First report of fluazuron resistance in rhipicephalus microplus: a field tick population resistant to six classes of acaricides

Contents lists available at Veterinary Parasitology First report of fluazuron resistance in Rhipicephalus microplus: A field tick population resistant to six classes of acaricides José Reck , Guilherme Marcondes Klafke , Anelise Webster, Bruno Dall'Agnol, Ramon Scheffer, Ugo Araújo Souza, Vivian Bamberg Corassini, Rafael Vargas, Julsan Silveira dos Santos, João Ricardo de Souza Martins Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Fundac¸ão Estadual de Pesquisa Agropecuária (FEPAGRO), Eldorado do The control of the cattle tick Rhipicephalus microplus is based mainly on the use of chem- Received 11 September 2013 ical acaricides, which has contributed to the emerging problem of selection of resistant Received in revised form 7 January 2014 tick populations. Currently, there are six main classes of acaricides commercially avail- Accepted 19 January 2014 able in Brazil to control cattle ticks, with fluazuron, a tick growth regulator with acaricidal properties, being the only active ingredient with no previous reports of resistance. Ticks (designated the Jaguar strain) were collected in a beef cattle ranch located at Rio Grande do Sul state, Southern Brazil, after a complaint of fluazuron treatment failure. To characterise the resistance of this strain against acaricides, larval tests were performed and showed that Multiple resistance the Jaguar strain was resistant to all of the drugs tested: cypermethrin (resistance ratio, RR = 31.242), chlorpyriphos (RR = 103.926), fipronil (RR = 4.441), amitraz (RR = 11.907) and ivermectin (3.081). A field trial was conducted to evaluate the efficacy of fluazuron treat- ment in heifers that had been experimentally infested with the Jaguar or a susceptible strain. Between 14 and 28 days after treatment, the average efficacy in cattle experimen- tally infested with the susceptible strain was 96%, while for the Jaguar strain the efficacy was zero. Additionally, the Jaguar strain response to fluazuron was evaluated in vitro using a modified adult immersion test (AIT) and the artificial feeding assay (AFA). With the AIT, 50 ppm of fluazuron inhibited 99% of larvae hatching in the susceptible strain (POA) and less than 50% in the Jaguar strain. Results of the AFA showed a larval hatching rate of 67% at 2.5 ppm of fluazuron with the Jaguar strain; conversely, only 3% of larvae of the susceptible strain hatched at the same fluazuron concentration. The results showed here demonstrated the first case of fluazuron resistance in R. microplus and the first tick population resistant to six classes of acaricides in Brazil.
2014 Elsevier B.V. All rights reserved.
∗ Corresponding author at: Laboratório de Parasitologia, Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Fundac¸ão Estadual de Pesquisa Agropecuária (FEPAGRO), Estrada do Conde, 6000, Eldorado do Sul 92990-000, RS, Brazil. Tel.: +55 51 3481 3711; fax: +55 51 3481 3711.
E-mail addresses: (G.M. Klafke).
These authors contributed equally for the conduction of this study.
0304-4017/$ – see front matter 2014 Elsevier B.V. All rights reserved.
J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136 tick-resistant populations, particularly in Southern Brazil The cattle tick Rhipicephalus microplus is a major cause of to describe the first report of fluazuron resistance in R.
concern for cattle breeding in the tropical and subtropical microplus, and also report the first tick strain that was world, particularly in Latin America. Its parasitism reduces multi-resistant to six classes of acaricides.
weight gain, causes anaemia, increases the risk for myia- sis occurrence, and can also result in the transmission of 2. Materials and methods
Babesia bovis, B. bigemina and Anaplasma marginale, which are the causative agents of cattle tick fever ( 2.1. Tick strains Currently, tick control is mainly, if not exclusively, based on the use of chemical 2.1.1. Jaguar strain Ticks were collected from beef cattle at a ranch located The control of cattle ticks with chemical compounds in the municipality of Eldorado do Sul, RS, Southern Brazil.
started at the end of the nineteenth century with the use This farm only breeds Bos taurus taurus cattle, and apart of arsenic The use of this compound was from raising beef cattle, it breeds bulls for sale. Addition- followed by the use of organochlorines (OC) in the 1940s, ally, it has an intense animal trade activity with other and a few years later, resistance to this class was reported.
