Vol. 42, no. 1 Journal of Vector Ecology 171

Efficacy of a fipronil bait in reducing the number of fleasOropsylla ( spp.) infesting wild black-tailed prairie dogs

David M. Poché1*, Daniel Hartman2, Larisa Polyakova1, and Richard M. Poché1

1Genesis Laboratories, Inc., Wellington, CO, U.S.A., [email protected] 2Colorado State University, Fort Collins, CO, U.S.A.

Received 12 December 2016; Accepted 28 March 2017

ABSTRACT: Bubonic plague (Yersinia pestis) is a deadly zoonosis with black-tailed prairie dogs (Cynomys ludovicianus) as a reservoir host in the United States. Systemic insecticides are a promising means of controlling the vectors, Oropsylla spp. , infesting these prairie dogs, subsequently disrupting the Y. pestis cycle. The objective of this study was to conduct a field trial evaluating the efficacy of a grain bait containing fipronil (0.005%) against fleas infesting prairie dogs. The study was performed in Larimer County, CO, where bait was applied to a treatment area containing a dense prairie dog population, three times over a three-week period. Prairie dogs were captured and combed for fleas during four study periods (pre-, mid-, st1 post-, and 2nd post-treatment). Results indicated the use of bait containing fipronil significantly reduced burden. The bait containing fipronil was determined to reduce the mean number of fleas per prairie dog> 95% for a minimum of 52 days post-initial treatment application and 31 days post-final treatment application. These results suggest the potential for this form of treatment to reduce flea population density on prairie dogs, and subsequently plague transmission, among mammalian hosts across the United States and beyond. Journal of Vector Ecology 42 (1): 171-177. 2017.

Keyword Index: Bubonic plague, Cynomys ludovicianus, systemic insecticides, fleas, southwestern United States.

