|Year : 2018 | Volume
| Issue : 4 | Page : 258-264
Approaches for the successful isolation and cell culture of American Rickettsia species
R Tello-Martin, K Dzul-Rosado, J Zavala-Castro, C Lugo-Caballero
Emerging and Re-emerging Diseases Laboratory, CIR Hideyo Noguchi, Universidad Autónoma de Yucatán (UADY), Mérida, México
|Date of Submission||20-Oct-2017|
|Date of Acceptance||03-May-2018|
|Date of Web Publication||18-Apr-2019|
Emerging and Re-emerging Diseases Laboratory, CIR Hideyo Noguchi. Av. Itzáes #490 x 59th Street, Postal Code 97000, Mérida, Yucatán
Source of Support: None, Conflict of Interest: None
Rickettsia are intracellular vector-borne bacteria, which are the etiologic agent of severe infections that could inflict death to their host. The intracellular behaviour of Rickettsia makes the study of its genetics, proteomics and cellular processes very difficult. Hence, isolation remains an important experimental technique that permits the obtention of important yields of bacteria, useful for a broad range of experiments. Isolation of Rickettsia using passages in animals or embryonated eggs has been described for long time; however, it was until the 1990s that faster and more feasible approaches for cell culture were developed. Current isolation approaches are mainly based on shell vial culture, that varies according to the media, atmosphere or temperature conditions. These variations have allowed the establishment of isolates from different pathogenic and non-pathogenic Rickettsia species, using arthropod, animal or human samples. Purification method of bacteria has also witnessed changes alongside the quantification of its load in the resulting isolates, from the laborious and time consuming plaque assays, to the routinary use of real-time polymerase chain reaction (qPCR), which is faster and more accurate. This review discusses various approaches that have been used for the isolation and purification of different Rickettsia species along with the mention of some successful examples. It indicated that a successful strategy for the isolation of Rickettsia requires a careful selection of media, cell lines and culture conditions which now are not as time consuming as used to be.
Keywords: Cell culture; clinical samples; isolation; Rickettsia; rickettsiosis
|How to cite this article:|
Tello-Martin R, Dzul-Rosado K, Zavala-Castro J, Lugo-Caballero C. Approaches for the successful isolation and cell culture of American Rickettsia species. J Vector Borne Dis 2018;55:258-64
|How to cite this URL:|
Tello-Martin R, Dzul-Rosado K, Zavala-Castro J, Lugo-Caballero C. Approaches for the successful isolation and cell culture of American Rickettsia species. J Vector Borne Dis [serial online] 2018 [cited 2019 Nov 18];55:258-64. Available from: http://www.jvbd.org/text.asp?2018/55/4/258/256560
| Introduction|| |
The Rickettsia genus (Family : Rickettsiaceae) includes several gram-negative, intracellular bacteria, that according to their membrane proteins are categorized into two main groups: Spotted fever group (SFG), and typhus group (TG). The members of the SFG are transmitted by ticks (Rickettsia rickettsii), fleas (R. felis) and mites (R. akari); are able to express the surface or outer membrane proteins OmpA and OmpB, have cross-reactivity with OX2 (Proteus vulgaris) and cause exanthematic fever in humans. The members of the TG include Rickettsia's that are transmitted by fleas (R. typhi) or louse (R. prowazekii); are able to express only OmpB, have cross-reactivity with OX19 (P. vulgaris) and cause typhus in humans.
The life cycle of these bacteria involves different species of hosts (mainly mammals) and arthropod vectors (ticks, fleas, louse mites),. Transmission is accidental for humans, and it occurs through the bite of an infected vector, blood transfusion or aerosol contamination, ,. Differential diagnosis is confirmed with several molecular techniques, including polymerase chain reaction (PCR) and serologic techniques like indirect immunofluorescence assay (IFA); however, isolation of the bacteria remains the definitive technique for diagnosis. Since, the obtention of highly pure bacterial culture is essential to study its virulence and pathogenicity, its genetic and proteomic characteristics and its antibiotic susceptibility; isolation can be considered a foundation stone for infectious diseases research, particularly for intracellular pathogens. In this review, different strategies that have allowed the culture and isolation of different Rickettsia species in America from different sample sources are described. It is important to note that laboratory accidents associated with the cultures of Rickettsia sp. marks the importance of proper training of the staff, adequate infrastructure and good practices in the laboratory safety and contention procedures,.
