Studies in Cryptosporidium: Maintenance of stable populations through in vivo propogation and molecular detection strategies.

摘要

Cryptosporidiosis, an infection caused by several genotypically and phenotypically diverse Cryptosporidium species, is a serious enteric disease of animals and humans worldwide. The current understanding of cryptosporidiosis, transmission, diagnosis, treatment and prevention measures for this disease is discussed. Contaminated water represents the major source of Cryptosporidium infections for humans. Manure from cattle can be a major source of Cryptosporidium oocysts. Oocysts transport to surface water can occur through direct fecal contamination, surface transport from land-applied manure or leaching through the soil to groundwater. Identification of Cryptosporidium species and genotypes facilitates determining the origin of the oocysts and to recognize sources of infection in outbreak situations and the risk factors associated with transmission. Very few studies have applied isolation methods to field samples because of difficulties with detection of oocysts in environmental samples. The objective of this study was to develop an easy method that can be applied to field samples to rapidly detect the presence of Cryptosporidium oocysts and identify their species. A molecular detection system that included an oocyst recovery method combined with spin column DNA extraction, followed by PCR-hybridization for detection and a Real-Time PCR-melting curve analysis for species assignment. Due to its versatility and capability of rapid high-throughput analysis of multiple targets, an oligonucleotide microarray was also designed to identify Cryptosporidium parasites and discriminate between species. The detection assay was then used to assess Cryptosporidium contamination in swine and poultry samples and to study the transport of Cryptosporidium oocysts through disturbed (tilled) and nondisturbed (no-till) soil during simulated rainfall. The results of the study demonstrated the potential of the assay for the detection of the parasite in environmental samples.; In vitro cultivation systems that permit Cryptosporidium development and propagation are still under development; therefore clonal reference strains are not available. Using micromanipulation, single-cell clones of C. hominis were derived and maintained in a gnotobiotic pig model. Genetic stability of each subsequent generation was monitored through microsatellite fingerprinting and sequence analysis. The results indicated that these single oocyst derivatives led to the expansion and maintenance of genetically and phenotypically homogeneous populations.