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Haemophagocytic syndrome (HS) is a rare disease with a severe prognosis that is defined by clinical, laboratory and histopathological criteria. Infections represent the classical cause of HS. HS secondary to Mediterranean spotted fever (MSF) is rare with only a few cases being reported in the literature.We report two cases of HS secondary to MSF in 2 men aged 77 and 63 years presenting a febrile maculo-purpuric eruption with inoculation ulcer associated with laboratory abnormalities (cytopenia, elevated ferritin, hypertriglyceridaemia). Haemophagocytosis was present in 2 cases. Serology and PCR for Rickettsia conorii were positive and militated in favour of recent infection responsible for the diagnosis of MSF.The first case of HS was described in 1979. Sixteen cases of HS secondary to MSF are described in the literature. Cytopenia associated with hyperferritinaemia and hypertriglyceridaemia strongly suggests MSF complicated by HS. The prognosis depends on the time elapsed since diagnosis and host-specific factors. Immunosuppressants and antibiotics may be necessary to ensure healing.Rickettsioses can induce HS, and this potential complication with a severe prognosis must be known.

La fièvre boutonneuse méditerranéenne (FBM) est une rickettsiose du groupe boutonneux due à rickettsia conorii. Cette zoonose est réputée d'évolution bénigne mais peut se compliquer dans les formes sévères d'une atteinte neurologique qui en font parfois toute la gravité. Nous rapportons l'observation d'une patiente âgée de 49 ans ayant présenté au cours de son hospitalisation en dermatologie pour une rickettsiose, une hémiplégie droite massive d'installation brutale. L'angio-IRM cérébrale a objectivé un accident vasculaire cérébral ischémique sylvien profond gauche. Le diagnostic de rickettsiose de type conorii a été retenu sur l'aspect des lésions dermatologiques et la positivité des sérologies par immuno-fluorescence indirecte. L'évolution était favorable sous anti-biothérapie (doxycycline et fluoroquinolone). Exceptionnellement rapporté dans le cadre des manifestations neurologiques des rickettioses, l'infarctus cérébral reste une complication à ne pas méconnaitre surtout en regard d'un bilan étiologique en particulier cardiovasculaire demeuré négatif.

We report a case of a 43-year-old Israeli male who presented with an intermittent fever associated with a gradual appearance of diffusely scattered erythematous non-pruritic maculopapular lesions, generalized body malaise, muscle aches, and distal extremity weakness. He works in the Israeli military and has been exposed to dogs that are used to search for people in tunnels and claimed that he had removed ticks from the dogs. In the hospital, he presented with fever, a diffuse maculopapular rash, and an isolated round black eschar. He was started on doxycycline based on suspected Mediterranean spotted fever (MSF) in which he improved significantly with resolution of his clinical complaints. His immunoglobulin G (IgG) MSF antibody came back positive.

INTRODUCTION: Mediterranean spotted fever (MSF) is a zoonotic disease caused by Rickettsia conorii. In Spain, deficiencies in the official reporting result in misreporting of this disease. This study aims to describe the clinical and temporal-spatial characteristics of MSF hospitalizations between 1997 and 2014. MATERIALS AND METHODS: We performed a retrospective descriptive study using the Hospitalization Minimum Data Set (CMBD). All CMBD's hospital discharges with ICD-9 CM code 082.1 were analyzed. Hospitalization rates were calculated and clinical characteristics were described. Spatial distribution of cases and their temporal behavior were also assessed. RESULTS: A total of 4,735 hospitalizations with MSF diagnosis were recorded during the study period, out of which 62.2% were male, mean age of 48. Diabetes mellitus, alcohol dependence syndrome, and chronic liver disease occurred in 10.8%, 2.4% and 2.8% hospitalizations, respectively. The median annual hospitalization rate showed a decreasing trend from a maximum of 12.9 in 1997 to a minimum rate of 3.1 in 2014. Most admissions occurred during the summer, showing a significant annual seasonal behavior. Important regional differences were found. DISCUSSION: Although MSF hospitalization rates have decreased considerably, it remains a public health problem due to its severity and economic impact. Therefore, it would be desirable to improve its oversight and surveillance. This study was supported by Project “PI15/01398”, funded by Instituto de Salud Carlos III and co-funded by European Union (ERDF/ESF, “Investing in your future”) and the Network Biomedical Research on Tropical Diseases (RICET in Spanish) RD12/0018/0001, supported by the European Regional Development Fund (FEDER) from the European Comission. Sí

The main clinical signs and symptoms caused by a rickettsial infection typically begin 6-10 days after the bite and are accompanied by nonspecific findings such as fever, headache and muscle pain. The diagnosis is mainly based on serological tests, however antibody presentation may be delayed, at least at the early stages of the disease, while seroconversion is usually detected 10-15 days after disease onset. Culture is difficult, requires optimized facilities and often proves negative. Under this scope, the presence of a characteristic inoculation eschar at the bite site may prove a useful clinical tool towards the early suspicion and diagnosis/differential diagnosis of tick-borne rickettsioses, even before the onset of rash and fever or serological confirmation. We describe herein the presence of skin lesions and/or an inoculation eschar at the tick bite site in 17 patients diagnosed, by molecular means, as suffering from spotted fever group rickettsioses. The detection of the pathogen's DNA in biopsy samples proved to be a useful means for early rickettsiae detection and identification. Moreover, the presence of an infiltrated erythema always seemed to precede the appearance of an eschar by 2-5 days and the initiation of fever by 1-10 days; these two signs might also prove useful in the context of the final diagnosis.

