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Table of Contents
REVIEW ARTICLE
Year : 2018  |  Volume : 55  |  Issue : 4  |  Page : 258-264

Approaches for the successful isolation and cell culture of American Rickettsia species


Emerging and Re-emerging Diseases Laboratory, CIR Hideyo Noguchi, Universidad Autónoma de Yucatán (UADY), Mérida, México

Date of Submission20-Oct-2017
Date of Acceptance03-May-2018
Date of Web Publication18-Apr-2019

Correspondence Address:
C Lugo-Caballero
Emerging and Re-emerging Diseases Laboratory, CIR Hideyo Noguchi. Av. Itzáes #490 x 59th Street, Postal Code 97000, Mérida, Yucatán
México
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.256560

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  Abstract 


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 Top


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)[1]. 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[1].

The life cycle of these bacteria involves different species of hosts (mainly mammals) and arthropod vectors (ticks, fleas, louse mites)[2],[3]. Transmission is accidental for humans, and it occurs through the bite of an infected vector, blood transfusion or aerosol contamination[1], [4],[5]. 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[1]. 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[6]. 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[7],[8].

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[9],[10]. 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)[11]. 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[12].

Blood samples should be carefully collected to avoid haemolysis; and stored in tubes containing heparin as an anticoagulant, avoiding EDTA or sodium citrate[7]. 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[6]. 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[6] [Figure 1].
Figure 1: General steps/workflow involved in the isolation of Rickettsia sp from different samples.

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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[8],[13]. Different cell lines have been used for initial isolation tests, including Vero ATCC-CCL81 (Cercopithecus aethiops kidney)[14], L929 (Mus musculus fibroblasts)[15], HEL (Human embryonic lung)[16] [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[7]. Tick cell lines have also been used to study differences in the proteomic and genomic expression among the vector and host[6],[17]. Plaque formation by different Rickettsia species could be employed as a tool to choose a proper cell line for an isolation protocol[6],[13],[18]. 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[8],[9],[19]. Though, 0.2% penicillin-streptomycin plus 1% fungizone (amphotericin B) effectively works to limit contamination[6],[8]. 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[20],[21]. 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[22].
Table 1: Culture requirements for isolation of different Rickettsia species

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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[16],[23],[24],[25]. 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[6],[7]. The only alternative method that has been proposed, requires serial passages on 24 well plates to obtain R. typhi isolates[19]. 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[19]. 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”[38] [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[8],[39]. 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[40],[41],[42],[43]. 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[41],[42],[43]. 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)[1]. The lethality caused by this bacteria could vary from 10%, as reported in USA[1], to 30% as in developing countries like Mexico[44]. Several tick species are implied as vectors of R. rickettsii, including Dermacentor andersoni and D. variabilis in USA[45]; Amblyomma cajennense in South America[19] and Rhipicephalus sanguineus in Mexico[46]. 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)[28],[47],[48]. Arthropod cell lines from ticks, like DAE3 (D. andersoni embryo), DALB3 (D. albipictus embryo), ISE6 (Ixodes scapularis embryo)[17]; or AeAl2 (Aedes albopictus), C636 (Ae. albopictus clone) from mosquitoes have also been successfully employed[6]. 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[49]. 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[50]. 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[8]. 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[9].

Rickettsia felis: It is an emerging rickettsial pathogen whose main vector and reservoir is Ctenocephalides felis, the cat flea[51]. This pathogen is found in several arthropods, such as Achaeopsylla erinacei, Xeopsylla cheopis, Pulex irritans, R. sanguineus, and A. cajennense[52],[53],[54],[55],[56] however, it has only been isolated from C. felis and Liposcelis bostrychophila[29],[30]. 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[29],[30]. 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[31], 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[32]. 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[30].

Rickettsia parkeri: The transmission of this rickettsial pathogen has been associated to A. maculatum along the gulf of Mexico coast[9]; whereas in south America it has additionally been linked to A. triste[34],[57]. Isolates of R. parkeri have been established using Vero E6 in MEM culture media at 34.5 °C for A. triste samples[9], and at 28 °C using same culture conditions[34]. 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[35].

Rickettsia akari: Rickettsial pox is a disease caused by R. akari, which is a cosmopolitan member of the SFG[36]. Human cases have been registered in USA, Mexico and several European countries[36],[37],[38],[39],[40],[41],[42],[43],[44],[45],[46],[47],[48],[49],[50],[51],[52],[53],[54],[55],[56],[57],[58]. 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[59]. 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[14],[36],[60].

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[1],[61]. The mortality rate for murine typhus ranges near 4%, when no antibiotic therapy is administered[1]. This pathogen is mainly transmitted by Xenopsylla cheopis, a rat flea, whose main reservoirs are Rattus norvegicus and R. rattus[1]; however, its possible transmission by the flea, C. felis and the tick R. sanguineus has also been described[62],[63]. Different isolates of R. typhi have been obtained from rodent, arthropod and human samples[64]. 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[26].

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[65]. Humans are considered as the reservoirs of R. prowazekii; however, there are reports of other potential reservoirs like squirrels[37],[65],[66]. 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[16]. 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[16],[67].

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[26],[27],[33],[68]. There is no evidence of human pathogenicity from R. bellii, that exhibit a slow growth in chicken embryo being innocuous for mice[68]. 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[33]. Isolates in the study were obtained with BHI and MEM media supplemented with FBS (5%), at 28 °C[33].


  Conclusion Top


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 Top


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.



 
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