First detection of spotted fever group Rickettsiae in Ixodes ricinus from Italy - Dispatches

Emerging Infectious Diseases, Sept, 2002 by Tiziana Beninati, Nathan Lo, Hiroaki Noda, Fulvio Esposito, Annapaola Rizzoli, Guido Favia, Claudio Genchi

Ixodes ricinus from Italy were examined for the first time to detect whether rickettsiae were present. Using molecular methods, we detected three different spotted fever group rickettsiae, including Rickettsia helvetica. Our results raise the possibility that bacteria other than R. conorii are involved in rickettsial diseases in Italy.

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The genus Rickettsia comprises obligately intracellular, gram-negative bacteria. Before sequence-based classification methods were introduced, the genus was divided into two groups: the typhus group (TG), which included R. prowazekii, R. typhi, and R. canada, and the spotted fever group (SFG), which comprised all others. Recent phylogenetic studies of genes such as gltA, ompA, "gene D," and that encoding the 17-kDa protein (hereafter referred to as "17kDa") have shown that these two groupings are not consistent with species relationships; consequently, they have been modified (summarized in [1]). The TG now comprises only R. prowazekii and R. typhi, while the SFG contains seven divergent lineages: the R. rickettsii group, R. japonica, R. montana, the R. massiliae group, R. helvetica, R. felis, and the R. akari group. The AB bacterium, R. bellii, and R. canada cluster outside both the TG and SFG in most analyses (1).

Members of the SFG rickettsiae are usually associated with ixodid ticks, which transfer them to vertebrates via salivary secretions and between themselves transtadially and transovarially. Several tick-borne rickettsiae are causative agents of human or animal diseases. The prevalences of these diseases are primarily dependent on the geographic distribution of host ticks, which act as both vector and reservoir. Among rickettsiae found in Europe, R. conorii is probably the most well known. This bacterium, transmitted by Rhipicephalus sanguineus, causes "boutonneuse" or Mediterranean spotted fever (MSF), an endemic disease in several countries. Until recently, MSF was thought to be the only rickettsial disease prevalent in Europe, but in recent years some new human rickettsioses have been attributed to bacteria previously considered of unknown pathogenicity (2). An example is Rickettsia helvetica, which was originally isolated in 1979 from Ixodes ricinus but was shown to have pathologic relevance only in 1999, when it was associated with fatal perimyocarditis in two Swedish men (3). In addition, R. helvetica stimulated a specific antibody response in a man in France who had low-grade fever, headache, and myalgia (4).

In Italy, the only rickettsia isolated from humans and ticks thus far has been R. conorii. Since its host, Rh. sanguineus, favors warm climates, MSF is more common in central and southern Italy (5,6). In the years 1992-1998, approximately 8,500 cases of human rickettsioses presumed to be MSF were reported to the Italian Ministry of Health. Regarding the distribution of cases in different parts of Italy, some central (Lazio) and southern (Sardinia, Sicily, and Calabria) regions of the country have a particularly high morbidity rate, reaching an average of 11.9 cases for every 100,000 inhabitants in Sardinia, compared with the national average of 2.1.

The diagnosis of MSF in Italy usually depends on clinical evidence supported by serologic confirmation, mainly by the microimmunofluorescence (MIF) technique. A major limitation of MIF is cross-reactivity, which renders it unable to differentiate between various SFG rickettsiae (4). Thus, some cases of MSF, in Italy, especially where the disease is not endemic, may in fact be due to other rickettsiae.

I. ricinus is found with high prevalence in the Italian Alps and Apennines (reaching 96% of all ticks collected in some areas) and in almost all other Italian regions that contain humid, forested habitats (7). While all life stages of Rh. sanguineus are mainly associated with dogs, I. ricinus can feed on >200 host species, primarily wild rodents and ruminants. In a survey in Liguria of ticks recovered from people, most ticks (89.3%) were I. ricinus; Rh. sanguineus was recorded less frequently (9.8%) (8).

To date, no studies have been conducted on potential rickettsiae in Italian ticks, other than Rhipicephalus spp. Recently, various Rickettsia species have been found in I. ricinus from other European countries, including R. helvetica in Switzerland, France, Sweden, Slovenia, and Portugal (4) and Rickettsia spp. IRS3/4 in Slovakia (9). To check whether such bacteria are also present in Italian I. ricinus, we studied specimens from three regions. We used molecular-sequence-based identification techniques, which offer high sensitivity and specificity compared with serologic tests and circumvent the need for bacterial culturing.

The Study

A total of 109 I. ricinus specimens were collected in northern and central Italy (Figure 1), identified by using standard taxonomic keys, and stored at-20[degrees]C. Specifically, 89 ticks (70 adults and 19 nymphs) were collected by dragging vegetation in different parts of Trentino Province in April-October 1997 and 1999, and 10 ticks (7 adults and 3 nymphs) by dragging in Feltre (Veneto Region) in March 2000. Ten more ticks (7 adults and 3 nymphs) were collected from a patient at the Ospedale di Careggi in Firenze in May 1997. The man had been bitten in Parco Nazionale delle Foreste Casentinesi (Toscana Region; see Figure 1) a number of hours earlier but did not display any illness. M1F tests with R. conorii antigens were performed on his arrival at the hospital and again 4 weeks later: results were negative in each instance. Tick samples were placed in 50 [micro]L of 10 mM Tris*HCl (pH 8.0), heated at 90[degrees]C for 10 min, crushed with a sterile plastic homogenizer, and treated with 10 [micro]g of proteinase K at 50[degrees]C for 3 h. Polymerase chain reaction (PCR) of a 341-bp portion of gltA was performed by using the primers Rp CS.877p and Rp CS.1258n under conditions previously described (10). These primers were chosen for an initial screening because they are known to amplify all rickettsiae (11).