Nucleic acid approaches for detection and identification of biological warfare and infectious disease agents

Biological warfare agents are the most problematic of the weapons of mass destruction and terror. Both civilian and military sources predict that over the next decade the threat from proliferation of these agents will increase significantly. In this review we summarize the state of the art in detection and identification of biological threat agents based on PCR technology with emphasis on the new technology of microarrays. The advantages and limitations of real-time PCR technology and a review of the literature as it applies to pathogen and virus detection are presented. The paper covers a number of issues related to the challenges facing biological threat agent detection technologies and identifies critical components that must be overcome for the emergence of reliable PCR-based DNA technologies as bioterrorism countermeasures and for environmental applications. The review evaluates various system components developed for an integrated DNA microchip and the potential applications of the next generation of fully automated DNA analyzers with integrated sample preparation and biosensing elements. The article also reviews promising devices and technologies that are near to being, or have been, commercialized.     

Recombinant antibodies: a new reagent for biological agent detection

Antibodies are critical reagents used in several biodetection platforms for the identification of biological agents. Recent advances in phage display technology allow isolation of high affinity recombinant antibody fragments (Fabs) that may bind unique epitopes of biological threat agents. The versatility of the selection process lends itself to efficient screening methodologies and can increase the number of antigen binding clones that can be isolated. Pilot scale biomanufacturing can then be used for the economical production of these immunoglobulin reagents in bacterial fermentation systems, and expression vectors with hexahistidine tags can be used to simplify downstream purification. One such Fab reagent directed against botulinum neurotoxin A/B has been shown to be sensitive in a variety of assay formats including surface plasmon resonance (SPR), flow cytometry, enzyme linked immunosorbent assay (ELISA), and hand-held immunochromatographic assay. Recombinant antibodies can provide another source of high quality detection reagents in our arsenal to identify or detect pathogens in environmental samples.     

Density-dependent interactions between the nematode Meloidogyne incognita and the biological control agent Agasicles hygrophila on invasive Alternanthera philoxeroides and its native congener Alternantera sessilis

BioControl - Biological invasions are a great threat to biodiversity. Invasive plants targeted for control with introduced biological control agents are also exposed to indigenous herbivores in the...     

Rapid identification of biological warfare agents using an instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system

An instrument employing a light addressable potentiometric sensor and a flow-through immunofiltration-enzyme assay system has been developed for the rapid and specific identification of biological warfare (BW) agents. The system has been designed to assay for up to eight agents simultaneously and provides an indication of the absence or presence of a threat within 15 min. Parameters affecting the mixing of the reagents within the instrument's fluidic lines were investigated and optimized. Measurements of blank samples and samples containing Bacillus subtilis spores in the concentration range of 10(4) to 10(6) cfu/ml indicate the limit of detection (LOD) is 3 x 10(3) cfu/ml for B. subtilus. Although the LOD is higher than that of several technologies currently under development, this instrument offers an immediate interim approach for addressing the need to rapidly detect biological warfare agents in the field.     

Biological threat characterization research: a critical component of national biodefense

Biological warfare (BW) threat assessments identify and prioritize BW threats to civilian and military populations. In an ideal world, they provide policymakers with clear and compelling guidance to prioritize biodefense research, development, testing, evaluation, and acquisition of countermeasures. Unfortunately, the biodefense community does not exist in an ideal world. National security professionals responsible for crafting BW threat assessments often are challenged by factors that limit the clarity and/or timeliness of those assessments. Moreover, the potential for life science advances to enhance threats enabled by state programs and the possibility that non-state actors may pursue crude but effective BW methodologies will drastically expand the scope of the perceived threat. Appropriate investment of federal biodefense funds will require some mechanism for validating and prioritizing present and future threats. Ideally, such a mechanism will incorporate empirical data targeted to elucidate actual hazards. In this regard, the Department of Homeland Security's creation of a Biological Threat Characterization Program for the technical validation of threat agents will be a valuable addition to the nation's overall biodefense strategy. This article articulates the need for a coordinated national biological threat characterization program, discusses some of the principal challenges associated with such research, and suggests a few options for their resolution.     

