© University of Pretoria Prevalence of Babesia species and associated ticks (Acari: Ixodidae) in captive cheetah (Acinonyx jubatus) populations in South Africa By Habib Golezardy Submitted in partial fulfillment of the requirments for the degree of Philosophiae Doctor in the Department of Veterinary Tropical Diseases Faculty of Veterinary Science University of Pretoria 2011 © University of Pretoria This work is dedicated to all those who have laboured before me and to those who endurced my many moods while I composed. Without their wisdom, perseverance, patient, and understanding, this study would not have been possible. I present this work to our peers and students, who continually challanged me to learn, to rethink, and to explain My efforts were inspired by my love of the God; my father, mother and sister and my profession. i © University of Pretoria Declaration Apart from the assistance received that has been reported in the acknowledgements and in the appropriate places in the text, this thesis represents the original work of the author. No part of this thesis has been presented for any other degree at any other university. Candidate …Habib Golezardy………… Date.....February 2012……. ii © University of Pretoria Summary Prevalence of Babesia species and associated ticks (Acari: Ixodidae) in captive cheetah (Acinonyx jubatus) populations in South Africa By Habib Golezardy Supervisors: Prof. B.L. Penzhorn Co-supervisors: Prof. I.G. Horak and Prof. M.C. Oosthuizen Due to prevailing environmental and climatic conditions South Africa hosts one cheetah subspecies (Acinonyx jubatus jubatus) and a wide range of tick-borne protozoa such as Babesia. Blood samples collected from 143 cheetahs at four study sites, namely the Ann van Dyk Cheetah Breeding Center-De Wildt (Brits and Shingwedzi), the Cheetah Outreach and the Hoedspruit Endangered Species Centre, were examined for Babesia infection. The V4 hypervariable region of 18S rRNA gene was amplified and subjected to the Reverse Line Blot (RLB) hybridisation assay. Hybridisation of the parasite DNA with Babesia genus and species-specific probes was evident. The results showed a predominance of Babesia lengau (n=63, 44.1%), followed by Babesia felis (n=3, 2.1%) and Babesia canis rossi (n=7, 4.8%). Unfed ixodid ticks (n=10,432), collected from the vegetation by drag-sampling, represented five species: Amblyomma hebraeum, Amblyomma marmoreum, Haemaphysalis elliptica, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus simus and Rhipicephalus zambeziensis,. The monthly occurrence of ixodid ticks at the De Wildt Cheetah Breeding Centre (Brits) showed a higher activity in the warm months of the year. Recovery of ticks decreased during the warm iii © University of Pretoria hours of the day, suggesting that free-living ticks are humid dependent. The presence of birds, rodents, free-ranging antelopes such as nyalas (Tragelaphus angasii), kudus (Tragelaphus strepsiceros), bushbucks (Tragelaphus scriptus) and impalas (Aepyceros melampus), as well as Burchell’s zebras (Equus burchellii) and leopard tortoises (Geochelone pardalis) can contribute to the availability of various tick species at the breeding centres. Mice as the host for immature instars of ixodid tick species and unfed ixodid ticks were studied for presence of Babesia species. Babesia lengau was detected in 22 (39.2%) mice as well as in Haemaphysalis elliptica larvae, nymphs and adults. The presence of B. lengau in mice suggests a long-term association since the host preference of B. lengau for mice remains unclear. However, the presence of this parasite in unfed imature and adult H. elliptica is indicative of a transstadial transmission suggesting that this tick species may be a potential vector for B. lengau. The correlation between Babesia infection and various parameters such as gender, age, tick burdens and location, in two different breeding farms belonging to the De Wildt Cheetah Breeding Centre was analysed using the Fisher’s exact test analysis. The prevalence of Babesia species in cheetahs was associated with tick burden suggesting a strong positive correlation between the prevalence of infection and presence of suspected vector ticks. Regardless of tick burden, age could be related to prevalence of infection, meaning that the fact that older cheetahs had a higher prevalence of infection with Babesia species. These findings were of considerable interest especially since at the time of study the cheetahs in both populations did not show clinical signs of infection with Babesia species. iv © University of Pretoria Table of contents Topics Pages Declaration ii Summary iii Table of contents v List of tables x List of figures xiii List of personal communications xix Acknowledgements xxiii Chapter 1: 1 General introduction 1 1. The Ann Van Dyk Cheetah Breeding Centre - De Wildt/Brits 3 2. The Ann Van Dyk Cheetah Breeding Centre - De Wildt/Shingwedzi 4 3. The Hoedspruit Endangered