INTRODUCTION plague in humans, controlling fleas can disrupt the disease cycle in prairie dogs and in turn increase prairie dog survivorship, in Bubonic plague is a deadly zoonosis caused by the bacterium non-epizootic cases, and survivorship of other species Yersinia pestis, most notably responsible for the Black Death dependent on them (Jachowski et al. 2011). epidemic in Europe during the 14th century. It is primarily To control fleas, prairie dog burrows are often dusted with maintained in nature by fleas acting as vectors and serving deltamethrin (Seery et al. 2003, Tripp et al. 2016), an insecticide as hosts and is commonly reported in the southwestern U.S. states to which fleas reportedly show resistance (Boyer et al. 2014). of New Mexico, Arizona, Colorado, and California. Black-tailed Additionally, the use of insecticidal spray is reported to have prairie dogs (Cynomys ludovicianus) are highly susceptible to negative effects on non-target species (Dinsmore 2013), plague, with epizootics occurring periodically within populations, suggesting the need for alternative control measures. Borchert resulting in up to 100% mortality in some instances (Pauli et and Poché (2011) discussed detailed methods for controlling al. 2006). In general, prairie dog populations in North America ectoparasites infesting , such as using systemic insecticides have been influenced by epizootic plague outbreaks since its to control ticks and fleas on rodents. Systemic insecticides, such introduction (Jachowski et al. 2011). The two primary flea species as fluazuron, lufenuron, and imidacloprid (Borchert et al. 2009, among prairie dogs are Oropsylla hirsuta and O. tuberculata Davis et al. 2008, Slowick et al. 2001) that can be administered cynomuris (Abbott and Rocke 2012). orally to host species, have shown promise in flea control in Humans typically become infected with plague through the that they can be applied at low concentrations with the route of bite of infected fleas that they are exposed to through contact with administration posing less risk to non-target insect and animal flea-infested wildlife, or pets that encounter wildlife (Walsh and species. Imidacloprid has proven effective against a wide variety of Haseeb 2015). In 2015, 16 cases of plague were reported in the pest including fleas (Borchert et al. 2009, Jachowski et al. United States, four of which resulted in death, an increase from 2011). Fipronil, an insecticide used topically on domestic animals ten cases reported in 2014 and four reported in 2013. There have (Nichols et al. 2014, Hutchinson et al. 1998), has recently shown been three confirmed human cases of plague in the United States promise as an orally administered drug against phlebotomine sand in 2016 (as of December 2), all from New Mexico (ProMED-mail flies Phlebotomus( argentipes) blood-feeding on roof rats (Rattus 2016). In 2015, two fatal cases were reported in Colorado (http:// rattus), proving more efficacious and faster acting when compared www.healthmap.org/site/diseasedaily/article/black-death-back- to imidacloprid, eprinomectin, and diflubenzuron (Ingenloff et second-plague-death-reported-colorado-81615). In addition to al. 2013). Orally-administered fipronil also shows high efficacy infecting humans, plague among prairie dog populations impairs against cattle-feeding sand flies (Poché et al. 2013) and mosquitos endangered black-footed ferret (Mustela nigripes) conservation (Anopheles spp.) (Poché et al. 2015) and rodent-feeding ticks efforts (Matchett et al. 2010), because the species is dependent (Borchert and Poché 2011). However, the use of fipronil has not on prairie dogs for habitat and prey (Roelle et al. 2006). Thus, been extensively reported as an oral systemic insecticide for flea intervention, such as vector control through use of insecticides, is control (Leirs et al. 2001). of paramount importance. In addition to preventing instances of The objective of this study was to determine the field 172 Journal of Vector Ecology June 2017 efficacy of orally applied grain rodent bait with fipronil (0.005%) prairie dogs were observed until sufficiently recovered, at which against fleas infesting black-tailed prairie dogs. The United point they were released at the point of capture. Fleas collected States Environmental Protection Agency (EPA) considers a flea- from each prairie dog were counted and placed back into the control product efficacious if a minimum of 90% reduction of mouth of a burrow at the same location of capture. At the end of fleas infesting the host is met under natural conditions (OPPTS the study, to confirm the presence of a plague vector, a sub-sample 810.3300, Product Performance Guidelines EPA 712-C-98-411). of fleas was collected and saved for species identification using Sufficient efficacy could potentially lead to reduction in instances methods described by Furman and Catts (1982) and Hubbard of plague among humans, black-tailed prairie dogs, and black- (1947). footed ferrets. Bait application MATERIALS AND METHODS The test bait evaluated during this study was an oat-based grain bait formulated with fipronil with a nominal concentration Study area of 0.005% (50 