A retrospective analysis by molecular-sequence-based techniques was performed to correctly identify the etiological agent of 24 Mediterranean spotted fever cases occurring in Western Sicily, Italy, from 1987 to 2001. Restriction analysis of a 632-bp PCR-amplified portion of the ompA gene allowed presumptive identification of five clinical isolates as belonging to Rickettsia conorii subsp. israelensis, the etiological agent of Israeli spotted fever (ISF). The remaining 19 rickettsial isolates were Rickettsia conorii subsp. conorii, the only pathogenic rickettsia of the spotted fever group reported in Italy until the present. Sequence analysis of the ompA gene confirmed the identification of all the R. conorii subsp. israelensis isolates and demonstrated that rickettsiosis caused by R. conorii subsp. israelensis can be traced back to 1991 in Sicily. The recorded clinical data of the five ISF patients support the idea that these strains could correlate to more-severe forms of human disease. Three of five patients experienced severe disease, and one of them died.
Rickettsia conorii subsp. israelensis is the agent of Israeli spotted fever. The present study reports the draft genome of Rickettsia conorii subsp. israelensis strain ISTT CDC1, isolated from a Rhipicephalus sanguineus tick collected in Israel.
Rickettsia spp. are an underrecognized cause of undifferentiated febrile illness.
Rickettsial diseases have not been described previously from Laos, but in a prospective study, acute rickettsial infection was identified as the cause of fever in 115 (27%) of 427 adults with negative blood cultures admitted to Mahosot Hospital in Vientiane, Laos. The organisms identified by serologic analysis were Orientia tsutsugamushi (14.8%), Rickettsia typhi (9.6%), and spotted fever group rickettsia (2.6% [8 R. helvetica, 1 R. felis, 1 R. conorii subsp. indica, and 1 Rickettsia "AT1"]). Patients with murine typhus had a lower frequency of peripheral lymphadenopathy than those with scrub typhus (3% vs. 46%, p<0.001). Rickettsioses are an underrecognized cause of undifferentiated febrile illnesses among adults in Laos. This finding has implications for the local empiric treatment of fever.
Laos; typhus; fever; Orientia tsutsugamushi; Rickettsia typhi; Rickettsia helvetica; Rickettsia felis; Rickettsia conorii; Rickettsia AT1
Rickettsia conorii, an obligate intracellular bacterium that infects vascular endothelial cells, is the etiologic agent of Mediterranean spotted fever. We correlated the results of 205 R. conorii blood and skin cultures for 157 patients and the results of 48 detections of R. conorii in circulating endothelial cells (CEC) for 41 patients with relevant serological, clinical, and therapeutic data. R. conorii was cultured from 40% of patients and 29.8% of samples. R. conorii was detected in CEC in 50% of samples, representing 46.2% of patients. When these calculations were limited to the samples from untreated patients prior to their seroconversion to R. conorii, the sensitivity of culture was 59%, whereas it remained at 50% for detection in CEC. We also performed PCRs for the detection of R. conorii on eight shell vial supernatants from positive cultures and on 43 blood samples. Only nonfrozen supernatants from fresh cultures were positive. The methods described in this report are suitable for use in all laboratories. Our findings suggest that for samples to be suitable for culture they must be collected prior to the initiation of an antibiotic regimen, as early as possible in the course of the disease, and be inoculated onto shell vials with minimal delay, if R. conorii is to be successfully isolated. For patients who have been treated or who have a delayed diagnosis, detection of R. conorii in CEC remains helpful.
To report the clinical features and management of seven cases of intraocular inflammation caused by Rickettsia infection and review published literature.
Rickettsia conorii or Rickettsia spp. infection was diagnosed based on the following criteria: (1) positive serology according to the European Guidelines, (2) titer normalization after specific treatment, and (3) complete resolution of ophthalmic disease and accompanying symptoms after antibiotic therapy.
