• View in gallery

    Scattering of Xenopsylla cheopis (A) and Leptopsylla segnis (B) collected fleas from Cyprus. Rickettsia typhi- and R. felis-positive fleas are also demonstrated. This figure appears in color at www.ajtmh.org.

  • View in gallery

    Incidence map showing human Rickettsia typhi cases in Cyprus during 2000–2003. This figure appears in color at www.ajtmh.org.

  • 1.

    Azad AF, 1990. Epidemiology of murine typhus. Annu Rev Entomol 35: 553569.

  • 2.

    Chaniotis B, Psarulaki A, Chaliotis G, Gozalo Garcia G, Gozadinos T, Tselentis Y, 1994. Transmission cycle of murine typhus in Greece. Ann Trop Med Parasitol 88: 645647.

    • Search Google Scholar
    • Export Citation
  • 3.

    Fergie JE, Purcell K, Wanat D, 2000. Murine typhus in south Texas children. Pediatr Infect Dis J 19: 535538.

  • 4.

    Azad AF, Radulovic S, Higgins JA, Noden BH, Troyer JM, 1997. Flea-borne rickettsioses: ecologic considerations. Emerg Infect Dis 3: 319327.

  • 5.

    Azad AF, 1988. Relationship of vector biology and epidemiology of louse and flea-borne rickettsioses. David W, ed. Biology of Rickettsial Diseases. Boca Raton, FL: CRC Press, Inc., 5161.

    • Search Google Scholar
    • Export Citation
  • 6.

    Schriefer ME, Sacci JB Jr, Dumler JS, Bullen MG, Azad AF, 1994. Identification of a novel rickettsial infection in a patient diagnosed with murine typhus. J Clin Microbiol 32: 949954.

    • Search Google Scholar
    • Export Citation
  • 7.

    Adams JR, Schmidtmann ET, Azad AF, 1990. Infection of colonized cat fleas, Ctenocephalides felis (Bouche), with a Rickettsia-like microorganism. Am J Trop Med Hyg 43: 400409.

    • Search Google Scholar
    • Export Citation
  • 8.

    Parola P, Davoust B, Raoult D, 2005. Tick- and flea-borne rickettsial emerging zoonoses. Vet Res 36: 469492.

  • 9.

    Renvoise A, Joliot AY, Raoult D, 2009. Rickettsia felis infection in man, France. Emerg Infect Dis 15: 11261127.

  • 10.

    Perez-Osorio CE, Zavala-Velazquez JE, Arias Leon JJ, Zavala-Castro JE, 2008. Rickettsia felis as emergent global threat for humans. Emerg Infect Dis 14: 10191023.

    • Search Google Scholar
    • Export Citation
  • 11.

    Koliou M, Psaroulaki A, Georgiou C, Ioannou I, Tselentis Y, Gikas A, 2007. Murine typhus in Cyprus: 21 paediatric cases. Eur J Clin Microbiol Infect Dis 26: 491493.

    • Search Google Scholar
    • Export Citation
  • 12.

    Antoniou M, Psaroulaki A, Toumazos P, Mazeris A, Ioannou I, Papaprodromou M, Georgiou K, Christophi N, Patsias A, Loucaides F, Moschandreas J, Tsatsaris A, Tselentis Y, 2010. Rats as indicators of the presence and dispersal of pathogens involved in zoonoses in Cyprus. Vector Borne Zoonotic Dis Apr 6 [Epub ahead of print].

    • Search Google Scholar
    • Export Citation
  • 13.

    Horta MC, Chiebao DP, de Souza DB, Ferreira F, Pinheiro SR, Labruna MB, Schumaker TT, 2006. Prevalence of Rickettsia felis in the fleas Ctenocephalides felis and Ctenocephalides canis from two Indian villages in Sao Paulo Municipality, Brazil. Ann N Y Acad Sci 1078: 361363.

    • Search Google Scholar
    • Export Citation
  • 14.

    Roux V, Rydkina E, Eremeeva M, Raoult D, 1997. Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae. Int J Syst Bacteriol 47: 252261.

    • Search Google Scholar
    • Export Citation
  • 15.

    Roux V, Raoult D, 2000. Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB). Int J Syst Evol Microbiol 50: 14491455.

    • Search Google Scholar
    • Export Citation
  • 16.

