DURATION OF REPELLENCY OF SELECTED AGENTS AGAINST CULICOIDES SPECIES WHEN APPLIED TO POLYESTER MESH

DURATION OF REPELLENCY OF SELECTED AGENTS AGAINST CULICOIDES SPECIES WHEN APPLIED TO POLYESTER MESH by Patrick Collin Page Submitted in partial fulfilment of the requirements for the degree of MMedVet (Equine Medicine) in the Faculty of Veterinary Science, University of Pretoria Pretoria September 2009 University of Pretoria

TABLE OF CONTENTS ACKNOWLEDGEMENTS... 5 ABBREVIATIONS... 6 LIST OF FIGURES... 7 LIST OF TABLES... 8 LIST OF APPENDICES... 9 SUMMARY... 10 CHAPTER 1: GENERAL INTRODUCTION... 11 CHAPTER 2: LITERATURE REVIEW... 13 2.1 CULICOIDES....13 2.2 AFRICAN HORSE SICKNESS... 15 2.3 EQUINE ENCEPHALOSIS... 16 2.4 EQUINE INSECT HYPERSENSITIVITY... 16 2.5 CONTROL MEASURES... 17 2.5.1 Insect repellents... 18 2.5.2 N,N-diethyl-3-methylbenzamide (DEET)... 19 2.5.3 Pyrethrins and synthetic pyrethroids... 20 2.5.4 Essential oils... 22 CHAPTER 3: MATERIALS AND METHODS... 23 3.1 MODEL SYSTEM... 23 3.2 EXPERIMENTAL DESIGN... 23 3.3 ETHICAL CONSIDERATIONS... 25 3.4 EXPERIMENTAL PROCEDURES... 25 3.4.1 Test preparations... 25 3.4.2 Polyester mesh treatments... 26 3.4.3 Light trap operation... 27 3.4.4 Collection of Culicoides... 27 3.4.5 Sorting and counting... 27 3.4.6 Climatic data... 28 3.5 OBSERVATIONS... 28 3.6 STATISTICAL ANALYSIS... 28 2

CHAPTER 4: RESULTS... 30 4.1 CULICOIDES... 30 4.2 CLIMATE... 39 4.3. IMPREGNATED POLYESTER MESH... 41 CHAPTER 5: DISCUSSION... 42 CHAPTER 6: CONCLUSIONS... 45 REFERENCES... 46 APPENDICES... 53 3

To Louise and Connor Page and My parents, Harold and Kate Page 4

ACKNOWLEDGEMENTS I thank the following: Professor Alan Guthrie, project promoter, for his motivation, guidance, and overall support. Dr Gert Venter, project co-promoter, for his motivation, guidance, and entomological support. Miss Karien Labuschagne, for entomological support. Miss Jane Nurton, for her practical inputs with project logistics. Mr Roehann Sutherland and Dr Laura Lee for assistance with the sample collections. Miss Stellest de Villiers and Mr Cliffie Matjiane for assistance with the horse management. Dr Koos van den Berg, for his mentorship and kindling my interest in insect repellents, Culicoides midges and African horse sickness. Professor Peter Thompson for assistance with the statistical analysis. Professor Piet Stadler, for his motivation and guidance. Dr s Cynthia Donnellan, Martin Schulman, and Johan Marais for friendship, advice and motivation. The Faculty of Veterinary Science Research Fund. Mr Alan Boisragon, Director: Textile Dynamics, for sponsoring the polyester mesh. Mr Peter Harrison, Director: Maccallum and Associates, for sponsoring the DEET. 5

ABBREVIATIONS AHS AHSV ANOVA BT DEET EE EEV OVAH PMD UV WHO African horse sickness African horse sickness virus analysis of variance bluetongue N,N-diethyl-3-methylbenzamide equine encephalosis equine encephalosis virus Onderstepoort Veterinary Academic Hospital p-menthane-3,8-diol ultraviolet World Health Organisation 6

LIST OF FIGURES Figure 1: Diagram of the Onderstepoort light trap a....23 Figure 2: The location of the five camps (A to E) and the light traps at the study site...24 Figure 3: Polyester mesh (a) attached to the Onderstepoort light trap a, and (b) representative section of mesh....26 Figure 4: Total number of Culicoides midges collected per camp, over 15 nights in February 20, at the OVAH...32 Figure 5: Mean number of Culicoides midges collected per camp, over 15 nights in February 20, at the OVAH...32 Figure 6: Mean number of Culicoides midges collected hourly over 15 nights in February 20 at the OVAH with light traps fitted with polyester mesh treated with DEET, citronella, cypermethrin, ethanol and a control...34 Figure 7: Mean number of C. imicola collected hourly over 15 nights in February 20 at the OVAH with light traps fitted with polyester mesh treated with DEET, citronella, cypermethrin, ethanol and a control....35 Figure 8: Mean number of Culicoides midges collected each hour on 15 nights in February 20 at the OVAH with light traps fitted with (a) DEET, (b) citronella, (c) cypermethrin, (d) ethanol and (e) control mesh....38 7

LIST OF TABLES Table 1: Number of Culicoides midges collected during the preliminary phase on four consecutive nights in January 20 with five light traps operated simultaneously at the OVAH....30 Table 2: Number of Culicoides midges collected during the treatment phase on 15 nights in February 20 with five light traps operated simultaneously at the OVAH....31 Table 3: Culicoides species identification (alphabetical), number and percentage of the total Culicoides collected over 15 nights in February 20, with five light traps operated simultaneously at the OVAH....33 Table 4: Mean number of Culicoides midges, collected hourly over 15 nights in February 20 at the OVAH with light traps fitted with polyester mesh treated with DEET, citronella, cypermethrin, ethanol and a control, compared between treatment groups at each time point and overall....36 Table 5: Mean number of C. imicola, collected hourly over 15 nights in February 20 at the OVAH with light traps fitted with polyester mesh treated with DEET, citronella, cypermethrin, ethanol and a control, compared between treatment groups at each time point and overall...37 Table 6: Effects of temperature, maximum wind speed, humidity and rainfall on number of Culicoides collected over 15 nights in February 20 at the OVAH: multiple regression model....39 Table 7: Effects of temperature, maximum wind speed, humidity and rainfall on number of C. imicola collected over 15 nights in February 20 at the OVAH: multiple regression model....40 Table 8: Mean mesh weights and concentration of DEET, citronella oil and cypermethrin before treatment, after treatment (dry), 14 hours after treatment, and residual % of active on mesh after treatment...41 8

LIST OF APPENDICES APPENDIX A: Random allocation of Equine Research Centre horses to camps...53 APPENDIX B: Randomised locations of the light traps...54 APPENDIX C: Details and preparation of repellent solutions...55 APPENDIX D: Standardised sheet for recording Culicoides numbers...56 APPENDIX E: Preliminary phase Culicoides midge counts...57 APPENDIX F: Culicoides midge counts with light traps fitted with polyester mesh treated with (a) DEET, (b) citronella, (c) cypermethrin, (d) ethanol and (e) control...61 APPENDIX G: Culicoides imicola counts with light traps fitted with polyester mesh treated with (a) DEET, (b) citronella, (c) cypermethrin, (d) ethanol and (e) control...66 APPENDIX H: Sexed and age graded subsample of Culicoides collected on 24-25 February 20 (Day 12), at the OVAH...71 APPENDIX I: Climatic data (a) temperature, (b) relative humidity, (c) maximum wind speed and (d) rain recorded....72 APPENDIX J: Pre- and post-treatment weights of polyester meshes treated with (a) DEET, (b) citronella, (c) cypermethrin, (d) ethanol and (e) control....76 APPENDIX K: Article published in Veterinary Parasitology...81 9

SUMMARY Culicoides biting midges (Diptera: Ceratopogonidae) are of economic and veterinary significance worldwide. Of principal importance to equids in sub-saharan Africa are Culicoides (Avaritia) imicola Kieffer and Culicoides (Avaritia) bolitinos Meiswinkel which have been implicated in the transmission of African horse sickness virus (AHSV) and equine encephalosis virus (EEV). Various species of Culicoides are associated with equine insect hypersensitivity, a common equine skin allergy. Recommended measures to prevent diseases associated with Culicoides in equids include vaccination for African horse sickness (AHS), stabling at night, meshing of stables, and application of insect repellents both to the animal and its stable environment. The effects of repellents against Culicoides on humans have been documented, with most studies reporting the repellency of compounds against that of N,N-diethyl-3-methylbenzamide (DEET). DEET is registered for human use in South Africa, whilst citronella oil and cypermethrin are included in topical ectoparasiticides registered for use on horses. The aim of this study was to determine and compare repellent efficacy of 15% DEET, 0.6% citronella oil, and 0.3% α-cyanocypermethrin against Culicoides species when applied to polyester mesh under South African conditions. The repellent efficacy against Culicoides species was compared in three 5 X 5 Latin squares (15 replicates). DEET, citronella oil or α-cyano-cypermethrin was applied to polyester meshes that were fitted to down-draught suction 220 V ultraviolet (UV) light traps which were operated overnight. A total of 107,2 Culicoides midges was collected in hourly light-trap collections made over 15 nights from five light traps operated simultaneously. Of 34 Culicoides species collected C. imicola was the most abundant and comprised 79.1% of midges collected, followed by C. bolitinos which comprised 5.3%. The mean number of Culicoides midges as well as the mean number of C. imicola collected hourly with DEET was significantly (P < 0.05) lower than for all other treatments at all times except the first (19h00) and the last (06h00) sampling points. The study concluded that DEET had a significant repellent effect against Culicoides species, including C. imicola, for all catches made from after sunset to before sunrise, when applied to polyester mesh as tested with a down-draught suction light trap. No significant repellent effect against Culicoides was found for the citronella oil or the α-cyano-cypermethrin treatments. 10

CHAPTER 1: GENERAL INTRODUCTION Due to the viruses they transmit Culicoides biting midges (Diptera: Ceratopogonidae) are of economic and veterinary significance worldwide 1,2. Of principal importance to equids in sub-saharan Africa are Culicoides (Avaritia) imicola Kieffer and Culicoides (Avaritia) bolitinos Meiswinkel which have been implicated in the transmission of African horse sickness virus (AHSV) 3-6. Both C. imicola and C. bolitinos can also become infected with and permit replication of equine encephalosis virus (EEV) 7. Various species of Culicoides have been associated with equine insect hypersensitivity, the most common skin allergy affecting horses 8,9. The recent spread of bluetongue (BT) into northern Europe has highlighted the risk of introduction and rapid spread of vector-borne diseases outside their traditional boundaries 10-13. Culicoides imicola and members of the Culicoides obsoletus and Culicoides pulicaris complexes have been implicated in the outbreaks of BT in Europe 12, whilst both C. imicola and C. bolitinos have been implicated as vectors of the BT virus in South Africa 14. In endemic areas, vaccination is the primary African horse sickness (AHS) control measure 15. As part of an integrated control program other recommended measures to prevent diseases associated with Culicoides in equids include stabling at night, meshing of stables, and application of insect repellents both to the animal and its stable environment 9,16,17. Whilst stabling is recommended for control it has been shown that Culicoides midges do enter stables, and horses are protected from Culicoides bites only if the stables are adequately closed 18,19. The effects of repellents against Culicoides and the duration of their activity on humans have been documented, with most studies reporting the repellency of various compounds against that of N,N-diethyl-3-methylbenzamide (DEET) 20. Assessment of efficacy of repellents applied to horses against Culicoides and especially C. imicola is hampered by their relatively small size and their nocturnal activity which make direct observation difficult. A novel method for preliminary field screening of potential repellents to protect animals against Culicoides has been reported 21. Studies using this method, which utilizes light 11

traps and repellent impregnated polyester mesh, have provided an indication of the duration of repellency of the various compounds tested 20,22,23. Citronella oil and cypermethrin are included in various topical ectoparasiticides registered for use on horses in South Africa, whilst DEET is registered for human use. Carpenter et al 13 suggested that studies investigating ways of protecting horses from biting midges is of high priority, with emphasis on the potential risk of an outbreak of AHS in Europe. No published data is available on the efficacy and duration of repellency of insect repellents against Culicoides in sub-saharan Africa 2. The aims of this study were to determine and compare repellent efficacy of 15% DEET, 0.6% citronella oil, and 0.3% α-cyano-cypermethrin against Culicoides species when applied to polyester mesh under South African conditions, using the method described by Braverman and Chizov-Ginzburg 21, and to investigate the effect of climatic variables on duration of repellency. 12