mg/kg) (Scimetrics Ltd. Corp., Wellington, The study was conducted on private land in Larimer County, CO). To confirm fipronil concentration in bait prior to field CO, near Wellington. Two test plots (1 treatment, 1 control), application, a high-performance liquid chromatography (HPLC) separated by ~725 m with two hills serving as natural barriers, were reverse phase method with UV detection was used, following selected after close observation of colonies (occurring January- methods referenced in the 2012 CIPAC handbook (Collaborative February, 2016) confirmed a dense prairie dog population was International Pesticides Analytical Council, United Kingdom. present. Additionally, two of the primary fleas infesting prairie http://www.cipac.org/cipacpub.htm) modified for rodent bait. The dogs, O. hirsuta and O. tuberculata cynomuris, had been observed mean fipronil concentration in the grain bait was determined to in the area (Salkeld and Stapp 2008). The treatment and control be 54.9 + 1.5 mg/kg (n=4, recovery = 98.3%, CV = 2.64%). plot occupied areas ~0.027 and ~0.023 km2, respectively. A buffer The bait was scattered within 1 m of each prairie dog burrow zone extending ~70 m in all directions from the perimeter of on the treatment plot and within the buffer zone, three times the treatment plot was established. Treatment and Control plot during the study over a three-week period: on Day 0 (200g/active perimeters and the buffer zone were marked with flagging and burrow) (February 22), Day 7 (100g/ active burrow) (February 29), GPS coordinates (Garmin Etrex 30, Olathe, KS). Land-owner and Day 21 (100g/ active burrow) (March 14). Day 0 was defined consent was received prior to beginning the study. as the day when bait was first applied to the treatment plot. All animal activities in the field portion of this study followed Collection of prairie dogs and fleas Animal Welfare Act regulations and were approved by Genesis Wild prairie dogs were captured February-April, 2016 Laboratories, Inc. Institutional Animal Care and Use Committee using Tomahawk live traps (7.6x7.6x25cm) (Tomahawk Live (USDA Animal Welfare Act, 9 CFR Parts 1-3) on December 7, Trap Company, Hazelhurst, WI) placed within 1 m of active 2015. A Colorado Department of Natural Resources Colorado burrows within the treatment and control plot. To maximize Parks and Wildlife Scientific Collection License (16TR2069) was trapping success, traps were first wired open and pre-baited for obtained permitting the trapping of black-tailed prairie dogs. two days using oats mixed with peanut butter, after which the wires were removed. Prairie dogs were captured during four Data analysis periods: 1) Pre-treatment: performed before initial treatment Infested prairie dogs were defined as individuals having application (February 15-19), 2) Mid-treatment: within initial and a minimum of one flea. The flea index was defined as the mean final treatment applications (March 2-9), 3) 1st Post-treatment: number of fleas collected per captured prairie dog per plot during immediately following final treatment application (March 15- a single sampling period. Pre-, mid-, 1st post-, and 2nd post- 21), and 4) 2nd Post-treatment: ~1 month after final treatment treatment mean flea indices were used to calculate the percent application (April 12-14). Trap numbers were somewhat variable, efficacy of grain rodent bait containing fipronil (0.005%) adjusting with ~40-50 traps being positioned on each test plot, relative to for potential flea reduction within the control plot (Henderson trapping success. Traps were assigned unique numbers and their and Tilton 1955). Differences between and within pre-, mid-, st1 locations marked via flagging and GPS (Garmin Etrex 30, Olathe post-, and 2nd post-treatment flea indices within the treatment and KS). Traps were opened daily at ~7:00 and checked two to three control plot were further assessed using a student’s t-test followed times per day. Captured prairie dogs were individually marked by Tukey’s W procedure. Treatment was considered successful with unique ear tags and the trap location was noted. Prairie dogs if the flea index within the treatment plot was reduced by> 90% recaptured during the same collection period were immediately relative to the control, if flea indices within the treatment plot released at the point of capture. during the pre-treatment period were significantly higher than Captured prairie dogs and their fleas were processed using during the mid-, 1st post-, and 2nd post-treatment periods (p=0.05), methods similar to those described by Davis (1999). To reduce and if flea indices within the treatment plot were significantly animal stress, all captured prairie dogs were anesthetized with lower than the control plot during the mid-, 1st post-, and 2nd post- isoflurane (Piramal Enterprises Ltd., India) in an induction treatment periods (p=0.05). chamber connected to an oxygen driven vaporizer (Summit Medical Equipment Company, Salem, OR). Fleas were collected by suspending the anesthetized prairie dogs above a white plastic container and brushing them with a flea comb. After flea-combing, Vol. 42, no. 1 Journal of Vector Ecology 173

