(Boophilus) microplus (Acari - BioOne

VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS

Efficacy and Blood Sera Analysis of a Long-Acting Formulation of Moxidectin Against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) on Treated Cattle RONALD B. DAVEY,1,2 J. MATHEWS POUND,3 JEROME A. KLAVONS,3 KIMBERLY H. LOHMEYER,3 JEANNE M. FREEMAN,3 ADALBERTO A. PEREZ DE LEON,3 1 AND ROBERT J. MILLER

J. Med. Entomol. 48(2): 314Ð321 (2011); DOI: 10.1603/ME10154

ABSTRACT The therapeutic and persistent efÞcacy of a single subcutaneous injection of a longacting formulation of moxidectin at a concentration of 1 mg/kg body weight was determined against Rhipicephalus (Boophilus) microplus (Canestrini), along with the concentration-time blood sera proÞle in treated cattle. The therapeutic efÞcacy against ticks of all parasitic stages on cattle at the time of treatment was ⬎99.9%, and the mean tick number, index of fecundity, engorgement weight, and egg mass weight of ticks recovered from treated animals were all signiÞcantly lower than ticks from untreated animals. The index of fecundity, engorgement weight of females, and egg mass weight of ticks recovered from treated animals infested at weekly (7-d) intervals between 14 and 63 d posttreatment were signiÞcantly lower than for ticks on untreated animals, whereas the number of ticks per animal recovered from treated cattle remained lower than that of untreated cattle for up to 49 d posttreatment. The percentage control remained ⬎99% at weekly intervals between 14 and 49 d posttreatment, which is the minimum level of efÞcacy considered acceptable for use in the United States Cattle Fever Tick Eradication Program. The serum concentration of moxidectin in treated cattle increased to 25.6 ppb (parts per billion) within 1 d after treatment, and peaked at 47.3 ppb at 8 d posttreatment. Moxidectin sera levels remained above the estimated 100% threshold level for elimination of feeding ticks (5Ð 8 ppb) for 44 Ð53 d after treatment. The label claim of 50 d of prevention against reinfestation for the long-acting moxidectin formulation used in the study was supported by the efÞcacy and sera concentration data obtained. Based on these results, cattle could be treated at 63-d intervals with minimal risk of viable ticks detaching from treated animals. This treatment interval would be 4.5-fold longer than the presently required treatment interval of 14 d, thus leading to ⬇75% reduction in gathering and handling costs of cattle incurred by producers. KEY WORDS moxidectin, control, cattle fever tick, endectocide, macrocyclic lactone

Within the federally managed Cattle Fever Tick Eradication Program (CFTEP), rising labor and handling costs associated with treatment of cattle have created This article represents the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation by United States Department of Agriculture for its use. In conducting the research described in this report, the investigations adhered to the Guide for the Care and Use of Animals in Agricultural Research, as required and approved by the Institutional Animal Care and Use Committee of the Knipling-Bushland United States Livestock Insects Research Laboratory (Kerrville, TX). 1 United States Department of AgricultureÐAgricultural Research Service, Cattle Fever Tick Research Laboratory, Moore Air Base, Building 6419, 22675 N. MooreÞeld Road, Edinburg, TX 78541. 2 Corresponding author: United States Department of AgricultureÐ Agricultural Research Service, Southern Plains Area, Cattle Fever Tick Research Laboratory, Moore Air Base, Building 6419, 22675 N. MooreÞeld Road, Edinburg, TX 78541 (e-mail: ronald.davey@ ars.usda.gov). 3 United States Department of AgricultureÐAgricultural Research Service, Knipling-Bushland United States Livestock Insects Research Laboratory, 2700 Fredericksburg Road, Kerrville, TX 78029.

a serious impediment in preventing the re-establishment and dispersal of Rhipicephalus (Boophilus) spp. ticks into the United States. When cattle fever ticks are detected on cattle within the United States, the premises upon which the cattle are pastured are quarantined and the owner/producer has two options, as follows: 1) treating 100% of the cattle on the premises with an approved acaricide every 14 d for a period of 6 Ð9 mo, or 2) removing all livestock from the infested premises for 6 Ð9 mo, an option referred to as “pasture vacation.” By removing all livestock, which are the primary hosts, the ticks are eliminated through starvation. However, the pasture vacation option has, upon numerous occasions, failed to eliminate cattle fever tick infestations within the duration of the quarantine period. The increasing incidence of pasture vacation failures has been attributed to an enormous increase in the white-tailed deer, Odocoileus virginianus (Zimmermann), in Texas, which in 1991 was estimated to be 3.1 million animals (Texas Tech Uni-