    Psaroulaki A, Antoniou M, Papaeustathiou A, Toumazos P, Loukaides F, Tselentis Y, 2006. First detection of Rickettsia felis in Ctenocephalides felis fleas parasitizing rats in Cyprus. Am J Trop Med Hyg 74: 120122.

    • Search Google Scholar
    • Export Citation
  • 17.

    Bitam I, Parola P, De La Cruz KD, Matsumoto K, Baziz B, Rolain JM, Belkaid M, Raoult D, 2006. First molecular detection of Rickettsia felis in fleas from Algeria. Am J Trop Med Hyg 74: 532535.

    • Search Google Scholar
    • Export Citation
  • 18.

    Marquez FJ, Muniain MA, Rodriguez-Liebana JJ, Del Toro MD, Bernabeu-Wittel M, Pachon AJ, 2006. Incidence and distribution pattern of Rickettsia felis in peridomestic fleas from Andalusia, southeast Spain. Ann N Y Acad Sci 1078: 344346.

    • Search Google Scholar
    • Export Citation
  • 19.

    Venzal JM, Perez-Martinez L, Felix ML, Portillo A, Blanco JR, Oteo JA, 2006. Prevalence of Rickettsia felis in Ctenocephalides felis and Ctenocephalides canis from Uruguay. Ann N Y Acad Sci 1078: 305308.

    • Search Google Scholar
    • Export Citation
  • 20.

    Stevenson HL, Labruna MB, Montenieri JA, Kosoy MY, Gage KL, Walker DH, 2005. Detection of Rickettsia felis in a New World flea species, Anomiopsyllus nudata (Siphonaptera: Ctenophthalmidae). J Med Entomol 42: 163167.

    • Search Google Scholar
    • Export Citation
  • 21.

    De Sousa R, Edouard-Fournier P, Santos-Silva M, Amaro F, Bacellar F, Raoult D, 2006. Molecular detection of Rickettsia felis, Rickettsia typhi and two genotypes closely related to Bartonella elizabethae. Am J Trop Med Hyg 75: 727731.

    • Search Google Scholar
    • Export Citation
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    Gilles J, Just FT, Silaghi C, Pradel I, Passos LM, Lengauer H, Hellmann K, Pfister K, 2008. Rickettsia felis in fleas, Germany. Emerg Infect Dis 14: 12941296.

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    Jiang J, Soeatmadji DW, Henry KM, Ratiwayanto S, Bangs MJ, Richards AL, 2006. Rickettsia felis in Xenopsylla cheopis, Java, Indonesia. Emerg Infect Dis 12: 12811283.

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    Loftis AD, Reeves WK, Szumlas DE, Abbassy MM, Helmy IM, Moriarity JR, Dasch GA, 2006. Surveillance of Egyptian fleas for agents of public health significance: Anaplasma, Bartonella, Coxiella, Ehrlichia, Rickettsia, and Yersinia pestis. Am J Trop Med Hyg 75: 4148.

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    Azad AF, Traub R, 1987. Transmission of murine typhus rickettsiae by Leptopsylla segnis (Siphonaptera: Leptopsyllidae). J Med Entomol 24: 689693.

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    Tselentis Y, Psaroulaki A, Maniatis J, Spyridaki I, Babalis T, 1996. Genotypic identification of murine typhus Rickettsia in rats and their fleas in an endemic area of Greece by the polymerase chain reaction and restriction fragment length polymorphism. Am J Trop Med Hyg 54: 413417.

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    Raoult D, La Scola B, Enea M, Fournier PE, Roux V, Fenollar F, Galvao MA, de Lamballerie X, 2001. A flea-associated Rickettsia pathogenic for humans. Emerg Infect Dis 7: 7381.