RESULTS main vectors of the plague bacterium in prairie dog populations (Brinkerhoff et al. 2006). Bait application While application of insecticidal dusts such as deltamethrin The grain rodent bait containing fipronil (0.005%) was can potentially reduce the proportion of prairie dogs and burrows applied to the treatment plot three times (Day 0: 2/22, Day 7: infested with fleas up to 84 days post-treatment (Seery et al. 2/29, Day 21: 3/14) during the experiment, with a total of ~230 2003), the process would be more costly and cumbersome than kg bait applied, averaging 76.7 kg bait per treatment, at a rate systemic insecticide bait treatment if performed on a larger scale 2 2 of 1.92 g/m , amounting to 0.0958 mg fipronil/m (Table 1). We (Jachowski et al. 2011). Additionally, resistance to deltamethrin observed no abnormal animal behavior, primary or secondary has been observed in fleas (Boyer et al. 2014). Our study suggests non-target hazards, or other signs of negative impacts of fipronil that fipronil treatment results in a more rapid decline of on-host bait application. flea numbers than deltamethrin spraying, with 100% efficacy being observed Day 9 post-initial application. Also, given the relative Rodent and flea collection ease of reapplying systemic insecticide baits when compared to A total of 77 prairie dogs were captured within the test plots: spraying, the efficacy of systemic insecticide treatment at monthly 16 (pre-treatment), 20 (mid-treatment), 21 (1st post-treatment), or bimonthly frequencies should exceed the 84-day efficacy of and 20 (2nd post-treatment) (Table 2). Within the treatment plot, deltamethrin previously mentioned. after initializing treatment application, the proportion of prairie Potential resistance of flea populations is of concern when dogs infested with fleas and mean number of fleas infesting prairie applying insecticides in the field. However, topical applications of dogs decreased markedly. The percentages of prairie dogs infested fipronil and imidacloprid have suggested no resistance in strains with fleas during the pre-, mid-, st1 post-, and 2nd post-treatment of cat fleas (Ctenocephalides felis) (Rust et al. 2015), the flea species periods were 80, 0, 0, and 10% within the treatment plot and historically most associated with resistance (Bossard et al. 1998) 100, 90, 70, and 90% on the control plot, respectively, and are and known to show resistance to multiple insecticides (Coles and represented as proportions in Figure 1a. The mean flea indices Dryden 2014). Due to the relative infrequency of the proposed during the pre-, mid-, 1st post-, and 2nd post-treatment periods method of field application, we predict that insecticide resistance were 8.6, 0, 0, and 0.3 within the treatment plot and 10.5, 4.9, 4.8, will be of minimal concern. However, monitoring and surveillance and 7.4 within the control plot, respectively (Figure 1b). Of 24 of treated field-populations of fleas should be initiated to prevent fleas retained for species identification, 23 (~96%) wereO. hirsuta, insecticide resistance (Rust 2016). one being O. tuberculata cynomuris. Unlike insecticidal dusting, the grain bait is orally applied No significant differences were observed between to the host, targeting only on-host ecoparasites, and therefore treatment and control flea indices during the pre-treatment posing minimal risk of exposure to non-target insects (Borchert period (p=0.7743). Flea indices within the treatment plot were et al. 2009). During our study, it was presumed that prairie dogs significantly lower than within the control plot during the mid- consumed all grain bait presented, suggesting the low fipronil (p=0.0058), 1st post-(p=0.0136), and 2nd post-treatment (p=0.047) concentration in the bait to be palatable to the host, with no periods. Additionally, flea indices within the treatment plot were primary or secondary non-target hazards being observed. significantly higher during the pre-treatment phase than during However, our observations were made exclusively during the the mid- (p=0.0006), 1st post- (p=0.0008), and 2nd post-treatment day and above ground, so we cannot say with absolute certainty (p=0.0009) periods. For the control plot, no significant differences that no animals died within the burrow systems, off the plot, or in flea numbers were detected between the pre-treatment period that carcasses were not removed by scavengers. These caveats are and the mid- (p=0.1901), 1st post- (p=0.1038) and 2nd post- also mentioned by previous researchers conducting field trials treatment (p=0.2928) periods. The efficacy of the grain rodent involving insecticide-treated baits and ground-dwelling mammals bait containing fipronil (0.005%) in reducing flea indices on the (Borchert et al. 2009; Jachowski et al. 2011). However, the low treatment plot during the mid- and 1st and 2nd post-treatment fipronil concentration (0.005%) and application rate (0.0958 mg periods was calculated to be 100, 100, and 95.1%, respectively fipronil/m2) suggest that treatment did not negatively impact

