Latex agglutination tests for selected Escherichia coli enzymes

Rapid latex agglutination assays for identifying tryptophanase, glutamate decarboxylase, and glucuronidase from Escherichia coli cell lysates were developed by using rabbit polyclonal antibodies elicited to commercial E. coli enzyme preparations. The latex agglutination tests had sensitivities of 77% for tryptophanase, 83% for glutamate decarboxylase, and 70% for glucuronidase. Specificities were 61% for tryptophanase, 57% for glutamate decarboxylase, and 82% for glucuronidase. The sensitivities and specificities were too low to warrant continued development of the assays;Development of the latex agglutination tests required extensive studies in attempts to reduce or eliminate nonspecific agglutination reactions. The effects of a wide variety of assay conditions on assay performance were studied. Latex particles of different affinities and with different surface charges, different blocking agents, assay buffers of different compositions, pH values, and ionic strengths, chaotropic agents, and different antibody preparations were all examined. A combination of 0.22 [mu]m diameter latex particles sensitized with both 0.5 and 1.0 mg/ml immunoglobulin and blocked with a [beta]-casein and glycerol mixture resulted in the production of the best reactive latex reagents. Immunoglobulin adsorption studies yielded valuable information that was used to optimize the binding of the antibodies to the latex particles. Four different antibody purification methods had no significant effect in the elimination of nonspecific agglutination reactions. Antibody characterization by polyacrylamide gel electrophoresis, Ouchterlony immunodiffusion, enzyme immunoassays, and Western blot analysis revealed that the antibody preparations were heterologous and reacted with many different proteins. The cross-reactions among the antibody preparations and nontarget proteins indicated that better methods of antibody production must be used to provide more specific reagents for the latex agglutination assay

Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness

The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA MasterPLUS HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Realtime PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs

Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness

The Centers for Disease Control and Prevention and United States Army Research Institute for Infectious Diseases have developed real-time PCR assays for the detection of bioterrorism threat agents. These assays all rely on a limited number of approved real-time PCR master mixes. Because the availability of these reagents is a critical element of bioterrorism preparedness, we undertook a joint national preparedness exercise to address the potential surge needs resulting from a large-scale bio-emergency. We identified 9 commercially-available potential alternatives to an existing approved master mix (LightCycler FastStart DNA Master HybProbes): the TaqMan Fast Universal PCR master mix, OmniMix HS, FAST qPCR master mix, EXPRESS qPCR SuperMix kit, QuantiFast Probe PCR kit, LightCycler FastStart DNA MasterPLUS HybProbe, Brilliant II FAST qPCR master mix, ABsolute Fast QPCR Mix and the HotStart IT Taq master mix. The performances of these kits were evaluated by the use of real-time PCR assays for four bioterrorism threat agents: Bacillus anthracis, Brucella melitensis, Burkholderia mallei and Francisella tularensis. The master mixes were compared for target-specific detection levels, as well as consistency of results among three different real-time PCR platforms (LightCycler, SmartCycler and 7500 Fast Dx). Realtime PCR analysis revealed that all ten kits performed well for agent detection on the 7500 Fast Dx instrument; however, the QuantiFast Probe PCR kit yielded the most consistently positive results across multiple real-time PCR platforms. We report that certain combinations of commonly used master mixes and instruments are not as reliable as others at detecting low concentrations of target DNA. Furthermore, our study provides laboratories the option to select from the commercial kits we evaluated to suit their preparedness needs

A biologically relevant rapid quantification of physical and biological stress profiles on rocky shores.

Different combinations and intensities of physical (e.g. thermal) and biological (e.g.competition or predation) stress operate on organisms in different locations. Variation in these stresses can occur over small to medium spatial scales (cm to 10s m) in heterogeneous environments such as rocky shores, due to differences in sun and wave exposure, shore topography and/or recruitment. In this study we demonstrate how simple measurements can be taken that represent physical and biological stresses (stress profiles)in a given location. Using a bootstrapped principal component analysis, we identified significantly different stress profiles at four sites separated by only 10s to 100s of metres on the Shek O peninsula in Hong Kong. We then measured response to thermal stress, as determined by detachment temperature, in the limpet Cellana grata (which is known to be a sensitive indicator species to thermal stress) from each location. Significant differences in stress profile between locations were also seen in thermal stress tolerance of limpets from those locations. At locations where the major stresses are likely to be more physical or less biological in nature (e.g. southerly facing aspect or lower density of grazers), the mean detachment temperature was higher, whereas detachment temperature was lower at sites with more biological or less physical stress. This method is, therefore, able to determine biologically meaningful differences in stress profiles over small to medium spatial scales, and demonstrates that localised adaptation (i.e. post planktonic settlement) or acclimation of species may occur in response to these different stress profiles. The technique can be adapted to different environments and smaller or larger spatial scales as long as the stress experienced by the study species is relevant to these scales

