Development of a pair of real-time loop mediated isothermal amplification assays for detection of Yersinia pestis, the causative agent of plague.

Yersinia pestis, the causative agent of plague mainly infects rodents, while humans are the accidental host. The conventional diagnostic methods available for Y. pestis exhibit cross-reactivity with other enteropathogenic bacteria which makes its detection difficult. Rapid and reliable point-of-care detection of Y. pestis is essential for timely initiation of medical treatment. In the present study, a pair of loop mediated isothermal amplification (LAMP) assays has been developed for rapid detection of Y. pestis. Two sets of LAMP primers, each containing 6 primers were specifically designed targeting caf1 and 3a genes located on pFra plasmid and chromosome of Y. pestis, respectively. Isothermal amplification was accomplished at 65 °C for 40 min for caf1 target, and at 63 °C for 50 min for 3a choromosomal target. The analytical sensitivity of the assay for the caf1 and 3a targets was found to be 500 fg and 100 fg genomic DNA of Y. pestis, respectively. The caf1 and 3a LAMP assays detected as few as 100 copies of caf1 and 10 copies of 3a gene targets harboured in the respective recombinant plasmids. The amplified products were detected visually under visible and UV light using SYBR Green 1 dye. The assay pair was found to be highly specific as it did not cross-react with closely related and other bacterial species.

Quantification of low abundance Yersinia pestis markers in dried blood spots by immuno-capture and quantitative high-resolution targeted mass spectrometry.

Plague, caused by the bacterium Yersinia pestis, is still present in several countries worldwide. Besides, Y. pestis has been designated as Tier 1 agent, the highest rank of bioterrorism agents. In this context, reliable diagnostic methods are of great importance. Here, we have developed an original workflow based upon dried blood spot for simplified sampling of clinical specimens, and specific immuno-mass spectrometry monitoring of Y. pestis biomarkers. Targeted proteins were selectively enriched from dried blood spot extracts by multiplex immunocapture using antibody-coated magnetic beads. After accelerated on-beads digestion, proteotypic peptides were monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode. The DBS-IC-MS assay was designed to quantify both F1 and LcrV antigens, although 10-fold lower sensitivity was observed with LcrV. The assay was successfully validated for F1 with a lower limit of quantification at 5 ng·mL-1 in spiked blood, corresponding to only 0.1 ng on spots. In vivo quantification of F1 in blood and organ samples was demonstrated in the mouse model of pneumonic plague. The new assay could help to simplify the laboratory confirmation of positive point of care F1 dipstick.

Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System.

Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

Humoral and cellular immune responses to Yersinia pestis Pla antigen in humans immunized with live plague vaccine.

BACKGROUND: To establish correlates of human immunity to the live plague vaccine (LPV), we analyzed parameters of cellular and antibody response to the plasminogen activator Pla of Y. pestis. This outer membrane protease is an essential virulence factor that is steadily expressed by Y. pestis. METHODOLOGY/PRINCIPAL FINDINGS: PBMCs and sera were obtained from a cohort of naïve (n = 17) and LPV-vaccinated (n = 34) donors. Anti-Pla antibodies of different classes and IgG subclasses were determined by ELISA and immunoblotting. The analysis of antibody response was complicated with a strong reactivity of Pla with normal human sera. The linear Pla B-cell epitopes were mapped using a library of 15-mer overlapping peptides. Twelve peptides that reacted specifically with sera of vaccinated donors were found together with a major cross-reacting peptide IPNISPDSFTVAAST located at the N-terminus. PBMCs were stimulated with recombinant Pla followed by proliferative analysis and cytokine profiling. The T-cell recall response was pronounced in vaccinees less than a year post-immunization, and became Th17-polarized over time after many rounds of vaccination. CONCLUSIONS/SIGNIFICANCE: The Pla protein can serve as a biomarker of successful vaccination with LPV. The diagnostic use of Pla will require elimination of cross-reactive parts of the antigen.

A Bead-Based Flow Cytometric Assay for Monitoring Yersinia pestis Exposure in Wildlife.

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.

Continuous hypoxia reduces the concentration of streptomycin in the blood.

