Master Melly's Masters Teeeesis

105 Pages·2016·2.22 MB·English

by  Melly Manus

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

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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

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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.

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Dedication
For my parents: Thank you for always supporting my adventures, no matter where they
take me.

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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

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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

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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 ........................................................................................................................ 78
Appendix J – Unweighted UniFrac Distance Calculations for Non-Zebu Owners and
Zebu ........................................................................................................................................ 81
J.i Group ............................................................................................................................... 81
J.ii Body Site ........................................................................................................................ 82
Appendix K – Unweighted UniFrac Distance Calculations for Zebu Owners and
Non-Zebu Owners ................................................................................................................ 85
K.i Group ............................................................................................................................. 85
K.ii Body Site ....................................................................................................................... 85
References .................................................................................................................................... 87

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List of Tables
Table 1. Diversity of sample sites from zebu- owners and zebu. ......................................... 24
Table 2. Comparison of Faith's PD across zebu owners and zebu (one-way ANOVA &
Tukey's HSD). AN=ankle, AP=armpit, FA=forearm, HA=hand, Z=zebu. .......................... 25
Table 3. Comparison of number of OTUs across zebu owners and zebu (one-way
ANOVA & Tukey's HSD). AN=ankle, AP=armpit, FA=forearm, HA=hand, Z=zebu. ...... 25
Table 4. Diversity of sample sites from non-zebu owners and zebu. .................................. 28
Table 5. Comparison of Faith's PD across non-zebu owners and zebu (one-way ANOVA
&Tukey's HSD). AN=ankle, AP=armpit, FA=forearm, HA=hand, Z=zebu. ....................... 28
Table 6. Comparison of number of OTUs across non-zebu owners and zebu (one-way
ANOVA & Tukey's HSD). AN=ankle, AP=armpit, FA=forearm, HA=hand, Z=zebu. ...... 29
Table 7. Alpha diversity of zebu owners and non-zebu owners. ......................................... 32
Table 8. Comparison of Faith's PD and number of OTUs across zebu owners and non-
zebu owners (Kruskal-Wallis). AN=ankle, AP=armpit, FA=forearm, HA=hand............... 32
Table 9. Prevalence of a subset of bacteria of potential ecological or health significance.
Shaded values constitute 1% or more of that sample (ZAN=zebu owner ankle,
NZAN=non-zebu owner ankle, ZAP=zebu owner armpit, ZFA=zebu owner forearm,
ZHA=zebu owner hand, Z=zebu). ............................................................................................ 42

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