S ( ( W B F A A a C b c d e a A R R A K B C M R T D h 0 Preventive Veterinary Medicine 135 (2016) 74–86 Contents lists available at ScienceDirect Preventive Veterinary Medicine jo ur nal ho me pag e: www.elsev ier .com/ locate /prevetmed usceptibility of Rhipicephalus (Boophilus) microplus to fluazuron 2.5 mg/kg) and a combination of novaluron 2.0 mg/kg) + eprinomectin (0.36 mg/kg) in field studies in Brazil illian Giquelin Maciela, Welber Daniel Zanetti Lopesa,b,∗, Lucas Vinicius Costa Gomesa, reno Cayeiro Cruza, Gustavo Felippelli a, Isabella Barbosa Dos Santosa, ernando de Almeida Borgesc, Walter Antonio Gonç alves Juniord, lexandre Braga Scarpae, João Eduardo Nicarettab, Thiago Souza Azeredo Bastosb, lvimar José da Costaa CPPAR − Animal Health Research Center, Faculdade de Ciências Agrárias e Veterinárias, UNESP, Jaboticabal Campus, Access Route Prof. Paulo Donato astellane, 14884-900, Jaboticabal, São Paulo, Brazil Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás (IPTSP/EVZ/UFG), 74605-050, Goiânia, Goiás, Brazil Universidade Federal do Mato Grosso do Sul (UFMS), Campo Grande, Mato Grosso do Sul, Brazil Universidade Estadual de Maringá, Umuarama Regional Campus, Paraná, Brazil Universidade Federal de Goiás, Regional de Jataí, Brazil r t i c l e i n f o rticle history: eceived 5 June 2016 eceived in revised form 23 October 2016 ccepted 25 October 2016 eywords: enzoylphenyl urea attle acrocyclic lactone esistance icks a b s t r a c t The present study aimed to determine the susceptibility of 32 R. (B.) microplus populations from South- east, Midwest and South regions of Brazil, to fluazuron (2.5 mg/kg), administered topically (pour-on). Additionally, five populations (Southeast and Midwest regions) of the southern cattle tick were eval- uated using in vivo field studies, regarding their susceptibility to a new combination of novaluron (2.0 mg/kg) + eprinomectin (0.36 mg/kg), administered subcutaneously, compared with two positive con- trols (fluazuron 2.5 mg/kg and eprinomectin 0.5 mg/kg), both administered topically (pour-on). Selected bovines were allocated to treatment groups on day 0, and block formation was based on arithmetic means of female ticks (4.5–8.0 mm long) counted on three consecutive days (−3, −2 and −1). To evaluate ther- apeutic and residual efficacies of these formulations, tick counts (females ranging from 4.5 to 8.0 mm long) were performed on days 3, 7 and 14 post-treatment, continuing on a weekly basis until the end of each experiment. Results obtained throughout this study, utilizing field efficacy trials, allowed us to conclude that four R. (B.) microplus populations (including two in the Southeast and two in the Midwest regions) could be diagnosed as resistant, or with low susceptibility, to fluazuron (2.5 mg/kg). Such fact was detected in farms where owners applied products containing this active component on cattle for at least five years, with treatment intervals of 30–55 days during the rainy season. Nonetheless, in vitro studies should be performed in order to reinforce in vivo results obtained on the present study. Regarding efficacy indexes obtained by the association of eprinomectin and the novel molecule novaluron against R. (B.) microplus, none of the trials managed to obtain efficacies superior to 48%. Such results, allied to data obtained by different researchers and previously published in literature, reinforce the perception that maybe these formulations containing novaluron, in the administered dosages and treatment routes, may not be effective tools for controlling R. (B.) microplus. However, future studies must be conducted in order to support such hypothesis. Additionally, all five R. (B.) microplus populations were diagnosed as resistant, or with low susceptibility, to eprinomectin (0.5 mg/kg) as well. Even though fluazuron, adminis- tered topically (pour on), is still an excellent active principle to be used against R. (B.) microplus, resistance management strategies should minimum level for this compo ∗ Corresponding author at: CPPAR − Animal Health Research Center, Faculdade de Ciê onato Castellane, 14884-900, Jaboticabal, São Paulo, Brazil. E-mail address: wdzlopes@hotmail.com (W.D.Z. Lopes). ttp://dx.doi.org/10.1016/j.prevetmed.2016.10.019 167-5877/© 2016 Elsevier B.V. All rights reserved. be quickly implemented in order to keep selection pressure in Brazil at a und. © 2016 Elsevier B.V. All rights reserved. ncias Agrárias e Veterinárias, UNESP, Jaboticabal Campus, Access Route Prof. Paulo dx.doi.org/10.1016/j.prevetmed.2016.10.019 http://www.sciencedirect.com/science/journal/01675877 http://www.elsevier.com/locate/prevetmed http://crossmark.crossref.org/dialog/?doi=10.1016/j.prevetmed.2016.10.019&domain=pdf mailto:wdzlopes@hotmail.com dx.doi.org/10.1016/j.prevetmed.2016.10.019 eterina 1 i h s a a c e l w a p t o i C p r ( a n 2 d r i C d a a G a i ( g m a w H a m a s 2 a n i a t o a i a t a r ( s W.G. Maciel et al. / Preventive V . Introduction Rhipicephalus (Boophilus) microplus is considered one of the most mportant ectoparasites in cattle industry (Cruz et al., 2014). It as a wide geographical distribution, comprehending tropical and ubtropical regions situated between latitude parallels 32◦ north nd 35◦ south, comprising countries of Latin America, Africa, Asia nd Oceania (Wharton, 1974). It is estimated that, in Brazil, losses aused by this tick are close to 3.24 billion dollars per year (Grisi t al., 2014). Amongst the main tools used for controlling such ectoparasite ately, synthetic chemical compounds stand out, though it is note- orthy that control of R. (B.) microplus based on the use of chemical caricides is mostly done in an indiscriminate manner, without revious knowledge of biological and ecological aspects related o life cycles of ticks (Cruz et al., 2015a). Because of this, reports f tick resistance against several active components are increas- ng (Castro-Janer et al., 2011; Lopes et al., 2014; Reck et al., 2014; orrea et al., 2015). For several years, R. (B.) microplus control using chemical com- ounds was done mainly with macrocyclic lactones. However, the ising number of tick populations resistant to this acaricide family Cruz et al., 2015a) caused the use of such compounds to diminish nd, consequently, treatments of animals with different compo- ents, such as the benzoylphenyl ureas, to increase (Cruz et al., 015b; Gomes et al., 2015), which motivated even further the con- uction of the present study. Benzoylphenyl ureas, selective acaricides called insect growth egulators (IGR), act by inhibiting chitin synthesis, making it mpossible for larvae and nymphs of ticks to promote ecdysis. onsequently, parasites lose hemolymph and death occurs by ehydration. They show high specificity, low toxicity for mammals nd efficacy in low concentrations with a long period of action gainst adult R. (B.) microplus (Retnakaram and Whight, 1987; raf, 1993; Bull et al., 1996). Within that group stands out flu- zuron, first commercialized in Brazil in 1994, which assumes an mportant role for the control of the aforementioned ectoparasite Santos et al., 2010). Another IGR belonging the benzoylphenyl urea roup is novaluron (1-[3-chloro-4-(1, 1,2-trifuloro-2-trifluoro- ethoxy-ethoxy) phenyl]-3-(2, 6-difluorobenzoyl) urea). This