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1 Red de Revistas Científicas de América Latina, el Caribe, España y Portugal Sistema de Información Científica Pazinato, Rafael; Klauck, Vanderlei; Kharyna Grosskopf, Rhayana; Dalla Rosa, Luciana; Volpato, Andréia; Baretta, Dilmar; Moura Stefani, Lenita; Schafer Da Silva, Aleksandro Antiparasitic Resistance of Different Populations of ticks (Rhipicephalus microplus) in the Western of Santa Catarina State, Brazil Acta Scientiae Veterinariae, vol. 42, núm. 1, enero, 2014, pp. 1-6 Universidade Federal do Rio Grande do Sul Porto Alegre, Brasil Available in: Acta Scientiae Veterinariae, ISSN (Printed Version): ActaSciVet@ufrgs.br Universidade Federal do Rio Grande do Sul Brasil How to cite Complete issue More information about this article Journal's homepage Non-Profit Academic Project, developed under the Open Acces Initiative

2 Acta Scientiae Veterinariae, : RESEARCH ARTICLE Pub ISSN Antiparasitic Resistance of Different Populations of ticks (Rhipicephalus microplus) in the Western of Santa Catarina State, Brazil Resistência antiparasitária de diferentes populações de carrapatos (Rhipicephalus microplus) no oeste do estado de Santa Catarina, Brasil Rafael Pazinato 1, Vanderlei Klauck 1, Rhayana Kharyna Grosskopf 1, Luciana Dalla Rosa 2, Andréia Volpato 1, Dilmar Baretta 1, Lenita Moura Stefani 1 & Aleksandro Schafer Da Silva 1 ABSTRACT Background: In Brazil, the tick Rhipicephalus microplus is a serious problem for beef and milk cattle due to the damage caused directly to the animal. Ticks are responsible for major economic losses related to production, especially in breeds of European origin, prevalent in Southern Brazil. The use of most acaricides may allow the survival of some tick that transmits this characteristic to future generations, consequently each successive treatment is a process of selection. The lack of technical guidance on how to control ticks may contribute to the emergence of new resistant strains of ticks. The objective of this study was to test the efficacy of antiparasitic drugs on tick populations (Rhipicephalus microplus) from the Western region of Santa Catarina, Brazil. Materials, Methods & Results: Thirty-one farms participated in this study and 70 engorged female ticks were collect from each property. They were divided into seven groups of 10 specimens each. Ticks were exposed to six commercial drugs: amitraz (A); cypermethrin, chlorpyrifos, and citronellal (B); diazinon (C); diclovós and chlorpyrifos (D); cypermethrin (E); cypermethrin, chlorpyrifos, and piperonyl butoxide (F); and one group was used as a control to validate the test (G: no drug). Tests with acaricides followed standard protocols, recommended for in vitro testing. The effectiveness of the product was calculated based on egg laying and hatching. The drug tested is considered effective if the result was more than 95%. The number of farms with populations of resistant ticks were 13 (41.9% - group A), 2 (6.45% - group B), 15 (48.38% - group C), 6 (19.35% - group D) and 12 (38.7% - group E). Ticks from the group F did not show resistance to the tested products. i.e., all farms obtained 100% efficiency. Among the products tested that showed the highest efficacy were the combinations of three or two active principle as cypermethrin, chlorpyrifos and piperonyl butoxide (group F - 100%), cypermethrin, chlorpyrifos and citronella (group B %), and dichlorvos and chlorpyrifos (group D %). However, products with only one active principle showed lower efficiency such as amitraz (group A %), diazinon (group C %), and cypermethrin (group E %). Discussion: These results are very similar to other studies conducted recently in different Brazilian regions, but unknown in Western Santa Catarina, which currently has the largest dairy herd in the Santa Catarina State. In this study it was found that farmers are unaware of important tests to control tick, like the immersion test of engorged females that contributes to parasite control, identifies ineffective drugs and reduces labor costs. As researchers reported the inappropriate use of antiparasitic drugs like subdosage or superdosage, and the high frequency of acaricide use, may select