Comparative analysis of virulence factors secreted by Bacillus anthracis Sterne at host body temperature

Aims: For the analysis of virulence factors produced and secreted by Bacillus anthracis vegetative cells during mammalian host infection, we evaluated the secretome of B. anthracis Sterne exposed to host‐specific factors specifically to host body temperature. Methods and Results: We employed a comparative proteomics‐based approach to analyse the proteins secreted by B. anthracis Sterne under host‐specific body temperature conditions. A total of 17 proteins encoded on a single chromosome and the pXO1 plasmid were identified by peptide mass fingerprinting. Multiple algorithms were used to predict the secretion mechanisms of the detected proteins in B. anthracis. Conclusions: Several putative virulence factors and known factors responsible for sporulation were differentially regulated, including CodY, pXO1‐130 and BA1952, revealing insights into temperature cues in the B. anthracis secretome. Significance and Impact of the Study: This study identified temperature‐regulated proteins. Further studies aimed at understanding the physical and functional roles of these proteins in infection and control by elevated temperatures will contribute to detection, diagnostics and prophylaxis.     

Importance of dose‐response model form in probabilistic risk assessment: A case study of health effects from methylmercury in fish

We compared the effect of uncertainty in dose‐response model form on health risk estimates to the effect of uncertainty and variability in exposure. We used three different dose‐response models to characterize neurological effects in children exposed in utero to methylmercury, and applied these models to calculate risks to a native population exposed to potentially contaminated fish from a reservoir in British Columbia. Uncertainty in model form was explicitly incorporated into the risk estimates. The selection of dose‐response model strongly influenced both mean risk estimates and distributions of risk, and had a much greater impact than altering exposure distributions. We conclude that incorporating uncertainty in dose‐response model form is at least as important as accounting for variability and uncertainty in exposure parameters in probabilistic risk assessment.     

Dose-response time modelling for highly pathogenic avian influenza A (H5N1) virus infection

Aims: To develop time‐dependent dose–response models for highly pathogenic avian influenza A (HPAI) of the H5N1 subtype virus. Methods and Results: A total of four candidate time‐dependent dose–response models were fitted to four survival data sets for animals (mice or ferrets) exposed to graded doses of HPAI H5N1 virus using the maximum‐likelihood estimation. A beta‐Poisson dose–response model with the N₅₀ parameter modified by an exponential‐inverse‐power time dependency or an exponential dose–response model with the k parameter modified by an exponential‐inverse time dependency provided a statistically adequate fit to the observed survival data. Conclusions: We have successfully developed the time‐dependent dose–response models to describe the mortality of animals exposed to an HPAI H5N1 virus. The developed model describes the mortality over time and represents observed experimental responses accurately. Significance and Impact of the Study: This is the first study describing time‐dependent dose–response models for HPAI H5N1 virus. The developed models will be a useful tool for estimating the mortality of HPAI H5N1 virus, which may depend on time postexposure, for the preparation of a future influenza pandemic caused by this lethal virus.     

Bayesian Analysis for Linearized Multi-Stage Models in Quantal Bioassay

Bayesian methods for estimating dose response curves from linearized multi-stage models in quantal bioassay are studied. A Gibbs sampling approach with data augmentation is employed to compute the Bayes estimates. In addition, estimation of the “relative additional risk” and the “risk specific dose” is studied. Model selection based on conditional predictive ordinates from cross-validated data is developed. Model adequacy is addressed by means of a posterior predictive tail-area test.     

A simple and sensitive method for detection of Bacillus anthracis by loop‐mediated isothermal amplification

Aims: To develop a rapid and simple system for detection of Bacillus anthracis using a loop‐mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection. Methods and Results: A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30–40 min under isothermal conditions at 63°C. No cross‐reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3·6 CFU per test. Conclusions: The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis. Significance and Impact of the Study: The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.     

