Adaptation of archival egg-cultured Orientia tsutsugamushi to L929 mammalian cells enables persistent qPCR-detectable growth

Abstract

Rickettsial infections are a leading cause of febrile illness in Southeast Asia and Malaysia, although they are often underreported. Rickettsial pathogens largely fall within the genera Rickettsia and Orientia and classified within the Rickettsiaceae family. In Malaysia, scrub typhus, caused by Orientia tsutsugamushi, is the most frequently reported rickettsial infection. Traditionally, rickettsial organisms have been isolated and cultured from embryonated eggs. However, this method is labor and skill intensive, has limited scalability, and requires specialized equipment, making it less accessible to many laboratories. An alternative approach is to use mammalian/amphibian/invertebrate cell culture as a host for rickettsial propagation. In this study, we evaluated the potential for culturing rickettsial pathogens previously adapted and maintained in embryonated eggs in mammalian cell lines. Two mammalian cell lines (Vero E6 and L929) were inoculated with rickettsial strains previously identified as O. tsutsugamushi (strains Karp [n=8], Kato [n=4], and Gilliam [n=2]). The presence of O. tsutsugamushi was assessed by quantitative qPCR at 14-day intervals. After 90 days of culture, only one of the fifteen isolates (GL94) showed evidence of propagation in L929 cells, whereas O. tsutsugamushi DNA remained below the qPCR detection limit in Vero E6 cells for all isolates tested. Most of the other isolates showed little to no growth, with some exhibiting the presence of other bacteria. The identification and morphology of GL94 were confirmed via transmission electron microscopy (TEM), followed by full-length 16S sequencing. This study highlights the challenges of transitioning rickettsial culture from embryonated eggs to mammalian cell cultures.