Efficacy and toxicity of thirteen plant leaf acetone extracts used in ethnoveterinary medicine in South Africa

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Background

Globally, the sheep production industry provides major employment, animal protein, manure and wool for clothing from an estimated one billion animals. To the African small holder farmer, sheep rearing is a store of wealth and a cultural identity which is jealously guided as an inheritance. The sheep industry in South Africa is also a major part of the economy as it worth hundred millions of US dollar, particularly in wool production. Production is however hampered by constraints such as competition for scarce land resources, diseases, of which Haemonchus is the single most important constraint to sheep production globally.
Currently, there is a widespread resistance to available anthelmintic drug, with the exception of monepantel and derquantel. The development of resistance by Haemonchus requires alternative control strategy (MacGlafin et al., 2011). Options available include vaccine development, use of the pathogenic fungus Duddingtonia flagrans or the use of plant herbal preparations. Plant herbal preparations have beEN used for treatment of different ailments in animals for a very long time and this constitutes the ever growing field of ethnoveterinary medicine (EVM). Ethnoveterinary medicine (EVM) comprises a complex system of beliefs, skills, knowledge and practices relating to animal husbandry and general animal care (McCorkle, 1986). This all important field has not received the desired attention until the last thirty years. Currently, several plant species use in ethnoveterinary medicine are been subjected to scientific testing for efficacy and toxicity. In South Africa, only about 13 % of plants used for EVM have been evaluated scientifically for activity and toxicity. This requires more plant extracts to be evaluated for their efficacy and toxicity. This will improve our understanding of EVM and possibly lead to extracts or compounds with potent activity against pathogens that cause diseases in animals. With an estimated 87% of the global medications used against cancer, microbial and parasitic infections being derived from natural products, particularly higher plants the benefits of EVM in primary animal healthcare is clearly evident (Martin et al., 2001).

Hypothesis

The leaves of the thirteen selected plant species used for this study have bioactive compounds against Haemonchus contortus.

Statement of the problem

Haemonchus contortus is a major cause of diseases in sheep with high morbidity and mortality. Anthelmintic resistance is a serious setback to available anthelmintic. The exploration and bio prospecting of plants with anthelmintic activity is a possible solution to the above mentioned problem.

Justification of the study

Result generated from this study will add to the increasing data base of knowledge on EVM, there by contributing to the safety and efficacy of plant extracts used in treatment of diseases. This study can also lead to the identification of new therapeutic skeletons from medicinal plants or whole extracts that can be used for treatment of resistant Haemonchus strains and other pathogens.

Aim

The aim of this study is to screen and possibly identify lead compound(s) or plant extracts that may be useful as anthelmintic agents.

Overview of sheep in Production

The genus Ovis includes all sheep, with domesticated sheep belonging to the species Ovis aries. While the origin of the modern domestic sheep remains uncertain, sheep are believed to be the second species domesticated by ancient man and as a group is made up of more breeds than other domesticated species. Interesting more argument exists on the ancestry and zoological classification of sheep than any other farm animal (Ryder, 1984). A total of 7 distinct wild forms with 40 different varieties have been recognized. Several wild sheep species or subspecies have been proposed as the ancestors of domestic sheep (Ryder, 1984) or are believed to have contributed to specific breeds. Among the wild species that have contributed to the present day modern sheep are the Argali (Ovis ammon) of Central Asia, the Urial (Ovis vignei), also of Asia and the Moufflon (Ovis musimon) of Asia Minor and Europe (Davendra and McElroy, 1982). Nonetheless nine thousand BC is probably the most widely accepted time of domestication of sheep on the borders of present day Iran – Iraq (Figure, 2.1). From this place of origin sheep spread to the remaining part of the continental masses of Asia, Africa and Europe. The most recent and possibly the greatest migration took place 100-200 years ago, with the settlement of the southern hemisphere by Europeans.
They took the Merino breed and the British breeds to South America, South Africa, Australia and New Zealand. Today these places account for 35-40% of the world sheep population (Zygoyiannis, 2006). In South Africa, the Namaque Afrikaner is the oldest breeds of sheep in the area. It appears they were brought into South Africa by the Khoi-Khoin people, who migrated from the central lake area of Africa bringing their sheep with them (Anon, 2010). Sheep are multiparous animals, kept for various producing purposes of which their primary worth is in meat and wool production, with some regional differences as countries such as Turkey, Iran and southern and central Europe place more value in milk production (Zygoyiannis, 2006). The World total sheep population is estimated at approximately 1.024 billion with Africa contributing 23.8% second only to Asia with 40.6% (Anon, 2005, George and McKellar, 2006). South Africa is second only to Sudan within the Africa continent, with a sheep population of about 25.3 million and 10th on the world ranking (Anon, 2005). Sheep production is an important aspect of South Africa agriculture and makes substantial contribution to the economy. The contributions include granting employment opportunities to thousands, source of clothing and feeding millions with good quality protein. Sheep wool production contributed 1, 616, 701,669 Rands in the 2007/2008 season and 2.27 billion Rands for the 2011/2012 (Anon, 2012).

