Cryptosporidium parvum, belonging to the phylum Apicomplexa, is a major cause of waterborne gastroenteritis throughout the world. The sporozoites are thought to invade host enterocytes using an active process termed gliding motility. However, the biological and morphological changes within the sporozoites during this process are not fully understood. In the present study, excysted sporozoites of C. parvum were analysed ultrastructurally in vitro and their viability was evaluated using fluorescent dyes. The sporozoites excysted from oocysts changed morphologically from banana-shaped to rod-shaped and finally to a rounded shape, in culture media in 3 h. Transmission microscopy revealed that the distance between the apical end and the nucleus was markedly reduced, dense granules were present close to the rhoptry in the apical region, amylopectin granules were absent, and membranes of round sporozoites were less clear. A fluorescent assay showed that the rate of survival decreased from 89% to 56% at 0–3 h (84·3% for banana-shaped and 49·2% for rod-shaped sporozoites). Therefore, post-excysted sporozoites in vitro underwent morphological changes and a rapid loss of viability. This staining method is useful, inexpensive and provides an alternative to more costly and intensive flow cytometric assays or infectivity assays with host cells in vitro.

Cryptosporidium parvum are protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring within Cryptosporidium sporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37°C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.

Sporozoites of Eimeria tenella were disrupted by sonication and subcellular fractions were separated by sucrose gradient ultracentrifugation. Fractions from gradients were characterized by electron microscopical appearance and their polypeptide and antigenic profiles determined by PAGE and Western blotting with antisera to sporozoites and 1st- and 2nd-generation merozoites. Fractions containing micronemes, rhoptries or membranes showed markedly different polypeptide content and antigenic reactivity. Microneme epitopes were strongly conserved between sporozoites and 2nd-generation merozoites whereas the majority of rhoptry epitopes and many membrane epitopes were sporozoite specific. The only polypeptide of sporozoites which was strongly recognized by antisera raised to 1st generation merozoites was a microneme antigen of molecular weight approximately 100 kDa.

Protocols for in vitro excystation of oocysts of Cryptosporidium parvum, including different chemical pre-incubation steps, were compared to examine some of the biochemical triggers involved in excystation and to define an in vitro excystation protocol of a reproducibly high efficiency. Pre-incubation steps which increased the permeability of the oocysts were found to enhance excystation dynamics and pre-treatment of oocysts with saliva was found to decrease the permeability and reduce excystation. Although excystation was maximal after incubation for 4 h, sporozoites tended to lyse over this period, and maximum sporozoite recovery occurred after 30 min. The results obtained are discussed in relation to excystation protocols adopted by different research groups and a number of recommendations are given for in vitro excystation of C. parvum oocysts.

In an area of holoendemic malaria in Northern Tanzania, Anopheles gambiae s.l. females were age-graded by Polovodova's method and dissected for sporozoites. Age-specific sporozoite rates implied that mosquitoes acquired new infections at all ages. The extrinsic period lasted just over 3 gonotrophic cycles (9–11 days). Very high sporozoite rates in the oldest females implied the absence or rarity of genetic refractoriness to infection. A method is described for estimating the proportion of bloodmeals which result in mosquito infection. This method makes relatively few assumptions about mosquito behaviour, and could be useful for evaluating transmission-blocking interventions. Overall, it is estimated that about 21% of meals are infectious. This is much higher than previous estimates derived either from experimental mosquito feeding studies or from similar age-grading data collected from the same area in 1962. Various alternative explanations are considered, and it is concluded that there has been a 2.5-fold increase in human infectiousness in the last 25 years. This is partly attributable to suppression of human infectiousness by widespread chloroquine usage during the 1960s, followed by removal of this effect by drug resistance. It is argued that chloroquine would be expected to select for increased infectivity in the parasite, and this may also have contributed to the observed increase.

