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Evaluation of MALDI Biotyper for Identification of Taylorella Equigenitalis and Taylorella Asinigenitalis Kristina Lantz Iowa State University

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Evaluation of MALDI Biotyper for Identification of Taylorella Equigenitalis and Taylorella Asinigenitalis Kristina Lantz Iowa State University Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2017 Evaluation of MALDI Biotyper for identification of Taylorella equigenitalis and Taylorella asinigenitalis Kristina Lantz Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Microbiology Commons, and the Veterinary Medicine Commons Recommended Citation Lantz, Kristina, "Evaluation of MALDI Biotyper for identification of Taylorella equigenitalis and Taylorella asinigenitalis" (2017). Graduate Theses and Dissertations. 16520. https://lib.dr.iastate.edu/etd/16520 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Evaluation of MALDI Biotyper for identification of Taylorella equigenitalis and Taylorella asinigenitalis by Kristina Lantz A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Veterinary Microbiology Program of Study Committee: Ronald Griffith, Major Professor Steve Carlson Matthew Erdman Timothy Frana The student author and the program of study committee are solely responsible for the content of this thesis. The Graduate College will ensure this thesis is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2017 ii TABLE OF CONTENTS Page LIST OF FIGURES ................................................................................................... iii LIST OF TABLES ..................................................................................................... iv ACKNOWLEDGMENTS ......................................................................................... v ABSTRACT………………………………. .............................................................. vi CHAPTER 1 REVIEW OF LITERATURE ........................................................ 1 The genus Taylorella ........................................................................................... 1 MALDI-TOF bacterial identification .................................................................. 12 CHAPTER 2 INTRODUCTION ......................................................................... 22 CHAPTER 3 MATERIALS AND METHODS .................................................... 26 CHAPTER 4 RESULTS ....................................................................................... 36 CHAPTER 5 DISCUSSION ................................................................................. 42 REFERENCES .......................................................................................................... 67 iii LIST OF FIGURES Page Figure 1 Comparison of MALDI scores for all Taylorella organisms vs. day ......... 49 Figure 2 Direct comparison of NECLC and Bruker tube formic acid methods for protein extraction.................................................................................. 49 Figure 3 MALDI score data of Taylorella isolates on Bruker direct transfer and Bruker tube formic acid ...................................................................... 50 Figure 4 Media comparison study ............................................................................ 50 Figure 5 Custom library versus BDAL library ......................................................... 51 Figure 6 Custom library versus abbreviated custom library. ................................... 51 Figure 7 Biotyper 3 phylogenetic tree of all Taylorella MSPs in the custom library 52 Figure 8 Zoomed image of Taylorella phylogenetic tree. ........................................ 52 Figure 9 Phylogenetic tree of T. equigenitalis genomes generated using KSnp. ..... 53 Figure 10 Biotyper 3 phylogenetic tree of T. equigenitalis Eugon MSPs without replicates .................................................................................................... 54 Figure 11 Biotyper 3 phylogenetic tree of T. equigenitalis Eugon replicate MSPs. .. 54 Figure 12 Biotyper 3 phylogenetic tree of T. equigenitalis Eugon MSPs including replicate MSPs. .......................................................................... 55 Figure 13 CCI analysis of CEM 1978 isolates. .......................................................... 56 Figure 14 PCA dendrogram of CEM 1978 isolates. .................................................. 56 Figure 15 CCI analysis of CEM 2008-2010 isolates with 3 unrelated T. equigenitalis isolates. ............................................................................. 57 Figure 16 PCA dendrogram of CEM 2008-2010 isolates plus outliers. .................... 57 iv LIST OF TABLES Page Table 1 Taylorella isolates used for analysis .......................................................... 58 Table 2 Non-Taylorella isolates used for analysis .................................................. 61 Table 3 Extraction methods .................................................................................... 63 Table 4 Media comparison isolates ......................................................................... 63 Table 5 Additional media tested ............................................................................. 64 Table 6 Replicate T. equigenitalis MSPs for phylogenetic tree evaluation ............ 64 Table 7 Paired mean scores by method and media type ......................................... 65 Table 8 Comparison of custom and commercial BDAL library scores for T. asinigenitalis .......................................................................................... 65 Table 9 Comparison of custom and commercial BDAL library scores for T. equigenitalis ........................................................................................... 65 Table 10 Comparison of custom and abbreviated custom library scores for T. asinigenitalis .......................................................................................... 66 Table 11 Comparison of custom and abbreviated custom library scores for T. equigenitalis ........................................................................................... 66 v ACKNOWLEDGMENTS I would like to thank my major professor Dr. Ronald Griffith and my committee members Dr. Tim Frana, and Dr. Steve Carlson for this opportunity to work with them. I would especially like to thank my committee member Dr. Matt Erdman, whose knowledge and support through the years have played a major role in my professional development. In addition, I would like to thank Dr. Linda Schlater for allowing me the opportunity to work on this project and for her endless depth of knowledge on the history of CEM. Two statisticians assisted me on this project; Chris Tong provided invaluable support with study design, statistics and plotting and Marie Vendettuoli provided feedback on phylogenetic trees, CCI and PCA. I would like to thank the members of my lab, Linda Cox, Maria Munoz, and Keira Stuart, for their patience and willingness to assist with day-to-day matters while I was working on this project. Maria in particular was always there with willing hands and a smile. I would like to thank my family for their support through the years. My parents have always encouraged and supported me, even when it meant I hardly ever made it home to visit them. And finally and most of all, thank you to Nick, who showed endless patience while I was working on this project. vi ABSTRACT Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF) has become an increasingly popular method for bacterial identification in recent years. Contagious Equine Metritis is a venereal disease of equids of international significance caused by the gram negative organism, Taylorella equigenitalis. The prescribed test is by bacteriological culture, however, due to the slow-growing and fastidious nature of the bacteria, culture requires specialized media and extended culture time. In addition, differentiation of T. equigenitalis and the closely related Taylorella asinigenitalis from each other and from common contaminants of the equine genital tract is difficult and has required advanced diagnostics with specialized reagents. This study evaluates the use of MALDI-TOF identification for detecting and differentiating individual colonies of T. equigenitalis and T. asinigenitalis directly from culture plates, significantly decreasing the time and cost of diagnosis. The study compared the effects of varying extraction methods, types of culture media and day of culture on the ability to identify these species. This method represents an inexpensive, rapid, and accurate new diagnostic tool for Contagious Equine Metritis diagnosis. 1 CHAPTER I. REVIEW OF LITERATURE The genus Taylorella Taylorella equigenitalis is the causative organism of the equine disease Contagious Equine Metritis (CEM). Along with the only other known member of the genus, Taylorella asinigenitalis, these host adapted bacterial organisms are found only in the genital tract of animals of the family Equidae [1, 2]. Taylorella equigenitalis causes endometritis and reproductive failure in mares. Many mares clear the infection rapidly and completely while others may develop an asymptomatic carrier state in which the bacteria persist on the external genitalia [3]. Antibody response
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