Complete development of Cryptosporidium parvum in MDBK cells

Abstract Sporozoites of Cryptosporidium parvum excysted in vitro from bovine oocysts were incubated with monolayers of Madin-Darby bovine kidney cells. The extent of parasite colonisation was monitored by light microscopy and immunofluorescence. Electron microscopy confirmed the complete development and replication of C. parvum within Madin-Darby bovine kidney cells.     

Characterization of a major sporozoite surface glycoprotein of Cryptosporidum parvum

We have identified S60, a new Cryptosporidium parvum sporozoite surface glycoprotein. S60 was cleaved into two subunits, S16 and S45, approximately 16–18 and 45–47 kDa respectively, with cleavage occurring at an SRSRR motif likely to be sensitive to trypsin in vivo. Analysis by surface biotinylation, lectins and monoclonal antibodies suggests S60 is an abundant sporozoite surface glycoprotein that is shed by migrating sporozoites. The major glycosylation on S60 was identified as single O-linked N-acetylgalactosamine sugars on threonine and serine residues. The gene encoding the S60 protein was identified and revealed a C-terminal consensus sequence for the addition of a glycosylphosphatidyl inositol anchor. Antisera raised against recombinant S60 produced in Escherichia coli reacted with the surface of sporozoites and the inner wall of excysted oocysts. Electronic Publication     

Antiparasitic activity of flavonoids and isoflavones against Cryptosporidium parvum and Encephalitozoon intestinalis

Flavonoids, polyphenolic compounds found in plants, have demonstrated activity against several parasites and can augment the efficacy of other drugs by either increasing the uptake or decreasing the efflux of these drugs. We evaluated 11 of these compounds alone or in combination in order to test the hypothesis that flavonoids are effective against Cryptosporidium parvum and Encephalitozoon intestinalis. Using in vitro cell culture assays, HCT-8 cells or E6 cells were infected with C. parvum and E. intestinalis, respectively, and treated with compounds at doses ranging from 1 to 200 microM. We found that six compounds were active against C. parvum. Naringenin and genistein had the greatest activities with EC(50) of 15 and 25 microM, respectively. Two compounds, quercetin and apigenin, had activity against E. intestinalis at EC(50) of 15 and 50 microM, respectively. The EC(50) of trifluralin, a dinitroaniline compound, was decreased significantly when combined with genistein in an in vitro assay, suggesting that compounds may be used alone on in combination with other moderately active drugs to increase efficacy. In addition, induction of apoptosis by these compounds was studied but not observed to be a significant mechanism of action.     

Die-off of Cryptosporidium parvum in soil and wastewater effluents

AIMS: To determine the effect of biotic and abiotic components of soil on the viability and infectivity of Cryptosporidium parvum, and evaluate the suitability of viability tests as a surrogate for oocyst infectivity under various environmental settings. METHODS AND RESULTS: The die-off of C. parvum in saturated and dry loamy soil was monitored over time by immunofluorescence assay (IFA) and PCR to estimate oocysts viability and by cell culture to estimate oocysts infectivity. Pseudomonas aeruginosa activity resulted in digestion of the outer layer of the oocysts, as demonstrated by loss of the ability to react in IFA. Whereas, P. aeruginosa activity did not affect the DNA amplification by PCR. A 1-log reduction in the oocysts infectivity was observed at 30 degrees C in distilled water and in saturated soil while oocysts viability was unchanged. Incubation for 10 days in dry loamy soil at 32 degrees C resulted in a 3-log(10) reduction in their infectivity while no change of oocysts viability was recorded. CONCLUSIONS: Under low temperature, C. parvum oocysts may retain their infectivity for a long time. Soil desiccation and high temperatures enhance the die-off rate of C. parvum. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous die-off studies of C. parvum used viability tests that do not necessarily reflect the oocyst infectivity. Under low temperatures, there was an agreement observed between viability and infectivity tests and oocysts retained their infectivity for a long time. Desiccation and high temperatures enhance the loss of infectivity of C. parvum. The presented die-off data have significant implications on the management of wastewater reuse in warm environments.     

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.     

Current progress in the fatty acid metabolism in Cryptosporidium parvum

Cryptosporidium parvum is one of the apicomplexans that can cause severe diarrhea in humans and animals. The slow development of anti-cryptosporidiosis chemotherapy is primarily due to the poor understanding on the basic metabolic pathways in this parasite. Many well-defined or promising drug targets found in other apicomplexans are either absent or highly divergent in C. parvum. The recently discovered apicoplast and its associated Type II fatty acid synthetic enzymes in Plasmodium, Toxoplasma, and Eimeria apicomplexans are absent in C. parvum, suggesting this parasite is unable to synthesize fatty acids de novo. However, C. parvum possesses a giant Type I fatty acid synthase (CpFAS1) that makes very long chain fatty acids using mediate or long chain fatty acids as precursors. Cryptosporidium also contains a Type I polyketide synthase (CpPKS1) that is probably involved in the production of unknown polyketide(s) from a fatty acid precursor. In addition to CpFAS1 and CpPKS1, a number of other enzymes involved in fatty acid metabolism have also been identified. These include a long chain fatty acyl elongase (LCE), a cytosolic acetyl-CoA carboxylase (ACCase), three acyl-CoA synthases (ACS), and an unusual "long-type" acyl-CoA binding protein (ACBP), which allows us to hypothetically reconstruct the highly streamlined fatty acid metabolism in this parasite. However, C. parvum lacks enzymes for the oxidation of fatty acids, indicating that fatty acids are not an energy source for this parasite. Since fatty acids are essential components of all biomembranes, molecular and functional studies on these critical enzymes would not only deepen our understanding on the basic metabolism in the parasites, but also point new directions for the drug discovery against C. parvum and other apicomplexan-based diseases.     

