title: In vitro cultivation and cryopreservation of Babesia bigemina sporokinetes in hemocytes of Rhipicephalus microplus.
abstract: Cultures of tick hemocytes represent alternative cell lines for the isolation and cultivation of a variety of hemoparasites. The present study reports the development and evaluation of methods for the in vitro culture and maintenance of sporokinetes of Babesia bigemina in association with hemocytes of the tick Rhipicephalus microplus. Hemolymph, from engorged females infected with B. bigemina sporokinetes, was incubated at 28 °C in L15 culture medium supplemented with 40% fetal bovine serum. Adherence of hemocytes to flask surfaces and the development of B. bigemina sporokinetes commenced on the first day of cultivation. The protozoa demonstrated clear motility and the capacity to adhere to hemocyte membranes for up to 25 days, at which time the hemocytes began to show signs of degeneration. Examination of Giemsa stained hemocyte cultures, revealed the presence of pyriformis forms, as well as mature and immature sporokinetes with dark red nuclei, centralized or near the apical extremities. Sporokinetes harvested from culture supernatants were cryopreserved in liquid nitrogen. Inoculation of parasite-free hemocyte cultures with defrosted sporokinetes, demonstrated the viability and interaction of the protozoa with the hemocytes over 21 days. Cultured hemocytes of R. microplus hold potential for development as a tool in the study of host parasite interactions and as a substrate for the in vitro maintenance of B. bigemina sporokinetes.
authors.affiliation: Universidade Federal Rural do Rio de Janeiro Seropédica, BR 465, Km 7, CEP 23897-000, Seropédica, RJ, Brazil. Electronic address: rezendejania@gmail.com.
authors.forename: Jania
authors.initials: J
authors.lastname: de Rezende
authors.affiliation: Universidade Federal Rural do Rio de Janeiro Seropédica, BR 465, Km 7, CEP 23897-000, Seropédica, RJ, Brazil.
authors.forename: Charles P
authors.initials: CP
authors.lastname: Rangel
authors.affiliation: Universidade Federal Rural do Rio de Janeiro Seropédica, BR 465, Km 7, CEP 23897-000, Seropédica, RJ, Brazil.
authors.forename: Douglas
authors.initials: D
authors.lastname: McIntosh
authors.affiliation: Universidade Federal de Minas Gerais, Departamento de Parasitologia, ICB/UFMG, Belo Horizonte, Brazil.
authors.forename: Júlia A G
authors.initials: JA
authors.lastname: Silveira
authors.affiliation: Universidade Federal Rural do Rio de Janeiro Seropédica, BR 465, Km 7, CEP 23897-000, Seropédica, RJ, Brazil.
authors.forename: Nathalie C
authors.initials: NC
authors.lastname: Cunha
authors.affiliation: Universidade Federal de Mato Grosso do Sul, Faculdade Medicina Veterinária e Zootecnia, Campo Grande, MS, Brazil.
authors.forename: Carlos A N
authors.initials: CA
authors.lastname: Ramos
authors.affiliation: Universidade Federal Rural do Rio de Janeiro Seropédica, BR 465, Km 7, CEP 23897-000, Seropédica, RJ, Brazil.
authors.forename: Adivaldo H
authors.initials: AH
authors.lastname: Fonseca
mesh_terms: D000818:Animals; D001403:Babesia / Q000302:isolation & purification*; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D006434:Hemocytes / Q000469:parasitology*; D008297:Male; D048494:Rhipicephalus / Q000469:parasitology*
keywords: Babesia spp.; Cryopreservation; Tick hemocytes