(Table 3). prairie dogs or non-target wildlife. The acute oral LD50 of fipronil for mammals (Rattus norvegicus) is reported to be 97 mg/kg body DISCUSSION weight. Given the concentration of fipronil in the bait we applied (0.005%, or 50 mg/kg), an adult C. ludovicianus weighing on During this study, grain rodent bait containing fipronil average ~0.916 kg (Roelle et al. 2006) would need to eat >1.75 kg

(0.005%) was effective in reducing the mean number of fleas of bait in a single sitting to meet the acute oral LD50, a feat which infesting prairie dogs by 95-100%, maintaining the efficacy for> 31 would not be feasible given prairie dogs consume ~0.9kg/week on days post-application, likely resulting in a reduction in potential average (Hygnstrom and Virchow 1994). human and animal Y. pestis infections. In total, efficacy of the bait, In future field trials, the post-treatment sample period should when applied three times over the course of 21 days, was >95% for be increased considerably to determine the specific duration in a minimum of 52 days post-initial treatment application and 31 which >90% efficacy of grain rodent bait containing fipronil days post-final treatment application, exceeding the 90% efficacy (0.005%) can be maintained. Jachowski et al. (2011) found required by the EPA for product registration (OPPTS 810.3300). imidacloprid-treated grain bait to have longer efficacy (up to 90 Flea species were confirmed asO. hirsuta and O. tuberculata days) against fleas on C. ludovicianus. A study performed by cynomuris, which are specialist parasites of prairie dogs and the Ingenloff et al. (2013) found fipronil, orally administered toR. 174 Journal of Vector Ecology June 2017

Table 1. Rate of bait application within the treatment plot and buffer area (~9.75 acres) for treatment with grain rodent bait containing fipronil (0.005%) performed three times between February 22-March 14, 2016. Treatment No. (Date Amount of Bait Applied Bait Application Rate (g/ Fipronil Application Applied) (kg) m2) Rate (mg/m2) Treatment 1 (2/22) 106.69 2.69 0.1345 Treatment 2 (2/29) 57.13 1.38 0.0690 Treatment 3 (3/14) 66.26 1.68 0.0840 Average 76.70 1.92 0.0958

Table 2. Number of black-tailed prairie dogs captured, within two test plots, during four trapping periods performed from February 15-April 14, 2016.

Trapping Period Test Plot No. Prairie Dogs (Date Range) Identification Captured Pre-Treatment Treatment 10 (2-15/2-19) Control 6 Mid-Treatment Treatment 10 (3-2/3-9) Control 10 1st Post-Treatment Treatment 9 (3-15/3-21) Control 12 2nd Post-treatment Treatment 10 (4-12/4-14) Control 10

Table 3. Efficacy of grain rodent bait containing fipronil (0.005%) against fleas infesting black-tailed prairie dogs captured during three trapping periods (Mid-Treatment, 1st Post-Treatment, 2nd Post-Treatment) performed between March 2-April 14, 2016.

Trapping Period Efficacy1 (%) (Date Range) Mid-Treatment 100 (3-2/3-9) 1st Post-Treatment 100 (3-15/3-21) 2nd Post-Treatment 95.1 (4-12/4-14) 1 Efficacy calculated using the method described by Henderson and Tilton (1955) that adjusts for potential reduction in the control population. Vol. 42, no. 1 Journal of Vector Ecology 175