General considerations for the culture of Rickettsia sp.
Screening: Clinical samples and arthropod vectors have been widely used for isolation experiments once found positive by PCR,. When a single gene is used for diagnosis, sensitivity is low (33.3% with gltA); hence, two or more genes are used for amplification. The most used genes for diagnosis or screening include 16S, htrA, gltA, OmpA and OmpB. In such cases, sensitivity increases up to values between 93 (OmpA semi-nested + OmpB nested) and 100% (OmpA semi-nested + OmpB nested + gltA). A qualitative alternative for screening of positive arthropods and cultures is Giménez stain technique, which involves the processing of haemolymph samples with basic fuchsin stain against a counterstain of the background with malachite green.
Blood samples should be carefully collected to avoid haemolysis; and stored in tubes containing heparin as an anticoagulant, avoiding EDTA or sodium citrate. When a delay is expected in the processing of the sample, it should be stored at –80 °C. Otherwise, storage at 4 °C should work within the next 24–48 h after its proper shipment to the laboratory. Vector (arthropods) samples should be rinsed in 10% bleach (for 10 min) and then in 70% ethanol (for 5 min) before its dissection; if a delay is expected, samples should be frozen-rinsed in sucrose-phosphateglutamate buffer (pH 7.0) at –80 °C until its processing [Figure 1].
|Figure 1: General steps/workflow involved in the isolation of Rickettsia sp from different samples.|
Click here to view
Cell lines: Several successful attempts have been made for the isolation of Rickettsia species from yolk sacs of embryonated chicken eggs, however experimental results have shown that tissue culture cells provide more bacteria and are easier to purify,. Different cell lines have been used for initial isolation tests, including Vero ATCC-CCL81 (Cercopithecus aethiops kidney), L929 (Mus musculus fibroblasts), HEL (Human embryonic lung) [Table 1]. Particularly, Vero CCL-81 and L929 allows a fast isolation for highly infected samples; whereas the other mentioned cell types can sustain a prolonged incubation, with poorly infected samples. Tick cell lines have also been used to study differences in the proteomic and genomic expression among the vector and host,. Plaque formation by different Rickettsia species could be employed as a tool to choose a proper cell line for an isolation protocol,,. Contamination risk can be managed with proper antibiotic and antifungal use in the first 24–72 h of infection, however, its use is not recommended, as such compounds could inhibit several biochemical functions of animal cells (Vero, HEL and others) affecting the yield and quality of the isolate,,. Though, 0.2% penicillin-streptomycin plus 1% fungizone (amphotericin B) effectively works to limit contamination,. The optimal growth temperature for TG members is 35 °C whereas for SFG members it is 32 °C, with an average replication time of 8–10 h; replication gets remarkably slow under 32 °C and over 40 °C,. Additionally, an atmosphere of 5% CO2 is required for the optimal growth of Rickettsia during the log phase, as its lower concentration retards the bacteria proliferation which is useful for culture synchronization.
|Table 1: Culture requirements for isolation of different Rickettsia species|
Click here to view
Isolation techniques: Shell vial technique, a method originally employed for the isolation of cytomegalo virus, has been the gold standard for Rickettsia isolation and culture due to its versatility and efficiency,,,. In shell vial technique, only a small volume of cell and inocula are required, which are put in close contact through centrifugation, a critical step to enhance the penetration of the bacteria into the cells,. The only alternative method that has been proposed, requires serial passages on 24 well plates to obtain R. typhi isolates. This method is slower than shell vial, but is cheaper and guarantees higher success than shell vial technique, due to larger volume of cells and inocula; besides, the resulting isolate is more useful for subsequent experiments, for example, genomic analysis. Typically, Rickettsia can grow on several media optimized for the particular cell line with the corresponding supplementation. Nevertheless, several species with different requirements have been isolated using specific cell types. Obtaining >90% infection rate for the cells in a flask after three passages, is considered the standard for a good isolate, and regarded as “established” [Figure 1].