A Multicompartment Approach - Diatoms,Macrophytes, Benthic Macroinvertebrates and Fish - To Assess the Impact of Toxic Industrial Releases on a Small French River

The River Luzou flows through a sandy substrate in the South West of France. According to the results of two assessment surveys, the Water Agency appraised that this river may not achieve the good ecological status by 2015 as required by the Water Framework Directive (2000/60/EC). This ecosystem is impacted by industrial effluents (organic matter, metals and aromatic compounds). In order to assess and characterize the impact, this study aimed to combine a set of taxonomic and non-taxonomic metrics for diatoms, macrophytes, macroinvertebrates and fish along the up- to downstream gradient of the river. Diversity metrics, biological indices, biological and ecological traits were determined for the four biological quality elements (BQE). Various quantitative metrics (biomass estimates) were also calculated for diatom communities. The results were compared to physicochemical analysis. Biological measurements were more informative than physicochemical analysis, in the context of the study. Biological responses indicated both the contamination of water and its intensity. Diversity metrics and biological indices strongly decreased with pollution for all BQE but diatoms. Convergent trait selection with pollution was observed among BQE: reproduction, colonization strategies, or trophic regime were clearly modified at impaired sites. Taxon size and relation to the substrate diverged among biological compartments. Multiple anthropogenic pollution calls for alternate assessment methods of rivers'' health. Our study exemplifies the fact that, in the case of complex contaminations, biological indicators can be more informative for environmental risk, than a wide screening of contaminants by chemical analysis alone. The combination of diverse biological compartments provided a refined diagnostic about the nature (general mode of action) and intensity of the contamination.     

Development of species-, strain- and antibiotic biosynthesis-specific quantitative PCR assays for Pantoea agglomerans as tools for biocontrol monitoring

Pantoea agglomerans is a cosmopolitan plant epiphytic bacterium that includes some of the most effective biological antagonists against the fire blight pathogen Erwinia amylovora, a major threat to pome fruit production worldwide. Strain E325 is commercially available as Bloomtime Biological? in the USA and Canada. New quantitative PCR (qPCR) assays were developed for species- and strain -specific detection in the environment, and for detection of indigenous strains carrying the biocontrol antibacterial peptide biosynthesis gene paaA. The qPCR assays were highly specific, efficient and sensitive, detecting fewer than three cells per reaction or 700 colony forming units per flower, respectively. The qPCR assays were tested on field samples, giving first indications to the incidence of P. agglomerans E325 related strains, total P. agglomerans and pantocin A producing bacteria in commercial orchards. These assays will facilitate monitoring the environmental behavior of biocontrol P. agglomerans after orchard application for disease protection, proprietary strain-tracking, and streamlined screening for discovery of new biocontrol strains.     

Analysis of host response to bacterial infection using error model based gene expression microarray experiments

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples.     

A general framework for detecting disease associations with rare variants in sequencing studies

Biological and empirical evidence suggests that rare variants account for a large proportion of the genetic contributions to complex human diseases. Recent technological advances in high-throughput sequencing platforms have made it possible for researchers to generate comprehensive information on rare variants in large samples. We provide a general framework for association testing with rare variants by combining mutation information across multiple variant sites within a gene and relating the enriched genetic information to disease phenotypes through appropriate regression models. Our framework covers all major study designs (i.e., case-control, cross-sectional, cohort and family studies) and all common phenotypes (e.g., binary, quantitative, and age at onset), and it allows arbitrary covariates (e.g., environmental factors and ancestry variables). We derive theoretically optimal procedures for combining rare mutations and construct suitable test statistics for various biological scenarios. The allele-frequency threshold can be fixed or variable. The effects of the combined rare mutations on the phenotype can be in the same direction or different directions. The proposed methods are statistically more powerful and computationally more efficient than existing ones. An application to a deep-resequencing study of drug targets led to a discovery of rare variants associated with total cholesterol. The relevant software is freely available.     

The NYC native air sampling pilot project: using HVAC filter data for urban biological incident characterization

Native air sampling (NAS) is distinguished from dedicated air sampling (DAS) devices (eg, BioWatch) that are deployed to detect aerosol disseminations of biological threat agents. NAS uses filter samples from heating, ventilation, and air conditioning (HVAC) systems in commercial properties for environmental sampling after DAS detection of biological threat agent incidents. It represents an untapped, scientifically sound, efficient, widely distributed, and comparably inexpensive resource for postevent environmental sampling. Calculations predict that postevent NAS would be more efficient than environmental surface sampling by orders of magnitude. HVAC filter samples could be collected from pre-identified surrounding NAS facilities to corroborate the DAS alarm and delineate the path taken by the bioaerosol plume. The New York City (NYC) Native Air Sampling Pilot Project explored whether native air sampling would be acceptable to private sector stakeholders and could be implemented successfully in NYC. Building trade associations facilitated outreach to and discussions with property owners and managers, who expedited contact with building managers of candidate NAS properties that they managed or owned. Nominal NAS building requirements were determined; procedures to identify and evaluate candidate NAS facilities were developed; data collection tools and other resources were designed and used to expedite candidate NAS building selection and evaluation in Manhattan; and exemplar environmental sampling playbooks for emergency responders were completed. In this sample, modern buildings with single or few corporate tenants were the best NAS candidate facilities. The Pilot Project successfully demonstrated that in one urban setting a native air sampling strategy could be implemented with effective public-private collaboration.     

