Marina E. Eremeeva

Quantitative Analyses of Variations in the Injury of Endothelial Cells Elicited by 11 Isolates of Rickettsia 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.

Rickettsia and Orientia.

Publisher Summary The term rickettsia has, for many years, been loosely applied to a very wide range of gram-negative bacteria simply because of their obligate association with arthropods or other hosts, their size, and their intracellular habitat. These microorganisms are, however, now known to be a highly polyphyletic group. Molecular approaches to the phylogeny of rickettsiae are used to demonstrate that most species in the genera Rickettsia, Orientia, Ehrlichia, Anaplasma, Wolbachia, Cowdria and Neorickettsia have similar evolutionary origins and that all these genera belong to the α subdivision of Proteobacteria. Micro-organisms in the genera Rickettsia and Orientia are obligately intracellular, gram-negative bacteria. The genus Rickettsia is classically separated into the typhus group and the spotted fever group based on the presence of distinct group lipopolysaccharide antigens. Improvements in isolation techniques for rickettsiae and widespread application of molecular tools for the identification of these fastidious micro-organisms have resulted in the recognition of some new species and other as yet unnamed genotypes. Orientia tsutsugamushi is the aetiological agent of scrub typhus, also known as tsutsugamushi disease. It is a single species that contains many antigenic and genotypic variants. Immunological techniques—such as complement fixation and fluorescent antibody tests with guinea pig or rabbit antisera; biological tests, including in vivo and in vitro neutralisation assays; and cross-vaccination protection tests—were originally used to define antigenic prototypes.