Modeling the inactivation kinetics of bacillus spores by glutaraldehyde

Aims: Our goal was to develop a mathematical kinetic model to predict the sporicidal activity of glutaraldehyde, which is an active ingredient frequently used in commercial products employed for liquid disinfection and decontamination. Methods and Results: We used our previously published data on spore inactivation by glutaraldehyde to develop a predictive model obtained by calculating multiple independent modifying functions. The model was then validated by comparing model predicted values to new experimental data. For model validation, quality‐controlled spores of Bacillus athrophaeus (previously and generally known as Bacillus subtilis globigii) were exposed under conditions where several physicochemical variables were modified simultaneously, and the spore surviving fractions were measured by titration. Conclusions: The model predicted within one order of magnitude variations in sporicidal effectiveness due to changes in main parameters (glutaraldehyde concentration, temperature or time‐duration of the treatment). Other parameters such pH, salinity and the effect of serum concentration were also addressed, albeit with less accuracy. Significance and Impact of the study: The model should be useful to quantitatively estimate the effectiveness of glutaraldehyde‐based disinfectants, decontaminants, and germicides under the described conditions, particularly when limited data are available or when spore virulence (like that of Bacillus anthracis) precludes extensive experimentation. A similar approach could predict the effectiveness of a variety of decontaminant and disinfecting agents.     

Laboratory studies on surface sampling of Bacillus anthracis contamination: summary,gaps and recommendations

This article summarizes previous laboratory studies to characterize the performance of methods for collecting, storing/transporting, processing and analysing samples from surfaces contaminated by Bacillus anthracis or related surrogates. The focus is on plate culture and count estimates of surface contamination for swab, wipe and vacuum samples of porous and nonporous surfaces. Summaries of the previous studies and their results were assessed to identify gaps in information needed as inputs to calculate key parameters critical to risk management in biothreat incidents. One key parameter is the number of samples needed to make characterization or clearance decisions with specified statistical confidence. Other key parameters include the ability to calculate, following contamination incidents, the (i) estimates of B. anthracis contamination, as well as the bias and uncertainties in the estimates and (ii) confidence in characterization and clearance decisions for contaminated or decontaminated buildings. Gaps in knowledge and understanding identified during the summary of the studies are discussed. Additional work is needed to quantify (i) the false‐negative rates of surface‐sampling methods with lower concentrations on various surfaces and (ii) the effects on performance characteristics of: aerosol vs liquid deposition of spores, using surrogates instead of B. anthracis, real‐world vs laboratory conditions and storage and transportation conditions. Recommendations are given for future evaluations of data from existing studies and possible new studies.     

A Bayesian modelling framework to estimate Campylobacter prevalence and culture methods sensitivity: application to a chicken meat survey in Belgium

Aims: To estimate the true prevalence of Campylobacter and the diagnostic sensitivity of routine detection methods by applying a Bayesian modelling approach. Methods and Results: Results from a Belgium‐wide survey of Campylobacter contamination in chicken meat preparations (n = 656 samples) showed that Campylobacter was detected in 24·2% of the samples by enrichment, compared with 41% detected by direct plating. Combining positive results from both methods increased the apparent prevalence to 48·02%. Bayesian model was set up in WinBUGS software, the model estimates Campylobacter prevalence as 60% (95% Credibility interval (CI): 47–82%), and the sensitivity of enrichment culture and direct plating as 41% (95% CI: 31–52%) and 69% (95% CI: 50–85%), respectively. Conclusions: The parallel use of direct plating and enrichment culture adds value for Campylobacter detection from chicken meat preparations, but the false‐negative results from each culture method must be taken into account. Significance and Impact of the Study: Monitoring data could be strongly biased by the microbiological techniques used to generate it. To circumvent this bias, we describe an applied Bayesian framework for better interpretation of Campylobacter survey data in view of the imperfect test characteristics of routine culture methods.     

