Confirmation of Rickettsia conorii Subspecies indica Infection by Next-Generation Sequencing, Shandong, China.

We describe 3 similar cases of rickettsial disease that occurred after tick bites in a mountainous rural area of Shandong Province, China. Next-generation sequencing indicated the etiologic agent of 1 patient was Rickettsia conorii subspecies indica. This agent may be more widely distributed across China than previously thought.

Assessment of real-time PCR assay for detection of Rickettsia spp. and Rickettsia rickettsii in banked clinical samples.

Two novel real-time PCR assays were developed for the detection of Rickettsia spp. One assay detects all tested Rickettsia spp.; the other is specific for Rickettsia rickettsii. Evaluation using DNA from human blood and tissue samples showed both assays to be more sensitive than nested PCR assays currently in use at the CDC.

Genome sequences of the human body louse and its primary endosymbiont provide insights into the permanent parasitic lifestyle.

As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever, and trench fever. Here, we present genome sequences of the body louse and its primary bacterial endosymbiont Candidatus Riesia pediculicola. The body louse has the smallest known insect genome, spanning 108 Mb. Despite its status as an obligate parasite, it retains a remarkably complete basal insect repertoire of 10,773 protein-coding genes and 57 microRNAs. Representing hemimetabolous insects, the genome of the body louse thus provides a reference for studies of holometabolous insects. Compared with other insect genomes, the body louse genome contains significantly fewer genes associated with environmental sensing and response, including odorant and gustatory receptors and detoxifying enzymes. The unique architecture of the 18 minicircular mitochondrial chromosomes of the body louse may be linked to the loss of the gene encoding the mitochondrial single-stranded DNA binding protein. The genome of the obligatory louse endosymbiont Candidatus Riesia pediculicola encodes less than 600 genes on a short, linear chromosome and a circular plasmid. The plasmid harbors a unique arrangement of genes required for the synthesis of pantothenate, an essential vitamin deficient in the louse diet. The human body louse, its primary endosymbiont, and the bacterial pathogens that it vectors all possess genomes reduced in size compared with their free-living close relatives. Thus, the body louse genome project offers unique information and tools to use in advancing understanding of coevolution among vectors, symbionts, and pathogens.

Two pathogens and one disease: detection and identification of flea-borne Rickettsiae in areas endemic for murine typhus in California.

Results of an environmental assessment conducted in a newly emergent focus of murine typhus in southern California are described. Opossums, Didelphis virginiana Kerr, infested with cat fleas, Ctenocephalides felis Buché, in the suburban area were abundant. Animal and flea specimens were tested for the DNA of two flea-borne rickettsiae, Rickettsia typhi and Rickettsia felis. R. felis was commonly detected in fleas collected throughout this area while R. typhi was found at a much lower prevalence in the vicinity of just 7 of 14 case-patient homes identified. DNA of R. felis, but not R. typhi, was detected in renal, hepatic, and pulmonary tissues of opossums. In contrast, there were no hematologic polymerase chain reaction findings of R. felis or R. typhi in opossums, rats, and cats within the endemic area studied. Our data suggest a significant probability of human exposure to R. felis in the area studied; however, disease caused by this agent is not recognized by the medical community and may be misdiagnosed as murine typhus using nondiscriminatory serologic methods.

Presence, genetic variability, and potential significance of "Candidatus Midichloria mitochondrii" in the lone star tick Amblyomma americanum.

