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(Qpcr) Assay for the Beta- Proteobacterium BK-BJC, and Its Application in Lake Trout (Salvelinus Namaycush) During an Epitheliocystis-Associated Mortality Event

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(Qpcr) Assay for the Beta- Proteobacterium BK-BJC, and Its Application in Lake Trout (Salvelinus Namaycush) During an Epitheliocystis-Associated Mortality Event Development of a Quantitative Real-Time PCR (qPCR) Assay for the Beta- Proteobacterium BK-BJC, and Its Application in Lake Trout (Salvelinus namaycush) During an Epitheliocystis-Associated Mortality Event by Doran Witherspoon Kirkbright A Thesis presented to The University of Guelph In partial fulfillment of the requirements for the degree of Doctor of Veterinary Science Guelph, Ontario, Canada © Doran Witherspoon Kirkbright, August, 2016 ABSTRACT Development of a Quantitative Real-Time PCR (qPCR) Assay for the Beta- Proteobacterium BK-BJC, and Its Application in Lake Trout (Salvelinus namaycush) During an Epitheliocystis-Associated Mortality Event Doran Witherspoon Kirkbright Advisors: University of Guelph, 2016 Dr. John Lumsden and Dr. Brandon Lillie Epitheliocystis is a multi-etiologic gill condition in which a single cell is expanded by an intracytoplasmic inclusion filled with gram negative bacteria. My research focused on characterizing one purported agent, Blue Jay Creek Burkholderia (BK-BJC), and its relationship to epitheliocystis-associated annual mortality events and histopathologic gill lesions in economically valuable Ontario reared lake trout. Based on the 1503 bp 16S rRNA gene sequence of BK-BJC identified by Contador et al. (2016), two primer sets were developed for detection of this bacterium. From the first primer set (BKBJCV8F/R) with hydrolysis probe, a qPCR assay was developed and used to quantify BK- BJC amounts in the gills of Blue Jay Creek Fish Culture Station lake trout and tank water during an epitheliocystis-associated mortality event in 2013 and a non-outbreak winter in 2015. A second primer set (BKBJCV3) was created to differentiate BK-BJC from ‘Candidatus Branchiomonas cysticola’. In addition, BK-BJC-infected gills were streaked onto a variety of agar plates. Colony growth was assessed by Gram stain, MALDI-TOF mass spectrometry, and qPCR. The BKBJCV8 was found to be non-specific in silico and experimentally, potentially amplifying at least 33 non-target bacteria from GenBank. Conventional PCR with the BKBJCV3 confirmed that all BKBJCV8 qPCR positive samples were positive for BK-BJC. BK-BJC was present in 1 % of the samples collected from fish in 2015. There was a significant direct relationship (p=0.035) between BK-BJC and BK-BJC-like, non-target bacterial loads and mortality rates, and there was an observable direct relationship with interlamellar hyperplasia and single cell necrosis of the gills. No relationship was discovered between epitheliocystis inclusions and bacteria loads in either 2013 or 2015 lake trout. Further, BK-BJC or BK-BJC-like bacteria were not found in the water samples, nor did they grow on any culture medium. In conclusion, we developed two PCR assays and confirmed that BK-BJC was present in all lake trout from an EP outbreak at Blue Jay Creek in 2013. Future research should focus on the development of a more sensitive and specific, qPCR assay for the rapid diagnosis of BK-BJC and further investigations into the ability of BK-BJC to cause EP. ACKNOWLEDGEMENTS I would like to express my gratitude to Drs. John Lumsden and Brandon Lillie, and committee members Drs. Salvatore Frasca Jr and Niels Bols for all their invaluable advice, support, and effort in formulating and executing this DVSc project. Deepest thanks also go to Drs. Foster, Caswell, Plattner, Hayes, Lillie, Smith, Turner, and Susta for the countless hours teaching me pathology at the microscope and on the post mortem floor, as well as supporting my (sometimes) wild etiologic diagnoses and helping me craft reports while maintaining my unique voice. To all my pathology co-residents, I would like to say thank you for being great friends and colleagues. I am so grateful that our paths crossed and we went on this journey together. You will all remain my closest friends and confidants for life. You made living and working in Canada a wonderful experience. There are no words that can truly express how thankful I am to laboratory technicians—Leah Read, Jutta Hammermueller, Paul Huber, and Pat Bell Rogers—and AHL histology department. Thank you for your sagacious advice, patiently teaching me how laboratories work, and allowing me to run by with questions at all times of day. Your expertise and generosity is humbling. iv I would also like to thank my semi-aquatic labmates—Elena, Ehab, Juan Ting, Maureen, Ryan, Jaramar, Paige and Lowia for showing me new technologies and how to gracefully deal with the inevitable delays and set-backs that come with