Aloe Vera Polysaccharides and Proteins As Biological Response Modifiers and Their Therapeutic Efficacy Against Coccidiosis in Chickens

Aloe vera polysaccharides and proteins as biological response modifiers and their therapeutic efficacy against coccidiosis in chickens

KASHFA KHALIQ M.Sc (Hons) Microbiology

Dissertation submitted in partial fulfillment of the requirements for the award of the degree of

DOCTOR OF PHILOSOPHY

PARASITOLOGY

DEPARTMENT OF PARASITOLOGY FACULTY OF VETERINARY SCIENCES UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2015

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)

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

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Dedicated

To My

Sweet and Loving

Parents, Teachers, Brothers, Sisters,

Husband

My cute kids

Ayesha, Hassan and Jannat

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

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.

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

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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

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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

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

ABSTRACT

There are several natural biological products, which are effective to trigger immune responses in animals and human beings. In this regard, various plant species are considered to be potent natural biological products and their efficacy has been reported in various animal models. In the present study, Aloe (A.) vera derived biomolecules including polysaccharides and proteins were isolated and characterized as biological response modifiers and their subsequent protective effects against coccidiosis in chickens. A total of 640 chicks were randomly divided into two main treatment groups namely A (polysaccharides) and B (proteins), each containing 320 chicks. Each group was administered orally with the graded doses of polysaccharides and proteins for three consecutive days i.e. day 5th, 6th and 7th of age. Cellular immune responses were assessed for in vivo and in vitro lymphoproliferative responses to Phytohemagglutinin-P (PHA-P), Concanavalin-A (Con-A) respectively and Carbon particle clearance assay. Humoral immune responses were detected by microplate haemagglutination assay on 7th and 14th days post primary and secondary injections of sheep red blood cells. Weekly weight gains and feed conversion ratios were also calculated. For therapeutic studies chickens were challenged with mixed Eimeria species on 14th day post administration of A. vera biomolecules. Results revealed significantly higher (p<0.05) lymphoproliferative response to PHA-P and Con-A in chickens administered with A. vera biomolecules as compared to control. Carbon particle clearance assay showed significantly higher clearance index (K) in control group than all biomolecules administered groups and phagocytic index (α) showed significantly higher response in all three biomolecules administered groups as compared to control. Significantly higher total Igs, IgG and IgM titers were also detected in groups administered with A. vera biomolecules. Biomolecules administered chickens showed better feed conversion ratios and significantly higher (p<0.05) weekly weight gains as compared to control. In challenge experiment maximum protection 70% and 57.5% were observed in polysaccharides and proteins administered groups, respectively. Significantly lower oocysts per gram of droppings, lesser lesion scores, better weight gains and higher anticoccidial index were observed in biomolecules administered groups. From the current study, it was concluded that A. vera derived biomolecules have the potential to be used as immunotherapeutic agent(s) in poultry and can be commercialized.

CHAPTER 1 INTRODUCTION

The use of plants as therapeutic agents has a long traditional history; although isolation of their active compounds did not gain much attention till 19th century (Phillipson and Anderson, 2001). According to World Health Organization, 80% population of world used herbal medicines for differnt human diseases (Serrentino, 1991). In this regard, many plants and their constituents including Angelica gigas, Cissampelos pareira, Astragalus membranaceus, Mangifera indica, Ganoderma lucidum, Ocimum sanctum, Zingiber officinale, Phyllanthus emblica, Allium sativum, Curcuma longa, Nyctanthus arbor-tritis, Saccharum officinarum and Aloe vera (Minja, 1989; Waller et al., 2001; Fajimi and Taiwo, 2005; Githiori et al., 2006; Athanasiadou et al., 2007; Awais et al., 2011; Akhtar et al., 2012; Awais et al., 2013) have been reported for different medicinal properties.

Among these, Aloe (A.) vera (Aloe barbadensis Miller) is the most commonly used medicinal plant having historical importance. It is a succulent plant found in tropical and subtropical areas of many continents including Pakistan. Modern therapeutic use of A. vera started in 1920s and now it is found in many journals, topic of numerous researches and commercial literature on internet. It has been reported to cure variety of conditions including mild fever, burns, wounds, gastrointestinal disorders, sexual vitality, fertility problems, inflammation, arthritis, cancer, and acquired immunodeficiency syndrome (Bashir et al., 2011; Manvitha and Bidya, 2014). Now a day, A. vera is extensively used in many manufacturing industries of foods, pharmaceuticals and cosmetics (Yang et al., 2003).

A. vera is unique in nature having many biological active components including proteins, carbohydrates, vitamins, enzymes, minerals, sugar, lignin, saponins and salicylic acids (Surjushe et al., 2008). These compounds have numerous pharmacological activities including wounds healing, antinflammatory, antiarthritis, antioxidative, antidiabetic and anticarcinogen (Waihenya et al., 2002; Saritha et al., 2010; Iji et al., 2010). A. vera polysaccharides, anthraquinones and lectins have immunomodulatory effects (Leung et al., 2004; Akev et al., 2007; Liu and Wang, 2007). Its immunomodulatory activities include stimulation of macrophages, which produced nitric oxide and thus showed

effects on the antigen presenting cells (APC) to release cytokine(s) (Strickland, 2001). The most important component of A. vera gel is acemannan (a linear carbohydrate polymer containing acetylated mannose). It is a storage polysaccharide, present in parenchyma protoplast (Rodriguez et al., 2010) and has potential to act against viral infections, which reduced opportunistic infections and stimulate wound healing (Ni et al., 2004; Christiaki and Florou-Paneri, 2010). Its immunomodulatory effects had been reported in different animal models (Krishnan, 2006). It stimulates the natural and adaptive defense mechanism(s) including cytokines and help the body to protect itself (Alamgir and Uddin, 2010). Due to its biological response modifying activities it shows beneficial prophylactic and therapeutic effects against pathogens. It can be successfully used in oncology, inflammatory diseases, transplantation medicine and autoimmune disorders. Many types of colony stimulating factors, interferons, monoclonal antibodies, interleukins, tumor necrosis factor and erythropoietin are also being used as biological response modifiers (immunomodulators); body can produce them but in small quantity (Murthy et al., 2010). These can be prepared from certain species of plants, fungi (cell wall and cytoplasm) and bacteria (cell wall lipopolysaccharides); although they are not successfully used (Leung et al., 2004). As far as therapeutic efficacy of A. vera and its component(s) is concerned, it had been reported in different animal models and human beings with promising results (Strickland, 2001; Agarry et al., 2005), although limited work had been conducted on poultry (Mwale et al., 2006; Djeraba and Quere, 2000; Akhtar et al., 2012). Keeping in view the diverse biological functions of A. vera, the present study was conducted with following objectives:

1. Isolation and characterization of A. vera biomolecules including polysaccharides and proteins 2. Evaluation as biological response modifiers in terms of cellular and humoral immune responses in chickens 3. Assessment of their immunotherapeutic effects against avian coccidiosis in chickens. The general objectives of these studies were to investigate the potential of A. vera biomolecules as alternative therapeutic and immunomodulatory agents in chickens.

CHAPTER 2 REVIEW OF LITERATURE

Aloe (A.) vera is a hard, tropical, perennial, succulent, xerophytic and drought resistance plant (Surjushe et al., 2008; Ernst, 2000; Manvitha and Bidya, 2014). It belongs to kingdom Plantae, order Asparagales, family Liliaceae/Asphodelaceae and genus Aloe (Agarwal and Sharma, 2011). More than 500 species of A. vera has been reported so far among, which A. vera is most common (Bawankar et al., 2012).

The height and weight of mature A. vera plant (4-6 years old) ranges from 80-100 cm and 1.5-2 kg respectively. Under favorable conditions, it can survive for more than 50 years (Sharma et al., 2012). A. vera flowers are yellow tubular and fruit contains many seeds (Shelton, 1991). It has long, triangle, spiked and fleshy leaves, which has 20 inches length and 5 inches width (Moghaddasi and Verma, 2011). The leaves are made of three layers, upper layer termed as rind that synthesizes proteins and carbohydrates and have xylem, phloem and vascular bundles for transporting substances (water and starch). Lower layer termed as sap that contains bitter fluid, which protect it from being consumed by animals. Middle layer contains bitter yellow substance containing glycosides and anthraquinones. Inside these layers, a jelly like substance named mucilage gel is present. It is a clear substance made of 99 per cent water and glucomannans, vitamins, lipids, aminoacids and sterols (Balasubramanian and Narayanan, 2013).

Owing to variety of medicinal uses, A. vera is commonly known as Barbados Aloe, burn plant, Chinese Aloe, first aid plant, lily of desert, Indian Aloe, medicine plant and true Aloe (Liao et al., 2004). The synonyms are Aloe barbadensis Miller, A. elongate Murray, A. vera L chinensis Berger, A. rubescens DC, A. vulgaris Lam, A. indica Royle, A. perfoliata L. var. (Newton, 1979). The name of A. vera is driven from two words alloeh and vera; alloeh means shining bitter substance and vera means true (Surjushe et al., 2008; Joseph and Raj, 2010). Mostly, its four species are commonly used for medicinal purposes including Aloe barbadensis Miller (Chandana et al., 2007), Aloe arborescens (Morita et al., 2007), Aloe ferox (Zahn et al., 2007) and Aloe perryi baker (Eshun and He, 2004). Commercially Aloe barbadensis Miller and Aloe arborescens are grown today (Manvitha and Bidya, 2014). Of

3 these two, Aloe barbadensis Miller (Aloe vera) is most commonly used and studied species due to its diverse clinical efficacy against various health conditions including laxative, analgesic, antifungal, antitumour, antibacterial, immunomodulator, antidiabetic etc (Biswas and Mukherjee, 2003).

Summarizing these reports, A. vera is a plant of medicinal importance. Plants have become major basic source for new medicines, due to increased knowledge of their composition. About 25% medicines are plant origin (Irshad et al., 2011). Plants also have comparatively low adverse reactions as compared to conventional chemical based medicines (Ahmed and Hussain, 2013). Moreover, its juice has proven to be nontoxic (Manvitha and Bidya, 2014).

2.1: History of medicinal uses of Aloe vera

A. vera use as medicinal plant is as old as history of man. Mesopotamians and Egyptians had been using this plant since 1750 BC (Shelton, 1991). It has a traditional role in medicine like Ayurveda, Unani, Siddha and homeopathy. It has been used as medicine in many cultures from millennia including Egypt, China, India, Mexico, Greece and Japan. Greek scientists considered it a universal panacea and Egyptians regarded it as immortality plant (Marshall, 1990).

The famous beauty queens of Egypt Cleopatra (69-30 BCE) and Nefertiti (1353 BCE) used A. vera in their beauty products (Haller, 1990; Manvitha and Bidya, 2014). According to Bible, Jesus covered his hand with A. vera for soothing the pain and wound healing when he was hung on the cross. Arab physicians and Hippocrates also used it. Spanish explorers brought A. vera to western hemisphere. The Alexander the Great (333 B.C.) occupied Indian island named Socotra to secure A.vera supply, which was used to treat wounded soldiers. Its first English reference was translated by John Goodyew AD. 1655 Dioscorides Medical treatise De Materia Medica (Surjushe et al., 2008). The old well known physicians like Galen, Dioscsordes, Charaka and Surushta used A. vera in their medicines. In early 1800, it has been used as a laxative throughout United States. In 1930, it gained popularity in United States as it was used to treat X-ray burns (Rowe et al, 1941; Tyler et al., 1981). It was reported as a medicine in 1935 (Collin and Collin, 1935). Crewe used its gel locally for

4 treating pruritus vulvae and eczema in 1937 and found very effective. A. vera was also reported an effective agent against thermal burns in combination with mineral oil (Crewe, 1939). Its pulp was used for treating ulcers induced by radiation, which increased healing rate (Rowe et al., 1941). Barnes (1947) used its leaves with petrolatum for treating finger abrasions. The results showed that A. vera healed better than petrolatum mixed base (Barnes, 1947). In 1953, Lushbaugh and Hale studied its effects on beta irradiation induced wounds in rabbits. Histopathology of lesions showed a rapid healing of treated sites by increasing repair and degeneration rate. The treated wounds also have more collagen deposition (Lushbaugh and Hale, 1953). History of A. vera use has been summarized in table 2.1.

Table 2.2: A tabulated presentation of history of medicinal uses of Aloe vera Era/Year Scientist/Researcher/Publications Medicinal uses

50 B.C. Celsius  Laxative 41-68 A.D. Greek Herbal book  Wound healing  Heals tonsil  Sleep inducing agent  Boils treatment  Prevents hair loss  Cleanses stomach  Mouth and eye diseases 200 European physicians Aretaces,  Medicinal plant for clinical Galen and Antyllus purposes 700-800 Chinese  Fever  Convulsions  Sinus treatment 1300-1500 English  Purgative  Wound healing 1700-1900 In 1720, Carl Von Linne  Skin protecting agent Written in pharmacopeia of United  Purgative States in 1820.  Ulcers, dermatitis, radiation injuries and burns 1950 Amico and Luisa  Antibiotic property

1975 Galal et al.  Skin allergies  Cyst inflammation  Abscesses  Laceration 1979 Suzuki et al.  Mitogenic activity  Hemagglutination activity 1985 Bland  Indigestion  Infections 1987 Davis et al.  Wound healing 1992 Swaim et al.  Antibiotic activity  Wound healing 1994 Koo  Antiulcer  Antidiabetic 1997 Yagi et al.  Propagation and cellular growth of normal human dermal cells 2000 Pecere  Antineuroectodermal action in vivo and in vitro 2001 Yagi et al.  Wound healing

2002 Choi et al.  Angiogenic effect on embryonic chorioallantoic membranes  Reduced treatment related side effects 2003 Singh et al.  Liver diseases  Improve liver enzymes function, helpful in carcinogen metabolism 2004 Talmadge et al.  Hematopoietic and hematologic Rajasekaran et al. activity regulation of liver lung cytokines levels  Carbohydrate metabolizing enzymes regulation  Glucose homeostasis 2005 Paulsen et al.  Significantly reduced psoriasis lesions including erythema 2006 Davis and Rosenthal  Gel topical and oral administration Mwale et al. has anti-inflammatory effect  against avian coccidiosis 2007 Vaidya and Devasagayam  Antioxidant activity Bajwa et al.  Antimicrobial activity 2008 He and Wang  Proliferation of stem cells Madan et al.  increased white blood cells 2009 Takahashi et al.  Skin wounds  Immunomodulatory effect

2010 Kim et al.  Increased levels of immunoglobulin E and inflammatory cytokines in spontaneous atopic dermatitis

2011 Das et al.  Antiinflammatory and antifungal properties

2012 Akhtar et al.  Immunomodulator Kedarnath et al.  Immunotherapeutic efficacy against Halder et al. coccidiosis in chickens  Antifungal agent  Antimicrobial agent 2013 Thu et al.  Antimicrobial agent Arora et al.  Antimycoplasmic agent 2014 Suganya et al.  Antiinflammatory Ibe et al.  Antioxidant

2.2: Constituents of Aloe vera The leaves of A. vera have a yellow sap called Aloin, which is used by pharmaceutical industry as an active ingredient for laxative medicines. The intact fleshy part (inside leaf) contains cell walls and organelles, and is called parenchyma tissue or pulp. The other component is gel or mucilage. It is a clear viscous, semisolid, transparent and colorless gel inside the parenchyma tissues and is a main component of various skin products as moisturizer and nutritional agent since early 50s (Ni and Tizard, 2004).

A. vera plants contain more than 75 bioactive compounds, usually present in its gel and latex (Eshun and He, 2004; Joseph and Raj, 2010; Ajmera et al., 2014). Gel is mainly water (about 99%) and remaining portion contains monosaccharides (mannose-6-phosphate) and polysaccharides (gluco-mannans) (Shelton, 1991). It also contains a major glucomannan named as acemannan (commercially available), glycoprotein named alprogen (antiallergic) and C-glucosyl chromone (anti-inflammatory) (Hutter and Salmon, 1996; Ro et al., 2000).

A. vera gel extract contains 38% carbohydrates containing 4% glucose, 41% galacturonic acid, 6% xylose, 39% mannose, 1% rhamnose, 6% galactose, 1% fucose, 1% arabinose and 2% proteins (Luta et al., 2009; Marzorati et al., 2010). It contains eight essential amino acids, which are protein building blocks and influence the functioning of brain. Human body needs 22 amino acids, which can be synthesized except 8 essential amino acids, acquired through daily food intake. These all essential amino acids are present in A. vera including valine, isoleucine, methionine, tryptophan, leucine, threonine, phenylalanine, and lysine. Moreover, non-essential amino acids including aspartic acid, alanine, glutamine, arginine, tyrosine, serine, asparagines, histidine, cysteine, glutamic acid, glycine and proline are also present in the A. vera (Moghaddasi and Verma, 2011).

Phytochemicals present in A. vera include lectins, acetylated mannans, anthraquinones, C-glycosides, emodin, anthrones and polymannans (King et al., 1995; Boudreau and Beland., 2006); whereas, its qualitative phytochemical analysis have evinced presence of flavonoids, saponins, resins, tannins, glycosides, acidic compounds, anthraquinone and terpenoids. A. vera gel also contains sterols, reducing sugars and carbohydrates (Mariappan and Shanthi, 2012).

