EVALUATION OF THREE IN VITRO BIOASSAYS FOR MEASURING THE ANTHELMINTIC ACTIVITY OF PLANT EXTRACTS CONTAINING CONDENSED TANNINS by JANET SUE HOWELL (Under the Direction of Ray M. Kaplan) ABSTRACT Certain forages high in condensed tannins (CT) demonstrate anthelmintic activity, and appear to be a useful non-chemical adjunct to parasite control in small ruminants. In vitro bioassays have been used to evaluate this antiparasitic effect against several species of trichostrongyle nematodes. However, it remains unclear which in vitro bioassay is the most appropriate evaluation tool. The goal of this research is to evaluate different in vitro methods, and determine which are the most suitable for measuring the effective concentration (EC ) of 50 these extracts. In this research, 3 in vitro methods (egg hatch assay (EHA), larval development assay (LDA) and larval migration inhibition assay (LMIA) were performed using CT extracts from 4 plants with Haemonchus contortus eggs or larvae. Of the 3 bioassays, the LDA yielded the most consistent results, and therefore, appears to be the most appropriate bioassay for measuring the antiparasitic activity of CT plant extracts in small ruminants. INDEX WORDS: anthelmintic resistance, condensed tannins, Haemonchus contortus, in vitro bioassays, plant extracts, Sericea lespedeza, small ruminants EVALUATION OF THREE IN VITRO BIOASSAYS FOR MEASURING THE ANTHELMINTIC ACTIVITY OF PLANT EXTRACTS CONTAINING CONDENSED TANNINS by JANET SUE HOWELL B.S., University of Alabama at Birmingham, 1992 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2009 © 2009 Janet Sue Howell All Rights Reserved EVALUATION OF THREE IN VITRO BIOASSAYS FOR MEASURING THE ANTHELMINTIC ACTIVITY OF PLANT EXTRACTS CONTAINING CONDENSED TANNINS by JANET SUE HOWELL Major Professor: Ray M. Kaplan Committee: James Noe Michael Yabsley Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2009 DEDICATION I would like to dedicate this work to all my family, past and present, who instilled an interest in education, and encouraged me along the way. I thank everyone who has supported me through this challenging process, particularly Bill and Jerry. I could never have accomplished this without you. iv ACKNOWLEDGEMENTS I would like to thank all of those who were instrumental in helping me obtain this challenging but rewarding degree. I would especially like to thank my mentor, Dr. Kaplan, whose patience and guidance these past years has been invaluable. I also want to thank my committee members, Dr. James Noe and Dr. Michael Yabsley for their guidance during this research. I would like to thank the “lab team”, particularly Bob Storey and Dr. Andy Moorhead, as well as, various undergraduate and graduate students, whose assistance has been greatly appreciated. I could never have completed this without you. I would like to recognize the USDA Sustainable Agriculture Research and Education (SARE) Grants Program for the funding of this project, and the SCSRPC members, in particular, Drs Tom Terrill, Joan Burke, and Jorge Mosjidis for their comments on the thesis. I would also like to recognize Dr. Jim Muir, Dr. B.R. Min and Richard Wolfe (Texas A & M) for the various plant extracts. I appreciated the comments and information regarding various aspects of the thesis work from Drs Jennifer Gill and Anand Vidyashankar. I would particularly like to thank my family, especially Bill and Jerry, who not only graciously prepared countless meals for me, but listened endlessly to my conversations about goats and parasites through many of those meals. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 LITERATURE REVIEW ..............................................................................................1 Introduction to Haemonchus contortus .....................................................................1 Haemonchus life cycle and behavior .........................................................................2 Haemonchus morphology and structures ..................................................................5 Clinical importance and pathogenicity of H. contortus ..........................................10 History of anthelmintic use and the development and diagnosis of resistance .......11 Alternatives to drug use in grazing ruminants .........................................................18 Introduction to plant tannins ....................................................................................19 Experimental plant extracts used in this study ........................................................26 Conclusions .............................................................................................................29 2 INTRODUCTION .......................................................................................................31 3 MATERIALS AND METHODS .................................................................................34 Parasite isolate .........................................................................................................34 Condensed tannin extracts .......................................................................................34 Egg Hatch/Larval Development Assays ..................................................................35 vi Larval Migration Inhibition Assay ..........................................................................37 Data analysis ............................................................................................................40 4 RESULTS ....................................................................................................................44 Egg Hatch Assay .....................................................................................................44 Larval Development Assay .....................................................................................44 Larval Migration Inhibition Assay ..........................................................................45 Parasite isolate and plate replicates .........................................................................45 5 DISCUSSION ..............................................................................................................51 REFERENCES ..............................................................................................................................58 vii LIST OF TABLES Page Table 1.1: Percent condensed tannin present in various trees and plants…………………….… 21 Table 3.1: Summary of assays performed……………………………………………………… 42 Table 4.1: 1-way ANOVA results for assays…………………………………………………... 47 viii LIST OF FIGURES Page Figure 1.1: Light microscopy image of adult female H. contortus ..................................................7 Figure 1.2: Scanning electron microscopy image of adult female H. contortus ..............................7 Figure 1.3: Light microscopy image of adult male H. contortus with bursa and spicules ..............7 Figure 1.4: Light microscopy image of adult female H. contortus with vulvar flap .......................8 Figure 1.5: Polarized microscopy image of H. contortus eggs ........................................................8 Figure 1.6: Light microscopy image of 1st stage free living larvae of H. contortus ........................8 Figure 1.7: Light microscopy image of 3rd stage larvae of H. contortus ........................................9 Figure 1.8: Scanning electron microscopy image of 3rd stage larvae of H. contortus .....................9 Figure 1.9: Scanning electron microscopy image of 3rd stage larvae of H. contortus .....................9 Figure 3.1: Schematic for the Larval Migration Inhibition Assay (LMIA) ...................................43 Figure 4.1: Graph of thiabendazole for the Egg Hatch Assay (EHA) ...........................................46 Figure 4.2: Graphs of condensed tannin extracts for the EHA ......................................................46 Figure 4.3: Graph of thiabendazole for the Larval Development Assay (LDA) ...........................48 Figure 4.4: Graphs of condensed tannin extracts for the LDA ......................................................48 Figure 4.5: Graph of the EC means of the dose responses for all 4 CT extracts for the LDA ... 49 50 Figure 4.6: Graph of levamisole for the LMIA..............................................................................50 Figure 4.7: Graphs of condensed tannin extracts for the LMIA ....................................................50 ix