We used next generation sequencing to detect the bacterium "Candidatus Midichloria mitochondrii" for the first time in lone star ticks (Amblyomma americanum) from the eastern United States. 177 individuals and 11 tick pools from seven sites in four states were tested by pyrosequencing with barcoded 16S rRNA gene eubacterial primers targeting variable regions 5-3. Average infection prevalence was 0.15 across all surveyed populations (range 0-0.29) and only the site with the smallest sample size (n = 5) was negative. Three genotypes differing by 2.6-4.1 % in a 271 bp region of 16S rRNA gene were identified. Two variants co-occurred in sites in North Carolina and New York, but were not observed in the same tick at those sites. The third genotype was found only in Georgia. Phylogenetic analysis of this fragment indicated that the three variants are more closely related to "Candidatus Midichloria mitochondrii" genotypes from other tick species than to each other. This variation suggests that multiple independent introductions occurred in A. americanum which may provide insight into bacterial spread within its ecosystem and parasitism on this tick. Whether the presence of this bacterium affects acquisition or maintenance of other pathogens and symbionts in A. americanum or the survival, biology and evolution of the tick itself is unknown.

Genetic typing of isolates of Rickettsia typhi.

Murine typhus, which is caused by Rickettsia typhi, has a wide range of clinical manifestations. It has a low mortality rate but may result in meningoencephalitis and interstitial pneumonia in severe cases. Comparisons of complete genome sequences of R. typhi isolates from North Carolina, USA (Wilmington), Myanmar (B9991PP), and Thailand (TH1527) identified only 26 single nucleotide polymorphism (SNP) and 7 insertion-deletion (INDEL) sites in these highly syntenic genomes. Assays were developed to further define the distribution of these variant sites among 15 additional isolates of R. typhi with different histories from Asia, the USA, and Africa. Mismatch amplification mutation assays (MAMA) were validated for 22 SNP sites, while the 7 INDEL sites were analyzed directly on agarose gels. Six SNP types, 9 INDEL types, 11 total types were identified among these 18 isolates. Replicate DNA samples as well as comparisons of isolates with different passage and source histories gave consistent genetic typing profiles. Comparison of the SNP and INDEL markers to R. typhi's nearest neighbor Rickettsia prowazekii demonstrated that the majority of the SNPs represent intra-species variation that arose post divergence of these two species while several INDEL sites also exhibited intraspecies variability among the R. prowazekii genomes that have been completely sequenced. The assays for the presence of these SNP and INDEL sites, particularly the latter, comprise a low technology gel method for consistently distinguishing R. typhi and R. prowazekii as well as for differentiating genetic types of R. typhi.

Analytically sensitive Rickettsia species detection for laboratory diagnosis.

Clinical and laboratory diagnosis of rickettsial diseases is challenging because of the undifferentiated symptoms (commonly fever, headache, and malaise) and low bacteremia (< 100 genomic copies [gc]/mL) during the early acute stage of illness. Early treatment with doxycycline is critical for a positive outcome, especially in Rickettsia rickettsii (Rocky Mountain spotted fever) infections where cases may be fatal within 5 to 10 days from symptom onset, emphasizing the need for more sensitive diagnostics. A real-time reverse transcriptase polymerase chain reaction (PCR) assay, RCKr, was developed and validated for Rickettsia spp. nucleic acid detection in human clinical samples. The limit of detection for RCKr was determined to be 20 gc/mL, compared with our 2013 (Kato et al.) laboratory developed test, PanR8 at 1,800 to 2,000 gc/mL. Inclusivity, exclusivity, accuracy, and precision results correlated as expected. From an evaluation of 49 banked clinical samples, RCKr detected 35 previously positive samples, as well as two specimens that were PanR8 real-time PCR negative yet clinically diagnosed as possible rickettsiosis. Ct values from RCKr clinical sample testing show a 100-fold increase relative to PanR8. Additional testing is needed to understand the clinical sensitivity of RCKr; however, this study demonstrates RCKr to have high analytical specificity and sensitivity for Rickettsia detection.

Molecular Characterization of Rickettsial Agents in Ticks (Acari: Ixodidae) from Sri Lanka.

