Focus: DNA Isolation
Using the Wizard® Plus SV Minipreps DNA Purification System to isolate Rikettsia DNA
Rickettsia conorii subspecies were isolated and studied to determine the cause of 24 cases of Mediterranean spotted fever occurring in Western Sicily, Italy, from 1987 to 2001.
From the article: Giammanco, G.M. et al. (2005) J. Clin. Microbiol. 43, 6027–31.
Published in January 2008
Introduction
Mediterranean spotted fever (MSF) is caused by Rickettsia conorii, an intracellular obligate,
slow-growing gram-negative bacterium. The disease is usually benign, but in 6% of cases, MSF becomes severe and has an overall death rate of 2.5% (1).
Rickettsia conorii was thought to be the only pathogenic
rickettsia of the spotted fever group (SFG) in Italy (2). Rickettsia strains related to
R. conorii caused similar diseases and were sorted into four subspecies based on genetic and serological methods (3). The causative agent
of Israeli spotted fever (ISF), R. conorii subsp. israelensis, was initially isolated in Israel in 1974, but also has been found in Portugal.
The recent isolation of R. conorii subsp. israelensis from a Rhipicephalus sanguineus tick, which is the main vector
for MSF in Sicily, indicated that ISF may be more widely distributed than believed. Therefore, a sequence-based identification was undertaken to
study clinical isolates of MSF from a 15-year period.
Methods
Twenty four Rickettsia isolates were obtained from patient blood samples taken during 1987–2001, and used to infect Vero cell monolayers. Bacterial DNA was isolated from 200µl of infected Vero cell suspensions using the Wizard® Plus SV Minipreps DNA Purification System (Cat.# A1330). A 632bp portion of the ompA gene was amplified for 35 cycles and the PCR product subjected to restriction digestion and sequence analysis.
Results and Conclusion
A 3-band PstI
restriction profile was seen for some of the amplimers, identifying five of the
clinical isolates as belonging to R. conorii subsp. israelensis; the remaining 19 isolates were confirmed as
R. conorii
subsp. conorii. Of note was the earliest R. conorii subsp. israelensis sample was from 1991. Analysis of the amplified and sequenced ompA fragments of
the five clinical R. conorii subsp. israelensis isolates demonstrated their complete
identity, and comparison with GenBank® sequences showed
100% identity to the reference R. conorii subsp. israelensis. Thus, R. conorii subsp. israelensis has been present in Italy from at least 1991 as an
etiological agent of MSF.
Reference
- Raoult, D. et al. (1986) Mediterranean spotted fever: clinical, laboratory and epidemiological features of 199 cases. Am. J. Trop. Med. Hyg. 35, 845–50.
- Tringali, G. et al. (1986) Epidemiology of boutonneuse fever in western Sicily. Distribution and prevalence of spotted fever group rickettsial infection in dog ticks (Rhipicephalus sanguineus). Am. J. Epidemiol. 123, 721–7.
- Zhu, Y. et al. (2005) Proposal to create subspecies of Rickettsia conorii based on multilocus sequence typing and an emended description of Rickettsia conorii. BMC Microbiol. 5, 11.
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