Species Centre 4 4. The Cheetah Outreach 4 Breeding management and husbandry at the centers 4 References 7 Chapter 2: 10 Literature review 10 1. Cheetah 10 1.1. The cheetah in history 10 1.2. Classification of cheetahs 11 1.3. Distribution of cheetahs 12 1.4. Threats and status 13 v © University of Pretoria 1.5. Diseases in cheetahs 14 2. Babesia 15 2.1. Babesia species 15 2.2. Background of Babesia species in felids 16 2.3. Life cycle of Babesia species 17 2.4. Babesiosis in felids 19 2.5. Epidemiology of feline babesiosis in South Africa 19 2.6. Diagnostic tests 21 2.6.1. Microscopic identification 21 2.6.2. Serological test 23 2.6.3. Nucleic acid detection 23 2.7. Chemotherapy and control of babesiosis in felids 24 3. Vector 24 3.1. Vector-borne diseases 24 3.2. Tick (Acari: Ixodidae) 25 3.2.1. Classification of ticks 26 3.2.2. Life cycle of ticks 26 3.3. Effect of environmental variables on tick populations in a 27 region 3.4. Effect of environmental variables on tick populations on a host 28 3.5. Ixodid ticks as potential vectors for Babesia species 29 References 31 Chapter 3: 42 Species diversity and diurnal and seasonal patterns of activity of questing ticks (Acari: Ixodidae) associated with captive cheetah (Acinonyx jubatus) 42 populations in South Africa Abstract 42 Introduction 43 vi © University of Pretoria Materials and methods 45 1. Survey localities and period 45 2. Tick recovery 45 2.1. Drag sampling 45 2.2. Cheetahs 46 2.3. Murid rodents 47 Results 49 Discussion 69 References 77 Chapter 4: 85 Detection of Babesia species in captive cheetah (Acinonyx jubatus) populations, associated field-collected ticks (Acari: Ixodidae), mice and their 85 related ticks in South Africa Abstract 85 Introduction 86 Materials and Methods 88 1. Survey localities and period 88 2. Samples collection 89 3. Preparation of blood smears 89 4. DNA isolation 90 4.1. Blood samples from cheetahs 90 4.2. Tick specimens 90 4.3. Blood samples from mice 91 4.4. Tick specimens from the trapped mice 92 5. PCR reactions 92 6. Agarose gel electrophoresis 93 7. Reverse line blot (RLB) hybridization assay 93 vii © University of Pretoria 7.1. Babesia species-specific probes 93 7.2. Preparation of the plasmid control 94 7.3. Preparation of the RLB membrane 95 7.4. Hybridization 95 Results 97 Discussion 106 References 110 Chapter 5: 116 Phylogeny of Babesia species detected in captive cheetahs and 116 Haemaphysalis elliptica (Acari: Ixodidae) in South Africa Abstract 116 Introduction 117 Materials and Methods 119 1. DNA samples 119 2. PCR reaction and PCR product purification 120 3. Cloning and plasmid extraction 121 4. Sequence analysis 122 5. Phylogenic tree construction 124 Results 124 Discussion 131 References 133 Chapter 6: 138 Phylogeny of Haemaphysalis elliptica (Acari: Ixodidae) using mitochondrial 138 12S and 16S rRNA gene sequence analysis Abstract 138 Introduction 139 Materials and Methods 141 1. Sample collection and localities 141 viii © University of Pretoria 2. Genomic DNA extraction 141 3. PCR amplification and PCR product purification 141 4. Sequencing and alignment 143 5. Phylogenetic analysis 145 Results 146 Discussion 162 References 164 Chapter 7: 168 Risk factors for infection with Babesia species at various cheetah breeding 168 centres in South Africa Abstract 168 Introduction 171 Materials and methods 171 1. Study localities and period 171 2. Tick sampling 171 3. Blood sampling and molecular analysis 171 4. Data analysis 172 Results 173 Discussion 180 References 183 Chapter 8: 187 General discussion 187 Conclusion 189 References 191 ix © University of Pretoria List of tables Tables Pages 2.1. Classification of the cheetah 11 2.2. Classification of Babesia 15 3.1. Diversity and numbers of all the ixodid tick species collected throughout the study by drag-sampling the vegetation at two cheetah 51 breeding centres in South Africa 3.2. Seasonality of the mean numbers of all stages of development combined of three ixodid tick species questing from vegetation at the 51 Ann van Dyk Cheetah Breeding Centre _ De Wildt/Brits 3.3. Combined number (and standard deviation) of all stages of development of all ixodid ticks collected per drag-sample in each month from March 2008 to February 2009 at the Ann van Dyk Cheetah 52 Breeding Center – De Wildt/Brits 3.4. The hourly number of immature ixodid ticks collected per drag-sample from vegetation at the Ann van Dyk Cheetah Breeding Centre – De 53 Wildt/Brits (June 2008) 3.5. The hourly number of immature ixodid ticks collected per drag-sample from vegetation at the Ann van Dyk Cheetah Breeding Center – De 54 Wildt/Brits (December 2008) 3.6. Ixodid ticks collected from vegetation at the Hoedspruit Endangered Species Centre (July 2008) 55 3.7. Ixodid ticks collected from vegetation at the Hoedspruit Endangered Species Centre (November 2008) 55 3.8. Ixodid tick species associated with cheetahs at the Ann van Dyk Cheetah Breeding Centre - De Wildt/Brits 56 3.9.