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Cryptosporidium parvum is an apicomplexan parasite that infects a wide range of hosts including humans. Due to the parasite's quasi-intracellular, intermembrane location on the host cell, it is difficult to purify parasites from in vitro and in vivo infections for molecular studies. We have developed a method to greatly enrich in vitro C. parvum merozoites from host cells. The efficiency of the protocol was assessed with C. parvum (KSU-1 isolate) parasites of different developmental stages isolated following a synchronized infection of HCT-8 host cells. Total RNA was extracted from the samples and used to evaluate the quantity of host cell contamination in enriched parasite fractions. The quality of the RNA was verified using an Agilent BioAnalyzer. cDNA libraries of RNA isolated from 24 and 48 h C. parvum in vitro preparations isolated via this protocol were sequenced at the Broad Institute via an NIH Microbial Sequencing (GSCID) Contract. Cryptosporidium sequences comprised 30% of the cDNA reads, demonstrating significant enrichment.

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The family Partitiviridae includes plant and fungal viruses with bisegmented dsRNA genomes and isometric virions in which the two genome segments are packaged separately and used as templates for semiconservative transcription by the viral polymerase. A new genus, Cryspovirus, has been approved for this family. Its name is based on that of the host genus, Cryptosporidium, which encompasses several species of apicomplexan parasites that infect a wide range of mammals, birds, and reptiles, and are a major cause of human diarrheal illness worldwide. The type species of the new genus is Cryptosporidium parvum virus 1. Distinguishing characteristics include infection of a protozoan host, a smaller capsid protein than found in other members of the family Partitiviridae, and sequence-based phylogenetic divergence.

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During August 2003 and August 2004, 11 adult eastern red bats, Lasiurus borealis, were collected and their feces examined for coccidian parasites. Bats were obtained in August 2003 from Garland, Montgomery, and Yell counties, Arkansas (n=6) and in August 2004 from Anson and Montgomery counties, North Carolina (n=5). Seven (63.6%) of the bats were passing oocysts of 2 undescribed species of Eimeria. Oocysts of Eimeria dowleri n. sp. were subspherical to ellipsoidal, 24.7 x 22.0 (23-26 x 20-23) microm, with a bilayered wall, externally moderately pitted, internally smooth, and with a shape index of 1.1. Micropyle and oocyst residuum were absent, but a polar granule was present. Sporocysts were ovoidal, 13.4 x 9.2 (12-14 x 8-9) pm; shape index was 1.5; Stieda and sub-Stieda bodies were present. A sporocyst residuum consisting of homogeneous granules was scattered among the sporozoites; sporozoites were elongate, with a subspherical anterior refractile body and an elongate posterior refractile body; a nucleus was not discernable. Oocysts of Eimeria sealanderi n. sp. were subspherical to ellipsoidal, 16.7 x 14.4 (15-18 x 13-16) microm, with a bilayered wall, externally lightly pitted, internally smooth, and with a shape index of 1.2. A micropyle was absent, but the oocyst residuum and polar granule were present. Oocyst residuum consisted of a single, membrane-bound homogenous granule. Sporocysts were ovoidal, 8.9 x 5.7 (8-10 x 5-6) microm, with a shape index of 1.6; Stieda and sub-Stieda bodies were present. The sporocyst residuum consisted of 10, to several dozen, homogeneous granules of various sizes loosely clustered among the sporozoites, which were elongate and without obvious refractile bodies and nucleus. This is the first time any coccidian has been reported from this host and the first instance of a bat coccidian reported from North Carolina.

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Between December 2002 and June 2004, 10 marbled salamanders, Ambystoma opacum, were examined for coccidian parasites. Salamanders were collected in Bradley (n = 2), Little River (n = 1), Miller (n = 1), and Sevier (n = 1) Counties, Arkansas; Webster Parish, Louisiana (n = 2); and Bowie (n = 1) and Nacogdoches (n = 2) Counties, Texas. Two of 10 (20%) A. opacum from Louisiana harbored an undescribed species of Eimeria. Oocysts of Eimeria trauthi n. sp. were ellipsoidal, 36.6 x 33.1 (33-40 x 29-37) microm, with a thin, single-layered wall; shape index 1.1. Polar granule(s) and micropyle were absent. Oocyst residuum was composed of hundreds of loosely packed homogenous granules of various sizes enclosing a vacuole. Sporocysts were elongate-ellipsoidal, 20.8 x 8.1 (19-22 x 7-9) microm; shape index 2.6. Sporocyst residuum was spherical and composed of a cluster of granules often membrane-bound. This is the first time a coccidium has been reported from an amphibian species in Louisiana and the second time a coccidium has been described from this salamander host. In addition, the following 26 salamanders from various counties in Arkansas, Oklahoma, and Texas were surveyed during the study period and were negative for coccidia: Ambystomatidae, 4 spotted salamanders (Ambystoma maculatum) and 7 mole salamanders (Ambystoma talpoideum); Cryptobranchidae, 4 Ozark hellbenders (Cryptobranchus alleganiensis bishopi); Plethodontidae, 6 spotted dusky salamanders (Desmognathus conanti) and 3 many-ribbed salamanders (Eurycea multiplicata multiplicata); and Salamandridae, 2 central newts (Notophthalmus viridescens louisianensis).

