Clemson University TigerPrints All Theses Theses 5-2019 Humoral Immune Responses to Select Bacterial Pathogens in the American Alligator, Alligator mississippiensis Bailey Marie Alston Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_theses Recommended Citation Alston, Bailey Marie, "Humoral Immune Responses to Select Bacterial Pathogens in the American Alligator, Alligator mississippiensis" (2019). All Theses. 3135. https://tigerprints.clemson.edu/all_theses/3135 This Thesis is brought to you for free and open access by the Theses at TigerPrints. It has been accepted for inclusion in All Theses by an authorized administrator of TigerPrints. For more information, please contact [email protected]. HUMORAL IMMUNE RESPONSES TO SELECT BACTERIAL PATHOGENS IN THE AMERICAN ALLIGATOR, Alligator mississippiensis A Thesis Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Master of Science Environmental Toxicology by Bailey M. Alston May 2019 Accepted by: Dr. Charles D. Rice, committee chair Dr. Thomas R. Rainwater Dr. Thomas E. Schwedler i Abstract The American alligator, Alligator mississippiensis, is widely distributed and abundant throughout the southeastern United States. Despite their abundance, these reptiles have not been examined for their role in environmental pathogen distribution, as a sentinel for the presence of various p athogens, or other humoral immune responses in individuals from different habitats. This study investigates the humoral immune responses of alligators to select bacterial pathogens. Immunoglobulin Y (IgY), an immunoglobulin molecule found in amphibians, birds and reptiles, similar to IgG in higher vertebrates. IgY was purified from pooled alligator serum collected in coastal South Carolina. Mouse polyclonal antisera (pAb) was then generated against IgY to develop a sensitive ELISA to quantify serum antibody responses and relative titers. Serum samples from alligators collected from multiple localities in Florida were screened for bacteria-specific antibodies to the following nine aquatic bacteria: Vibrio cholera, Escherichia coli , V. anguillarum , V. vulnificus, V. parahaemolyticus, Brevundimonas vesicularis, Mycobacterium marinum , Erysipelothrix rhusiopathiae and Streptococcus agalactiae. C-reactive protein (CRP) is an ancient acute phase protein, participating in complement activation and opsonisation of pathogens, and is usually indicative of relative levels of systemic inflammation. Alligator CRP was purified and used to generate a specific monoclonal antibody to develop ELISA-based approaches to semi- quantifying circulating CRP in individuals. Finally, serum lysozyme enzymatic activity was also quantified in individual samples. Humoral immune responses to various pathogens, along with CRP and lysozyme activity, may be correlated with environmental ii health. This study provides the first baseline data and proof of concept, to compare responses of wild alligators to pathogens throughout their distribution. This information will allow A. mississippiensis to be used as a sentinel of pathogen occurrence and environmental quality in future studies. iii Dedication To the Academy of the Holy Names, Ms. Clancy and Mr. Triller, for instilling a love of science in me at an early age. To my grandfather, “Daddy Max” for his inspirational and lifelong pursuit of education. iv Acknowledgements First, I would like to thank Dr. Rice for providing me with such a wonderful opportunity to get my Master’s in his lab and for being so supportive along the way. Thank you for your mentorship and for setting me up for success. I came out of this program having learned not only about research, but also important life lessons. Two of the most important life lessons I have learned from Dr. Rice are that you shouldn’t take yourself too seriously and that not every experimental error is the end of the world. Thank you to Dr. Schwedler and Dr. Rainwater for serving on my committee. I really appreciate your time, expertise, guidance and understanding throughout this process. Thank you to Dr. Rainwater for providing samples and putting us in touch with Dr. John Bowden at the University of Florida, and to Dr. Bowden for providing alligator samples. To Kursten and Delia – the “angels on my shoulder”. Thank you for being such supportive lab-mates and friends. I know I can always count on you two to have my back, help clean up messes (both in my life and in the lab (i.e. the centrifuge incident)) and celebrate achievements. To my “ride or die”, Lindsey Louise Barnett Jr. - thank you for always keeping it real and for hyping me up when I needed it (and when I don’t). Natalie, thank you for being such a supportive and understanding roomie, cat mom and friend this past year. Last but not least, I would also like to thank my family. My parents, George and Jennifer, for the opportunities and life experiences you’ve given me. For teaching me the importance of education, always pushing me to do my best and the continuous support and unconditional love over the past 25 years. Ashley, for showing me the importance of v always being yourself. Grace, for being my voice of reason and best friend, and Will and Christian for being the best little brothers (making me look good) and always having my back. vi Table of Contents Page Title Page ...........................................................................................................................i Abstract ............................................................................................................................ ii Dedication ........................................................................................................................iv Acknowledgements ........................................................................................................... v List of Figures ............................................................................................................... viii Chapters I. Literature Review............................................................................................ 1 Alligator mississippiensis : Species background ............................................. 1 Effects of temperature ..................................................................................... 3 Threats to the American alligator ................................................................... 4 Alligator immunology ..................................................................................... 4 Reptilian innate immunity............................................................................... 5 Reptilian adaptive immunity ........................................................................... 8 Potential role as sentinel species and amplifier host ..................................... 13 II. Chapter One .................................................................................................. 15 Introduction ................................................................................................... 15 Materials and Methods .................................................................................. 20 Results ........................................................................................................... 28 Discussion ..................................................................................................... 41 References ....................................................................................................................... 45 vii List of Figures Figure Page 1. American Alligator Habitat Range ....................................................................... 2 2. IgY Structure ....................................................................................................... 12 3. Alligator serum average lysozyme activity ......................................................... 30 4. SDS PAGE Gel of American Alligator Purified IgY ......................................... 31 5. Western Blot of Bacterial Lysates Probed with Alligator Whole Serum ........... 32 6. E.coli titers in serum ........................................................................................... 33 7. M.marinum titers in serum .................................................................................. 34 8. S.agalactae titers in serum .................................................................................. 35 9. V.vulnificus titers in serum .................................................................................. 36 10. V.anguillarium titers in serum ............................................................................ 37 11. V.parahaemoliticus titers in serum ..................................................................... 38 12. V.cholerae titers in serum ................................................................................... 39 13. Alligator relative CRP levels in serum ............................................................... 40 viii Chapter I: Literature Review Alligator mississippiensis: Species Background The American alligator, Alligator mississippiensis, is the largest reptile and only alligator native to the United States. These carnivorous reptiles are found primarily throughout the Southeast U.S., from the Virginia-North Carolina border to South Florida and as far west as Texas (Figure 1 ) (United States Geographical Services). Once considered an endangered species