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versity 1997). Although deer, as host animals, do not support large numbers of cattle fever ticks (Cooksey et al. 1989, Davey 1990), they can successfully produce enough offspring to sustain a tick population for a period of time that exceeds the presently prescribed quarantine period of 6 Ð9 mo, especially when the deer population is high at the onset of the quarantine period (R.B.D., unpublished data). The CFTEP has long held that the maintenance and systematic treatment of cattle in infested premises is the best method for achieving eradication of cattle fever ticks because the continual treatment of the cattle, which are the preferred host, will eventually reduce the tick population to the point in which deer are no longer a factor in sustaining the tick population. However, it has been difÞcult to convince owners/producers to choose the dipping option because of associated costs of repeated gathering, handling, and treating cattle on a 14-d schedule for 6 Ð9 mo. The organophosphate acaricide, coumaphos, has been the only agent approved for use in the CFTEP since 1968 (Graham and Hourrigan 1977). However, coumaphos, like virtually all of the other chemical agents (pyrethroids, formamidines, and macrocyclic lactone endectocides) used in the United States and Mexico, has a rather short persistent effectiveness (⬍2-wk protective period against reinfestation) (Wharton et al. 1970; Roy-Smith 1975; Nolan et al. 1979; Davey et al. 1983, 1984, 2005; Khan and Srivastava 1988; Singh and Chhabra 1992; George and Davey 2004). The short duration of persistent efÞcacy of these acaricides requires owners/producers to treat cattle every 2 wk while maintained on tick-quarantined premises. There is a critical need to provide owners/producers a practical means to maintain cattle on infested premises when vacating the premises is not an option. This may be achieved through the development of acaricide treatment technologies that substantially reduce the need for frequent gathering and dipping of cattle to obtain eradication. During the past 10 Ð12 yr, the evaluation and/or development of several new chemical treatment technologies have provided encouraging results in the effort to extend the treatment intervals in eliminating cattle fever ticks. The use of a bioabsorbable injectable microsphere technology with the endectocide, ivermectin, produced remarkable results, showing that a natural cattle fever tick population could be eradicated within 11 wk after a single subcutaneous treatment of microspheres (Miller et al. 1999). Unfortunately, more than a decade after the demonstration of the potential beneÞt of this technology, the product remains unregistered for several reasons, and there has been little progress in obtaining a registration. More recently, long-acting (LA) formulations of ivermectin and moxidectin, which are registered for use in other countries, including Mexico, have shown prolonged activity against internal and external parasites (Bridi et al. 2001; Rehbein et al. 2002; Geurden et al. 2004; Cleale et al. 2004a, b). Recently, a LA formulation of ivermectin, which is registered for use in Mexico, but not in the United States, was evaluated for

315

potential use in the CFTEP as a technology for extending the treatment interval beyond the present 14-d requirement (Davey et al. 2010). Results were disappointing because activity fell far short of the 75-d label claim for protection against reinfestation. Whereas the LA formulation of moxidectin, which also is registered for use in Mexico, but not in the United States, has never been evaluated in the context of an eradication program, it has a label claim of 50 d of protection against reinfestation for cattle fever ticks, which makes it a potential candidate for use in the CFTEP. The objective of the current study was to evaluate the therapeutic and persistent efÞcacy of a LA formulation of moxidectin against larvae of Rhipicephalus (Boophilus) microplus (Canestrini) released on cattle at various intervals before and after treatment. Results obtained from this study could provide the CFTEP with an alternative treatment strategy that would reduce the number of treatments necessary to eliminate cattle fever ticks from an infested premise. Positive results could also provide the necessary Þnancial incentive for owners/producers to maintain cattle on the infested premise during the quarantine period rather than choosing the pasture vacation option, as the means of eradicating the tick population. Materials and Methods This study was conducted at the United States Department of Agriculture, Agricultural Research Service, Cattle Fever Tick Research Laboratory (Edinburg, TX). Twelve Angus calves, weighing ⬇200 kg each and with no prior exposure to Rhipicephalus ticks, were randomly divided into two groups of six animals per group. One group of six calves was designated as an untreated group, and a second group of calves, to which the candidate acaricide was administered, was designated as the treated group. The material used in the study was a LA-injectable formulation containing 10% active ingredient of moxidectin, Cydectin Onyx 10% (Fort Dodge Animal Health, Mexico City, Mexico), with a recommended dosage rate of 1 ml/100 kg body weight, which produced a concentration of 1 mg of moxidectin/kg body weight. The acaricide is registered and commercially available for use in Mexico on cattle for control of internal and external parasites. The product was purchased in Mexico, declared and passed through United States Customs into the United States, and brought to the Cattle Fever Tick Research Laboratory for experimental testing. At ⱕ1 wk before treatment, the calves were weighed on certiÞed scales to ensure proper dosing. All animals were held in an open-sided barn under ambient conditions, except that a roof prevented direct sunlight or rainfall from reaching the animals. Each animal was held throughout the study in an individual stanchion within a 3.3 ⫻ 3.3-m stall, and stalls were separated from each other by 1.7-m-high cinder block walls to prevent crossover contamination of ticks between animals. Therapeutic Efficacy. At ⫺19, ⫺12, and ⫺5 d before treatment, all calves from both groups were artiÞcially

