Bacterial zoonoses are established causes of severe febrile illness in East Africa. Within a fever etiology study, we applied a high-throughput 16S rRNA metagenomic assay validated for detecting bacterial zoonotic pathogens. We enrolled febrile patients admitted to 2 referral hospitals in Moshi, Tanzania, during September 2007-April 2009. Among 788 participants, median age was 20 (interquartile range 2-38) years. We performed PCR amplification of V1-V2 variable region 16S rRNA on cell pellet DNA, then metagenomic deep-sequencing and pathogenic taxonomic identification. We detected bacterial zoonotic pathogens in 10 (1.3%) samples: 3 with Rickettsia typhi, 1 R. conorii, 2 Bartonella quintana, 2 pathogenic Leptospira spp., and 1 Coxiella burnetii. One other sample had reads matching a Neoerhlichia spp. previously identified in a patient from South Africa. Our findings indicate that targeted 16S metagenomics can identify bacterial zoonotic pathogens causing severe febrile illness in humans, including potential novel agents.

Francisella tularensis is the causative agent of tularemia. We tested the susceptibility of 278 F. tularensis isolates from the United States received during 2009-2018 to 8 antimicrobial drugs (ciprofloxacin, levofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, chloramphenicol, and erythromycin). All isolates were susceptible to all tested drugs.

BACKGROUND: Certain associations observed in the National Birth Defects Prevention Study (NBDPS) contrasted with other research or were from areas with mixed findings, including no decrease in odds of spina bifida with periconceptional folic acid supplementation, moderately increased cleft palate odds with ondansetron use and reduced hypospadias odds with maternal smoking. OBJECTIVES: To investigate the plausibility and extent of differential participation to produce effect estimates observed in NBDPS. METHODS: We searched the literature for factors related to these exposures and participation and conducted deterministic quantitative bias analyses. We estimated case-control participation and expected exposure prevalence based on internal and external reports, respectively. For the folic acid-spina bifida and ondansetron-cleft palate analyses, we hypothesized the true odds ratio (OR) based on prior studies and quantified the degree of exposure over- (or under-) representation to produce the crude OR (cOR) in NBDPS. For the smoking-hypospadias analysis, we estimated the extent of selection bias needed to nullify the association as well as the maximum potential harmful OR. RESULTS: Under our assumptions (participation, exposure prevalence, true OR), there was overrepresentation of folic acid use and underrepresentation of ondansetron use and smoking among participants. Folic acid-exposed spina bifida cases would need to have been ≥1.2× more likely to participate than exposed controls to yield the observed null cOR. Ondansetron-exposed cleft palate cases would need to have been 1.6× more likely to participate than exposed controls if the true OR is null. Smoking-exposed hypospadias cases would need to have been ≥1.2 times less likely to participate than exposed controls for the association to falsely appear protective (upper bound of selection bias adjusted smoking-hypospadias OR = 2.02). CONCLUSIONS: Differential participation could partly explain certain associations observed in NBDPS, but questions remain about why. Potential impacts of other systematic errors (e.g. exposure misclassification) could be informed by additional research.

Tick-borne relapsing fever (TBRF) spirochetes are likely an overlooked cause of disease in Latin America. In Panama, the pathogens were first reported to cause human disease in the early 1900s. Recent collections of Ornithodoros puertoricensis from human dwellings in Panama prompted our interest to determine whether spirochetes still circulate in the country. Ornithodoros puertoricensis ticks were collected at field sites around the City of Panama. In the laboratory, the ticks were determined to be infected with TBRF spirochetes by transmission to mice, and we report the laboratory isolation and genetic characterization of a species of TBRF spirochete from Panama. Since this was the first isolation of a species of TBRF spirochete from Central America, we propose to designate the bacteria as Borrelia puertoricensis sp. nov. This is consistent with TBRF spirochete species nomenclature from North America that are designated after their tick vector. These findings warrant further investigations to assess the threat B. puertoricensis sp. nov. may impose on human health.

