Neurological manifestation of Brazilian spotted fever in childhood.

Rocky Mountain Spotted Fever is a rickettsial disease caused by the bacteria Rickettsia rickettsii. In Brazil, the disease is known as Brazilian spotted fever (BSF), being the most significant tick-borne disease in the country. Among the affected patients, only 5% of cases occur in children aged one to nine years. Typical symptoms of the disease are fever, rash, headache and digestive symptoms. Neurological manifestations such as seizures, aphasia and hemiparesis have been described in few patients. This study aimed to describe the case of an infant diagnosed with BSF who presented severe signs of neurological manifestation.

Density and behavior of capybara (Hydrochoerus hydrochaeris) ticks (Acari: Ixodidae) Amblyomma sculptum and Amblyomma dubitatum with notes on Rickettsia bellii infection: Assessing human exposure risk.

In several urban and peri­urban areas of Brazil, populations of Amblyomma sculptum and Amblyomma dubitatum ticks are maintained by capybaras (Hydrochoerus hydrochaeris). In some of these areas, this host and these tick species are associated with Brazilian spotted fever (BSF), a lethal human disease caused by the bacterium Rickettsia rickettsii. In this work, we evaluated the risk of human exposure to these tick species using four collection techniques to discern host-seeking behavior. The study was carried out in 10 urban sites inhabited by capybaras in Uberlândia, a BSF-free municipality in southeastern Brazil. Ticks were collected in areas of 400 m2 at each site and at three seasons. Within the same municipality, the distance and speed of A. sculptum nymphs moving towards the CO2 traps were evaluated. In a sample of ticks Rickettsia DNA was investigated. During the study period, 52,953 ticks were collected. Among these, 83.4 % were A. sculptum (1,523 adults, 10,545 nymphs and 32,104 larvae) and 16.6 % were A. dubitatum (464 adults, 2,153 nymphs and 6,164 larvae). An average annual questing tick density of 4.4/m² was observed, with the highest density recorded at one site in autumn (31.8/m²) and the lowest in summer at another site (0.03/m²). The visual search yielded the highest proportion of A. sculptum larvae, constituting 47 % of the total and 63.6 % of all A. sculptum larvae. In contrast, CO2 traps collected a greater proportion of nymphs and adults of A. sculptum ticks. In the case of A. dubitatum, the CO2 trap was the most efficient technique with 57.7 % of captures of this species, especially of nymphs (94.5 % of captures) and adults (97.8 % of captures). Ticks' ambush height on vegetation (9 to 77 cm), observed by visual search 30 times, yielded a total of 20,771 ticks. Of these, 28 (93 %) were A. sculptum ticks, with only two (7 %) identified as A. dubitatum ticks. Among A. sculptum ticks, the nymph was the most attracted stage to humans and larva in the case of A. dubitatum. Amblyomma sculptum adults and nymphs were significantly more attracted to humans than those of A. dubitatum, but A. dubitatum larvae were significantly more attracted than the same stage of A. sculptum. The maximum distance and speed of horizontal displacement for A. sculptum nymphs were five meters and 2.0 m/h, respectively. The only species of Rickettsia detected in ticks, exclusively in A. dubitatum, was R. bellii. Importantly, it was observed that the higher the proportion of A. sculptum in the community of ticks, the lower the rate of infection of A. dubitatum by R. bellii. In conclusion, host-seeking behavior differed between the two tick species, as well as between stages of the same species. A greater restriction of A. dubitatum ticks to the soil was observed, while larvae and nymphs of A. sculptum dispersed higher in the vegetation. The behavior presented by A. sculptum provides greater opportunities for contact with the hosts, while A. dubitatum depends more on an active search for a host, the hunter behavior. Taken together, these observations show that a human being crossing an area infested with A. sculptum and A. dubitatum ticks will have almost exclusive contact with A. sculptum larvae and/or nymphs. Humans in a stationary position (sitting, lying or immobile) are exposed to both tick species, but they are more attractive to adults and mainly nymphs of A. sculptum compared to the corresponding stages of the tick A. dubitatum. The negative effect of A. sculptum on A. dubitatum infection by R. bellii deserves further studies.

