Spotted Fever Group

Proteobacteria of the family Rickettsiaceae, order Rickettsia- les, are made up of highly specialized obligate intracellular, gram-negative bacteria that survive freely within the cytosol of the host cell. Members of the genus Rickettsia are divided into two genetically similar groups, the spotted fever group (SFG) and the typhus group (TG), on the basis of host specificity, intracellular location, in vitro growth conditions, antigenic characteristics, the molecular sequences of conserved genes, clinical features, and epidemiology (15, 16, 37, 38). Seventeen species of the genus Rickettsia are categorized within the SFG rickettsiae. With the exception of Rickettsia akari (mite-borne) and R. felis (flea-borne), the remaining SFG rickettsia species are recognized as tick-borne rickettsiae that are passed to subsequent generations or stages transovarially and transtadi- ally (21). While the members of the SFG rickettsiae are adapted to existence within specific hosts, they are capable of infecting humans after humans are bitten by infected arthro- pods. The TG contains two species, R. prowazekii and R. typhi. The former scrub typhus group has been moved into its own genus, Orientia tsutsugamushi, following analysis of the 16S rRNA gene (47).

Objective: Murine typhus has been increasingly reported on Reunion island, Indian ocean, following documenta- tion of eight autochthonous infections in 2012–2013. We conducted a serosurvey to assess the magnitude of the seroprevalence of rickettsioses in the population. Two hundred and forty-one stored frozen sera taken from the 2009 Copanflu-RUN cohort were analysed using an immunofluorescence assay allowing to distinguish typhus group (TGR) and spotted fever group Rickesttsiae (SFGR). Seropositivity was defined for a dilution titre of Rickettsia IgG antibod- ies ≥ 1:64. Seroprevalence was weighted to account for the discrepancy between the Copanflu-RUN subset and the general population, as to infer prevalence at community level. Prevalence proportion ratios (PPR) were measured using log-binomial models.

Increasing entomologic and epidemiologic evidence suggests that spotted fever group rickettsiae (SFGR) other than Rickettsia rickettsii are responsible for spotted fever rickettsioses in the United States. A retrospective seroepidemiologic study was con- ducted on stored acute- and convalescent-phase sera that had been submitted for Rocky Mountain spotted fever testing to the North Carolina State Laboratory of Public Health. We evaluated the serologic reactivity of the paired sera to R. rickettsii, Rickett- sia parkeri, and Rickettsia amblyommii antigens. Of the 106 eligible pairs tested, 21 patients seroconverted to one or more anti- gens. Cross-reactivity to multiple antigens was observed in 10 patients, and seroconversions to single antigens occurred in 11 patients, including 1 against R. rickettsii, 4 against R. parkeri, and 6 against R. amblyommii. Cross-absorption of cross-reactive sera and/or Western blots identified two presumptive cases of infection with R. parkeri, two presumptive cases of infection with R. rickettsii, and one presumptive case of infection with R. amblyommii. These findings suggest that species of SFGR other than R. rickettsii are associated with illness among North Carolina residents and that serologic testing using R. rickettsii antigen may miss cases of spotted fever rickettsioses caused by other species of SFGR.

A spotted fever group rickettsia isolated from the common tick, Ixodes ricinus, was genetically characterized by PCR and genomic sequencing. This study was performed with nymphal and adult ticks collected in southern and central Sweden. I. ricinus is the only North European tick species of medical importance which is regularly collected from humans. No species of the genus Rickettsia has previously been found in Scandinavian ticks, nor has any case of domestic rickettsial infection in humans or animals been reported. According to the nucleotide sequencing, the present Rickettsia sp. belongs to the spotted fever group of rickettsiae. Ticks are the most common arthropod reservoirs and vectors of the rickettsiae of this group. Among 748 ticks investigated, 13 (1.7%) were positive for a Rickettsia sp. Borrelia burgdorferi was detected in 52 (7%) of the ticks, a prevalence similar to or somewhat lower than that previously been recorded in other Swedish studies. There was no evidence of ehrlichial or chlamydial DNA in these ticks. The Rickettsia sp. was further characterized by 16S ribosomal DNA (rDNA) sequencing and restriction fragment length polymorphism (RFLP). The 16S rDNA sequencing resulted in a sequence identical to that described for Rickettsia helvetica, but the pattern obtained with RFLP of the citrate synthetase gene diverged from previously known patterns. The rickettsial agent of one tick which was positive by PCR was confirmed by transmission electron microscopy. The morphology of this rickettsia was similar to that of the spotted fever and typhus group rickettsiae. This represents the first documented isolate of a Rickettsia sp. from Swedish ticks.

