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Characterization of Mollicutes and Other

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Characterization of Mollicutes and Other CHARACTERISATION, PATHOGENICITY AND ANTIBIOTIC RESISTANCE OF MYCOPLASMAS INVOLVED IN VULVOVAGINITIS IN GOATS. BY OHIOMAH JOHN IZEBERE MATRIC NO: 125430. DVM (ABU), M.Sc (IBADAN). A THESIS IN THE DEPARTMENT OF VETERINARY MICROBIOLOGY AND PARASITOLOGY SUBMITTED TO THE FACULTY OF VETERINARY MEDICINE IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A PH.D DEGREE. APRIL 2013. i ABSTRACT Vulvovaginitis is an inflammation of the vulva and vagina caused by bacteria, fungi, viruses, parasites and allergy. The condition results in reduction in mating ability, infertility, abortion and death of the affected animals with resultant economic loss to the livestock industry. Although mycoplasmas have been isolated from cases of vulvovaginitis, their role as sole causative agents and in the pathogenicity of the disease have not been investigated in goats in Nigeria. The aim of this study was to characterise mycoplasma isolates from cases of vulvovaginitis and also and determine the pathogenicity of mycoplasma-induced vulvovaginitis in goats. Two hundred and twenty-one vaginal swabs were obtained from identified cases of vulvovaginitis in goats aged 8- 11 months from markets in and around Lagos metropolis. Samples were analyzed bacteriologically and mycoplasmotologically. Mycoplasmas were identified biochemically using standard procedures and conventional polymerase chain reaction with specific primers. Antisera raised in rabbits using 0.5ml of 4.0 x107 CFU/ml of selected Mycoplasma species including M. bovis, M. capri, M. capricolum and M. arginini were used to group the mycoplasmas serologically by growth inhibition method. Sensitivity of the isolated Mycoplasmas and other bacteria to aminoglycosides, fluoroquinolones, cephalosporins and nitrofurans was carried out. Pathogenicity of the isolated M. bovis, M. capri, M. capricolum and M. arginini was evaluated using 2ml inoculum containing 4.0 x 10 7 CFU/ml of each isolate per vulva to reproduce vulvovaginitis over a six week experimental period. Four goats were used for each experimental group and the control group. Animals were observed for symptoms of vulvovaginitis such as hyperemia, vulva swelling and vaginal mucus discharges. Post-mortem gross and histopathological examination was carried out and findings reported. Other data were analysed using descriptive statistics. Two hundred and fifty-seven bacterial isolates were recovered from the 221 field samples as follows: Mycoplasma capricolum (1.6%), Mycoplasma arginini (1.6%), Mycoplasma capri (1.2%), Mycoplasma bovis (0.78%), Ureaplasma species (1.2%), Acholeoplasma species (0.8%). Other bacteria were Escherichia coli (35.4%), Streptococcus species (29.2%) and Staphylococcus species (28.4%). Mycoplasma isolates were confirmed with the production of specific 280bp bands. Isolated Mycoplasma species and other bacteria were sensitive to ciprofloxacin and nitrofurantoin. Six different combinations of antibiotic resistant patterns were observed with amoxicillin, norfloxacin and ampicillin having the highest level of resistance (100.0%) and nitrofurantoin the least (33.0%). Clinical symptoms, which included hyperemia, swollen vulva, vaginal mucus discharges with pyrexia (38.9 – 39.60 C), were first observed in the M. bovis- infected group on day three, ten and thirteen. Mortality was recorded on days 30, 33, 38 and 41 post- inoculation in the M. bovis, M. capricolum, M. arginini and M. capri- infected groups, respectively. M. bovis produced the most severe lesions marked by lymphoid necrosis of the vulva tissue, diffuse hyperaemia in the lung alveolar septa and massive alveolar infiltration with neutrophils while the mildest lesions were observed in the M. arginine-infected group. Vulvovaginitis was reproduced in goats with all the Mycoplasma species as the major infective agent and Mycoplasma bovis as the most pathogenic. Ciprofloxacin and nitrofurantoin were the most effective antibiotics against Mycoplasmas and other bacteria isolated. Keywords: Vulvovaginitis, Mycoplasma species, Antibiotic sensitivity Word count: 498 ii CERTIFICATION I certify that this study has been carried out by Ohiomah John Izebere in the Department of Veterinary Microbiology and Parasitology , University of Ibadan, Ibadan, Nigeria. ................................................................................................................. SUPERVISOR PROF. A. I. ADETOSOYE DVM (ABU), Dr. MED. VET (VIENNA), CERT. ANIM. MANAG. AND REPROD. BIOL. (VIENNA), CERT. IN DIAGN. AND SERO. OF MYCOPLASMA (IEMVT, FRANCE); PH.D (IBADAN) PROFESSOR, DEPT. OF VETERINARY MICROBIOLOGY AND PARASITOLOGY, UNIVERSITY OF IBADAN, NIGERIA. iii DEDICATION This project work is dedicated to the Almighty GOD, my FATHER and CREATOR who is the pivot on whom my entire destiny revolves. iv ACKNOWLEDGEMENTS I want to use this opportunity to deeply appreciate my Supervisor