ctive principle has very low toxicity to mammals, birds and earth- orms (lshaaya and Horowitz, 1998; Barazani, 2001; Ishaaya and orowitz, 2002), and is widely used in agriculture (Kostyukovsky nd Trostanetsky, 2006; Beuzelin et al., 2010). In 2015, a new com- ercial formulation containing novaluron + eprinomectin became vailable in Brazilian market, being indicated for treatments against outhern cattle ticks, Rhipicephalus (Boophilus) microplus (Sindan, 015). Considering that fluazuron is the most used active principle gainst R. (B.) microplus in some regions of the world, and there are ew active components belong to the same chemical group becom- ng available commercially to control the southern cattle tick, such s novaluron, information about R. (B.) microplus susceptibility to hese compounds is essential in order to create strategies for devel- ping a progressive and more rational chemical control strategy gainst this ectoparasite in cattle (Lopes et al., 2014). Therefore, the present study aimed to determine the susceptibil- ty of 32 R. (B.) microplus populations, from the Southeast, Midwest nd Southern regions of Brazil, to fluazuron (2.5 mg/kg), adminis- ered topically (pour on). Additionally, five populations (Southeast nd Midwest regions) of the southern cattle tick were evaluated egarding their susceptibility to the new molecule of novaluron 2.0 mg/kg) associated to eprinomectin (0.36 mg/kg), administered ubcutaneously, compared with two positive controls (fluazuron ry Medicine 135 (2016) 74–86 75 2.5 mg/kg and eprinomectin 0.5 mg/kg), both administered topi- cally (pour-on), by means of in vivo field studies. 2. Materials and methods 2.1. Study locations, division of groups, dose determination procedures and tick counts Between February 2012 and March 2016, experiments were conducted in different rural properties of Brazil. All studies were performed between the months of October and May, in order to ensure that the challenge of animals with R. (B.) microplus would be in accordance with population dynamic data of this ectopara- site in the regions where all 32 studies were conducted. Such data, obtained by several different authors, demonstrate that this tick species present an average of thre to four tick generations dur- ing this period of the year, considered to be three rainy season in Brazil (Kasai et al., 2000; Martins et al., 2002; Pereira et al., 2008; Gomes et al., 2016). A total of 32 R. (B.) microplus popula- tions were evaluated regarding their susceptibility to fluazuron, administered topically (pour on) at a dose of 2.5 mg/kg. Five pop- ulations were tested against a novel combination of novaluron (2.0 mg/kg) + eprinomectin (0.36 mg/kg), as well as a formulation of eprinomectin (0.50 mg/kg), administered by subcutaneous and pour on routes, respectively. For fluazuron, thirteen tick popula- tions were selected on the state of São Paulo and ten came from the state of Minas Gerais, both located on the Southeast region of Brazil. Three tick strains originated at the state of Mato Grosso do Sul and four from Goiás, states from the Midwest region of Brazil. The final two tick populations were from the state of Paraná, South region of Brazil. Regarding novaluron + eprinomectin and eprinomectin, three tick populations from the state of São Paulo and two from Goiás were evaluated regarding the efficacy of these formulations. The chemical compounds used at each property, as well as the his- tory of fluazuron usage on each herd since the beginning of its administration are described in Table 1. For experiments 1–27, 20 mixed breed bovines (Bos taurus x Bos indicus) naturally infested by R. (B.) microplus, with ages ranging from 8 to 18 months, were utilized. In each of these experiments, animals were distributed into two groups of ten animals each. Treatment 01 received saline solution, while treatment 02 received fluazuron topically (pour on) at a dosage of 2.5 mg/kg. In exper- iments 28–32, 40 mixed breed animals (Bos taurus x Bos indicus) naturally infested by R. (B.) microplus, with ages ranging from 8 to 18 months, were selected. In this case, bovines were divided into four groups of ten animals each. Treatment 01 received saline solution; treatment 02 received 2.0 mg/kg novaluron + 0.36 mg/kg eprinomectin subcutaneously; animals belonging to treatment 03 were treated with 2.5 mg/kg fluazuron topically (pour on); and treatment 04 received 0.5 mg/kg of eprinomectin topically (pour on). Due to the fact that novaluron is a novel active component, recently marketed in Brazil against southern cattle ticks, fluazuron (2.5 mg/kg) and eprinomectin (0.5 mg/kg) were used as reference products. Counting all studies, the total number of experimental animals was 740. For each of the trials, the same category of ani- mals was selected, so that body weight of animals did not differ statistically between treated and control groups. Only clinically healthy bovines with a good nutritional condition were selected for all experiments. Cattle chosen for all studies had not been med- icated for a period of at least 90 days before the beginning of each experiment. All animals were maintained on a grazing regimen, following the one already in use on each rural property. Bovines were allocated to experimental groups on day 0, being randomly designated to treatments according to a masked com- plete block design. Block formation was based on arithmetic means 76 W.G. Maciel et al. / Preventive Veterinary Medicine 135 (2016) 74–86 Table 1 Usage history of fluazruon in the properties where the studies were performed. Study Region of Brazil Farm city − State Month and year of begining the study Product1 used Route How long uses the product? (years) Frequence of use of fluazuron 1 Southeast Pimenta − MG January 2013 fluazuron pour-on two at least two times per year 2 Southeast Campo Florido − MG Febuary, 2012 fluazuron pour-on three at least two times per year 3 Southeast Passos − MG November of 2014 fluazuron pour-on two at least two times per year 4 Southeast Poç os de Caldas − MG January 2015 fluazuron pour-on three the owner could not say 5 Southeast Formiga − MG October 2015 fluazuron pour-on eigth at least two times per year 6 Southeast São Sebastião do Paraíso − MG Febuary, 2012 fluazuron pour-on four at least tree times per year 7 Southeast Arcos − MG December 2014 fluazuron pour-on five each 40–45 days of interval 8 Southeast Divinópolis- MG January 2016 fluazuron pour-on four at least three times per year 9 Southeast Pains- MG Febuary, 2014 fluazuron pour-on five at least three times per year 10 Southeast Caldas − MG March 2013 fluazuron pour-on five at least four times per year 11 Southeast Franca − SP November 2014 fluazuron pour-on never used not applied 12 Southeast Casa Branca − SP Febuary, 2013 fluazuron pour-on one two times of use 13 Southeast Aguas da Prata − SP December 2013 fluazuron pour-on one one time 14 Southeast Itirapuã − SP March 2014 fluazuron pour-on two three to six times since the begin 15 Southeast Gastão Vidgal − SP January 2013 fluazuron pour-on six each 30–40 days of interval 16 Southeast São João da Boa Vista − SP December 2015 fluazuron pour-on seven three to six times since the begin 17 Southeast São José do Rio Pardo − SP January 2015 fluazuron pour-on five at