BIOLOGICAL ACTIVITY OF EXTRACTS FROM COMMIPHORA SWYNNERTONII AGAINST MICROBES OF VETERINARY IMPORTANCE IN CHICKENS GAYMARY GEORGE BAKARI A THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF SOKOINE UNIVERSITY OF AGRICULTURE. MOROGORO, TANZANIA. 2013 i ABSTRACT Studies were carried out to establish ethno-botanical information and biological activities of crude extracts from Commiphora swynnertonii against selected microbes of veterinary importance in chickens. Initially, a questionnaire survey was conducted to gather information on practices and knowledge on ethno-botanical uses of C. swynnertonii in the study area. Then extracts from different morphological parts of the study plant were tested against selected bacteria and fungi in vitro using agar well diffusion assay. Resin and root bark extracts showed significant activities (P < 0.001) against S. pyogenes, E. coli and B. subtilis compared to other extracts. The fungi, C. albicans and A. niger, were moderately inhibited. Antiviral activity of the resin and root bark extract was tested in ovo using embryonated chicken eggs inoculated with Newcastle disease virus (NDV). Both extracts significantly (P < 0.001) and effectively reduced virus titres. An animal trial was carried out using the resin and chickens experimentally infected with NDV. Results revealed significant reduction (P < 0.05) in clinical signs and mortality rates following administration of the resin before and after the infection. Prophylactic administration of the extract was found to be more effective than the therapeutic approach. HI titres decreased significantly (P < 0.001) in resin and root bark treated groups and in all chickens treated with resin irrespective of dose given and on whether the extract was administered before or after infection suggesting that the plant materials were capable of destroying the NDV before stimulating the developing chick‘s immunity. Another animal trial investigated the effect of the resin against ii experimental coccidiosis in chickens. Results showed that oral administration of the resin significantly (P < 0.001) reduced mortality rate. Safety margin of the resin was also investigated by determining its effects on selected physiological and biochemical parameters in chickens. The results revealed a good margin of safety provided that the dosage ranges between 200 to 800 mg resin/kg body weights. A phytochemical study was also carried to determine major bioactive compounds in the resin and root bark extracts. With these studies, it is concluded that extracts from C. swynnertonii especially resin, has significant antibacterial, antifungal, antiviral and anticoccidial effect against the selected microbes. Further research is required to test and validate the extract against other pathogens of medical and veterinary importance. iii DECLARATION I, Gaymary George Bakari do hereby declare to the Senate of Sokoine University of Agriculture that this dissertation is my own original work and that it has neither been submitted nor being concurrently submitted for degree award in any other institution. ________________________________ _______________________ Gaymary George Bakari (PhD. Candidate) Date The above declaration is confirmed ___________________________________ _______________________ Prof. M.M.A Mtambo (Supervisor) Date ___________________________________ _______________________ Prof. . R.A. Max (Supervisor) Date _______________________________ _______________________ Prof. E.C.J. Phiri (Supervisor) Date iv COPYRIGHT No part of this dissertation may be reproduced, stored in any retrieval system or transmitted in any form or by any means without prior written permission of the author or Sokoine University of Agriculture in that behalf. v ACKNOWLEDGEMENT First and foremost, I thank God for owing me with good health and patience during the whole period of this study. It is my pleasure to acknowledge and express my sincere gratitude to a number of organisations and individuals through whose efforts contributed to the success of this study. To begin with, I wish to give special thanks to Prof. R. H. Mdegela, in his capacity as the Node coordinator of the CR AFFNET, for all his assistance and for the offer that enabled me to carry out my PhD studies. To my sponsor, CR-AFFNET, I am also grateful. . I also thank the RISE- AFNNET for the financial support of this work. My gratitude is due to my supervisors Prof. A. M Mtambo, Prof. R. A. and Prof. E. C. Phiri for their valuable guidance, suggestions, constructive criticism and devotion to the work throughout the study. Their support, consideration and loyalty have contributed greatly to the successful accomplishment of this dissertation. Special thanks to my colleagues: Ms. F. S. Mabiki, Dr. S. Mshamu and E. Barnabas, for the support and encouragement during the whole period of the study. I will also not forget Dr. C. J. Kasanga, Dr. H. Nonga, Prof. A. E. Pereka and Prof. S. P. Bittegeko for assistance on the improvement and shaping of this work. vi I would like to express my thanks to Mr. J. Fitwangile, A. Mwanyika, A J Ramadhani, P. Mkuchu, L. Ndaki, P. Jingu, D. Mwangoka and L. Msalilwa at the Faculty of Veterinary Medicine, Sokoine University of Agriculture for their valuable assistance during data collection and sample analysis. The technical assistance from Mr. K. Muganizi and Ms. P. Kalinga for taking care of my experimental chickens is highly appreciated. Mr K. Kayombo, the botanist at Olmotonyi Forest Institute is highly acknowledged for his assistance during the field work and identification of the plant species. Last but not the least; I extend my sincere thanks to my husband, Mr. Daudi P. Siarra, my son Aaron and my daughter, Mary-glory, my parents Dr. George S.M. Bakari and my mother Mrs. Mariam J. Bakari, my sisters and brothers for their patience, prayers, encouragement, moral, financial and material support during the whole period of the study. It is not possible to mention all individuals who assisted this work in one way or another, I sincerely express my gratitude. May the Almighty God bless all abundantly vii DEDICATION I would like to dedicate this work to my beloved husband (Daudi P.N. Siarra), children (Aaron and Mary-glory) and mostly, to my parents, Dr. George and Mrs. Mariam Bakari who laid the foundation of my education. viii TABLE OF CONTENTS ABSTRACT ........................................................................................................................ i DECLARATION ............................................................................................................. iii COPYRIGHT .................................................................................................................... iv ACKNOWLEDGEMENT ................................................................................................. v DEDICATION ................................................................................................................ vii CHAPTER ONE ................................................................................................................ 1 1. INTRODUCTION.......................................................................................................... 1 1.1 Importance of medicinal plants ................................................................................ 1 1.2 Research problem and justification of study ............................................................ 6 1.3 Objectives ................................................................................................................. 7 1.3.1 General objective .............................................................................................. 7 1.3.2 Specific objectives ............................................................................................ 7 1.4 Significance of this research .................................................................................... 8 CHAPTER TWO ............................................................................................................... 