A projection of ozone‐induced wheat production loss in China and India for the years 2000 and 2020 with exposure‐based and flux‐based approaches

Using a high‐resolution (40 × 40 km) chemical transport model coupled with the Regional Emission inventory in Asia (REAS), we simulated surface ozone concentrations ([O₃]) and evaluated O₃‐induced wheat production loss in China and India for the years 2000 and 2020 using dose–response functions based on AOT40 (accumulated [O₃] above 40 ppb) and POD_Y (phytotoxic O₃ dose, accumulated stomatal flux of O₃ above a threshold of Y nmol m^?2 s^?1). Two O₃ dose metrics (90 days AOT40 and POD₆) were derived from European experiments, and the other two (75 days AOT40 and POD₁₂) were adapted from Asian studies. Relative yield loss (RYL) of wheat in 2000 was estimated to be 6.4–14.9% for China and 8.2–22.3% for India. POD₆ predicted greater RYL, especially for the warm regions of India, whereas the 90 days AOT40 gave the lowest estimates. For the future projection, all the O₃ dose metrics gave comparable estimates of an increase in RYL from 2000 to 2020 in the range 8.1–9.4% and 5.4–7.7% for China and India, respectively. The lower projected increase in RYL for India may be due to conservative estimation of the emission increase in 2020. Sensitivity tests of the model showed that the POD_Y‐based estimates of RYL are highly sensitive to perturbations in the meteorological inputs, but that the estimated increase in RYL from 2000 to 2020 is much more robust. The projected increase in wheat production loss in China and India in the near future is substantially larger than the uncertainties in the estimation and indicates an urgent need for curbing the rapid increase in surface [O₃] in these regions.     

Dose-response model for Burkholderia pseudomallei (melioidosis)

Aims: The objective of this study was development of a dose–response model for exposure to Burkholderia pseudomallei in different animal hosts and analysis of the results. The data sets with which the model was developed were taken from the open literature. Methods and Results: All data sets were initially tested for a trend between dose and outcome using the Cochran–Armitage test. Only data showing a statistically significant trend were subjected to further analysis (fitting with parametric dose–response relationships). Dose–response relationships (exponential, beta-Poisson and log-probit) were fit to data using the method of maximum likelihood estimation. Conclusions: Dose–response analysis of BALB/c mice, C57BL/6 mice, guinea pigs and diabetic rats showed that BALB/c mice exposed intranasally (i.n.) and guinea pigs exposed intraperitoneally (i.p.) are significantly more sensitive to B. pseudomallei than C57BL/6 mice exposed i.n. and diabetic rats exposed i.p. Significance and Impact of the Study: The results confirmed the findings of a study of outbreak data that the diabetic population is more susceptible to infection with B. pseudomallei than the general population. The low dose prediction from best fit dose–response models can be used to draw guidelines for public health decision making processes, including consideration of sensitive subpopulations.     

Circulating lethal toxin decreases the ability of neutrophils to respond to Bacillus anthracis

Polymorphonuclear leucocytes (PMNs) play a protective role during Bacillus anthracis infection. However, B. anthracis is able to subvert the PMN response effectively as evidenced by the high mortality rates of anthrax. One major virulence factor produced by B. anthracis, lethal toxin (LT), is necessary for dissemination in the BSL2 model of mouse infection. While human and mouse PMNs kill vegetative B. anthracis, short in vitro half‐lives of PMNs have made it difficult to determine how or if LT alters their bactericidal function. Additionally, the role of LT intoxication on PMN's ability to migrate to inflammatory signals remains controversial. LF concentrations in both serum and major organs were determined from mice infected with B. anthracis Sterne strain at defined stages of infection to guide subsequent administration of purified toxin. Bactericidal activity of PMNs assessed using ex vivo cell culture assays showed significant defects in killing B. anthracis. In vivo PMN recruitment to inflammatory stimuli was significantly impaired at 24 h as assessed by real‐time analysis of light‐producing PMNs within the mouse. The observations described above suggest that LT serves dual functions; it both attenuates accumulation of PMNs at sites of inflammation and impairs PMNs bactericidal activity against vegetative B. anthracis.     

