Survival of Rickettsia conorii in artificially contaminated whole and leukoreduced canine blood units during the storage period.

BACKGROUND: The ability of tick-borne agents to survive in stored blood bags is a key factor for their transmissibility by blood transfusion. The aim of this study was to evaluate the survival and potential infectivity of Rickettsia conorii (RC) in artificially contaminated canine whole blood (WB) and in leukoreduced whole blood (LR-WB) during the storage period. METHODS: RC was cultured on L929 cells. We used a one-week 25-cm2 flask with 70-80% of L929 infected cells to prepare the bacterial inoculum by pelleting cells and suspending the pellet in the donors' serum. We infected five 100 ml WB units with RC within 2 h from the collection and maintained it at room temperature for 4 h prior to refrigeration. We filtered 50 ml of each WB bag to obtain leukoreduced WB (LR-WB) at day 1 post-infection (dpi). We checked WB and LR-WB bags at 1, 4, 7, 14, 21, 28, 35 dpi for RC presence and viability through real-time PCR (rPCR) for DNA and mRNA, respectively, and by isolation. Identification of isolates was confirmed by indirect immunofluorescence and rPCRs. RESULTS: RC survived for the entire storage period in both whole and leukoreduced blood. All bags contained viable bacteria until 7 dpi; RC viability generally decreased over time, particularly in LR-WB bags where the isolation time was longer than in WB. Viable bacteria were still isolated at 35 dpi in 3 WB and 3 LR-WB. CONCLUSIONS: Leukoreduction reduced but did not eliminate RC in infected units. The survival and infectivity of RC in canine blood during the storage period may represent a threat for recipients.

Spatial and temporal trends of Mediterranean spotted fever in Spain, 2005-2015.

BACKGROUND: Mediterranean spotted fever (MSF) is a zoonotic disease caused by Rickettsia conorii and transmitted by Rhipicephalus sanguineus sensu lato. The aim of this study is to understand the epidemiology and trends regarding the disease in Spain, based on notifications to the Spanish National Epidemiology Surveillance Network (RENAVE) and the National Hospital Discharge Database (CMBD) between 2005 and 2015. METHODS: We carried out a retrospective cross-sectional study of the cases and the outbreaks reported to the RENAVE and of those found in the CMBD between January 1st, 2005 and December 31st, 2015. We studied the characteristics of the cases and analyzed their spatio-temporal distribution. RESULTS: 1603 cases notified to the RENAVE and 1789 cases registered in the CMBD were analyzed. The most affected group were men between 45 and 64. There were 8 MSF outbreaks during the study period. RENAVE registered lower rates until 2012, when it was decided that MSF in Spain would become a notifiable disease. Across the temporal series we saw that there was seasonality with an increase in cases in summer, and an overall upward trend according to the RENAVE data and descending according to the CMBD. The geographic distribution was heterogeneous throughout the territory, with maximum rates in La Rioja at 1.87 cases and 2.01 cases per 100,000 inhabitants according to the RENAVE and the CMBD, respectively. CONCLUSIONS: It is of great importance to continue monitoring the disease since it appears to be endemic throughout Spain. There is a need for a common strategy on monitoring and reporting, which would facilitate a more accurate picture on the MSF epidemiological scenario. Entomological information will be of added value.

The Rickettsia conorii Adr1 Interacts with the C-Terminus of Human Vitronectin in a Salt-Sensitive Manner.