Purification and quantification of isolates: The purification and quantification of the isolates for its further use in experimentation or propagation is highly recommended. Typically, bacteria are released from cell cultures through sonication, and then purified by isopycnic density gradient centrifugation using sucrose or renografin for obtaining good yields of purified bacteria,. New methodologies to release the bacteria prior to purification, have described the use of digitonin instead of sonication to eliminate the risk of infection by aerosolized bacteria,,,. After the purification steps, the amplification of rOmpA and 16S genes by qPCR have been widely used to detect several species of Rickettsia in a variety of samples, including tissue, blood, cell cultures and ectoparasites, with a sensitivity and specificity similar or better to plaque assays,,. Therefore, qPCR can be considered as the optimal choice for diagnosis, detection or to quantify purified bacteria [Figure 1].
Isolation of spotted fever group members
Rickettsia rickettsii: Rickettsia rickettsii is the etiologic agent of the rocky mountain spotted fever (RMSF), which is one of the most severe human infection in the western hemisphere (USA, Mexico, South of Canadá, Central and South America). The lethality caused by this bacteria could vary from 10%, as reported in USA, to 30% as in developing countries like Mexico. Several tick species are implied as vectors of R. rickettsii, including Dermacentor andersoni and D. variabilis in USA; Amblyomma cajennense in South America and Rhipicephalus sanguineus in Mexico. There are several reports of experimental approaches for its cultivation and isolation, where in Vero or L929 cells have been the most used cells at a temperature ranging from 28 °C (blood specimens) to 34 °C (ticks specimens),,. Arthropod cell lines from ticks, like DAE3 (D. andersoni embryo), DALB3 (D. albipictus embryo), ISE6 (Ixodes scapularis embryo); or AeAl2 (Aedes albopictus), C636 (Ae. albopictus clone) from mosquitoes have also been successfully employed. For the cell line selection, it is important to note that temperature has a clear impact in the protein expression of R. rickettsii, as some proteins expressed at higher temperatures (34 °C) are absent at lower temperatures (28 °C) and vice versa. The replication time takes between 9–12 h and almost a week to have a vastly infected monolayer, in the Vero and L929 cell lines that have the fastest results; however HEL or MRC5 cell lines allows prolonged incubations which could be useful for some experiments. The infection with R. rickettsii can become highly pathogenic for cells, therefore, it must be closely monitored for acidification in the media (DMEM goes from red to orange or yellow) or cytopathic damage. As it has been mentioned, an alternative method using 24 well plates instead of shell vial has been successfully employed with A. parvum eggs for the isolation of R. rickettsii.
Rickettsia felis: It is an emerging rickettsial pathogen whose main vector and reservoir is Ctenocephalides felis, the cat flea. This pathogen is found in several arthropods, such as Achaeopsylla erinacei, Xeopsylla cheopis, Pulex irritans, R. sanguineus, and A. cajennense,,,, however, it has only been isolated from C. felis and Liposcelis bostrychophila,. The isolation assays of R. felis have been optimally performed on BHI medium and subconfluent XTC-2 or C6/26 cells, at 28 or 25 °C, respectively, using a shell vial method,. To date, there are no reports of human isolates. Alternatively, ISE6 cells in glucose/tryptose supplemented L15 medium can be used to infect samples from I. scapularis, at 32 °C for obtaining isolates on Day 14. It has also been reported than Sua5B (Anopheles gambiae) and Aa23 (An. albopictus) cell lines, can sustain the infection by R. felis, in the Schneider medium at 34 °C; however, this process is trickier, slower and more difficult to achieve. A similar difficulty is observed when using Vero cells at 28 and 32 °C, which results in a slower growth of the bacteria and more time consumption to obtain an isolate.
