Polyclonal Fab phage display libraries with a high percentage of diverse clones to Cryptosporidium parvum glycoproteins

The protozoan parasite Cryptosporidium parvum is regarded as a major public health problem world-wide, especially for immunocompromised individuals. Although no effective therapy is presently available, specific immune responses prevent or terminate cryptosporidiosis and passively administered antibodies have been found to reduce the severity of infection. Therefore, as an immunotherapeutic approach against cryptosporidiosis, we set out to develop C. parvum-specific polyclonal antibody libraries, standardised, perpetual mixtures of polyclonal antibodies, for which the genes are available. A combinatorial Fab phage display library was generated from the antibody variable region gene repertoire of mice immunised with C. parvum surface and apical complex glycoproteins which are believed to be involved in mediating C. parvum attachment and invasion. The variable region genes used to construct this starting library were shown to be diverse by nucleotide sequencing. The library was subjected to one round of antigen selection on C. parvum glycoproteins or a C. parvum oocyst/sporozoite preparation. The two selected libraries showed specific reactivity to the glycoproteins as well as to the oocyst/sporozoite preparation, with 50-73% antigen-reactive members. Fingerprint analysis of individual clones from the two antigen-selected libraries showed high diversity, confirming the polyclonality of the selected libraries. Furthermore, immunoblot analysis on the oocyst/sporozoite and glycoprotein preparations with selected library phage showed reactivity to multiple bands, indicating diversity at the antigen level. These C. parvum-specific polyclonal Fab phage display libraries will be converted to libraries of polyclonal full-length antibodies by mass transfer of the selected heavy and light chain variable region gene pairs to a mammalian expression vector. Such polyclonal antibody libraries would be expected to mediate effector functions and provide optimal passive immunity against cryptosporidiosis.     

Pyrrhocoricin as a potential drug delivery vehicle for Cryptosporidium parvum

This study analysed the intracellular delivery capacity of insect derived pyrrhocoricin with a peptide cargo in Cryptosporidium parvum in vitro using fluorescence microscopy. Results revealed that pyrrhocoricin was capable of acting as a delivery vehicle in transducing peptides across the parasite cell membrane for multiple life-cycle stages. The successful transduction may aid in target validation and the delivery of future peptide-based drugs against this important human pathogen.     

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.     

Infection status of pigs with Cryptosporidium parvum

To investigate the infection status of pigs with Cryptosporidium parvum, 589 fecal samples were collected from pigs raised at farm in Chungcheongbuk-do and Chungcheongnam-do. Of the 589 pig fecal samples, 62 (10.5%) were positive for C. parvum. The area showing the highest positive rate was Dangjin-gun, Chungcheongnam-do (14.0%), and the lowest (0%) Salmi-myon, Chungcheongbuk-do. The positive rate of C. parvum in Judok-eup increased from 12.7% in the winter to 22.1% in the summer. The results of this study suggest that the pigs may be a source of human C. parvum infection.     

Cryptosporidium parvum: the course of Cryptosporidium parvum infection in C57BL/6 mice co-infected with the nematode Heligmosomoides bakeri

The effects of Heligmosomoides bakeri infection on the course of a concurrent Cryptosporidium parvum infection were studied in C57BL/6 mice. Mice were initially infected with 80 L₃ of H. bakeri and then challenged with 10⁴ oocysts of C. parvum, administered during the patent period of the nematode infection (28 day post H. bakeri infection). The number of C. parvum oocysts excreted in the feces and the number of adult H. bakeri in the small intestine were monitored during the experiment. Concurrent H. bakeri infection resulted in a prolonged course of infection with C. parvum. The intensities of both parasite infections were higher in co-infections. We also investigated the cellular immune response at 14 and 42 days post infection C. parvum. During infection with C. parvum there was an increase in production of IFN-γ and IL-12 but co-infection with H. bakeri inhibited IFN-γ secretion. The present study is the first to demonstrate that infection with H. bakeri markedly exacerbates the intensity of a concurrent C. parvum infection in laboratory mice and also affects immune effectors mechanisms in co-infection with H. bakeri.     