Spotted fever group (SFG) Rickettsia species are inoculated into the mammalian bloodstream by hematophagous arthropods. Once in the bloodstream and during dissemination, the survival of these pathogens is dependent upon the ability of these bacteria to evade serum-borne host defenses until a proper cellular host is reached. Rickettsia conorii expresses an outer membrane protein, Adr1, which binds the complement inhibitory protein vitronectin to promote resistance to the anti-bacterial effects of the terminal complement complex. Adr1 is predicted to consist of 8 transmembrane beta sheets that form a membrane-spanning barrel with 4 peptide loops exposed to the extracellular environment. We previously demonstrated that Adr1 derivatives containing either loop 3 or 4 are sufficient to bind Vn and mediate resistance to serum killing when expressed at the outer-membrane of E. coli. By expressing R. conorii Adr1 on the surface of non-pathogenic E. coli, we demonstrate that the interaction between Adr1 and vitronectin is salt-sensitive and cannot be interrupted by addition of heparin. Additionally, we utilized vitroenctin-derived peptides to map the minimal Adr1/vitronectin interaction to the C-terminal region of vitronectin. Furthermore, we demonstrate that specific charged amino acid residues located within loops 3 and 4 of Adr1 are critical for mediating resistance to complement-mediated killing. Interestingly, Adr1 mutants that were no longer sufficient to mediate resistance to serum killing still retained the ability to bind to Vn, suggesting that Adr1-Vn interactions responsible for resistance to serum killing are more complex than originally hypothesized. In summary, elucidation of the mechanisms governing Adr1-Vn binding will be useful to specifically target this protein-protein interaction for therapeutic intervention.

Mediterranean spotted fever in Spain, 1997-2014: Epidemiological situation based on hospitalization records.

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.

Nonselective Persistence of a Rickettsia conorii Extrachromosomal Plasmid during Mammalian Infection.

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.

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections.

Molecular detection of Rickettsia conorii and other zoonotic spotted fever group rickettsiae in ticks, Romania.

The diverse tick fauna as well as the abundance of tick populations in Romania represent potential risks for both human and animal health. Spotted fever group (SFG) rickettsiae are recognized as important agents of emerging human tick-borne diseases worldwide. However, the epidemiology of rickettsial diseases has been poorly investigated in Romania. In urban habitats, companion animals which are frequently exposed to tick infestation, play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Therefore, the aim of the present study was to investigate the occurrence of SFG rickettsiae in ticks infesting dogs in a greater urban area in South-eastern Romania. Adult ixodid ticks (n=205), including Rhipicephalus sanguineus sensu lato (n=120), Dermacentor reticulatus (n=76) and Ixodes ricinus (n=9) were collected from naturally infested dogs and were screened for SFG rickettsiae using conventional PCR followed by sequencing. Additionally, ticks were screened for DNA of Babesia spp., Hepatozoon spp., Ehrlichia canis, and Anaplasma platys. Four zoonotic SFG rickettsiae were identified: Rickettsia raoultii (16%) and Rickettsia slovaca (3%) in D. reticulatus, Rickettsia monacensis (11%) in I. ricinus, and Rickettsia conorii (0.8%) in Rh. sanguineus s.l. Moreover, pathogens of veterinary importance, such as B. canis (21%) in D. reticulatus and E. canis (7.5%) in Rh. sanguineus s.l. were identified. The findings expand the knowledge on distribution of SFG rickettsiae as well as canine pathogens in Romania. Additionally, this is the first report describing the molecular detection of R. conorii in ticks from Romania.

Rickettsia massiliae and Rickettsia conorii Israeli Spotted Fever Strain Differentially Regulate Endothelial Cell Responses.

Rickettsiae primarily target microvascular endothelial cells. However, it remains elusive how endothelial cell responses to rickettsiae play a role in the pathogenesis of rickettsial diseases. In the present study, we employed two rickettsial species with high sequence homology but differing virulence to investigate the pathological endothelial cell responses. Rickettsia massiliae is a newly documented human pathogen that causes a mild spotted fever rickettsiosis. The "Israeli spotted fever" strain of R. conorii (ISF) causes severe disease with a mortality rate up to 30% in hospitalized patients. At 48 hours post infection (HPI), R. conorii (ISF) induced a significant elevation of IL-8 and IL-6 while R. massiliae induced a statistically significant elevated amount of MCP-1 at both transcriptional and protein synthesis levels. Strikingly, R. conorii (ISF), but not R. massiliae, caused a significant level of cell death or injury in HMEC-1 cells at 72 HPI, demonstrated by live-dead cell staining, annexin V staining and lactate dehydrogenase release. Monolayers of endothelial cells infected with R. conorii (ISF) showed a statistically significant decrease in electrical resistance across the monolayer compared to both R. massiliae-infected and uninfected cells at 72 HPI, suggesting increased endothelial permeability. Interestingly, pharmacological inhibitors of caspase-1 significantly reduced the release of lactate dehydrogenase by R. conorii (ISF)-infected HMEC-1 cells, which suggests the role of caspase-1 in mediating the death of endothelial cells. Taken together, our data illustrated that a distinct proinflammatory cytokine profile and endothelial dysfunction, as evidenced by endothelial cell death/injury and increased permeability, are associated with the severity of rickettsial diseases.