Seven patients were referred for uveitis of unknown etiology. All came from regions where Mediterranean spotted fever is prevalent. One patient met the European guidelines criteria for Rickettsia spp. infection, while the other six cases met the criteria for R. conorii infection. The main symptoms were visual loss, floaters, eye redness, photophobia, and ocular pain. Predominant ophthalmic signs included vasculitis, choroiditis, vitritis, and macular edema. All patients required antibiotic treatment that resulted in the remission of the infection. Doxycycline was the first choice and the only antibiotic used to treat four patients. One patient needed ciprofloxacin as a second antibiotic after not responding to doxycycline. Two patients had doxycycline as a second antibiotic after not responding primarily to sulfonamides (which had been given after 2–3 days of doxycycline gastric intolerance); one of these patients needed ciprofloxacin as a third antibiotic.
Intraocular inflammation can occur as the main manifestation of Rickettsia conorii or Rickettsia spp. infection. It should be considered as a differential diagnosis for uveitis especially for patients living in countries where this infection is endemic in the world. Antibiotic treatment remains effective in the management of Rickettsia infection.
intraocular inflammation; Mediterranean spotted fever; Rickettsia conorii; uveitis
The genomes of spotted fever group rickettsiae isolated in different geographical areas of Israel (two from ticks and four from humans, obtained over a span of 20 years) were studied by polymerase chain reaction (PCR) and restriction endonuclease fragment length polymorphism (RFLP) analysis. The human isolates were obtained from patients suffering from rickettsial disease of different degrees of severity. The PCR products obtained with five pairs of oligonucleotide primers (two primer sets derived from the 190-kDa polypeptide gene and three from the 120-kDa polypeptide gene) and cleaved with restriction endonucleases were used to study the Israeli isolates and reference Rickettsia conorii isolates. Subtle differences between the PCR-RFLP patterns of Israeli isolates and the two R. conorii reference strains (Moroccan and no. 7) were seen when the PCR products derived from the 190-kDa gene-derived primer sets were digested. All of the Israeli isolates were identical by RFLP analysis using all of the primer sets. This study showed that the Israeli spotted fever group isolates (from both ticks and humans) were genetically homogeneous by the criteria used in this study, despite the time and location differences in their original isolation, and different as a group from R. conorii.
Rickettsiae closely related to the Malish strain, the reference Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness.
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 rDNA, gltA, ompA, ompB, and sca4 genes, respectively. Representatives of the 4 MLST types were also classified within four types using MST genotyping as well as mouse serotyping.
Although homogeneous genotypically, strains within the R. conorii species show MST genotypic, serotypic, and epidemio-clinical dissimilarities. We, therefore, propose to modify the nomenclature of the R. conorii species through the creation of subspecies. We propose the names R. conorii subsp. conorii subsp. nov. (type strain = Malish, ATCC VR-613), R. conorii subspecies indica subsp. nov. (type strain = ATCC VR-597), R. conorii subspecies caspia subsp. nov. (type strain = A-167), and R. conorii subspecies israelensis subsp. nov. (type strain = ISTT CDC1). The description of R. conorii is emended to accomodate the four subspecies.
Spotted fever group rickettsiae cause life-threatening human infections worldwide. Until now, the immune regulatory mechanisms involved in fatal rickettsial infection have been unknown. C3H/HeN mice infected with 3 × 105 PFU of Rickettsia conorii developed an acute progressive disease, and all mice succumbed to this infection. A sublethal infection induced protective immunity, and mice survived. Compared to splenic T cells from sublethally infected mice, splenic T cells from lethally infected mice produced significantly lower levels of interleukin-2 (IL-2) and gamma interferon (IFN-γ) and a higher level of IL-10, but not of IL-4 or transforming growth factor β, and there was markedly suppressed CD4+ T-cell proliferation in response to antigen-specific stimulation with R. conorii. Furthermore, lethal infection induced significant expansion of CD4+ CD25+ Foxp3− T cells in infected organs compared to the levels in naïve and sublethally infected mice. In a lethal infection, splenic CD4+ CD25+ Foxp3− T cells, which were CTLA-4high T-bet+ and secreted both IFN-γ and IL-10, suppressed the proliferation of and IL-2 production by splenic CD4+ CD25− Foxp3− T cells in vitro. Interestingly, depletion of CD25+ T cells in vivo did not change the disease progression, but it increased the bacterial load in the lung and liver, significantly reduced the number of IFN-γ-producing Th1 cells in the spleen, and increased the serum levels of IFN-γ. These results suggested that CD4+ CD25+ T cells generated in acute murine spotted fever rickettsiosis are Th1-cell-related adaptive T-regulatory cells, which substantially contribute to suppressing the systemic immune response, possibly by a mechanism involving IL-10 and/or cytotoxic T-lymphocyte antigen 4.
