BIOLOGICAL CHARACTERISTICS OF KATAHDIN SHEEP SELECTED FOR FOOTROT RESISTANCE _______________________________________ A Dissertation presented to the Faculty of the Graduate School at the University of Missouri-Columbia _______________________________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy _____________________________________________________ by SAMANEH AZARPAJOUH Dr. Tumen Wuliji and Dr. William R. Lamberson, Dissertation Supervisors DECEMBER 2014 The undersigned, appointed by the dean of the Graduate School, have examined the dissertation entitled BIOLOGICAL CHARACTERISTICS OF KATAHDIN SHEEP SELECTED FOR FOOTROT RESISTANCE presented by Samaneh Azarpajouh, a candidate for the degree of doctor of philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. Dr.Tumen Wuliji Dr. William Lamberson Dr. Duane Keisler Dr. Tim Safranski Dr. Bruce Shanks Dr. Heide Schatten “Read: In the name of your Lord who created. Who created man from a clinging entity. Read! Your Lord is the most Noble, Who taught by the pen. Who taught man what he did not know.” (Qur’an, 96:1-5) “Call to me and I will answer you, and will tell you great and hidden things that you have not known.” (Jeremiah 33:3) “How great are your works, O Lord, how deep are your thoughts.” (Psalms 92:6) To my beloved family who taught me human excellence is in seeking the truth. ACKNOWLEDGEMENT There are many people that have played an important role over the past several years in helping me reach this point in life. While it is impossible to thank everyone, I would like to express my appreciation to several individuals for their assistance and guidance. I would like to gratefully and sincerely thank my graduate committee, Dr. Tumen Wuliji, Dr. William Lamberson, Dr. Duane Keisler, Dr. Tim Safranski, Dr. Bruce Shanks, and Dr. Heide Schatten for their guidance, patience, knowledge and support throughout my graduate experience. I would like to thank USDA and Lincoln University of Missouri for providing the source of funding for this research project. A special thanks goes to Lincoln University George Washington Carver Farm, Lincoln University of New Zealand and Electron Microscopy Core staff at University of Missouri – Columbia for their assistance to collect data. I must mention Lincoln University Cooperative Research faculty and staff, all the faculty and graduate students at Division of Animal Science, University of Missouri –Columbia for their friendships and assistance. I would like to express my deepest gratitude to Dr. Ahmadreza Mohamdnia, Dr. Marzieh Mehdizadeh, Dr. Charlotte Clifford-Rathert, Amy Bax, Heather Hilsenbeck, Cindy Thompson, Cindy Borgwordt, and Luke Wilber for their friendship and assistance in all aspects of my program. I have been far from my family in Iran, but have been fortunate to be part of Farley, Rollins and Kaplan families. Thank you for your love and support. The work presented in this dissertation would not have been possible without each and every one of you. Thank you. ii TABLE OF CONTENTS ACKNOWLEDGEMENT .......................................................................................................... ii LIST OF TABLES ................................................................................................................... vi LIST OF FIGURES ................................................................................................................. vii ABSTRACT .......................................................................................................................... viii Chapter 1. INTRODUCTION ........................................................................................................ 1 2. LITERATURE REVIEW ................................................................................................ 4 a. Genetics of disease resistance…………………………………………………………………….4 b. Marker assisted selection……………………………………………………………………………6 c. Footrot in small ruminants…………………………………………………………………………11 d. Etiology of footrot……………………………………………………………………………………..13 e. Footrot scores……………………………………………………………………………………………16 f. Heritability of footrot resistance………………………………………………………………..17 g. Phenotypic and genetic variation in footrot resistance……………………………..19 h. Selection for resistance to footrot……………………………………………………………..21 i. Phenotypic and genetic assessment for resistance to footrot…………………….23 j. Footrot gene-marker screening test…………………………………………………………..24 iii 3. COMPARISON OF SELECTED RESISTANT AND SUSCEPTIBLE KATAHDIN SHEEP TO OVINE FOOTROT USING A GENE MARKER SCREENING TEST………………………...26 a. Abstract………………………………………………………………………………………………….26 b. Introduction……………………………………………………………………………………………28 c. Materials and methods…………………………………………………………………………..30 i. Footrot gene marker test (FGMT) screening…………………………….……….30 ii. Footrot lesion scoring, swabbing and bacteria inoculants preparation……………………………………………………………………………………………..…31 iii. Footrot bacteria inoculants preparation……………………………………….….32 iv. Bacteria D. nodosus protease thermostability determination.............33 v. Footrot pathogenic challenge…………………………………………………...........34 vi. White blood cell count………………………………………................................36 vii. Red blood cell count………………...………………………................................37 viii. Packed cell volume (PCV) measurement………………….........................37 ix. WBC differential test……………...………………………...................................38 x. Immune response assay…………...………………………..................................38 d. Statistical analysis ………………………………………………………………………………..…39 e. Results.........................................................................................................39 f. Discussion....................................................................................................42 4. COMPARISON OF WHITE BLOOD CELL PHAGOCYTIC EFFICIENCY IN FOOTROT RESISTANT VS. SUSCEPTIBLE GENOTYPES OF KATAHDIN SHEEP......................55 iv a. Abstract……………………………………………………………………………………………......55 b. Introduction……………………………………………………………………………………………56 c. Materials and methods…………………………………………………………………………..58 d. Results........................................................................................................61 e. Discussion...................................................................................................63 5. COMPARISON OF ULTRASTRUCTURAL CHANGES CAUSED BY DICHELOBACTER NODOSUS IN STRATUM CORNEUM OF HOOF TISSUE OF SHEEP .....................71 a. Abstract……………………………………………………………………………………………......71 b. Introduction……………………………………………………………………………………………72 c. Materials and methods…………………………………………………………………………..74 d. Results.........................................................................................................75 e. Discussion....................................................................................................76 6. GENERAL DISCUSSION............................................................................................83 LITERATURE CITED ............................................................................................................ 87 VITA ................................................................................................................................. 101 v LIST OF TABLES Table Page 1. Footrot scores by hoof color, gene marker (low, medium and high resistance) and breed in treatment (D) group ............................................................................. 52 2. Average RBC count, WBC count, and PCV in C and D groups at the beginning (CB and DB) and the end of trial (CE and DE) ........................................................... 53 3. WBC differential count in C and D groups at the beginning (CB and DB) and the end of trial (CE and DE) ....................................................................................... 54 vi LIST OF FIGURES Figure Page 1. Comparison of average body condition score at the beginning (BCS1) and the end (BCS2) of trial between control (C) and treatment(D) groups ........................... 46 2. Comparison of average body weight at the beginning (WT1) and the end (WT2) of trial between control (C) and treatment (D) groups .......................................... 47 3. Comparison of IgG level in sera between control (C) and treatment (D) groups ... 48 4. Clear zones of proteolysis around inoculated wells with D. nodosus in the gelatin test before incubation ........................................................................................ 49 5. Clear zones of proteolysis around inoculated wells with D. nodosus in the gelatin test 8 min incubation .......................................................................................... 50 6. Clear zones of proteolysis around inoculated wells with D. nodosus in the gelatin test 16 min incubation ........................................................................................ 51 7. Comparison of the numbers of bacterial colonies after addition of neutrophils and incubation periods in the low resistant gene marker group vs. the high resistant gene marker group ............................................................................................. 68 8. Comparison of the numbers of bacterial colonies after addition of neutrophils and incubation periods in ewes vs. rams .................................................................. 69 9. Comparison of the number of bacterial colonies after addition of neutrophils and after 20,