Isolation, in Vitro Excystation, and in Vitro Development of Cryptosporidium Sp from Calves Douglas Byron Woodmansee Iowa State University
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Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1986 Isolation, in vitro excystation, and in vitro development of Cryptosporidium sp from calves Douglas Byron Woodmansee Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Zoology Commons Recommended Citation Woodmansee, Douglas Byron, "Isolation, in vitro excystation, and in vitro development of Cryptosporidium sp from calves " (1986). Retrospective Theses and Dissertations. 8128. https://lib.dr.iastate.edu/rtd/8128 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 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Other University Microfilms International Isolation, in vitro excystation, and in vitro development of Cryptosporidium sp. from calves by Douglas Byron Woodmansee A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Zoology Major; Zoology (Parasitology) Approved: Signature was redacted for privacy. In Charge of Maj^r Work Signature was redacted for privacy. For the Major Department Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1986 ii TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 Explanation of Dissertation Format 3 LITERATURE REVIEW 4 Historical Aspects 4 Taxonomy 10 Life Cycle and Morphology 15 Diseases Associated with Cryptosporidium 29 SECTION I. DEVELOPMENT OF CRYPTOSPORIDIUM SP. IN CULTURED CELLS 46 Abstract 46 Introduction 46 Materials and Methods 47 Results 49 Discussion 51 Literature Cited 52 iii SECTION II. AN IN VITRO STUDY OF SPORULATION IN CRYPTOSPORIDIUM SP. 71 Abstract 71 Introduction 71 Materials and Methods 73 Results 74 Discussion 75 Literature Cited 75 SECTION III. TAUROCHOLIC ACID AFFECTS MOTILITY AND MORPHOLOGY OF CRYPTOSPORIDIUM SPOROZOITES IN VITRO 84 Abstract 84 Introduction 84 Materials and Methods 85 Results 88 Discussion 89 Literature Cited 91 iv SECTION IV. STUDIES OF IN VITRO EXCYSTATION OF CRYPTOSPORIDlUM SP. FROM CALVES 106 Abstract 106 Introduction 106 Materials and Methods 107 Results 112 Discussion 115 Literature Cited 118 SECTION V. TECHNIQUES FOR THE RECOVERY OF CRYPTOSPORIDIUM OOCYSTS AND SPOROZOITES FROM CALF FECES 142 Abstract 142 Introduction 142 Materials and Methods 143 Results and Discussion 146 Literature Cited 149 GENERAL SUMMARY 151 LITERATURE CITED 154 ACKNOWLEDGMENTS 184 1 GENERAL INTRODUCTION The genus Cryptosporidium consists of intracellular parasitic protozoa which infect the apical portions of epi thelial cells of several organs. The implication of infec tion by Cryptosporidium as a cause of disease (particularly diarrhea) has attracted substantial interest from research ers in human and veterinary medicine. In cattle and sheep, the infection appears to be a cause of the neonatal diarrhea syndrome known as scours. In humans, infections are associ ated with severe, protracted diarrhea in immunosuppressed patients and with less severe, self resolving diarrhea in immunologically normal people (43a, 199). Most of the recent research on Cryptosporidium has been done by pathologists "and clinicians rather than parasitolo gists. Therefore, the research emphasis has been heavily weighted toward studies of the pathological, clinical and epidemiological features of the disease rather than toward the taxonomy, physiology and comparative biology of the parasite. As a result of this emphasis, there is strong evidence that Cryptosporidium is a primary pathogen and the clinical aspects of the disease are well documented. We have a poorer understanding of the taxonomy, life history and basic biology of the parasite. 2 Studies of the biology of Cryptosporidium in comparison to other coccidian parasites are few, despite a number of unusual features of Cryptosporidium which are quite inter esting. The most common reason given for this neglect is that Cryptosporidium is difficult to work with. It is the smallest coccidian, with oocysts only about half the size of most other species. Furthermore, growth of the parasite in cultured cells (difficult for any coccidian) has not yet been achieved in a manner conducive to physiological studies. Finally, isolation of Cryptosporidium life cycle stages from in vivo sources has been far less efficient than the isolation of similar stages of other species. This dissertation reports efforts to address some of these problems. Specifically, efforts to grow Cryp tosporidium in cell culture, an examination of the sporula tion phase of the life cycle, a study of coccidian excysta- tion using Cryptosporidium as a model organism, and new techniques for the isolation of oocysts and sporozoites are reported. The variety of topics approached in this disser tation is a reflection of the breadth of the work that still needs to be done with this sometimes fascinating, and some times frustrating organism. 3 Explanation of Dissertation Format This dissertation is written in the alternate format. Five papers are presented in addition to a general introduc tion, a general literature review, and a general summary. References for the general introduction and literature re view follow the general summary. Portions of the paper en titled "Development of Cryptosporidium sp. in cultured cells" were published in the Proceedings of the Fourth International Symposium on Neonatal Diarrhea, 1983, Veteri nary Infectious Disease Organization, Saskatoon, Sask., Canada, pp. 306-319. The paper entitled "An in vitro study of sporulation in Cryptosporidium sp." has been accepted for publication in the Journal of Parasitology. All parts of this dissertation have been written to conform to the style required by the Journal of Protozoology. 4 LITERATURE REVIEW Historical Aspects The genus Cryptosporidium was first erected by Tyzzer in 1907 in a preliminary description of Cryptosporidium muris (172); a complete report followed in 1910 (173). Tyz zer found large numbers of protozoans infecting the gastric glands of laboratory mice- He thoroughly described these parasites with light microscopy using both stained and un stained preparations, and experimentally infected weanling mice with mucosal scrapings from naturally infected mice. His work demonstrated that these parasites were not develop mental stages of other murine protozoan parasites and that reports by other investigators (34, 194) describing