The role of Rhipicephalus sanguineus sensu lato saliva in the dissemination of Rickettsia conorii in C3H/HeJ mice.

Animal models have been developed for the study of rickettsial pathogenesis. However, to understand what occurs during the natural route of rickettsial transmission via the tick bite, the role of tick saliva should be considered in these models. To address this, we analysed the role of tick saliva in the transmission of Rickettsia conorii (Rickettsiales: Rickettsiaceae) in a murine host by intradermally (i.d.) inoculating two groups of susceptible C3H/HeJ mice with this Rickettsia, and infesting one group with nymphal Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks. Quantification of bacterial loads and mRNA levels of interleukin-1ß (IL-1ß), IL-10 and NF-κB was performed in C3H/HeJ lung samples by real-time quantitative polymerase chain reaction (PCR) and real-time reverse transcriptase PCR, respectively. Lung histology was examined to evaluate the pathological manifestations of infection. No statistically significant difference in bacterial load in the lungs of mice was observed between these two groups; however, a statistically significant difference was observed in levels of IL-1ß and NF-κB, both of which were higher in the group inoculated with rickettsiae but not infected with ticks. Lung histology in both groups of animals revealed infiltration of inflammatory cells. Overall, this study showed that i.d. inoculation of R. conorii caused infection in the lungs of C3H/HeJ mice and tick saliva inhibited proinflammatory effects.

Increased expression of the homeostatic chemokines CCL19 and CCL21 in clinical and experimental Rickettsia conorii infection.

BACKGROUND: Based on their essential role in concerting immunological and inflammatory responses we hypothesized that the homeostatic chemokines CCL19 and CCL21 may play a pathogenic role in rickettsiae infection. METHODS: Serum levels of CCL19 and CCL21 in patients with R. africae and R. conorii infection were analyzed by enzyme immunoassays. Lungs from R. conorii infected mice were examined for CCL19, CCL21 and CCR7 expression by immunohistochemistry. RESULTS: We found that patients with R. africae infection (n = 15) and in particular those with R. conorii infection (n = 16) had elevated serum levels of CCL19 on admission, with a decline during follow-up. While a similar pattern was seen for CCL21 in R. africae infection, patients with R. conorii infection showed persistently increased CCL21 levels during follow-up. In experimental R. conorii infection, we found strong immunostaining of CCL19 and CCL21 in the lungs, particularly in individuals that had received lethal doses. Immunofluorescence showed co-localization of CCR7 to endothelial cells, macrophages and fibroblasts within the lung tissue of R. conorii infected mice. CONCLUSIONS: Our findings suggest that the CCL19/CCL21/CCR7 axis is up-regulated during R. africae and in particular during R. conorii infection, which may potentially contribute to the pathogenesis of these disorders.

Effects of homologous and heterologous immunization on the reservoir competence of domestic dogs for Rickettsia conorii (israelensis).

A number of spotted fever group (SFG) rickettsiae cause serious infections in humans. Several antigenically related rickettsial agents may coexist within the same geographical area, and humans or vertebrate hosts may be sequentially exposed to multiple SFG agents. We assessed whether exposure of a vertebrate reservoir to one SFG Rickettsia will affect the host's immune response to a related pathogen and the efficiency of transmission to uninfected ticks. Two pairs of dogs were each infected with either Rickettsia massiliae or Rickettsia conorii israelensis, and their immune response was monitored twice weekly by IFA. The four immunized dogs and a pair of naïve dogs were each challenged with R. conorii israelensis-infected Rhipicephalus sanguineus nymphs. Uninfected Rh. sanguineus larvae were acquisition-fed on the dogs on days 1, 7, and 14 post-challenge. These ticks were tested for the presence of rickettsial DNA after molting to the nymphal stage. The naive dogs became infected with R. conorii israelensis and were infectious to ticks for at least 3 weeks, whereas reservoir competence of dogs previously infected with either R. massiliae or R. conorii was significantly diminished. This opens an opportunity for decreasing the efficiency of transmission and propagation of pathogenic Rickettsia in natural foci by immunizing the primary hosts with closely related nonpathogenic SFG bacteria. However, neither homologous immunization nor cross-immunization significantly affected the efficiency of R. conorii transmission between cofeeding infected nymphs and uninfected larvae. At high densities of ticks, the efficiency of cofeeding transmission may be sufficient for yearly amplification and persistent circulation of a rickettsial pathogen in the vector population.