A. vera is also a natural source of vitamins A, B12, C, E, folic acid, thiamine and niacin. Minerals present in it are chromium, sodium, calcium, potassium, iron, magnesium, copper, manganese and zinc (Shelton, 1991). Glycosides named anthraquinones and aloin (A, B) are rich in A. vera latex (Tyler, 1994). Twelve different anthraquinones are present in A. vera and the most important are aloin and emodin, which have analgesic, antibacterial and antiviral activities. These compounds aid in breaking dead cells and resultant cell residues attract blood to the affected area and expel materials from ulcers and wounds. Recently, eighteen phenolic compounds had been isolated and identified from A. vera by reverse phase high performance liquid chromatography (HPLC) (Lopez et al., 2013).

Enzymes mainly present in A. vera are amylase, which degrades starches and sugars; bradykinase have analgesic, immunostimulant, and anti-inflammatory effects; creatinine phosphokinase effects on metabolism; cellulase digests cellulose; lipase digests fats and catalase avoids water accumulation in body and proteolyase causes hydrolysis of proteins into its constituents. Alkaline phosphatase, oxidase and carboxypeptidase present in A. vera act as biochemical catalysts. Enzymes also enable body cells to work efficiently (Nandal and Bhardwaj, 2012). A. vera contains proteolytic enzymes named glutathione peroxidase, carboxypeptidase and many isozymes of superoxide dismutase (Klein and Penneys, 1988; Sabeh et al., 1996).

The most important component of A. vera gel is acemannan (a linear polymer containing acetylated mannose). It is a storage polysaccharide (average molecular weight of 1000kDa) and present in parenchyma protoplast (Femenia et al., 1999; Rodriguez et al., 2010). It act as immunomodulator, antiviral, reduces opportunistic infections and stimulates healing (Ni et al., 2004; Christiaki and Florou-Paneri, 2010). HPLC analysis of gel and leaf extract showed presence of chromones, glucomannose, vitamin C, galactoglucoaralimannone, anthraquinone, gluconic acid and phenols (Mariappan and Shanthi, 2012).

Different constituents isolated from A. vera in several studies along with their pharmacological activities are summarized in table 2.2.

Table 3.2: A tabular presentation of Aloe vera constituents and their pharmacological activities

Constituents Processig Types of constituents General activities Reference type (compounds) Carbohydrates Gel  Acetylated mannan Antiallergic Ni et al.,  Acetylated glucomannan Antiinflammatory 2004;  Glucogalactomannn  Pure mannan Immunomodulation Jani et al.,  Xylan 2007  Galactogalacturan  Galactan  Arabinogalactan  Cellulose  Pectic substances  Hemicellulose  Arabinan  Arabinorhamnogaln  Glucuronic acid Proteins Leaf  Aloctin A & B Antibacterial Ni and Tizard,  ATF 11 Antiinflammatory 2004;  Salicylic acid Hemagglutination Davis and Maro, 1989 Aminoacids Leaf  Eight essential amino enzyme production Agarwal and acids including Muscle building Sharma, 2011  Valine  Isoleucine  Methionine  Tryptophan  Leucine  Threonine  Phenylalanine  lysine. Enzymes Leaf  Alkaline phosphatase Improve digestion Obata, 1993;  Peroxidase Antiinflammatory Shelton, 1991;

 Catalase Balance stomach Ro et al.,  Aliiase pH 2000; Nandal  Lipase  Cellulase and Bhardwaj,  Carboxypeptidase 2012  Amylase

Vitamins Gel  A, B, C, E, F, Folic acid Red blood cells Surjushe et and choline but vitamin production and al., 2008; D is not present development Agarwal and Amino acids Sharma, 2011; metabolism Moghaddasi Antioxidants and Verma, 2011 Anthraquinons Rind  12 anthraquinones Laxative Mckeon,1987; Latex present Antibacterial Lee et al., Sap  Anthrol Pain killer 2001; Yang et  Isobarbaloin Antifungal al., 2003; Yeh  Barbaloin Analgesic et al., 2003;  Arborescens Antibacterial Davis and  Emodin Antiviral Rosenthal, Antitumor 2006 Hormones Gel  Gibberellins Antiinflammatory Surjushe et  Auxins wound healing al., 2008 Chromones Leaf  8-C-glucosyl-(2’-O- Anti-tyrosine Okamura et cinnamoyl)-7-O- activity al., 1998; methylaloediol A Hamman,  8-C-glucosyl-(S)-aloesol 2008  8-C-glucosyl-7-O- methyl-(S)-aloesol  8-C-glucosyl-7-O- methylaloediol

 8-C-glucosyl-noreugenin  isoaloeresin D  isorabaichromone  neoaloesin A Sugars Gel  Monosaccharides Antiviral Nandal and including glucose and Immunomodulator Bhardwaj, fructose Antiinflammatory 2012  Polysaccharides including glucomannans and polymannose Sterols Gel  Cholesterol Antiseptic Saritha and Steroids  Lupeol Analgesic activities Anilakumar.,  Campesterol Antiinflammatory 2010;  Sitosterol Edema reduction Nandal and Bhardwaj, 2012 Saponins Leaf  Glycosides Antiseptic Narsih et al., CNS depressant 2012 Minerals Gel  Calcium Metabolic function Surjushe et  Zinc of many enzymes al., 2008  Magnesium Antioxidants  Chromium  Iron  Potassium  Manganese  Copper  Sodium. Salicylic acid Gel  Salicylic acid Analgesic Nandal and Antipyretic effects Bhardwaj, 2012

Phenolic Flower  Gentisic acid Antibacterial Lopez et al., compounds  Quercitrin Antimycoplasmic 2013  Epicatechin Miscellaneous Leaf  Arachidonic acid Shelton, 1991; compounds Peel  γ-linolenic acid Narsih et al.,  Uric acid  Potassiumsorbate 2012  Triglicerides  Carvone  Squalene  Limonene  Comaric  7-tetradecane  n-hexadecanoic acid  Eicosane

2.3: General uses of Aloe vera

The use of A. vera is common in many manufacturing industries of foods (drinks, jellies), medicines (pills, capsules), cosmetics and insecticides (Yang et al., 2003). Cosmetics include makeup, shampoo, soaps, moisturizers, sunscreens, shaving cream, sprays, lotions, creams and lip balms. It can penetrate deeply into dermal layers. Its gel has same antiaging effect as vitamin A (Danhof, 1993). Its juice contain oligoelements (manganese and selenium) act as antioxidant and antiaging agent, which strengthen cells to combat negative effects of oxygen and radiations. Daily gel application on skin has shown refreshing, regenerative, cleansing, blood supply stimulation, oxygenation and toxins elimination effects, which results smooth, elastic and moisturized skin (Byung, 1993). A. vera is also helpful to solve hair problems like hair loss and scalp. Its pH (6) is close to skin pH so it allows easy penetration in skin, which strengthens hair follicle and promotes hair growth. Bathfoam containing A. vera has reported superior cleansers, leave protective film on skin, which prevent dirt invasion and gives pleasant, clean and fresh sensation. Presently, A. vera is most widely and actively used ingredient in sun blocks skin preparations and cosmetics (Grindlay and Reynolds, 1986).

It is used as water conservator in small animal farms. It also played a role in fresh food preservation (Serrano et al., 2006). A. vera consumed along with vitamins C and E has improved their absorption for maximum 2-4 hours more than control. It has slowed vitamins absorption and they remained on plasma membranes for long time. It is only supplement, which increased absorption of these vitamins and also complement them (Vinson et al., 2005). It has unique benefits including penetration (ability to reach deepest layers of body tissues), cleanses (normalizes and detoxifies body metabolism) antiseptic (six agents to kill viruses, bacteria and fungi), stimulate cell growth and settle nerves by clearing effect on nervous system (Manvitha and Bidya, 2014). A. vera even in higher doses trials on rats, dogs and mice has showed no signs of intoxication or death. Its extract containing acemannan (at the dose rate of 2,000 mg/kg/day) to Beagle dogs for 6 consecutive months remained innocuous without any toxicity (Fogleman et al., 1992).

Table 2.4: Tradional medicinal uses of Aloe vera in Ayurveda

Medicinal uses References

Healing (Yim et al., 2011; Hamman, 2008)

Inflammation (Langmead et al., 2004; Sahu et al., 2013)

Dermatitis (Olsen et al., 2001; Kim et al., 2010)

Arthritis (Joseph and Raj, 2010; Kaur et al., 2012)

Bacterial infections (Waihenya et al., 2002; Agarry et al., 2005)

Viral infection (Sheets et al., 1991; Lee et al., 2001)

Fungal infections (Rodriguez et al., 2005; Das et al., 2011)

Parasitic infections (Dutta et al., 2008; Akhtar et al., 2012)

Anti-oxidant (Lopez et al., 2013; Ibe et al., 2014)

Cancers (El-Shemy et al., 2010; Harlev et al., 2012)

Diabetes mellitus (Yagi et al., 2009; Agarwal and Sharma, 2011)

HIV (McDaniel et al., 1990; Urch and David, 1999)

Constipation (Ishii et al., 1994; He and Wang, 2008)

Hepatitis (Neall, 2004; Bottenberg et al., 2007)

Heart diseases (Balch and James, 2000; Verma et al., 2013)

Antiseptic (Surjushe et al., 2008; Jain and Rai, 2014)

Immunomodulation (Ro et al., 2000; Akhtar et al., 2012)

2.4: Aloe vera as a biological response modifier and immunotherapeutic agent

Immune system involves several organs having complex cascade of mechanisms to protect against diseases. Biological response modifiers (BRMs) mean to modulate these immune mechanisms by using various synthetic or natural substances. Phytotherapeutic substances provide an alternative to conventional chemotherapy for several diseases, especially when host defense activation is required either in case of impaired immune response against any disease, or an immunosuppression required against autoimmune disorders. These phytotherapeutic agents have been advocated as promising alternatives to antibiotic therapy in many disease conditions. Several different types of BRMs (immunomodulators) have been identified so far, including natural sources (plants) and purified from different microorganisms. They may be defined as any substance of biological or synthetic origin, which can modulate, stimulate or suppress any of the components of innate or adaptive arms of the immune system (Kumar et al., 2012). Immunology is the most important developing area of medical research. It plays a basic role in prevention and treatment of various diseases including central organs, digestive tract, skin, respiratory system and joints. Presently infectious diseases are considered as immunological disorders. Immunomodulators are playing a central part in 21st century medicines. They normalize weak/overactive immune system (Patil et al., 2011).

Various constituents of A. vera have shown biological response modifying activities especially acemannan and alprogen. Acemannan (acetylated β1-4 linked glucomannan), a major component of its gel (Femenia et al., 1999; Gowda, 1979) showed both immunoregulatory and immunostimulatory effects including stimulation of humoral arm, increased phagocytosis and oxidative effects (Rajasekaran et al., 2005; Altug et al., 2010). Acemannan is a medium sized polysaccharide of molecular weight (200,000-1,000,000) (Zhang and Tizard, 1996; Liu et al., 2006; Zhang et al., 2006), maximum BRM activities have shown between 5 kDa to 400 kDa (Im et al., 2005). Acemannan (800mg/day) has significantly increased macrophages and circulating monocytes; and showed clinical improvements in acquired immunodeficiency syndrome patients (McDaniel et al., 1990). Acemannan also found effective against spontaneously evolved feline and canine

16 fibrosarcomas (King et al., 1995). Acemannan associated macrophage activation led to an increased phagocytosis and release of inflammatory cytokines including interleukin-6 and tumour necrosis factor (Zhang and Tizard, 1996), increased nitric oxide production (Karaca et al., 1995; Djeraba and Quere, 2000), hematopoiesis and induced immature dendritic cell proliferation (Lee et al., 2001). Moreover, stimulation of human lymphocytes antigenic response and all types leucocytes formation from bone marrow and spleen have also been documented (Singh et al., 2011). Alprogen, another BRM of A. vera origin, possesses inhibitory effect on the release of leukotriene, histamine and calcium influx from mast cells (Ro et al., 2000). A. vera glycoproteins (lectins) have also shown BRM activities and act as carrier proteins and activate T cells (Harlev et al., 2012).These activities occur by modulation of DNA damage activated signal transduction pathways. A. vera polysaccharides effect on cytokine cascade and antigen presenting cells (Strickland, 2001; Singh et al., 2011).

Several investigators reported that its gel components including acetylated mannan, glucomannan and galactogalacturan, significantly reduced tumor cells upon intraperitoneal administration in mice (Peng et al., 1991; Yagi et al., 1997). Acetylated mannan stimulated an increase in splenic cellularity, white blood cells, lymphocytes, monocytes and neutrophils in mice (Davis et al., 1989; Egger et al., 1996). A. vera potentiated both humoral and cell mediated immunity in mice, at the dose of 100mg/kg and suppressed delayed type hypersensitivity. The researchers suggested A. vera as an immunotherapeutic agent in immunologic diseases (Chandu et al., 2011). These immune regulatory activities have been shown due to presence of polysaccharides and glycoproteins (Dua et al., 1989). Polysaccharides have anticomplement activity (Hart et al., 1989). Its proteins and enzymes act as chemotactic agent, increase white blood motility (leucocytes) into stressed area (Fijita and Teradaira, 1976; Heggers and Robson, 1985; Reynolds and Dweck, 1999). A. vera carbohydrates showed hematopoietic activities (Talmadge et al., 2004). In another study, immunomodulation of its gel was assessed in mice (at dose rate of 150mg/kg and 300mg/kg body weight) for five days and total white blood cells count, antibody production, plaque forming cells and peritoneal macrophages phagocytic activity were assessed. Gel administration at the dose rate of 300 mg/kg showed significant increase in white blood cells (WBC) count, higher antibody titers, higher number of plaque forming cells and an increased phagocytic activity of peritoneal macrophages than control. It stimulated proliferation of

17 stem cells and humoral immunity, which is indicated by increased total white blood cell counts, plaque forming cells and circulating antibodies (Halder et al., 2012). Madan et al. (2008) suggested A. vera use as immunomodulator in immunosuppressed clinical diseases. Its polysaccharides are plant origin immunopotentiator (Sun et al., 2011) and activate T cell mediated immunity (Strickland, 2001), however immune regulation pathways are not completely understood (Kim et al., 1998). Beta-glucan polysaccharide in it acts as nonspecific immunopotentiating biologic response modifier (Pelley and Strickland, 2000). Beta- glucan and acemannan stimulate macrophage by binding mannose receptors (Weis et al., 1998). Orally taken polysaccharides are partially hydrolyzed by intestinal bacteria into fragments. Further they can bind gut epithelium and show localized effects on immune system. They can also be absorbed in blood stream and show systemic effects (Ramberg et al., 2010).

Kwon and his coworkers (2011) investigated levels of Th1, Th2, IL-2, IL-4, IL-10, IgA and IFN-γ in mice. A. vera medicated groups produced significantly high concentration of IL-4, IL-10 and IgA. However, IFN-γ and IL-2 concentration showed non-significant response. IgA represented as an important mucosal surface antibody. IL-4 (cytokine) showed very important effects in humoral immunity through precursor T cells differentiation to Th2 helper cells. It also increased IgG1 production from B-lymphocytes (Nicola, 1994). Increased IL-4 concentration produced more antibodies in medicated groups. IL-10 (an antiinflammatory cytokine produced by Th2 cells) antagonized Th1 and inhibited IL-2, IL-6, and IFN-γ production from macrophages (Florquin et al., 1994); whereas, Th1cytokines (TNF-α, IFN- γ, IL-1, IL-2, and IL-6) involved in septic shock (Bayston and Cohen, 1990; Rongione et al., 1997). Cytokines act as biological response modifiers by modulation of immune responses, haematopoiesis and inflammation in innate and adaptive immunity. Consequently, induced cytokines were balanced by A. vera peel extract. Its active anthraquinones (aloin and emodin) components showed immunomodulatory effects (Pandey, 2010) by reducing Th1cytokines and promoting Th2 (Liu et al., 2009). A. vera peel medication also showed elevated levels of IgG and IgA (Kwon et al., 2011).

A. vera administration has showed marked increase in proliferative and phagocytic activity of reticuloendothelial system (Im et al., 2005). It stimulated humoral and cell

18 mediated immunity by stimulating cells forming erythroid and myeloid colonies, granulocyte macrophage colony-forming cells and murine pluripotent hematopoietic stem cells (Egger et al., 1996; Boudreau and Beland, 2006). Various scientists reported that these immunomodulatory effects were due to innate immune cells activation (NK cells, macrophages, lymphocytes and neutrophils), production and release of cytokines (IL-1, IL-2, IL-6, IL-8, IFN-γ and TNF-α), increased nitric oxide production and induced antibodies production (Yates et al., 1992; Pugh et al., 2001; Brown and Gordon, 2003; Boudreau and Beland, 2006).

Vahedi et al. (2011) investigated effects of A. vera extract on cellular and humoral immune responses in rabbits. Results showed a significant increase in CD4+(after 14 and 21 days) and CD8+lymphocytes (after 7, 14 and 21 days) in administered groups as compared to control. CD4+ (white blood cells) have essential role in combating against infections. Its count in blood indicated functioning of immune system (Shirwan et al., 2003). These CD8+cells (transmembrane glycoprotein), function as co receptor of T cell receptors. They bind to major histocompatibility complex class 1 present on natural killer cells and cytotoxic T cells (Vecchione et al., 2002). Various researchers showed that A. vera administration enhanced IgG, CD4+ and CD8+concentrations. Therefore, immune enhancing action might be due to increased synthesis and release of cytokines (IL-1, IL-2, IL-6, IL-8, IFN-γ and TNF-α) and production of specific and nonspecific antibodies (Hanaue et al., 1989; Qiu et al., 2000; Sampedro et al., 2004; Leung et al., 2004). CD8+cells have a significant role in cellular immune responses and CD4+ cells take part in humoral and cellular immune responses. CD4+ according to their function (cytokines release) divided into two types of cells, Th1 and Th2. Th1 stimulated cells activate macrophages and induce T cell cytotoxicity by release of cytokines (TNF-β, IL-2, and IFN-γ). These stimulated Th2 cells produce B cells, stimulate cytokines (IL-4, IL-5, IL-10 and IL-13), which further increased B cells production (Chinnah et al., 1992).