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infested with ⬇5,000 larval R. microplus ticks (as determined by egg weight, i.e., 250 mg of eggs ⫽ ⬇5,000 larvae) that were 2Ð 4 wk old. A 16 ⫻ 70-mm shell vial (2-dram) with a cotton stopper to prevent escape of the larvae was glued to the midline of the back of each animal with branding cement, and the cotton plug was removed to allow the larvae to disperse over the body of the animal. This infestation regime allowed for evaluation of the LA moxidectin against all parasitic developmental stages of the tick (larva, nymph, and adult) on the host at the time of the treatment (therapeutic efÞcacy). At the time of treatment (day 0), each treated calf was injected subcutaneously with the test material at the manufacturerÕs recommended concentration according to individual body weights of the animals. Beginning on the day after treatment (day 1) and continuing through day 34 posttreatment, engorged female ticks that detached from each animal within each daily 24-h interval were collected from the ßoor of the stall, counted, and recorded. A random sample of up to 10 engorged females (whenever possible) was saved from each animal on each day of the evaluation period (day 1Ð34 posttreatment) to obtain oviposition and fertility data. Ticks from each daily sample obtained from each animal were weighed collectively, placed in a coded 9-cm-diameter plastic petri dish, and held in an incubator at 27 ⫾ 2⬚C, 92.5% RH, under a 12:12 L:D cycle for 20 d. After 20 d, eggs from each individual sample were harvested, weighed, placed in a coded shell vial, and returned to the incubator, and spent females were discarded. At 4 wk after eggs were weighed, the hatch rate of each sample was visually estimated by comparing the proportion of unhatched eggs with the proportion of egg shells present in the vial, as described by Davey et al. (2005). Data for tick counts, egg mass weights, and estimated percentage egg hatch for each animal over the entire 34 d posttreatment evaluation period were used to calculate the index of fecundity (IF) of each animal on each day using the following formula reported by Davey et al. (2001): IF ⫽ number of 씸씸 collected ⫻ (weight of eggs (g)/number of 씸씸 saved) ⫻ egg hatch (%) Calculation of IF provided a method of estimating the fecundity and fertility of the ticks recovered from each calf on each of the 34 d after treatment. The percentage control was determined Þrst by summing the daily IF values of ticks from each calf over the entire evaluation period, and then the summed IF value for each treated calf was compared with the mean of the summed IF values of ticks from untreated calves using the following modiÞed AbbottÕs (1925) formula: % control ⫽ (mean IF of untreated ⫺ summed IF of each treated calf/mean IF of untreated) ⫻ 100

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Table 1. Relationship between posttreatment larval infestation and subsequent collection of detached engorged females recovered at 21–27 d after infestation Posttreatment day of larval infestation

Posttreatment collection daysa

14 21 28 35 42 49 56 63

35Ð41 42Ð48 49Ð55 56Ð62 63Ð69 70Ð76 77Ð83 84Ð90

a Days posttreatment at which recovered detaching engorged females correspond to 21Ð27 d after infestation.

