, sent extensions into the developing sporozoites. Sporozoites escaped into the gut tissue, leaving a large oocyst residuum with the remains of a refractile body. These sporozoites invaded a variety of connective tissue cells, endothelial cells and circulatory leucocytes in the lamina propria. Sporozoites caused lysis of the host cell cytoplasm at their perimeter and multiple sporozoite infections led to complete degradation of the host cell.     

Vesicular ATPase-overexpressing cells determine the distribution of malaria parasite oocysts on the midguts of mosquitoes

In Plasmodium-infected mosquitoes, oocysts are preferentially located at the posterior half of the posterior midgut. Because mosquitoes rest vertically after feeding, the effect of gravity on the ingested blood has been proposed as the cause of such a biased distribution. In this paper, we examined the oocyst distribution on the midguts of mosquitoes that were continuously rotated to nullify the effect of gravity and found that the typical pattern of oocyst distribution did not change. Invasion of the midgut epithelium by ookinetes was similarly found to be biased toward the posterior part of the posterior midgut. We examined whether the distribution of oocysts depends on the distribution of vesicular ATPase (V-ATPase)-overexpressing cells that Plasmodium ookinetes preferentially use to cross the midgut epithelium. An antiserum raised against recombinant Aedes aegypti V-ATPase B subunit indicated that the majority of V-ATPase-overexpressing cells in Ae. aegypti and Anopheles gambiae are localized at the posterior part of the posterior midgut. We propose that the typical distribution of oocysts on the mosquito midgut is attributable to the presence and the spatial distribution of the V-ATPase-overexpressing cells in the midgut epithelium.     

The role of Plasmodium berghei ookinete proteins in binding to basal lamina components and transformation into oocysts.

The ookinete is a motile form of the malaria parasite that travels from the midgut lumen of the mosquito, invades the epithelial cells and settles beneath the basal lamina. The events surrounding cessation of ookinete motility and its transformation into an oocyst are poorly understood, but interaction between components of the basal lamina and the parasite surface has been implicated. Here we report that interactions occur between basal lamina constituents and ookinete proteins and that these interactions inhibit motility and are likely to be involved in transformation to an oocyst. Plasmodium berghei ookinetes bound weakly to microtitre plate wells coated with fibronectin and much more strongly to wells coated with laminin and collagen IV. A 1:1 mixture of collagen and laminin significantly enhanced binding. Binding increased with time of incubation up to 10 h and different components showed different binding profiles with time. Two parasite molecules were shown to act as ligands for basal lamina components. Western blots demonstrated that the surface molecule Pbs21 bound strongly to laminin but not to collagen IV whereas a 215 kDa molecule (possibly PbCTRP) bound to both laminin and collagen IV. Furthermore up to 90% inhibition of binding of ookinetes to collagen IV/laminin combination occurred if parasites were pre-incubated with anti-Pbs21 monoclonal antibody 13.1. Some transformation of ookinetes to oocysts occurred in wells coated with laminin or laminin/collagen IV combinations but collagen IV alone did not trigger transformation. No binding or transformation occurred in uncoated wells. Our data support the suggestion that ookinete proteins Pbs21 and a 215 kDa protein may have multiple roles including interactions with midgut basal lamina components that cause binding, inhibit motility and trigger transformation.     

The establishment of Plasmodium berghei in mosquitoes of a refractory and a susceptible line of Anopheles atroparvus

The events between the ingestion of Plasmodium berghei-infected mouse blood and the establishment of the ookinetes in the epithelium of the midgut in refractory (R) and susceptible (S) Anopheles atroparvus are described. Simultaneously fed, fully engorged female mosquitoes were randomly assigned to dissection at 22, 28, 32, 48 h and 10 days (controls) after the infective feed (post-infection: p.i.). Serial transverse sections of 6 micron were cut. Every 10th section was studied. The maturation of ookinetes was monitored at 16, 19 and 22 h p.i. The infections in R and S mosquitoes developed similarly with regard to the maturation of ookinetes and the number of mature ookinetes in the lumen of the midgut. The semiquantitative evaluation of the envelopment of the food bolus by the peritrophic layer showed that this layer cannot function as a physical barrier against migrating ookinetes. In the midgut epithelium the number of ookinetes decreased significantly with time in both R and S mosquitoes, but a similar number of penetrations was recorded for both types of mosquito. In S mosquitoes maximal 1% of the ookinetes present in the midgut formed an oocyst. In both R and S mosquitoes a substantial loss of parasites was found, first in the lumen of the midgut and second after penetration of the midgut epithelium by the mature ookinetes. Relatively few parasites develop into oocysts in S, but hardly any do so in R individuals. The factors in control of refractoriness are likely to operate on early oocyst development.     

Plasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites

Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites.     

Development of Plasmodium berghei ookinetes to young oocysts in vitro.