CHAPTER 2: LITERATURE REVIEW 2.1 CULICOIDES Culicoides species (Diptera: Ceratopogonidae) 24 are small biting flies, one to three millimetres in size 2. They are also referred to as biting midges, no-see-ums, punkies, and are sometimes improperly referred to as sandflies a term more accurately used for members of the genus Phlebotomus 24. More than 1,200 species are known worldwide 2 and approximately 120 species of Culicoides are known to occur in South Africa 4. These midges are abundant during the warm summer months and feed mainly during twilight periods and at night. Dependent on the species, fertilised females lay up to 450 eggs 24 on a moist substrate, such as cow dung or mud containing a high concentration of organic matter, in which the larvae feed and eventually pupate 2. The complete life cycle may take three to four weeks under favourable conditions, so several generations can develop in a single season. Only the females are blood-suckers 2,24. Culicoides midges are of economic and veterinary significance worldwide 1, mainly due to transmission of viruses in the families Bunyaviridae, Reoviridae and Rhabdoviridae 2. The viruses are transmitted biologically and not mechanically 15. Thus, the viruses first replicate in the vector before they can be transmitted. Of particular importance to equids are AHSV and EEV, both of which are orbiviruses falling under the family Reoviridae 2. It was anticipated in 1944 that the transmitter of AHS would be found within the genus Culicoides 3. Subsequently, in 1970, successful transmission of horse sickness from an infected to a susceptible animal via Culicoides bite was cited 25. In South Africa, C. imicola is the most abundant livestock-associated Culicoides species; comprising 71.4% of over three million Culicoides collected in a geographical distribution study 26, and AHS virus has been repeatedly isolated from it 2. To date, despite overwhelming evidence, further proof that C. imicola is able to infect susceptible horses by transmitting AHSV, after an appropriate incubation period, is however lacking 4. 13

Recently C. bolitinos has also been implicated as a vector of AHSV following an outbreak in the eastern Free State, South Africa 19,27,28. This species, whose larval habitat includes cattle dung as well as African buffalo and blue wildebeest dung 29, is common in cold as well as hot areas and may be more important than C. imicola as a disease vector in certain regions 2. Ten character states have been described to separate the two species C. imicola and C. bolitinos morphologically. The juxtaposition of a pale and dark area on the anterior distal third of vein M 2, also referred to as a pale preapical excision, is the single most diagnostic character when the identification of the C. imicola female is based on wing pattern alone 29. In C. bolitinos the median third of both the posterior and anterior margins of vein M 2 are broadly and entirely dark, but taper and fade simultaneously leaving the apex of vein M 2 pale 29. The number of Culicoides caught in a single suction light trap at Onderstepoort during the period 1963 to 1970 was recorded daily and plotted together with the total monthly rainfall and mean monthly maximum and minimum temperatures 30. Mean nightly light trap catches during the month of January varied from 21,623 Culicoides in 1967 to 1,789 Culicoides in 1970 30. Increases in catches were linked to good rains during the previous month whilst temperatures did not differ markedly enough from year to year to account for sudden changes in Culicoides numbers, except during winter 30. Other factors which may influence the number of Culicoides caught on a specific night include the presence of breeding sites, other light sources in the vicinity of the light trap, the height of the trap above ground level, wind speed and relative humidity 26. Temperatures below 18 ºC can reduce overnight light trap catches of Culicoides (Avaritia) brevitarsis Kieffer 31. Wind speeds above eight km/h also reduce catches whilst the close proximity of hosts can greatly increase catches 31,32. Culicoides are attracted to vertebrate host odours such as carbon dioxide and at least some species respond to lactic acid and 1-octen-3-ol 23. Moon phase also has significant effects on trap catches of C. brevitarsis with fewest caught at full moon 31. This is likely due to changes in the effective limits of traps rather than a behavioural response to changes in light intensity 31. The importance of humidity is unclear and further investigation is required, especially in relation to the effect of saturated air conditions on flight behaviour 31. Studies have also suggested that higher wind speeds increase the mortality rates of adult C. imicola and that this may lead to lower population sizes 33. 14

2.2 AFRICAN HORSE SICKNESS African horse sickness is a peracute, acute, subacute or mild infectious disease of equids characterised by fever, inappetance, oedema of the subcutaneous and intermuscular tissues, transudation into the body cavities and haemorrhages, particularly of the serosal surfaces 15. The mortality rate in susceptible horses may be as high as 95%, with donkeys and mules being less susceptible 15. In South Africa the disease usually first appears in the north-eastern parts of the country and then spreads southwards depending on the time of year that the disease first makes its appearance and the extent of favourable climatic conditions for the breeding of Culicoides midges 15. The most serious outbreaks of disease commonly occur during the months of March and April 15. In contrast to previous results, indicating AHSV to be exclusively vectored by C. imicola, present oral susceptibility results demonstrate that AHSV could persist for at least 10 days in nine Culicoides species, represented by at least six different subgenera. Cumulative oral susceptibility and field infection results indicate that susceptibility to AHS virus is restricted to certain Culicoides species, however, this characteristic is widespread in the genus Culicoides and not restricted to the subgenus Avaritia 34. In endemic areas, vaccination is the primary AHS control measure 15. A polyvalent, live attenuated AHS vaccine is commercially available for compulsory vaccination of horses in South Africa. Until 1990 this attenuated live-virus vaccine comprised two quadrivalent vaccines, one with serotypes -1, -3, -4 and -5 and the other serotypes -2, -6, -7 and -8 28. The vaccine strain of AHSV-5 was discontinued in 1990 due to safety concerns 28. Recently, positive preliminary results were reported with a recombinant canarypox virus vectored (ALVAC ) vaccine for protective immunization of equids against AHSV infection 35. 15

2.3 EQUINE ENCEPHALOSIS Equine encephalosis (EE) is a viral disease that, like AHS, occurs particularly in late summer and autumn in southern Africa in those years in which climatic conditions favour the spread of insect-borne viruses 36. Most infected animals are asymptomatic, or only show mild signs of illness characterised by fever, congestion and icterus 36,37. Less commonly, signs of supraorbital swelling, signs referable to central nervous system involvement, abortions, or cardiac failure may occur 36. In contrast to AHS the mortality rate due to EE is much lower 36. Equine encephalosis virus was first isolated from a mixed pool of Culicoides (mainly C. imicola) collected at Onderstepoort 2. Subsequent serological and virus isolation surveys have indicated that EE is present throughout South Africa, unlike the distribution of AHS 37,38. Both C. imicola and C. bolitinos can become infected with and permit replication of EEV 7. Oral susceptibility studies in the laboratory shows that at least four non-avaritia Old World species, Culicoides (Meijerehelea) leucostictus Kieffer, Culicoides (Culicoides) magnus Colaço, Culicoides (Hoffmania) zuluensis de Meillon and Culicoides (Unassigned) onderstepoortensis Fiedler may be susceptible to infection with EEV and may therefore be regarded as potential natural vectors 7,39. 2.4 EQUINE INSECT HYPERSENSITIVITY In addition to transmission of viral disease, Culicoides midges are associated with equine insect hypersensitivity, the most common skin allergy affecting horses 8,9. The seasonal, pruritic dermatitis caused by hypersensitivity to the saliva of Culicoides midges has been identified in many parts of the world and has been given various names such as sweet itch, summer itch, Queensland itch, summer seasonal recurrent dermatitis, kasen, and sommerekzem 40-42. Certain breeds such as the Shire, Icelandic and Welsh ponies appear to be particularly sensitive, suggesting a genetic basis to the hypersensitivity 8. A Type-I (immediate) hypersensitivity mediated by circulating immunoglobulin E 9, as well as involvement of Type-IV (delayed) hypersensitivity mediated by allergen-specific T cells, 16

has been demonstrated 16,41,43. Recently, a specific salivary allergen of the North American midge Culicoides (Monoculicoides) sonorensis Wirth and Jones, capable of inducing typical allergic reactions in horses, has been described 43. Equine insect hypersensitivity is characterised clinically by papules, tufted hair and hyperaesthesia, followed by intense pruritis and self-excoriation. This leads to serous effusion, localised alopecia and the development of secondary skin lesions 8,9. Sites classically involved are the ears, poll, mane, withers, tail head and ventral abdomen, with the predominant area of involvement probably being related to the particular species of midge involved 16,41. Hypersensitivity is more common when insects target the dorsal parts of the body 8. C. imicola was considered the most likely principal agent causing sweet itch in Israel, and this species showed a clear preference for the dorsal aspect of the body 44. 2.5 CONTROL MEASURES Recommended measures to prevent diseases associated with Culicoides in equids include vaccination in the case of AHS 15, stabling at night, meshing of stables, and application of insect repellents both to the animal and its stable environment 2,9,13,16. Whilst stabling is recommended to reduce biting risk it has been shown that Culicoides do enter stables, and horses are protected from Culicoides bites only if the stables are adequately closed 18,19. Closing stables by meshing with 80% shadecloth resulted in a 14-fold reduction in the numbers of Culicoides entering 19. The most important control measure in preventing Culicoides hypersensitivity is the protection of the horse from further contact with Culicoides insects 8,9. Moving the horse to a new area away from Culicoides breeding habitats such as ponds, marshes, and irrigation canals, and regular cleaning of stagnant water troughs is advised 42. As the use of insectproof stables is rarely achievable, the use of a fine screen mesh that may be sprayed with insect repellents, combined with the use of protective rugs is recommended 8,42. Frequent application of insect repellents to the horse has also been recommended to decrease Culicoides exposure 42. Products that contain pyrethrins with synergists, and repellents should ideally be applied in the late afternoon before the insects peak feeding 17

time 42. Specific immunotherapy in the treatment of Culicoides hypersensitive horses using an extract of whole, unfed Culicoides (Monoculicoides) variipennis Coquillett (= C. sonorensis) was not successful 45. Smoking of stables is a farmers remedy applied in an effort to repel midges, however light traps that have been operated in the palls of smoke have still yielded enormous catches of Culicoides, with these catches as large as any made at smokeless stables 4. 2.5.1 Insect repellents Chemical barriers to insects comprise the use of natural and synthetic repellents applied to the skin or to protective screens and fabrics. The ideal insect repellent would repel multiple species of biting arthropods, remain effective for at least eight hours, cause no irritation to the skin or mucous membranes, cause no systemic toxicity, be resistant to abrasion and rub-off, and be greaseless and odourless 46. Insect repellents are tested via biological assays to determine whether the candidate material is repellent, the quantity of material required for repellency, and the duration of repellency. Repellent bioassays are grouped according to whether they use in vitro or in vivo methods, with in vitro systems having the benefits of being inexpensive, safe and fast, and may be used to test many repellents regardless of toxicity 47. Repellents do not all share a single mode of action and surprisingly little is known about how repellents act on their target insects 46. To be effective a repellent must show an optimal degree of volatility, making it possible for an effective repellent vapour concentration to be maintained at the skin surface without evaporating so quickly that it loses its effectiveness 46. Five possible modes of action have been proposed for repellents: inhibition of response to an otherwise attractive signal; switching of the sensory message from attraction to repulsion; activation of a receptor system that controls a competing behavior; activation of a noxious odour receptor; activation of different receptor types simultaneously, causing loss of the specific signal for host location 47. Assessment of efficacy of repellents applied to horses against Culicoides species and especially C. imicola is hampered by their relatively small size and their nocturnal activity which make direct observation difficult. In previous studies utilizing light traps and repellent 18

