Isolate-Dependent Differences in Clinical, Pathological, and Transcriptional Profiles following In Vitro and In Vivo Infections with Rickettsia rickettsii.

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 SmithT, 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.

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.

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.

Survey of vector-borne agents in feral cats and first report of Babesia gibsoni in cats on St Kitts, West Indies.

BACKGROUND: As there is little data on vector-borne diseases of cats in the Caribbean region and even around the world, we tested feral cats from St Kitts by PCR to detect infections with Babesia, Ehrlichia and spotted fever group Rickettsia (SFGR) and surveyed them for antibodies to Rickettsia rickettsii and Ehrlichia canis. RESULTS: Whole blood was collected from apparently healthy feral cats during spay/ neuter campaigns on St Kitts in 2011 (N = 68) and 2014 (N = 52). Sera from the 52 cats from 2014 were used to detect antibodies to Ehrlichia canis and Rickettsia rickettsii using indirect fluorescent antibody tests and DNA extracted from whole blood of a total of 119 cats (68 from 2011, and 51 from 2014) was used for PCRs for Babesia, Ehrlichia and Rickettsia. We could not amplify DNA of SFG Rickettsia in any of the samples but found DNA of E. canis in 5% (6/119), Babesia vogeli in 13% (15/119), Babesia gibsoni in 4% (5/119), mixed infections with B. gibsoni and B. vogeli in 3% (3/119), and a poorly characterized Babesia sp. in 1% (1/119). Overall, 10% of the 52 cats we tested by IFA for E. canis were positive while 42% we tested by indirect fluorescent antibody (IFA) for R. rickettsii antigens were positive. CONCLUSIONS: Our study provides the first evidence that cats can be infected with B. gibsoni and also indicates that cats in the Caribbean may be commonly exposed to other vector-borne agents including SFGR, E. canis and B. vogeli. Animal health workers should be alerted to the possibility of clinical infections in their patients while public health workers should be alerted to the possibility that zoonotic SFGR are likely circulating in the region.

Fatal Rocky Mountain Spotted Fever along the United States-Mexico Border, 2013-2016.

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.

Genetic identification of rickettsial isolates from fatal cases of Brazilian spotted fever and comparison with Rickettsia rickettsii isolates from the American continents.

Fifteen bacterial isolates from spotted fever group rickettsiosis in Brazil were genetically identified as Rickettsia rickettsii. In a phylogenetic analysis with other R. rickettsii isolates from GenBank, the Central/South American isolates showed low polymorphism and formed a clade distinct from two North American clades, with the North American clades having greater in-branch polymorphism.

Phylogeography of Rickettsia rickettsii genotypes associated with fatal Rocky Mountain spotted fever.

Rocky Mountain spotted fever (RMSF), a tick-borne zoonosis caused by Rickettsia rickettsii, is among the deadliest of all infectious diseases. To identify the distribution of various genotypes of R. rickettsii associated with fatal RMSF, we applied molecular typing methods to samples of DNA extracted from formalin-fixed, paraffin-embedded tissue specimens obtained at autopsy from 103 case-patients from seven countries who died of RMSF. Complete sequences of one or more intergenic regions were amplified from tissues of 30 (29%) case-patients and revealed a distribution of genotypes consisting of four distinct clades, including the Hlp clade, regarded previously as a non-pathogenic strain of R. rickettsii. Distinct phylogeographic patterns were identified when composite case-patient and reference strain data were mapped to the state and country of origin. The phylogeography of R. rickettsii is likely determined by ecological and environmental factors that exist independently of the distribution of a particular tick vector.

Molecular characterization of Rickettsia rickettsii isolated from human clinical samples and from the rabbit tick Haemaphysalis leporispalustris collected at different geographic zones in Costa Rica.

