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.     

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.     

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.     

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.     

Ultrastructural Localization of Cryptosporidium parvum Antigen Using Human Patients Sera

The antigen location of Cryptosporidium parvum, which stimulates antibody formation in humans and animals, was investigated using infected human sera. Immuno-electron microscopy revealed that antigenicity-inducing humoral immunity was located at various developmental stages of parasites, including asexual, sexual stages, and oocysts. The amount of antigen-stimulating IgG antibodies was particularly high on the oocyst wall. The sporozoite surface was shown to give stimulation on IgG and IgM antibody formation. Trophozoites implicated the lowest antigenicity to humoral immunity, both IgG and IgM, by showing the least amount of gold labeling. Immunogold labeling also provided clues that antigens were presented to the host-cell cytoplasm via feeder organelles and host-parasite junctions.     

Genotype and animal infectivity of a human isolate of Cryptosporidium parvum in the Republic of Korea

Cryptosporidium parvum oocysts were isolated from a child suffering from acute gastroenteritis and successfully passaged in a calf and mice (designated hereafter SNU-H1) in the Republic of Korea; its molecular genotype has been analyzed. The GAG microsatellite region was amplified by a polymerase chain reaction (PCR), with a 238 base pair product, which is commonly displayed in C. parvum. The isolate was shown to be a mixture of the genotypes 1 (anthroponotic) and 2 (zoonotic). To study its infectivity in animals, 2 calves and 3 strains of mice were infected with the SNU-H1; in these animals, the propagation of both genotypes was successful. In immunosuppressed (ImSP) BALB/c and C57BL/6 mice the number of oocysts decreased after day 10 post-infection (PI); but in ImSP ICR mice, they remained constant until day 27 PI. The results show that both the C. parvum genotypes 1 and 2 can be propagated in calves and ImSP mice.     

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 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.     

Cryptosporidium hominis Infection Diagnosed by Real-Time PCR-RFLP

There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections.     

Detection of Plasmodium vivax by Nested PCR and Real-Time PCR

Malaria is endemic in the Cukurova region while it is sporadic in other regions of Turkey. Therefore, the laboratory and clinical diagnosis of malaria is important for the treatment of malaria. In this study, 92 blood samples that were taken from the suspected malaria patients for routine diagnosis in a period of 10 years between 1999 and 2009 were analyzed. All of these blood samples were examined by microscopic examinations using Giemsa-stained thick blood films, nested PCR, and real-time PCR. The sensitivity-specificity and positive-negative predictive values for these diagnostic tests were then calculated. It was found that the positive predictive values of microscopic examination of thick blood films, nested PCR, and real-time PCR were 47.8%, 56.5%, and 60.9% for malaria, respectively. The real-time PCR was found to have a specificity of 75% and sensitivity of 100%, while specificity and sensitivity of nested PCR was found 81.2% and 97.7% according to the microscopic examination of thick blood films, respectively.     

Role of murine Peyer's patch lymphocytes against primary and challenge infections with Cryptosporidium parvum

In order to determine the role of Peyeros patch lymphocytes (PPL) in self-clearing of Cryptosporidium parvum infection in murine models, changes in PPL subsets, their cytokine expression, and in vitro IgG1 and IgA secretions by PPL were observed in primary- and challenge-infected C57BL/6 mice. In primary-infected mice, the percentages of CD4+ T cells, CD8+ T cells, sIgA+ B cells, IL-2+ T cells, and IFN-gamma+ T cells among the PPL, increased significantly (P < 0.05) on day 10 post-infection (PI). Secretion of IgG1 and IgA in vitro by PPL also increased on day 10 PI. However, all these responses, with the exception of IgG1 and IgA secretions, decreased in challenge-infected mice on day 7 post-challenge (= day 13 PI); their IgG1 and IgA levels were higher (P > 0.05) than those in primaryinfected mice. The results suggest that murine PPL play an important role in self-clearing of primary C. parvum infections through proliferation of CD4+, CD8+, IL-2+, and IFN-gamma+ T cells, and IgG1 and IgA-secreting B cells. In challenge infections, the role of T cells is reduced whereas that of B cells secreting IgA appeared to be continuously important.     