Rickettsia rickettsii virulence determinants RARP2 and RapL mitigate IFN-ß signaling in primary human dermal microvascular endothelial cells.

We compared the growth characteristics of a virulent Rickettsia rickettsii strain (Sheila Smith) to an attenuated R. rickettsii stain (Iowa) and a non-pathogenic species (R. montanensis) in primary human dermal microvascular endothelial cells (HDMEC). All replicated in Vero cells, however, only the Sheila Smith strain productively replicated in HDMECs. The Iowa strain showed minimal replication over a 24-h period, while R. montanensis lost viability and induced lysis of the HDMECs via a rapid programmed cell death response. Both the virulent and attenuated R. rickettsii strains, but not R. montanensis, induced an interferon-1 response, although the response was of lesser magnitude and delayed in the Sheila Smith strain. IFN-ß secretion correlated with increased host cell lysis, and treatment with anti-IFNAR2 antibody decreased lysis from Iowa-infected but not Sheila Smith-infected cells. Both Sheila Smith- and Iowa-infected cells eventually lysed, although the response from Sheila Smith was delayed and showed characteristics of apoptosis. We, therefore, examined whether reconstitution of the Iowa strain with two recently described putative virulence determinants might enhance survival of Iowa within HDMECs. Reconstitution with RARP2, which is inhibitory to anterograde trafficking through the Golgi apparatus, reduced IFN-ß secretion but had no effect on cell lysis. RapL, which proteolytically processes surface exposed autotransporters and enhances replication of Iowa in Guinea pigs, suppressed both IFN-ß production and host cell lysis. These findings suggest distinct mechanisms by which virulent spotted fever group rickettsiae may enhance intracellular survival and replication.IMPORTANCEWe examined a naturally occurring non-pathogenic rickettsial species, R. montanensis, a laboratory-attenuated R. rickettsii strain (Iowa), and a fully virulent R. rickettsii strain (Sheila Smith) for growth in human dermal microvascular endothelial cells. The two avirulent strains replicated poorly or not at all. Only the virulent Sheila Smith strain replicated. IFN-ß production correlated with the inhibition of R. rickettsii Iowa. Reconstitution of Iowa with either of two recently described putative virulence determinants altered the IFN-ß response. A rickettsial ankyrin repeat protein, RARP2, disrupts the trans-Golgi network and inhibits IFN-ß secretion. An autotransporter peptidase, RapL, restores proteolytic maturation of outer membrane autotransporters and diminishes the IFN-ß response to enhance cell survival and permit replication of the recombinant strain. These studies point the way toward discovery of mechanisms for innate immune response avoidance by virulent rickettsia.

An impedimetric immunosensor for diagnosis of Brazilian spotted fever in blood plasma.

Brazilian spotted fever (BSF) is a serious disease of medical importance due to its rapid evolution and high lethality. The effectiveness of the treatment mainly depends on the rapid diagnosis, which is currently performed by indirect immunofluorescence and PCR tests, which require high costs and laboratory structure. In order to propose an alternative methodology, we sought to develop an impedimetric immunosensor (IM) based on the immobilization of specific IgY antibodies for IgG anti Rickettsia rickettsii, using blood plasma from capybara (Hydrochoerus hydrochaeris), for characterization, validation and applications of the ready IM. IM selectivity was observed when comparing capybara reagent IgG (IgGcr) readings with non-reagent IgG (IgGnr). A reagent IgG calibration curve was obtained, from which the limits of detection (LOD) and quantification (LOQ) of 1.3 ng mL-1 and 4.4 ng mL-1 were calculated, respectively. The accuracy tests showed that different concentrations of IgGcr showed a maximum deviation of 20.0%, with CI between 90.00% and 95.00%. Intermediate precision tests showed a relative standard deviation of 2.09% for researcher 1 and 2.61% for researcher 2, and the F test showed no significant difference between the recovery values found between the two analysts, since Fcal 1.56 < 5.05 and P-value 0.48 > 0, 05. Therefore, an impedimetric immunosensor was developed to detect anti BSF IgG in capybara blood plasma, which greatly contributes to the improvement of diagnostic tests, cost reduction and ease of execution.

