Aloe vera polysaccharides and proteins as biological response modifiers and their therapeutic efficacy against coccidiosis in chickens By KASHFA KHALIQ M.Sc (Hons) Microbiology Dissertation submitted in partial fulfillment of the requirements for the award of the degree of DOCTOR OF PHILOSOPHY In PARASITOLOGY DEPARTMENT OF PARASITOLOGY FACULTY OF VETERINARY SCIENCES UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2015 i To, The Controller of Examination(s), University of Agriculture, Faisalabad. We, the supervisory committee, certify that the contents and form of thesis submitted by Mrs. Kashfa Khaliq (93-ag-697) have been found satisfactory and recommend that it be processed for evaluation by external Examiner(s) for the award of degree. SUPERVISORY COMMITTEE: Chairman _______________________________ (Prof. Dr. Masood Akhtar) Member _______________________________ (Prof. Dr. Zafar Iqbal) Member ________________________________ (Prof. Dr. Iftikhar Hussain) ii UNIVERSITY OF AGRICULTURE, FAISALABAD DEPARTMENT OF PARASITOLOGY DECLARATION I, hereby declare that the contents of thesis, “Aloe vera polysaccharides and proteins as biological response modifiers and their therapeutic efficacy against coccidiosis in chickens” are product of my own research and no part has been copied from any published source (except the reference, standard mathematical or genetic models/ formulae /protocols etc.). I further declare that this work has not been submitted for award of any other diploma/ degree. The University may take action if the information provided is found inaccurate at any stage. (Kashfa Khaliq) Reg. No. 93-ag-697 iii Dedicated To My Sweet and Loving Parents, Teachers, Brothers, Sisters, Husband & My cute kids Ayesha, Hassan and Jannat iv TABLE OF CONTENTS Chapters Title Page No. List of Tables ii List of Figures iv Abstract vi 1 Introduction 01 2 Review of Literature 03 3 Materials and Methods 21 4 Results 33 4.1 Isolation and characterization of Aloe vera polysaccharides 33 4.2 Isolation and characterization of Aloe vera proteins 56 5 Discussion 81 6 Summary 95 References 98 Annexures 120 v ACKNOWLEDGEMENTS I am thankful to Most Gracious, Merciful and Almighty “ALLAH” who gave me the health, thoughts and opportunity to complete the work. I bow my compassionate endowments to the Holy Prophet “MUHAMMAD” (Peace Be Upon Him) who is ever, a torch of guidance and knowledge for humanity as a whole. I wish to express my profound gratitude to the members of my supervisory committee for their keen interest, inspiring guidance and valuable suggestions in planning and writing this manuscript. I pay special thanks to my worthy supervisor Prof. Dr. Masood Akhtar for his cooperative attitude, help, keen interest and provision of favorable environment for productive research. It is a great pleasure for me to thank Prof. Dr. Zafar Iqbal and Prof. Dr. Iftikhar Hussain, who enlightened my path to success by their kind guidance throughout my research work. I also like to thank Prof. Dr. Laeeq Akbar Lodhi, Dean Faculty of Veterinary Sciences for their moral support. I would like to thank Higher Education Commission (HEC) to provide funds for the current project and without which, it was almost impossible to complete this project. With deep sense of gratitude, I wish to thanks Dr. Mian Muhammad Awais, Dr. Muhammad Shahid, Prof. Dr. Amer Jamil, Dr. Mudassar and Prof. Dr. Shahbaz Talib Sahi for their technical help and cooperation during the research and also for their permission to conduct part of the research in their labs. I wish to extend my worthy thanks to my husband Mr. Mansoor Zafar and colleagues Dr. Qari Kaleem, Dr. Mateen Humayun, Dr. Rizwan, Dr. Tanweer, Dr. Irfan Ullah and Dr. Muhammad Saqib who supported me a lot for completion of my research project. Finally, I am immensely grateful to my parents, in-laws, brothers, sisters, kids and other family members for their moral and countless prayers for my success during the studies. May ALLAH grant all of the above mentioned personalities with success and honour. vi LIST OF TABLES Table Title Page No. No. 2.1 A tabulated presentation of history of medicinal uses of Aloe 6 vera 2.2 A tabular presentation of Aloe vera constituents and their 10 pharmacological activities 2.3 Tradional Medicinal Uses of Aloe vera in Ayurveda 15 4A Quantitative analysis of monosaccharides detected in the 39 hydrolyzed sample of Aloe vera polysaccharides 4.1 In vivo lymphoproliferative response to PHA-P in 40 experimental and control chickens 4.2 In vitro lymphoproliferative response to Concanavalin-A in 41 experimental and control chickens 4.3 Carbon particle clearance assay in experimental and control 42 chickens 4.4 Antibody response to sheep red blood cells in experimental 44 and control chickens 4.5 Organ-body weight ratios on 14th day post administration of 47 Aloe vera polysaccharides in experimental and control chickens 4.6 Weekly (1st-6th) weight gains in experimental and control 48 groups 4.7 Results of weekly FCR post administration of Aloe vera 49 polysaccharides in experimental and control chickens 4.8 Oocysts