Prevalence and Genetic Relatedness of Besnoitia Besnoiti Isolates from Different Geographical Regions of South Africa

Prevalence and genetic relatedness of Besnoitia besnoiti isolates from different geographical regions of South Africa By Mokgadi Pulane Malatji 214585800 [email protected] Submitted in fulfilment of the academic requirements for degree of Master of Science School of Life Sciences College of Agriculture Engineering and Science University of KwaZulu-Natal Durban 400 December 2015 Supervisor: Prof. S. Mukaratirwa Co-supervisor: Dr. S. Chitanga As the candidate’s supervisor I have approved this thesis/dissertation for submission. Signed: Name: Prof. S. Mukaratirwa Date: 30 November 2015 Signed: Name: Dr. S. Chitanga Date: 30 November 2015 i ABSTRACT Bovine besnoitiosis is a protozoan disease caused by an apicomplexan parasite Besnoitia besnoiti and is reportedly re-emerging in Europe and occurring in many other countries including South Africa. The disease has long been neglected and has only recently started getting attention due to its increasing geographical distribution. This parasite causes significant economic losses due to reduced body condition, declined milk production, irreversible sterility in males and mortality. This study was conducted to determine the prevalence of Besnoitia besnoiti infection in cattle, as well as to establish the phylogenetic relationship among parasite isolates in different geographical regions of South Africa, where the disease was previously reported. A total of 688 cattle (688 blood and 376 skin samples) were randomly sampled from farms located in Limpopo, Gauteng, KwaZulu-Natal and Eastern Cape provinces of South Africa. Based on the analyses of DNA sequences of the nuclear ribosomal internal transcriber spacer 1 (ITS1), it was observed that 15.7% (108/688) of the sampled animals were positive, with 5.3% (20/376) and 14.4% (99/688) of the animals being positive on skin and blood samples, respectively. 2.9% (11/376) of the animals were positive on both blood and skin samples. The difference between in prevalence between the areas sampled were not significant, χ2 = 0.263. The parasite was more prevalent in communal farms (30.8 %) and in exotic breeds (35.3 %) than local or mixed breeds, and the difference in prevalence between farming and breed type were not significant (χ2 = 0.199, χ2 = 0.227 respectively). Aligned sequences were analysed by Maximum parsimony, neighbour joining and maximum likelihood and phylogenetic analysis of the isolates was carried out. Results showed that, based on the ITS1 region, our isolates were closely related to the wildebeest strain which is currently used for the manufacture of the vaccine, forming a clade which is separate from the European strains. One of the isolates from Gravelotte, Limpopo province, was closely related to the European strains, forming a sister clade for the European strains from GenBank. This is the first report on molecular characterisation of the parasite in South Africa and will aid in disease surveillance and control in the studied areas. ii PREFACE The research contained in this thesis was completed by the candidate, from September 2014 to October 2015, while based in the Discipline of Parasitology, School of Life Sciences, University of KwaZulu-Natal, Westville Campus, under the supervision of Professor S. Mukaratirwa and Dr S. Chitanga. The research was financially supported by National Research Foundation (NRF). The contents of this study represent original work by the author, and have not been submitted in any form to another tertiary institution, except where the work of others is acknowledged in the text. Signed: Prof. S. Mukaratirwa Date: 30 November 2015 iii DECLARATION I Mokgadi Pulane Malatji, declare that: 1. The research reported in this thesis, except where otherwise indicated, and is my original research. 2. This thesis has not been submitted for any degree or examination at any other university. 3. This thesis does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This thesis does not contain other persons’ writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written but the general information attributed to them has been referenced b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This thesis does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References sections. Signature: _____________________ Signed: Malatji M.P Date: __________________ iv ACKNOWLEDGEMENTS Firstly I would like to thank the almighty God for his love and mercy, for his protection and helping me endure every challenge I encountered in my life. Thanks for granting me with the wisdom, courage and strength to pull through this research project. My sincere gratitude to my supervisor Prof. S. Mukaratirwa, and co-supervisor Dr Chitanga for accepting me as their student at the University of Kwa-Zulu-Natal, and for providing the support, encouragement, and guidance