The anal pore route is efficient to infect Amblyomma spp. ticks with Rickettsia rickettsii and allows the assessment of the role played by infection control targets.

Adult Amblyomma sculptum and Amblyomma aureolatum ticks are partially refractory to Rickettsia rickettsii when fed on infected hosts, hindering the functional characterization of potentially protective targets in the bacterial acquisition. In the current study, we used the anal pore route to infect adult A. sculptum and A. aureolatum ticks with R. rickettsii and to assess the effects of the knockdown of microplusin in infection control. The anal pore route was efficient to infect both species, resulting in a prevalence of around 100% of infected ticks. Higher loads of R. rickettsii were detected in microplusin-silenced A. aureolatum in relation to the control, as previously obtained when microplusin-silenced ticks were fed on R. rickettsii-infected rabbits. This is the first report showing R. rickettsii infection through the anal pore in Amblyomma ticks, highlighting this route as a powerful tool to assess the role played by additional targets in the control of pathogens.

First molecular evidence of Wolbachia occurrence in Amblyomma sculptum (Acari: Ixodidae).

As the main vector for the bacterium Rickettsia rickettsii in Brazil, the tick Amblyomma sculptum is a parasite of great public health importance in this country. Wolbachia is an endosymbiont bacterium highly widespread among invertebrates and because of its impact on its hosts' biology, form a powerful alternative for pests and disease control. The aim of this study was to investigate the occurrence of this bacterium in A. sculptum. For this, 187 adult ticks collected in two municipalities in the interior of the state of São Paulo, Brazil, were analyzed using molecular techniques and bioinformatics tools. A total of 15 ticks were positive for the presence of Wolbachia. Phylogenetic analysis on the 16S rRNA gene indicated that the Wolbachia DNA sequences obtained in this investigation belonged to different clades, probably in supergroups B and F. This was the first study to report the occurrence of Wolbachia in A. sculptum and it enriches knowledge about the susceptibility of ticks to this bacterium. Now that we know that Wolbachia can be found in A. sculptum, the objective for a next study must be to investigate Wolbachia's possible origin in this tick.

Genetic sequencing of a 1944 Rocky Mountain spotted fever vaccine.

Rocky Mountain spotted fever (RMSF) is a rapidly progressive and often fatal tick-borne disease caused by Rickettsia rickettsii. Its discovery and characterization by Howard Ricketts has been hailed as a remarkable historical example of detection and control of an emerging infectious disease, and subsequently led to the establishment of the Rocky Mountain Laboratories (RML). Here, we examined an unopened bottle of a vaccine, labeled as containing RMSF inactivated by phenol-formalin of infected ticks, developed prior to 1944 at RML by DNA analysis using Illumina high throughput sequencing technology. We found that it contains DNA from the Rocky Mountain wood tick (Dermacentor andersoni), the vector of RMSF, the complete genome of Rickettsia rickettsii, the pathogen of RMSF, as well as the complete genome of Coxiella burnetii, the pathogen of Q-fever. In addition to genomic reads of Rickettsia rickettsii and Coxiella burnetii, smaller percentages of the reads are from Rickettsia rhipicephali and Arsenophonus nasoniae, suggesting that the infected ticks used to prepare the vaccine carried more than one pathogen. Together, these findings suggest that this early vaccine was likely a bivalent vaccine for RMSF and Q-fever. This study is the among the first molecular level examinations of an historically important vaccine.

Evaluation of a mimotope of the Rickettsia outer membrane protein A (OmpA) as an antigen in enzyme-linked immunosorbent assay to detect rickettsiosis in capybaras (Hydrochoerus hydrochaeris), horses (Equus caballus), and opossums (Didelphis sp.).

Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever, which is an important tick-borne zoonosis and, in Brazil, it causes Brazilian spotted fever, which has high lethality rate. This study aimed to evaluate a synthetic peptide corresponding to a segment of the outer membrane protein A (OmpA) as an antigen in a serological test for the diagnosis of rickettsial infections. The amino acid sequence of the peptide was selected by predicting B cell epitopes using B Cell Epitope Prediction (Immune Epitope Database and Analysis Resource) and Epitopia and OmpA sequences of Rickettsia rickettsii strain 'Brazil' and Rickettsia parkeri strains 'Maculatum 20' and 'Portsmouth'. A peptide with amino acid sequence common to both Rickettsia species was synthesized and arbitrarily named OmpA-pLMC. To evaluate this peptide in enzyme-linked immunosorbent assay (ELISA), serum samples of capybara (Hydrochoerus hydrochaeris), horse (Equus caballus), and opossum (Didelphis albiventris) that had been previously tested by indirect immunofluorescence assay (IFA) for rickettsial infection were separated into IFA-positive and IFA-negative groups and used in the assay. There were no significant differences in ELISA optical density (OD) values between IFA-positive and IFA-negative groups with horse samples. The mean OD values were significantly higher in the IFA-positive capybara serum samples (IFA-pos vs. IFA-neg = 2.389 ± 0.761 vs. 1.760 ± 0.840). However, receiver operating characteristic (ROC) curve analysis did not show significant diagnostic parameters. On the other hand, 12 out of 14 (85.7%) opossum samples of the IFA-positive group showed reactivity in ELISA, and this was significantly higher than of the IFA-negative group (0.7196 ± 0.440 vs. 0.2318 ± 0.098, respectively; 85.7% sensitivity, 100% specificity). Therefore, our results show that OmpA-pLMC has a potential to be used in immunodiagnostic assays to detect spotted fever group rickettsial infections.

Clinical manifestations of Rickettsia rickettsii in a familial outbreak in Panama.

We report an isolated outbreak of Rickettsia rickettsii in the Ngäbe-Buglé indigenous region, located 750 m (tropical wet) above sea level, in a jungle and mountainous area of Western Panama. Seven members of a family were infected simultaneously, resulting in four deaths. Family outbreaks have been previously described and are responsible for 4-8% of the cases described [1-4]. The simultaneous onset of symptoms in the affected population group is extremely unusual [1,5], but it should not dissuade the clinician from considering the possibility of Rickettsia rickettsii infection.

Fatal Brazilian spotted fever in a healthy military man during field training in Rio de Janeiro city, southeastern Brazil.

Brazilian spotted fever, a zoonotic disease transmitted by ticks, is caused by Rickettsia rickettsii. We report a fulminant case of this zoonosis in a healthy 46-year-old military man in the urban region of Rio de Janeiro city, in October, 2021. Ticks and capybaras (Amblyomma sculptum, Hydrochoerus hydrochaeris, respectively) were identified in the military fields, pointing to the participation of this large synanthropic rodent, recognized as an efficient amplifier host of Rickettsia rickettsii in Brazil. As the military population is considered a risk group for spotted fever, it is necessary to alert health professionals to the importance of the early detection of the disease and its adequate management, mainly in populations that are particularly at risk of exposure to ticks, in order to avoid fatal outcomes.

Detection of Rickettsia montanensis in Dermacentor variabilis in Northern Wisconsin.

Background: Spotted fever rickettsiosis is caused by a group of closely related bacteria that includes Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever. Recently, Rickettsia montanensis has been reported to cause clinical and subclinical symptoms in both humans and animal models. Materials and Methods: In this study, we collected ticks in Ashland County, located in northern Wisconsin, and tested 16 ticks identified as Dermacentor variabilis for the presence of rickettsial bacteria using PCR techniques. Results: Four positive results identified using gel electrophoresis were then sequenced to determine the rickettsiae species. Of the samples sequenced, three matched for R. montanensis (∼19% of the 16 ticks tested). Conclusion: In this study, we report the presence and prevalence of R. montanensis in northern Wisconsin.

Procedure for spotted fever group Rickettsia isolation from limited clinical blood specimens.

