Shell-vial culture and real-time PCR applied to Rickettsia typhi and Rickettsia felis detection
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Published on: October 11, 2022
Ferran Segura1, Immaculada Pons1, Júlia Pla2
1Corporació Sanitària Parc Taulí, Institut Universitari Parc Taulí - Universitat Autonoma de Barcelona, 1st Parc Taulí, 08208, Sabadell, Spain.
Abstract:
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.
Insights
Diagnosing Rickettsia typhi and Rickettsia felis infections is challenging. This study optimized shell-vial culture with real-time PCR for early detection and identified effective antibiotics for isolating these flea-borne zoonotic agents.
Area of Science:
- Microbiology
- Infectious Diseases
- Veterinary Entomology
Background:
- Murine typhus, caused by Rickettsia typhi, and Rickettsia felis infections present similar symptoms, complicating diagnosis.
- Early diagnosis of these intracellular bacterial infections is crucial to prevent severe outcomes and inappropriate treatment.
- Serological methods are ineffective in the early stages of infection due to the requirement for seroconversion.
Purpose of the Study:
- To optimize the shell-vial (SV) culture assay for detecting Rickettsia species using real-time PCR.
- To identify effective antibiotic combinations for isolating Rickettsia from fleas, minimizing bacterial contamination.
- To apply optimized methods for isolating R. felis from fleas obtained from a veterinary clinic.
Main Methods:
- Shell-vial cultures inoculated with R. typhi and R. felis were incubated at various temperatures and monitored using real-time PCR, Gimenez staining, and indirect immunofluorescence assay.
- Antibiotic efficacy for Rickettsia isolation from fleas was assessed by incubating microorganisms and fleas with different antibiotic concentrations.
- Optimized SV culture conditions were used to attempt R. felis isolation from field-collected flea samples.
Main Results:
- R. typhi grew at all tested temperatures, while R. felis grew optimally at 28 and 32°C.
- Real-time PCR demonstrated higher sensitivity and earlier detection of Rickettsia compared to classical methods.
- Specific antibiotics were identified as effective for isolating R. typhi (gentamicin, sulfamethoxazole, trimethoprim) and R. felis (gentamicin, streptomycin, penicillin, amphotericin B).
Conclusions:
- Optimized shell-vial culture coupled with real-time PCR offers a sensitive and rapid method for Rickettsia detection.
- The identified antibiotic regimens aid in the successful isolation of R. typhi and R. felis from fleas.
- Challenges remain in establishing R. felis cultures from field samples, likely due to suboptimal collection or storage conditions.
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