A Sustained Release Gel Formulation of Doramectin for Control of Lone Star Ticks (Acari: Ixodidae) and Horn Flies (Diptera: Muscidae) on Cattle

VETERINARY ENTOMOLOGY A Sustained Release Gel Formulation of Doramectin for Control of Lone Star Ticks (Acari: Ixodidae) and Horn Flies (Diptera: Muscidae) on Cattle

1 K. H. LOHMEYER, J. A. MILLER, J. M. POUND, AND J. A. KLAVONS

USDAÐARS, Knipling Bushland U.S. Livestock Insect Research Laboratory, 2700 Fredericksburg Road, Kerrville, TX 78028

J. Econ. Entomol. 102(2): 804Ð808 (2009) ABSTRACT A gel formulation formed by incorporating technical doramectin into a 10% hydroxypropyl methylcellulose aqueous solution was used to subcutaneously inject steers at varying dosages. Doramectin serum concentration of steers receiving 600 ␮g (AI)/kg body weight declined from 21.9 ppb at 0.5 wk to below detectable at 8 wk postinjection. The 1,200 ␮g (AI)/kg injection resulted in serum concentrations of 29.1 ppb at 0.5 wk and declined to 0.5 ppb at 8 wk postinjection. Both the 600 and 1,200 ␮g (AI)/kg injections provided 100% inhibition of index of fecundity (IF) in adult lone star ticks, Amblyomma americanum L. (Acari: Ixodidae) through week 8, after which inhibition declined to 79.4 and 45.3%, respectively, during the 12th week posttreatment. For steers treated at 600 ␮g (AI)/kg, mortality of adult horn ßies, Hematobia irritans L. (Diptera: Muscidae), declined from 16.9% during week 2 to 3.1% during week 7 postinjection. The blood from steers treated at 1,200 ␮g (AI)/kg resulted in a similar decline in mortality of blood fed adult horn ßies from 29.4% during week 1 to 4.0% during week 7. The 600 ␮g (AI)/kg treatment provided complete control of larval horn ßies in the manure for 9 wk, whereas the 1,200 ␮g (AI)/kg injection gave complete control for 14 wk posttreatment. The doramectin gel formulation provided long-lasting delivery of doramectin to cattle and extended control of lone star ticks and larval horn ßies. Such a simple and inexpensive formulation could be useful in tick eradication programs by reducing the frequency of gathering cattle.