Rickettsia parkeri: The transmission of this rickettsial pathogen has been associated to A. maculatum along the gulf of Mexico coast; whereas in south America it has additionally been linked to A. triste,. Isolates of R. parkeri have been established using Vero E6 in MEM culture media at 34.5 °C for A. triste samples, and at 28 °C using same culture conditions. Its successful isolation has also been reported from a patient's eschar using Vero E6 in L-glutamine supplemented RPMI-1640 media at 34.5 °C.
Rickettsia akari: Rickettsial pox is a disease caused by R. akari, which is a cosmopolitan member of the SFG. Human cases have been registered in USA, Mexico and several European countries,,,,,,,,,,,,,,,,,,,,,,. Isolates have been obtained from whole blood samples of febrile patients, infected with R. akari MK (Kaplan) strain, after successful cell passages in chicken embryo. Recently, isolates have been obtained from a mite Allodermanyssus sanguineus (L. sanguineus) whose main reservoir is Mus musculus; and from patients' eschars in Vero E6 cells with RPMI supplemented with 5% fetal bovine serum (FBS) and 2 mM L-glutamine at 34.5 °C in presence of 5% CO2,,.
Isolation of typhi group members
Rickettsia typhi: Rickettsia typhi is the etiologic agent of murine typhus, which is a less severe disease compared to RMSF, yet capable of producing complications, like spleen rupture, endocarditis, neuropathies, etc that could lead a patient to death,. The mortality rate for murine typhus ranges near 4%, when no antibiotic therapy is administered. This pathogen is mainly transmitted by Xenopsylla cheopis, a rat flea, whose main reservoirs are Rattus norvegicus and R. rattus; however, its possible transmission by the flea, C. felis and the tick R. sanguineus has also been described,. Different isolates of R. typhi have been obtained from rodent, arthropod and human samples. The best strategy to obtain clinical isolates of R. typhi, requires the use of Vero cells growth in MEM complemented with FBS at 5%, at 33 °C with 5% CO2 atmosphere.
Rickettsia prowazekii: Rickettsia prowazekii, the causative agent of epidemic typhus is transmitted by the louse Pediculus humanus corporis. Infection is due to contamination of the bite site with arthropod faeces containing the bacteria. Humans are considered as the reservoirs of R. prowazekii; however, there are reports of other potential reservoirs like squirrels,,. The first isolates of this rickettsial pathogen have been obtained through an expensive and time consuming process that involved the use of embryonated chicken egg yolk sacs. To overcome these difficulties, this process of Rickettsia culture was later replaced by shell-vial isolation technique, which has been optimized to obtain clinical isolates from human blood using HEL and L929 cells, in MEM culture media at 34 °C, generally with the aid of antibiotics,.
Rickettsia bellii: Tick species infected with R. bellii includes D. variabilis and D. andersonii in the United States; A. cooperii, A. ovale and A. aureolatum in Brazil,,,. There is no evidence of human pathogenicity from R. bellii, that exhibit a slow growth in chicken embryo being innocuous for mice. However, isolates can cause cytopathic damage, and destroy Vero cell monolayers in few days (depending on the temperature of incubation); hence, indicating a possibility for human pathogenicity. Isolates in the study were obtained with BHI and MEM media supplemented with FBS (5%), at 28 °C.
| Conclusion|| |
Isolation of Rickettsia is a powerful experimental technique not only for diagnosis, but for the obtention of pure bacteria, which is a critical step for the success of genetic and proteomic experiments. Special conditions for the workflow with different species have been established, for an easier adjustment on a particular situation. The time consuming and difficult steps in these workflows have been minimized with more affordable techniques, that are within reach of many laboratories. To date, shell vial technique remains the standard for rickettsia isolation; however, serial dilutions in 24 well plaques could be considered as an interesting alternative due to its cheaper cost and the higher yield of bacteria. Vero cells on the other hand, supplemented with MEM at 28–34 °C are the most common elements on the isolation strategy for a given rickettsial pathogen.
