Isolation of spotted fever group rickettsias from triturated ticks using a modification of the centrifugation-shell vial technique

Kelly, Patrick J.; Raoult, Didier; Mason, Peter R.

doi:10.1016/0035-9203(91)90303-g

Cited by 62 publications

(56 citation statements)

References 6 publications

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“…A further droplet of hemolymph was mixed with 500 pl of brain heart infusion and inoculated into two shell vials containing monolayers of L929 cells. The vials were centrifuged at 700 X g for 1 h, and the supernatant was discarded and replaced with 1 ml of Earle's minimum essential medium (MEM) containing 4% fetal calf serum and 2 mM L-glutamine (28,33). After 6 days of incubation in a C 0 2 incubator at 32"C, the cells were gently scraped from the bottom of the shell vial and stained to detect rickettsiae (20).…”

Section: Methodsmentioning

confidence: 99%

Rickettsia aeschlimannii sp. nov., a New Spotted Fever Group Rickettsia Associated with Hyalomma marginatum Ticks

Béati

Meskini

Thiers

et al. 1997

International Journal of Systematic Bacteriology

155

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We formally propose the name Rickettsia aeschlimannii sp. nov. for a new spotted fever group (SFG) rickettsia, strain MCMT, isolated from Hyalumma marginatum marginatum ticks collected in Morocco. This organism shows a typical rickettsial morphology when analyzed by electron microscopy. After characterization by serotyping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western immunoblotting, PCR-restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis, and 16s rDNA sequencing, this organism was found to be different from all of the recognized SFG rickettsiae. Identical PCR-FWLP profiles have, however, been found in H. marginatum marginatum from Portugal and H. marginatum rujipes from Zimbabwe, which suggests that the distribution of this rickettsia reaches from the Mediterranean to southern Africa.Although the first descriptions of Mediterranean spotted fever and its ecology were made in northern Africa (13) and one of the reference strains of Rickettsia conorii is called the Moroccan strain, there have been few recent reports about the ecology and epidemiology of rickettsioses in this area (31). In early studies, all three African spotted fever group (SFG) rickettsioses, Mediterranean spotted fever, South African tick bite fever, and Kenyan tick typhus (25), were attributed to infection with R. conorii. The recognized vectors were dog ticks: Rhipicephalus sanguineus in the Mediterranean area (11) and Rhipicephalus simus or Haemaphysalis leachi in the eastern and southern regions of Africa (22,27). When other tick species were found to be infected with rickettsia-like organisms (RLO), they were considered to be "secondary" vectors of R. conorii (21). More recently, however, it has been shown that other SFG rickettsiae occur throughout Africa and that several tick species carry rickettsiae different from R. conorii (7,41).The presence of RLO in Hyalomma ticks of Morocco and Sudan was first reported in the 1950s (21). RLO have been detected by PCR and direct immunofluorescence in H. impeltatum, H. dromedari, and H. anatolicum in Egypt; however, they have not been identified (30). During a large-scale field survey in Zimbabwe, hemolymph tests showed that 11% of Hyalomma marginatum rufipes ticks were infected with RLO (7). Although isolation attempts were unsuccessful, PCR-restriction fragment length polymorphism (RFLP) analysis showed that these ticks contained SFG rickettsiae genotypically similar to a strain (PoTiR8) isolated from H. marginatum marginaturn ticks from Portugal (2). Strain PoTiR8 has, however, not been further characterized.In Morocco, H. marginatum marginaturn is one of the most widely distributed tick species, possibly representing up to 42% of the tick burden of cattle (3, 32). Although in its juvenile stages it usually bites birds, under particular circumstances it may also infest humans (23). We present here the first description of an SFG rickettsial strain (MC16T) isolated from Moroccan H. marginaturn marginaturn. This isolate was compared to the recogn...

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Section: Methodsmentioning

confidence: 99%

Rickettsia aeschlimannii sp. nov., a New Spotted Fever Group Rickettsia Associated with Hyalomma marginatum Ticks

Béati

Meskini

Thiers

et al. 1997

International Journal of Systematic Bacteriology

155

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“…The use of antibiotics in the culture medium cannot explain such a difference because those used were not bacteriostatic. 35,36 Although the microimmunfluorescence test remains the reference method for the identification of new rickettsiae, the advent of molecular methods has recently enabled the development of useful and rapid methods, such as sequence analysis of PCR products, 11 for the identification of rickettsiae. Identification strategies based on recognition of sequences within the gene encoding for the 16S rRNA gene, 37 the citrate synthase-encoding gene, 38 the rOmpB-encoding gene, 39 the 17-kD protein-encoding gene, 40 or the 190-kD protein rOmpA 11 as used in this work, have been described.…”

Section: Amblyomma Variegatummentioning

confidence: 99%

Tick-borne rickettiosis in Guadeloupe, the French West Indies: isolation of Rickettsia africae from Amblyomma variegatum ticks and serosurvey in humans, cattle, and goats.

