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Rocky mountain spotted fever (RMSF) causes significant illness and death in children. Although historically rare in California, USA, RMSF is endemic in areas of northern Mexico that border California. We describe 7 children with RMSF who were hospitalized at a tertiary pediatric referral center in California during 2017-2023. Five children had recent travel to Mexico with presumptive exposure, but 2 children did not report any travel outside of California. In all 7 patients, Rickettsia rickettsii DNA was detected by plasma microbial cell-free next-generation sequencing, which may be a useful diagnostic modality for RMSF, especially early in the course of illness, when standard diagnostic tests for RMSF are of limited sensitivity. A high index of suspicion and awareness of local epidemiologic trends remain most critical to recognizing the clinical syndrome of RMSF and initiating appropriate antimicrobial therapy in a timely fashion.

Tick-borne diseases are important for animal and human health, because they can cause death if not diagnosed and treated early. Canine monocytic ehrlichiosis (CME) can cause high morbidity in dog populations. Rocky Mountain Spotted Fever (RMSF) is among the most virulent infectious in humans; dogs are also susceptible to infection. The aims of this study were to evaluate the presence of Ehrlichia canis and Rickettsia spp. infections in domestic dogs, and to identify tick species parasitizing dogs among urban areas of two municipalities (Sobral and Alcântaras) in the Ceará State, Northeastern Brazil. A total of 208 domiciled dogs was sampled. After clinical evaluation, blood samples and ticks were collected and submitted to Real-Time Polymerase Chain Reaction (RT-PCR) targeting E. canis DNA. Serum samples were screened by Indirect Immunofluorescence Assays (IFA) for antibodies against different strains of Rickettsia spp. previously recognized in Brazil. The results of this study indicate the molecular detection of E. canis in the state of Ceará, Brazil, where the proportion of canine infection in Sobral (9.9%) was higher than in Alcântaras (5.6%). Rhipicephalus sanguineus sensu lato was the prevalent tick species infesting the dogs in both municipalities (43.5 and 53.3%, respectively). Our serological results indicate that dogs of the study area were at low risk of exposure to these tick-borne Rickettsia spp. of the spotted fever group. Our study offers epidemiological data of these diseases to better understanding Rickettsiales epidemic and enzootic cycles in the Brazilian semiarid region, improving prevention and control measures., Competing Interests: Declaration of competing interest There were no conflicting interests that could have influenced the conduction of this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The incidence of spotted fever group (SFG) rickettsioses in the United States has tripled since 2010. Rocky Mountain spotted fever, the most severe SFG rickettsiosis, is caused by Rickettsia rickettsii. The lack of species-specific confirmatory testing obfuscates the relative contribution of R. rickettsii and other SFG Rickettsia to this increase. We report a newly recognized rickettsial pathogen, Rickettsia sp. CA6269, as the cause of severe Rocky Mountain spotted fever-like illness in 2 case-patients residing in northern California. Multilocus sequence typing supported the recognition of this pathogen as a novel Rickettsia genotype most closely related to R. rickettsii. Cross-reactivity observed for an established molecular diagnostic test indicated that Rickettsia sp. CA6269 might be misidentified as R. rickettsii. We developed a Rickettsia sp. CA6269-specific real-time PCR to help resolve this diagnostic challenge and better characterize the spectrum of clinical disease and ecologic epidemiology of this pathogen.

Rickettsioses have been reported in parts of Mexico since the last century, with Rocky Mountain spotted fever (RMSF) being one of the most prevalent in northern states. Unfortunately, fatality rates for RMSF in Mexico are higher than in other countries, like the USA. The reason for this difference in fatality rates is currently unknown and could be associated with a genotype of the bacterium, but no comparative molecular typing has been conducted in Mexico to date. The purpose of this study was to analyze 47 RMSF samples with different outcomes from several states in northern Mexico to know the genetic variability of Rickettsia rickettsii, as well as to reconstruct its phylogeny, for which the following intergenic regions were sequenced: RR0155-rpmB, cspA-ksgA, RR1240-tlc5, and Spo0J-abc T1, as well as the following partial genes: ompA, ompB, and gltA. We identified 8 genotypes with different distribution and prevalence among the states analyzed, as well as a different association with case outcome; these genotypes were clustered in 2 clades and 5 lineages were revealed, some of them probably exclusive from Mexico., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

In several urban and peri‑urban 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 m 2 at each site and at three seasons. Within the same municipality, the distance and speed of A. sculptum nymphs moving towards the CO 2 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, CO 2 traps collected a greater proportion of nymphs and adults of A. sculptum ticks. In the case of A. dubitatum, the CO 2 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., Competing Interests: Declaration of competing interest The authors declared that no competing interests exist., (Copyright © 2024. Published by Elsevier GmbH.)