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 μl 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.
The pathophysiologic mechanisms of severity of Mediterranean spotted fever (MSF) and the host and microbial risk factors for a fatal outcome are incompletely determined.
In a prospective study of 140 patients with documented identification of the causative rickettsial strain admitted to 13 Portuguese hospitals during 1994−2006, univariate and multivariate analyses determined the risk factors for a fatal outcome.
Seventy one (51%) patients were infected with Rickettsia conorii Malish strain and 69 (49%) with Israeli spotted fever (ISF) strain. Patients were admitted to ICU (29%), hospitalized as routine inpatients (67%), or managed as outpatients (4%). Deaths occurred in 29 (21%) adults. Fatal outcome was significantly more likely for patients with ISF strain infection, and alcoholism was a risk factor. The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, confusion/obtundation, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated hepatic enzymes and creatine kinase. Some but not all these were observed more often in ISF strain-infected patients.
Although fatalities and similar clinical manifestations occurred with both strains, ISF strain was more virulent than Malish strain. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strongly associated with a fatal outcome.
Rickettsia conorii; alcoholism; Israeli spotted fever strain; Malish strain; eschar
Rickettsia conorii conorii is the etiological agent of Mediterranean spotted fever, which is transmitted by the brown dog tick, Rhipicephalus sanguineus. The relationship between the Rickettsia and its tick vector are still poorly understood one century after the first description of this disease.
An entomological survey was organized in Algeria to collect ticks from the houses of patients with spotted fever signs. Colonies of R. conorii conorii-infected and non-infected ticks were established under laboratory conditions. Gimenez staining and electron microscopy on the ovaries of infected ticks indicated heavy rickettsial infection. The transovarial transmission of R. conorii conorii in naturally infected Rh. sanguineus ticks was 100% at eleven generations, and the filial infection rate was up to 99% according to molecular analyses. No differences in life cycle duration were observed between infected and non-infected ticks held at 25°C, but the average weight of engorged females and eggs was significantly lower in infected ticks than in non-infected ticks. The eggs, larvae and unfed nymphs of infected and non-infected ticks could not tolerate low (4°C) or high (37°C) temperatures or long starvation periods. R. conorii conorii-infected engorged nymphs that were exposed to a low or high temperature for one month experienced higher mortality when they were transferred to 25°C than non-infected ticks after similar exposure. High mortality was observed in infected adults that were maintained for one month at a low or high temperature after tick-feeding on rabbits.
These preliminary results suggest that infected quiescent ticks may not survive the winter and may help explain the low prevalence of infected Rh. sanguineus in nature. Further investigations on the influence of extrinsic factors on diapaused R. conorii-infected and non-infected ticks are required.
The bacterium Rickettsia conorii conorii is the etiological agent of Mediterranean spotted fever (MSF), which is a life-threatening infectious disease that is transmitted by Rhipicephalus sanguineus, the brown dog tick. Rh. sanguineus-R. conorii conorii relationships in the wild are still poorly understood one century after the discovery of the disease. In this study, we collected naturally infected ticks from the houses of people afflicted by MSF in Algeria. Colonies of both infected and non-infected ticks were maintained in our laboratory, and we studied the effect of temperature variations on the infected and non-infected ticks. We did not observe any major differences between the biological life cycle of the infected and non-infected ticks held at 25°C. However, a comparatively higher mortality relative to the control group was noticed when R. conorii conorii-infected engorged nymphs and adults were exposed to a low temperature (4°C) or high temperature (37°C) for one month and transferred to 25°C. R. conorii conorii-infected Rh. sanguineus may maintain and serve as reservoirs for the Rickettsia if they are not exposed to cold temperatures. New populations of ticks might become infected with Rickettsiae when feeding on a bacteremic animal reservoir.