A previously healthy 42-year-old man from the upper midwestern USA presented with a 1-day history of fever, fatigue, headache, myalgias and arthralgias. He reported removing a ‘wood tick’ the day of admission. His temperature was 38.4 °C. No swollen joints, rash or neurological abnormalities were noted on physical examination. | | Testing revealed lymphopenia (0.24 × 109/L; reference range 0.95 × 109/L to 3.07 × 109/L) and slightly elevated alanine transaminase (59 U/L; reference range 7–55 U/L). Tick-borne pathogen PCR on blood for Borrelia burgdorferi sensu lato [1], Anaplasma phagocytophilum, Borrelia miyamotoi, Ehrlichia and Babesia species was positive only for Borrelia mayonii. Several spirochaetes were observed on Giemsa-stained thin blood films (Fig. 1). Borrelia mayonii spirochaetes were recovered in culture initiated with 0.5 mL of blood (see Supplementary material, Video S1), with subsequent confirmation by genome sequencing. The patient was treated with an initial dose of intravenous ceftriaxone and doxycycline, followed by 21 days of oral doxycycline and experienced resolution of symptoms.

During a recent outbreak of Bartonella quintana disease in Denver, 15% of 241 persons experiencing homelessness who presented for severe acute respiratory syndrome coronavirus 2 testing were seroreactive for Bartonella. Improved recognition of B quintana disease and prevention of louse infestation are critical for this vulnerable population.

Bacterial vector-borne diseases, including Borrelia species, present a significant diagnostic, clinical, and public health challenge due to their overlapping symptoms and the breadth of causative agents and arthropod vectors. The relapsing fever (RF) borreliae encompass both established and emerging pathogens and are transmitted to humans by soft ticks, hard ticks, or lice. We developed a real-time semi-multiplex PCR assay that detects multiple RF borreliae causing human illness and classifies them into one of three groups. The groups are based on genetic similarity and include agents of soft-tick relapsing fever (B. hermsii and others), the emerging hard tick transmitted pathogen B. miyamotoi, and the agent of louse-borne relapsing fever (B. recurrentis) The real-time PCR assay uses a single primer pair designed to amplify all known pathogenic RF borreliae, and multiple TaqMan probes to allow for detection of and differentiation among the three groups. The assay detects all RF borreliae tested with an analytical limit of detection below 15 genome equivalents per reaction. Thirty isolates of RF borreliae encompassing six species were accurately identified. Thirty-nine of 41 residual specimens (EDTA whole blood, serum, or plasma) from patients with RF were detected and correctly classified. None of 42 clinical samples from patients with other infections and 46 culture specimens from non-RF bacteria were detected. The development of a single assay real-time PCR approach will help to improve diagnosis of RF by simplifying the selection of tests to aid in clinical management of acutely ill RF patients.

Reported cases of tick-borne diseases have steadily increased for more than a decade. In the United States, a majority of tick-borne infections are caused by bacteria. Clinical diagnosis may be challenging as tick-borne diseases can present with similar symptoms. Laboratory diagnosis has historically relied on serologic methods, which have limited utility during the acute phase of disease. Pathogen-specific molecular methods have improved early diagnosis, but can be expensive when bundled together and miss unexpected or novel pathogens. To address these shortcomings, we developed a 16S ribosomal RNA (rRNA) gene PCR with next-generation sequencing approach to detect tick-borne bacteria in whole blood. A workflow was optimized by comparing combinations of two extractions platforms and two primer sets, ultimately pursuing DNA extraction from blood with the MagNA Pure 96 and PCR amplification using dual-priming oligonucleotide primers specific to the V1-V3 region of the 16S rRNA gene. The amplified product underwent modified Illumina 16S metagenomics sequencing library preparation and sequencing on a MiSeq V2 Nano flow cell, with data analysis using Pathogenomix RipSeq NGS software. Results with the developed method were compared to those from a V1-V2 16S rRNA gene primer set described by the Centers for Disease Control and Prevention (CDC). The V1-V3 assay demonstrated equivalent performance to the CDC assay, with each method showing concordance with targeted PCR results in 31 of 32 samples, and detecting 22 of 23 expected organisms. These data demonstrate the potential for using a broad-range bacterial detection approach for diagnosis of tick-borne bacterial infection from blood.

