Spotted Fever Group Rickettsiae in Ticks, Morocco

Identified rickettsiae were 4 pathogens, 2 suspected pathogens, and 1 incompletely described species.

T ick-borne rickettsioses are infections caused by obligate intracellular gram-negative bacteria of the spotted fever group (SFG) in the genus Rickettsia and the order Rickettsiales. These zoonoses are now recognized as emerging vector-borne infections worldwide (1,2). They share characteristic clinical features, including fever, headache, rash, and occasional eschar formation at the site of the tick bite. Although these diseases have been known for a long time, they have been poorly investigated in northern Africa, including Morocco (2).
Two human tick-borne SFG rickettsioses are known to occur in Morocco. Mediterranean spotted fever, caused by Rickettsia conorii conorii, is transmitted by the brown dog tick, Rhipicephalus sanguineus, which is well adapted to urban environments and is endemic to the Mediterranean area (2). In Morocco, clinicians usually consider patients with spotted fever as having Mediterranean spotted fever. However, in 1997, Beati et al. isolated a new rickettsia, R. aeschlimannii, from Hyalomma marginatum marginatum ticks collected in Morocco (3). In 2002, human infection with this rickettsia was reported in a patient returning from Morocco to France (4).
To date, all studies on rickettsioses conducted in Morocco have been based on only clinical and serologic features. However, the number of representatives of the genus Rickettsia and the number of newly described rickettsioses have increased in recent decades because of improved cell culture isolation techniques and extensive use of bacterial detection and identifi cation by molecular biologic techniques (2). Comparison of the sequences of PCR-amplifi ed fragments of genes encoding 16S rRNA, citrate synthase (gltA), or outer membrane protein (ompA) has become a reliable method for identifying rickettsiae in arthropods, including ticks (1). Therefore, our aim was to detect and characterize rickettsiae in hard ticks collected in Morocco by using PCR and sequence analysis of amplifi ed products and to discuss their potential threat for humans and animals. Ticks were rinsed with distilled water for 10 min, dried on sterile fi lter paper in a laminar fl ow hood, and crushed individually in sterile Eppendorf (Hamburg, Germany) tubes. DNA was extracted by using the QIAamp Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Rickettsial DNA was detected by PCR by using primers Rp CS.409p and Rp CS.1258n (Eurogentec, Seraing, Belgium), which amplify a 750-bp fragment of the gltA gene of Rickettsia spp. as described (5). All ticks positive for gltA were tested for the ompA gene of Rickettsia spp. by using primers Rr. 190.70 and Rr. 190.701, which amplify a 629-632-bp fragment (5). A negative control (distilled water instead of tick DNA template) and a positive control (DNA from R. montanensis) were included in each test. All PCRs were conducted in Marseille by using the GeneAmp PCR System 2400 and 9700 thermal cyclers (PerkinElmer, Waltham, MA, USA). Amplifi cation products were analyzed after electrophoresis on a 1% agarose gel stained with ethidium bromide. To identify detected Rickettsia spp., PCR products were purifi ed and sequencing was performed as described (5). All sequences obtained were assembled and edited with Auto Assembler software version 1.4 (PerkinElmer). Sequences were analyzed by BLAST (www.ncbi.nlm.nih. gov/blast/Blast.cgi) sequencing analysis of sequences in the GenBank database.

Molecular Identifi cation of Ticks
To help identify the ticks at the species level, molecular tools were used for some ticks that had not been morphologically identifi ed at the species level and that were positive for rickettsiae. Amplifi cation by PCR with T1B and T2A primers and sequencing of a 338-bp amplifi ed fragment of the 12S rRNA gene of the ticks were performed as described (6).

Discussion
Before this study, only 2 SFG rickettsiae pathogenic to humans had been described in Morocco, R. conorii conorii, the agent of Mediterranean spotted fever, and the recently described R. aeschlimannii (2,3). In our study, in addition to R. aeschlimannii, we identifi ed 3 other SFG pathogenic rickettsiae in Morocco: R. massiliae, R. slovaca, and R. monacensis. Furthermore, 2 tick-borne SFG Rickettsia spp. presumptively associated with human illnesses, R. helvetica and R. raoultii, and an undescribed bacterium have been identifi ed.
DNA extraction and PCR were performed in different locations (Morocco and France), and all results were supported by 2 sets of primers. The gltA primers used in the fi rst screening are known to amplify all known tick-borne rickettsiae (7). A second set of primers targeting the ompA gene was used to confi rm positive results, although some rickettsia (e.g., R. helvetica) cannot be amplifi ed by using this set. There were no cases in which multiple species of rickettsiae were detected in an infected tick, as in most of the similar molecular surveys published (1,2). Our results did not address prevalence and distribution of rickettsiae detected. Systematic sampling was not conducted. Also, some tick samples tested with rickettsial primers have not been tested with tick primers in parallel. Therefore, inhibitors that could be responsible for false-negative results and underestimation of infection rates cannot be ruled out.