least three times per year 18 Southeast Santo Antônio de Posse- SP Febuary, 2014 fluazuron pour-on five at least five times per year 19 Southeast Vargem Grande do Sul- SP March 2013 fluazuron pour-on two three to six times since the begin 20 Southeast São Sebastião da Grama- SP October, 204 fluazuron pour-on five the owner could not say 21 Midwest Bandeirantes − MS December 2012 fluazuron pour-on more than five at least four times per year 22 Midwest Terenos − MS December 2012 fluazuron pour-on more than five at least four times per year 23 Midwest Jaguari − MS Febuary, 2014 fluazuron pour-on more than five at least four times per year 24 Midwest Caiapônia − GO March 2015 fluazuron pour-on three the owner could not say 25 Midwest Jataí − GO January 2015 fluazuron pour-on never used not applied 26 South Icaraíma − PR January 2015 fluazuron pour-on four the owner could not say 27 South Umuarama − PR December 2014 fluazuron pour-on three at least four times per year 28 Southeast Espirito Santo do Pinhal − SP November of 2015 fluazuron, eprinimectin and noval- uron + eprinomectin subcutaneous five three to eight times since the begin 29 Southeast São João da Boa Vista − SP November of 2015 fluazuron, eprinimectin and noval- uron + eprinomectin subcutaneous five at least four times per year 30 Southeast Tambaú − SP January 2016 fluazuron, eprinimectin and noval- uron + eprinomectin subcutaneous three the owner could not say 31 Midwest Jataí − GO Febuary, 2016 fluazuron, eprinimectin and noval- uron + eprinomectin subcutaneous three months two times of use 32 Midwest Acreúna − GO March 2016 fluazuron, eprinimectin and noval- n + ep subcutaneous four at least four times per year 2 o c a i d uro Commercial formulation purchased in the local market. f female ticks (measuring 4.5 to long 8.0 mm) counted on three onsecutive days (−3, −2 and −1), as recommended by Wharton nd Utech (1970). In each experiment, animals were distributed nto ten blocks containing two or four bovines, and these were ran- omly placed in one of the treatment groups inside each block. The rinomectin number of bovines per group used in all experiments (10 animals per group) was determined in accordance with recommendations described by the Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento − MAPA), Ordinance number 48 (Brazil, 1997). eterina a v i 2 c 0 p e t r t i a s l t e a t p r t f o c r t ( d m m T r s u c f t f i t e w g a s c w t d f w e t t t p e M s W.G. Maciel et al. / Preventive V Acaricide compounds used for treatment of experimental nimals were commercial products available in the Brazilian eterinary market that contained 2.5 mg/kg fluazuron, admin- stered at pour-on route (Acatak® − Novartis Animal Health); .0 mg/kg novaluron + 0.36 mg/kg eprinomectin, administered sub- utaneously (Novatak Gold® − Clarion Animal Health); and .5 mg/kg eprinomectin (Eprinex® − Merial Animal Health). All roducts were stored and used according to label specifications. In ach experiment, cattle were individually weighed one day prior o treatment (day −1) in order to allow calculations of the cor- ect dosage. It is important to reinforce that scales were previously ested and verified for accuracy. Each treatment was performed ndividually in each animal, and the administration of products was lways performed by a veterinarian. To ensure dosing techniques were conducted using similar tandards, volumes administered in each experiment were calcu- ated using the measured individual weight of each animal before reatment and, if necessary, were rounded down in 0.1 mL. For xample: an animal of 176 kg, which would receive 17.6 mL using pour-on product, therefore received 17.5 mL, and an animal of he same weight, which would receive 3.52 mL using an injectable roduct, therefore received 3.5 mL. All bovines were appropriately estrained in a chute during treatment. It is important to report hat cattle treated with pour-on formulations did not suffer inter- erence of rain in the first 72 h post-treatment, with the exception f studies 1 (Pimenta city), 2 (Campo Florido city), 8 (Divinópolis ity), 13 (Águas da Prata city) and 25 (Jataí city), where presence of ain (approximately 15 mm3) was registered in the first 24 h after reatment of animals. Additional effects of fluazuron (2.5 mg/kg) and the novaluron 2.0 mg/kg) + eprinomectin (0.36 mg/kg) association on the repro- uctive parameters of field populations of Rhipicephalus (Boophilus) icroplus were not evaluated on the present study, since the ethodology recommended for such analysis is adapted for Stall est studies, using experimentally infested bovines. According to available works in literature, there is only one eport of R. (B.) microplus resistant to fluazuron (Reck et al., 2014) o far. In Brazil, the number of products containing benzoylphenyl reas, such as fluazuron and novaluron, the last molecule to become ommercially available in the country, are widely increasing. Such actor further motivated the conduction of all 32 experiments using hese actives compounds. During all experimental periods of each trial, in all selected arms, untreated control groups and groups treated with the njectable formulation (novaluron + eprinomectin) were main- ained in the same paddock, formed from natural pastures of ach location. In specific instances when pour-on formulations ere applied (fluazuron and eprinomectin), animals from different roups were kept in separate paddocks during the first seven days fter treatment, allowing the products to be totally absorbed, con- equently preventing any possibility of contaminating untreated ontrol groups. Drinkable water and mineral supplementation ere provided for all experimental cattle ad libitum during all of he experiments, following previously established handling proce- ures at each property. To evaluate therapeutic and residual efficacy indexes of each ormulation, tick counts (females between 4.5 and 8.0 mm long) ere performed on days 3, 7 and 14 post-treatment, continuing very 7 days thereafter until the end of each trial, according to the echnique described by Wharton and Utech (1970). To ensure that ick counting procedures were conducted consistently between ime points and study locations, counts in each experiment were erformed at the same time of day and by the same person (a vet- rinarian), on all experimental dates before and after treatment. oreover, each person carried a metal card containing holes mea- uring 4.5 and 8.0 mm long (scale tested), to ensure that only ticks ry Medicine 135 (2016) 74–86 77 from the same size range were counted. Animals treated with injectable solutions and untreated controls were counted first, fol- lowed by groups which received pour-on topical treatments, in order to prevent any possible contamination. In all studies, dispos- able gloves were used and changed between tick counts on each animal. All experiments were blinded; thus, completely reliable data were obtained. Before the implementation of the present study, all proce- dures using animals were approved by the Ethical Committee for Animal Welfare of IPESA, under protocol number 121/12, being, therefore, considered in compliance with the Ethical Principles in Animal Research, adopted by the College of Animal Experimenta- tion (COBEA). 