Pathogenic Rickettsia species acquire vitronectin from human serum to promote resistance to complement-mediated killing.

Bacteria of the genus Rickettsia are transmitted from arthropod vectors and primarily infect cells of the mammalian endothelial system. Throughout this infectious cycle, the bacteria are exposed to the deleterious effects of serum complement. Using Rickettsia conorii, the etiologic agent of Mediterranean spotted fever (MSF), as a model rickettsial species, we have previously demonstrated that this class of pathogen interacts with human factor H to mediate partial survival in human serum. Herein, we demonstrate that R. conorii also interacts with the terminal complement complex inhibitor vitronectin (Vn). We further demonstrate that an evolutionarily conserved rickettsial antigen, Adr1/RC1281, interacts with human vitronectin and is sufficient to mediate resistance to serum killing when expressed at the outer-membrane of serum sensitive Escherichia coli. Adr1 is an integral outer-membrane protein whose structure is predicted to contain eight membrane-embedded ß-strands and four 'loop' regions that are exposed to extracellular milieu. Site-directed mutagenesis of Adr1 revealed that at least two predicted 'loop' regions are required to mediate resistance to complement-mediatedkilling and vitronectin acquisition. These results demonstrate that rickettsial species have evolved multiple mechanisms to evade complement deposition and that evasion of killing in serum is an evolutionarily conserved virulence attribute for this genus of obligate intracellular pathogens.

Compartmentalized, functional role of angiogenin during spotted fever group rickettsia-induced endothelial barrier dysfunction: evidence of possible mediation by host tRNA-derived small noncoding RNAs.

BACKGROUND: Microvascular endothelial barrier dysfunction is the central enigma in spotted fever group (SFG) rickettsioses. Angiogenin (ANG) is one of the earliest identified angiogenic factors, of which some are relevant to the phosphorylation of VE-cadherins that serve as endothelial adherens proteins. Although exogenous ANG is known to translocate into the nucleus of growing endothelial cells (ECs) where it plays a functional role, nuclear ANG is not detected in quiescent ECs. Besides its nuclear role, ANG is thought to play a cytoplasmic role, owing to its RNase activity that cleaves tRNA to produce small RNAs. Recently, such tRNA-derived RNA fragments (tRFs) have been shown to be induced under stress conditions. All these observations raise an intriguing hypothesis about a novel cytoplasmic role of ANG, which is induced upon infection with Rickettsia and generates tRFs that may play roles in SFG rickettsioses. METHODS: C3H/HeN mice were infected intravenously with a sublethal dose of R. conorii. At days 1, 3, and 5 post infection (p.i.), liver, lung and brain were collected for immunofluorescence (IF) studies of R. conorii and angiogenin (ANG). Human umbilical vein endothelial cells (HUVECs) were infected with R. conorii for 24, 48, and 72 hrs before incubation with 1µg/ml recombinant human ANG (rANG) in normal medium for 2 hrs. HUVEC samples were subjected to IF, exogenous ANG translocation, endothelial permeability, and immunoprecipitation phosphorylation assays. To identify small non-coding RNAs (sncRNAs) upon rickettsial infection, RNAs from pulverized mouse lung tissues and HUVECs were subjected to library preparation and deep sequencing analysis using an Illumina 2000 instrument. Identified sncRNAs were confirmed by Northern hybridization, and their target mRNAs were predicted in silico using BLAST and RNA hybrid programs. RESULTS: In the present study, we have demonstrated endothelial up-regulation of ANG, co-localized with SFG rickettsial infection in vivo. We also have provided direct evidence that rickettsial infection sensitizes human ECs to the translocation of exogenous ANG in a compartmentalized pattern at different times post-infection. Typically, exogenous ANG translocates into the nucleus at 24 hrs and to the cytoplasm at 72 hrs post-infection. The ANG cytoplasmic translocation enhances phosphorylation and destabilization of VE-cadherin and attenuates endothelial barrier function. Of note, deep sequencing analysis detected tRFs, mostly derived from the 5'-halves of host tRNAs, that are induced by ANG. Northern hybridization validates the two most abundantly cloned tRFs derived from tRNA-ValGTG and tRNA-GlyGCC, in both mouse tissues and human cells. Bioinformatics analysis predicted that these tRFs may interact with transcripts associated with the endothelial barrier, the host cell inflammatory response, and autophagy. CONCLUSIONS: Our data provide new insight into the role of compartmentalized ANG during SFG rickettsioses, and highlight its possible mediation through tRFs.