Borrelia spirochetes are the causative agents of Lyme borreliosis (LB) and relapsing fever (RF). Despite the steady rise in infections and the identification of new species causing human illness over the last decade, isolation of borreliae in culture has become increasingly rare. A modified Barbour-Stoenner-Kelly (BSK) media formulation, BSK-R, was developed for isolation of the emerging RF pathogen, Borrelia miyamotoi. BSK-R is a diluted BSK-II derivative supplemented with Lebovitz's L-15, mouse and fetal calf serum. Decreasing the concentration of CMRL 1066 and other components was essential for growth of North American B. miyamotoi. Sixteen B. miyamotoi isolates, originating from Ixodes scapularis ticks, rodent and human blood collected in the eastern and upper midwestern United States, were isolated and propagated to densities > 10(8) spirochetes/mL. Growth of five other RF and ten different LB borreliae readily occurred in BSK-R. Additionally, primary culture recovery of 20 isolates of Borrelia hermsii, Borrelia turicatae, Borrelia burgdorferi and Borrelia mayonii was achieved in BSK-R using whole blood from infected patients. These data indicate this broadly encompassing borreliae media can aid in in vitro culture recovery of RF and LB spirochetes, including the direct isolation of new and emerging human pathogens.

Here, we present the 2,139,666-bp circular chromosome of Francisella sp. strain LA11-2445 (FDC406), a proposed novel species of Francisella that was isolated from a human cutaneous lesion and is related to Francisella species from marine environments.

The western blacklegged tick, Ixodes pacificus, an important vector in the western United States of two zoonotic spirochetes: Borrelia burgdorferi (also called Borreliella burgdorferi), causing Lyme disease, and Borrelia miyamotoi, causing a relapsing fever-type illness. Human cases of Lyme disease are well-documented in California, with increased risk in the north coastal areas and western slopes of the Sierra Nevada range. Despite the established presence of B. miyamotoi in the human-biting I. pacificus tick in California, clinical cases with this spirochete have not been well studied. To assess exposure to B. burgdorferi and B. miyamotoi in California, and to address the hypothesis that B. miyamotoi exposure in humans is similar in geographic range to B. burgdorferi, 1,700 blood donor sera from California were tested for antibodies to both pathogens. Sampling was from high endemic and low endemic counties for Lyme disease in California. All sera were screened using the C6 ELISA. All C6 positive and equivocal samples and nine randomly chosen C6 negative samples were further analyzed for B. burgdorferi antibody using IgG western blot and a modified two ELISA test system and for B. miyamotoi antibody using the GlpQ ELISA and B. miyamotoi whole cell sonicate western blot. Of the 1,700 samples tested in series, eight tested positive for antibodies to B. burgdorferi (0.47%, Exact 95% CI: 0.20, 0.93) and two tested positive for antibodies to B. miyamotoi (0.12%, Exact 95% CI: 0.01, 0.42). There was no statistically significant difference in seroprevalence for either pathogen between high and low Lyme disease endemic counties. Our results confirm a low frequency of Lyme disease and an even lower frequency of B. miyamotoi exposure among adult blood donors in California; however, our findings reinforce public health messaging that there is risk of infection by these emerging diseases in the state.

Plague, an acute zoonosis caused by Yersinia pestis, is endemic in the West Nile region of northwestern Uganda and neighboring northeastern Democratic Republic of the Congo (DRC) (1-4). The illness manifests in multiple clinical forms, including bubonic and pneumonic plague. Pneumonic plague is rare, rapidly fatal, and transmissible from person to person via respiratory droplets. On March 4, 2019, a patient with suspected pneumonic plague was hospitalized in West Nile, Uganda, 4 days after caring for her sister, who had come to Uganda from DRC and died shortly thereafter, and 2 days after area officials received a message from a clinic in DRC warning of possible plague. The West Nile-based Uganda Virus Research Institute (UVRI) plague program, together with local health officials, commenced a multipronged response to suspected person-to-person transmission of pneumonic plague, including contact tracing, prophylaxis, and education. Plague was laboratory-confirmed, and no additional transmission occurred in Uganda. This event transpired in the context of heightened awareness of cross-border disease spread caused by ongoing Ebola virus disease transmission in DRC, approximately 400 km to the south. Building expertise in areas of plague endemicity can provide the rapid detection and effective response needed to mitigate epidemic spread and minimize mortality. Cross-border agreements can improve ability to respond effectively.