impregnated polyester netting, repellency has been assessed by comparison of the numbers of Culicoides midges caught in the light traps over a period of time 22. Netting made of polyester or nylon fibers is preferred; monofilament polyethylene does not retain deposits for as long as polyester, whilst cotton is not as durable and requires higher doses of some compounds 48. A few controlled studies have been done to document effects of repellents against Culicoides and the duration of the repellent activity on humans, with most comparing the repellency of various compounds with that of N,N-diethyl-3- methylbenzamide (DEET) 20. Three studies utilizing a novel method with light traps and repellent impregnated polyester netting for preliminary field screening of potential Culicoides repellents, including DEET; a Meliaceae-derived plant extract named Ag1000; Oregano; Herbipet, a mixture of plant extracts comprising oils of sage, rosemary and oregano; Tri-Tec14, containing cypermethrin and pyrethrins; Pyrethroid-T, a type II pyrethroid containing the α-cyano,3- phenoxybenzyl moiety; Stomoxin, containing permethrin; Mosi-guard with Eucalyptus extract containing p-methane-3,8-diol (PMD) plus isopulegol and citronellol; and Lice Free containing plant extracts, have recently been reported 20,22,23. These three studies included some assessment of the duration of repellency of the various compounds, tested. 2.5.2 N,N-diethyl-3-methylbenzamide (DEET) N,N-diethyl-3-methylbenzamide, formerly known as N,N-dimethyl-m-toluamide, was patented by the United States army in 1946 and remains the principal synthetic repellent compound in use today 46. It is classified by the World Health Organization (WHO) as a selected-spectrum, non-cumulative substituted toluamide insect repellent of slight toxicity to mammals, with residual activity 49. It is believed to repel insects by providing a layer of vapour above the region to which it is applied 50. The United States Environmental Protection Agency estimates that worldwide use of DEET-based insect repellents exceeds 200 million people annually 46. When DEET-based repellents are applied in combination with permethrin-treated clothing nearly 100% protection against mosquito bites can be achieved in humans 46. Repellents containing DEET should be carefully applied because they can damage plastics, synthetic fabrics, leather and painted and varnished surfaces; DEET does not damage natural fibres such 19

as cotton or wool and has no effect on nylon 46. When DEET-treated garments are stored in plastic bags the repellent effect can last for many weeks 48. There have been concerns about the safety of DEET due to reports of central nervous system toxicity in man 46,47. Multiple toxicity studies conducted as a result of these reports have indicated that the risk of serious medical side-effects following the normal use of DEET-containing insect repellents is low 46,47. DEET is practically insoluble in water, but is miscible with ethanol and other organic solvents 49. Cyclodextrins have been investigated as an alternative to the commonly used co-solvent ethanol in topical DEET formulations to reduce the release rate of DEET from the formulation and thereby reduce toxic potential 50. Horses sprayed with DEET during a 60 day study developed hypersteatosis and dermatoses when DEET was applied in a concentration above 15% 51. While little research has been done on the direct benefits of repellents applied to equines, the application of DEET to draught horses pulling timber in forests was reported to result in a 58% increase in working capacity compared to an untreated control group 52. DEET is often used as the gold standard in repellency trials. However, a recent study using an in vitro method of assessing the duration of repellency of various synthetic and natural compounds indicated that a synthetic pyrethroid compound, containing the α-cyano-3-phenoxybenzyl moiety, named Pyrethroid-T, was superior to DEET in repelling Culicoides midges 22. This synthetic pyrethroid showed repellency for up to nine hours, while DEET and a plant-derived compound, Ag1000, exerted a repellent effect for four hours 22. 2.5.3 Pyrethrins and synthetic pyrethroids Pyrethrins are natural insecticides derived from Chrysanthemum cinerariaefolium and related species, while pyrethroid insecticides are synthetic analogues of the pyrethrins developed to increase insecticidal stability and efficacy 53. Natural pyrethrins and synthetic pyrethroids are fat-soluble neurotoxicants that affect organisms by altering the activity of sodium ion channels of nerves 53. Permethrin is a pyrethroid that works as a contact insecticide, causing nervous system toxicity leading to death or knockdown of the insect 46. The mode of action of synthetic pyrethroids appears to be an interference with sodium channels of the parasite nerve axons resulting in delayed repolarisation and eventual paralysis 54. Type I compounds interfere with the axonal sodium gate and result in delayed 20

repolarisation and repetitive discharge of the nerve whilst Type II compounds also act on the sodium gate, but do so without resulting in repetitive discharge 54. The lethal activity of the synthetic pyrethroids seems to involve both central and peripheral neurons, whilst the knockdown effect is probably due to peripheral neuron effects only 54. Being highly lipophilic, pyrethroids readily pass through cell membranes and may be absorbed via the skin, inhalation or ingestion 53. Their rapid metabolism however greatly lowers the magnitude of any resultant toxicity in humans. Toxicologically these compounds have a useful characteristic, the production of skin paraesthesia, which gives an early indication of exposure 55. Pyrethroids may be applied to mosquito nets or other types of fabric where they act to repel or kill biting insects, retain activity for several months even after laundering, are relatively safe to use if applied properly, are inexpensive and effective at low-concentrations 47. A 4% permethrin compound resulted in an 86% improvement in Culicoides hypersensitivity in 43 horses when applied once to three times a week as a dorsal midline pour-on 56. Cypermethrin is a synthetic pyrethroid insecticide, containing the α-cyano-3- phenoxybenzyl moiety, and has been used as a barrier insect repellent in horses 57. It is classified as a moderately toxic material by dermal absorption or ingestion, has no reported adverse effects on reproduction, is not teratogenic, nor mutagenic, is classified as a possible human carcinogen, and may cause adverse effects on the central nervous system 57. The insecticidal efficacy of cyfluthrin 58 and alphacypermethrin 59, when applied as a pour on to cattle and sheep, against Culicoides has been demonstrated. Cypermethrin is an active ingredient in various topical ectoparasiticide products registered for use on horses in South Africa. Cyfluthrin is registered for use on cattle and sheep in South Africa. Whilst repellents on skin last for a few hours, pyrethroids (especially α-cyano-pyrethroids) have been reported to last for six to 12 months when applied to fabric 47. 21

2.5.4 Essential oils Plants whose essential oils have been reported to have insect repellent activity include citronella, cedar, verbena, pennyroyal, geranium, lavender, pine, cajeput, cinnamon, rosemary, basil, thyme, allspice, garlic and peppermint 46. Unlike synthetic insect repellents, plant-derived repellents have been poorly studied, and when tested most of these essential oils tended to give short-lasting protection, usually less than two hours 46. Oil of citronella is a volatile, liquid oil derived from dried, cultivated grasses that has been used for over 50 years as an insect repellent and as an animal repellent 60. No adverse effects of concern, other than skin irritation have been reported 60. Citronella is known to be generally less repellent to mosquitoes than DEET 47. Citronella oil is used alone or in combination with other plant oils in a number of commercial insect repellent products 47. An extraction process for citronellol from an indigenous plant, Pelargonium graveolens, for potential use by resource limited animal owners in southern Africa has been described 61. Citronellol (3,7-dimethyl-6-octen-8-ol) is a constituent of rose, geranium and citronella oil, that may exert an insect repellent effect via its strong sweet smell, that masks body odours and carbon dioxide which are considered to attract biting insects to humans and animals 61. An extraction process utilizing lemon eucalyptus oil has been used to produce PMD, the repellent effect of which is more persistent than citronella and nearly equal to that of DEET 47. Against Culicoides (Culicoides) impunctatus Goetghebuer in Scotland both PMD and DEET afforded 98% protection from bites up to eight hours after application in human landing catches 62. 22

CHAPTER 3: MATERIALS AND METHODS 3.1 MODEL SYSTEM An in vitro model, using repellent-impregnated polyester mesh and Onderstepoort downdraught suction ultraviolet (UV) light traps a was used according to the method reported by Braverman and Chizov-Ginsburg 21. A diagram of the Onderstepoort a light trap is shown in Figure 1. Figure 1: Diagram of the Onderstepoort light trap a. 3.2 EXPERIMENTAL DESIGN A randomised, blinded, field experiment was conducted at the height of the Culicoides season, during February 20, at the Onderstepoort Veterinary Academic Hospital (OVAH), Faculty of Veterinary Science, Onderstepoort (25º 38 51.42 S, 28º 10 45.96 E, 1,238 m). Figure 2 shows the location at the study site. a ARC-Institute for Agricultural Engineering, South Africa 23

Figure 2: The location of the five camps (A to E) and the light traps at the study site. The preliminary part of the experiment entailed pre-treatment light trap catches of insects in the experimental area to confirm the presence of Culicoides species in the area and to confirm that each trap position was effective in collecting sufficient numbers of Culicoides. Five downdraught suction light traps were operated from 18h00 to 06h00 for four nights. A horse in each camp, along with the UV light of the trap served as an attractor for Culicoides midges. Each horse was randomly allocated to a camp by drawing a name from a hat (Appendix A, page 53). The camps were identified as camp A, camp B, camp C, camp D, and camp E. Insect samples were collected hourly on the four nights. Each nightly catch was sorted and the Culicoides in each catch counted. The total and mean nightly pre-treatment Culicoides count was determined from the above collections. Thereafter the treatment phase consisted of five polyester mesh applications: (1) 15% DEET, (2) 0.6% citronella oil, (3) 0.3% α-cyano-cypermethrin, (4) 70% ethanol solvent, (5) untreated control, applied to five 220 V down-draught suction light traps equipped with 8 W 23 cm UV-light tubes. The locations of the light traps in the five outside camps were 24

randomised for 15 nights in three 5 X 5 Latin square designs (15 replicates) (Appendix B, page 54) 63. A horse in each camp, along with the UV light of the trap served as an attractor for Culicoides midges. The light traps were operated from 18h00 to 06h00 for 15 week nights (Monday to Friday). The light trap catches were collected hourly from 19h00 until 06h00, and stored in 70% ethanol. Each catch was marked with the camp identification, date and time of sample collection only. Each catch was sorted and the Culicoides species in each catch counted and identified to species level. The person counting the midges was blinded as to the identity of the compound applied to each light trap. 3.3 ETHICAL CONSIDERATIONS Materials used in the experiment posed no undue health risk to researchers and no animals were harmed. The horses involved in the study were fully vaccinated against AHS. The horses involved usually spent the night in an outside paddock, adjacent to the camps where the study was done. No abnormal clinical signs were noted in any of the horses during the trial. Protective gloves were worn during preparation of the repellent impregnated polyester mesh and necessary precautions taken as prescribed by WHO guidelines 47,48. Residual repellent impregnated polyester meshes were disposed of via sealed medical waste containers. Residual repellent preparation was stored in sealed containers until disposed of according to WHO guidelines 48,49. Approval from the Faculty of Veterinary Science Research Committee and the University of Pretoria Institutional Animal Use and Care Committee were obtained prior to initiation of the project. 3.4 EXPERIMENTAL PROCEDURES 3.4.1 Test preparations The test preparations were prepared as solutions made up of the active ingredient and 70% ethanol solvent (Appendix C, page 55). Ethanol was utilised as solvent as DEET is insoluble in water, but miscible with ethanol 49, and a mixture of 70:30 (v/v) ethanol/water was reported to result in the fastest release of DEET from a test formulation 50. 25

