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Tick-Borne Rickettsioses around the World: Emerging Diseases Challenging Old Concepts

Unité des Rickettsies, CNRS UMR 6020, IFR 48, Université de la Méditerranée, Faculté de Médecine, 13385 Marseille Cedex 5, France,¹ Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Mailstop G-32, 1600 Clifton Road, Atlanta, Georgia²

SUMMARY

INTRODUCTION

RECENT DEVELOPMENTS AND CONTINUING GAPS IN RICKETTSIOLOGY

Microbiology and Taxonomy: What Defines a Rickettsia sp.?

The Genome Era

Tick-Rickettsia Relationships

Pathogenicity of Tick-Borne Rickettsiae

TICK-BORNE RICKETTSIAE IDENTIFIED AS HUMAN PATHOGENS

Pathogens Described Prior to 1984

Rickettsia rickettsii (Rocky Mountain spotted fever)

''Rickettsia conorii subsp. conorii'' (Mediterranean spotted fever)

''Rickettsia conorii subsp. israelensis'' (Israeli spotted fever)

''Rickettsia sibirica subsp. sibirica'' (Siberian tick typhus or North Asian tick typhus)

Rickettsia australis (Queensland tick typhus)

Emerging Pathogens (1984 to 2004)

Rickettsia japonica (Japanese or Oriental spotted fever)

''Rickettsia conorii subsp. caspia'' (Astrakhan fever)

Rickettsia africae (African tick bite fever)

Rickettsia honei (Flinders Island spotted fever)

''Rickettsia sibirica subsp. mongolitimonae.''

Rickettsia slovaca

Rickettsia heilongjiangensis

Rickettsia aeschlimannii

Rickettsia parkeri

Rickettsia massiliae

''Rickettsia marmionii.''

TICK-BORNE SFG RICKETTSIAE PRESUMPTIVELY ASSOCIATED WITH HUMAN ILLNESSES

''Rickettsia conorii subsp. indica'' (Indian Tick Typhus)

Rickettsia canadensis

''Rickettsia amblyommii''

''Rickettsia texiana''

Rickettsia helvetica

RICKETTSIAE ISOLATED FROM OR DETECTED IN TICKS ONLY

NEW APPROACHES TO DIAGNOSIS

Serology

Culture

Histochemical and Immunohistochemical Methods

Molecular Tools

TREATMENT

CONCLUSION

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) of the genus Rickettsia within the family Rickettsiaceae in the order Rickettsiales (276). These zoonoses are among the oldest known vector-borne diseases. In 1899, Edward E. Maxey reported the first clinical description of Rocky Mountain spotted fever (RMSF), the prototypical tick-borne rickettsiosis (198). In 1906, Howard T. Ricketts reported the role of the wood tick in the transmission of the causative agent, subsequently named Rickettsia rickettsii (283, 284, 365). In 1919, S. Burt Wolbach provided definitive experimental evidence that R. rickettsii, referred to as "Dermacentroxenus rickettsii" at that time, was maintained by ticks and also described the fundamental histopathologic lesions of RMSF (365). For approximately the next 90 years, R. rickettsii would be the only tick-borne rickettsia conclusively associated with disease in humans in the Western Hemisphere. During the 20th century, many other formally described or incompletely characterized SFG rickettsiae were detected in North American ticks, including Rickettsia parkeri in 1939, Rickettsia montanensis (formerly R. montana) in 1963, and Rickettsia rhipicephali in 1978. However, these rickettsiae were generally considered nonpathogenic (267, 276).

Distinctions between the occurrences of a single pathogenic tick-borne rickettsia and the various other nonpathogenic rickettsiae that resided in ticks were also made by investigators from other continents. In 1910, the first case of Mediterranean spotted fever (MSF) was reported in Tunis (72). The typical inoculation eschar was described in 1925 in Marseille (223). In the 1930s, the roles of the brown dog tick, Rhipicephalus sanguineus, and the causative agent Rickettsia conorii were described (43). For several decades, R. conorii was considered to be the only agent of tick-borne SFG rickettsioses in Europe and Africa. In a similar manner, Rickettsia sibirica (in the former USSR and China) and Rickettsia australis (in Australia) were generally believed to be the sole tick-borne rickettsial agents associated with these respective locations (276).

Until relatively recently, the diagnosis of tick-borne SFG rickettsioses was confirmed almost exclusively by serologic methods (174, 276). The Weil-Felix test, the oldest but least specific serological assay for rickettsioses, is still used in many developing countries. This test is based on the detection of antibodies to various Proteus antigens that cross-react with each group of rickettsiae, including the SFG. This assay lacks sensitivity and specificity and can suggest only possible spotted fever group rickettsiosis in a patient. Even with the microimmunofluorescence (MIF) assay, the current reference method in rickettsial serology, there are wide antigenic cross-reactions among SFG rickettsiae (276). In this context, when only one antigen is used (i.e., the agent known to be pathogenic for humans in the considered location), a positive serologic reaction does not necessarily imply that the patient's illness was caused by the rickettsial species used as the antigen in the assay. Inferences made from the results of relatively nonspecific serologic assays have likely hampered the correct identification of several novel SFG rickettsioses.

The recognition of multiple distinct tick-borne SFG rickettsioses during the last 20 years has been greatly facilitated by broad use of cell culture systems and the development of molecular methods for the identification of rickettsiae from human samples and ticks (267). As a consequence, during 1984 through 2005, 11 additional rickettsial species or subspecies were identified as emerging agents of tick-borne rickettsioses throughout the world (267, 276). In 1984, an emerging SFG rickettsiosis was identified in Japan (183). Its agent was isolated from a patient in 1989 and subsequently named Rickettsia japonica (342, 343). Thereafter emerging pathogens throughout the world were described, including "Rickettsia conorii subsp. caspia" (proposed name) in Astrakhan, Africa, and Kosovo; Rickettsia africae in sub-Saharan Africa and the West Indies; Rickettsia honei in Flinders Island (Australia), Tasmania, Thailand, and perhaps the United States; Rickettsia slovaca in Europe; "Rickettsia sibirica subsp. mongolitimonae" (proposed name) in China, Europe, and Africa; "Rickettsia heilongjanghensis" (proposed name) in China and the Russian Far East; Rickettsia aeschlimannii in Africa and Europe; "Rickettsia marmionii" (proposed name) in Australia (N. Unsworth, J. Stenos, and J. Graves, Abstr. 4th Int. Conf. Rickettsiae Rickettsial Dis., abstr. O-50, 2005), and R. parkeri in the United States (267). The last rickettsia is probably the best illustration, as R. parkeri was considered a nonpathogenic rickettsia for more than 60 years. Furthermore, the pathogenicity of Rickettsia massiliae has been recently demonstrated, 13 years after its isolation from ticks (349). Other recently described rickettsiae, including Rickettsia helvetica strains in Europe and Asia, have been presented as possible pathogens (267).