In addition to calculated IF values, the biological data (female engorgement weight and egg mass weight) of daily sampled ticks from the untreated group were compared with data obtained from treated calves to determine any measurable effect on weight and fecundity of females that survived the treatment. Persistent Efficacy. Cattle used in the therapeutic portion of the study also were used to evaluate the persistent efÞcacy (duration of the protective period against larval reinfestation) of the test material. Determination of the duration of the protective period against larval reinfestation after treatment was evaluated based on a series of larval infestations applied to all cattle (both groups) at various intervals after treatment. To determine the persistent efÞcacy beginning on 14 d posttreatment, each calf was artiÞcially infested with ⬇2,500 larval R. microplus ticks, as described in the therapeutic portion of the trial. Subsequently, to assess the persistent efÞcacy, all calves were infested with ⬇2,500 larvae at 21, 28, 35, 42, 49, 56, and 63 d posttreatment, as described previously. A system based on the detachment pattern of R. microplus reported by Hitchcock (1955), which showed ⱖ95% of a cohort of ticks infested at the same time will detach from the animal at 21Ð27 d after infestation, was used to establish the posttreatment interval at which recovered engorged females were infested on the cattle as larvae (Table 1). Thus, the protective period (persistent efÞcacy) was based on tick counts, fecundity, and fertility data collected at 21Ð27 d after each posttreatment infestation. Beginning at 35 d posttreatment and continuing through 90 d posttreatment, daily tick collection and sampling of each animal were conducted, as described in the therapeutic efÞcacy portion of the study. A daily IF of each calf on each day between 35 and 90 d after treatment was calculated using the previously described formula. To determine the persistent (residual) efÞcacy at each posttreatment infestation interval, the mean summed IF value for the untreated control group at each posttreatment classiÞcation interval was compared with the summed IF values for each calf in the treated group having the same posttreatment classiÞcation interval using the previously described formula. In addition to calculating IF values and percentage control levels at each posttreatment infestation interval, biological data (fe-

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317

Fig. 1. Mean ⫾ SE concentration of moxidectin in serum of cattle treated with a single subcutaneous injection of a LA formulation of moxidectin at 1 mg/kg body weight.

male engorgement weight and egg mass weight) obtained from the sampled females collected from both untreated and treated calves at each classiÞcation interval were compared to determine any measurable adverse effects on female weight and fecundity through time that could be attributed to the treatment. Pharmacokinetic Evaluation. To evaluate the concentration of moxidectin in the blood sera of the treated cattle, blood samples were obtained at various posttreatment intervals. Using 12.5 ml of SST Vacutainers (Tyco Healthcare Group LP, MansÞeld, MA), blood samples were taken from the jugular vein of each animal at 1, 5, 8, 12, 15, 19, 22, 26, 29, 35, 42, 49, 56, 63, and 70 d posttreatment. Blood was centrifuged at 2,000 rpm for 30 min to obtain serum, which was poured into individually coded 14-ml polypropylene round-bottom tubes (BD Biosciences, Franklin Lakes, NJ) and frozen at ⫺80⬚C for later analysis. At the time of analysis, sera samples were thawed and analyzed by high performance liquid chromatography to determine concentration of the test material in the serum (Oehler and Miller 1989). The technique enables quantiÞcation of ⱖ2 ppb (parts per billion) of the test material in 5 ml of serum. Data Analysis. Data on tick number, IF, female engorgement weight, and egg mass weight obtained for untreated and treated females in the therapeutic portion of the study (ticks on the host at the time of treatment) were analyzed by Mann-Whitney rank sum test to determine differences for each measured parameter (Systat Software 2006). Data from the persistent efÞcacy portion of the study were analyzed by two methods. First, the tick number, IF, engorgement weight, and egg mass weight value for untreated and treated ticks at each posttreatment infestation interval were subjected to a t test to determine differences

between the two groups. Secondly, the tick number, IF, engorgement weight, egg mass weight, and percentage control of only treated ticks were analyzed by repeated measures analysis of variance across all posttreatment infestation intervals, and differences among means were determined by the Holm-Sidak pairwise method (Systat Software 2006).

Results Pharmacokinetic Evaluation. The moxidectin level in the sera of treated cattle reached 25.6 ⫾ 3.7 ppb the day after treatment (day 1), and a maximum concentration of 47.3 ⫾ 3.8 ppb occurred at 8 d posttreatment (Fig. 1). The mean concentration of moxidectin in sera remained ⬎20 ppb during the Þrst 22 d posttreatment, and concentrations remained ⬎10 ppb through day 40 posttreatment. The mean concentration remained ⬎8 ppb (estimated minimum level for 99% control) for 44 d posttreatment, and ⬎5 ppb until 53 d posttreatment. No levels at ⬍2.0 ppb were obtained in any individual sample until 63 d posttreatment. Therapeutic Efficacy. SigniÞcantly fewer (T ⫽ 57.0, n ⫽ 6, P ⬍ 0.002) ticks infested on the treated cattle at the time of treatment subsequently reached repletion than the same cohort of ticks infested on untreated cattle (Table 2). Likewise, the IF of females that survived to repletion on treated cattle was also signiÞcantly lower (T ⫽ 57.0, n ⫽ 6, P ⬍ 0.002) than that of untreated ticks, thus providing ⬎99.9% control. The engorgement weight of females recovered from treated animals was signiÞcantly less (T ⫽ 1651.0; n ⫽ 54, 161; P ⬍ 0.001) than untreated females, as was the mean weight of egg masses produced by treated females (T ⫽ 1780.0; n ⫽ 54, 161; P ⬍ 0.001).