KEY WORDS Amblyomma americanum, Hematobia irritans, endectocide, delivery system

The availability of highly potent endectocides pro- Miller et al. (1983) described a sustained release vides unique opportunities for development of novel implant for delivery of ivermectin with extended ef- delivery systems for management of livestock pests. Þcacy against larval horn ßies, Hematobia irritans L. The avermectins such as ivermectin (Campbell 1989), (Diptera: Muscidae), and tick pests on livestock. doramectin (Goudie et al. 1993), eprinomectin Boyce et al. (1992) demonstrated that the implant was (Shoop et al. 1996), and the closely related milbemy- effective against Psoroptes mites in free-ranging big- cins such as moxidectin (Webb et al. 1991, Scholl et al. horn sheep. Although implants are commonly used in 1992, Miller et al. 1994) are the most typical of this the livestock industry, an injectable formulation is group that have found signiÞcant utility in the animal considered more desirable because of reduced trauma health industry. EfÞcacy at dosages in the microgram to the animal and greater ease of administration. Con- per kilogram range has been shown effective against sequently, Miller et al. (1998) developed bioabsorb- a variety of livestock pests (Drummond 1985, Lasota able, injectable microspheres for delivery of ivermec- and Dybas 1991). Despite the potential of using the tin over an extended period (Ͼ12 wk) to cattle and potency of these compounds to develop long-lasting demonstrated long-lasting effectiveness against adult formulations, few such products have become avail- lone star ticks, Ambylomma americanum L. (Acari: able, with the exception of the Ivomec SR Bolus Ixodidae), feeding on cattle. They later showed that (Merck & Co., Rahway, NJ) and a long-acting inject- this formulation could be used to eliminate Boophilus able formulation of moxidectin (Soll et al. 1989, annulatus (Say) (Acari: Ixodidae) from treated cattle Zingerman et al. 1997, Miller et al. 2001, Cleale et al. and the infested pasture where they were held (Miller 2004, Yazwinski et al. 2006). et al. 1999). Similar formulations of both ivermectin and moxidectin have been used to deliver low dosages to dogs for the prevention of heartworm for at least 6 This article represents the results of research only. Mention of a mo (Clark et al. 2004, Lok et al. 2001). proprietary product does not constitute an endorsement or a recom- mendation by USDA for its use. To reduce the cost of the polymers, solvents, and 1 Corresponding author, e-mail: [email protected]. labor for production of the injectable microsphere April 2009 LOHMEYER ET AL.: DORAMECTIN GEL FOR CONTROL OF TICKS AND FLIES 805 formulations, we conducted research to develop a Tick Bioassay. Standard lone star tick (LST) bioas- simpler formulation for extended delivery of an aver- says were conducted to determine efÞcacy of the mectin. The objective of the research reported here treatment against feeding adults. A stockinet sleeve was to 1) develop a bioabsorbable gel formulation for was glued onto the side of each animal by using con- sustained release of doramectin, 2) determine the tact cement at least 24 h before infestations. At each drug-serum proÞle over time for such a formulation, 3) infestation, 30 mating pair of laboratory-reared LST determine the persistence of the formulation and eval- adults were placed in the stockinet sleeve on each uate its ability to inhibit reproduction of adult lone star animal. Infestations were initiated on the day of treat- ticks feeding on cattle, and 4) determine the efÞcacy ment and at 2-wk intervals. The ticks were examined and persistence of the formulation against adult horn periodically to determine percentage of attachment ßies feeding on cattle and immature horn ßies devel- and to collect detached engorged females. The num- oping in the manure of treated cattle. ber of engorged females, the weight of each female, the weight of the egg mass produced, and the estimated hatch of eggs were used to determine percentage of inhibition of the index of fecundity (IF) (Davey Methods and Materials et al. 2001). Calculation of the IF provided a means of Formulation. Hydroxypropyl methylcellulose estimating the reproductive capacity of ticks recov- (HPMC) (Methocel, The Dow Chemical Company; ered from each steer by incorporating the number of