title: In vitro establishment of ivermectin-resistant Rhipicephalus microplus cell line and the contribution of ABC transporters on the resistance mechanism.
abstract: The cattle tick Rhipicephalus microplus is one of the most economically damaging livestock ectoparasites, and its widespread resistance to acaricides is a considerable challenge to its control. In this scenario, the establishment of resistant cell lines is a useful approach to understand the mechanisms involved in the development of acaricide resistance, to identify drug resistance markers, and to develop new acaricides. This study describes the establishment of an ivermectin (IVM)-resistant R. microplus embryonic cell line, BME26-IVM. The resistant cells were obtained after the exposure of IVM-sensitive BME26 cells to increasing doses of IVM in a step-wise manner, starting from an initial non-toxic concentration of 0.5 μg/mL IVM, and reaching 6 μg/mL IVM after a 46-week period. BME26-IVM cell line was 4.5 times more resistant to IVM than the parental BME26 cell line (lethal concentration 50 (LC50) 15.1 ± 1.6 μg/mL and 3.35 ± 0.09 μg/mL, respectively). As an effort to determine the molecular mechanisms governing resistance, the contribution of ATP-binding cassette (ABC) transporter was investigated. Increased expression levels of ABC transporter genes were found in IVM-treated cells, and resistance to IVM was significantly reduced by co-incubation with 5 μM cyclosporine A (CsA), an ABC transporter inhibitor, suggesting the involvement of these proteins in IVM-resistance. These results are similar to those already described in IVM-resistant tick populations, and suggest that similar resistance mechanisms are involved in vitro and in vivo. They reinforce the hypothesis that ABC transporters are involved in IVM resistance and support the use of BME26-IVM as an in vitro approach to study acaricide resistance mechanisms.
authors: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre 91501-970, RS, Brazil; Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Avenida Professor Lineu Prestes, 1374, São Paulo 05508-900, SP, Brazil.
authors: Paula C
authors: PC
authors: Pohl
authors: Departamento de Análises Especiais, SD&W Modelagem e Soluções Estratégicas Ltda., Rua João de Lacerda Soares, 316, São Paulo 04707-010, SP, Brazil.
authors: Danielle D
authors: DD
authors: Carvalho
authors: Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Avenida Professor Lineu Prestes, 1374, São Paulo 05508-900, SP, Brazil.
authors: Sirlei
authors: S
authors: Daffre
authors: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre 91501-970, RS, Brazil; Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9090, Porto Alegre 91540-000, RS, Brazil. Electronic address: itabajara.vaz@ufrgs.br.
authors: Itabajara da Silva
authors: Ida S
authors: Vaz
authors: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre 91501-970, RS, Brazil; Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre 91501-970, RS, Brazil.
authors: Aoi
authors: A
authors: Masuda
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D056810:Acaricides / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology*; D002460:Cell Line; D004351:Drug Resistance; D007559:Ivermectin / Q000494:pharmacology*; D048494:Rhipicephalus / Q000187:drug effects / Q000235:genetics*; D013984:Tick Infestations / Q000469:parasitology / Q000662:veterinary*
chemical_list: D018528:ATP-Binding Cassette Transporters; D056810:Acaricides; D007559:Ivermectin
keywords: ATP-binding cassette transporter; Ivermectin resistance; Rhipicephalus microplus; Tick cell line