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.

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 etal.) 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.

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.

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.

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 akari, an obligately intracellular bacterium, is the causative agent of the cosmopolitan urban disease rickettsialpox. R. akari is an atypical representative of spotted fever group rickettsiae (SFG) as it is associated with rodent mites rather than ticks or fleas; however, only limited information is available about the degree of genetic variability found among isolates of R. akari. We examined 13 isolates of R. akari from humans, rodents and mites in the USA, the former Soviet Union, and the former Yugoslavia made between 1946 and 2003 for diversity in their tandem repeat regions (TR) and intergenic regions (IGR). The 1.23 Mb genome of R. akari strain Hartford CWPP was analyzed using Tandem Repeat Finder software (http://tandem.bu.edu) and 374 different TRs were identified, with size variation from 1 to 483 bp and with TR copy numbers ranging between 21 and 1.9, respectively. No size polymorphisms were detected among the 11 TR regions examined from 5 open reading frames and 6 IGR. Eighteen non-TR IGR’s were amplified and sequenced for the same isolates comprising a total of 5.995 bp (0.49%) of the Hartford CWPP strain chromosome. Three single nucleotide polymorphism (SNP) sites were detected in two IGR’s which permitted separation of the five R. akari isolates from Ukraine SSR from the other eight isolates. In conclusion, this is the first study reporting genetic heterogeneity among R. akari isolates of different geographic origins. Further exploration of this genetic diversity is needed to understand better the geographic distribution of R. akari and the epidemiology of rickettsialpox. The potential of mites as hosts for other rickettsial agents also needs further investigation.

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.

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 (Bouche) 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.

Orientia tsutsugamushi (Ots) frequently causes severe scrub typhus infections in the Asia-Pacific region. Korean investigators have demonstrated that Ots encodes five different autotransporter domain (ATD) proteins (ScaA-ScaE). ScaA functions as an adhesin and confers protective immunity in a lethal mouse model of Ots infection. Specific antibodies are detected against ScaA and ScaC in Korean scrub typhus patients. However, there is limited data on the distribution of the Sca protein genes in diverse isolates of Ots. By BLAST analysis with the conserved beta barrel autotransporter domain (ATD) regions of the sca proteins, we discovered a sixth gene scaF among 3 of 10 new partial Ots genome sequences available at NCBI GenBank (Sido, Karp, AFSC7). We designed two to seven specific TaqMan assays to detect the ATD for each of the six sca genes. The TaqMan assays among those for each sca gene which gave the greatest sensitivity and linearity with DNA log dilutions were then used for screening DNAs from Ots isolates grown in L929 mouse cells for sca genes. The sca prevalence survey was performed for all six sca genes with 178 DNAs from isolates from 12 countries. The survey results were confirmed by conventional PCR with primers from conserved regions of the passenger domains (PD) and ATD of the sca proteins. The ATD was highly conserved between the DNAs of different genotypes compared to the sca PD but each TaqMan assay was sca specific. The percentage positivity for 56 kDa and scaA genes in the 178 DNAs using Ha primers was 59.6% and 62.4%, respectively. Our scaA conventional ATD PCR assay was positive in 98.3% but scaA was present in all 178 DNAs (100%) by ATD TaqMan. scaB, scaC, scaD, scaE and scaF were detected in 33.7%, 97.8%, 93.8%, 97.2% and 43.3% isolates by ATD TaqMan, respectively. The ATDs of Ots sca genes are thus sufficiently conserved between different genotypes for molecular assay design. Four sca genes are widely distributed among diverse Ots isolates from diverse geographical areas. scaB and scaF were detected in fewer Ots isolates and absent from some available genome sequences. Whether the utility of the ScaA, ScaC, ScaD, and ScaE antigenic passenger protein domains exceeds that of the mixed 56 kDa type surface antigens of Ots now used in combination diagnostic assays needs to be determined before they can be considered as suitable alternative serological antigens for diagnosis of scrub typhus.