per gram of droppings post challenge in 50 experimental and control chickens 4.9 Daily weight gains from day 3rd to 12th post challenge in 51 experimental and control chickens 4.10 Per cent protection and mortality post challenge in 52 experimental and control chickens 4.11 Lesion scoring and per cent protection against lesions in 53 experimental and control chickens 4.12 Anticoccidial index for polysaccharides 54 4.13 Organ-body weight ratios post challenge in experimental and 55 control chickens 4B Bradford assay for protein quantification 62 4B1 Results of protein sample by Bradford assay 62 4B2 Profile of proteins isolated from Aloe vera by Automated 63 Electrophoresis system 4.14 In vivo lymphoproliferative response to PHA-P in 65 experimental and control chickens vii Table Title Page No. No. 4.15 In vitro lymphoproliferative response to Concanavalin-A in 66 experimental and control chickens 4.16 Carbon particle clearance assay in experimental and control 67 chickens 4.17 Antibody response to sheep red blood cells in experimental 69 and control chickens 4.18 Organ-body weight ratios on day 14th post administration of 72 Aloe vera proteins in experimental and control chickens 4.19 Weekly (1st-6th) weight gains in experimental and control 73 chickens 4.20 Results of weekly FCR post administration of Aloe vera 74 proteins in experimental and control chickens 4.21 Oocysts per gram of droppings post challenge in 75 experimental and control chickens 4.22 Daily weight gains in chickens from day 3rd to 12th post 76 challenge in experimental and control chickens 4.23 Per cent protection and mortality post challenge in 77 experimental and control chickens 4.24 Lesion scores and per cent protection against lesions in 78 experimental and control chickens 4.25 Anticoccidial index for proteins 79 4.26 Organ-body weight ratios post challenge in experimental and 80 control chickens viii LIST OF FIGURES Fig No. Title Page No. 4 A1 Chromatogram showing the peaks of monosaccharides in Aloe 39 vera polysaccharides at different retention times 4.1 In vivo lymphoproliferative response to PHA-P in experimental 40 and control chickens 4.2 In vitro lymphoproliferative response to Concanavalin-A in 41 experimental and control chickens 4.3a Carbon particle clearance assay (Clearance Index K) 42 4.3b Carbon particle clearance assay (Phagocytic Index α) 43 4.4a Antibody response to sheep red blood cells in experimental and 45 control chickens (At day 7th post primary injection) 4.4 b At day 14th post primary injection 45 4.4 c At day 7th post secondary injection 46 4.4d At day 14th post secondary injection 46 4.5 Organ-body weight ratios on 14th day post administration of Aloe 47 vera polysaccharides in experimental and control chickens 4.6 Weekly (1st-6th) weight gains in experimental and control groups 48 4.7 Results of weekly FCR post administration of Aloe vera 49 polysaccharides in experimental and control chickens 4.8 Oocysts per gram of droppings post challenge in experimental 50 and control chickens 4.9 Daily weight gains from day 3rd to 12th post challenge in 51 experimental and control chickens 4.10 Per cent protection post challenge in experimental and control 52 chickens 4.11 Per cent protection against lesions in experimental and control 53 chickens 4.12 Anticoccidial index for polysaccharides 54 4.13 Organ-body weight ratios post challenge in experimental and 55 control chickens 4 B Standard curve for protein quantification by Bradford assay 62 4 C1 Profile of proteins isolated from Aloe vera 64 4 C2 Profile of proteins isolated from Aloe vera 64 4.14 In vivo lymphoproliferative response to PHA-P in experimental 65 and control chickens 4.15 In vitro lymphoproliferative response to Concanavalin-A in 66 experimental and control chickens ix Fig No. Title Page No. 4.16a Carbon particle clearance assay (Clearance Index) 67 4.16b Carbon particle clearance assay (Phagocytic Index) 68 4.17a Antibody response to sheep red blood cells in experimental and 70 control chickens (At day 7th post primary injection) 4.17b At day 14th post primary injection 70 4.17c At day 7th post secondary injection 71 4.17d At day 14th post secondary injection 71 4.18 Organ-body weight ratio on day 14th post administration of Aloe 72 vera proteins in experimental and control chickens 4.19 Weekly (1st-6th) weight gains in experimental and control 73 chickens 4.20 Results of weekly FCR post administration of Aloe vera proteins 74 in experimental and control chickens 4.21 Oocysts per gram of droppings post challenge in experimental 75 and control chickens 4.22 Daily weight gains in chickens from day 3rd to 12th post challenge 76 in experimental and control chickens 4.23 Per cent protection post challenge in experimental and control 77 chickens 4.24 Per cent protection against lesions in experimental and control 78 chickens 4.25 Anticoccidial index for proteins 79 4.26 Organ-body weight ratios post challenge in experimental and 80 control chickens x ABSTRACT There are several natural biological products, which are effective to trigger immune responses in animals and human beings.