needed on completing the dissertation process. Your assistance, knowledge and dedication greatly contributed to my success and helped me persevere even through the hard times. Your patience is appreciated. A huge thank you to my research team Lorna Gcanga and Dr Nandipha Ndudane for the hard and good work we have put in this project. Dr Oliver Zishiri and Prof, Jenny Lamb: thank you for dedicating your time assisting me with molecular laboratory wok and phylogenetic analysis. I also acknowledge the different farmers and their respective animal health technicians/veterinarians for their co-operation. A special thanks to my parents and my siblings Modjadji, Mokhenete and Kabelo for their unconditional love, support, encouragement and understanding. You are the pillars of my strength v LIST OF ABBREVIATIONS µl – Microliter µm – Micrometre DNA – Deoxyribonucleic acid EDTA – Ethylenediaminetetraacetic acid ELISA – Enzyme-linked immunosorbent assay H2O – Water IFAT – Immunofluoroscence antibody test ITS – Internal transcribed spacer PCR – Polymerase chain reaction Taq - Thermus aqueous TBE – Tris/ Borate/ EDTA UV – Ultraviolet MEGA – Molecular evolutionary genetics analysis DnaSP - DNA Sequence Polymorphism TCS – Transitive consistency score vi TABLE OF CONTENTS Abstract ii Preface iii Declaration iv Acknowledgements v List of Abbreviations vi Table of content vii List of tables x List of figures xi Chapter 1: Introduction 1 1.1. Background 1 1.2. Objectives 2 Chapter 2: Literature review 3 2.1. Introduction 3 2.2. Description of pathogen 3 2.3. Mode(s) of transmission 3 2.4. Life cycle 5 2.5. Pathogenesis, clinical signs and pathological findings 6 2.5.1. Febrile acute phase 6 2.5.2. Chronic or scleroderma phase 7 2.6. Diagnosis 8 2.7. Economic impact and welfare 8 vii 2.7.1. Effect of productivity 8 2.7.2. Economic impact 9 2.8. Epidemiology 10 2.8.1 Geographical distribution 10 2.8.2 Risk factors 11 2.8.3 Prevalence of B. besnoiti infection 13 2.9. Control 15 Chapter 3: Materials and Methods 18 3.1. Study site and sample collection 18 3.2. DNA extraction 19 3.2.1. Blood DNA extraction 19 3.2.2. Skin DNA extraction 19 3.3. PCR and electrophoresis 19 3.4. Sequencing 20 3.5. Molecular analysis 21 3.5.1. Phylogenetic analyses 21 3.5.2. Haplotype analysis 21 3.6. Statistical analysis 21 Chapter 4: Results 22 4.1. Prevalence of B. besnoiti infection in cattle 22 4.2. Phylogenetic relationship of parasite isolates 24 viii Chapter 5: Discussion 28 Conclusion 31 References 32 Appendices 41 ix LIST OF TABLES AND APPENDICES Table 1: Prevalence of B. besnoiti infection in cattle from selected provinces of South Africa 22 Table 2: Prevalence of B. besnoiti infection in cattle by husbandry systems in South Africa 23 Table 3: Prevalence of B. besnoiti infection in cattle by breed in four provinces of South Africa 23 Appendix 1: Summary of molecular analysis and vaccination history 41 Appendix 2: P-distance matrix between Besnoitia besnoiti isolates as shown in figure 3 42 Appendix 3: P-distance matrix between Besnoitia isolates as shown in figure 4 43 x LIST OF FIGURES Fig 1: Life cycle and transmission of Besnoitia besnoiti 5 Fig 2: Schematic diagram of management measures coupled to diagnosis for the control of bovine besnoitiosis 16 Fig 3: Agarose gel-electrophoretic analysis (2%) of amplification products from conventional Besnoitia besnoiti ITS1 rDNA PCR. 20 Fig 4: Phylogenetic tree based on 200 nucleotides of the ITS-1 region, showing the relationship between cattle sample, Genbank Besnoitia besnoiti and outgroup sequences. 25 Fig 5: Statistical parsimony haplotype network on 200 nucleotides of the ITS-1 region of the cattle sample and Genbank Besnoitia besnoiti sequences. 26 Fig 6: Phylogenetic tree based on 55 nucleotides of the nuclear ribosomal ITS-1 region of the parasite, showing the relationship between cattle samples, Genbank Besnoitia besnoiti and outgroups. 27 xi CHAPTER 1: INTRODUCTION 1.1 Background Bovine besnoitiosis is a chronic and debilitating disease of cattle, caused by Besnoitia besnoiti (B. besnoiti) and reported to be endemic in sub-Saharan Africa, Middle East and France (Bigalke and Prozesky, 1994; Chatikobo et al., 2013). The causative agent is a cyst-forming coccidial parasite belonging to the family Sarcocystidae, subfamily Toxoplasmatidae and phylum Apicomplexa. The disease manifests cutaneously and systemically and in its chronic form is referred to as elephant skin disease (Jacquiet et al., 2010; Alvarez-Garcia et al., 2013). B. besnoiti is regarded as a parasite of economic importance through mortality of infected animals, loss of income caused by reduced value of hides and downgrading of carcass quality, and permanent or temporary sterility in breeding bulls that survived the acute and chronic stages of the infection (Bigalke, 1968; Basson et al., 1970; Sekoni et al., 1992; Cortes et al., 2005).

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