Because the majority of spotted fever group rickettsiae are transmitted to humans by tick bites, it is important to understand which ticks might play a role in transmission of rickettsial pathogens in Sri Lanka. The purpose of our study was to conduct molecular surveillance of 847 ticks collected in different locations in central Sri Lanka to determine which were infected with Rickettsia and Anaplasmataceae. Molecular methods were used to identify the ticks and the agents detected. Most ticks (Amblyomma, Haemaphysalis, and Rhipicephalus) were collected by flagging, and lower number was collected from dogs, cattle, pigs, a pangolin, and tortoises. Five spotted fever genotypes were identified: a Rickettsia africae-like agent in Amblyomma larvae, Rhipicephalus massiliae and a related genotype identified in association with the tropical type of Rhipicephalus sanguineus from dogs and Rhipicephalus haemaphysaloides from dogs and cattle, and Candidatus R. kellyi and another novel genotype (SL94) in R. haemaphysaloides. Twenty-three ticks were positive for Anaplasmataceae, including one Anaplasma and two Ehrlichia genotypes. Because the sequence database for both ticks and rickettsial agents from Sri Lanka and southern India is not extensive, additional molecular characterization of the tick species of Sri Lanka and their rickettsial agents is required to understand their pathogenic potential more completely. However, several of the agents we identified in this survey may well be pathogenic for humans and domestic animals, and should be considered as a part of epidemiological surveillance and patient management.

Eschar-associated spotted fever rickettsiosis, Bahia, Brazil.

In Brazil, Brazilian spotted fever was once considered the only tick-borne rickettsial disease. We report eschar-associated rickettsial disease that occurred after a tick bite. The etiologic agent is most related to Rickettsia parkeri, R. africae, and R. sibirica and probably widely distributed from Sao Paulo to Bahia in the Atlantic Forest.

Rickettsia rickettsii in Rhipicephalus ticks, Mexicali, Mexico.

Circulation of a unique genetic type of Rickettsia rickettsii in ticks of the Rhipicephalus sanguineus complex was detected in Mexicali, Baja California, Mexico. The Mexican R. rickettsii differed from all isolates previously characterized from the endemic regions of Rocky Mountain spotted fever in northern, central, and southern Americas. Rhipicephalus ticks in Mexicali are genetically different from Rh. sanguineus found in the United States.

Detection of Rickettsia Species, and Coxiella-Like and Francisella-Like Endosymbionts in Amblyomma americanum and Amblyomma maculatum from a Shared Field Site in Georgia, United States of America.

Two abundant species of aggressive ticks commonly feed on humans in Georgia: the Gulf Coast tick (Amblyomma maculatum) and the Lone Star tick (A. americanum). A. maculatum is the primary host of Rickettsia parkeri, "Candidatus Rickettsia andeanae," and a Francisella-like endosymbiont (AmacFLE), whereas A. americanum is the primary host for R. amblyommatis, Ehrlichia chaffeensis, E. ewingii, and a Coxiella-like endosymbiont (AamCLE). Horizontal transmission of R. parkeri from A. maculatum to A. americanum by co-feeding has been described, and R. amblyommatis has been found infrequently in A. maculatum ticks. We assessed the prevalence of these agents and whether exchange of tick-associated bacteria is common between A. maculatum and A. americanum collected from the same field site. Unengorged ticks were collected May-August 2014 in west-central Georgia from a 4.14 acre site by flagging and from humans and canines traversing that site. All DNA samples were screened with quantitative PCR assays for the bacteria found in both ticks, and the species of any Rickettsia detected was identified by species-specific TaqMan assays or sequencing of the rickettsial ompA gene. Only R. amblyommatis (15) and AamCLE (39) were detected in 40 A. americanum, while the 74 A. maculatum only contained R. parkeri (30), "Candidatus Rickettsia andeanae" (3), and AmacFLE (74). Neither tick species had either Ehrlichia species. Consequently, we obtained no evidence for the frequent exchange of these tick-borne agents in a natural setting despite high levels of carriage of each agent and the common observance of infestation of both ticks on both dogs and humans at this site. Based on these data, exchange of these Rickettsia, Coxiella, and Francisella agents between A. maculatum and A. americanum appears to be an infrequent event.