The technical grade DEET b was diluted with 70% ethanol to a concentration of 15% active ingredient before application to the mesh. The citronella oil c was diluted with 70% ethanol to a concentration of 0.6% active ingredient before application to the mesh. The cypermethrin d was diluted with 70% ethanol to a concentration of 0.3% active ingredient before application to the mesh. The 70% ethanol was applied undiluted to the solvent test mesh. The test compounds were applied to the polyester mesh, and the amount of preparation absorbed per m 2 of net was calculated based on the weight of the net before and after application of the test preparation according to the method of Schreck and Self 64. 3.4.2 Polyester mesh treatments The test preparations were impregnated onto pieces of white, non-resin-impregnated, polyester mesh e (area 0.07 m 2 ; mesh size 3-4 mm) allowing free entrance to Culicoides midges. The polyester mesh attached to the light trap, and a representative section of the mesh is shown in Figures 3a and 3b, respectively. (a) (b) Figure 3: Polyester mesh (a) attached to the Onderstepoort light trap a, and (b) representative section of mesh. b Diethylytoluamide, Lot 17177, McClaughlin Gormley King Company, USA c Citronella, Product Code P268, Nicola-J Flavours and Fragrances, South Africa d Polytrin 200EC, Batch 2410068,Villa Crop Protection (Pty) Ltd, South Africa e N159 polyester mesh, Textile Dynamics, South Africa 26

Each piece of mesh was weighed, immersed in the solution of test preparation for 30 minutes, air-dried for 30 minutes, and then weighed again. Meshes were kept individually wrapped in tin foil inside sealed Ziploc f plastic bags after preparation on the afternoon prior to the start of the experiment. A new mesh was prepared each afternoon for each of the light traps, i.e. each impregnated mesh was used only once, for one test article application. The mesh was fixed to the entrance portal of the suction light trap with elastic bands. 3.4.3 Light trap operation Onderstepoort downdraught, 220V suction light traps a with 8W 23 cm UV-light tubes were operated from 18h00 to 06h00. The light traps were suspended at the northern periphery of each camp, from the roof, with the entrance portal at a height of 1.8 m, and a distance of 5.6 m between traps. 3.4.4 Collection of Culicoides Insects entering the light trap were collected into labelled 500 ml plastic beakers g containing 200 ml 0.5% Savlon (chlorhexidine gluconate 0.3 g/100 ml, cetrimide 3.0 g/100 ml) and water solution to break the surface tension and help preserve the specimens. The beakers from each trap were collected hourly from 19h00 until 06h00. The excess Savlon solution was decanted through a gauze filter and the insect residue collected was placed into labelled, sealed plastic containers with 70% ethanol until sorted, identified and counted. The plastic containers were labelled with the camp identification, date and time of collection. The residual Savlon solution was reused in the collecting beakers. Torches covered with red cellophane were used to enable beaker changes during the night, as this wavelength of light is reported to not disturb the activity of the insects 44. 3.4.5 Sorting and counting The Culicoides midges were separated from other insects collected, counted and identified to species level. The results of the counts were recorded on a standardised Excel computer spreadsheet (Appendix D, page 56). No subsampling was done. f S.C. Johnson and Son, South Africa g Plastpro Scientific, South Africa 27

3.4.6 Climatic data An automatic weather station, Weather Monitor II h and Weatherlink h data logger were operated from the start of the experiment. The weather station was placed in a central location between the camps. Temperature, relative humidity, daily rainfall and wind speed were recorded hourly and plotted on a daily basis using the Weatherlink h 3.01 computer program. Time of sunset, sunrise, and moon phase were also recorded. 3.5 OBSERVATIONS The following data were collected: The number of Culicoides midges and C. imicola, the most abundant species, collected each hour from each trap. Hourly outside temperature, relative humidity, wind speed and rainfall during the collection period. 3.6 STATISTICAL ANALYSIS Statistical analyses were done using Stata version 8.2 i and NCSS 20 j. Because the outcomes were not normally distributed, various logarithmic and power transformations were attempted. Logarithmic transformation failed to achieve near-normality. However, cube root transformation of midge counts produced near-normality, both overall and within treatment groups, and was therefore used in further analysis. Mean numbers of Culicoides species and of C. imicola were compared between treatment groups while controlling for the effects of camp, time and day, using analysis of variance (ANOVA). This was more powerful than one way ANOVA, as variation due to camp, time and day was accounted for. Separate ANOVAs were also done at each time point while controlling for camp and day. Where F-ratios were significant, all pairwise comparisons were done between treatment groups using Tukey s test. h Davis, USA i StataCorp, College Station, TX j NCSS, Kaysville, UT 28

In order to estimate the effect of temperature, wind speed, humidity and rainfall on counts, multiple regression models were used. However, since several outliers were present in the data, and assumptions of normality were somewhat violated, a robust regression technique (Huber s method) was used. This regression technique down-weights the effect of outliers, providing better regression coefficient estimates. P < 0.05 was considered significant. 29

CHAPTER 4: RESULTS 4.1 CULICOIDES 4.1.1 Preliminary phase During the four night preliminary phase a total of 29,237 Culicoides midges were collected in the hourly light-trap collections from the five light traps operated simultaneously (Appendix E, pages 57-60). The total number of Culicoides collected each night, in each trap, and the total and mean numbers of Culicoides midges collected per night during the preliminary phase is shown in Table 1. Table 1: Number of Culicoides midges collected during the preliminary phase on four consecutive nights in January 20 with five light traps operated simultaneously at the OVAH. Date Camp A B C D E Total Mean 27 January 20 1,813 1,252 758 2,380 2,060 8,263 1,653 28 January 20 1,835 1,054 766 2,246 1,433 7,334 1,467 29 January 20 15 23 16 34 34 122 24 30 January 20 2,934 2,334 1,612 3,947 2,691 13,518 2,7 Total 6,597 4,663 3,152 8,607 6,218 29,237 5,847 Mean 1,649 1,166 788 2,152 1,555 7,309 1,462 Wind overnight. The mean Culicoides collection for the trap sited in Camp C, the centre camp, was lower than for the two outer camps on either side. This result further justifies the use of the Latin square design used. 30

4.1.2 Treatment phase During the treatment phase a total of 107,2 Culicoides midges were collected in the hourly light-trap collections made over the 15 nights from the five light traps operated simultaneously (Appendix F, pages 61-65). The number of Culicoides caught each day, in each trap, and the total and mean numbers of Culicoides midges caught per day is shown in Table 2. Table 2: Number of Culicoides midges collected during the treatment phase on 15 nights in February 20 with five light traps operated simultaneously at the OVAH. Day Camp A Camp B Camp C Camp D Camp E Total Mean 1 679 494 376 1,121 836 3,506 701 2 618 1,110 867 1,193 1,479 5,267 1,053 3 843 855 1,536 589 1,541 5,364 1,073 4 748 319 863 1,191 1,376 4,497 899 5 1,6 994 1,217 2,882 359 7,054 1,411 6 1,943 2,719 795 3,197 1,7 10,358 2,072 7 3,840 839 1,660 1,573 1,3 9,216 1,843 8 2,033 1,831 3,372 1,161 2,373 10,770 2,154 9 1,225 1,935 1,637 2,693 2,208 9,698 1,940 10 654 476 993 1,382 537 4,2 808 11 851 2,180 1,595 1,819 1,928 8,373 1,675 12 754 746 861 1,863 6 4,828 966 13 2,251 682 2,379 2,839 2,932 11,083 2,217 14 1,081 1,561 412 2,483 1,783 7,320 1,464 15 1,8 935 1,689 882 1,274 5,828 1,166 Total 20,170 17,676 20,252 26,868 22,238 107,2 21,441 Mean 1,345 1,178 1,350 1,791 1,483 7,147 1,429 31

The total number of Culicoides and the mean number of Culicoides midges caught per camp are shown in Figures 4 and 5 respectively. There was no significant difference between camp means (P = 0.29). 30,000 26,868 25,000 22,238 Number Culicoides midges 20,000 15,000 10,000 20,170 17,676 20,252 5,000 0 Camp A Camp B Camp C Camp D Camp E Figure 4: Total number of Culicoides midges collected per camp, over 15 nights in February 20, at the OVAH. 2,000 1,800 1,791 1,600 1,483 Number Culicoides midges 1,400 1,200 1,000 800 600 1,345 1,178 1,350 400 200 0 Camp A Camp B Camp C Camp D Camp E Figure 5: Mean number of Culicoides midges collected per camp, over 15 nights in February 20, at the OVAH. 32

Of the 34 Culicoides species caught C. imicola was the most abundant (Appendix G, pages 66-70) and comprised 79.1% of midges collected, followed by C. bolitinos which comprised 5.3%. Of the 107,2 total Culicoides midges, 106,786 (99.6%) were identified to species. The number of each species identified, and percentage of the total identified is compared in Table 3. Table 3: Culicoides species identification (alphabetical), number and percentage of the total Culicoides collected over 15 nights in February 20, with five light traps operated simultaneously at the OVAH. Culicoides specie Number % of Total #54 (d/f) 3 < 0.01 #75 1 < 0.01 #107 4 < 0.01 C. bedfordi 265 0.25 C. bolitinos 5,616 5.26 C. brucei 25 0. C. coarctatus 20 0. C. cornutus 1 < 0.01 C. dekeyseri 4 < 0.01 C. enderleini 1,913 1.79 C. engubandei 2 < 0.01 C. exspectator 231 0.22 C. glabripennis 3 < 0.01 C. gulbenkiani 122 0.11 C. imicola 84,470 79.10 C. leucostictus 2,069 1.94 C. loxodontis 10 0.01 C. magnus 12 0.01 C. neavei 82 0.08 C. near angolensis 1 < 0.01 C. nevilli 15 0.01 C. nigripennis group 8 0.01 C. nivosus 3,789 3.55 C. pretoriensis 1 < 0.01 C. punctithorax 1 < 0.01 C. pycnostictus 3,586 3.36 C. ravus 66 0.06 C. schultzei 9 0.01 C. similis 175 0.16 C. subschultzei 95 0.09 C. trifasciellus 9 0.01 C. tropicalis 53 0.05 C. tuttifrutti 4 < 0.01 C. zuluensis 4,121 3.86 = undescribed species, numbered according to the system of R. Meiswinkel, formerly from the ARC-Onderstepoort Veterinary Institute. 33

The mean number of Culicoides midges (Fig. 6) as well as the mean number of C. imicola (Fig. 7) collected hourly with DEET was significantly (P < 0.05) lower than for all other treatments, overall and at all times except the first (19h00) and the last (06h00) sampling points. During the period of collection sunset fell between 18h40 and 18h58, and sunrise between 05h44 and 06h00. Figure 6: Mean number of Culicoides midges collected hourly over 15 nights in February 20 at the OVAH with light traps fitted with polyester mesh treated with DEET, citronella, cypermethrin, ethanol and a control. = sunset, = sunrise. * = P < 0.05 34