Two isolates of a Gram-negative, non-spore-forming coccobacillus cultured from the blood and cerebrospinal fluid of immunocompromised patients in the United States were described previously. Biochemical and phylogenetic analyses revealed that they belong to a novel species within the Francisella genus. Here we describe a third isolate of this species, recovered from blood of a febrile patient with renal failure, and formally name the Francisella species. Whole genome comparisons indicated the three isolates display greater than 99.9 % average nucleotide identity (ANI) to each other and are most closely related to the tick endosymbiont F. persica, with only 88.6-88.8 % ANI to the type strain of F. persica. Based on biochemical, metabolic and genomic comparisons, we propose that these three isolates should be recognized as Francisella opportunistica sp. nov, with the type strain of the species, PA05-1188(T), available through the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 107100) and the American Type Culture Collection (ATCC BAA-2974).

BACKGROUND: Women with a previous neural tube defect (NTD)-affected pregnancy are recommended to consume 4,000 mug daily folic acid (FA) for prevention (10 times the general-population recommendation). Protection from doses between 400 and 4,000 mug for this and other higher risk groups is unclear. METHODS: In the case-control Slone Birth Defects Study (1988-2015), we examined the associations between periconceptional FA doses and NTDs among four higher risk groups: NTD family history, periconceptional antiepileptic drug exposure (AED), pregestational diabetes, and prepregnancy obesity. Mothers completed standardized interviews about pregnancy events and exposures. FA categorizations were based on (a) supplements only and (b) supplements and diet ("total folate"). We estimated odds ratios (ORs) and 95% confidence intervals (CIs) (adjusted for age and study center) using logistic regression. RESULTS: Cases and controls included: 45 and 119 with family history, 25 and 108 with AED exposure, 12 and 63 with pregestational diabetes, 111 and 1,243 with obesity. Daily FA supplementation was associated with lower NTD risk compared to no supplementation (adjusted ORs were 0.33 [95% CI 0.13, 0.76] for family history, 0.31 [0.09, 0.95] for AED exposure, 0.25 [0.04, 1.05] for pregestational diabetes, 0.65 [0.40, 1.04] for obesity). Though estimates were imprecise, as total folate increased stronger point estimates were observed, notably among family history. No mothers with a prior NTD-affected pregnancy supplemented with 4,000 mug. CONCLUSIONS: Our findings reinforce that all women of childbearing potential should consume at least 400 mug FA/day to protect against NTDs. Higher risk groups may benefit from higher doses.

In July 2017, fever and sepsis developed in 3 recipients of solid organs (1 heart and 2 kidneys) from a common donor in the United States; 1 of the kidney recipients died. Tularemia was suspected only after blood cultures from the surviving kidney recipient grew Francisella species. The organ donor, a middle-aged man from the southwestern United States, had been hospitalized for acute alcohol withdrawal syndrome, pneumonia, and multiorgan failure. F. tularensis subsp. tularensis (clade A2) was cultured from archived spleen tissue from the donor and blood from both kidney recipients. Whole-genome multilocus sequence typing indicated that the isolated strains were indistinguishable. The heart recipient remained seronegative with negative blood cultures but had been receiving antimicrobial drugs for a medical device infection before transplant. Two lagomorph carcasses collected near the donor's residence were positive by PCR for F. tularensis subsp. tularensis (clade A2). This investigation documents F. tularensis transmission by solid organ transplantation.

Tularemia is a bacterial zoonosis caused by Francisella tularensis. We conducted a serosurvey of black-tailed prairie dogs ( Cynomys ludovicianus) in Devils Tower National Monument, Wyoming, following an epizootic in voles ( Microtus spp.) due to F. tularensis. Only 1 of 44 (2%) sampled prairie dogs was seropositive for F. tularensis, providing evidence of survival and potentially limited spread among free-ranging prairie dogs.

