Ticks and Tick-borne Diseases 5 (2014) 446–448

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Original article

Rickettsia conorii israelensis in Rhipicephalus sanguineus ticks, Sardinia, Italy Valentina Chisu a , Giovanna Masala a,∗ , Cipriano Foxi a , Cristina Socolovschi b , Didier Raoult b , Philippe Parola b a

Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar), Inserm 1095, WHO Collaborative Center for Rickettsioses and Other Arthropod-Borne Bacterial Diseases, Marseille, France b

a r t i c l e

i n f o

Article history: Received 2 December 2013 Received in revised form 4 February 2014 Accepted 5 February 2014 Available online 19 May 2014 Keywords: Vector tick Pathogen Tick-borne disease Rickettsia

a b s t r a c t The presence of tick-borne Rickettsia spp. was examined by PCR using DNA samples extracted from 254 ticks collected from mammals originating from northern and eastern Sardinia, Italy. The spotted fever group rickettsial agent Rickettsia conorii israelensis was detected in 3 Rhipicephalus sanguineus ticks from a dog for the first time in this geographical area. In addition, Ri. massiliae, Ri. slovaca, and Ri. aeschlimannii were detected in Rh. turanicus, Rh. sanguineus, Dermacentor marginatus, and Hyalomma marginatum marginatum ticks from dogs, goats, wild boar, and horse. Moreover, Candidatus Rickettsia barbariae was detected in 2 Rh. turanicus ticks from goats. The detection of Ri. conorii israelensis, an emergent agent which causes Israeli spotted fever, increases our knowledge on tick-borne rickettsioses in Sardinia. © 2014 Elsevier GmbH. All rights reserved.

Introduction

Materials and methods

Spotted fever group (SFG) rickettsioses include both old and emerging tick-borne diseases. Mediterranean spotted fever (MSF) is caused by Rickettsia conorii conorii and transmitted by the brown dog tick Rhipicephalus sanguineus (Parola et al., 2013). This zoonosis is prevalent in Italy, where it is a reportable disease, mostly in the southern regions and on the islands. In Sardinia, the number of notified clinical cases of SFG rickettsiosis, presumably MSF, has been 11.9 for every 100 000 inhabitants, in contrast to the national average of 2.1 (Beninati et al., 2002). Although Rh. sanguineus is the most represented tick species in Sardinia, the detection of Ri. conorii conorii in ticks and in patients has not yet been described for this island. In recent years, 3 other tick-borne pathogens, Ri. massiliae, Ri. aeschlimannii, and Ri. slovaca, and a rickettsia of unknown pathogenicity, Candidatus Rickettsia barbariae, have been identified from ticks collected in Sardinia (Mura et al., 2008; Masala et al., 2012). In this study, we aim to update the occurrence of tickborne rickettsiae in Sardinia by using molecular-based methods for detection of rickettsiae in ticks.

Ticks have been fortuitously collected from vertebrate hosts by veterinary practitioners in 3 municipalities of Sassari province (Sassari: N 40◦ 43 , E 8◦ 33 ; Usini: N 40◦ 40 , E 8◦ 32 ; Anela: N 40◦ 26 , E 9◦ 03 ) and 2 municipalities of Ogliastra province (Talana: N 40◦ 02 , E 9◦ 30 ; Urzulei: N 40◦ 06 , E 9◦ 30 ) in June 2010. The ticks were removed from their hosts with tweezers, placed in vials at room temperature, and stored at −20 ◦ C. The ticks were then identified using a binocular microscope (10–50×) and were classified into their family, genus, and species using taxonomic keys and morphometric tables (Manilla, 1998). The ticks were immersed in distilled water for 10 min, dried on sterile filter paper, and crushed with a sterile scalpel in microtubes (Eppendorf, Hilden, Germany). Genomic DNA was extracted from 1000 ␮l of homogenized ticks using QIAgen columns (QIAamp tissue kit, Qiagen, Hilden, Germany), according to the manufacturer’s instructions. All DNA samples were screened by PCR using the oligonucleotide primers Rp CS.409D and Rp CS.1258R (Eurogentec, Seraing, Belgium), which amplify a 750-bp fragment of the citrate synthase gene (gltA) present in all Rickettsia species (Masala et al., 2012). Positive samples were confirmed by a second PCR amplification using the Rr 190.70, Rr 190.180, and Rr 190.701 primers for the ompA gene; these primers amplify a 629–632-bp fragment present in almost all spotted fever group rickettsiae (Masala et al., 2012). A negative control using DNA extracted from uninfected laboratory

∗ Corresponding author at: Istituto Zooprofilattico Sperimentale della Sardegna, Via Duca degli Abruzzi 8, 07100 Sassari, Italy. Tel.: +39 079 2892325; fax: +39 079 2892324. E-mail address: [email protected] (G. Masala). http://dx.doi.org/10.1016/j.ttbdis.2014.02.003 1877-959X/© 2014 Elsevier GmbH. All rights reserved.