Cryptosporidium parvum: identification of a new surface adhesion protein on sporozoite and oocyst by screening of a phage-display cDNA library

Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. The specific molecules that mediate C. parvum-host interaction and the molecular mechanisms involved in the pathogenesis are unknown. In this study we described a novel phage display method to identify surface adhesion proteins of C. parvum. A cDNA library of the sporozoite and oocyst stages of C. parvum expressed on the surface of T7 phage was screened with intestinal epithelial cells (IECs) from the newborn Cryptosporidium-free Holstein calves. Proteins that selectively and specifically bound to IECs were then enriched using a multi-step panning procedure. Two proteins of C. parvum were selected, one was previously reported (p23), which was an important surface adhesion protein; the other was a novel surface adherence protein (CP12). Sequence analysis showed that CP12 has a N-terminal signal peptide, a transmembrane region, a N-glycosylation site, a casein kinase II phosphorylation site and two N-myristoylation sites. Immunofluorescence assay (IFA) using antibody specific for rCP12 demonstrated that the antibody can specifically bind the surface of sporozoite and oocyst, especially apical region of sporozoite. The surface localization of CP12 and its involvement in the host-parasite interaction suggest that it may serve as an effective target for specific preventive and therapeutic measures for cryptosporidiosis.     

Ultrastructural changes in Cryptosporidium parvum oocysts by gamma irradiation

Cryptosporidium parvum is known as one of the most highly resistant parasites to gamma irradiation. To morphologically have an insight on the radioresistance of this parasite, ultrastructural changes in C. parvum sporozoites were observed after gamma irradiation using various doses (1, 5, 10, and 25 kGy) following a range of post-irradiation incubation times (10 kGy for 6, 12, 24, 48, 72, and 96 hr). The ultrastructures of C. parvum oocysts changed remarkably after a 10-kGy irradiation. Nuclear membrane changes and degranulation of dense granules were observed with high doses over 10 kGy, and morphological changes in micronemes and rhoptries were observed with very high doses over 25 kGy. Oocyst walls were not affected by irradiation, whereas the internal structures of sporozoites degenerated completely 96 hr post-irradiation using a dose of 10 kGy. From this study, morphological evidence of radioresistance of C. parvum has been supplemented.     

Molecular targets for detection and immunotherapy in Cryptosporidium parvum

Cryptosporidium parvum is an obligate protozoan parasite responsible for the diarrheal illness cryptosporidiosis in humans and animals. Although C. parvum is particularly pathogenic in immunocompromised hosts, the molecular mechanisms by which C. parvum invades the host epithelial cells are not well understood. Characterization of molecular-based antigenic targets of C. parvum is required to improve the specificity of detection, viability assessments, and immunotherapy (treatment). A number of zoite surface (glyco)proteins are known to be expressed during, and believed to be involved in, invasion and infection of host epithelial cells. In the absence of protective treatments for this illness, antibodies targeted against these zoite surface (glyco)proteins offers a rational approach to therapy. Monoclonal, polyclonal and recombinant antibodies represent useful immunotherapeutic means of combating infection, especially when highly immunogenic C. parvum antigens are utilized as targets. Interruption of life cycle stages of this parasite via antibodies that target critical surface-exposed proteins can potentially decrease the severity of disease symptoms and subsequent re-infection of host tissues. In addition, development of vaccines to this parasite based on the same antigens may be a valuable means of preventing infection. This paper describes many of the zoite surface glycoproteins potentially involved in infection, as well as summarizes many of the immunotherapeutic studies completed to date. The identification and characterization of antibodies that bind to C. parvum-specific cell surface antigens of the oocyst and sporozoite will allow researchers to fully realize the potential of molecular-based immunotherapy to this parasite.     

Prevalence of diarrhea caused by Cryptosporidium parvum in non-HIV patients in Jeollanam-do, Korea

The present study investigated the prevalence rate of Cryptosporidium parvum as a cause of diarrhea. We examined 942 stools of unidentified reasons occurring in patients in whom no immunosuppression had been detected. We examined the stools for Cryptosporidium parvum via modified acid-fast staining. The clinical records of all of the positive patients were then analyzed. Nine (1%) of the stools among the 942 diarrheal patients were positive for C. parvum. The positive rate in the males was 1.1% (6/522) and the positive rate of the females was 0.7% (3/420). Age distribution revealed that the highest positive rates were in patients in their sixties, with a positive rate of 2.5% (4/158). In the clinical tests, levels of c-reactive protein, erythrocyte sedimentation rates, and neutrophil proportions were normally increased in the peripheral blood, whereas the lymphocyte proportion exhibited a tendency towards decrease. The pathological findings were compatible with an inflammatory reaction in the host.     