Five strains of spotted fever group (SFG) rickettsiae previously isolated from human clinical cases and from the tick Haemaphysalis leporispalustris were used for molecular characterization in this study to establish their genetic relationship compared with the prototype Rickettsia rickettsii strain Sheila Smith. Samples were tested by polymerase chain reaction (PCR) targeting the rickettsial genes gtlA, ompA, and ompB. PCR products of the latter two genes were DNA sequenced and compared with available sequences in GenBank. The ompA partial sequences of the five Costa Rican isolates showed 100% identity to several R. rickettsii sequences available in GenBank, including the sequence of the virulent reference strain Sheila Smith, whereas the ompB partial sequences of the five Costa Rican isolates showed 99.8-100% identity to R. rickettsii sequences from GenBank. This study showed the first molecular detection of R. rickettsii isolates from Rocky Mountain Spotted Fever patients and from the rabbit tick H. leporispalustris in different geographical zones in Costa Rica.

Detection of a spotted fever group Rickettsia in the tick Ixodes tasmani collected from koalas in Port Macquarie, Australia.

Four species of Rickettsia are recognized as endemic to Australia. This study reports the detection of a new spotted fever group Rickettsia in the common marsupial tick Ixodes tasmani Neumann collected from koalas (Phascolarctos cinereus) in Port Macquarie, NSW, Australia. Based on the results of polymerase chain reaction (PCR) amplification of extracted tick DNA with primers targeting the citrate synthase gene (gltA) and the outer membrane proteins A and B (ompA. ompB), Rickettsiae were detected in 22 of 78 I. tasmani tick samples (28.2%). Sequence data obtained for the three genes displayed the closest degree of similarity to Rickettsia heilongjiangiensiss for gltA (99.4%; 331/333 bp), Rickettsia amblyommii for the ompA gene (94.8%; 417/440 bp), and both Rickettsia massiliae and Rickettsia rhipicephali for the ompB gene (97%; 770/803 bp). BLAST and phylogenetic analysis of partial sequences obtained for the three genes were found to have sufficient nucleotide variation from the current recognized Australian species to be considered a distinct spotted fever group Rickettsia.

Evaluation of conventional and real-time PCR assays for detection and differentiation of Spotted Fever Group Rickettsia in dog blood.

Spotted Fever Group Rickettsia is important cause of emerging and re-emerging infectious disease in people and dogs. Importantly, dogs can serve as sentinels for disease in people. Sensitive and specific diagnostic tests that differentiate among species of infecting Rickettsia are needed. The objective of this study was to develop a sensitive and specific PCR that differentiates SFG Rickettsia infecting dog blood. Conventional and real-time PCR assays were developed using primers that targeted a small region of the ompA gene. Their sensitivity, determined by testing a cloned target sequence in the presence of host DNA, was 15-30 and 5 copies of DNA, respectively. Testing of Rickettsia cultures and analysis of Rickettsia gene sequences deposited in GenBank verified DNA could be amplified and used to differentiate species. DNA from the blood of infected dogs was also tested. Importantly, Rickettsia DNA was detected before seroconversion in some dogs. The species of infecting Rickettsia was also identified. We conclude these assays may assist in the timely diagnosis of infection with SFG Rickettsia. They may also facilitate the discovery of novel SFG Rickettsia infecting dogs, and in the investigation of dogs as sentinels for emerging rickettsioses.

Molecular typing of isolates of Rickettsia rickettsii by use of DNA sequencing of variable intergenic regions.

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is found throughout the Americas, where it is associated with different animal reservoirs and tick vectors. No molecular typing system currently exists to allow for the robust differentiation of isolates of R. rickettsii. Analysis of eight completed genome sequences of rickettsial species revealed a high degree of sequence conservation within the coding regions of chromosomes in the genus. Intergenic regions between coding sequences should be under less selective pressure to maintain this conservation and thus should exhibit greater nucleotide polymorphisms. Utilizing these polymorphisms, we developed a molecular typing system that allows for the genetic differentiation of isolates of R. rickettsii. This typing system was applied to a collection of 38 different isolates collected from humans, animals, and tick vectors from different geographic locations. Serotypes 364D, from Dermacentor occidentalis ticks, and Hlp, from Haemaphysalis leporispalustris ticks, appear to be distinct genotypes that may not belong to the species R. rickettsii. We were also able to differentiate 36 historical isolates of R. rickettsii into three different phylogenetic clades containing seven different genotypes. This differentiation correlated well, but not perfectly, with the geographic origin and likely tick vectors associated with the isolates. The few apparent typing discrepancies found suggest that the molecular ecology of R. rickettsii needs more investigation.

