DIAGNOSIS OF MYCOPLASMA MASTITIS: VALIDATION AND DEVELOPMENT By SUKOLRAT BOONYAYATRA A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY College of Veterinary Medicine DECEMBER 2010 To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of SUKOLRAT BOONYAYATRA find it satisfactory and recommend that it be accepted. ______________________________ Lawrence K. Fox, Ph.D., Chair ______________________________ Thomas E. Besser, Ph.D. _____________ ______ ___________ Ashish Sawant, Ph.D. ______________________________ John M. Gay, Ph.D. ii ACKNOWLEDGMENTS I would like to thank Dr. Larry Fox, committee chair, for his guidance, support and encouragement of working with mycoplasma mastitis. Additionally, I am very grateful for his acceptance when I transferred from Tennessee. I thank my other committee members, Dr. John Gay, Dr. Ashish Sawant, and Dr. Tom Besser for their guidance and suggestions on the various aspects and helping me solve many problems with my work. This thesis dissertation would not have been possible without many people who provided technical assistance in the laboratory. I am very thankful to Dorothy Newkirk for her expert assistance. I also thank to other FDIU staffs for their suggestion and helping me go through all lab work smoothly. I want to thank Dr. Ziv Raviv and Dr. Amy Wetzel for their kindness of teaching me everything about real-time PCR when I was in the Ohio State University. I am grateful to Dr. Stephen Oliver, other professers and staffs in the University of Tennessee in Knoxville for providing me a wonderful time in my life when I was in this volunteer state. I want to sincerely thank to the Royal Thai government for the scholarship which allowed me to have this great experience in the United States. I thank my husband, Veerasak Punyapornwithaya, for helping me solve many problems and being such a good husband and friend. I want to thank my daughter, Minnie, for being my biggest motivation of finishing the research. I am deeply grateful to my parents and sisters for their encouragement and understanding for everything I have done. Finally, I would like to thank all people who contributed to the successful of all the work in this thesis dissertation. iii DIAGNOSIS OF MYCOPLASMA MASTITIS: VALIDATION AND DEVELOPMENT Abstract by Sukolrat Boonyayatra, Ph.D. Washington State University December 2010 Chair: Lawrence K. Fox Microbiological culture of milk samples has been used as a standard diagnosis for mycoplasma mastitis. It is suggested to perform with fresh samples for optimum diagnosis. Submission of fresh samples is often difficult given the logistics of collection and shipping of milk samples from farm to laboratory. Therefore , milk samples are usually chilled or frozen before culture. A study of the effects of storage methods on the recovery of Mycoplasma species from milk samples was performed. The results indicated that holding milk at refrigerated temperatures (5°C) for 5 days and freezing milk samples (-20°C) lowers the number of recovered Mycoplasma species . Moreover the addition of glycerol prior freezing to achieve 10% and 30% (v/v) solutions was found to improve the recovery of Mycoplasma species from frozen milk samples. Even though a Mycoplasma -like-colony is observed by a standard culture method, the diagnosis can be misinterpreted as Acholeplasma species which is indistinguishable by culture. To validate and suggest techniques used to discriminate between these two genera, a study on the discrimination between Mycoplasma and Acholeplasma using digitonin and nisin disc diffusion assays, and PCR was performed. Findings revealed a high and comparable efficiency of using iv nisin and digitonin disc diffusion assays and PCR to distinguish Mycoplasma and Acholeplasma species. Given the fastidious nature of Mycoplasma species, and the time-consuming nature of the standard culture method, a diagnostic test of mycoplasma mastitis that is more sensitive, less time consuming, and can speciate mycoplasma mastitis pathogens would be valuable. The development of real-time PCR assays to detect 3 major mycoplasma mastitis pathogens: M. bovis, M. californicum and M. bovigenitalium was investigated to provide an alternative diagnostic tool for mycoplasma mastitis. The results given by the novel real-time PCR assays showed a perfect agreement with the gold standard, and the results were obtained within 4-5 hours. The overall goal of studies reported herein was to investigate improvements in the efficiency of the diagnosis of mycoplasma mastitis in dairy cows. Findings indicate that both phenotypic and genotypic diagnostic techniques can be applied, and in conjunction with current standard techniques, will better identify cows with mycoplasma mastitis. v TABLE OF CONTENTS Page ACKNOWLEDGMENT.................................................................................................... iii ABSTRACT ....................................................................................................................... iv LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ............................................................................................................ x CHAPTER 1 1.1. INTRODUCTION ............................................................................................1 1.2. REFERENCES .................................................................................................4 CHAPTER 2 (LITERATURE REVIEW) 2.1. MYCOPLASMA SPECIES ................................................................................7 2.2. MYCOPLASMA MASTITIS IN DAIRY CATTLE ........................................8 2.3. EFFECT OF STORAGE METHODS ON SURVIVAL OF MYCOPLASMA 10 2.4. DISCRIMINATION BETWEEN MYCOPLASMA AND ACHOLEPLASMA 12 2.5. DIGITONIN ....................................................................................................13 2.6. NISIN ..............................................................................................................14 2.7. NESTED PCR TECHNIQUE .........................................................................16 2.8. SPECIATION OF MYCOPLASMA SPECIES ...............................................17 2.9. REAL-TIME PCR .........................................................................................19 2.10. CONCLUSION AND HYPOTHESIS ..........................................................23 vi CHAPTER 3 3.1. INTRODUCTION ......................................................................................................44 3.2. MATERIALS AND METHODS ................................................................................46 3.3. RESULTS ...................................................................................................................47 3.4. DISCUSSION .............................................................................................................48 3.5. ACKNOWLEDGMENT.............................................................................................51 3.6. REFERENCES ...........................................................................................................52 CHAPTER 4 4.1. INTRODUCTION ......................................................................................................56 4.2. MATERIALS AND METHODS ................................................................................58 4.3. RESULTS ...................................................................................................................62 4.4. DISCUSSION .............................................................................................................65 4.5. ACKNOWLEDGMENT.............................................................................................68 4.6. REFERENCES ...........................................................................................................68 CHAPTER 5 5.1. INTRODUCTION ......................................................................................................80 5.2. MATERIALS AND METHODS ................................................................................81 5.3. RESULTS ...................................................................................................................87 5.4. DISCUSSION .............................................................................................................89 5.5. ACKNOWLEDGMENT.............................................................................................92 5.6. REFERENCES ...........................................................................................................93 CHAPTER 6 CONCLUSIONS..................................................................................................106 vii APPENDIX A. RAW DATA A.1: Chapter 3 (Refrigeration experiment) ....................................................108 A.2: Chapter3 (Freezing experiment) ............................................................118 A.3: Chapter 4 and 5 ......................................................................................131 B. METHODS B.1: Cloning and filtration techniques for Mycoplasmas .............................143 B.2: DNA extraction and purification using Purelink Genomic DNA kit 144 B.3: PCR to amplify 16S-23S rRNA intergenic spacer region of Mycoplasma species and Acholeplasma