First Isolation and Identification of Rickettsia conorii from Ticks Collected in the Region of Fokida in Central Greece

Abstract

Three different spotted-fever group rickettsiae-Rickettsia conorii, R. massiliae, and R. rhipicephali-were detected and identified by PCR-restriction fragment length polymorphism analysis in Rhipicephalus ticks collected from domestic animals in the Fokida region of Greece, where a high seroprevalence of antibodies to R. conorii was previously demonstrated. The infection rate of ticks was 1.6%. Moreover, R. conorii was isolated from one Rhipicephalus sanguineus tick.Rickettsia conorii, the causative agent of Mediterranean spotted fever, is an obligate intracellular, gram-negative bacterium that infects humans. It is transmitted by the bite of infected arthropods (1, 9). The presence of the spotted-fever group (SFG) rickettsiae in ticks has been reported in several countries (2, 6-11, 13). Many rickettsiae remain poorly characterized due to the inability to be maintained in mammalian cell culture, embryonated chicken eggs, or small rodents, which have traditionally been the standard maintenance hosts used in rickettsiology. The advents of new culture techniques, such as the shell vial-centrifugation technique, and the detection of rickettsial DNA have increased the number of rickettsial species identified (6).A seroepidemiological survey, using an immunofluorescence assay and a Western blot method, was conducted in three villages (Leukaditi, Vounichora, and Makrini) of the Greek province of Fokida in 1991. This study reported a high seroprevalence of antibodies to R. conorii in humans (46%) (1). In a culture survey of ticks from the same area, strain GS (for "Greek strain") was isolated. This strain is genetically similar to Rickettsia massiliae; moreover, R. conorii was not found (2). As a follow-up to previous studies, we focused our efforts on determining the presence and infection rate of R. conorii in field-collected ticks from the three study villages of the Fokida (Fig. 1).In the summer of 1998 (May to August), a total of 439 ticks collected from goats, sheep, and dogs were examined for the presence of SFG rickettsiae. According to standard taxonomic keys, 207 ticks were identified as Rhipicephalus sanguineus, 94 were identified as Rhipicephalus turanicus, and 138 were identified as Rhipicephalus bursa (8). Each tick was triturated in 500 l of minimum essential medium supplemented with 4% fetal calf serum and 2 mM L-glutamine. Half of the above suspension was used for the detection of rickettsial DNA by PCR, and the rest was used for the isolation of rickettsiae by the shell vial technique (3, 4, 9, 11-13). The DNA extraction from the ticks was performed using the QIAamp tissue kit (Qiagen, Hilden, Germany), according to the instructions of the manufacturer. The primer set Rp.CS.877-Rp.CS.1258n was used to amplify a 381-bp sequence of the citrate synthase gene (9). As a negative control for each tick sample, pure PCR buffer treated in the same way as the tick samples was included. As a positive control, purified DNA from R. conorii (Moroccan strain) was used. The DNA from ticks that were positive for the c...

Detección molecular de Rickettsia massiliae y Anaplasma platys en garrapatas Rhipicephalus sanguineus y caninos domésticos del municipio de Bahía Blanca (Argentina)

“…Rickettsia massiliae ha sido detectada en garrapatas del género Rhipicephalus con diversos niveles de infección, de 2,5-18% en Europa 25,26 , 5-6% en África 27,28 , 25% en Israel 29 hasta más de 25% en Estados Unidos de América 9,30 . En Sudamérica, sólo había sido detectada en CABA, con un nivel de infección de 3,4 16 a 20% 31 .…”

Prevalence of tick species in sheep and goat flocks in areas of southern Greece

“…All these studies had been conducted mainly in central and northern Greece (Papazahariadou et al, 1995;Papadopoulos et al, 1996;Psaroulaki et al, 2003;Psaroulaki et al, 2006;Pavlidou et al, 2008;Kahrimanidou et al, 2010;Kahrimanidou et al, 2011). According to the typhus (Wall and Shearer, 2001).…”

“…On the contrary, in these regions the species with the highest prevalence was I. ricinus, (Pavlidou et al, 2008), I. gibbosus (Papazahariadou et al, 1995) and R. bursa (Papadopoulos et al, 1996). Rhipicephalus sanguineus was mainly found in central Greece and R. turanicus and R. bursa in Fokida and the island of Cephalonia (Psaroulaki et al, 2003;Psaroulaki et al, 2006). Moreover, in the island of Cephalonia also Hyalomma, Dermacentor and Haemaphysalis species were identified in sheep and goat flocks (Psaroulaki et al, 2006).…”

“…The studies on ticks of mammals in Greece have been mainly conducted in areas of the Northern country (Papazahariadou et al, 1995;Papadopoulos et al, 1996;Pavlidou et al, 2008;Kachrimanidou et al, 2010;Kachrimanidou et al, 2011) and in a less extend in central and islands of Greece (Psaroulaki et al, 2003;Psaroulaki et al, 2006).…”

Update on tick-borne bacterial diseases in Europe

“…turanicus from Portugal, the Greek Islands, Spain, and Italy; in I. ricinus from Spain and Germany; and in Rh. pusillus from Spain (Bernasconi et al, 2002;Psaroulaki et al, 2003;Psaroulaki et al, 2006;Mura et al, 2008). The first human case of infection with R. massiliae was confirmed in 2005, 20 years after the isolate was obtained from the patient.…”