AN EVALUATION OF THE ORAL MICROBIOME AND POTENTIAL ZOONOSES OF TWO UBIQUITOUS SOUTH AFRICAN STREPSIRRHINES, OTOLEMUR CRASSICAUDATUS AND GALAGO MOHOLI. BY MICHAEL JOHN STRINDEN B.A. University of North Dakota, 2011 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirement for the degree of Master of Arts. Department of Anthropology 2014 This thesis entitled: An Evaluation of the Oral Microbiome and Potential Zoonoses of Two Ubiquitous South African Strepsirrhines, Otolemur crassicaudatus and Galago moholi. written by Michael John Strinden has been approved for the Department of Anthropology Dr. Michelle L. Sauther (Committee Chair) Dr. Herbert H. Covert Dr. Frank P. Cuozzo Date ______________ The final copy of this thesis has been examined by the signatories, and we Find that both the content and the form meet acceptable presentation standards Of scholarly work in the above mentioned discipline. iii Strinden, Michael John (M.A. Anthropology) An Evaluation of the Oral Microbiome and Potential Zoonoses of Two Ubiquitous South African Strepsirrhines, Otolemur crassicaudatus and Galago moholi. Thesis directed by Professor Michelle L. Sauther As Otolemur crassicaudatus and Galago moholi are ubiquitous throughout part of South Africa they are thought to be at little conservation risk. However, this has resulted in a situation where these species are seldom studied. This thesis addresses this lack of knowledge about these two species by investigating the oral microbiome of each for potential zoonoses. This is an important undertaking due to the widespread nature of these species which could provide an avenue for potential zoonoses to spread. In order to assess the oral microbiome, two genetic sequencing methods were utilized. Real- time PCR was utilized to search for mycobacterial contributors to the oral microbiome such as Mycobacterium tuberculosis and Mycobacterium leprae. Next-gen Illumina sequencing of the 16s region was chosen as an effective method to get an overall quantification of the amount of different bacterial contributors. Through these two methods major bacterial contributors of each tested oral microbiome were isolated and those with potential for causing illness in humans or domestic animals were identified. The results illustrated a potential core microbiome for all Otolemur crassicaudatus consisting of three separate bacterial contributors; Mannheimia caviae, Porphyromonas catonia, and Gemella cunicula. While all tested O. crassicaudatus exhibited these three contributors the rest of the oral microbiome was made of different contributors in varying amounts. The qPCR (real-time PCR) for mycobacteria (tuberculosis and leprosy) yielded negative results despite several potential initial amplifications. Despite qPCR findings several potentially pathogenic bacterial strains were discovered through next-gen 16s sequencing. These included Fusobacterium naviforme, Streptococcus bovis, and Gemella cunicula; all of which are known to cause disease in human hosts. A number of other bacterial strains with both human and non-human health risks were discovered in wild O. crassicaudatus as well as in captive sampled Galago moholi. These findings thus form the basis for several future avenues of potential research to assess any real zoonotic risks from these galagos to the broader human and animal populations of southern Africa. Dedication This thesis is dedicated to my family without whose encouragement, inspiration, and support this work would not have been possible. v Acknowledgements Growing up in a house with a medical professional and a former serology technician as parents instilled in me a curiosity for health and how the body reacts to diseases. Through continued study into epidemiology and immunology I have furthered my understanding of how populations as well as the human body react in the presence of both known and novel pathogens. What I could not understand however is how new diseases emerge and how we can seemingly do so little to prevent the spread in the early stages of a truly novel pathogen. In order to better fill in the gaps in my knowledge, as well as some of those of the medical field, I have decided to focus my studies on the emergence of diseases from non-human primate sources and how these can contribute to epidemics of known and novel pathogens. Whether the great epidemics from history or the recurring pandemic scares which happen every few years I have become fascinated by how some diseases manage to appear from nowhere and can ravage an entire population while others stay endemic to a group without causing any significant long lasting damage to the population as a whole. Diseases which we are not accustomed to can have devastating effects as we have