Pathogenic species of the spotted fever group Rickettsia are subjected to repeated exposures to the host complement system through cyclic infections of mammalian and tick hosts. The serum complement machinery is a formidable obstacle for bacteria to overcome if they endeavor to endure this endozoonotic cycle. We have previously demonstrated that that the etiologic agent of Mediterranean spotted fever, Rickettsia conorii, is susceptible to complement-mediated killing only in the presence of specific monoclonal antibodies. We have also shown that in the absence of particular neutralizing antibody, R. conorii is resistant to the effects of serum complement. We therefore hypothesized that the interactions between fluid-phase complement regulators and conserved rickettsial outer membrane-associated proteins are critical to mediate serum resistance. We demonstrate here that R. conorii specifically interacts with the soluble host complement inhibitor, factor H. Depletion of factor H from normal human serum renders R. conorii more susceptible to C3 and membrane attack complex deposition and to complement-mediated killing. We identified the autotransporter protein rickettsial OmpB (rOmpB) as a factor H ligand and further demonstrate that the rOmpB β-peptide is sufficient to mediate resistance to the bactericidal properties of human serum. Taken together, these data reveal an additional function for the highly conserved rickettsial surface cell antigen, rOmpB, and suggest that the ability to evade complement-mediated clearance from the hematogenous circulation is a novel virulence attribute for this class of pathogens.
The impact of climate on the vector behaviour of the worldwide dog tick Rhipicephalus sanguineus is a cause of concern. This tick is a vector for life-threatening organisms including Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, R. conorii, the agent of Mediterranean spotted fever, and the ubiquitous emerging pathogen R. massiliae. A focus of spotted fever was investigated in France in May 2007. Blood and tissue samples from two patients were tested. An entomological survey was organised with the study of climatic conditions. An experimental model was designed to test the affinity of Rh. sanguineus for biting humans in variable temperature conditions. Serological and/or molecular tools confirmed that one patient was infected by R. conorii, whereas the other was infected by R. massiliae. Dense populations of Rh. sanguineus were found. They were infected with new genotypes of clonal populations of either R. conorii (24/133; 18%) or R. massiliae (13/133; 10%). April 2007 was the warmest since 1950, with summer-like temperatures. We show herein that the human affinity of Rh. sanguineus was increased in warmer temperatures. In addition to the originality of theses cases (ophthalmic involvements, the second reported case of R. massiliae infection), we provide evidence that this cluster of cases was related to a warming-mediated increase in the aggressiveness of Rh. sanguineus, leading to increased human attacks. From a global perspective, we predict that as a result of globalisation and warming, more pathogens transmitted by the brown dog tick may emerge in the future.
The impact of climate on the behaviour of the worldwide dog tick Rhipicephalus sanguineus is a cause of concern. This tick is a vector for life-threatening organisms including Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, R. conorii, the agent of Mediterranean spotted fever, and the ubiquitous emerging pathogen R. massiliae. A focus of spotted fever was investigated in France in May 2007. One patient was found to be infected by R. conorii, whereas the other was infected by R. massiliae. Theses cases were original because of ophthalmic involvements, and the report of the second case of R. massiliae infection in the scientific literature. During an entomological survey, dense populations of Rh. sanguineus were found in the house where the patient had been bitten by ticks. Ticks were infected with either R. conorii or R. massiliae. Interestingly, April 2007 was the warmest since 1950, with summer-like temperatures. In this work, we show that the human affinity of Rh. sanguineus is increased in warmer temperatures, and provide evidence that this cluster of cases was related to a warming-mediated increase in the aggressiveness of Rh. sanguineus, leading to increased human attacks. From a global perspective, we predict that as a result of globalisation and warming, more pathogens transmitted by the brown dog tick may emerge in the future.
We report a fatal case of rickettsiosis in a woman from the United States living in Kenya, who had a history of tick exposure. Immunohistochemical staining of skin, kidney, and liver demonstrated spotted fever group rickettsiae. The clinical findings, severity, and fatal outcome are most consistent with Rickettsia conorii infection.