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Cryptosporidosis is a severe opportunistic infection of immuno-compromised individuals for which no reliable therapy exists. The parasite scavenges host-derived polyamines, particularly spermine, which is then converted to the lower polyamines by the combined action of spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine oxidase (PAO). We have isolated and expressed the Cryptosporidium parvum SSAT for kinetic and molecular comparison with the host enzyme. The CpSSAT is a homotetramer with a subunit molecular mass of 18 kDa and low sequence similarity to higher eukaryotes but maintains the critical arginine residues in the active site. The CpSSAT had an activity of 299 nmol(-1)min(-1)(mg of protein)(-1) and exhibits an ordered Bi-Bi kinetics with preferred substrate specificity for spermine. Polyamine analogues having unsaturated central carbons were found to exhibit mixed inhibition kinetics of the CpSSAT. The cis-analogues were more effective inhibitors of the CpSSAT with lower K(i) values than the trans-analogues. Experiments aimed at determining the ratio of the time of the analogue in the enzyme active site to that spent out (in-out time: delta ln E/deltat) confirmed the higher efficiency of the cis-analogues as inhibitors of the CpSSAT. The results of this study reveal that the C. parvum SSAT may provide a rational target for drug design.

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The in vivo effectiveness of a series of conformationally restricted polyamine analogues alone and selected members in combination with DL-alpha-difluoromethylarginine against Cryptosporidium parvum infection in a T-cell receptor alpha-deficient mouse model was tested. Polyamine analogues were selected from the extended bis(ethyl)-sym-homospermidine or bis(ethyl)-spermine backbone having cis or trans double bonds at the center of the molecule. The cis isomers were found to have significantly greater efficacy in both preventing and curing infection in a mouse model than the trans polyamine analogues when tested in a T-cell receptor alpha-deficient mouse model. When tested in combination with DL-alpha-difluoromethylarginine, the cis-restricted analogues were found to be more effective in preventing oocyst shedding. This study demonstrates the potential of polyamine analogues as anticryptosporidial agents and highlights the presence of multiple points in polyamine synthesis by this parasite that are susceptible to inhibition resulting in growth inhibition.

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We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (C(T)([P18S])) and host cells (C(T)([H18S])), and evaluates the relative expression between parasite and host rRNA levels (i.e., deltaC(T) = C(T)([P18S]) - C(T)([H18S])) to minimize experimental and operational errors. The choice of qRT-PCR over quantitative PCR (qPCR) in this study is based on the observations that (i) the relationship between the logarithm of infected parasites (log[P]) and the normalized relative level of rRNA (deltadeltaC(T)) is linear, with a fourfold dynamic range, by qRT-PCR but sigmoidal (nonlinear) by qPCR; and (ii) the level of RNA represents that of live parasites better than that of DNA, because the decay of RNA (99% in approximately 3 h) in dead parasites is faster than that of DNA (99% in approximately 24 to 48 h) under in vitro conditions. The reliability of the qRT-PCR method was validated by testing the efficacies of nitazoxanide and paromomycin on the development of two strains of C. parvum (IOWA and KSU-1) in HCT-8 cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC50 values for nitazoxanide and paromomycin were 0.30 to 0.45 micro/ml and 89.7 to 119.0 microg/ml, respectively, comparable to the values reported previously. Using the qRT-PCR assay, we have also observed that pyrazole could inhibit C. parvum development in vitro (MIC50 = 15.8 mM), suggesting that the recently discovered Cryptosporidium alcohol dehydrogenases may be explored as new drug targets.