R. aeschlimannii was isolated from H. marginatum marginatum ticks collected in Morocco in 1997 (3). This rickettsia has also been detected in H. marginatum rufi pes ticks in Zimbabwe, Niger, and Mali; in H. marginatum marginatum in Portugal, Croatia, Spain, Greece, Algeria, and Egypt; and in both ticks in Corsica (2,8,9). H. marginatum marginatum is also known as the Mediterranean Hyalomma and may represent up to 42% of ticks found on cattle in Morocco. This tick is also a suspected reservoir of R. aeschlimannii because transstadial and transovarial transmission have been reported (8). As a result, the distribution of R. aeschlimannii may parallel that of H. marginatum marginatum.
In 2002, the pathogenic role of infection with R. aeschlimannii was demonstrated by PCR and serologic testing in a patient who returned to France from Morocco (4). Clinical signs in this 36-year-old man were fever, generalized maculopapular rashes, and a vesicular lesion of the ankle that became necrotic and resembled the typical tache noire of Mediterranean spotted fever. A second case was identifi ed in a patient in South Africa in 2002 (10). This pa-tient had an eschar around the attachment site. No additional symptoms developed, and treatment with antimicrobial drugs may have prevented progression of the syndrome.
A total of 4.7% of the Rh. sanguineus ticks tested were infected by R. massiliae. This rickettsia was isolated from Rh. sanguineus ticks collected near Marseille, France, in 1992 (11). It has been also found in Rh. sanguineus and Rh. turanicus in Greece, Spain, Portugal, Switzerland, central Africa, and Mali (2,12,13). Eremeeva et al. (14) recently reported detection and isolation of R. massiliae from 2 of 20 Rh. sanguineus ticks collected in eastern Arizona in the United States. R. massiliae may be commonly associated with these ticks, which are distributed worldwide. Transstadial and transovarial transmission of rickettsia in ticks has been reported (13).
In 2003, serologic fi ndings from Spain showed that in 5 of 8 serum samples titers against R. massiliae were higher than those against R. conorii, the agent of Mediterranean spotted fever (12). The authors analyzed clinical symptoms of patients with strong serologic reactions against R. massiliae antigens but did not fi nd relevant clinical differences between these patients and those with Mediterranean spotted fever. However, it is generally recognized that there are relatively few clinical differences among the different spotted fever diseases, and these differences are occasionally not taken into account by clinicians when reporting clinical data of patients (12). The only confi rmed case of a person infected with R. massiliae was a patient hospitalized in Sicily, Italy. This patient had fever, a maculopapular rash on the palms of his hands and the soles of his feet, an eschar, and hepatomegaly. The strain of R. massiliae was isolated in Vero cells in 1985 and stored for 20 years in Sicily, but was not defi nitively identifi ed until 2005 at the Unité de Rickettsies in Marseille, France (15).
The third SFG pathogenic rickettsia found in our study was R. slovaca in 5 (45.5%) of 11 D. marginatus. R. slovaca, which was identifi ed in Dermacentor spp. ticks in Slovakia in 1968, has been subsequently found in D. marginatus and D. reticulatus in France, Switzerland, Portugal, Spain, Armenia, Poland, Bulgaria, Croatia, Russia, and Germany (2,16). These ticks may act as vectors and reservoirs of R. slovaca, which is maintained in ticks through transstadial and transovarial transmission (17). Human infection with R. slovaca was reported in France in 1997. Patients with similar clinical signs were observed in Spain, Bulgaria, and Hungary, where the syndrome was known as tick-borne lymphadenopathy or Dermacentor-borne necrosis erythema lymphadenopathy because of eschar at the tick bite site in the scalp and cervical lymphadenopathy (2,(18)(19)(20). The incubation period ranges from 4 to 15 days. Low-grade fever and rash were present. The acute disease can be followed by fatigue and residual alopecia at the bite site (16,21). Recently, Gouriet et al. reported 14 new cases with tick-borne lymphadenopathy and Dermacentor-borne necrosis erythema lymphadenopathy in southern France during January 2004-May 2005 (22). In this group, tickborne lymphadenopathy occurred mainly in young children and women and during the colder months (22). Overall, data in our study indicate that clinicians should be aware that this tick-related disorder may be found in Morocco.