Altug et al. (2010) investigated A. vera and β-Glucan effects on immune system in polyvalent vaccinated dogs. Their findings match with previous researchers as A. vera medication stimulated cellular and humoral immune responses, increased platelets count and restored thrombocytopenia.

The effects of ethanolic and aqueous extracts of A. vera on immune functions of broiler birds with induced coccidiosis has been investigated in our laboratory. Results of study showed a significantly higher lymphoproliferative response in chickens administered with ethanolic extracts. Birds showed 60 % protection against coccidiosis challenge. It was concluded that A. vera may have a potential to stimulate immune responses and can be successfully used as immunotherapeutic agent against coccidiosis in industrial broiler chickens (Akhtar et al., 2012).

CHAPTER 3 MATERIALS AND METHODS

3.1. Procurement and processing of Aloe vera

Aloe (A.) vera (Aloe barbadensis Miller) used in the present study were procured from Botanical garden, University of Agriculture, Faisalabad (UAF), Pakistan and got authenticated by a botanist in the Department of Botany, UAF, Pakistan. The plant specimens were kept in the Ethno-veterinary Research and Development Centre, Department of Parasitology, UAF as voucher No. 0173. Immediately after harvesting, leaves were washed with water containing chlorine (5-10 parts per million; ppm) and then with a solution containing formalin (37 % formaldehyde; 0.001-0.005 ppm) and finally with distilled water (Femenia et al., 1999). 3.1.1. Preparation of Aloe vera leaf gel Mucilaginous leaf gel was collected from the cleaned A. vera leaves within three to four hours post-harvesting to minimize any deterioration. For this purpose, one leaf was taken at a time, placed its flat side on a cutting wooden board and epidermis was removed by using sharp knife. Afterwards, serrated edges of both sides of the leaf were cut off to make slices of the leaf lengthwise. With the help of a wooden spatula, gel was scrapped out, thoroughly homogenized followed by filtration through cheese cloth and stored in screw capped glass bottles at 4°C in a refrigerator for further use. 3.2. Isolation and Characterization of Aloe vera biomolecules 3.2.1. Polysaccharides Extraction

Polysaccharides from the A. vera gel were extracted according to the method described by Chang et al. (2011). Briefly, A. vera gel, was vigorously mixed with four volume of 95% (v/v) ethanol and kept overnight at 4 °C. The precipitate thus obtained was centrifuged at 6500 g for 10 minutes and supernatant was discarded. The precipitate dissolved in double distilled water and kept overnight. It was again precipitated with four volume of 95% (v/v) ethanol. The precipitation and solubilization procedures were conducted repeatedly to remove all the colored materials. Finally, the precipitate thus obtained was

21 mixed in doubled distilled water and treated with Sevag reagent {1:4 (v/v) (1-butanol: 4- chloroform)}. Free proteins were removed by repeated oscillation and centrifugation (Staub, 1965). This solution was mixed with three volumes of ethanol (95%, v/v) which precipitated polysaccharides. The crude polysaccharides thus obtained were further purified by washing twice with absolute ethanol followed by acetone and ethyl-ether. Then, it was dried completely at 40°C for 48 hours (Chang et al., 2011). 3.2.2 Characterization 3.2.2.a. Hydrolysis

The polysaccharides were hydrolysed to get the monosaccharides according to the procedure described by Osborn et al. (1999) with minor modifications. Briefly, the polysaccharides were refluxed in 2M Trifloroacetic acid (TFA) (Sigma-Aldrich®, USA) at 100°C for 2 hours in a round-bottomed flask equipped with a reflux condenser. The TFA was then removed by evaporation at 75°C and the water was removed by freeze drying (-65 °C) overnight.

3.2.2.b. High Performance Liquid chromatography (HPLC) analysis for determination of monosaccharides

The monosaccharides were analysed on a Shimadzu-10A HPLC workstation (Japan) supplied with a quaternary gradient pump unit and a refractive index detector (RID). The analytical columns to get absorption spectra included Rezex RCM-Monosaccharide Ca+2 and Phenomenex (80°C temperature and a wavelength of 235 nm). Isocratic double distilled water (DD H2O) was used as mobile phase. For analysis 20μl injection volume was taken for every sample and standards.

3.3. Aloe vera proteins extraction Proteins from A. vera gel were extracted according to the method adopted by He and Wang (2008) with minor modifications. Briefly, A. vera leaves were ground with liquid nitrogen. The powdered leaf tissue (0.2 g) was washed with acetone (1mL) containing 0.2% dithiothreitol. Then it was vortexed for 1 minute in an eppendorf tube and centrifuged at 15,000 g for 20 minutes at 4°C followed by drying on ice. Powdered tissue (2 g) was homogenized in 2 ml of 2X extraction buffer (20mM Tris-HCl; pH 7.5, 250 mM sucrose, 10

22 mM ethylene glycol tetraacetic acid, 1mM phenylmethylsulfonyl fluoride, 1% Triton X-100 and 2% β-mercaptoethanol). The proteins thus precipitated were dissolved in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS (3-[(3 Cholamidopropyl) dimethylammonio]-1- propanesulfonate), 2% ampholine (pH 3–10) and 1% dithiothreitol). Protein concentration was measured by Bradford method (Bradford, 1976). 3.3.1.a. Quantification of protein

The method involves the binding of Coomassie Brilliant Blue G-250 to protein. This dye binding caused changes in absorption from 465-595 nm, and increased in absorption at 595 nm was monitored by spectrophotometer.

Coomassie Brilliant Blue G-250 (Sigma) 100mg was dissolved in 50 mL of 95% ethanol. To this solution 100 mL 85% (w/v) phosphoric acid was added. The resulting solution was diluted to final volume of one liter. Final concentrations in the reagent were 0.01% (w/v) Coomassie Brilliant Blue G-250, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid.

3.3.1.b. Protein assay (standard method)

Protein solution containing 10 to100µg protein in a volume up to 0.1 mL was pipetted into 12 x 100 mm test tubes. The volume in the test tube was adjusted to 0.1 mL with appropriate buffer. Five milliliters of protein reagent was added to the test tube and the contents mixed either by inversion. The absorbance at 595 nm was measured after 2 minutes and before 1 hour in 3 ml cuvettes against a reagent blank prepared from 0.1 mL of the appropriate buffer and 5 mL of protein reagent. The weight of protein was plotted against the corresponding absorbance resulting in a standard curve used to determine the protein in unknown samples.

3.3.1.c. Microprotein assay

Protein solution (extracted from A. vera) containing 1- 10 µg protein in a volume up to 0.1 ml was pipetted into 12 x 100 mm test tubes. The volume in the test tubes was adjusted to 0.1 mL with the appropriate buffer. One milliliter of protein reagent was added to the test tube and the contents mixed as in the standard method. Absorbance at 595 nm was measured as in the standard method except in 1 mL cuvettes against a reagent blank prepared from 0.1

23 mL of the appropriate buffer and 1 mL of protein reagent. Standard curves were prepared and used as in the standard method.

3.3.2. Characterization of proteins Proteins isolated from A. vera leaves were subjected to automated electrophoresis system (BioRad®, USA) for their quantification and determination of molecular masses according to the instructions of the manufacturer.

3.4. Infective material for challenge experiment

3.4.1. Collection and processing of chicken guts

Chicken guts suspected to be naturally infected with Eimeria species were collected from different commercial poultry sale points and outbreak cases of poultry farms of Faisalabad district. All the collected guts were brought and investigated in the Immunoparasitology Laboratory, Department of Parasitology, University of Agriculture, Faisalabad, Pakistan. Guts were opened by giving longitudinal incision and contents were examined by direct microscopy (Soulsby, 1982). The contents from the positive guts were placed in 2.5% sodium hypochlorite (Speer et al., 1973). The suspension was diluted twice with distilled water (4:1) to remove sodium hypochlorite and the sediment thus obtained was subjected to sporulation process.

3.4.2. Sporulation and purification of oocysts

The contents from the positive samples were subjected to sporulation in 2.5% potassium dichromate (K2Cr2O7) solution (upto 6 mm thickness) in petri dishes. The sporulation was confirmed by microscopic examination after an interval of every 12 hours. After the completion of sporulation, suspension was thoroughly mixed with the help of a glass rod and passed through a muslin cloth followed by filtration through a sieve. The filtrate was centrifuged at 1500g for 10 minutes and sediment thus obtained was subjected to

Zinc Sulphate (ZnSO4) Floatation technique following the method of Levine (1961). Briefly,

ZnSO4 floatation solution (Annexure I) was mixed with equal quantity of the suspension of sporulated oocysts and centrifuged (1500 g for 10 minutes). The supernatant having sufficient number of sporulated oocysts was aspirated by using the pippeting system and collected in a separate jar. The sediment was processed in the same way until no sporulated

24 oocysts remained in the supernatant. Sporulated oocysts were washed thrice with phosphate buffered saline (PBS; pH 7.2), placed in a sterilized screw capped bottle and stored at 4°C till further use.

Sporulated oocysts, mixed species of genus Eimeria, were identified on the basis of their predilection site, morphology and size according to Reid and Long (1979) (Annexure II).

3.4.3. Quantification of Sporulated oocysts

Purified suspension of sporulated oocysts (mixed species; local isolates) was subjected to McMaster counting technique to quantify the number of sporulated oocysts per mL of suspension and to adjust the infective dose (Ryley et al., 1976). The final concentration was adjusted to 6.5×104-7.0×104 sporulated oocysts per 2mL of PBS.

3.5. Experimental design

The whole study was divided into two experiments viz. Experiment I and II. Experiment-I was meant for the evaluation of polysaccharides from A. vera gel for their biological response modifier (BRM) and immunotherapeutic activities; whereas, Experiment- II was conducted to evaluate the BRM and immunotherapeutic activities of proteins isolated from A. vera leaves.

3.5.1. Experiment-I

A total of 320 day old industrial broiler chicks (Hubbard) purchased from local hatchery were reared in coccidia-free environment at Experimental Station, Department of Parasitology, University of Agriculture, Faisalabad, Pakistan. All the chicks were fed withdrawal feed and water ad libitum. After 5 days of acclimatization, chicks were randomly divided into four main treatment groups namely A1, A2 and A3 each containing 80 chicks; whereas, A4 served as control group. Each treatment group was further divided into two sub- groups each containing 40 chicks and were administered orally with the following treatments by using oral gavages for three consecutive days i.e. day 5th, 6th and 7th of age as per schedule.

Groups Treatment Dose/ Kg of body weight/day

A1 Polysaccharides from A. vera gel 100 mg

A2 (Each conc. dissolved in 3 mL PBS) 200mg

A3 300 mg

A4 Control group (PBS) 3 mL

3.5.2. Experiment-II

For experiment-II, 320 day old industrial broiler chicks procured from local hatchery were reared in the same conditions like that of Experiment-I at Experimental Station, Department of Parasitology, University of Agriculture, Faisalabad, Pakistan. After 5 days of acclimatization, chicks were randomly divided into four main treatment groups namely B1,

B2, and B3 containing 80 chicks. The group B4 served as control group. Each treatment group was further sub-divided into two sub-groups each containing 40 chicks and were administered orally with the following treatments by using oral gavages for three consecutive days i.e. day 5th, 6th and 7th of age as per schedule.

Groups Treatment Dose /Kg of body weight/day

B1 Proteins from A. vera leaves 100 mg

B2 (Each conc. dissolved in 3 mL PBS) 200 mg

B3 300 mg

B4 Control group (PBS) 3mL

In both the experiments, chickens in all the groups were routinely vaccinated according to the routine schedule (Annexure III) (Awais, 2010).

On day 14th post-administration of A. vera bio-molecules, half of the chickens from each group were used for immunological evaluation and the remaining half for therapeutic evaluation

3.6. Immunological evaluation

The effect of A. vera bio-molecules on the cellular and humoral immune responses were investigated. Cell mediated immune response was evaluated by in vivo and in vitro lymphoproliferative responses to Phytohemagglutinin-P (PHA-P), Carbon particle clearance

assay and Concanavalin-A (Con-A), respectively; whereas, antibody responses to sheep red blood cells (SRBCs) were used to detect the humoral immune responses.

3.6.1. Cell mediated Immune responses 3.6.1.a. In vivo lymphoproliferative response to PHA-P

Classic toe-web assay was used to quantify the in vivo lymphoblastogenic response to PHA-P as described by Corrier (1990).

Test Procedure

i. On day 14th post-administration of A. vera bio-molecules, experimental and control chickens were injected PHA-P (Sigma®, USA) intradermally (100µg/100µL/chicken) between the third and fourth digits of the right foot. ii. The left foot was injected with PBS (100µL) to serve as control. iii. The thickness of the inter-digital skin was measured at 24, 48 and 72 hours post injection with the help of a pressure sensitive micrometer screw gauge. iv. Lymphoproliferative response to PHA-P was calculated by using the formula. Lymphoproliferative response = (PHA-P response, right foot) – (PBS response, left foot)

3.6.1.b. In vitro lymphoproliferative response to Concanavalin-A (Con-A)

Peripheral blood lymphocytes blastogenesis assay was used to evaluate the in vitro lymphoproliferative response in both treated and untreated chickens according to the method described by Qureshi et al. (2000).

Procedure

i. On day 7th and 14th post administration of A. vera bio-molecules, birds from each experimental and control groups were randomly assigned for in vitro lymphoblastogenesis assay. ii. Chickens were killed humanely and blood samples (5mL/chicken) were collected by heart puncture with a heparinized syringe and diluted with equal volume of PBS. iii. The peripheral blood lymphocytes (PBLs) were separated by layering the heparinized blood on Histopaque-1077 (Sigma®, USA) followed by centrifugation at 400g for 30 minutes at room temperature (26°C).

iv. Mononuclear cells migrate during centrifugation, forming a distinct, opaque layer at the plasma- Histopaque-1077 interface. Plasma phase (top layer) was removed carefully and lymphocytes layer was collected by aspiration. v. The cells were washed thrice with PBS and adjusted to a concentration of 3x106/mL of RPMI-1640 growth medium (Sigma-Aldrich®, USA), supplemented with fetal calf serum (Sigma-Aldrich®, USA) (7%) inactivated at 56 °C and 1μg/mL gentamicin. vi. PBLs (100µL suspension), containing 0.3x106 cells from each group were added to five wells (in duplicate) of a flat bottomed microtitration plate (96-wells; Medium binding, polystyrene, Flow Labs., UK) followed by the addition of Con-A (Sigma®, USA) (25 µg/100µL) to five wells; whereas, other five wells served as unstimulated controls.

vii. The plates were incubated at 41°C in an incubator in the presence of 5% CO 2 and 50- 70% humidity. viii. After 22-24 hours of incubation, 50µL of a 0.1mg/mL stock of MTT (3-[5,5- dimethylthiazol-2-yl]-2,5-[diphenyltetrazolium]) (Sigma®, USA) was added to each well and the plates were re-incubated for another 4 hours. ix. After that, liquid from all the wells was carefully removed and 150 µL of MTT acid (200 mL 2-propanol [isopropyl alcohol] + 1.32 mL conc. HCl + 100 mL PBS) was added to each well and Trypan blue crystals (Merck®, Germany) were dissolved by repeated pipetting. x. The optical density (OD) was read on a plate reader (BioTek-MQX200, USA) at 540 nm wavelength. xi. The mean OD values from each group were used to calculate the stimulation indices by using the formula: Mean OD value = Con A stimulated – unstimulated unstimulated 3.6.1.c. Carbon particle clearance assay

The phagocytic ability of chickens blood cells was determined by carbon particle clearance assay. On day 14th post administration of A. vera polysaccharides (experiment I) and proteins (experiment II) carbon particle clearance assay was performed as described by Zhang (2007). Briefly, India ink was centrifuged to get supernatant. A 0.05ml/10g/body

weight, India ink was injected into wing vein of chickens and blood was collected from other wing vein at 3 and 15 minutes post injection of India ink. The blood samples were placed in tubes containing 2ml of 0.1 % sodium carbonate and optical density values were measured at 600nm in a spectrophotometer.

The clearance index (K), phagocytic index (α) and immune organ index (organ-body weight ratios) were calculated by following formulas (Hou et al., 2010).

K = (logOD3–logOD7) t2-t1

α = ______Body weight______(spleen weight+liver weight) 3√K

Immune organ index= immune organ weight (mg) Body weight (g)

3.7. Humoral Immune responses 3.7.1. Antibody response to sheep red blood cells (SRBCs)

Sheep red blood cells were used as T-dependent antigens to demonstrate the antibody response.

Preparation of SRBCs

i. Blood sample (5mL) was collected from a healthy sheep maintained by the Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan, through jugular vein in a sterile tube containing anti-coagulant EDTA (10mg EDTA/5mL of blood). ii. Blood collected was centrifuged at 1500g for 15 minutes, plasma and buffy coat layers were decanted and pelleted RBCs were washed thrice with PBS. iii. The concentration of washed RBCs was adjusted to 5% (v/v) with PBS. Haemagglutination Test Anti-SRBC antibody titers were detected by using microplate haemagglutination test according to the method of Yamamoto and Glick (1982) with minor modifications suggested by Qureshi and Havenstein (1994).