Orientia tsutsugamushi (Ots) is an obligate, intracellular, mite-transmitted human pathogen which causes scrub typhus. Understanding the diversity of Ots antigens is essential for designing specific diagnostic assays and efficient vaccines. The protective immunodominant type-specific 56 kDa antigen (TSA) of Ots varies locally and across its geographic distribution. TSA contains four hypervariable domains. We bioinformatically analyzed 345 partial sequences of TSA available from India, most of which contain only the three variable domains (VDI-III) and three spacer conserved domains (SVDI, SVDII/III, SVDIII). The total number (152) of antigenic types (amino acid variants) varied from 14-36 in the six domains of TSA that we studied. Notably, 55% (787/1435) of the predicted CD4 T-cell epitopes (TCEs) from all the six domains had high binding affinities (HBA) to at least one of the prevalent Indian human leukocyte antigen (HLA) alleles. A surprisingly high proportion (61%) of such TCEs were from spacer domains; indeed 100% of the CD4 TCEs in the SVDI were HBA. TSA sequences from India had more antigenic types (AT) than TSA from Korea. Overall, >90% of predicted CD4 TCEs from spacer domains were predicted to have HBA against one or more prevalent HLA types from Indian, Korean, Asia-Pacific region or global population data sets, while only <50% of CD4 TCEs in variable domains exhibited such HBA. The phylogenetically and immunologically important amino acids in the conserved spacer domains were identified. Our results suggest that the conserved spacer domains are predicted to be functionally more important than previously appreciated in immune responses to Ots infections. Changes occurring at the TCE level of TSA may contribute to the wide range of pathogenicity of Ots in humans and mouse models. CD4 T-cell functional experiments are needed to assess the immunological significance of these HBA spacer domains and their role in clearance of Ots from Indian patients.

We report a partial genome sequence for the Coxiella-like endosymbiont strain CLE-RmD, assembled from metagenomics data obtained from the southern cattle tick (Rhipicephalus microplus) Deutsch strain.

The rabbit tick, Haemaphysalis leporispalustris Packard, is known for its association with Rickettsia rickettsii as it harbors both virulent and avirulent strains of this pathogen. In this manuscript we report findings and preliminary characterization of a novel spotted fever group rickettsia (SFGR) in rabbit ticks from California, USA. Rickettsia sp. CA6269 (proposed "Candidatus Rickettsia lanei") is most related to known R. rickettsii isolates but belongs to its own well-supported branch different from those of all R. rickettsii including strain Hlp2 and from Rickettsia sp. 364D (also known as R. philipii) and R. peacockii. This SFGR probably exhibits both transovarial and transstadial survival since it was found in both questing larvae and nymphs. Although this rabbit tick does not frequently bite humans, its role in maintenance of other rickettsial agents and this novel SFGR warrant further investigation.

Rickettsia philipii (type strain "Rickettsia 364D"), the etiologic agent of Pacific Coast tick fever (PCTF), is transmitted to people by the Pacific Coast tick, Dermacentor occidentalis. Following the first confirmed human case of PCTF in 2008, 13 additional human cases have been reported in California, more than half of which were pediatric cases. The most common features of PCTF are the presence of at least one necrotic lesion known as an eschar (100%), fever (85%), and headache (79%); four case-patients required hospitalization and four had multiple eschars. Findings presented here implicate the nymphal or larval stages of D. occidentalis as the primary vectors of R. philipii to people. Peak transmission risk from ticks to people occurs in late summer. Rickettsia philipii DNA was detected in D. occidentalis ticks from 15 of 37 California counties. Similarly, non-pathogenic Rickettsia rhipicephali DNA was detected in D. occidentalis in 29 of 38 counties with an average prevalence of 12.0% in adult ticks. In total, 5,601 ticks tested from 2009 through 2015 yielded an overall R. philipii infection prevalence of 2.1% in adults, 0.9% in nymphs and a minimum infection prevalence of 0.4% in larval pools. Although most human cases of PCTF have been reported from northern California, acarological surveillance suggests that R. philipii may occur throughout the distribution range of D. occidentalis.