The last major review on rickettsioses was published in 1997 (276). Since that time, rickettsiology has undergone a significant evolution. Some SFG rickettsiae detected or isolated from ticks only and presented as potential pathogens in 1997 are now formally described and recognized as emerging pathogens. Many previously unrecognized rickettsiae of unknown pathogenicity have been recently detected in or isolated from ticks. The use of PCR and sequencing methods for the identification of SFG rickettsiae in ticks has led to new questions regarding the geographical distribution of tick-borne rickettsiae and the tick-rickettsia association. We present here an overview of the various tick-borne rickettsioses described to date and focus on some epidemiological circumstances that have contributed to the emergence of these newly recognized diseases. We also discuss some of the questions remaining to be resolved in the future.

RECENT DEVELOPMENTS AND CONTINUING GAPS IN RICKETTSIOLOGY

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During the last decade, the taxonomy of rickettsiae has undergone extensive reorganization (134, 276). The family Bartonellaceae (including Bartonella quintana, the agent of trench fever) as well as Coxiella burnetii, the agent of Q fever, were removed from the order Rickettsiales, which includes now two families, the Anaplasmataceae and Rickettsiaceae. The classification of this order continues to be modified as new data become available. Currently, all tick-associated rickettsiae (with the exception of Rickettsia bellii and Rickettsia canadensis) belong to the spotted fever group of the genus Rickettsia within the Rickettsiaceae (Fig. 1).

View larger version (35K): [in this window] [in a new window]   FIG. 1. Phylogenetic organization of tick-transmitted rickettsiae based on the comparison of gltA, ompA, ompB, and gene D sequences by using the parsimony method.

The comparison of 16S rRNA sequences is not useful for the taxonomy of rickettsiae because greater than 97% similarity exists between any two taxa. Several other genes can be used including gltA, ompA, ompB, and gene D. By use of molecular tools, one of the difficulties in rickettsiology has been the determination of a cutoff in the percent divergence among gene sequences that define a species, subspecies, or strain within the Rickettsia genus. Recent genetic guidelines for the classification of rickettsial isolates at the genus, group, and species levels, using the sequences of five rickettsial genes, including a 16S rRNA (rrs) gene, gltA, ompA, ompB, and gene D, have been proposed (104). This work was done using universally recognized species. According to these guidelines, to be classified as a new Rickettsia species, an isolate should not have more than one of the following degrees of nucleotide similarity, with the most homologous validated species: 99.8 and 99.9% for the rrs and gltA genes, respectively, and, when amplifiable, 98.8, 99.2, and 99.3% for the ompA and ompB genes and gene D, respectively (104). However, these guidelines may later be updated by the introduction of additional genetic or phenotypic characteristics and of new Rickettsia species.

The utility of multiple-gene sequencing for taxonomy has been discussed for the reevaluation of species definitions in bacteriology. Further, polyphasic taxonomy, which integrates phenotypic and phylogenetic data, seems to be particularly useful for rickettsial taxonomy, as demonstrated for other bacteria (326, 346). However, experts in the field of rickettsiology frequently do not agree on defining a species. One example concerns the closely related rickettsiae of the so-called R. conorii complex, including R. conorii strain Malish (the agent of MSF), Israeli spotted fever rickettsia (ISFR), R. conorii strain Indian (Indian tick typhus rickettsia [ITTR]), and Astrakhan spotted fever rickettsia (AFR).

In 1978, Philip et al., using mouse MIF serotyping, concluded that R. conorii isolates Malish, Moroccan, and Kenya belonged to the same serotype as ITTR (252). Using complement fixation, Bozeman et al. were also unable to distinguish ITTR from R. conorii isolates (F. M. Bozeman, J. W. Humphries, J. M. Campbell, and P. L. O'Hara, Symp. Spotted Fever Group Rickettsiae, p. 7-11, 1960). In contrast, Goldwasser et al. (R. A. Goldwasser, M. A. Klingberg, W. Klingberg, Y. Steiman, and T. A. Swartz, 12th Int. Congr. Intern. Med., p. 270-275, 1974), using mouse polyclonal antibodies, and Walker et al. (355), using monoclonal antibodies, observed that ITTR differed substantially from other R. conorii isolates. Regarding ISFR and AFR, we reported that PCR-restriction fragment length polymorphism (RFLP) allowed differentiation of these rickettsiae (292), and we demonstrated that AFR was different from ISFR and R. conorii on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and pulsed-field gel electrophoresis profiles (95). In 1995, Walker et al., using serotyping, Western blotting (WB), monoclonal antibody reactivity, and PCR amplification of the tandem repeats within ompA, concluded that ISFR belonged to the R. conorii species (352). However, in 1998, Dasch and colleagues differentiated R. conorii from ISFR by using PCR-RFLP and then proposed the names "R. sharonii" and "R. caspii" for ISFR and AFR, respectively (77, 149). However, phylogenetically, these rickettsiae constitute a homogeneous cluster supported by significant bootstrap values and are distinct from other Rickettsia species. In 2003, using the combination of genotypic criteria described above, we demonstrated that ITTR, AFR, and ISFR were not genetically different enough to be considered new species but belonged to the R. conorii species (104). Moreover, these rickettsiae exhibit differentiable serotypes and cause diseases with distinct clinical features in defined geographic locations.

To clarify the situation, we recently considered the report of the ad hoc committee on reconciliation of approaches to bacterial systematics which proposed that bacterial isolates within a species could be considered distinct subspecies if they were genetically close but diverged in phenotype (359). Therefore, we estimated the degrees of genotypic variation among 31 isolates of R. conorii, 1 isolate of ITTR, 2 isolates and 3 tick amplicons of AFR, and 2 isolates of ISFR by using multilocus sequence typing (MLST). Also, 16S rRNA and gltA genes, as well as three membrane-exposed protein-encoding genes, ompA, ompB, and sca4 (formerly gene D), were incorporated in MLST. To further characterize the specificities of distinct MLST types, we incorporated a prototype isolate from each of these into a multispacer typing (MST) assay, which we have previously demonstrated to be more discriminant than MLST at the strain level for R. conorii (see below). Furthermore, mouse serotypes were obtained for each of these MLST types. It is important to emphasize that this work was not a pure sequence-based classification. Among the 39 isolates or tick amplicons studied, four MLST genotypes were identified: (i) the Malish type, (ii) the ITTR type, (iii) the AFR type, and (iv) the ISFR type. Among these four MLST genotypes, the pairwise similarity in nucleotide sequence varied from 99.8 to 100%, 99.4 to 100%, 98.2 to 99.8%, 98.4 to 99.8%, and 99.2 to 99.9% for 16S rRNA genes, gltA, ompA, ompB, and sca4, respectively. Representatives of the four MLST types were also classified within four types by using MST genotyping as well as mouse serotyping. By using these results, we proposed to modify the nomenclature of the R. conorii species through the creation of the following subspecies: "R. conorii subsp. conorii subsp. nov." (type strain Malish, ATCC VR-613), "R. conorii subspecies indica subsp. nov." (type strain ATCC VR-597) (formerly Indian tick typhus rickettsia), "R. conorii subspecies caspia subsp. nov." (type strain A-167) (formerly Astrakhan fever rickettsia), and "R. conorii subspecies israelensis subsp. nov." (type strain ISTT CDC1) (formerly Israeli spotted fever rickettsia) (374). The description of R. conorii has been emended to accommodate the four subspecies (for detailed descriptions of the four subspecies, see reference 374 ). The same approach has been recently proposed for R. sibirica, for which two subspecies have been proposed, including "R. sibirica subsp. sibirica" and "R. sibirica subsp. mongolitimonae" (P. E. Fournier, Y. Zhu, and D. Raoult, Abstr. 4th Int. Conf. Rickettsiae Rickettsial Dis., abstr. P-180, 2005). Nonetheless, rickettsial taxonomy remains an evolving and controversial field; in this context, there is no universal consensus on the current classification, and some rickettsiologists believe that there are too many described species of Rickettsia.