BACKGROUND: Due to their close relationship with the environment, Alaskans are at risk for zoonotic pathogen infection. One way to assess a population's disease burden is to determine the seroprevalence of pathogens of interest. The objective of this study was to determine the seroprevalence of 11 zoonotic pathogens in people living in Alaska. METHODS: In a 2007 avian influenza exposure study, we recruited persons with varying wild bird exposures. Using sera from this study, we tested for antibodies to Cryptosporidium spp., Echinococcus spp., Giardia intestinalis, Toxoplasma gondii, Trichinella spp., Brucella spp., Coxiella burnetii, Francisella tularensis, California serogroup bunyaviruses, and hepatitis E virus (HEV). RESULTS: Eight hundred eighty-seven persons had sera tested, including 454 subsistence bird hunters and family members, 160 sport bird hunters, 77 avian wildlife biologists, and 196 persons with no wild bird exposure. A subset (n = 481) of sera was tested for California serogroup bunyaviruses. We detected antibodies to 10/11 pathogens. Seropositivity to Cryptosporidium spp. (29%), California serotype bunyaviruses (27%), and G. intestinalis (19%) was the most common; 63% (301/481) of sera had antibodies to at least one pathogen. Using a multivariable logistic regression model, Cryptosporidium spp. seropositivity was higher in females (35.7% vs. 25.0%; p = 0.01) and G. intestinalis seropositivity was higher in males (21.8% vs. 15.5%; p = 0.02). Alaska Native persons were more likely than non-Native persons to be seropositive to C. burnetii (11.7% vs. 3.8%; p = 0.005) and less likely to be seropositive to HEV (0.4% vs. 4.1%; p = 0.01). Seropositivity to Cryptosporidium spp., C. burnetii, HEV, and Echinococcus granulosus was associated with increasing age (p </= 0.01 for all) as was seropositivity to >/=1 pathogen (p < 0.0001). CONCLUSION: Seropositivity to zoonotic pathogens is common among Alaskans with the highest to Cryptosporidium spp., California serogroup bunyaviruses, and G. intestinalis. This study provides a baseline for use in assessing seroprevalence changes over time.

We aimed to investigate associations between individual and concurrent (>/=2) intakes of one-carbon cofactors vitamins B6 and B12, choline, betaine, and methionine and neural tube defect (NTD) outcomes among mothers meeting the folic acid recommendations. In the Slone Birth Defects Study (case-control design; North America, 1998-2015), mothers of 164 NTD cases and 2,831 nonmalformed controls completed food frequency questionnaires and structured interviews. Estimated intakes of one-carbon cofactors were dichotomized (high vs. low) for all except betaine (low or middle vs. high). We used logistic regression models to estimate odds ratios and 95% confidence intervals adjusted for center, age, and race. The analysis was restricted to mothers with estimated daily total folate intake of >/=400 mug during periconception. Fewer cases, compared with controls, had high intakes for each one-carbon cofactor except betaine, where the starkest contrast occurred in the middle group. Women with concurrent high intakes of B6, B12, choline, and methionine and moderate intake of betaine had approximately half the risk of an NTD-affected pregnancy (odds ratio = 0.49, 95% confidence interval: 0.23, 1.08). These findings suggest that, in the presence of folic acid, one-carbon cofactors-notably when consumed together-might reduce NTD risk. Additional research should inform any changes to clinical recommendations.

Yersinia pestis is the causative agent of plague, and is considered a category A priority pathogen due to its potential for high transmissibility and the significant morbidity and mortality it causes in humans. Y. pestis is endemic to the Western United States and much of the world, necessitating programs to monitor for this pathogen on the landscape. Elevated human risk of plague infection has been spatially correlated with spikes in seropositive wildlife numbers, particularly rodent-eating carnivores which are frequently in contact with the enzootic hosts and the associated arthropod vectors of Y. pestis In this study, we describe a semi-automated bead-based flow cytometric assay developed for plague monitoring in wildlife called the F1-Luminex Plague Assay (F1-LPA). Based upon Luminex/Bio-Plex technology, the F1-LPA targets serological responses to the F1 capsular antigen of Y. pestis and was optimized to analyze antibodies eluted from wildlife blood samples preserved on Nobuto filter paper strips. In comparative evaluations using wildlife samples with passive hemagglutination, the gold standard tool for wildlife plague serodiagnosis, the F1-LPA demonstrated as much as 64x improvement in analytical sensitivity to F1-specific IgG detection, and allowed for unambiguous classification of IgG status. The functionality of the F1-LPA was demonstrated for coyotes and other canids, which are the primary sentinels in wildlife plague monitoring, as well as felids and raccoons. Additionally, assay formats that do not require species-specific immunological reagents, which are not routinely available for several wildlife species used in plague monitoring, were determined to be functional in the F1-LPA.

