Chulalongkorn Medical Journal


Objective : The biological roles of Wolbachia, a filarial intracellular bacteria, have promoted applied researches in lymphatic filariasis. Analysis of Wolbachia proteins will provide complementary information on the biology of Wolbachia, as well as the immune mechanisms against filarial nematodes. We therefore developed a Wolbachia isolation method from Dirofilaria immitis (dog heartworm) for protein analysis. Methods : Wolbachia bacteria were homogenized and isolated by fractioned centrifugation by using 0.85% NaCl supplemented with varying concentration of Nonidet P-40 (NP-40) as the homogenization buffer. The purity of Wolbachia extracts were analyzed by sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE), and by quantitative polymerase chain reaction (qPCR) targeting the single copy of the Wolbachia surface protein (wsp) gene. The contaminations from filarial extracts were analyzed by qPCR targeting the filarial glutathione-S-transferase (gst) gene. Results : We were able to isolate Wolbachia bacteria from D. immitis with high purity. The higher concentration of NP-40 could increase the yield of bacterial isolation and decrease the host parasite contaminations. Conclusion : The homogenization buffer which provided the highest Wolbachia protein concentration, but the lowest filarial contamination, was 0.85% NaCl + 0.08% NP-40. This technique is likely to provide the most suitable methods for Wolbachia isolation to facilitate the proteomic studies. However, our purification procedures do need validation in other applications, which require intact Wolbachia DNA materials, and/or viable cells.



First Page


Last Page




To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.