Introducere. Febra butonoasă (FB) este o boală infecţioasă emergentă în România, evoluând în sudul ţării, după cum reiese din rata incidenţei înregistrată în ultimii ani, conform studiilor noastre anterioare de epi- demiologie descriptivă. Ne-am propus să evaluăm prevalenţa anticorpilor specifici IgG Rickettsia conorii într-un eşantion de populaţie generală din aria afectată. Metode. S-a efectuat un studiu de seroprevalenţă prin metoda ELISA pe 301 probe de ser colectate din aria de sud a României; aria cea mai afectată de FB. Unul din criteriile de selecţie a fost absenţa semnelor şi simptomelor caracteristice bolii în perioada de evoluţie sezonieră a FB. Analiza statistică. Datele au fost prelucrate statistic cu softul SPSS V.19 (indicatori tendinta centrala, dispersie, frecventa si teste de comparare). Rezultate. Rezultatele serologice au arătat o rata de pozitivitate generală de 25.2% cu cea mai mare rată de pozitivitate la grupul de populaţie de peste 60 de ani 34,21% din totalul probelor de ser (63,6% pentru judeţul Tulcea şi 55.56% pentru Bucureşti). Concluzii. Confirmând ipoteza că FB îmbracă un caracter endemic în sudul României, bazată pe studiile noastre anterioare, studiul de faţă a adus argumente în sprijinul necesităţii de a introduce un program naţional de supraveghere în scopul cunoaşterii reale a incidenţei, managementului şi controlului FB în ţara noastră. După cunoştintele noastre, acesta este primul studiu de seroprevalenţă a anticorpilor specifici IgG Rickettsia conorii în România.

Summary Objectives To study the epidemiology, clinical features, and changing pattern of rickettsial infections on the western slopes of the hilly Central Province of Sri Lanka over 6 years. Methods All patients with rickettsial infections who presented to the Teaching Hospital, Peradeniya were studied prospectively from January 2002 to December 2007. An immunofluorescent antibody assay (IFA) was used to confirm the diagnosis. Results Of the 371 clinical cases of rickettsial infection, 122 underwent IFA to confirm the diagnosis. Species-specific IgG antibodies were positive in 105/122 (86.1%) cases: 43/105 (40.9%) to Rickettsia conorii and 6/105 (5.7%) to Orientia tsutsugamushi , with mixed antibody reactivity to more than one antigen in 56/105 (53.3%) cases, including Rickettsia typhi in 27/105 (25.7%). Among those with mixed IgG reactivity, IgM assays were used to detect pathogens responsible for acute infections. Finally, a total of 55 spotted fever group (SFG) infections, seven scrub typhus infections, and one case of murine typhus were identified. Of the 105 positive cases, 53 (50.5%) were male and 52 (49.5%) were female, and the mean age was 40 years (range 11–83 years). In the SFG patients, 13/55 (24%) had severe vasculitis with fern leaf type skin necrosis and 17/55 (31%) had arthritis. Three patients (5%) had an altered level of consciousness. A patient with scrub typhus had transient deafness. None of the 105 patients had an eschar. Conclusions It appears that SFG rickettsioses are on the rise in the hilly Central Province of Sri Lanka, whilst murine typhus and scrub typhus are present at a lower rate.

INTRODUCTION: Mediterranean spotted fever (MSF) is a tick-borne disease caused by Rickettsia conorii subspp. conorii. It is transmitted by the bite of the tick Rhipicephalus sanguineus. Modified by the rickettsial invasion, the micro-vascular endothelium acquires an activated inflammatory phenotype and initiates secretion of cytokines and expression of cell adhesion molecules (CAMs) and chemoattractans. AIM: This study aims at investigating the alterations in the soluble cellular adhesion molecules (sCAMs) and chemokine MCP-1 levels in patients with MSF of varying severity in the acute and convalescence stage in order to assess their diagnostic and prognostic value. MATERIALS AND METHODS: The soluble forms of cellular adhesion molecules (sCAMs) - sЕ- selectin and sР-selectin, the intercellular (sICAM-1) and vascular (sVCAM-1) adhesion molecules as well as the monocyte chemoattractant protein-1 (MCP-1) were studied in the sera of 80 patients with MSF. The presence of MSF was confirmed serologically by indirect fluorescence assay (IFA). In order to study disease dynamics, serum samples from 80 patients were drawn on day 1 following the onset of rash; in 60 patients (part of the surveyed 80) a second sample was taken in the convalescence period - 14 days post hospital discharge. The investigation was focused on mild, moderate and severe forms of MSF. Enzyme linked immune-sorbent assay was used for sCAMs determination (Quantikine IVD colorimetric ELISA). RESULTS: Overall, in the acute stage, patients presented with increased levels of sЕ-selectin, sICAM-1, sVCAM-1 and MCP-1, whereas sР-selectin level was decreased. The levels of sЕ-selectin, sICAM-1 and sVCAМ-1 were significantly elevated in mild, moderate and severe forms of the disease with sE-selectin level exhibiting a plateau tendency and sICAM and sVCAM levels demonstrating an upward trend from mild towards severe MSF forms. MCP-1 level was elevated only in severe MSF. In all forms of MSF, in the convalescence period, sICAM-1, sVCAM-1 and МСР-1 concentration returned to reference levels whereas sE-selectin level persisted elevated. In the convalescence stage, sP- selectin concentration also showed an upward tendency, which in severe forms of MSF slightly exceeded the level in controls. sP-selectin levels correlated directly with platelet count, whereas sICAM-1 and sVCAM-1 levels showed a reverse correlation. sE-selectin, sICAM-1 and MCP-1 levels directly correlated with aminotransferase activity (ALT and/or AST). CONCLUSION: The soluble forms of CAMs reflect the endothelial inflammatory potential. There is evidence that endothelium activation is more potent in severe forms of MSF. Assessment of the endothelial response in the course of the disease is an important predictor of the outcome, the choice of therapeutic approach and disease prognosis.