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Vol. 48, no. 2

Table 2. Mean ⴞ SE tick number per calf, female weight, egg mass weight, IF, and percentage control of Rhipicephalus (Boophilus) microplus recovered from untreated and treated cattle infested at 19, 12, and 5 d before a single subcutaneous injection of a LA moxidectin formulation at 1 mg/kg body weight Treatment

No. ticks per calf

Female weight (mg)

Egg mass weight (mg)

IF

% control

Untreated Treated

1,768 ⫾ 365a 27 ⫾ 8b

263 ⫾ 6a 44 ⫾ 6b

101 ⫾ 4a 3 ⫾ 2b

196.738 ⫾ 47.483a 0.036 ⫾ 0.036b

Ñ ⬎99.9 ⫾ 0.0

Means tested by Mann-Whitney rank sum test; means within the same column followed by a different letter are signiÞcantly different (P ⬍ 0.05). Number of ticks per calf, T ⫽ 57.0; df ⫽ 6, 6; P ⫽ 0.002; female weight, T ⫽ 1651.0; df ⫽ 54, 161; P ⬍ 0.001; egg mass weight, T ⫽ 1780; df ⫽ 54, 161; P ⬍ 0.001; IF, T ⫽ 57.0; df ⫽ 6, 6; P ⫽ 0.002.

Persistent Efficacy. Comparison between untreated and treated calves at each posttreatment larval infestation interval showed that signiÞcantly fewer engorged females per calf (t test: t ⫽ 3.2Ð10.5, df ⫽ 10, P ⬍ 0.01) were recovered from treated cattle than from untreated cattle at 14 Ð 49 d posttreatment (Table 3). Conversely, treated cattle produced more ticks per animal than untreated animals at 56 and 63 d posttreatment, although in both instances differences were not signiÞcant. The IF of engorged females subsequently produced from each of the larval infestation intervals (14 Ð 63 d posttreatment) was signiÞcantly lower (t test: t ⫽ 2.7Ð14.5, df ⫽ 10, P ⬍ 0.02) for ticks recovered from treated cattle than that of ticks recovered from untreated cattle. The number of adult ticks recovered per animal across all larval infestation intervals for treated cattle showed signiÞcant differences (F ⫽ 20.7; df ⫽ 7, 35; P ⬍ 0.001) among infestation intervals (Table 3). The number of recovered engorged females per calf that developed from larvae released 63 d posttreatment was signiÞcantly higher (P ⬍ 0.05) than at all other infestation intervals. Similarly, the number of female ticks per calf that developed from larvae infested at 56 d posttreatment was signiÞcantly higher (P ⬍ 0.05) than all earlier posttreatment infestations, except for larvae infested at 49 d posttreatment. There was no difference (P ⬎ 0.05) in the number of ticks recovered from reinfested calves at 14 Ð 49 d posttreatment. The IF of ticks across all posttreatment larval infestations for treated cattle also differed signiÞcantly

(F ⫽ 5.2; df ⫽ 7, 40; P ⬍ 0.001) among infestation intervals (Table 3). There was no difference (P ⬎ 0.05) in the IF of ticks developed from larvae infested at 14 Ð56 d posttreatment. However, the IF of engorged females developed from larvae infested 63 d posttreatment was signiÞcantly higher (P ⬍ 0.05) than that of females recovered at all other postinfestation intervals, except for larvae infested at 56 d posttreatment. There were signiÞcant differences (F ⫽ 13.4; df ⫽ 7, 40; P ⬍ 0.001) in the percentage control among the various posttreatment larval infestation intervals (Table 3). The percentage control remained ⱖ99.2% for all posttreatment infestation intervals through 49 d posttreatment with no signiÞcant differences among the means (P ⬎ 0.05). However, the level of control achieved against ticks infested at 63 d posttreatment (90.5%) was signiÞcantly lower (P ⬍ 0.05) than all other infestation intervals, except for ticks infested on cattle at 56 d posttreatment, which was not different from ticks infested at either 49 or 63 d posttreatment. Comparison of the mean female engorgement weight of ticks recovered from untreated and treated cattle at all of the posttreatment larval infestation intervals showed that ticks from treated cattle weighed signiÞcantly less (t test: t ⫽ 6.2Ð18.6, df ⫽ 38 Ð75, P ⬍ 0.001) than ticks recovered from untreated cattle at each interval (Table 4). Likewise, the mean weight of the egg mass produced by females recovered from treated cattle at all of the posttreatment infestation intervals was also signiÞcantly less (t test: t ⫽