The mosquito stage of Plasmodium berghei was cultivated in vitro, with special attention to ookinete transformation into early oocyst. The ookinetes were obtained by in vitro culture of gametocytes taken from infected mice, purified by density gradient of metrizoic acid or a lymphocyte separation medium, and incubated either in acellular culture or in co-cultivations with mosquito cells. In acellular culture, the ookinetes were found to aggregate with each other and transformed from banana to round shapes. Their inner pellicular membranes and subpellicular microtubules partially disappeared, indicating that development to early oocyst had occurred. Co-cultivation wtih Aedes albopictus cells (C6/36 clone) revealed that ookinetes transformed into early oocyst in the medium, or invaded the cells and then transformed to early oocysts within the cell cytoplasm as well. However all of these transformed cells failed to develop further, i.e., neither deposition of the oocyst capsule nor nuclear division was observed. Many ookinetes which failed to penetrate the Aedes cells were phagocytized within three days of culture. A significant difference between invaded and transformed oocysts and phagocytized ookinetes was seen in that the former lacked vacuole membrane. Co-cultivation with Toxorhynchites amboinensis cells (TRA-284-SFG clone) permitted transformation of ookinetes into early oocysts in the medium as in the acellular culture, but no ookinete invasion nor phagocytosis by the cell was observed.     

Fine Structure of Oocyst Transformation and the Sporozoites of Leucocytozoon dubreuili*

SYNOPSIS. The sporogonic stages of Leucocytozoon dubreuili in the midgut and salivary glands of the simuliid vectors was studied by electron microscopy. Young uninucleate oocysts have a pellicle that initially resembles that of the ookinetc. Numerous electron-dense bodies and microtubules in the peripheral cytoplasm may be involved in the formation of the cyst wall. The dense bodies appear to give rise to the amorphous material of the wall. The tubules which run circumferentially beneath the oocyst's boundary probably serve as a skeletal support for the cell surface during deposition of the wall material. A subcapsular “space” which provides area for expansion of the developing sporozoites is formed in early multinucleate oocysts. The subcapsular “space” appears to be formed through a condensation of the peripheral cytoplasm, resulting in an osmotic gradient across the oocyst's limiting membrane. Consequently water diffuses out, creating a fluid-filled space. Sporozoite formation begins with localized thickenings on the oocyst's limiting membrane. Subsequent extension of the thickened regions into the subcapsular “space” marks the onset of sporozoite budding. The process is highly synchronized, and culminates with the production of up to 150 sporozoites about the sporoblastoid body. The structure of sporozoites from mature oocysts and of the salivary glands of the vector is basically similar, although salivary gland sporozoites are more elongate and have numerous electron-dense micronemes. The paired rhoptries in the latter sporozoites are more elongate and uniformly electron-dense than in oocyst sporozoites.     

Distinct malaria parasite sporozoites reveal transcriptional changes that cause differential tissue infection competence in the mosquito vector and mammalian host

The malaria parasite sporozoite transmission stage develops and differentiates within parasite oocysts on the Anopheles mosquito midgut. Successful inoculation of the parasite into a mammalian host is critically dependent on the sporozoite's ability to first infect the mosquito salivary glands. Remarkable changes in tissue infection competence are observed as the sporozoites transit from the midgut oocysts to the salivary glands. Our microarray analysis shows that compared to oocyst sporozoites, salivary gland sporozoites upregulate expression of at least 124 unique genes. Conversely, oocyst sporozoites show upregulation of at least 47 genes (upregulated in oocyst sporozoites [UOS genes]) before they infect the salivary glands. Targeted gene deletion of UOS3, encoding a putative transmembrane protein with a thrombospondin repeat that localizes to the sporozoite secretory organelles, rendered oocyst sporozoites unable to infect the mosquito salivary glands but maintained the parasites' liver infection competence. This phenotype demonstrates the significance of differential UOS expression. Thus, the UIS-UOS gene classification provides a framework to elucidate the infectivity and transmission success of Plasmodium sporozoites on a whole-genome scale. Genes identified herein might represent targets for vector-based transmission blocking strategies (UOS genes), as well as strategies that prevent mammalian host infection (UIS genes).     

Development of Plasmodium berghei Ookinetes to Young Oocysts In Vitro

ABSTRACT. The mosquito stage of Plasmodium berghei was cultivated in vitro, with special attention to ookinete transformation into early oocyst. The ookinetes were obtained by in vitro culture of gametocytes taken from infected mice, purified by density gradient of metrizoic acid or a lymphocyte separation medium, and incubated either in acellular culture or in co-cultivations with mosquito cells. In acellular culture, the ookinetes were found to aggregate with each other and transformed from banana to round shapes. Their inner pellicular membranes and subpellicular microtubules partially disappeared, indicating that development to early oocyst had occurred. Co-cultivation with Aedes albopictus cells (C6/36 clone) revealed that ookinetes transformed into early oocyst in the medium, or invaded the cells and then transformed to early oocysts within the cell cytoplasm as well. However, all of these transformed cells failed to develop further, i.e. neither deposition of the oocyst capsule nor nuclear division was observed. Many ookinetes which failed to penetrate the Aedes cells were phagocytized within three days of culture. A significant difference between invaded and transformed oocysts and phagocytized ookinetes was seen in that the former lacked vacuole membrane. Co-cultivation with Toxorhynchites amboinensis cells (TRA-284-SFG clone) permitted transformation of ookinetes into early oocysts in the medium as in the acellular culture, but no ookinete invasion nor phagocytosis by the cell was observed.     