Evidence for mitochondrial-derived alternative oxidase in the apicomplexan parasite Cryptosporidium parvum: a potential anti-microbial agent target

The observation that Plasmodium falciparum possesses cyanide insensitive respiration that can be inhibited by salicylhydroxamic acid (SHAM) and propyl gallate is consistent with the presence of an alternative oxidase (AOX). However, the completion and annotation of the P. falciparum genome project did not identify any protein with convincing similarity to the previously described AOXs from plants, fungi or protozoa. We undertook a survey of the available apicomplexan genome projects in an attempt to address this anomaly. Putative AOX sequences were identified and sequenced from both type 1 and 2 strains of Cryptosporidium parvum. The gene encodes a polypeptide of 336 amino acids and has a predicted N-terminal transit sequence similar to that found in proteins targeted to the mitochondria of other species. The potential of AOX as a target for new anti-microbial agents for C. parvum is evident by the ability of SHAM and 8-hydroxyquinoline to inhibit in vitro growth of C. parvum. In spite of the lack of a good candidate for AOX in either the P. falciparum or Toxoplasma gondii genome projects, SHAM and 8-hydroxyquinoline were found to inhibit the growth of these parasites. Phylogenetic analysis suggests that AOX and the related protein immutans are derived from gene transfers from the mitochondrial endosymbiont and the chloroplast endosymbiont, respectively. These data are consistent with the functional localisation studies conducted thus far, which demonstrate mitochondrial localisation for some AOX and chloroplastidic localization for immutans. The presence of a mitochondrial compartment is further supported by the prediction of a mitochondrial targeting sequence at the N-terminus of the protein and MitoTracker staining of a subcellular compartment in trophozoite and meront stages. These results give insight into the evolution of AOX and demonstrate the potential of targeting the alternative pathway of respiration in apicomplexans.     

An epidemiological survey on Cryptosporidium parvum infection of inhabitants in Chorwon-gun, Kangwon-do.

The present study was undertaken to know the infection status of Cryptosporidium parvum among the residents of Chorwon-gun, Kangwon-do in 1993. Total 461 fecal samples were collected from the inhabitants residing in Chorwon-gun during the period of August 12 to September 14, 1993. Fecal smears were prepared by formalin-ether sedimentation, and examined after modified acid fast staining. Of the 461 fecal samples, 9 (1.9%) were positive for C. parvum oocysts. The positive cases were limited to thirties (4) patients, forties (3), and sixties (2), and no oocyst was detected in other age groups. The oocyst positive rate for male was 1.4% and that of female was 2.6%.     

In vitro infection of Cryptosporidium parvum to four different cell lines

To determine a suitable condition for in vitro infection model of Cryptosporidium parvum, four different cell lines, AGS, MDCK, HCT-8 and Caco-2, were used as host cell lines which were cultured at various concentrations of added supplements. These supplement include fetal bovine serum (FBS), sodium choleate, ascorbic acid, folic acid, calcium pantothenate, para-aminobenzoic acid and pyruvate and their effects on the cell lines which were infected with C. parvum were evaluated. The results of this study showed that the AGS cell line was most susceptible to C. parvum whereas the Caco-2 cells appeared to be least susceptible to C. parvum. In regards to the serum condition, 10% FBS was suitable for the growth of AGS and HCT-8 cells, and 1% FBS was good for the growth of the MDCK cells when they were inoculated with C. parvum. Vitamins had a positive effect on the AGS cells, and pyruvate also showed positive effects on all of the cell lines except for Caco-2. Modified medium for each cell line was prepared by adding appropriate amounts of each supplement which resulted in the highest parasite infection number. Modified media increased the number of parasites infected on AGS cells to 2.3-fold higher when compared to the control media. In this study, we found that the AGS cell line was a suitable host model for evaluating C. parvum in vitro study and the media contents for the optimal infection conditions were suggested.     