Table 3. Mean ⴞ SE number of ticks per calf, IF, and percentage control of Rhipicephalus (Boophilus) microplus females that survived to repletion from larval infestations applied to untreated and treated cattle at various intervals after a single subcutaneous injection of a LA moxidectin formulation at 1 mg/kg body weight No. engorged females per animal

Day larvae infested

Untreated

14 21 28 35 42 49 56 63

55 ⫾ 18 102 ⫾ 21 535 ⫾ 89 409 ⫾ 37 257 ⫾ 43 213 ⫾ 42 88 ⫾ 20 82 ⫾ 27

IF

Treated

Between treatments

Untreated

Treated

Between treatments

% control

0 ⫾ 0a ⬍1 ⫾ 0a 1 ⫾ 1a 13 ⫾ 6a 22 ⫾ 9a 65 ⫾ 14ab 119 ⫾ 17b 189 ⫾ 43c

* * * * * * NS NS

5.7 ⫾ 2.0 9.1 ⫾ 2.4 55.2 ⫾ 14.0 41.4 ⫾ 7.9 23.0 ⫾ 4.8 20.5 ⫾ 5.6 8.7 ⫾ 2.9 9.9 ⫾ 4.9

0.00 ⫾ 0.00a 0.00 ⫾ 0.00a 0.03 ⫾ 0.03a 0.03 ⫾ 0.03a 0.06 ⫾ 0.03a 0.17 ⫾ 0.08a 0.42 ⫾ 0.16ab 0.95 ⫾ 0.37b

* * * * * * * *

100 ⫾ 0.0a 100 ⫾ 0.0a ⬎99.9 ⫾ 0.1a 99.9 ⫾ 0.1a 99.8 ⫾ 0.1a 99.2 ⫾ 0.4ab 95.2 ⫾ 1.8bc 90.5 ⫾ 3.7c

Means for untreated and treated groups in each row for number of ticks per calf and IF tested by t test or Mann-Whitney rank test (P ⬍ 0.05); asterisks in each row of columns 4 and 7 indicate signiÞcant difference; NS indicates no difference. Within columns 3, 6, and 8, means for treated cattle only, tested by repeated measures analysis of variance; means within the same column followed by a different letter are signiÞcant by the Holm-Sidak method (P ⬍ 0.05). Number of ticks per animal, F ⫽ 20.7; df ⫽ 7, 35; P ⬍ 0.001; IF, F ⫽ 5.2; df ⫽ 7, 40; P ⬍ 0.001; percentage control, F ⫽ 13.3; df ⫽ 7, 40; P ⬍ 0.001.

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Table 4. Mean ⴞ SE female engorgement weight and egg mass weight of Rhipicephalus (Boophilus) microplus that survived to repletion from larval infestations applied to untreated and treated cattle at various intervals after a single subcutaneous injection of a LA moxidectin formulation at 1 mg/kg body weight Female engorgement weight (mg)

Day larvae infested

Untreated

14 21 28 35 42 49 56 63

261 ⫾ 13 249 ⫾ 9 262 ⫾ 7 267 ⫾ 7 259 ⫾ 7 250 ⫾ 12 245 ⫾ 11 227 ⫾ 15

Egg mass weight (mg)

Treated

Between treatments

Untreated

Treated

Between treatments

No ticks 55 ⫾ 0ab 104 ⫾ 51ab 55 ⫾ 2ab 62 ⫾ 4ab 64 ⫾ 3a 73 ⫾ 3ab 86 ⫾ 6b

NT * * * * * * *

110 ⫾ 9 92 ⫾ 9 99 ⫾ 7 109 ⫾ 7 109 ⫾ 8 83 ⫾ 8 106 ⫾ 9 92 ⫾ 10

No ticks 0 ⫾ 0a 20 ⫾ 20b 2 ⫾ 1a 3 ⫾ 1a 4 ⫾ 1a 5 ⫾ 1a 7 ⫾ 1a

NT * * * * * * *

Means for untreated and treated groups in each row for female engorgement weight and egg mass weight tested by t test or Mann-Whitney rank test (P ⬍ 0.05); in each row of columns 4 and 7, astericks indicate signiÞcant difference; NT indicates not tested. Within columns 3 and 6, means for treated cattle only, tested by repeated measures analysis of variance; means within the same column followed by a different letter are signiÞcantly different by the Holm-Sidak method (P ⬍ 0.05). Female engorgement weight, F ⫽ 4.4; df ⫽ 6, 113; P ⬍ 0.001; egg mass weight, F ⫽ 4.1; df ⫽ 6, 113; P ⬍ 0.001.