Plasmodium malariae: distribution of circumsporozoite protein in midgut oocysts and salivary gland sporozoites

The distribution of the circumsporozoite protein within developing Plasmodium malariae oocysts and salivary gland sporozoites was examined by immunoelectron microscopy using protein A-gold and a monoclonal antibody specific for the CS protein of P. malariae. Gold particles were found along the capsule of immature oocysts but rarely within the cytoplasm. Gold label was detected on the inner surface of peripheral vacuoles during oocyst maturation and the plasma membrane of the sporoblast. Salivary gland sporozoites and budding sporozoites in mature oocysts were labeled uniformly on the outer surface of their plasma membranes. The surface of sporozoites that ruptured into midgut epithelial cells were entirely covered with gold particles. No label was seen on the surface of sporozoites which ruptured into the midgut lumen. In addition, a rabbit polyclonal antibody against repeat a region of P. brasilianum CS protein reacted with P. malariae sporozoites.     

Fine Structure of Differentiating Oocysts and Mature Sporozoites of Haemoproteus metchnikovi in its Intermediate Host Chrysops callidus*

The fine structure of the sporogonic stages of Haemoproteus metchnikovi has been investigated by electron microscopy. Young oocysts are found beneath the basement membrane of midgut epithelial cells. These eventually protrude outward into the haemocoel space and are surrounded by a distinct oocyst capsule. Sporozoite formation begins with a subcapsular vacuolation. Evagination of the oocyst cytoplasm occurs in regions of membrane thickenings and 100–200 sporozoites are formed about a single sporoblastoid body. Remnants of the ookinete pellicle can be observed in maturing oocysts and always are found in the residual body. The fine structure of the mature sporozoite is essentially similar to that which has been described for other haemosporidia and a spherical body is described in association with the mitochondrion of the sporozoite. The sporogonic stages of H. metchnikovi have features common to the sporogonic stages of Plasmodium and Leucocytozoon that are not held in common by the latter 2 genera, including pattern of sporozoite formation and number of sporozoites formed, the presence of a cytostome and of “crystalloid” in the sporozoite.     

Mutational analysis of the GPI-anchor addition sequence from the circumsporozoite protein of Plasmodium

The plasma membrane of Plasmodium sporozoites is uniformly covered by the glycosylphosphatidylinositol (GPI)-anchored circumsporozoite (CS) protein. Sporozoites form in the mosquito midgut through a budding process that occurs within a multinucleate oocyst underneath the basal lamina of the gut. Earlier genetic studies established that normal sporozoite development requires CS. Mutant parasites lacking CS [CS (-)] do not form sporozoites. Ultrastructural analysis of the oocysts from these parasites revealed that there is an early block in the cytokinesis that occurs within the multinucleate oocysts to generate individual sporozoites. Parasites that are hypomorphic for CS expression gave rise to sporozoites with abnormal morphology. These results proved that CS plays a direct role in the maturation of oocysts and in the normal budding of sporozoites. In this article, we examined if the membrane localization of CS via a GPI-anchor, is crucial for its function during sporozoite formation. We generated three mutants in Plasmodium berghei CS, CS-DeltaGPI, CS-TM1 and CS-TM2. In CS-DeltaGPI, we deleted the signal sequence required for the addition of a GPI-anchor to CS. The resulting protein was found only in the cytoplasm of the oocyst. In CS-TM1 and CS-TM2, the GPI-anchor addition sequence of CS was substituted by the transmembrane domain and truncated (to different degrees) cytoplasmic tail of Plasmodium thrombospondin-related anonymous protein (TRAP). The resulting CS protein was detected on the plasma membrane of the oocysts. The amount of CS in the mutants was similar to that of wild type. The sporozoite budding and development were abrogated in both CS-DeltaGPI and CS-TM mutants. The ultrastructure of the mutant oocysts was indistinguishable from that of the CS (-) parasites. Our results suggest that the GPI-anchor of the CS protein is required for sporogenesis.     

Early events in the life cycle of the mouse coccidium Eimeria falciformis (Eimer, 1870) Schneider, 1875 in naive and immune hosts

The initial phases of invasion of mammalian coccidia of the genus Eimeria into host tissue are still poorly known. This process, including the passage of oocysts through the intestinal lumen, excystation of sporozoites, their penetration into epithelial cells and migration to the target site was studied in both naive and immune mice infected with Eimeria falciformis. After oral infection, the intact oocysts were transported with enteral contents to the large intestine, where the excystation of sporozoites and their penetration into superficial epithelium took place. The sporozoites subsequently migrated into the epithelium of crypts, which is the specific site of asexual multiplication. The immune status of the hosts did not affect the passage of oocysts, excystation and penetration of sporozoites. However, the migration of sporozoites towards their target site (crypts) was impeded in immune mice and sporozoites tended to remain in superficial mucosa rather than migrate to the crypts.