research. This project would not have been possible without the fish collection and processing assistance of Paul Methner, Michael Burke, and the staff at BJC Fish Culture Station and Alma Research Station. Also, thank you goes to Mykolas Kamaitis for taking the long journey to Manitoulin Island to collect fish with me. I would like to thank my funding sources—NSERC, OMNR, and the Ontario Veterinary College (OVC)—with whom this project would not have been possible. A personal debt of gratitude goes to Donna Kangas who always ensured that I stayed on the right side of registrar’s office and for our spontaneous chats that I will remember fondly. Last, but not least, I thank my family and boyfriend, Alex Zaleznik, for their constant support and cheerleading. I could not have made it this far without you. v DECLARATION OF WORK PERFORMED The majority of the work was performed by Doran Witherspoon Kirkbright under the supervision of Drs. John Lumsden, Brandon Lillie, and the advisory committee members of Dr. Niels Bols and Dr. Salvatore Frasca Jr., except for the following: The development of the initial primer set, BKBJCV8, and PCR optimization of that primer set was done by Elena Contador. Collection and processing of 45 lake trout from Blue Jay Creek Fish Culture Station for DNA extraction and histopathologic examination was performed by Mykolas Kamaitis, a summer and veterinary student (University of Guelph, OVC). All slides were prepared by Susan Lapos and the staff of the Animal Health Laboratory at the University of Guelph. vi TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iv DECLARATION OF WORK PERFORMED ............................................................................... vi TABLE OF CONTENTS .............................................................................................................. vii LIST OF TABLES ........................................................................................................................ xii LIST OF FIGURES ..................................................................................................................... xiv LIST OF ABBREVIATIONS ...................................................................................................... xvi GENERAL INTRODUCTION ....................................................................................................... 1 1. LITERATURE REVIEW ........................................................................................................... 3 1.1. EPITHELIOCYSTIS ............................................................................................................ 3 1.2. CLINICAL SIGNS AND GROSS FINDINGS ................................................................... 6 1.3.1. HISTOLOGY - SPECIAL STAINS .............................................................................. 8 1.4. DISEASE AND RELATIONSHIP WITH HOST DEATH ............................................... 10 1.5. CO-INFECTIONS .............................................................................................................. 12 1.6. ADVANCED DIAGNOSTICS .......................................................................................... 14 1.6.1. TRANSMISSION ELECTRON MICROSCOPY ....................................................... 14 1.6.2. BACTERIAL CULTURE ........................................................................................... 16 1.6.3. POLYMERASE CHAIN REACTION ........................................................................ 19 1.6.4. In situ HYBRIDIZATION........................................................................................... 20 1.6.5. IMMUNOHISTOCHEMISTRY ................................................................................. 22 1.7. BACTERIAL PATHOGENS ............................................................................................. 24 1.7.1. CHLAMYDIAE .......................................................................................................... 24 1.7.2. BURKHOLDERIALES............................................................................................... 26 1.7.3. ENDOZOICIMONAS ELYSICOLA ............................................................................. 30 1.8. TREATMENTS ................................................................................................................. 31 1.9. FURTHER RESEARCH .................................................................................................... 32 2. RATIONALE, PURPOSE and HYPOTHESES ....................................................................... 34 2.1. RATIONALE ..................................................................................................................... 34 2.2. PURPOSE .........................................................................................................................
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