Complete development of Cryptosporidium parvum in host cell-free culture

The present study describes the complete in vitro development of Cryptosporidium parvum (cattle genotype) in RPMI-1640 maintenance medium devoid of host cells. This represents the first report in which Cryptosporidium is shown to multiply, develop and complete its life cycle without the need for host cells. Furthermore, cultivation of Cryptosporidium in diphasic medium consisting of a coagulated new born calf serum base overlaid with maintenance medium greatly increased the total number of Cryptosporidium stages. Type I and II meronts were detected giving rise to two morphologically different merozoites. Type I meronts, which appear as grape-like clusters as early as 48 h post culture inoculation, release merozoites, which are actively motile, and circular to oval in shape. Type II meronts group in a rosette-like pattern and could not be detected until day 3 of culturing. Most of the merozoites released from type II meronts are generally spindle-shaped with pointed ends, while others are rounded or pleomorphic. In contrast to type I, merozoites from type II meronts are less active and larger in size. Sexual stages (micro and macrogamonts) were observed within 6-7 days of culturing. Microgamonts were darker than macrogamonts, with developing microgametes, which could be seen accumulating at the periphery. Macrogamonts have a characteristic peripheral nucleus and smooth outer surface. Oocysts at different levels of sporulation were seen 8 days post culture inoculation. Cultures were terminated after 4 months when the C. parvum life cycle was still being perpetuated with the presence of large numbers of excysting and intact oocysts. Culture-derived oocysts obtained after 46 days p.i. were infective to 7- to 8-day-old ARC/Swiss mice. The impact of C. parvum developing in cell-free culture is very significant and will facilitate many aspects of Cryptosporidium research.     

Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species

A study was undertaken to compare the performance of five different molecular methods (available in four different laboratories) for the identification of Cryptosporidium parvum and Cryptosporidium hominis and the detection of genetic variation within each of these species. The same panel of oocyst DNA samples derived from faeces (n=54; coded blindly) was sent for analysis by: (i) DNA sequence analysis of a fragment of the HSP70 gene; (ii) DNA sequence analysis and the ssrRNA gene in laboratory 1; (iii) single-strand conformation polymorphism analysis of part of the ssrRNA; (iv) SSCP analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA region in laboratory 2; (v) 60 kDa glycoprotein (gp60) gene sequencing with prior species determination using PCR with restriction fragment length polymorphism analysis of the ssrRNA gene in laboratory 3; and (vi) multilocus genotyping at three microsatellite markers in laboratory 4. For detecting variation within C. parvum and C. hominis, SSCP analysis of ITS-2 was considered to have superior utility and determined 'subgenotypes' in samples containing DNA from both species. SSCP was also most cost effective in terms of time, cost and consumables. Sequence analysis of gp60 and microsatellite markers ML1, ML2 and 'gp15' provided good comparators for the SSCP of ITS-2. However, applicability of these methods to other Cryptosporidium species or genotypes and to environmental samples needs to be evaluated. This trial provided, for the first time, a direct comparison of multiple methods for the genetic characterisation of C. parvum and C. hominis samples. A protocol has been established for the international distribution of samples for the characterisation of Cryptosporidium. This can be applied in further evaluation of molecular methods by investigation of a larger number of unrelated samples to establish sensitivity, typability, reproducibility and discriminatory power based on internationally accepted methods for evaluation of microbial typing schemes.     

Two distinct oxysterol binding protein-related proteins in the parasitic protist Cryptosporidium parvum (Apicomplexa)

Two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) have been identified from the parasitic protist Cryptosporidium parvum (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans including Plasmodium, Toxoplasma, and Eimeria possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.     

Cryptosporidium in farmed animals: the detection of a novel isolate in sheep.