Until recently, there was no formal genotypic method to describe rickettsiae at the strain level. In 2004, Fournier et al. tested the hypothesis that the most suitable sequences for genotyping bacterial strains are those that are found to be most variable when the genomes of two closely related bacteria are aligned (114). Using the nearly perfectly colinear genomes of R. conorii (a spotted fever group rickettsia) and R. prowazekii (a typhus group rickettsia), they found that the most variable sequences at the species level were variable intergenic spacers, which are significantly more than conserved genes, split genes, remnant genes, and conserved spacers (P values of <10^–2 in all cases). These spacers were also the most variable at the strain level. Using a combination of sequences from three highly variable spacers in a multispacer tool, they identified 27 genotypes among 39 strains of R. conorii subsp. conorii strain Malish (Seven). Further, this technique, which was named multispacer typing (MST), appeared to be a valuable tool for tracing rickettsial isolates from a single source with a difference in culture history of at least 60 passages. It was also found to be more discriminatory for strain genotyping than multiple-gene sequencing (P < 10^–2) (114). The advantages of MST include high discrimination, reproducibility, simplicity of interpretation, and ease of incorporation of the data obtained into accessible databases. MST could be used for tracking isolates from a wide variety of sources, including isolates from a single strain with different passage histories, and even be applied to clinical specimens.

Questions regarding the specificity of associations among rickettsiae and a particular tick species are unresolved, in part because specific characterizations of species and subspecies in both phyla may lack sensitivity. Consequently, it is difficult to determine how long a tick species has been associated with a rickettsial species and if coevolution has occurred. Some rickettsiae, such as R. rickettsii, may be associated with several different tick vectors from several different genera. This contrasts with other rickettsiae, such as R. conorii, which appear to be associated with only one tick vector (276). Between these extremes, there are certain rickettsiae which are associated with several species within the same genus, such as R. africae and R. slovaca with various Amblyomma spp. and Dermacentor spp., respectively (245). Finally, there is some evidence to indicate that some typhus group rickettsiae, particularly Rickettsia prowazekii, may in certain circumstances be associated with ticks (A. Medina-Sanchez, D. H. Bouyer, C. Mafra, J. Zavala-Castro, T. Whitworth, V. L. Popov, I. Fernandez-Salas, and D. H. Walker, Abstr. 4th Int. Conf. Rickettsiae Rickettsial Dis., abstr. O-53, 2005) (54).

In the 1980s, Burgdorfer et al. showed that ticks infected with the SFG rickettsia Rickettsia peacockii were refractory to infection with and maintenance of R. rickettsii (51). Recent studies of interspecies competition between different rickettsiae in the same tick, using cohorts of R. montanensis-infected and R. rhipicephali-infected D. variabilis organisms, have demonstrated similar inhibitory effects between rickettsiae: rickettsia-infected ticks exposed to the other rickettsial species by capillary feeding were incapable of maintaining both rickettsial species transovarially. It was suggested that rickettsial infection of tick ovaries may alter the molecular expression of the oocytes and cause interference or blocking of the second infection (182). The process of rickettsial "interference," i.e., one species of SFG rickettsia successfully outcompeting another for the microenvironment inside the tick, may have profound implications regarding the distribution and frequency of various pathogenic rickettsiae and the specific diseases they cause (F. M. Bozeman, J. W. Humphries, J. M. Campbell, and P. L. O'Hara, Symp. Spotted Fever Group Rickettsiae).

The life cycles of most tick-borne rickettsiae are also incompletely known. In natural vertebrate hosts, infections may result in a rickettsemia that allows noninfected ticks to become infected and for the natural cycle to be perpetuated. For example, R. rickettsii has been isolated from various small mammals, and some, including meadow voles, golden-mantled ground squirrels, and chipmunks, develop rickettsemias of sufficient magnitude and duration to infect laboratory-reared ticks. However, other wild and domesticated animals susceptible to infection with R. rickettsii, including dogs, cotton rats, and wood rats, produce rickettsemias too low or too transiently to routinely infect ticks (50, 219).

Ticks may also acquire rickettsiae through transovarial passage (transfer of bacteria from adult female ticks to the subsequent generation of ticks via the eggs). Because ixodid ticks feed only once at each life stage (245), rickettsiae acquired during blood meal acquisition from a rickettsemic host or through tranovarial route can be transmitted to another host only when the tick has molted to its next developmental stage and takes its next blood meal. This so-called transstadial passage (transfer of bacteria from stage to stage) is a necessary component for the vectorial competence of the ticks. When rickettsiae are transmitted efficiently both transstadially and transovarially in a tick species, this tick will serve as a reservoir of the bacteria and the distribution of the rickettsiosis will be identical to that of its tick host (245). R. slovaca multiplies in almost all organs and fluids of its tick host, particularly in the salivary glands and ovaries, which enables transmission of rickettsiae during feeding and transovarially, respectively (279). Two other methods for acquiring rickettsiae have been reported. Sexual transmission of R. rickettsii from infected males to noninfected female ticks has been described, but this process is unlikely to significantly propagate the infection in tick lineages, as venereally infected females do not appear to transmit rickettsiae transovarially (307). A second suggested method of acquisition of rickettsiae by ticks is the process of cofeeding, which occurs as several ticks feed in proximity on the host. In this circumstance, direct spread of bacteria from an infected tick to an uninfected tick may occur during feeding at closely situated bite sites, as demonstrated with R. rickettsii and D. andersoni (250).

Although tick-rickettsia relationships were a focus of interest by many pioneering rickettsiologists, most early studies concentrated on the role of ticks as vectors. Considerably less attention was directed to the relationships of rickettsiae with various tick cells, tissues, and organs and with specific physiologic processes of acarines. Transovarial transmission of rickettsiae in their recognized vectors has been demonstrated for several SFG species, including R. rickettsii (307), R. slovaca (279), R. sibirica (297), R. africae (154), R. helvetica (46), and R. parkeri (124). In some instances, transovarial transmission of a particular Rickettsia species may occur in a particular tick but cannot be sustained for more than one generation (182).

