Newly Recognized Spotted Fever Group Rickettsia as Cause of Severe Rocky Mountain Spotted Fever-Like Illness, Northern California, USA.

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.

Identification of an autotransporter peptidase of Rickettsia rickettsii responsible for maturation of surface exposed autotransporters.

Members of the spotted fever group rickettsia express four large, surface-exposed autotransporters, at least one of which is a known virulence determinant. Autotransporter translocation to the bacterial outer surface, also known as type V secretion, involves formation of a ß-barrel autotransporter domain in the periplasm that inserts into the outer membrane to form a pore through which the N-terminal passenger domain is passed and exposed on the outer surface. Two major surface antigens of Rickettsia rickettsii, are known to be surface exposed and the passenger domain cleaved from the autotransporter domain. A highly passaged strain of R. rickettsii, Iowa, fails to cleave these autotransporters and is avirulent. We have identified a putative peptidase, truncated in the Iowa strain, that when reconstituted into Iowa restores appropriate processing of the autotransporters as well as restoring a modest degree of virulence.

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.

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.

Report of Amblyomma sculptum in a House in a Rickettsia rickettsii Circulation Area.

Amblyomma sculptum is a tick that has medical and veterinary importance as, in Brazil, it is the main vector of Rickettsia rickettsii, a disease affecting humans. The presence of ticks was observed outside a residence in a peri-urban area of the Atlantic Forest region in Brazil, as well as on two dogs that lived there. Eighteen A. sculptum adults were seen walking on a cemented pillar at the porch of the house and sheltering inside the pillar's crevices; meanwhile on the dogs, only Rhipicephalus sanguineus sensu lato ticks were found. It is hypothesized that as the dogs circulated in the forest regions, they might have carried A. sculptum to the residence. This situation highlights the role of dogs as possible carriers of Brazilian spotted fever (BSF) tick vectors into human habitation. Strategies for the prevention and control of BSF should consider the hypothesis that ticks infected with R. rickettsii can be harbored in human dwellings in peri-urban areas.

New Jersey-Wide Survey of Spotted Fever Group Rickettsia (Proteobacteria: Rickettsiaceae) in Dermacentor variabilis and Amblyomma americanum (Acari: Ixodida: Ixodidae).

For the last decade, the New Jersey (NJ) Department of Health has reported between 42 and 144 new cases each year of "spotted fever group rickettsiosis" (SFGR), a statistic that reflects uncertainty regarding which rickettsial agents (Proteobacteria: Rickettsiaceae: Rickettsia) are infecting NJ residents. To identify the Rickettsia circulating in NJ ticks, we used a combination of conventional and real time PCR approaches to screen 560 Dermacentor variabilis Say and 245 Amblyomma americanum L. obtained from a 1-day state-wide surveillance in May 2018 and an additional 394 D. variabilis collected across NJ in 2013-2018. We found zero D. variabilis infected with Rickettsia rickettsii, the agent of Rocky Mountain spotted fever and, on average, 1.3% infected with presumed nonpathogenic Rickettsia montanensis. We also found zero A. americanum infected with R. rickettsii, and 20% infected with Rickettsia amblyommatis, a prevalence somewhat lower than in more southern states. Overall, we conclude that it is unlikely that R. rickettsii vectored by D. variabilis is a primary cause of SFGR cases in NJ and discuss our findings in the context of known facts and current limitations. We conclude that understanding the causes of SFGR east of the Mississippi will require collaboration among medical doctors, public health authorities, and medical entomologists to follow up presumptive human cases of SFGR with detailed histories of exposure, species-specific molecular assays, and active surveillance of putative vectors and the pathogens they may carry.

Needlestick-Associated Rocky Mountain Spotted Fever, Brazil.

We report a fatal case of Rocky Mountain spotted fever (RMSF) in a man in Brazil without recent history of tick bites or environmental exposure. He received an accidental needlestick while working as a nurse. The nurse and his patient died. Both cases were confirmed as RMSF by molecular methods.