Evaluation of a PCR assay for quantitation of Rickettsia rickettsii and closely related spotted fever group rickettsiae.

A spotted fever rickettsia quantitative PCR assay (SQ-PCR) was developed for the detection and enumeration of Rickettsia rickettsii and other closely related spotted fever group rickettsiae. The assay is based on fluorescence detection of SYBR Green dye intercalation in a 154-bp fragment of the rOmpA gene during amplification by PCR. As few as 5 copies of the rOmpA gene of R. rickettsii can be detected. SQ-PCR is suitable for quantitation of R. rickettsii and 10 other genotypes of spotted fever group rickettsiae but not for R. akari, R. australis, R. bellii, or typhus group rickettsiae. The sensitivity of SQ-PCR was comparable to that of a plaque assay using centrifugation for inoculation. The SQ-PCR assay was applied successfully to the characterization of rickettsial stock cultures, the replication of rickettsiae in cell culture, the recovery of rickettsial DNA following different methods of extraction, and the quantitation of rickettsial loads in infected animal tissues, clinical samples, and ticks.

[Study on spotted fever group Rickettsiae in Guangdong province].

OBJECTIVE: To explore the existence of spotted fever group Rickettsiae (SFGR) in Guangdong province. METHODS: Sera were tested to find the SFGR in population and host animals. The target samples were screened by polymerase chain reaction (PCR), and Rickettsiae was isolated with embryonated hen eggs and identified by serological tests. RESULTS: Eight hundred and sixty people in natural condition and 321 of mice were determined. The mean positive rate of healthy population was 3.84%. To compare results among elected places, Fisher's exact test was applied. The difference was suggestive (P < 0.01), and there was no significant difference between mountain and plain areas. There was also no significant difference between mountain and plain areas (P > 0.05). Positive rate of mice was 4.67%, with Rattus fulvescens, Rattus edwardsi, Bandicota indica 11.59%, 12.90%, 3.13% respectively. It was the first time that SFGR antibodies in Rattus fulvescens, Rattus edwardsi, Bandicota indica were reported. A total number of 321 mice spleens and 394 ticks from the surface of mice body were collected. Two strains of SFGR, GDFK58-2000 and GDFK59-2000, were isolated in the ticks from the body surface of 2 Rattus fulvescens. They were identified as Rickettsia sibirica by serological tests. Five hundred thirty-three bp OmpA gene fragments of the two strains were cloned and sequenced. Compared with other relevant strains in Genbank, the rates of homology of nucleotide sequences of GDFK58-2000 and GDFK59-2000 and other Rickettsia sibirica strains were from 99.6% to 100%, and the homology of amino acid speculated was 100%. CONCLUSION: It has been proved that epidemic areas of north Asia tick-transmitted SFGR, did exist in Guangdong province confirmed by hostanimals, transmission vectors and aetiology.

Genetic analysis of isolates of Rickettsia rickettsii that differ in virulence.

We examined the degree of intraspecies genetic polymorphisms present among 13 R. rickettsii strains isolated from the blood of patients and ixodid ticks from North and South America. Preliminary confirmation of these isolates as R. rickettsii was done by evaluating RsaI and PstI restriction fragment length polymorphic (RFLP) sites in a 631 bp fragment of the rOmpA gene amplified with primers encompassing the Rr190.70-701 nt region. All isolates had the same profile, which was identical to that previously described for strain Bitterroot. The AluI RFLP analysis of the rOmpA fragment differentiated only Hlp#2 isolate from the other strains. Two types of RFLP patterns were obtained for a 381 bp gltA fragment. Strains Bitterroot, Sheila Smith, Lost Horse Canyon, and Morgan contained an additional AluI fragment of 40 bp, compared to the four-band profile of 122, 87, 81, and 43 bp determined for other isolates. These differences correlated with the absence of a CGC insert in the former isolates as determined by comparative DNA sequence analysis. To measure the levels of deletion/mutation events among isolates of R. rickettsii, DNA sequence analysis was performed on a portion of a 2,672 bp region spanning the 3'-end of polA to the 5'-end of dnaE. Except for Hlp#2 strain these isolates were highly conserved in the regions sequenced.