and propagate the occurrence of resistance selection pressure. Therefore, it is concluded that the resistance of ticks is a problem present in the majority of the Western Santa Catarina properties. The current study shows that 81.7% of the tested tick population of parasitic resistance presented to one or more drugs. This study found that the cattle farmers were not aware of tests that detect ticks susceptibility to acaricides and did not adopt proper technical procedure when using chemicals. These indicate the need to educate cattle farmers on considering resistance as criteria, considering efficiency and cost of the treatment. Keywords: Boophilus microplus, acaricides, parasite resistance, ticks. Received: 27 April 2014 Accepted: 28 August 2014 Published: 11 September Departamento de Zootecnia, Universidade do Estado de Santa Catarina (UDESC), Chapecó, SC, Brazil. 2 Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil. CORRESPONDENCE: A.S. Da Silva [aleksandro_ss@yahoo.com.br - Fax: +55 (49) ]. Laboratório de Pesquisa em Parasitologia Animal (LPPA), Centro de Educação Superior do Oeste, Rua Beloni Trombeta Zanin, 680-E, Bairro Santo Antonio. CEP Chapecó, SC, Brazil. 1

3 R. Pazinato, V. Klauck, R. K. Grosskopf, et al Antiparasitic Resistance of Different Populations of ticks (Rhipicephalus microplus) INTRODUCTION In Brazil, the tick Rhipicephalus microplus is a serious problem for beef and milk cattle due to the damage caused directly to the animal. Ticks are responsible for major economic losses related to production, especially in breeds of European origin, prevalent in Southern Brazil [1,16]. The parasite can easy multiply due to the climatic conditions of this region, and in general it is difficult to control, causing high infestation of pastures, reduced milk production, poor feed conversion, and consequently poor weight gain, and elevated cost in diseases treatment [14]. In order to face this problem, farmers use acaricides more often, which favors the selection pressure resulting in the emergence of resistant parasites. According to literature, the use of most acaricides may allow the survival of some tick that transmits this characteristic to future generations, consequently each successive treatment is a process of selection [6]. The lack of technical guidance on how to control ticks may contribute to the emergence of new resistant strains of ticks. Therefore, error handling associated with improper use of acaricides causes tick-resistance to the available drugs, thus contributing to the worsening of the problem [7]. Researchers have estimated that the damage caused by this tick in Brazil, considering direct and indirect damages, may exceed two billion dollars per year [8]. These facts stimulate the evaluation of acaricide efficacy and the occurrence of resistance. Therefore, the objective of this study was to test the efficacy of antiparasitic drugs on tick populations from the Western region of Santa Catarina, Brazil. MATERIALS AND METHODS Ticks used were from 31 naturally infestated milk (30) and beef (1) farms that did not use any acaricide treatment for at least 30 days. In each farm were collected manually at least 70 ticks, stored in plastic containers, and transported to the Laboratory of Animal Parasitology of Universidade do Estado de Santa Catarina for in vitro tests to evaluate tick resistance to acaricides. The viable engorged females were evenly distributed in petri dishes (n = 10 ticks). Females that showed no mobility were discarded. After these procedures, engorged females were immersed as described by Drummond and cols [3]. Ticks from each farm were divided into seven groups in order to test the effectiveness of six acaricides marketed in the region. The antiparasitic drugs were tested 2 as recommended by the manufacturers on the following groups: A (amitraz 1 ), B (cypermethrin, chlorpyrifos and citronellal 2 ), C (diazinon 3 ), D (dichlorvos and chlorpyrifos 4 ) E (cypermethrin 5 ), and F (cypermethrin, chlorpyrifos and piperonyl butoxide 6 ). The group G was used as control (only used distilled water) to validate the test. In each group, the ticks were submerged for a period of 30 seconds (average time a cattle takes to pass an