The supernatants were tested in the IIF assay for binding to the MAVI strain and four spotted fever group rickettsia strains isolated from Sicilian ticks two virulent and two nonpathogen[r]

container was checked for the same. For one of these patients, the skin biopsy sent for histopathological examination was inadequate. In four cases both the skin biopsy and whole blood was positive by 17-kDa PCR. An additional 19 skin biopsies were positive for spotted fever group rickettsial DNA by 17-kDa PCR. In one case diagnosed as spotted fever (as per the case definition) serology and molecular tests for spotted fever were negative. This patient presented on the fifth day of illness and defervescence of fever was observed within 28 hours of initiating treatment with doxycycline. Scrub typhus IgM antibodies could not be detected by ELISA in this individual. According to our case definition this is a case of spotted fever which is false negative by the serological and molecular assays used in the current study. Other rickettsial illness such as typhus fever and Ehrlichiosis where a similar response to specific therapy is seen are also possible diagnosis. Sequencing performed on three skin biopsy amplicons each for the gltA gene and 17-kDa gene followed by BLAST analysis provided confirmatory evidence for specificity of target amplification. The three amplicons sequenced (of each target) showed the same sequence but differed by three base pairs (gltA) and one base pair from the nearest match/best match, when compared with existing/published sequences in the GenBank. This suggests that probably one strain or species (which could be novel) of spotted fever group rickettsiae is circulating in our area. This assumption based on the available data needs to be assessed further by using other targets such as ompA and ompB genes.

Background: Spotted fever group (SFG) rickettsiae have recently been identified for the first time in UK ticks. This included the findings of Rickettsia helvetica in Ixodes ricinus and Rickettsia raoultii in Dermacentor reticulatus. This paper further investigates the occurrence of SFG rickettsiae in additional geographically distinct populations of D. reticulatus, and for the first time, investigates the occurrence of SFG rickettsiae in UK populations of Haemaphysalis punctata ticks. Methods: Questing D. reticulatus and H. punctata were collected at a number of sites in England and Wales. DNA from questing ticks was extracted by alkaline lysis and detection of rickettsiae DNA was performed, in addition to detection of A. phagocytophilum, N. mikurensis, C. burnetii and B. burgdorferi sensu lato.

Rickettsiae are Gram-negative obligate intracellular bac- teria, closely associated with blood-feeding arthropods and subdivided in two groups: typhus group (TGR) and spotted-fever group (SFGR) [1]. They are responsible for many human infections resulting in mild to severe dis- eases, causing public health problems in many countries around the world. To our knowledge, there have been no confirmed reports of human cases of acute rickettsial infection from Madagascar. However, recent studies have revealed a high prevalence of Rickettsia africae, a SFGR, in Amblyomma ticks collected from cattle [2] and tortoises [3], as well as evidence of low rates of previous exposure to SFGR in pregnant women [2]. Most SFGR

There are two major bird migratory routes in eastern Europe, connecting the north-eastern part of the contin- ent, as well western Siberia to the Mediterranean region and these two join into the Eastern Mediterranean Fly- way on the territory of Romania (in the Danube Delta). This region is one of the most important migratory stop-over sites in Europe, with more than 300 species of migratory birds being recorded in most years. Not only the diversity, but also the numbers passing and stopping-over is high, with an estimated two million birds using the diverse habitats of the region twice a year [14]. In addition, 15 tick species were recorded in this area [9, 15]. Thus, the region provides a unique setting to assess the importance of birds in the cycling of Spotted Fever Group Rickettsia (SFG Rickettsia). There are no data on rickettsial infections of birds or their ticks from Romania. The aim of this study was to investigate the presence of Rickettsia spp. in ticks collected from birds at a migratory hot- spot in the Danube Delta.