Ancestral divergence of Rickettsia bellii from the spotted fever and typhus groups of Rickettsia and antiquity of the genus Rickettsia.

The eubacterial genus Rickettsia belongs to the alpha subgroup of the phylum Proteobacteria. This genus is usually divided into three biotypes on the basis of vector host and antigenic cross-reactivity characteristics. However, the species Rickettsia bellii does not fit into this classification scheme; this organism has characteristics common to both the spotted fever group and the typhus group biotypes and also exhibits some unique features. Sequences of the 16S rRNA and 23S rRNA genes from Rickettsia rickettsii (spotted fever group), Rickettsia prowazekii (typhus group), and R. bellii were studied to determine the position of R. bellii in the rickettsial classification scheme. The 23S rRNA gene sequences described in this paper are the first 23S rRNA sequences reported for any member of the Rickettsiaceae. The 23S rRNA gene contains substantially more phylogenetic information than is contained in the 16S rRNA sequences, and the 23S rRNA gene sequence has diverged about 1.9 times faster in the three Rickettsia species which we studied. Taken together, the molecular data obtained from the two genes indicate that R. bellii is not a member of either the spotted fever group or the typhus group; rather, this organism appears to be the product of a divergence which predates the separation of the genus into the spotted fever group and the typhus group. Consequently, different combinations of the ancestral characteristics retained by R. bellii have been retained in the more derived lineages of the genus.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenic relationships among the rickettsiae of the spotted fever and typhus groups.

Using immunoblots to analyze antigenic relationships among the pathogenic spotted fever and typhus group rickettsiae, I found that the rickettsial lipopolysaccharide (LPS) was a group-specific antigen. All the rickettsiae examined had 135-kDa and 58-kDa protein antigens. The spotted fever rickettsiae and Rickettsia canada had, in addition, 190-kDa protein antigens which were antigenic analogs of previously described protective antigens of R. conorii and R. rickettsii.

DNA base composition of Rickettsia tsutsugamushi determined by reversed-phase high-performance liquid chromatography.

The DNA base composition of Rickettsia tsutsugamushi was determined by reversed-phase high-performance liquid chromatography and compared with that of Rickettsia rickettsii. The G+C contents were 28.1 to 30.5 mol% for R. tsutsugamushi and 32.1 mol% for R. rickettsii.

Causative agent of spotted fever group rickettsiosis in Japan.

Since 1984, it has been known that spotted fever group rickettsiosis exists in Japan. We isolated three strains of the causative rickettsiae, designated Katayama, Misaka, and Abe, from patients with the disease and studied the characteristics of the isolates. Nude mice and cyclophosphamide-treated mice died after infection with the isolates. However, infected normal mice recovered and acquired immunity. Infected adult male guinea pigs had fever, a scrotal reaction, and seroconversion. The isolates propagated well in tissue-cultured Vero cells. Analysis by the cross-immunofluorescence antibody method showed that these isolates were closely related serologically. To reveal their immunological properties in detail, we produced 21 anti-Katayama monoclonal antibodies. Seven of these antibodies reacted with all representative strains of spotted fever group rickettsiae used in this study, and five others reacted only with the homologous strain, revealing that the Katayama strain has a strain-specific antigen(s) different from those of other spotted fever group rickettsiae. Moreover, these strain-specific antibodies also reacted with the Misaka and Abe strains. These results demonstrate that the causative agent of spotted fever group rickettsiosis in Japan is a new serotype of spotted fever group rickettsiae.

Isolation and identification of Rickettsia rickettsii from the rabbit tick (Haemaphysalis leporispalustris) in the Atlantic zone of Costa Rica.

During the ecological studies done in Costa Rica on Rocky Mountain spotted fever, we have been able to isolate 2 strains of rickettsiae from the rabbit tick Haemaphysalis leporispalustris, indistinguishable from R. rickettsii isolated from humans in our country. Moreover, serological evidence of this agent was demonstrated in convalescent guinea pigs and in the sera of the wild rabbit (Sylvilagus braziliensis).