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-β 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-β 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-β 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-β production correlated with the inhibition of R. rickettsii Iowa. Reconstitution of Iowa with either of two recently described putative virulence determinants altered the IFN-β response. A rickettsial ankyrin repeat protein, RARP2, disrupts the trans- Golgi network and inhibits IFN-β secretion. An autotransporter peptidase, RapL, restores proteolytic maturation of outer membrane autotransporters and diminishes the IFN-β 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., Competing Interests: The authors declare no conflict of interest.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nassar, Pavanelo, Labruna, Daffre, Esteves and Fogaça.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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., (Copyright © 2023. Published by Elsevier Ltd.)

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., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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&#95;06520 ( roaM [ r egulator o f a ctin-based m otility]) 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.

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.

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.

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., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Rickettsia rickettsii , the etiological agent of Rocky Mountain spotted fever (RMSF), a life-threatening tick-borne disease that affects humans and various animal species, has been recognized in medicine and science for more than 100 years. Isolate-dependent differences in virulence of R. rickettsii have been documented for many decades; nonetheless, the specific genetic and phenotypic factors responsible for these differences have not been characterized. Using in vivo and in vitro methods, we identified multiple phenotypic differences among six geographically distinct isolates of R. rickettsii , representing isolates from the United States, Costa Rica, and Brazil. Aggregate phenotypic data, derived from growth in Vero E6 cells and from clinical and pathological characteristics following infection of male guinea pigs ( Cavia porcellus ), allowed separation of these isolates into three categories: nonvirulent (Iowa), mildly virulent (Sawtooth and Gila), and highly virulent (Sheila Smith T , Costa Rica, and Taiaçu). Transcriptional profiles of 11 recognized or putative virulence factors confirmed the isolate-dependent differences between mildly and highly virulent isolates. These data corroborate previous qualitative assessments of strain virulence and suggest further that a critical and previously underappreciated balance between bacterial growth and host immune response could leverage strain pathogenicity. Also, this work provides insight into isolate-specific microbiological factors that contribute to the outcome of RMSF and confirms the hypothesis that distinct rickettsial isolates also differ phenotypically, which could influence the severity of disease in vertebrate hosts.

Background and Objectives: Rocky Mountain spotted fever (RMSF) is a rapidly progressive and potentially fatal tickborne disease caused by Rickettsia rickettsii. Despite current recommendations and information on the severity of RMSF, studies show that delayed recognition and treatment continues to occur., Methods: A literature search was performed on cases published in English between 1990-2017. The frequencies for demographic, clinical, and treatment variables was calculated., Results: A total of 340 cases from 34 articles were included. Data on rash were available for 322 patients, and 261 (80%) noted rash. Mortality was 4% (2) in those who received doxycycline within the first five days of illness, and 35% (18) when treatment was delayed beyond Day five. Twenty-four (16%) reported chronic sequelae, including speech impairment (7, 5%) and ataxia (5, 3%)., Interpretation and Conclusion: These data highlight the importance of early treatment, and add to our understanding of long-term sequelae. Early recognition by providers will facilitate appropriate treatment and reduction in morbidity and mortality., Competing Interests: None

Background: Since 2000, the reported prevalence of tick-borne spotted fever rickettsiosis has increased considerably. We compared the level of antibody reactivity among healthy blood donors from 2 widely separated regions of the United States and evaluated the impact of antibody prevalence on public health surveillance in one of these regions., Methods: Donor serum samples were evaluated by indirect immunofluorescence antibody assay to identify immunoglobulin G (IgG) antibodies reactive with Rickettsia rickettsii. The Georgia Department of Public Health (GDPH) analyzed characteristics of cases from 2016 surveillance data to evaluate the utility of laboratory surveillance for case assessment., Results: Of the Georgia donors (n = 1493), 11.1% demonstrated antibody titers reactive with R. rickettsii at titers ≥64, whereas 6.3% of donors from Oregon and Washington (n = 1511) were seropositive. Most seropositive donors had a titer of 64; only 3.1% (n = 93) of all donors had titers ≥128. During 2016, GDPH interviewed 243 seropositive case patients; only 28% (n = 69) met inclusion criteria in the national case definition for spotted fever rickettsiosis., Conclusions: These findings suggest that a single IgG antibody titer is an unreliable measure of diagnosis and could inaccurately affect surveillance estimates that define magnitude and clinical characteristics of Rocky Mountain spotted fever and other spotted fever rickettsioses., (Published by Oxford University Press for the Infectious Diseases Society of America 2019.)