The percentage of infected eggs obtained from females of the same tick species infected with the same rickettsial strain may vary for as yet unknown reasons (47, 55). For some rickettsia-tick relationships, such as R. montanensis in D. variabilis (182), R. slovaca in Dermacentor marginatus (279), and R. massiliae in Rhipicephalus sanguineus group ticks (196), maintenance of rickettsiae via transovarial transmission may reach 100% and have no effect on the reproductive fitness and viability of the tick host. In contrast, transovarial transmission of R. rickettsii in D. andersoni diminishes survival and reproductive capacity of tick filial progenies. Recent experiments have shown that R. rickettsii is lethal for the majority of experimentally and transovarially infected D. andersoni ticks. In one study, most nymphs infected as larvae by feeding on rickettsemic guinea pigs died during the molt into adults, and most of adult female ticks infected as nymphs died prior to feeding. Rickettsiae were vertically transmitted to 39.0% of offspring, and significantly fewer larvae developed from infected ticks (214). The lethal effect of R. rickettsii on its acarine host, coupled with the competitive interactions among different rickettsiae that inhabit the tick microenvironment, may influence the low prevalence of ticks infected with R. rickettsii in nature and affect its enzootic maintenance (214).

Interestingly, basic questions about the tick-rickettsia relationship remain for MSF, one of the oldest recognized tick-borne rickettsioses. In this context, we are unaware of any well-documented demonstration of transovarial transmission of R. conorii in Rhipicephalus sanguineus. In 1932, Blanc and Caminopetros demonstrated that larvae, nymphs, and adults could act as vectors of MSF. Furthermore, over winter, unfed males and females were shown to be able to transmit the agent (38). It was also shown that when eggs or larvae obtained from infected Rhipicephalus sanguineus females were crushed and inoculated into humans, MSF was obtained. These data suggest that transovarial transmission of the MSF agent occurs in ticks (38). However, neither the transovarial transmission rate (the proportion of infected females giving rise to at least one positive egg or larva) nor the filial infection rate (proportion of infected eggs or larvae obtained from an infected female), which would be useful for comparison with infection rates in nature, is known to our knowledge. It is not known if transovarial transmission of R. conorii is maintained from generation to generation of Rhipicephalus sanguineus. In a similar manner, the effect of R. conorii on its tick host and potential interactions with other rickettsiae associated with the same tick species, such as R. massiliae, are unknown (25).

Female D. andersoni ticks infected with R. rickettsii and incubated at 4°C show a lower mortality rate than infected ticks at 21°C (214). This discrepancy may be linked with the long-recognized but poorly explained phenomenon known as reactivation (325). In nature, stress conditions encountered by rickettsiae within the tick include starvation and temperature shifts. As an example, as ticks enter diapause, weeks to months may pass until they obtain their next blood meal. In the laboratory, R. rickettsii in D. andersoni ticks loses its virulence for guinea pigs when the ticks are subjected to physiological stress, such as low environmental temperature or starvation. However, subsequent exposure of these same ticks to 37°C for 24 to 48 h or the ascertainment of a blood meal may restore the original virulence of the bacteria. During tick blood-feeding, rickettsiae undergo various physiological changes and proliferate intensively as they reactivate from a dormant avirulent state to a pathogenic form (47, 325, 363, 364).

The precise molecular mechanisms responsible for the adaptation of rickettsiae to different host conditions and for reactivation of virulence are unknown. However, the stress adaptation in some gram-negative bacteria, also called the stringent response, has been shown to be mediated by the nucleotide guanosine-3,5(bis)pyrophosphate(ppGpp), which is modulated by spoT genes. Interestingly, annotation of R. conorii genome reveals five spoT paralogs, and environmental stress conditions are accompanied by a variable spoT1 transcription in R. conorii (296). This phenomenon could play a role in adaptation of rickettsiae to ticks and during the process of reactivation. It has also been hypothesized that changes in outer surface proteins occur during alternating infection in ticks and in mammals (296). Further studies of the molecular dynamics of between rickettsiae and ticks, similar to work on molecular interactions at the tick ovary-rickettsia interface recently published by Mulenga et al. (210), will be needed to better understand these processes.

The first component for a rickettsia to be a potential pathogen of humans is the likelihood of the bacterium to be transmitted through the tick bite, which generally implies that the rickettsia can localize to the salivary glands of the tick. Some SFG rickettsiae, including R. peacockii, produce heavy infections in the ovaries but do not invade the salivary glands of their tick hosts, precluding subsequent transmission to potential vertebrate hosts during blood meal acquisition (215). A potential pathogen must also be associated with a tick with some proclivity to bite a human host. In this context, tick-host specificity is a key component of the epidemiology of tick-borne diseases. Certain tick species rarely, if ever, bite humans, and even if these ticks are associated with highly pathogenic rickettsiae, disease in humans will rarely, if ever, be associated with these species. However, it may also be possible that tick-borne rickettsiae that are excreted in tick feces might initiate infection via abraded skin. For example, viable R. slovaca have been isolated from the feces of D. marginatus collected in nature (279). The abundance of a particular tick vector, the prevalence of the infection within ticks, and the prevalence of natural hosts of the ticks that come in contact with humans are other elements that affect the frequency of tick-borne rickettsioses that have been discussed elsewhere (245).

When transmitted to a susceptible human host, pathogenic tick-borne SFG rickettsiae localize and multiply in endothelial cells of small- to medium-sized blood vessels, causing a vasculitis which is responsible for the clinical and laboratory abnormalities that occur in tick-borne rickettsioses (276). Molecular characteristics and the expression of particular rickettsial gene products likely contribute to differences in pathogenicity among various species of spotted fever group rickettsiae. The expression of OmpA by R. rickettsii allows adhesion of and entry into host endothelial cells by this pathogen (178). Despite its close phylogenetic placement to R. rickettsii, R. peacockii (another SFG rickettsia found in D. andersoni) possesses an ompA gene that contains three premature stop codons and is unable to express the OmpA protein; this rickettsia is considered a nonpathogen (18). Also, it has been suggested that the OmpB plays a role in the adherence to and invasion of host cells by R. japonica (344).

After phagocytosis and internalization, the phagocytic vacuole is rapidly lysed and rickettsiae escape the phagocytic digestion to multiply freely in the host cell cytoplasm and nucleus, the latter a characteristic specific for bacteria in the spotted fever group of the genus Rickettsia (276). Rickettsiae can move from cell to cell by actin mobilization (357). Escape from vacuole is suspected to be mediated by an enzyme, possibly phospholipase A2 (351). However, the presence of a gene encoding a phospholipase D has been recently shown, and this gene may be a key factor for virulence (281). More recently, a R. conorii surface protein, RickA, was identified in vitro as an activator of the Arp2/3 complex, which is essential in actin polymerization (126).

Many aspects of rickettsial pathogenesis remain unknown. Animal models have been used to predict the pathogenicity to humans of other symbionts found in arthropods; however, this technique is unreliable with the rickettsiae. For example, the T-type strain of R. rickettsii causes only a mild illness in guinea pigs but is highly pathogenic in humans. In the past, pathogenicity of various rickettsiae in guinea pigs was considered an indication of the pathogenicity of the agent in humans; however, the pathogenic role of a tick-borne rickettsia can only be determined conclusively by isolating or detecting the organisms from patients with signs of disease. In this context, nonpathogenic rickettsiae are better characterized as rickettsiae of unknown pathogenicity until clear evidence exists to show that the particular bacterium does not cause disease in humans.

In this review, the rickettsiae designated as human pathogens have been isolated in cell culture or detected by molecular methods from blood or tissues from patients with illnesses clinically compatible with spotted fever rickettsioses. When cases are documented solely by serologic methods, the pathogenicity of the rickettsia used as an antigen can only be presumed, particularly when a limited number of rickettsial antigens are used in the evaluation. Other rickettsiae can be considered potential pathogens, particularly if they have been detected in the salivary glands of tick species readily biting people.