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Rickettsia typhi and Rickettsia felis in Xenopsylla cheopis and Leptopsylla segnis Parasitizing Rats in Cyprus

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  • Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University of Crete, Heraklion, Greece; Veterinary Services, Nicosia, Cyprus; Medical and Public Health Services, Ministry of Health, Nicosia, Cyprus

Fleas collected from rats during a three-year period (2000–2003) in 51 areas of all provinces of Cyprus were tested by molecular analysis to characterize the prevalence and identity of fleaborne rickettsiae. Rickettsia typhi, the causative agent of murine typhus, was detected in Xenopsylla cheopis (4%) and in Leptopsylla segnis (6.6%). Rickettsia felis was detected in X. cheopis (1%). This is the first report of R. typhi in X. cheopis and L. segnis from rats, in Cyprus, and the first report of R. felis in X. cheopis in Europe. The role of fleas (mainly X. cheopis) was confirmed in the epidemiologic cycle of murine typhus in Cyprus by interrelation of current results with those of previous studies. The geographic distribution of fleas coincided with the geographic distribution of the pathogen they can harbor, which emphasizes the potential risk of flea-transmitted infections in Cyprus.

Introduction

Murine typhus, sometimes referred to as endemic typhus, is a zoonotic infectious disease caused by Rickettsia typhi (R. mooseri), an obligate intracellular bacterium. Rickettsia typhi is a member of the typhus group rickettsiae. The disease is characterized by headache, rash, and fever and occurs worldwide in a variety of environments.1

The pathogen is maintained in nature by a cycle involving vertebrate hosts and their ectoparasites. The classic cycle of murine typhus involves rats (Rattus norvegicus and R. rattus) as reservoirs, and their fleas; the oriental rat flea Xenopsylla cheopis is the main vector.1,2 However, other vertebrate hosts, such as house mice, shrews, opossums, skunks, and cats, which live in or enter rat-infested buildings and human habitations, may be involved in the epidemiology of murine typhus.3,4 Infection of the vertebrate host presumably results from contamination of broken skin, the respiratory tract, or the conjunctivae of the host with infected flea feces or flea tissues.1 Although X. cheopis is considered the major vector of murine typhus, natural infection with R. typhi has been reported for nine flea species.5

Rickettsia felis is the causative agent of an emerging disease known as fleaborne spotted fever. This pathogenic agent is a recently described flea-transmitted Rickettsia that was first detected in 1990 in Ctenocephalides felis and described as the ELB agent.6,7 To date, R. felis has been identified worldwide, mainly in the cat flea C. felis, which is regarded as its main vector. However, evidence of R. felis in other flea species and in ticks and mites suggests a variety of arthropod hosts.8,9 Nevertheless, the occurrence of R. felis infections in humans is relatively rare: 69 human cases had been reported worldwide during 1994–2009.9,10 Because clinical signs of the disease are similar to those of murine typhus and other febrile illnesses, the infection in humans may easily be underestimated and remain poorly characterized.

The prevalence of R. typhi infections among humans in Cyprus was unknown until 1996, when an outbreak of 35 clinical cases of murine typhus occurred in an area near Nicosia, the capital of Cyprus. Murine typhus was diagnosed by immunofluorescent antibody testing. The predominant clinical manifestations of patients were fever, headache, and rash; acute renal failure also developed in two patients. During the summer of 1997 24 additional cases were reported, most from the same area. After this outbreak, a serologic survey was conducted in 1997, in which a prevalence of 47.3% for IgG and 14.4% for IgM to R. typhi was found (Psaroulaki A and others, unpublished data). Recently, 21 pediatric cases of murine typhus were reported.11 However, the actual incidence of murine typhus and the prevalence of R. felis infections among humans in Cyprus are unknown.

The current study is part of a survey that involved capturing rats and their fleas and using them as indicators of the presence and dispersal of zoonotic agents in Cyprus by using geographic information system technology (Psaroulaki A and others, unpublished data).12 Fleas collected from 622 captured rats were tested by molecular means for Rickettsia spp. The results were compared with those of previous studies for human cases of murine typhus in Cyprus.

Materials and Methods

Study area and rats and their fleas.

During a three-year survey (2001–2003), fleas were collected from 622 wild rats (220 R. rattus frugivorus and 402 R. norvegicus) captured at 51 localities in all five prefectures of Cyprus.

Fleas were counted and identified by using accepted morphologic criteria and were kept frozen at –80°C in sterile tubes until they were sent to the Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, Heraklion, Greece, for further analysis.

A total of 1.035 fleas were collected after combing the animals and identified as X. cheopis (70.34%, n = 728), C. felis (24.22%, n = 250) C. canis (0.48%, n = 5), Leptopsylla segnis (4.36%, n = 45), and Nosophylla fasciatus (0.67%, n = 7). All C. felis collected, had already been tested in a previous study.13 In the present study, 457 fleas were tested: 400 X. cheopis randomly selected from 728 animals, 45 L. segnis, 7 N.s. fasciatus, and 5 C. canis.

