Clemson University TigerPrints All Theses Theses 12-2017 An Evaluation of Methodologies for Measuring Antibacterial Activity of the Epithelial Mucosa of Farmed Tilapia (Oreochromis niloticus) Against Streptococcus agalactiae and Streptococcus iniae Betsy Virginia White Presgraves Clemson University Follow this and additional works at: https://tigerprints.clemson.edu/all_theses Recommended Citation White Presgraves, Betsy Virginia, "An Evaluation of Methodologies for Measuring Antibacterial Activity of the Epithelial Mucosa of Farmed Tilapia (Oreochromis niloticus) Against Streptococcus agalactiae and Streptococcus iniae" (2017). All Theses. 2775. https://tigerprints.clemson.edu/all_theses/2775 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]. AN EVALUATION OF METHODOLOGIES FOR MEASURING ANTIBACTERIAL ACTIVITY OF THE EPITHELIAL MUCOSA OF FARMED TILAPIA (OREOCHROMIS NILOTICUS) AGAINST STREPTOCOCCUS AGALACTIAE AND STREPTOCOCCUS INIAE A Thesis Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Master of Science Animal and Veterinary Science by Betsy Virginia White Presgraves December 2017 Accepted by: Dr. Thomas R. Scott, Chair Dr. Lance Beecher Dr. Amy Messersmith-Love ABSTRACT Tilapiine cichlids are the third largest farmed fish worldwide and are among the easiest and most profitable fish to farm. The most common pathogens affecting farmed tilapia are Streptococcus agalactiae and Streptococcus iniae, which together account for losses of more than 150 million dollars annually. Fish are at the highest risk for developing a Streptococcus infection when they are in high density stock conditions. Research indicates that the secondary effects of high stock density such as low dissolved oxygen and high ammonia levels are of less significance to infection and mortality than the damage that over stocking causes to the mucosal immune system. However, assays used to determine the effectiveness of the tilapiine mucosal immune system have proven unreliable. The present work has been undertaken to evaluate mucus collection procedures and antibacterial assay effectiveness. One “spot-on-lawn” assay, four disk diffusion assays, and four micro titer plate assays were chosen based upon common levels of use in the literature. The assays were executed; however, each of the disk diffusion assays failed to accurately measure antibacterial activity when controlled for the antibacterial activity of additives. The microtiter plate assays successfully measured limited antibacterial activity at lower growth reduction levels of 10-30%. Additionally, these assays were administered on samples which were collected from tilapia in an actual aquaponics facility in contrast to the majority of experiments which are conducted on fish that have been purposely housed in clean and controlled conditions within research facilities. This deviation from standard methods introduced additional variables which influence assay outcomes; therefore, future research should address these discrepancies. ii ACKNOWLEDGEMENTS I would like to thank John Abercrombie and Ian Cummings of the Clemson Microbiology Prep Lab for their constant instruction and support. Without their selfless teaching, none of this would be possible. I would also like to show my gratitude to my committee, Dr. Thomas Scott, Dr. Lance Beecher, and Dr. Amy Messersmith for their guidance and advice. I am also grateful to my colleagues in the Animal and Veterinary Science graduate student offices for daily collaboration, intellectual challenge, and laughter. Finally, I would like to thank my family for the incalculable ways in which they sacrifice so that I may continue to forge my own unique career path. iii TABLE OF CONTENTS Page TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................ iv LIST OF FIGURES ....................................................................................................... vii LIST OF TABLES .......................................................................................................... ix CHAPTER 1. REVIEW OF LITERATURE Introduction ............................................................................................. 1 The Teleost Immune System Overview .................................................. 3 Tilapiine Immune Anatomical Structures ............................................... 4 Tilapiine Immune Cellular Components ................................................. 5 Tilapiine Mucosal Immune System ........................................................ 7 Relevent Fish Diseases Streptococcus agalactiae and Streptococcus iniae……………..9 Aeromonas salmonicida……………………………………….12 Yersinia ruckeri..........................................................................13 Importance of Mucosa in Preventing Streptococcus infection……….14 2. RESEARCH DESIGN AND METHODS Collection Methods ............................................................................... 16 Quantification of Protein....................................................................... 17 Standardizing of Bacterial Cultures ...................................................... 18 Contamination Prevention .................................................................... 18 Antibacterial Assays Spot on Lawn Assay………………………………………….19 Disk Diffusion Assay with Lyophilization…………………...21 Disk Diffusion Assay /UV Sterilization of Liquid Sample…..21 Disk Diffusion Assay /UV sterilization of Crude Mucus…… 22 Disk Diffusion Assay/ Chloroform Methanol Precipitation….22 Microtiter Broth Dilution Assay/Minimal Processing………. 23 Microtiter Broth Dilution Assay with Sodium Acetate………24 Microtiter Broth Dilution Assay for Buffer Effectiveness…. 25 Microtiter Broth Dilution Assay with PBS and Nanosep Centrifugal Filter ............................................................ 25 iv Page 3. RESULTS AND DISCUSSION Collection Method ................................................................................. 27 Protein Quantification ............................................................................ 29 Contamination Prevention ..................................................................... 35 Spot on Lawn Assay .............................................................................. 39 Disk Diffusion Assay with Lyophilization ............................................ 41 Disk Diffusion Assay/UV Sterilization of Liquid Sample .................... 46 Disk Diffusion Assay/UV sterilization of Crude Mucus ....................... 51 Disk Diffusion Assay/Chloroform Methanol Precipitation ................... 53 Microtiter Broth Dilution Assay/Minimal Sample Processing .............. 55 Microtiter Broth Dilution Assay with Sodium Acetate ......................... 57 Microtiter Broth Dilution Assay for Buffer Effectiveness .................... 58 Microtiter Broth Dilution Assay with PBS and Nanosep Centrifugal Filter ........................................................................ 60 4. CONCLUSION .................................................................................................. 64 APPENDICES A: Raw Data ................................................................................................................... 74 B: Statistical Analysis .................................................................................................. 101 REFERENCES ............................................................................................................ 111 v LIST OF FIGURES Figure Page 1.1 Anatomical structures of the teleost immune system (Evolution 2016) ......................................................................... 4 1.2 Diagram of immunoglobulin production in teleosts (Flajnik 2005)............................................................................... 5 1.3 Tail line ulceration on tilapia infected with Streptococcus agalactiae ........................................................... 11 1.4 Streptococcus agalactiae colonies on sheep’s blood agar…………......11 1.5 Streptococcus iniae colonies on sheep’s blood agar……………….......12 1.6 Aeromonas salmonicida colonies on sheep’s blood agar……………....13 1.7 Yersinia ruckeri colonies on sheep’s blood agar…………………….....14 3.1 Images of mucus smears for each collection method………………......28 3.2 Curve representing density of crude protein sample per absorbance 280 for three trials ................................................... 30 3.3 Curve representing density of retentate sample per absorbance 280 for three trials ................................................... 31 3.4 Curve representing density of filtrate sample per absorbance 280 for three trials ................................................... 32 3.5 Ladder for the blue standard used from Biorad Precision Protein Plus (left). Electrophoresis results with sample in columns 1 and 3 with blue standard in column 2 (right) ............................ 34 4.1 S. agalactiae growth measured after 12 hours and 24 hours using the minimally processed sample microtiter