Figure 7: Mean number of C. imicola collected hourly over 15 nights in February 20 at the OVAH with light traps fitted with polyester mesh treated with DEET, citronella, cypermethrin, ethanol and a control. = sunset, = sunrise. * = P < 0.05 A subsample of 164 Culicoides midges, caught on Day 12, was identified to species, sexed and age graded according to the method of Dyce 65 (Appendix H, page 71). Females comprised 95.1% of the total Culicoides midges and 4.9% were males. Of the females, 53.8% were nulliparous, 33.3% parous, 10.3% gravid, and 2.6% blood-fed. Culicoides imicola was the most abundant species identified in the subsample, totalling 130 midges; 99.2% were females and 0.8% were males. The ratio of the numbers of Culicoides midges collected to other insects in the subsample was 1:5. 35

Results of the ANOVAs for Culicoides midges are shown in Table 4, and for C. imicola in Table 5. Table 4: Mean number of Culicoides midges, collected hourly over 15 nights in February 20 at the OVAH with light traps fitted with polyester mesh treated with DEET, citronella, cypermethrin, ethanol and a control, compared between treatment groups at each time point and overall. Treatment group mean Time DEET Citronella Cypermethrin Ethanol Control 19h00 0.0 a 0.5 a 0.2 a 0.2 a 0.3 a 20h00 33.6 a 115.7 c 93.0 bc 73.2 b 96.9 bc 21h00 87.4 a 256.2 c 176.6 b 166.1 b 198.4 bc 22h00 91.0 a 228.3 c 190.9 bc 155.3 b 218.4 c 23h00 77.8 a 229.9 c 166.6 bc 138.2 b 193.4 bc 24h00 64.5 a 184.6 c 121.5 b 118.0 b 151.4 bc 01h00 65.7 a 2.7 c 119.3 b 130.9 b 158.9 bc h00 47.9 a 137.4 b 94.5 b 97.1 b 109.7 b 03h00 32.6 a 100.9 c 56.8 b 72.1 bc 82.3 bc h00 35.7 a 91.5 c 67.2 bc 61.4 b 75.7 bc 05h00 22.4 a 55.5 b 40.6 b 45.3 b 51.3 b 06h00 2.0 a 10.6 b 2.8 a 8.4 b 8.9 b Overall 34.5 a 1.5 c 68.8 b 68.3 b 85.3 bc Back-transformation of the mean of the transformed values. a,b,c Values in same row with no superscripts in common differ significantly (P < 0.05). 36

Table 5: Mean number of C. imicola, collected hourly over 15 nights in February 20 at the OVAH with light traps fitted with polyester mesh treated with DEET, citronella, cypermethrin, ethanol and a control, compared between treatment groups at each time point and overall. Treatment group mean Time DEET Citronella Cypermethrin Ethanol Control 19h00 0.0 a 0.2 a 0.0 a 0.1 a 0.2 a 20h00 17.0 a 69.1 b 47.9 b 47.1 b 58.0 b 21h00 65.6 a 189.1 c 127.6 bc 118.6 b 135.7 bc 22h00 72.5 a 175.7 c 141.9 bc 116.1 b 164.5 bc 23h00 61.3 a 185.3 c 129.2 bc 108.8 b 149.9 bc 24h00 51.6 a 151.3 c 95.4 b 94.1 b 117.9 bc 01h00 54.0 a 164.7 c 91.7 b 1.4 b 127.2 bc h00 41.0 a 115.4 b 76.9 b 79.9 b 89.8 b 03h00 25.6 a 79.5 c 45.5 b 54.6 bc 63.0 bc h00 28.1 a 69.7 b 50.7 b 48.2 b 61.3 b 05h00 15.9 a 40.4 b 30.5 b 33.7 b 39.2 b 06h00 1.5 a 8.4 c 1.8 a 3.8 ab 5.9 bc Overall 26.1 a 78.1 c 49.7 b 50.9 b 63.5 bc Back-transformation of the mean of the transformed values. a,b,c Values in same row with no superscripts in common differ significantly (P < 0.05). Summary charts of the mean number of Culicoides midges collected hourly over the 15 nights with light traps fitted with polyester mesh treated with DEET, citronella, cypermethrin, ethanol and a control are shown in Figure 8. 37

(a) DEET (b) Citronella (c) Cypermethrin (d) Ethanol (e) Control Figure 8: Mean number of Culicoides midges collected each hour on 15 nights in February 20 at the OVAH with light traps fitted with (a) DEET, (b) citronella, (c) cypermethrin, (d) ethanol and (e) control mesh. 38

4.2 CLIMATE Temperature, relative humidity, wind speed and rainfall were recorded hourly (Appendix I, pages 72-75). Light trap catches of the total number of Culicoides and C. imicola increased with increasing temperature (P < 0.001), and decreased with increasing wind speed and rainfall (P < 0.001). Humidity had little effect, although there was a tendency for increasing humidity to decrease C. imicola catches. The outputs of the multiple regression models to estimate the effects of temperature, maximum wind speed, humidity and rainfall on Culicoides catches and on C. imicola catches are shown in Tables 6 and 7 respectively. Table 6: Effects of temperature, maximum wind speed, humidity and rainfall on number of Culicoides collected over 15 nights in February 20 at the OVAH: multiple regression model. Variable b SE (b) 95% confidence interval P-value Temperature 0.158 0.3 0.112 to 0.2 <0.001 Maximum wind speed -0.149 0.013-0.174 to -0.124 <0.001 Humidity 0.000 0.003-0.006 to 0.005 0.914 Rainfall -0.364 0.062-0.484 to -0.243 <0.001 Treatment group <0.001 Camp <0.001 Time <0.001 Unit increase in cube root of Culicoides catch per unit increase in predictor variable 39

Table 7: Effects of temperature, maximum wind speed, humidity and rainfall on number of C. imicola collected over 15 nights in February 20 at the OVAH: multiple regression model. Variable b SE (b) 95% confidence interval P-value Temperature 0.119 0.3 0.074 to 0.164 <0.001 Maximum wind speed -0.157 0.013-0.181 to -0.133 <0.001 Humidity -0.005 0.003-0.010 to 0.000 0.066 Rainfall -0.356 0.060-0.474 to -0.239 <0.001 Treatment group <0.001 Camp <0.001 Time <0.001 Unit increase in cube root of C. imicola catch per unit increase in predictor variable 40

4.3. IMPREGNATED POLYESTER MESH Pre- and post-treatment weights of polyester meshes treated with DEET, citronella, cypermethrin, ethanol and control were recorded (Appendix J, pages 76-80). The amounts of active ingredient absorbed by the meshes before application to the light-traps were 11.0, 0. and 0. g/m 2 for the DEET, citronella oil and α-cyano-cypermethrin respectively. The residual percentage of active ingredient remaining on the meshes 14 hours after application to the light traps were 69.1, 10.4 and 40.6% for the DEET, citronella oil, and α-cyano-cypermethrin respectively (Table 8). Table 8: Mean mesh weights and concentration of DEET, citronella oil and cypermethrin before treatment, after treatment (dry), 14 hours after treatment, and residual % of active on mesh after treatment. Mean mesh weight (g) Concentration active on mesh (g/m 2 ) Residual % active Pretreatment Posttreatment (dry) 14 h posttreatment Pretreatment 14 h posttreatment 14 h posttreatment DEET 13.94 19.16 17.55 11.00 7.6 69.1 Citronella 13.99 14.55 14. 0. 0.0039 10.4 Cypermethrin 14.11 14.55 14.29 0. 0.0076 40.6 Solvent 14.03 14.38 14.06 Control 13.98 Subjectively, a characteristic DEET odour could be smelt throughout the night up to approximately 1 m from the trap with the DEET impregnated mesh. A citronella odour could be smelt in the area where the citronella impregnated mesh was drying, but was less obvious when the mesh was dry. 41

CHAPTER 5: DISCUSSION DEET had a significant repellent effect against Culicoides species, and C. imicola for all catches made from after sunset to before sunrise, when applied to polyester mesh as tested with a down-draught suction UV light trap. The relatively strong attractant effect of UV light traps for Culicoides species and C. imicola reported by Venter and Hermanides 68 is a potential disadvantage of using light traps with repellent-impregnated mesh for screening of compounds for repellency. Despite this severe limitation a significant repellent effect of DEET when applied to polyester mesh was confirmed against Culicoides species and C. imicola. A DEET-treated mesh in the absence of competition from a UV source should provide an even greater barrier to Culicoides entering a stable, if correctly fitted to all openings. A finer mesh size than that used in this study, treated with repellent may be even more effective in keeping Culicoides out of stables. Previous studies in Israel using light-traps and repellent-impregnated polyester mesh have documented durations of repellency of two hours 23 and four hours for DEET 15% 22. The amount of DEET absorbed per m 2 polyester mesh used in the present study was greater than previously reported 22,23 and may have prolonged the duration of repellency. The meshes treated with DEET retained a high residual percentage of DEET after overnight application in comparison to the citronella oil and cypermethrin treated meshes. However, residual efficacy of the used meshes was not investigated further. DEET is the principal active ingredient in human insect repellent formulations 47 and remains the gold standard of currently available insect repellents 46. The effect of six, topically-applied, DEET aerosol formulations to horses has been investigated by Palmer 51, who noted no adverse effect after single application of 3.75 to 75% concentrations. However, after 60 daily applications, excess of oil in the hair coat considered due to hypersteatosis was seen when concentrations of 15% or greater were applied, and cracking of the skin and ulceration was noted in two horses exposed to 50 and 75% concentrations 51. Similar exfoliative lesions were reported in a horse after repeated exposure to 50 and 75% DEET concentrations 66. The concentration of DEET in commercial products registered for human use in South Africa is below the threshold at 42

which adverse effects have been reported in horses. DEET applied to mesh screens would present no direct risk of exposure to high concentrations for horses in the stable area. Other compounds previously evaluated via repellent-impregnated mesh have included a Meliaceae-derived plant extract named Ag1000; Oregano; Herbipet, a mixture of plant extracts comprising oils of sage, rosemary and oregano; Tri-Tec14, containing cypermethrin and pyrethrins; Pyrethroid-T, a type II pyrethroid containing the α-cyano,3- phenoxybenzyl moiety; Stomoxin, containing permethrin; Mosi-guard with Eucalyptus extract containing PMD plus isopulegol and citronellol; and Lice Free, containing plant extracts 20,22,23. Of these compounds the type II pyrethroid was superior to DEET in one study and demonstrated duration of repellency of nine hours 22. A 4% permethrin pour-on provided an 86% positive response in clinical signs due to Culicoides hypersensitivity in horses 56. No significant repellency was demonstrated for the cypermethrin synthetic pyrethroid used in this study. The difference may be due to the formulation of pyrethroid used, low percentage of cypermethrin applied to the mesh or other factors which were not investigated in the study. No significant repellency was demonstrated for the citronella oil in the present study. A citronella fragrance was noted while the citronella oil was drying on the mesh, but was less obvious when the mesh had dried. Any repellent effect due to the fragrance may have thus been reduced prior to mesh application to the light trap. In contrast, a characteristic DEET odour could be smelt throughout the night up to approximately 1 m from the trap fitted with the DEET impregnated mesh. Most of the plant derived essential oils tend to give short protection, usually less than two hours 46. Oil of neem has recently been evaluated against C. impunctatus, with significant distance repellency demonstrated for a 1% concentration and significant reduction in blood feeding on membranes treated with neem 67. Ninety five percent of the Culicoides subsample identified to species level and sexed were females. Similarly, females made up the greater percentage of the C. imicola subsample. Light traps appear not to attract male and/or blood-fed and gravid females. The apparent low percentage of these groups in light trap catches may be the result of a difference in the physiology of these groups and that they are not searching for a host to feed on, rather than smaller populations. 43