V. Chisu et al. / Ticks and Tick-borne Diseases 5 (2014) 446–448 Table 1 Tick species collected on vertebrate hosts from Sassari and Ogliastra Province in Sardinia, Italy.

Sassari Province Rh. sanguineus

Ogliastra Province Rh. turanicus Hyal. m. marginatum

Table 2 Detection and identification by PCR and sequencing of spotted fever group rickettsiae from ticks collected in Sardinia, Italy.

No. of ticks

Sex

Host (n)

Collection sites

Tick species

No. of positive ticks and their sex

Rickettsial identification

GenBank accession number

13

6 males 7 females 2 males 2 females 9 males 5 females

Dog (2)

Sassari

Rh. turanicus

1♂–1♀

EU272186

Dog (1)

Usini

Candidatus Rickettsia barbariae Ri. massiliae

Wild boar (1)

Anela

94 males 117 females 9 males 3 females

Goat (15)

Talana

Horse (1)

Urzulei

4 D. marginatus

447

14

211 12

ticks and a positive control using Ri. rickettsii DNA were included in each test. The reactions were performed in automated DNA thermal cyclers (GeneAmp PCR Systems 2400 and 9700; Applied Biosystems, Courtaboeuf, France). The PCR products were verified by electrophoresis in 1.5% agarose gels stained with ethidium bromide and examined under UV transillumination. The DNA samples positive for the ompA gene were then delivered to the WHO Collaborative Center for Rickettsial and Arthropod-Borne Bacterial Diseases in Marseille, France. The PCR products were purified using a QIAquick Spin PCR purification kit (Qiagen) and sequenced using a DNA sequencing kit (dRhodamine Terminator Cycle Sequencing Ready Reaction; Applied Biosystems), according to the manufacturer’s instructions. The sequences were assembled and edited with the ChromasPro software (version 1.34; Technelysium Pty Ltd., Tewantin, Queensland, Australia) and compared to those found in the GenBank database. Results A total of 254 adult ticks (134 females and 120 males partially or completely engorged) were removed from 3 domestic dogs, 15 goats, one horse, and one wild boar. The collected ticks were identified morphologically as Rhipicephalus turanicus (211 specimens), Rh. sanguineus (17 specimens), Dermacentor marginatus (14 specimens), and Hyalomma marginatum marginatum (12 specimens). Moreover, tick species, their sex, and hosts from both geographic areas are detailed in Table 1. Rickettsial DNA was found in 13 of the 254 ticks (5%) examined using the 2 specific-target genes by PCR. Two Rh. turanicus ticks collected from goats (1%; 95% CI 0.16–3.75) contained rickettsial DNA with 100% similarity to the 630-bp ompA fragment of Candidatus Rickettsia barbariae. Moreover, one Rh. turanicus tick from goats (0.5%; 95% CI 0.02–3.02) contained rickettsia with an ompA gene 99.8% (620/621) similar to that of Ri. massiliae. Three Rh. sanguineus ticks (17.6%; 95% CI 4.67–44.20) from dogs possessed an ompA gene with 100% nucleotide similarity to that of Ri. conorii israelensis. In addition, in 2 Rh. sanguineus ticks (11.8%; 95% CI 2.06–37.75), Ri. massiliae was identified with 100% identity. The sequence analyses of Rickettsia DNA detected in 4 D. marginatus ticks (28.6%; 95% CI 9.58–58.00) from wild boar showed 100% similarity with the 630bp ompA fragment of Ri. slovaca. One H. m. marginatum tick (8.3%; 95% CI 0.44–40.25) from horse contained rickettsial DNA with 99.8% (559/560) similarity to the GenBank Ri. aeschlimannii ompA sequence (Table 2). Candidatus Rickettsia barbariae, Ri. slovaca, and Ri. aeschlimannii were only detected from ticks collected in Ogliastra Province. Rickettsia conorii israelensis was found in Sassari Province, while Ri. massiliae was recovered in both geographical areas.