ranches, exchanging cattle with several other properties Subsequently, these were replaced by organophosphates in in RS and others states of Brazil. Since 2010, there has been the 1950s (According to the Brazilian Com- a suspicion of fluazuron treatment failure as reported by pendia of Veterinary Products (there are property staff. In the past years, cattle have been heavily currently six main classes of chemical acaricides marketed infested with ticks at this location, particularly during the for tick control in Brazil: (i) organophosphates (OP), (ii) for- late summer and mid-autumn (February to May). Tick con- mamidines (amitraz), (iii) synthetic pyrethroids (SP), (iv) trol had been based on fluazuron treatments (Acatak® macrocyclic lactones (ML), (v) phenylpyrazoles (fipronil), Pour On, Novartis Saúde Animal, Barueri, SP, Brazil), and (vi) benzoylphenyl ureas (fluazuron). The large scale pour-on application and dipping vats with a mixture of and frequent use of acaricides aiming to control ticks on chlorpyrifos and cypermethrin (Colosso® Pour On and cattle has favoured the selection of tick populations that are Colosso® Pulverizac¸ão, Ourofino Saúde Animal, Cravinhos, resistant to several active ingredients (AI) of commercial SP, Brazil), and the periodic use of long-acting formula- acaricides. In recent years, there have been an increasing tions of avermectins. This property had a history of a high number of products in the Brazilian market that are mix- level of tick resistance to cypermethrin, deltamethrin, ami- tures of two or more AIs traz, mixture of cypermethrin + chlorpyriphos, and mixture Reports of resistance against the main classes of aca- of cypermethrin + ethion, according to data from previous ricides, particularly for OP, SP, and amitraz, have been adult immersion tests (AIT) conducted at IPVDF (data not published in the regions in which the cattle tick is found shown). On February 11th, 2011, ticks from this ranch were sampled to establish a laboratory colony. At that time, the As a result, the use of ML, fipronil, frequency of treatment with fluazuron was shorter than fluazuron, and mixtures of SP and OP has become common.
every six weeks, and the frequency of treatments using The first report of OP resistance was registered in Australia mixtures of organophosphate and pyrethroid was less than (and eight years later in Brazil every three weeks. Even with these practices to attempt (Amitraz started to be used in 1975 in control, the majority of cattle was observed to be highly Australia and in 1977 in Brazil, and acaricide resistance infested with all stages of R. microplus. Some bulls had more to this chemical class was reported in Australia by 1981 than 700 adult ticks. Ticks referred to as "Jaguar R" were (Twelve years later, amitraz resistance obtained by the selection of ticks which survived the first was documented in Brazil SPs were AIT bioassays, as described below.
introduced in the early 1980s, and resistance was simul- taneously detected by 1989 in both Australia and in the 2.1.2. São Gabriel (SG) strain state of Rio Grande do Sul (RS), Brazil Ticks were obtained from cattle at FEPAGRO Experimen- MLs became commercially available tal Station of São Gabriel, municipality of São Gabriel, RS, in 1981 and resistance in Brazil was first reported in 2001, Brazil (30◦20 S, 54◦15 W). This experimental station has once again in the state of RS ( a closed herd, i.e. it was maintained without the introduc- Fipronil has been on the market since 1996, and within tion of cattle from other farms for more than 10 years. The a decade, between 2004 and 2006, the first report of in tick population from this herd has a history of resistance vitro resistance was published in RS, Brazil against synthetic pyrethroids, amitraz and macrocyclic lac- One year later, fipronil resistance was confirmed in tones however, it was never a stall test in Uruguay Fluazuron is a exposed to fluazuron, so it was considered a susceptible benzoylphenylurea derivative that regulates tick growth field strain for this study.
by inhibiting chitin incorporation into the tick's cuticle, acts systemically, and has acaricidal properties; this was 2.1.3. Porto Alegre (POA) strain released in the market in 1994, and to date had been These ticks were collected from a ranch along the considered the only AI without any evidence of resistance, border of Brazil and Uruguay in 1992. It has been kept at despite the remarkable increase in its use to control cattle Porto Alegre municipality, RS, Brazil, in the facilities of J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136 Universidade Federal do Rio Grande do Sul (UFRGS). The 0.00195, 0.0039, 0.0078, 0.0156, 0.0312, 0.0625, 0.125, 0.25 POA strain has been widely used as a susceptible reference tick strain since its isolation more than twenty years ago, and has been maintained without exposure to acaricides.
