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D. G. Dance, E. Simmons, B. Appukuttan, J. T. Stout, G. Punkosdy, K. Winthrop; Ocular Onchocerciasis (River Blindness): Can Wolbachia Infect Retinal Pigment Epithelial Cells in vitro?. Invest. Ophthalmol. Vis. Sci. 2008;49(13):830.
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The purpose of this study is to establish whether Wolbachia endobacteria can invade and survive within retinal pigment epithelial cells. To our knowledge, no study has assessed Wolbachia’s role in the pathogensesis and progression of chorioretinitis. In Vitro demonstration of Wolbachia survivability in a human retinal cell line would provide evidence for a potential role for Wolbachia in the pathogenesis of chorioretinitis.
Wolbachia were purified from adult female Brugia malayi worms. We used Baclight live-dead staining to demonstrate Wobachia viability. Purified Wolbachia were introduced to ARPE-19 cells and the cell lines were maintained for 2-10 days. Uninfected ARPE-19 cells and heat-killed Wolbachia treated ARPE-19 cultures served as a negative contols. Wolbachia infected ARPE-19 cells and control slides were prepared for confocal microscopy by staining actin myofilaments with AlexaFluor 594 to create a 3-D framework of RPE cell architecture and by using antibodies against Wolbachia-surface protein (WSP) to stain for Wolbachia. The ability of a confocal microscope to make serial, optical cross-sections through the stained framework of actin myofilaments was used to determine the location of the Wolbachia (intra-vs-extracellular).
Viable Wolbachia were successfully extracted from Brugia malayi as demonstrated by Baclight assay. Confocal microscopy of Wolbachia infected ARPE-19 cells demonstrated foci of intracellular Wolbachia in cells fixed on days 2 and 9. No intracellular bacteria were demonstrated in the heat-killed Wolbachia-infected cell cultures. All cell cultures exposed to Wolbachia remained healthy without evidence of cytotoxicity by the bacteria.
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