immersion bath). Then, the ticks were dried to remove the product, and again housed into Petri dishes, and incubated for 7-15 days on BOD (27±1 C; RH 80±10%), to perform the posture. At the end of laying, egg mass was weighed and placed in test tubes closed with cotton plugs to allow air circulation and maintained on BOD (27±1 C, and RH= 80±10%) until larvae hatching. After 30 days it was performed the analysis of egg hatching with the help of stereoscopic to confirmed the presence of larvae. The 31 properties were evaluated individually. Data analysis took into account the weight of engorged females, number of postures, posture weight, and the eggs hatched using the methodology described by Drummond et al. [3]. In Brazil, for a new acaricidal drug to be licensed, it must show at least 95% of efficiency. RESULTS The results of the in vitro tests for tick susceptibility to all different chemical compounds (groups A-F) are presented in Table 1. The tests were valid for all farms, because the ticks in the control group (G) performed posture and eggs were fertile and able to hatch. The table shows that there was a variation from 0 to 100% in the test effectiveness between farms. The number of farms with populations of resistant ticks (efficacy < 95%) were 13 (41.9% - group A), 2 (6.45% - group B), 15 (48.38% - group C), 6 (19.35% - group D) and 12 (38.7% - group E). Ticks from the group F did not show resistance to the tested products. i.e., all farms obtained 100% efficiency. Each treatment had 31 replications with different tick populations and the results are presented as averages in Table 1. Among the products tested that showed the highest efficacy were the combinations of three or two active principle as cypermethrin, chlorpyrifos and piperonyl butoxide (group F - 100%), cypermethrin, chlorpyrifos and citronella (group B %), and dichlorvos and chlorpyrifos (group D %). However, products with only one active principle showed lower efficiency such as amitraz (group A %), diazinon (group C %), and cypermethrin (group E %).

4 R. Pazinato, V. Klauck, R.K. Grosskopf, et al Antiparasitic Resistance of Different Populations of ticks (Rhipicephalus microplus) Table 1. Degree of efficiency of acaricides against Rhipicephalus microplus collected from 31 different properties in the Santa Catarina State, Brazil. Degree of efficacy of products against ticks Farm Group A Group B Group C Group D Group E Group F MEAN % % % % % % Degree of efficiency calculated using the average efficiency obtained from different commercial formulations. Composition of groups:group A - amitraz, Group B - cypermethrin + chlorpyrifos + citronellal, Group C - diazinon, Group D -chlorpyrifos + dichlorvos, Group E - cypermethrin, Group F - cypermethrin + chlorpyrifos + piperonyl butoxide. The tests were reliable, because the control group (G) presented the results at the recommended technique. Results in bold (<95%), corresponding to anti-ticks ineffective and/or low efficacy on the population of ticks tested. 3

5 R. Pazinato, V. Klauck, R. K. Grosskopf, et al Antiparasitic Resistance of Different Populations of ticks (Rhipicephalus microplus) DISCUSSION These results are very similar to other studies conducted recently in different Brazilian regions [1,10,18], but unknown in Western Santa Catarina, which currently has the largest dairy herd in the Santa Catarina State. In this study it was found that farmers are unaware of important tests to control tick, like the immersion test of engorged females that contributes to parasite control, identifies ineffective drugs and reduces labor costs. As researchers reported the inappropriate use of antiparasitic drugs like subdosage or superdosage, and the high frequency of acaricide use, may select and propagate the occurrence of resistance selection pressure [5]. According to the literature, commercial products with only one active principle may have lower costs to farmers, but they promote quicker tick resistance [14,15]. Similarly to other studies, commercial products with a single active principle have higher rates of resistance such as amitraz, cypermethrin and diazinon, drugs commonly used in the farms. In the present study, amitraz obtained efficiency