BACKGROUND: A high incidence and mortality of plague in the past two decades occurred in the Qinghai-Tibet Plateau, China. High dose streptomycin (6-8 g/d) remained the first practical strategy for controlling the progressive, vicious clinical circumstances for patients with pneumonic plague in the Plateau, as opposed to the routine dosage recommended by the World Health Organization. To investigate whether patients with pneumonic plague truly required a large dosage of streptomycin in the hypoxic environment of the Tibetan Plateau, we investigated the hypothesis that hypoxic environment would change the pharmacokinetics of streptomycin in vivo. METHODS: (1) We retrospectively analyzed the data of pneumonic plague patients administered streptomycin from January 1, 2000 to December 31, 2012 in these areas, which came from the database of the Qinghai Center for Disease Control; and (2) We used a persistent hypoxia chamber to simulate the plateau hypoxic environment and fed Sprague Dawley rats in the chambers for one month. Then, we continuously administered hypoxic rats a single loading dose (200 mg/kg) of streptomycin and analyzed its concentrations by high performance liquid chromatography. The pharmacokinetic profiles were analyzed using a non-compartmental method in the Phoenix WinNonlin program. RESULTS: (1) There were 32 cases of patients with pneumonic plague in the past two decades totally and 9 of them died (all-cause mortality 28.125%, 9/32), including 7 cases died of delayed diagnosis without treatment of streptomycin, and the only 2 patients received normal dose of streptomycin. (2) The pharmacokinetic behaviors of streptomycin were different between the hypoxic and normal rats. Administration in a hypoxic state resulted in 74.81% and 29.28% decreases in maximum plasma concentration and area under the concentration-time curve from time zero to infinity compared with those values under normal condition for streptomycin. CONCLUSIONS: These results indicated that hypoxic condition could significantly decrease the absorption rate and extent of streptomycin. Therefore, patients with pneumonic plague require higher doses of streptomycin to maintain effective drug concentrations in Qing Hai and the Tibetan Plateau.

Simultaneous Immunodetection of Anthrax, Plague, and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays.

Multiplexed detection technologies are becoming increasingly important given the possibility of bioterrorism attacks, for which the range of suspected pathogens can vary considerably. In this work, we describe the use of Luminex MagPlex magnetic microspheres for the construction of two multiplexed diagnostic suspension arrays, enabling antibody-based detection of bacterial pathogens and their related disease biomarkers directly from blood cultures. The first 4-plex diagnostic array enabled the detection of both anthrax and plague infections using soluble disease biomarkers, including protective antigen (PA) and anthrax capsular antigen for anthrax detection and the capsular F1 and LcrV antigens for plague detection. The limits of detection (LODs) ranged between 0.5 and 5 ng/ml for the different antigens. The second 2-plex diagnostic array facilitated the detection of Yersinia pestis (LOD of 1 × 106 CFU/ml) and Francisella tularensis (LOD of 1 × 104 CFU/ml) from blood cultures. Inoculated, propagated blood cultures were processed (15 to 20 min) via 2 possible methodologies (Vacutainer or a simple centrifugation step), allowing the direct detection of bacteria in each sample, and the entire assay could be performed in 90 min. While detection of bacteria and soluble markers from blood cultures using PCR Luminex suspension arrays has been widely described, to our knowledge, this study is the first to demonstrate the utility of the Luminex system for the immunodetection of both bacteria and soluble markers directly from blood cultures. Targeting both the bacterial pathogens as well as two different disease biomarkers for each infection, we demonstrated the benefit of the multiplexed developed assays for enhanced, reliable detection. The presented arrays could easily be expanded to include antibodies for the detection of other pathogens of interest in hospitals or labs, demonstrating the applicability of this technology for the accurate detection and confirmation of a wide range of potential select agents.

Infectious blood source alters early foregut infection and regurgitative transmission of Yersinia pestis by rodent fleas.

