Vol. 34, no. 1 Journal of Vector Ecology 161

Scientific Note

Effects of ivermectin on the susceptibility of sonorensis (Diptera: ) to bluetongue and epizootic hemorrhagic disease viruses

Will K. Reeves1,4, Pauline Nol2, Myrna M. Miller1, and Guinevere Z. Jones3

1Arthropod-Borne Diseases Laboratory-Agricultural Research Service, Laramie, WY 82071, U.S.A. 2Animal Plant Health Inspection Service, National Wildlife Research Center, Fort Collins, CO 80521, U.S.A. 3Department of Renewable Resources, 1000 E. University Ave., Laramie, WY 82071, U.S.A. 4Present address: USAG-J, Unit 45013, Box 2582, APO, AP 96338

Received 25 January 2009; Accepted 30 March 2009

Ivermectin and related avermectins are among the questions. most commonly used antiparasitic drugs in both pets All were verified to be antibody negative for and livestock (Campbell 1989). Its toxicity to BTV and EHDV prior to experimentation. Six Shetland and arachnids involves the hyperpolarization of nerve ponies were treated with oral ivermectin paste at a dose and muscle cells and increases in the permeability of cell of 200 µg/kg. Blood was drawn from two ponies prior to membranes to chloride ions, causing muscular paralysis and treatment, two at 24 h post-treatment, and two at four days death (Turner and Schaffer 1989). Blood-feeding insects are post-treatment. A total of six were treated with 400 exposed to ivermectin when they ingest blood from treated µg/kg of injectable ivermectin. Blood was drawn from two animals. Some Culicoides spp. are susceptible to ivermectin sheep prior to treatment, two at 24 hours post treatment, and and suffer up to 99% mortality when fed on animals treated two at 11 days post treatment. Five elk were treated with an with 200 µg/kg of ivermectin (Standfast et al. 1984). Based injectable subcutaneous dose of 200 µg/kg ivermectin and on laboratory bioassays, Culicoides sonorensis Jones and 2000 µg/kg clorsulon (Ivomec Plus). Blood was drawn from Wirth (Diptera: Ceratopogonidae) is tolerant to ivermectin all five elk immediately prior to treatment and 24 h post- when it is mixed directly with blood in the laboratory and treatment. Blood from the ponies and sheep was citrated when fed on treated (Holbrook 1994, Holbrook and with sodium citrate (Becton Dickinson, Franklin Lakes, Mullens 1994). However, animals treated with ivermectin NJ). Blood from the elk was treated with sodium heparin can be less attractive to some Culicoides spp. than untreated (Becton Dickinson, Franklin Lakes, NJ). animals (Sollai et al. 2007). Insecticidal treatments of animals Colonies of C. sonorensis were continuously maintained with ivermectin have been proposed as an alternative to at the USDA--Borne Animal Diseases Research slaughter or quarantine when the vectors of disease are Laboratory as described by Hunt (1994). Teneral adult susceptible (Standfast et al. 1984). The bioavailability of midges were held in a cage without a sugar meal for three ivermectin varies among formulations, route of entry, and days prior to feeding. Females of C. sonorensis were fed animal species, and interactions between ivermectin and control or treated blood with membrane feeders described vector competence to is poorly documented by Hunt and McKinnon (1990). Mortality was assessed (Lo et al. 1985, Marriner et al. 1987). three days post-feeding (dpf) for control and treatment In North America, C. sonorensis is a vector of bluetongue . After blood feeding, midges were maintained on a (BTV) and epizootic hemorrhagic disease (EHDV) viruses 10% sugar water diet for 12 days at 24o C. Females of C. (Foster et al. 1977, 1963). It is a potential vector of exotic sonorensis fully digest their blood meal and oviposit in 3-4 arboviruses such as Akabane and dpf (Hunt 1994).

viruses (Jennings and Mellor 1989, Martin et al. 1997). The Aliquots of 7 log10 TCID50 of BTV-17 and EHDV-2 in effects of ivermectin on vector competence to BTV and cell culture media were mixed 1:1 with treated or untreated EHDV are unknown and the toxicity of ivermectin in blood blood and fed to females of C. sonorensis (Ausman and AK of elk and ponies to C. sonorensis has not been assessed. In colonies). After virus feeding, midges were maintained on a addition, Ivomec Plus (Merial, Duluth, GA), used to control sugar water diet for 12 days at 24° C. Positive control midges parasites in cervids and cattle, contains clorsulon that kills (five to six per feeding) were fed virus as described above Fasciola hepatica (Linnaeus) (Echinostomida: Fasciolidae) and then frozen immediately after the 2 h feeding. All insects by inhibiting enzymes in the glycolytic pathway (Martin were sorted on a chill table, decapitated, and the heads et al. 1997). The toxicity of ivermectin with clorsulon to and bodies were macerated separately in homogenization C. sonorensis is unknown. Our goal was to address these buffer with tungsten beads (Spirit River, Roseburg, OR)