Mediterranean spotted fever (MSF) is widely prevalent in many endemic regions in Bulgaria. The disease is still not quite thoroughly studied as to some aspects of its pathogenesis and especially to issues that concern the crucial signals for apoptosis in the target microvascular endothelial cells. To study the expression of Bcl-2 family proteins and Caspase-3 in the dermal capillary endothelial cells from skin papules and in the eschar (tache noire) epidermal layers of patients with MSF so that we can establish apoptotic processes and the time of their occurrence and deployment. Immunohistochemical reactions for Bcl-2, Bax and Caspase-3 were obtained in slices of punch-biopsies taken from papules of the skin rash and from the eschars of eight patients with MSF. The average intensity of the reactions was compared with that in control punch-biopsy slices from four healthy subjects. MSF was etiologically confirmed in all patients by positive antibody response to a specific antigen, Rickettsia conorii, with indirect immunofluorescent assay performed by the Rickettsial Reference Laboratory. The immune reaction for Bcl-2 was found to be poorly expressed in the capillary endothelial cells of skin papules of patients without any differences from controls. The expression of Bax and Caspase-3 was strongly upregulated in comparison with the controls. The Bcl-2/Bax ratio was significantly decreased. Microvascular endothelial cells of the eschar showed similar changes. While the Bcl-2/Bax ratio decreased in the epidermal layers of the eschar "tache noire", there were no changes in the intensity of the immunoreactivity of Caspase-3 as compared with controls. The upregulation of Bax and Caspase-3 is an indication of ongoing apoptotic processes in the dermal microvascular endothelial cells of MSF patients. The epidermal layers of the eschar showed increased sensitivity to apoptosis, however, executive phase of apoptosis did not occur.

A study on tick fauna and tick-borne pathogens was undertaken in Pianosa, an island in the Tuscany Archipelago that constitutes an important stopping and nesting point for migratory birds. Ticks were removed from feral cats and a few terrestrial birds, and host-seeking ticks were collected by dragging. A total of 89 ticks were found on animals: 57 Ixodes ventalloi Gil Collado, 1936 and 32 Ixodes acuminatus Neumann, 1901. Host-seeking ticks were 354 Hyalomma spp. larvae and 18 Hyalomma spp. adults, identified as Hyalomma marginatum C.L. Koch, 1844 (n=11) and 7 Hyalomma detritum Schulze, 1919 (n=7). A sample of adult ticks was subjected to molecular analyses to look for Rickettsia spp. and Borrelia burgdorferi sensu lato (s.l.). Sequence analysis of the 5S-23S intergenic spacer region and OspA gene of B. burgdorferi s.l.-positive samples showed the presence of Borrelia spielmanii (n=3; 3.7%, 95% confidence interval [CI] 0.08-10.4) and Borrelia valaisiana (n=13; 13.6%, 95% CI 7.0-23.0) in Ixodes ticks from cats and terrestrial birds. Ixodes spp. were also infected by Rickettsia helvetica (n=19; 23.4%, 95% CI 14.7-34.2). Finally, we detected Rickettsia aeschlimannii in 3 out of 12 host-seeking Hyalomma spp. adults tested (25%, 95% CI 5.5-57.2). Our study shows the presence of several tick-borne pathogens in Pianosa. Hyalomma spp. and Ixodes ticks other than I. ricinus seem to be involved in their epidemiological cycle, and birds could contribute to the pathogen dispersal along their migration routes. This is the first finding of B. spielmanii in Italy. We hypothesize the involvement of peridomestic rodents or hedgehogs in its maintenance in Pianosa.

Small regulatory RNAs comprise critically important modulators of gene expression in bacteria, yet very little is known about their prevalence and functions in Rickettsia species. R. conorii, the causative agent of Mediterranean spotted fever, is a tick-borne pathogen that primarily infects microvascular endothelium in humans. We have determined the transcriptional landscape of R. conorii during infection of Human Microvascular Endothelial Cells (HMECs) by strand-specific RNA sequencing to identify 4 riboswitches, 13 trans-acting (intergenic), and 22 cis-acting (antisense) small RNAs (termed ‘Rc_sR’s). Independent expression of four novel trans-acting sRNAs (Rc_sR31, Rc_sR33, Rc_sR35, and Rc_sR42) and known bacterial sRNAs (6S, RNaseP_bact_a, ffs, and α-tmRNA) was next confirmed by Northern hybridization. Comparative analysis during infection of HMECs vis-à-vis tick AAE2 cells revealed significantly higher expression of Rc_sR35 and Rc_sR42 in HMECs, whereas Rc_sR31 and Rc_sR33 were expressed at similar levels in both cell types. We further predicted a total of 502 genes involved in all important biological processes as potential targets of Rc_sRs and validated the interaction of Rc_sR42 with cydA (cytochrome d ubiquinol oxidase subunit I). Our findings constitute the first evidence of the existence of post-transcriptional riboregulatory mechanisms in R. conorii and interactions between a novel Rc_sR and its target mRNA.