Whilst the success of live attenuated AHS vaccines as the primary AHS control measure in endemic situations is evident, secondary control measures to reduce the Culicoides biting rate and thereby limit dissemination of virus amongst horses during high risk periods are important. Application of proven Culicoides repellents, such as DEET, to horses and their stable environment, including mesh screens and solid surfaces, for this purpose is justified. Under epidemic situations, where the use of live attenuated AHS vaccines may not be appropriate and no commercial inactivated or sub-unit vaccines are available 17 protection of horses by reducing biting rates is vital. Under such conditions, application of proven Culicoides repellents to horses and their stable environment, along with stabling and meshing of stables, is of primary importance. 44

CHAPTER 6: CONCLUSIONS It was concluded that DEET had a significant repellent effect against Culicoides species, including C. imicola, for all catches made from after sunset to before sunrise, when applied to polyester mesh as tested with a down-draught suction light trap. No significant repellent effect against Culicoides was found for the citronella oil or the α-cyano-cypermethrin treatments. Applications of DEET, a proven Culicoides repellent to horses and/ or their stable environment is justified as a secondary control measure to reduce the Culicoides biting rate and thereby limit transmission of equine orbivirus infections between horses during high risk periods. 45

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APPENDICES APPENDIX A: Random allocation of Equine Research Centre horses to camps Camp Camp A (east): Camp B: Camp C: Camp D: Camp E (west): Horse Lipstick Cassandra Cassadian Blue Amigo 53

APPENDIX B: Randomised locations of the light traps Camp A Camp B Camp C Camp D Camp E Day 1 Cypermethrin Citronella DEET Control Solvent Day 2 DEET Control Solvent Cypermethrin Citronella Day 3 Solvent Cypermethrin Citronella DEET Control Day 4 Citronella DEET Control Solvent Cypermethrin Day 5 Control Solvent Cypermethrin Citronella DEET Day 6 Cypermethrin Citronella DEET Control Solvent Day 7 Citronella DEET Control Solvent Cypermethrin Day 8 Solvent Cypermethrin Citronella DEET Control Day 9 DEET Control Solvent Cypermethrin Citronella Day 10 Control Solvent Cypermethrin Citronella DEET Day 11 DEET Control Solvent Cypermethrin Citronella Day 12 Control Solvent Cypermethrin Citronella DEET Day 13 Citronella DEET Control Solvent Cypermethrin Day 14 Cypermethrin Citronella DEET Control Solvent Day 15 Solvent Cypermethrin Citronella DEET Control 54

APPENDIX C: Details and preparation of repellent solutions DEET technical grade, McClaughlin Gormley King Company, USA, Lot 17177 Require 15% Deet 7.5 ml Deet 100% + 42.5 ml ethanol 70% per night 50 ml/mesh/night Cypermethrin 20%, Polytrin 200 EC, Villa Crop Protection (Pty) Ltd, South Africa, Batch 2410068 200 mg/ml = 20% Require 0.3% Cypermethrin 0.75 ml Cypermethrin 20% + 49.25 ml ethanol 70% per night 50 ml/mesh/night Citronella oil, Nicola-J Flavours and Fragrances, South Africa, Product Code P268 Require citronella oil 0.6% 0.3 ml of Citronella oil + 49.7 ml ethanol 70% per night 50 ml/mesh/night 55

APPENDIX D: Standardised sheet for recording Culicoides numbers UNIVERSITY OF PRETORIA, FACULTY OF VETERINARY SCIENCE HOURLY COLLECTIONS CULICOIDES PROTOCOL 36-5-621 DR PC PAGE Day CAMP E Ave CAMP D CUL. NO RATIO CAMP A CAMP B CAMP C CUL. NO RATIO DATE TIME CUL. NO RATIO CUL. NO RATIO CUL. NO RATIO Total Total 5 trap Ave total 56

APPENDIX E: Preliminary phase Culicoides midge counts Number of Culicoides midges Date Time Camp A Camp B Camp C Camp D Camp E Total Mean 27 January 20 19h00 0 1 0 0 0 1 0.2 27 January 20 20h00 96 72 42 197 81 488 97.6 27 January 20 21h00 264 164 85 264 187 964 192.8 27 January 20 22h00 137 54 33 109 65 398 79.6 27 January 20 23h00 198 148 100 238 207 891 178.2 27 January 20 24h00 231 169 126 452 347 1,325 265.0 28 January 20 01h00 363 251 143 4 365 1,526 305.2 28 January 20 h00 188 146 60 216 336 946 189.2 28 January 20 03h00 55 51 28 79 123 336 67.2 28 January 20 h00 157 1 62 203 191 715 143.0 28 January 20 05h00 121 86 76 205 153 641 128.2 28 January 20 06h00 3 8 3 13 5 32 6.4 Total 1,813 1,252 758 2,380 2,060 8,263 1,652.6 57

Number of Culicoides midges Date Time Camp A Camp B Camp C Camp D Camp E Total Mean 28 January 20 19h00 0 0 0 0 0 0 0.0 28 January 20 20h00 52 60 27 205 70 414 82.8 28 January 20 21h00 256 129 109 382 123 999 199.8 28 January 20 22h00 334 186 173 438 1 1,235 247.0 28 January 20 23h00 322 174 161 342 123 1,122 224.4 28 January 20 24h00 315 145 107 281 295 1,143 228.6 29 January 20 01h00 335 188 94 283 290 1,190 238.0 29 January 20 h00 38 43 9 32 32 154 30.8 29 January 20 03h00 28 17 19 43 53 160 32.0 29 January 20 h00 88 68 41 155 243 595 119.0 29 January 20 05h00 59 38 23 81 91 292 58.4 29 January 20 06h00 8 6 3 4 9 30 6.0 Total 1,835 1,054 766 2,246 1,433 7,334 1,466.8 58

Number of Culicoides midges Date Time Camp A Camp B Camp C Camp D Camp E Total Mean 29 January 20 19h00 0 0 0 0 0 0 0.0 29 January 20 20h00 3 1 5 4 3 16 3.2 29 January 20 21h00 9 16 3 15 21 64 12.8 29 January 20 22h00 0 0 0 3 1 4 0.8 29 January 20 23h00 0 0 0 0 0 0 0.0 29 January 20 24h00 0 3 1 2 1 7 1.4 30 January 20 01h00 0 0 0 0 0 0 0.0 30 January 20 h00 0 1 0 1 0 2 0.4 30 January 20 03h00 1 1 2 3 3 10 2.0 30 January 20 h00 0 0 0 1 3 4 0.8 30 January 20 05h00 1 0 2 4 2 9 1.8 30 January 20 06h00 1 1 3 1 0 6 1.2 Total 15 Note. Wind gusting 29 January 20 night. 23 16 34 34 122 24.4 59

Number of Culicoides midges Date Time Camp A Camp B Camp C Camp D Camp E Total Mean 30 January 20 19h00 0 0 0 0 0 0 0.0 30 January 20 20h00 230 294 134 525 255 1,438 287.6 30 January 20 21h00 625 371 237 866 288 2,387 477.4 30 January 20 22h00 329 283 588 118 2 1,520 3.0 30 January 20 23h00 225 139 87 399 93 943 188.6 30 January 20 24h00 336 263 135 524 259 1,517 303.4 31 January 20 01h00 142 161 62 216 255 836 167.2 31 January 20 h00 183 197 98 382 535 1,395 279.0 31 January 20 03h00 408 310 114 474 569 1,875 375.0 31 January 20 h00 331 223 116 366 163 1,199 239.8 31 January 20 05h00 2 87 38 72 68 267 53.4 31 January 20 06h00 123 6 3 5 4 141 28.2 Total 2,934 2,334 1,612 3,947 2,691 13,518 2,703.6 60

APPENDIX F: Culicoides midge counts with light traps fitted with polyester mesh treated with (a) DEET, (b) citronella, (c) cypermethrin, (d) ethanol and (e) control (a) DEET Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 0 0 0 0 0 0 0 0 0 1 2 1 1 2 0 20h00 2 32 36 17 31 152 72 86 16 12 28 28 19 40 67 21h00 25 84 110 32 20 198 298 307 482 23 37 21 48 93 85 22h00 113 130 85 44 47 52 195 121 181 45 28 146 69 57 227 23h00 90 57 67 79 125 62 117 240 164 25 11 93 65 51 86 24h00 50 87 93 79 52 54 42 142 55 71 7 96 138 25 91 01h00 26 68 92 12 22 119 40 79 135 144 37 96 120 46 88 h00 12 27 35 8 20 99 20 100 59 84 255 39 77 30 58 03h00 13 30 15 33 17 29 18 34 37 43 145 23 56 19 58 h00 13 83 32 14 17 13 23 30 57 49 191 8 53 23 83 05h00 30 20 23 1 6 16 12 20 38 38 98 30 30 17 32 06h00 2 0 1 0 2 1 2 2 1 2 12 23 6 9 7 Total 376 618 589 319 359 795 839 1,161 1,225 537 851 6 682 412 882 61

(b) Citronella Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 0 0 0 0 2 1 1 0 5 2 4 2 4 2 0 20h00 11 186 76 60 205 609 360 192 142 39 1 37 68 125 65 21h00 21 366 263 83 285 466 1,981 593 605 62 119 70 158 298 190 22h00 134 219 178 57 422 222 559 331 405 138 52 337 214 198 385 23h00 85 84 155 181 740 197 401 857 230 242 44 251 355 186 154 24h00 82 121 275 159 617 174 180 423 106 157 18 457 419 54 143 01h00 39 214 3 34 161 468 155 239 268 299 1 433 358 236 153 h00 16 73 81 12 203 360 45 395 140 122 536 93 296 144 166 03h00 24 49 59 107 108 117 47 152 88 150 398 52 151 73 184 h00 11 115 70 45 105 55 68 130 124 127 303 26 122 138 153 05h00 62 45 71 9 26 44 33 45 93 43 220 33 76 88 77 06h00 9 7 4 1 8 6 10 15 2 1 28 72 30 19 19 Total 494 1,479 1,536 748 2,882 2,719 3,840 3,372 2,208 1,382 1,928 1,863 2,251 1,561 1,689 62

(c) Cypermethrin Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 0 0 0 1 0 1 0 2 0 0 1 3 1 1 1 20h00 4 143 48 91 69 505 168 96 79 280 82 29 97 112 32 21h00 33 287 1 152 82 644 611 206 595 40 78 42 235 247 121 22h00 178 179 81 233 148 137 303 225 885 116 52 172 237 141 241 23h00 151 98 1 410 236 103 136 620 198 111 24 158 397 126 1 24h00 106 85 154 301 393 85 24 217 77 72 14 214 538 31 85 01h00 75 164 196 15 68 167 9 122 429 136 86 126 587 109 63 h00 18 80 57 13 115 215 21 181 131 48 620 30 297 94 83 03h00 32 43 26 108 33 33 3 72 77 58 232 28 251 29 95 h00 13 78 47 41 57 18 19 69 118 95 428 13 165 120 79 05h00 64 36 41 11 14 34 10 17 1 36 181 26 1 60 28 06h00 5 0 1 0 2 1 0 4 2 1 21 20 25 11 5 Total 679 1,193 855 1,376 1,217 1,943 1,3 1,831 2,693 993 1,819 861 2,932 1,081 935 63

