Coccidian parasites are transmitted via a fecal oocyst stage that is exceptionally resistant to environmental stress and harsh chemical treatments, which allows parasites to stably persist outside a host. Because of its oocyst durability Cryptosporidium parvum is a significant water-and food-borne pathogen of humans, as well as animals of agricultural importance. To date, only one apicomplexan oocyst membrane protein has been identified, Cryptosporidium oocyst wall protein 1 (COWP1). COWP1 has a highly cysteine-rich periodicity due to arrays of two apicomplexan-specific motifs, designated the type I and type II domains. In this study, exhaustive BLAST screening of a complete C. parvum genome sequence database resulted in identification of eight additional genes encoding similar arrays of cysteine-rich type I and/or type II domains. Transcript expression analysis revealed that all COWP genes are abundantly expressed at a time when developing oocysts are observed, roughly 48 to 72 h after inoculation of in vitro cultures. A monoclonal antibody recognizing COWP8 specifically localized to the C. parvum oocyst wall, supporting the hypothesis that multiple COWPs play a role in the oocyst wall structure. BLAST screening of the Toxoplasma gondii genome sequence database resulted in identification of a gene encoding at least one COWP homolog (TgOWP1), and this multiexon sequence information was used to isolate a full-length cDNA. Exhaustive screening of Plasmodium sp. genome sequence databases by using COWP genes as BLAST queries failed to detect similar proteins in Plasmodium. We therefore propose that the COWP family of proteins have a structural role in apicomplexan species that produce durable shed cysts capable of surviving environmental stress.Cryptosporidium parvum is an obligate intracellular parasite that causes severe diarrhea via infection of the lower gastrointestinal tract epithelium of humans and other mammals. Cryptosporidium is one of several genera in the phylum Apicomplexa that have common life cycle and morphological stages, collectively referred to as coccidia (reviewed in reference 5). Coccidian oocyst stages are highly resistant to environmental stress and chemical disinfection (9), and this is attributed to a durable oocyst wall, a complex protective barrier consisting of a double layer of a protein-lipid-carbohydrate matrix (5). Ultrastructural studies have revealed that coccidian oocyst walls appear to form as a result of the interaction of wall-forming bodies and pellicular membranes (7,14). During oocyst maturation wall-forming bodies degranulate in the cytoplasm, and the vesicular material is delivered into the intermembrane space. Once the construction of the outer layer of the oocyst wall nears completion, a similar process begins for the inner layer. In the case of a related coccidian parasite, Eimeria nieschulzi, seven membranes and two types of wallforming bodies that appear to be involved in oocyst wall formation have been identified (14).Despite numerous studies of this unique structure...

The existence of a large number of single nucleotide polymorphisms (SNPs) provides opportunities and challenges to screen DNA variations affecting complex traits using a candidate gene analysis. In this article, four types of epistasis effects of two candidate gene SNPs with Hardy-Weinberg disequilibrium (HWD) and linkage disequilibrium (LD) are considered: additive ϫ additive, additive ϫ dominance, dominance ϫ additive, and dominance ϫ dominance. The Kempthorne genetic model was chosen for its appealing genetic interpretations of the epistasis effects. The method in this study consists of extension of Kempthorne's definitions of 35 individual genetic effects to allow HWD and LD, genetic contrasts of the 35 extended individual genetic effects to define the 4 epistasis effects, and a linear model method for testing epistasis effects. Formulas to predict statistical power (as a function of contrast heritability, sample size, and type I error) and sample size (as a function of contrast heritability, type I error, and type II error) for detecting each epistasis effect were derived, and the theoretical predictions agreed well with simulation studies. The accuracy in estimating each epistasis effect and rates of false positives in the absence of all or three epistasis effects were evaluated using simulations. The method for epistasis testing can be a useful tool to understand the exact mode of epistasis, to assemble genome-wide SNPs into an epistasis network, and to assemble all SNP effects affecting a phenotype using pairwise epistasis tests.single nucleotide polymorphism; quantitative trait; linkage disequilibrium; Hardy-Weinberg disequilibrium THE SIGNIFICANCE OF EPISTASIS in complex traits has been well recognized (3,12,16,17). It has been hypothesized that epistasis is ubiquitous in determining susceptibility to common human diseases. Observations supporting this hypothesis include deviations from Mendelian ratios, molecular interactions in gene regulation and biochemical and metabolic systems, nonrepeatable positive effects of single polymorphisms, and gene interaction effects commonly found when properly investigated (16). Large epistasis networks found in yeast (19, 21) also point to the ubiquitous nature of epistasis. The presence of a large number of single nucleotide polymorphisms (SNPs) provides opportunities and challenges to identify DNA variations associated with complex traits using SNPs as candidate genes. A number of studies on SNP-disease association have been reported (2,7,8,20,24), but most current candidate gene studies focus on single gene effects. Ongoing studies of geneotyping up to 500,000 SNPs on 2,000 individuals have been reported (1). Such large-scale SNP studies would be capable of detecting certain epistasis effects. 1For two bi-allelic genes unaffected by the environment, a total of 512 disease models are possible, but the number of nonredundant models is in the range of 50 -102 (12). For two bi-allelic genes such as two SNP loci with quantitative measures, epistasis effects are typica...

