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.
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Ben-Zvi I, Meltzer E, Nogueras MM, Segura F, Bank I (2010) First detection of human infection with Rickettsia felis in Israel. Am J Med Sci 340:343
Centers for Disease Control and Prevention (CDC) (2009) Outbreak of Rickettsia typhi infection—Austin, Texas 2008. Morb Mortal Wkly Rep 58:1267–1270
Dzul-Rosado K, González-Martínez P, Peniche-Lara G, Zavala-Velázquez J, Zavala-Castro J (2013) Murine typhus in humans, Yucatan, Mexico. Emerg Infect Dis 19:1021–1022
Gillespie JJ, Ammerman NC, Beier-Sexton M, Sobral BS, Azad AF (2009) Louse- and flea-borne rickettsioses: biological and genomic analyses. Vet Res 40:12
Henry KM, Jiang J, Rozmajzl PJ, Azad AF, Macaluso K, Richards AL (2007) Development of quantitative real-time PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of Murine typhus and flea-borne spotted fever. Mol Cell Probes 21:17–23
Hii SF, Kopp SR, Thompson MF, O’Leary CA, Rees RL, Traub RJ (2011) Molecular evidence of Rickettsia felis infection in dogs from Northern Territory, Australia. Parasit Vectors 4:198
Horta MC, Labruna MB, Durigon EL, Schumaker TT (2006) Isolation of Rickettsia felis in the mosquito cell line C6/36. Appl Environ Microbiol 72:1705–1707
Hun L, Troyo A, Taylor L, Barbieri AM, Labruna MB (2011) First report of the isolation and molecular characterization of Rickettsia amblyommii and Rickettsia felis in Central America. Vector Borne Zoonotic Dis 11:1395–1397
Jiang J, Chan TC, Temenak JJ, Daxh GA, Ching W, Richards AL (2004) Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi. Am J Trop Med Hyg 70:351–356
La Scola B, Raoult D (1996) Diagnosis of Mediterranean spotted fever by cultivation of Rickettsia conorii from blood and skin samples using the centrifugation-shell vial technique and by detection of R. conorii in circulating endothelial cells: a 6-year follow-up. J Clin Microbiol 34:2722–2727
Marrero M, Raoult D (1989) Centribufacion-shell vial technique for rapid detection of Mediterranean spotted fever Rickettsia in blood culture. Am J Trop Med Hyg 40:197–799
McLeod MP, Qin X, Karpathy SE, Gioia J, Highlander SK, Fox GE, McNeill TZ, Jiang H, Muzny D, Jacob LS, Hawes AC, Sodergren E, Gill R, Hume J, Morgan M, Fan G, Amin AG, Gibbs RA, Hong C, Yu X, Walker DH, Weinstock GM (2004) Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae. J Bacteriol 186:5842–5855
Nilsson K, Wallménius K, Hartwig S, Norlander T, Påhlson C (2014) Bell’s palsy and sudden deafness associated with Rickettsia spp. infection in Sweden. A retrospective and prospective serological survey including PCR findings. Eur J Neurol 21:206–214
Nogueras MM, Pons I, Ortuño A, Segura F (2009) Seroprevalence of Rickettsia typhi and Rickettsia felis in dogs of Northeastern Spain. Clin Microbiol Infect 15:237–238
Nogueras MM, Pons I, Ortuño A, Lario S, Segura F (2011) Rickettsia felis in fleas from Catalonia. Northeastern Spain. Vector Borne Zoonotic Dis 11:479–483
Nogueras MM, Pons I, Ortuño A, Miret J, Pla J, Castella J, Segura F (2013a) Molecular detection of Rickettsia typhi in cats and fleas. PLoS One 8:e71386
Nogueras MM, Pons I, Pla J, Ortuño A, Miret J, Sanfeliu I, Segura F (2013b) The role of dogs in the eco-epidemiology of Rickettsia typhi, etiological agent of Murine typhus. Vet Microbiol 163:97–102