TICK-BORNE RICKETTSIAE IDENTIFIED AS HUMAN PATHOGENS

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RMSF remains the most severe of all tick-borne rickettsioses. Prior to the discovery of effective antibiotics and appropriate supportive therapy, persons with RMSF frequently succumbed to the infection: from 1873 to 1920, 283 (66%) of 431 reported cases resulted in death (68). RMSF also claimed the lives of many early investigators, including entomologists and laboratorians, who worked with R. rickettsii (261). This disease continues to cause significant mortality in the United States. Five to 39 deaths were reported annually to public health authorities during 1983-1998; however, the magnitude of underreporting may be profound, and it is estimated that approximately 400 additional RMSF deaths were not reported during this same interval (229). Despite its name, RMSF has been reported throughout most of the continental United States, except for Maine and Vermont (194).

Although most cases are associated with rural or semirural areas, autochthonous cases have been described in large urban centers, including New York City, where rickettsia-infected D. variabilis were found in parks and vacant lots (302). The disease is most prevalent in the southeastern and midwestern United States, with the largest number of reported cases originating from North Carolina, Oklahoma, Tennessee, Arkansas, South Carolina, Maryland, and Virginia (194, 337). Because of the seasonal activity associated with the tick vectors of R. rickettsii, RMSF demonstrates a similar pattern, with a peak in cases observed during mid-spring through late summer in the United States. From 1997 through 2002, 3,649 cases of RMSF were reported to the Centers for Disease Control and Prevention, and approximately 90% of confirmed cases occurred from April through September. The average annual incidence of RMSF for this period was 2.2 cases per million persons (67).

Multiple and diverse factors contribute to the incidence rates of complex zoonoses, including RMSF and other tick-borne SFG rickettsioses, and annual case counts are generally subject to wide regional and temporal variabilities. The annual number of cases of RMSF in the United States, as determined by passive surveillance, has fluctuated markedly since the beginning of systematic collection of these data in 1920. These numbers may have been affected by one or more of the following: changes in surveillance affected by improved recognition and disease reporting, cyclic changes in the transmission caused by competition or interference with other tick-borne rickettsiae, diminished tick populations caused by widespread use of pesticides (particularly dichlorodiphenyltrichloroethane), and increased human contact with tick-infested habitats through recreational activities (68). For example, the average annual incidence of RMSF in the United States fluctuated from a low of 1.4 cases per million persons in 1998 to a high of 3.8 cases per million in 2002, representing the lowest and highest incidence rates, respectively, recorded since 1993 (67).

The primary vector of RMSF for most of the United States is the American dog tick D. variabilis (Fig. 2). This tick inhabits the Great Plains region, the Atlantic Coast, California, and southwestern Oregon. It has also been described in southeastern Saskatchewan Province in Canada and as far south as northern Mexico. Adult and nymphal activity generally begins in March or April and extends through August or September. The ticks at immature stages feed almost exclusively on small rodents. D. andersoni (the Rocky Mountain wood tick) is an important vector in the Rocky Mountain states and Canada. The distribution of this tick occurs in the mountainous regions of the western United States and the southern parts of British Columbia. Adults feed primarily on large animals such as horses, cattle, sheep, coyotes, deer, and bear. Immature stages feed largely on small mammals. The Rocky Mountain wood tick is most abundant in areas where small rodent share habitats with large wild and domestic animals. This situation was prevalent in the Bitterroot valley of Western Montana during early investigations of RMSF, when a large population of Columbian ground squirrels (Citellus columbianus columbianus) lived in close association with humans and domestic animals (44).

View larger version (45K): [in this window] [in a new window]   FIG. 2. Dermacentor variabilis, the primary vector of Rocky Mountain spotted fever in most of the United States. From left to right, male, female, nymph, and larva. Bar scale, 1 cm.

In Central and South America, natural infections with R. rickettsii have been identified in Amblyomma cajennense (the Cayenne tick) specimens collected in Mexico (59), Panama (84), and Brazil (86) and in Amblyomma aureolatum specimens in Brazil (303). Considerable evidence accumulated by investigators in Mexico during the early to mid-1940s convincingly demonstrated a role of Rhipicephalus sanguineus in the transmission cycle of a severe spotted fever rickettsiosis (presumably RMSF) to humans in several northern and central states of that country, including Coahuila, Durango, San Luis Potosí, Sinaloa, Sonora, and Veracruz (58, 59, 189). Surprisingly, despite historical data on natural infections in and vector competency of Rhipicephalus sanguineus (235), and a generally ubiquitous and peridomestic distribution of this tick, similar studies to conclusively incriminate the brown dog tick as an important vector of RMSF in other regions of the Western Hemisphere were absent until 2002-2004, when 15 cases of RMSF were identified in two rural communities in eastern Arizona. In both locales, only Rhipicephalus sanguineus ticks were found in the areas frequented by case patients, typically in peridomestic settings associated with abundant pet and stray dogs. Rhipicephalus sanguineus ticks were also found occasionally attached to individuals in the community, most often children, and infesting the local dog population. R. rickettsii was identified by using culture and PCR in ticks collected at case households (82). It is likely that similar ecologic scenarios exist in other areas of the Western Hemisphere and that investigators will subsequently identify other peridomestic cycles of RMSF that involve Rhipicephalus sanguineus.

The mean incubation period of RMSF following tick bite is 7 days (range, 2 to 14 days). Only approximately 60% of patients recall a tick bite (194, 317), as these bites are generally painless and the tick may attach in places of the body difficult to observe, including the scalp, axillae, and inguinal areas (245). In contrast with most other tick-borne SFG rickettsiae, R. rickettsii does not generally elicit an eschar at the tick bite site. The onset of the disease includes high fever and a headache that may be associated with malaise, myalgias, nausea, vomiting, anorexia, generalized or focal abdominal pain, and diarrhea. When such nonspecific symptoms dominate the clinical presentation, misdiagnosis and treatment delay can occur. The rash of RMSF is usually not apparent until the third day of fever or later and begins as small, irregular, pink macules that typically appear first on wrists, ankles, and forearms. The rash may later evolve to papules or petechiae. The characteristic spotted rash of RMSF is generally observed in persons on or after the fifth day of illness and heralds progression of the infection to more severe disease (313, 317). In approximately 10% of patients, the rash may be absent, which may delay diagnosis and therapy (317).