Fatal Brazilian Spotted Fever Associated with Dogs and Amblyomma aureolatum Ticks, Brazil, 2013.

In São Paulo metropolitan area, Brazil, Amblyomma aureolatum ticks are the main vector of Rickettsia rickettsii, which causes Brazilian spotted fever. In 2013, a boy in São Paulo died of Brazilian spotted fever associated with household dogs and A. aureolatum ticks. Prompt recognition and treatment of this illness might prevent deaths.

Seroprevalence of spotted fever group rickettsiae in canines along the United States-Mexico border.

Portions of northern Mexico are experiencing a re-emergence of Rocky Mountain spotted fever (RMSF), a tickborne disease caused by Rickettsia rickettsii, a member of the spotted fever group of rickettsiae (SFGR). Infection with R. rickettsii can result in serious and life-threatening illness in people and dogs. Canine seroprevalence has been used as a sentinel for human RMSF in previous studies. This study aims to quantify SFGR seroprevalence in canines in three northern Mexican states and identify risk factors associated with seropositivity. A total of 1,136 serum samples and 942 ticks were obtained from dogs participating in government sterilization campaigns and from animal control facilities in 14 Mexican cities in three states. SFGR antibodies were detected using indirect immunofluorescence antibody assays at titre values ≥1/64. Six per cent (69 dogs) showed antibodies to SFGR, with the highest seroprevalence reported in Baja California (12%), Coahuila (4%) and Sonora (4%). Dogs from Baja California had three times higher odds of having SFGR antibodies compared to dogs from Sonora (OR = 3.38, 95% CI, 1.81-6.37). Roughly one quarter (25%) of surveyed dogs were parasitized by ticks (Rhipicephalus sanguineus sensu lato) at the time of sample collection. A portion of collected ticks were tested for rickettsial DNA using polymerase chain reaction. Positive samples were then sequenced, showing evidence of SFGR including R. massiliae, R. parkeri and R. rickettsii. Dogs that spent the majority of time on the street, such as free-roaming or community-owned dogs, showed a greater risk of tick infestation, seropositivity, bearing seropositive ticks, and may play a pivotal role in the spread of SFGR among communities. Estimating the seroprevalence of SFGR in the canine population can help public health campaigns target high-risk communities for interventions to reduce human RMSF cases.

Development of a rapid and visual detection method for Rickettsia rickettsii combining recombinase polymerase assay with lateral flow test.

OBJECTIVES: Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever, which is the most severe spotted fever group (SFG) rickettsiosis. Developing a simple and reliable detection method is required. METHODS: A detection method for R. rickettsii was established based on a recombinase polymerase amplification (RPA) assay and the lateral flow (LF) test. A specific target sequence was screened, and corresponding primers and probes were designed, synthesized, and screened for establishing an RPA assay with high amplification efficiency. Reagent concentrations, amplification time, and loading volume for strip development were optimized. The detection limit, analytic sensitivity and specificity were evaluated. RESULTS: A rapid, visual, sensitive and specific method for the detection of R. rickettsii based on RPA and the LF test was successfully established. The novel method had a limit of detection of 10 to 50 copies/reaction without recognizing other organisms. Analytical sensitivity and specificity were ≥90% and 100%, respectively, as evaluated by animal and simulative human samples. CONCLUSIONS: Using the established method, detection could be completed in 30 min with visually detectable results by the naked eye, without requirement of any instrument except a constant temperature equipment. The technique shows superior detection performance and is promising for wide use in the field as well as resource-limited areas for R. rickettsii detection.

Molecular Confirmation of Rocky Mountain Spotted Fever Epidemic Agent in Mexicali, Mexico.

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.

Rickettsia Lipid A Biosynthesis Utilizes the Late Acyltransferase LpxJ for Secondary Fatty Acid Addition.