| Acknowledgements|| |
This study was supported by the grant CB-2015-253559 given to Dr César Lugo Caballero from the National Council of Science and Technology (Conacyt), México.
| References|| |
Biggs HM, Behravesh CB, Bradley KK, Dahlgren FS, Drexler NA, Dumler JS, et al
. Diagnosis and management of tick borne rickettsial diseases: Rocky mountain spotted fever and other spotted fever group Rickettsioses, Ehrlichioses, and Anaplasmosis—United States. MMWR Recomm Rep
Raoult D, Roux V. Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev
1997; 10(4): 694–719.
Rizzo M, Mansueto P, Di Lorenzo G, Morselli S, Mansueto S, Rini GB. Rickettsial disease: Classical and modern aspects. New Microbiol
2004; 27(1): 87–103.
Oster CN, Burke DS, Kenyon RH, Ascher MS, Harber P, Pedersen CE Jr. Laboratory-acquired rocky mountain spotted fever: The hazard of aerosol transmission. N Engl J Med
Wells GM, Woodward TE, Fiset P, Hornick RB. Rocky mountain spotted fever caused by blood transfusion. JAMA
Lagier JC, Edouard S, Pagnier I, Mediannikov O, Drancourt M, Raoult D. Current and past strategies for bacterial culture in clinical microbiology. Clin Microbiol Rev
La Scola B, Raoult D. Laboratory diagnosis of rickettsioses: Current approaches to diagnosis of old and new rickettsial diseases. J Clin Microbiol
1997; 35(11): 2715–27.
Ammerman NC, Beier-Sexton M, Azad AF. Laboratory maintenance of Rickettsia rickettsii. Curr Protoc Microbiol
2008; Chapter 3: Unit–3A.5.
Paddock CD, Fournier PE, Sumner JW, Goddard J, Elshenawy Y, Metcalfe MG, et al
. Isolation of Rickettsia parkeri
and identification of a novel spotted fever group Rickettsia
sp. from Gulf Coast ticks (Amblyomma maculatum)
in the United States. Appl Environ Microbiol
2010; 76(9): 2689–96.
Sardelic S, Fournier PE, Punda Polic V, Bradaric N, Grgic D, Ivic I, et al
. First isolation of Rickettsia conorii
from human blood in Croatia. Croat Med J
2003; 44(5): 630–4.
Santibanez S, Portillo A, Santibanez P, Palomar AM, Oteo JA. Usefulness of rickettsial PCR assays for the molecular diagnosis of human rickettsioses. Enferm Infecc Microbiol Clin
Gimenez DF. Staining rickettsiae in yolk-sac cultures. Stain Technol
Weiss E. Growth and physiology of rickettsiae. Bacteriol Rev
1973; 37(3): 259–83.
Radulovic S, Feng HM, Morovic M, Djelalija B, Popov V, Crocquet-Valdes P, et al
. Isolation of Rickettsia akari
from a patient in a region where Mediterranean spotted fever is endemic. Clin Infect Dis
1996; 22(2): 216–20.
Turco J, Winkler HH. Isolation of Rickettsia prowazekii
with reduced sensitivity to gamma interferon. Infect Immun
1989; 57(6): 1765–72.
Birg ML, La Scola B, Roux V, Brouqui P, Raoult D. Isolation of Rickettsia prowazekii
from blood by shell vial cell culture. J Clin Microbiol
1999; 37(11): 3722–4.
Bell-Sakyi L, Zweygarth E, Blouin EF, Gould EA, Jongejan F. Tick cell lines: Tools for tick and tick-borne disease research. Trends Parasitol
2007; 23(9): 450–7.
Wike DA, Tallent G, Peacock MG, Ormsbee RA. Studies of the rickettsial plaque assay technique. Infect Immun
1972; 5(5): 715–22.