DNA extraction.

Each flea was immersed for 5 minutes in a solution of 70% ethanol/0.2% iodine, washed 3 times (5 minutes/wash) in sterile distilled water, and dried on sterile filter paper before being crushed in a sterile plastic tube. DNA was extracted by using the QIAamp Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. A sample of sterile water was included as negative control for every 10 flea samples. DNA extracts were stored at –20°C until further processed.

Polymerase chain reaction amplifications and sequence analysis.

DNA extracts were tested by PCR with the primer pair CS.Rp877p/CS.Rp1258n, which amplifies a 381-basepair fragment of the citrate synthase gene (gltA) of rickettsiae, as described.14 To confirm positive PCR results, a second PCR assay was conducted with the primer pair 120-M59/120-807, which amplifies a 833-bp fragment of the rompB protein gene (ompB) of Rickettsia species, as described.15

The PCR amplicons were purified (QIAquick Spin PCR Purification Kit; Qiagen) and sequenced (sequencer CEQ 8000; Beckman Coulter, Athens, Greece). BLASTn (http://www.ncbi.nlm.nih.gov/BLAST), Chromas version 1.49 (Technelysium Pty. Ltd., Holland Park, Queensland, Australia), ClustalW37, and Lasergene version 7.1 (DNASTAR Inc., Madison, WI) software was used for processing identified sequences.

Statistical analysis.

Statistical analysis was performed by using SPSS version 16 (SPSS Inc., Chicago, IL). Analysis of variance and chi-square tests were performed at a significance level of P < 0.05. The geographical distribution of fleas and of the PCR-positive fleas was investigated by using geographic information system (GIS) technology (ArcGis 8.1; Environmental Systems Research Institute, Redlands, CA) and Microsoft (Redmond, WA) Access. Coordinates (provided by the Veterinary Services of Cyprus) of each sampling site were entered on a map of the island that was placed into a GIS database. The map was linked to a database that enabled detailed epidemiologic studies. A software program was developed that allowed contemporary visualization of the name of the village, its population, altitude, the number of animals, and other relative epidemiologic information.

Correlation of results with previous data.

During the same study period (2001–2003), human cases of murine typhus were recorded by using data from the reporting system established in the Department of Medical and Public Health Services of the Ministry of Health. The geographic distribution of the murine typhus cases was investigated by using the GIS technology and areas were correlated with area where positive fleas were detected.

Results

Twenty-three (5%) of 457 fleas tested were positive by both PCRs. Of these 23 fleas, 20 were X. cheopis and 3 were L. segnis. A sequence with 100% similarity to the gltA (AE017197) and ompB (AE017197) genes of R. typhi was detected in 16 (4%) of 400 X. cheopis and in 3 (6.6%) of 45 L. segnis. A sequence with 100% similarity to the gltA (CP000053) and ompB (AF182279) genes of R. felis was identified in 4 (1%) of 400 X. cheopis tested.

The proportion of fleas infected by R. typhi or R. felis was significantly higher (P < 0.001) in the Nicosia Prefecture, where 13 X. cheopis and 3 L. segnis harbored R. typhi and 2 of 4 X. cheopis harbored R. felis (Table 1).

Table 1

Fleas collected from rats and tested for Rickettsia spp. by PCR, Cyprus*

Flea speciesNo. fleas testedNo. (%) PCR positiveRickettsia spp. (no. fleas)Host (no.)Province localities
Xenopsylla cheopis40020 (5)R. typhi (16)Rattus rattus (2), Rattus norvegicus (12)10
R.felis (4)R. rattus (1), R. norvegicus (2)3
Leptopsylla segnis453 (6.7)R. typhi (3)R. rattus (2)2
Ctenocephalides canis5
Nosphylla fasciatus7
Total45723 (5)

PCR = polymerase chain reaction.

Distribution of tested and positive fleas and human cases of murine typhus is shown in Figure 1. Areas with R. typhi-infected fleas and human cases of murine typhus were identified and characterized as high-risk areas for R. typhi (Figure 2).

Figure 1.
Figure 1.

