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(Boophilus) Microplus and Rhipicephalus (Boophilus) Decoloratus on a Farm in the Eastern Cape Province, South Africa

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(Boophilus) Microplus and Rhipicephalus (Boophilus) Decoloratus on a Farm in the Eastern Cape Province, South Africa The distribution of Rhipicephalus (Boophilus) microplus and Rhipicephalus (Boophilus) decoloratus on a farm in the Eastern Cape Province, South Africa By MICHELLE POTTINGER Submitted in fulfillment of the requirements in respect of the Master’s Degree, ZOOLOGY in the DEPARTMENT OF ZOOLOGY AND ENTOMOLOGY in the FACUILTY OF NATURAL AND AGRICULTURAL SCIECNES at the UNIVERSITY OF THE FREE STATE January 2019 Supervisor: Ms. EMSP van Dalen DECLARATION I, Michelle Pottinger, declare that the Master’s degree research dissertation that I herewith submit for the Master’s degree qualification, MSc Zoology, at the University of the Free State is my independent work, and that I have not previously submitted it for a qualification at another institution of higher education. Signed: Date: 23-01-2019 TABLE OF CONTENTS Table Index pg. I Figure Index pg. II Abbreviations pg. V Ethical Statement pg. VI Acknowledgement’s pg. VII Abstract pg. VIII Chapter 1: General Introduction & Literature Review 1.1. Tick Classification pg. 2 1.2. General Background pg. 3 1.2.1. Life Cycles pg. 3 1.2.2. Host Detection pg. 5 1.2.3. Feeding pg. 5 1.2.4. Habitat and Distribution pg. 6 1.2.5. Tick and Tick-borne Diseases pg. 7 1.3. Focus Species pg. 8 1.3.1. Rhipicephalus (Boophilus) decoloratus (Koch, 1844). pg. 8 1.3.2. Rhipicephalus (Boophilus) microplus (Canestrini, 1888). pg. 9 1.4. Introduction and Invasion of Rhipicephalus (Boophilus) microplus pg. 10 1.4.1. Factors Which Influence Distribution and Abundance pg. 10 1.4.1.1. Climate Change pg. 10 1.4.1.2. Uncontrolled Movement of Hosts pg. 11 1.4.1.3. Resource Limited Communal Farming pg. 12 1.4.2. Distribution of Rhipicephalus (Boophilus) microplus on the African Continent pg. 13 1.4.2.1. Africa pg. 13 1.4.2.2. South Africa pg. 14 1.4.2.3. Eastern Cape pg. 14 1.5. Tick Control Measures pg. 15 1.5.1. Chemical Control and Acaricide Resistance pg. 15 1.5.2. Currents Acaricides Used in South Africa pg. 15 1.5.3. How Acaricide Resistance develops pg. 18 1.5.4. The History of Acaricide Resistance pg. 19 1.6. Alternative Control Methods pg. 21 1.6.1. Vaccinations pg. 21 1.6.2. Pasture Management pg. 21 1.6.2.1. Rotational Grazing pg. 21 1.6.2.2. Pasture Burning pg. 22 1.6.2.3. Artificial Grazing and Use of Fertilizers pg. 22 1.6.3. Biological control pg. 22 1.6.3.1. Bacteria pg. 22 1.6.3.2. Fungi pg. 23 1.6.3.3. Nematodes pg. 23 1.6.3.4. Insects pg. 24 1.6.3.5. Birds pg. 24 1.6.4. Botanicals pg. 25 1.6.5. Genetically Resistant Hosts pg. 25 1.7. Justification pg. 26 1.8. Main Objectives pg. 27 References Chapter 2: Study Location 2.1. Introduction pg. 34 2.2. Farm location pg. 34 2.2.1. Camps pg. 35 2.2.2. Vegetation Type pg. 39 2.2.3. Temperature & Rainfall pg. 40 2.2.4. Host Animals & Husbandry pg. 40 2.3. Laboratory Work pg. 42 2.3.1. Laboratory Safety & Waste Disposal pg. 42 References Chapter 3: Distribution and Composition of Tick Species 3.1. Introduction pg. 44 3.2. Methods & Materials 3.2.1. Sample collections pg. 47 3.2.1.1. On-host: Adult Ticks pg. 47 3.2.1.2. Selected Herd pg. 49 3.2.1.3. Off-host: Collection of Larvae pg. 50 3.2.2. Morphological Identification pg. 51 3.2.2.1. Adults pg. 51 3.2.2.2. Larvae pg. 53 3.2.3. Environmental Parameters pg. 54 3.2.4. Data Analysis pg. 54 3.3. Results 3.3.1. Overall Composition of All Tick Species Collected pg. 55 3.3.2. Rhipicephalus (Boophilus) decoloratus Collection pg. 56 3.3.3. Rhipicephalus (Boophilus) microplus Distribution pg. 58 3.3.4. Population Trend of Rhipicephalus (Boophilus) microplus 2016-2018. pg. 63 3.3.4.1. Movement of Selected Herd pg. 66 3.3.5. Effect of Temperature and Humidity on Larval Collection pg. 69 3.4. Discussion pg. 71 3.5. Conclusion pg. 83 3.6. Recommendations pg. 83 References Chapter 4: Genetic Identification of Larvae 4.1. Introduction pg. 89 4.2. Methods & Materials 4.2.1. DNA Extraction pg. 90 4.2.2. PCR – Polymerase Chain Reaction pg. 93 4.2.2.1. Preparation of master mix pg. 93 4.2.2.2. Set up of individual reactions pg. 93 4.2.2.3. Polymerase Chain Reaction pg. 94 4.2.3. Restriction Enzymes pg. 94 4.2.4. Gel