International audience; Mediterranean spotted fever caused by Rickettsia conorii is a potentially lethal disease characterized by vascular inflammation affecting multiple organs. Studies of R. conorii so far have focused on activation of inflammatory cells and their release of inflammatory cytokines, but complement activation has not been investigated in R. conorii-infected patients. Here, we performed a comprehensive analysis of complement activation markers and the soluble cross-talking co-receptor CD14 (sCD14) in plasma from R. conorii-infected patients. The clinical data were supplemented with ex vivo experiments where the cytokine response was characterized in human whole blood stimulated with R. conorii. Complement activation markers at the level of C3 (C3bc, C3bBbP) and terminal pathway activation (sC5b-9), as well as sCD14, were markedly elevated (p < 0.01 for all), and closely correlated (p < 0.05 for all), in patients at admission compared with healthy matched controls. All tested markers were significantly reduced to baseline values at time of follow up. Rickettsia conorii incubated in human whole blood was shown to trigger complement activation accompanied by release of the inflammatory cytokines interleukin-1 beta (IL-1 beta), IL-6, IL-8 and tumour necrosis factor. Whereas inhibition of either C3 or CD14 had only a minor effect on released cytokines, combined inhibition of C3 and CD14 resulted in significant reduction, virtually to baseline levels, of the four cytokines (p < 0.05 for all). Our data show that complement is markedly activated upon R. conorii infection and complement activation is, together with CD14, responsible for a major part of the cytokine response induced by R. conorii in human whole blood. K. Otterdal, (C) 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Background: Rickettsial infections are re-emerging. In India, they are now being reported from several areas where they were previously unknown. Objectives: The objective of this study was to describe the epidemiology, clinical profile and outcome of serologically-confirmed scrub typhus and spotted fever among children in a tertiary care hospital in Bengaluru. Materials and Methods: Hospitalised children aged rickettsial disease admitted between January 2010 and October 2012 were included prospectively. Diagnosis was based on scrub typhus and spotted fever-specific IgM and IgG by enzyme-linked immunosorbent assay (ELISA). Results: Of 103 children with clinical features suggestive of rickettsial illness, ELISA test confirmed 53 cases for scrub typhus, 23 cases for spotted fever group and 14 with mixed infection. The average age was 7.3 (±3.9) years and 44 (71.0%) children were male. Majority of cases were from Karnataka (50%), Andhra Pradesh (32.3%) and Tamil Nadu (17.7%). Common clinical features included fever (100%, average duration 11 days), nausea and vomiting (44%), rash (36%); eschar was rare. Compared to the ELISA test, Weil-Felix test (OX-K titre of 1:80) had a sensitivity and specificity of 88.7% and 43.9%, respectively. Treatment with chloramphenicol or doxycycline was given to the majority of the children. Complications included meningoencephalitis (28%), shock (10%), retinal vasculitis (10%) and purpura fulminans (7%). Conclusions: These findings suggest that the burden of rickettsial infection among children in India is high, with a substantially high complication rate. Rickettsial-specific ELISA tests can help in early diagnosis and early institution of appropriate treatment that may prevent life-threatening complications.

La fièvre boutonneuse méditerranéenne est une maladie infectieuse du groupe des rickettsioses due à une bactérie intracellulaire: Rickettsia Conorii. Les formes pauci symptomatiques et bénignes sont prédominantes. Les formes graves sont rares et de plus en plus rapportées dans la littérature récente avec une atteinte multi systémique sévère pouvant menacer le pronostic vital. Nous rapportons dans ce travail un cas très rare d’un patient de 52 ans admis en réanimation pour convulsions, état de choc septique et insuffisance rénale aiguë. Devant la découverte au deuxième jour de la prise en charge de lésions de type « chancre d’inoculation » et boutonneuses, le diagnostic de rickettsiose grave avec une atteinte multi viscérale sévère a été évoqué puis confirmé par une sérologie. Une antibiothérapie par doxycycline a été alors introduite. Le patient va développer cependant une nécrose tubulaire aiguë nécessitant des séances d’épuration extra-rénale. Il va développer rapidement une défaillance multi viscérale mortelle. La rickettsiose, par la vascularite microcirculatoire qu’elle engendre, peut donner des tableaux aussi sévères. Les mécanismes impliqués et les facteurs pronostiques éventuels sont discutés dans ce travail à travers une revue de la littérature.

International audience; Background: The pathophysiological hallmark of Rickettsia conorii (R. ă conorii) infection comprises infection of endothelial cells with ă perivascular infiltration of T-cells and macrophages. Although ă interferon (IFN)-gamma-induced protein 10 (IP-10)/CXCL10 is induced ă during vascular inflammation, data on CXCL10 in R. conorii infection is ă scarce. ă Methods: Serum CXCL10 was analyzed in two cohorts of southern European ă patients with R. conorii infection using multiplex cytokine assays. The ă mechanism of R. conorii-induced CXCL10 release was examined ex vivo ă using human whole blood interacting with endothelial cells. ă Results: (i) At admission, R. conorii infected patients had excessively ă increased CXCL10 levels, similar in the Italian (n = 32, similar to ă 56-fold increase vs controls) and the Spanish cohort (n = 38, 68-fold ă increase vs controls), followed by a marked decrease after recovery. The ă massive CXCL10 increase was selective since it was not accompanied with ă similar changes in other cytokines. (ii) Heat-inactivated R. conorii ă induced a marked CXCL10 increase when whole blood and endothelial cells ă were co-cultured. Even plasma obtained from R. conorii-exposed whole ă blood induced a marked CXCL10 release from endothelial cells, comparable ă to the levels found in serum of R. conorii-infected patients. Bacteria ă alone did not induce CXCL10 production in endothelial cells, macrophages ă or smooth muscle cells. ă Conclusions: We show a massive and selective serum CXCL10 response in R. ă conorii-infected patients, likely reflecting release from infected ă endothelial cells characterized by infiltrating T cells and monocytes. ă The CXCL10 response could contribute to T-cell infiltration within the ă infected organ, but the pathologic consequences of CXCL10 in clinical R. ă conorii infection remain to be defined. (C) 2016 Elsevier Ltd. All ă rights reserved.