Scattering of Xenopsylla cheopis (A) and Leptopsylla segnis (B) collected fleas from Cyprus. Rickettsia typhi- and R. felis-positive fleas are also demonstrated. This figure appears in color at www.ajtmh.org.

Citation: The American Society of Tropical Medicine and Hygiene 83, 6; 10.4269/ajtmh.2010.10-0118

Figure 2.
Figure 2.

Incidence map showing human Rickettsia typhi cases in Cyprus during 2000–2003. This figure appears in color at www.ajtmh.org.

Citation: The American Society of Tropical Medicine and Hygiene 83, 6; 10.4269/ajtmh.2010.10-0118

Discussion

Cyprus is an island ecosystem located in the southeastern part of the Mediterranean Sea in which climatic and ecologic conditions are favorable for maintaining reservoirs and vectors of fleaborne rickettsiae. Nevertheless, little data are available on the presence and distribution of fleaborne rickettsiae in ectoparasitic fleas that might transmit the disease to humans. Murine typhus caused by R. typhi had been considered to be the only fleaborne rickettsiosis present in Cyprus until 2006, when R. felis was detected in C. felis.16 We report molecular detection and identification of R. typhi in X. cheopis and L. segnis collected from rats in Cyprus and the occurrence of R. felis in X. cheopis.

Rickettsia felis has primarily been associated with C. felis parasitizing cats, dogs, or opossums.4 However, the only recognized vector is the cat flea.8 Other flea species such as C. canis,1719 Anomiopsyllus nudata,20 Archaeopsylla erinacei,16,21,22 Ctenophthalmus sp.,21 X. cheopis,22,23 Pulex irritans,18 and ticks and mites8 have been implicated as alternative competent vectors of R. felis.

Animal hosts of infected ectoparasites include cats, dogs, rodents, opossums, hedgehogs, horses, sheep, goats, gerbils, and monkeys.8 However, the role of vertebrates as reservoirs of this emerging pathogen has not been determined.

The presence of R. felis in X. cheopis-infested rats (R. rattus and R. norvegicus), as demonstrated in the present study, corroborates results of other studies.23,24 The same rat species also harbored R. felis-infected C. felis as reported.17 These data highlight two rodent flea species that can harbor R. felis in Cyprus. Other investigators also reported rodent-associated R. felis-infected flea species, such as A. nudata,20 A. erinacei maura,17,21 Ctenophtalmus sp.,21 which suggests a role of rats and their fleas in the epidemiology of R. felis.

Rickettsia typhi was identified in L. segnis (collected from R. rattus and R. norvegicus) in Cyprus. Leptopsylla segnis fleas harboring R. typhi have been reported in Egypt (2.7%)24 and Portugal (23.8%).21 Laboratory studies have shown that L. segnis is more effective than X. cheopis in transmitting R. typhi to rats.25 However, L. segnis has not been considered as an important vector of R. typhi regarding the transmission of murine typhus humans because it does not bite humans. Nevertheless, the dense accumulation of infective feces on certain sites of the fur of the host raises the possibility of air-borne transmission to humans or rodents.26

Rickettsia typhi was also identified in X. cheopis collected from R. rattus and R. norvegicus throughout Cyprus. The high abundance of X. cheopis and the high infection rate (4%), similar to that reported in Greece27 and Egypt,24 indicate that this flea species plays a key role in the epidemiology of murine typhus in Cyprus.

The results of the present study showed a correlation with data from previous studies regarding murine typhus and other zoonoses in Cyprus (Psaroulaki A and others, unpublished data),12 in which 48.6% of rats (the same rats from which the fleas in this study were removed) were seropositive for R. typhi. The seropositivity was strongly associated with region (the highest number of seropositive cases was found in the Larnaca area), season (more seropositive cases in autumn), and the presence of fleas. Seropositivity against R. typhi was strongly associated with X. cheopis (P < 0.0001). The geographic distribution of X. cheopis appeared to coincide with the geographic distribution of R. typhi-seropositive rats (mainly R. norvegicus) (Psaroulaki A and others, unpublished data). Moreover, the geographic distribution of R. typhi-positive X. cheopis correlated with the geographic distribution of the recorded human cases of murine typhus during the same study period (2001–2003) (Figure 2).

