| Both Cryptosporidium spp. and Cyclospora spp. are emerging foodborne and waterborne pathogens and they distribute widely in environment. There are multiple species/genotypes within each genus. The majority of species/genotypes have host specificity while a few zoonotic species with broad host range are exceptional. In addition, even with the same species, the virulence is different among different isolates. However, little is known about the molecular basis for the host difference of different species, the driving force for the emergence of high virulent isolates, the zoonotic feature of some emerging species/genotypes infecting humans, and the geographic segregation of some species cuasing massive outbreaks. To address the above issues, we conducted the following studies:1. Since the scarcity of whole genome sequence for Cryptosporidium and lack of sequence for Cyclospora is casued by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens, we firstly developed a strategy for isolation and enrichment of genomic DNA followed by verification of DNA purity for Cryptosporidium and Cyclospora respectively. The strategy combined sucrose and cesium chloride density-gradient separation and immunomagnetic separation (Cryptosporidium) or flow cytometry sorting (Cyclospora) for purification of oocysts from fecal specimens, and then used whole genome amplification (WGA) to increase the quantity of the extracted genomic DNA from purified oocysts. qPCR analysis was initially used to assess the quality of WGA products, and Sanger sequencing of cloned products was subsequently used to assess the purity of WGA products. The use of these strategies has led to the sequencing of near complete genomes of 20 specimens of six Cryptosporidium species/genotype and one Cyclospora cayetanensis specimen, which proved the efficiency of the strategies.2. Using the above strategy, we sequenced the genomes of two field specimens each of the two virulent Cryptosporidium hominis subtypes IbA10G2 and IaA28R4, which caused numerous outbreaks, and conducted a comparative genomic analysis of the obtained sequences with those from the published C. hominis (IaA25R3) genome. The four genomes of virulent subtypes were divergent from the reference C. hominis IaA25R3 genome in the highly polymorphic regions across all eight chromosomes, while the sequence differences among them were mainly in the 5’and 3’ends and gp60 region of chromosome 6. Especially in the 5’and 3’ends of chromosome 6, the genomes of virulent subtypes had the same nucleotide sequence type with the reference C. hominis IaA25R3 genome, which is largely the result of genetic recombination. In contrast, at the gp60 locus, the genomes of virulent subtypes and the reference IaA25R3 had their own unique nucleotide sequence type respectively. Thus, genetic recombination possibly plays a potential role in the emergence of virulent C. hominis subtypes.3. Cryptosporidium parvum and C. hominis are extremely divergent from each other in host specificity. The former is zoonotic and infects both humans and ruminants, while the latter is largely human-specific. To better understand the molecular basis for this difference, we conducted a comparative analysis of the genomes of C. hominis (including the obtained genomes of virulent subtypes in this study and the published C. hominis genome) with the published C. parvum genome. The analysis revealed that the genomes of these two species have 97% similarity in nucleotide sequence, however, they both have species-specific genes. The C. parvum-specific genes are located in three chromosomes (5,6 and 8), and most of them were members of multi-copy gene families, including MEDLE family of secreted proteins and the insulinase-like proteases. In contrast, C. hominis appears to only have one unique gene in chromosome 3. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of multi-copy gene families indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum.4. Cryptosporidium chipmunk genotype I is an emerging zoonotic pathogen in humans. It was initially found in rodents and occasionally in water, but has been subsequently reported in sporadic cases in humans in the United States and Europe. To identify whether human infections with this pathogen are zoonotically transmitted, we conducted whole genome sequencing of one chipmunk genotype I specimen and developed subtyping tools based on the gp60 gene and a mucin protein gene. Using these tools, genetic similarity among chipmunk genotype I specimens from humans, wildlife and water were observed. At the gp60 locus,15 subtypes were identified and they all belonged to one subtype family. At the mucin locus, the two subtypes identified differed from each other only in the number of a 30-bp minisatellite repeat. The genetically similarity of chipmunk genotype I from humans and wildlife indicates it is zoonotic in nature. Therefore, the transmission of chipmunk genotype I to humans was possibly originated from rodents and transmitted, via a waterborne transmission.5. Of the known Cyclospora spp., Cyclospora cayetanensis is the only one infecting humans and has caused plenty of cyclosporiasis outbreaks recently. To develop a molecular tool for tracking teh infection/contamination sources, we conducted whole genome sequencing of a C. cayetanensis specimen from China and developed a multilocus sequence typing (MLST) tool targeting five polymorphic loci. Using this tool,25 MLST types were detected from 64 C. cayetanensis specimens from different countries, and geographical segregation was found in these MLSTs. Thus, the developed MLST tool may be useful in the tracking of infection sources.In conclusion, we developed a strategy to isolate and enrich Cryptosporidium and Cyclospora genomic DNA for whole genome sequencing, the obtained genome sequence data have improved our understanding of the genetic basis for the differences in host specificity and virulence of Cryptosporidium, and facilitated the development of high resolution molecular typing tools for Cryptosporidium and Cyclospora. The findings of these studies will facilitate the understanding of the genetic determinats underlining other phenotypic traits and population genetics structure of Cryptosporidium and Cyclospora, the whole genome sequencing of other pathogens which is difficult to be cultured, and thus provide a basis for making effective strategies of disease control and prevention of cryptosporidiosis and cyclosporiasis. |
PDF Full Text Request