rates ranging from 41.5 to 100%. In this study, 18 farms showed R. microplus with 95% of sensitivity. On the other hand 13 farms had problems of amitraz resistance, which could be expected. That is because of the vast use of this drug by most farmers, which increases the selection pressure favoring the development of resistant ticks. As reported in the literature, the amitraz is one of the most popular acaricides used to control ticks on cattle in Australia, Latin America, and South Africa [9]. The resistance or low susceptibility of R. microplus to amitraz was also reported in the Brazilian states, such as Rio de Janeiro, Paraná, Minas Gerais, Rio Grande do Sul, and São Paulo [1,4,5,18,20]. In a study conducted in e Santa Catarina (plateau region) in the late 90`s, researchers found that amitraz was the only effective acaricide [17], a situation different from the observed in this study. In the West of Santa Catarina more than 50% of the tick populations were resistant to diazinon with ratios ranging from 0-100%. The results observed here are also expected because the organophosphates were the first commercially available acaricide. The resistance or low efficiency of diazinon against ticks were observed in other studies [13,19], as well as other organophosphorated drugs used in commercial products alone or associated [1,10,15,18]. In this study when using dichlorvos associated to chlorpyrifos, only six properties (19.3%) showed tick resistance, a much lower result than in São Paulo that reported 60% of tick resistance to this combination of active principles [18]. Researchers comparing acaricide-susceptible and acaricide-resistant tick s populations indicated an adaptation less of susceptible strain compared to resistant, so the use of acaricides should exercise caution while preserving its effectiveness for longer periods in order to delay resistance [2]. The susceptibility of ticks to cypermethrin was found in 18/31 farms, a product widely used in the control of ticks due to its low price. Among the tick populations was found efficiency index between 0 and 100%, and the average efficiency was 73.15% for cypermethrin. This result can be explained by the wide use of pyrethroids, a drug that is irreversible according to literature for R. microplus [1]. Resistance to pyrethroids is reported from the 80 s in Brazilian states [11,12]. However, R. microplus susceptibility was higher in the current study, when was combined with cypermethrin and organophosphates (chlorpyrifos, cypermethrin, and citronellol), where it was found that only two properties (6.4%) had less than 95% of efficiency, lower results than those reported by researchers in Southern Brazil (39% of farms) [1]. Another product based on this active principle when combined (cypermethrin, chlorpyrifos, and piperonyl butoxide) was 100%, proved to be the anti-tick of choice for farms with great problem of resistance to different drugs, as it was the case in this study where tick populations from farms 6 and 7 where the drug was only effective in vitro tests (>95%). The results showed, in vitro tests, which tick populations of the species R. microplus resistant to different active principles in the Western of Santa Catarina State, Brazil. Products with only one active principle as amitraz, cypermethrin and diazinon were those with lower efficacy (also interpreted as resistance), when compared to products that used a combination of two or three active principles. The current study shows that 81.7% of the tested tick population of parasitic resistance presented to one or more drugs. This study found that the cattle farmers were not aware of tests that detect ticks susceptibility to acaricides and did not adopt proper technical procedure when using chemicals. These indicate the need to educate cattle farmers on considering resistance as criteria, considering efficiency and cost of the treatment. 4