Brazilian spotted fever (BSF), caused by Rickettsia rickettsii, is the most lethal tick-borne disease in the western hemisphere. In Brazil, Amblyomma sculptum ticks are the main vector. Capybaras (Hydrochoerus hydrochaeris), the largest living rodents of the world (adults weighing up to 100 Kg), have been recognized as amplifying hosts of R. rickettsii for A. sculptum in BSF-endemic areas; i.e., once primarily infected, capybaras develop bacteremia for a few days, when feeding ticks acquire rickettsial infection. We conducted experimental infections of five capybaras with an A. sculptum-derived strain of R. rickettsii and performed clinical and bacteremia evaluation during primary and subsequent infections. Bacteremia was detected in all capybaras during primary infection, but not in subsequent infections. All animals seroconverted to R. rickettsii (titres range: 64-32,768), and remained seropositive throughout the study. Primary infection resulted in clinical spotted fever illness in four capybaras, of which two had a fatal outcome. Subsequent infections in seropositive capybaras resulted in no clinical signs. Capybaras developed a sustained immune response that prevented a second bacteremia. This condition may imply a high reproduction rate of capybaras in BSF-endemic areas, in order to continuously generate capybaras susceptible to bacteremia during primary infection.

The bacterium Rickettsia rickettsii is the agent of Brazilian spotted fever (BSF), a highly fatal disease that is transmitted in Brazil mainly by the tick Amblyomma sculptum Berlese, which uses capybaras (Hydrochoerus hydrochaeris Linnaeus) as major hosts. In 2015, we captured nine capybaras in a BSF-endemic area of southeastern Brazil. From each capybara, we collected blood sera that were tested through the immunofluorescence assay using Rickettsia spp. antigens, and A. sculptum ticks, processed for isolation of R. rickettsii through guinea pig inoculation. All capybaras (100%) were seroreactive to Rickettsia spp., with highest titers to R. rickettsii. A total of 166 A. sculptum ticks were macerated and inoculated into nine guinea pigs, from which only one presented high fever and seroconverted to R. rickettsii. Blood from this febrile animal was inoculated into additional guinea pigs (guinea pig passages), which also became febrile and seroconverted, or when euthanized during the fever period, their internal organs (spleen, lung) were shown to contain R. rickettsii DNA. The present rickettsial isolate has been maintained cryopreserved as infected guinea pig organs. There was at least one R. rickettsii-infected tick among the 166 macerated ticks, giving a minimal infection rate of 0.6% (1/166). This infection rate is within the range of previous studies, which reported that only 0.05% to at most 1.28% A. sculptum ticks were infected by R. rickettsii in other BSF-endemic areas. These low infection rates support the low incidence of BSF, despite of A. sculptum being the most frequent human-biting tick in southeastern Brazil., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Dynamics of infection by Bartonella and Rickettsia species, which are epidemiologically associated in dogs, have not been explored in a controlled setting., Objectives: Describe an outbreak investigation of occult Bartonella spp. infection among a group of dogs, discovered after experimentally induced Rickettsia rickettsii (Rr) infection., Animals: Six apparently healthy purpose-bred Beagles obtained from a commercial vendor., Methods: Retrospective and prospective study. Dogs were serially tested for Bartonella spp. and Rr using serology, culture, and PCR, over 3 study phases: 3 months before inoculation with Rr (retrospective), 6 weeks after inoculation with Rr (retrospective), and 8 months of follow-up (prospective)., Results: Before Rr infection, 1 dog was Bartonella henselae (Bh) immunofluorescent antibody assay (IFA) seroreactive and 1 was Rickettsia spp. IFA seroreactive. After inoculation with Rr, all dogs developed mild Rocky Mountain spotted fever compatible with low-dose Rr infection, seroconverted to Rickettsia spp. within 4-11 days, and recovered within 1 week. When 1 dog developed ear tip vasculitis with intra-lesional Bh, an