Rickettsiae in ixodid ticks, Sicily.

To the Editor: Members of the spotted fever group rickettsiae are intracellular bacteria usually associated with ixodid ticks, which are transferred to vertebrates by salivary secretions and within ticks transtadially and transovarially. Several tickborne rickettsiae cause human or animal diseases and, in the last 10 years, the increased use of molecular-based identification methods has resulted in new spotted fever group rickettsiae being characterized in ixodid ticks throughout Europe (1). Until recently, no rickettsiae, other than Rickettsia conorii, were reported in Italy. Since 2002, R. helvetica and Israeli spotted fever Rickettsia (R. conorii complex) have been detected in Ixodes ricinus and Rhipicephalus sanguineus, respectively (2-4). In Italy, Mediterranean spotted fever is endemic. This disease appears to occur more commonly in some central and southern regions (5); in 2002, more than half (498 of 890) of the cases of Mediterranean spotted fever identified in Italy and reported to the Ministry of Health came from Sicily.

Because of the relatively high prevalence of rickettsial diseases in southern regions, we analyzed tick samples collected during 2001 and 2002 from herbivores (bovines, ovines, donkeys) from Sicilian farms in Corleone (Palermo Province) to determine the diversity of spotted fever group rickettsiae in various ixodid tick species. DNA from 238 tick samples from various genera (Dermacentor, Rhipicephalus, Hyalomma, Haemaphysalis, Ixodes) was extracted; in some cases, individual ticks of the same species collected from the same animal were pooled. Polymerase chain reaction screening and sequencing with primers for the gene encoding the cell surface antigen (sca4) (previously known as "gene D") and the 17-kDa antigen gene were performed as previously reported (6,7). A total of 7 positives were found, and the sequences obtained were compared to other bacterial sequences present in the GenBank database (Table). A 469-bp fragment with 100% identity to the R. slovaca sca4 sequence (AF155054) was obtained from 2 Dermacentor marginatus and 1 Haemaphysalis punctata. A 403-bp fragment with 99.75% identity (1-bp difference) to the sca4 sequence from R. africae (AF151724) was found from 1 Hyalomma marginatum, and a 423-bp fragment with 100% similarity to R. conorii sca4 sequence (AE008626) was found from Rhipicephalus turanicus. Finally, a 489-bp fragment with 99.79% identity (1-bp difference) to R. aeschlimannii sca4 sequence (AF163006) was obtained from 2 H. marginatum samples. The levels of identity between the 17-kDa antigen sequences (ranging in length from 351 to 419 bp) obtained during this study and those in GenBank were generally lower than those for sca4 because the 17-kDa antigen gene has not been sequenced for most of the Rickettsia spp. identified here on the basis of sca4 sequences. One exception was the fragment obtained from the Rh. turanicus sample, which had 100% identity with the R. conorii 17-kDa antigen sequence (AE008675). All sca4 and 17-kDa antigen gene sequences described in this study have been deposited in the EMBL database (accession no. AJ781411-AJ781420).

Among the numerous rickettsia species recently described in Europe, R. africae and R. slovaca are known as human pathogens (8), and the first case of R. aeschlimannii infection in humans has recently been reported (9). African tick bite fever caused by R. africae is known as an imported disease in patients returning from sub-Saharan Africa or the West Indies (8), but our report raises the possibility that the rickettsial agent is actually present in European ticks from genera other than Amblyomma. R. slovaca has been shown to be responsible for a human disease known as tick-borne lymphadenopathy (10). Considering the problem of cross-reaction between different spotted fever group rickettsiae during serologic tests, our findings underscore the importance of using antigens from other spotted fever group rickettsiae, in addition to that of R. conorii, to obtain a more specific diagnosis of rickettsioses in Italy (10). Considering the large number of tick species present in Italy, and their infection with different spotted fever group rickettsiae, identifying the tick species responsible for a bite could be helpful for accurate diagnosis.

Table. Identification of Rickettsia spp. in tick samples collected
from herbivores, Corleone (Palermo Province), Italy, 2001-2002

No. of ticks infected/total no. of           Minimum-maximum
ticks examined (tick species)               infection rate (%)

Dennacentormarginatus (2/7)                       28.5
Haemaphysafs punctata (1/15)                       6.6
Hyalomma marginatum (2 */24)                     8.3-20.8
Hyalomma marginatum (1 /24)                        4.1
Rhipicephalus turanicus (1(/52)                  1.9-7.6

                                      Rickettsia spp. identified (%
No. of ticks infected/total no. of    identity with sca4 of spotted
ticks examined (tick species)            fever group rickettsiae)

Dennacentormarginatus (2/7)                 R. slovaca (100) p
Haemaphysafs punctata (1/15)                 R. sloveca (100)
Hyalomma marginatum (2 */24)             R. aeschlimannii (99.79)
Hyalomma marginatum (1 /24)                 R. africae (99.75)
                                              Rickettsia sp.
                                             strain S (99.25)
                                             R. honei (99.0)
Rhipicephalus turanicus
   (1 ([dagger])/52)                          R. conorii (1001

* Two pools of 2 and 3 ticks were positive.

([dagger]) One pool of 4 ticks was positive.

Acknowledgments

This work was funded by the Istituto Zooprofilattico della Sicilia "A. Mirri."

Nathan Lo was supported by Ministry of Education, University and Research of the Italian government.

Tiziana Beninati, * Claudio Genchi, * Alessandra Torina, ([dagger]) Santo Caracappa, ([dagger]) Claudio Bandi, * and Nathan Lo *

* Universita' degli Studi di Milano, Milano, Italy; and ([dagger]) Istituto Zooprofilattico della Sicilia, Palermo, Italy

References

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(7.) Noda H, Munderloh UG, Kurtti TJ. Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals. Appl Environ Microbiol. 1997;63:3926-32.

(8.) Parola P, Raoult D. Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin Infect Dis. 2001;32:897-928.

(9.) Raoult D, Fournier PE, Abboud P, Caron F. First documented human Rickettsia aeschlimannii infection. Emerg Infect Dis. 2002;8:748-9.

(10.) Raoult D, Lakos A, Fenollar F, Beytout J, Brouqui P, Fournier PE. Spotless rickettsiosis caused by Rickettsia slovaca and associated with Dermacentor ticks. Clin Infect Dis. 2002;34:1331-6.

Address for correspondence: Tiziana Beninati, Dipartimento di Patologia Animale, Igiene e Sanita' Pubblica Veterinaria, Universita' degli Studi di Milano, via Celoria 10, Milan 20133, Italy; fax: [phone]; email: [e-mail]