Rickettsia; Kenya; tick typhus; African tick-bite fever
Rickettsia conorii, the causative agent of the Mediterranean spotted fever, is transmitted to humans by the bite of infected ticks Rhipicephalus sanguineus. The skin thus constitutes an important barrier for the entry and propagation of R. conorii. Given this, analysis of the survival strategies used by the bacterium within infected skin is critical for our understanding of rickettsiosis.
Here, we report the first genome-wide analysis of R. conorii gene expression from infected human skin biopsies. Our data showed that R. conorii exhibited a striking transcript signature that is remarkably conserved across patients, regardless of genotype. The expression profiles obtained using custom Agilent microarrays were validated by quantitative RT-PCR. Within eschars, the amount of detected R. conorii transcripts was of 55%, this value being of 74% for bacteria grown in Vero cells. In such infected host tissues, approximately 15% (n = 211) of the total predicted R. conorii ORFs appeared differentially expressed compared to bacteria grown in standard laboratory conditions. These genes are mostly down-regulated and encode proteins essential for bacterial replication. Some of the strategies displayed by rickettsiae to overcome the host defense barriers, thus avoiding killing, were also pointed out. The observed up-regulation of rickettsial genes associated with DNA repair is likely to correspond to a DNA-damaging agent enriched environment generated by the host cells to eradicate the pathogens. Survival of R. conorii within eschars also involves adaptation to osmotic stress, changes in cell surface proteins and up-regulation of some virulence factors. Interestingly, in contrast to down-regulated transcripts, we noticed that up-regulated ones rather exhibit a small nucleotide size, most of them being exclusive for the spotted fever group rickettsiae.
Because eschar is a site for rickettsial introduction, the pattern of rickettsial gene expression observed here may define how rickettsiae counteract the host defense.
Mediterranean spotted fever (MSF) is an acute febrile, zoonotic disease caused by Rickettsia conorii and transmitted to humans by the brown dogtick Rhipicephalus sanguineus. Nearly four hundred cases are reported every year (mainly from June to September) on the Italian island of Sicily. The aim of the study was to analyze the clinical and laboratory characteristics of patients with MSF and the efficacy of the drugs administered.
Our study was carried out on 415 children with MSF, during the period January 1997 – December 2004, at the "G. Di Cristina" Children's hospital in Palermo, Sicily, Italy. On admission patients' clinical history, physical and laboratory examination and indirect immunofluorescence antibody test (IFAT) for Rickettsia conorii were performed. Diagnosis was considered confirmed if the patients had an MSF diagnostic score greater than or equal to 25 according to the Raoult's scoring system. All patients were treated with chloramphenicol or with macrolides (clarithromycin or azithromycin).
Fever, rash and tache noire were present in 386 (93%), 392 (94.5%) and 263 (63.4%) cases respectively. Eighteen (4.6%) children showed atypical exanthema. Chloramphenicol and newer macrolides all appeared to be effective and safe therapies.
Clinical features of 415 children with MSF were similar to those reported by other authors except for a lower incidence of headache, arthralgia and myalgia and a higher frequency of epato-splenomegaly. Concerning therapy, clarithromycin can be considered a valid alternative therapy to tetracyclines or chloramphenicol especially for children aged < eight years.
Mediterranean spotted fever (caused by Rickettsia conorii) is one of the tick‐borne rickettsioses. It is prevalent in southern Europe, Africa and central Asia and may also be seen in travellers returning from these areas. It presents with various non‐specific symptoms, including fever, maculopapular rash, headache, myalgia or diarrhoea and vomiting. A visible eschar at the site of the tick bite is characteristic but not present in all cases. There is no test that reliably confirms the disease in its early stages and diagnosis is often made on clinical grounds. Delay in diagnosis and in providing correct antibiotic treatment increases the mortality rate of this condition. Emergency clinicians should be aware of the possible diagnosis in travellers returning from endemic areas in order to start the correct treatment as early as possible and minimise subsequent complications and mortality.