Keyword Index: Bluetongue virus, epizootic hemorrhagic disease virus, Culicoides sonorensis, ivermectin, vector competence. 162 Journal of Vector Ecology June 2009 using a Tissue Lyser (Qiagen, Valencia, CA) with previously ivermectin and demonstrate that the failure to control was described protocols (Kato and Mayer 2007). A sterile scalpel not related to factors in the blood of cattle. was used for each decapitation to avoid contamination Injectable ivermectin at a dose of 400 µg/kg significantly and all tools were sterilized with 4% bleach and 70% reduced the susceptibility of C. sonorensis to BTV-17. The ethanol between each transfer. A non-template control was reason for the reduced infection rate could include cellular included for each extraction. RNA was extracted from the inhibition or toxicity to midgut cells. While there was homogenate using an RNeasy kit (Qiagen, Valencia, CA). no significant mortality between treatment and control BTV was detected by infrared-RT-PCR using previously insects, there could be subclinical toxicity or physiological described protocols (Kato and Mayer 2007). EHDV was responses of the midgut cells that inhibited the virus or detected using the same reaction conditions with primers the infection process. Standfast et al. (1984) suggested that EHDv 63-F1 (5’-AACAGTTACTACGCAAATCA-3) ivermectin could be used to control bluetongue because it and EHDv245-R1 (5’-AGCCATTTCAGCCAATCT-3’) could kill potential vectors and block transmission. Our synthesized with a 5′ IR-Dye 800 chromophore (LI-COR, data do not support this assertion, but they do suggest that Inc., Lincoln, NE). These primers were designed to detect ivermectin can inhibit the infection of C. sonorensis by EHDV-1 and EHDV-2. BTV-17. The benefits of reduced vector susceptibility are Mortality of treated and control insects was analyzed unlikely to be financially significant to ranchers. However, with a chi square test. Treatments were statistically analyzed the data support the use of ivermectin for other purposes, with a Randomized Complete Block Design of a Factorial such as parasite control, during the transmission season of Analysis (RCBD ANOVA) using SAS version 9.1. BTV-17, because it does not increase the susceptibility of C. There were no significant differences (P>0.05) in sonorensis to BTV-17. mortality between C. sonorensis fed on controls or treated Our data do not support the use of ivermectin blood. Negative controls performed as expected in both and clorsulon to control EHDV-2 in elk. There was no the BTV-17 and EHDV-2 assay. Both BTV-17 and EHDV- significant effect of the combined drugs. However, there 2 were detected in the bodies but not heads of positive was no indication that injectable ivermectin and clorsulon control flies. This indicated that virions did not stick to the increased the susceptibility of C. sonoresnis to EHDV-2. mouthparts or head of the after feeding. All heads that Thus, there is no contraindication to using these drugs tested positive for BTV-17 or EHDV-2 were associated with during the transmission season. bodies that also tested positive. There were no PCR positive heads without positive bodies. Acknowledgments Of the midges fed control blood, 40% of the bodies were positive for EHDV-2 and 38% of the treatment bodies were We thank P.K. Bryant, L. DeBrey, A. Fabian, J. Kempert, positive. Virus was detected in 14% and 12% of the control L. McHolland, C. Stith, and W. Yarnell for laboratory and treatment heads. There was no significant difference assistance, cell culture, maintenance of the colonies of C. (P>0.05) in the infection rate between the midges fed on sonorensis, and other assistance with this project. The use of Ivomec Plus-treated elk blood mixed with EHDV-2 and trade names in this document does not constitute an official control blood. In the BTV-17 treatment, 60% of the bodies endorsement or approval of the use of such commercial of the midges fed control blood were positive for virus, hardware or software. Do not cite this document for while 18% of the bodies from midges fed treated blood were advertisement. positive. Virus was detected in 43% of the heads from midges fed control blood, while 14% of the heads from treated blood REFERENCES CITED fed midges were positive. There was a significant difference (P<0.05) in the infection rate of midges fed BTV-17 mixed Campbell, W.C. 1989. Preface. In: W.C. Campbell (ed.) with sheep blood treated with ivermectin and control blood. Ivermectin and Abamectin. Springer-Verlag. 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