Midland, MI) was prepared as a 10% solution in deion- ticks collected, the egg mass weight of sampled ticks, ized water. Two 10-ml plastic syringes were attached and the estimated percentage of hatch of the eggs to each end of an 18-gauge ϫ 2Ð7/8-in. micro-emul- produced by females into the formula. Records were sifying needle (Popper & Sons, Inc., New Hyde Park, kept on each tick from each animal. The engorged NY). One syringe contained 5 ml of prepared HPMC females and subsequent egg masses were held in an gel along with the required amount of technical do- environment of 27ЊC, 85% RH, and a photoperiod of ramectin (PÞzer Animal Health, Exton, PA). The sus- 14:10 (L:D) h. The percentage of inhibition of IF due pension was emulsiÞed by conducting a back-and- to treatment was calculated using a modiÞed AbbottÕs forth motion of the two syringe pistons until no formula (Abbott 1925) reported by Davey et al., 2001. doramectin particles were visible. The required dos- Horn Fly Bioassay. Adult horn ßy bioassays were age for each animal was prepared just before injection conducted by feeding 100 laboratory-reared horn ßies along with syringes containing 5 ml of the HPMC gel in 1.4-liter acrylic cages with a screened top and bot- only to be used for the control steers. tom by using the inverted blood vial technique de- Treatment. Six Ϸ200 kg Hereford steers were di- scribed by Miller et al. (1986). Flies had been reared vided into three groups of two animals each and held at the Kerrville facility by using techniques described in stanchions in an enclosed research barn. The steers by Lohmeyer and Kammlah (2006). Flies were held in were fed twice daily with alfalfa cubes at the rate of a rearing room at 27ЊC, 85% RH, and a photoperiod of Ϸ3% of body weight and were provided water ad 14:10 (L:D) h. The mortality of the ßies fed on blood libitum. Two steers were treated with a 5-ml subcu- from each animal was recorded at 96 h, and percentage taneous injection at a dose of 600 ␮g doramectin/kg of control was calculated using AbbottÕs formula (Ab- body weight, two steers were given an injection at a bott 1925). In addition, manure samples collected dose of 1,200 ␮g doramectin/kg body weight, and the weekly from each animal were used for larval horn ßy remaining two were given a 5-ml injection of the gel bioassays (Miller et al. 1986). For this bioassay, 100 only. The injections were administered in the pres- horn ßy eggs were placed on 100-g samples of manure capular area on the left side of the neck of each animal in waxed paper cups for each dose. Cups of manure in a downward direction. In conducting the research were held in a rearing room at 27ЊC, 85% RH, and a described in this report, we adhered to the Guide for photoperiod of 14:10 (L:D) h. After 7 d, the pupae the Care and Use of Laboratory Animals, as promul- were extracted using a ßotation technique, placed in gated by the Institutional Animal Use and Care Com- small paper cups, and the adults allowed to emerge. mittee (IAUCC) of the Knipling-Bushland U.S. Live- Corrected percentage of inhibition of larval horn ßies stock Insects Research Laboratory, Kerrville, TX. was determined using AbbottÕs formula (Abbott Blood Sampling. Blood samples were collected from 1925). All doses were assayed in triplicate. the jugular vein of each animal before treatment and Data Analyses. The doramectin concentration in weekly postinjection. Two 13-ml Vacutainers (BD serum, percentage of inhibition of IF of ticks, mortality Biosciences, Franklin Lakes, NJ) containing sodium of adult horn ßies, and larval horn ßy mortality were citrate were drawn from each calf for feeding adult analyzed using an analysis of variance (ANOVA) horn ßies. In addition, two 13-ml Vacutainers were mixed model procedure with repeated measures (SAS taken from each animal for high-performance liquid Institute 2004). chromatography (HPLC) analysis of doramectin in serum. Samples were analyzed using an HPLC method Results and Discussion developed in our laboratory (Oehler and Miller 1989, Miller and Oehler 1996). The technique enables quan- Doramectin in Serum. The concentration of dor- tiÞcation of as little as 2 ppb ivermectin in 5 ml of amectin in the serum of the treated steers is shown in serum. Fig. 1. For the 600 ␮g (AI)/kg injection, the serum 806 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 102, no. 2