6 R. Pazinato, V. Klauck, R.K. Grosskopf, et al Antiparasitic Resistance of Different Populations of ticks (Rhipicephalus microplus) SOURCES AND MANUFACTURERS 1 Amitraz, Laboratório Bio-Vet S/A - Vargem Grande Paulista, SP, Brazil. 2 Couro Limpo, Noxon do Brasil química e farmacêutica LTDA, Cravinhos, SP, Brazil. 3 Diazinon, Laboratório Agener União- Embu-Guaçu, SP, Brazil. 4 Ectofós, Laboratório Vallée, Montes Claros, MG, Brazil. 5 Ectic, Tortuga Companhia Zootécnica Agrária, São Paulo, SP, Brazil. 6 Ciclorfós, Laboratório Alvet, Londrina, PR, Brazil. Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. REFERENCES 1 Camillo G., Vogel F.F., Sangioni L.A., Cadore G.C. & Ferrari R Eficiência in vitro de acaricidas sobre carrapatos de bovinos no Estado do Rio Grande do Sul, Brasil. Ciência Rural. 39(3): Dalla Rosa L., Moura A.B., Souza A.P., Bellato V., Sartor A.A., Trevisani N. & Scott F.B Biological parameters of two strains (acaricide resistant and susceptible) of the tick Rhipicephalus microplus. Semina: Ciências Agrárias. 34(6): Drummond R.O., Ernst S.T., & Trevino J.L Boophilus annulatus and Boophilus microplus: laboratory tests for insecticides. Journal of Economic Entomology. 66(1): Faustino M.A.G., Pena E.J.M. & Gurgel A.E.B Eficiência in vitro de produtos carrapaticidas em fêmeas ingurgitadas de cepas de Boophilus microplus da sub-região da Zona da Mata de Pernambuco. Revista Brasileira de Parasitologia Veterinária. 4(1): Furlong J. & Martins J.R.S Resistência dos carrapatos aos carrapaticidas. Juiz de Fora: Cnpgl-Embrapa 59: Gonzáles J.C O controle do carrapato dos bovinos. Porto Alegre: Editora Sulina, 103p. 7 Gauss C.L.B. & Furlong J Comportamento de larvas infestantes de Boophilus microplus em pastagem de Brachiaria decumbens. Ciência Rural. 32(3): Grisi L., Massard C.L., Moya-Borja G.E. & Pereira J.B Impacto das principais ectoparasitoses em bovinos no Brasil. Hora Veterinária. 21(1): Jonsson N.N. & Hope M Progress in the epidemiology and diagnosis of amitraz resistance in the cattle tick Boophilus microplus. Veterinary Parasitology. 146(2): Landim V.J.C., Silva E.A., Da Paes J.M.V., Fernandes L.O., Couto G.S., Fidalgo E.D.E.L., Silva N.L. & Furlong J Diagnóstico da situação da resistência do carrapato Boophilus microplus a carrapaticidas em bovinos de corte e leite na região de Uberaba. FAZU Revista. 3(1): Laranja R.J., Martins J.R. & Ceresér V.H Identificação de uma estirpe de Boophilus microplus resistente a carrapaticidas piretróides no Estado do Rio Grande do Sul. In: Seminário Brasileiro de Parasitologia Veterinária (Rio de Janeiro, Brasil). p Leite R.C Boophilus microplus (Canestrini, 1887): susceptibilidade, uso atual e retrospectivo de carrapaticidas em propriedades das regiões fisiografias da Baixada do Grande Rio e Rio de Janeiro. Uma abordagem epidemiológica. 122f. Tese (Doutorado em Parasitologia) - Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro. 13 Li A.Y., Davey R.B., Miller R.J. & George J.E Resistance to coumaphos and diazinon in Boophilus microplus (Acari: Ixodidae) and evidence for the involvement of an oxidative detoxification mechanism. Journal of Medical Entomology. 40(3): Martins J.R Manejo da resistência aos carrapaticidas. Revista Brasileira de Parasitologia Veterinária. 13(1): Monteiro S.G Parasitologia Veterinária. São Paulo: Roca, 352p. 16 Santos F.C.C. & Vogel F.S.F Resistência do carrapato Rhipicephalus (Boophilus) microplus frente ao amitraz e cipermetrina em rebanhos bovinos no Rio Grande do Sul de 2005 a Revista Portuguesa de Ciências Veterinária. 111(1): Souza A.P., Bellato V., Sartor A.A. & Kolling A Comparação da eficácia de carrapaticidas em testes a campo com o tempo de imersão in vitro. Revista de Ciências Agroveterinárias. 3(1):

7 R. Pazinato, V. Klauck, R. K. Grosskopf, et al Antiparasitic Resistance of Different Populations of ticks (Rhipicephalus microplus) 18 Ueno T.E.H., Mendes E.E.B., Pomaro S.H.K., Lima C.K.P., Guilloux A.G.A. & Mendes M.C Sensitivity profile of Rhipicephalus (boophilus) microplus ticks of dairy cattle to acaricides in small farms in the northwestern São Paulo state, Brazil. Arquivos do Instituto de Biologia. 79(1): Vargas M.S., Céspedes N.S., Sánches H.F., Martins J.R. & Céspedes C.O.C Avaliação in vitro de uma cepa de campo de Boophilus microplus (Acari: Ixodidae) resistente à amitraz. Ciência Rural. 33(5): Yamamura M.H. & Merlini L.S Estudo in vitro da resistência de Boophilus microplus a carrapaticidas na pecuária leiteira do norte do Estado do Paraná. Semina: Ciências Agrárias. 19(1):