We estimated the seroprevalence and determined the frequency of acute infections with Neorickettsia sennetsu, spotted fever group rickettsiae, Rickettsia typhi, and Orientia tsutsugamushi among 2,225 febrile patients presenting to community hospitals in three rural Thailand provinces during 2002-2005. The seroprevalence was 0.2% for sennetsu neorickettsiosis (SN), 0.8% for spotted fever group (SFG) rickettsiae, 4.2% for murine typhus (MT), and 4.2% for scrub typhus (ST). The frequency of acute infections was 0.1% for SN, 0.6% for SFG, 2.2% for MT, and 1.5% for ST. Additional studies to confirm the distribution of these pathogens and to identify animal reservoirs and transmission cycles are needed to understand the risk of infection.

The Ehrlichia muris-like agent (EMLA) is a newly recognized human pathogen found in Wisconsin and Minnesota. Ecological investigations have implicated both the blacklegged tick, Ixodes scapularis, and the white-footed mouse, Peromyscus leucopus, as playing roles in the maintenance of EMLA in nature. The work presented here shows that I. scapularis is an efficient vector of EMLA in a laboratory mouse model, but that Dermacentor variabilis, another frequent human biting tick found in EMLA endemic areas, is not. Additionally, I. scapularis larvae are able to acquire EMLA through co-feeding with infected nymphs. As EMLA only persists in mouse blood for a relatively short period of time, co-feeding transmission may play an important role in the maintenance of EMLA in ticks, and subsequently may play a role in limiting the geographic distribution of this pathogen in areas where co-feeding of larvae and nymphs is less common.

Amblyomma americanum is an abundant tick in the southeastern, midwestern, and northeastern United States. It is a vector of multiple diseases, but limited genomic resources are available for it. We sequenced the complete mitochondrial genome of a single female A. americanum collected in Georgia using the Illumina platform. The consensus sequence was 14,709bp long, and the mean coverage across the assembly was >12,000x. All expected tick genomic features were present, including two "Tick-Box" motifs, and in the expected order for the Metastriata. Heteroplasmy rates were low compared to the most closely related tick for which data are available, Amblyomma cajennense. The phylogeny derived from the concatenated protein coding and rRNA genes from the 33 available tick mitochondrial genomes was consistent with those previously proposed for the Acari. This is the first complete mitochondrial sequence for A. americanum, which provides a useful reference for future studies of A. americanum population genetics and tick phylogeny.

Rickettsiae are obligately intracellular bacteria that are transmitted to vertebrates by a variety of arthropod vectors, primarily by fleas and ticks. Once transmitted or experimentally inoculated into susceptible mammals, some rickettsiae may cause febrile illness of different morbidity and mortality, and which can manifest with different types of exhanthems in humans. However, most rickettsiae circulate in diverse sylvatic or peridomestic reservoirs without having obvious impacts on their vertebrate hosts or affecting humans. We have analyzed the key features of tick-borne maintenance of rickettsiae, which may provide a deeper basis for understanding those complex invertebrate interactions and strategies that have permitted survival and circulation of divergent rickettsiae in nature. Rickettsiae are found in association with a wide range of hard and soft ticks, which feed on very different species of large and small animals. Maintenance of rickettsiae in these vector systems is driven by both vertical and horizontal transmission strategies, but some species of Rickettsia are also known to cause detrimental effects on their arthropod vectors. Contrary to common belief, the role of vertebrate animal hosts in maintenance of rickettsiae is very incompletely understood. Some clearly play only the essential role of providing a blood meal to the tick while other hosts may supply crucial supplemental functions for effective agent transmission by the vectors. This review summarizes the importance of some recent findings with known and new vectors that afford an improved understanding of the eco-epidemiology of rickettsiae; the public health implications of that information for rickettsial diseases are also described. Special attention is paid to the co-circulation of different species and genotypes of rickettsiae within the same endemic areas and how these observations may influence, correctly or incorrectly, trends, and conclusions drawn from the surveillance of rickettsial diseases in humans.

