A SURVEY OF POTENTIAL BACTERIAL ZOONOTIC PATHOGENS IN SHREW FECES by Logan M. Gray Thesis submitted in partial fulfilment of the requirements for the Degree of Bachelors of Science with Honours in Biology Acadia University March, 2012 © Copyright by Logan M. Gray, 2012 i This thesis by Logan M. Gray is accepted in its present form by the Department of Biology as satisfying the thesis requirements for the degree Bachelor of Science with Honours (Biology). Approved by the Thesis Supervisor ________________________ __________ Dr. Don Stewart Date Approved by the Head of Department ________________________ __________ Dr. Soren Bondrup-Nielsen Date Approved by the Honours Committee ________________________ __________ Date ii I, Logan M. Gray, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in microform, paper or electronic formats on a non-profit basis. I, however, retain the copyright in my thesis. ______________________________ Logan M. Gray ______________________________ Date iii Acknowledgements First off I would like to thank both of my supervisors, Dr. Don Stewart and Helene d‟Entremont for their guidance during this project. Both were irreplaceable in their respective fields and without them, this project would have been impossible. I have their guidance, knowledge, and most of all patience to thank for being able to complete this study for my Honours degree in Biology. I‟d also like to thank Dr. Brian Wilson for sitting on my review committee and for his help in editing this thesis. Secondly, I‟d like to thank the Biology Faculty of Acadia University for all of their help and use of facilities. I‟d like to especially thank Alanna Maynard for her help with the autoclave and use of microbial materials. I‟d also like to thank my fellow lab mate and friend Mathieu Gregoire for his help with everything as well as Bounie and Fen for the “donation” of a deceased shrew they found in Gaspereau. I‟d like to thank my closest friends here at Acadia, Ryo Ishigami, Andrew McMillan, and Stuart Lang for all of the fun times we had and for their “motivation” for me to do my research during the summer. Last, but not least, I would like to thank my parents for all their love and support during my entire undergraduate degree. Without them I would not have been able to pursue my goals as a scientific researcher. Thank you all. iv Table of Contents Acknowledgements ............................................................................................................iv Table of Contents ................................................................................................................v List of Tables .....................................................................................................................vi List of Figures ...................................................................................................................vii Abstract ............................................................................................................................viii Literature Review ................................................................................................................1 Introduction .........................................................................................................................6 Materials and Methods ......................................................................................................12 Feces Collection Method 1: Intestinal Samples ....................................................12 Feces Collection Method 2: Feeding Tubes ..........................................................12 Serial Dilutions ......................................................................................................13 Plating: Spread Plates and T-streaks .....................................................................13 Biochemical Testing .............................................................................................14 DNA Extraction ....................................................................................................17 Polymerase Chain Reaction ..................................................................................18 Gel Electrophoresis ...............................................................................................20 Sequencing from McGill University .....................................................................21 BLAST and Genbank Database ............................................................................22 Results ...............................................................................................................................23 Discussion .........................................................................................................................32 Conclusion ....................................................................................................................... 49 Literature Cited .................................................................................................................51 v List of Tables Results Table 1. PCR ingredients and relative volumes for 50 µl reactions..................................20 Table 2. Isolated bacterial colonies with sources, morphologies, and other observations........................................................................................................................25 Table 3. Biochemical test results for all isolated bacterial colonies..................................27 Table 4. BLAST results of bacterial isolates.....................................................................32 Table 5. Pathogenicity of identified bacterial samples as stated by scientific literature...33 Table 6. Various pathogenic bacteria found in shrews as reported in the scientific literature.............................................................................................................................35 vi List of Figures Results Figure 1. Agarose gel of 16s rRNA PCR products for samples 1-W, 2, 3, 4, 6-W, and 7- W. Bright band in ladder (L) marks 500 base pairs...........................................................28 Figure 2. Agarose gel of 16s rRNA PCR products for samples 1-O, 1-C, 5, 7-Y, and 7-B. Bright band in ladder (L) marks 500 base pairs. ...............................................................29 Figure 3. Agarose gel of 16s rRNA PCR products for samples 6-B, 6-Y, and 6-S. Bright band in ladder (L) marks 500 base pairs. ..........................................................................30 vii Abstract Zoonoses are diseases transmittable to humans by vertebrate animals. They are estimated to be the cause of 75% of emerging diseases worldwide. We are concerned about the possibility of transmission of pathogens in small mammal feces as part of a conservation survey focused on the maritime shrew, Sorex maritimensis (Stewart et al., 2003), and the white-bearded water shrew, Sorex albibarbis (Mycroft et al., 2012). The survey makes use of community members (e.g., school and naturalist groups) by having them set and tend feeding tube stations. The volunteers will forward to Acadia University any fecal samples collected from the feeding tubes. Handling the feces of a vertebrate sets the stage for possible zoonotic transmission. The purpose of this study was to explore possible zoonoses by isolating bacterial species from the feces. Fecal samples were collected using the same feeding tubes that the conservation survey will use and by dissecting shrews that were found dead in the wild. Bacteria were isolated from the feces and tested biochemically. DNA was also extracted from the bacteria. The polymerase chain reaction was used to amplify and sequence a portion of the 16S rRNA gene. The resulting sequences were compared to those in the GenBank database using the basic local alignment search tool. Matches from GenBank identified a number of possible species matches for each sample. These possible identities were compared to the biochemical characteristics which allowed the identities to be narrowed down with more accuracy. Most bacteria identified from the feces were listed in the literature as opportunistic pathogens. Based on the results of this study, field and lab protocols can be modified to help prevent bacterial zoonotic transmission. viii Literature Review Zoonoses are infectious diseases transmittable between humans and vertebrate animals (Krauss, 2003; World Health Organisation, 2010; Vaughan et al., 2011). Diverse arrays of pathogenic agents are involved in zoonoses which are in turn, largely involved with emerging human disease worldwide (Taylor et al., 2000). The emergence of zoonotic disease depends on many complex and interacting factors including the evolving ecology, human populations, and international travel (Taylor et al., 2000; Chomel et al., 2007). Zoonotic disease and its correlation with emerging disease have considerable effects on humans, which is made distinct when the entirety of human disease is further analyzed. A review of scientific literature identifies that over 1400 organisms, both prokaryotic and eukaryotic, are thought to be pathogenic to humans. Of these organisms, 868 (61%) are zoonotic pathogens (Taylor et al., 2000). In other words, of all the known diseases in the word caused by pathogens, approximately 61% are zoonotic i.e. transferrable to humans through vertebrate animals, sometimes including an invertebrate vector. Select species of bacteria,