Rickettsia 364D: a newly recognized cause of eschar-associated illness in California.

BACKGROUND: Four spotted fever group rickettsiae (SFGR) are known to infect humans in the United States. A member of the SFGR designated 364D and detected in Dermacentor occidentalis ticks has not previously been identified as a human pathogen. METHODS: An 80-year-old man from a rural northern California community presented with an eschar on his forearm. A skin punch biopsy of the lesion was evaluated by immunohistochemistry and molecular analysis. Serum specimens obtained from the patient and 3 other area residents with similar illnesses were tested by immunofluorescence and Western immunoblot for antibodies to SFGR. Ticks were collected near the patient's residence and tested for SFGR. RESULTS: Abundant intracellular rickettsiae and fragmented rickettsial antigens were observed in the mononuclear inflammatory infiltrates of the biopsy. Nucleotide sequences of DNA fragments amplified from the biopsy were identical to those of 364D. Convalescent sera from all four patients exhibited high immunoglobulin G titers to Rickettsia rickettsii, Rickettsia rhipicephali, and 364D antigens. Three adult D. occidentalis were positive for 364D, R. rhipicephali, and an unidentified Rickettsia species. CONCLUSIONS: This is the first confirmation of human disease associated with the SFGR 364D, which was likely transmitted by D. occidentalis. Although the patients described here presented with a single cutaneous eschar as the principal manifestation, the full spectrum of illness associated with 364D has yet to be determined. Possible infection with 364D or other SFGR should be confirmed through molecular techniques in patients who present with "spotless" Rocky Mountain spotted fever or have serum antibodies to R. rickettsii with group-specific assays.

Bacteremia, fever, and splenomegaly caused by a newly recognized bartonella species.

Bartonella species cause serious human infections globally, including bacillary angiomatosis, Oroya fever, trench fever, and endocarditis. We describe a patient who had fever and splenomegaly after traveling to Peru and also had bacteremia from an organism that resembled Bartonella bacilliformis, the causative agent of Oroya fever, which is endemic to Peru. However, genetic analyses revealed that this fastidious bacterium represented a previously uncultured and unnamed bartonella species, closely related to B. clarridgeiae and more distantly related to B. bacilliformis. We characterized this isolate, including its ability to cause fever and sustained bacteremia in a rhesus macaque. The route of infection and burden of human disease associated with this newly described pathogen are currently unknown.

Novel PCR exclusion assay to detect spotted fever group rickettsiae in the lone star tick (Amblyomma americanum).

The lone star tick (Amblyomma americanum) is the most common and abundant human-biting tick in the southeastern United States where spotted fever rickettsioses frequently occur. However, the role of this tick in transmitting and maintaining pathogenic and non-pathogenic spotted fever group rickettsiae (SFGR) remains poorly defined. This is partially due to the high prevalence and abundance of Rickettsia amblyommatis in most populations of A. americanum. Many molecular assays commonly employed to detect rickettsiae use PCR primers that target highly conserved regions in the SFGR so low abundance rickettsia may not be detected when R. amblyommatis is present. It is costly and inefficient to test for low abundance rickettsial agents with multiple individual specific assays even when they are multiplexed, as most samples will be negative. Real time PCR assays may also be hampered by inadequate limits of detection (LODs) for low abundance agents. We exploited the absence of an otherwise relatively SFGR-conserved genome region in R. amblyommatis to design a hemi-nested PCR-assay which has a sensitivity of 10 copies in detecting the presence of most SFGR, but not R. amblyommatis in DNA of infected lone star ticks. This deletion is conserved in 21 isolates of R. amblyommatis obtained from multiple states. We demonstrated the assay's utility by detecting a pathogenic SFGR, Rickettsia parkeri, in 15/50 (30 %) of field collected A. americanum ticks that were previously screened with conventional assays and found to be positive for R. amblyommatis. These co-infected ticks included 1 questing female, 6 questing nymphs, and 8 attached males. The high prevalence of R. parkeri among host-attached ticks may be due to several variables and does not necessarily reflect the risk of disease transmission from attached ticks to vertebrate hosts. This novel assay can provide accurate estimates of the prevalence of less common SFGR in A. americanum and thus improve our understanding of the role of this tick in the maintenance and transmission of the SFGR commonly responsible for human rickettsioses.