In vitro development of Cryptosporidium parvum in serum-free media

AIM: To compare commercially available serum-free media with common, standard, growth medium for their ability to support growth of Cryptosporidium parvum in HCT-8 cell cultures. METHODS AND RESULTS: Twelve serum-free media formulations with or without additional supplements were tested against a standard growth medium containing 2% FBS in HCT-8 cell cultures. After a 48-h incubation period, the level of parasite development was determined by ELISA. The extent of development in the serum-free media was determined as a percentage of infections compared with those obtained using a standard growth medium. CONCLUSIONS: Several of the serum-free media formulations, which included MDCK, UltraMDCK, PC-1, UltraCHO and UltraCulture, compared favourably with a traditional, standard growth medium. Moreover, increasing FBS concentrations to 10% actually resulted in an overall decrease in development in many cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Several serum-free medium formulations are available which allow development of C. parvumin vitro at levels comparable with standard media employing FBS. These serum-free media are particularly useful for applications, which may require a more defined medium without the presence of FBS. Moreover, the elimination of FBS as a variable allows investigators the ability to more closely regulate their experimental systems when growing C. parvum in cell cultures.     

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%.     

PCR-restriction fragment length polymorphism analysis of a diagnostic 452-base-pair DNA fragment discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum isolates of human and animal origin

Genomic DNAs from human Cryptosporidium isolates previously typed by analysis of the 18S ribosomal DNA locus (Cryptosporidium parvum bovine genotype, C. parvum human genotype, Cryptosporidium meleagridis, and Cryptosporidium felis) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous Cryptosporidium wrairi sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate Cryptosporidium species and C. parvum genotypes.     

PCR-Restriction Fragment Length Polymorphism Analysis of a Diagnostic 452-Base-Pair DNA Fragment Discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum Isolates of Human and Animal Origin

Genomic DNAs from human Cryptosporidium isolates previously typed by analysis of the 18S ribosomal DNA locus (Cryptosporidium parvum bovine genotype, C. parvum human genotype, Cryptosporidium meleagridis, and Cryptosporidium felis) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous Cryptosporidium wrairi sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate Cryptosporidium species and C. parvum genotypes.     

A simple and reliable method of producing in vitro infections of cryptosporidium parvum (apicomplexa)

Abstract A variety of techniques have been used to infect cell monolayers in culture with the protozoan, Cryptosporidium parvum . However, most of these methods rely on the use of trypsin and/or bile salts to excyst sporozoites in vitro, followed by washing sporozoites free of excystation solution prior to their addition to subconfluent monolayers. This method not only increases the amount of time required to establish infections in vitro, but also results in prolonged exposure of free sporozoites to environmental conditions. Here we report a simple, fast, and efficient method of obtaining consistent infections of C. parvum in cell monolayers. This technique relies on the ability of the parasite to excyst at 37°C but not at room temperature following pretreatment with sodium hypochlorite. By adding surface-sterilized oocysts directly to monolayers, sporozoites have access to host cells immediately upon excystation.     

Role of Corynebacterium parvum in the activation of peritoneal macrophages: I. Association between intracellular C. parvum and cytotoxic macrophages

We have investigated the association between intracellular C. parvum (CP) and macrophage (Mφ) cytotoxicity. Mouse peritoneal Mφs were activated by ip administration of CP and were subjected to a combination of fractionation techniques to study this. Velocity sedimentation demonstrated that only the largest cells were cytotoxic. These same cells contained CP and suggested an association between the two variables. Further separation of the largest Mφs using a BSA equilibrium buoyant density gradient demonstrated that cytotoxicity was due to Mφs and further substantiated the strong correlation between intracellular CP and cytotoxicity. Various fluorochrome tagged CP preparations were also used to activate Mφs and to isolate CP-containing Mφs using fluorescence-activated cell sorting. When velocity-enriched Mφs were sorted on the basis of the presence or absence of fluorescent CP, only the Mφ fractions which contained CP were cytotoxic. The results indicate that most cytolytic macrophages present at the peak of the response contain CP. Thus, a convenient probe with which to follow macrophage activation at the single cell level was provided.     

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.     

C. parvum in germ-free mice

Summary The effect of previous sensitization to C. parvum, by cross-reacting antigens from other bacteria, on the immunostimulatory effects of C. parvum treatment were studied in germ-free and conventional mice. It was found that the development of splenomegaly and specific delayed hypersensitivity following C. parvum injection were similar in both germ-free mice and conventional mice.Supported by U.S. Public Health Service Grant No. 5S07 RR05705 and NIH Grant no. AM 18530Visiting Investigator. Present address: Department of Experimental Immunobiology, The wellcome Research Laboratories Beckenham, Kent, England.Recipient of a post-doctoral fellowship from the National Foundation for Ileitis and Colitis Inc.Recipient of Research Career Development Award No. AM 0073 from the National Institute of Arthritis, Metabolism and Digestive Diseases     

Immunological characterization of a 17-kDa antigen from Cryptosporidium parvum recognized early by mucosal IgA antibodies

Cryptosporidium parvum antigens were characterized by immunoblot analysis of sera and intestinal secretions of BALB/c mice orally infected with 10(5) oocysts. A major band at 17 kDa under non-reduced conditions and at 18 kDa under reduced conditions was recognized by anti-C. parvum IgA and IgG in serum and intestinal secretions from day 15 post-infection. This recognition persisted throughout the experiment (day 30). Mouse-serum antibodies raised against the 17-kDa purified antigen (P17) showed no cross-reactivity with other C. parvum antigens. Immunofluorescence study revealed that this antigen is located on the sporozoite. It is suggested that this antigen could be a good candidate for studies of mucosal immune response to C. parvum and for vaccination.