Inter-Species Microbial Sharing in Rural Madagascar A Study of Environmental Influences on the Skin Microbiome by Melissa B Manus Duke Global Health Institute Duke University Date:_______________________ Approved: ___________________________ Charles Nunn, Supervisor ___________________________ Lawrence Park ___________________________ Julie Horvath Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the Duke Global Health Institute in the Graduate School of Duke University 2016 i v ABSTRACT Inter-Species Microbial Sharing in Rural Madagascar A Study of Environmental Influences on the Skin Microbiome by Melissa B Manus Duke Global Health Institute Duke University Date:_______________________ Approved: ___________________________ Charles Nunn, Supervisor ___________________________ Lawrence Park ___________________________ Julie Horvath An abstract of a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the Duke Global Health Institute in the Graduate School of Duke University 2016 Copyright by Melissa B Manus 2016 Abstract The skin is home to trillions of microbes, many of which are recently implicated in immune system regulation and various health conditions (33). The skin is continuously exposed to the outside environment, inviting microbial transfer between human skin and the people, animals, and surfaces with which an individual comes into contact. Thus, the aim of this study is to assess how different environmental exposures influence skin microbe communities, as this can strengthen our understanding of how microbial variation relates to health outcomes. This study investigated the skin microbial communities of humans and domesticated cattle living in rural Madagascar. The V3 region of the 16S rRNA gene was sequenced from samples of zebu (the domesticated cattle of Madagascar), zebu owners, and non-zebu owners. Overall, human armpits were the least diverse sample site, while ankles were the most diverse. The diversity of zebu samples was significantly different from armpits, irrespective of zebu ownership (one-way ANOVA and Tukey’s HSD, p<0.05). However, zebu owner samples (from the armpit, ankle forearm, and hand) were more similar to other zebu owner samples than they were to zebu, yet no more similar to other zebu owner samples than they were to non-zebu owner samples (unweighted UniFrac distances, p<0.05). These data suggest a lack of a microbial signature shared by zebu owners and zebu, though further taxonomic analysis is required to explain the role of additional environmental variables iv in dictating the microbial communities of various samples sites. Understanding the magnitude and directionality of microbial sharing has implications for a breadth of microbe-related health outcomes, with the potential to explain mosquito host preference and mitigate the threats of vector-borne diseases. v Dedication For my parents: Thank you for always supporting my adventures, no matter where they take me. vi Contents Abstract ......................................................................................................................................... iv List of Tables .................................................................................................................................. x List of Figures ............................................................................................................................... xi Acknowledgements .................................................................................................................. xiii 1. Introduction ............................................................................................................................... 1 1.1 Background .................................................................................................................. 1 1.2 Mosquito host preference ........................................................................................... 4 1.3 Ecological and evolutionary applications ................................................................ 8 1.4 Applications to Madagascar ...................................................................................... 9 2. Methods .................................................................................................................................... 13 2.1 Setting ............................................................................................................................... 13 2.2 Participants ...................................................................................................................... 13 2.3 Field procedures ............................................................................................................. 15 2.3.1 Participant recruitment ............................................................................................. 15 2.3.2 Sample collection ....................................................................................................... 16 2.3.3 Sample storage ........................................................................................................... 19 2.3.4 Importation and other permits ................................................................................ 19 2.4 Laboratory procedures .................................................................................................. 20 2.5 Measures .......................................................................................................................... 20 2.5.1 Measure 1 – Alpha diversity .................................................................................... 20 vii 2.5.2 Measure 2 – Beta diversity ....................................................................................... 21 2.6 Analysis ............................................................................................................................ 22 3. Results ....................................................................................................................................... 24 3.1 General results ................................................................................................................ 24 3.1.1 Alpha diversity of zebu owners and non-zebu owners compared to zebu ...... 24 3.2.1 Beta diversity of zebu owners and non-zebu owners .......................................... 33 3.2 Results specific to predictions ....................................................................................... 34 3.2.1 Prediction 1: Similarity of zebu owners to zebu ................................................... 34 3.2.2 Prediction 2: Similarity of non-zebu owners to zebu ........................................... 36 3.2.3 Prediction 3: Similarity of a zebu to his owner ..................................................... 37 3.3 Taxonomic results ........................................................................................................... 40 4. Discussion ................................................................................................................................ 43 4.1 Alpha diversity ............................................................................................................... 43 4.2 Beta diversity ................................................................................................................... 45 4.3 Taxonomic trends ........................................................................................................... 49 4.4 Implications for policy and practice ............................................................................ 51 4.5 Implications for further research .................................................................................. 53 4.6. Study strengths and limitations .................................................................................. 54 5. Conclusion ............................................................................................................................... 58 Appendices .................................................................................................................................. 59 Appendix A – General Health Survey ............................................................................... 59 Appendix B – General Health Consent ............................................................................. 61 viii Appendix C – Skin Microbiome Survey ............................................................................ 61 Appendix D – Consent for Skin Swab ............................................................................... 62 Appendix E – Consent for Zebu Owners .......................................................................... 63 Appendix F – DNA Extraction Protocol ............................................................................ 65 Appendix G – DNA Sequencing Protocol......................................................................... 66 Appendix H – QIIME Pipeline ........................................................................................... 76 Appendix I – Unweighted UniFrac Distance Calculations for Zebu Owners and Zebu ................................................................................................................................................. 77 I.i Group ............................................................................................................................... 77 I.ii Body Site