9 2. LITERATURE REVIEW .............................................................................................. 9 2.1 The genus Commiphora ........................................................................................... 9 2.1.1 Features of Commiphora species ...................................................................... 9 2.1.3 History of Commiphora spp ............................................................................ 12 2.1.4 Phytochemistry of Commiphora plants ........................................................... 12 2.1.5 Uses of Commiphora plants ............................................................................ 16 2.2 Local chickens in Tanzania .................................................................................... 25 2.3 Newcastle disease .................................................................................................. 26 2.3.1 Aetiology ......................................................................................................... 27 2.3.2 Host range and transmission ........................................................................... 27 2.3.4 Clinical symptoms ........................................................................................... 28 2.3.5 Gross and pathological lesions ........................................................................ 29 2.3.6 Control and prevention measures .................................................................... 29 2.4 Poultry coccidiosis ................................................................................................. 31 2.4.1 Aetiology and host range ................................................................................ 31 2.4.3 Transmission ................................................................................................... 32 2.4.4 General life cycle of Eimeria spp. .................................................................. 32 2.4.6 Gross and pathological lesions ........................................................................ 34 2.4.7 Management and treatment of avian coccidiosis ............................................ 35 CHAPTER THREE .......................................................................................................... 37 3. MATERIALS AND METHODS ................................................................................. 37 3.1 Study area ............................................................................................................... 37 3.2 Questionnaire design and administration ............................................................... 38 3.3 Sample collection ................................................................................................... 39 3.3.1 Preparation and extraction of plant materials .................................................. 39 3.3.2 Preparation resin extract for clinical trials ...................................................... 40 3.4. Investigating the effect C. swynnertonii on selected bacteria and fungi ............... 40 3.4.1. Agar well diffusion method ........................................................................... 40 ix 3.4.2 Minimum inhibitory concentration (MIC) ...................................................... 41 3.5 Investigating the cytotoxicity effect of C. swynnertonii on brine shrimps ............ 42 3.5.1 Hatching the brine shrimp ............................................................................... 42 3.5.2 The Bioassay ................................................................................................... 43 3.6 Investigating the effect C. swynnertonii on Newcastle disease virus .................... 43 3.6.1 Preparation of the inoculums and in-ovo antiviral assay ................................ 43 3.6.2 Estimation of virus in allantoic fluid using haemaglutination assay ............... 45 3.6.3 Haemaglutination Inhibition (HI) titres against NDV in hatched chicks ........ 46 3.6.4 Preparation of NDV inoculums for in vivo trial ............................................. 46 3.6.5 In vivo studies in experimental chickens ........................................................ 47 3.6.6 Experimental animals ...................................................................................... 48 3.6.7 Experimental design ........................................................................................ 48 3.6.8 Clinical observations ....................................................................................... 50 3.7. Investigating the effect of C. swynnertonii on coccidial parasite ......................... 51 3.7.1 In vivo studies in experimental chickens ........................................................ 51 3.7.2 Experimental animals ...................................................................................... 51 3.7.3 Isolation and identification of Eimeria spp. oocysts ....................................... 52 3.7.4 Grouping and treatments allocation ................................................................ 53 3.7.5 Observations and measurements ..................................................................... 54 3.8 Investigating the effect of C. swynnertonii on physiological parameters .............. 55 3.8.1 Experimental animals ...................................................................................... 55 3.8.2 Experimental design ........................................................................................ 56 3.8.3 Collection of blood for haematological and biochemical parameters ............. 56 3.8.4 Determination of haematological parameters ................................................. 57 3.8.5 Determination of biochemical parameters ...................................................... 59 3.9 Post mortem examination ....................................................................................... 62 3.10 Phytocemical screening of rootbark and resin extracts ........................................ 62 3.10.1 Test for tannins .............................................................................................. 62 3.10.2 Test for phlobatannins ................................................................................... 63 3.10.3 Test for terpenoids (Salkowski test) .............................................................. 63 3.10.4 Test for flavonoids ........................................................................................ 63 3.10.5 Test for cardiac glycosides (Keller-Killani test) ........................................... 64 3.10.6 Test for steroids ............................................................................................. 64 3.10.7 Test for saponins ........................................................................................... 64 3.10.8 Test for anthraquinones ................................................................................. 65 3.11 Data analysis ........................................................................................................ 65 CHAPTER FOUR ............................................................................................................ 67 4. RESULTS .................................................................................................................... 67 4.1 Ethnobotanical survey on traditional uses of C. swynnertonii in Simanjiro .......... 69 4.1.1 Demographic information ............................................................................... 69 4.1.2 Peoples‘ knowledge about the plant ................................................................ 69 4.1.3 Uses of C. swynnertonii .................................................................................. 70 4.2 Effect of C. swynnertonii on brine shrimps, bacteria and fungi in vitro ................ 72 4.3 Effect of resin from C. swynnertonii against Newcastle infection in ovo ............. 77