The effect of microfilament inhibitor on the Cryptosporidium infection in vitro

This study was focused on the effects of microfilament inhibitor, Cytochalasin D (CD) on the invasiveness of sporozoites of Cryptosporidium spp. into the host cells. MDCK and AGS cell lines were used as host cells for C. parvum and C. muris, respectively. When MDCK cells were pretreated with CD for 1 hr before inoculation of the sporozoites, C. parvum infection was significantly inhibited when compared to the control cells. These inhibitory effects of CD on the rate of infection were dose-dependent. In addition, C. muris infection was hampered when AGS cell lines were pretreated with CD. However, the capability of invasiveness of the sporozoites into the host cells was not greatly influenced by the pretreatment of sporozoites with CD before infection. These results suggest that microfilaments of host cells, rather than parasites, play an important role for the invasion of Cryptosporidium spp.     

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.     

Detection and Molecular Characterization of Cryptosporidium spp. from Wild Rodents and Insectivores in South Korea

In order to examine the prevalence of Cryptosporidium infection in wild rodents and insectivores of South Korea and to assess their potential role as a source of human cryptosporidiosis, a total of 199 wild rodents and insectivore specimens were collected from 10 regions of South Korea and screened for Cryptosporidium infection over a period of 2 years (2012-2013). A nested-PCR amplification of Cryptosporidium oocyst wall protein (COWP) gene fragment revealed an overall prevalence of 34.2% (68/199). The sequence analysis of 18S rRNA gene locus of Cryptosporidium was performed from the fecal and cecum samples that tested positive by COWP amplification PCR. As a result, we identified 4 species/genotypes; chipmunk genotype I, cervine genotype I, C. muris, and a new genotype which is closely related to the bear genotype. The new genotype isolated from 12 Apodemus agrarius and 2 Apodemus chejuensis was not previously identified as known species or genotype, and therefore, it is supposed to be a novel genotype. In addition, the host spectrum of Cryptosporidium was extended to A. agrarius and Crosidura lasiura, which had not been reported before. In this study, we found that the Korean wild rodents and insectivores were infected with various Cryptosporidium spp. with large intra-genotypic variationa, indicating that they may function as potential reservoirs transmitting zoonotic Cryptosporidium to livestock and humans.     

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.     

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.     

A Waterborne Outbreak and Detection of Cryptosporidium Oocysts in Drinking Water of an Older High-Rise Apartment Complex in Seoul

From May to June 2012, a waterborne outbreak of 124 cases of cryptosporidiosis occurred in the plumbing systems of an older high-rise apartment complex in Seoul, Republic of Korea. The residents of this apartment complex had symptoms of watery diarrhea and vomiting. Tap water samples in the apartment complex and its adjacent buildings were collected and tested for 57 parameters under the Korean Drinking Water Standards and for additional 11 microbiological parameters. The microbiological parameters included total colony counts, Clostridium perfringens, Enterococcus, fecal streptococcus, Salmonella, Shigella, Pseudomonas aeruginosa, Cryptosporidium oocysts, Giardia cysts, total culturable viruses, and Norovirus. While the tap water samples of the adjacent buildings complied with the Korean Drinking Water Standards for all parameters, fecal bacteria and Cryptosporidium oocysts were detected in the tap water samples of the outbreak apartment complex. It turned out that the agent of the disease was Cryptosporidium parvum. The drinking water was polluted with sewage from a septic tank in the apartment complex. To remove C. parvum oocysts, we conducted physical processes of cleaning the water storage tanks, flushing the indoor pipes, and replacing old pipes with new ones. Finally we restored the clean drinking water to the apartment complex after identification of no oocysts.