R. raoultii is a recently described SFG rickettsia (23). In 1999, three new rickettsial genotypes, RpA4, DnS14, and DnS28, were identifi ed in ticks collected in Russia by using PCR amplifi cation and sequencing of 16S rDNA, gltA, and ompA genes. Genotypes identical to DnS14, DnS28, and RpA4 were thereafter detected in various areas in Russia and Kazakhstan in D. reticulatus, D. marginatus, and D. silvarum (24), in Germany and Poland in D. reticulatus (25,26), and in Spain, France, and Croatia in D. marginatus (23). Recently, cultivation of 2 rickettsial isolates genetically identical to Rickettsia sp. genotype DnS14, two rickettsial isolates genetically identical to Rickettsia sp. genotype RpA4, and 1 rickettsial isolate genetically identical to Rickettsia sp. genotype DnS28 was described (23). These isolates have been shown to fulfi ll the requirements for their classifi cation within a new species, R. raoultii, by using multigene sequencing (16S rDNA, gltA, ompA, ompB, sca4, ftsY, and rpoB genes) and serotyping techniques (23,27). In our study, we detected R. raoultii in D. marginatus in Morocco. This tick is found in the cooler and more humid areas of the Mediterranean region associated with the Atlas Mountains. It is restricted to small areas of Morocco and Tunisia (28). Detection of R. raoultii in Morocco is of clinical relevance because it is suspected to be a human pathogen. In 2002, it was detected in D. marginatus obtained from a patient in France in whom typical clinical symptoms of tick-borne lymphadenopathy developed (23).
R. helvetica is another species identifi ed in Morocco in this study. It is one of the few SFG species in which a commonly used ompA primer set does not amplify a PCR product (7,29). However, sequencing gltA enabled defi nitive identifi cation. R. helvetica was isolated in Switzerland from I. ricinus in 1979 and has been identifi ed in many European countries, where the tick is both a vector and a reservoir (2). The distribution of R. helvetica is not limited to Europe but extends into Asia (30). Our data show that the distribution of this bacterium extends into northern Africa. A small population of I. ricinus is present in Tunisia, Algeria, and Morocco. Our study was conducted in Taza, a humid area in the middle of the Atlas Mountains, which was the only site in Morocco that contained I. ricinus ticks (2).
R. helvetica was considered to be a nonpathogenic rickettsia for ≈20 years after its discovery. However, in 1999 it was implicated in fatal perimyocarditis in patients in Sweden (31). The authors of this study subsequently reported a controversial association between R. helvetica and sarcoidosis in Sweden (32) and found R. helvetica DNA in human aortic valves (33). However, the validity of these associations has been questioned by some rickettsiologists (2), and additional studies did not detect antibodies to rickettsia in a group of Scandinavian sarcoidosis patients (34). In 2000, seroconversion for R. helvetica was described in a patient in France with a nonspecifi c febrile illness (35). Serologic data, including cross-absorption and Western blotting, supported R. helvetica as the cause of disease. During 2003-2007, serologic fi ndings in tickbite patients or in patients with fever of unknown origin from Switzerland, Italy, France, and Thailand were suggestive of acute or past R. helvetica infection (5,36). The few patients with a serology-based diagnosis had relatively mild, self-limited illnesses associated with headache and myalgias, and had a rash or eschar less frequently. Additional evaluation and isolation of the bacterium from clinical samples are needed to confi rm the pathogenicity of R. helvetica.
We have detected in I. ricinus ticks a bacterium known as R. monacensis that was isolated from I. ricinus collected in 1998 in a park in Munich, Germany (37). This rickettsia is also found in the literature by other names such as the Cadiz agent found in Spain and Rickettsia IRS3 and IRS4, detected in Slovakia and Bulgaria. More recently, it has been identifi ed in I. ricinus in Hungary (38). Recently, 2 human cases of infection with R. monacensis were documented in Spain (39). Investigators isolated this agent from the blood of 2 patients with Mediterranean spotted feverlike illnesses. The fi rst patient was an 84-year-old man from La Rioja, Spain. He had fever and maculopapular rash without any inoculation eschar. The second patient was a 59-year-old woman from the Basque region of Spain. She had a history of a tickbite, fever, and a rash at the tickbite site (39). With our results, R. monacensis joins the list of autochthonous Rickettsia spp. confi rmed as human pathogens in Morocco.
A total of 69% of Haemaphysalis spp. ticks tested harbored an incompletely described rickettsia. A closely related gltA sequence was found in GenBank as Rickettsia endosymbiont of Haemaphysalis sulctata. Duh et al. detected this bacterium in Ha. sulcata ticks collected from sheep and goats in southern Croatia (40). Using molecular analysis of the complete gltA gene and a portion of ompB, these authors detected this bacterium in 795 (22.8%) ticks tested. Similar to our fi ndings, these researchers could not amplify DNA by PCR for the ompA gene with the primers Rr. 190.70-Rr. 190.701. Identifi cation and isolation of this bacterium are needed until the name provisionally proposed by Duh et al, "R. kastelanii" (40), is accepted (41).
These fi ndings demonstrate that species of ticks and several pathogens causing tick-transmitted diseases may be prevalent in the same area. Our study also detected R. slovaca, R. helvetica, R. monacensis, R. raoultii, and an incompletely described rickettsia in Morocco. Clinicians in Morocco and those who may see patients returning from this country should be aware that many species of rickettsiae are present in this region and should consider a range of spotted fever rickettsial diseases in differential diagnosis of patients with febrile illnesses. Our data increase information on distribution of SFG rickettsiae in Morocco. Additional studies are needed to determine the epidemiologic and clinical relevance of different rickettsioses in this region.