investigation of Bartonella spp. infection was undertaken. All dogs had seroconverted to 1-3 Bartonella spp. between 7 and 18 days after Rr inoculation. Between 4 and 8 months after Rr inoculation, Bh DNA was amplified from multiple tissues from 2 dogs, and Bartonella vinsonii subsp. berkhoffii (Bvb) DNA was amplified from 4 of 5 dogs' oral swabs., Conclusions and Clinical Importance: Vector-borne disease exposure was demonstrated in research dogs from a commercial vendor. Despite limitations, our results support the possibilities of recrudescence of chronic subclinical Bartonella spp. infection after Rr infection and horizontal direct-contact transmission between dogs., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Rocky Mountain spotted fever (RMSF), caused by Rickettsia rickettsii , is a severe tick-borne infection endemic to the Americas. Oral doxycycline is effective, but during severe life-threatening disease, intravenous therapy is recommended. Unfortunately, intravenous formulations of doxycycline are not always available. Therefore, we aimed to determine the susceptibility of R. rickettsii to an alternative parenteral agent, tigecycline, in vitro and in vivo. To determine the minimum inhibitory concentration of tigecycline, R. rickettsii -inoculated Vero cells were incubated with medium containing tigecycline. At various time points, monolayers were collected and R. rickettsii was quantified via real-time polymerase chain reaction (PCR). The growth of R. rickettsii was inhibited in the presence of ≥ 0.5 µg/mL of tigecycline. To determine the effectiveness of tigecycline in vivo, guinea pigs were inoculated with R. rickettsii. Five days after inoculation, they were treated twice daily with subcutaneous tigecycline 3.75 mg/kg or subcutaneous doxycycline 5 mg/kg. Treated animals improved, whereas untreated controls remained ill. Tissues were collected for quantitative PCR-determined bacterial loads on day 8. Median bacterial loads in the tigecycline group were less than those in untreated animals: liver (0 versus 2.9 × 10 4 copies/mg), lung (0 versus 8.3 × 10 3 copies/mg), skin (2.6 × 10 2 versus 2.2 × 10 5 copies/mg), spleen (0 versus 1.3 × 10 4 copies/mg), and testes (0 versus 1.0 × 10 5 copies/mg, respectively). There were no significant differences in the bacterial loads between doxycycline-treated versus tigecycline-treated guinea pigs. These data indicate that tigecycline is effective against R. rickettsii in cell culture and in an animal model of RMSF.

As a result of migrations and globalization, people may face a possible increase in the incidence of central nervous system rickettsial infections (CNS R). These diseases, caused by Rickettsia species and transmitted to humans by arthropod bites, are putatively lethal. However, the diagnosis of CNS R is challenging and often delayed due to their nonspecific clinical presentation and the strict intracellular nature of rickettsiae. Furthermore, transfer of rickettsiae to the brain parenchyma is not yet understood. The aim of this review is to analyze and summarize the features and correlated findings of CNS R in order to focus attention on these intriguing but frequently neglected illnesses. We also incorporated data on CNS infections caused by Rickettsia-related microorganisms., Competing Interests: The authors have declared that no competing interests exist.

This report describes a new lethal case of Rickettsia rickettsii infection in a child from Northwestern Colombia, after ten years of the last outbreaks in the same region. Colombian public-health authorities should consider to include this severe rickettsiosis in the compulsory-reporting diseases, with the aim of knowing its burden in the country., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Amblyomma sculptum is an important vector of Rickettsia rickettsii , causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host's blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E 2 (PGE 2 ) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE 2 , a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE 2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE 2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii -DC interactions both in vitro and in vivo .

