This study reports on the validation of a real-time polymerase chain reaction test targeting the vomp region of Bartonella quintana. The assay displayed 100% sensitivity and specificity for the 52 bloods and 159 cultures tested. Molecular diagnosis of Bartonella quintana can aid clinical treatment during acute infection.

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.

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.

Introduction: Tularemia is a zoonotic infection caused by the highly infectious bacterium Francisella tularensis. Persons having outdoor professions are more likely than others to be exposed to F. tularensis through increased contact with arthropods, infected animals, and contaminated aerosols. Materials and Methods: After a tularemia epizootic during July and August 2015 at Devils Tower National Monument and an associated tularemia infection in a park employee, we assessed seroprevalence of F. tularensis antibodies, risk factors for F. tularensis seropositivity, and use of protective measures among park employees. Results: Seroprevalence among participating employees was 13% (3/23). Seropositive employees reported multiple risk factors for F. tularensis exposure through both job-related and recreational activities. Activities reported by more seropositive than seronegative employees included using a power blower (67% vs. 5%, p = 0.03), collecting animal carcasses (100% vs. 30%, p = 0.047), and hunting prairie dogs recreationally (67% vs. 5%, p = 0.03). Seropositive employees reported exposure to more ticks (median 30, range 25-35) than seronegative employees (median 6, range 0-25, p = 0.001). Most employees used protective measures (e.g., insect repellent) inconsistently but increased use after receiving educational materials. Conclusions: Educating and enabling at-risk employees to use protective measures consistently, both at work and during recreational activities, can reduce exposure during epizootics.

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, US, 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.

ABSTRACT 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 semiautomated 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 with passive hemagglutination, the gold standard tool for wildlife plague serodiagnosis, the F1-LPA demonstrated as much as 64× improvement in analytical sensitivity for 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.

Multiple publications have reported the use of coyotes (Canis latrans) in animalbased surveillance efforts for the detection of Yersinia pestis. Coyotes are likely exposed via flea bite or oral routes and are presumed to be resistant to the development of clinical disease. These historic data have only been useful for the evaluation of the geographic distribution of Y. pestis in the landscape. Because the canid immunologic response to Y. pestis has not been thoroughly characterized, we conducted experimental inoculation of captive-reared, juvenile coyotes (n58) with Y. pestis CO92 via oral or intradermal routes. We measured the humoral response to Y. pestis fraction 1 capsular protein (anti-F1) and found a significant difference between inoculation groups in magnitude and duration of antibody production. The anti-F1 titers in animals exposed intradermally peaked at day 10 postinoculation (PI; range51:32 to 1:128) with titers remaining stable at 1:32 through week 12. In contrast, orally inoculated animals developed higher titers (range51:256 to 1:1,024) that remained stable at 1:256 to 1:512 through week 6. No clinical signs of disease were observed, and minimal changes were noted in body temperature, white blood cell counts, and acute phase proteins during the 7 days PI. Gross pathology was unremarkable, and minimal changes were noted in histopathology at days 3 and 7 PI. Rechallenge at 14 wk PI via similar dosage and routes resulted in marked differences in antibody response between groups. Animals in the orally inoculated group produced a striking increase in anti-F1 titers (up to 1:4,096) within 3 days, whereas there was minimal to no increase in antibody response in the intradermal group. Information gathered from this experimental trial may provide additional insight into the spatial and temporal evaluation of coyote plague serology.

Using filter paper to collect blood from wildlife for antibody analysis can be a powerful technique to simplify the collection, transport, and storage of blood samples. Despite these advantages, there are limited data that detail how long these samples can be stored and how storage conditions affect antibody longevity. We used blood samples collected on filter paper from coyotes experimentally infected with Yersinia pestis to determine optimum sample storage conditions over time. Blood samples collected on filter paper were stored for 454 d or more in four groups: 1) at ambient temperature and at ambient relative humidity, 2) at ambient temperature with desiccant, 3) at 4 C with desiccant, and 4) at -20 C with desiccant. Samples stored at 4 C or -20 C with desiccant had detectable antibody for a longer period of time than the samples stored at room temperature.

We examined sera from snowshoe hares (Lepus americanus) livetrapped in the northern Greater Yellowstone Area (GYA), US, for antibodies to Brucella abortus, Francisella tularensis, and snowshoe hare virus (SSHV). Zero of 90, 0 of 67, and 40 of 100 samples were antibody positive for B. abortus, F. tularensis, and SSHV, respectively. Hares were trapped from 2009 to 2012, and of the six animals that were captured twice with at least 1 yr between captures, four developed antibody to SSHV, indicating active exposure to the agent. These findings suggest snowshoe hares in the GYA do not play a significant role as a reservoir of B. abortus, but do maintain the zoonotic, encephalitic SSHV in the population.