On day 14th post administration of A. vera bio-molecules, 10 chickens from each experimental and control groups were injected with 1 mL of 5% saline suspension of SRBCs via intramuscular route. At day 14th post-first injection, a booster injection of SRBCs (1 mL) was injected in 5 chickens out of the ten chickens initially injected with a primary injection of SRBCs. Blood samples from all the chickens were collected on 7th and 14th days post primary and booster injections to separate the sera samples that were used to detect the anti- SRBC antibody titers by haemagglutination test, carried out in 96 well round bottom microtitration plates (Flow Labs., UK).

Test Procedure i. PBS (50 µl) was added in all the wells of a round bottom microtitration plate (Flow Labs., UK). ii. Test sera (50 µl) was added in first well and after proper mixing 50 µl sample was taken out from 1st well and transferred to the next one and so on up to the 11th well to make the two fold serial dilutions as 1:2, 1:4, 1:8,…………1:2048; whereas, last well was kept as negative control. iii. Suspension of 5% (v/v) SRBCs (50 µl) was added in each well of the microtitration plate and mixed gently. iv. The plates were incubated at room temperature (26 °C) for half an hour and results were recorded. v. The titer of the well containing 50% agglutination and 50% reticulum settling (clumping) was considered as the total anti-SRBC antibody titer of the test sera. vi. Same protocol was adopted for IgG titers except in the first step 50 µl of 0.01M 2- mercaptoethanol (2-ME) (Riedel-de Haen, Germany) was added in PBS in each well that destroyed the IgM immunoglobulins and the remaining titer was of IgG. vii. IgM titer was calculated by subtracting the IgG titer from total antibody titer of the respective samples. viii. Results were expressed in terms of Geo-mean titer (GMT) (Burgh, 1978). 3.8. Relative weight of lymphoid organs

Chickens from all the experimental and control groups were individually weighed for live weight and sacrificed at day 21st (fourteen days post administration of A. vera bio-

30 molecules). Lymphoid organs including bursa of fabricius, thymus, spleen and caecal tonsils were incised out and weighed. Per cent organ weight ratio relative to the live body weight (immune organ index) was calculated by using the formula as described by Giambrone and Closser (1990):

Organ weight (gm) × 100 Organ-body weight ratio = Live body weight gain (gm)

3.9. Feed conversion ratios (FCR)

Chickens from all the groups were weighed on weekly basis post administration of A. vera extracts. Feed given to each group was also recorded on weekly basis and the data thus obtained was used to calculate the weekly FCR by using the formula as described by Singh and Panda (1992):

Feed consumption (gm) Feed conversion ratio = Body weight gain (gm) 3.10. Therapeutic evaluation

Forty chickens from each group were challenged with 6.5×104-7×104 sporulated oocysts of mixed species of genus Eimeria (local isolates mainly, E. tenella, E. acervulina, E. maxima and E. necatrix) on day 14th post administration of A. vera bio-molecules. Chickens of each group were monitored for body weight gains per day from day 3rd to 12th post challenge. Droppings from each group were collected and examined on alternate day from day 4th to 12th post challenge to determine the oocysts per gram of droppings by McMaster counting technique (Ryley et al., 1976).

During challenge experiment, chickens of each group were monitored for mortality. Dead and survived chickens in all the groups were sacrificed and scored for lesion scoring (Johnson and Reid, 1970). Further, the per cent protection against lesions was calculated by using the formula described by Singh and Gill (1975):

Per cent Protection against lesions = Average lesion score (IUC) – Average lesion score (IMC) ×100 Average lesion score (IUC)

3.11. Anticoccidial index (ACI)

Anticoccidial index was calculated by using formula as described by Shah et al. (2009):

ACI= (relative rate of weight gain + survival rate)-(lesion value + oocyst value)

Relative rate of weight gain was calculated by subtracting weight gain at the time of challenge from the body weight at the end of experiment. Survival rate was estimated by the number of surviving chickens divided by the initial number of chickens.

Lesion score of the chickens from all groups were calculated by method of Johnson and Reid (1970); and oocyst value was calculated by using the formula described by Peek and Landman (2003) as (the number of oocysts from the positive control chickens-Treated chickens)/positive control chickens x 100%).

3.12. Effect on the development of lymphoid organs of chickens

Chickens from each group were individually weighed and slaughtered on day 12th post challenge. Lymphoid organs including bursa of fabricius, thymus, spleen and caecal tonsils were removed surgically and weighed to demonstrate the correlation of disease with their development, if any, in experimental and control chickens. The data thus collected was expressed as per cent organ-body weights ratio relative to the live body weight by using the formula as described earlier.

3.13. Statistical analysis

One way analysis of variance (ANOVA) and Duncan’s multiple range (DMR) tests were used for the determination of statistical significance using statistical analysis software ® (SAS , 2004). Data on per cent mortality and protection were analyzed using the Chi square test. Differences were considered statistically significant at p<0.05.

CHAPTER 4 RESULTS

In the present study, Aloe (A.) vera polysaccharides and proteins were isolated and characterized; and evaluated as biological response modifiers and therapeutic agents against coccidiosis in chickens.

Experiment-I 4.1. Isolation and characterization of Aloe vera polysaccharides 4.1.1. High Performance Liquid Chromatography (HPLC) analysis

A. vera monosaccharides quantitative HPLC analysis resulted in three distinct peaks on chromatogram at different retention time. These peaks were compared with standards, which showed three different monosaccharides including maltose, glucose and mannose in hydrolyzed solution of A. vera at retention times 9.423, 11.080 and 12.55, respectively. Molar concentration percentage quantities of these detected monosaccharides in hydrolyzed solution of A. vera polysaccharides are presented in Table 4 A.

4.1.2. Immunological evaluation

For immunological evaluation, both cell mediated and humoral immune responses were detected. Cell mediated immune responses were detected by in vivo lymphoproliferative response to PHA-P and carbon particle clearance assay. In vitro cell mediated immune responses were detected by lymphoproliferative response to Concanavalin-A (Con-A). Humoral immune responses were demonstrated by antibody response to sheep red blood cells.

4.1.2a. Cell mediated immune (CMI) responses i. In vivo lymphoproliferative response (mm) to Phytohaemagglutinin-P (PHA-P)

To demonstrate in vivo CMI response, lymphoproliferative response to PHA-P was calculated. For this purpose, the amplitude of toe-web swelling was measured at 24, 48 and 72 hours after PHA-P injection in all experimental and control groups. A. vera polysaccharides administered groups showed significantly higher (p<0.05)

33 lymphoproliferative response in all experimental groups as compared to control. Maximum swelling was observed at 24 hours post PHA-P injection in all groups followed by 48 and 72 hours. Maximum swelling after 24 hrs (1.13±0.11) was noticed in group A2, which was administered with A. vera polysaccharides at the dose rate of 200mg/kg body weight. Results of group A2 were followed by A3 (1.09±0.15), A1 (1.00±0.12) and A4 (0.68±0.03). The difference was statistically non significant (p>0.05) between experimental groups A3 and A1 administered with A. vera polysaccharides at the dose rate of 300 and 100 mg/kg body weight, respectively. At 48 hours post administration of A. vera polysaccharides, group A2 showed significantly higher response (0.92±0.02) followed by group A3 (0.89±0.02), A1

(0.76±0.04) and A4 (0.62±0.01). Group A3 showed statistically similar response to group A1 and A2. At 72 hours group A2 (0.81±0.07) showed significantly higher response (p<0.05) as compared to all groups including A3 (0.78±0.05), A1 (0.61±0.03) and A4 (0.40±0.03), respectively. On the whole, groups A2 and A3 showed higher responses to PHA-P as compared to A1 and A4 (control) groups (Table 4.1; Figure 4.1). ii. In vitro lymphoproliferative response to Concanavalin-A (Con-A)

In vitro lymphoproliferative response to concanavalin-A was detected at 7th and 14th day post administration of A. vera polysaccharides at graded doses in all groups by lymphoblastogenic response of chickens peripheral blood lymphocytes (PBLs) to Con-A. All the experimental groups showed significantly higher (p<0.05) response in the PBLs of chickens as compared to those of control group, both on day 7th and 14th post administration of A. vera polysaccharides. However, among the treatment groups, chickens administered with A. vera polysaccharides at the dose rate of 200 and 300 mg/kg of body weight showed significantly higher (p<0.05) response as compared to those administered with A. vera polysaccharides at dose rate of 100 mg/kg of body weight (Table 4.2; Figure 4.2). iii. Carbon particle clearance assay

On 14th day post administration of A. vera polysaccharides, carbon particle clearance assay was performed to check the phagocytic activity of chicken macrophages. The clearance index (K), phagocytic index (α) and immune organ index were calculated at 3 and 15 minutes post injection of India ink.

The results of clearance index showed significantly higher (p<0.05) clearance index in control group A4 (0.0185±0.0040) and other experimental groups showed low clearance index values indicating less carbon in blood. Among the experimental groups, group A2 showed lowest K value (0.0073±0.0024) followed by group A1 (0.0106±0.0039) and A3

(0.0124±0.0019). A3 response was statistically similar to group A1 and A4 (Table 4.3; Figure 4.3 a)

The results of phagocytic index (α) showed significantly higher (p<0.05) response in all three experimental groups as compared to control. Maximum response was observed in group A2 (94.8283±9.3211), which was administered with A. vera polysaccharides at the dose rate of 200mg/kg body weight, followed by A1 (69.3054±13.6106) and A3

(51.2677±10.2013). Statistically, group A2 showed significantly higher (p<0.05) phagocytic index followed by group A1; whereas, A3 showed response, which was similar to group A1 and A4 (Table 4.3; Figure 4.3 b). 4.1.2b. Humoral Immune responses i. Antibody response to sheep red blood cells (SRBCs) Humoral immune responses were detected by performing haemagglutination test in chickens of experimental and control groups on day 7th and 14th post primary and secondary injections of SRBCs; and serum antibody response to sheep RBCs were calculated as total immunoglobulins Igs, IgM and IgG titers. The results were explained in terms of geomean titers (GMT) (Table 4.4; Fig.4.4 a-d).

In the present study, all the experimental groups showed higher Igs, IgM and IgG titers as compared to control. However, at day 7th post primary injection (PPI), polysaccharides administered group A2 (at the dose rate of 200mg/kg body weight) showed maximum Igs, IgM and IgG titers (73.52, 52.4, 21.11) followed by group A3 (64, 48, 16) th group A1 (42.22, 28.29, 13.92) and A4 (32, 19.87, 12.12). On day 14 PPI, geomean titer for total anti-SRBC antibodies was highest in group A2 (55.72, 18.95, 36.75) followed by A3

(42.22, 14.36, 27.85), A1 (42.22, 17.97, 24.25) and A4 group (24.25, 8.25, 16). A similar trend was observed on day 7th and 14th post-secondary injections (PSI).

4.1.3. Effect on the development of lymphoid organs of chickens

The organ-body weight ratio (immune organ index) of lymphoid organs was calculated on day 14th post administration of A. vera polysaccharides including bursa of fabricius, spleen, thymus and caecal tonsils. Experimental groups showed apparently higher per cent organ-body weight ratios as compared to control group; whereas, the statistical difference was non-significant (p>0.05) in immune organs of all groups except thymus of experimental group A2 showed statistically significant response as compared to control (Table 4.5; Figure 4.5).

4.1.4. Effects of polysaccharides on weight gains and feed conversion ratio (FCR)

Weekly weights and feed consumed were monitored as indicator of performance in all the groups and results expressed in terms of weekly weight gains and weekly FCR from st th 1 to 6 week. Experimental groups (A2 and A3) showed significantly higher (p<0.05) weight gains as compared to group A1 and A4, control. Maximum weight gains (gm±SE) were observed at 6th week in group of chickens administered with A. vera polysaccharides. Among the experimental groups, maximum weight gains were observed in group A3 (1753.33±174.7) followed by A2 (1683.33±104.0), A1 (1566.67±28.87) and control group A4 (1500.00±152.7) (Table 4.6; Figure 4.6).

Similarly, all experimental groups administered A. vera polysaccharides showed better FCR as compared to control. However, FCR was better in group A3 (1.95) followed by group A2 (1.96), A1 (2.01) and control group A4 (2.16) (Table 4.7; Figure 4.7).

4.1.5. Challenge experiment a. Oocyst Count

Experimental and control groups administered with mixed Eimeria species (6.5×104- 7.0×104 sporulated oocysts) were monitored for oocysts per gram (OPG) of droppings from day 4th to 12th post challenge. All the experimental groups showed significantly lower OPG than control (p<0.05) and maximum OPG were calculated on 9th day post infection in all groups. OPG count was lower in group A2 and A3 as compared to A1 and control A4; although difference between groups A2 and A3 was non-significant (Table 4.8; Figure 4.8).

36 b. Daily weight gains post challenge

The average daily weight gains (gm±SE) of all the groups were calculated from day 3rd to 12th post challenge (Table 4.9; Figure 4.9). Weight gains were significantly higher

(p<0.05) in chickens of experimental group A2 administered with A. vera polysaccharides at dose rates of 200 mg/kg of body weight as compared to chickens of A3, A1 and control groups; whereas, difference in weight gains in chickens of groups A3 and A1 was statistically non-significant (p>0.05). c. Per cent protection and mortality post challenge in experimental and control groups

Maximum protection (70%) was observed in groups administered with A. vera polysaccharides at the dose rate of 200mg/kg body weight followed by group A3 (60%) and group A1 (55%). Chickens in control group A4 gave protection (30%). The protection difference among experimental groups was statistically significant (p<0.05) (Table 4.10; Figure 4.10).

Further, chickens in experimental groups A2 and A3 were comparatively active than control, took normal feed and water intake as compared to control group A4, which were depressed with ruffled feathers and took less feed and water. d. Lesion scoring and per cent protection against lesions

From day 6th to 12th post challenge, All survived and dead chickens were monitored for lesion scoring of caeca and intestine on a scale from 0 to 4. Experimental groups showed lesser lesions as compared to control and similarly showed more protection against lesions.

Among experimental groups, chickens of group A2 showed least severe caecal lesion scores

(2.55) followed by A1 (3.05) and A3 (3.2) as compared to chickens in control group, which showed severe lesion scores (3.87).

Per cent protection against caecal lesions was calculated to estimate the protective efficacy of A. vera polysaccharides in experimental and control groups against lesions caused by Eimeria challenge. Chickens of group A2 showed maximum per cent protection against caecal lesions (36.25%) followed by group A1 (23.75%), A3 (20%) and control group A4 (3.25%) (Table 4.11; Figure 4.11).

Chickens of experimental groups showed minimum lesion scores in intestines for group A2 (2.025) followed by group A3 (2.325) and A1 (2.675); whereas, control group A4 showed highest mean lesion score of (3.5). Similarly, results of per cent protection against intestinal lesions showed maximum protection of 49.375 % in group A2, followed by group

A3 (41.875%), A1 (33.125%) and control group showed least protection of 12.5%. Maximum (85%) chickens in control group showed severe lesions (3.0-4.0); moderate lesions (2.0) were also observed in 15 per cent chickens. Among experimental groups, chickens of group A2 developed least severe lesions (50%) followed by A3 (60%) and A1 (80%). A similar trend was observed in case of intestinal lesion scores. e. Anticoccidial index for polysaccharides Relative rate of weight gain, survival rate, lesion value and oocyst clearance rate were determined to calculate anticoccidial index (AI). Maximum AI was calculated in chickens of group A2 (239.63) administered with A. vera polysaccharides (200mg/kg body weight) followed by group A3 (195.31), A1 (159.75) and control A4 (36.57) (Table 4.12; Figure 4.12).

4.1.6. Organ-body weight ratios post challenge

The organ-body weight ratios of immune organs including spleen, thymus, caecal tonsils and bursa of fabricius were calculated on day 12th post challenge with mixed Emeria species. All groups showed statistically similar organ-body weight ratios (p>0.05) except spleen. Spleen of control group showed higher organ-body ratio as compared to groups administered with A. vera polysaccharides and difference was statistically significant (p<0.05) (Table 4.13; Figure 4.13).

