Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites

Sporozoites of the apicomplexan parasite Cryptosporidium parvum were subjected to cell disruption and subcellular fractionation using a sucrose density step gradient. With this procedure, highly enriched preparations of the parasite membrane, the micronemes, dense granules and amylopectin granules were produced. No separate fraction containing rhoptries was obtained, however this organelle was found in defined fractions of the gradient, still associated with the apical tip of the sporozoites. Using negative staining, the internal structure of the micronemes was revealed by transmission electron microscopy. Micronemes and dense granules showed characteristic protein compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The micronemes contained three major proteins of approximately 30, 120 and 200 kDa and the dense granules contain five major proteins in the 120-180 kDa range.     

Localization of pyruvate:NADP+ oxidoreductase in sporozoites of Cryptosporidium parvum

Cryptosporidium parvum contains a unique fusion protein pyruvate:NADP+ oxidoreductase (CpPNO) that is composed of two distinct, conserved domains, an N-terminal pyruvate:ferredoxin oxidoreductase (PFO) and a C-terminal cytochrome P450 reductase (CPR). Unlike a similar fusion protein that localizes to the mitochondrion of the photosynthetic protist Euglena gracilis, CpPNO lacks an N-terminal mitochondrial targeting sequence. Using two distinct polyclonal antibodies raised against CpPFO and one polyclonal antibody against CpCPR, Western blot analysis has shown that sporozoites of C. parvum express the entire CpPNO fusion protein. Furthermore, confocal immunofluorescence and transmission electron microscopy confirm that CpPNO is localized within the cytosol rather than the relict mitochondrion of C. parvum. The distribution of this protein is not, however, strictly confined to the cytosol. CpPNO also appears to localize posteriorly within the crystalloid body.     

Immunolocalization of an enterotoxic glycoprotein exoantigen on the secretory organelles of Cryptosporidium parvum sporozoites

In this study, the fine ultrastructures of the secretory organelles of C. parvum sporozoites were demonstrated using transmission electron microscopy (TEM). Meanwhile, a previously identified enterotoxic 18-20 kDa copro-antigen (18-20 kDa CCA), associated with cryptosporidiosis in both human and calves, was isolated and immunolocalized on C. parvum sporozoites. Using immunoelectron microscopy and anti-18-20 kDa monospecific antibody demonstrated marked existence of the 18-20 kDa CCA on the apical organelles and at the trilaminar pellicles. An anterior extrusion-of this protein was demonstrated around the excysted and released sporozoites. However, non excysted sporozoites did not show this protein. Affinity blotting, with biotinylated jacalin, demonstrated the O-linked oligosaccharide moiety of this protein. The potential role of this protein in the host cell invasion and/or gliding motility remains unelucidated. However, its enterotoxicity, location and secretory nature suggest that it may be a target for neutralization or invasion inhibition of Cryptosporidium.     

Immunofluorescent microscopical visualization of trails left by gliding Cryptosporidium parvum sporozoites

Cryptosporidium parvum sporozoites that exhibited gliding motility in vitro were examined by immunofluorescence with anticryptosporidial monoclonal antibodies (Mabs) for surface antigen deposition on poly-L-lysine-coated glass microscope slides. The Mabs that revealed trails are specific for an immunodominant 23-kDa antigen previously localized to the sporozoite surface.     

Neutralization-sensitive epitopes are exposed on the surface of infectious Cryptosporidium parvum sporozoites

Cryptosporidiosis is a diarrheal disease of humans, calves, and other mammals caused by the coccidian parasite Cryptosporidium parvum. Immune bovine serum and two surface-reactive antisporozoite mAb with neutralizing activity were used to identify sporozoite surface Ag by radioimmunoprecipitation/SDS-PAGE and immunoblotting. When isolated sporozoites were incubated with mAb 18.44, 12 to 25 times the ID50 for mice was completely neutralized. This mAb binds diffusely to the sporozoite surface and recognizes a sporozoite surface Ag that eluted in the void volume of a Bio Gel A column with an exclusion limit of 500,000 daltons. The Ag recognized by mAb 18.44 was not radiolabeled with 125I or [35S] methionine, migrated with the dye front in SDS-PAGE, and was insensitive to proteinase K digestion, suggesting a non-protein composition. mAb 17.41 significantly neutralized 25 times the ID50 of sporozoites for mice. This mAb binds multifocally to the sporozoite surface and recognizes [35S] methionine-labeled sporozoite surface Ag of 28,000 m.w., 55,000 m.w., and 98,000 m.w. Immune bovine serum immunoprecipitated [35S] methionine- or 125I-labeled sporozoite Ag ranging from less than 14,300 m.w. to greater than 200,000 m.w., including surface Ag of 28,000 m.w. and 55,000 m.w. The results indicate that two different molecules capable of inducing neutralizing antibody are exposed on the surface of C. parvum sporozoites.     

Hemolytic properties of lytic peptides active against the sporozoites of Cryptosporidium parvum.

Cryptosporidium parvum is a protozoan parasite that causes mildto-severe diarrheal disease in animals and humans. There are currently no effective chemotherapeutic agents available for the treatment of cryptosporidiosis. Recently, small, naturally occurring antimicrobial lytic peptides with anti-protozoal activities have been described. In the present study, we compare the in vitro anti-cryptosporidial activities of synthetic lytic peptides and their corresponding hemolytic activities after a 30 min incubation at 37 degrees C. Sporozoite viability was assessed microscopically by the uptake of the vital dyes fluorescein diacetate (FDA) and propidium iodide (PI). Hemolysis was assessed spectrophotometrically by the release of soluble hemoglobulin. The most active peptide, Hecate-1, reduced sporozoite viability by 85.5% with a corresponding hemolytic activity of 21.5% at a concentration of 10 microM.     

Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from water samples

AIMS: Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from tap water. METHODS AND RESULTS: Ferric sulphate, aluminium sulphate and calcium carbonate were compared for their recovery efficiency of C. parvum oocysts from tap water. Lower mean recovery was achieved by calcium carbonate (38.8%) compared with ferric sulphate (61.5%) and aluminium sulphate (58.1%) for the recovery of 2.5 x 10(5) oocysts l(-1); 2.5 oocysts l(-1) and 1 oocyst l(-1) were adequately purified using ferric sulphate flocculation. In vitro excystation experiments showed that ferric sulphate flocculation does not markedly reduce the viability of oocysts. CONCLUSIONS: Ferric sulphate flocculation is a simple and effective tool for the purification of C. parvum oocysts from tap water. SIGNIFICANCE AND IMPACT OF THE STUDY: The high recovery rates and low impact on oocyst viability provided by ferric sulphate flocculation might be useful for the detection of Cryptosporidium oocysts in environmental water samples.     

Ultrastructure of Cryptosporidium parvum oocysts and excysting sporozoites as revealed by high resolution scanning electron microscopy

Cryptosporidium parvum oocysts isolated from calf feces were examined by scanning electron microscopy during excystation. Intact C. parvum oocysts were spheroid to ellipsoid, approximately equal to 3.5 X 4.0 micron, with length : width ratio = 1.17. The oocyst wall had a single suture at one pole, which spanned 1/3 to 1/2 the circumference of the oocyst. During excystation the suture dissolved, resulting in a slit-like opening, which the sporozoites used to exit the oocyst. Sporozoites were 3.8 X 0.6 micron and had a rough outer surface.     

Effect of bovine manure on Cryptosporidium parvum oocyst attachment to soil

The objective of this work was to assess the effect of dilute bovine manure (1.0% and 0.1%) versus that of no manure on attachment and subsequent detachment of Cryptosporidium parvum oocysts to soil. Manure enhanced the attachment of oocysts to soil particles; the maximum attachment was observed with 0.1% manure. Oocyst attachment was partially reversible; maximum detachment was observed with dilute manure. These results indicate that oocyst attachment to soil is substantially affected by bovine manure in a complex manner and should have implications for how oocysts may be transported through or over soils.     

Characterization of the thioredoxin peroxidase from Cryptosporidium parvum

Cryptosporidium parvum can survive exposure to harsh environmental conditions, various disinfectants, and high doses of γ-radiation. Recently, it was found that the expression of thioredoxin peroxidase (CpTPx) in C. parvum increased after a high dose of γ-irradiation to the parasite. CpTPx is a two-cysteine peroxiredoxin that contains cysteines at positions 49 and 170. Recombinant CpTPx fused to an N-terminal hexahistidine sequence, (His)₆-CpTPx, exhibited substantial thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation in vitro. (His)₆-CpTPx was used to screen sera from C. parvum-infected mice and humans for antibodies against CpTPx. In Western blots, 10% of the mouse sera and 20% of the human sera reacted with (His)₆-CpTPx, suggesting that after infection by C. parvum CpTPx can induce a host-immune reaction but is not a major antigen. Immunolocalization studies revealed that CpTPx is expressed mainly in the cytoplasm of C. parvum at various developmental stages.     

Molecular identification of Cryptosporidium parvum from avian hosts

Cryptosporidium species are protozoan parasites that infect humans and a wide variety of animals. This study was aimed at identifying Cryptosporidium species and genotypes isolated from avian hosts. A total of 90 samples from 37 different species of birds were collected throughout a 3-month period from April 2008 to June 2008 in the National Zoo of Kuala Lumpur, Malaysia. Prior to molecular characterization, all samples were screened for Cryptosporidium using a modified Ziehl-Neelsen staining technique. Subsequently samples were analysed with nested-PCR targeting the partial SSU rRNA gene. Amplicons were sequenced in both directions and used for phylogenetic analysis using Neighbour-Joining and Maximum Parsimony methods. Although 9 (10%) samples were positive for Cryptosporidium via microscopy, 8 (8.9%) produced amplicons using nested PCR. Phylogenetic trees identified all the isolates as Cryptosporidium parvum. Although C. parvum has not been reported to cause infection in birds, and the role of birds in this study was postulated mainly as mechanical transporters, these present findings highlight the significant public health risk posed by birds that harbour the zoonotic species of Cryptosporidium.     

Effect of hyperimmune bovine colostrum raised against Cryptosporidium parvum on infection of guinea pigs by Cryptosporidium wrairi.

Oocysts shedding was markedly reduced in guinea pigs inoculated intraintestinally with Cryptosporidium wrairi sporozoites that had been incubated with hyperimmune bovine colostrum raised to C. parvum when compared with shedding in guinea pigs inoculated with sporozoites incubated in either non-immune bovine colostrum or buffered saline. However oocyst shedding was apparently not reduced in guinea pigs inoculated by gavage with oocysts of C. wrairi and subsequently treated twice daily per os with hyperimmune bovine colostrum. Similarly, oocyst shedding was apparently not reduced by oral treatment with hyperimmune bovine colostrum when treatment was begun simultaneously with inoculation of C. wrairi oocysts.