Fig. 1. Mean Ϯ SD concentration of doramectin in serum Fig. 3. Mean Ϯ SD corrected mortality of adult horn ßies of steers as a result of the treatment with the gel formulation feeding on the blood from steers treated with the doramectin at either 600 or 1,200 ␮g (AI)/kg. gel formulation at either 600 or 1,200 ␮g (AI)/kg.

concentration declined from 21.9 Ϯ 5.4 ppb at 0.5 wk tick IF in the 14th week after treatment. Percentage of to below detectable at 8 wk postinjection. The 1200 ␮g inhibition of tick IF was similar for the two injection ϭ ϭ ϭ (AI)/kg injection resulted in doramectin concentra- dosages (F 0.09; df 1, 2; P 0.79). As expected, ϭ ϭ Ͻ tions in the serum of 29.1 Ϯ 24.2 ppb at 0.5 wk, which there was a signiÞcant (F 12.78; df 7, 14; P declined to 0.5 Ϯ 0.7 ppb at 8 wk postinjection. Serum 0.0001) effect of time postinjection. No signiÞcant ϭ ϭ ϭ concentrations in both treatments seemed to peak (F 1.43; df 7, 14; P 0.27) interaction of time and within the Þrst week after treatment, similar to that group was observed. observed by Toutain et al. (1997) for the conventional Adult Horn Fly Bioassays. The corrected percent- 200 ␮g (AI)/kg injections of doramectin and ivermec- age of mortality of adult horn ßies feeding on the blood tin. However, it is noteworthy that even with these taken from the treated steers is shown in Fig. 3. For ␮ higher dosages of 600 and 1,200 ␮g (AI)/kg, the gel steers treated at 600 g (AI)/kg, corrected mortality Ϯ mediated the availability of the doramectin absorption of horn ßies feeding on blood declined from 16.9 Ϯ in the blood and increased the time that drug could be 1.5% during week 2 to 3.1 2.3% during week 7 detected in the serum. postinjection. The blood from steers treated at 1,200 ␮ Tick Bioassays. Percentage of inhibition of IF for g (AI)/kg resulted in a similar decline in mortality Ϯ Ϯ each infestation of lone star adults on the steers from 29.4 16.7% during week 1 to 4.0 5.7% during through the 14th wk postinjection is represented in week 7. The mortality of adult horn ßies feeding on ␮ Fig. 2. Both 600 and 1200 ␮g (AI)/kg injections pro- blood of steers treated at 1200 g (AI)/kg was signif- ϭ ϭ ϭ vided 100% inhibition of IF through the eighth week, icantly greater (F 33.13, df 1, 2; P 0.029) than ␮ after which there was a decline in inhibition to 79.4 Ϯ that of ßies feeding on the steers treated at 600 g ϭ ϭ 28.1 and 45.3 Ϯ 0.9%, respectively, during the 12th (AI)/kg. There was a signiÞcant (F 11.16, df 8, 16; Ͻ week posttreatment. The data from week 10 are not P 0.0001) decline in efÞcacy with an increasing time ϭ included in Fig. 2 because rats invaded the stockinet postinjection. There was also a signiÞcant (F 3.76, ϭ ϭ sleeves and consumed the ticks. The higher mean df 8, 16; P 0.012) interaction between treatment inhibition of IF observed in the 600 ␮g (AI)/kg treated level and time postinjection. steers was the result of one of the steers providing Larval Horn Fly Bioassays. Both dosages of the gel nearly 100% inhibition of IF, even in the 12th week formulation were effective in inhibiting larval horn posttreatment. Both groups showed no inhibition of ßies in the manure of treated cattle (Fig. 4). The 600 ␮g (AI)/kg treatment provided complete control of larval horn ßies in the manure for 9 wk, whereas the

Fig. 2. Mean Ϯ SD corrected percentage of inhibition of fecundity of adult lone star ticks feeding on steers treated Fig. 4. Mean Ϯ SD corrected mortality of larval horn ßies with the doramectin gel formulation at either 600 or 1,200 ␮g reared in the manure of steers treated with the doramectin (AI)/kg. gel formulation at either 600 or 1,200 ␮g (AI)/kg. April 2009 LOHMEYER ET AL.: DORAMECTIN GEL FOR CONTROL OF TICKS AND FLIES 807