(85 mm × 75 mm – Whatman No. 1, Whatman Inc., All tick strains/colonies were maintained in stall con- Maldstone, England), or six nylon fabrics in the case of ditions at the Isolation Unit of Instituto de Pesquisas amitraz (85 mm × 75 mm – Type 2320, Cerex Advanced Veterinárias Desidério Finamor (IPVDF), Eldorado do Sul, Fabrics, Pensacola, FL), were prepared (three for each RS, Brazil, without exposure to other contaminant tick strain). Filter papers were impregnated with 0.67 ml each populations or acaricides as previously described of the solutions using a micropipette. The material was left to dry for 2 h inside a fume hood to allow trichloroethylene evaporation. After drying, the filter papers were folded 2.2. Larval bioassay in the middle and sealed on the sides with a metal clip to form the packets. Approximately 100 larvae were Determination of the magnitude of resistance to differ- transferred to each packet using a No. 2 paintbrush. The ent classes of acaricides was accomplished using a larval packets were sealed with a third clip and incubated at bioassay with the Jaguar and POA strains. For cypermethrin, 27–28 ◦C and 80–90% relative humidity. The control group chlorpyriphos and fipronil, the larval packet test (LPT) was was exposed to filter paper treated with acaricide-free performed according to procedures established by TCE-OO. After 24 h, larvae mortality was determined by toxicity was evaluated with the larval counting the total dead and alive individuals. Larvae that immersion test (LIT) as described by were paralysed or moving their legs without the capability The modified LPT was conducted according to to walk were considered dead.
amitraz. All of the larval tests were performed simultaneously with the susceptible POA strain.
2.5. Larval immersion test The tests were conducted with technical grade cyper- methrin, chlorpyriphos, ivermectin (Sigma Chemical Co., Initially, a solution of 2% Triton X-100 (Sigma Chemical St. Louis, MO, USA), and fipronil (BASF Chemicals, Paulínia, Co., St. Louis, MO, USA) was prepared in absolute ethanol SP, Brazil). The tests with amitraz were done using a com- (Merck, Darmstadt, Germany) (ETH-TX2%). Technical iver- mercial formulation at 12.5% (Triatox®, MSD Saúde Animal, mectin was diluted to 1% in 10 ml of the ETH-TX2% solution São Paulo, Brazil).
in order to prepare a stock solution. At the time of testing, 100 ␮L of the stock solution was added to 9.9 ml distilled 2.3. Preparation of ticks water to obtain the following final concentrations in the mixture: 0.01% ivermectin, 1% ethanol, and 0.02% Triton Engorged females of the POA and Jaguar strains were X-100. This mixture was serially diluted 10 times at a collected following normal detachment from the host.
30% rate in a diluent composed of 1% ethanol and 0.02% Ticks were processed in the laboratory according to FAO Triton X-100, in order to obtain the final immersion solu- procedures (Briefly, after being washed with tions with the following concentrations (in % of AI): 0.01, water and dried with paper towels, the ticks were incu- 0.007, 0.0049, 0.00343, 0.0024, 0.00168, 0.00117, 0.00082, bated in plastic Petri dishes (90 mm diameter × 22 mm 0.00057, 0.0004 and 0.00028. Diluent without acaricide high) inside an environmental chamber, in the dark at was used as a control. Five hundred microlitres of each temperatures between 27 and 28 ◦C and relative humid- immersion solution was distributed in three 1.5 ml micro- ity between 85 and 90% for two weeks to allow oviposition.
centrifuge tubes (Axygen, Union City, CA, USA). Using a No.
Egg masses were thoroughly mixed and then placed in glass 2 paintbrush, approximately 100 larvae were transferred vials (5 ml) closed with a cotton lid to allow air and humid- to each tube, which was then closed and shaken vigorously ity passage. Eggs in the vials were incubated under the same to ensure sinking of the larvae. After 10 min of immer- conditions as the adult females to allow the emergence of sion, the larvae were removed from the tube with a clean larvae. Larvae used for testing were between 14 and 21 days paintbrush and allowed to dry on a piece of paper towel, before being transferred to a packet of filter paper folded in the middle and closed on the sides with metal clips. After 2.4. Larval packet test adding the larvae, the packet was sealed with a third clip and incubated in an environmental chamber at 27–28 ◦C Stock solutions of cypermethrin, chlorpyriphos, fipronil and 85–90% relative humidity in the dark. After 24 h, larvae and amitraz were prepared in a mixture containing two mortality was determined by counting the total number of parts trichloroethylene (Synth, Diadema, Brazil) and one dead and alive individuals. Larvae that were paralysed or part commercial olive oil (TCE-OO). These stock solutions moving only their legs without the capability to walk were were used to prepare the following impregnation solutions considered dead.
