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Studies in Cryptosporidium STUDIES IN CRYPTOSPORIDIUM: MAINTENANCE OF STABLE POPULATIONS THROUGH IN VIVO PROPOGATION AND MOLECULAR DETECTION STRATEGIES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Norma E. Ramirez, M.P.H. ! ! ! ! The Ohio State University 2005 Dissertation Committee: Approved by Dr. Srinand Sreevatsan, Adviser Dr. Y.M. Saif ______________________________ Dr. Roger W. Stich Adviser Dr. Lucy A. Ward Graduate Program in Veterinary Preventive Medicine ABSTRACT 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 ii 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 non- disturbed (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. iii Dedicated to My parents, Danilo and Norma and My husband, Juan iv ACKNOWLEDGMENTS I would like to express my appreciation and gratitude to everyone who collaborated in my doctoral training and research providing guidance, support and friendship: My advisor, Dr. Srinand Sreevatsan The members of my committee, Dr. Y. M. Saif, Dr. Roger W. Stich and Dr. Lucy A. Ward for her role as my advisor during the first part of my training. My friends in the laboratory, Chris, Sonia, Matt, Mohamed, Ellen, Megan, Mike, Alifiya, Harish, David, Kaori, Sukhbir, and Zhu. The members of the Food Animal Health Research Program Dr. Raymond Clarke and Mrs. Gloria Clarke My family and friends My husband ¡Muchas Gracias! v VITA August 3, 1973 …… Born - San Juan, Puerto Rico 1995 ………………. Bachelor of Sciences, Biology. Department of Biology, School of Arts and Sciences, University of Puerto Rico, Mayaguez Campus, Mayaguez, Puerto Rico. Magna Cum Laude. 1995 – 1996 ………. Biologist I, Puerto Rico Department of Agriculture, Agrological Laboratory, Dorado, Puerto Rico. 1997 ………………. Master in Public Health, Epidemiology. Department of Biostatistics and Epidemiology, School of Public Health, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico. 1997 – 1999 ………. Research Assistant, Department of Biostatistics and Epidemiology, School of Public Health, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico. 2000 – 2001 ………. Research Assistant, Department of Veterinary Preventive Medicine and Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio. 2001-present ……… PhD Candidate. Department of Veterinary Preventive Medicine and Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio. vi PUBLICATIONS Peer Review Journals 1. Pereira SJ, Ramirez NE, Xiao L, Ward LA. Pathogenesis of human and bovine Cryptosporidium parvum in gnotobiotic pigs. J. Infect. Dis. 2002 Sep 1;186(5):715-8. 2. Ramirez NE, Ward LA, Sreevatsan S. A review of the biology and epidemiology of cryptosporidiosis in humans and animals. Microbes Infect. 2004 Jul;6(8):773-85. FIELD OF STUDY Major Field: Veterinary Preventive Medicine Studies in Parasitology and Molecular Epidemiology vii TABLE OF CONTENTS Page Abstract …………………………………………………………………………... ii Dedication ………………………………………………………………………... iv Acknowledgments ……………………………………………………………….. v Vita ………………………………………………………………………………. vi List of Tables …………………………………………………………………….. x List of Figures ………………………………………………………………........ xii Chapters: 1. Introduction ………………………………………………………………....... 1 Cryptosporidium biology …………………………………………………….. 4 Cryptosporidium taxonomy and strain variation …………………………….. 5 Cryptosporidium infection and cryptosporidiosis…………………………….. 8 Cryptosporidium diagnosis and molecular detection methods ………………. 19 Treatment …………………………………………………………………….. 22 Prevention and control ……………………………………………………….. 24 Conclusion …………………………………………………………………… 29 References ……………………………………………………………………. 31 2. Development of a sensitive detection system for Cryptosporidium in environmental samples ………………………………………………………... 51 Introduction …………………………………………………………………… 51 Materials and methods………………………………………………………… 53 Results…………………………………………………………………………. 61 Discussion……………………………………………………………………... 63 References …………………………………………………………………...... 68 viii 3. Cryptosporidium detection by oligonucleotide microarray …………………... 83 Introduction …………………………………………………………………… 83 Materials and methods………………………………………………………… 85 Results…………………………………………………………………………. 89 Discussion……………………………………………………………………... 90 References …………………………………………………………………...... 94 4. Effect of soil tillage and rainfall on the transport of Cryptosporidium through soil …………………………………………………………………... 104 Introduction …………………………………………………………………… 104 Materials and methods………………………………………………………… 107 Results…………………………………………………………………………. 111 Discussion……………………………………………………………………... 113 References ……………………………………………………………………. 117 5. Derivation of Cryptosporidium hominis progeny in the gnotobiotic pig model …………………………………………………………………………. 127 Introduction …………………………………………………………………… 127 Materials and methods………………………………………………………… 129 Results…………………………………………………………………………. 133 Discussion…………………………………………………………………...… 137 References …………………………………………………………………..… 144 Appdendix A: Swine and poultry operations sample description …………….….. 153 Bibliography…………………………………………………………………….... 163 ix LIST OF TABLES Page Table 1.1 Valid taxonomic nomenclature of Cryptosporidium species and their 49 host range. Table 1.2 Cryptosporidium species and genotypes reported in human 50 infections. Table 2.1 Primers used for the construction of the Internal Positive Control. 71 Table 2.2 Mean absorbance at 450nm of PCR-Hybridization products from 72 Cryptosporidium spiked soil samples. Table 2.3 Mean absorbance at 450nm of PCR-Hybridization products from 72 Cryptosporidium spiked water samples. Table 2.4 Melting temperature of the hybridization probes for different 73 Cryptosporidium species in five independent assays. Table 2.5 Procedures for Cryptosporidium oocysts purification and detection 74 for different types of environmental samples. Table 2.6 Number of Cryptosporidium-positive samples from swine 74 operations under different waste management technologies. Table 2.7 Number of Cryptosporidium-positive samples from poultry 74 operations under different waste management technologies. Table 2.8 Melting temperatures (Tm) of hybridization probes for swine and 75 poultry positive samples. Table 3.1 Primer sequences for PCR amplification of Cryptosporidium 18s 96 rDNA, hsp70, and β-tubulin genes and universal 16s rRNA primers. Table 3.2 Sequences of microarray capture probes. 97 x Table 3.3 18s rDNA nucleotide sequences used for the design of the five 98 Cryptosporidium species specific capture probes. Table 4.1 Presence of Crytposporidium parvum oocysts in the block sections of tilled (T) and no-tilled (NT) soil after rainfall treatments (T2, T3, 121 T5, T6). Table 5.1 Summary of Cryptosporidium hominis oocyst passages performed in 148 gnotobiotic pigs. Table 5.2 Frequency of nucleotide base substitutions among cloned C. hominis 149 β-tubulin and GP60 genes sequences. Table 5.3 Summary of microsatellite polymorphisms by Cryptosporidium 150 hominis oocyst passages. xi LIST OF
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