RMSF may result in various neurological manifestations, including deafness, convulsions, and hemiplegia. Other manifestations of severe disease include pulmonary and renal failure, myocarditis, and necrosis and gangrene of the fingers, toes, earlobes, and external genitalia. The case fatality rate of untreated RMSF is 10 to 25%, depending on patient's age, and approximately half of the deaths occur on or before the eighth day of illness (194). Recently, risk factors for death of 6,388 RMSF-confirmed (81%) and probable RMSF cases reported during 1981-1998, including 213 deaths (average annual case fatality rate, 3.3%), were studied. Older patient age, onset-to-treatment interval of 5 days, lack of tetracycline treatment, and chloramphenicol-only treatment remained significantly associated with fatal outcome (137). Although chloramphenicol and tetracyclines were considered effective antibiotic therapies for RMSF, the results of this study indicate that tetracylines are superior to chloramphenicol for the treatment of this disease. Doxycycline is currently considered the drug of choice for nearly all patients with RMSF, including young children (137, 194, 265). Unfortunately some physicians are not aware of this: among 84 primary care physicians in Mississippi who participated in a 2002 survey examining the knowledge, attitudes, and practices regarding diagnosis and treatment of RMSF, only 21% of family practice physicians and only 25% of emergency medicine physicians correctly identified doxycycline as the antibiotic of choice for treating children with RMSF (224).

RMSF is likely underdiagnosed and underreported in the United States, particularly in states where physicians are less aware of the disease (350). Surprisingly few research teams in the United States currently work with SFG rickettsiae, even though many questions posed by Ricketts and others in 1909 are still unanswered (350). Early investigators commented on differences between case fatality rates of RMSF identified among patients residing in certain areas in the United States (i.e., 5% in Idaho versus 65 to 80% in the Bitterroot Valley of Montana) (284, 365). In a similar manner, several contemporary studies have commented on the frequency of antibodies reactive with R. rickettsii among persons with no history of an illness of the severity generally associated with RMSF. Using these data, some have inferred that mild or subclinical infections with R. rickettsii may occur. For example, serum specimens of 32 (9.1%) of 352 children showed immunoglobulin G (IgG) titers of 64 to R. rickettsii antigen when tested by indirect fluorescent-antibody assay (IFA); however, only 8 of these children had experienced a febrile illness accompanied by rash or headache in the previous year, and none had ever been hospitalized or treated for RMSF (335). Another recent study identified IgG titers of 64 to R. rickettsii in the sera of 239 (12%) 1999 children 1 to 17 years of age, collected from various medical facilities in six southeastern and south-central states in the United States. These investigators suggested that at least some of the seroreactivity identified in this study could be directed against other spotted fever group rickettsiae not previously considered pathogenic and that most infections with SFG rickettsia may be relatively mild (192).

More compelling are recent prospective evaluations of individuals who seroconvert to R. rickettsii following tick bites and for whom mild or no illness is reported. In two recent studies involving military personnel exposed to ticks during training exercises in rural areas, only 20 to 44% of persons with recent evidence of spotted fever rickettsial infection by IFA or enzyme immunoassay tests developed symptoms compatible with rickettsiosis (e.g., fever, rash, myalgia, or headache), and none of the 67 individuals from these studies who seroconverted to R. rickettsii developed an illness severe enough to require hospitalization (199, 370). These findings, particularly viewed in context with the historically recognized severity of RMSF and the known cross-reactivity of spotted fever group rickettsial antigens, strongly suggest that many serologically confirmed cases of RMSF in the United States represent infections with spotted fever group rickettsiae other than R. rickettsii.

RMSF has also been identified in several provinces of Canada (201), several states in Mexico (56, 57), and in Panama (83), Costa Rica (115), Colombia (247), Brazil (86), and Argentina (286). Outside of the United States, RMSF has been most extensively described in Brazil, where R. rickettsii has been associated of with various synonymous diseases termed "São Paulo exanthematic typhus," "Minas Gerais exanthematic typhus," and "Brazilian spotted fever" since the early 1930s (86, 118, 232). A primary vector of R. rickettsii in Brazil is A. cajennense, a tick that feeds on various medium to large wild and domesticated animals, including tapirs, capybaras, horses, and dogs (168). Despite the extensive investigation of this disease by South American scientists during the years shortly following its discovery, relatively little attention was directed to the study of spotted fever in Brazil, and few cases were identified until the mid-1980s. During the last 20 years, a resurgence in identified cases, accompanied by increasing interest in rickettsioses by Brazilian investigators and others, have intensified the study of these diseases in this region (79, 80, 117, 207, 318). Cases of spotted fever have been documented by serology or immunostaining of tissues in several states, particularly in the southeast region of the country, including Minas Gerais, São Paulo, Rio de Janeiro, and Espirito Santo (79, 118), where most cases occur between July and December. Recently, Angerami et al. reported 23 patients with a confirmed diagnosis of Brazilian spotted fever either by isolation of R. rickettsii from blood or skin (13 patients) or a fourfold rise in MIF titers (8 patients). They were admitted with fever at the Hospitalas das Clinicas da Unicamp, São Paulo, Brazil. Relevant clinical features included myalgias (80%), headache (66%), icterus (52%), exanthema (47%), consciousness impairment (43%), vomiting (42%), abdominal pain (38%), respiratory distress (37.5%), acute renal insufficiency (35.3%), and hypotension and shock (33%). Hemorrhagic manifestations, including petechiae and suffusions, were frequent (69.5%). The case fatality rate was 30% (R. N. Angerami, M. R. Resende, S. B. Stuchi Raquel, G. Katz, E. Nascimento, and L. J. Silva, Abstr. 4th Int. Conf. Rickettsiae Rickettsial Dis., abstr. P-160, 2005).

The first confirmed cases of spotted fever rickettsiosis in Argentina were described in 1999. Between November 1993 and March 1994 in Jujuy Province in northwestern Argentina, six children with fever, rash, and a history of recent tick bite were evaluated for rickettsial infections. Immunohistochemical staining of tissues obtained at an autopsy of one fatal case confirmed a spotted fever group rickettsiosis, and the serum of another patient convalescing from the illness showed high antibody titers to R. rickettsii when tested by MIF. A. cajennense ticks were collected from dogs and pets in the area (286). The recent identification of spotted fever rickettsiosis in Peru (37) indicates that R. rickettsii or other related rickettsiae are also endemic in this country (36). The incidence and distribution of RMSF and other tick-borne rickettsioses in Latin America are undoubtedly underestimated and await the collaborative efforts of physicians, rickettsiologists, entomologists, and epidemiologists to characterize the magnitude and public health impact of these infections in these regions (350).

"Rickettsia conorii subsp. conorii" (Mediterranean spotted fever) In 1910, the first case of MSF was reported in Tunis (72). The disease was thereafter also known as "boutonneuse fever" because of a papular rather than macular rash. The typical inoculation eschar at the tick bite site, the hallmark of many SFG rickettsioses, was described in 1925 in Marseille by Boinet and Pieri (223, 276). In the 1930s, the role of Rhipicephalus sanguineus and the causative agent subsequently named R. conorii were described (43). As discussed previously, these isolates may be collectively identified as Rickettsia conorii subsp. conorii subsp. nov. (104, 374). Three strains of R. conorii subsp. conorii include (i) Seven or Malish (the most common strain identified in our laboratory from France, Portugal, and northern Africa), (ii) Kenyan, and (iii) Moroccan, which is apparently a unique isolate (D. Raoult, unpublished data).