in TCE-OO (in % of AI): (i) Cypermethrin 0.01, 0.015, 0.02, 0.03, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, 2, 3 and 4; (ii) Chorpyriphos 0.003, 0.006, 0.01, 0.02, 0.03, 0.04, 0.06, 0.3, 0.6, 0.8, 1, 2, 3 and 4; (iii) Fipronil 0.00008, 0.00016, A total of 20 heifers (Bos taurus taurus, Angus breed), 0.00032, 0.0006, 0.0012, 0.0025, 0.005 and 0.01; (iv) Ami- aged 18–20 months, were used. Cattle were divided traz 0.000061, 0.000122, 0.000244, 0.000488, 0.000976, into four groups (five animals each) maintained in four J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136 paddocks (≈1 ha each) separated by electric fences at the in demineralised sterile water) to give four additional test IPVDF facilities (30◦0306 S; 51◦1844 W), Eldorado do concentrations (in ppm of fluazuron): 50, 5, 0.5, and 0.05.
Sul, RS, Brazil. Cattle were allowed to graze freely on nat- Thirty engorged females of each strain (POA, Jaguar and ural pasture and had free access to water. The cattle were Jaguar R) were immersed in 30 mL of each solution for handled in accordance with local institutional guidelines 1 min. The treated ticks were then dried with a paper and all procedures were in accordance with international towel and incubated individually in glass tubes (5 mL) at guidelines for animal farm experimentation 27 ± 1 ◦C and 80% relative humidity for six weeks. Follow- At days −21, −14, −7 and −1, all animals were infested ing egg hatching, the proportion of larvae was estimated with approximately 20,000 larvae (14–21 days old) of by visual inspection using a stereomicroscope performed Jaguar (resistant) or SG (susceptible) strains, as described by the same trained operator.
below. Four homogeneous groups were formed by a ran- domised block design (higher to lower tick count) based 2.8. Artificial feeding assay (AFA) on the average number of adult ticks on the left side of each animal counted at days As fluazuron is a systemic acaricide and ticks become −2, −1 and zero. The four experimental groups were: exposed when they feed on blood, experiments of arti- (1) Jaguar-infested heifers (control); (2) Jaguar-infested ficial feeding were conducted as an alternative in vitro heifers treated with fluazuron; (3) SG-infested heifers (con- assay to characterise the toxicity of fluazuron between trol); (4) SG-infested heifers treated with fluazuron.
tick strains. Artificial feeding was performed as described On day 0, animals from groups 2 and 4 were treated by Briefly, partially engorged female with fluazuron (Acatak®, Novartis Saúde Animal, Barueri, ticks (between 30 and 80 mg) from the POA and Jaguar SP, Brazil), according to the manufacturer's instructions, i.e.
strains were manually collected 19 days after larvae were 2.5 mg/kg (5 ml/50 kg). No rain was recorded in the days used to experimentally infest heifers housed in individ- after the treatments.
ual pens. Blood for artificial feeding was obtained from a Adult tick counts (female ticks >4.5 mm) on the left side pool of samples collected with sodium citrate from three of heifers, done according to a previously established pro- heifers maintained without previous acaricide exposure.
tocol were determined on days Blood was placed in a microhaematocrit capillary tube 7, 14, 21 and 28. Adult tick counts are reported as the (without heparin) that was placed at the tick's hypos- mean ± S.E. of five animals.
tome. Treatments included: (i) blood, (ii) blood + fluazuron For analysis of treatment efficacy, the reproductive solution (final concentrations 2.5, 5 and 10 ppm), and (iii) parameters of collected engorged females were assessed blood + vehicle (0.02% Triton X-100 in 1% acetone in dem- according to WAAVP guidelines for evaluation of the effi- ineralised sterile water). Twenty ticks were used for each cacy of acaricides Briefly, at least treatment. Fluazuron solution preparation was performed 10 fully engorged female ticks were collected on days 7, as described above. Ticks were fed for approximately 24 h.