Background Birds are important hosts and dispersers of parasitic arthropods and vector-borne zoonotic pathogens. Particularly migratory species may carry these parasites over long distances in short time periods. Migratory hotspots present ideal conditions to get a snapshot of parasite and pathogen diversity of birds migrating between continents. The aim of this study was to investigate the presence and diversity of Rickettsia spp. in ticks collected from birds at a migratory hot-spot in the Danube Delta, Romania, eastern Europe. Methods DNA was extracted from ticks that were collected from migratory birds in the Danube Delta during migratory seasons in 2011–2012. Two 360 bp fragments of the 16S ribosomal RNA gene and a 381 bp fragment Gene gltA were PCR amplified and analyzed by sequence analysis (performed at Macrogen Europe, Amsterdam, The Netherlands). Nucleotide sequences were compared to reference sequences available in the GenBank database, using Basic Local Alignment Search Tool. Results Four hundred ticks of four different species were found on 11 bird species. The prevalence of Rickettsia spp. infection was 14 % (56/400, CI: 11.7–29.1), with significantly more nymphs hosting rickettsial infection compared to larvae (48 vs 7; P

Background The Gulf Coast tick (Amblyomma maculatum) is an arthropod vector of Rickettsia parkeri, the causative agent of American boutonneuse fever and an infectious agent of public health significance. In this study, we evaluated the biological significance of the superoxide dismutases (SODs) of A. maculatum in hematophagy and R. parkeri colonization within the tick host. Methods An RNA interference approach was used to measure the functional roles of tick SODs (Cu/Zn-SOD and Mn-SOD) in R. parkeri colonization of the tick vector. Total microbial load, R. parkeri infection rate, and compensatory mechanisms by tick genes were examined using quantitative polymerase chain reaction (PCR) and quantitative reverse-transcriptase PCR assays. SOD enzymatic activity assays and malondialdehyde (MDA) lipid peroxidation were employed to determine the redox states in the tick tissues. Results Knockdown of the Cu/Zn-SOD gene caused the upregulation of Mn-SOD in transcript levels. Single and dual knockdowns of the SOD genes caused an increase in MDA lipid peroxidation while SOD enzymatic activities did not show a significant change. Mn-SOD knockdown resulted in a substantial increase in the microbial load; however, Cu/Zn-SOD transcript depletion prompted an upsurge in the midgut bacterial load, and significantly decreased the bacterial load in salivary gland tissues. Additionally, Cu/Zn-SOD transcript silencing led to significantly fewer R. parkeri DNA copy numbers in both tick tissues (midguts and salivary glands). Conclusions SOD enzymes play an important function in the regulation of bacterial communities associated with tick vectors and also in the defense mechanisms against the damage caused by reactive oxygen species within the tick. Knockdown experiments increased the levels of total oxidative stress in ticks, revealing the interplay between SOD isozymes that results in the transcriptional regulation of tick antioxidants. Moreover, the tick's Cu/Zn-SOD aids in the colonization of R. parkeri in tick tissues providing evidence of A. maculatum's vectorial success for a spotted fever group rickettsial pathogen. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1579-1) contains supplementary material, which is available to authorized users.

Scientific analysis of the genus Rickettsia is undergoing a rapid period of change with the emergence of viable genetic tools. The development of these tools for the mutagenesis of pathogenic bacteria will permit forward genetic analysis of Rickettsia pathogenesis. Despite these advances, uncertainty still remains regarding the use of plasmids to study these bacteria in in vivo mammalian models of infection, namely, the potential for virulence changes associated with the presence of extrachromosomal DNA and nonselective persistence of plasmids in mammalian models of infection. Here, we describe the transformation of Rickettsia conorii Malish 7 with the plasmid pRam18dRGA[AmTrCh]. Transformed R. conorii stably maintains this plasmid in infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth kinetics in cell culture similar to those of nontransformed R. conorii . Using a well-established murine model of fatal Mediterranean spotted fever, we demonstrate that R. conorii (pRam18dRGA[AmTrCh]) elicits the same fatal outcomes in animals as its untransformed counterpart and, importantly, maintains the plasmid throughout infection in the absence of selective antibiotic pressure. Interestingly, plasmid-transformed R. conorii was readily observed both in endothelial cells and within circulating leukocytes. Together, our data demonstrate that the presence of an extrachromosomal DNA element in a pathogenic rickettsial species does not affect either in vitro proliferation or in vivo infectivity in models of disease and that plasmids such as pRam18dRGA[AmTrCh] are valuable tools for the further genetic manipulation of pathogenic rickettsiae.

Rickettsioses represent a group of emerging infectious diseases in Europe. Climate changes and the anthropization of rural environment have favored vectors' biological cycle and geographic spread. In Sardinia, Mediterranean spotted fever (MSF) is endemic and represents an important public health problem.We investigated the etiology and the clinical presentation of MSF-like illness in northern Sardinia by enrolling patients admitted to the Infectious Disease Unit of the University of Sassari.Diagnostic tests included ELISA, Indirect immunofluorescence (IFI), DNA isolation from blood and from eschar samples with real-time PCR and genotyping. Eighty-seven patients with a mean age of 53 ± 14 years, of whom 65 (75 %) males, were included in the study. The most common diagnosis was MSF (79 %), followed by Q fever (8 %), and anaplasmosis (2 %). A tache noire was found in 58 % of rickettioses and 28 % of Coxiella burnetii infections. MSF was confirmed in 47 % of the cases by IFI and 43 % by ELISA antibody tests. The isolation of rickettsial DNA from the eschar was positive in 10/13 (77 %) of the cases due to Rickettsia conorii. Using this method, we identified the first case of R. monacensis infection in Italy.In conclusion, antibody-based tests confirmed the diagnosis in less than 50 % of the cases, whereas DNA isolation confirmed the diagnosis in 77 % of tested cases and allowed the identification of a new pathogenic species in Italy. Therefore, DNA isolation should be implemented to better identify the etiology of MSF-like illnesses and help the clinician in the management of patients.