2.2. Data analysis Raw counts of partially engorged female ticks on experimen- tal cattle were log transformed, using the equation ln (x + 1), prior to statistical analysis, and underwent the UNIVARIATE procedure. Subsequently, all data were analyzed by the GLM procedure using the REPEATED command, to test sphericity and orthogonality of data, which determined that the sphericity condition of the matrix should not be rejected (SAS, 1996). Differences between treatments were evaluated at a significance level of 5% (P ≤ 0.05). Efficacy percentages obtained against R. (B.) microplus were cal- culated using arithmetic means, according to a formula proposed by Roulston et al. (1968) and adopted by the Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento − MAPA), Ordinance number 48 (Brazil, 1997), as described below: Efficacy percentage= [ 1 − TaxCb TbxCa ] x100 In this equation, Ta represents the mean number of partially engorged female ticks counted on treated animals after medica- tion; Tb is the mean number of partially engorged female ticks counted on treated animals during the three days that preceded treatment; Ca is the mean number of partially engorged female ticks counted on the control group after the experiment was ini- tiated; and Cb is the mean number of partially engorged females counted on untreated animals (control) during the three days that preceded treatment. 2.3. Criteria for diagnosis of susceptibility or resistant/low susceptibility of a tick population to fluazuron, novaluron and eprinomectin According to EMEA (2004) and Holdsworth et al. (2006), with some exceptions, the control of a tick population by a specific for- mulation is considered unsatisfactory when efficacy values of such compound are inferior to 90%. As described by Holdsworth et al. (2006), for testing products with a slower onset of action, such as benzoylphenyl ureas, selec- tive acaricides (IGR), draft product labels must indicate the period of time required to kill a designated percentage of ticks on treated ruminants, based on valid scientific data. Under such criteria, and considering previous studies which demonstrated that 2.5 mg/kg fluazuron takes about 14 days to present its satisfactory effects against this cattle tick (Cruz et al., 2014; Gomes et al., 2015), R. (B.) microplus populations subjected to fluazuron and novaluron were considered susceptible when these compounds showed an efficacy greater than 90% (EMEA, 2004; Holdsworth et al., 2006) after the 14th day post treatment. For eprinomectin, the Brazilian Ministry of Agriculture, Live- stock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento − MAPA), Ordinance number 48 (Brazil, 1997), 7 eterin s l s d w l 4 i d t l ( 3 o o 3 t i a t b 2 m o m t d r t 4 3 s t u f V I b m f F c b o 3 r e t d b u ( 8 W.G. Maciel et al. / Preventive V tates the importance of considering mean efficacies of a formu- ation between days 7 and 14 post-treatment. A mean efficacy uperior to 90% between these dates was the criteria adopted to esignate R. (B.) microplus as susceptible to eprinomectin, coupled ith the fact that peak plasma levels of this same active formu- ation (1.0 and 1.5 mg/kg) in treated cattle occurs between 2 and days after administration (Lifschitz et al., 2016). Therefore, populations of R. (B.) microplus where the efficacy ndexes of different compounds were inferior 90%, on experimental ates cited above, were classified as resistant, or with low suscep- ibility, to such compounds. Studies indicate that arithmetic means should be used to calcu- ate the efficacy indexes of a formulation against target parasites Dobson et al., 2009; Vercruysse et al., 2011; Cruz et al., 2015b). . Results No signs of abnormalities or systemic intoxication were bserved in experimental animals before or after administration f tested formulations in all 32 studies. .1. Fluazuron 2.5 mg/kg (Minas gerais state) Efficacy values obtained by fluazuron on studies conducted at he state of Minas Gerais (Southeast region of Brazil) are described n Table 2. Based on such results, it is possible to verify that, amongst ll ten evaluated R. (B.) microplus populations, only one, located on he city of Arcos, was diagnosed as resistant, or with low suscepti- ility, to fluazuron, administered topically (pour-on) at a dosage of .5 mg/kg. At this trial, the aforementioned formulation reached a aximum efficacy index of 87.1% on the 21st DPT (Table 2). In three ther experiments conducted on the same state, fluazuron reached aximum efficacy values (100%) against R. (B.) microplus between he 14th and 21st DPT. When analyzing the residual period, in days, uring which this formulation maintained efficacy levels supe- ior or equal to 90% after the 14th DPT, it was possible to verify hat residual efficacy lasted 21 days in the city of Caldas and up to 2 days on the cities of Pimenta, Campo Florido and Passos (Table 2). .2. Fluazuron 2.5 mg/kg (São paulo state) Considering the criteria, previously established on the present tudy, for diagnosing susceptibility of R. (B.) microplus populations o fluazuron (2.5 mg/kg), it was possible to diagnose one tick pop- lation as resistant, or at least with low susceptibility, to such ormulation on the state of São Paulo (Southeast Brazil), at Gastão idigal city, in a similar way to what happened in Minas Gerais. n this particular experiment, the highest efficacy index obtained y fluazuron was 83.2%, 5 days after treatment of bovines. Maxi- um efficacy (100%) against R. (B.) microplus was obtained by this ormulation in two other trials on the same state, at the cities of ranca and Vargem Grande do Sul. Residual acaricide periods (effi- acies superior or equal to 90% after the 14th DPT) were observed y fluazuron for up to 14 and 63 days after treatment on the cities f Santo Antonio de Posse and Franca, respectively (Table 3). .3. Fluazuron 2.5 mg/kg (Mato grosso do sul and goiás states) On the states of Mato Grosso do Sul and Goiás (Center-West egion of Brazil), between all seven R. (B.) microplus populations valuated, two could be diagnosed as resistant, or with low suscep- ibility, to fluazuron (2.5 mg/kg), both on the state of Mato Grosso o Sul. In one of these populations, maximum efficacy obtained y such formulation was 40.8% on the 14th DPT (Table 4). Resid- al efficacies against R. (B.) microplus were presented by fluazuron indexes ≥ 90% after the 14th DPT), with values of 28 and 42 days ary Medicine 135 (2016) 74–86 post treatment, on the cities of Icaraíma and Umuarama, respec- tively (Table 4). 3.4. Fluazuron 2.5 mg/kg and Novaluron 2.0 mg/kg + Eprinomectin 0.36 mg/kg (São Paulo and Goiás States) Regarding efficacy values obtained on all five trials, that evaluated indexes obtained by the novaluron + eprinomectin com- bination, in comparison to fluazuron and eprinomectin, it was possible to verify that all analyzed tick populations were suscep- tible to fluazuron (2.5 mg/kg). On the other hand, when looking at results obtained by the novel combination of novaluron asso- ciated to eprinomectin, it can be observed that, in all tested R. (B.) microplus populations, efficacy indexes of such combination were always inferior to 48.0%. Similar results were demonstrated by the pour-on eprinomectin formulation, since such drug, admin- istered alone, reached maximum efficacy of 57.8% (14th DPT) in the city of Espírito Santo do Pinhal (Table 5). Based on obtained data, and considering criteria previously established for diagnosing susceptibility of R. (B.) microplus populations to a benzoylphenyl urea and eprinomectin, all tested tick populations were diag- nosed as resistant, or with low susceptibly, to the novaluron (2.0 mg/kg) + eprinomectin (0.36 mg/kg) combination and to epri- nomectin (0.5 mg/kg), administered through subcutaneous and pour on routes, respectively (Table 5). 