Response of Danaus plexippus to pollen of two new Bt corn events via laboratory bioassay

Laboratory bioassays were conducted to evaluate the response of first instar larvae of the monarch butterfly, Danaus plexippus L. (Lepidoptera: Danaidae), a non‐target species, to pollen from corn, Zea mays L. (Commelinales: Poaceae), from two new corn hybrids genetically modified to express different types of insecticidal proteins derived from the bacterium Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt). One hybrid expresses both Cry1Ab and Cry2Ab2 proteins (MON 810 × MON 84006), active against lepidopteran pests, and the other expresses Cry3Bb1 protein (MON 863), targeted against coleopteran pests. First instar larvae were placed on milkweed leaves (Asclepias syriaca L.) (Gentianales: Asclepiadaceae) dusted with doses of either Bt pollen or its nonexpressing (isoline) pollen counterpart ranging from 50 to 3200 grains cm^?2 of milkweed leaves, or no pollen at all. Larvae were exposed to pollen for 4 days, then moved to pollen‐free leaves and observed for another 6 days. Survival was observed after 2, 4, and 10 days. Weight gain was estimated after 4 and 10 days, leaf consumption after 2 and 4 days, and larval development after 10 days. Exposure to pollen of the Cry1Ab/Cry2Ab2‐Bt expressing hybrid reduced larval survival approximately 7.5–23.5% at the dose ranges tested relative to a no pollen control. Larval weight gain and consumption were reduced for larvae exposed to pollen of this hybrid and a small minority of larvae (3.1%) never developed past the third instar after 10 days of observation. Exposure to pollen of the Cry3Bb1‐Bt expressing hybrid had no negative effects on larval mortality, weight gain, consumption, or development relative to the consumption of Bt‐free corn pollen. The relevance of these findings to the risk that these Bt corn hybrids pose to monarch populations is discussed.     

Species richness, area and climate correlates

Aim Species richness–area theory predicts that more species should be found if one samples a larger area. To avoid biases from comparing species richness in areas of very different sizes, area is often controlled by counting the numbers of co‐occupying species in near‐equal area grid cells. The assumption is that variation in grid cell size accrued from working in a three‐dimensional world is negligible. Here we provide a first test of this idea. We measure the surface area of c. 50 × 50 km and c. 220 × 220 km grid cells across western Europe. We then ask how variation in the area of grid cells affects: (1) the selection of climate variables entering a species richness model; and (2) the accuracy of models in predicting species richness in unsampled grid cells. Location Western Europe. Methods Models are developed for European plant, breeding bird, mammal and herptile species richness using seven climate variables. Generalized additive models are used to relate species richness, climate and area. Results We found that variation in the grid cell area was large (50 × 50 km: 8–3311 km²; 220 × 220: 193–55,100 km²), but this did not affect the selection of variables in the models. Similarly, the predictive accuracy was affected only marginally by exclusion of area within models developed at the c. 50 × 50 km grid cells, although predictive accuracy suffered greater reductions when area was not included as a covariate in models developed for c. 220 × 220 km grid cells. Main conclusions Our results support the assumption that variation in near‐equal area cells may be of second‐order importance for models explaining or predicting species richness in relation to climate, although there is a possibility that drops in accuracy might increase with grid cell size. The results are, however, contingent on this particular data set, grain and extent of the analyses, and more empirical work is required.     

Genetic isolation within the malaria mosquito Anopheles melas

Anopheles melas is a brackish water–breeding member of the Anopheles gambiae complex that is distributed along the coast of West Africa and is a major malaria vector within its range. Because little is known about the population structure of this species, we analysed 15 microsatellite markers and 1161 bp of mtDNA in 11 A. melas populations collected throughout its range. Compared with its sibling species A. gambiae, A. melas populations have a high level of genetic differentiation between them, representing its patchy distribution due to its fragmented larval habitat that is associated with mangroves and salt marsh grass. Populations clustered into three distinct groups representing Western Africa, Southern Africa and Bioko Island populations that appear to be mostly isolated. Fixed differences in the mtDNA are present between all three clusters, and a Bayesian clustering analysis of the microsatellite data found no evidence for migration from mainland to Bioko Island populations, and little migration was evident between the Southern to the Western cluster. Surprisingly, mtDNA divergence between the three A. melas clusters is on par with levels of divergence between other species of the A. gambiae complex, and no support for monophyly was observed in a maximum‐likelihood phylogenetic analysis. Finally, an approximate Bayesian analysis of microsatellite data indicates that Bioko Island A. melas populations were connected to the mainland populations in the past, but became isolated, presumably when sea levels rose after the last glaciation period (≥10 000–11 000 bp ). This study has exposed species‐level genetic divergence within A. melas and also has implications for control of this malaria vector.     