Electrophoresis pg. 95 4.2.4.1. Preparation of 2% Agarose gel pg. 95 4.2.4.2. Preparation of gel tray pg. 95 4.2.4.3. Running of Gel pg. 95 4.2.5. Observation of Bands pg. 96 4.3. Results pg. 96 4.4. Discussion pg. 98 4.5. Conclusion pg. 99 References Chapter 5: Acaricide Resistance Profiles 5.1. Introduction pg. 102 5.2. Method & Materials 5.2.1. Treatment Strategies Used by the Producer pg. 104 5.2.1.1. Milbatraz pg. 104 5.2.1.2. Drastic Deadline Extreme pg. 104 5.2.1.3. Ivermax pg. 105 5.2.2. Application Methods pg. 105 5.2.2.1. Spray Race pg. 105 5.2.2.2. Hand Spraying pg. 106 5.2.2.3. Pour-on pg. 106 5.2.2.4. Injectable pg. 107 5.2.3. Tick Collections pg. 107 5.2.4. Acaricide Preparation pg. 107 5.2.5. Shaw Larval Immersion Test pg. 109 5.2.5.1. Set-up pg. 109 5.2.5.2. Exposure to Acaricides pg. 110 5.2.5.3. Post Exposure Treatment pg. 110 5.2.5.4. Mortality Counts pg. 111 5.3. Results 5.3.1. One-host Tick Resistance pg. 112 5.3.1.1. Amidine Resistance pg. 112 5.3.1.2. Pyrethroid & Organophosphate pg. 119 Resistance 5.3.2. Multi Host Ticks pg. 121 5.3.2.1. Amblyomma hebraeum Resistance Status pg. 121 5.3.2.2. Rhipicephalus evertsi evertsi Resistance Status pg. 122 5.4. Discussion pg. 123 5.4.1. Comparison of the Two and Three-host Tick Resistance Status pg. 128 5.5. Conclusion pg. 130 5.6. Recommendations pg. 131 References Appendix 1. Ethical Clearance pg. 138 2. Producers Consent pg. 139 TABLE INDEX Chapter 2: Study Location Table 2.1: List of names of each Camp represented in Figure 2.2 pg. 37 Chapter 3: Distribution and Composition of tick species Table 3.1: All tick species collected on Claypits over the study period, presented from most to least abundant pg. 55 Table 3.2: The total abundance of Rhipicephalus (Boophilus) decoloratus specimens collected during the field collections pg.57 Table 3.3: The number of Rhipicephalus (Boophilus) microplus specimens collected in selected camps during field collections pg. 62 Table 3.4: The percentage of Rhipicephalus (Boophilus) microplus collected in each camp during the field collection trips pg. 65 Table 3.5: The schedule followed by the producer for the selected herd which was tracked from April 2017- April 2018 with the number of blue ticks collected pg. 68 Table 3.6: Statistical analysis of the relationship between the temperature and relative humidity to the number of larvae collected. pg. 71 Chapter 4: Genetic Identification of Larvae Table 4.1: Solutions used in the extraction of DNA during the CTAB method pg. 92 Table 4.2: Components used to make up master mix pg. 93 Table 4.3: The volume of components added to each sample of extracted DNA pg. 94 Table 4.4: The PCR cycling protocol pg. 94 Table 4.5: Table of the base pairs identified from the bands seen in Figure 4.1. pg. 98 Chapter 5: Acaricide Resistance Profiles Table 5.1: Chemicals used for resistance testing pg. 107 Table 5.2: Dilution tables for resistance testing pg. 108 Table 5.3: The resistance ranges used in the representation of the resistance results pg. 112 Table 5.4: A summary of the Amidine results over the study period pg. 119 I | P a g e FIGURE INDEX Chapter 1: General Introduction & Literature Review Figure 1.1: Dorsal view of a (A) Female and (B) Male hard tick pg. 3 Chapter 2: Study Location Figure 2.1: The location of the farm, relative to the town of Grahamstown pg. 35 Figure 2.2: Map of the farm divided into the 53 camps pg. 36 Figure 2.3: Camps which are not only used for grazing activities. A: Lands and Old Oranges camp containing fields used for the plantation of sorghum. B: Quarry camp contains a quarry used by the producer pg. 38 Figure 2.4: Camps on the farm either contain natural waters sources as seen in A: Barbers Dam camp while others require water to be pumped to the surface like in B: Bushalt camp pg. 38 Figure 2.5: Dense sweet veld found in the lower lying camps on the farm such as in A: Kens and B: Morne Fir camps pg. 39 Figure 2.6: Open sour veld found on the higher areas of the farm such as A: Gumtree dam and B: Gavin Hill camps pg. 40 Figure 2.7: Appearance of South Devon cattle found on the farm, A: bulls are large and muscular, B: cows have a more feminine appearance with thick woolly hair pg. 41 Chapter 3: Distribution and Composition of tick species Figure 3.1: The location on the cattle of which collections of adult ticks occurred.
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