Dzul-Rosado K, Peniche-Lara G, Tello-Martin R, Zavala-Velazquez J, Pacheco Rde C, Labruna MB, et al. Rickettsia rickettsii
isolation from naturally infected Amblyomma parvum
ticks by centrifugation in a 24-well culture plate technique. Open Vet J
2013; 3(2): 101–5.
Weiss E, Moulder JW. The rickettsias and chlamydias. Order I Rickettsiales. In: Krieg NR, Holt JG, editors. Bergey's manual of systematic bacteriology
. Baltimore: Williams & Wilkins 1984; p. 687–729.
Roux V, Raoult D. Phylogenetic analysis of the genus Rickettsia
by 16S rDNA sequencing. Res Microbiol
1995; 146(5): 385–96.
Kopmans-Gargantiel AI, Wisseman CL Jr. Differential requirements for enriched atmospheric carbon dioxide content for intracellular growth in cell culture among selected members of the genus Rickettsia. Infect Immun
1981; 31(3): 1277–80.
Gouriet F, Fenollar F, Patrice J-Y, Drancourt M, Raoult D. Use of shell-vial cell culture assay for isolation of bacteria from clinical specimens: 13 years of experience. J Clin Microbiol
2005; 43(10): 4993–5002.
Quesada M, Sanfeliu I, Cardenosa N, Segura F. Ten years' experience of isolation of Rickettsia
spp. from blood samples using the shell-vial cell culture assay. Ann N Y Acad Sci
Walker DH. Rocky mountain spotted fever: A disease in need of microbiological concern. Clin Microbiol Rev
1989; 2(3): 227–40.
Zavala-Castro JE, Dzul-Rosado KR, Peniche-Lara G, Tello-Martin R, Zavala-Velazquez JE. Isolation of Rickettsia typhi
from human, Mexico. Emerg Infect Dis
2014; 20(8): 1411–2.
Pinter A, Labruna MB. Isolation of Rickettsia rickettsii
and Rickettsia bellii
in cell culture from the tick Amblyomma aureolatum
in Brazil. Ann N Y Acad Sci
Pacheco RC, Moraes-Filho J, Guedes E, Silveira I, Richtzenhain LJ, Leite RC, et al
. Rickettsial infections of dogs, horses and ticks in Juiz de Fora, southeastern Brazil, and isolation of Rickettsia rickettsii
from Rhipicephalus sanguineus
ticks. Med Vet Entomol
Horta MC, Labruna MB, Durigon EL, Schumaker TT. Isolation of Rickettsia felis
in the mosquito cell line C6/36. Appl Environ Microbiol
2006; 72(2): 1705–7.
Raoult D, La Scola B, Enea M, Fournier PE, Roux V, Fenollar F, et al
. A flea-associated Rickettsia
pathogenic for humans. Emerg Infect Dis
2001; 7(1): 73–81.
Pornwiroon W, Pourciau SS, Foil LD, Macaluso KR. Rickettsia felis
from cat fleas: Isolation and culture in a tick-derived cell line. Appl Environ Microbiol
2006; 72(8): 5589–95.
Sakamoto JM, Azad AF. Propagation of arthropod-borne Rickettsia
spp in two mosquito cell lines. Appl Environ Microbiol
Labruna MB, Whitworth T, Horta MC, Bouyer DH, McBride JW, Pinter A, et al. Rickettsia
species infecting Amblyomma cooperi
ticks from an area in the state of Sao Paulo, Brazil, where Brazilian spotted fever is endemic. J Clin Microbiol
Silveira I, Pacheco RC, Szabo MP, Ramos HG, Labruna MB. Rickettsia parkeri
in Brazil. Emerg Infect Dis
2007; 13(7): 1111–3.
Paddock CD, Sumner JW, Comer JA, Zaki SR, Goldsmith CS, Goddard J, et al. Rickettsia parkeri:
A newly recognized cause of spotted fever rickettsiosis in the United States. Clin Infect Dis
2004; 38(6): 805–11.
