Improved plaque assays for Rickettsia prowazekii in Vero 76 cells

Abstract

Typhus group rickettsiae, including Rickettsia prowazekii and R. typhi, produce visible plaques on primary chick embryo fibroblasts and low-passage mouse embryo fibroblasts but do not form reproducible plaques on continuous cell culture lines. We tested medium overlay modifications for plaque formation of typhus group rickettsiae on the continuous fibroblast cell line Vero76. A procedure involving primary overlay with medium at pH 6.8, which was followed 2 to 3 days later with secondary overlay at neutral pH containing 1 g of emetine per ml and 20 g of NaF per ml, resulted in visible plaques at 7 to 10 days postinfection. A single-step procedure involving overlay with medium containing 50 ng of dextran sulfate per ml also resulted in plaque formation within 8 days postinfection. These assays represent reproducible and inexpensive methods for evaluating the infectious titers of typhus group rickettsiae, cloning single plaque isolates, and testing the susceptibilities of rickettsiae to antibiotics.

Immune response against rickettsiae: lessons from murine infection models

“…Rickettsiae can enter nearly all tissues and organs. Apart from ECs they also infect monocytes/MΦ [ 3 , 6 , 17 ] and non-immune cells including hepatocytes [ 18 , 19 ], smooth muscle cells [ 20 ], neurons [ 21 ] and fibroblasts that are commonly used for in vitro culture of rickettsiae [ 22 – 25 ]. Humans develop disease upon rickettsial infections within 10–14 days after inoculation.…”

Comparative analysis of host-cell signalling mechanisms activated in response to infection with Rickettsia conorii and Rickettsia typhi

“…This resulted in host-cell infection with a mean of 2.57±0.18 organisms (n53) for R. conorii and 3.22±0.12 organisms (n53) for R. typhi. The slightly higher rate of infection with R. typhi was probably due to underestimation of the R. typhi titre in stock preparations, as plaques generated by TG Rickettsia species tend to be smaller and comparatively less distinct than those resulting from infection with SFG organisms (Hackstadt, 1996;Policastro et al, 1996).…”

“…ml 21 ) (Rydkina et al, 2004). For R. typhi, the plaque assay procedure was modified to enhance the formation of resultant plaques by inclusion of dextran sulphate (50 ng ml 21 ; Sigma) in the agarosecontaining overlay medium and by the performance of all incubations at 35 uC (Policastro et al, 1996). The determination of extent of infection for different Rickettsia species was performed by real-time PCR using primer pair Rp877p and Rp1258n for the rickettsial citrate synthase gene (Regnery et al, 1991) and a TaqMan-based internal probe with 100 % homology to the target gene.…”

Rickettsia Rickettsii Infection of the Ea.Hy 926 Endothelial Cell Line: Morphological Response To Infection and Evidence for Oxidative Injury

“…Secondary 0·5 % agarose overlay in a volume equal to the first overlay and containing 2 pg emetine ml −1 and 40 pg NaF ml −1 (both from Sigma) was applied on day 2 p.i. as recommended by Policastro et al (1996). The effects of R. rickettsii were examined for 10 d p.i.…”

“…One millilitre of serial tenfold dilutions of rickettsial suspension was inoculated in triplicate into 12-well plates (Costar) and rickettsiae were allowed to adhere for 2 h at 35 "C. The inoculum was aspirated, infected cell monolayers were overlaid with 0.5 '/o agarose prepared in culture medium and plates were cultivated at 35 "C. Secondary 0.5 % agarose overlay in a volume equal to the first overlay and containing 2 pg emetine ml-l and 40 pg NaF ml-l (both from Sigma) was applied on day 2p.i. as recommended by Policastro et al (1996). The effects of R. rickettsii were examined for 10 d p i .…”