A 3-step procedure for cloning Theileria parva parasites was developed. The first step involved the in vitro infection of a fixed number of bovine lymphocytes with titrated sporozoites. The cell lines obtained from infections initiated using sporozoite/lymphocyte ratios below 1:100 were then selected for cloning as these contained schizont-infected cells, each of which was derived from infection with a single sporozoite. In the second step, these cell lines were cloned by limiting dilution. As sporozoites infect lymphocytes and transform to induce clonal multiplication, this step produced infected cell lines containing both cloned parasites and cloned lymphocytes. In the third step, the cloned cell lines were used to infect cattle and isolation of the parasite in ticks was made during piroplasm parasitaemia. Finally, sporozoites were harvested from infected ticks and used for further characterization. Sporozoites derived from cloned cell lines of T. parva Muguga, Marikebuni, Boleni, Uganda and buffalo-derived 7014 were characterized using monoclonal antibody profiles, DNA restriction fragment length polymorphism detected using repetitive and telomeric probes, in vivo infectivity and, in one case, cross-immunity studies. Additionally, several distinct schizont-infected lymphocyte clones were isolated from the Muguga, Mariakani and buffalo-derived 7014 stocks. The combined results of the characterization revealed that the cloning procedure selected clones of T. parva from the parental stocks which were known to contain a mixture of genetically different parasite populations. The cloning method and the clones generated will be of value in studies of the biology of the parasite and in elucidating the strain specificity of immune responses in cattle.

Mass-scale production of Plasmodium vivax sporozoites in Anopheles stephensi was achieved using the chimpanzee (Pan troglodytes) as a source of infective blood. Membrane feeding was as successful as feeding mosquitoes directly on the animal so long as the time between drawing the blood and feeding was restricted to 45 min. Longer delays such as 2–3 h resulted in loss of infectivity in terms of oocyst production. The selected strain of A. stephensi was highly susceptible to P. vivax (Chesson strain). A strain of A. stephensi relatively refractory to P. falciparum showed no cross-refractoriness to P. vivax. Mixed infections of P. falciparum and P. vivax did not interfere with each other in their development in A. stephensi. A second normal blood meal to mosquitoes infected with P. vivax increased the yield of salivary gland sporozoites.

Within the genus Cryptosporidium 2 lineages have evolved, one adapted to the acid environment of the stomach and abomasum, the other comprising parasites that multiply in the small intestine. We tested whether the release of sporozoites from oocysts, a process known as excystation, is triggered by conditions which mimic the site of infection. Specifically, we exposed oocysts from gastric and intestinal Cryptosporidium species to acid conditions or to a neutral solution of taurocholic acid, at 37°C. We found that oocysts from the gastric species C. muris and C. andersoni excysted in both conditions, whereas the intestinal species C. parvum and C. hominis did not respond to acid. When the effect of temperature alone was tested on C. muris and C. parvum, only oocysts from the former species excysted in significant numbers. Oocysts from intestinal species did not respond to temperature alone, nor to acidity. These observations are consistent with the need of gastric species to rapidly excyst and release the sporozoites upon ingestion, and indicate that Cryptosporidium oocysts have evolved to maximize delivery of sporozoites to the region of the gastro-intestinal tract where the parasite multiplies.