Fleas can transmit Yersinia pestis by two mechanisms, early-phase transmission (EPT) and biofilm-dependent transmission (BDT). Transmission efficiency varies among flea species and the results from different studies have not always been consistent. One complicating variable is the species of rodent blood used for the infectious blood meal. To gain insight into the mechanism of EPT and the effect that host blood has on it, fleas were fed bacteremic mouse, rat, guinea pig, or gerbil blood; and the location and characteristics of the infection in the digestive tract and transmissibility of Y. pestis were assessed 1 to 3 days after infection. Surprisingly, 10-28% of two rodent flea species fed bacteremic rat or guinea pig blood refluxed a portion of the infected blood meal into the esophagus within 24 h of feeding. We term this phenomenon post-infection esophageal reflux (PIER). In contrast, PIER was rarely observed in rodent fleas fed bacteremic mouse or gerbil blood. PIER correlated with the accumulation of a dense mixed aggregate of Y. pestis, red blood cell stroma, and oxyhemoglobin crystals that filled the proventriculus. At their next feeding, fleas with PIER were 3-25 times more likely to appear partially blocked, with fresh blood retained within the esophagus, than were fleas without PIER. Three days after feeding on bacteremic rat blood, groups of Oropsylla montana transmitted significantly more CFU than did groups infected using mouse blood, and this enhanced transmission was biofilm-dependent. Our data support a model in which EPT results from regurgitation of Y. pestis from a partially obstructed flea foregut and that EPT and BDT can sometimes temporally overlap. The relative insolubility of the hemoglobin of rats and Sciurids and the slower digestion of their blood appears to promote regurgitative transmission, which may be one reason why these rodents are particularly prominent in plague ecology.

[Dynamics of F1 antibody responses to Yersinia pestis infection in Rhombomys opimus].

Objective: To observe the dynamics of antibody response in great gerbils infected with Yersinia pestis in experiment. Method: A total of 211 great gerbils were captured in the southern margin of plague natural focus of Junggar Basin of the Xinjiang Uygur Autonomous Region in 2011. Among them, there were 167 great gerbils without infection of Y. pestis and 44 great gerbils infected by Y.pestis. Y.pestis No. 2504 was employed for this experimental strain, which was strong toxic strain with negativity in the reduction experiment of nitrate. 35 great gerbils without the infection of Y. pestis were divided randomly and averagely into 7 groups including 6 experimental groups and 1 control group. Great gerbils in the 1st to 6th experimental groups were exposed first with 1 × 10(6)-1 × 10(11) CFU/ml of bacterial fluid with 10 times of gradient dilution; groin areas of great gerbils in the control group were injected subcutaneously with physiological saline; and the amount of infection was all 1 ml. 17 great gerbils infected with Y. pestis and the first detection of F1-antibody titer in 1∶256-1∶4 096 were grouped according to F1-antibody titer: group 1∶4 096 (n=4), group 1∶2 048 (n=4), group 1∶1 024 (n=3), group 1∶512 (n=3) and group 1∶256 (n=3); and blood in caudal regions was collected in asepsis for the detection of F1-antibody, with a total of 5 times. 9 great gerbils which were selected from the remaining great gerbils infected with Y. pestis and detected F1-antibody negative 2 times were exposed 1×10(6) CFU/ml of bacterial fluid for the second infection, with the amount of infection being 1 ml. Blood in caudal regions of great gerbils after the first and second infection were collected for the detection of plague F1-antibody on the 3rd, 5th, 7th, 15th, 30th, 60th, 90th and 120th day after infection. Declined regression models for great gerbils' antibodies were established with unary linear regression equation; declined change diagrams for the antibodies were drawn to observe the declined F1-antibody after great gerbils were exposed to Y. pestis. Results: In great gerbils with the first infection of Y. pestis, antibodies were detected in the 1 × 10(6)-1 × 10(8) CFU/ml of group on the 30th, 15th and 15th day, respectively; the positive rates of antibody were 1/4, 3/4 and 4/5, respectively; the group 1×10(7) and 1× 10(8) CFU/ml reached to the highest antibody titer with 1∶256 on the 120th day; antibodies were revealed in the group 1×10(9), 1×10(10) and 1×10(11) CFU/ml from the 5th to 7th day when the seroconversion of all antibodies was observed; group 1×10(11) CFU/ml reached to the highest antibody titer on the 120th day with 1∶4 096. In the great gerbils with the second exposure to Y.pestis, positive antibodies were detected on the 3rd day with the positive rate being 2/9; and the highest antibody titer with 1∶2 048 was noted on the 90th day. Unary linear regression equation of declined F1 antibody of great gerbils was y=0.045x- 0.321 (F=115.40, P< 0.001), and the shortest duration for F1-antibody titer declining from 1∶4 096 to 0 was 140 d and the longest duration 200 d. Conclusion: Great gerbils infected with the high concentration of Y. pestis fluid show shorter duration in producing F1-antibody, the antibody positive rate is also higher, and the highest antibody titer can reach 1∶4 096. The great gerbils could hold the plague F1 antibodies for a long time which was about 140 to 200 days from the highest titer.