BACKGROUND: Current isolation techniques for spotted fever group Rickettsia from clinical samples are laborious and are limited to tissue, blood and blood derivatives with volumes ideally greater than 1 mL. We validated the use of simplified methodologies for spotted fever group Rickettsia culture isolation that overcome sample volume limitations and provide utility in clinical diagnostics and research studies. METHODOLOGY/PRINCIPAL FINDINGS: A modified cell culture method is evaluated for the isolation of Rickettsia ssp. from human diagnostic samples. Culture sampling method, culture platform, and growth phase analysis were evaluated to determine best practices for optimal culture isolation conditions. Rickettsial isolates (R. conorii, R. rickettsii, and R. parkeri) were grown in Vero E6 cells over a course of 5 to 7 days at low inoculum treatments (~40 bacterial copies) to standardize the sampling strategy at a copy number reflective of the bacteremia in acute diagnostic samples. This methodology was verified using small volumes (50 µL) of 25 unprocessed clinical whole blood, plasma, and serum samples from acute samples of patients suspected of having Rocky Mountain Spotted Fever, of which 10 were previously confirmed positive via the PanR8 qPCR assay, 13 had no detectable Rickettsia DNA by the PanR8 qPCR assay, and 2 were not previously tested; these samples resulted in the cultivation of 7 new R. rickettsii isolates. CONCLUSIONS/SIGNIFICANCE: We observed that rickettsial isolate growth in culture is reproducibly identified by real-time PCR testing of culture media within 72 hours after inoculation. Additionally, specimen sedimentation prior to isolation to remove red blood cells was found to decrease the amount of total organism available in the inoculum. A small volume culture method was established focusing on comparative qPCR detection rather than bacterial visualization, taking significantly shorter time to detect, and requiring less manipulation compared to traditional clinical isolate culture methods.

Rickettsia-Host-Tick Interactions: Knowledge Advances and Gaps.

Ticks are hematophagous ectoparasites capable of transmitting multiple human pathogens. Environmental changes have supported the expansion of ticks into new geographical areas that have become the epicenters of tick-borne diseases (TBDs). The spotted fever group (SFG) of Rickettsia frequently infects ticks and causes tick-transmitted rickettsioses in areas of endemicity where ixodid ticks support host transmission during blood feeding. Ticks also serve as a reservoir for SFG Rickettsia. Among the members of SFG Rickettsia, R. rickettsii causes Rocky Mountain spotted fever (RMSF), the most lethal TBD in the United States. Cases of RMSF have been reported for over a century in association with several species of ticks in the United States. However, the isolation of R. rickettsii from ticks has decreased, and recent serological and epidemiological studies suggest that novel species of SFG Rickettsia are responsible for the increased number of cases of RMSF-like rickettsioses in the United States. Recent analyses of rickettsial genomes and advances in genetic and molecular studies of Rickettsia provided insights into the biology of Rickettsia with the identification of conserved and unique putative virulence genes involved in the rickettsial life cycle. Thus, understanding Rickettsia-host-tick interactions mediating successful disease transmission and pathogenesis for SFG rickettsiae remains an active area of research. This review summarizes recent advances in understanding how SFG Rickettsia species coopt and manipulate ticks and mammalian hosts to cause rickettsioses, with a particular emphasis on newly described or emerging SFG Rickettsia species.

Increasing Incidence of Spotted Fever Group Rickettsioses in the United States, 2010-2018.

Spotted fever group Rickettsia species are intracellular bacteria transmitted by tick or mite vectors and that cause human diseases referred to as spotted fever group rickettsioses, or spotted fevers. In the United States, the most recognized and commonly reported spotted fevers are Rocky Mountain spotted fever (RMSF) (Rickettsia rickettsii), Rickettsia parkeri rickettsiosis, Pacific Coast tick fever (Rickettsia species 364D), and rickettsialpox (Rickettsia akari). In this study, we summarize and evaluate surveillance data on spotted fever cases reported to the Centers for Disease Control and Prevention (CDC) through the National Notifiable Diseases Surveillance System from 2010 to 2018. During this period, there were 36,632 reported cases of spotted fevers with 95.83% (N = 35,104) reported as meeting the case definition as probable and 4.17% (N = 1528) reported as meeting the case definition as confirmed. The average national incidence of total cases, both probable and confirmed, was 12.77 cases per million persons per year. The highest statewide incidence was in Arkansas, with 256.84 per million per year, whereas the lowest incidence occurred in California, with 0.32 per million per year (note that spotted fevers were not notifiable in Hawaii and Alaska). Cases of spotted fevers were reported more frequently among males by gender, White by race, and non-Hispanic by ethnicity. The incidence of spotted fevers increased significantly from 2010 to 2018, but it is uncertain how many of the reported cases were RMSF and how many developed from more moderate spotted fevers. Improvement of the ability to differentiate between spotted fever group Rickettsia species is needed.