14, 21 and 28 from each heifer for evaluation of egg mass The capillary tubes were replaced with new ones when weight and larvae hatching. Ticks collected from experi- empty (approximately every 2 h). During artificial feeding, mental animals were washed with water, dried in a paper ticks were maintained in incubators at 27 ± 1 ◦C and 80% towel, and placed in Petri dishes inside an environmen- relative humidity. Upon repletion, ticks were weighed and tal chamber at 27 ± 1 ◦C and 80% relative humidity for two held individually in glass tubes at 27 ± 1 ◦C and 80% relative weeks. Thereafter, eggs were removed and transferred to humidity for six weeks. The proportion of larvae emerg- glass tubes sealed with cotton plugs and maintained under ing from hatched eggs was estimated by visual inspection the same conditions described for females until all the using a stereomicroscope performed by the same trained larvae hatched, which occurred approximately 4–6 weeks later. Larvae were enumerated by visual inspection using a stereomicroscope performed by the same trained operator.
2.9. Statistical analysis 2.7. Adult immersion test (AIT) with fluazuron Statistical significance of the treatments in the field trial was analysed by the Student's t test. Statistical analyses for the AIT and AFA were calculated using one-way analysis procedure to evaluate the relative sensitivity of R. microplus of variance (ANOVA) followed by Tukey's multiple com- to fluazuron. The FAO Guidelines for Resistance Manage- parison test. P values < 0.05 were considered statistically ment and Integrated Parasite Control in Ruminants ( different. Student's t test and ANOVA were performed using suggest that this method could be improved and GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA, adapted for testing resistance. Briefly, technical grade flu- azuron (Sigma Chemical Co., St. Louis, MO, USA) was diluted For the larvae tests, a probit analysis was performed on in 2% Triton X-100 in technical grade acetone (Merck, the mortality results using the software Polo-Plus (LeOra Darmstadt, Germany) to produce a 5% fluazuron solution Software, 2003). The following parameters were deter- (50,000 ppm). The 5% fluazuron solution was diluted 1:100 mined for each test: lethal concentrations for 50% (LC50), in demineralised sterile water to give the top dose solu- confidence intervals of 95% (CI 95%), and the slope of the tion for the test: 0.05% (500 ppm) fluazuron in 0.02% Triton regression line. Resistance ratios (RR) and their CI 95% were X-100 and 1% acetone. This top dose solution was seri- generated with the software Polo-Plus using the formula ally diluted in vehicle (0.02% Triton X-100 in 1% acetone described by The significance of J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136 Results of the larval tests with cypermethrin, chlorpyriphos, fipronil, amitraz and ivermectin conducted with the Jaguar (resistant) and Porto Alegre (POA, LC50 (%) (CI 95%) 0.026 (0.021–0.29) 0.798 (0.751–0.848) 31.242 (28.789–33.903) 0.014 (0.009–0.019) 1.458 (1.372–1.546) 0.0008 (0.0006–0.0009) 0.0034 (0.0028–0.0044) 4.441 (3.849–5.123) 0.0015 (0.001–0.0019) 0.0177 (0.0149–0.0205) 11.907 (9.552–14.843) 0.0007 (0.0006–0.0008) 0.0022 (0.002–0.0026) 3.081 (2.852–3.328) n, number of larvae; SE, standard error; 2, Chi-square; df, degrees of freedom; LC50, median lethal concentration in % of active ingredient; CI, confidence interval; RR, resistance ratio.
a Larval packet test.
b Larval immersion test.
each comparison was determined when the calculated con- supplementary table 1). Treatment efficacy against the fidence intervals did not overlap.
Jaguar strain was zero at 14, 21 and 28 days p.t. Additionally, in vitro tests were conducted to charac- 3. Results
terise fluazuron resistance in the Jaguar strain. The adult immersion test (AIT) with five concentrations (0.05, 0.5, The Jaguar strain showed resistance to all the acari- 5, 50 and 500 ppm) of technical grade fluazuron was per- cides evaluated in vitro. Results of larval tests are shown in formed with the POA (susceptible), Jaguar, and Jaguar R Resistance ratios (RR) were: 31.242 for cyperme- strains. Fluazuron treatment of the POA strain resulted in a thrin, 103.926 for chlorpyriphos, 4.441 for fipronil, 11.907 reduction in larval hatch, ranging from 86% at 0.05 ppm to for amitraz and 3.081 for ivermectin.