[Experimental observation on the histopathological and ultrastructural pathology of Great Gerbils (Rhombomys opimus) in the Junggar Basin by subcutaneous injecting of Yersinia pestis].

Objective: To understand the histopathological and ultrastructural pathology changes of great gerbils in the Junggar Basin to Yersinia pestis infection. Methods: Forty captured great gerbils from the Junggar Basin that tested negative for anti-F1 antibodies were infected. The Y. pestis strain 2504, isolated from a live great gerbil in the natural plague foci of the Junggar Basin in 2005 with a median lethal dose (LD(50)) of <10 CFU/ml, was used in this study. Forty great gerbils were divided into seven infection groups and were subcutaneously infected with 7.4×10(5), 7.4×10(6), 7.4×10(7), 7.4×10(8), 7.4×10(9), 7.4×10(10), or 3.0×10(11) CFU/ml of 2504. One milliliter of physiological saline was injected in the noninfected group as a control. We collected the liver, spleen, heart, and lung from all animals for histopathologic and ultrastructural pathology examination. Results: Great gerbils in the 7.4×10(8)-3.0×10(11) CFU/ml groups did not survive and exhibited pathological changes and altered ultrastructural pathology. The liver tissue of infected great gerbils showed spotty necrosis and fatty degeneration, intranuclear canaliculi with increased hepatocytes, and uneven distribution of organelles. Additionally, reactive proliferation of lymphoid tissue in the spleen, blood sinusoid lacunae with neutrophil infiltration, and phagocytosed bacteria in phagocyte cells were observed. Myocardial fiber hypertrophy and interstitial indistinction, nuclear matrices decreased in cardiac myocytes, and loose arrangement of myogenic fibers in myocardial cells were also observed. Angiectasia, capillary congestion, and tissue necrosis were found in the lung. No significant difference in histopathological and ultrastructural pathology in the parenchymal organ was observed between the 7.4×10(5)-7.4×10(7) CFU/ml groups and the 7.4×10(8)-3.0×10(11) CFU/ml groups, and no specific death caused by Y. pestis infection was apparent in the 7.4×10(5)-7.4×10(7) CFU/ml groups. Conclusion:Y. pestis infection altered tissue and ultrastructural pathology in the parenchyma apparatus of great gerbils. In particular, the liver and spleen appeared to be the primary site of Y. pestis infection in great gerbils.

Infection Prevalence, Bacterial Loads, and Transmission Efficiency in Oropsylla montana (Siphonaptera: Ceratophyllidae) One Day After Exposure to Varying Concentrations of Yersinia pestis in Blood.

Unblocked fleas can transmit Yersinia pestis, the bacterium that causes plague, shortly (≤4 d) after taking an infectious bloodmeal. Investigators have measured so-called early-phase transmission (EPT) efficiency in various fleas following infection with highly bacteremic blood (≥108 cfu/ml). To date, no one has determined the lower limit of bacteremia required for fleas to acquire and transmit infection by EPT, though knowing this threshold is central to determining the length of time a host may be infectious to feeding fleas. Here, we evaluate the ability of Oropsylla montana (Baker) to acquire and transmit Y. pestis after feeding on blood containing 103 to 109 cfu/ml. We evaluated the resulting infection prevalence, bacterial loads, and transmission efficiency within the early-phase time period at 1 d postinfection. Fleas acquired infection from bacteremic blood across a wide range of concentrations, but transmission was observed only when fleas ingested highly bacteremic blood.