Detection of Rickettsia asembonensis in Fleas (Siphonaptera: Pulicidae, Ceratophyllidae) Collected in Five Counties in Georgia, United States.

We conducted a molecular survey of Rickettsia in fleas collected from opossums, road-killed and live-trapped in peridomestic and rural settings, state parks, and from pet cats and dogs in Georgia, United States during 1992-2014. The cat flea, Ctenocephalides felis (Bouché) was the predominant species collected from cats and among the archival specimens from opossums found in peridomestic settings. Polygenis gwyni (Fox) was more prevalent on opossums and a single cotton rat trapped in sylvatic settings. Trapped animals were infested infrequently with the squirrel flea, Orchopeas howardi (Baker) and C. felis. TaqMan assays targeting the BioB gene of Rickettsia felis and the OmpB gene of Rickettsia typhi were used to test 291 flea DNAs for Rickettsia. A subset of 53 C. felis collected from a cat in 2011 was tested in 18 pools which were all bioB TaqMan positive (34% minimum infection prevalence). Of 238 fleas tested individually, 140 (58.8%, 95% confidence interval [CI]: 52.5-64.9%) DNAs were bioB positive. Detection of bioB was more prevalent in individual C. felis (91%) compared to P. gwyni (13.4%). Twenty-one (7.2%) were ompB TaqMan positive, including 18 C. felis (9.5%) and 3 P. gwyni (3.2%). Most of these fleas were also positive with bioB TaqMan; however, sequencing of gltA amplicons detected only DNA of Rickettsia asembonensis. Furthermore, only the R. asembonensis genotype was identified based on NlaIV restriction analysis of a larger ompB fragment. These findings contribute to understanding the diversity of Rickettsia associated with fleas in Georgia and emphasize the need for development of more specific molecular tools for detection and field research on rickettsial pathogens.

Identification of cross-reactive epitopes on the conserved 47-kilodalton antigen of Orientia tsutsugamushi and human serine protease.

Orientia tsutsugamushi is the causative agent of scrub typhus. One of the protein antigens of this species, the conserved 47-kDa protein (HtrA), has been shown to induce an antibody response in patients and can provide protective immunity against live challenge by Orientia in mice. Pepscan experiments identified many peptide epitope clusters in different parts of this protein. The majority of the most reactive epitopes are located at the C terminus of the protein (from amino acid 333 to amino acid 430). Protein sequence analysis revealed that the 47-kDa protein contains a trypsin domain and has sequence homology to human serine protease HtrA1 (hHtrA1). As the 47-kDa protein is a potential vaccine candidate and its ability to induce autoimmunity is a concern, the reactivity of scrub typhus patient sera with purified recombinant 47-kDa and hHtrA1 proteins was tested. A significant percentage (>20%) of scrub typhus patient sera reacted strongly with recombinant hHTRA1 and two of the antigenic polypeptide epitopes in hHtrA1. These findings suggest that the safety of the full-length 47-kDa antigen as a vaccine candidate is a significant issue due to its cross-reactivity with a human protein, which may also contribute to autoimmune responses or enhanced pathology in some scrub typhus patients.

Cluster of sylvatic epidemic typhus cases associated with flying squirrels, 2004-2006.