Members of the Rickettsia genus are obligate intracellular, Gram-negative coccobacilli that infect mammalian and arthropod hosts. Several rickettsial species are human pathogens and are transmitted by blood-feeding arthropods. In Gram-negative parasites, the outer membrane (OM) sits at the nexus of the host-pathogen interaction and is rich in lipopolysaccharide (LPS). The lipid A component of LPS anchors the molecule to the bacterial surface and is an endotoxic agonist of Toll-like receptor 4 (TLR4). Despite the apparent importance of lipid A in maintaining OM integrity, as well as its inflammatory potential during infection, this molecule is poorly characterized in Rickettsia pathogens. In this work, we have identified and characterized new members of the recently discovered LpxJ family of lipid A acyltransferases in both Rickettsia typhi and Rickettsia rickettsii, the etiological agents of murine typhus and Rocky Mountain spotted fever, respectively. Our results demonstrate that these enzymes catalyze the addition of a secondary acyl chain (C14/C16) to the 3'-linked primary acyl chain of the lipid A moiety in the final steps of the Raetz pathway of lipid A biosynthesis. Since lipid A architecture is fundamental to bacterial OM integrity, we believe that rickettsial LpxJ may be important in maintaining membrane dynamics to facilitate molecular interactions at the host-pathogen interface that are required for adhesion and invasion of mammalian cells. This work contributes to our understanding of rickettsial outer membrane physiology and sets a foundation for further exploration of the envelope and its role in pathogenesis.IMPORTANCE Lipopolysaccharide (LPS) triggers an inflammatory response through the TLR4-MD2 receptor complex and inflammatory caspases, a process mediated by the lipid A moiety of LPS. Species of Rickettsia directly engage both extracellular and intracellular immunosurveillance, yet little is known about rickettsial lipid A. Here, we demonstrate that the alternative lipid A acyltransferase, LpxJ, from Rickettsia typhi and R. rickettsii catalyzes the addition of C16 fatty acid chains into the lipid A 3'-linked primary acyl chain, accounting for major structural differences relative to the highly inflammatory lipid A of Escherichia coli.

The Rickettsial Ankyrin Repeat Protein 2 Is a Type IV Secreted Effector That Associates with the Endoplasmic Reticulum.

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.

[A fatal case series of Rocky Mountain spotted fever in Sonora, México].

INTRODUCTION: Rocky Mountain spotted fever is a highly lethal infectious disease, particularly if specific treatment with doxycycline is given belatedly. OBJECTIVE: To describe the clinical profile of fatal Rocky Mountain spotted fever cases in hospitalized patients in the state of Sonora, México. MATERIALS AND METHODS: We conducted a cross-sectional study on a series of 47 deaths caused by Rickettsia rickettsii from 2013 to 2016. The diagnosis of Rocky Mountain spotted fever was confirmed in a single blood sample by polymerase chain reaction (PCR) or by a four-fold increase in immunoglobulin G measured in paired samples analyzed by indirect immunofluorescence. Clinical and laboratory characteristics were compared stratifying subjects into two groups: pediatric and adult. RESULTS: There were no differences in clinical characteristics between groups; petechial rash was the most frequent sign (96%), followed by headache (70%) and myalgia (67%). Although that doxycycline was administered before the fifth day from the onset of symptoms, death occurred in 55% of patients. In clinical laboratory, thrombocytopenia, and biomarkers of liver acute failure and acute kidney failure were the most frequent. CONCLUSION: Rocky Mountain spotted fever remains as one of the most lethal infectious diseases, which may be related not only to the lack of diagnostic suspicion and delayed administration of doxycycline, but to genotypic characteristics of Rickettsia rickettsii that may play a role in the variability of the fatality rate that has been reported in other geographical regions where the disease is endemic.

