title: Establishment of transient and stable transfection systems for Babesia ovata.
abstract: BACKGROUND
  Bovine babesiosis is a tick-borne disease caused by several species of Babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. Babesia ovata is a benign species widespread in east Asian countries and causes anemia, particularly in cattle which are co-infected with Theileria orientalis. The development of genetic manipulation methods is necessary to improve our understanding of the basic biology of protozoan pathogens toward a better control of disease. Such tools have not been developed for B. ovata, and are the aim of this study.
  
  
  METHODS
  In this study we transfected constructs that were designed to evaluate the ability of several B. ovata promoter candidates to drive expression of a reporter luciferase. We found that the elongation factor-1 alpha intergenic region (ef-1α IG) and the actin 5' non-coding region (NR) had highest promoter activities. To establish a stable transfection system, we generated a plasmid construct in which the ef-1α IG promoter drives gfp expression, and the actin 5' NR mediates expression of the selectable marker hdhfr. The plasmid was designed for episomal transfection, as well as to integrate by double cross-over homologous recombination into the ef-1α locus. Circular or linearized plasmid was transfected by electroporation into in vitro cultured B. ovata and retention of the plasmid was facilitated by drug selection with 5 nM WR99210 initiated 48 h after transfection.
  
  
  RESULTS
  After one-week cultivation with WR99210, GFP-expressing parasites were observed by fluorescence microscopy. Integration of the plasmid construct into the ef-1α locus was confirmed by PCR, Southern blot analysis, and sequencing of recombination sites. These results confirm successful development of a stable transfection system for B. ovata.
  
  
  CONCLUSIONS
  The current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata.
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto 1-12-4, Nagasaki, 852-8523, Japan.
authors.forename: Hassan
authors.initials: H
authors.lastname: Hakimi
authors.affiliation: Research Center for Zoonosis Control, Hokkaido University, Sapporo, 001-0020, Japan.|Global Station for Zoonosis Control, GI-CoRE, Hokkaido University, Sapporo, 001-0020, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Yamagishi
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto 1-12-4, Nagasaki, 852-8523, Japan.|Leading Program, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, 852-8523, Japan.
authors.forename: Yuto
authors.initials: Y
authors.lastname: Kegawa
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto 1-12-4, Nagasaki, 852-8523, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Kaneko
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan. skawazu@obihiro.ac.jp.
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Kawazu
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto 1-12-4, Nagasaki, 852-8523, Japan. masahitoasada@nagasaki-u.ac.jp.
authors.forename: Masahito
authors.initials: M
authors.lastname: Asada
mesh_terms: D019677:Artificial Gene Fusion; D001403:Babesia / Q000235:genetics*; D020869:Gene Expression Profiling; D018390:Gene Targeting; D017930:Genes, Reporter; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D059765:Homologous Recombination; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014162:Transfection / Q000379:methods*
keywords: Babesia ovata; Bovine babesiosis; Double cross-over homologous recombination; Stable transfection