We describe the discovery of polymorphisms in the Cryptosporidium oocyst wall protein (COWP) gene conferring a novel restriction fragment length polymorphism (RFLP) pattern in 26/60 (43%) isolates from a flock of sheep sampled following a waterborne outbreak of human cryptosporidiosis. The sheep isolates showed identical PCR-RFLP patterns to each other by COWP genotyping but different from those of most currently recognised genotypes, including the major Cryptosporidium parvum genotypes 1 and 2. Sequence analysis of the 550bp amplicon from the COWP gene was compared with a DNA coding region employed in previous studies and showed the novel isolate to differ from other Cryptosporidium species and C. parvum isolates by 7-21%. The sheep-derived isolates were compared at this and further three Cryptosporidium gene loci with isolates from other farmed animals. The loci employed were one in the thrombospondin related adhesive protein (TRAP-C2) gene and two in the 70kDa heat shock protein (HSP70) gene (CPHSP1 and 2). Other animal samples tested in our laboratory were from clinically ill animals and all contained C. parvum genotype 2. The sheep in which the novel isolate was identified were healthy and showed no symptoms of cryptosporidiosis, and the novel sheep isolate could represent a non-pathogenic strain. Our studies suggest that a previously undetected Cryptosporidium sub-type may exist in sheep populations, reflecting the increasingly recognised diversity within the parasite genus.     

Direct evidence for cyanide-insensitive quinol oxidase (alternative oxidase) in apicomplexan parasite Cryptosporidium parvum: phylogenetic and therapeutic implications

Cryptosporidium parvum is a parasitic protozoan that causes the diarrheal disease cryptosporidiosis, for which no satisfactory chemotherapy is currently available. Although the presence of mitochondria in this parasite has been suggested, its respiratory system is poorly understood due to difficulties in performing biochemical analyses. In order to better understand the respiratory chain of C. parvum, we surveyed its genomic DNA database in GenBank and identified a partial sequence encoding cyanide-insensitive alternative oxidase (AOX). Based on this sequence, we cloned C. parvum AOX (CpAOX) cDNA from the phylum apicomplexa for the first time. The deduced amino acid sequence (335 a.a.) of CpAOX contains diiron coordination motifs (-E-, -EXXH-) that are conserved among AOXs. Phylogenetic analysis suggested that CpAOX is a mitochondrial-type AOX, possibly derived from mitochondrial endosymbiont gene transfer. The recombinant enzyme expressed in Escherichia coli showed quinol oxidase activity. This activity was insensitive to cyanide and highly sensitive to ascofuranone, a specific inhibitor of trypanosome AOX.     

Heterogeneous expression and functional analysis of two distinct replication protein A large subunits from Cryptosporidium parvum

Replication protein A is a single stranded DNA-binding protein that has multiple roles in eukaryotic DNA metabolism. Typically, eukaryotic replication protein A is a stable heterotrimeric complex with three subunits of 70 kDa (RPA1), 32 kDa (RPA2) and 14 kDa (RPA3). We have previously cloned and characterised an RPA1 subunit from Cryptosporidium parvum, which shares high homology with other eukaryotic replication protein A 1 proteins, but lacks an N-terminal domain. Here, we have identified a second replication protein A 1 (termed CpRPA1B) from the ongoing C. parvum genome-sequencing project. The deduced protein sequence to CpRPA1B shows only 16% sequence identity with CpRPA1, indicating that two different types of RPA1 subunits are present in C. parvum. The CpRPA1B gene predicts a 75.5 kDa peptide similar in size to those of higher eukaryotes, but in contrast to the 53.9 kDa N-terminal short-type CpRPA1 protein. However, western blot analysis suggested that, although the entire CpRPA1B open reading frame might be translated, the protein may be cleaved by posttranslational modification, similar to that observed with the replication protein A 1 gene product in Plasmodium falciparum. Indirect immunofluorescence studies indicated a diffused pattern for both proteins in sporozoites. However, differential localisation was observed with CpRPA1 to the anterior region that contains the apical-complex and CpRPA1B to the central region in/or around the nuclei of the sporozoites. Both CpRPA1 and CpRPA1B full-length open reading frames were expressed for functionality assays. The CpRPA1 and CpRPA1B recombinant proteins were expressed in bacterial Escherichia coli as maltose-binding protein fusion proteins and the entire fusion proteins were assayed for their DNA-binding properties. Studies indicate that CpRPA1B binds ssDNA of >or=5 nucleotides (dT), while CpRPA1 only binds ssDNA >or=20 nucleotides (dT). This study indicates that C. parvum possesses two different types of replication protein A large subunits (replication protein A 1), both differing significantly from their hosts.