The efficacy of seven southern African ethnoveterinary medicinal plants in the mitigation of cutaneous myiasis Submitted in fulfilment of the requirements for the degree of Philosophiae Doctor (PhD) in Paraclinical Sciences by Lillian Mukandiwa (MSc. Animal Science, University of Zimbabwe) Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria Supervisor: Prof. V. Naidoo Co-Supervisor: Prof. J.N. Eloff 2012 © University of Pretoria Declaration The experimental work and results described in this thesis is my original work (except where the input of others is acknowledged), conducted in the Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria. This thesis has not been submitted in any other form to another University or academic institution for consideration. I, Lillian Mukandiwa, declare the above statement to be true. ...................................................................... L. Mukandiwa ii © University of Pretoria Acknowledgements I would like to express my sincere gratitude to the following organizations and people: Supervisors, Prof. Vinasan (Vinny) Naidoo and Prof. Jacobus (Kobus) Eloff, for their guidance, invaluable input and constructive criticism that made this work a success. German Academic Exchange Services (DAAD), for the scholarship that allowed my enrolment for this study. National Research Foundation (NRF), for providing some of the research funds that made these studies possible. Magret McNamara Memorial Fund (MMMF), for providing some of the research funds that made possible these studies. The Department of Paraclinical Sciences, for allowing me an opportunity to undertake my studies and providing the required facilities as well as financial support. The South African National Biodiversity Institute (SANBI), for providing the required plant material from the Pretoria National Botanical Garden. And a special mention goes to Khangela Baloyi, the garden guide, for his help in tree identification and collection of plant material. Lyndy McGaw, for her expertise on the in vitro cell toxicity testing. Kerri Wolter, for the supply of blowfly larvae that were used to establish the laboratory fly colony. Thank you for your patience and bearing with my emails when yet more larvae were required. Tharien De Winnar, for the support and encouragement throught my studies. For coping with my “unusual” research requirements, I appreciate all those trips to the butchery, making sure I had enough meat and liver for the larvae. iii © University of Pretoria Friends and Colleagues, namely Nana B, Tshepiso M, Donald S, Matthew for their help and advice with different experiments and critiquing my draft manuscripts. My boys, Tanaka, Axikhesiwe and Donald, for the support and understanding. You made it easy; the trips to the farm became family outings. And finally to my late Mama, Emelda Mukandiwa. You were the inspiration for this study through your steadfast belief that Aloe was better than Terramycin®. Thank you for the support and encouragement Mama. You believed in me, and gave me the courage to undertake this study. You were my best supporter, always taking time to enthusiastically ask me how my study was going and always asking for an update on the results. I am deeply saddened that we cannot celebrate the completion of my PhD, the way we should have. However I believe you are looking down and smiling, and celebrating with the other angels, thank you Mama. iv © University of Pretoria List of Abbreviations EVM Ethnoveterinary medicine NWS New World Screwworm OWS Old World screwworm PSW Palaearctic Screwworm RH Relative humidity OP Organophosphate AChE Acetylcholinesterase Ach Acetylcholine NTE Neuropathy target esterase OPIDN Organophosphate-induced delayed neurotoxicity IGRs Insect growth regulators JH Juvenile hormone PTTH Prothoracicotropic hormone CSIs Chitin Synthesis Inhibitors JHAs Juvenile hormone agonists GABA Gamma-aminobutyric acid CNS Central nervous system MIC Minimum inhibitory concentrations v © University of Pretoria Publications Full articles prepared from thesis: Published 1. Mukandiwa, L., Naidoo, V. Eloff, J.N. 2012. In vitro antibacterial activity of seven plants used traditionally to treat wound myiasis in animals in southern Africa. Journal of Medicinal Plants Research, 6, 4379-4388. 2. Mukandiwa, L., Eloff, J.N., Naidoo, V. 2012. Screening of plants used traditionally to treat myiasis for effect on the survival and development of Lucilia Cuprina and Chrysomya Marginalis (Diptera: Calliphoridae). Veterinary Parasitology, 190, 566-572. 