Decontamination of a hard surface contaminated with Bacillus anthracisΔSterne and B. anthracis Ames spores using electrochemically generated liquid-phase chlorine dioxide (eClO2)

Aims: To evaluate the inactivation of Bacillus anthracisΔSterne and Ames spores using electrochemically generated liquid‐phase chlorine dioxide (eClO₂) and compare two sporulation and decontamination methods with regard to cost, safety and technical constraints. Methods and Results: Spores were prepared via agar and broth methods and subsequently inoculated and dried onto clean, autoclave‐sterilized glass coupons. Bacillus anthracis spore inactivation efficacy was evaluated using the modified three‐step method (AOAC 2008.05) and a single‐tube extraction method. Spores (7·0 ± 0·5 logs) were inactivated within 1 min at room temperature using freshly prepared eClO₂. Bacillus anthracisΔSterne spores decreased in size after eClO₂ treatment as measured using a Beckman Coulter Multisizer. Conclusions: eClO₂ saturation of a hard surface was an effective B. anthracis sporicide. Broth sporulation and the single‐tube extraction method required less time and fewer steps, yielded a higher percentage of phase‐bright spores and showed higher spore recovery efficiency compared with AOAC 2008.05, making it more amenable to biosafety level 3 (BSL3) testing of virulent spores. Significance and Impact of the Study: Two test methods demonstrated the sporicidal efficacy of eClO₂. A new single‐tube extraction test protocol for decontaminants was introduced.     

The effects of subinhibitory concentrations of costus oil on virulence factor production in Staphylococcus aureus

Aim: To determine the antimicrobial activity of costus (Saussurea lappa) oil against Staphylococcus aureus, and to evaluate the influence of subinhibitory concentrations of costus oil on virulence‐related exoprotein production in staph. aureus. Methods and Results: Minimal inhibitory concentrations (MICs) were determined using a broth microdilution method, and the MICs of costus oil against 32 Staph. aureus strains ranged from 0.15 to 0.6 μl ml^?1. The MIC₅₀ and MIC₉₀ were 0.3 and 0.6 μl ml^?1, respectively. Western blot, haemolytic, tumour necrosis factor (TNF) release and real‐time RT‐PCR assays were performed to evaluate the effects of subinhibitory concentrations of costus oil on virulence‐associated exoprotein production in Staph. aureus. The data presented here show that costus oil dose dependently decreased the production of α‐toxin, toxic shock syndrome toxin 1 (TSST‐1) and enterotoxins A and B in both methicillin‐sensitive Staph. aureus (MSSA) and methicillin‐resistant Staph. aureus (MRSA). Conclusion: Costus oil has potent antimicrobial activity against Staph. aureus, and the production of α‐toxin, TSST‐1 and enterotoxins A and B in Staph. aureus was decreased by costus oil. Significance and Impact of the Study: The data suggest that costus oil may deserve further investigation for its potential therapeutic value in treating Staph. aureus infections. Furthermore, costus oil could be rationally applied in food products as a novel food preservative both to inhibit the growth of Staph. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.     

Rapid evolution of parasitoids when faced with the symbiont-mediated resistance of their hosts

Insects harbour a wild diversity of symbionts that can spread and persist within populations by providing benefits to their host. The pea aphid Acyrthosiphon pisum maintains a facultative symbiosis with the bacterium Hamiltonella defensa, which provides enhanced resistance against the aphid parasitoid Aphidius ervi. Although the mechanisms associated with this symbiotic‐mediated protection have been investigated thoroughly, little is known about its evolutionary effects on parasitoid populations. We used an experimental evolution procedure in which parasitoids were exposed either to highly resistant aphids harbouring the symbiont or to low innate resistant hosts free of H. defensa. Parasitoids exposed to H. defensa gained virulence over time, reaching the same parasitism rate as those exposed to low aphid innate resistance only. A fitness reduction was associated with this adaptation as the size of parasitoids exposed to H. defensa decreased through generations. This study highlighted the considerable role of symbionts in host–parasite co‐evolutionary dynamics.