1200 ␮g (AI)/kg injection provided complete control Siebenaler for assistance with ßy bioassays, data analysis, and for 14 wk posttreatment. By the 16th week, the cor- preparation of Þgures. rected larval mortality had declined to 16.7 Ϯ 19.9% in the manure of the steers treated at 600 ␮g (AI)/kg and 54.6 Ϯ 41.9% in the manure of those treated at 1,200 ␮g References Cited (AI)/kg. However, there was no signiÞcant difference Abbott, W. S. 1925. A method for computing effectiveness (F ϭ 1.37, df ϭ 1, 2; P ϭ 0.36) in the effect of the dosage of an insecticide. J. Econ. Entomol. 18: 265Ð267. on mortality of larval horn ßies. The time postinjection Boyce, W. M., J. A. Miller, D. A. Jessup, and R. K. Clark. 1992. signiÞcantly (F ϭ 16.91, df ϭ 16, 32; P Ͻ 0.0001) Use of ivermectin implants for the treatment of psoroptic affected larval survival in the manure of treated steers. scabies in free-ranging Bighorn sheep. J. Zoo Wildl. Med. No signiÞcant interaction (F ϭ 0.79, df ϭ 16, 32; P ϭ 23: 211Ð213. 0.69) between time and dosage was observed. Campbell, W. C. [ed.]. 1989. Ivermectin and abamectin. Springer, New York. All animals were examined at 4 and 24 h after the Clark, S. L., A. J. Crowley, P. G. Schmidt, A. R. Donoghue, subcutaneous injection and weekly thereafter and C. Piche. 2004. Long-term delivery of ivermectin by throughout the study. No adverse reaction at the in- use of poly(D,L-lactic-co-glycolic)acid microparticles in jection site was observed. At the 4-h inspection, an dogs. Am. J. Vet. Res. 65: 752Ð757. enlargement at the injection site due to the 5-ml in- Cleale, R. M., J. E. Lloyd, L. L. Smith, M. A. Grubs, S. T. jection volume could still be seen, but it had disap- Grubbs, R. Kumar, D. M. Amodie. 2004. Persistent ac- peared by the 24-h inspection. tivity of moxidection long-acting injectable formulations A few interesting, although unexplainable, obser- against natural and experimentally enhanced populations of lice infesting cattle. Vet. Parasitol. 120: 215Ð227. vations are worth noting in these data. Although the Davey, R. B., J. A. Miller, and J. E. George. 2001. EfÞcacy of serum concentration of doramectin declined to below daily oral treatments of ivermectin administered to cattle detectable by the seventh week postinjection, the infested with Boophilus microplus (Acari: Ixodidae). J. effect on the adult lone star ticks on the steers per- Agric. Urban Entomol. 18: 127Ð137. sisted for 12 wk. Perhaps this is an indication of ac- Drummond, R. O. 1985. Effectiveness of ivermectin for quired immunity on the part of the treated animals. control of arthropod pests of livestock. Southwest. En- However, no obvious immune reactions were ob- tomol. Suppl. 7: 34Ð42. served and the untreated control animals continued to Goudie, A. C., N. A. Evans, K.A.F. Gration, B. F. Bishop, S. P. yield expected numbers of engorged females through- Gibson, K. S. Holdom, B. Kaye, S. R. Wicks, D. Lewis, A. J. Weatherley, C. I. Bruce, A. Herbert, and D. J. Seymour. out the study. In addition, doramectin continued to be 1993. DoramectinÑa potent novel endectocide. Vet. excreted in the manure for up to 16 wk postinjection Parasitol. 49: 5Ð15. as reßected by the larval horn ßy mortality. The results Lasota, J. A., and R. A. Dybas. 1991. Avermectins, a novel of feeding adult horn ßies on the blood of the treated class of compounds: implications for use in arthropod pest steers were also unexpected. Miller et al. (1986) re- control. Annu. Rev. Entomol. 36: 91Ð117. Lohmeyer, K. H., and D. M. Kammlah. 2006. Improved mass ported that the LC50 value for male and female horn ßies was 3.2 and 6.6 ppb, respectively, for ivermectin rearing techniques for the horn ßy, Haematbia irritans in blood. However, even the considerably higher lev- (L.) (Diptera: Muscidae). Southwest. Entomol. 31: 83Ð85. els of doramectin observed in the blood of these Lok, J. B., D. H. Knigt, G. T. Wang, M. E. Doscher, T. J. treated steers did not result in 50% mortality of adult Nolan, M. J. Hendrick, W. Steber, and K. Heaney. 2001. horn ßies. Activity of an injectable, sustained-release formulation From this study, we conclude that a hydroxypropyl of moxidectin administered prophylactically to mixed- methylcellulose formulation can provide long-lasting breed dogs to prevent infection with Diroﬁlaria immi- delivery of doramectin to cattle for extended control tis. Am. J. Vet. Res. 62: 1721Ð1726. of lone star ticks and larval horn ßies. The gel formu- Miller, J. A., and D. D. Oehler. 1996. Concentrations of lation seems nearly as persistent as the injectable mi- ivermectin in bovine serum as a function of treatment. J. crosphere formulation described by Miller et al. Agric. Urban Entomol. 13: 29Ð39. Miller, J. A., R. O. Drummond, and D. D. Oehler. 1983. A (1998), yet it offers the advantage of being consider- sustained release ivermectin implant for livestock pest ably simpler and less expensive to formulate than the control, pp. 223Ð236. In T. J. Roseman and S. Z. Mansdorf microspheres. These results indicate that other hy- [eds.], Controlled release delivery systems. Marcel Dek- drogel formulations should be investigated for deliv- ker Inc., New York. ery of avermectin for livestock pest control. Such Miller, J. A., D. D. Oehler, A. J. Siebenaler, and S. E. Kunz. formulations could be useful in eradication programs, 1986. Effect of ivermectin on the survival and fecundity such as the Cattle Fever Tick Program by reducing the of horn ßies and stable ßies (Diptera: Muscidae). J. Econ. expense of frequent gathering of cattle. Entomol. 79: 1564Ð1569. Miller, J. A., D. D. Oehler, and P. J. Scholl. 1994. Moxidec- tin: pharmacokinetics and activity against horn ßies (Diptera: Muscidae) and trichostrongle nematode egg Acknowledgments production. Vet. Parasitol. 53: 133Ð143. Miller, J. A., D. D. Oehler, and J. M. Pound. 1998. Delivery We thank Gary Earl, Wayne Ryan, Keith Shelley, and the of ivermectin by injectable microspheres. J. Econ. Ento- late Chris Santos for invaluable assistance in handling of mol. 91: 655Ð659. cattle and assistance with tick bioassays. In addition, we Miller, J. A., R. B. Davey, D. D. Oehler, J. M. Pound, J. E. thank Matt Waldon for help with ßy rearing and Alfred George, and E. H. Ahrens. 1999. Control of Boophilus 808 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 102, no. 2