1♀ 2♂–1♀

CP000683

1♂–1♀

Ri. conorii israelensis Ri. massiliae

HNM050294

D. marginatus

4♂

Ri. slovaca

CP003375

Hyal. m. marginatum

1♀

Ri. aeschlimannii

JQ691734

Rh. sanguineus

AY197564

Discussion In this study, we identified the presence of one more pathogen, Ri. conorii israelensis, to add to the 4 human pathogenic rickettsiae previously found in Sardinia, Ri. massiliae, Ri. aeschlimannii, Ri. slovaca, and Ri. monacensis (Mura et al., 2008; Madeddu et al., 2012; Masala et al., 2012). To date, Ri. conorii israelensis was detected in human and tick specimens in Israel, Italy (Sicily), and Portugal, and in addition, human cases were recently reported in Tunisia and Libya (Giammanco et al., 2005; De Sousa et al., 2007; Weinberger et al., 2008; Parola et al., 2013). The comparison of Israeli spotted fever (ISF) and MSF is difficult, as the strains involved in these cases are not generally verified. However, ISF-causing strains are more virulent than others, but cause fewer inoculation eschars than MSF strains (Sousa et al., 2008; Parola et al., 2013). Levin et al. (2012) suggested that Ri. conorii israelensis is vectored by the tick Rh. sanguineus, in which bacteria are successfully transmitted transstadially from the nymphal to the adult stage. Moreover, the same authors demonstrated that dogs are a competent reservoir of Ri. conorii israelensis. Our data show that the geographic distribution of ISF is wider than previously reported and includes Sardinia. Rickettsia massiliae is an emerging pathogen that has a worldwide distribution. This SFG rickettsia was detected in Rh. sanguineus, Rh. turanicus, Rh. pusillus, Rh. bursa, and Ixodes ricinus in several European countries and collected from domestic and wild hosts such as dogs, house sparrows, horses, cats, hedgehogs, red foxes, hares, goats, and asymptomatic humans. The reported infection rates of ticks in the field range from 2% to 92%. In addition, 2 Italian human cases were diagnosed using serological and molecular tools in patients with MSF signs (Cascio et al., 2013; Parola et al., 2005, 2013). The detection of Ri. massiliae in Rh. sanguineus and Rh. turanicus ticks collected from dogs and goats was in accordance with a previous study carried out by Mura et al. (2008) in Sardinia. Rickettsia aeschlimannii was detected in Hyalomma ticks in several European countries. In particular, it was detected in Hyalomma ticks collected from several bird species, such as Acrocephalus schoenbaenus and Hirundo rustica in Corsica (Parola et al., 2005) and Luscinia megarhynchos and Acrocephalus scirpaceus in southern France and, in the latter species, also in Germany (Rumer et al., 2011; Socolovschi et al., 2012; Parola et al., 2013). Finally, adult host-seeking Hyalomma ticks resulted positive for Ri. aeschlimannii in Pianosa Island (Tomassone et al., 2013). In Italy, this pathogenic SFG rickettsia was found in H. m. marginatum ticks collected from bovines, ovines, and donkeys on Sicilian farms (Beninati et al., 2005), and it has already been found in ticks removed from horses in Sardinia (Mura et al., 2008). The first human infection caused by Ri. aeschlimannii was reported for a French patient who became ill with

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symptoms similar to those of MSF after returning from Morocco (Parola et al., 2005). Rickettsia slovaca is associated with a syndrome characterized by scalp eschars and neck lymphadenopathy following tick bites. The term SENLAT was proposed for this clinical entity in 2010 (Angelakis et al., 2010). Initially, this syndrome was named TIBOLA (tick-borne lymphadenopathy) or DEBONEL (Dermacentor-borne necrotic erythema and lymphadenopathy). Rickettsia slovaca has been found in D. marginatus, D. reticulatus, and Haemaphysalis punctata ticks in a many European countries, and it is responsible for at least two thirds of all TIBOLA cases in Europe (Parola et al., 2009; Torina et al., 2012; Parola et al., 2013). In accordance to those data, Ri. slovaca had already been found in D. marginatus ticks collected from wild boar in Tuscany and Sardinia (Selmi et al., 2009; Masala et al., 2012). Rickettsia monacensis, recently considered pathogenic, has been detected in I. ricinus ticks in Europe and in 2005, it was identified as a human pathogen in patients in Spain and in Italy. In addition to fever and flu-like symptoms, the inoculation eschar was identified in only one Italian patient. A generalized rash including the palms and soles was identified only in Spanish patients (Jado et al., 2007; Madeddu et al., 2012; Parola et al., 2013). Candidatus Rickettsia barbarie was found in Rh. turanicus taken from sheep in Ogliastra Province (Mura et al., 2008), and now it was detected in Rh. turanicus taken from goats of the same geographical site. Although the samples were fortuitously collected, a new rickettsia detected in Rh. sanguineus ticks can be added to the list of pathogenic rickettsiae already found in Sardinia. Clinicians in Sardinia should be informed that several pathogenic rickettsiae are prevalent on the island, even virulent ones. Further clinical studies are needed to better understand the prevalence of tick-borne rickettsioses in Sardinia. References Angelakis, E., Pulcini, C., Waton, J., Imbert, P., Socolovschi, C., Edouard, S., Dellamonica, P., Raoult, D., 2010. Scalp eschar and neck lymphadenopathy caused by Bartonella henselae after tick bite. Clin. Infect. Dis. 50, 549–551. Beninati, T., Lo, N., Noda, H., Esposito, F., Rizzoli, A., Favia, G., Genchi, C., 2002. First detection of spotted fever group rickettsiae in Ixodes ricinus from Italy. Emerg. Infect. Dis. 8, 983–986. Beninati, T., Genchi, C., Torina, A., Caracappa, S., Bandi, C., Lo, N., 2005. Rickettiae in ixodid ticks, Sicily. Emerg. Infect. Dis. 11, 509–511. Cascio, A., Torina, A., Valenzise, M., Blanda, V., Camarda, N., Bombaci, S., Iaria, C., De Luca, F., Wasniewska, M., 2013. Scalp eschar and neck lymphadenopathy caused by Rickettsia massiliae. Emerg. Infect. Dis. 19, 836–837.

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