Rickettsia conorii subsp. caspia is the agent of Astrakhan fever, a spotted fever group rickettsiosis endemic to Astrakhan, Russia. The present study reports the draft genome of Rickettsia conorii subsp. caspia strain A-167.
Multiplex-nested PCR and sequencing analysis indicated rickettsialike agents in serum specimens from febrile patients.
The presence of the nucleic acid of the spotted fever group (SPG) and typhus group (TG) rickettsiae was investigated in 200 serum specimens seropositive for SFG rickettsiae by multiplex-nested polymerase chain reaction with primers derived from the rickettsial outer membrane protein B gene. The DNA of SFG, TG, or both rickettsiae was amplified in the 24 serum specimens, and sequence analysis showed Rickettsia conorii, R. japonica, and R. felis in the specimens. R. conorii and R. typhi were found in 7 serum specimens, which indicated the possibility of dual infection in these patients. These findings suggest that several kinds of rickettsial diseases, including boutonneuse fever, rickettsialpox, R. felis infection, and Japanese spotted fever, as well as scrub typhus and murine typhus, are occurring in Korea.
molecular detection; nested PCR; RFLP; Spotted fever group rickettsiosis; Typhus group rickettsiosis; research
The emergence and reemergence of a serious infectious disease are often associated with a high case-fatality rate because of misdiagnosis and inappropriate or delayed treatment. The current reemergence of spotted fever rickettsiosis caused by Rickettsia rickettsii in Brazil has resulted in a high proportion of fatal cases. We describe two familial clusters of Brazilian spotted fever in the state of Minas Gerais, involving six children 9 months to 15 years of age; five died. Immunohistochemical investigation of tissues obtained at necropsy of a child in each location, Novo Cruzeiro and Coronel Fabriciano municipalities, established the diagnosis by demonstration of disseminated endothelial infection with spotted fever group rickettsiae. The diagnosis in the two fatal cases from Coronel Fabriciano and the surviving patient from Novo Cruzeiro was further supported by immunofluorescence serologic tests.
Mediterranean spotted fever due to infection by Rickettsia conorii, is characterized by a general vasculitis. This vasculitis is thought to be due to a direct injury to endothelial cells induced by R. conorii. However, production and activity of cytokines on endothelial cells is an important pathway in inflammation, and part of the underlying mechanism of vasculitis. In the present studies, human umbilical vein endothelial cells (HUVEC) infected with R. conorii actively secrete high levels of IL-8 and IL-6 (P < 0.002, and P < 0.03, respectively, compared with uninfected cells). IL-1alpha, IL-1beta, or TNFalpha were not detected in the culture supernates. Nevertheless, IL-6 and IL-8 production was due, in a large part, to a cell-associated form of IL-1 alpha expressed on R. conorii-infected HUVEC, since production of these cytokines was suppressed by 80% (P = 0.0001) and 85% (P < 0.04) by the addition of IL-1 receptor antagonist, or anti-IL-1alpha antibodies (60% inhibition, P < 0.01 and 65% inhibition, P < 0.05, respectively) and IL-1alpha was measured after lysis of R. conorii-infected HUVEC but not in uninfected cells (P < 0.01). Rickettsial lipopolysaccharide does not seem to be involved, since polymyxin B did not reduce cytokine secretion. On the contrary, infection by intracellular R. conorii appears to be necessary to induce IL-1alpha and subsequently IL-8, since formalin-fixed R. conorii did not induce cytokine production. These observations demonstrate that R. conorii-infected HUVEC secrete IL-6 and IL-8 via the induction of cell-associated IL-1alpha, providing a possible mechanism for the vasculitis observed in Mediterranean spotted fever.