Una serie de casos fatales de fiebre manchada de las Montañas Rocosas en Sonora, México / A fatal case series of Rocky Mountain spotted fever in Sonora, México

Resumen Introducción. La fiebre manchada de las Montañas Rocosas es una infección muy letal, particularmente si no se diagnostica y se trata oportunamente. Objetivo. Describir el perfil clínico de los casos fatales de pacientes con fiebre manchada de las Montañas Rocosas, hospitalizados en Sonora, México. Materiales y métodos. Se analizó una serie de 47 defunciones por fiebre manchada de las Montañas Rocosas en el periodo de 2013 a 2016. El diagnóstico se confirmó mediante reacción en cadena de la polimerasa (PCR) o la cuadruplicación de los títulos de inmunoglobulina G (IgG) en muestras de suero pareadas analizadas mediante inmunofluorescencia indirecta. Se compararon las características clínicas y de laboratorio, estratificando a los sujetos en dos grupos: pediátricos y adultos. Resultados. No hubo diferencias en las manifestaciones clínicas entre los grupos; el exantema petequial fue el signo más frecuente (96 %), seguido por cefalea (70 %) y mialgias (67 %). La muerte ocurrió en el 55 % de los sujetos a pesar de haber recibido doxiciclina antes del quinto día del inicio de los síntomas. Los marcadores de laboratorio más frecuentes fueron trombocitopenia, falla hepática e insuficiencia renal. Conclusión. La fiebre manchada de las Montañas Rocosas es una enfermedad muy letal, lo cual puede estar relacionado con la ausencia de sospecha del diagnóstico y el retraso en la administración de doxiciclina, pero también con características atribuibles a Rickettsia rickettsii que inciden en la variabilidad de los resultados adversos que se han observado en regiones donde la enfermedad es frecuente.

Abstract Introduction: Rocky Mountain spotted fever is a highly lethal infectious disease, particularly if specific treatment with doxycycline is given belatedly. Objective: To describe the clinical profile of fatal Rocky Mountain spotted fever cases in hospitalized patients in the state of Sonora, México. Materials and methods: We conducted a cross-sectional study on a series of 47 deaths caused by Rickettsia rickettsii from 2013 to 2016. The diagnosis of Rocky Mountain spotted fever was confirmed in a single blood sample by polymerase chain reaction (PCR) or by a four-fold increase in immunoglobulin G measured in paired samples analyzed by indirect immunofluorescence. Clinical and laboratory characteristics were compared stratifying subjects into two groups: pediatric and adult. Results: There were no differences in clinical characteristics between groups; petechial rash was the most frequent sign (96%), followed by headache (70%) and myalgia (67%). Although that doxycycline was administered before the fifth day from the onset of symptoms, death occurred in 55% of patients. In clinical laboratory, thrombocytopenia, and biomarkers of liver acute failure and acute kidney failure were the most frequent. Conclusion: Rocky Mountain spotted fever remains as one of the most lethal infectious diseases, which may be related not only to the lack of diagnostic suspicion and delayed administration of doxycycline, but to genotypic characteristics of Rickettsia rickettsii that may play a role in the variability of the fatality rate that has been reported in other geographical regions where the disease is endemic.

Comparative genomic analysis of Rickettsia rickettsii for identification of drug and vaccine targets: tolC as a proposed candidate for case study.

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.

Confirming Rickettsia rickettsii as the etiological agent of lethal spotted fever group rickettsiosis in human patients from Espírito Santo state, Brazil.

Although Espírito Santo state is considered an endemic area for Brazilian spotted fever (BSF) with related lethal cases, it also constitutes the only state of southeastern Brazil that currently lacks a specific confirmation of the specific rickettsial agent. In an attempt to a species level confirmation of the etiological agent of fatal rickettsiosis cases in Espírito Santo state, in this study we tested human sera obtained between 2015 to 2017 by means of qPCR and subsequent conventional PCR protocols targeting gltA (citrate synthase) and ompA (190-kDA outer membrane protein) rickettsial genes. All samples were found to contain rickettsial DNA through the citrate synthase qPCR protocol. By conventional PCR, rickettsial gltA and ompA specific DNA fragments were detected in 25% (one sample) and 50% (2 samples) of the screened sera, respectively. Obtained consensuses for each gene partial sequences were 100% identical to Rickettsia rickettsii gltA and ompA genes. The present study confirms for the first time R. rickettsii as the etiological agent of a lethal spotted fever group rickettsiosis in human patients from Espírito Santo state.