99% at 50 and 500 ppm However, fluazuron treat- Results from the comparison of efficacy under field ment of the Jaguar strain reduced larval hatch by only 21% conditions between the susceptible SG strain and the at 0.05 ppm to 52% at 500 ppm (Fluazuron efficacy Jaguar strain confirmed suspicions by local produc- was further reduced after selection (using ticks obtained ers of fluazuron treatment failure. Treatment of cattle by the selection of ticks from Jaguar strain which survived experimentally infested with the SG strain (susceptible) first AIT tests), with larval hatch reduced by approximately resulted in a significant reduction in engorged female tick 9% at 0.05 ppm and 20% at 500 ppm count 14 and 21 days post-treatment (p.t.) respectively The pattern of fluazuron efficacy upon tick strains using supplementary table 1). Treatment efficacy in the artificial feeding assay (AFA) was similar to the AIT. Off- cattle experimentally infested with SG strain was 94.09, spring of the POA strain (susceptible) ticks did not hatch 98.4 and 100% at 14, 21 and 28 days p.t., respectively when ticks were fed 5 and 10 ppm of fluazuron; only 3% hatch was observed at 2.5 ppm However, the lar- Steers in the treated and control groups infested val hatch of eggs collected from treated Jaguar engorged with the ticks from the Jaguar strain had equivalent female ticks was 67% at 2.5 ppm, 43% at 5 ppm, and 8% at engorged female tick counts through day 28 p.t. ( Fig. 1. Engorged female ticks count from heifers treated with fluazuron under field conditions. Panel A, São Gabriel (SG) (susceptible) tick strain infested
heifers. Panel B, Jaguar (resistant) tick strain infested heifers. For both panels, open symbols indicate non-treated heifers and black symbols represent fluazuron-treated heifers. Results shown are the mean ± S.E.M. of five animals. Statistical significance was analysed by the Student's t test (*P < 0.05, **P < 0.01).
J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136 Fig. 2. Adult immersion test (AIT) with technical grade fluazuron with the
Porto Alegre (POA) (susceptible, black triangles), Jaguar (resistant, open squares) and Jaguar R (resistant, open diamonds) strains. Statistical signif- icance was analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (***P < 0.001 comparing POA with both Jaguar and Jaguar R strains; #P < 0.05, ##P < 0.01, ###P < 0.001 comparing Jaguar and Jaguar R strains).
As a result of the indiscriminate use of acaricides to con- trol ticks infesting livestock, tick resistance to acaricides is one of the biggest challenges and a cause of concern for cattle production wherever R. microplus is endemic or in areas prone to invasion by this injurious ectoparasite species. Recently, R. microplus populations with multiple resistances to acaricides have been identified in several ranches, particularly in Brazil and Mexico Currently, there are six classes of acaricides available on the Brazilian market to control cattle ticks with fluazuron being the only AI with no previous reports of resistance. Here, we report for the first time an R. microplus tick strain that is resistant to fluazuron.
This strain is also the first with documented resistance to Fig. 3. Artificial feeding assay (AFA) with technical grade fluazuron with
Porto Alegre (POA) (susceptible, open bars) and Jaguar (resistant, black bars) strains. Statistical significance was analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Differ- ent letters above the bars indicate a statistical difference among analysed groups; P < 0.05.
J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136 the six classes of acaricides used to control cattle tick in past field exposure of tick populations to fluazuron and, whenever possible, include a comparison between in vitro In 2010, ticks were collected for the same population and in vivo results.
from which the Jaguar strain was sourced for the first time, Although the AIT has been questioned as a precise tool as part of the survey system for cattle tick acaricide resis- for resistance diagnosis in ticks, particularly for systemic tance in Rio Grande do Sul state, Brazil, performed by IPVDF.
acaricides it is important to note At that time, no in vitro test for fluazuron resistance was that in contrast to ML, a larval test cannot be employed available, as there was no suspicion of fluazuron resistance.
to investigate fluazuron resistance. This situation is a con- Subsequently, an investigation concerning fluazuron resis- sequence of the mechanism of action of fluazuron, which tance began after ranch employees reported that they could inhibits chitin synthase activity and subsequently pre- no longer control ticks infesting cattle even using fluazuron.
vents larval ecdysis As a consequence, To investigate whether the observation in the field was due fluazuron is a developmental inhibitor and does not kill to resistance, ticks were sampled and the Jaguar colony was adult engorged females or larvae as other acaricides do.
established at IPVDF.