4. Discussion Regarding fluazuron effects against Rhipicephalus (Boophilus) microplus, Santos et al. (2010), using artificial infestation tri- als known as stall tests, detected a residual acaricide effect (efficacy ≥ 90%) of 95 and 92 days post treatment for 2.5 mg/kg fluazuron and 3.0 mg/kg fluazuron + 0.5 mg/kg abamectin, respec- tively. In another southern cattle tick population, Gomes et al. (2015), in two studies of natural and artificial (stall test) infesta- tions, detected efficacies superior or equal to 90%, that lasted for 49 and 77 days after treatment of animals, respectively. Using field trials on the present study, residual acaricide effects of fluazuron 2.5 mg/kg ranged between 14 and 63 days, when these formu- lations presented efficacy values ≥ 90%. Bull et al. (1996), in an article on R. (B.) microplus, observed that practically no adult tick was recorded between 3 and 12 weeks after treatment of cattle with fluazuron. However, it is important to mention that R. (B.) microplus populations from Australia were recently reclassified as Rhipicephalus (Boophilus) australis (Estrada-Peña et al., 2012; Ali et al., 2016), which means that, probably, the tick species involved in the study of Bull et al. (1996) is actually R. (B.) australis instead of R. (B.) microplus. In the present study, the fact that efficacy values of fluazuron took approximately 15 days to overcome 90% levels can be justi- fied by action mechanisms of this particular acaricide class, since such compounds are inhibitors of chitin synthesis. Consequently, it takes more time until larvae and nymphs are affected by this active principle (Retnakaram and Whight, 1987; Holdsworth et al., 2006). The first report of a R. (B.) microplus population resistant to flu- azuron was recently published by Brazilian researchers (Reck et al., 2014). This specific strain originated from the Rio Grande do Sul State, South region of Brazil, and is also the first with documented resistance to all six classes of acaricides used to control cattle tick in this country: organophosphates, formamidines (amitraz), syn- thetic pyrethroids, macrocyclic lactones, phenylpyrazoles (fipronil) and benzoylphenyl ureas (fluazuron). Based on results obtained by the present study, it is possible to verify that there are also R. (B.) microplus populations resistant, or presenting low susceptibility, to W.G. Maciel et al. / Preventive Veterinary Medicine 135 (2016) 74–86 79 Table 2 Mean counts of female R. (B.) microplus (4.5–8.0 mm long) for control and treated cattle groups; results of variance analysis of tick counts and efficacy percentages in experiments performed in Southeast region of Brazil (Minas Gerais State). Experiment 1 − Pimenta city Day of study T01: Control (Saline solution) T02: Fluazuron (2.5 mg/kg)2 Efficacy (%) Classification of R. (B.) microplus strain to fluazuron3 Residual efficacy of fluazrunon to R. (B.) microplus4 Mean tick count1 Range Mean tick count1 Range Arithmetic mean 0 26.8 A 10.0 – 31.7 26.9 A 10.3 – 33.7 – Susceptible 42 3 28.1 A 10 – 54 26.6 A 14 – 38 14.3 7 28.6 A 18 – 53 15.1 A 3 – 40 52.2 14 48.7 A 24 – 106 1.7 B 0 – 6 96.8 21 29.0 A 18 – 55 0.0 B 0 – 0 100.0 28 25.5 A 16 – 54 0.0 B 0 – 0 100.0 35 25.5 A 18 – 36 0.3 B 0 – 2 98.9 42 21.9 A 16 – 29 1.4 B 0 – 3 94.2 49 23,2 A 8 – 38 9.7 B 4 – 16 62,1 Experiment 2 − Campo Florido city 0 27.7 A 14.0 – 72.3 25.2 A 14.3 – 41.0 – Susceptible 42 3 26.0 A 11 – 58 17.7 A 3 – 51 25.2 7 32.3 A 7 – 87 18.7 B 2 – 117 36.4 14 32.2 A 5 – 91 0.6 B 0 – 3 98.0 21 25.3 A 6 – 65 0.2 B 0 – 1 99.1 28 20.2 A 8 – 49 0.6 B 0 – 4 96.7 35 19.1 A 8 – 36 0.6 B 0 – 2 96.5 42 18.5 A 7 – 33 1.5 B 0 – 11 91.1 49 24.8 A 6 – 57 5.7 B 0 – 26 74.7 56 18.1 A 9 – 42 8.7 B 0 – 38 47.2 Experiment 3 − Passos city 0 40.1 A 23.7 – 40.0 40.4 A 20.3 – 41.3 – Susceptible 42 3 40.7 A 26 – 55 42.7 A 15 – 61 0.0 7 42.3 A 28 – 68 37.6 A 10 – 45 11.9 14 39.8 A 26 – 59 2.5 B 0 – 8 93.8 21 37.3 A 26 – 51 0.0 B 0 – 0 100.0 28 36.8 A 23 – 53 0.8 B 0 – 1 97.8 35 31.8 A 18 – 45 1.7 B 0 – 3 94.8 42 31.5 A 21 – 43 3.0 B 1 – 5 90.6 49 31.3 A 17 – 41 8.5 B 7 – 21 73.1 Experiment 4 − Poç os de Caldas city 0 21.9 A 9.7 – 45.7 21.4 A 13.7 – 34.0 – Susceptible 28 3 32.6 A 8 – 59 25.6 B 4 – 54 19.5 7 19.1 A 9 – 43 14.4 B 1 – 54 22.7 14 18.2 A 8 – 34 5.6 B 0 – 16 68.5 21 21.1 A 7 – 82 1.4 B 0 – 6 93.2 28 21.7 A 7 – 84 0.2 A 0 – 1 99.1 35 25.0 A 5 – 89 6.1 B 3 – 12 75.0 Experiment 5 −Formiga city 0 26.8 A 12.0 – 60.0 26.5 A 12.3 – 54,0 – Susceptible 28 3 30.6 A 15 – 55 26.4 A 12 – 51 12.7 7 31.6 A 13 – 58 20.1 A 2 – 34 35.6 14 28.3 A 16 – 42 1.7 B 0 – 6 93.9 21 35.4 A 10 – 68 0.1 B 0 – 1 99.7 28 41.4 A 11 – 79 0.7 B 0 – 5 98.3 35 43.4 A 13 – 81 12.3 B 0 – 78 71.4 42 47.9 A 9 – 87 15.5 B 3 – 114 67.4 Experiment 6 − São Sebastião do Paraíso city 0 46.1 A 23.8 – 78.7 46.3 A 25.7 – 79.8 – Susceptible 35 3 47.3 A 26 – 76 25.2 A 13 – 37 47.1 7 44.5 A 28 – 81 16.0 A 12 – 23 64.2 14 46.3 A 26 – 98 1.0 B 0 – 3 97.9 21 42.8 A 21 – 90 0.8 B 0 – 1 99.6 28 42.5 A 23 – 87 1.0 B 0 – 4 97.7 35 37.8 A 18 – 81 1.33 B 0 – 5 96.5 42 38.7 A 21 – 86 4.7 B 2 – 12 88.0 49 31.3 A 17 – 41 11.0 B 3 – 21 65.1 Experiment 7 − Arcos city 0 50.2 A 17.3 – 138.0 59.1 A 18.1 – 138.7 – Resistant or with low susceptibility Not apllied 3 56.7 A 18 – 108 50.0 A 12 – 117 25.1 7 59.0 A 14 – 137 31.8 B 3 – 101 54.2 14 88.1 A 22 – 227 24.6 B 2 – 72 76.3 80 W.G. Maciel et al. / Preventive Veterinary Medicine 135 (2016) 74–86 Table 2 (Continued) Experiment 1 − Pimenta city Day of study T01: Control (Saline solution) T02: Fluazuron (2.5 mg/kg)2 Efficacy (%) Classification of R. (B.) microplus strain to fluazuron3 Residual efficacy of fluazrunon to R. (B.) microplus4 Mean tick count1 Range Mean tick count1 Range Arithmetic mean 21 83.8 A 28 – 180 12.7 B 1 – 78 87.1 28 87.7 A 33 – 168 15.9 B 1 – 38 84.6 35 83.4 A 38 – 143 23.6 B 2 – 69 76.0 42 79.0 A 41 – 135 13.0 B 1 – 36 86.0 49 43.0 A 13 – 86 13.7 B 0 – 31 73.0 Experiment 8 − Divinópolis city 0 46.8 A 32.1 – 67.7 46.6 A 32.3 – 67.3 – Susceptible 28 3 39.8 A 30 – 69 39.8 A 31 – 43 0.0 7 43.2 A 27 – 76 39.7 A 21 – 36 7.6 14 44.5 A 25 – 79 0.5 B 0 – 4 98.9 21 43.4 A 23 – 81 0.0 B 0 – 0 100.0 28 46.7 A 22 – 81 1.3 B 0 – 7 97.2 35 44.5 A 26 – 86 4.5 B 0 – 21 89.8 42 48.7 A 32 – 84 15.6 B 1 – 45 67.8 Experiment 9 − Pains city 0 37.3 A 18.7 – 78.3 37.7 A 18.3 – 77.7 – Susceptible 35 3 32.5 A 21 – 77 32.0 A 14 – 71 2.6 7 38.7 A 19 – 79 31.1 A 15 – 54 20.5 14 39.8 A 21 – 77 2.1 B 0 – 7 94.8 21 37.6 A 17 – 67 1.4 B 0 – 6 96.3 28 41.5 A 18 – 69 1.7 B 0 – 9 95.9 35 42.5 A 18 – 74 3.5 B 0 – 11 91.9 42 42.9 A 19 – 78 13.5 B 0 – 78 68.9 49 40.5 A 18 – 74 21.6 B 2 – 79 47.2 Experiment 10 − Caldas city 0 53.6 A 21.6 – 71.3 53.3 A 21.3 – 71.3 – Susceptible 21 3 56.7 A 20 – 78 56.7 A 19 – 78 0.0 7 59.8 A 19 – 76 51.0 A 10 – 56 14.2 14 61.7 A 23 – 74 5.6 B 3 – 9 90.9 21 63.5 A 21 – 79 5.8 B 1 – 7 90.8 28 65.5 A 23 – 81 10.5 B 3 – 31 83.9 35 67.8 A 22 – 83 23.6 B 8 – 45 65.0 1 Means values followed by the same letter on the same line do not differ significantly at a 95% reliability level. 