Mediterranean spotted fever, a tick-borne rickettsiosis caused by Rickettsia conorii, may lead to small-vessel or deep-vein thrombosis. In order to evaluate the role of endothelial cell alteration in this lesion, we infected human endothelial cells derived from umbilical veins with R. conorii. We report the induction of two previously unreported prothrombotic mechanisms in rickettsial disease: (i) a progressive decline in thrombomodulin antigen and (ii) early expression of tissue factor, and, as described for R. rickettsii infection, later release of von Willebrand factor from Weibel-Palade bodies. Thrombomodulin expression in infected endothelial cells, measured by the thrombin-dependent activation of protein C or flow cytometric analysis, decreased steadily between 4 and 24 h after inoculation with rickettsiae. R. conorii infection induced tissue factor expression, measured by clotting assay and flow cytometric analysis, which was detectable 2 h postinoculation, reached its maximum 4 h postinoculation, and progressively decreased thereafter. Infection resulted in a relatively late release of von Willebrand factor antigen into the culture medium. A double-label immunofluorescence assay for the simultaneous evaluation of von Willebrand factor and R. conorii showed that the depletion of cytoplasmic von Willebrand factor stored in Weibel-Palade bodies was due to a direct effect of the intracellular R. conorii. These disturbances of endothelial function observed with R. conorii-infected cells may provide a paradigm for the elucidation of thrombotic pathobiology with Mediterranean spotted fever.
Rickettsia conorii, an obligate intracellular bacterium and the causative agent of Mediterranean spotted fever, preferentially infects microvascular endothelial cells of the mammalian hosts leading to onset of innate immune responses, characterized by the activation of intracellular signaling mechanisms, release of pro-inflammatory cytokines and chemokines, and killing of intracellular rickettsiae. Our recent studies have shown that interferon (IFN)-β, a cytokine traditionally considered to be involved in antiviral immunity, plays an important role in the autocrine/paracrine regulation of host defense mechanism and control of R. conorii growth in the host endothelial cells. Here, we show that R. conorii infection induces the expression of ISG15 (an interferon-stimulated gene coding a protein of 17 kD) and UBP43 (an ISG15-specific protease) at the levels of mRNA and protein and report the evidence of ISGylation of as yet unidentified target proteins in cultured human microvascular endothelium. Infection-induced expression of ISG15 and UBP43 requires intracellular replication of rickettsiae and production of IFN-β, because treatment with tetracycline and presence of an antibody capable of neutralizing IFN-β activity resulted in near complete attenuation of both responses. Inhibition of R. conorii-induced ISG15 by RNA interference results in significant increase in the extent of rickettsial replication, whereas UBP43 knockdown yields a reciprocal inhibitory effect. In tandem, these results demonstrate the stimulation of interferon-β-mediated innate immune mechanisms capable of perturbing the growth and replication of pathogenic rickettsiae and provide first evidence for ISG!5-mediated post-translational modification of host cellular proteins during infection with an intracellular bacterium.
Endothelial cells; Interferon; ISG15; Rickettsia conorii; UBP43
Establishment of infection by spotted fever group rickettsial species is dependent on the ability of these bacteria to adhere to and invade the host endothelium. Recent studies have attributed these processes to a handful of rickettsial surface proteins from the surface cell antigen (sca) family of autotransporters. A rickettsial autotransporter from Rickettsia conorii, Sca2, has been shown to be sufficient to mediate both adherence and invasion of human endothelial cells and to participate in intracellular actin-based motility. Here we identify a region of Sca2 capable of interacting with the mammalian cell surface and show that this function of Sca2 is independent and separable from its actin nucleation activity. Furthermore, pre-incubation of mammalian cells with the Sca2 mammalian association region prior to R. conorii infection can competitively inhibit rickettsial invasion, suggesting that Sca2 plays an important role in the initial interaction with mammalian cells. Together our results demonstrate that the Sca2 autotransporter protein in R. conorii contains distinct functional domains that likely are involved in mediating cellular interactions at the plasma membrane and the host cytosol.
Three different spotted-fever group rickettsiae—Rickettsia conorii, R. massiliae, and R. rhipicephali—were detected and identified by PCR-restriction fragment length polymorphism analysis in Rhipicephalus ticks collected from domestic animals in the Fokida region of Greece, where a high seroprevalence of antibodies to R. conorii was previously demonstrated. The infection rate of ticks was 1.6%. Moreover, R. conorii was isolated from one Rhipicephalus sanguineus tick.