Our results showed that two fleaborne rickettsiae, R. felis, and R. typhi, circulate in Cyprus. Rickettsia felis in two flea species, C. felis,16 which often feed on humans and animals, and X. cheopis, which are highly abundant on rats, amplifies the risk for rickettsial transmission in Cyprus. However, to date, no R. felis infections in humans have been documented, and the prevalence of R. felis infections among humans in Cyprus is unknown. Because the clinical symptoms of R. felis infection (fever, headache, and rash) are similar to those of other rickettsioses and antibodies against R. felis cross-react with those against R. typhi and other spotted fever group Rickettsia antigens,27 R. felis infection in humans may be misdiagnosed or missed. When cross-reactivity between R. felis, R. conorii, and R. typhi is observed, the infection is probably with R. felis, whereas when cross-reactivity between R. felis and R. typhi is observed, the causative agent likely belongs to the typhus group.28 Because all cases of murine typhus in Cyprus were diagnosed only by serologic tests, it is difficult to exclude or confirm the cause of infection as R. felis or R. typhi.

Additional studies are needed to investigate the clinical manifestations of these two fleaborne infections, their epidemiology, and their natural maintenance and transmission in mammals to assess the possible role of X. cheopis in the epidemiology of R. felis and to determine whether humans and animals are exposed to the pathogen. Rickettsia felis infection should be considered an emergent threat to human health.

Acknowledgments:

We thank the technical staff of the Veterinary Services and the Department of Agriculture of Cyprus and Papadopoulos Byron and Christofi Nikos for valuable assistance.

  • 1.

    Azad AF, 1990. Epidemiology of murine typhus. Annu Rev Entomol 35: 553569.

  • 2.

    Chaniotis B, Psarulaki A, Chaliotis G, Gozalo Garcia G, Gozadinos T, Tselentis Y, 1994. Transmission cycle of murine typhus in Greece. Ann Trop Med Parasitol 88: 645647.

    • Search Google Scholar
    • Export Citation
  • 3.

    Fergie JE, Purcell K, Wanat D, 2000. Murine typhus in south Texas children. Pediatr Infect Dis J 19: 535538.

  • 4.

    Azad AF, Radulovic S, Higgins JA, Noden BH, Troyer JM, 1997. Flea-borne rickettsioses: ecologic considerations. Emerg Infect Dis 3: 319327.

  • 5.

    Azad AF, 1988. Relationship of vector biology and epidemiology of louse and flea-borne rickettsioses. David W, ed. Biology of Rickettsial Diseases. Boca Raton, FL: CRC Press, Inc., 5161.

    • Search Google Scholar
    • Export Citation
  • 6.

    Schriefer ME, Sacci JB Jr, Dumler JS, Bullen MG, Azad AF, 1994. Identification of a novel rickettsial infection in a patient diagnosed with murine typhus. J Clin Microbiol 32: 949954.

    • Search Google Scholar
    • Export Citation
  • 7.

    Adams JR, Schmidtmann ET, Azad AF, 1990. Infection of colonized cat fleas, Ctenocephalides felis (Bouche), with a Rickettsia-like microorganism. Am J Trop Med Hyg 43: 400409.

    • Search Google Scholar
    • Export Citation
  • 8.

    Parola P, Davoust B, Raoult D, 2005. Tick- and flea-borne rickettsial emerging zoonoses. Vet Res 36: 469492.

  • 9.

    Renvoise A, Joliot AY, Raoult D, 2009. Rickettsia felis infection in man, France. Emerg Infect Dis 15: 11261127.

  • 10.

    Perez-Osorio CE, Zavala-Velazquez JE, Arias Leon JJ, Zavala-Castro JE, 2008. Rickettsia felis as emergent global threat for humans. Emerg Infect Dis 14: 10191023.

    • Search Google Scholar
    • Export Citation
  • 11.

    Koliou M, Psaroulaki A, Georgiou C, Ioannou I, Tselentis Y, Gikas A, 2007. Murine typhus in Cyprus: 21 paediatric cases. Eur J Clin Microbiol Infect Dis 26: 491493.

    • Search Google Scholar
    • Export Citation
  • 12.