Background: Tick-transmitted Borrelia species fall into two heterogeneous bacterial complexes comprised of multiple species, the relapsing fever (RF) group and the Borrelia burgdorferi sensu lato group, which are the causative agents of Lyme borreliosis (LB), the most common tickborne disease in the northern hemisphere. Geographic expansion of human LB in the United States and discovery of emerging Borrelia pathogens underscores the importance of surveillance for disease causing Borrelia. Methods: De-identified clinical specimens, submitted by providers throughout the United States, for patients suspected of LB, anaplasmosis, ehrlichiosis, or babesiosis, were screened using a Borrelia genus level TaqMan PCR. Borrelia species and sequence types (STs) were characterized by multi-locus sequence typing (MLST) utilizing next generation sequencing. Results: Among the 7,292 tested specimens tested, five different Borrelia species were identified: two causing LB, B. burgdorferi (n=25) and B. mayonii (n=9), and three RF borreliae, B. hermsii (n=1), B. miyamotoi (n=8), and CandidatusB. johnsonii (n=1), a species previously detected only in the bat tick, Carios kelleyi. ST diversity was greatest for B. burgdorferi positive specimens, with new STs identified primarily among synovial fluids. Conclusion: These results demonstrate broad PCR screening followed by MLST is a powerful surveillance tool for uncovering the spectrum of Borrelia species causing human disease, improving understanding of their geographic distribution, and investigating the correlation between B. burgdorferi STs and joint involvement. Detection of CandidatusB. johnsonii in a patient with suspected tickborne disease suggests this species may be a previously undetected cause of illness in humans with exposure to bat ticks.

Plague is a zoonotic disease (transmitted mainly by fleas and maintained in nature by rodents) that causes severe acute illness in humans. We present a human plague case who became infected by the bite of a wild Gunnison's prairie dog, and a good practical example of the One Health approach that resulted in a rapid public health response. The exposure occurred while the animal was being transported for relocation to a wildlife refuge after being trapped in a plague enzootic area. This is the first report of a human plague case resulting from the bite of a Gunnison's prairie dog. Additionally, we present an observation of a longer incubation period for plague in captive prairie dogs, leading to a recommendation for a longer quarantine period for prairie dogs during translocation efforts.

A total of 6,255 ticks belonging to three genera and six species (Haemaphysalis flava Neumann, Haemaphysalis longicornis Neumann, Haemaphysalis phasiana Saito, Ixodes nipponensis Kitaoka & Saito, Ixodes persulcatus Schulze, and Amblyomma testudinarium Koch) collected from May-August, 2013, at four southwestern provinces in the Republic of Korea (ROK) were submitted to the Armed Forces Research Institute of Medical Sciences and assayed for selected tick-borne pathogens. One pool each of H. flava and H. phasiana was positive by PCR and sequencing for a Francisella-like endosymbiont, while all pools were negative for Francisella tularensis, the causative agent of tularemia.

Borrelia mayonii is a newly described member of the Borrelia burgdorferi sensu lato complex that is vectored by the black-legged tick (Ixodes scapularis Say) and a cause of Lyme disease in Minnesota and Wisconsin. Vertebrate reservoir hosts involved in the enzootic maintenance of B. mayonii have not yet been identified. Here, we describe the first isolation of B. mayonii from naturally infected white-footed mice (Peromyscus leucopus Rafinesque) and an American red squirrel (Tamiasciurus hudsonicus Erxleben) from Minnesota, thus implicating these species as potential reservoir hosts for this newly described spirochete.

Borrelia miyamotoi, of the relapsing-fever spirochete group, is an emerging tick-borne pathogen causing human illness in the northern hemisphere. Here, we present the chromosome, eight extrachromosomal linear plasmids, and a draft sequence for five circular and one linear plasmid of a Borrelia miyamotoi strain isolated from an Ixodes sp. tick from Connecticut, USA.