3. Mukandiwa, L., Eloff, J.N., Naidoo, V. 2012. Extracts of four plant species used traditionally to treat myiasis influence pupation rate, pupal mass and adult blowfly emergence of Lucilia cuprina and Chrysomya marginalis (Diptera: Calliphoridae). Journal of Ethnopharmacology, 143, 812-818. Submitted 1. Mukandiwa, L., Eloff, J.N, Sibanda, D.R., Naidoo, V. : Field evaluation of acetone extracts of Clausena anisata as a blowfly population control product for management of myiasis in livestock. Submitted to Veterinary Parasitology, November 2012. 2. Mukandiwa, L., Ahmed, A., Naidoo, V. Eloff, J.N. Isolation of seselin from Clausena anisata (Rubiaceae) leaves and its effects on the feeding and development of blowfly larvae may explain its use in ethnoveterinary medicine. Submitted to Journal of Ethnopharmacology, November 2012. Published conference abstracts from thesis 1. Mukandiwa, L., Naidoo, V., Eloff, J.N., 2011. The rationale for plant species used traditionally to treat or manage myiasis. South African Journal of Botany 77, 545- 546. vi © University of Pretoria 2. JN Eloff, L. Mukandiwa, V Naidoo. 2010. Evaluation of some plants species traditionally used to treat myiasis in southern africa for antibacterial and larval growth inhibition. Herbal Remedies in Animal Farming. ZooBioDi, Biodinamica. Conference presentations from thesis a) International Mukandiwa, L., Eloff, J.N. Evaluation of plants used traditionally to protect animals against myiasis. Presented at the 14th NAPRECA Symposium and AAMPS Ethnoveterinary Medicine Symposium held in Nairobi, Kenya, 8 – 12 August 2011. Mukandiwa, L., Naidoo, V., Eloff, J.N. In vitro antibacterial activity of seven plants used traditionally to treat wound myiasis in animals in southern Africa. Presented at the Regional Symposium on Plants of Economic Use held in Lusaka, Zambia, 21 – 23 June 2010. b) National Mukandiwa, L., Naidoo, V., Eloff, J.N., 2011. The rationale for plant species used traditionally to treat or manage myiasis. Presented at the South African Association of Botanists (SAAB) Conference, held at Rhodes University, Grahamstown, South Africa, 14-19 January 2011. Mukandiwa, L., Naidoo, V., Eloff, J.N. The effects of plants used traditionally to treat myiasis on the survival and development of blowfly (Calliphoridae). Presented at the Indigenous Plant Use Forum (IPUF), held in Keimos, South Africa, 28 June – 01 July 2010. vii © University of Pretoria Abstract Myiasis, the infestation of the skin of mammals by larvae of a variety of fly species, has been recognised as a major disease from ancient times. Despite being well known as a disease, it remains poorly controlled in the animal production industry with severe economic losses resulting thereof. The control of the myiasis-causing flies relies heavily on the use of pharmaceutical chemicals such as the organophosphates, pyrethroids or insect growth regulators. Unfortunately these chemicals are characterised by a high potential to cause human or animal toxicity in addition to being environmental contaminants. Another problem resulting from their continued use has been the development of resistance in the treated flies. Newer products need to be discovered. One source of these compounds could be the ethnoveterinary plants already in use by subsistence farmers where insecticides are either unavailable or unaffordable. This study focused on seven plant species used as such in South Africa and Zimbabwe: Aloe marlothii A. Berger, Aloe zebrina Baker, Calpurnia aurea (Aiton) Benth, Psydrax livida (Hiern) Bridson (Canthium huillense), Clausena anisata (Willd), Erythrina lysistemon Hutch, and Spirostachys africana Sond. In the first step of evaluation, the selected plant species were screened for their activity against selected wound contaminating bacteria, which are recognised as the inciting factor attracting myiasis-causing flies due to the characteristic odour they produce. Using the microdilution and bioautography methods, all plants had inhibitory activity with the acetone extracts being superior with MICs ranging from 0.04 to 0.6 mg/ml. Using the surface area of a general wound and the density of the extract, it was demonstrated that wounds treated with these extracts could reach these MIC levels. It was concluded that these plants could be beneficial in managing myiasis, in part, by reducing secondary infections and fly attraction. Two studies were undertaken to determine the in vitro effects of these plant species on the behaviour and development of blowfly larvae. For the first study, larvicidal activity of the seven plant species was determined on third instar larvae fed on a combination of viii © University of Pretoria meat (30 g) baited with the acetone leaf extracts (1 ml at 10 mg/ml). Aloe zebrina, C. anisata, E. lysistemon and S.africana, induced developmental anomalies in the larvae including paralysis, prolongation