Intestinal protozoan parasites in Northern India – investigations on transmission routes Philosophiae Doctor (PhD) Thesis Kjersti Selstad Utaaker Department of Food Safety and Infection Biology Faculty of Veterinary Medicine Norwegian University of Life Sciences Adamstuen (2017) Thesis number 2018:10 ISSN 1894-6402 ISBN 978-82-575-1750-2 1 2 To Jenny, Vilmer, Viljar and Ivo. “India can do it. People of India can do it.” – PM Modi on Swachh Bharat Abhiyan 3 4 Contents Acknowledgements ................................................................................................................................. 7 Abbreviations ........................................................................................................................................ 10 List of research papers .......................................................................................................................... 12 List of additional papers ........................................................................................................................ 14 Summary ............................................................................................................................................... 15 Sammendrag (Norwegian summary) .................................................................................................... 18 सारा車श (Hindi summary) ......................................................................................................................... 21 1. Introduction ................................................................................................................................... 24 1.1 Background .............................................................................................................................. 24 1.2 Giardia and Cryptosporidium in developing countries ............................................................ 25 1.3 General presentation of Cryptosporidium and Giardia .......................................................... 30 1.4 Giardia and Cryptosporidium as waterborne pathogens ........................................................ 46 1.5 Fresh produce as vehicles of infection for Giardia and Cryptosporidium ............................... 51 1.6 Domestic animals as potential sources of environmental contamination with Giardia and Cryptosporidium in the Indian context .......................................................................................... 58 1.7 Knowledge gaps ....................................................................................................................... 70 2. Aims of study ................................................................................................................................. 72 2.1 Main objectives ....................................................................................................................... 75 3. Summary of papers ....................................................................................................................... 77 4. Materials and Methods ................................................................................................................. 83 4.1 Diagnostic tools for environmental samples ........................................................................... 83 4.2 Diagnostic tools for detection of Cryptosporidium oocysts and Giardia cysts in faeces......... 88 4.3 Viability assessment methods ................................................................................................. 89 4.4 Material and methods used in this study ................................................................................ 93 4.5 Completion of sample processing at the Parasitology Lab at NMBU .................................... 103 4.6. Molecular methods .............................................................................................................. 108 5 4.7 Statistics ................................................................................................................................. 113 5. Results and general discussion .................................................................................................... 115 5.1 Experimental studies ............................................................................................................. 115 5.2 Survey studies ........................................................................................................................ 117 5.3 Limitations and challenges experienced in the study ........................................................... 126 6. Concluding remarks and future perspectives.............................................................................. 129 7. References ................................................................................................................................... 134 8. Compilation of papers…………………………………………………………………………………………..…………………161 6 Acknowledgements This present doctorate was carried out at the Department of Food Safety and Infection Biology at the Norwegian University of Life Sciences, Campus Adamstuen, and the Department of Medical Parasitology, at the Postgraduate Institute of Medical Education and Research, Chandigarh, India during the period of 2013 – 2017. It was financed by the Norwegian Research Council, project number 227965. Lucy J. Robertson, There sure were some marvellous moments. Thank you for all the support, from nicking blankets on the plane to India to proof-reading and making these studies come through. You have been a fantastic supervisor, role model, and mentor through the years of this thesis, and this work would never have seen the light of day without your enthusiasm and guidance. You are a person and professional I strive to be. Eystein Skjerve, for always being ready to help me with the statistics, and for your good advice and encouragement to turn every problem on the way into a challenge. Valeria Letkova, who supported me all the way through the writing of my in-depth study in parasitology during my Veterinary degree, and for her enthusiasm and encouragement which made me follow my dream to go into research. Ingrid and Arne Utaaker, my dear parents to whom I owe all my achievements. This thesis would never see light of day if it wasn`t for your continuous support. I deeply love and appreciate you. Camilla, Hedvig, Sveinung and Oscar-Torjus, my beloved siblings who I was lucky to grow up in the middle of. I would never be able to do my PhD without your continuous support, love and the friendship we have. Special thanks to Sveinung, who was always ready to help me with tables, statistics and outlay in some of the articles. Per Gunnar Karlsen, for being my mentor, colleague and support during my veterinary practice days both before and during my PhD, for showing a great interest in my research work and always inspiring me. 7 Bror Jonathan Myhren and Nina Myhr, for cheering me on from beginning till end of this work, sharing my concerns when results were absent, as well as celebrated the small victories along the way. You really have showed what true friendship is. Kristoffer Relling Tysnes, The support you have given me through my PhD years have been more than I can ever begin to elaborate. you are a great friend, researcher, mentor and person. Thanks for all the talks (and runs!) we`ve had over the years, and I look forward to our continuing scientific journeys ahead! NMBU labmates; Jemere Bekele Harito, Birgitte Kasin Hønsvall, John James Debenham, Teresa Hagen, Anna Barzcak, Hanne Landuyt, Sophie Kosters, Ane Holbø and Gabriel Gati, your enthusiasm, drive and skills have been a great inspiration to me. Thanks for always being ready to help and for the good advice, laughs and inspirations along the way. Dr. Rajinder Singh Bajwa, in my opinion, Indias finest Veterinarian. thanks for helping with the collection of samples, your good advice and willingness to cooperate. Veterinarians rock! Sandhya Khunger, Kirti Megha, Suman Chaudhary, and Harpreet Singh. We have danced, we have worked and we have laughed and cried through some amazing times together in India, and there is yet more to come. Paramvir Singh and Kavita, Thanks for the whisky, flying foxes and some crazy scooter-rides! Himanshu Joshi and Anil Kumar. We had some good times collecting the samples, thanks for all the help in both collection and logistics. PGIMER staff and labmates, you welcomed me as one of your own. Thanks for all the good times we have shared together! My hostel mates in Sector 11, for making the hostel stays so much more enjoyable. Thanks for invading my room when I thought I wanted to be alone. I believe and hope all your plans and dreams for this life comes true. Aman Khurana, your gym was my safe haven during my stays in Chandigarh! You are a great trainer and an even better person. 8 Mera bhedh, I`ll always hate you the least. 9 Abbreviations 18S rDNA: 18S ribosomal DNA AR: Attributable Risk. the portion of disease incidence in the exposed that is due to the exposure. cowp: Cryptosporidium oocyst wall protein. CPG: Number of Giardia cysts per gramme feces. DAPI: 4´, 6-diamino-2-phenylindole. DNA: Deoxyribonucleic acid. DAPI: 4′6 diamidino-2-phenylindole, a non-specific fluorescent stain that binds to double- stranded DNA. ELISA: Enzyme-linked immunosorbent assay. gdh: Glutamate dehydrogenase. GP60: 60kDa glycoprotein. IFAT: Indirect fluorescent antibody test. Immuno-magnetic separation (IMS): the separation of oocysts and cyst by para-magnetic