Table of Contents :

• Title page
• Declaration
• Dedication
• Acknowledgements
• Publications
• Conferences
• Abstract
• Table of Contents
• List of Figures
• List of Tables
• List of abbreviations
• CHAPTER 1: INTRODUCTION
  • 1.1 Background
  • 1.2 Hypothesis
  • 1.3 Statement of the problem
  • 1.4 Justification of the study
  • 1.5 Aim
  • 1.6 Objectives
• CHAPTER 2: LITERATURE REVIEW
  • 2.1 Overview of sheep in Production
    • 2.2 Parasitic gastroenteritis (PGE) complex
    • 2.3 Haemonchus contortus (Rudolphi, 1803)
    • 2.3.1 Morphology
    • 2.3.2 Life cycle
    • 2.3.3 Epidemiology of Haemonchosis
      • 2.3.3.1 Environmental factors
      • 2.3.3.2 Nutrition
      • 2.3.3.3 Breed
      • 2.3.3.4 Age, sex and reproductive status
    • 2.3.4 Clinical signs
      • 2.3.4.1 Anorexia
      • 2.3.4.2 Anaemia
      • 2.3.4.3 Hypoproteinaenia
      • 2.3.4.4 Weight loss
    • 2.3.5 Pathology
    • 2.3.6 Immunity of sheep to Haemonchus
    • 2.3.7 Diagnosis of Haemonchus
      • 2.3.7.1 Clinical diagnosis
      • 2.3.7.2 Laboratory Diagnosis
    • 2.3.8 Post-mortem examination
    • 2.3.9 Serology
  • 2.4 Treatment and control
  • 2.4.1 Anthelmintics
  • 2.4.1.1 Problems with current anthelmintic
  • 2.5 Alternative Treatment Strategies
    • 2.5.1 Vaccination
    • 2.5.2 Grazing management
    • 2.5.3 Biological control
    • 2.5.4 Herbal preparations
  • 2.6 Plant selection
    • 2.6.1 Brachylaena discolor DC
    • 2.6.2 Apodytes dimidiata E. Mey. ex Arn
    • 2.6.3 Clerodendrum glabrum E. Mey
    • 2.6.4 Clausena anisata (Wild.) Hook.f. ex Benth
    • 2.6.5 Cyathea dregei Kunze
    • 2.6.6 Heteromorpha trifoliata (Spreng.) Cham. & Schltdl. var. abyssinica (A.Rich.)
    • 2.6.7 Indigofera frutescens
    • 2.6.8 Leucosidea sericea Eckl. & Zeyh
    • 2.6.9 Milletia grandis E.Mey
    • 2.6.10 Melia azedarach
    • 2.6.11 Maesa lanceolata Forssk
    • 2.6.12 Strychnos mitis S. Moore
    • 2.6.13 Zanthoxylum capense (Thunb). Harv
  • 2.7 General Conclusion
• Chapter 3: Efficacy and toxicity of thirteen plant leaf acetone extracts used in ethnoveterinary medicine in South Africa on egg hatching and larval development of Haemonchus contortus
  • 3.1 Background
  • 3.2 Methods
    • 3.2.1 Plant collection
    • 3.2.2 Plant extraction
    • 3.2.3 Recovery and preparation of eggs
    • 3.2.4 Egg hatch assay (EHA)
    • 3.2.5 Larval development test (LDT)
    • 3.2.6 Cytotoxicity assay using MTT
    • 3.2.7 Data analysis
  • 3.3 Results
    • 3.3.1 Yield
    • 3.3.2 Egg hatch assay
    • 3.3.3 Larval development test
    • 3.3.4 Cytotoxicity
  • 3.4 Discussion
  • 3.4.1 Conclusion
• Chapter 4: Some southern African plant species used to treat helminth infections in ethnoveterinary medicine have excellent antifungal activities
  • 4.1 Background
  • 4.2 Methods
    • 4.2.1 Plant collection
    • 4.2.2 Plant extraction
    • 4.2.3 Chromatographic analysis
    • 4.2.4 Antifungal activity
    • 4.2.5 Cytotoxicity assay using MTT
