The aim of this study was, firstly, to detect the presence of Rickettsial DNA by PCR and, then, to identify

the Rickettsiae species using restriction endonuclease fragment length polymorphism (RFLP) on two amplified

genes

Longheu, Carla

Masala, Giovanna

Mura, A. M.

Piras, Pierpaola

Satta, Giuseppe

Tola, Sebastiana

UnissResearch

182 SOIPA XXIV ABSTRACTS Parassitologia 48, 2006 
Detection and characterization of Rickettsial strains in 
ticks from Sardinia, Italy 
Mura A.M., Longheu C., Piras P., Satta G., Masala G., Tola S. 
Istituto Zooprofilattico Sperimentale della Sardegna "G.Pegreffi", 07100 Sassari. 
Ticks are of considerable medicaI and veterinary importance, because they harm the host through their feed-
ing action and so transmit a lot of pathogens. Tick-trasmitted pathogens are quite diverse and include organ-
isms belonging to the genera Rickettsia, Ehrlichia, Anaplasma, Borrelia, Francisella, Cowdria and Coxiella. 
In Sardinia, the cases of tick bite related fever are considerably higher than in the rest of Italy: an average 
rate of 11.9 cases year for every 100,000 inhabitants versus a national average of 2.1 cases year for 100,000 
inhabitants has been reported (Beninati T et al, 2002, Emerging Infectious Diseases, 9: 983-986). 
The aim of this study was, first1y, to detect the presence of Rickettsial DNA by PCR and, then, to identify 
the Rickettsiae species using restriction endonuclease fragment length polymorphism (RFLP) on two ampli-
fied genes. 
Tick collection: Between December 2004 to December 2005 a total of 1770 aduIt ticks was colIected from 
different animals (muflon, deer, hedgehog, sheep, goat, horse, cattle, dog , cat, etc.) and vegetation in two 
provinces (Ogliastra and Cagliari). Ticks were stored in vials containing 700/0 ethanol and identified using 
basic taxonomic schemes. 
DNA extraction and PCR amplification: ticks from each animaI were before pooled according to their species 
and sex and after homogenized in STE buffer. DNA was extracted and purified following the procedure 
described by Wu-Chun Cao et al (2000, Joumal of Clinical Microbiology, 38: 2778-2780). 
PCR amplification was performed by using oligonucleotide primers derived from the 120 kDa outer mem-
brane protein (ompB) gene of Rickettsia rickettsii as described by Noda H et al (1997, Applied Environ 
Microbiol, 63: 3929-3932). 
PCR amplification of DNA was verified by electrophoresis in 1 % agarose gel. Positive samples were sub-
jected to two further amplifications using primer sets from ompA and citrate syntase (gItA) genes respec-
tively (Regnery R et al, 1991, J of Bact, 173: 1576-1589). 
Amplicon from ompA (536 bp) was digested with Pstl and Rsal endonucleases whereas amplicon from gItA 
(353 bp) was cleaved with Alul restriction enzyme at 37°C for 16 hours. The digested reactions were loaded 
on 1 mm thick, 12% polyacrylamide vertical gelso Gels were run at 100 V for 4 hours, stained with ethidi-
um bromide and examined by UV lamp in an ImageMaster VDS-CL (Amersham). DNA molecular mass 
marker VIII (fragment mixture prepared by cleavage of pUCBM21 DNA with restriction endonucleases Hpa 
II and Dra I plus Hind III - Roche) was run with the samples to determine the molecular masses of the DNA 
fragments. 
ResuIts: Preliminary electrophoretic analysis of uncut PCR products suggested that alI rickettsial ompB PCR 
amplified products were similar. Of a total of 210 DNA samples 14 (6.7%) were PCR-positive for ompB 
gene. Six of these samples were subjected to RFLP analysis. The resuIts have permitted to identify only one 
strain as R. africae. Further analyses are in progress to study the other species. 


Detection and characterization of Rickettsial strains in ticks from Sardinia, Italy

Abstract

Similar works

Full text

Available Versions

UnissResearch