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Ultraviolet (UV) light is being considered as a disinfectant by the water industry because it appears to be very effective for controlling potential waterborne pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms such as nucleotide excision repair and photolyase enzymes for repairing UV-induced DNA damage and regaining preirradiation levels of infectivity or population density. Genes encoding UV repair proteins exist in C. parvum, so the parasite should be able to regain infectivity following exposure to UV. Nevertheless, there is an increasing body of evidence that the organism is unable to reactivate following UV irradiation. This paper describes the effective inactivation of C. parvum by UV light, identifies nucleotide excision repair genes in the C. parvum and Cryptosporidium hominis genomes and discusses the inability of UV-exposed oocysts to regain infectivity.

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Ultraviolet light is being considered as a disinfectant by the water industry because it appears to be very effective for inactivating pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms for repairing ultraviolet light-induced DNA damage, which may limit the utility of this disinfection technology. Inactivation of C. parvum was assessed by measuring infectivity in cells of the human ileocecal adenocarcinoma HCT-8 cell line, with an assay targeting a heat shock protein gene and using a reverse transcriptase polymerase chain reaction to detect infections. Oocysts of five different isolates displayed similar sensitivity to ultraviolet light. An average dosage of 7.6 mJ/cm2 resulted in 99.9% inactivation, providing the first evidence that multiple isolates of C. parvum are equally sensitive to ultraviolet disinfection. Irradiated oocysts were unable to regain pre-irradiation levels of infectivity, following exposure to a broad array of potential repair conditions, such as prolonged incubation, pre-infection excystation triggers, and post-ultraviolet holding periods. A combination of data-mining and sequencing was used to identify genes for all of the major components of a nucleotide excision repair complex in C. parvum and Cryptosporidium hominis. The average similarity between the two organisms for the various genes was 96.4% (range, 92-98%). Thus, while Cryptosporidum spp. may have the potential to repair ultraviolet light-induced damage, oocyst reactivation will not occur under the standard conditions used for storage and distribution of treated drinking water.

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There has been an explosion of descriptions of new species of Cryptosporidium during the last two decades. This has been accompanied by confusion regarding the criteria for species designation, largely because of the lack of distinct morphologic differences and strict host specificity among Cryptosporidium spp. A review of the biologic species concept, the International Code of Zoological Nomenclature (ICZN), and current practices for Cryptosporidium species designation calls for the establishment of guidelines for naming Cryptosporidium species. All reports of new Cryptosporidium species should include at least four basic components: oocyst morphology, natural host specificity, genetic characterizations, and compliance with the ICZN. Altogether, 13 Cryptosporidium spp. are currently recognized: C. muris, C. andersoni, C. parvum, C. hominis, C. wrairi, C. felis, and C. cannis in mammals; C. baïleyi, C. meleagridis, and C. galli in birds; C. serpentis and C. saurophilum in reptiles; and C. molnari in fish. With the establishment of a framework for naming Cryptosporidium species and the availability of new taxonomic tools, there should be less confusion associated with the taxonomy of the genus Cryptosporidium. The clarification of Cryptosporidium taxonomy is also useful for understanding the biology of Cryptosporidium spp., assessing the public health significance of Cryptosporidium spp. in animals and the environment, characterizing transmission dynamics, and tracking infection and contamination sources.

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Cryptosporidium muris, predominantly a rodent species of Cryptosporidium, is not normally considered a human pathogen. Recently, isolated human infections have been reported from Indonesia, Thailand, France, and Kenya. We report the first case of C. muris in a human in the Western Hemisphere. This species may be an emerging zoonotic pathogen capable of infecting humans.

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A series of benzindazole-4,9-quinones was tested for growth-inhibitory effects on Cryptosporidium parvum in vitro and in vivo. Most compounds showed considerable activity at concentrations from 25 to 100 micro M. For instance, at 25 micro M the derivatives 5-hydroxy-8-chloro-N1-methylbenz[f]-indazole-4,9-quinone and 5-chloro-N2-methylbenz[f]indazole-4,9-quinone inhibited growth of C. parvum 78-100%, and at 50 micro M seven of the 23 derivatives inhibited growth > or = 90%. The activity of the former two compounds was confirmed in a T-cell receptor alpha (TCR-alpha)-deficient mouse model of chronic cryptosporidiosis. In these mice, the mean infectivity scores (IS) in the caecum were 0.63-0.20, whereas in sham-treated mice the score was 1.44 (P < 0.05). There were similar differences in IS in the ileum, where the score for treated mice was 1.12-0.20 and that for mice receiving no drug was 1.32. There was no acute or chronic toxicity for any compound tested in vivo.

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