    Antoniou M, Psaroulaki A, Toumazos P, Mazeris A, Ioannou I, Papaprodromou M, Georgiou K, Christophi N, Patsias A, Loucaides F, Moschandreas J, Tsatsaris A, Tselentis Y, 2010. Rats as indicators of the presence and dispersal of pathogens involved in zoonoses in Cyprus. Vector Borne Zoonotic Dis Apr 6 [Epub ahead of print].

    • Search Google Scholar
    • Export Citation
  • 13.

    Horta MC, Chiebao DP, de Souza DB, Ferreira F, Pinheiro SR, Labruna MB, Schumaker TT, 2006. Prevalence of Rickettsia felis in the fleas Ctenocephalides felis and Ctenocephalides canis from two Indian villages in Sao Paulo Municipality, Brazil. Ann N Y Acad Sci 1078: 361363.

    • Search Google Scholar
    • Export Citation
  • 14.

    Roux V, Rydkina E, Eremeeva M, Raoult D, 1997. Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae. Int J Syst Bacteriol 47: 252261.

    • Search Google Scholar
    • Export Citation
  • 15.

    Roux V, Raoult D, 2000. Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB). Int J Syst Evol Microbiol 50: 14491455.

    • Search Google Scholar
    • Export Citation
  • 16.

    Psaroulaki A, Antoniou M, Papaeustathiou A, Toumazos P, Loukaides F, Tselentis Y, 2006. First detection of Rickettsia felis in Ctenocephalides felis fleas parasitizing rats in Cyprus. Am J Trop Med Hyg 74: 120122.

    • Search Google Scholar
    • Export Citation
  • 17.

    Bitam I, Parola P, De La Cruz KD, Matsumoto K, Baziz B, Rolain JM, Belkaid M, Raoult D, 2006. First molecular detection of Rickettsia felis in fleas from Algeria. Am J Trop Med Hyg 74: 532535.

    • Search Google Scholar
    • Export Citation
  • 18.

    Marquez FJ, Muniain MA, Rodriguez-Liebana JJ, Del Toro MD, Bernabeu-Wittel M, Pachon AJ, 2006. Incidence and distribution pattern of Rickettsia felis in peridomestic fleas from Andalusia, southeast Spain. Ann N Y Acad Sci 1078: 344346.

    • Search Google Scholar
    • Export Citation
  • 19.

    Venzal JM, Perez-Martinez L, Felix ML, Portillo A, Blanco JR, Oteo JA, 2006. Prevalence of Rickettsia felis in Ctenocephalides felis and Ctenocephalides canis from Uruguay. Ann N Y Acad Sci 1078: 305308.

    • Search Google Scholar
    • Export Citation
  • 20.

    Stevenson HL, Labruna MB, Montenieri JA, Kosoy MY, Gage KL, Walker DH, 2005. Detection of Rickettsia felis in a New World flea species, Anomiopsyllus nudata (Siphonaptera: Ctenophthalmidae). J Med Entomol 42: 163167.

    • Search Google Scholar
    • Export Citation
  • 21.

    De Sousa R, Edouard-Fournier P, Santos-Silva M, Amaro F, Bacellar F, Raoult D, 2006. Molecular detection of Rickettsia felis, Rickettsia typhi and two genotypes closely related to Bartonella elizabethae. Am J Trop Med Hyg 75: 727731.

    • Search Google Scholar
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Author Notes

*Address correspondence to Anna Psaroulaki, Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine (World Health Organization Collaborating Center), Medical School, University of Crete, Heraklion, Greece. E-mail: annapsa@med.uoc.gr
†These authors contributed equally to this article.

Financial support: This study was partially supported by the Cyprus Research Promotion Foundation.

Authors' addresses: Christos Christou, Medical and Public Health Services, Ministry of Health, Nicosia, Cyprus, E-mail: cchristou@mphs.moh.gov.cy. Anna Psaroulaki, Maria Antoniou, Dimosthenis Chochlakis, and Yannis Tselentis, Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University of Crete, Heraklion, Greece, E-mails: annapsa@med.uoc.gr, antoniou@med.uoc.gr, surreydimos@hotmail.com, tselendi@med.uoc.gr. Pavlos Toumazos, Ioannis Ioannou, and Apostolos Mazeris, Veterinary Services, Nicosia, Cyprus, E-mails: director@vs.moa.gov.cy, gioannou@vs.moa.gov.cy, and amazeris@vs.moa.gov.cy.

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