Figure 4A1: Chromatogram showing the peaks of monosaccharides in Aloe vera polysaccharides at different retention times

[mV] 20

7.560 7.560

Voltage

12.550 12.550

2.687 2.687

8.890 8.890

11.080 11.080

10.243 10.243

9.423 9.423 0

0 2 4 6 8 10 12 14 [min.] Time Chromatograms of monosaccharides standards are shown in annexure IV

A1 AMPLIFIED

[mV]

2.0 7.560

1.5

1.0

12.550 12.550

2.687 2.687

8.890 8.890 Voltage 0.5

11.080 11.080

10.243 10.243

0.0 9.423

-0.5

2 4 6 8 10 12 14 [min.] Time

Table 5A: Quantitative analysis of monosaccharides detected in the hydrolyzed sample of Aloe vera polysaccharides

Monosaccharide Retention Time Area Height Quantity (minutes) (mV.s) (mV) (molar %) Maltose 9.423 11.837 0.540 0.04

Glucose 11.080 35.252 0.605 0.11

Mannose 12.550 36.885 0.612 0.02

Table 4.1: In vivo lymphoproliferative response to PHA-P in experimental and control chickens Groups Increase in thickness

24 hrs. 48 hrs. 72 hrs. (mm±SE) (mm±SE) (mm±SE)

ab b bc A1 1.00±0.12 0.76±0.04 0.61±0.03

a a a A2 1.13±0.11 0.92±0.02 0.81±0.07

ab ab a A3 1.09±0.15 0.89±0.02 0.78±0.05

b c c A4 0.68±0.03 0.62±0.01 0.40±0.03

Means sharing similar letters in a column are statistically non-significant (p>0.05) A1=A. vera polysaccharides @ 100mg/kg of BW A2=A. vera polysaccharides @ 200mg/kg of BW A3=A. vera polysaccharides @ 300mg/kg of BW A4=Control

Figure 4.1: In vivo lymphoproliferative response to PHA-P in experimental and control chickens

Table 4.2: In vitro lymphoproliferative response to Concanavalin-A in experimental and control chickens Groups Stimulation index post administration of Aloe vera polysaccharides Day 7th Day 14th (MeanOD+SE) (MeanOD+SE) b b A1 0.38±0.04 0.39±0.03 a a A2 0.74±0.02 0.75±0.02 a a A3 0.75±0.01 0.74±0.02

c c A4 0.28±0.01 0.32±0.01 Means sharing similar letters in a column are statistically non-significant (p>0.05) A1=A. vera polysaccharides @ 100mg/kg of BW A2=A. vera polysaccharides @ 200mg/kg of BW A3=A. vera polysaccharides @ 300mg/kg of BW A4=Control

Figure 4.2: In vitro lymphoproliferative response to Concanavalin-A in experimental and control chickens

Table 4.3: Carbon particle clearance assay in experimental and control chickens Groups Carbon particle clearance assay post administration of Aloe vera polysaccharides Clearance index (K) Phagocytic index (α) (MeanOD±SE) (MeanOD±SE) b b A1 0.0106±0.0039 69.3054±13.6106 c a A2 0.0073±0.0024 94.8283±9.3211

ab bc A3 0.0124±0.0019 51.2677±10.2013 a c A4 0.0185±0.0040 48.5554±9.8143 Means sharing similar letters in a column are statistically non-significant (p>0.05) A1= A. vera polysaccharides @ 100mg/kg of BW A2=A. vera polysaccharides @ 200mg/kg of BW A3=A. vera polysaccharides @ 300mg/kg of BW A4=Control Figure 4.3 (a): Carbon particle clearance assay (Clearance Index K)

0.025

0.02

0.015 Clearance Index (K) 0.01

0.005

0 A1 A2 A3 A4

Groups

Figure 4.3 (b): Carbon particle clearance assay (Phagocytic Index α)

120

100

Phagocytic index (α) 40

0 A1 A2 A3 A4

Groups

A1= A. vera polysaccharides @ 100mg/kg of BW A2=A. vera polysaccharides @ 200mg/kg of BW A3=A. vera polysaccharides @ 300mg/kg of BW A4=Control

Table 4.4: Antibody response to sheep red blood cells in experimental and control chickens ------Total anti-SRBCs antibody titer----

Group Day 7 PPI Day 14 PPI Day 7 PSI Day 14 PSI

A1 42.22 42.22 64 48.50

A2 73.52 55.72 84.45 64

A3 64 42.22 73.51 55.71

A4 32 24.25 27.85 13.92

------Immunoglobulin-M------

A1 28.29 17.97 36.14 11.74

A2 52.40 18.95 47.69 15.49

A3 48 14.36 45.65 13.49

A4 19.87 8.25 15.73 4.73

------Immunoglobulin-G-----

A1 13.92 24.25 27.85 36.75

A2 21.11 36.75 36.75 48.50

A3 16 27.85 27.85 42.22

A4 12.12 16 12.12 9.18

A1=A. vera polysaccharides @ 100mg/kg of BW A2=A. vera polysaccharides @ 200mg/kg of BW A3=A. vera polysaccharides @ 300mg/kg of BW A4=Control

Figure 4.4 (a-d): Antibody response to sheep red blood cells in experimental and control chickens

a. At day 7th post primary injection

b. At day 14th post primary injection

c. At day 7th post secondary injection

d. At day 14th post secondary injection

Table 4.5: Organ-body weight ratios on 14th day post administration of Aloe vera polysaccharides in experimental and control chickens

Groups Thymus Spleen Bursa Caecal tonsils (Mean±SE) (Mean±SE) (Mean±SE) (Mean±SE)

b A1 0.36±0.011 0.23±0.011 0.26±0.008 0.09±0.008

a A2 0.38±0.008 0.23±0.008 0.26±0.006 0.09±0.010

ab A3 0.37±0.0158 0.23±0.007 0.26±0.0100 0.09±0.007

b A4 0.35±0.008 0.22±0.006 0.25±0.016 0.08±0.007

Figure 4.5: Organ-body weight ratios on 14th day post administration of Aloe vera polysaccharides in experimental and control chickens

A1 A2 A3 A4 0.45 0.4 0.35 0.3 0.25 0.2

0.15 body body weight ratios - 0.1 0.05 Organ 0 Thymus Spleen Bursa C. tonsils

Lymphoid organs

Table 4.6: Weekly (1st-6th) weight gains in experimental and control groups

A1 A2 A3 A4 Weeks (Mean±SE) (Mean±SE) (Mean±SE) (Mean±SE)

1st 123.33±15.28b 118.00±7.21c 126.67±15.28a 110.00±10.00d

2nd 220.00±20.00c 246.67±35.12b 296.67±15.28a 246.67±15.28b

3rd 356.67±15.28c 446.67±15.28a 426.67±25.17ab 350.00±50.00c

4th 843.33±45.09c 950.00±50.00b 1010.00±36.06a 816.67±76.38d

5th 1166.67±76.38c 1276.67±25.17ab 1383.33±76.38a 1200.0±132.9bc

6th 1566.67±28.87c 1683.33±104.0b 1753.33±174.7a 1500.00±152.7c

Figure 4.6: Weekly (1st-6th) weight gains in experimental and control groups

A1 A2 A3 A4 2500 2000 1500 1000 500

0 Weekly Weekly weight gains 1st 2nd 3rd 4th 5th 6th

Weeks post administration of Aloe vera polysccharides

Table 4.7: Results of weekly FCR post administration of Aloe vera polysaccharides in experimental and control chickens

Weeks A1 A2 A3 A4

Feed Conversion Ratios

1st 2.21 2.17 2.07 2.03

2nd 2.16 2.01 2.03 2.12

3rd 2.04 1.97 1.98 2.15

4th 2.05 1.96 1.97 2.19

5th 2.01 1.98 1.94 2.17

6th 2.01 1.96 1.95 2.16

A1=A. vera polysaccharides @ 100mg/kg of BW A2=A. vera polysaccharides @ 200mg/kg of BW A3=A. vera polysaccharides @ 300mg/kg of BW A4=Control

Figure 4.7: Results of weekly FCR post administration of Aloe vera polysaccharides in experimental and control chickens

2.2

2.15

2.1

2.05

1.95

1.9 Feed Conversion ratios 1.85

1.8 A1 A2 A3 A4 Groups

Table 4.8: Oocysts per gram of droppings post challenge in experimental and control chickens Groups

Days A1 A2 A3 A4 (Mean±SE) (Mean±SE) (Mean±SE) (Mean±SE)

4th 9843.33±577.09b 8519.33±800.18d 8862.00±236.25c 34933.33±1866.58a

5th 13838.00±2170.67b 11906.67±674.86dc 12290.00±121.24c 57629.33±5783.97a

7th 45273.33±3896.29b 40940.00±1989.07d 41986.67±1364.89c 227135.33±23038.51a

9th 95462.67±3354.00b 66083.33±48356.33dc 91125.33±6720.55bc 383179.33±20736.35a

11th 70242.67±5000.97b 64996.00±4152.12c 63432.67±4626.43d 128292.67±25891.74a

12th 38113.33±4266.72b 34766.67±4467.33dc 36326.00±2228.15c 66797.33±8571.50a

Table 4.9: Daily weight gains from day 3rd to 12th post challenge in experimental and control chickens Days Groups A A A A 1 2 3 4 (gm±SE) (gm±SE) (gm±SE) (gm±SE) 3rd 16.89±0.05b 19.61±1.50a 16.55±0.62b 16.72±0.92b 4th 16.72±0.56b 19.78±0.75a 17.55±0.48ab 15.22±0.33c 5th 17.31±0.82b 19.44±0.93a 15.89±0.50c 13.22±0.44d 6th 15.83±0.67b 18.44±0.84a 16.05±0.59b 11.56±0.52c 7th 13.39±0.46c 16.78±0.79a 14.89±0.42b 11.89±0.60d 8th 12.39±0.60c 16.11±0.66b 14.39±1.00a 9.55±0.41d 9th 12.09±1.02b 15.78±1.06a 13.39±0.85b 9.55±0.58c 10th 13.05±1.01b 16.61±0.63a 13.39±0.56b 10.55±0.50c 11th 12.89±0.40b 16.78±0.71a 13.89±0.71b 11.55±0.43c 12th 14.39±0.82b 16.94±0.45a 15.12±0.24ab 12.89±0.52c Means sharing similar letters in a column are statistically non-significant (p>0.05) A1=A.vera polysaccharides @ 100mg/kg of BW A2=A.vera polysaccharides @ 200mg/kg of BW A3=A. vera polysaccharides @ 300mg/kg of BW A4=Control Figure 4.9: Daily weight gains from day 3rd to 12th post challenge in experimental and control chickens

A1 A2 A3 A4

SE) 20 ±

5 Daily Daily weight gains (gm

0 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th Days post Challenge

Table 4.10: Per cent protection and mortality post challenge in experimental and control chickens

A1 A2 A3 A4

Protection (%) 55 70 60 30

Mortality (%) 45 30 40 70

A1= A. vera polysaccharides @ 100mg/kg of BW A2=A. vera polysaccharides @ 200mg/kg of BW A3=A. vera polysaccharides @ 300mg/kg of BW A4=Control

Figure 4.10: Per cent protection post challenge in experimental and control chickens

A1 A2 A3 A4

30% 55%

60%

70%

Table 4.11: Lesion scoring and per cent protection against lesions in experimental and control chickens Groups Lesion Scoring of Birds Protection against lesions 0 1 2 3 4 (%) Caeca

A1 0 5 6 11 18 23.75

A2 0 8 14 6 12 36.25

A3 0 0 8 16 16 20

A4 0 0 5 11 28 3.25 Intestine

A1 0 11 6 8 15 33.125

A2 0 12 9 5 9 49.375

A3 0 16 7 5 12 41.875

A4 0 0 2 16 22 12.5

A1 =A. vera polysaccharides @ 100mg/kg of BW A2=A. vera polysaccharides @ 200mg/kg of BW A3=A. vera polysaccharides @ 300mg/kg of BW A4=Control Figure 4.11: Per cent protection against lesions in experimental and control chickens

Table 4.12: Anticoccidial index for polysaccharides

Groups Relative rate of wt. Survival Lesion Oocyst ACI gains rate value clearance rate

A1 162.567 0.55 2.675 0.696 159.75

A2 241.696 0.7 2.025 0.746 239.63

A3 197.756 0.6 2.325 0.717 195.31

A4 40.14 0.3 3.5 0 36.57

A1=A. vera polysaccharides @ 100mg/kg of BW A2=A. vera polysaccharides @ 200mg/kg of BW A3=A. vera polysaccharides @ 300mg/kg of BW A4=Control

Figure 4.12: Anticoccidial index for polysaccharides

300 250 200 150 100

Anticoccidial Anticoccidial Index 50 0 A1 A2 A3 A4 Groups

Table 4.13: Organ-body weight ratios post challenge in experimental and control chickens

Groups Thymus Spleen Bursa Caecal tonsils (Mean±SE) (Mean±SE) (Mean±SE) (Mean±SE)

c A1 0.37±0.01 0.27±0.02 0.26±0.01 0.09±0.01

b A2 0.38±0.01 0.28±0.01 0.25±0.01 0.08±0.01

b A3 0.37±0.01 0.28±0.01 0.26±0.01 0.08±0.01

a A4 0.36±0.01 0.29±0.01 0.25±0.01 0.07±0.01

Figure 4.13: Organ-body weight ratios post challenge in experimental and control chickens

0.45 A1 A2 A3 A4

0.4

0.35

0.3

0.25

0.2

body body weight ratios 0.15 -

0.1 Organ 0.05

0 Thymus Spleen Bursa C. tonsils Lymphoid organs

Experiment-II 4.2. Isolation and characterization of Aloe vera proteins Proteins extraction from A. vera were quantified in duplicate by Bradford assay and concentration was ranged from 22.6-26 µg/ml. (Table 4B, B1; Figure 4B). Automated electrophoretic analysis of the isolated proteins revealed the presence of proteins of molecular weight 1 and 6kD. Three samples were analyzed, which showed similar results (Table 4B2; Figure 4C1, C2).

4.2.1. Immunological evaluation For immunological evaluation both cell mediated and humoral immune responses were detected. lymphoproliferative response to PHA-P and carbon particle clearance assay were used to demonstrate in vivo cell mediated immune responses; whereas, in vitro cell mediated immune response was detected by lymphoproliferative response to Concanavalin-A (Con-A). Humoral immune response was detected by antibody response to sheep red blood cells. 4.2.2a. Cell mediated immune responses i. In vivo lymphoproliferative response (mm) to Phytohaemagglutinin-P (PHA-P) The amplitude of toe-web swelling was measured at 24, 48 and 72 hours post PHA-P injection (mm±SE) in experimental and control groups. A. vera proteins administered groups showed significantly higher response in all experimental groups as compared to control (p<0.05). Maximum swelling was observed at 24 hours post PHA-P injection in all the groups (treated and control) followed by 48 and 72 hours. Group B2, which was administered with A. vera proteins at the dose rate of 200mg/kg body weight showed maximum swelling

(0.81±0.04) followed by group B3 (0.78±0.02), B1 (0.72±0.05) and control (0.63±0.04). The difference between groups was significantly higher in group B2 but it was similar to group

B3. At 48 hours post PHA-P injection, groups B2 and B3 showed PHA-P response of 0.73±0.03 and 0.72±0.03. Their response was significantly higher response as compared to

B1 (0.65±0.06) and B4 (0.58±0.03). The difference between groups B2 and B3 were statistically similar. At 72 hours post PHA-P injection, maximum swelling was observed in chickens of group B2 (0.63±0.03) followed by group B3 (0.61± 0.02), B1 (0.57±0.07) and B4 (0.40±0.03). Statistically results at 24 hours were similar to observed at 72 hours post PHA-P injection (Table 4.14; Figure 4.14). 56 ii. In vitro lymphoproliferative response to Concanavalin-A in experimental and control chickens In vitro lymphoproliferative response to concanavalin-A was detected at 7th and 14th day post administration of A. vera proteins at graded doses in all groups by lymphoblastogenic response of chicken peripheral blood lymphocytes (PBLs) to Con-A. All treated groups showed significantly higher (p<0.05) response in the chickens PBLs as compared to control group on both day 7th and 14th post administration of A. vera proteins. However, among the treated groups, chickens administered with A. vera proteins at the dose rate of 200 and 300 mg/kg of body weight showed significantly higher (p<0.05) response as compared to those administered with A. vera proteins at dose rate of 100 mg/kg of body th weight. Statistically group B2 at 14 day showed response similar to group B3 and group B1 (Table 4.15; Figure 4.15). iii. Carbon particle clearance assay in experimental and control chickens

On 14th day post administration of A. vera proteins, carbon particle clearance assay was performed to check the phagocytic activity of chicken macrophages. The clearance index (K), phagocytic index (α) and immune organ index (organ-body weight ratios) were calculated at 3 and 15 minutes post injection of India ink.

The results of clearance index showed significantly higher clearance index in control group B4 (0.027±0.002) and all the other medicated groups showed low clearance index values indicating less carbon particles in blood. Among treated groups with A. vera proteins at graded doses group B2 showed lowest K value (0.006±0.001) followed by group B1

(0.017±0.002) and B3 (0.019±0.001). B3 and B1 response was statistically similar (Table 4.16; Figure 4.16 a).

The results of phagocytic index (α) showed significantly higher response in all three medicated groups as compared to control. Maximum response was observed in group B2 (89.635±7.649), which was administered with A. vera proteins at the dose rate of 200mg/kg body weight, followed by B1 (66.734±12.245) and B3 (50.481±8.477). Statistically group B2 showed significantly higher phagocytic index followed by group B1, whereas B3 showed response, which was similar to group B1 and B4 (Table 4.16; Figure 4.16 b).

4.2.2b. Humoral Immune responses i. Antibody response to sheep red blood cells (SRBCs) Humoral immune responses were detected by microplate haemagglutination assay in chickens of experimental and control groups on day 7th and 14th post primary and secondary injections of SRBCs; and results were calculated in terms of geomean titers (GMT) (Table 4.17; Figure 4.17 a-d).

Total anti-SRBCs antibody titer

Highest geomean anti-SRBCs antibody titer (GMT 64.0) was detected in chickens administered with A. vera extracted proteins @ 300 mg/kg body weight followed by chickens th in group B2 (GMT 55.72), B1 (GMT 32) and control group B4 (GMT 27.86) at day 7 PPI. At day 14th PPI, chickens in all the groups showed similar trend of antibody titer. Moreover, total anti-SRBCs Igs titers at day 7th and 14th PSI were also at the same pattern as observed on day 7th and 14th PPI.