Since fluazuron is a systemic acaricide, the artificial feed- Results from the field trial presented here clearly ing technique provided a more similar condition to which demonstrated that fluazuron treatment was unable to the parasitic life stages are exposed to fluazuron than the control ticks of the Jaguar strain, neither by reducing AIT. Pharmacokinetic studies conducted with fluazuron tick count or larvae hatching. This lack of efficacy was showed that after the administration of 1.5 mg/kg of live more noticeable when comparing fluazuron performance weight, the serum concentration increased to more than against the SG strain. Field trial studies conducted in South- 1.3 ppm in cattle As the fluazuron label rate ern Brazil when fluazuron was released on the market is 2.5 mg/kg of live weight, the concentration of AI added achieved more than 99% efficacy at 21 days after treat- to blood fed to ticks in the AFA here ranged from 2.5 to ment, when using 2 mg/kg of this AI ( 10 ppm. Additionally, the marked differences in mortality In another field trial, performed in Australia, the adult tick after exposure to fluazuron between resistant and suscep- reduction was 100 and 98% at three and six weeks after tible tick strains used in this study lend support to the treatment, respectively Recently, some resistance status of the Jaguar strain. In this sense, the authors have shown that fluazuron still gives significant AFA is a technique that is well suited for the characteri- results under field conditions in Brazil. sation of resistance, especially for systemic drugs with the in a study using artificial and natural understanding that, in some cases, biotransformation of the tick infestations that fluazuron (2.5 mg/kg) efficacy ranged parent drug may be required to produce the pharmaco- from 96% to 100% between 14 and 49 days after treatment.
logically active agent that fluazuron (2.5 mg/kg) treat- ment reduced more than 99% of natural tick infestation Investigation of tick resistance and constant survey of between 14 and 28 days after treatment.
population susceptibility status are essential to (i) recog- Scientific literature concerning the validation and use nise if acaricide resistance is the cause of tick control of in vitro tests to evaluate fluazuron susceptibility is lack- failure; (ii) determine the most efficient acaricide for ing. This can be attributed to several factors, such as the each location/ranch; (iii) understand the epidemiology and absence of a resistant reference strain, and the apparent spread of resistance; and (iv) develop control strategies lack of need, since it was assumed that there were no that minimise the selection of resistant genotypes cases of field resistance to fluazuron. Results from the AIT RS state, located in Southern Brazil, has a demonstrated a difference in susceptibility to fluazuron remarkable history of tick resistance to acaricides as it was between the POA and Jaguar strains. Moreover, the abil- the first region in Brazil to report resistance to OP and SP, ity of fluazuron to decrease larval hatch was even lower and the first location in the world to report resistance to in the Jaguar R selected strain, which indicates there is ML, mixtures of OP and SP, and in vitro resistance to fipronil a genetic basis for the trait that could be increased and selected for in the resistant tick population. Together with The breeds of cattle, which are the in vivo results, the AIT has proven to be a useful tool predominantly Bos taurus taurus, and high stocking rates to detect resistance to fluazuron in tick populations. Previ- are two factors that contribute to the problem of acaricide ously published data showed that a 200 ppm concentration resistance among populations of R. microplus in RS. These of fluazuron could be used as a discriminating dose (DD) to factors lead to heavy tick loads, which, in turn, lead to more induce total, or near complete inhibition of larvae hatching treatments per year, and consequently more resistance in the Parkhurst, Yeerongpilly, and Ulam reference strains selection. There may be other factors contributing to the from Australia In our tests, 50 ppm induced resistance problem in RS requiring further epidemiologi- more than 99% larvae hatching inhibition in the POA strain, cal studies for its full identification and characterisation.
and less than 50% in the Jaguar strain. Larval hatch in the In this sense, information regarding resistance in RS could AIT was close to 50% with the Jaguar strain and >80% with facilitate the understanding of resistance in other regions of the Jaguar R strain using fluazuron at 500 ppm. The results the world where R. microplus impacts livestock production presented here highlight the need for comprehensive stud- ies with the AIT and fluazuron, particularly concerning test The resistance mechanism for fluazuron in the Jaguar reproducibility and the establishment of a discriminating strain remains to be fully described. A similar situa- dose to diagnose resistance. Such studies must consider tion exists with amitraz, ML, and fipronil. Tick resistance J. Reck et al. / Veterinary Parasitology 201 (2014) 128–136 against drugs can be due to target site insensitivity and/or metabolic resistance, including molecular efflux pumps.