Phongmany S, Rolain JM, Phetsouvanh R, Blacksell SD, Soukkhaseum V, Rasachack B, Phiasakha K, Soukkhaseum S, Frichithavong K, Chu V, Keolouangkhot V, Martinez-Aussel B, Chang K, Darasavath C, Rattanavong O, Sisouphone S, Mayxay M, Vidamaly S, Parola P, Thammavong C, Heuangvongsy M, Syhavong B, Raoult D, White NJ, Newton PN (2006) Rickettsial infections and fever, Vientiane, Laos. Emerg Infect Dis 12:256–262
Pornwiroon W, Pourciau SS, Foil LD, Macaluso KR (2006) Rickettsia felis from cat fleas: isolation and culture in a tick-derived cell line. Appl Environ Microbiol 72:5589–5595
Quesada M, Sanfeliu I, Cardeñosa N, Segura F (2006) Ten years’ experience of isolation of Rickettsia spp. from blood samples using the shell-vial cell culture assay. Ann NY Acad Sci 1078:578–581
Radulovic S, Higgins JA, Jaworski DC, Dasch GA, Azad AF (1995) Isolation, cultivation, and partial characterization of the ELB agent associated with cat fleas. Infect Immun 63:4826–4829
Rahman MS, Ammerman NC, Sears KT, Ceraul SM, Azad AF (2010) Functional characterization of a phospholipase A(2) homolog from Rickettsia typhi. J Bacteriol 192:3294–3303
Raoult D, La Scola B, Enea M, Fournier PE, Roux V, Fenollar F, Galvao MA, de Lamballerie X (2001) A flea-associated Rickettsia pathogenic for humans. Emerg Infect Dis 7:73–81
Stevenson HL, Labruna MB, Montenieri JA, Kosoy MY, Gage KL, Walker DH (2005) Detection of Rickettsia felis in a New World flea species, Anomiopsyllus nudata (Siphonaptera: Ctenophthalmidae). J Med Entomol 42:163–167
Thepparit C, Sunyakumthorn P, Guillotte ML, Popov VL, Foil LD, Macaluso KR (2011) Isolation of a rickettsial pathogen from a non-hematophagous arthropod. PLoS One 6:e16396
Trilar T, Radulovic S, Walker DH (1994) Identification of a natural cycle involving Rickettsia typhi infection of Monopsyllus sciurorum sciurorum fleas from the nests of the fat dormouse (Glis glis). Eur J Epidemiol 10:757–762
Uchiyama T, Ogawa M, Kishi M, Yamashita T, Kishimoto T, Kurane I (2009) Restriction of the growth of typhus group rickettsiae in tick cells. Clin Microbiol Infect 15:332–333
Vestris G, Rolain JM, Fournier PE, Birg ML, Enea M, Patrice JY, Raoult D (2003) Seven years’ experience of isolation of Rickettsia spp. from clinical specimens using the shell vial cell culture assay. Ann NY Acad Sci 990:371–374
Weiss E, Coolbaugh JC, Williams JC (1975) Separation of viable Rickettsia typhi from yolk sac and L cell host components by renografin density gradient centrifugation. Appl Microbiol 30:456–463
Zavala-Castro J, Zavala-Velázquez J, Walker D, Pérez-Osorio J, Peniche-Lara G (2009) Severe human infection with Rickettsia felis associated with hepatitis in Yucatan, Mexico. Int J Med Microbiol 299:529–533
Znazen A, Hammami B, Mustapha AB, Chaari S, Lahiani D, Maaloul I, Jemaa MB, Hammami A (2013) Murine typhus in Tunisia. A neglected cause of fever as a single symptom. Med Mal Infect 43:226–229
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This study was supported by the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), and the Instituto de Salud Carlos III (Grant PI12/02308).
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Segura, F., Pons, I., Pla, J. et al. Shell-vial culture and real-time PCR applied to Rickettsia typhi and Rickettsia felis detection. World J Microbiol Biotechnol 31, 1747–1754 (2015). https://doi.org/10.1007/s11274-015-1925-z
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DOI: https://doi.org/10.1007/s11274-015-1925-z