"Leopards do not change their spots:" tick borne disease symptomology case report.

BACKGROUND: Human Monocytic Ehrlichiosis is caused by infection with the bacteria Ehrlichia chaffeensis through the bite of an infected lone star tick (Amblyomma americanum). Patients infected with Human Monocytic Ehrlichiosis often present with symptoms including fever, headache, myalgia, and occasionally a macular rash. The presence of other endemic tick-borne diseases with similar symptoms, such as Rocky Mountain Spotted Fever, complicate the diagnosis of Human Monocytic Ehrlichiosis. CASE PRESENTATION: A patient developed a fever, diffuse myalgia, headache, and a non-productive cough 5 days after a fishing trip in late May in central North Carolina. Over the course of the illness the patient's symptoms worsened, with arthralgia, bilateral lower extremity erythema and edema, and a developing bilateral rash on the palms. With testing that revealed elevated liver enzymes, a potential for recent tick exposure (e.g., fishing trip), presentation during tick season, and the development of a rash, Rocky Mountain Spotted Fever and Human Monocytic Ehrlichiosis were considered. The patient was prescribed a seven-day course of oral doxycycline and cefalexin, which would provide coverage from Rickettsia, Ehrlichia and gram-positive bacteria typically responsible for cellulitis. Many of the patient's symptoms resolved or improved, although the right shoulder remained painful to active movement. The patient was prescribed another seven-day course of doxycycline due to his perceived incomplete response to the first course. Approximately 5 weeks after symptom onset (D0 + 36), the patient followed up with a provider for convalescent testing and counseling. Convalescent Ehrlichia and Rickettsia serological tests were ordered. The acute Ehrlichia serology and acute Rickettsia serology were originally non-reactive with both titers measured at < 1:64. Convalescent serology, ordered 28 days after the acute sample collection, showed a greater than four-fold increase in the Ehrlichia IgG titer (1:256), satisfying clinical and laboratory case definitions for ehrlichiosis. In follow-up, 3 weeks later (D0 + 57), the patient reported that most of his pain had subsided, though he still occasionally got shooting nerve pain when exercising. CONCLUSION: This case of Human Monocytic Ehrlichiosis in North Carolina exemplifies the need for a knowledge of spatial epidemiological patterns and clinical manifestations in the diagnosis of tick-borne diseases.

Fluazuron orally administered to guinea pigs: pharmacokinetic and efficacy against Amblyomma sculptum.

BACKGROUND: Brazilian spotted fever (BSF), the most lethal tick-borne disease in the Western Hemisphere, is caused by the bacterium Rickettsia rickettsii and transmitted by the bite of Amblyomma sculptum. Capybaras are considered primary hosts of this tick and amplifier hosts of R. rickettsii, generating new infected lineages of A. sculptum in BSF-endemic areas. To define a possible treatment regimen for controlling the tick A. sculptum in capybaras, the aim of this study was to establish an effective fluazuron (FLU) dose to control A. sculptum larvae in artificially infested guinea pigs. METHODS: In Study I (pharmacokinetic and pharmacodynamic analysis), 24 guinea pigs were divided into four equal groups: control group (CG; untreated) and treated groups receiving FLU administered by gavage in three doses: G1-1 mg/kg, G2-5 mg/kg and G3-10 mg/kg, once a day for 15 days (d0 to d + 14). Blood samples were collected from the animals of the treated groups before and at d + 1, + 2, + 4, + 7, + 15 and + 21. The guinea pigs were artificially infested at d + 7 with A. sculptum larvae, and specimens were recovered at d + 11 to d + 14 and kept in a climatized chamber for 14 days. In Study II (evaluation of pharmacokinetic parameters), one group of eight animals received FLU administered by gavage in a single dose of 10 mg/kg, and blood samples were collected before and on day 0 (8 h after treatment), + 1, + 4, + 7, + 15, + 21 and + 28 after single FLU administration. FLU was analyzed in plasma samples by high-performance liquid chromatography with ultraviolet detection. RESULTS: FLU plasma concentrations increased quickly, indicating rapid absorption, and decreased slowly. Some larvae from all treated groups exhibited morphological and behavioral changes. FLU interfered in molting, and the efficacy obtained was 100% for all treated groups. CONCLUSIONS: The results offer promising perspectives for the development of a palatable feed cube containing FLU for free-living capybaras to control A. sculptum and also to prevent BSF in areas where capybaras have been shown to play a primary role.

