University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2016 Distribution of Staphylococcus non-aureus isolated from bovine milk in Canadian herds Condas, Larissa Condas, L. (2016). Distribution of Staphylococcus non-aureus isolated from bovine milk in Canadian herds (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/25729 http://hdl.handle.net/11023/3441 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Distribution of Staphylococcus non-aureus isolated from bovine milk in Canadian herds by Larissa Anuska Zeni Condas A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES CALGARY, ALBERTA OCTOBER, 2016 © Larissa Anuska Zeni Condas 2016 Abstract The Staphylococci non-aureus (SNA) species are among the most prevalent isolated from bovine milk. However, the role of each species within the SNA group still needs to be fully understood. Knowing which SNA species are most common in bovine intramammary infections (IMI), as well as their epidemiology, is essential to the improvement of udder health on dairy farms worldwide. This thesis is comprised of two studies on the epidemiology of SNA species in bovine milk, and used molecular methods to identify of isolates obtained from the Canadian Bovine Mastitis and Milk Quality Research Network. The first study focused on the prevalence of SNA species on Canadian dairy farms and potential associations of SNA positive mammary quarters with bulk milk somatic cell count (BMSCC), barn type, parity, month of lactation and quarter location. Overall SNA represented 9% of the isolates from culture positive mammary quarters and the most common species were S. chromogenes, S. simulans, S. xylosus, S. haemolyticus, and S. epidermidis. Province and barn type were associated with SNA species distribution; Albertan bedded-packs were mostly affected by S. chromogenes, Maritimes free- stall herds by S. epidermidis, and Ontario and Quebec tie-stalls by S. xylosus. Staphylococcus arlettae, S. cohnii, and S. gallinarum were isolated from quarters of herds with high BMSCC. Fresh heifers and cows in later lactation were most frequently infected by S. chromogenes. The second study focused on the distribution of the same species in SNA positive-quarters according to udder inflammation status, classified according to low and high SCC and clinical mastitis. Average somatic cell count (SCC) for the SNA as a group was 70,000 cells/mL, driven mostly by S. chromogenes, S. haemolyticus, S. xylosus and S. epidermidis. Species-specific prevalence of SNA-positive quarters was higher in high (≥ 200,000 cells/mL) than in low SCC (< 200,000 ii cells/mL) samples for the 11 most frequently isolated SNA species. Staphylococcus sciuri was more frequently isolated from clinical mastitis samples. Considering SNA as a group will misrepresent the role of individual species on farms. Ultimately, adopting molecular identification of SNA species along with future research in species-specific risk factors are necessary to fully elucidate the importance of of the different SNA species on udder health and possible species-specific interventions. iii Acknowledgements Firstly, I would like to thank my supervisors Herman Barkema and Jeroen De Buck for their support and patience guiding me through such an ambitious project. Since my first year in Calgary, Herman encouraged me to persevere in research and I have undoubtedly learned a lot. Also, special thank you to Jeroen for encouraging me to improve my presentation skills in weekly meetings and lab group presentations. I also would like to thank my committee members, whose experience and insights contributed for making this thesis and publications undeniably better. I would like specially thank Dr. John Kastelic for his astonishing writing skills translated into edits of manuscripts and scientific writing courses at UofC and for all the encouraging words in the worst moments, when they were so needed. This research was only possible with the collaboration of the Natural Sciences and Engineering Research Council of Canada (NSERC) Industrial Research Chair in Infectious Diseases of Dairy Cattle. Also, thank you all of the dairy producers, animal health technicians, and Canadian Bovine Mastitis and Milk Quality Research Network (CBMQRN) regional coordinators (Trevor De Vries, University of Guelph, Canada; Jean-Philippe Roy and Luc Des Côteaux, University of Montreal, Canada; Kristen Reyher, University of Prince Edward Island, Canada; and Herman Barkema, University