(d) Ethanol Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 1 1 1 0 0 0 0 0 0 0 3 3 2 0 1 20h00 11 101 53 103 55 410 248 86 43 25 70 6 85 158 37 21h00 40 182 243 221 63 431 598 246 388 15 82 34 206 322 107 22h00 174 170 72 213 122 100 370 183 373 38 41 97 316 143 247 23h00 54 77 61 288 213 83 182 623 176 52 19 159 330 207 73 24h00 145 110 160 200 268 56 63 167 70 51 12 184 453 82 101 01h00 103 93 142 21 95 286 35 230 210 77 57 136 436 327 108 h00 35 36 42 14 69 203 15 310 147 48 446 34 462 150 87 03h00 82 32 20 87 34 61 16 69 64 81 256 34 274 124 95 h00 31 41 22 34 42 27 32 84 84 68 345 12 149 136 80 05h00 154 22 23 10 29 41 11 29 76 19 232 22 108 105 32 06h00 6 2 4 0 4 6 3 6 6 2 32 25 18 29 80 Total 836 867 843 1,191 994 1,7 1,573 2,033 1,637 476 1,595 746 2,839 1,783 1,8 64

(e) Control Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 0 0 0 0 2 1 0 0 0 2 3 4 2 1 3 20h00 9 108 101 68 99 720 225 101 49 24 101 18 75 236 108 21h00 56 170 216 113 93 740 582 5 498 19 75 56 188 426 168 22h00 272 212 159 105 144 299 412 247 419 121 74 107 332 385 279 23h00 256 99 152 209 210 222 194 796 188 107 30 120 245 346 180 24h00 183 167 208 180 635 147 74 261 86 63 8 231 384 99 80 01h00 85 138 354 33 175 520 62 153 278 125 72 122 368 218 99 h00 24 41 91 18 76 363 30 161 54 47 661 40 372 349 103 03h00 71 58 71 101 58 82 16 77 95 60 393 11 208 112 103 h00 27 84 79 26 75 42 36 51 182 63 487 8 101 147 101 05h00 127 29 1 9 27 58 21 19 83 18 244 15 84 130 41 06h00 11 4 8 1 8 3 8 5 3 5 32 22 20 34 9 Total 1,121 1,110 1,541 863 1,6 3,197 1,660 2,373 1,935 654 2,180 754 2,379 2,483 1,274 65

APPENDIX G: Culicoides imicola counts with light traps fitted with polyester mesh treated with (a) DEET, (b) citronella, (c) cypermethrin, (d) ethanol and (e) control. (a) DEET Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 0 0 0 0 0 0 0 0 0 1 1 1 0 1 0 20h00 0 25 14 6 24 98 58 49 2 3 10 15 11 27 57 21h00 12 65 79 20 15 136 249 276 396 18 22 12 39 78 67 22h00 66 121 62 35 42 36 173 97 151 37 21 100 61 48 190 23h00 70 44 54 60 121 48 93 205 138 18 7 64 54 37 63 24h00 35 80 76 50 50 40 37 122 49 67 4 68 114 18 69 01h00 19 54 77 9 22 101 38 63 122 128 21 71 99 40 72 h00 11 23 31 6 19 86 20 91 56 77 196 31 58 25 45 03h00 6 27 12 21 17 23 17 31 33 35 100 17 48 12 47 h00 9 61 26 4 15 11 23 28 53 42 148 6 45 18 62 05h00 22 17 16 0 6 14 8 17 35 30 77 20 21 11 20 06h00 2 0 0 0 2 1 2 2 0 2 8 17 5 7 6 Total 252 517 447 211 333 594 718 981 1,035 458 615 422 555 322 698 66

(b) Citronella Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 0 0 0 0 2 0 1 0 5 0 3 1 2 1 0 20h00 4 119 42 18 164 372 314 100 106 19 69 11 44 73 40 21h00 10 287 165 40 252 356 1,659 481 438 44 94 40 119 234 140 22h00 73 166 134 33 391 166 503 282 336 103 38 188 181 153 338 23h00 59 59 123 128 710 164 344 769 190 206 30 180 289 138 123 24h00 58 85 227 107 600 138 157 376 83 137 15 347 382 40 118 01h00 26 166 263 25 157 391 144 212 223 269 62 324 296 170 123 h00 12 55 59 8 201 328 42 365 132 101 433 74 239 114 132 03h00 20 36 43 56 99 92 46 141 79 1 3 39 128 50 157 h00 8 83 50 18 100 44 62 114 1 93 257 19 91 89 118 05h00 46 32 51 2 23 38 33 36 83 34 178 13 57 61 50 06h00 5 6 4 1 8 4 10 14 2 1 24 46 23 12 14 Total 321 1,094 1,161 436 2,707 2,093 3,315 2,890 1,779 1,111 1,507 1,282 1,851 1,135 1,353 67

(c) Cypermethrin Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 0 0 0 0 0 0 0 0 0 0 0 3 0 1 1 20h00 3 117 32 50 53 278 123 42 56 15 44 8 58 69 21 21h00 15 259 56 124 56 470 5 150 442 25 55 20 170 192 99 22h00 91 142 48 211 136 93 252 169 770 93 37 87 186 105 170 23h00 108 76 59 363 225 86 107 532 164 94 16 100 326 82 66 24h00 74 72 124 248 376 63 15 176 66 59 11 151 467 13 66 01h00 41 123 137 12 62 140 9 111 377 117 59 76 481 68 46 h00 12 61 38 10 114 192 20 160 118 38 512 22 226 76 59 03h00 23 30 15 79 31 20 3 67 66 40 172 23 196 18 78 h00 9 55 25 27 54 12 17 63 1 71 350 10 119 79 58 05h00 34 27 24 8 12 28 10 16 91 25 163 17 75 33 24 06h00 4 0 0 0 2 1 0 4 1 1 18 11 20 7 3 Total 414 962 558 1,132 1,121 1,383 1,060 1,490 2,253 578 1,437 528 2,324 743 691 68

(d) Ethanol Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 1 1 1 0 0 0 0 0 0 0 2 2 1 0 1 20h00 6 78 29 68 48 258 219 51 25 14 38 2 53 84 30 21h00 25 153 143 163 46 296 513 198 301 11 58 9 160 250 66 22h00 119 136 45 187 99 56 333 142 335 29 28 38 270 98 190 23h00 37 65 38 246 197 64 154 532 150 40 15 97 291 140 52 24h00 1 92 111 153 258 39 56 138 63 43 10 124 392 58 79 01h00 79 77 103 8 91 240 31 210 190 61 43 81 380 278 80 h00 29 32 32 13 65 177 13 279 131 39 346 15 411 117 66 03h00 52 23 11 70 31 51 15 62 58 52 168 17 230 106 72 h00 23 31 16 19 41 19 31 75 76 52 276 5 128 101 66 05h00 108 17 14 4 27 36 10 25 72 9 188 13 84 78 21 06h00 5 1 1 0 3 3 3 5 4 0 26 9 14 23 5 Total 588 706 544 931 906 1,239 1,378 1,717 1,405 350 1,198 412 2,414 1,333 728 69

(e) Control Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 0 0 0 0 1 1 0 0 0 1 2 2 2 0 2 20h00 4 75 56 44 71 532 190 36 29 12 59 2 48 142 77 21h00 26 141 117 76 72 540 485 390 386 10 44 10 151 340 117 22h00 147 179 114 85 128 221 366 190 372 90 50 35 299 311 211 23h00 191 70 118 170 203 191 151 655 162 82 19 57 208 269 127 24h00 118 129 164 120 619 112 62 217 77 48 6 149 332 75 52 01h00 65 103 293 23 170 442 55 139 244 109 48 79 320 148 65 h00 16 36 75 14 71 317 30 141 43 36 517 24 316 308 72 03h00 48 45 56 63 54 75 15 70 90 34 294 5 178 78 71 h00 23 69 67 13 72 28 33 43 167 49 455 7 77 116 63 05h00 85 21 83 3 24 53 20 16 77 10 208 8 68 1 27 06h00 4 4 7 0 6 2 7 4 3 3 23 12 16 28 7 Total 727 872 1,150 611 1,491 2,514 1,414 1,901 1,650 484 1,725 390 2,015 1,919 891 70

APPENDIX H: Sexed and age graded subsample of Culicoides collected on 24-25 February 20 (Day 12), at the OVAH. Culicoides specie Nulliparous Parous Females Blood fed Gravid Total Female Total Male Total C. imicola 74 48 3 4 129 1 130 C. nivosus 0 1 0 8 9 2 11 C. bolitinos 5 0 1 0 6 0 6 C. zuluensis 1 2 0 0 3 1 4 C. enderleini 1 0 0 1 2 1 3 C. leucostictus 1 0 0 0 1 2 3 C. bedfordi 0 1 0 1 2 0 2 C. pycnostictus 1 0 0 1 2 0 2 C. exspectator 0 0 0 1 1 0 1 C. engubandei 0 0 0 0 0 1 1 C. nevilli 1 0 0 0 1 0 1 Total 84 52 4 16 156 8 164 (%) (53.8%) (33.3) (2.6%) (10.3%) (95.1%) (4.9%) 71

APPENDIX I: Climatic data (a) temperature, (b) relative humidity, (c) maximum wind speed and (d) rain recorded. (a) Temperature ( C) Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 27-26- 25-24- 23-13- 12-11- 10-9- 6-5- 4-3- 2- Date 27.7 20.4 25.3 25.0 23.9 26.7 23.3 19.2 19.7 23.6 22.2 23.6 19h00 22.1 - - 25.6 19.3 22.7 22.7 23.1 24.9 21.4 19.3 18.9 22.2 21.3 22.2 20h00 21.3 - - 24.9 17.8 20.9 21.6 22.3 23.2 20.3 19.2 18.9 21.4 21.0 21.5 21h00 20.3 - - 24.1 17.5 19.9 19.3 21.8 21.1 20.3 19.0 18.7 21.0 20.7 20.7 22h00 19.6 - - 23.5 17.6 19.3 18.8 21.6 20.2 20.3 18.3 18.7 20.8 20.7 19.6 23h00 19.3 - - 22.5 16.4 19.3 17.3 20.2 19.9 20.2 18.0 18.4 20.9 20.7 19.1 24h00 19.8 - - 21.8 15.3 19.2 16.4 19.5 20.2 19.9 18.1 18.3 21.0 20.8 18.4 01h00 20.0 - - 20.8 15.1 19.2 15.7 18.7 19.7 19.2 18.3 18.2 20.7 20.7 18.2 h00 20.0 - - 20.2 14.8 18.8 15.3 17.5 20.1 19.1 18.3 18.3 20.3 20.2 17.7 03h00 19.2 - - 19.6 14.9 18.2 15.4 17.1 20.8 19.4 18.4 18.2 19.8 19.6 17.3 h00 18.6 - - 18.9 14.3 17.6 14.8 16.9 20.0 19.1 18.2 18.3 19.4 19.6 16.7 05h00 18.2 - - 18.5 15.1 17.3 14.6 16.7 19.6 19.1 18.2 18.1 19.6 19.7 16.7 06h00 17.3 - - No data recorded. Weatherlink error. 72

