Culture of rickettsial organisms

Organisms that fall under the classification of rickettsial organisms typically fall within the genera Rickettsia, Orientia and Coxiella are obligate intracellular organisms, that require a host cell for culture. Other organisms that are sometimes similarly classified require similar culture techniques. As such the culture methods are like that of viral culture. In recent times Coxiella has been grown axenically, however this method is not recommended for primary isolation as not all Coxiella group types will amplify.

Rickettsial culture is usually employed for the purposes of diagnostics (live pathogen amplification), maintenance of rickettsial stocks and growth of rickettsial antigens. Culture must be done in a suitable laboratory environment that is able to contain Risk Group 3 (RG3) organisms. In many places, the allowed laboratory classification is a minimum of Biosafety Level 3 (BSL3). However, there has been a push to reduce the laboratory containment requirement to Biosafety Level 2 (BSL2) for low concentration amplification which will include primary cultures for certain rickettsial types/species.

Culture of rickettsial organisms utilizing viral culture techniques is usually conducted in tissue culture flasks with a suitable adherent cell line acting as the host. Antibiotic free media is utilized for these cultures as rickettsial organisms are bacteria and are thus susceptible to antibiotics traditionally used in viral culture. Host cell lines can be any cell line that is vulnerable to rickettsial infection at their preferred growth temperature and can come from a mammal (Vero), amphibian (XTC-2), insect (C6/36) or tick source. Another criterion for host cell selection is their susceptibility to cytopathic effect (CPE) by rickettsial organisms, for easy visualization when the rickettsial culture is ready for harvesting for subsequent processes. However, CPE is not commonly seen in primary isolations and a secondary screening technique will need to be implemented. This can be an immunofluorescence technique to visualize the rickettsiae directly or a PCR to detect their DNA.

Choice of host cell line is also determined by the target rickettsial organism's optimal growth temperature. Example for many members of Rickettsia spp, the optimal growth temperature is 28°C, and suitable cell lines are XTC-2, C6/36 or most tick cell lines. For rickettsial organisms that require a higher temperature, Veros are used for temperatures between 32-35°C. It is thus advisable to attempt isolation of a novel or unknown rickettsial organism from a source material in different host cell lines, to determine their host cell affinity and optimal growth temperature.

Source material can come from a range of different sources. The most common that are used include buffy coat, serum, tick hemolymph and vector/biopsy homogenate. Depending on source, additional steps to “clean” the source material (especially vector and biopsy) maybe needed to prevent contamination of culture. This may include cleaning the material in an appropriate disinfectant or adding an additional step such as filtration. To induce entry of rickettsial organisms into the host monolayer, an additional step of centrifugation at a low speed (10-15min at <500g) is recommended. The rickettsial primary culture should then be incubated in media containing 2-4x the normal concentration of antibiotics (pen/strep and gentamycin) for 24 hours before washing the cell layer with phosphate buffered saline (PBS) and changing the media to one without any antibiotics. The initial “antibiotic shock” in the first 24 hours is optional, as it helps to reduce the likelihood of contamination by unwanted microorganisms at the risk of lowering initial rickettsial inoculation numbers.

Rickettsial organisms have a long doubling time of approximately 8 hours and thus the culture needs to be maintained for at least 6 weeks, with media changes every 7 days. It is also recommended to test for rickettsial presence after 2 weeks of culture by doing an immunofluorescence (IFA) or PCR test of the media on a tiny scraping of the monolayer. Overgrowth of the monolayer is common, and the culture can be passaged as per normal into a fresh flask. Maintaining isolation culture attempts up to 16 weeks is not uncommon.