title: Use of Percoll gradients to purify Anaplasma marginale (Rickettsiales: Anaplasmataceae) from tick cell cultures.
abstract: Anaplasma marginale (Rickettsiales: Anaplasmataceae) is an obligate intracellular bacterium that multiplies exclusively within membrane-bound vacuoles in the cytoplasm of host cells. A number of A. marginale isolates can be propagated in the Ixodes scapularis IDE8 tick cell line, which provides a reliable source of antigens for a wide variety of studies. However, because of its intracellular nature, separation of bacteria from host cell materials remains an important constraint for researchers. In the present study, we evaluated the use of Percoll gradients for purification of two Brazilian strains of A. marginale grown in IDE8 tick cells. The purified A. marginale monitored in Giemsa-stained smears contained only minimal amounts of IDE8 cell stroma. The total protein yields were 1.2mg and 1.7mg, while the DNA titers quantified with real-time PCR were 6.4×10(9) for UFMG1 and 4.87×10(9) for UFMG2 copies in the purified material, respectively. Additionally, we confirmed the viability of purified bacteria by infecting tick cells after being freshly purified and after retrieval from long-term storage. Importantly, the viability of the organisms is preserved after use of this separation method, and therefore the purified organisms can be used in enzymatic assays and other research approaches where live organisms would be preferred.
authors.affiliation: Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstrasse 5, 80802 Munich, Germany.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Lis
authors.affiliation: Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstrasse 5, 80802 Munich, Germany.
authors.forename: Nour
authors.initials: N
authors.lastname: Najm
authors.affiliation: SaBio, Instituto de Investigación de Recursos Cinegéticos, IREC-CSIC-UCLM-JCCM, 13005 Ciudad Real, Spain; Department of Veterinary Pathobiology, Center for Veterinary Health Sciences Oklahoma State University, Stillwater, OK 74078, USA.
authors.forename: José
authors.initials: J
authors.lastname: de la Fuente
authors.affiliation: SaBio, Instituto de Investigación de Recursos Cinegéticos, IREC-CSIC-UCLM-JCCM, 13005 Ciudad Real, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Fernández de Mera
authors.affiliation: Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstrasse 5, 80802 Munich, Germany.
authors.forename: Erich
authors.initials: E
authors.lastname: Zweygarth
authors.affiliation: Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstrasse 5, 80802 Munich, Germany.
authors.forename: Kurt
authors.initials: K
authors.lastname: Pfister
authors.affiliation: Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstrasse 5, 80802 Munich, Germany; Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte 30.161-970, Brazil. Electronic address: lygiapassos@yahoo.com.
authors.forename: Lygia M F
authors.initials: LM
authors.lastname: Passos
mesh_terms: D000709:Anaplasma / Q000502:physiology*; D000818:Animals; D001431:Bacteriological Techniques; D002460:Cell Line; D011205:Povidone / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry*; D013987:Ticks / Q000166:cytology*
chemical_list: C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone
keywords: Anaplasma marginale; Intracellular bacteria; Percoll gradients; Purification