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Introduction: The aim of this study was to describe the epidemiological patterns of Mediterranean spotted fever (MSF) as well as its treatment and outcomes in children in south Jordan. Methodology: A retrospective observational study was conducted from June 2013 to December 2015. Data regarding demographics, clinical presentation, laboratory findings, treatment, and outcomes were collected. Results: In total, 35 male and 20 female patients (mean age: 6 years ± 3.6) were included. The incidence of MSF was 7.9 cases/100,000 inhabitants/year; MSF affected 89% of individuals in the summer, 74.5% of those living in a rural area with tent housing, and 100% of those who had contact with animals. All cases presented with fever, and 94.5% had a skin rash. Serological tests were positive in 87.2% of cases, and Rickettsia conorii (the Moroccan strain) was present in all positive cases. All cases had thrombocytopenia, but none had leukocytosis. Hyponatremia was present in 71% of cases, and 49%, 61.8%, and 72.7% had increased urea, alanine transaminase, and aspartate aminotransferase levels, respectively. Doxycycline was administered to all patients, with a cure rate of 96.4% and mortality rate of 3.6%. Conclusions: MSF caused by R. conorii (the Moroccan strain) is prevalent in Jordan, and contact with animals is the route of transmission. The patients’ responses to doxycycline were excellent. A high index of suspicion, early diagnosis, and specific treatment considerably decrease mortality. MSF should be considered as a possible cause of febrile disease in those with a rash and in those living in rural areas.

The paper analyzes data on the prevalence and possible epidemiological importance of Rickettsia raoultii. The possible risk of infection in the population was assessed from the results of a serological study of patients after tick suction and from those of molecular biological determination of contamination of removed carriers. The place of Rickettsia raoultii among other rickettsia causing rickettsioses of a tick-borne spotted fever group is assessed in Russia.

Summary Aim To review the current state of knowledge concerning rickettsiae and rickettsioses in Croatia and to discuss their implications for travellers. Methods The PubMed database was searched from 1991 to 2015 by combining the words “rickettsia,” “rickettsiosis”, “travellers” and “Croatia”. Results Since 1969, Croatia appears to be free of epidemic typhus (ET) caused by Rickettsia prowazekii and the last case of Brill-Zinsser disease was recorded in 2008. Mediterranean spotted fever (MSF) caused by Rickettsia conorii is the most frequent human rickettsial infection in Croatia, followed by murine typhus caused by Rickettsia typhi . Human cases of MSF and murine typhus have been predominantly observed along the eastern Adriatic coast from Zadar to Dubrovnik and between Zadar and Split, respectively. Rickettsia akari , etiologic agent of rickettsialpox, was isolated from blood of a patient diagnosed with MSF in Zadar, but no cases of rickettsialpox were reported. Several species of pathogenic ( Rickettsia slovaca , Rickettsia aeschlimannii , Ricketsia helvetica , and Ricketsia raoultii ) and species of undetermined pathogenicity ( Ricketsia hoogstraalii sp. nov.) rickettsiae were identified in ticks collected in different ecological regions of Croatia. A search of the literature revealed no evidence of rickettsial infection in travellers visiting Croatia. Three imported cases of Rickettsia africae were observed in travellers returning from South Africa. Conclusion Rickettsiae and rickettsial diseases continue to be present in Croatia. As they can be acquired while travelling, physicians should consider rickettsial infection in the differential diagnosis of patients returning from Croatia and presenting with febrile illness.

Pathogenic rickettsiae of the spotted fever group (SFG) induce skin lesions called "eschars" at the sites of arthropod bites. We recently described a guinea pig model based on eschar formation to predict the eventual pathogenicity of new Rickettsia species, and in this study, we used the model to study cross-protection among SFG Rickettsia species and subspecies. We showed that the intradermal inoculation of guinea pigs with Rickettsia conorii subsp. conorii significantly reduced, but not completely prevented, the number of eschars after sub-challenges with the same pathogen (P=0.0004). The same effect was also observed for other subspecies of the R. conorii complex. Additionally, the bacterial load in the eschars was significantly lower in immunized animals than in naïve animals. No protection was observed when sub-challenges were performed with other Rickettsia species, such as R. africae, R. sibirica subsp. mongolitimonae, R. aeschlimannii and R. massiliae. Our data suggest that patients may experience several episodes of infection with related or with the same species of Rickettsia. Moreover, the absence of cross-protection between Rickettsia species may explain the co-existence of two or more tick-borne rickettsioses in the same geographic areas.

Summary Background Spotted fever group (SFG) rickettsial infections are increasingly detected in Sri Lanka. We describe 17 patients with SFG who developed neurological manifestations. Methods The cases were studied prospectively from 2008 at the Teaching Hospital, Peradeniya. An immunofluorescent antibody assay (IFA) was used to confirm the diagnosis. Results All had an IFA IgG titer ranging from 1/64 to 1/4096 and a positive IFA IgM titer against Rickettsia conorii antigen; in 10 (59%) cases the IgG titers were ≥1/256 (definitive cases). The median age of the patients was 62 years (range 26–82 years); 10 were male and seven female. The median duration of fever was 12 days (range 4–35 days). Neurological manifestations on admission were drowsiness or confusion in 14 (82%) and a semi-comatose state in three (18%). Rigidity of the limbs occurred in 14 (82%), bradykinesia and resting tremors in 12 (71%), which persisted after defervescence, neck stiffness in seven (42%), weakness of the limbs in five (29%), deafness in two (12%), and stupor in three (18%). Electroencephalograms in three (18%) showed generalized slow waves. Cerebrospinal fluid examination showed a cellular reaction, predominantly lymphocytes, in three cases. Two patients died (fatality rate 12%). Conclusion We have documented for the first time the neurological features of SFG rickettsioses in the Central Province, Sri Lanka. These were predominantly extrapyramidal features in patients of older age.