2 Commercial formulation purchased in the local market. 3 ean. 3 n. 4 fl M e b t t A ( Á o t i ( D s r R f w m p i w Rating susceptible performed based on the efficacy (>90%) using the arithmetic m Rating resistant performed based on the efficacy (≤90%) using the arithmetic mea The last day of the study that the chemical compund show efficacy ≥90%. uazuron (2.5 mg/kg) on the states of Minas Gerais, São Paulo and ato Grosso do Sul (Southeast and Center-West regions of Brazil). The therapeutic efficacy of a formulation can be defined as the ffect a certain product causes against ticks present on the entire ody surface of an animal on treatment day. Residual efficacy, on he other hand, can be defined as the effects presented by a formula- ion against new infestations of the treated animal with more ticks. t this study, even though rain was present in some experiments trial #1 in Pimenta city, trial #8 in Divinópolis city, trial #13 in guas da Prata city and trial #25 in Jataí city), 24 h after treatment f animals with pour on fluazuron, such fact did not interfere on herapeutic efficacies of the aforementioned product since, even n these trials where rain was present, fluazuron reached anti-R. B.) microplus efficacy indexes between 98.7% and 100% on the 21st PT. According to Silva et al. (2015), rainfall, or even incidence of unlight and dew over animals’ coats, can eventually interfere on esidual efficacy indexes of a topically administered formulation. esults obtained on studies conducted by such researchers rein- orce the aforementioned inference. Silva et al. (2015), in studies ith R. (B.) microplus, using artificially and naturally infested ani- als kept in pasture conditions, report that animals treated with our on formulations and maintained in the stall test did not suffer nterference of rain or direct sunlight. Unlike those, animals treated ith the same compound and maintained in field conditions, in turn, suffered interference in the residual action period of a com- pound. Therefore, differences in residual efficacy periods (during which the product maintained efficacies ≥ 90% after the 14th DPT) of fluazuron, observed on artificial infestation studies (stall tests) by Santos et al. (2010) and Gomes et al. (2015), in comparison to those observed on the present study, can be justified by reports described above. Other relevant aspect, that must be mentioned in this article is that R. (B.) microplus populations diagnosed as resistant, or at least less susceptible, against fluazuron (2.5 mg/kg) were the ones present in farms where owners used such formulation on herds for more than five years, with treatment intervals of 30–55 days between them during the rainy season. Novaluron is also a chitin synthesis inhibitor, that acts by both ingestion and contact, particularly targeting immature stages that actively synthesize chitin. Previous works have shown that noval- uron is effective against a variety of insects, including Coleoptera, Homopterta, Hymenoptera, Lepidoptera and Diptera (Malinowski and Pawinska, 1992; Glowacka and Malinowski, 1994; Pluciennik et al., 1999; Barazani, 2001; Ishaaya and Horowitz, 1998, 2002; Cutler et al., 2005; Mascari et al., 2007; Arredondo-Jimenez and Valdez-Delgado, 2006). This active component showed satisfactory efficacy values against insects parasitizing grains and sugarcane (Kostyukovsky and Trostanetsky, 2006; Beuzelin et al., 2010). The W.G. Maciel et al. / Preventive Veterinary Medicine 135 (2016) 74–86 81 Table 3 Mean counts of female R. (B.) microplus (4.5–8.0 mm long) for control and treated cattle groups; results of variance analysis of tick counts and efficacy percentages in experiments performed in Southeast region of Brazil (São Paulo State). Experiment 11- Franca city Day of study T01: Control (Saline solution) T02: Fluazuron (2.5 mg/kg)2 Efficacy (%) Classification of R. (B.) microplus strain to fluazuron3 Residual efficacy of fluazrunon to R. (B.) microplus4 Mean tick count1 Range Mean tick count1 Range Arithmetic mean 0 52,3 A 23.7 – 93.0 51,9 A 27 – 82 – Susceptible 63 3 46.5 A 24 – 85 40,7 A 17 – 69 11.8 7 44.7 A 22 – 76 27,9 B 9 – 59 37.1 14 32.3 A 18 – 57 5,3 B 0 – 27 91.9 21 25.7 A 8 – 76 0,1 B 0 – 1 99.6 28 22.2 A 10 – 34 0,0 B 0 – 0 100.0 35 24.4 A 12 – 30 0,2 B 0 – 2 99.2 42 18.6 A 6 – 27 0,8 B 0 – 3 98.2 49 21.8 A 13 – 32 0,9 B 0 – 8 98.5 56 37.0 A 21 – 61 4,6 B 0 – 28 94.3 63 30.3 A 20 – 43 3,3 B 0 – 11 93.4 70 31.3 A 15 – 57 13,6 B 1 – 38 63.3 77 31.9 A 17 – 52 19,6 B 9 – 34 35.1 Experiment 12 − Casa Branca city 0 40.5 A 26.3 – 55.6 40.6 A 25.7 – 57.6 – Susceptible 49 3 52.9 A 18 – 101 49.4 A 31 – 82 6.8 7 64.2 A 31 – 134 50.3 A 31 – 69 21.8 14 63.5 A 33 – 123 3.5 B 0 – 10 94.5 21 61.3 A 23 – 100 0.3 B 0 – 2 99.5 28 51.6 A 16 – 87 0.1 B 0 – 1 99.8 35 50.6 A 13 – 80 0.2 B 0 – 1 99.6 42 49.3 A 10 – 75 0.5 B 0 – 1 99.0 49 46.5 A 12 – 69 2.6 B 0 – 2 94.4 56 43.3 A 13 – 69 8.2 B 3 – 23 81.1 63 44.1 A 25 – 59 12.0 B 3 – 25 72.9 Experiment 13 − Aguas da Prata city 0 56.1 A 32.7 – 86.7 55.6 A 32,0 – 83.7 – Susceptible 28 3 63.8 A 32 – 105 54.2 B 31 – 98 14.3 7 58.9 A 19 – 127 49.8 B 10 – 101 14.7 14 46.2 A 16 – 135 1.0 B 0 – 4 97.8 21 45.6 A 12 – 135 0.4 B 0 – 2 99.1 28 52.0 A 11 – 148 0.8 B 0 – 3 98.4 35 27.5 A 13 – 59 4.1 B 0 – 20 85.0 42 28.7 A 18 – 51 10.3 B 1 – 26 63.8 49 28.1 A 16 – 52 12.3 B 3 – 28 55.8 Experiment 14 − Itirapuã city 0 36.9 A 20.3 – 154.7 36.9 A 19.7 – 158.7 – Susceptible 35 3 38.4 A 18 – 169 30.0 A 14 – 127 21.9 7 43.4 A 14 – 188 26.7 B 16 – 103 38.5 14 32.8 A 10 – 150 4.4 B 0 – 14 86.6 21 28.8 A 8 – 127 1.7 B 0 – 11 94.1 28 28.1 A 9 – 121 0.9 B 0 – 7 96.8 35 29.9 A 7 – 97 3.0 B 0 – 17 90.0 42 30.6 A 8 – 102 4.7 B 0 – 21 84.7 49 35.2 A 12 – 103 10.4 B 3 – 30 70.5 56 29.0 A 14 – 87 12.3 B 6 – 29 57.6 Experiment 15 − Gastão Vidgal city 0 68.1 A 22.0 – 146.7 68.2 A 20.67 – 173.7 – Resistant or with low susceptibility Not applied 3 67.5 A 41 – 141 51.9 A 17 – 103 23.2 7 67.1 A 48 – 134 30.8 B 15 – 60 54.1 14 58.6 A 44 – 76 12.3 B 8 – 32 79.0 21 48.6 A 33 – 74 9.9 B 2 – 18 79.7 28 42.7 A 29 – 61 7.5 B 0 – 23 82.6 35 40.4 A 18 – 67 6.8 B 3 – 25 83.2 42 47.2 A 21 – 72 15.6 B 6 – 32 67.0 Experiment 16 − São João da Boa Vista city 0 33.1 A 13.7 – 64.7 34.3 A 12.3 – 71.7 – Susceptible 42 3 28.1 A 12 – 65 24.9 A 9 – 48 14.5 7 28.0 A 17 – 51 15.5 B 3 – 58 46.6 14 33.0 A 15 – 57 2.3 B 0 – 5 93.3 21 30.0 A 10 – 51 0.1 A 0 – 1 99.7 28 30.4 A 8 – 65 1.3 B 0 – 5 95.9 35 34.5 A 7 – 73 1.9 B 0 – 6 94.7 82 W.G. Maciel et al. / Preventive Veterinary Medicine 135 (2016) 74–86 Table 3 (Continued) Experiment 11- Franca city Day of study T01: Control (Saline solution) T02: Fluazuron (2.5 mg/kg)2 Efficacy (%) Classification of R. (B.) microplus strain to fluazuron3 Residual efficacy of fluazrunon to R. (B.) microplus4 Mean tick count1 Range Mean tick count1 Range Arithmetic mean 42 41.3 A 9 – 69 2.3 B 3 – 9 94.6 49 39.8 A 10 – 66 9.8 B 6 – 21 76.2 Experiment 17 − São José do Rio Pardo city 0 35.8 A 21.7 – 69.0 35.4 A 22.3 – 68.0 – Susceptible 28 3 46.1 A 17 – 89 34.6 