ABSTRACT Serological assays and a two-tiered test algorithm are recommended for laboratory confirmation of Lyme disease. In the United States, the sensitivity of two-tiered testing using commercially available serology-based assays is dependent on the stage of infection and ranges from 30% in the early localized disease stage to near 100% in late-stage disease. Other variables, including subjectivity in reading Western blots, compliance with two-tiered recommendations, use of different first- and second-tier test combinations, and use of different test samples, all contribute to variation in two-tiered test performance. The availability and use of sample sets from well-characterized Lyme disease patients and controls are needed to better assess the performance of existing tests and for development of improved assays. To address this need, the Centers for Disease Control and Prevention and the National Institutes of Health prospectively collected sera from patients at all stages of Lyme disease, as well as healthy donors and patients with look-alike diseases. Patients and healthy controls were recruited using strict inclusion and exclusion criteria. Samples from all included patients were retrospectively characterized by two-tiered testing. The results from two-tiered testing corroborated the need for novel and improved diagnostics, particularly for laboratory diagnosis of earlier stages of infection. Furthermore, the two-tiered results provide a baseline with samples from well-characterized patients that can be used in comparing the sensitivity and specificity of novel diagnostics. Panels of sera and accompanying clinical and laboratory testing results are now available to Lyme disease serological test users and researchers developing novel tests.

Abstract Multiple publications have reported the use of coyotes (Canis latrans) in animal-based surveillance efforts for the detection of Yersinia pestis. Coyotes are likely exposed via flea bite or oral routes and are presumed to be resistant to the development of clinical disease. These historic data have only been useful for the evaluation of the geographic distribution of Y. pestis in the landscape. Because the canid immunologic response to Y. pestis has not been thoroughly characterized, we conducted experimental inoculation of captive-reared, juvenile coyotes (n = 8) with Y. pestis CO92 via oral or intradermal routes. We measured the humoral response to Y. pestis fraction 1 capsular protein (anti-F1) and found a significant difference between inoculation groups in magnitude and duration of antibody production. The anti-F1 titers in animals exposed intradermally peaked at day 10 postinoculation (PI; range = 1∶32 to 1∶128) with titers remaining stable at 1∶32 through week 12. In contrast, orally inoculated animals developed higher titers (range = 1∶256 to 1∶1,024) that remained stable at 1∶256 to 1∶512 through week 6. No clinical signs of disease were observed, and minimal changes were noted in body temperature, white blood cell counts, and acute phase proteins during the 7 days PI. Gross pathology was unremarkable, and minimal changes were noted in histopathology at days 3 and 7 PI. Rechallenge at 14 wk PI via similar dosage and routes resulted in marked differences in antibody response between groups. Animals in the orally inoculated group produced a striking increase in anti-F1 titers (up to 1∶4,096) within 3 days, whereas there was minimal to no increase in antibody response in the intradermal group. Information gathered from this experimental trial may provide additional insight into the spatial and temporal evaluation of coyote plague serology.

Plague, caused by the bacterium Yersinia pestis, causes die-offs of colonies of prairie dogs (Cynomys ludovicianus). It has been argued that other small rodents are reservoirs for plague, spreading disease during epizootics and maintaining the pathogen in the absence of prairie dogs; yet there is little empirical support for distinct enzootic and epizootic cycles. Between 2004 and 2006, we collected blood from small rodents captured in colonies in northern Colorado before, during, and for up to 2 yr after prairie dog epizootics. We screened 1,603 blood samples for antibodies to Y. pestis, using passive hemagglutination and inhibition tests, and for a subset of samples we cultured blood for the bacterium itself. Of the four species of rodents that were common in colonies, the northern grasshopper mouse (Onychomys leucogaster) was the only species with consistent evidence of plague infection during epizootics, with 11.1-23.1% of mice seropositive for antibody to Y. pestis during these events. Seropositive grasshopper mice, thirteen-lined ground squirrels (Spermophilus tridecemlineatus), and deer mice (Peromyscus maniculatus) were captured the year following epizootics. The appearance of antibodies to Y. pestis in grasshopper mice coincided with periods of high prairie dog mortality; subsequently, antibody prevalence rates declined, with no seropositive individuals captured 2 yr after epizootics. We did not detect plague in any rodents off of colonies, or on colonies prior to epizootics, and found no evidence of persistent Y. pestis infection in blood cultures. Our results suggest that grasshopper mice could be involved in epizootic spread of Y. pestis, and possibly, serve as a short-term reservoir for plague, but provide no evidence that the grasshopper mouse or any small rodent acts as a long-term, enzootic host for Y. pestis in prairie dog colonies.