IgM anti-SRBCs antibody titer

th At day 7 PPI, highest IgM anti-SRBC antibody titers were detected in group B3

(GMT 39.74), followed by B2 (GMT 37.34), B1 (GMT 21.44) and control group B4 (GMT th 18.69). At day 14 PPI, chickens in group B3 (GMT 20.64) showed the highest antibody titer followed by those of group B2 (GMT 17.97), B1 (GMT 9.48) and control group (GMT 7.18); and showed similar trend on day 7th and 14th PSI.

IgG anti-SRBCs antibody titer

Anti-SRBCs IgG titers showed a similar trend to total anti-SRBCs Igs titers as detected on both days 7th and 14th post primary and post secondary injections of SRBCs (Table 4.17; Figure 4.17 a-d).

4.2.3. Effect on the development of lymphoid organs of chickens

The organ-body weight ratios of lymphoid organs (immune organ index) were calculated on day 14th post administration of A. vera proteins. Immune organs observed were bursa of fabricius, spleen, thymus and caecal tonsils. Experimental groups showed higher per cent organ-body weight ratios except spleen as compared to control group; whereas the

58 statistical difference was non-significant (p>0.05) in immune organs of all groups except thymus of experimental groups showed statistically significant response as compared to control (Table 4.18; Figure 4.18).

4.2.4. Effect on body weight gains and feed conversion ratios (FCR)

Chickens from all groups were weighed from week 1st to 6th; feed consumed by each group was also recorded. Experimental groups (B1, B2 and B3) showed statistically significant weight gains as compared to control (p<0.05). Maximum weight gains (gm±SE) at 6th week were observed in group administered with A. vera proteins (Table 4.19; Figure

4.19). Among experimental groups, maximum weight gain was observed in group B2

(1700.00±50.00) followed by B3 (1630.00±81.85), B1 (1556.67±11.55) and control group B4 (1503.33±20.2). Similarly, all experimental groups administered with A. vera proteins showed better FCR as compared to control. FCR was calculated on weekly basis for all experimental and control groups from week 1st to 6th. On the whole, results of FCR was better in group B3 (A. vera proteins @ 300 mg/kg body weight) followed by group B2, B1 and control group B4 (Table 4.20; Figure 4.20).

4.2.5. Challenge experiment a. Oocysts per gram (OPG) of droppings post-challenge in experimental and control chickens

Experimental groups administered with A. vera proteins showed significantly lower OPG as compared to control in challenge experiment. OPG values of control group were significantly higher (p<0.05) than experimental groups. Among experimental groups, lowest

OPG was observed in group B2 (proteins @ 200mg/kg body weight) followed by B3, B1 and control; although statistically response was similar between group B2 and B3 (Table 4.21; Figure 4.21). b. Daily weight gains post challenge

Daily weight gains (gm±SE) of all the groups were calculated from day 3rd to 12th post challenge. The average body weight gains were significantly higher (p<0.05) in chickens of experimental groups B2 administered with A. vera proteins (@ 200 mg/kg of body weight) as compared to chickens of group B3, B1 and control groups; whereas, difference in weight

59 gains in chickens of groups B3 and B1 was statistically non-significant (p>0.05) (Table 4.22; Figure 4.22). c. Per cent protection post challenge in experimental and control groups

Chickens of experimental groups showed higher protection against coccidiosis post challenge as compared to control. Maximum protection (57.5 %) was observed in chickens administered with A. vera proteins @ 200 mg/kg body weight (group B2) followed by group

B3 (52.5%), B1 (50%) and control group B4 (25%). The chickens of experimental groups were comparatively active and took more feed and water as than control group. The per cent protection among experimental groups was statistically significant (p<0.05) (Table 4.23; Figure 4.23). d. Lesion scoring and per cent protection against lesions

Lesion score and per cent protection against lesions were calculated in all groups challenged with mixed Eimeria species (local isolates). All survived and dead chickens were monitored for lesion scoring of caeca and intestine on a scale from 0 to 4. Experimental groups showed lesser lesion as compared to control and showed higher protection against lesions. Among experimental groups, chickens of group B2 (3.175) showed least severe caecal lesion score followed by B3 (3.325), B1 (3.4). Further, chickens in control group showed severe lesion score (3.5) (Table 4.24; Figure 4.24).

Per cent protection against caecal lesions was calculated to demonstrate the protective efficacy of A. vera proteins in experimental and control groups against lesions caused by mixed species Eimeria challenge. Chickens of group B2 showed maximum per cent protection against lesions (20.625%) followed by group B3 (16.875%), B1 (15%) and control group B4 (12.5%) (Table 4.24; Figure 4.24).

Chickens of experimental groups showed minimum lesion scores in intestines for group B2 (2.425) followed by group B3 (2.775) and B1 (3.075); whereas, control group B4 showed highest mean lesion score of (3.475). Similarly, results of per cent protection against intestinal lesions showed maximum protection (39.376%) in group B2, followed by group B3

(30.625%), B1 (23.125%), and control group (13.125%). In control group, maximum chickens (90%) showed severe (3.0-4.0); whereas 10 % chicken showed moderate (2.0)

60 intestinal lesions. In group B2, 42.5% chickens develop severe lesions; 30% moderate and 27.5% showed mild to moderate lesions (1.0-2.0). e. Anticoccidial index for proteins

Maximum anticoccidial index was calculated for group B2 (172.26) followed by group B3 (162.37), B1 (103.26) and control group B4 (17.85) (Table 4.25; Figure 4.25).

4.2.6. Organ-body weight ratios post challenge in experimental and control groups

The organ-body weight ratios of immune organs including thymus, spleen, bursa of fabricius and caecal tonsils were calculated. All the groups showed statistically similar (p>0.05) response (Table 4.26; Figure 4.26).

Table 4B: Bradford assay for protein quantification

Standards Concentration 100 200 300 400 500 600 700 800 900 1000 Absorbance 0.498 0.526 0.54 0.572 0.581 0.59 0.592 0.617 0.657 0.663

Figure 4B: Standard curve for protein quantification by Bradford assay

Table 4B1: Results of protein sample by Bradford assay

Absorbance 0.180 0.193 Concentration (µg/ml) 22.6 26.0

Table 4B2: Profile of proteins isolated from Aloe vera by Automated Electrophoresis system

Peak Number Migration Time (sec) Mol. Wt. Corrected Area (kDa) 1. 12.20 0.00 34.59 2. 13.55 1.00 27.35 3. 14.55 0.00 1835.10 4. 17.55 0.00 11.00 5. 19.90 6.00 2515.88 6. 43.20 0.00 1.07 7. 44.00 0.00 22.22 8. 54.85 0.00 1.54 9. 59.60 0.00 2.36

Figure 4C1: Profile of proteins isolated from Aloe vera

Figure 4C2: Profile of proteins isolated from Aloe vera

Table 4.14: In vivo lymphoproliferative response to PHA-P in experimental and control chickens Groups Increase in thickness (mm) 24 hrs. 48 hrs. 72 hrs. (mm±SE) (mm±SE) (mm±SE)

b b b B1 0.72±0.05 0.65±0.06 0.57±0.07

a a a B2 0.81±0.04 0.73±0.03 0.63±0.03

ab a ab B3 0.78±0.02 0.72±0.03 0.61±0.02

c c c B4 0.63±0.04 0.58±0.03 0.40±0.03

Means sharing similar letters in a column are statistically non-significant (p>0.05) B1=A. vera proteins @ 100mg/kg of BW B2=A. vera proteins @ 200mg/kg of BW B3=A.vera proteins @ 300mg/kg of BW B4=Control

Figure 4.14: In vivo lymphoproliferative response to PHA-P in experimental and control chickens

Table 4.15: In vitro lymphoproliferative response to Concanavalin-A in experimental and control chickens Groups Stimulation index post administration of Aloe vera proteins Day 7th Day 14th (MeanOD+SE) (MeanOD+SE) b b B1 0.364±0.02 0.380±0.02 a ab B2 0.382±0.02 0.388±0.04

a a B3 0.378±0.04 0.394±0.03 c c B4 0.260±0.04 0.280±0.04 Means sharing similar letters in a column are statistically non-significant (p>0.05) B1=A. vera proteins @ 100mg/kg of BW B2=A. vera proteins @ 200mg/kg of BW B3=A.vera proteins @ 300mg/kg of BW B4=Control

Figure 4.15: In vitro lymphoproliferative response to Concanavalin-A in experimental and control chickens

0.45 B1 B2 B3 B4

0.4 SE) + 0.35 0.3 0.25 0.2 0.15 0.1

Stimulation Index (Mean 0.05 0 7th 14th Days post administration of Aloe vera proteins

Table 4.16: Carbon particle clearance assay in experimental and control chickens Groups Carbon particle clearance assay post administration of Aloe vera proteins Clearance index (K) Phagocytic index (α) (MeanOD±SE) (MeanOD±SE) b b B1 0.017±0.002 66.734±12.245

c a B2 0.006±0.001 89.635±7.649

b bc B3 0.019±0.001 50.481±8.477

a c B4 0.027±0.002 47.812±8.150 Means sharing similar letters in a column are statistically non-significant (p>0.05) B1=A. vera proteins @ 100mg/kg of BW B2=A. vera proteins @ 200mg/kg of BW B3=A. vera proteins @ 300mg/kg of BW B4=Control

Figure 4.16 (a): Carbon particle clearance assay (Clearence Index)

Figure 4.16 (b): Carbon particle clearance assay (Phagocytic Index)

120

100

20 Phagocytic index Phagocytic(α) index

0 B1 B2 B3 B4 Groups

B1=A. vera proteins @ 100mg/kg of BW B2=A. vera proteins @ 200mg/kg of BW B3=A. vera proteins @ 300mg/kg of BW B4=Control

Table 4.17: Antibody response to sheep red blood cells in experimental and control chickens ------Total anti-SRBCs antibody titer------Groups Day 7 PPI Day 14 PPI Day 7 PSI Day 14 PSI

B1 32 27.86 55.72 48.50

B2 55.72 42.22 73.52 64

B3 64 48.50 84.45 73.52

B4 27.86 21.11 32 27.86 ------Immunoglobulin-M------

B1 21.44 9.48 31.46 16.50

B2 37.34 17.97 45.66 21.78

B3 39.74 20.64 47.69 25.01

B4 18.69 7.18 21.44 6.74 ------Immunoglobulin-G-----

B1 10.56 18.37 24.25 32

B2 18.37 24.25 27.85 42.22

B3 24.25 27.85 36.76 48.50

B4 9.19 13.93 10.56 21.11

B1=A. vera proteins @ 100mg/kg of BW B2=A. vera proteins @ 200mg/kg of BW B3=A. vera proteins @ 300mg/kg of BW B4=Control

Figure 4.17(a-d): Antibody response to sheep red blood cells in experimental and control chickens

a. At day 7th post primary injection

b. At day 14thpost primary injection

c. At day 7th post secondary injection

d. At day 14thpost secondary injection

Table 4.18: Organ-body weight ratios on day 14th post administration of Aloe vera proteins in experimental and control chickens Groups Thymus Spleen Bursa Caecal tonsils (Mean±SE) (Mean±SE) (Mean±SE) (Mean±SE)

B1 0.36±0.03a 0.20±0.01 0.24±0.02 0.09±0.01

B2 0.37±0.02a 0.22±0.02 0.25±0.02 0.08±0.01

B3 0.36±0.01a 0.22±0.02 0.25±0.01 0.08±0.01

B4 0.34±0.01b 0.23±0.00 0.24±0.01 0.08±0.01

Means sharing similar letters in a column are statistically non-significant (p>0.05) B1=A.veraproteins @ 100mg/kg of BW B2=A.vera proteins @ 200mg/kg of BW B3=A.vera proteins @ 300mg/kg of BW B4=Control

Figure 4.18: Organ-body weight ratios on day 14th post administration of Aloe vera proteins in experimental and control chickens

B1 B2 B3 B4 0.4 0.35 0.3 0.25 0.2

0.15 body body weight ratios

- 0.1 0.05 Organ 0 Thymus Spleen Bursa C. tonsils

Lymphoid organs

Table 4.19: Weekly (1st-6th) weight gains in experimental and control chickens

B1 B2 B3 B4 Weeks (Mean±SE) (Mean±SE) (Mean±SE) (Mean±SE) b a ab b 1st 120.00±10.00 130.00±10.00 128.33±7.64 121.67±7.64 c a b bc 2nd 231.67±18.93 271.67±2.89 265.00±5.00 263.33±12.5

b ab a b 3rd 360.00±20.00 416.67±12.58 426.67±25.17 350.00±50.00 c a ab d 4th 856.67±28.87 985.00±22.91 920.00±30.0 836.67±55.08

bc a ab c 5th 1136.67±32.1 1360.00±52.9 1270.00±34.6 1063.33±77.6 b a ab b 6th 1556.67±11.5 1700.00±50.0 1630.00±81.85 1503.33±20.2

Figure 4.19: Weekly (1st-6th) weight gains in experimental and control chickens

B1 B2 B3 B4 2000 1800 1600 1400 1200 1000 800 600 Weekly weight gains 400 200 0 1st 2nd 3rd 4th 5th 6th

Weeks post administration of Aloe vera proteins

Table 4.20: Results of weekly FCR post administration of Aloe vera proteins in experimental and control chickens B B Weeks B1 B2 3 4

Feed Conversion Ratios

1st 2 2.01 2.01 2.12

nd 2 2.2 1.99 2.01 2.11

rd 3 2.17 1.98 1.98 2.12

th 4 2.14 1.97 1.97 2.13

5th 2.11 1.97 1.98 2.14

th 6 2.08 1.95 1.94 2.14

B1=A. vera proteins @ 100mg/kg of BW B2=A. vera proteins @ 200mg/kg of BW B3=A. vera proteins @ 300mg/kg of BW B4=Control

Figure 4.20: Results of weekly FCR post administration of Aloe vera proteins in experimental and control chickens

2.2

2.15

2.1

2.05

1.95

FeedConversion ratios 1.9

1.85

1.8 B1 B2 B3 B4 Groups

Table 4.21: Oocysts per gram of droppings post challenge in experimental and control chickens Groups

Days B1 B2 B3 B4 (Mean±SE) (Mean±SE) (Mean±SE) (Mean±SE)

4th 9900.33±577.09b 8576.67±799.80d 8919.67±235.09c 34991.33±1867.55a

5th 13894.33±2169.60b 11964.33±673.80dc 12347.67±122.15c 58685.67±5150.63a

7th 45329.67±3896.07b 40996.67±1988.66d 42043.33±1365.42c 227193.33±23039.01a

9th 95519.33±3354.86b 66138.67±48360.06dc 91183.67±6719.70c 386569.67±15535.00a

11th 70299.67±5000.97b 65053.00±4152.12c 63494.33±4634.43d 128683.00±25796.56a

12th 38168.67±4267.31b 34824.33±4466.23dc 36382.33±2228.25c 67187.67±8379.42a

Means sharing similar letters in a column are statistically non-significant (p>0.05) B1=A.vera proteins @ 100mg/kg of BW B2=A.vera proteins @ 200mg/kg of BW B3=A. vera proteins @ 300mg/kg of BW B4= Control

Figure 4.21: Oocysts per gram of droppings post challenge in experimental and control chickens

B1 B2 B3 B4 1000000 100000 10000 1000 100 10 1

Oocysts pergramof droppings 4th 5th 7th 9th 11th 12th Days post challenge

Table 4.22: Daily weight gains in chickens from day 3rd to 12th post challenge in experimental and control chickens Days Groups B B B B 1 2 3 4 (gm±SE) (gm±SE) (gm±SE) (gm±SE) 3rd 15.00±1.00a 15.75±0.66a 14.67±0.76b 14.83±0.76b

4th 14.83±0.52bc 16.00±1.00a 15.67±0.76b 13.33±1.53c

5th 15.42±0.52a 15.67±0.58a 14.00±1.00b 11.33±0.58c

6th 13.92±0.38b 14.67±0.76a 14.17±1.04a 9.67±0.58c

7th 11.50±0.50b 13.00±1.00a 13.00±1.00a 10.00±1.00c

8th 10.50±0.50b 12.33±1.04a 12.50±0.50a 7.67±0.58c

9th 10.50±0.50b 12.00±1.00a 11.50±0.50a 7.67±1.53c

10th 11.17±0.76b 12.83±0.76a 11.50±0.50b 8.67±1.15c

11th 11.00±1.00b 13.00±0.50a 12.00±0.50ab 9.67±0.58c

12th 12.50±0.50b 13.17±1.26a 12.50±0.87b 11.00±1.00c Means sharing similar letters in a column are statistically non-significant (p>0.05) B1=A. vera proteins @ 100mg/kg of BW B2=A. vera proteins @ 200mg/kg of BW B3=A. vera proteins @ 300mg/kg of BW B4= Control

Figure 4.22: Daily weight gains in chickens from day 3rd to 12th post challenge in experimental and control chickens

Table 4.23: Per cent protection and mortality post challenge in experimental and control chickens

Mortality and B1 B2 B3 B4 Chi square protection

4.698 Birds Died 20 17 19 30 (p<0.195) % Mortality 50 42.5 47.5 75

Birds Protected 20 23 21 10 5.459 (p<0.141) % Protection 50 57.5 52.5 25 Means sharing similar letters in a column are statistically non-significant (p>0.05) B1=A. vera proteins @ 100mg/kg of BW B2=A. vera proteins @ 200mg/kg of BW B3=A. vera proteins @ 300mg/kg of BW B4= Control

Figure 4.23: Per cent protection post challenge in experimental and control chickens

B1 B2 B3 B4

25% 50%

52.50%

57.50%

Table 4.24: Lesion scores and per cent protection against lesions in experimental and control chickens Groups Lesion Scoring of Birds Protection against lesions