In February 2006, a diagnosis of sylvatic epidemic typhus in a counselor at a wilderness camp in Pennsylvania prompted a retrospective investigation. From January 2004 through January 2006, 3 more cases were identified. All had been counselors at the camp and had experienced febrile illness with myalgia, chills, and sweats; 2 had been hospitalized. All patients had slept in the same cabin and reported having seen and heard flying squirrels inside the wall adjacent to their bed. Serum from each patient had evidence of infection with Rickettsia prowazekii. Analysis of blood and tissue from 14 southern flying squirrels trapped in the woodlands around the cabin indicated that 71% were infected with R. prowazekii. Education and control measures to exclude flying squirrels from housing are essential to reduce the likelihood of sylvatic epidemic typhus.

Detection and identification of rickettsial agents in ticks from domestic mammals in eastern Panama.

Several outbreaks of Rocky Mountain spotted fever have occurred in recent years in Colombian communities close to the border with Panama. However, little is known about rickettsiae and rickettsial diseases in eastern Panamanian provinces, the Darien Province and the Kuna Yala, located north of the endemic area in Colombia. In 2007, 289 ticks were collected in several towns from dogs, horses, mules, cows, and pigs. DNA was extracted from 124 Dermacentor nitens, 64 Rhipicephalus sanguineus, 43 Amblyomma ovale, 35 A. cajennense, 10 Boophilus microplus, 4 A. oblongoguttatum, and 9 A. cajennense nymphs. SYBR-Green polymerase chain reaction assays targeting a fragment of the OmpA and 16S rRNA genes were used for detection of DNA of the spotted fever group rickettsiae (SFGR) and Anaplasmataceae (Anaplasma and Ehrlichia), respectively. In total, 37.4% ticks were positive for SFGR, including 20.3% R. sanguineus, 27.9% A. ovale, 25.8% D. nitens, 50% B. microplus, 50% A. oblongoguttatum, and 100% A. cajennense. The presence of Rickettsia amblyommii DNA was confirmed by sequencing in A. cajennense, A. oblongoguttatum, A. ovale, B. microplus, and R. sanguineus. DNA of R. rickettsii was only detected in one D. nitens collected from a horse in Santa Fe, Darien Province. Prevalence of Anaplasmataceae varied from 6.3% in R. sanguineus to 26.5% in A. cajennense. DNA of Ehrlichia chaffensis was found in three D. nitens and three A. cajennense from horses. This is the first study providing molecular characterization and prevalence information on SFGR in ticks from these areas and thus will be helpful for future evaluations of the risk of rickettsial diseases for individuals living in this region.

Rocky Mountain spotted fever from an unexpected tick vector in Arizona.

BACKGROUND: Rocky Mountain spotted fever is a life-threatening, tick-borne disease caused by Rickettsia rickettsii. This disease is rarely reported in Arizona, and the principal vectors, Dermacentor species ticks, are uncommon in the state. From 2002 through 2004, a focus of Rocky Mountain spotted fever was investigated in rural eastern Arizona. METHODS: We obtained blood and tissue specimens from patients with suspected Rocky Mountain spotted fever and ticks from patients' homesites. Serologic, molecular, immunohistochemical, and culture assays were performed to identify the causative agent. On the basis of specific laboratory criteria, patients were classified as having confirmed or probable Rocky Mountain spotted fever infection. RESULTS: A total of 16 patients with Rocky Mountain spotted fever infection (11 with confirmed and 5 with probable infection) were identified. Of these patients, 13 (81 percent) were children 12 years of age or younger, 15 (94 percent) were hospitalized, and 2 (12 percent) died. Dense populations of Rhipicephalus sanguineus ticks were found on dogs and in the yards of patients' homesites. All patients with confirmed Rocky Mountain spotted fever had contact with tick-infested dogs, and four had a reported history of tick bite preceding the illness. R. rickettsii DNA was detected in nonengorged R. sanguineus ticks collected at one home, and R. rickettsii isolates were cultured from these ticks. CONCLUSIONS: This investigation documents the presence of Rocky Mountain spotted fever in eastern Arizona, with common brown dog ticks (R. sanguineus) implicated as a vector of R. rickettsii. The broad distribution of this common tick raises concern about its potential to transmit R. rickettsii in other settings.