Co-feeding as a route for transmission of Rickettsia conorii israelensis between Rhipicephalus sanguineus ticks.

Rickettsia conorii is widely distributed in Europe, Asia, and Africa. The brown dog tick, Rhipicephalus sanguineus, is the recognized vector of R. conorii. In this study, we assessed the efficiency of R. conorii israelensis transmission between co-feeding Rh. sanguineus ticks. Infected Rh. sanguineus adults and uninfected nymphs were fed simultaneously upon either naïve dogs or a dog previously exposed to this agent. When ticks were placed upon naïve dogs, 92-100% of nymphs acquired the infection and 80-88% of infected engorged nymphs transmitted it transstadially. When ticks were placed upon a seropositive dog, only 8-28.5% of recipient nymphs became infected. Our results establish the first evidence for efficient natural transmission of R. conorii israelensis between co-feeding ticks upon both naïve and seropositive dogs. This route of transmission can ensure continuous circulation of R. conorii israelensis in tick vectors even in the absence of naïve reservoir hosts.

Deciphering the relationships between Rickettsia conorii conorii and Rhipicephalus sanguineus in the ecology and epidemiology of Mediterranean spotted fever.

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.

Incongruent effects of two isolates of Rickettsia conorii on the survival of Rhipicephalus sanguineus ticks.

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever is widely distributed in Southern Europe, the Middle East, Africa, India and the Caspian region. In the Mediterranean region, the brown dog tick, Rhipicephalus sanguineus, is the recognized vector of R. conorii. To study tick-pathogen relationships and pathogenesis of infection caused in model animals by the bite of an infected tick, we attempted to establish a laboratory colony of Rh. sanguineus persistently infected with R. conorii. Rhipicephalus sanguineus ticks of North American and Mediterranean origin were exposed to R. conorii isolates of African (R. conorii conorii strain Malish) and Mediterranean (R. conorii israelensis strain ISTT) origin. Feeding of ticks upon infected mice and dogs, intra-hemocoel inoculation, and submersion in suspensions of purified rickettsiae were used to introduce the pathogen into uninfected ticks. Feeding success, molting success and the longevity of molted ticks were measured to assess the effects of R. conorii on the survival of Rh. sanguineus. In concordance with previously published results, Rh. sanguineus larvae and nymphs from both North American and Mediterranean colonies exposed to R. conorii conorii Malish experienced high mortality during feeding and molting or immediately after. The prevalence of infection in surviving ticks did not exceed 5%. On the other hand, exposure to ISTT strain had lesser effect on tick survival and resulted in 35-66% prevalence of infection. Rh. sanguineus of Mediterranean origin were more susceptible to infection with either strain of R. conorii than those from North America. Previous experimental studies had demonstrated transovarial and transstadial transmission of R. conorii in Rh. sanguineus; however, our data suggest that different strains of R. conorii may employ different means of maintenance in nature. The vertebrate host may be a more important reservoir than previously thought, or co-feeding transmission between different generations of ticks may obviate or lessen the requirement for transovarial maintenance of R. conorii.

Rickettsial outer-membrane protein B (rOmpB) mediates bacterial invasion through Ku70 in an actin, c-Cbl, clathrin and caveolin 2-dependent manner.