Host-seeking Ixodes ricinus (L.) ticks were collected systematically, from May to September 2006, at selected sites in Southern Germany, including a large city park in Munich. Polymerase Chain Reactions for amplification of genes of the rickettsial citrate synthase (gltA), the outer membrane proteins A and B (ompA and ompB), and the 16S rDNA were used to investigate 2,861 specimen (adults and nymphs). GltA sequences of spotted fever group rickettsiae were detected in 151 of all samples (5.3%; 95% CI 4.3% to 6.2%). Sequencing revealed Rickettsia helvetica in 91.4% of them and R. monacensis in 8.6%. Amplification of ompA was not possible for R. helvetica, but in all except one of the R. monacensis. The results were analyzed statistically to test the effects of season, location, developmental stage and gender of the tick on prevalence of Rickettsia spp. Although rickettsial DNA was detected in all investigated sites, sites in natural forest areas had significantly higher prevalences than sites in landscaped city parks (p<0.001). Adult female and male ticks had a similar prevalence and were significantly more often infected than nymphs (p<0.001). Monthly differences were not statistically significant. These results indicate that R. helvetica might lead to a public health threat to humans, especially after tick exposure in areas of high prevalence.

Although quantitative PCR assays have been developed and applied to studies with rickettsial relatives in the family Anaplasmataceae (13, 14), a comparable procedure has not been developed and tested on diverse rickettsial samples. Ro- lain et al. (16) developed a quantitative PCR assay using the Roche LightCycler based on the conserved rickettsial gltA primers 877F and 1258R. The assay was used to monitor the growth kinetics and antibiotic susceptibility of R. conorii, R. typhi, and R. felis in Vero or XTC-2 cells. However, although both probe- and SYBR Green-based assays were mentioned, Rolain et al. did not specify the probe used or completely describe the methods for calibration and optimization of the assay. We describe here the detailed characterization of an alternative rOmpA-based spotted fever rickettsia quantitative PCR (SQ-PCR) assay that we found to be more suitable for quantifying R. rickettsii and other closely related spotted fever group (SFG) rickettsiae. The SQ-PCR assay was compared with the plaque assay for the ability to quantify R. rickettsii. Its utility for detection and quantitation of rickettsiae in experi- mental samples from cell cultures, animal tissues, and tick and clinical samples was determined.

In Spain, several cases of the disease have been described from all over the country, and serosurveys have been con- ducted in different areas (16, 19, 20, 22, 25, 31, 42, 44, 45). These studies indicated that epidemiological data recorded in the Catalan region differ from those recorded in other Spanish or Mediterranean regions. For instance, these surveys showed that the seroprevalence of antibodies reactive with spotted fever group (SFG) rickettsiae in human and canine popula- tions was significantly lower in Catalonia than in the center of Spain (16, 26). Catalan patients presented mild forms of MSF compared with those reported from other regions (43, 46). Furthermore, children infected with SFG rickettsiae in Cata- lonia showed an unusual unresponsiveness to treatments with rifampin (8). The reasons for these epidemiological peculiari- ties have not been established yet.

were tested for amplification that included seven species of the spotted fever group (R. sibirica, R. conorii, R. rickettsii, R. africa, R. parkeri, R. japonicai, and R. heilongjiangensis). All seven SFGR tested by this method were positive and other common pathogens were not detected by LAMP. These results indicate that the ompB LAMP assay was specific for SFGR. The LAMP assay developed here did not detect R. prowazekii and R. typhi even though both possess an ompB gene but no conserved sequences where primers were design are present in these bacteria.

A nested PCR assay was developed for the detection of spotted fever group (SFG) rickettsiae in serum samples. The assay was based on specific primers derived from the rickettsial outer membrane protein B gene (rompB) of Rickettsia conorii. An SFG rickettsia-specific signal is obtained from R. akari, R. japonica, R. sibirica, and R. conorii. Other bacterial species tested did not generate any signal, attesting to the specificity of the assay. As few as seven copies of the rompB gene of R. conorii could be detected in 200 ␮ l of serum sample. The assay was evaluated with a panel of sera obtained from patients with acute-phase febrile disease tested by immunofluorescent antibody assay (IFA). The SFG rickettsia-specific DNA fragment was detected in 71 out of 100 sera, which were proven to have immunoglobulin M antibodies against SFG rickettsial antigen by IFA. The results were further confirmed by restriction fragment length polymorphism and sequencing analysis of the DNA fragments. The results indicated that this PCR assay is suitable for the diagnosis of spotted fever group rickettsiosis in Korea.