` 0 1 2 3 4 (%) Caeca

B1 0 0 4 16 20 15

B2 0 0 10 13 17 20.625

B3 0 0 6 15 19 16.875

B4 0 0 5 10 25 12.5 Intestine

B1 0 0 15 3 18 23.125

B2 0 11 12 6 11 39.756

B3 0 0 14 6 15 30.625

B4 0 0 4 13 23 13.125

B1=A. vera proteins @ 100mg/kg of BW B2=A. vera proteins @ 200mg/kg of BW B3=A. vera proteins @ 300mg/kg of BW B4= Control

Table 4.24: Per cent protection against lesions in experimental and control chickens

B1 B2 B3 B4 45 40 35 30 25 20 15 10 5 0 Per cent protection against lesions Caeca Intestine Pre-dilaction sites

Table 4.25: Anticoccidial index for proteins Oocyst Relative rate of wt. Survival Lesion Groups clearance ACI gains rate value rate

B1 106.508 0.525 3.075 0.697 103.26

B2 176.178 0.575 2.425 0.748 172.26

B3 165.397 0.5 2.775 0.718 162.37

B4 21.4 0.25 3.475 0 17.85

B1=A. vera proteins @ 100mg/kg of BW B2=A. vera proteins @ 200mg/kg of BW B3=A. vera proteins @ 300mg/kg of BW B4= Control

Figure 4.25: Anticoccidial index for proteins

200 180 160 140 120 100 80

Anticoccidial Anticoccidial Index 60 40 20 0 B1 B2 B3 B4 Groups

Table 4.26: Organ-body weight ratios post challenge in experimental and control chickens Groups Thymus Spleen Bursa Caecal tonsils (Mean±SE) (Mean±SE) (Mean±SE) (Mean±SE)

B1 0.35±0.02 0.24±0.02 0.25±0.01 0.094±0.01

B2 0.36±0.01 0.25±0.02 0.27±0.02 0.096±0.02

B3 0.37±0.02 0.24±0.02 0.25±0.01 0.094±0.004

B4 0.35±0.02 0.25±0.02 0.24±0.02 0.092±0.01

Figure 4.26: Organ-body weight ratios post challenge in experimental and control chickens

0.4 B1 B2 B3 B4

0.35

0.3

0.25

0.2

0.15

bodyweight ratios -

0.1 Organ 0.05

0 Thymus Spleen Bursa C. tonsils Lymphoid organs

CHAPTER 5 DISCUSSION

Many plants and their products have shown biological response modifying (BRM) activities including Angelica gigas, Cissampelos pareira, Astragalus membranaceus, Mangifera indica, Ganoderma lucidum, Ocimum sanctum, Zingiber officinale, Phyllanthus emblica, Allium sativum, Curcuma longa, Nyctanthus arbor-tritis, Vernonia amygdalina, Azadirachta indica, Musa paradisiacal and Aloe (A.) vera (Minja, 1989; Nundkumar and Ojewole, 2002; Fajimi and Taiwo, 2005; Githiori et al., 2006; Athanasiadou et al., 2007; Akhtar et al., 2008; Ismail and Asad, 2009; Anosa and Okoro, 2011; Patil et al., 2011; Akhtar et al., 2012). Keeping in view the encouraging results of A. vera as BRM in previous studies conducted in our laboratory (Akhtar et al., 2012), the current study was focused on the evaluation of specific A. vera derived biomolecules responsible for such biological activities. Biological response modifiers (BRMs) are compounds, which induce modifications in the functioning of the immune system and produce effects on the physiology of man and animals (Murthy et al., 2010). These include synthetic and natural preparations, which increase host responses to proliferative lesions and pathogens (Moore, 2004). BRMs either stimulate or suppress the immune responses of an organism against foreign bodies/antigens. The innate immune responses, considered to be first line of defense, include skin (physical component); complement, lysozyme and interferon (biochemical component); and neutrophils, macrophages and monocytes (cellular components). When these innate responses of immune system are inadequate against any infection then adaptive responses play their role by antibodies production and T-lymphocytes activation that are effector cells of cell mediated immunity. B-cell or humoral immunity produces circulating antibodies, which effect invading agents. Cell mediated immunity produce large number of activated lymphocytes to kill the invaders (Rang et al., 2003; Patil et al., 2011; Saroj et al., 2012). BRMs also reported to reduce the immunosuppressive effects due to medicines and simultaneously increased their effectiveness (Thakur et al., 2012 ). These modulate immune responses by stimulation or suppression, which prevents the development of diseases (Ghule et al., 2006).

Due to the encouraging results of A. vera as BRM and therapeutic agent in our previous studies, the current study was focused on A. vera (Aloe barbadensis Miller), which is one of the most ancient and extensively used medicinal plant (Davis et al., 1989). It has been used as folk and traditional medicines for centuries to treat and cure many diseases. It is native of northern parts of Africa but can be found across the world; and more than 500 species of A. vera so far have been reported (Bawankar et al., 2012; Ahmed and Hussain, 2013). Modern therapeutic use of A. vera was started in 1930s and up till now considerable research has been reported in many reputed journals. A. vera has been reported to cure variety of conditions including fever, burns and wound healing, gastrointestinal disorders, sexual vitality and fertility problems, inflammation, ulcer, arthritis, cancer, immunosupression, acquired immune deficiency syndrome and coccidiosis (Swaim et al., 1992; Davis et al., 1994; Koo, 1994; Mwale et al., 2005; Bashir et al., 2011; Akhtar et al., 2012). It was also considered as an effective tool to enhance immunity in broiler chicks and increasing microvilli density (Jinag et al., 2005; Yim et al., 2011; Akhtar et al., 2012).

Moreover, various herbal preparations have showed immunomodulatory properties by stimulating immune responses (Chopra and Doiphode, 2002; Benny and Vanitha, 2004; Govindarajan et al., 2005; Spelman et al., 2006; Changkang et al., 2007; Kumar et al., 2012; Awais et al., 2011, 2013; Akhtar et al., 2012; Ramesh et al., 2012; Hussain et al., 2013). In this regard, A. vera is considered as one of the promising candidate having biological response modifying effects in different animal models (Djeraba and Quere, 2000; Lee et al., 2001; Krishnan, 2006; Liu and Wang, 2007; Hamman, 2008; Madan et al., 2008; Sikarwar et al., 2010; Patil et al., 2011; Chandu et al., 2011; Thakur et al., 2012; Kumar et al., 2012; Farahnejad et al., 2013). A. vera leaves contain three layers including rind, sap and gel (inner most layer). Its raw pulp contains about 98.5% water. Its gel is a clear substance made of 99-99.5% water (Eshun and He, 2004). Remaining portion contains glucomannans, vitamins, minerals, phenolic compounds, polysaccharides, lipids, proteins, amino acids, enzymes, saponins, salicylic acids, sugars and sterols (Boudreau and Beland, 2006; Surjushe et al., 2008; Balasubramanian and Narayanan, 2013).

Clinical analysis of A. vera revealed that its pharmacological activities are mainly concentrated in leaf gel and rind portions. A. vera plants contained >75 bioactive compounds (Eshun and He, 2004; Joseph and Raj, 2010). Its filet is 99.5 % water; whereas, 60% of remaining solids are polysaccharides. Most of the previous studies have focused on the evaluation of biological activities of two important components isolated from its leaves including anthraquinones (cathartic effect) and polysaccharides (effect on skin burns and incised wounds) (Zhang and Tizard, 1996; Chithra and Sjithlal, 1998); although only few reports are available on the elaboration of pharmacological mechanisms involved in such activities (Yao et al., 2009). In this regard, therapeutic efficacy of A. vera components had been reported in different animal models and human beings with promising results (Strickland, 2001; Agarry et al., 2005; Mwale et al., 2006), although limited work has been conducted on poultry (Mwale et al., 2005; Durrani et al., 2008; Akhtar et al., 2012; Yim et al., 2011). Keeping in view, the present project was designed to investigate the biological response modifying and therapeutic efficacy of A. vera polysaccharides and proteins in chickens.

The whole current study was divided into two experiments; one for isolation and characterization of polysaccharides and its evaluation as biological response modifiers and therapeutic efficacy against avian coccidiosis. The second experiment was meant for protein isolation and quantification followed by their effects as biological response modifiers and therapeutic efficacy against coccidiosis.

Polysaccharides are carbohydrates, containing monosaccharide units joined to each other by glycosidic linkages; their structure ranges from linear to highly branched (Varki et al., 2008). They have diverse macromolecules structure, which enable them to hold vast biological information. Polysaccharides are different from nucleic acids and proteins by having repetitive structural features of monosaccharide polymers joined by glycosidic linkages (Ooi and Liu, 2000). Polysaccharides have shown biological response modifying activities (Xia and Cheng, 1988; Zhang and Tizard, 1996; Ishizuka et al., 2004; Ramberg et al., 2010) by modulating both humoral and cellular immune responses (Xue and Meng, 1996; Tzianabos, 2000; Vahedi et al., 2011). Polysaccharides have variable potential to influence various biological mechanisms and can modulate immune cells (Vilcek and Lee, 1991;

Duerksen-Hughes et al, 1992; Fidler and Kleinerman, 1993; Klostergaard, 1993; Gordon, 2002). Mostly higher plant origin polysaccharides are relatively non-toxic and do not produce significant side effects, which are mostly associated with bacterial and synthetic immunomodulatory polysaccharide compounds. So, plant derived polysaccharides can be ideally used as immunomodulators, wound-healing and anti-tumor agents (Ovodov, 1998; Sherenesheva et al., 1998; Lazareva et al., 2002; Speranza et al., 2005; Mythilypriya et al., 2008). Polysaccharides extracted from A. vera contributed a major role to its ethnomedicinal properties. A few reports regarding its polysaccharides constituents and their characterization are available (Chen et al., 2002; Yan et al., 2003; Vinson et al., 2005; Ai et al., 2008). Further, a little information about polysaccharides structure is reported; so, mostly it is not possible to determine how specifically polysaccharides influence macrophage activity for immunomodulation (Schepetkin and Quinn, 2006).

A. vera is a rich source of many carbohydrate units including polysaccharide, mannose-6-phosphate and acemannan (Davis et al., 1994; Hamman, 2008). Acemannan (β- (1, 4)-linked acetylated mannose) has shown immunomodulatory activities by macrophages activation; enhanced cytokine release, stimulated macrophages interactions and increased production of cytotoxic T-lymphocytes (Womble and Helderman, 1992; Roberts and Travis, 1995). It also regulated macrophages phagocytosis and candidicidal activity (Stuart et al., 1997); and potentiated antibody production against coxackie virus (Roberts and Travis, 1995). Alcoholic precipitation of A. vera polysaccharides have shown significant radiation damage repair (Talmadge et al., 2004), wound healing (Tizard et al., 1994), pressure ulcers (Thomas et al., 1998), alveolar osteitis treatment after tooth extraction, modulate immune responses (Kahlon et al., 1991; Chinnah et al., 1992; Strickland et al., 1994; Karaca et al., 1995; Pugh et al., 2001), antineoplastic (Harris et al.,1991; King et al., 1995; ) and antiviral (Montaner et al., 1996) activities.

In the current study, purified polysaccharides from A. vera were analyzed by using high performance liquid chromatography (HPLC). Sugars were identified by comparing retention times with the monosaccharide standards. Results showed the presence of three different monosaccharides including maltose, glucose and mannose in hydrolyzed solution of A. vera at retention times of 9.423, 11.080 and 12.55, respectively.

Major polysaccharide containing Mannose named Acemannan has been isolated by various researchers (Hart et al., 1989). Acemannan is a linear polysaccharide made of (1, 4)- linked mannosyl residues (Femenia et al., 1999). Mandal and Das (1980) isolated polysaccharides including arabinan, galactan and glucomannan. Ni et al. (2004) and Choi and Chung (2003) isolated polysaccharides from A. vera inner leaf gel including arabinan, glucogalactomannan, glucuronic acid, galactoglucoarabinomannan, arabinorhamnogalactan, galactogalacturan and galactan. Mannose and glucose were major isolated sugars between total 55% and 75% of total monosaccharides. The mannose residues probably came from acemannan, which has been found in large amounts in A. vera gel and filet (Fogleman et al., 1992). Similar results were observed in the current study. Yao et al. (2009) isolated monosaccharide residues from A. vera including D-glucose (45.6%), d-6-deoxy-mannose (0.2%), d-galactose (44.1%), d-fructose (0.1%) and a new polysaccharide from gel named galactoglucan (molecular weight 150 kDa). Moghaddasi and Verma (2011) described that A. vera contained two polysaccharides acemannan and glucomannan; whereas, saccharides were mannose, aldopentose, L-rhamnose and glucose. Acemannan is a major carbohydrate present in gel. It is water soluble and contains long chains of mannose polymer. It accelerates wound healing process. It modulates immune system by particularly activating macrophages and cytokines production (Peng et al., 1991). Chang et al. (2010) isolated mannose glucose and galactose from gel juice. A. vera gel extract contains 38% carbohydrates containing 4% glucose, 41% Galacturonic acid, 6% xylose, 39% mannose, 1% rhamnose, 6% galactose, 1% fucose, 1% arabinose (Luta et al., 2009). Present study indicated the presence of glucose, maltose and manose, which were also reported by Luta et al. (2009). In some other studies, different polysaccharide of variable molecular sizes have been isolated from A. vera (Leung, 1978; Femenia et al., 1999; Reynolds and Dweck, 1999; Chow et al., 2005) and this variability may be due to seasonal and cultivational variation as reported by Leung (1978). In another study, Pierce (1983) studied differences in cultivation and seasonal conditions that resulted in difference in A. vera gel nutritional contents. In some studies, technical differences during isolation and endogenous enzymes mediated polysaccharide degradation have been reported to be the cause of different polysaccharide molecular sizes (Chang et al., 2006).

As for as A. vera proteins are concerned, many of the researchers used whole leaf as a starting material to isolate proteins (Fujita et al., 1978; Yoshimoto et al., 1987; Koike et al., 1995); and isolated glycoproteins of 29 kDa and 14kDa subunits, showed effects on wound healing (Fujita et al., 1978; Yagi et al., 1997; Choi et al., 2001), enhanced proliferation of human fibroblasts and hamster kidney cells (Yagi et al., 1997). In another study, glycoproteins of 5.5kDa, mainly of mannose (up to 70 %), had also been isolated, which also showed effects on wound healing and cells proliferation, both in vivo and in vitro (Choi et al., 2001). Lectin proteins from Aloe arborescence of 35 kDa, composed of 9kDa subunits had also been reported, which showed hemagglutinating and mitogenic effects (Winters et al., 1981). In the current study, proteins extracted from A. vera gel were quantified and its concentration was ranged from 22.6-26 µg/ml. Automated electrophoretic analysis revealed the presence of proteins of molecular weight 1 and 6kD. Variability in proteins molecular weight in current and previous studies might be due to seasonal conditions/variations, techniques used to isolate proteins etc.

In biological evaluation, immunological responses were determined both for cellular and humoral immune responses. Cell mediated immune responses were determined by in vivo lymphoproliferative response to PHA-P and carbon particle clearance assay. In vitro cell mediated immune responses were detected by lymphoproliferative response to Concanavalin- A (Con-A). Humoral immune responses were quantified by antibody response to sheep red blood cells.

Results revealed significantly higher in vivo cellular immune responses in chickens administered with A. vera polysaccharides and proteins. During body defenses, phagocytosis immediately affects invading foreign materials; whereas, T cells take time to be stimulated and proliferated before responding to invading agent (Lamont, 1986). PHA-P is a T-cell mitogen, which induces proliferation of T-lymphocytes. Its injection at a particular site induces localized in vivo T-cell lymphoproliferation (Cheema et al., 2003). These higher responses in experimental groups might be due to A. vera polysaccharides (Acemannan). Acemannan activates macrophages to inflammatory cytokines including IL-1, IL-6 and TNF- α (Tan and Vanitha, 2004). Acemannan also induces nitric oxide production through macrophage mannose receptors and activates macrophages, which cause immunomodulation

86 in chickens (Karaca et al., 1995). From these results, it can be speculated that A. vera polysaccharides induced cellular immune response that might play role in controlling and clearing the organism (Kougt et al., 1994) thus enhanced resistance against coccidiosis in chickens (Parmentier et al., 2001).

Concanavalin-A was used to assess in vitro cell mediated immune responses in experimental and controlled chickens at 7th and 14th day post administration of A. vera polysaccharides and proteins. Chickens administered with A. vera polysaccharides and proteins showed higher lymphoproliferation responses as compared to control group. It indicates that T-lymphocytes contained CD4 and CD8 cells. These cells repopulated peripheral blood leukocytes (PBLs) during any infection and helped in immune responses amplification including antibody production by producing different cytokines (Qureshi et al., 2000). This increased stimulation index in treated groups was due to direct contact of T- lymphocytes mitogen receptors, came in direct contact with Con-A (T-cell mitogen). It caused lymphocytes cell division, which resulted in increased response to Con-A (Qureshi et al., 2000). These stimulated PBLs by Con-A, produced interleukin-1 by monocytes, which stimulated lymphocyte proliferation (Abbas et al., 1991). In the current study, carbon particle clearance test was used as an indicator of phagocytic activity of reticuloendothelial system, generally measured by clearance of carbon particles from blood stream (Neha and Mishra, 2011). The test was performed to evaluate drug effects on reticuloendothelial system. It involved phagocytic cells, fixed tissues and mobile macrophages. These cells have an important role in particle clearance from blood (Ismail and Asad, 2009). Phagocytosis is a defensive mechanism, which showed its effects immediately after invasion of foreign particles (Lamont, 1986). Macrophages phagocytic activity is an important marker of immune reactivity, which can be increased by herbal polysaccharides (Shin et al., 2002; Moretao et al., 2003; Chen et al., 2010).