Regulator of Actin-Based Motility (RoaM) Downregulates Actin Tail Formation by Rickettsia rickettsii and Is Negatively Selected in Mammalian Cell Culture.

The etiological agent of Rocky Mountain spotted fever, Rickettsia rickettsii, is an obligately intracellular pathogen that induces the polymerization of actin filaments to propel the bacterium through the cytoplasm and spread to new host cells. Cell-to-cell spread via actin-based motility is considered a key virulence determinant for spotted fever group rickettsiae, as interruption of sca2, the gene directly responsible for actin polymerization, has been shown to reduce fever in guinea pigs. However, little is known about how, or if, motility is regulated by the bacterium itself. We isolated a hyperspreading variant of R. rickettsii Sheila Smith that produces actin tails at an increased rate. A1G_06520 (roaM [regulator of actin-based motility]) was identified as a negative regulator of actin tail formation. Disruption of RoaM significantly increased the number of actin tails compared to the wild-type strain but did not increase virulence in guinea pigs; however, overexpression of RoaM dramatically decreased the presence of actin tails and moderated fever response. Localization experiments suggest that RoaM is not secreted, while reverse transcription-quantitative PCR (RT-qPCR) data show that various levels of RoaM do not significantly affect the expression of the known rickettsial actin-regulating proteins sca2, sca4, and rickA. Taken together, the data suggest a previously unrecognized level of regulation of actin-based motility in spotted fever group rickettsiae. Although this gene is intact in many isolates of spotted fever, transitional, and ancestral group Rickettsia spp., it is often ablated in highly passaged laboratory strains. Serial passage experiments revealed strong negative selection of roaM in Vero 76 cells. IMPORTANCE The mechanism of actin-based motility of spotted fever group Rickettsia has been studied extensively, but here, we provide genetic evidence that motility is a regulated process in R. rickettsii. The findings also suggest that serial passage of rickettsial strains in cell culture may cause the bacteria to lose essential genes that are no longer conserved under natural selective pressure. These findings are likely relevant to the interpretation of studies concerning virulence determinants of rickettsiae.

The Retropepsin-Type Protease APRc as a Novel Ig-Binding Protein and Moonlighting Immune Evasion Factor of Rickettsia.