The Brazilian state of Goiás, untouched by spotted fever (SF) until 2012, has subsequently reported cases of the disease in several regions. This study aimed to survey the diversity of potential vectors and rickettsia in areas of Goiás under environmental surveillance or case investigation for SF. Collected specimens were assayed with molecular biology technology using DNA extraction, amplification and sequencing of fragments of the genes gltA, ompA, ompB and sca4 to detect rickettsia in ticks and fleas. Amplification of cytochrome oxidase subunit II and 16S rRNA was performed to assist tick identification. Rickettsia felis (Rickettsiales: Rickettsiaceae) was found in Ctenocephalides felis (Bouché, 1835) (Siphonaptera: Pulicidae). Rickettsia bellii was found in Amblyomma rotundatum Koch, 1844 (Ixodida: Ixodidae) and in Amblyomma cajennense sensu lato. Rickettsia sp. strain NOD was found in Amblyomma nodosum Neumann, 1899. Of the Amblyomma cajennense complex, Amblyomma sculptum Berlese, 1888 was confirmed in the northern, northeast, midwest and southeast regions of Goiás, whereas Amblyomma cajennense sensu stricto (Fabricius, 1787) was found only in the northern region of the state. Amblyomma dubitatum Neumann, 1899 associated with a species of the A. cajennense complex was the most common epidemiological finding, although Rickettsia rickettsii was not detected. This is the first report of Rickettsia sp. strain NOD in Goiás., (© 2018 The Royal Entomological Society.)

Although Dermacentor spp. ticks are considered the primary vectors of Rickettsia rickettsii in the United States, other North American tick species are also capable of transmitting the agent, including the lone star tick-Amblyomma americanum. The lone star tick is an aggressive human-biting tick abundant in the South, Central, and Mid-Atlantic United States, which has been shown to be a competent vector of R. rickettsii in laboratory studies. However in nature, A. americanum frequently carry Rickettsia amblyommatis-another member of the spotted fever group-with the prevalence of infection reaching 84% in some populations. It has been postulated that the presence of an endosymbiotic Rickettsia in a significant proportion of a vector population would diminish or even block transmission of pathogenic Rickettsia in ticks from generation to generation due to transovarial interference. We measured the ability of R. amblyommatis-infected A. americanum to acquire R. rickettsii from an infected host with a bloodmeal, and transmit it transstadially, horizontally (to a susceptible host), and vertically to the next generation. Larvae from both the R. amblyommatis-infected and R. amblyommatis-free cohorts acquired R. rickettsii from infected guinea pigs, but the presence of the symbiont diminished the ability of coinfected engorged larvae to transmit R. rickettsii transstadially. Conversely, acquisition of R. rickettsii by cofeeding was unaffected in R. amblyommatis-infected nymphs and adults; prevalence of R. rickettsii in engorged adults reached 97% in both R. amblyommatis-infected and R. amblyommatis-free cohorts. In guinea pigs exposed to dually infected nymphs, R. rickettsii infection was milder than in those fed upon nymphs infected with R. rickettsii only. The frequency of transovarial transmission of R. rickettsii in the R. amblyommatis-infected cohort (31%) appeared lower than that in the R. amblyommatis-free cohort (48%), but the difference was not statistically significant. Larval progenies of dually infected A. americanum females transmitted R. rickettsii to naïve guinea pigs confirming viability of the pathogen. Thus, the vector competence of A. americanum for R. rickettsii was not significantly affected by R. amblyommatis.

Amblyomma aureolatum ticks are vectors of Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever in Brazil. Maintenance of R. rickettsii in nature depends on horizontal transmission along tick generations. Although such transmission is known to occur when uninfected and infected ticks feed simultaneously on susceptible animals (co-feeding systemic transmission), we investigated co-feeding nonsystemic transmission, which was based on R. rickettsii-infected and -uninfected A. aureolatum ticks feeding simultaneously on guinea pigs immune to R. rickettsii. Our acquisition and transmission infestations demonstrated that horizontal transmission of R. rickettsii by co-feeding ticks on immune hosts with no systemic infection did not occur when uninfected larvae fed distantly from infected nymphs but did occur in a few cases when uninfected larvae fed side-by-side with infected nymphs, suggesting that they shared the same feeding site. The co-feeding nonsystemic transmission type might have no epidemiologic importance for Rocky Mountain spotted fever.

Since 2008, a large epidemic of Rocky Mountain spotted fever has been emerging among humans and dogs in Mexicali, adjacent to the United States in Baja California, Mexico. We molecularly confirmed the causative agent; this information can be used to study the origin and dynamics of the epidemic.