A Conceptual Framework for the Interpretation of Biological Markers for Environmental Exposure Assessment

Human exposure to environmental contaminants occurs via air, water, soil, dust, food, and other environmental media. Given this multitude of sources, environmental exposure assessment is moving away from single route exposure assessment to more integrated measures of exposure. Biological markers are frequently advocated as appropriate exposure assessment tools since they provide a measure of internal dose integrated over all routes of exposure. However, contributing sources may be difficult to identify through use of biological markers, and thus, have had limited utility in the regulatory community. To explore the different perspectives on the use and application of biological markers for exposure assessors, epidemiologists, and regulatory personnel, we have developed a biological marker conceptual framework. This framework is developed as a paradigm for the interpretation of biological markers for environmental exposure assessment linking the exposure assessment and the health effects assessment perspectives regarding biological markers. Further, it incorporates issues of source-specific exposures, aggregate exposure assessment, route-specific contributions, and biological variation in response to exposure. This structure provides an approach to explore the current constraints in using biological markers to evaluate source-specific exposures. This framework is discussed in the context of currently available biological markers for lead, carbon monoxide, and toluene. Biological markers represent a complex tool to assess human exposures to environmental contaminants; the biological marker framework presents a structure for their interpretation recognizing that many of the determinants of exposure, bioavailablity, and toxicokinetics are still being evaluated. The conceptual framework presented here provides another tool for the researcher in assessing the utility of biological markers in exposure assessment and epidemiology.     

Perspectives on the bacterial control of vectors in the tropics

Biological agents have not found wide application in the control of insects that transmit agents of human disease. Fungal pathogens may have a potential for field use, but environmental constraints, lack of knowledge of their life cycle, or dependence on production in vivo, have prevented their widespread use. In fact, sporogenous bacteria are the only biological agents used on a large scale for the control of mosquitoes and blackflies that transmit diseases in the tropics. Two bacteria, Bacillus thuringiensis sp. israelensis and B. sphaericus are very effective for the control of these vectors. In this article, Jean Marc Hougard and Christian Back discuss various factors associated with the use of bacteria for vector control, and the prospects for further developments.     

Production and use of biological pest control agents

Biological pest control agents are gaining prominence for the control of insect pests in agriculture and forestry. The shift from chemical control has been due to environmental concerns and recent innovations in biotechnology. Production and use of biological insect control agents is the challenge of the future for pest management.     

Considerations when using discriminant function analysis of antimicrobial resistance profiles to identify sources of fecal contamination of surface water in Michigan

The goals of this study were to (i) identify issues that affect the ability of discriminant function analysis (DA) of antimicrobial resistance profiles to differentiate sources of fecal contamination, (ii) test the accuracy of DA from a known-source library of fecal Escherichia coli isolates with isolates from environmental samples, and (iii) apply this DA to classify E. coli from surface water. A repeated cross-sectional study was used to collect fecal and environmental samples from Michigan livestock, wild geese, and surface water for bacterial isolation, identification, and antimicrobial susceptibility testing using disk diffusion for 12 agents chosen for their importance in treating E. coli infections or for their use as animal feed additives. Nonparametric DA was used to classify E. coli by source species individually and by groups according to antimicrobial exposure. A modified backwards model-building approach was applied to create the best decision rules for isolate differentiation with the smallest number of antimicrobial agents. Decision rules were generated from fecal isolates and applied to environmental isolates to determine the effectiveness of DA for identifying sources of contamination. Principal component analysis was applied to describe differences in resistance patterns between species groups. The average rate of correct classification by DA was improved by reducing the numbers of species classifications and antimicrobial agents. DA was able to correctly classify environmental isolates when fewer than four classifications were used. Water sample isolates were classified by livestock type. An evaluation of the performance of DA must take into consideration relative contributions of random chance and the true discriminatory power of the decision rules.     

Biological control agents elevate hantavirus by subsidizing deer mouse populations

Biological control of exotic invasive plants using exotic insects is practiced under the assumption that biological control agents are safe if they do not directly attack non-target species. We tested this assumption by evaluating the potential for two host-specific biological control agents ( Urophora spp.), widely established in North America for spotted knapweed ( Centaurea maculosa ) control, to indirectly elevate Sin Nombre hantavirus by providing food subsidies to populations of deer mice ( Peromyscus maniculatus ), the primary reservoir for the virus. We show that seropositive deer mice (mice testing positive for hantavirus) were over three times more abundant in the presence of the biocontrol food subsidy. Elevating densities of seropositive mice may increase risk of hantavirus infection in humans and significantly alter hantavirus ecology. Host specificity alone does not ensure safe biological control. To minimize indirect risks to non-target species, biological control agents must suppress pest populations enough to reduce their own numbers.     

