Surprisingly, unlike most Apicomplexa, Cryptosporidium parvum appears to lack a plastid genome. Primers based upon the highly conserved plastid small- or large-subunit rRNA (SSU/LSU rRNA) and the tufA-tRNAPhe genes of other members of the phylum Apicomplexa failed to amplify products from intracellular stages of C. parvum, whereas products were obtained from the plastid-containing apicomplexans Eimeria bovis and Toxoplasma gondii, as well as the plants Allium stellatum and Spinacia oleracea. Dot-blot hybridization of sporozoite genomic DNA (gDNA) supported these PCR results. A T. gondii plastid-specific set of probes containing SSU/LSU rRNA and tufA-tRNAPhe genes strongly hybridized to gDNA from a diverse group of plastid-containing organisms including three Apicomplexa, two plants, and Euglena gracilis, but not to those without this organelle including C. parvum, three kinetoplastids, the yeast Saccharomyces cerevisiae, mammals and the eubacterium Escherichia coli. Since the origin of the plastid in other apicomplexans is postulated to be the result of a secondary symbiogenesis of either a red or a green alga, the most parsimonious explanation for its absence in C. parvum is that it has been secondarily lost. If confirmed, this would indicate an alternative evolutionary fate for this organelle in one member of the Apicomplexa. It also suggests that unlike the situation with other diseases caused by members of the Apicomplexa, drug development against cryptosporidiosis targeting a plastid genome or metabolic pathways associated with it may not be useful.

We have performed a whole-genome-sequence survey for the gregarine, Ascogregarina taiwanensis and herein describe both features unique to this early diverging apicomplexan and properties that unite it with Cryptosporidium, the Coccidia, and the Apicomplexa. Phylogenetic trees inferred from a concatenated protein sequence comprised of 10,750 amino acid positions, as well as the large subunit rRNA genes, robustly support phylogenetic affinity of Ascogregarina with Cryptosporidium at the base of the apicomplexan clade. Unlike Cryptosporidium, Ascogregarina possesses numerous mitochondrion-associated pathways and proteins, including enzymes within the Krebs cycle and a cytochrome-based respiratory chain. Ascogregarina further differs in the capacity for de novo synthesis of pyrimidines and amino acids. Ascogregarina shares with Cryptosporidium a Type I fatty acid synthase and likely a polyketide synthase. Cryptosporidium and Ascogregarina possess a large repertoire of multidomain surface proteins that align it with Toxoplasma and are proposed to be involved in coccidian-like functions. Four families of retrotransposable elements were identified, and thus, retroelements are present in Ascogregarina and Eimeria but not in other apicomplexans that have been analyzed. The sum observations suggest that Ascogregarina and Cryptosporidium share numerous molecular similarities, not only including coccidian-like features to the exclusion of Haemosporidia and Piroplasmida but also differ from each other significantly in their metabolic capacity.

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., ΔC_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 (ΔΔC_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 ∼3 h) in dead parasites is faster than that of DNA (99% in ∼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 μg/ml and 89.7 to 119.0 μg/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.