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Culture and Phenotypic Characterization of a Wolbachia pipientis Isolate

Unité des Rickettsies, Université de la Méditerranée, Faculté de Médecine, CNRS UPRESA 6020, 13385 Marseille, France,¹ Department of Pathology, The John Hopkins University School of Medicine, Baltimore, Maryland 21287,² Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom³

Received 19 May 2003/ Returned for modification 18 August 2003/ Accepted 8 September 2003

The recent isolation of Wolbachia pipientis in the continuous cell line Aa23, established from eggs of a strain of the Asian tiger mosquito Aedes albopictus, allowed us to perform extensive characterization of the isolate. Bacterial growth could be obtained in C6/36, another A. albopictus cell line, at 28°C and in a human embryonic lung fibroblast monolayer at 28 and 37°C, confirming that its host cell range is broader than was initially thought. The bacteria were best visualized by Diff-Quik and May-Grünwald-Giemsa staining. Proteins from 213 to 18 kDa with two major protein bands of 65 and 25 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By Western blotting with specific polyclonal mouse and rabbit antisera, dominant immunoreactive antigens were found at approximately 100, 80, and 30 kDa. The genome size was calculated to be 1,790 ± 17 kb by pulsed-field gel electrophoresis. The sequence of the citrate synthase gene (gltA) of W. pipientis was determined by gene walking. Its position in the phylogenetic tree constructed with gltA confirmed that found in a phylogenetic tree constructed with 16S rRNA genes and that it belongs in the subgroup of the class Proteobacteria and that it is closely related to but independent from the genera Ehrlichia, Anaplasma, and Neorickettsia.

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