3.5Ð11.9, df ⫽ 38 Ð75, P ⬍ 0.001) than females obtained from untreated cattle at each interval. The female engorgement weight of ticks from treated cattle across all posttreatment infestations showed signiÞcant differences (F ⫽ 4.4; df ⫽ 6, 113; P ⬍ 0.001) among the infestation intervals (Table 4). The engorgement weight of females recovered from the larval infestation applied at 49 d posttreatment (64 mg) was signiÞcantly less (P ⬍ 0.05) than the engorgement weight of females recovered from ticks infested at 63 d posttreatment (86 mg). Females recovered from all other posttreatment infestations were not signiÞcantly different (P ⬎ 0.05) in engorgement weight from ticks recovered from either the 49 or 63 d posttreatment infestations. The mean egg mass weight of females obtained from treated cattle across all posttreatment infestation intervals also showed signiÞcant differences (F ⫽ 4.1; df ⫽ 6, 113; P ⬍ 0.001) among the various infestation intervals (Table 4). With one exception, the mean egg mass weight produced by ticks from all posttreatment infestation intervals was ⬍10 mg, with no differences (P ⬎ 0.05) among any of the means. However, the mean egg mass weight of ticks recovered from the infestation applied at 28 d posttreatment (20 mg) was signiÞcantly greater (P ⬍ 0.05) than that of ticks from all other posttreatment infestation intervals. Discussion Although the therapeutic efÞcacy of the LA moxidectin formulation applied at a concentration of 1 mg kg⫺1 provided virtually complete control (⬎99.9%), these results were not surprising considering results of another study that reported 99.1% control using a 1% formulation of moxidectin administered at a concentration of 200 ␮g/kg, which was 5 times lower than the dosage used in this study (Davey et al. 2005). However, in comparison with results reported by Davey et al. (2005) using the 1% moxidectin formulation at the same infestation rate, the LA moxidectin formulation resulted in 6.1 times fewer engorged females per calf

(27 ⫾ 8 ticks) than the 1% formulation (165 ⫾ 35 ticks), and the engorgement weight of females and egg mass weight for ticks treated with the LA formulation were 2.3 and 5.7 times lower, respectively, than that of ticks treated with the 1% formulation. Therefore, even though the traditional 1% formulation and the LA formulation both provided the 99% level of therapeutic control considered to be the minimum acceptable level for use in the United States CFTEP, the LA formulation demonstrated a clear advantage over the 1% formulation as a candidate for use in the CFTEP. It has been reported that a level of 5Ð 8 ppb of an endectocide in the sera of cattle would be the threshold level at which elimination of feeding ticks could be expected (Nolan et al. 1985, Pound et al. 1996, Miller et al. 1999). By this standard, moxidectin levels were well above the 100% kill rate within 1 d posttreatment (25.6 ⫾ 3.7 ppb) and remained there until 44 Ð53 d posttreatment (Fig. 1). A previous study reported a peak concentration of LA moxidectin that was somewhat higher (55.71 ⫾ 15.59 ppb) with a time interval for peak concentration that was 2.4 times shorter (3.40 ⫾ 3.36 d posttreatment) than was determined in this study (Dupuy et al. 2007). However, another investigative study reported that Angus cattle absorbed moxidectin at a slower rate with a delayed peak concentration interval as compared with Holstein animals (Sallovitz et al. 2002). Consequently, it is possible that the use of Angus cattle in this study may have accounted for the somewhat lower peak concentration (47.3 ⫾ 3.8 ppb) and the delay in time to peak concentration (8 d posttreatment). In contrast to this study, pharmacokinetic data of the traditional 1% injectable formulation of moxidectin at a concentration of 200 ␮g/kg body weight showed that sera concentration peaked 6.7 times sooner (1.2 d posttreatment) in water buffaloes (Dupuy et al. 2008) and 26.7 times sooner (8 h) in cattle (Lanusse et al. 1997). In addition, the maximum concentration of moxidectin obtained in water buffalo never reached the 5Ð 8 ppb threshold level for 100% kill, and was 88.4% lower (5.46