Strains of Rickettsia rickettsii , the tick-borne agent of Rocky Mountain spotted fever, vary considerably in virulence. Genomic comparisons of R. rickettsii strains have identified a relatively small number of genes divergent in an avirulent strain. Among these is one annotated as Rickettsia ankyrin repeat protein 2 (RARP-2). Homologs of RARP-2 are present in all strains of R. rickettsii , but the protein in the avirulent strain Iowa contains a large internal deletion relative to the virulent Sheila Smith strain. RARP-2 is secreted in a type IV secretion system-dependent manner and exposed to the host cell cytosol. RARP-2 of Sheila Smith colocalizes with multilamellar membranous structures bearing markers of the endoplasmic reticulum (ER), whereas the Iowa protein shows no colocalization with host cell organelles and evidence of proteolytic degradation is detected. Overexpression of Sheila Smith RARP-2 in R. rickettsii Iowa converts this avirulent strain's typically nonlytic or opaque plaque type to a lytic plaque phenotype similar to that of the virulent Sheila Smith strain. Mutation of a predicted proteolytic active site of Sheila Smith RARP-2 abolished the lytic plaque phenotype but did not eliminate association with host membrane. RARP-2 is thus a type IV secreted effector and released from the rickettsiae into the host cytosol to modulate host processes during infection. Overexpression of Sheila Smith RARP-2 did not, however, restore the virulence of the Iowa strain in a guinea pig model, likely due to the multifactorial nature of rickettsial virulence. IMPORTANCE Members of the genus Rickettsia are obligate intracellular bacteria that exhibit a range of virulence from harmless endosymbionts of arthropods to the etiologic agents of severe disease. Despite the growing number of available genomes, little is known regarding virulence determinants of rickettsiae. Here, we have characterized an ankyrin repeat-containing protein, RARP-2, which differs between a highly virulent and an avirulent strain of R. rickettsii , the agent of Rocky Mountain spotted fever. RARP-2 is secreted by a type IV secretion system into the cytosol of the host cell, where it interacts with and manipulates the structure of the endoplasmic reticulum. RARP-2 from the avirulent strain is truncated by the loss of seven of 10 ankyrin repeat units but, although secreted, fails to alter ER structure. Recognition of those rickettsial factors associated with virulence will facilitate understanding of regional and strain-specific variation in severity of disease.

Background: Antibiotic resistance is increasing rapidly in pathogenic organisms, creating more complications for treatment of diseases. Rocky Mountain spotted fever (RMSF) is a neglected tropical disease in humans caused by Rickettsia rickettsii for which no effective therapeutic is available. Subtractive genomics methods facilitate the characterization of non-homologous essential proteins that could be targeted for the discovery of potential therapeutic compounds against R. rickettsii to combat RMSF. Present study followed an in-silico based methodology, involving scanning and filtering the complete proteome of Rickettsia rickettsii by using several prioritization parameters in the search of potential candidates for drug development. Further the putative targets were subjected to series of molecular dockings with ligands obtained from PDB ligand database to identify suitable potential inhibitors. The comparative genomic analysis revealed 606 non-homologous proteins and 233 essential non-homologous proteins of R. rickettsii. The metabolic pathway analysis predicted 120 proteins as putative drug targets, out of which 56 proteins were found to be associated with metabolic pathways unique to the bacteria and further subcellular localization analysis revealed that 9 proteins as potential drug targets which are secretion proteins, involved in peptidoglycan biosynthesis, folate biosynthesis and bacterial secretion system. As secretion proteins are more feasible as vaccine candidates, we have selected a most potential target i.e. tolC, an outer membrane efflux protein that belongs to type I secretion system and has major role in pathogen survival as well as MDR persistence. So for case study, we have modelled the three dimensional structure of tolC (tunnel protein). The model was further subjected to virtual screening and in-silico docking. The study identified three potential inhibitors having PDB Id 19V, 6Q8 and 39H. Further we have suggested that the above study would be most important while considering the selection of candidate targets and drug or vaccine designing against R. rickettsii., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The clinical and pathologic characterisation of two fatal cases of tick-borne rickettsiosis in rural (El Valle) and urban (City of Panama) Panama are described. Clinical and autopsy findings were non-specific, but the molecular analysis was used to identify Rickettsia rickettsii in both cases. No ticks were collected in El Valle, while in the urban case, R. rickettsii was detected in Rhipicephalus sanguineus s.l., representing the first molecular finding in this tick in Panama and Central America.