Rickettsiae are obligate intracellular Gram-negative bacteria transmitted by arthropod vectors. Despite their reduced genomes, the function(s) of the majority of rickettsial proteins remains to be uncovered. APRc is a highly conserved retropepsin-type protease, suggested to act as a modulator of other rickettsial surface proteins with a role in adhesion/invasion. However, APRc's function(s) in bacterial pathogenesis and virulence remains unknown. This study demonstrates that APRc targets host serum components, combining nonimmune immunoglobulin (Ig)-binding activity with resistance to complement-mediated killing. We confirmed nonimmune human IgG binding in extracts of different rickettsial species and intact bacteria. Our results revealed that the soluble domain of APRc is capable of binding to human (h), mouse, and rabbit IgG and different classes of human Ig (IgG, IgM, and IgA) in a concentration-dependent manner. APRc-hIgG interaction was confirmed with total hIgG and normal human serum. APRc-hIgG displayed a binding affinity in the micromolar range. We provided evidence of interaction preferentially through the Fab region and confirmed that binding is independent of catalytic activity. Mapping the APRc region responsible for binding revealed the segment between amino acids 157 and 166 as one of the interacting regions. Furthermore, we demonstrated that expression of the full-length protease in Escherichia coli is sufficient to promote resistance to complement-mediated killing and that interaction with IgG contributes to serum resistance. Our findings position APRc as a novel Ig-binding protein and a novel moonlighting immune evasion factor of Rickettsia, contributing to the arsenal of virulence factors utilized by these intracellular pathogens to aid in host colonization. IMPORTANCE Many Rickettsia organisms are pathogenic to humans, causing severe infections, like Rocky Mountain spotted fever and Mediterranean spotted fever. However, immune evasion mechanisms and pathogenicity determinants in rickettsiae are far from being resolved. We provide evidence that the highly conserved rickettsial retropepsin-type protease APRc displays nonimmune immunoglobulin (Ig)-binding activity and participates in serum resistance. APRc emerges then as a novel Ig-binding protein from Gram-negative bacteria and the first to be identified in Rickettsia. Bacterial surface proteins capable of Ig binding are known to be multifunctional and key players in immune evasion. We demonstrate that APRc is also a novel moonlighting protein, exhibiting different actions on serum components and acting as a novel evasin. This work strengthens APRc as a virulence factor in Rickettsia and its significance as a potential therapeutic target. Our findings significantly contribute to a deeper understanding of the virulence strategies used by intracellular pathogens to subvert host immune responses.

Nitric Oxide Inhibition of Rickettsia rickettsii.

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is an enzootic, obligate, intracellular bacterial pathogen. Nitric oxide (NO) synthesized by the inducible NO synthase (iNOS) is a potent antimicrobial component of innate immunity and has been implicated in the control of virulent Rickettsia spp. in diverse cell types. In this study, we examined the antibacterial role of NO on R. rickettsii. Our results indicate that NO challenge dramatically reduces R. rickettsii adhesion through the disruption of bacterial energetics. Additionally, NO-treated R. rickettsii cells were unable to synthesize protein or replicate in permissive cells. Activated, NO-producing macrophages restricted R. rickettsii infections, but inhibition of iNOS ablated the inhibition of bacterial growth. These data indicate that NO is a potent antirickettsial effector of innate immunity that targets energy generation in these pathogenic bacteria to prevent growth and subversion of infected host cells.

Small mammals, ticks and rickettsiae in natural and human-modified landscapes: Diversity and occurrence of Brazilian spotted fever in Brazil.

We studied communities of small mammals and their ticks in endemic (E) and non-endemic (NE) areas for Brazilian spotted fever (BSF), aiming to infer if diversity parameters of parasites and hosts could be related to occurrence and prevalence of rickettsial infection, especially Rickettsia rickettsii. We compared E and NE areas in human-modified landscapes (HMLs) and natural areas (BIO) with no report of BSF cases. Composition and equitability were important components of diversity explaining differences among areas. The marsupial Didelphis albiventris was dominant in HMLs, but not in natural areas, and this opossum was the main host for the tick Amblyomma sculptum, principal vector of R. rickettsii, especially in E areas. Communities of ticks were dominated by A. sculptum, followed by Amblyomma dubitatum in E areas. In NE areas, this dominance was inverted, with more A. dubitatum than A. sculptum infesting small mammals, but the numbers of ticks were much lower than in E areas. Composition and abundance of ticks in natural areas were very dissimilar from HMLs, with the lowest tick burdens. Didelphis albiventris in E areas presented higher seroprevalence and endpoint titres against R. rickettsii than in other areas. At least three Rickettsia species, non-pathogenic to humans, were detected in natural areas (Rickettsia bellii, Rickettsia amblyommatis and 'Candidatus Rickettsia andeanae'), and only one non-pathogenic species in HMLs (R. bellii). Our results suggest that higher diversity of ticks, hosts and rickettsiae could be relevant factors in buffering the effect in BSF occurrence. Particularly for D. albiventris, its importance has to be quantified in further studies considering the epidemiological scenario of BSF.