Paddock CD, Koss T, Eremeeva ME, Dasch GA, Zaki SR, Sumner JW. Isolation of Rickettsia akari
from eschars of patients with rickettsial pox. Am J Trop Med Hyg
2006; 75(4): 732–8.
Sonenshine DE, Bozeman FM, Williams MS, Masiello SA, Chadwick DP, Stocks NI, et al
. Epizootiology of epidemic typhus (Rickettsia prowazekii)
in flying squirrels. Am J Trop Med Hyg
1978; 27(2): 339–49.
Pacheco R, Rosa S, Richtzenhain L, Szabó MPJ, Labruna MB. Isolation of Rickettsia bellii
from Amblyomma ovale
and Amblyomma incisum
ticks from southern Brazil. Rev MVZ Córdoba
Aniskovich LP, Eremeeva ME, Balaeva NM, Ignatovich VF, Artemiev MI, Emelyanov VV, et al
. Methods for purification of Rickettsia prowazekii
separated from the host tissue: A step-by-step comparison. Acta Virol
1989; 33(4): 361–70.
Valarikova J, Sekeyova Z, Skultety L, Bohacsova M, Quevedo-Diaz M. New way of purification of pathogenic rickettsiae reducing health risks. Acta Virol
2016; 60(2): 206–10.
Eremeeva ME, Dasch GA, Silverman DJ. Evaluation of a PCR assay for quantitation of Rickettsia rickettsii
and closely related spotted fever group rickettsiae. J Clin Microbiol
2003; 41(12): 5466–72.
Giulieri S, Jaton K, Cometta A, Trellu LT, Greub G. Development of a duplex real-time PCR for the detection of Rickettsia
spp and typhus group Rickettsia
in clinical samples. FEMS Immunol Med Microbiol
2012; 64(1): 92–7.
Znazen A, Sellami H, Elleuch E, Hattab Z, Ben Sassi L, Khrouf F, et al
. Comparison of two quantitative realtime PCR assays for Rickettsia
detection in patients from Tunisia. PLoS Negl Trop Dis
2015; 9(2): e0003487.
Alvarez-Hernandez G, Roldan JFG, Milan NSH, Lash RR, Behravesh CB, Paddock CD. Rocky mountain spotted fever in Mexico: Past, present, and future. Lancet Infect Dis
2017; 17(6): e189–96.
Dumler JS, Walker DH. Rocky mountain spotted fever changing ecology and persisting virulence. N Engl J Med
2005; 353(6): 551–3.
Eremeeva ME, Zambrano ML, Anaya L, Beati L, Karpathy SE, Santos-Silva MM, et al. Rickettsia rickettsii
ticks, Mexicali, Mexico. J Med Entomol
2011; 48(2): 418–21.
Labruna MB, Ogrzewalska M, Soares JF, Martins TF, Soares HS, Moraes-Filho J, et al
. Experimental infection of Amblyomma aureolatum
ticks with Rickettsia rickettsii. Emerg Infect Dis
2011; 17(5): 829–34.
Eremeeva ME, Bosserman E, Zambrano M, Demma L, Dasch GA. Molecular typing of novel Rickettsia rickettsii
isolates from Arizona. Ann N Y Acad Sci
Policastro PF, Munderloh UG, Fischer ER, Hackstadt T. Rickettsia rickettsii
growth and temperature-inducible protein expression in embryonic tick cell lines. J Med Microbiol
1997; 46(10): 839–45.
Kelly PJ, Raoult D, Mason PR. Isolation of spotted fever group rickettsias from triturated ticks using a modification of the centrifugation-shell vial technique. Trans R Soc Trop Med Hyg
1991; 85(3): 397–8.
Perez-Osorio CE, Zavala-Velazquez JE, Arias Leon JJ, Zavala-Castro JE. Rickettsia felis
as emergent global threat for humans. Emerg Infect Dis
2008; 14(7): 1019–23.