annulatus (Acari: Ixodidae) on cattle using injectable multihost tick species with sustained-release ivermectin. microspheres containing ivermectin. J. Econ. Entomol. Exp. Appl. Acarol. 7: 121Ð130. 92: 1142Ð1146. Toutain, P. L., D. W. Upson, T. N. Terhune, and M. E. Miller, J. A., R. B. Davey, D. D. Oehler, J. M. Pound, and J. E. McKenzie. 1997. Comparative pharmacokinetics of do- George. 2001. The Ivomec SR bolus for control of ramectin and ivermectin in cattle. Vet. Parasitol. 72: 3Ð8. (Acari: Ixodidae) on cattle in South Texas. J. Econ. En- Webb, J. D., J. G. Burg, and F. W. Knapp. 1991. Moxidectin tomol. 94: 1622Ð1627. evaluation against Solenopotes capillatus (Anoplura: Li- Oehler, D. D., and J. A. Miller. 1989. Liquid chromato- nognathidae), Bovicola bovis (Mallophaga: Trichodecti- graphic determination of ivermectin in bovine serum. J. dae) and Musca autumnalis (Diptera: Muscidae) on cat- Assoc. Off. Anal. Chem. 72: 59. tle. J. Econ. Entomol. 84: 1266Ð1269. SAS Institute. 2004. SAS/STAT userÕs guide, version 9.1. Yazwinski, T. A., J. C. Williams, L. L. Smith, C. Tucker, A. F. SAS Institute, Cary, NC. Loyacano, A. DeRosa, P. Peterson, D. J. Bruer, and R. L. Scholl, P. J., F. S. Guillot, and G. T. Wang. 1992. Moxidectin: Delay. 2006. Dose determination of the persistent activ- systemic activity against common cattle grub (Hypo- ity of moxidectin long-acting injectable formulations derma lineatum) (Diptera: Oestridae) and tricho- against various nematode species of cattle. Vet. Parasitol. strongyle nematodes in cattle. Vet. Parasitol. 41: 203Ð209. 137: 273Ð285. Shoop, W. L., J. R. Egerton, C. H. Eary, H. W. Haines, B. F. Zingerman, J. L., J. R. Cardinal, R. T. Chern, J. Holste, J. B. Micheal, H. Mrozik, P. Eskola, M. H. Fisher, L. Slayton, Williams, B. Eckenhoff, and J. T. Wright. 1997. The in D. A. Ostlind, et al. 1996. Eprinomectin: A novel aver- vitro and in vivo performance of an osmotically con- mectin, for use as a topical endectocide for cattle. Int. J. trolled delivery systemÐIvomec SR᭨ bolus. J. Control. Parasitol. 26: 1237Ð1242. Rel. 47: 1Ð11. Soll, M. D., I. H. Carmichael, G. E. Swan, and S. J. Gross. 1989. Control of induced infestations of three African Received 28 August 2008; accepted 19 December 2008.