Quantitative Analyses of Variations in the Injury of Endothelial Cells Elicited by 11 Isolates ofRickettsia rickettsii

Abstract

Eleven isolates of spotted fever group rickettsiae from the blood of patients or ixodid ticks from North and South America were characterized. All isolates were identified as Rickettsia rickettsii using restriction fragment length polymorphism analysis of a 532-bp rOmpA gene fragment obtained by PCR. The ability of the R. rickettsii isolates to elicit cytopathic effects and parameters of oxidative injury were examined in cultured human EA.hy 926 endothelial cells. Cytopathic effects were determined by direct observation of infected cultures, by measuring the release of cytoplasmic lactate dehydrogenase (LDH), and by determination of intracellular pools of peroxide and reduced glutathione. Four biotypes of R. rickettsii were defined. Group I included two highly cytopathic isolates from Montana, Bitterroot and Sheila Smith, and three isolates from Maryland, North Carolina, and Brazil. These isolates rapidly damaged cells, released large amounts of cytoplasmic LDH, caused accumulation of intracellular peroxide, and depleted intracellular pools of reduced glutathione. Group II contained three isolates, two from Montana, Hlp#2 and Lost Horse Canyon, and an isolate from Colombia, which were similar to group I but caused either lower responses in LDH release or smaller changes in intracellular peroxide levels. The group III isolates, Sawtooth from Montana and 84JG from North Carolina, caused lower cellular injury by all measures. Group IV isolate Price T from Montana was the least cytopathic and caused minimal alterations of all parameters measured. Understanding the molecular basis for the varied cellular injury caused by different isolates of R. rickettsii may contribute to improved treatment of Rocky Mountain spotted fever and to the rapid identification of those isolates which are more likely to cause fulminant disease.

Molecular Typing of Isolates ofRickettsia rickettsiiby Use of DNA Sequencing of Variable Intergenic Regions

“…rickettsii isolates and DNA preparation. R. rickettsii isolates (Table 1) were cultivated in Vero cells (strain C1008; green monkey kidney cells) as described elsewhere (17). DNAs for isolates Brazil-A, 84JG, Hlp#2-A, Bitterroot, Colombia, Lost Horse Canyon, Morgan, PriceT, and Sheila Smith were prepared by phenol-chloroform extraction from partially purified R. rickettsii cells as described previously (16).…”

“…During his early work on the virulence of R. rickettsii, Price classified isolates recovered from D. andersoni ticks collected in Montana into four categories (R, S, T, and U) based on the different degrees of pathology observed in a guinea pig model of infection (38). An in vitro endothelial cell system for differentiating virulence properties of R. rickettsii has also been described (17). Anacker and colleagues also reported differences in the virulence of R. rickettsii isolates in the guinea pig model, grouping their isolates into three categories: isolates with highest virulence, lesser virulence, and lowest virulence (2,3).…”

Evaluation of a PCR Assay for Quantitation of Rickettsia rickettsii and Closely Related Spotted Fever Group Rickettsiae

“…Rickettsiae were grown in Vero cell monolayers, purified by centrifugation through a sucrose cushion, and stored in aliquots at Ϫ80°C in sucrose potassium glutamate buffer supplemented with 5 mM MgCl 2 and 1% Renografin as described elsewhere (2,5). The characteristics of R. rickettsii strains Bitterroot, Sheila Smith, Long Horse Canyon, 84JG, Morgan, Hlp#2, Brazil, Colombia, and Price T have been previously reported (6). The viable titer of purified R. rickettsii was determined by PFU assay according to the method of Wike and Burgdorfer (23).…”

Passive surveillance as an instrument to identify risk factors for fatal Rocky Mountain spotted fever: is there more to learn?

“…23 Epidemiologic explanations for some of the geographic differences in CFRs are explored later; however, significant genetic and phenotypic variation among isolates of Rickettsia rickettsii presumably contributes to the observed differences in severity of disease. 24 From 1935 through 1944, the annual CFR for RMSF was consistently above 20% and averaged 22%. 25 Beginning in 1944, the CFR declined steadily to less than 10% in 1949, a drop attributed to improvements in supportive therapy derived from a more complete understanding of the pathophysiology of RMSF and to the introduction of para-aminobenzoic acid as therapy for RMSF.…”