Whole genome single nucleotide polymorphism based phylogeny of Francisella tularensis and its application to the development of a strain typing assay

<p>Abstract</p> <p>Background</p> <p>A low genetic diversity in <it>Francisella tularensis </it>has been documented. Current DNA based genotyping methods for typing <it>F. tularensis </it>offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates.</p> <p>Results</p> <p>We have generated a high-resolution phylogenetic tree from 40 <it>Francisella </it>isolates, including 13 <it>F. tularensis </it>subspecies <it>holarctica </it>(type B) strains, 26 <it>F. tularensis </it>subsp. <it>tularensis </it>(type A) strains and a single <it>F. novicida </it>strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip^®resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of <it>F. tularensis </it>subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional <it>F. tularensis </it>strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2).</p> <p>Conclusion</p> <p>Whole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of <it>F. tularensis </it>subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of <it>F. tularensis </it>to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.</p

A rhesus macaque (Macaca mulatta) model of aerosol-exposure brucellosis (Brucella suis): pathology and diagnostic implications

The US Centers for Disease Control and Prevention lists Brucella as a potential bioterrorism threat requiring enhanced diagnostic capacity and surveillance (http://emergency.cdc.gov/bioterrorism/). Successful treatment and management of patients after exposure to biological threat agents depends on accurate and timely diagnosis, but many biothreat agents present with similar, vague clinical signs – commonly referred to as ‘flu-like illness’. Diagnosis of brucellosis is notoriously challenging, especially early in infection, and definitive diagnosis may require invasive methods, e.g. bone marrow biopsy. We studied the pathogenesis of Brucella suis aerosol infection in rhesus macaques in an effort to guide the diagnostic algorithm in case of possible intentional exposure of humans. Rhesus proved to be an excellent model for human brucellosis; the data showed that PCR DNA amplification testing of non-invasive diagnostic samples has the potential to definitively detect a point-source outbreak immediately and for several days after exposure

Avian cholera in a southern giant petrel (Macronectes giganteus) from Antarctica

A southern giant petrel (Macronectes giganteus) was found dead at Potter Peninsula, King George Island, South Shetland, Antarctica. The adult male was discovered approximately 48 hr after death. Macroscopic and microscopic lesions were compatible with avian cholera and the bacterium Pasteurella multocida subsp. gallicida, serotype A1 was isolated from lung, heart, liver, pericardial sac, and air sacs. In addition, Escherichia coli was isolated from pericardial sac and air sacs. This is the first known report of avian cholera in a southern giant petrel in Antarctica.Facultad de Ciencias Veterinaria

Repositories for Taxonomic Data: Where We Are and What is Missing

AbstractNatural history collections are leading successful large-scale projects of specimen digitization (images, metadata, DNA barcodes), thereby transforming taxonomy into a big data science. Yet, little effort has been directed towards safeguarding and subsequently mobilizing the considerable amount of original data generated during the process of naming 15,000–20,000 species every year. From the perspective of alpha-taxonomists, we provide a review of the properties and diversity of taxonomic data, assess their volume and use, and establish criteria for optimizing data repositories. We surveyed 4113 alpha-taxonomic studies in representative journals for 2002, 2010, and 2018, and found an increasing yet comparatively limited use of molecular data in species diagnosis and description. In 2018, of the 2661 papers published in specialized taxonomic journals, molecular data were widely used in mycology (94%), regularly in vertebrates (53%), but rarely in botany (15%) and entomology (10%). Images play an important role in taxonomic research on all taxa, with photographs used in &amp;gt;80% and drawings in 58% of the surveyed papers. The use of omics (high-throughput) approaches or 3D documentation is still rare. Improved archiving strategies for metabarcoding consensus reads, genome and transcriptome assemblies, and chemical and metabolomic data could help to mobilize the wealth of high-throughput data for alpha-taxonomy. Because long-term—ideally perpetual—data storage is of particular importance for taxonomy, energy footprint reduction via less storage-demanding formats is a priority if their information content suffices for the purpose of taxonomic studies. Whereas taxonomic assignments are quasifacts for most biological disciplines, they remain hypotheses pertaining to evolutionary relatedness of individuals for alpha-taxonomy. For this reason, an improved reuse of taxonomic data, including machine-learning-based species identification and delimitation pipelines, requires a cyberspecimen approach—linking data via unique specimen identifiers, and thereby making them findable, accessible, interoperable, and reusable for taxonomic research. This poses both qualitative challenges to adapt the existing infrastructure of data centers to a specimen-centered concept and quantitative challenges to host and connect an estimated $\le$2 million images produced per year by alpha-taxonomic studies, plus many millions of images from digitization campaigns. Of the 30,000–40,000 taxonomists globally, many are thought to be nonprofessionals, and capturing the data for online storage and reuse therefore requires low-complexity submission workflows and cost-free repository use. Expert taxonomists are the main stakeholders able to identify and formalize the needs of the discipline; their expertise is needed to implement the envisioned virtual collections of cyberspecimens. [Big data; cyberspecimen; new species; omics; repositories; specimen identifier; taxonomy; taxonomic data.]</jats:p

