Developing Methodologies for the Investigation of Free-living Amoeba as a Tool for Pathogen Surveillance on Dairy Farms and Aquaculture by John McLean A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Molecular and Cellular Biology Guelph, Ontario, Canada © John McLean, April, 2014 ABSTRACT DEVELOPING METHODOLOGIES FOR THE INVESTIGATION OF FREE-LIVING AMOEBA AS A TOOL FOR PATHOGEN SURVEILLANCE ON DAIRY FARMS AND FISHERIES John M. McLean Advisor: University of Guelph, 2014 Dr. Lucy Mutharia Free-living amoeba are phagocytic protozoans that act as environmental reservoirs, a protective niche, and a vehicle for transmission for amoeba-resistant bacterial pathogens. Many amoeba-resistant bacteria have been identified using only laboratory-adapted Acanthamoeba. We isolated resident amoeba from target environments of dairy farms and aquaculture settings to evaluate their use as a pathogen detection tool. Amoeba were only isolated from 3 of 23 (13%) environmental samples using established methods. A two-step sample decontamination protocol was developed and led to the isolation of 14 additional amoeba. An amoeba co-culture method was developed to assess the survival of 12 mycobacterial species within environmental and laboratory-adapted amoeba. Major strain differences were observed at the amoeba level which had drastic effects on the survival of different bacterial species within individual amoeba. Targeted isolation of resident bacteria from soils and feces using amoebal enrichment protocols were unsuccessful. However, the methodologies developed in this study provide a valid technical starting point for future studies. Acknowledgments First and foremost I would like to thank my advisor, Dr. Lucy Mutharia for her guidance and continued support throughout the course of this work. I am ever grateful for her willingness to provide direction and assistance at both the undergraduate and graduate level. I would also like to thank my committee members, Dr. R.M.W. Stevenson and Dr. Cezar Khursigara for their assistance and expertise during the course of my project, it would not have been possible without you. Many thanks must also be given to the members of the Mutharia and Stevenson labs, especially Tony Facciuolo and Melinda Raymond for being an ever present source of advice and direction. I must also thank Steve Lord and members of the lab of Dr. Dave Kelton for obtaining many of the environmental samples that made this work possible. Recognition must also go to my office mates Daniel Jeffery, Kate Murphy, Alison Berezuk and Michelle Daigneault as a constant source of both entertainment and advice. Finally I'd like to thank my family for their unwavering support throughout this process. iii Table of Contents List of Figures………………………………………………………………………………......vii List of Tables………………………………………………………………………………….....ix Chapter 1.0: Introduction….........……….....……………………………………………….......1 1.1 Free-living amoeba……………………………………………………………………........2 1.2 Cyst Resistance………………………………………………………………………........10 1.3 Amoeba-resistant Microbes………………………………………………………….........11 1.3.1 Pathogen Persistence…………………………………………………………….....17 1.3.2 Amoeba as a Bacterial Training Ground for Intracellular Pathogens.......................19 1.3.3 A Protective Environment.........................................................................................21 1.3.4 Pathogen Transmission.............................................................................................23 1.4 Research Rationale and Objectives......................................................................................25 Chapter 2.0: Materials and Methods.........................................................................................28 2.1. Bacterial Strains and Growth Conditions...........................................................................28 2.1.1 Preparation of NNA-E. coli Media...........................................................................30 2.1.2 Culture of Amoeba....................................................................................................30 2.1.3 Long-term Storage of Amoeba.................................................................................31 2.2 Development of Methodologies for Amoeba Isolation.......................................................32 2.2.1 Determining Cyst Resistance....................................................................................32 2.2.2 Primary Amoeba Isolation from Dairy Farms and Aquaculture...............................33 2.2.3 Methods Developed for Amoeba Isolation from Dairy Farms and Aquaculture.....34 2.2.4 Conversion of Xenically Culturable Amoeba to Axenically Culturable Forms.......36 2.3 Amoebal Enrichment and Co-culture..................................................................................37 iv 2.3.1 Preparation of Amoebal Microplates........................................................................37 2.3.2 Screening of Amoeba-resistant Bacteria by the Co-culture Protocol.......................38 2.3.3 Isolation of Environmental Amoeba-resistant Bacteria............................................39 2.3.4 MAP isolation from Cow Feces................................................................................40 2.3.5 MAP Isolation from Cow Feces by Amoebal Enrichment......................................41 2.4 DNA methods......................................................................................................................42 2.4.1 PCR conditions.........................................................................................................44 2.5 Microscopy..........................................................................................................................45 2.5.1 Staining of Amoeba Trophozoites and Amoeba-resistant Bacteria..........................45 2.5.2 Transmission Electron Microscopy..........................................................................46 Chapter 3.0: Results.....................................................................................................................48 3.1 Developing Methodologies for the Isolation of Free-living Amoeba from Environmental Samples.......................................................................................................48 3.2 Molecular Identification of Free-living Amoeba.................................................................54 3.2.1 Diversity of Free-living Amoeba..............................................................................63 3.3 Axenic Culture of Free-living Amoeba...............................................................................67 3.4 Developing Methodologies for Identification of Amoeba-resistant Bacteria......................70 3.4.1 Identifying Amoeba-resistant Bacteria Resident in Free-living Amoeba.................75 3.4.2 Enriching for MAP from Feces................................................................................78 3.4.3 Persistence of MAP within Amoebal Cysts..............................................................82 Chapter 4.0: Discussion...............................................................................................................86 v 4.1 Conclusions..........................................................................................................................96 4.2 Future Direction....................................................................................................................97 5.0 REFERENCES.......................................................................................................................98 6.0 APPENDICES......................................................................................................................108 6.1 Appendix 1: Media and Buffers..........................................................................................109 6.2 Appendix 2: 18S rRNA Sequences....................................................................................114 vi List of Figures Figure 1.1 Locomotive form of an amoebal trophozoite................................................................3 Figure 1.2 Overview of phagocytosis of bacteria by free-living amoeba.......................................6 Figure 1.3 Cross-section of an Aca. polyphaga cyst showing cyst-wall structure..........................9 Figure 1.4 Mechanisms used by amoeba-resistant bacteria to resist phagocytic killing..............14 Figure 3.1 NNA-E. coli plate during primary amoeba isolation showing fungal overgrowth..........................................................................................................................50 Figure 3.2 Amoebal walk-out.......................................................................................................51 Figure 3.3 Schematic overview of the developed methods for primary amoeba isolation..........................................................................................................................................56 Figure 3.4 Morphology of different environmental amoeba isolated...........................................58 Figure 3.5 Representation of the amoebal 18S rRNA gene showing variable regions.................59 Figure 3.6 Agarose gel of amplified amoebal DNA using the JDP1/JDP2 primer set.................60 Figure 3.7 Agarose gel of amplified amoebal DNA using the P-FLA primer set........................61 Figure 3.8 Agarose gel of products