Fibrinolytic and procoagulant activities of Yersinia pestis and Salmonella enterica.

Pla of the plague bacterium Yersinia pestis and PgtE of the enteropathogen Salmonella enterica are surface-exposed, transmembrane ß-barrel proteases of the omptin family that exhibit a complex array of interactions with the hemostatic systems in vitro, and both proteases are established virulence factors. Pla favors fibrinolysis by direct activation of plasminogen, inactivation of the serpins plasminogen activator inhibitor-1 and α2-antiplasmin, inactivation of the thrombin-activable fibrinolysis inhibitor, and activation of single-chain urokinase. PgtE is structurally very similar but exhibits partially different functions and differ in expression control. PgtE proteolysis targets control aspects of fibrinolysis, and mimicry of matrix metalloproteinases enhances cell migration that should favor the intracellular spread of the bacterium. Enzymatic activity of both proteases is strongly influenced by the environment-induced variations in lipopolysaccharide that binds to the ß-barrel. Both proteases cleave the tissue factor pathway inhibitor and thus also express procoagulant activity.

Survival advantage of heterozygous factor V Leiden carriers in murine sepsis.

BACKGROUND: The high allelic frequency of the prothrombotic Leiden polymorphism in human blood coagulation factor V (FV) has been speculated to reflect positive selection during evolution. Heterozygous Leiden carriers enrolled in the placebo arm of the PROWESS sepsis trial and heterozygous Leiden mice challenged with endotoxin both showed reduced mortality, whereas homozygous Leiden mice were not protected from lethal endotoxemia. Follow-up analyses of clinical outcomes and of mouse models of infection with various pathogens remained inconclusive. OBJECTIVE: To establish whether activated protein C resistance of FV Leiden modifies the outcome of bacterial infection in murine sepsis models. METHODS: Homozygous and heterozygous FV Leiden mice were subjected to gram-positive (S. aureus) or gram-negative (Y. pestis; E. coli) septic peritonitis or polymicrobial, focal septic peritonitis induced by cecal ligation and puncture. The effect of FV Leiden on 7-day survival and bacterial dissemination was assessed. Outcomes were compared with the sepsis survival of mice with genetically impaired hemostasis (hemophilia A, thrombocytopenia, thrombin receptor PAR4 [protease activated receptor 4] deficiency, endothelial protein C receptor [ProcR/EPCR] deficiency). RESULTS: Heterozygous, but not homozygous, Leiden mice were protected from lethal infection with highly virulent S. aureus and Y. pestis strains. FV Leiden did not affect the outcome of sepsis induced by cecal ligation and puncture, staphylokinase-deficient S. aureus, Pla-deficient Y. pestis, or E. coli. Thrombocytopenia, deficiency of PAR1 or PAR4 did not affect S. aureus sepsis survival, whereas hemophilia A increased mortality. ProcR deficiency selectively abolished the survival advantage of heterozygous Leiden mice. CONCLUSIONS: In mice, heterozygous FV Leiden carriers are protected from sepsis mortality after infection with clinically relevant human bacterial pathogens.

Hexa-acylated LPS-lipid A deploys the appropriate level of fibrin to confer protection through MyD88.

OBJECTIVES: Fibrin has been demonstrated to function protectively against pathogens in our previous studies, but we observed that a very high level of fibrin played a negative role during infection. We performed this research to address the complication. METHODS: After infection, mice were monitored daily and harvested on day 4. The fibrin levels within the tissue samples were quantified by Western-blot. The in situ assay was used to detect plasminogen activators, protein C-ase and prothrombinase activation. PT-PCR was used to test coagulation factors expression. RESULTS: Mice treated with Coumadin showed that the protection correlates with fibrin levels. By interacting with Toll-like receptor 4, the hexa-acylated lipopolysaccharide, although not the tetra-acylated lipopolysaccharide, activates coagulation and regulates plasminogen activator inhibitor 1, thrombin activatable fibrinolysis inhibitor and thrombomodulin expression through myeloid differentiation factor 88, leading to plasminogen activators, protein C-ase and prothrombinase activation and fibrin formation. Because of the regulation, fibrin formation was controlled to deposit appropriate levels and confer protection. CONCLUSIONS: We demonstrated that the appropriate level of fibrin formation was deployed by hexa-acylated LPS-lipid A through myeloid differentiation factor 88 to confer protection.