The lone star tick (Amblyomma americanum) is an abundant and aggressive biter of humans, domestic animals, and wildlife in the southeastern-central USA and an important vector of several known and suspected zoonotic bacterial pathogens. However, the biological drivers of bacterial community variation in this tick are still poorly defined. Knowing the community context in which tick-borne bacterial pathogens exist and evolve is required to fully understand the ecology and immunobiology of the ticks and to design effective public health and veterinary interventions. We performed a metagenomic survey of the bacterial communities of questing A. americanum and tested 131 individuals (66 nymphs, 24 males, and 41 females) from five sites in three states. Pyrosequencing was performed with barcoded eubacterial primers targeting variable 16S rRNA gene regions 5-3. The bacterial communities were dominated by Rickettsia (likely R. amblyommii) and an obligate Coxiella symbiont, together accounting for 6.7-100% of sequences per tick. DNAs from Midichloria, Borrelia, Wolbachia, Ehrlichia, Pseudomonas, or unidentified Bacillales, Enterobacteriaceae, or Rhizobiales groups were also detected frequently. Wolbachia and Midichloria significantly co-occurred in Georgia (p<0.00001), but not in other states. The significance of the Midichloria-Wolbachia co-occurrence is unknown. Among ticks collected in Georgia, nymphs differed from adults in both the composition (p = 0.002) and structure (p = 0.002) of their bacterial communities. Adults differed only in their community structure (p = 0.002) with males containing more Rickettsia and females containing more Coxiella. Comparisons among adult ticks collected in New York and North Carolina supported the findings from the Georgia collection despite differences in geography, collection date, and sample handling, implying that the differences detected are consistent attributes. The data also suggest that some members of the bacterial community change during the tick life cycle and that some sex-specific attributes may be detectable in nymphs.

Sylvatic typhus is an infrequent, potentially life-threatening emerging zoonotic disease. In January of 2009, the New York State Department of Health was notified of a familial cluster of two suspected cases. Due to the paucity of typhus cases in New York, epidemiologic and environmental investigations were conducted to establish rickettsial etiology and determine potential sources of infection. Patients presented with symptoms consistent with typhus, and serologic testing of each patient confirmed infection with typhus group rickettsiae. Serologic analysis of blood obtained from southern flying squirrels (Glaucomys volans) captured from the attic crawlspace above an enclosed front porch of the cases' residence indicated evidence of infection with Rickettsia prowazekii, with 100% seroprevalence (n=11). Both patients reported spending significant time on the porch and hearing animal activity above the ceiling prior to onset of illness, implicating these flying squirrels as the likely source of infection.

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 Buche, 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.

OBJECTIVES: We describe an outbreak of spotted fever group (SFG) rickettsiosis that occurred in 2007 in a farming community in southeastern Guatemala. We identified 17 cases of an acute febrile illness, among which 10, including two fatalities, were confirmed or probable cases of rickettsial disease (case-fatality proportion 12%). METHODS: PCR, a microimmunofluorescence assay (IFA), and Western blotting were performed on patient samples, and PCR was performed on ticks. RESULTS: Using an indirect IFA, seven of 16 (44%) ill persons tested had both IgM and IgG antibodies reacting with one or more Rickettsia spp antigens; the other nine (56%) had only IgM titers or were seronegative. Antibodies to SFG protein and lipopolysaccharide were detected by Western blotting with antigens of Rickettsia typhi, Rickettsia rickettsii, and Rickettsia akari. Only one sample, from an ill person who died, tested positive by PCR for a SFG Rickettsia. PCR analysis of Amblyomma cajennense ticks from domestic animals in the area detected the presence of SFG Rickettsia DNA in one of 12 ticks collected. CONCLUSIONS: Further studies in Guatemala are warranted to establish the prevalence of rickettsioses and to fully characterize the identity of the etiologic agents and vectors.