little to no inherent resistance to them, an example being the introduction of smallpox to the New World and the massive death toll this caused in the native populations. To this end I have decided to study how new diseases emerge, where they come from, and how to alleviate and prevent new pandemics from occurring in the modern era. For their theoretical, instructional, and material contributions to the research behind this thesis I would like to thank Drs. Michelle Sauther, Frank Cuozzo, Krista Fish, Adrian Tordiffe, Anne Stone, Helene Brettschneider, Steven Strinden, and Ms. Genevieve Hausman. Without their expertise and guidance this work would not have been possible. Dr. Ian Gaigher, director of Lajuma Research Centre deserves special acknowledgement for his creation of the research area and his support of our project within it. I would also like to thank the staff of the National Zoological Gardens of South Africa, especially Dr. Antoinette Kotze, Mark Howitt and Lee Smith for their assistance in locating, sampling, and sequencing samples from Galago moholi. Dr. Adrian Tordiffe also deserves another acknowledgement for his help in the field as well as his assistance in procuring laboratory contacts and lab assistance and instruction. In recognition of their financial support allowing for the acquisition of supplies, materials, transport, and other ancillary needs I also thank the CU Museum, the University of North Dakota, Colorado College, and the Beverly Sears grant committee. Finally, for their support and encouragement I would finally like to thank the employees of Lowe’s store 0220, Derek Mihm, and James Millette who helped me through the frustrations of delving into personally unknown territories in order to author this work. vi CONTENTS CHAPTER 1. INTRODUCTION…………………………………………………………………………………………………… 1 Zoonoses and Emerging Pathogens……………………………………………………………….. 1 Non-Human Primates as Important Disease Reservoirs for Zoonotic Diseases………………….......................................................................................... 4 Microbiomes...................................................................................................... 5 The Importance of Africa for Understanding Zoonotic Diseases Linked to Primates…………………………………………………………………………………………………….. 5 Study Species…………………………………………………………………………………………………. 6 Rationale for this Study………………………………………………………………………………….. 8 2. METHODS……………………………………………………………………………………………………………… 10 Project Framework………………………………………………………………………………………… 10 Environmental and Ecological Assay………………………………………………………………. 11 Specimen Capture and Sample Collection for Wild Samples……………………….... 12 Humoral Analysis………………………………………………………………………………………...... 14 Sampling of Captive Galago moholi……………………………………………………………….. 14 Salivary DNA Storage……………………………………………………………………………………... 15 16s Illumina sequencing.……………………………………………………………………………….. 15 Tuberculosis and Leprosy…………………………………………………………………...…………. 17 Leprosy and Tuberculosis DNA Extraction…………………………………………. 17 qPCR Testing......................................................................................... 19 3. RESULTS………………………………………………………………………………………………………………… 22 vii 16s……………………………………………………………………………………………………………....... 22 qPCR…………………………………………………………………………………………………………….... 22 Humoral and Fecal Analysis……………………………………………………………..………….... 23 Next-Gen Illumina sequencing…………………………………………………………………...….. 23 4. DISCUSSION…………………………………………………………………………………………………...……. 32 5. ONGOING RESEARCH AND CONCLUSION…………………………………………………………….. 38 6. BIBLIOGRAPHY………………………………………………………………………………………………....... 40 APPENDIX 1. CUSTOM CELL LYSIS BUFFER INGREDIENTS……………………………………………………. 45 2. NANODROP CONCENTRATIONS AND WORKING DILUTION VOLUMES………….. 46 3. FORWARD PRIMERS, REVERSE PRIMERS, AND FLUORESCENT PROBES USED FOR MYCOBACTERIAL qPCR…………………………………………………………………………… 47 4. OPTIMIZED qPCR MASTER MIX RATIOS PER REACTION (uL)…………………………… 48 5. qPCR AMPLIFICATION SDS STANDARDS AND RESULTS FOR MYCOBACTERIA…...................................................................................... 49 6. 16s BACTERIAL CONTRIBUTOR RATIOS BY VOLUME (# of bacterial reads per sample/total microbial reads per sample).……………… 59 viii TABLES 1. Number of distinct bacterial contributors to each specimen’s oral microbiome as well as top 8 contributors by volume (Number of reads identified/total number of reads)… ............................. 29 2. Values and results of Mann-Whitney U-tests run to investigate potential differences in bacterial load between sex classifications of specimens tested……………………………………………………………………………………………………... 30 3. Identified bacterial pathogens and their known effects............................. 31 ix FIGURES 1. Two Galago moholi kept as