Serologic survey for cross-species pathogens in urban coyotes (Canis latrans), Colorado, USA.

Abstract As coyotes (Canis latrans) adapt to living in urban environments, the opportunity for cross-species transmission of pathogens may increase. We investigated the prevalence of antibodies to pathogens that are either zoonotic or affect multiple animal species in urban coyotes in the Denver metropolitan area, Colorado, USA, in 2012. We assayed for antibodies to canine parvovirus-2, canine distemper virus, rabies virus, Toxoplasma gondii, Yersinia pestis, and serotypes of Leptospira interrogans. Overall, 84% of the animals had antibodies to canine parvovirus-2, 44% for canine distemper virus, 20% for T. gondii (IgG), 28% for Y. pestis, and 4% for L. interrogans serotype Grippotyphosa. No neutralizing antibodies were detected to rabies virus, T. gondii (IgM), or L. interrogans serotypes other than Grippotyphosa. With 88% of animals exposed to at least one pathogen, our results suggest that coyotes may serve as important reservoirs and sentinels for etiologic agents.

[Effect of immune modulation on immunogenic and protective activity of a live plague vaccine].

AIM: Comparative evaluation of the effect of polyoxidonium and betaleukin on immunogenic and protective activity of a live plague vaccine in model animal experiments. MATERIALS AND METHODS: Plague vaccine EV, polyoxidonium, betaleukin, erythrocytic antigenic diagnosticum for determination of F1 antibodies and immune reagents for detection of lymphocytes with F1 receptors (LFR) in adhesive test developed by the authors were used. The experiments were carried out in 12 rabbits and 169 guinea pigs. RESULTS: Immune modulation accelerated the appearance and disappearance of LFR (early phase) and ensured a more rapid and intensive antibody formation (effector phase). Activation by betaleukin is more pronounced than by polyoxidonium. The more rapid and intensive was the development of early phase, the more effective was antibody response to the vaccine. Immune modulation in the experiment with guinea pigs significantly increased protective activity of the vaccine. CONCLUSION: The use of immune modulators increased immunogenic (in both early and effector phases of antigen-specific response) and protective activity of the EV vaccine. A connection between the acceleration of the first phase of antigen-specific response and general intensity of effector phase of immune response to the EV vaccine was detected. ,

The innate immune response may be important for surviving plague in wild Gunnison's prairie dogs.

Prairie dogs (Cynomys spp.) are highly susceptible to Yersinia pestis, with ≥99% mortality reported from multiple studies of plague epizootics. A colony of Gunnison's prairie dogs (Cynomys gunnisoni) in the Aubrey Valley (AV) of northern Arizona appears to have survived several regional epizootics of plague, whereas nearby colonies have been severely affected by Y. pestis. To examine potential mechanisms accounting for survival in the AV colony, we conducted a laboratory Y. pestis challenge experiment on 60 wild-caught prairie dogs from AV and from a nearby, large colony with frequent past outbreaks of plague, Espee (n = 30 per colony). Test animals were challenged subcutaneously with the fully virulent Y. pestis strain CO92 at three doses: 50, 5,000, and 50,000 colony-forming units (cfu); this range is lethal in black-tailed prairie dogs (Cynomys ludovicianus). Contrary to our expectations, only 40% of the animals died. Although mortality trended higher in the Espee colony (50%) compared with AV (30%), the differences among infectious doses were not statistically significant. Only 39% of the survivors developed moderate to high antibody levels to Y. pestis, indicating that mechanisms other than humoral immunity are important in resistance to plague. The ratio of neutrophils to lymphocytes was not correlated with plague survival in this study. However, several immune proteins with roles in innate immunity (VCAM-1, CXCL-1, and vWF) were upregulated during plague infection and warrant further inquiry into their role for protection against this disease. These results suggest plague resistance exists in wild populations of the Gunnison's prairie dog and provide important directions for future studies.

