Shell-vial culture and real-time PCR applied to Rickettsia typhi and Rickettsia felis detection.
Murine typhus is a zoonosis transmitted by fleas, whose etiological agent is Rickettsia typhi. Rickettsia felis infection can produces similar symptoms. Both are intracellular microorganisms. Therefore, their diagnosis is difficult and their infections can be misdiagnosed. Early diagnosis prevents severity and inappropriate treatment regimens. Serology can't be applied during the early stages of infection because it requires seroconversion. Shell-vial (SV) culture assay is a powerful tool to detect Rickettsia. The aim of the study was to optimize SV using a real-time PCR as monitoring method. Moreover, the study analyzes which antibiotics are useful to isolate these microorganisms from fleas avoiding contamination by other bacteria. For the first purpose, SVs were inoculated with each microorganism. They were incubated at different temperatures and monitored by real-time PCR and classical methods (Gimenez staining and indirect immunofluorescence assay). R. typhi grew at all temperatures. R. felis grew at 28 and 32 °C. Real-time PCR was more sensitive than classical methods and it detected microorganisms much earlier. Besides, the assay sensitivity was improved by increasing the number of SV. For the second purpose, microorganisms and fleas were incubated and monitored in different concentrations of antibiotics. Gentamicin, sufamethoxazole, trimethoprim were useful for R. typhi isolation. Gentamicin, streptomycin, penicillin, and amphotericin B were useful for R. felis isolation. Finally, the optimized conditions were used to isolate R. felis from fleas collected at a veterinary clinic. R. felis was isolated at 28 and 32 °C. However, successful establishment of cultures were not possible probably due to sub-optimal conditions of samples.
Corporació Sanitària Parc Taulí, Institut Universitari Parc Taulí - Universitat Autonoma de Barcelona, 1st Parc Taulí, 08208, Sabadell, Spain.
Ferran
F
Segura
Corporació Sanitària Parc Taulí, Institut Universitari Parc Taulí - Universitat Autonoma de Barcelona, 1st Parc Taulí, 08208, Sabadell, Spain.
Immaculada
I
Pons
Veterinary Clinic "Centre", 53th Colom St., 08201, Sabadell, Spain.
Júlia
J
Pla
Corporació Sanitària Parc Taulí, Institut Universitari Parc Taulí - Universitat Autonoma de Barcelona, 1st Parc Taulí, 08208, Sabadell, Spain. mnogueras@tauli.cat.
María-Mercedes
MM
Nogueras
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002522:Chlorocebus aethiops; D042241:Early Diagnosis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012282:Rickettsia Infections / Q000175:diagnosis / Q000382:microbiology; D041161:Rickettsia felis / Q000187:drug effects / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D012286:Rickettsia typhi / Q000187:drug effects / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D005423:Siphonaptera / Q000382:microbiology; D013696:Temperature; D014437:Typhus, Endemic Flea-Borne / Q000175:diagnosis / Q000382:microbiology; D014709:Vero Cells
D000900:Anti-Bacterial Agents
Cellular culture; Fleas; Real-time PCR; Rickettsia felis; Rickettsia typhi; Shell vial assay