Brazilian spotted fever (BSF) is a potentially lethal human disease caused by Rickettsia rickettsii transmitted by ticks, including Amblyomma sculptum. However, in Southeast Brazil, where most BSF cases occur, capybaras are key hosts for both A. sculptum and Amblyomma dubitatum. We therefore compared the risk of human exposure to these ticks at a non-endemic anthropogenic site in Southeast Brazil where both tick species are maintained by capybaras and occur at high abundance. Cloth dragging, human baits and CO 2 traps were used to assess tick abundance and risk for human exposure. The two tick species displayed profound differences in behavior and microhabitat use. Notably, A. sculptum but not A. dubitatum quested for hosts openly from vegetation (ambush behavior) and infested human baits. Furthermore, A. dubitatum was more aggregated at a specific site whereas A. sculptum was more widespread along differing and drier microhabitats. Adults and nymphs of both species were infected with Rickettsia bellii. Overall, the results indicate that even though both species co-existed in the same area, A. sculptum posed a greater risk for biting humans and thus also for transmitting tick-borne pathogens., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Ticks are obligate blood feeding ectoparasites that transmit a wide variety of pathogenic microorganisms to their vertebrate hosts. Amblyomma sculptum is vector of Rickettsia rickettsii , the causative agent of Rocky Mountain spotted fever (RMSF), the most lethal rickettsiosis that affects humans. It is known that the transmission of pathogens by ticks is mainly associated with the physiology of the feeding process. Pathogens that are acquired with the blood meal must first colonize the tick gut and later the salivary glands (SG) in order to be transmitted during a subsequent blood feeding via saliva. Tick saliva contains a complex mixture of bioactive molecules with anticlotting, antiplatelet aggregation, vasodilatory, anti-inflammatory, and immunomodulatory properties to counteract both the hemostasis and defense mechanisms of the host. Besides facilitating tick feeding, the properties of saliva may also benefits survival and establishment of pathogens in the host. In the current study, we compared the sialotranscriptome of unfed A. sculptum ticks and those fed for 72 h on rabbits using next generation RNA sequencing (RNA-seq). The total of reads obtained were assembled in 9,560 coding sequences (CDSs) distributed in different functional classes. CDSs encoding secreted proteins, including lipocalins, mucins, protease inhibitors, glycine-rich proteins, metalloproteases, 8.9 kDa superfamily members, and immunity-related proteins were mostly upregulated by blood feeding. Selected CDSs were analyzed by real-time quantitative polymerase chain reaction preceded by reverse transcription (RT-qPCR), corroborating the transcriptional profile obtained by RNA-seq. Finally, high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis revealed 124 proteins in saliva of ticks fed for 96-120 h. The corresponding CDSs of 59 of these proteins were upregulated in SG of fed ticks. To the best of our knowledge, this is the first report on the proteome of A. sculptum saliva. The functional characterization of the identified proteins might reveal potential targets to develop vaccines for tick control and/or blocking of R. rickettsii transmission as well as pharmacological bioproducts with antihemostatic, anti-inflammatory and antibacterial activities.

In Brazil, two pathogenic Rickettsia species have been identified causing tick-borne spotted fever (SF). The aetiological agent Rickettsia rickettsii causes serious illness, particularly in the south-eastern region of the country. Moreover, the Rickettsia sp. strain Atlantic Rainforest cause milder clinical manifestations in south-eastern, south and north-east regions. This study has sought to analyse predictive factors for fatal SF. A case-control study was performed using disease notification records in Brazil. The cases included were individuals with laboratory confirmation and fatal progression of SF, while the controls included individuals with SF who were cured. A total of 386 cases and 415 controls were identified (1 : 1.1), and the cases and controls were similar in age. The factors identified as being protective against death were reported presence of ticks (odds ratio [OR], 0.60; 95% confidence interval [CI], 0.41-0.88), residing in urban areas (OR, 0.47, 95% CI, 0.31-0.74) and presenting lymphadenopathy (OR, 0.43; 95% CI, 0.23-0.82). Males exhibited a greater chance of death (OR, 1.57; 95% CI, 1.13-2.18), as did patients who were hospitalized (OR, 10.82; 95% CI, 6.38-18.35) and who presented hypotension or shock (OR, 10.80; 95% CI, 7.33-15.93), seizures (OR, 11.24; 95% CI, 6.49-19.45) and coma (OR of 15.16; 95% CI, 8.51-27.02). The study demonstrates the severity profile of the SF cases, defined either as the frequency of hospitalization (even in cases that were cured) or as the increased frequency of the clinical complications typically found in critical patients. Opportune clinical diagnosis, a careful evaluation of the epidemiological aspects of the disease and adequate care for patients are determining factors for reducing SF fatality rates., (© 2017 Blackwell Verlag GmbH.)