To the Editor: Francisella tularensis, the etiologic agent of tularemia, is highly infectious and considered a potential bioweapon (1–3). Although 4 subspecies of F. tularensis are recognized, most cases of tularemia are due to infection by subsp. tularensis (type A) or holarctica (type B). North America is the only region where both type A and type B cause human disease. Subspecies novicida is also found in North America, but it is of reduced virulence. Disease incidence attributable to either type A or type B is essentially unknown because the traditional method for classification of these subspecies is glycerol fermentation, which requires culture recovery (4). F. tularensis is fastidious and slow growing, with isolates recovered in a small percentage of cases. We developed real-time TaqMan PCR assays for classification of F. tularensis type A and type B after F. tularensis is identified by culture or, in the absence of culture, by a PCR method such as the F. tularensis multitarget TaqMan assay (5). The type A TaqMan assay targets pdpD, which is present in type A, almost entirely absent from type B, and contains a 144-bp insert in novicida (6,7) (F: 5´-GAGACATCAATTAAAAGAAGCAATACCTT-3´; R: 5´-CCAAGAGTACTATTTCCGGTTGGT-3´; probe: 5´-AAAATTCTGCTCAGCAGGATTTTGATTTGGTT-3´). The type B assay targets a junction between ISFtu2 and a flanking 3´ region (GenBank AY06) (F: 5´- CTTGTACTTTTATTTGGCTACTGAGAAACT-3´; R: 5´- CTTGCTTGGTTTGTAAATATAGTGGAA-3´; probe: 5´- ACCTAGTTCAACCTCAAGACTTTTAGTAATGGGAATGTCA-3´). In type A and novicida, ISFtu2 is absent from this position (8). Oligonucleotides were designed with Primer Express version 2.0 (Applied Biosystems, Foster City, CA, USA). Probes were synthesized with a 5´ 6-carboxy-fluorescein reporter and an internal quencher (either BHQ1 [type A] or QSY-7[type B]) at the nucleotide position indicated by the quotation marks. Assays were optimized by using 1 ng of type A (strain SchuS4) or type B (strain LVS) DNA on the LightCycler 1.2 (Roche Applied Science, Indianapolis, IN, USA). Optimized concentrations (20 μL final volume) were 1× LightCycler Fast Start DNA Master Hybridization Probe mix (Roche), 750 nmol/L primers, 200 nmol/L probe, 5 mmol/L MgCl2 and 0.5 U uracil-DNA glycosylase. PCR conditions were 50°C for 2 min, 95°C for 10 min, 45 cycles of 95°C for 10 s and 65°C for 30 s, then 45°C for 5 min. Cycle threshold (Ct) values were calculated by using the second derivative maximum method with the y-axis at F1/F3 (LightCycler software version 3.5). Sensitivity of each assay was assessed by using 10-fold serial dilutions (100,000 to 1 genomic equivalents [GE]) of SchuS4 or LVS DNA. Testing was performed in triplicate, with a reproducible detection limit of 10 GE for both assays. Specificity of each assay was tested with 1 ng of DNA from a panel of 62 Francisella isolates (Table A1) and 22 non-Francisella isolates (Acinetobacter, Bacillus, Brucella, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Haemophilus, Klebsiella, Legionella, Proteus, Pseudomonas, Serratia, Staphylococcus, Streptococcus, and Yersinia species). Isolates were grown, DNA purified, and quantified as previously described (5). Specificity was also evaluated with DNA (2 μL) extracted as previously described from Francisella-like tick endosymbionts of Dermacentor variabilis and Francisella-like soil bacteria (Table A1) (9,10). The type A assay recognized all type A isolates with an average Ct value of 17.9 (n = 19). The type B assay detected all type B strains with an average Ct value of 17.1 (n = 21). Neither assay displayed cross-reactivity with F. tularensis subsp. novicida (n = 7), F. philomiragia (n = 15), Francisella-like tick endosymbionts (n = 3), Francisella-like soil bacteria (n = 7) (Table A1), or non-Francisella spp. (n = 22). To evaluate the ability of the type A and type B TaqMan assays, in conjunction with the multitarget assay, to identify F. tularensis and classify subspecies in primary specimens, human, animal, and tick samples were tested (Table ). DNA was extracted from 200 μL fluid, 25 mg liver, and 10 mg spleen or lung by using the QIAamp DNA MiniKit (Qiagen, Valencia, CA, USA) and 1 μL tested. Multitarget PCR conditions were as described (5). Table Comparison of standard diagnostic methods with the multitarget Francisella tularensis TaqMan assay and type A and type B assays using primary specimens The multitarget and subspecies-specific PCR assays accurately identified and classified F. tularensis in all specimens positive by standard diagnostic methods (Table). In addition, the type A and type B assays provided subspecies information for positive specimens in which an isolate was not recovered for glycerol fermentation testing (Table ). All specimens negative by standard diagnostic methods tested negative by PCR. These preliminary results suggest that a F. tularensis PCR identification method, in combination with the type A and type B assays, provides the capability to identify F. tularensis and determine subspecies in the absence of culture. We describe real-time PCR assays capable of classifying F. tularensis type A and type B and distinguishing these subspecies from the less virulent subsp. novicida. These assays are designed for use after F. tularensis has been identified by culture or by PCR. Supplemental use of these assays will allow laboratories to actively subtype F. tularensis isolates and primary specimens, thus providing subspecies information for a higher percentage of tularemia cases. Improved subspecies information will further understanding of the disease incidence and geographic distribution of F. tularensis type A and type B in North America.