We used the fluorescent dye monochlorobimane (MCB) which binds glutathione (GSH) to localize between 2 and 6 distinctly labelled nuclear and cytoplasmic GSH foci in recently excreted and aged, intact Cryptosporidium parvum oocysts and sporozoites. Buthionine sulfoximine (BSO), a potent and specific inhibitor of GSH, was used to determine whether GSH is synthesized in BSO-treated C. parvum oocysts, by labelling treated oocysts with MCB. Both visual and electronic quantifications were performed. At 5 mM BSO, a significant inhibition of MCB fluorescence, reflecting reduced MCB uptake, was observed in GSH-depleted oocysts (mean±S.D. 35±3·7) compared with controls (3·3±1·2, P=0). This clear reduction occurred only in viable oocysts. 1 mM BSO-treated oocysts exhibited weak or no MCB fluorescence, although they were viable (excluded propidium iodide, PI)), and intact and contained sporozoites by differential interference contrast microscopy (DIC). MCB was used in conjunction with PI to determine C. parvum oocyst viability. Oocysts labelled with MCB/PI or 4′6-diamidino-2-phenyl indole (DAPI)/PI produced comparable labelling patterns. Viable oocysts were labelled with MCB or DAPI whereas dead oocysts were labelled with PI only. The localization of GSH in viable, intact oocysts and excysted sporozoites and UV light-irradiated oocysts and sporozoites revealed no changes in MCB uptake at levels up to 40 mJ.cm−2 irradiation. Although GSH can be detected following MCB localization in both the nucleus and cytoplasm of sporozoites, and can be specifically depleted by BSO treatment, MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts.

The C57BL6 strain of mice is highly susceptible to Plasmodium berghei sporozoite infections and consequently requires repeated immunizations with irradiated sporozoites to obtain protective immunity. After a live sporozoite challenge in the immunized hosts, hepatic-stage parasites found in the liver after 48 h are of different sizes – small schizonts corresponding to blocked forms (derived from irradiated sporozoites), and schizonts of intermediate size (derived from live sporozoites). Large schizonts corresponding to mature hepatic forms are found only in unimmunized but challenged C57BL6 mice. Using monoclonal and polyclonal antibodies directed to liver-stage parasites, different patterns of binding reactivity to the above forms are observed. More than 20% of the irradiated sporozoites transform into blocked forms after immunization and persist in the liver. Upon sporozoite challenge in such immunized animals the rate of transformation of sporozoites into hepatic parasites is less than 2%. These observations shed light on the fate of live sporozoite development in irradiated sporozoite-immunized C57BL6 mice.

The BALB/c strain of mice is comparatively more resistant to sporozoite infections of Plasmodium berghei than the C57BL6 strain. Infection with live sporozoites results in the formation of small hepatic forms in the BALB/c liver that persist for as long as 6 days. Upon infection with small numbers of sporozoites, some of the parasites are destroyed in the liver whereas the rest persist as blocked forms. When larger numbers of sporozoites are injected the same process occurs but, in addition, a fraction of the liver-stage parasites complete full development and give rise to blood forms. Although blocked liver forms persist until day 6 post-infection they actually develop to only 24 h of maturity. The nature of these persistent forms is similar to those obtained from irradiated sporozoite immunization. There is a stronger cell proliferation to liver-stage antigens by spleen lymphocytes of irradiated sporozoite-immunized BALB/c mice in comparison to that of immunized C57BL6 mice suggesting that a stronger priming to liver-stage antigens, probably due to the presence of blocked hepatic forms in the liver for a longer period of time (as compared to C57BL6), occurs in the BALB/c mice. This could be a reason for the long-lasting protective memory observed in BALB/c mice.

Transmission electron microscopy was used to study Toxoplasma gondii infections in the small intestines of Swiss-Webster mice at 2–48 h post-feeding of oocysts (p.f.). Sporozoites passed through intestinal epithelial cells (enterocytes and goblet cells) and infected all cells except red blood cells in the lamina propria. Parasites in intestinal epithelial cells or in cells in the lamina propria were located within a single type of parasitophorous vacuole, which contained exocytosed electron-dense material and well-developed tubulovesicular membranous networks. Sporozoites did not infect intraepithelial lymphocytes (IELs), but at 48 h p.f. IELs had become infected with tachyzoites arising from those that had developed in the lamina propria. At 48 h p.f., the lamina propria contained numerous tachyzoites, much cellular debris, and few intact cells. The intestinal epithelium exhibited limited cytopathological changes except for villar fusion, slight vacuolation, and cell separation at the bases of enterocytes.