Reif KE, Macaluso KR. Ecology of Rickettsia felis:
A review. J Med Entomol
2009; 46(4): 723–36.
Blanco JR, Perez-Martinez L, Vallejo M, Santibanez S, Portillo A, Oteo JA. Prevalence of Rickettsia felis-like
spp in Ctenocephalides felis
and Ctenocephalides canis
from La Rioja (Northern Spain). Ann N Y Acad Sci
Bitam I, Parola P, De La Cruz KD, Matsumoto K, Baziz B, Rolain JM, et al
. First molecular detection of Rickettsia felis
in fleas from Algeria. Am J Trop Med Hyg
2006; 74(4): 532–5.
Eremeeva ME, Warashina WR, Sturgeon MM, Buchholz AE, Olmsted GK, Park SY, et al. Rickettsia typhi
and R. felis
in rat fleas (Xenopsylla cheopis)
, Oahu, Hawaii. Emerg Infect Dis
2008; 14(10): 1613–5.
Sackal C, Laudisoit A, Kosoy M, Massung R, Eremeeva ME, Karpathy SE, et al. Bartonella
spp and Rickettsia felis
in fleas, Democratic Republic of Congo. Emerg Infect Dis
2008; 14(12): 1972–4.
Nava S, Elshenawy Y, Eremeeva ME, Sumner JW, Mastropaolo M, Paddock CD. Rickettsia parkeri
in Argentina. Emerg Infect Dis
2008; 14(12): 1894–7.
Zavala-Castro JE, Zavala-Velazquez JE, Peniche-Lara GF, Sulu Uicab JE. Human rickettsial pox, southeastern Mexico. Emerg Infect Dis
2009; 15(10): 1665–7.
Shankman B. Report on an outbreak of endemic febrile illness, not yet identified, occurring in New York City. N Y State J Med
Huebner RJ, Jellison WL, Pomerantz C. Rickettsial pox, a newly recognized rickettsial disease; isolation of a Rickettsia
apparently identical with the causative agent of rickettsial pox from Allodermanyssus sanguineus
, a rodent mite. Public Health Rep
1946; 61(47): 1677–82.
Civen R, Ngo V. Murine typhus: An unrecognized suburban vector-borne disease. Clin Infect Dis
2008; 46(6): 913–8.
Blanton LS, Idowu BM, Tatsch TN, Henderson JM, Bouyer DH, Walker DH. Opossums and cat fleas: New insights in the ecology of murine typhus in Galveston, Texas. Am J Trop Med Hyg
2016; 95(2): 457–61.
Dzul-Rosado K, Lugo-Caballero C, Tello-Martin R, Lopez-Avila K, Zavala-Castro J. Direct evidence of Rickettsia typhi
infection in Rhipicephalus sanguineus
ticks and their canine hosts. Open Vet J
2017; 7(2): 165–9.
Liu WT. Isolation of typhus rickettsiae from rat mites, Liponyssus bacoti
, in Peiping. Am J Hyg
1947; 45(1): 58–66.
Bechah Y, Capo C, Mege JL, Raoult D. Epidemic typhus. Lancet Infect Dis
2008; 8(7): 417–26.
Duma RJ, Sonenshine DE, Bozeman FM, Veazey JM Jr, Elisberg BL, Chadwick DP, et al
. Epidemic typhus in the United States associated with flying squirrels. JAMA
1981; 245(22): 2318–23.
Rachek LI, Tucker AM, Winkler HH, Wood DO. Transformation of Rickettsia prowazekii
to rifampin resistance. J Bacteriol
1998; 180(8): 2118–24.
Philip RN, Casper EA, Anacker RL, Cory J, Hayes SF, Burgdorfer W, et al. Rickettsia bellii
sp nov.: A tick-borne Rickettsia
, widely distributed in the United States, that is distinct from the spotted fever and typhus biogroups. Int J Syst Evol Microbiol
1983; 33(1): 94–106.