The laboratory diagnosis of rickettsioses is based on serology (reference method), cell culture and/or molecular tools. However, the main drawback of serology is its incapacity to provide identification of Rickettsiae at the level of species. The aim of this study was to propose the versatile protein markers able to discriminate the patients with murine typhus from those with Mediterranean spotted fever. We have cloned and expressed 20 proteins of Rickettsia prowazekii and Rickettsia rickettsii, respectively, using the GATEWAY approach. These recombinant proteins were screened by ELISA with sera of infected patients with Rickettsia typhi and Rickettsia conorii, respectively. We identified several potential markers which allowed infection due to R. typhi to be discriminated from those due to R. conorii. However, the values of test-operating parameters were not sufficient for its 'routine' clinical use. Our diagnostic test requires further optimization for be applied as a point-of-care strategy in the management of patients with suspected cases of rickettsiosis.

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 mechanisms 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 17kD) 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 ISG15-mediated post-translational modification of host cellular proteins during infection with an intracellular bacterium.

Summary Objective The aim of this work was to evaluate the prevalence of past infection due to Rickettsia typhi and Rickettsia conorii in the Canary Islands (Spain). Methods A representative sample of the population of the seven islands, formed of 662 people aged between 5 and 75 years (368 females, 294 males), was analyzed. Epidemiological data were obtained by direct survey. The detection of serum IgG antibodies against both microorganisms was based on an indirect immunofluorescence test, considered positive if the titers were ≥1/80. Results Of the analyzed population 3.9% had IgG antibodies against R. typhi and 4.4% against R. conorii . Out of these positive samples, only three were positive for both species. The seroprevalence was similar in both sexes. Positive results were found in all age groups, but a higher rate was noticed in those aged 46 years and older ( p R. typhi was found to be more prevalent in rural areas of all islands, as well as in farmers. Conclusions Our results confirm the presence of antibodies against the causative agents of murine typhus and Mediterranean spotted fever in the Canary Islands. Indirect data suggest that the detection of antibodies to R. conorii might be due to a cross-reaction between these species.

OBJECTIVES To identify risk factors for malignant Mediterranean spotted fever (MSF) caused by Rickettsia conorii conorii. PATIENTS AND METHODS Epidemiological, clinical and biological characteristics as well as risk factors (including treatment regimens) for severe MSF cases were analysed retrospectively. A patient with two or more organ dysfunctions or patient death was defined as a severe case. RESULTS During the study period (January 1999 to December 2009), 161 MSF cases were referred to our centre for rickettsioses. Twenty-six cases (16.1%) were considered severe, which is 3-fold higher than in our previous studies. The clinical and laboratory findings were comparable to those reported elsewhere except that the type of antibiotic treatment was associated with disease severity. Doxycycline administration prior to deterioration of disease (in 31 patients) protected patients from development of severe MSF [relative risk (RR) 0.248, 95% confidence interval (CI) 0.08-0.76] and induced earlier defervescence compared with the other treatment regimens (3.02 ± 2.2 days versus 7.1 ± 6.57 days, P = 0.021). In contrast, fluoroquinolone treatment (in 21 patients) was significantly and independently associated with MSF severity (RR 2.53, 95% CI 1.40-4.55) and was associated with a significantly longer hospital stay. CONCLUSIONS In this retrospective study fluoroquinolone treatment was associated with increased MSF disease severity. Fluoroquinolones have been previously associated with treatment failure in typhus and scrub typhus cases. Thus, we do not recommend the use of fluoroquinolones to treat rickettsial diseases.

In summer 2008, two fatal cases were observed in Northeastern Greece: a Crimean-Congo hemorrhagic fever (CCHF) case (first report in Greece) and a Mediterranean spotted fever case. In total, 537 ticks removed from humans who referred for this reason to the two hospitals of the region during June-September 2008 were identified. The vast majority of them (81.5%) were Rhipicephalus sanguineus, which is the main vector of Rickettsia conorii, while Hyalomma marginatum, the main vector of CCHF virus, accounted for 5.2%. The increased aggressiveness of R. sanguineus might be related to the weather conditions occurred during 2007-2008, while a variety of factors, including climate, might play a role in CCHF emergence.

Haemophagocytic lymphohistiocytosis is a rare but potentially fatal disease resulting from dysregulated activation and proliferation of lymphocytes. We present a case of haemophagocytic syndrome occurring in a 5-year-old Italian boy as a complication of Mediterranean spotted fever. The characteristics of this case have been analysed and contextualized among those of another 15 cases of haemophagocytic syndrome associated with rickettsial diseases found through a systematic review of the international literature.

The diagnosis of multiple sclerosis (MS), despite well defined clinical criteria is not always simple. On many occasions it is difficult to differentiate MS from various non-MS idiopathic demyelinating disorders, specific and infectious inflammatory diseases or non-inflammatory demyelinating diseases. Clinicians should be aware of various clinical and MRI "red flags" that may point to the other diagnosis and demand further diagnostic evaluation. It is generally accepted that atypical clinical symptoms or atypical neuroimaging signs determine necessity for broad differential diagnostic work up. Of the infectious diseases that are most commonly mistaken for MS the clinician should take into account Whipple's disease, Lyme disease, Syphilis, HIV/AIDS, Brucellosis, HHV-6 infection, Hepatitis C, Mycoplasma and Creutzfeld-Jacob disease, among others. Cat scratch disease caused by Bartonella hensellae, Mediterranean spotted fever caused by Riketssia connore and Leptospirosis caused by different Leptospira serovars rarely cause focal neurological deficit and demyelinating MRI changes similar to MS. When atypical clinical and neuroimaging presentations are present, serology on rare infectious diseases that may mimic MS may be warranted. This review will focus on the infectious diseases mimicking MS with presentation of rare illustrative cases.