and coach – Professor A.I.Adetosoye for his immeasurable guide, interest, support, corrections and handling of this work, daddy you have been there since 2004 and your presence has been a plus all the way, I am grateful to you, Sir. Prof. O. A. Akinboade, a God sent – thank you very much for all the valuable guide and interest shown me, I am grateful to you, Sir. Dr. A.T.P. Ajuwape – a mentor and a brotherly - father, I do appreciate all your inputs, they are very rewarding, the good God bless you, Sir. To my ever accommodating Acting Head of Department- Dr. (Mrs) O. A. Adediran, I am indeed very grateful for all your helps at all time, God bless you richly. Drs. Sadiq, Adejimi, Alaka, Ademola, Oluwayelu, Ikheloa, Aiki-Raji, Fagbohun, Ogunleye and Amosun- thank you all for making me feel accepted and belonged, your various inputs are unquantifiable- Our God bless you all richly. Dr. Kunle Ayinmode – a brother and friend, the Heavens and even the earth will reward you richly, thank you for making things work, God bless you. I thank all the non- teaching staff of the Microbiology laboratory- Mrs Orioke and her team- for their immense cooperation. I also extend my profound appreciation to Mr. Odeseye of the Nigeria Institute of Laboratory Science Technology (NILST), Ibadan and all the staff of the Microbiology section of the same Institute, for their contribution to the success of this work, may God bless you all. To my God given pal – Mr. Tusin Olajide, the God of helps will visit you suddenly for all your unquantifiable support, you are blessed. To my friend-Olumide Adekanmi and all the staff of Ized Veterinary, thank you so much. My siblings –Philip, Lara, Beauty, Rita and Omozei- I thank my God for you all and to our mother and father who packaged us early in life with the sense of love and togetherness- I love you all, and to Rita who could not wait to see the end of this program, rest peacefully in the bosom of the Lord till we meet to part no more, Amen. v My God given family – my beloved and cherished wife, Olubunmi, I love and appreciate you forever, my children- great men in Zion Joseph, Joshua, Peter and Praise the sons of Ohiomah John- thank you all for coping with me and always remember- daddy loves you very dearly. To the Pastorate at Canaanland –past and present- thank you for all your goodwill towards me and your prayers always- Our God will reward you all richly and individually. To my father in the Christian Faith, Bishop David. O. Oyedepo, thank you so much daddy for all your love, care and concern in all areas that you have and still do show towards me and all that is mine, I enjoy serving God with you, daddy I am grateful for the way you have taken and accepted me as the beloved, thank you daddy. And now to our heavenly God and Father – the Creator and Giver of Life, to JESUS CHRIST my Savior and Lord and to The HOLY SPIRIT my comforter and Reliable Guide- You are the pivot on whom I revolve and the anchor of my sustenance, I submit to you completely and that the next phase of life after this is in your loving hands, in this I rest my hope, thank you Lord, Amen. John O. Izebere April 2013. vi TABLE OF CONTENTS Title i Abstract ii Certification page iii Dedication iv Acknowledgements v Table of contents vii Lists of tables xiii Lists of plates xiv Lists of maps xv CHAPTER ONE. Introduction 1 Justification of study 6 Objectives 7 CHAPTER TWO. Literature review 8 2.1 Historical background 8 2.2 Taxonomy of the class Mollicutes 11 2.3 Animal Mycoplasmosis 14 2.4 Plant and Insect Mycoplasmosis 15 2.5 Mycoplasma species 17 2.5.1 Mycoplasma agalactiae 17 2.5.2 Mycoplasma arginini 18 2.5.3 Mycoplasma bovirhinis 18 2.5.4 Mycoplasma bovis 18 2.5.5 Mycoplasma capricolum 19 2.5.6 Mycoplasma conjunctivae 22 2.5.7 Mycoplasma gallinarum 23 vii 2.5.8 Mycoplasma mycoides ssp capri 23 2.5.9 Mycoplasma mycoides ssp mycoides 24 2.5.10 Mycoplasma ovipneumonia 27 2.5.11 Mycoplasma putrefaciens 28 2.6 Undetermined Species of Mycoplasma 30 2.7 Acholeplasma 32 2.8 Ureaplasmas 33 2.9 Cell Wall Morphology and Ultrastructure of Mycoplasma 34 2.10 Pathogenicity of Mycoplasma 35 2.11 Identification and Isolation of Mycoplasma 36 2.12 Susceptibility of Mycoplasma to Antibiotics 37 2.13 Predisposing Factors to Mycoplasma Infection 39 2.14 Disease cases in Various Animals 41 2.16 Goat Breeds in Nigeria 42 2.16.1 Goats 42 2.16.1a Sahel or Desert Goat 42 2.16.1b Sokoto Red Goat 42 2.16.1c West African Dwarf Goat 43 2.17. Mycoplasma and Ureaplasma involvement in Vulvovaginitis of Goats 48 CHAPTER THREE: 3.0 Isolation and Microscopic Identification of Mycoplasma and Ureaplasma isolates 50 3.1 Introduction 50 3.2 Materials and Methods 51 3.3 Innoculation and Incubation of Solid Media 53 3.4 Isolation and Morphologic Identification 54 3.4.1 Mycoplasma 54 3.4.2 Microscopy 54 3.4.3 Cloning of Isolates 54 3.5 Digitonin Sensitivity Testing 54 viii 3.6 Urease Test 55 3.7 Results 58 3.8 Discussion 60 CHAPTER FOUR 4.0 Molecular identification of Mycoplasma spp.
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