Parola¹,

Vestris²,

Martinez³

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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Abstract. Twenty-seven rickettsiae were isolated and/or detected from 100 Amblyomma variegatum ticks collected on Guadeloupe in the French West Indies. In this study, the polymerase chain reaction procedure appeared to be more sensitive in detecting rickettsiae in ticks than the shell-vial technique. Sequencing a portion of the outer membrane protein A-encoding gene showed that these rickettsiae appeared to be identical to Rickettsia africae, a member of the spotted fever group rickettsiae recently described as an agent of African tick-bite fever occurring in sub-Sahelian Africa. A high seroprevalence of antibodies to R. africae was demonstrated among mammals, particularly humans, cattle, and goats. These results and a recently reported case of an infection due to R. africae on Guadeloupe demonstrate that R. africae is present on this island. Although this disease has been underdiagnosed there, it may be frequent and may exist on other Caribbean islands where A. variegatum has propagated dramatically over recent years.Bacteria of the genus Rickettsia are gram-negative intracellular bacilli associated with arthropods.

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“…In the present study, we tested a sample of H. juxtakochi ticks collected in the state of São Paulo (southeastern Brazil), attempting to isolate rickettsiae in cell culture, especially a possible strain of R. rhipicephali. Ticks were brought alive to the laboratory, where they were frozen at Ϫ80°C until processed by the shell vial technique for isolation of rickettsiae in cell culture, as previously described (20) with some modifications (22). Briefly, ticks were individually thawed and triturated in brain heart infusion broth, which was inoculated into shell vials previously seeded with approximately 200,000 Vero cells.…”

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confidence: 99%

Isolation of Rickettsia rhipicephali and Rickettsia bellii from Haemaphysalis juxtakochi Ticks in the State of São Paulo, Brazil

Labruna

Pacheco

Richtzenhain

et al. 2007

Appl Environ Microbiol

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In the present study, attempts to isolate Rickettsia in cell culture were performed individually in seven specimens of Haemaphysalis juxtakochi ticks collected in the state of São Paulo (southeastern Brazil). Rickettsia was successfully isolated by the shell vial technique and established in Vero cell culture from six ticks (six isolates). DNA extracted from infected cells of these isolates was tested by PCR and DNA sequencing, using genus-specific Rickettsia primers targeting the genes gltA, htrA, ompA, and ompB. After the generated sequences were compared with available sequences in GenBank, five out of the six isolates were identified as Rickettsia bellii (isolates HJ#1, HJ#2, HJ#3, HJ#4, and HJ#7). The sixth isolate (HJ#5) was closest to Rickettsia sp. strain R300, previously detected in H. juxtakochi in northern Brazil, and to Rickettsia rhipicephali, isolated from ticks in the United States. Following recent gene sequence-based criteria proposed for the identification of Rickettsia isolates, both isolate HJ#5 and strain R300 were identified as South American strains of R. rhipicephali, which was confirmed in this continent for the first time. Isolation of R. bellii from H. juxtakochi ticks, added to eight other tick species that have been reported to be infected with this bacterium in Brazil, indicates that R. bellii is indeed the most frequent Rickettsia species infecting ticks in Brazil. Currently, the role of both R. rhipicephali and R. bellii as human pathogens is regarded as unknown.

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Isolation of spotted fever group rickettsias from triturated ticks using a modification of the centrifugation-shell vial technique

Cited by 62 publications

References 6 publications

Rickettsia aeschlimannii sp. nov., a New Spotted Fever Group Rickettsia Associated with Hyalomma marginatum Ticks

Rickettsia aeschlimannii sp. nov., a New Spotted Fever Group Rickettsia Associated with Hyalomma marginatum Ticks

Tick-borne rickettiosis in Guadeloupe, the French West Indies: isolation of Rickettsia africae from Amblyomma variegatum ticks and serosurvey in humans, cattle, and goats.

Isolation of Rickettsia rhipicephali and Rickettsia bellii from Haemaphysalis juxtakochi Ticks in the State of São Paulo, Brazil

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