    • 4.2.6 Bioautographic investigations
  • 4.3 Results
    • 4.3.1 Plant species yield
    • 4.3.2 Phytochemical analysis
    • 4.3.3 Bioautography
    • 4.3.4 Antifungal activity of extracts
    • 4.3.5 Cytotoxicity and therapeutic index
  • 4.4 Discussion
  • 4.4.1 Conclusions
• Chapter 5: The antibacterial and antioxidant activity of thirteen South African plant species used in ethnoveterinary medicine to treat helminth infections
  • 5.1 Introduction
  • 5.2 Materials and Methods
    • 5.2.1 Plant collection
    • 5.2.2 Plant extraction
    • 5.2.3 Chromatographic analysis
    • 5.2.4 Antioxidant activity
    • 5.2.5 Antibacterial activity
    • 5.2.6 Bioautographic investigations
  • 5.3 Results
    • 5.3.1 Plant extracts yield
    • 5.3.2 Phytochemical profiling and Antioxidant activity
    • 5.3.3 Bioautography
    • 5.3.4 Minimal Inhibitory Concentrations and total activity against bacterial pathogens
    • 5.3.5 Therapeutic index
  • 5.4 Discussion
  • 5.4.1 Conclusions
• Chapter 6: Anthelmintic phloroglucinol derivatives and antifungal activity of fractions from Leucosidea sericea (Rosaceae)
  • 6.1. Introduction
  • 6.2. Materials and Methods
    • 6.2.2. Plant material
    • 6.2.3. Extraction and bio-guided isolation
      • 6.2.3.1. Agrimol G (2)
      • 6.2.3.2. Agrimol A (3)
    • 6.2.4. Anthelmintic assay
      • 6.2.4.1. Recovery and preparation of eggs
      • 6.2.4.2. Egg hatch assay (EHA)
      • 6.2.4.3. Larval development test (LDT)
    • 6.2.5. Antifungal assay
    • 6.2.6. Cytotoxicity assay using MTT
    • 6.2.7. Statistical analysis
  • 6.3. Results
  • 6.4. Discussion
  • 6.5. Conclusions
• Chapter 7: Establishing the effects of phloroglucinol derivatives isolated from Leucosidea sericea on the ultrastructure of Haemonchus contortus, using electron microscopy
  • 7.1 Introduction
  • 7.2 Material and Methods
    • 7.2.1 Study compounds
    • 7.2.2 Parasite
    • 7.2.3 Transmission Electron Microscopy
    • 7.2.4 Scanning Electron Microscopy
  • 7.3. Results
  • 7.4. Discussion
• Chapter 8: The anthelmintic effect of acetone leaf extract of Leucosidea sericea in sheep artificially infected with Haemonchus contortus
  • 8.1 Introduction
  • 8.2 Materials and methods
  • 8.2.1 Plant material and extract preparation
  • 8.2.2 Infective larvae
  • 8.2.3 Animals and experimental design
    • 8.2.4.1 Treatment procedure
  • 8.2.6 Faecal egg counts (FECs)
  • 8.2.7 Clinical pathology
  • 8.2.9 Statistical analysis
  • 8.3 Results
    • 8.3.1. General clinical observation
    • 8.3.2 Faecal egg count reduction test (FECRT) and abomasa worm counts
    • 8.3.3 Effect on haematology and liver enzymes following treatment with L. sericea and their controls
  • 8.4 Discussion
  • 8.5 Conclusions
• Chapter 9: General Discussions and Recommendations
  • 9.1 General Discussions
  • 9.2 Recommendations
  • References
  • Appendix

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