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ppb) than the maximum concentration observed in this study (Dupuy et al. 2008). The development of LA formulations was initially predicated on the perceived need for an extended period of protection against reinfestation and reducing treatment frequency (Lifschitz et al. 2007). The current study clearly demonstrated that in comparison with ticks recovered from untreated cattle, tick numbers from treated cattle were signiÞcantly reduced when larval ticks were infested for up to 49 d posttreatment, whereas the IF, engorgement weights, and egg mass weights were all dramatically adversely affected in ticks infested up to 63 d posttreatment. Consequently, these Þndings resulted in a level of control that was ⱖ99% against ticks infested on treated cattle for a period of up to 49 d posttreatment, which is the minimum percentage of control considered acceptable for use in the CFTEP. It is noteworthy that this 49-d period of ⱖ99% control was virtually identical to the label claim for cattle fever ticks of 50-d prevention of reinfestation for the Cydectin Onyx 10% LA formulation used in the study, and this product is currently registered for use in Mexico. Whereas no published information exists regarding the use of the LA formulation of moxidectin against cattle fever ticks, investigations conducted against gastrointestinal strongylid nematodes compared favorably with results obtained in this study (Cleale et al. 2004a, Geurden et al. 2004), showing that the formulation produced 96.7Ð100% reduction in egg output for a period of 55Ð56 d posttreatment. In another cattle study, LA moxidectin evaluated against the lice, Linognathus vituli and Solenopotes capillatus, provided 100% control for 28 d posttreatment, but provided ⬎97% protection against reinfestation for a period of 133 d posttreatment (Cleale et al. 2004b). The current study stands in stark contrast to an LA formulation of ivermectin evaluated against cattle fever ticks in which it was reported that 99% control was achieved only against ticks infested at 14 d posttreatment, after which control diminished to 70.4% against ticks infested at 28 d posttreatment (Davey et al. 2010). Results also contrasted with the traditional 1% formulation of moxidectin in which the persistent efÞcacy was 92.4% against ticks infested at only 1 wk posttreatment, and control decreased to only 19.5% against ticks infested at 4 wk (28 d) posttreatment (Davey et al. 2005). Because the therapeutic efÞcacy of the LA formulation of moxidectin provided ⬎99.9% control, it would be a strong candidate for use in the CFTEP for the following reasons. The 49-d period of protection against reinfestation (⬎99%) would make the formulation highly desirable by substantially increasing the interval between treatments beyond the presently required 14-d interval. Also, because it is known that cattle fever ticks are unable to oviposit in ⬍18 d (Davey et al. 1982), the 49-d interval between treatments could safely be lengthened by perhaps an additional 14 d. For example, if larval ticks successfully attained a treated host animal at 49 d posttreatment, it would still take a minimum of 18 additional days for

Vol. 48, no. 2

the ticks to reach ovipositional status. Thus, repeated treatments at 63 d (49 ⫹ 14 d) apart would pose virtually no risk of having viable ticks detach from infested animals and return to the Þeld to sustain the natural tick population. The use of LA moxidectin in the CFTEP with this treatment regime could reduce the number of required treatments, as well as gathering and handling costs, by ⬇75%, as compared with the presently required 14-d treatment interval, thereby providing great incentive for producers to maintain cattle on infested premises during the quarantine period. Although use of this LA endectocide formulation in the CFTEP could substantially reduce treatment number and gathering costs, nevertheless, there are factors that could adversely impact the use of the material. Because of the highly lipophilic nature of moxidectin, the material is extensively distributed from the bloodstream to different tissues, particular adipose tissue, which could act as a drug reservoir, thereby contributing to its long residence time (Lanusse et al. 1997, Dupuy et al. 2007). Repeated treatment of cattle, even at intervals of 63 d apart, could result in unacceptable levels of moxidectin in animal tissues. In fact, the label for the material used in the study stated that animals treated with the material could not be slaughtered for human consumption for a period of 73 d after the last treatment. Thus, the incentive for producers to maintain animals on an infested premise resulting from 75% fewer treatments might be offset by the fact that treated animals could not be marketed for 73 d after the last treatment. Another potentially adverse effect that could occur from the use of LA moxidectin is that repeated treatments over a long period of time, as would be necessary in the CFTEP, could increase selection pressure on off-target organisms, such as helminthes or other parasites, thereby leading to the emergence and development of endectocide resistance. These factors should be carefully considered before recommending the general use of LA moxidectin in the CFTEP.

Acknowledgments We thank Renata Miller for assistance in obtaining the product material used in this study. We also thank Michael Moses for assistance in executing the protocol for this study and collection and tabulation of the data, and Cesario Agado, James Hellums, Ruben Ramirez, Homero Vasquez, Joseph Sparks, and George Gonzalez for assistance in the handling and care of the animals used in this study. Additionally, we thank Jason Tidwell for assistance in testing all animals for the presence of disease (Babesia spp.) during this study.

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