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.

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.

Rickettsia felis is an emerging human pathogen associated primarily with the cat flea Ctenocephalides felis. In this study, we investigated the presence of Rickettsia felis in C. felis from Guatemala and Costa Rica. Ctenocephalides felis were collected directly from dogs and cats, and analyzed by polymerase chain reaction for Rickettsia-specific fragments of 17-kDa protein, OmpA, and citrate synthase genes. Rickettsia DNA was detected in 64% (55 of 86) and 58% (47 of 81) of flea pools in Guatemala and Costa Rica, respectively. Sequencing of gltA fragments identified R. felis genotype URRWXCal(2) in samples from both countries, and genotype Rf2125 in Costa Rica. This is the first report of R. felis in Guatemala and of genotype Rf2125 in Costa Rica. The extensive presence of this pathogen in countries of Central America stresses the need for increased awareness and diagnosis.

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.

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.

A recurrent focus of Rhipicephalus sanguineus infestation was investigated in a suburban area of southern California after reports of suspected Rocky Mountain spotted fever in two dogs on the same property. Abundant quantities of Rh. sanguineus were collected on the property and repeatedly from each dog, and Rickettsia massiliae DNA was detected by polymerase chain reaction (PCR). Whole blood and serum samples from four dogs were tested by using PCR and microimmunofluorescent assay for antibodies against spotted fever group rickettsiae. Serum samples from all four dogs contained antibodies reactive with R. massiliae, R. rhipicephali, R. rickettsii, and 364D Rickettsia but no rickettsial DNA was detected by PCR of blood samples. Serum cross-absorption and Western blot assays implicated R. massiliae as the most likely spotted fever group rickettsiae responsible for seropositivity. To our knowledge, this is the first detection of R. massiliae in ticks in California.

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.

To the Editor: A spay-neuter (sterilization) program for feral cats from Basseterre, the capital of the Caribbean Island St. Kitts, found that most (45/58; 66%) cats had antibodies to spotted fever group rickettsiae (SFGR). The antibodies were detected with Rickettsia rickettsii antigen in a standard microimmunofluorescence assay (1). Titers for 13 (20%) cats were ≥320.

Rickettsia conorii, the etiologic agent of Mediterranean spotted fever is widely distributed in Southern Europe, the Middle East, Africa, India and the Caspian region. In the Mediterranean region, the brown dog tick, Rhipicephalus sanguineus, is the recognized vector of R. conorii. To study tick-pathogen relationships and pathogenesis of infection caused in model animals by the bite of an infected tick, we attempted to establish a laboratory colony of Rh. sanguineus persistently infected with R. conorii. Rhipicephalus sanguineus ticks of North American and Mediterranean origin were exposed to R. conorii isolates of African (R. conorii conorii strain Malish) and Mediterranean (R. conorii israelensis strain ISTT) origin. Feeding of ticks upon infected mice and dogs, intra-hemocoel inoculation, and submersion in suspensions of purified rickettsiae were used to introduce the pathogen into uninfected ticks. Feeding success, molting success and the longevity of molted ticks were measured to assess the effects of R. conorii on the survival of Rh. sanguineus. In concordance with previously published results, Rh. sanguineus larvae and nymphs from both North American and Mediterranean colonies exposed to R. conorii conorii Malish experienced high mortality during feeding and molting or immediately after. The prevalence of infection in surviving ticks did not exceed 5%. On the other hand, exposure to ISTT strain had lesser effect on tick survival and resulted in 35-66% prevalence of infection. Rh. sanguineus of Mediterranean origin were more susceptible to infection with either strain of R. conorii than those from North America. Previous experimental studies had demonstrated transovarial and transstadial transmission of R. conorii in Rh. sanguineus; however, our data suggest that different strains of R. conorii may employ different means of maintenance in nature. The vertebrate host may be a more important reservoir than previously thought, or co-feeding transmission between different generations of ticks may obviate or lessen the requirement for transovarial maintenance of R. conorii.