of Calgary, Canada) who participated in the data collection. The bacterial isolates were provided by the CBMQRN. The CBMQRN pathogen and data collections were financed by the Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada), Alberta Milk (Edmonton, AB, Canada), Dairy Farmers of New Brunswick (Sussex, New Brunswick, Canada), Dairy Farmers of Nova Scotia (Lower Truro, NS, iv Canada), Dairy Farmers of Ontario (Mississauga, ON, Canada) and Dairy Farmers of Prince Edward Island (Charlottetown, PE, Canada), Novalait Inc. (Quebec City, QC, Canada), Dairy Farmers of Canada (Ottawa, ON, Canada), Canadian Dairy Network (Guelph, ON, Canada), Agriculture and Agri-Food Canada (Ottawa, ON, Canada), Public Health Agency of Canada (Ottawa, ON, Canada), Technology PEI Inc. (Charlottetown, PE, Canada), Université de Montréal (Montréal, QC, Canada) and University of Prince Edward Island (Charlottetown, PE, Canada), through the CBMQRN (Saint-Hyacinthe, QC, Canada). Thank you as well for Annik L’Esperance for her effort in organizing and shipping all the 6000 SNA isolates to Calgary and promptly answering gazillions of e-mails about barcodes. Moreover, I would like to express my appreciation to Dr. Simon Dufour for guiding me with the cohort dataset. I would like to thank Uliana Kanevets and Aaron Lucko for the great work done with PCRs. Their help turned my lab experience happier and less repetitive. This thesis was also possible due to effort and statistical knowledge of Diego Borin Nobrega. Thank you so much for the uncountable discussions about the cohort, and all the statistical input. Also, thank you to Domonique Autumn Carson, whose writing skills and support helped me throughout the countless prevalence tables and graphs. I cannot forget the remaining “CNS Gang”, Sohail Naushad, Ana Paula Alves Monteiro, Ali Naqvi and Liu Gang. I would also like to thank the awesome colleagues at my office Caroline C., Caroline R., Marija, Emily, Taya, Christina, Robert, Laura, Barbara, Vineet, Nidhi, Verocai, Casey, and Janneke. I certainly will have good memories of our laughs, and talks. v Further thank you to Dr. Karin Orsel, that provided continuous feedback related to several epidemiological topics throughout our regular Epi Journal Club. Certainly the meetings contributed in my improvement as a scientist. I would like to thank my friends in Calgary, which have been family to me (and Guilherme) for all these years. You are special and made this time so much more enjoyable. I will never forget such dearest moments with you. I’m so thankful to sweet Ana Carolina Rasera, Luis Saviolli, Christina Tse, Anderson Macedo Silva, Estela Costa, Alysson Macedo Silva, Ana Luisa Bras, Marina Chueiri, Diego, Larissa Ozeki, and Tulio Alcantara. My deep gratitude and love to my husband, Guilherme Borges Bond, who brought me to Canada in a new endeavour, shared all difficult times, and gave me all the strength necessary to conquer this outcome. We have certainly achieved many milestones together which lead us to amazing personal growth. Literally, a lovely lifetime experience at your side. Finally, my dearest family, from which I was physically distant for a good purpose, but with them always in my thoughts. Thank you so much for your support in every moment. “Sou um pouco de todos que conheci, um pouco dos lugares que fui, um pouco das saudades que deixei e sou muito de tudo que apreciei” (Antonie de Saint-Exupéry). vi Dedication To my family, in particular my husband Guilherme Borges Bond, and my parents Sara Jane Soares Zeni Condas and Luiz Carlos Condas who always supported my passion for learning vii Table of Contents Abstract .......................................................................................................................... ii Acknowledgements ........................................................................................................ iv Dedication ..................................................................................................................... vii Table of Contents ......................................................................................................... viii List of Tables .................................................................................................................. x List of Figures and Illustrations ...................................................................................... xi List of Symbols, Abbreviations and Nomenclature