MSF is endemic in the Mediterranean area, including northern Africa and southern Europe. Cases continue to be identified in new locations within this region, as some cases were recently described in Turkey (165). In Italy, approximately 1,000 cases are reported each year (8). MSF is a reportable disease in Portugal (14), where the annual incidence rate of 9.8 cases per 100,000 persons is the highest of the rates of all Mediterranean countries (85). As with all rickettsioses, this rate likely underestimates the true incidence, and some authors suggest that there are seven times more cases than officially reported (85). Some cases have also been sporadically reported in northern and central Europe, including Belgium (172), Switzerland (248), and northern France (312), where Rhipicephalus sanguineus can be imported with dogs and survive in peridomestic environments providing acceptable microclimatic conditions, including kennels and houses (245). MSF is also encountered infrequently in sub-Saharan Africa and around the Black Sea (276). Although an MSF-like disease was described in Vladivostok in the eastern part of Russia in 1966 (332), no direct evidence of R. conorii infection has been reported there since that time.

In Europe, cases are encountered in late spring and summer, when the tick vectors are most active. In France, most cases are diagnosed during July and August, because of increased outdoor activity associated with the peak of activity of immature ticks that are far smaller than adults and difficult to observe even when attached to the body (276). Similarly, most samples submitted for diagnostic evaluation in Portugal at the Center for Vectors and Infectious Disease Research, National Institute of Health, are received between July and September (14). In Croatia, >80% of cases occur between July and September, with a peak in August (263).

An increase in the numbers of MSF cases observed in France, Italy, Spain, and Portugal during the 1970s paralleled similar increases in RMSF observed in the United States during this same decade (188). This increase in incidence was correlated with higher temperatures and lower rainfall in Spain and with a decrease in the number of days of frost during the preceding year in France (121). Although Rhipicephalus sanguineus adapts well to urban environments, it is relatively host specific and rarely feeds on people unless its preferred host (the domestic dog) is not available. For this reason, the incidence of MSF is relatively low in southern France (approximately 50 cases per year per 100,000 persons), despite the fact that 5% to 12% of Rhipicephalus sanguineus ticks in the region are infected with spotted fever group rickettsiae. To our knowledge, a recent report describing 22 Rhipicephalus sanguineus (1 adult and 21 nymphs) attached to an alcoholic homeless man living with his dog near Marseille (135) was the first documentation of more than one Rhipicephalus sanguineus feeding on a human host (121). Because this infestation was associated with the highest summer temperatures noted in France during the past 50 years in France, it is possible that host-seeking and feeding behaviors of this tick were altered by unusual climactic circumstances (245). In this context, it is also likely that other homeless persons who live and sleep in proximity to Rhipicephalus sanguineus-infested dogs are at increased risk for MSF (248, 277). Most recently, another unusual case of MSF including three inoculation eschars has also been observed in southern France (D. Raoult, unpublished data).

After an asymptomatic incubation of 6 days, the onset of MSF is abrupt and typical cases present with high fever (>39°C), flu-like symptoms, a black eschar (tache noire) at the tick bite site (7, 276) (Fig. 3). In a few cases, the inoculation occurred through conjunctivae and patients presented with conjunctivitis. One to 7 days (median, 4 days) following the onset of fever, a generalized maculopapular rash that often involves the palms and soles but spares the face develops (Fig. 3). Usually, patients will recover within 10 days without any sequelae. However, severe forms, including major neurological manifestations and multiorgan involvement may occur in 5 to 6% of the cases (1, 274). In France, MSF involves mostly males under 10 years of age or older than 50 years. The mortality rate is usually estimated around 2.5% among diagnosed cases (1.50% in the last decade in Portugal, including 2.58% in 1997) (1, 14). Classic risk factors for severe forms include advanced age, immunocompromised situations, chronic alcoholism, glucose-6-phosphate-dehydrogenase deficiency, prior prescription of an inappropriate antibiotic, and delay of treatment (276). In 1997 in Beja, a southern Portuguese district, the case fatality rate in hospitalized patients with MSF was 32.3%, the highest ever obtained there since 1994. Interestingly, when risk factors for fatal outcome were studied in 105 patients hospitalized between 1994 and 1998, the risk of dying was significantly associated with diabetes, vomiting, dehydration, and uremia (85). Some differences in the severity of MSF in different areas, even in the same country, such as Catalonia in northern Spain, have been noted. There, the disease seems to be milder than elsewhere in the country (102). However, cases in this area could be caused by rickettsiae different than R. conorii. For example, a new spotted fever group rickettsial strain (R. massiliae Bar 29) of unknown pathogenicity for humans was isolated there in 1996 from Rhipicephalus sanguineus ticks. We know now that this rickettsia is pathogenic for humans (349).

View larger version (53K): [in this window] [in a new window]   FIG. 3. Inoculation eschar (top panel) and maculopapular rash (bottom panel) on a patient with Mediterranean spotted fever.

"Rickettsia conorii subsp. israelensis" (Israeli spotted fever) The first cases of rickettsial spotted fever in Israel were reported in the late 1940s (345), and the number of cases increased following the development of new settlements in the rural areas of this country (276). Clinically, the disease appeared milder and with a shorter duration than classical MSF, and the typical inoculation eschar was usually lacking. These preliminary clinical data led some investigators to suspect than the cause of this rickettsiosis was different from the agent of MSF. In 1971, the agent of Israeli spotted fever was isolated from a patient (R. A. Goldwasser, M. A. Klingberg, W. Klingberg, Y. Steiman, and T. A. Swartz, Front. Intern. Med., 12th Int. Congr. Intern. Med., p. 270-275, 1974). Two other antigenically identical agents were isolated from Rhipicephalus sanguineus ticks collected on the dogs of two patients with serologically documented Israeli spotted fever. These three isolates were characterized as rickettsiae closely related to but slightly different from R. conorii isolates obtained from patients with MSF (R. A. Goldwasser, M. A. Klingberg, W. Klingberg, Y. Steiman, and T. A. Swartz, Front. Intern. Med., 12th Int. Congr. Intern. Med., p. 270-275, 1974). This observation has been confirmed by recent molecular studies (111, 294, 295, 310), and it has been recently proposed that the agent of Israeli spotted fever constitutes a subspecies of R. conorii identified as Rickettsia conorii subsp. israelensis subsp. nov. (374).

Israeli spotted fever appears as a typical spotted fever, but the eschar at the inoculation site is absent in >90% of cases and resembles a small pinkish papule rather than a real eschar (130). Splenomegaly and hepatomegaly are seen in 30 to 35% of patients. The disease may be acquired even without direct contact with animals, through exposure to ticks in places frequented by dogs, as demonstrated in three grouped cases in children (319). Several fatal cases and severe forms have been described, especially in children and in people with glucose-6-phosphate dehydrogenase deficiency, and the prevalence of the disease seems to be increasing (130, 278, 369). Although asymptomatic infections have been described by seroconversion, the test used was not specific enough to ensure that the Israeli isolate was definitely the agent provoking the serologic response (306).

In 1999, R. conorii subsp. israelensis was isolated from three patients living in semirural areas along the River Tejo in Portugal (12). Of interest was the fact that none of the patients had traveled away from Portugal during the previous year, and none reported an eschar. All patients had severe disease, and two patients died with septic shock and multiorgan failure. More recently, Sousa et al. reported the clinical data of 44 patients infected with R. conorii subsp. israelensis in Portugal between 1994 and 2004. Cases were confirmed by isolation of the rickettsia from blood or by PCR on skin biopsy specimens. The absence of an eschar was noted for 54% of the patients. All but two patients presented with a rash. A total of 10 patients died. These clinical characteristics were not statistically different from those of 44 patients infected with R. conorii subsp. conorii at the same period (R. Sousa et al., Abstr. 4th Int. Conf. Rickettsiae Rickettsial Dis., abstr. O-22).