Rocky Mountain spotted fever (RMSF) is an emerging public health concern near the US-Mexico border, where it has resulted in thousands of cases and hundreds of deaths in the past decade. We identified 4 patients who had acquired RMSF in northern Mexico and subsequently died at US healthcare facilities. Two patients sought care in Mexico before being admitted to US-based hospitals. All patients initially had several nonspecific signs and symptoms, including fever, headache, nausea, vomiting, or myalgia, but deteriorated rapidly without receipt of a tetracycline-class antimicrobial drug. Each patient experienced respiratory failure late in illness. Although transborder cases are not common, early recognition and prompt initiation of appropriate treatment are vital for averting severe illness and death. Clinicians on both sides of the US-Mexico border should consider a diagnosis of RMSF for patients with rapidly progressing febrile illness and recent exposure in northern Mexico.

Ehrlichiosis and rickettsiosis are two common bacterial tick-borne diseases in the southeastern United States. Ehrlichiosis is caused by ehrlichiae transmitted by Amblyomma americanum and rickettsiosis is caused by rickettsiae transmitted by Amblyomma maculatum and Dermacentor variabilis. These ticks are common and have overlapping distributions in the region. The objective of this study was to identify Anaplasma, Ehrlichia, and Rickettsia species associated with questing ticks in a Rocky Mountain spotted fever (RMSF) hotspot, and identify habitats, time periods, and collection methods for collecting questing-infected ticks. Using vegetation drags and CO 2 -baited traps, ticks were collected six times (May-September 2012) from 100 sites (upland deciduous, bottomland deciduous, grassland, and coniferous habitats) in western Tennessee. Adult collections were screened for Anaplasma and Ehrlichia (simultaneous polymerase chain reaction [PCR]) and Rickettsia using genus-specific PCRs, and resulting positive amplicons were sequenced. Anaplasma and Ehrlichia were only identified within A. americanum (Ehrlichia ewingii, Ehrlichia chaffeensis, Panola Mountain Ehrlichia, and Anaplasma odocoilei sp. nov.); more Ehrlichia-infected A. americanum were collected at the end of June regardless of habitat and collection method. Rickettsia was identified in three tick species; "Candidatus Rickettsia amblyommii" from A. americanum, R. parkeri and R. andeanae from A. maculatum, and R. montanensis ( = montana) from D. variabilis. Overall, significantly more Rickettsia-infected ticks were identified as A. americanum and A. maculatum compared to D. variabilis; more infected-ticks were collected from sites May-July and with dragging. In this study, we report in the Tennessee RMSF hotspot the following: (1) Anaplasma and Ehrlichia are only found in A. americanum, (2) each tick species has its own Rickettsia species, (3) a majority of questing-infected ticks are collected May-July, (4) A. americanum and A. maculatum harbor pathogenic bacteria in western Tennessee, and (5) R. rickettsii remains unidentified.

Spotted fever is an acute febrile illness, which is considered severely underreported and misdiagnosed in the Brazilian Pampa, caused by tick-borne Rickettsiae. Here, we report an eco-epidemiological investigation of Rickettsia spp. in ticks from a spotted fever focus in Toropi, southern Brazil. Ticks were collected from capybara carcasses and processed individually to obtain genomic DNA. Rickettsia was investigated using PCR that amplified the rickettsial fragments of the gltA, ompA and htrA genes. DNA from Rickettsia parkeri was found in four of 14 Amblyomma dubitatum ticks collected from capybara carcasses in Toropi and the nearby municipality of Quevedos. We also tested 210A. dubitatum ticks obtained from road-killed capybaras of other localities from the Pampa biome; none of them were positive for Rickettsiae. Thus, in Rio Grande do Sul, two Rickettsia species can be potentially associated to spotted fever: Rickettsia sp. strain Atlantic Rainforest, associated with Amblyomma ovale ticks in the Atlantic Rainforest biome, and R. parkeri, associated both with Amblyomma tigrinum and A. dubitatum ticks in the Pampa biome. Our results reinforce that R. parkeri may be the agent associated with spotted fever in the Brazilian Pampa., (Copyright © 2017 Elsevier B.V. All rights reserved.)