A 18 – 77 24.2 7 41.8 A 17 – 63 33.4 A 9 – 126 19.3 14 35.0 A 21 – 65 1.0 B 0 – 5 97.1 21 32.0 A 20 – 60 0.8 B 0 – 4 97.5 28 34.2 A 20 – 59 0.7 B 0 – 5 97.9 35 44.5 A 19 – 117 15.5 B 0 – 20 64.8 42 61.0 A 20 – 139 37.0 B 0 – 128 38.7 49 93.2 A 41 – 223 66.0 B 9 – 145 28.5 Experiment 18 − Santo Antonio de Posse 0 23.4 A 12.1 – 49.3 23.5 A 12.3 – 50.1 – Susceptible 14 3 26.3 A 12 – 35 19.0 A 7 – 42 27.4 7 19.9 A 14 – 46 15.4 B 5 – 34 22.3 14 21.0 A 15 – 51 1.1 B 0 – 5 94.8 21 19.0 A 13 – 39 13.8 B 3 – 65 27.8 28 24.0 A 14 – 38 19.2 A 4 – 61 20.2 35 26.0 A 15 – 39 23.5 A 9 – 78 10.2 Experiment 19 − Vargem Grande do Sul 0 40.8 A 20.7 – 67.3 40.6 A 20.3 – 68.1 – Susceptible 42 3 42.9 A 23 – 65 21.5 B 12 – 45 49.6 7 45.0 A 18 – 68 20.4 B 8 – 49 54.4 14 46.9 A 21 – 71 1.2 B 0 – 3 97.4 21 41.4 A 22 – 78 1.0 B 0 – 1 97.6 28 38.2 A 21 – 56 0.0 B 0 – 0 100.0 35 38.0 A 23 – 62 0.0 B 0 – 0 100.0 42 44.0 A 23 – 65 3.1 B 0 – 18 92.9 49 46.0 A 17 – 69 14.4 B 0 – 29 68.5 56 52.0 A 18 – 57 32.0 B 3 – 78 38.1 Experiment 20 − São Sebastião da Grama 0 89.9 A 33.3 – 147.3 89.7 A 33.1 – 146.5 – Susceptible 21 3 86.4 A 46 – 178 65.3 A 13 – 101 24.3 7 101.5 A 41 – 198 71.3 B 17 – 112 29.7 14 99.3 A 54 – 213 4.5 B 0 – 21 95.5 21 98.7 A 55 – 206 3.2 B 0 – 14 96.8 28 105.6 A 68 – 187 21.4 B 0 – 55 79.7 35 110.4 A 72 – 198 43.1 B 1 – 85 60.9 1 Means values followed by the same letter on the same line do not differ significantly at a 95% reliability level. 2 rmed 3 n. 4 fi w u u b h t e t d c a o ( a t v i Commercial formulation purchased in the local market.3 Rating susceptible perfo Rating resistant performed based on the efficacy (≤90%) using the arithmetic mea The last day of the study that the chemical compund show efficacy ≥90%. rst trial conducted with such molecule against R. (B.) microplus as performed by USDA researchers (Lohmeyer et al., 2012), which sed different dosages (2.5 and 5.0 mg/kg) of an unregistered noval- ron formulation. Results obtained by such authors reveal that oth concentrations of pour on novaluron evaluated appear to ave modest effects on southern cattle ticks. Therapeutically, both ested dosages caused a small reduction in numbers of recovered ngorged female ticks, in comparison with amounts recovered from he untreated control group. In this case, Lohmeyer et al. (2012) escribed that some degree of residual efficacy was observed on the ontrol of larvae that were applied to animals one and two weeks fter their treatment. At the present study, results similar to those btained by Lohmeyer et al. (2012) were observed for the novaluron 2.0 mg/kg) + eprinomectin (0.36 mg/kg) combination in all evalu- ted R. (B.) microplus strains. In none of these tick populations, he novel novaluron + eprinomectin association reached efficacy alues superior to 50% against R. (B.) microplus females, measur- ng between 4.5 and 8.0 mm long, parasitizing bovines. Efficacy based on the efficacy (>90%) using the arithmetic mean. results obtained by this association on the present study are signifi- cantly inferior to indexes required by Brazilian legislation [Brazilian Ministry of Agriculture, Livestock and Food Supply (Ministério da Agricultura, Pecuária e Abastecimento − MAPA)] in order for a prod- uct to be licensed and commercialized as an acaricide in the country (Brasil, 1997). Such findings, allied to results obtained with this same molecule by Lohmeyer et al. (2012), reinforce the percep- tion that maybe formulations of novaluron, in utilized dosages and administration routes, may not be an effective tool for controlling R. (B.) microplus. However, future studies must be performed in order to prove and reinforce such hypothesis. Few studies were identified by present researchers, in con- sulted literature, which aimed to evaluate efficacy indexes of eprinomectin against R. (B.) microplus (Rangel, 2003; Aguirre et al., 2005; Lifschitz et al., 2016). Additionally, all studies report elevated efficacies (≥95%) of such active principle, administered topically (pour on), in different dosages, against the aforementioned ectopar- asite. Results obtained by eprinomectin, administered via pour on W.G. Maciel et al. / Preventive Veterinary Medicine 135 (2016) 74–86 83 Table 4 Mean counts of female R. (B.) microplus (4.5–8.0 mm long) for control and treated cattle groups; results of variance analysis of tick counts and efficacy percentages in experiments performed in Midwest and South regions of Brazil (Mato Grosso do Sul, Goiás and Paraná States). Experiment 21 − Bandeirantes city (Mato Grosso do Sul State) Day of study T01: Control (Saline solution) T02: Fluazuron (2.5 mg/kg)2 Efficacy (%) Classification of R. (B.) microplus strain to fluazuron3 Residual efficacy of fluazrunon to R. (B.) microplus4 Mean tick count1 Range Mean tick count1 Range Arithmetic mean 0 59.3 A 18.0 – 127.33 59.5 A 17.3 – 129.0 – Susceptible 35 3 46.8 A 13 – 72 56.8 A 43 – 101 0.0 7 41.1 A 12 – 79 33.7 A 3 – 78 18.3 14 30.9 A 14 – 63 6.9 B 1 – 35 77.8 21 68.9 A 20 – 107 2.1 B 0 – 6 97.0 28 19.3 A 17 – 22 0.8 B 0 – 3 95.9 35 22.4 A 17 – 30 0.5 B 0 – 1 97.8 42 39.1 A 14 – 66 26.8 A 7 – 162 31.7 49 36.3 A 10 – 60 34.1 A 8 – 158 6.4 Experiment 22 − Terenos city (Mato Grosso do Sul State) 0 33.2 A 8.3 – 110.0 33.7 A 8.7 – 110.7 – Resistant or with low susceptibility Not applied 3 42.7 A 8 – 144 36.3 A 6 – 112 16.3 7 64.1 A 9 – 214 34.1 B 7 – 90 47.6 14 52.1 A 7 – 195 31.3 B 7 – 115 40.8 21 22.4 A 8 – 80 13.7 B 0 – 45 39.7 28 14.4 A 7 – 36 5.1 A 0 – 21 65.1 Experiment 23 − Jaraguari city (Mato Grosso do Sul State) 0 68.9 A 13.3 – 240.7 69.0 A 14.7 – 267.0 – Resistant or with low susceptibility Not applied 3 62.9 A 14 – 183 74.9 A 24 – 161 0.0 7 68.4 A 9 – 145 74.2 A 12 – 234 0.0 14 97.8 A 7 – 378 32.3 B 8 – 71 67.0 21 105.7 A 16 – 274 19.0 B 2 – 71 82.1 28 112.0 A 17 – 365 43.9 B 5 – 106 60.9 35 101.7 A 10 – 317 115.2 A 30 – 342 0.0 Experiment 24 − Caiapônia city (Goiás State) 0 44.3 A 27.2 – 77.8 44.7 A 26.3 – 78.7 – Susceptible 35 3 39.7 A 21 – 78 32.1 A 16 – 56 19.9 7 44.5 A 24 – 119 29.8 B 15 – 43 33.6 14 47.6 A 18 – 87 2.7 B 0 – 3 94.4 21 51.3 A 21 – 91 1.1 B 0 – 5 97.9 28 43.5 A 26 – 58 0.2 B 0 – 4 99.5 35 44.3 A 23 – 67 0.3 B 0 – 3 99.3 42 47.8 A 21 – 66 13.7 B 8 – 28 71.6 49 49.7 A 20 – 69 17.6 B 8 – 32 64.9 Experiment 25 − Jataí city (Goiás State) 0 59.3 A 18.0 – 111.0 59.5 A 32.3 – 113.0 – Susceptible 35 3 46.8 A 13 – 78 57.1 A 25 – 101 0.0 7 41.1 A 12 – 78 33.7 A 3 – 74 18.3 14 30.9 A 9 – 63 6.9 B 1 – 35 77.8 21 68.9 A 20 – 105 0.9 B 0 – 6 98.7 28 19.3 A 10 – 32 0.8 B 0 – 3 95.9 35 22.4 A 15 – 30 0.7 B 0 – 2 96.9 42 39.1 A 4 – 73 26.8 B 7 – 162 31.7 Experiment 26 − Icaraíma city (Paraná State) 0 93.9 A 35.3 – 145.7 92.4 A 35.7 – 148.7 – Susceptible 28 3 159.1 A 32 – 156 110.5 A 21 – 101 29.4 7 136.7 A 31 – 154 101.7 B 17 – 98 24.3 14 133.2 A 30 – 143 0.0 B 0 – 5 99.8 21 115.6 A 21 – 134 0.0 B 0 – 0 100.0 28 109.5 A 23 – 121 0.0 B 0 – 0 100.0 35 103.8 A 19 – 101 23.5 B 0 – 18 77.0 42 93.7 A 19 – 98 40.9 B 4 – 78 55.6 Experiment 27 − Umuarama city (Paraná State) 0 21.0 A 13.3 – 44.2 21.7 A 11.3 – 46.7 – Susceptible 42 3 24.8 A 15 – 50 20.6 A 14 – 32 19.6 7 28.5 A 12 – 61 16.9 B 9 – 29 42.6 14 27.3 A 12 – 66 2.2 B 0 – 7 92.2 21 27.1 A 11 – 39 1.0 B 0 – 3 96.4 28 26.1 A 14 – 43 1.5 B 0 – 6 94.4 35 29.0 A 20 – 43 2.1 B 0 – 4 93.0 42 31.5 A 18 – 54 2.3 B 0 – 8 92.9 49 27.0 A 18 – 34 8.9 B 5 – 14 68.1 56 25.1 A 15 – 43 16.7 B 12 – 23 35.6 1 Means values followed by the same letter on the same line do not differ significantly at a 95% reliability level. 