Studies reported the key role of detoxification mecha- Alonso-Díaz, M.A., Rodríguez-Vivas, R.I., Fragoso-Sánchez, H., Rosario- nisms in resistance to several drugs ( It is possible that the inten- Alves-Branco, F.P.J., Sapper, M.F.M., Alves-Branco, L.R.F., Henrique, C.H., sive use of different acaricides against the Jaguar strain Sandoval, G.A.F., Cassol, D.M.S., Mello, I.A.S., Silva, L.M., Toma, S.B., Rizzi, V.G., Carneiro, R., 2010. in the field induced several non-specific pathways which may contribute to resistance to fluazuron. In fact, syner- gist bioassays and enzyme activity quantification studies with Musca domestica ( Angus, B.M., 1996. Lucilia cuprina and Cydia pomonella (revealed, at least in part, the role of mixed function oxidase enzymes as a mechanism of Arteche, C.C.P., 1972. resistance to diflubenzuron, a benzoylphenyl urea com- pound. The occurrence of chitin synthase alteration which Bloomquist, J.R., 2013. Insecticides: chemistries and characteristics.
could promote insensitivity to fluazuron also remains In: Radcliffe, E.B., Hutchison, W.D., Cancelado, R.E. (Eds.), Rad- cliffe's IPM World Textbook. University of Minnesota, St. Paul, MN as a possible resistance mechanism. These mechanisms need to be investigated further. Biological phenomena Bull, M.S., Swindale, S., Overend, D., Hess, E.A., 1996. underlying tick resistance against fluazuron remain to be Corrier, D.E., Vizcaino, O., Terry, M., Betancourt, A., Kuttler, K.L., Carson, Here, we documented the first case of fluazuron resis- C.A., Trevino, G., Ristic, M., 1979. tance in R. microplus and the first tick population resistant to six classes of acaricide. Assessing the efficacy of flu- Cuore, U., Trelles, A., Sanchis, J., Gayo, V., Solari, M.A., 2007. azuron against other populations of R. microplus subjected to high selection pressure with acaricides and the char- acterisation of resistance mechanisms in the Jaguar strain FAO (Food and Agriculture Organization), 1998. Residues of Some Veterinary Drugs in Animal and Foods, FAO Food and Nutrition require investigation. This study highlights the spreading Papers. Food and Agriculture Organization, Agriculture and Consumer and emerging issue of multiple resistance to acaricides in Protection Department, Available on-line at: R. microplus, which is a cause of concern worldwide. The FAO (Food and Agriculture Organization), 2004. inability to control cattle ticks by using only acaricides cur- rently available in an indiscriminate manner demonstrates the need for more research to establish an integrated strat- egy that includes chemical control, and the development FASS (Federation of Animal Science Societies), 1999. of alternative control tools.
Fernández-Salas, A., Rodríguez-Vivas, R.I., Alonso-Díaz, M.A., 2012. Conflict of interest statement
Authors declare no conflict of interest.
Pimpripkar, G.D., Georghiou, G.P., 1979. George, J.E., 2000. George, J.E., Pound, J.M., Davey, R.B., 2008. Authors would like to thank to Felipe Zeni, Marjana Traesel and Juliane Soares (IPVDF) for support in animal handling and AFA, and to Dr. Robert John Miller (ARS, USDA, Graf, J.F., Schmid, H.R., Hess, E.A., Friedel, T., 1994. Edinburg, TX, USA) for critical review of this manuscript.
This work was financially supported by Fundac¸ão Estad- Gloria, M.A., Flausino, J.R.N., Grisi, L., 1993. ual de Pesquisa Agropecuária (FEPAGRO), Edital 064/2008 (Conselho Nacional de Desenvolvimento Científico e Tec- nológico – CNPq and Ministério da Agricultura, Pecuária e Abastecimento – MAPA), Projeto SANIMARS (Finan- Guerrero, F.D., Bendele, K.G., Chen, A.C., Li, A.Y., Miller, R.J., Pleasance, E., ciadora de Estudos e Projetos – FINEP), and Fundac¸ão Varhol, R., Rousseau, M.E., Nene, V.M., 2007. de Amparo à Pesquisa do Estado do Rio Grande do Sul Guerrero, F.D., Lovis, L., Martins, J.R., 2012. Appendix A. Supplementary data
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