Distance decay in an old‐growth neotropical forest

Abstract. Questions: Does distance decay exist in an old‐growth neotropical forest? Is this distance decay stronger than expected due to environmental heterogeneity alone? At what spatial scales are distance decay manifested? Location: La Selva Biological Station, Costa Rica, Central America. Methods: An index of distance decay is applied appropriate for small quadrats (the probability of encountering a conspecific tree) to a grid of 1170 0.01‐ha plots. A null model is provided that accounts for environmental heterogeneity. Results: Significant, but weak, distance decay is found. After correcting for known patterns of environmental heterogeneity, the distance decay almost disappears, except for fine spatial scales. Conclusions: These results are inconsistent with models that predict distance decay at all spatial scales. However, biological processes leading to distance decay may be more relevant and detectable at scales broader than this study. Research utilizing objectively‐located samples over much broader scales is necessary to evaluate the generality and magnitude of distance decay.     

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH₃, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples.     

Characterization of airborne bacteria at an underground subway station

The reliable detection of airborne biological threat agents depends on several factors, including the performance criteria of the detector and its operational environment. One step in improving the detector's performance is to increase our knowledge of the biological aerosol background in potential operational environments. Subway stations are enclosed public environments, which may be regarded as potential targets for incidents involving biological threat agents. In this study, the airborne bacterial community at a subway station in Norway was characterized (concentration level, diversity, and virulence- and survival-associated properties). In addition, a SASS 3100 high-volume air sampler and a matrix-assisted laser desorption ionization-time of flight mass spectrometry-based isolate screening procedure was used for these studies. The daytime level of airborne bacteria at the station was higher than the nighttime and outdoor levels, and the relative bacterial spore number was higher in outdoor air than at the station. The bacterial content, particle concentration, and size distribution were stable within each environment throughout the study (May to September 2010). The majority of the airborne bacteria belonged to the genera Bacillus, Micrococcus, and Staphylococcus, but a total of 37 different genera were identified in the air. These results suggest that anthropogenic sources are major contributors to airborne bacteria at subway stations and that such airborne communities could harbor virulence- and survival-associated properties of potential relevance for biological detection and surveillance, as well as for public health. Our findings also contribute to the development of realistic testing and evaluation schemes for biological detection/surveillance systems by providing information that can be used to mimic real-life operational airborne environments in controlled aerosol test chambers.     

生物入侵的危害与防治对策

生物入侵是一个世界范围的生态学现象。入侵种通过竞争、捕食、改变生境和传播疾病等方式对本地种及其系统产生影响。生物入侵的危害表现为：造成巨大的经济损失,仅美国每年因外来种入侵造成的经济损失近1370亿美元;威胁到人类的健康和生存。成千上万的人被外来种传染疾病以致死亡;引起严重的社会恐慌和动荡,人们寝食不安;改变了生态系统的结构和功能,全球自然灾害频频爆发;导致生物多样性的急剧下降,威胁到子孙后代的生存和发展。防治生物入侵有3条途径：实行全面检疫,阻止外来种的偶然入侵;采取全面的生态评估和监测,防范引进品种的入侵灾难;对已入侵的外来种采用机械法、化学法和生物防治法进行根除和控制,应用生物防治法、筛选天敌和对其进行危害评估时要特别慎重。     

Rapid Method To Estimate the Presence of Secondary Metabolites in Microbial Extracts

Screening microbial secondary metabolites is an established method to identify novel biologically active molecules. Preparation of biological screening samples from microbial fermentation extracts requires growth conditions that promote synthesis of secondary metabolites and extraction procedures that capture the secondary metabolites produced. High-performance liquid chromatography (HPLC) analysis of fermentation extracts can be used to estimate the number of secondary metabolites produced by microorganisms under various growth conditions but is slow. In this study we report on a rapid (approximately 1 min per assay) surrogate measure of secondary metabolite production based on a metabolite productivity index computed from the electrospray mass spectra of samples injected directly into a spectrometer. This surrogate measure of productivity was shown to correlate with an HPLC measure of productivity with a coefficient of 0.78 for a test set of extracts from 43 actinomycetes. This rapid measure of secondary metabolite productivity may be used to identify improved cultivation and extraction conditions by analyzing and ranking large sets of extracts. The same methods may also be used to survey large collections of extracts to identify subsets of highly productive organisms for biological screening or additional study.