Tick-borne rickettsiae of the spotted fever group (SFG) cause disease worldwide (17). Australia has two rickettsial spotted fever illnesses: Queensland tick typhus, which is caused by Rickettsia australis (2, 14) and occurs eastern Australia, and Flinders Island spotted fever, which is caused by a unique SFG rickettsia (4). The clinical illnesses have been previously re- viewed (4, 6, 8, 18, 20); they have similar symptoms and signs, and both respond well to therapy with doxycycline. To localize the geographical ranges and distributions of these two ill- nesses, we compared eight rickettsial isolates obtained from patients with spotted fever on the mainland and Flinders Is- land. The comparison of these isolates was based on previously published differences in the genus-specific 17-kDa antigen gene (4) and 16S rDNA sequence analysis. We have compared six recent patient rickettsial isolates with the two remaining R. australis isolates in existence. These two strains of R. australis, designated PHS and JC, were isolated from humans in 1946 and 1955, respectively (2, 14).

Background: Spotted fever group rickettsioses (SFGR) transmitted mostly by ticks are increasingly discovered around the World and some of them are either re-emerging or emerging in Sri Lanka. Accidental human infections caused by these vector borne zoonotic diseases generally give rise to nonspecific acute febrile illnesses which can be complicated by multi organ involvement carrying high morbidity and mortality. Nonspecific clinical features and non-availability of early diagnostic facilities are known to result in delay in the diagnosis of rickettsial infections. Therefore, awareness of their prevalence and more importantly their clinical features would be help in the early diagnosis and institution of appropriate therapy.

Interestingly, R. massiliae-Bar29 seroprevalence was the highest, even if cats with antibodies exclusively against this strain were considered (15.6%). Moreover, highest titres were observed against R. massiliae-Bar29 antigen, most of them in cats without cross-reactions. The majority of studies are focused on the presence of R. massiliae strains in ticks. In fact, R. massiliae has been detected in Rhipicephalus sanguineus, R. turanicus, R. pusillus, R. guilhoni, R muhsamae, R. lunulatus, R. sulcatus collected on humans, dogs, cats, donkeys, cows, hedgehogs, horses, red foxes, roe deer, goat, cattle and sheep and wild boars or by flagging [6,8,12,14-21,24,25]. Moreover, it has been found in other genera such as Ixodes tasmani (collected on Tasmanian devils) [22], Ixodes ricinus [16], and Dermacentor marginatus (col- lected on dogs) [23]. Association between ticks and hu- man cases has been observed [12,16,24]. The transstadial and trasovarial transmission of Rickettsia massiliae-Bar29 have been observed in R. sanguineus group. In addition, this microorganism does not have any effect on viability of R. sanguineus and its reproductive fitness [6,25,40]. There- fore, these ticks are considered a reservoir.

Rickettsia spp. have been recognized as emerging or re- emerging pathogens of public health relevance [2]. With the completion of the complete genome sequences, new perspectives on rickettsial evolution have been acquired. Apart from the traditional classification based on their morphological, antigenic, and metabolic characteristics, phylogenomic studies showed that the genus Rickettsia was classified into four different groups, including the well-defined SFG and TG, the R. belli group and the R. canadensis group [2, 22]. Until now, 27 characterized and dozens of as yet uncharacterized strains had been recognized worldwide [2]. The availability of specific and sensitive molecular tools used for taxonomic purposes have allowed for the identification of new species of Rickettsia in places where no rickettsioses had been re- ported. In the current study, the prevalence and molecu- lar characterization of Rickettsia spp. was determined in H. qinghaiensis ticks, which was the dominant tick species and mainly recorded in northwestern China [23, 24].

ettsia (61, 68). In our study, strains of R. conorii, particularly the Indian tick typhus rickettsia, Kenya tick typhus rickettsia, M-1, and Seven strains, all showed very similar expression of epitopes. The Astrakhan fever rickettsia and the Israeli tick typhus rickettsia also expressed most of the R. conorii epitopes (68), concurring with their inclusion in an R. conorii subgroup (R. conorii complex) as proposed by genotypic comparative methods (20, 24, 60). Isolates of R. conorii from different geo- graphical regions as well as from both humans and ticks, have been shown to exhibit the extremely-well-conserved macror- estriction patterns (48). When ompA gene sequences of strains of R. conorii were compared, they also exhibited high similar- ities to each other (24, 47). Indeed, the gltA sequences of four strains (M-1, Indian tick typhus rickettsia, Moroccan, and Seven) were identical (51). Thus, the evolutionary homology of R. conorii as demonstrated by these studies is reflected in the high levels of antigenic similarity revealed by our work (Tables 2 and 3) (68).

The microbiological and molecular characteristics of the rickettsiae isolated from humans with Queensland tick typhus QTT caused by Rickettsia australis and the recently described Flinde[r]