The occurrence of R. conorii subsp. israelensis in Portugal indicates that the geographic distribution of Israeli spotted fever is wider than previously appreciated. This was confirmed more recently when R. conorii subsp. israelensis was detected in Sicilian Rhipicephalus sanguineus ticks (120).

"Rickettsia sibirica subsp. sibirica" (Siberian tick typhus or North Asian tick typhus) R. sibirica is the agent of Siberian tick typhus, a spotted fever group rickettsiosis that was first described in Primorye in the spring-summer season of 1934 to 1935 by Shmatikov (280). Human isolates obtained in 1946 were used as reference strains for molecular studies many years later (17). Siberian tick typhus is well documented in the former USSR, but relatively few descriptions are available in the English medical literature (280). Active foci of the disease are widely spread in Asiatic Russia, with more than 80% of the cases being observed in Altai (Western Siberia) and Krasnoyarsk regions. The disease is frequently reported during spring and summer months. Since 1979, a constant increase of the number of cases has been observed. Between 1979 and 1997, 23,891 cases were recorded (297).

R. sibirica has been found in several species of ticks, and some of them have been presented as the principal vectors Siberian tick typhus (297). These include Dermacentor nuttalli in the mountainous steppe of western and eastern Siberia, D. marginatus in the steppe and meadow regions of western Siberia and northern Kazakhstan, Dermacentor silvarum in forest shrubs, and Haemaphysalis concinna in swampy tussocks of some southern and far eastern territories of Siberia (17, 297). Isolates obtained from these species of ticks, respectively, in 1949, 1959, 1983, and 1986 are available at the Gamaleya Research Institute of Epidemiology and Microbiology in Moscow (17). These ticks may act as vectors but also reservoirs of R. sibirica which is maintained in ticks through transstadial and transovarial transmission, as demonstrated at least for D. nuttalli. More recently, a rickettsial strain that had been isolated from Ixodes persulcatus and maintained at the Omsk Research Institute of Natural Foci Infections was identified as R. sibirica (S. Shpynov, P. E. Fournier, N. Rudakov, I. Samoilenko, T. Reshetnikova, V. Yastrebov, M. Schaiman, I. Tarasevich, and D. Raoult, Abst. 4th Int. Conf. Rickettsiae Rickettsial Dis., abstr. P-179).

The incubation period is usually 4 to 7 days following a tick bite. Clinical features include a high fever associated with an inoculation eschar that is often accompanied by regional lymphadenopathy. Severe headache, myalgia, and digestive disturbances are concomitant symptoms and can last for 6 to 10 days without treatment. The rash, which may be purpuric, usually occurs 2 to 4 days after the onset of symptoms. Although central neurological involvement may occur, this disease is usually mild and is seldom associated with severe complications (297).

Infection due to R. sibirica is also prevalent in northern China, where it is known as North Asian tick typhus (98, 371). There an isolate was obtained from D. nuttalli in 1974, and from patients in 1984 when five patients with characteristic symptoms of spotted fever were seen in Xinjiang, China. D. nuttalli specimens were attached to four patients, and the last patient recalled a tick bite (99). About 20 strains of SFG rickettsiae in China have been identified as R. sibirica from patients, various species of ticks, rodents (Microtus fortis), and hedgehogs (99, 372). Recently, a distinct strain of R. sibirica, currently considered a subspecies (see "Rickettsia sibirica subsp. mongolotimonae" below), emerged as a pathogen for humans in Europe and Africa.

Rickettsial strains antigenically identical to R. sibirica have also been isolated from several species of ticks in Pakistan (289). However, because only serological methods have been used to characterize these strains, to our knowledge, there is no definitive evidence of the prevalence of R. sibirica in Pakistan.

Rickettsia australis (Queensland tick typhus) Queensland tick typhus has been clinically recognized since 1946. The first cases were observed among Australian troops training in the bush of northern Queensland State in eastern Australia. Rickettsiae were isolated from 2 of 12 infected soldiers (4). Using serological methods, this agent was found to be a new spotted fever group rickettsia (256) and was named R. australis in 1950 (249). Thereafter, Queensland tick typhus has been recognized along the entire eastern coast of Australia east of the Great Dividing Range (127, 314, 316). In regions south of Queensland, cases were recorded in the 1990s in the eastern coastal region of New South Wales, including its capital, Sydney (93). Spotted fevers with slight clinical and epidemiological differences were subsequently reported in Victoria and on Flinders Island (328). Although these cases were primarily assumed to be due to R. australis (316), it is now known that a different pathogenic rickettsia, R. honei, occurs at least in Flinders Island, where it was shown to be responsible for the cases (see below). To date, R. australis has been definitely isolated only from patients in Queensland (4, 257).

R. australis has been identified in Ixodes holocyclus, a common, human-biting tick in Queensland (60). This tick also feeds on a broad range of vertebrate hosts. It is distributed primarily in coastal regions but is also prevalent in the rain forests of Queensland (287). R. australis has also been isolated from Ixodes tasmani, a species that exists along the coast as well as in the interior regions of south and western Australia (287). This tick rarely bites humans but may play a role in the enzootic maintenance of R. australis in small animals (60, 127). An uncharacterized SFG rickettsia was recently identified in the hemolymph of an Ixodes cornuatus tick removed from a human in Victoria (129). This tick is prevalent in south costal New South Wales, eastern Victoria, and Tasmania (288). A number of vertebrates, including bush rats, bandicoots, and domestic dogs, are common hosts for all these ticks. In one study, antibodies reactive with R. australis were detected in 54 of 307 bandicoots and rodents trapped in northern Queensland; however, the precise role of vertebrates as reservoirs of R. australis is not known (74).

Although Queensland tick typhus is a notifiable disease in Australia, it is seldom reported. A review on 62 cases of spotted fever recorded in Australia between 1946 and 1989 (16) indicated that 37 of these cases that originated from Queensland and New South Wales could be considered infections due to R. australis. A total of 78% of the cases occurred between June and November, and cases from both urban and suburban areas were reported. Approximately 76% of patients recall an antecedent tick bite. The disease is characterized by a sudden onset characterized by fever, headache, and myalgia, followed within 10 days by maculopapular or vesicular rash. An inoculation eschar is identified in approximately 65% of cases, and lymphadenopathy is identified in 71% of cases. The disease ranges from mild to severe, but only two patients with fatal disease have been described (127, 315).

Since 1984, approximately 30 to 40 cases have been reported annually, mainly along the coast of southwestern and central Japan (184, 185). The disease occurs from April to October. High-risk areas for acquiring the infection include bamboo plantations, crop fields, and coastal hills and forests. Japanese spotted fever has an abrupt onset with headache, high fever (39 to 40°C), and chills. A macular rash appears after two or three days, all over the body, including t