Defining Anuran Malformations in the Context of a Developmental Problem

This paper summarizes terminology and general concepts involved in animal development for the purpose of providing background for the study and understanding of frog malformations. The results of our radiographic investigation of rear limb malformations in Rana pipiens provide evidence that frog malformations are the product of early developmental errors. Although bacteria, parasites and viruses were identified in these metamorphosed frogs, the relevant window to look for the teratogenic affect of these agents is in the early tadpole stage during limb development. As a result, our microbiological findings must be regarded as inconclusive relative to determining their contribution to malformations because we conducted our examinations on metamorphosed frogs not tadpoles. Future studies need to look at teratogenic agents (chemical, microbial, physical or mechanical) that are present in the embryo, tadpole, and their environments at stages of development that are relevant for the malformation type. The impact of these teratogenic agents then needs to be assessed in appropriate animal models using studies that are designed to mimic field conditions. The results of these laboratory tests should then be analyzed in such a way that will allow comparison with the findings in the wild-caught tadpoles and frogs

Effects of coastal urbanization on salt-marsh faunal assemblages in the northern Gulf of Mexico

Author Posting. © American Fisheries Society, 2014. This article is posted here by permission of American Fisheries Society for personal use, not for redistribution. The definitive version was published in Marine and Coastal Fisheries: Dynamics, Management, and Ecosystem Science 6 (2014): 89-107, doi:10.1080/19425120.2014.893467.Coastal landscapes in the northern Gulf of Mexico, specifically the Mississippi coast, have undergone rapid urbanization that may impact the suitability of salt-marsh ecosystems for maintaining and regulating estuarine faunal communities. We used a landscape ecology approach to quantify the composition and configuration of salt-marsh habitats and developed surfaces at multiple spatial scales surrounding three small, first-order salt-marsh tidal creeks arrayed along a gradient of urbanization in two river-dominated estuaries. From May 3 to June 4, 2010, nekton and macroinfauna were collected weekly at all six sites. Due to the greater abundance of grass shrimp Palaemonetes spp., brown shrimp Farfantepenaeus aztecus, blue crab Callinectes sapidus, Gulf Menhaden Brevoortia patronus, and Spot Leiostomus xanthurus, tidal creeks in intact natural (IN) salt-marsh landscapes supported a nekton assemblage that was significantly different from those in partially urbanized (PU) or completely urbanized (CU) salt-marsh landscapes. However, PU landscapes still supported an abundant nekton assemblage. In addition, the results illustrated a linkage between life history traits and landscape characteristics. Resident and transient nekton species that have specific habitat requirements are more likely to be impacted in urbanized landscapes than more mobile species that are able to exploit multiple habitats. Patterns were less clear for macroinfaunal assemblages, although they were comparatively less abundant in CU salt-marsh landscapes than in either IN or PU landscapes. The low abundance or absence of several macroinfaunal taxa in CU landscapes may be viewed as an additional indicator of poor habitat quality for nekton. The observed patterns also suggested that benthic sediments in the CU salt-marsh landscapes were altered in comparison with IN or PU landscapes. The amount of developed shoreline and various metrics related to salt marsh fragmentation were important drivers of observed patterns in nekton and macroinfaunal assemblages