(b) Relative humidity (%) Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 81 - - 46 64 36 41 46 42 75 92 97 74 89 80 20h00 83 - - 47 69 48 43 51 47 91 96 100 90 94 87 21h00 80 - - 53 78 58 53 53 53 90 95 100 92 96 92 22h00 86 - - 53 80 65 57 57 60 94 95 100 95 98 93 23h00 81 - - 54 83 69 66 57 65 96 100 99 96 100 96 24h00 81 - - 60 81 64 72 64 69 97 95 100 96 97 100 01h00 81 - - 63 91 66 78 66 67 100 93 100 96 97 100 h00 72 - - 62 87 70 83 73 74 97 92 100 99 96 100 03h00 72 - - 61 92 76 84 76 76 97 92 100 100 100 99 h00 74 - - 61 92 80 84 80 70 98 93 100 100 97 100 05h00 76 - - 65 88 81 82 80 73 99 95 100 100 98 100 06h00 80 - - 68 92 83 85 81 75 97 96 100 100 100 100 No data recorded. Weatherlink error. 73

(c) Maximum wind speed (km/h) Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 6.4 - - 3.2 6.4 1.6 1.6 12.9 1.6 3.2 8 3.2 0 1.6 0 20h00 24.1 - - 3.2 0 0 0 6.4 3.2 4.8 1.6 3.2 0 1.6 0 21h00 6.4 - - 1.6 0 0 0 1.6 0 1.6 1.6 1.6 0 0 0 22h00 0 - - 6.4 0 0 0 0 0 0 8 0 0 0 0 23h00 11.3 - - 4.8 0 0 0 3.2 0 1.6 3.2 0 0 0 0 24h00 1.6 - - 1.6 0 0 0 0 1.6 1.6 4.8 0 0 0 0 01h00 3.2 - - 11.3 0 0 0 0 0 1.6 6.4 0 0 0 0 h00 8 - - 8 0 0 0 0 0 3.2 1.6 0 0 4.8 0 03h00 8 - - 6.4 0 1.6 0 0 0 0 1.6 1.6 0 1.6 0 h00 8 - - 6.4 0 1.6 0 0 0 0 0 1.6 0 6.4 0 05h00 1.6 - - 3.2 0 3.2 0 0 0 0 1.6 3.2 0 0 0 06h00 0 - - 4.8 0 0 0 0 0 0 3.2 0 0 0 0 No data recorded. Weatherlink error. 74

(d) Rain (mm) Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Date 2-3- 4-5- 6-9- 10-11- 12-13- 23-24- 25-26- 27-19h00 0 - - 0 0 0 0 0 0 0 1 0 0 1 0 20h00 1 - - 0 0 0 0 0 0 0 0 0 0 0 0 21h00 0 - - 0 0 0 0 0 0 1 1 0 0 0 0 22h00 0 - - 0 0 0 0 0 0 0 1 0 0 0 0 23h00 0 - - 0 0 0 0 0 0 1 5 0 0 0 0 24h00 0 - - 0 0 0 0 0 0 0 1 0 0 0 0 01h00 0 - - 0 0 0 0 0 0 0 0 0 0 0 0 h00 0 - - 0 0 0 0 0 0 0 0 4 0 0 0 03h00 0 - - 0 0 0 0 0 0 0 0 3 0 0 0 h00 0 - - 0 0 0 0 0 0 0 0 1 0 0 0 05h00 0 - - 0 0 0 0 0 0 0 0 0 0 0 0 06h00 0 - - 0 0 0 0 0 0 0 0 0 0 3 0 No data recorded. Weatherlink error. 75

APPENDIX J: Pre- and post-treatment weights of polyester meshes treated with (a) DEET, (b) citronella, (c) cypermethrin, (d) ethanol and (e) control. (a) DEET Pre-treatment Mean mesh weight (g) Post-treatment (dry) 14 hours posttreatment Amount active on mesh (g) Day 1 13.60 18.43 16.94 0.72 Day 2 13.81 18.72 17.64 0.74 Day 3 13.71 18.69 16.77 0.75 Day 4 14.12 18.80 16.80 0.70 Day 5 13.84 18.80 17.74 0.74 Day 6 14.18 19.24 17.89 0.76 Day 7 14.14 19.14 17.93 0.75 Day 8 13.86 19.03 17.56 0.78 Day 9 14.05 19. 17.62 0.75 Day 10 14.13 19.46 18.24 0.80 Day 11 14.27 22.34 18.11 1.21 Day 12 13.76 18.86 17.30 0.77 Day 13 13.90 18.68 17.39 0.72 Day 14 14.10 18.90 17.95 0.72 Day 15 13.62 19.33 17.42 0.86 Mean 13.94 19.16 17.55 0.78 76

(b) Citronella Pre-treatment Mean mesh weight (g) Post-treatment (dry) 14 hours posttreatment Amount active on mesh (g) Day 1 13.77 13.77 13.81 0.0000 Day 2 13.47 13.47 13.78 0.0000 Day 3 14.55 14.56 14.57 0.0001 Day 4 14. 14.06 14.06 0.0001 Day 5 14. 14. 14. 0.0001 Day 6 13.54 13.54 13.55 0.0000 Day 7 13.59 13.59 13.61 0.0000 Day 8 14. 14. 14.03 0.0000 Day 9 14.4 14.4 14.46 0.0000 Day 10 14.27 14.42 14.3 0.0009 Day 11 14 19.33 14. 0.0320 Day 12 14.11 14.81 14.13 0. Day 13 13.84 13.85 13.84 0.0001 Day 14 14.51 14.52 14.6 0.0001 Day 15 13.73 14.1 13.75 0.02 Mean 13.99 14.43 14. 0.06 77

(c) Cypermethrin Pre-treatment Mean mesh weight (g) Post-treatment (dry) 14 hours posttreatment Amount active on mesh (g) Day 1 14.15 14.39 14.34 0.0007 Day 2 14.44 14.55 14.58 0.0003 Day 3 14.48 14.67 14.66 0.0006 Day 4 14.4 14.54 14.56 0.00 Day 5 13.95 14.09 14.17 0.00 Day 6 14.11 14.23 14.27 0.00 Day 7 13.20 13.34 13.36 0.00 Day 8 14.06 14.19 14.21 0.00 Day 9 14.05 14.18 14.23 0.00 Day 10 15.20 15.46 15.38 0.0008 Day 11 13.74 16.98 13.96 0.0097 Day 12 14.1 14.66 14.27 0.0017 Day 13 13.83 13.95 13.99 0.00 Day 14 14. 14.14 14.24 0.00 Day 15 13.93 14.91 14.13 0.09 Mean 14.11 14.55 14.29 0.0013 78

(d) Ethanol Pre-treatment Mean mesh weight (g) Post-treatment (dry) 14 hours posttreatment Amount active on mesh (g) Day 1 13.71 13.71 13.75 0.00 Day 2 13.61 13.62 13.66 0.01 Day 3 13.63 13.7 13.63 0.07 Day 4 14.01 14.06 14.09 0.05 Day 5 14.09 14.09 14.1 0.00 Day 6 13.95 13.94 13.96-0.01 Day 7 13.93 13.96 13.97 0.03 Day 8 13.93 13.94 13.95 0.01 Day 9 13.96 13.94 13.97-0. Day 10 13.95 13.95 13.99 0.00 Day 11 14.33 18.93 14.36 4.60 Day 12 14.33 14.53 14.33 0.20 Day 13 14.32 14.3 14.32-0. Day 14 14.31 14.31 14.38 0.00 Day 15 14.34 14.67 14.37 0.33 Mean 14.03 14.38 14.06 0.35 79

(e) Control Mean mesh weight (g) Pre-treatment Day 1 13.44 Day 2 13.47 Day 3 13.48 Day 4 13.47 Day 5 13.46 Day 6 13.86 Day 7 14.33 Day 8 14.31 Day 9 14.31 Day 10 14.32 Day 11 14.23 Day 12 14.25 Day 13 14.24 Day 14 14.25 Day 15 14.28 Mean 13.98 80

APPENDIX K: Article published in Veterinary Parasitology Page PC, Labuschagne K, Nurton JP, Venter GJ, Guthrie AJ. Duration of repellency of N,N-diethyl-3-methylbenzamide, citronella oil and cypermethrin against Culicoides species when applied to polyester mesh. Vet Parasitol. 2009;163:105 9. 81

Veterinary Parasitology 163 (2009) 105 109 Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Duration of repellency of N,N-diethyl-3-methylbenzamide, citronella oil and cypermethrin against Culicoides species when applied to polyester mesh P.C. Page a, *, K. Labuschagne b, J.P. Nurton c, G.J. Venter b, A.J. Guthrie c a Department of Companion Animal Clinical Studies, Faculty of Veterinary Science, University of Pretoria, Private Bag X, Onderstepoort 0110, South Africa b PVVD, ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort 0110, South Africa c Equine Research Centre, Faculty of Veterinary Science, University of Pretoria, Private Bag X, Onderstepoort 0110, South Africa ARTICLE INFO ABSTRACT Article history: Received 26 September 2008 Received in revised form 24 March 2009 Accepted 30 March 2009 Keywords: DEET Citronella oil Culicoides Cypermethrin Repellents The repellent efficacy of 15% N,N-diethyl-3-methylbenzamide (DEET), 0.6% citronella oil, and 0.3% a-cyano-cypermethrin against Culicoides species was compared in three 5 5 Latin squares (15 replicates) under South African field conditions. DEET, citronella oil or a- cyano-cypermethrin were applied to polyester meshes that were fitted to down-draught suction 220 V UV light traps which were operated overnight. No significant repellent effect against Culicoides was found for the citronella oil or the a-cyano-cypermethrin. DEET had a significant repellent effect against Culicoides species and C. imicola for all catches made from after sunset to before sunrise. ß 2009 Elsevier B.V. All rights reserved. 1. Introduction Due to the viruses they transmit Culicoides biting midges (Diptera: Ceratopogonidae) are of economic and veterinary significance worldwide (Mellor et al., 2000; Meiswinkel et al., 20). Of principal importance to equids in sub-saharan Africa are Culicoides (Avaritia) imicola Kieffer and Culicoides (Avaritia) bolitinos Meiswinkel which have been implicated in the transmission of African horse sickness (AHS) virus (Du Toit, 1944; Meiswinkel, 1998; Venter et al., 2000; Meiswinkel and Paweska, 2003). Both C. imicola and C. bolitinos can also become infected with and permit replication of equine encephalosis (EE) virus (Paweska and Venter, 20), which is associated with similar clinical signs, but markedly lower mortality than * Corresponding author. Present address: Bayer Healthcare, Animal Health, P.O. Box 143, Isando 1600, South Africa. Tel.: +27 11 921 5747; fax: +27 11 921 5745. E-mail address: patrick.page@bayerhealthcare.com (P.C. Page). AHS in horses (Paweska et al., 1999; Howell et al., 20). Various species of Culicoides have been associated with equine insect hypersensitivity, also referred to as summer seasonal recurrent dermatitis (Braverman, 1988; Greiner, 1995), the most common skin allergy affecting horses (Pascoe and Knottenbelt, 1999). The recent spread of bluetongue (BT) into northern Europe has highlighted the risk of introduction and rapid spread of vector-borne diseases outside their traditional boundaries (Purse et al., 2005; Thiry et al., 2006; Nolan et al., 2007). Culicoides imicola and members of the Culicoides obsoletus and Culicoides pulicaris complexes have been implicated in the outbreaks of BT in Europe (Nolan et al., 2007), whilst both C. imicola and C. bolitinos have been implicated as vectors of the BT virus in South Africa (Du Toit, 1944; Venter et al., 1998). In endemic areas, vaccination is the primary AHS control measure (Coetzer and Guthrie, 20). A polyvalent, live attenuated AHS vaccine is commercially available for compulsory vaccination of horses in South Africa. Until 1990 the attenuated live-virus vaccine comprised two 03-4017/$ see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.03.055 82