Borrelia mayonii, a Borrelia burgdorferi sensu lato (Bbsl) genospecies, was recently identified as a cause of Lyme borreliosis (LB) among patients from the upper midwestern United States. By microscopy and PCR, spirochete/genome loads in infected patients were estimated at 105 to 106 per milliliter of blood. Here, we present the full chromosome and plasmid sequences of two B. mayonii isolates, MN14-1420 and MN14-1539, cultured from blood of two of these patients. Whole genome sequencing and assembly was conducted using PacBio long read sequencing (Pacific Biosciences RSII instrument) followed by hierarchical genome-assembly process (HGAP). The B. mayonii genome is ~1.31 Mbp in size (26.9% average GC content) and is comprised of a linear chromosome, 8 linear and 7 circular plasmids. Consistent with its taxonomic designation as a new Bbsl genospecies, the B. mayonii linear chromosome shares only 93.83% average nucleotide identity with other genospecies. Both B. mayonii genomes contain plasmids similar to B. burgdorferi sensu stricto lp54, lp36, lp28-3, lp28-4, lp25, lp17, lp5, 5 cp32s, cp26, and cp9. The vls locus present on lp28-10 of B. mayonii MN14-1420 is remarkably long, being comprised of 24 silent vls cassettes. Genetic differences between the two B. mayonii genomes are limited and include 15 single nucleotide variations as well as 7 fewer silent vls cassettes and a lack of the lp5 plasmid in MN14-1539. Notably, 68 homologs to proteins present in B. burgdorferi sensu stricto appear to be lacking from the B. mayonii genomes. These include the complement inhibitor, CspZ (BB_H06), the fibronectin binding protein, BB_K32, as well as multiple lipoproteins and proteins of unknown function. This study shows the utility of long read sequencing for full genome assembly of Bbsl genomes, identifies putative genome regions of B. mayonii that may be linked to clinical manifestation or tissue tropism, and provides a valuable resource for pathogenicity, diagnostic and vaccine studies.

Lyme borreliosis (LB) is a multisystem disease caused by spirochetes in the Borrelia burgdorferisensu lato (Bbsl) genospecies complex. We previously described a novel Bbsl genospecies (type strain MN14-1420T) that causes LB among patients with exposures to ticks in the upper midwestern USA. Patients infected with the novel Bbsl genospecies demonstrated higher levels of spirochetemia and somewhat differing clinical symptoms as compared with those infected with other Bbsl genospecies. The organism was detected from human specimens using PCR, microscopy, serology and culture. The taxonomic status was determined using an eight-housekeeping-gene (uvrA, rplB, recG, pyrG, pepX, clpX, clpA and nifS) multi-locus sequence analysis (MLSA) and comparison of 16S rRNA gene, flaB, rrf-rrl, ospC and oppA2 nucleotide sequences. Using a system threshold of 98.3 % similarity for delineation of Bbsl genospecies by MLSA, we demonstrated that the novel species is a member of the Bbsl genospecies complex, most closely related to B. burgdorferisensu stricto (94.7-94.9 % similarity). This same species was identified in Ixodes scapularis ticks collected in Minnesota and Wisconsin. This novel species, Borrelia mayonii sp. nov, is formally described here. The type strain, MN14-1420, is available through the Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH (DSM 102811) and the American Type Culture Collection (ATCC BAA-2743).

Francisella tularensis (Ft) is a highly virulent zoonotic pathogen that causes tularemia, and because of weaponization efforts in past world wars, is considered a Tier 1 biothreat agent. Detection and surveillance of Ft may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 17 new Francisella genomes and a comparison of their characteristics to each other and to 14 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features - for example, when some are virulent pathogens in humans and animals, while others are non-pathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, LPS biosynthesis, pathogenicity island) and whole genome comparisons (nucleotide and protein) gave congruent results, but with different discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisellas IMPORTANCE: DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near-relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics and environmental surveillance, where response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisellas identified in clinical samples or environmental surveys.