Figure 1. Results for all black-tailed prairie dogs, captured during the pre-, mid-, 1st post-, and 2nd post-treatment periods from February 15-April 14, 2016, indicating a) the proportions of black-tailed prairie dogs infested with fleas within the control and treatment plot, and b) mean No. fleas per captured black-tailed prairie dogs, within the control and treatment plot. Vertical bars indicate the ± standard deviation. 176 Journal of Vector Ecology June 2017 rattus, to be more efficacious than imidacloprid in controlling Boyer, S., A. Miarinjara, and N. Elissa. 2014. Xenopsylla cheopis phlebotomine sand fly adults and larvae. Additionally, future field (Siphonaptera: Pulicidae) Susceptibility to deltamethrin in trials involving grain rodent bait with fipronil (0.005%) should Madagascar. PLoS ONE 9: 1-7. consider monitoring off-host flea dynamics (Krasnov et al. 2004) in Brinkerhoff, R.J., A.B. Markeson, J.H. Knouft, K.L. Gage, and addition to on-host. Given that fleas spend a portion of their lives J.A. Montenieri. 2006. Abundance patterns of two Oropsylla off-host, it would be useful to note a fipronil-induced reduction (: Siphonaptera) species on black-tailed prairie in the mean number of fleas infesting active burrows. The results dog (Cynomys ludovicianus) hosts. J. Vector Ecol. 31: 355-363. of an ongoing field study, in which great gerbils (Rhombomys Coles, T. B. and M. W. Dryden. 2014. Insecticide/acaricide opimus) in southern Kazakhstan were treated with a grain bait resistance in fleas and ticks infesting dogs and cats. Parasit. containing 0.005% fipronil, suggested that the mean number of Vectors 7: 8. fleas infesting gerbils and gerbil burrows could be reduced by Davis, R.M. 1999. Use of orally administered chitin inhibitor 100% for >75 days post application (Poché et al. unpublished (Lufenuron) to control flea vectors of plague on ground data). Tripp et al. (2016) looked at the differing effects of dusting squirrels in California. J. Med. Entomol. 36: 562-567. white-tailed prairie dog burrows with deltamethrin during spring Davis, R.M., E. Cleugh, R.T. Smith, and C.L. Fritz. 2008. Use of and autumn 2009, and reported that dusting in autumn appeared a chitin synthesis inhibitor to control fleas on wild rodents to suppress flea numbers for longer periods. Knowing the precise important in the maintenance of plague, Yersinia pestis, in rate of decline in the efficacy of fipronil-based treatment against California. J. Vector Ecol. 33: 278-284. fleas infesting prairie dogs and burrows, as well as knowing the Dinsmore, S.J. 2013. Mountain Plover responses to deltamethrin ideal season in which to initiate treatment, will provide managers treatments on prairie dog colonies in Montana. Ecotoxicology with knowledge necessary to determine the frequency and timing 22: 415-424. at which treatment should be applied. Furman, D.P. and E.P. Catts. 1982. Manual of Medical Entomology. In the southwestern United States, plague outbreaks amongst 4th Ed. Cambridge University Press, New York, NY. 207 pp. prairie dog populations still pose a risk to humans. Plague Henderson, C.F. and E.W. Tilton. 1955. Tests with acaricides burden is not limited to the United States, with the most recent against the brown wheat mite. J. Econ. Entomol. 48: 157-161. epidemics having occurred in Africa, Asia, and South America Hubbard, C.A. 1947. Fleas of Western North America: Their with approximately 1,000-2,000 cases being reported annually. Relation to the Public Health. Iowa State College Press, Ames, Given the high efficacy of the fipronil bait againstOropsylla IA. 533 pp. spp. fleas infesting C. ludovicianus, we recommend this form of Hutchinson, M.J., D.E. Jacobs, M.T. Fox, P. Jeannin, and J.M. treatment for large-scale application as a vector control tool which Postal. 1998. Evaluation of flea control strategies using may potentially reduce flea population density, and subsequently fipronil on cats in a controlled simulated home environment. plague transmission, among mammalian hosts across the United Vet. Rec. 142: 356-357. States and beyond. Hygnstrom, S.E. and D.R. Virchow. 1994. Prairie dogs. In: Prevention and control of wildlife damage. Cooperative Acknowledgments Extension Division, Institute of Agriculture and Natural Resources, University of Nebraska, Lincoln, NE, B-85–B-96. This study was supported by the Department of Defense Ingenloff, K., R. Garlapati, D. Poché, M.I. Singh, J.L. Remmers, and Deployed Warfighter Protection (DWFP) Program (Grant No. R.M. Poché. 2013. Feed-through insecticides for the control W911QY-15-1-0001). We thank Zaria Torres for assistance with of the sand flyPhlebotomus argentipes. Med. Vet. Entomol. the literature review, manuscript writing and formatting, and 27: 10-18. helpful discussion. 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