Cryptosporidium parvum is recognized as an enteropathogen of great worldwide medical and veterinary importance, yet understanding of its pathogenesis has been hampered in part by limited knowledge of the invasion machinery of this parasite. Recently, genes containing thrombospondin type 1 (TSP1) domains have been identified in several genera of apicomplexans, including thrombospondin-related adhesive proteins (TRAPs) that have been implicated as key molecules for parasite motility and adhesion onto host cell surfaces. Previously, a large-scale random survey of the C. parvum genome conducted in our laboratory revealed the presence of multiple genomic DNA sequences with a high degree of similarity to known apicomplexan TRAP genes. In the present study, TBLASTN screening of available C. parvum genomic sequences by using TSP1 domains as queries identified a total of 12 genes possessing TSP1-like domains. All genes have putative signal peptide sequences, one or more TSP1-like domains, plus additional extracellular protein modules such as Kringle, epidermal growth factor, and Apple domains. Two genes, putative paralogs CpTSP8 and CpTSP9, contain predicted introns near their amino termini, which were verified by comparing PCR products from cDNA versus genomic DNA templates. Reverse transcription-PCR analysis of transcript levels reveals that C. parvum TSP genes were developmentally regulated with distinct patterns of expression during in vitro infection. TRAPC1, CpTSP3, and CpTSP11 were expressed at high levels during both early and late stages of infection, whereas CpTSP2, CpTSP5, CpTSP6, CpTSP8, and CpTSP9 were maximally expressed during the late stages of infection. Only CpTSP4 was highly expressed solely at an early stage of infection.Although its association with human diseases was not perceived until 1976 (18, 27, 31), the apicomplexan parasite Cryptosporidium parvum has emerged as a significant pathogen both of humans and animals of veterinary importance. The parasite primarily infects the epithelial cells of the small intestine and causes gastrointestinal diseases in both immunocompetent and immunocompromised humans and animals. Typically self-limiting in immunologically healthy individuals, cryptosporidial infection can be persistent and life-threatening in hosts with impaired immune systems. Prolonged infection is compounded by the fact that there is currently no effective anticryptosporidial drug (19). Efforts to develop novel therapeutic strategies have been hampered by a lack of understanding of C. parvum pathogenesis and a paucity of stage-and organelle-specific markers with which to dissect the parasite's life cycle.Thrombospondin-related adhesive proteins (TRAPs) have been identified in several genera of apicomplexans, including TRAP (38,39,45,53) and CTRP (13, 54, 57, 60, 61) of Plasmodium, Etp100 of Eimeria (10, 32, 56), MIC-2 of Toxoplasma (2, 20), and NcMIC2 of Neospora (24, 46) spp. TRAPs are characterized by the presence of two adhesive modules: one or more von Willebrand factor A (vWA)-like domai...

A method based on direct and indirect counting is developed for rapid and accurate linkage analysis for codominant and dominant loci. Methods for estimating gender-specific recombination frequencies are available for cases where at least one of the two loci is multiallelic and for biallelic loci with mixed parental linkage phases where at least one locus is codominant. Most of the estimates of gender-average and gender-specific recombination frequencies required iterative solutions. The new method makes use of the full data set, yields exact estimates of the recombination frequencies when the observed and expected genotypic frequencies are equal, and are computationally efficient. Relative efficiency of various data types is affected by the inheritance mode and by parental linkage phases of biallelic loci, but unaffected by the locus polymorphism when using the full data set for linkage analysis. The ability to determine parental linkage phases is affected by the locus polymorphism as well as inheritance mode. Intercross (or F-2 design) is more efficient for mapping codominant loci, whereas backcross is more efficient if dominance is involved. Mixed parental linkage phases of biallelic loci are less efficient than coupling or repulsion linkage phases. Ignoring noninformative offspring results in biased estimates of recombination frequency for biallelic loci only and reduced LOD scores for all cases.