The modern system for epidemiological surveillance of tick-borne infections (TBIs) in the Varna region requires a systematic analysis of the epidemic process. Aim : The aim of this article is to trace the evolution in parameters of the parasitic system in Lyme Borreliosis, Boutonneuse fever, Crimean-Congo hemorrhagic fever, and Q-fever in the Varna region for the period 2001-2017 and the activities of the epidemiological surveillance system. Materials and Methods: Data from active epidemiological studies and analyses of TBIs in the region of Varna and Bulgaria for the period 2001-2017 were used; as well as epidemiological and entomological methods, statistical and graphic analyses, nosogeographic analyses and others. Results: Data from the epidemiological analysis determined the continuing health and social significance of registered TBIs in the Varna region during the considered period. High levels of morbidity in Lyme disease (LD) and Boutonneuse fever (BF) with characteristic summer seasonality, correlated with the activity of vectors - reservoirs (Ixodes ticks), have been established. The system of epidemiological surveillance includes events for effective dispensary monitoring of the bitten by a tick; collecting complete epidemiological information about the area where the bite has occurred; preparation of epidemiological analysis of the morbidity of transmissible infections on the territory of the Varna region with emphasis on Lyme borreliosis, Boutonneuse fever, Q fever, Crimean-Congo hemorrhagic fever (CCHF), surveillance over the species composition and territorial distribution of tick populations and expanding the tick control activities in the region. There is a timely diagnosis and treatment of the registered patients. It is necessary to expand and deepen the system of epidemiological surveillance with the proposed strategy for surveillance and control of socially significant TBIs in the Black Sea region of the Republic of Bulgaria.

Forty-five days after the first confirmed and fatal Crimean–Congo haemorrhagic fever (CCHF) case in Greece in 2008, a female patient with similar signs and symptoms (high fever, thrombocytopaenia) ‘nd resident of the same area, was admitted to the University General Hospital of Alexandroupolis. Before admission, she had visited a local hospital where a cephalosporin was prescribed. A rash manifested over subsequent days, which was misdiagnosed as an allergy to the drug. Upon admission to the University Hospital, she was given further antibiotics, including doxycycline; a few hours later, ribavirin was added because CCHF was suspected. After the patient's death, rickettsiosis caused by Rickettsia conorii conorii (Meditteranean spotted fever; MSF) was diagnosed. Extremely high values of interleukin (IL)-1ra, IL-6, interferon-γ-inducible protein-10, monocyte chemoattractant protein-1 and an absence of tumour necrosis factor-α were observed. MSF is a potentially severe and even fatal disease resembling viral haemorrhagic fevers that has to be included in the differential diagnosis of febrile syndromes combined with thrombocytopaenia, even when a tick bite is not reported, and an eschar is absent. Physicians have to be aware of MSF in patients with severe disease who are returning from the Mediterranean area.

We review two cases of ocular manifestations of Rickettsia conorii infection in children. A girl who presented unilateral visual loss with focal retinitis and macular oedema and a boy with unilateral central scotoma and bilateral anterior uveitis. Progressive functional and anatomic recovery was observed after oral antibiotics and steroids were initiated.

Background. Mediterranean spotted fever (MSF) is a tick-borne infection caused by Rickettsia conorii conorii mainly endemic in the Mediterranean Basin. Although usually considered as a benign disease, severe forms of MSF have been sporadically reported. Methods. We report on three patients who developed severe MSF complications after a stay in Morocco. Literature was reviewed to assess the frequency and pattern of MSF complications in the largest reported case series in endemic countries. Results. Each of our three patients diagnosed with MSF presented with a different complicated course: one with meningoencephalitis, one with lung embolism and one with septic shock and multi organ failure. In published series, rate of complications (defined as severe organ involvement) ranged from 1% to 20%. However, study designs and settings were highly variable and did not allow for relevant comparisons. Meningoencephalitis and shock with multi organ failure were the most frequently observed complications. Mortality of severe course was up to 20% in some series. Conclusion. Severe organ involvement is not infrequent in patients with Mediterranean spotted fever and fatal outcome is regularly reported. Because presentations of complicated course may be extremely diverse, a high index of suspicion is required in febrile patients with potential exposure, in particular if skin rash and/or eschar are found. Early appropriate antibiotherapy is crucial to improve outcome.

It may seem perplexing that there is any uncertainty in Australia about the existence of zoonotic tick-associated infections1–3. Outside this country, particularly in the northern hemisphere, tick-borne diseases such as human granulocytic anaplasmosis, babesiosis, Boutonneuse fever, ehrlichiosis, Lyme borreliosis, and tick-borne encephalitis, have well documented aetiologies, epidemiology, diagnostic methods, and treatments. Why is Australia different and what research is being conducted to address this issue? This article briefly addresses these questions and explains how high-throughput metagenomic analysis has started to shed light on bacterial microbiomes in Australian ticks, providing new data on the presence and distribution of potentially zoonotic microbial taxa.

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 × 105PFU ofRickettsia conoriideveloped 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 withR. 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-4highT-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.

Mediterranean spotted fever is the most important tick-borne disease occurring in Southern Europe and North Africa. The first case of this life-threatening zoonosis was reported in 1910. In the 1930s, the role of the brown dog tick, Rhipicephalus sanguineus, and the causative agent, Rickettsia conorii were described. However, basic questions regarding the relationships between the rickettsia and its tick vector are still unresolved, and the life cycle of R. conorii is incompletely known. There is a lack of knowledge associated with the role of Rh. sanguineus in the maintenance and transmission of R. conorii. The infectious rate of Rh. sanguineus ticks with R. conorii has been found low every time it has been tested; usually lower that 1%. The deleterious impact of R. conorii on ticks has been suggested in experimental models as a potential reason to explain a low prevalence in nature. The long-recognized phenomenon known as reactivation has been suggested as a cause of negative effects - that is, the change in temperature and physiology of the tick host induces the agent to emerge from dormancy and attain infectivity with bad effects on ticks. However, naturally infected colonies of ticks have been maintained in laboratory conditions over several generations. We discuss here several aspects that have been recently studied to better understand Rh. sanguineus-R. conorii relationships, including comparison between the fitness of infected and non-infected ticks in laboratory conditions and the role of external factors such as temperature and starvation.