2 Commercial formulation purchased in the local market. 3 Rating susceptible performed based on the efficacy (>90%) using the arithmetic mean. 3 Rating resistant performed based on the efficacy (≤90%) using the arithmetic mean. 4 The last day of the study that the chemical compund show efficacy ≥90%. 84 W .G . M aciel et al. / Preventive V eterinary M edicine 135 (2016) 74–86 Table 5 Mean counts of female R. (B.) microplus (4.5–8.0 mm long) for control and treated cattle groups; results of variance analysis of tick counts and efficacy percentages in experiments performed in Midwest and Southeast regions of Brazil (São Paulo and Goiás State). Experiment 28 − Espírito Santo do Pinhal city (São Paulo State) Day of study T01: Control (Saline solution) T03: Novaluron (2.0 mg/kg) + Eprinomectin (0.36 mg/kg)2 T02: Fluazuron (2.5 mg/kg)2 T02: Eprinomectin (0.5 mg/kg)2 Efficacy (%) Classification of R. (B.) microplus strain Residual efficacy of fluazrunon to R. (B.) microplus4 Mean tick count1 Range Mean tick count1 Range Mean tick count1 Range Mean tick count1 Range Novaluron + Eprinomectin Fluazuron Eprinomectin Novaluron + Eprinomectin3 Fluazuron3 Eprinomectin3 0 33.8 A 20.7 – 67.3 33.7 A 20.1 – 66.3 33.6 A 20.3 – 66.3 33.5 A 20.0 – 66.8 – – – Resistant or with low susceptibility Susceptible Resistant or with low susceptibility 42 3 35.9 A 18 – 68 33.9 A 17 – 63 29.5 A 14 – 56 34.5 A 21 – 54 5.3 17.3 3.0 7 38.0 A 21 – 61 30.7 A 9 – 32 27.7 A 11 – 45 26.5 A 9 – 21 19.0 26.6 29.6 14 39.9 A 22 – 78 24.0 B 5 – 33 0.7 C 0 – 5 16.7 B 3 – 29 39.7 98.2 57.8 21 34.4 A 21 – 56 20.2 B 2 – 33 0.0 C 0 – 0 21.5 B 4 – 36 41.1 100.0 36.9 28 31.2 A 24 – 62 19.3 B 4 – 32 0.1 C 0 – 1 23.4 B 7 – 43 38.0 99.7 24.3 35 31.0 A 21 – 65 20.3 B 9 – 32 1.3 C 0 – 5 25.6 B 12 – 41 34.3 95.8 16.7 42 37.0 A 21 – 69 20.6 B 7 – 34 3.2 C 0 – 8 41.5 A 23 – 61 44.2 91.3 0.0 49 39.0 A 18 – 57 24.7 B 9 – 37 8.5 C 0 – 13 39.8 A 18 – 65 36.5 78.1 0.0 56 45.0 A 28 – 61 27.2 B 4 – 48 26.7 B 1 – 56 45.7 A 24 – 68 39.4 40.3 0.0 Experiment 29 − São João da Boa Vista city (São Paulo State) 0 62.5 A 20.3 – 203.0 62.9 A 21.7 – 204.3 62.4 A 20.1 – 205.6 62.3 A 21.0 – 201.7 – – – Resistant or with low susceptibility Susceptible Resistant or with low susceptibility 21 3 73.1 A 39 – 215 63.0 A 18 – 201 61.2 A 18 – 187 64.5 A 21 – 197 14.3 16.2 11.5 7 81.3 A 31 – 209 54.7 B 9 – 139 58.7 B 15 – 132 45.7 B 9 – 123 33.1 27.7 43.6 14 94.7 A 45 – 198 55.2 B 15 – 117 2.3 C 0 – 7 38.7 B 14 – 98 42.1 97.6 59.0 21 95.8 A 43 – 201 58.5 B 12 – 132 1.3 C 0 – 5 49.9 B 16 – 110 39.3 98.6 47.8 28 102.0 A 25 – 204 64.5 B 9 – 138 20.1 C 0 – 45 61.4 B 11 – 169 37.1 80.3 36.9 35 104.3 A 32 – 201 71.6 B 16 – 136 45.6 C 4 – 67 83.8 B 19 – 187 31.8 56.2 19.4 Experiment 30 − Tambaú city (São Paulo State) 0 79.2 A 23.7 – 213.3 79.6 A 21.7 – 212.7 79.1 A 22.3 – 211.7 78.8 A 22.1 – 212.3 – – – Resistant or with low susceptibility Susceptible Resistant or with low susceptibility 35 3 87.5 A 23 – 256 65.7 B 17 – 202 81.5 A 24 – 244 60.1 B 21 – 212 25.3 6.8 31.0 7 84.5 A 28 – 244 59.7 B 15 – 198 78.9 A 21 – 232 72.3 B 19 – 231 29.7 6.5 14.0 14 93.2 A 32 – 267 61.4 B 13 – 141 3.4 C 3 – 7 67.8 B 15 – 167 34.4 96.3 26.9 21 101.7 A 31 – 256 65.3 B 14 – 132 5.4 C 1 – 8 98.9 A 23 – 239 36.1 94.7 2.3 28 112.2 A 33 – 298 71.3 B 11 – 145 4.3 C 0 – 12 105.4 A 32 – 278 36.7 96.2 5.6 35 108.3 A 37 – 302 78.7 B 10 – 149 7.6 C 0 – 16 112.3 A 31 – 278 27.7 93.0 0.0 42 110.5 A 35 – 298 81.2 B 14 – 184 12.6 C 1 – 25 123.7 A 30 – 298 26.9 88.6 0.0 49 115.2 A 37 – 313 84.6 B 16 – 189 23.5 C 3 – 35 119.8 A 32 – 309 26.9 79.6 0.0 Experiment 31 − Jataí city (Goiás State) 0 49.5 A 25.3 – 123.7 49.9 A 25.7 – 122.3 49.4 A 25.7 – 121.6 49.3 A 25.3 – 122.7 – – – Resistant or with low susceptibility Susceptible Resistant or with low susceptibility 35 3 46.7 A 23 – 145 43.5 A 20 – 101 41.6 A 21 – 118 40.2 A 21 – 101 7.5 10.8 13.5 7 44.3 A 22 – 149 29.4 B 15 – 78 32.7 A 18 – 101 38.7 A 18 – 113 34.1 26.1 12.3 14 41.4 A 21 – 141 28.6 B 13 – 67 0.3 C 0 – 4 27.6 B 8 – 72 31.4 99.3 33.1 21 48.9 A 18 – 156 28.7 B 12 – 64 0.0 C 0 – 0 31.7 B 9 – 78 41.7 100.0 34.9 28 49.3 A 16 – 165 29.1 C 11 – 56 3.5 D 0 – 7 39.8 B 8 – 76 41.4 92.9 18.9 35 50.2 A 18 – 178 28.7 B 9 – 49 4.7 C 0 – 16 45.7 A 12 – 143 43.2 90.6 8.6 42 52.1 A 17 – 179 32.5 B 9 – 58 24.5 C 1 – 34 46.9 A 11 – 155 38.1 52.9 9.6 49 47.8 A 21 – 145 31.4 B 8 – 73 34.6 B 9 – 101 44.5 A 18 – 123 34.8 27.5 6.5 Experiment 32 − Acreúna city (Goiás State) 0 25.6 A 14.3 – 44.7 25.3 A 14.7 – 44.1 25.6 A 14.4 – 44.5 25.3 A 14.8 – 44.1 – – – Resistant or with low susceptibility Susceptible Resistant or with low susceptibility 21 3 26.7 A 15 – 43 19.3 A 14 – 32 18,4 A 13 – 36 21.4 A 13 – 37 26.9 31.1 18.9 7 32.1 A 16 – 47 16.7 B 8 – 30 16.5 C 9 – 31 15.4 B 7 – 28 47.7 48.6 51.5 14 28.7 A 13 – 39 16.9 B 6 – 29 1.4 C 0 – 7 15.9 B 1 – 12 40.4 95.1 43.9 21 30.2 A 16 – 41 16.9 B 7 – 31 1.6 C 0 – 12 20.5 B 9 – 34 43.4 94.7 31.3 28 30.7 A 15 – 46 19.3 B 6 – 27 11.3 C 0 – 31 23.5 B 12 – 43 36.4 63.2 22.5 35 34.5 A 15 49 21.4 B 7 – 32 17.8 C 9 45 26.7 B 11 59 37.2 48.4 21.7 1 Means values followed by the same letter on the same line do not differ significantly at a 95% reliability level. 2 Commercial formulation purchased in the local market. 3 Rating susceptible performed based on the efficacy (>90%) using the arithmetic mean. 3 Rating resistant performed based on the efficacy (≤90%) using the arithmetic mean. 4 The last day of the study that the chemical compund show efficacy ≥90%. eterina t l o fl u ( p b o p t o i e c t ( l e ( l a f a t B C R A A B B B B C C C C C C W.G. Maciel et al. / Preventive V he dosage of 0.5 mg/kg, described here, make it clear that all popu- ations of R. (B.) microplus tested on the present study are resistant, r present low susceptibility, to such active component. Based on all results obtained using 32 field efficacy trials with uazuron (2.5 mg/kg), as well as five experiments with noval- ron (2.0 mg/kg) + eprinomectin (0.36 mg/kg) and eprinomectin 0.5 mg/kg), it is possible to conclude that four southern cattle tick opulations were diagnosed as resistant, or at least less suscepti- le, to fluazuron (2.5 mg/kg). Such fact was detected in farms where wners administered this compound in their herds for a minimum eriod of five years, with treatment intervals of 30 to 55 days during he rainy season. However, in vitro studies should be performed in rder to reinforce the results obtained in the present study. Regard- ng efficacies obtained by the novel molecule novaluron, allied to prinomectin, against such ixodidae, this combination showed effi- acy values inferior to 48.0% in all trials. These findings, allied o results obtained with the same molecule by Lohmeyer et al. 2012), reinforce the perception that perhaps novaluron formu- ations, in tested dosages and administration routes, may not be ffective tools for controlling R. (B.) microplus. Besides, all five R. B.) microplus strains tested were diagnosed as resistant, or with ow susceptibility, to eprinomectin (0.5 mg/kg). 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