Rickettsia conorii, an obligate intracellular tick-borne pathogen and the causative agent of Mediterranean spotted fever, binds to and invades non-phagocytic mammalian cells. Previous work identified Ku70 as a mammalian receptor involved in the invasion process and identified the rickettsial autotransporter protein, rOmpB, as a ligand; however, little is known about the role of Ku70-rOmpB interactions in the bacterial invasion process. Using an Escherichia coli heterologous expression system, we show here that rOmpB mediates attachment to mammalian cells and entry in a Ku70-dependent process. A purified recombinant peptide corresponding to the rOmpB passenger domain interacts with Ku70 and serves as a competitive inhibitor of adherence. We observe that rOmpB-mediated infection culminates in actin recruitment at the bacterial foci, and that this entry process relies in part on actin polymerization likely imparted through protein tyrosine kinase and phosphoinositide 3-kinase-dependent activities and microtubule stability. Small-interfering RNA studies targeting components of the endocytic pathway reveal that entry by rOmpB is dependent on c-Cbl, clathrin and caveolin-2. Together, these results illustrate that rOmpB is sufficient to mediate Ku70-dependent invasion of mammalian cells and that clathrin- and caveolin-dependent endocytic events likely contribute to the internalization process.

[Mediterranean spotted fever due to contact with dog-tick]. / Köpek kenesi ile temas sonrasinda gelisen akdeniz benekli atesi.

Mediterranean spotted fever (MSF) is one of the tick-borne rickettsial infections caused by Rickettsia conorii. It is transmitted to humans by brown dog ticks (Rhipicephalus sanguineus). In this case report, a 16-years-old male patient who was diagnosed as MSF after an exposure to dog-tick in Bartin province (located at middle Black Sea region of Turkey) has been presented. His history revealed that, five days before admission to the hospital (on June, 2007) he had cleaned dog-ticks from his dog, and after 12 hours he found a stucked tick on his leg and he took it out right away with a tweezer. High fever, headache and generalized maculopapular rash including soles and palms and a black-colored lesion at the tick bite site developed three days later. In clinical examination, there was a black escar circled with a red-purple colored halo in front of the right tibia at the site of the tick bite showing high similarity to "tache noire" which was specific to MSF. Indirect immunofluorescence assay (IFA) for Rickettsia yielded negative result in the serum sample collected on admission day, however, it was found positive at 1/512 titer in the serum sample collected 10 days after admission. The patient has recovered completely without any complication after 10 days of doxycycline therapy. The aim of this presentation is to point out that MSF should be considered for the differential diagnosis of a patient with a history of tick bite, fever, maculopapular rash, headache, myalgia, arthralgia and especially with black escar during summer months in our country where the incidence of tick-borne infections has been increasing since recent years.

Development of a method for recovering rickettsial RNA from infected cells to analyze gene expression profiling of obligate intracellular bacteria.

The Rickettsia genus is composed of Gram-negative bacteria responsible for Typhus and spotted fevers. Because of the limitations imposed by their obligate intracellular location, the molecular mechanisms responsible for their pathogenicity remain poorly understood. Several rickettsial genomes are now available, thus providing the foundation for a new era of post-genomic research. Here, using Rickettsia conorii as model, we developed a suitable method for microarray-based transcriptome analysis of rickettsiae. Total RNA was extracted from infected Vero cells using a protocol preserving its integrity, as observed by Bioanalyzer (Agilent) profiles. By a subtractive hybridization method, the samples were subsequently depleted of eukaryotic RNA that represents up to 90% of the whole extract and that hampers fluorochrome labeling of rickettsial nucleic acids. To obtain the amount of material required for microarray hybridization, the bacterial RNA was then amplified using random primers. Hybridizations were carried out on microarrays specific for R. conorii but containing a limited number of selected targets. Our results show that this method yielded reproducible signals. Transcriptional changes observed following exposure of R. conorii to a nutrient stress were verified by real-time quantitative PCR and by quantitative reverse transcription PCR starting from amplified cDNA and total RNA as templates, respectively. We conclude that this approach has great potential for the study of mechanisms behind the virulence and intracellular survival of members of the genus Rickettsia.