Blood meal identification in off-host cat fleas (Ctenocephalides felis) from a plague-endemic region of Uganda.

The cat flea, Ctenocephalides felis, is an inefficient vector of the plague bacterium (Yersinia pestis) and is the predominant off-host flea species in human habitations in the West Nile region, an established plague focus in northwest Uganda. To determine if C. felis might serve as a Y. pestis bridging vector in the West Nile region, we collected on- and off-host fleas from human habitations and used a real-time polymerase chain reaction-based assay to estimate the proportion of off-host C. felis that had fed on humans and the proportion that had fed on potentially infectious rodents or shrews. Our findings indicate that cat fleas in human habitations in the West Nile region feed primarily on domesticated species. We conclude that C. felis is unlikely to serve as a Y. pestis bridging vector in this region.

Dynamics of Yersinia pestis and its antibody response in great gerbils (Rhombomys opimus) by subcutaneous infection.

BACKGROUND: Rhombomys opimus (great gerbil) is a reservoir of Yersinia pestis in the natural plague foci of Central Asia. Great gerbils are highly resistant to Y. pestis infection. The coevolution of great gerbils and Y. pestis is believed to play an important role in the plague epidemics in Central Asia plague foci. However, the dynamics of Y. pestis infection and the corresponding antibody response in great gerbils have not been evaluated. In this report, animal experiments were employed to investigate the bacterial load in both the liver and spleen of infected great gerbils. The dynamics of the antibody response to the F1 capsule antigen of Y. pestis was also determined. METHODOLOGY: Captured great gerbils that tested negative for both anti-F1 antibodies and bacterial isolation were infected subcutaneously with different doses (10(5) to 10(11) CFU) of a Y. pestis strain isolated from a live great gerbil during routine plague surveillance in the Junggar Basin, Xinjiang, China. The clinical manifestations, changes in body weight, anal temperature, and gross anatomy of the infected animals were observed. The blood cell count, bacterial load, and anti-F1 antibody titers were determined at different time points after infection using a blood analyzer, plate counts, and an indirect hemagglutination assay, respectively. CONCLUSIONS/SIGNIFICANCE: The dynamics of bacterial load and the anti-F1 antibody concentration in great gerbils are highly variable among individuals. The Y. pestis infection in great gerbils could persist as long as 15 days. They act as an appropriate reservoir for plague in the Junggar Basin, which is part of the natural plague foci in Central Asia. The dynamics of the Y. pestis susceptibility of great gerbil will improve the understanding of its variable resistance, which would facilitate the development of more effective countermeasures for controlling plague epidemics in this focus.

Evaluation of two multiplex real-time PCR screening capabilities for the detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples generated from murine infection models.

Two multiplex PCR screening capabilities (TaqMan Array Cards and FilmArray) were evaluated for their ability to detect Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples obtained from respective murine infection models. Blood samples were obtained from infected mice at 24 h intervals after exposure. Multiplex PCR results were compared with standard blood culture and singleplex real-time PCR. Across all three models, 71 mice were tested in total, within which a subset of 43 samples was shown to contain an infecting agent by at least one of the detection technologies. Within this subset of positive samples, for each model studied, the detection rates of each technology were compared. The B. anthracis model blood culture (14 of 15 agent-containing samples tested) and FilmArray PCR (12 of 15) were shown to have equivalent detection rates, which were significantly higher (at the 95 % confidence level) than singleplex (five of 14) or Array Card (two of 14) PCRs. The F. tularensis model blood culture (12 of 12) was shown to have a significantly higher (at 95 % confidence level) detection rate than all PCR technologies, with FilmArray (seven of 11) and singleplex (seven of 12) PCRs shown to have significantly higher (at 95 % confidence level) detection rates than the Array Card PCR (two of 11). Within the Y. pestis model, there was no significant difference in detection rates between blood culture (10 of 16), singleplex PCR (14 of 16), Array Card PCR (10 of 16) and FilmArray PCR (10 of 13).