BACKGROUND: Plague is a life-threatening disease caused by the bacterium, Yersinia pestis. Since the 1990s, Africa has accounted for the majority of reported human cases. In Uganda, plague cases occur in the West Nile region, near the border with Democratic Republic of Congo. Despite the ongoing risk of contracting plague in this region, little is known about Y. pestis genotypes causing human disease. METHODOLOGY/PRINCIPAL FINDINGS: During January 2004-December 2012, 1,092 suspect human plague cases were recorded in the West Nile region of Uganda. Sixty-one cases were culture-confirmed. Recovered Y. pestis isolates were analyzed using three typing methods, single nucleotide polymorphisms (SNPs), pulsed field gel electrophoresis (PFGE), and multiple variable number of tandem repeat analysis (MLVA) and subpopulations analyzed in the context of associated geographic, temporal, and clinical data for source patients. All three methods separated the 61 isolates into two distinct 1.ANT lineages, which persisted throughout the 9 year period and were associated with differences in elevation and geographic distribution. CONCLUSIONS/SIGNIFICANCE: We demonstrate that human cases of plague in the West Nile region of Uganda are caused by two distinct 1.ANT genetic subpopulations. Notably, all three typing methods used, SNPs, PFGE, and MLVA, identified the two genetic subpopulations, despite recognizing different mutation types in the Y. pestis genome. The geographic and elevation differences between the two subpopulations is suggestive of their maintenance in highly localized enzootic cycles, potentially with differing vector-host community composition. This improved understanding of Y. pestis subpopulations in the West Nile region will be useful for identifying ecologic and environmental factors associated with elevated plague risk.

BACKGROUND: Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. It is a multisystem disease caused by Borrelia burgdorferi sensu lato genospecies and characterised by tissue localisation and low spirochaetaemia. In this study we aimed to describe a novel Borrelia species causing Lyme borreliosis in the USA. METHODS: At the Mayo clinic, from 2003 to 2014, we tested routine clinical diagnostic specimens from patients in the USA with PCR targeting the oppA1 gene of B burgdorferi sensu lato. We identified positive specimens with an atypical PCR result (melting temperature outside of the expected range) by sequencing, microscopy, or culture. We collected Ixodes scapularis ticks from regions of suspected patient tick exposure and tested them by oppA1 PCR. FINDINGS: 100 545 specimens were submitted by physicians for routine PCR from Jan 1, 2003 to Sept 30, 2014. From these samples, six clinical specimens (five blood, one synovial fluid) yielded an atypical oppA1 PCR product, but no atypical results were detected before 2012. Five of the six patients with atypical PCR results had presented with fever, four had diffuse or focal rash, three had symptoms suggestive of neurological inclusion, and two were admitted to hospital. The sixth patient presented with knee pain and swelling. Motile spirochaetes were seen in blood samples from one patient and cultured from blood samples from two patients. Among the five blood specimens, the median oppA1 copy number was 180 times higher than that in 13 specimens that tested positive for B burgdorferi sensu stricto during the same time period. Multigene sequencing identified the spirochaete as a novel B burgdorferi sensu lato genospecies. This same genospecies was detected in ticks collected at a probable patient exposure site. INTERPRETATION: We describe a new pathogenic Borrelia burgdorferi sensu lato genospecies (candidatus Borrelia mayonii) in the upper midwestern USA, which causes Lyme borreliosis with unusually high spirochaetaemia. Clinicians should be aware of this new B burgdorferi sensu lato genospecies, its distinct clinical features, and the usefulness of oppA1 PCR for diagnosis.

Human plague is a severe and often fatal zoonotic disease caused by Yersinia pestis. For public health investigations of human cases, nonintensive whole genome molecular typing tools, capable of defining epidemiologic relationships, are advantageous. Whole genome multilocus sequence typing (wgMLST) is a recently developed methodology that simplifies genomic analyses by transforming millions of base pairs of sequence into character data for each gene. We sequenced 13 US Y. pestis isolates with known epidemiologic relationships. Sequences were assembled de novo, and multilocus sequence typing alleles were assigned by comparison against 3979 open reading frames from the reference strain CO92. Allele-based cluster analysis accurately grouped the 13 isolates, as well as 9 publicly available Y. pestis isolates, by their epidemiologic relationships. Our findings indicate wgMLST is a simplified, sensitive, and scalable tool for epidemiologic analysis of Y. pestis strains.