Polysaccharide primarily target macrophages and induced immunomodulation and have diverse therapeutic activities. They specially bind to many cell surface receptors on macrophages, including Toll-like receptors, dectin-1, CD14 and CR3, which are similar to microbial polysaccharides binding receptors. Polysaccharides activated macrophages to secrete nitric oxide and many chemokines and cytokines including (IFN-γ, IL-1, IL-6, IL-8,

IL-12 and TNF-α). They also increased myelopoiesis, macrophage maturation and monocyte release from bone marrow. There is a little information about polysaccharides structure in reported studies; it is therefore mostly impossible to demonstrate that how specifically polysaccharides influenced macrophages activity for immunomodulation (Schepetkin and Quinn, 2006). In the current study, when India ink injected intravenously into systemic circulation of chickens, carbon particles were engulfed by macrophages. The clearance rate of carbon particles and phagocytic index was calculated. The results of present study showed significantly higher clearance index in control group and all the other medicated groups showed low clearance index values indicating less carbon particles in blood. The results of phagocytic index (α) showed significantly higher response in all experimental groups administered with A. vera polysaccharides and proteins as compared to control. Maximum response was observed in group A2 and B2, which were administered with A. vera polysaccharides and proteins (at the dose rate of 200mg/kg body weight, respectively). Results indicated higher phagocytic index of treated groups (at dose rate of 100 and 200 mg/kg body weight), which might be due to reticuloendothelial system stimulation (Ismail and Asad, 2009; Neha and Mishra, 2011; Hajra et al., 2012).

Humoral immune responses were calculated in terms of total immunoglobulins (Igs), IgG and IgM antibody titers at day 7th and 14th post primary and secondary injection of sheep red blood cells (RBCs) and results expressed by calculating geomean titers in experimental and control groups. Significantly higher total Igs, IgG and IgM titers in experimental groups administered either A. vera polysaccharides or proteins showed their effects on the humoral arms of the chicken immune response as A. vera polysaccharides/proteins might have the ability to enhance the immunity (Waihenya et al., 2002; Akhtar et al., 2012). Effect of A. vera polysaccharides to stimulate the humoral immune responses is reported in mice in some previous studies (Madan et al., 2008; Yu et al., 2009) as polysaccharides contained aloeride, a high molecular weight polysaccharide, which induced IL-6 (a potent B-cell stimulant) to produce antibodies (Tan and Vanitha, 2004). Present study showed consistent findings to those of other scientists, who reported an increased antibody titer after feeding A. vera against Newcastle Disease virus (Hafeez et al., 2003; Jinag et al., 2005). Present study results were similar to Yu et al. (2009), who concluded that A. vera polysaccharides administered

88 groups of rats showed significant increase in IgA, IgM and IgG levels. Polysaccharides also increased indices of spleen and thymus; and increased levels of TNF-α, IL-2 and IgG in mice (Chen et al., 2005). Further, highest antibody titer was observed in groups of chickens administered with A. vera proteins @ 300mg/kg and polysaccharides @ 200mg/kg body weight. Sheep RBCs act as T-dependent antigens required combined effect of macrophages, B and T lymphocytes (Benacerraf, 1978), which elevated the humoral immune responses in the current study.

The chicken immune system contains specialized organs, cells and glands, which work efficiently and accurately to protect the body from invading pathogens including bacteria, viruses, parasites and fungi. Immune system can be divided into three basic subsystems viz. cellular, humoral and phagocytosis; each having different modes of action to prevent the body against diseases. Impaired functions of lymphoid organs are important indicators to demonstrate body’s ability to protect against certain diseases/disorders and can be expressed in terms of organ-body weight ratio (Heckert et al., 2002). Cells of the immune system, T and B lymphocytes, developed and differentiated in spleen and bursa (Eerola et al., 1987). In the present study, immune organ index was calculated in terms of organ-body weight ratios. Experimental groups administered with A. vera polysaccharides showed apparently higher per cent organ-body weight ratios as compared to control group; although, the difference was statistically non-significant (p>0.05) except thymus of experimental group

A2, which showed significantly higher response as compared to control. Similar response of organ-body weight ratios except spleen was observed in experimental chickens administered with proteins.

Chickens of all the groups were weighed on weekly basis from 1st to 6th week of age and feed consumed by each group was also recorded as important parameters/indicators of performance of a particular group. Experimental groups showed significantly higher (p<0.05) weight gains as compared to control. Maximum weight gains (at 6th week of age) were observed in groups administered with A. vera polysaccharides and proteins. These results were in accordance with findings of Jinag et al. (2005), Guo et al. (2004), Mehmet et al. (2005) and Durrani et al. (2007). Increased body weight gain and FCR had also been reported in other studies (Jagadeeswaran, 2007; Durrani et al., 2008; Olupona et al., 2010);

89 however, A. vera combination with Curcuma longa showed non-significant difference in weight gains but numerically higher body weight gains (Mehala and Moorthy, 2008). In another study, Lin et al. (2005) reported non-significant difference in feed conversion ratio between A. vera fed chickens and control.

In the present study, the therapeutic efficacy of A. vera polysaccharides and proteins against avian coccidiosis was investigated. Chickens of all the experimental and control groups were challenged with mixed Eimeria species (local isolates; E. tenella, E. maxima, E. acervulina and E. necatrix) at 14th day post administration of A. vera biomolecules.

Significantly lower oocysts per gram of droppings in experimental groups were calculated as compared to control, which may be due to resistance induced by administration of polysaccharides and proteins. Moreover, chickens administered with A. vera biomolecules, (protein and polysaccharides) developed lesser lesions on the caeca and intestine, and showed more protection than control. Lesion score is most common method for assessing intestinal condition during coccidiosis (Arabkhazaeli et al., 2013). Less intestinal lesions may be due to the effects of A. vera on intestinal tract microflora and reduced bowel putrefaction that subsided/decreased inflammation (Reynolds and Dweck, 1999) or lining of intestine layer with A. vera biomolecules (Waihenya et al., 2002). Acemannan treated broiler birds showed improved intestinal microflora (Lin et al., 2005).

Intestinal health is very important in chickens for better performance and improved FCR (Montagne et al., 2003). In vivo and in vitro anti parasitic properties of A. vera has been reported earlier. Its larvicidal and ovicidal effects against Haemonchus contortus and Leishmania spp. has also been documented (Dutta et al., 2008; Maphosa et al., 2010). Recently, Yim et al. (2011) reported that A. vera extract can be used as a safe dietary supplement against coccidiosis.

Maximum protection (70%) in polysaccharides administered chickens may be due to presence of Acemannan (a linear polymer containing acetylated mannose) component present in the A. vera, which act as immunomodulator and have antiviral activity; reduce opportunistic infections and stimulate wound healing (Christiaki and Florou-Paneri, 2010; Rodriguez et al., 2010; Darabighane et al., 2011). Further, Aloe proteins included Aloctin A and B, ATF 11 and salicylic acid, which have been reported to show antibacterial, anti

90 inflammatory, hemagglutinating and anti-inflammation effects (Davis and Maro, 1989; Ni and Tizard, 2004); and in current studies proteins administered chickens showed protection up to 57.5 per cent and showed lesser lesions as compared to control and more protection against lesions. Protection in the current study may be due to two effects of A. vera biomolecules; one as immunostimulation and second as wound healer (Talmadge et al., 2004). A. vera polysaccharides contain mannose, which increase fibroblast proliferation and macrophages activities (Davis et al., 1989), which in turn act as wound healing agent against coccidiosis. Further, several carbohydrate polymers (glucomannans) present in A. vera play role in healing process (Shelton, 1991) and inhibit cyclooxygenase pathway resulting in decreased prostaglandin production from arachidonic acids (Grindlay and Reynolds, 1986). As a result, it acts as an analgesic, anti-inflammatory and wound healing agent (Ovodov, 1998).

The chickens in all experimental groups were comparatively active as compared to control, had normal feed and water intakes; whereas, control group chickens were depressed/dull with ruffled feathers and less feed and water intake; may be due to coccidial lesions on the intestine, which effects body metabolism due to malabsorption (Kettunen et al., 2001; Dalloul et al., 2006) that resulted in less feed and water intake, decreased weight gains (Adams et al., 1996; Jang et al., 2007). Results of present study showed similar response against coccidiosis in terms of higher survival percentage, reduction in oocyst scores, higher weight gains and improved FCR when administered with herbal complex (Zaman et al., 2012), sugarcane (Awais et al., 2011).

Anticoccidial index (ACI) reflects a comprehensive ability of anticoccidial compounds or drugs. It was calculated by growth rate, oocyst value, survival rate and lesion value (You et al., 2008). According to Pablos et al. (2010), when ACI value lower than 120 this product has no anticoccidial activity, values between 120-160 partially effective and very effective at ˃ 160. ACI values calculated in present study for A. vera polysaccharides administered groups were A1 159.75, A2 239.63 and A3 195.31. While results of proteins administered groups were B1 103.26, B2 172.21 and B3 162.37. So A. vera polysaccharides and proteins at the dose rate of 200 and 300 mg/kg body weight proved very effective against

91 coccidiosis. Results of present study are consistent with the findings of Pablos et al. (2010), which showed higher ACI values in infected chickens when administered with maslinic acid.

Organ-body weight ratios of immune organs including spleen, thymus, caecal tonsils and bursa of fabricius were calculated on day 12th post challenge with mixed Eimeria species. All the experimental groups showed statistically similar organ-body weight ratios (p>0.05) except spleen; spleen of control chickens showed significantly higher organ-body weight ratios as compared to experimental groups administered with A. vera polysaccharides. Variable results on the lymphoid organs due to coccidiosis has been reported including adverse effect (Akhtar and Zafar, 2003), no effect (Augustine and Thomas, 1979) or increased in their weight (Venkatratnam et al., 1985). On the other hand, A. vera protein administered groups showed significantly higher spleen to body weight ratio; this may be due to cellular infiltration and spleen hypertrophy due to coccidiosis (Bettsille, 1986).

Results of current study indicated that A. vera bio-molecules, polysaccharides and proteins, have the potential to stimulate the humoral and cellular arms of the immune responses that may release cytokines to induce localized inflammatory reactions. This in turn increased vascular permeability; caused vasodilation, activation and accumulation of macrophages; enhanced phagocytic activity and more lytic enzyme production for destruction of pathogen(s) (Dashputre and Naikwade, 2010; Neha and Mishra, 2011).

Conclusions Following conclusions were drawn from the present study.

1. A. vera contains bio-molecules including polysaccharides and proteins, which had potential to act as biological response modifiers in broiler chickens. 2. A. vera polysaccharides and proteins administered chickens showed significantly higher cell mediated and humoral immune responses as compared to control. 3. Production performance in terms of weekly weight gains and FCR were better in chickens administered with polysaccharides and proteins at dose rates of 300 mg/kg body weight. 4. In challenge experiments, experimental groups administered with A. vera biomolecules showed higher per cent protection; statistically higher (p<0.05) daily weight gains, lower (p<0.05) oocysts counts and lesion scores as compared to chickens in control groups. 5. Chickens of experimental groups administered with A. vera biomolecules showed higher anticoccidial indices as compared to those of control groups. 6. Organ-body weight ratios of all lymphoid organs except spleen were statistically similar in both control and A. vera biomolecules administered chickens.

On the whole, it was concluded that A. vera derived biomolecules have the potential to be used as immunotherapeutic agents in poultry and can be commercialized. Recommendations Further studies are needed on field evaluation of A. vera derived biomolecules and to demonstrate their possible mechanism(s) of action for their scientific validation. Further, feasibility should also be made for the commercialization of these biomolecules for their efficient use as biological response modifiers in poultry.

CHAPTER 6 SUMMARY

Current study focused to isolate the A. vera polysaccharides and proteins and to evaluate their efficacy as biological response modifiers followed by their immunotherapeutic effects against avian coccidiosis in industrial broiler chickens. Purified polysaccharides from A. vera were analyzed by using high performance liquid chromatography (HPLC). Results showed presence of three different monosaccharides including maltose, glucose and mannose in hydrolyzed solution at retention times 9.423, 11.080 and 12.55, respectively. Proteins extracted from A. vera were quantified by Bradford assay and concentration was ranged from 22.6-26 µg/ml. The isolated proteins were subjected to automated electrophoretic analysis, which showed presence of proteins of molecular weight 1 and 6kD.

For immunological evaluation of A. vera polysaccharides and proteins, cell mediated and humoral immune responses were determined. Cell mediated immune responses were calculated by in vivo lymphoproliferative response to PHA-P and carbon particle clearance assay. In vitro cell mediated immune responses were detected by lymphoproliferative response to Concanavalin-A (Con-A). Humoral immune responses were calculated by antibody response to sheep red blood cells. Results of in vivo lymphoproliferative response to PHA-P showed significantly higher (p<0.05) response in chickens administered with A. vera polysaccharides and proteins as compared to control. In polysaccharides administered group, chickens of group A2 (at dose rate of 200mg/kg body weight) revealed a significantly higher (p<0.05) response as compared to control at 24, 48 and 72 hours post PHA-P injection. Chickens of protein administered groups showed significantly higher toe-web swelling for group B2 (at dose rate of 200mg/kg body weight). Lymphoblastogenic response to Con-A at 7th and 14th day post administration of A. vera polysaccharides and proteins administered chickens showed higher response than control.

Carbon particle clearance assay showed significantly higher clearance index in control group and all the other medicated groups showed low clearance index values indicating less carbon particles in blood. The results of phagocytic index showed significantly higher response in all three medicated groups as compared to control. Maximum

94 response was observed in group A2 and B2, which were administered with A. vera polysaccharides and proteins, respectively (at the dose rate of 200mg/kg body weight).

Humoral immune response were calculated in terms of total immunoglobulins (Igs), IgG and IgM antibody titers at day 7th and 14th post primary and secondary injection of sheep red blood cells. Results were expressed by calculating geomean titers in experimental and control groups. Significantly higher total Igs, IgG and IgM titers in experimental groups administered either A. vera polysaccharide or proteins showed their effects on the humoral arms of the chicken immune system. Results indicated that A. vera polysaccharides/proteins may have the ability to enhance the immunity.

Experimental groups administered with A. vera polysaccharides showed apparently higher per cent organ-body weight ratios as compared to control group; although, the difference was statistically non-significant (p>0.05) except thymus of experimental group A2, which showed significant response as compared to control. Almost similar response of organ-body weight ratios was observed in experimental chickens administered with protein. Chickens of all the groups were weighed (week 1st to 6th) and feed consumed by each group recorded as important parameters/indicators of performance of a particular group. Experimental groups showed significantly higher (p<0.05) weight gains as compared to control. Maximum weight gains at 6th week were observed in groups administered with A. vera polysaccharides and proteins. All experimental groups administered with A. vera polysaccharides and proteins showed better FCR as compared to control.

In this study, therapeutic efficacy of A. vera polysaccharides and proteins were checked against avian coccidiosis. Chickens of all the experimental and control groups were challenged with mixed Eimeria species local isolates. Maximum protection of 70% and 57.5% were observed in polysaccharides and proteins administered groups (at the dose rate of 200mg/kg body weight) respectively. Significantly lower oocysts per gram of droppings and lesion scores were calculated in both polysaccharides and proteins administered groups as compared to control.

Anticoccidial index (ACI) values calculated for A. vera polysaccharides were 159.75,

239.63 and 195.31 for groups A1, A2 and A3, respectively; and for proteins 103.26, 172.26 and

162.37 for group B1, B2 and B3, respectively. It was concluded that A. vera biomolecules, polysaccharides and proteins, have the potential to be used against coccidiosis.

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ANNEXURES Annexure I Zinc Sulphate Floatation Solution Reagents Zinc Sulphate 386 gm Distilled water 1000 ml Specific gravity 1.200-1.300

Annexure II

Different Eimeria species infecting chickens (Reid and Long, 1979)

Species Region Infected Average oocyst size (l x w, μm) E. tenella Caeca 22.0 x 19.0 E. acervulina Duodenum 18.3 x 14.6 E. brunetti Lower intestine, rectum 24.6 x 18.8 E. maxima Upper intestine, ileum 30.5 x 20.7 E. mitis Upper intestine 16.2 x 16.0 E. praecox Duodenum, upper jejunum 21.3 x 17.1 E. necatrix Mid intestine, Cecae 20.4 x 17.2

Annexure III Vaccination Schedule adopted to immunize the experimental and control chickens Age of the chick Vaccine Route (Days) adopted 6 Newcastle disease vaccine (Merial®, France) Eye drop 10 Infectious bursal disease vaccine (Merial®, France) Oral 17 HPS vaccine (Sanna Labs., Pakistan) S/C 20 Infectious bursal disease vaccine (Booster) Oral (Merial®, France) 22 Newcastle disease vaccine (Booster) (Merial®, France) Oral

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Annexure IV: Chromatograms of standard monosaccharide solutions

a. Chromatogram of D-Arabinose

b. Chromatogram of Galactose

c. Chromatogram of D-Xylose

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d. Chromatogram of Maltose

e. Chromatogram of Mannose

f. Chromatogram of Fructose

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g. Chromatogram of Glucose

h. Chromatogram of Melezitose

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